genetic, physiological and environmental regulation …

The Pennsylvania State University

The Graduate School

Intercollege Program in Plant Physiology

GENETIC, PHYSIOLOGICAL AND ENVIRONMENTAL

REGULATION OF ROOT PLAGIOGRAVITROPISM

A Thesis in

Plant Physiology

by

Paramita Basu

2006 Paramita Basu

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

August 2006

The thesis of Paramita Basu was reviewed and approved* by the following:

Kathleen M. Brown Professor of Post-Harvest Physiology Thesis Advisor and Chair of Committee

Jonathan P. Lynch Professor of Plant Nutrition

Simon Gilroy Associate Professor of Biology

Paula McSteen Assistant Professor Biology

Teh-hui Kao Professor of Biochemistry and Molecular Biology Program Chair of the Intercollege Program in Plant Physiology

*Signatures are on file in the Graduate School

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ABSTRACT

Basal roots together with the primary root forms the scaffolding of the root

system architecture of common bean (Phaseolus vulgaris L.) which responds to gravity

in concert with various environmental cues like phosphorus and hormonal signals such as

ethylene and auxin. Basal roots are a type of secondary roots resembling adventitious

roots, and they arise from tissue with shoot anatomy. They appear in tetrarch pattern like

adventitious and lateral roots. The basal roots emerge from two-three distinct whorls

from a one-centimeter region at the root-shoot interface and exhibit plagiogravitropic

growth which changes over time. Gravitropic growth of roots determines the three-

dimensional root architecture which is essential for efficient acquisition of soil resources.

The growth angle of basal roots is a primary determinant of the roots which impacts

efficient acquisition of limited and immobile nutrients like phosphorus. Genotypes of

common bean vary substantially in the growth angle of basal roots and by altering growth

angles the plants are better adapted to low phosphorus availability. Shallow basal roots

not only aid in topsoil exploration but also reduce intra and interplant competition for

phosphorus.

Since ethylene has been implicated in both gravitropic and edaphic stress, we

studied the role of ethylene and its interaction with phosphorus availability in regulating

growth angles of basal roots. We measured endogenous ethylene production from the

basal roots and also analyzed the response of basal roots to exogenous application of

ethylene in terms of growth angle and root growth. In addition, we developed a new

image analysis program ‘KineRoot’ to study the spatio-temporal patterns of

plagiogravitropic growth of basal roots in response to ethylene and phosphorus

treatments in a reliable semi-automated way, while minimizing user intervention. The

new software allows us to measure the local patterns of basal root growth and

graviresponding zones of basal roots and how these zones are affected by ethylene and

phosphorus availability. Moreover, the software enables us to measure the root diameter

and root midline which was used in calculating root curvature. Since from the available

literature we already know that auxin and ethylene are potential candidates regulating

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graviresponse of roots, we studied the possible cross-talk between auxin and ethylene in

modulating graviresponse of basal roots. To test the hypothesis that ethylene modulates

auxin effect on root growth and plagiogravitropic curvature of basal roots, we employed

both parental genotypes and recombinant inbred lines of common bean with contrasting

basal roots traits for this study. Response of basal root angle and root growth to different

doses of auxin was measured. In addition, we examine the effect of application of

ethylene action inhibitor 1-methylcyclopropene (MCP) and ethylene synthesis inhibitor

aminovinylglycine (AVG) on growth angle and root growth in the presence of

phosphorus. Free Indole-3-acetic acid (IAA) content in the basal roots was analyzed and

in a separate experiment the basal roots were treated with tritiated IAA to determine the

transport of 3H-IAA in the basal roots of different whorls.

Our work shows that position of origin i.e. whorl has more influence on growth

angle of basal roots than previously reported effects of genotype and phosphorus

availability. Genotypes of common bean vary in basal root number in each whorl. The

diversity in root architecture is generated partly by variation in basal root number as well

as variation in growth angles of basal roots. Although endogenous ethylene production

from the basal roots did not explain variation in growth angles, tissue sensitivity to

exogenous ethylene application appears to be more important in determining the growth

angle. Our results show that there is a strong correlation between ethylene sensitivity and

growth angle which supports our hypothesis that growth of basal roots may be partially

regulated by ethylene and the difference in ethylene sensitivity might explain variation in

growth angle with whorl, genotype and phosphorus availability. Basal root growth was

also affected by ethylene treatment; however, higher sensitivity for root elongation was

not found consistently in all treatments. Our results indicate that ethylene may be a

modifier of root responses to nutrient availability and ethylene perception may be a

central aspect of root response to low phosphorus availability.

The kinematic analysis shows that the basal roots accelerate growth rate of the

upper whorls at the cost of lower growth rate in lower whorls in response to low

phosphorus. Moreover, study of spatio-temporal patterns of differential growth ratio of

the growing root allows identification and measurement of root bending zones and

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bending amount. We examine the effects of ethylene and MCP on root curvature and

observe that both of these treatments do not alter local root curvature, but alters the span

and duration of the bending of the root upward or downward and thereby produce

shallow and deep roots respectively.

Our study about the possible interaction of auxin and ethylene supports the

hypothesis that the effect of interaction between auxin and ethylene on regulation of

growth angles is dependent on phosphorus availability. Free IAA analysis in the basal

roots show that lower whorls of basal roots have higher free auxin and are more sensitive

to auxin inhibition of basal root growth compared to upper whorls. However,

radiolabeled IAA treatment to the root-shoot junction just above the basal rooting zone

shows more radiolabeled IAA transported to upper whorls than lower whorls. In addition

while application of AVG or MCP together with IAA increases root growth and reduces

shallowness in phosphorus sufficient conditions, AVG or MCP do not reverse IAA-

inhibition of growth in low phosphorus. These results point to a phosphorus dependent

interaction between ethylene and auxin in regulation of root elongation, but a

phosphorus-independent interaction for control of growth angle.

vi

TABLE OF CONTENTS

LIST OF FIGURES ..................................................................................................... ..x

LIST OF TABLES....................................................................................................... ..xii

ACKNOWLEDGEMENTS......................................................................................... xiii

CHAPTER 1. INTRODUCTION……………………………………………. 1

Gravitropic response of roots……………………………………………………1

Role of auxin in gravitropism…………………………………………………...3

Ethylene as a regulator of root gravitropism…………………………………….6

Role of other hormones in regulating gravitropism……………………………..8

Common bean selected as a model for studying root architecture……………....9

Phosphorus availability and root architecture of common bean………………..10

OVERVIEW OF RESEARCH PROJECTS………………………………………….11

REFERENCES……………………………………………………………………….14

CHAPTER 2. GENETIC, POSITIONAL, AND NUTRITIONAL

REGULATION OF ROOT PLAGIOGRAVITROPISM

MODULATED BY ETHYLENE…………………………………….24

Abstract………………………………………………………………………....25

Introduction……………………………………………………………………..26

Methods………………………………………………………………………....27

Results…………………………………………………………………………..31

1. Morphology of basal root production………………………………....31

2. Basal root angle depends on genotype and position of origin………...31

3. Range of basal root growth angles………………………………….....32

4. Effect of genotype, phosphorus and position of origin

on ethylene …………………………………………………………....33

5. Basal root growth depends only on root position of origin…………...33

6. Ethylene treatment alters basal root growth angles,

their range and root growth…………………………………………....34

Discussion……………………………………………………………………....35

vii

Acknowledgement……………………………………………………………..39

References……………………………………………………………………...40

Chapter 2 Appendix…………………………………………………………....55

CHAPTER 3. KINEMATIC ANALYSIS OF ROOT GROWTH AND

GRAVITROPISM USING SEMI AUTOMATED

IMAGE ANALYSIS………………………………………………….57

Abstract………………………………………………………………………....58

Introduction…………………………………………………………………….60

Methods………………………………………………………………………...64

1. Experimental method………………………………………………...64

Results………………………………………………………………………….66

1. Image analysis………………………………………………………..66

2. Measurements………………………………………………………..74

3. Example measurements……………………………………………...76

Discussion……………………………………………………………………...78

Acknowledgement……………………………………………………………...81

References……………………………………………………………………...82

CHAPTER 4. GROWTH AND CURVATURE OF BASAL ROOTS ANALYZED

USING KINEMATIC APPROACH………………………………...103

Abstract………………………………………………………………………..104

Introduction……………………………………………………………………105

Materials and methods…..…………………………………………………….108

1. Plant culture………..………………………………………………..108

2. Treatment with ethylene and inhibitors of ethylene action………….109

3. Imaging procedure…………………………………………………...109

4. Measurements………………………………………………………..110

Results………………………………………………………………………….112

1. Time history of root growth rate….………………………………….112

2. Root growth velocity…………………………………………………112

3. Relative elongation rate………………………………………………113

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4. Root curvature and differential growth……………………………….114

Discussion……………………………………………………………………....117

Acknowledgement……………………………………………………………....121

References……………………………………………………………………....122

Chapter 4 Appendix………………………………………………………….....134

CHAPTER 5. HORMONAL REGULATION OF GRAVITROPIC GROWTH OF

BASAL ROOTS- A CROSS-TALK BETWEEN ETHYLENE AND

AUXIN……………………………………………………………….135

Abstract………………………………………………………………………....136

Introduction……………………………………………………………………..137

Methods…..……………………………………………………………………..141

1. Plant material………..………………………………………………...141

2. Comparison of growth angle of genotypes……………………………141

3. Treatment with auxin and NPA……………………………………….142

4. Measurement of ethylene production ………………………………...143

5. Treatment with ethylene inhibitors……………………………………143

6. Quantification of endogenous auxin…………………………………..144

7. Auxin transport analysis………………………………………………145

8. Statistical analysis…………………………………………………….145

Results…………………………………………………………………………..145

1. Basal root angle depends on genotype and position of origin…..…….145

2. Treatment with auxin alters basal root growth angle

and root growth………………………………………………………..146

3. Ethylene production from auxin treated seedlings…………………….147

4. Effect of NPA on growth angle and growth of basal roots…………....147

5. Influence of ethylene inhibitors on BRGA and basal root growth…....148

6. Free IAA concentrations are increased by ethylene…………………..148

7. Basal root growth rate vs. free IAA content…………………………..149

8. 3H IAA transport………………………………………………………150

Discussion…………………………………………………………………….....150

ix

References……………………………………………………………………......156

Chapter 5 Appendix……………………………………………………………...179

CHAPTER 6. SUMMARY OF THE WORK…………………………………………185

References………………………………………………………………………190

x

LIST OF FIGURES

1.1 A macroscopic view of 2-d old germinated bean seedling…………………………..23

2.1 Effect of genotype and position of origin on basal root angle……………………….44

2.2 Endogenous ethylene production…………………………………………………….45

2.3 Growth rate of basal roots……………………………………………………………46

2.4 Effect of MCP and 0.6 ul L-1 ethylene on basal root angle…………………………..47

2.5 Ethylene sensitivity of basal root angles……………………………………………..48

2.6 Ethylene sensitivity of basal root growth angle as a function of genotype, whorl…..49

2.7 Correlation between ethylene sensitivity and growth angle…………………………50

2.8 Ethylene sensitivity of growth response of basal roots……………………………....51

2.9 Effect of exogenous ethylene on the range of growth angles………………………..52

3.1 Photo showing the root system………………………………………………………86

3.2 Photo showing sprinkling of graphite particles……………………………………...87

3.3 Photo of the experimental set up……………………………………………………..88

3.4 Screenshot of the graphical user interface of software ‘KineRoot’………………….89

3.5 Schematic showing pattern matching algorithm……………………………………..90

3.6 Schematic showing the weights for calculating color-weighted correlation…….…..91

3.7 Steps of automatic edge detection…………………………………………………...92

3.8 Schematic showing projection of tracked points on root centerline…………………93

3.9 Schematic illustrating the calculation of root growth velocity………………………94

3.10 Schematic showing the growth of a small segment of the root…………………….95

3.11 Montage of 8 images of a basal root………………………………………………..96

3.12 Root length map…………………………………………………………………….97

3.13 Root growth velocity and mean relative elongation rate…………………………...98

3.14 Colored isocontour of rate of relative elongation…………………………………..99

3.15 Mean root diameter………………………………………………………………..100

3.16 Mean root curvature and differential growth ratio………………………………...101

3.17 Two images of the root……………………………………………………………102

4.1 Time course of basal root growth rate………………………………………...……126

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4.2 Spatial profiles of growth velocity of basal roots…………………………………..127

4.3 Spatial profiles of relative elongation versus distance from root tip……………….128

4.4 Color isocontour plot of relative elongation rate of basal roots……………………129

4.5 Superimposed time lapse photos of a growing basal root………………………….130

4.6 Examples of spatio-temporal color isocontour plot of differential growth ratio…...131

4.7 Spatio-temporal comparison of bending of basal roots…………………………….132

5.1 Effect of genotype and position of origin on basal root angle……………………...162

5.2 Auxin sensitivity of growth angles and growth rate of basal roots…………………163

5.3 Auxin sensitivity of growth angle of basal roots………………………………...…164

5.4 Correlation between auxin sensitivity and growth angle of basal roots……………165

5.5 Auxin sensitivity of growth response of basal roots…………………………….….166

5.6 Endogenous ethylene production…………………………………………………...167

5.7 Combined effect of AVG and IAA on growth angle……………………………….168

5.8 Combined effect of AVG and IAA on the basal root growth………………………169

5.9 Combined effect of MCP and IAA on growth angle……………………………….170

5.10 Combined effect of MCP and IAA on the basal root growth………………..……171

5.11 Free IAA in common bean basal roots……………………………………………172

5.12 Free IAA in common bean basal roots of seedlings of a shallow genotype………173

5.13 Basal root growth rate vs. free IAA content………………………………………174

5.14 Auxin transport activity in roots……………………………………………….….175

5.15 Anatomical sections of basal root emergence zone……………….………………176

xii

LIST OF TABLES

2.1 Average number of basal roots per whorl…………………………………………....53

2.2 Range of growth angles of basal roots per plant……………………………………..53

2.3 ANOVA of growth angle and growth response of basal roots………………………54

4.1 Periodicity of the wavy motion of bean basal roots………………………………...133

5.1 ANOVA of growth angle and growth response of basal roots……………………..177

5.2 Effect of NPA treatment on the basal root growth angle and growth rate………….178

xiii

ACKNOWLEDGEMENTS

I would like to first acknowledge Dr. Kathleen Brown, my advisor, for guiding

me during my Ph.D. I am grateful to her for taking me as a student and supporting me for

the last four years. I have been fortunate to have a wide range of responsibilities as a

graduate student in the lab, while maintaining freedom of my own work. I express my

deepest gratitude to my committee members, Dr. Jonathan Lynch, Dr. Simon Gilroy and

Dr. Paula McSteen for their service on my doctoral committee and for their valuable

suggestions and review of my thesis. I am also inspired by Dr. Teh-hui Kao, Plant

Physiology program chair, who offered financial as well as moral support during my stay

in Penn State. Partial financial support for my thesis was also available from US-AID

Bean-Cowpea CRSP.

I feel fortunate to have my husband Dr. Anupam Pal, Mechanical Engineering

Department,. PSU, as my collaborator without whose help I would not have done

significant contribution through my research using my kinematic analysis. I acknowledge

my gratitude to Anupam for giving me moral support in the days of my frustration and

inspiring me always to carry on good research.

I would like to thank Dr. Jurgen Engelberth, Entomology Department, PSU, for

analyzing free auxin content in the basal roots of common bean.

I also acknowledge my lab mates Hye-Ji Kim, Catalina Posada, Amy Burton,

Tom Walk, Melissa Ho, Amelia Henry, Ivan Ochoa, Raul Jaramillo, Magalhaes Miguel,

Soares Xerinda, Soares Jochua and others for their helpful cooperation and genuine

friendship during my Ph.D work. My affection goes to Bob Snyder and Michele Brown,

the lab managers for helping me and for their organizing skills.

Lastly, but most importantly, I am grateful and respectful and indebted to my

parents, whose love and constant support have helped me reaching my goal.

CHAPTER 1: INTRODUCTION

The primary objective of this dissertation research is to explore root architecture in

common bean (Phaseolus vulgaris L.), specifically the roles of low phosphorus

availability and hormones in the plagiogravitropic response of basal roots. Low

phosphorus and ethylene interact to affect aerenchyma formation, growth angle of basal

roots, lateral root formation, and root hair development resulting in efficient phosphorus

acquisition (He et al. 1992; Lynch and Brown 1997; Borch et al. 1999; Fan et al. 2003;

Zhang et al. 2003). Low phosphorus has been demonstrated to alter the growth angle of

basal roots and efficiency of phosphorus acquisition is highly correlated with basal root

shallowness (Bonser et al. 1996; Liao et al. 2001). My research aims to produce a better

understanding of basal root architecture in determining direction of plagiogravitropic

growth in concert with various environmental cues like phosphorus and endogenous

signals like ethylene. This study increases the scope for selection and breeding of crops

with efficient adaptation to low phosphorus availability (Lynch 1998) and with greater

productivity in low-input subsistence agricultural systems.

Gravitropic response of roots

I. Overview

Plant growth and development depends on the capacity to perceive and respond to

an array of environmental stimuli like light, water availability and gravity. Gravitropism

is a process by which an individual plant can sense and respond to gravitational forces.

The response to gravity affects the direction of growth of an individual plant organ by

regulating the rate of differential cellular elongation on opposite sides of the elongation

zones of the stimulated organ. Gravitropic responses of roots affect plant anchorage,

acquisition of belowground resources like water and mineral nutrients and interplant

competition. The dynamic response of individual roots to gravity throughout plant growth

is a major component of root system architecture.

2

Directional growth response of roots to gravity is a well-coordinated process

which has four steps: 1) sensing the direction of gravity by specific gravi-sensing cells, 2)

production of signal in the gravity-sensing cells, 3) transduction of the gravity signal to

the responding tissue and 4) asymmetric elongation of cells between upper and lower

sides of the responding organs which results in bending (Tasaka et al. 1999). Perception

of the gravity stimulus is generally attributed to the root cap (Sack 1997). The gravity-

responding organ generates curvature in the elongation zone which is located in between

apical meristem and the maturation zone of the root (Ishikawa and Evans 1997).

According to Ishikawa and Evans (1995; 1997), the region between the apical meristem

and central elongation zone is referred to as the distal elongation zone (DEZ) and from

their computer-assisted study of maize primary root gravitropism, the authors suggested

that gravitropic curvature is initiated in this zone (DEZ), which is characterized by the

rapid elongation of the upper flank of the bending root. The central elongation zone and

distal elongation zone differ in their mechanism of responses to different environmental

signals (Mullen et al. 1998).

Gravitropism does not necessarily mean vertical upward growth of shoots and

vertical downward growth of roots. Each and every plant organ has distinct and specific

response to gravity, which often results in plagiogravitropic growth i.e. growth at an

angle other than 0o relative to the gravity vector. This stable angle was referred to as

gravitropic set-point angle (GSA) by Firn and Digby (1997). A gravitropic response is

characterized by the formation of a curvature by the stimulated organ and is initiated

when the plant organ deviates its growth vector from the GSA (Firn and Digby 1997).

According to Firn and Digby, the growth of most plant organs occurs at a stable angle

determined by various factors, including gravity itself. GSA is the equilibrium angle at

which there is no gravity-induced differential growth (Firn and Digby 1997).

II. Events associated with root gravitropism

The most widely accepted gravi-sensing model is the starch-statolith hypothesis

according to which statoliths containing starch-filled amyloplasts are the gravi-sensing

organelles. The following steps 1) gravity perception by the root cap cells by

3

reorientation of amyloplasts located within the columella cells of the root cap, 2)

generation of a stronger signal by the sedimentation of statoliths through the cytoplasm,

3) initiation of differential growth in the stimulated organ, finally lead to the curvature of

the gravi-responding organ (Kiss 2000). An alternate hypothesis, the protoplast pressure

hypothesis, evolved from studies with cytoplasmic streaming in internodal cells of

characean algae, has also been implicated in gravity perception in plants. The supporters

of the protoplast pressure theory pose strong arguments against the starch-statolith model

based on the study of gravitropism in starch-deficient mutants (Kiss 2000).

The cytoskeleton, consisting primarily of a network of actin filaments and

microtubules, has been reported to play a major role in root gravitropism, possibly

intercepting amyloplasts and transducing their sedimentation into a graviresponse

(Blancaflor and Masson 2003). There is circumstantial evidence implicating Ca2+ and

calmodulin (CaM) in gravity perception. High levels of Ca2+ and CaM have been

reported to be associated with statoliths (Rosen et al. 1999). Another candidate which is

believed to play a key role in the transduction of the gravity signal is pH (Fasano et al.

2001). Finally, the involvement of auxin is well-established as the most important

intercellular signal in regulating gravitropic response of roots.

Role of auxin in gravitropism

The role of auxin in gravitropism has been explained by the Cholodny and Went

theory, according to which auxin redistribution and altered auxin movement across the

elongation zone of a gravistimulated root results in differential cell elongation on the

opposite flanks of the stimulated organ, resulting in downward curvature, as reviewed in

(Blancaflor and Masson 2003). According to this model, asymmetric lateral auxin

transport is the key regulator of curvature. The fountain model of auxin transport, which

refined the Cholodny-Went theory, depends on the facts that shoot-derived auxin moves

into the roots and root cap is necessary for gravitropism, which redirects auxin into polar

transport streams towards the elongation zone through epidermis or cortex. (Wolverton

2002). Asymmetric distribution of auxin in the root cap of a gravistimulated root results

in asymmetric distribution of auxin in the elongation zone which leads to subsequent

4

bending in response to gravity. Since the optimal concentration of auxin necessary for

root growth is much lower than that for shoot growth (Eliasson et al. 1989), higher auxin

content would be inhibitory to root growth. Therefore, concentrations of auxin that

promote growth of shoots inhibit growth of roots i.e. roots are more sensitive to auxin

than shoots.

Two types of auxin transport within roots have been recognized: A fast and non-

polar transport, coupled with the movement of assimilates (e.g sugar) in the phloem

(Baker 2000; Ljung et al. 2001) and a comparatively slower, directional, polar transport

pathway. These two types of transport could be directly or indirectly linked (Cambridge

and Morris 1996). The polar transport of auxin occurs via two pathways in roots, an

acropetal path from the base of the root to the apex of the root through the stelar tissure,

and a basipetal path from the apex of the root toward the base through the outermost

epidermal and cortical cell layers (Muday 2001). Basipetal auxin movement is required

for graviresponse (Rashotte et al. 2000) presumably because the targets of auxin action

are the outer cell layers exhibiting differential growth. Auxin transport via phloem is

much faster (approximately 1 cm/min or more) than polar transport of auxin (0.5 – 2 cm/

hour). However, the distinction between the role of non-polar and polar transport of auxin

in production of the auxin pool in different tissues has not yet been clearly elucidated.

The cell-to-cell transport of auxin, indole-3-acetic acid (IAA), was postulated to

take place through specific carrier proteins or protein complexes which control the flux of

auxin into and out of the cell. Activity of both efflux and influx carriers can be inhibited

by several synthetic compounds like 1-N-naphthylphthalamic acid (NPA) (Lomax et al.

1995; Bennett et al. 1998; Morris 2000; Muday and DeLong 2001). Moreover, 1-

naphthoxyacetic acid and 3-chloro-4-hydroxyphenylacetic acids are reported to inhibit

auxin influx carrier activity and thereby disrupt root gravitropic response (Parry et al.

2001). Recent molecular genetic studies on Arabidopsis gravitropic mutants have

revealed that auxin-influx (AUX1 in Arabidopsis) and auxin-efflux (PIN gene family in

Arabidopsis) carriers, which are differentially expressed in different tissues and organs,

play significant role in polar auxin transport (Swarup et al. 2001). The auxin-efflux

protein coded by PIN3 permits lateral distribution of auxin within the gravisensing

5

columella cells of the root cap (Friml et al. 2002) which could be very important for

asymmetrical distribution of auxin moving from the acropetal to the basipetal transport

stream. In addition, the auxin influx carrier AUX1 has been shown to participate in lateral

auxin transport within the cells of the columella, lateral root cap, and elongation zone

(Swarup et al. 2001). Recent work has shown that p-glycoproteins mediate cellular and

long-distance auxin transport like the PIN proteins (Giesler and Murphy 2006).

According to the Cholodny-Went hypothesis, auxin redistribution results in the

formation of an auxin gradient, which ultimately drives differential growth resulting in

bending of the stimulated organ. This theory is highly debated. There is considerable

evidence to support the involvement of auxin gradients in differential cell elongation

during root and hypocotyl curvature, e.g. redistribution of radio-labeled IAA in the root

tips of gravistimulated maize roots (Young et al. 1990), the differential expression of

auxin responsive promoter (DR5) on the lower half of gravistimulated roots during root

curvature (Rashotte et al. 2001), and formation of lateral auxin gradients across hypocotyl

and roots of wild type Arabidopsis compared with arg1 mutants (Boonsirichai et al.

2003). On the other hand, it has been claimed that auxin gradients are not present in some

instances of tropic curvature and that these gradients do not take place as rapidly as

required for the regulatory involvement of auxin. According to Ishikawa and Evans

(1993), roots treated with high auxin concentrations to mask the internal gradients of

auxin maintained gravitropic curvature. However, recent work demonstrated auxin

gradients from the columella cells to the lateral root cap and towards the elongation zone,

even in the presence of exogenous auxin (Ottenschlager et al. 2003). Another argument

against the Cholodny-Went model comes from the study of coleoptile segments where

the lateral auxin gradient is believed to be smaller than the growth differential which

results in an equivalent increase in the growth rate of the lower side and decrease in the

growth rate of the upper side of a gravistimulated coleoptile segment (Gutjahr et al.

2005). According to the authors, there might be an additional gradient of responsiveness

to auxin since the response of two halves of a gravistimulated rice coleoptile is different

for the same amount of auxin, with reduced response in the upper flank, and normal

response in the lower flank. There could be an interaction between auxin redistribution

6

and time-dependent change in auxin sensitivity as suggested by Ishikawa et al. (1991)

which could account for some of the complexity of gravitropic response.

Ethylene as a regulator of root gravitropism

The role of ethylene in gravitropism is not yet fully clear but provocative. Earlier

research suggested that ethylene mediates gravitropic responses in roots as well as shoots

(Chadwick and Burg 1967; Wheeler and Salisbury 1980; Philosoph-Hadas et al. 1996;

Kiss et al. 1999; Madlung et al. 1999; Edelmann 2002). Ethylene treatment disrupts root

gravitropic responses in many, but not all species (Abeles et al. 1992). While

investigating the effects of ethylene on response of maize roots to gravistimulation, Lee

et al. (1990) demonstrated that pretreatment of roots with ethylene resulted in an increase

in the latent period of gravitropic response and an extension of the duration of curvature.

Ethylene synthesis inhibitors had the opposite effect, enhancing initial curvature but

inhibiting long-term response (Lee et al. 1990). However, it must be mentioned that Lee

et al., (1990) treated the maize seedlings with extremely high ethylene concentrations, 10

µl/L and 100 µl/L.

The importance of auxin in gravitropism, as well as the close interaction between

ethylene and auxin in various developmental processes including root development, has

already been illustrated by various authors. Extensive studies regarding the physiological

interaction between auxin (IAA) and ethylene have established that at least two kinds of

interactions might exist. A well-established auxin-ethylene interaction is that the

application of exogenous auxin stimulates ethylene production (Rahman et al. 2001), first

observed by Chadwick and Burg (1967) as elevated ethylene production in pea roots

within 15 to 30 minutes after treatment with exogenous auxin. The second potential

interaction is that ethylene inhibits polar and lateral auxin transport (Rahman et al. 2001).

However, this conclusion was initially drawn from the work by (Burg and Burg 1967)

where the seedlings of pea, Avena and maize were treated with a very high amount of

ethylene (1000 µl/L). Work by Suttle (1988) suggested that the reduction in auxin

transport capacity in ethylene treated pea stem tissues could be due to either disruption of

the activity of efflux machinery or decrease in the concentration of the efflux proteins.

7

This work also confirmed that pretreatment of pea seedlings with very low amount of

ethylene reduced auxin transport (inhibition was found at 0.01 µl/L ethylene). Also

ethylene treatment in pea hypocotyl reduced the amount of auxin transport up to 95%,

and the number of NPA binding sites, affecting auxin efflux machinery (Burg and Burg

1967; Ruegger et al. 1997). Therefore, in roots by reducing auxin transport, ethylene

could cause auxin depletion in the root apex, thereby reducing root elongation or

retarding the polar auxin transport stream from the root tip to the elongation zone,

producing insufficient auxin pool in the elongation zone and reducing root elongation, as

reviewed by Casson and Lindsey (2003). In addition, it has been shown in citrus leaves

that ethylene treatment reduces endogenous IAA level by increasing conjugation of IAA

(Riov et al. 1982) and the increased auxin conjugation lowers movement of auxin through

the tissue. Increased IAA catabolism is another mechanism by which ethylene reduces

IAA content (Sagee et al. 1990). On the other hand, Madlung et al. (1999), suggested that

exogenous application of ethylene induces a signal which either stimulates asymmetric

redistribution of auxin or alters auxin sensitivity of the cells of a gravistimulated organ,

thereby regulating graviresponse.

Various reports demonstrate the direct role of ethylene in gravitropism of shoots,

roots, and cut-flower stems (Chadwick and Burg 1967; Wheeler and Salisbury 1980;

Philosoph-Hadas et al. 1996; Kiss et al. 1999; Madlung et al. 1999; Edelmann 2002;

Friedman et al. 2003a). It has been observed that an asymmetric ethylene gradient forms

in the lower half of snapdragon spikes following the asymmetric auxin gradient and prior

to bending of the spike (Philosoph-Hadas et al. 1996; Friedman et al. 2003b). However,

the role of an ethylene gradient across the graviresponding organ in the signal

transduction mechanism leading to the gravitropic response is still controversial

(Madlung et al. 1999; Friedman et al. 2005; Woltering et al. 2005). However, it seems

apparent that even a low concentration of ethylene could be required for regulating

gravitropic response (Harrison and Pickard 1986; Philosoph-Hadas et al. 1996; Madlung

et al. 1999).

8

Role of other hormones in regulating gravitropism

Other hormones which are reported to be involved in gravitropism are cytokinin,

abscisic acid (ABA), brassinosteroids and jasmonate. Cytokinin is considered to regulate

root and shoot gravitropism (Chen et al. 1999; Aloni et al. 2004). According to Aloni et

al., (2004), the root cap regulates the initiation of gravitropic bending by producing an

asymmetric distribution not only of auxin, but of cytokinin (as manifested by cytokinin

promoter- GUS expression) with decreased concentration on the upper side, and

increased concentration on the lower side of a gravistimulated root. Since cytokinin is

known to be a root growth inhibitor, increased concentration on the lower flank would

result in reduced elongation of the lower side, while promotion of elongation occurs on

the upper flank (Aloni et al. 2004).

Abscisic acid is also believed to a potential candidate in regulation of

gravireaction, possibly through a cross-talk between auxin and ethylene (Pilet 1991;

Hansen and Grossmann 2000; Grossmann and Hansen 2001). Asymmetric distribution of

auxin in a gravistimulated root increases ethylene-triggered ABA which results in

inhibition of growth on the lower side leading to downward bending of root (Grossmann

and Hansen 2001).

Jasmonate (JA) is reported to be a regulator of auxin responsiveness involved in

gravitropism (Gutjahr et al. 2005). Gravistimulation of rice coleoptiles leads to the

production of JA gradient between the upper and lower side the coleoptile; however, the

JA gradient appears even when production of the auxin gradient is blocked by NPA.

Gravitropic curvature was reduced if the JA gradient was eliminated by flooding with

exogenous JA and in a JA-deficient rice mutant. The authors conclude that although JA is

not solely required for curvature, its spatial distribution could be influencing the

graviresponse through its effect on auxin signal transduction processes (Gutjahr et al.

2005).

Brassinosteroids (BR), steroidal plant growth hormones, have been observed to

regulate shoot and root gravitropism in some species e.g. bean, tomato, maize, Brassica,

Arabidopsis and pea, (Meudt 1987; Park 1998; Kim et al. 2000; Chang et al. 2004; Li et

al. 2005; Amzallag and Vaisman 2006). BRs accelerate the upward curvature of

9

gravistimulated hypocotyls in bean and tomato (Meudt 1987; Park 1998) and promote

gravitropic curvature in maize primary root (Kim et al. 2000). There have been reports

stating that BR may interact with auxin and ethylene in regulating gravitropic response

(Kim et al. 2000; Chang et al. 2004). Other recent evidence shows that BRs initiate

graviresponse by promoting PIN2 activity and influence both acropetal and basipetal

transport of auxin, while regulating the distribution pattern of endogenous auxin through

modification of expression of PIN genes (Li et al. 2005).

From the available literature it seems that there is network of interactions between

different hormones and auxin in regulating gravitropism. Gravitropic reorientation is

caused by lateral IAA accumulation and there may be a relationship between hormones

like ABA, cytokinin, brassinosteroids and jasmonate and the production of auxin gradient

along the gravistimulated primary root. However, the relationship also depends on the

type of root tissue as different studies have been conducted in different plant species and

different species vary substantially in their behavior and response to auxin gradient.

Common bean selected as a model for studying root architecture

Common bean (Phaseolus vulgaris L.) has been used as a model system for

understanding the role of root architecture for soil resource acquisition. It is the most

important food legume in the world with a global production exceeding 23 million metric

tons, providing protein and important nutrients for over 500 million people in developing

nations (FAO 1991). Soil infertility, especially P deficiency, and drought stress are the

primary constraints to crop production in developing countries, affecting at least 80% of

the area planted to beans (CIAT 1992). Phosphorus, being a non-renewable and diffusion

limited nutrient, is the most important mineral limitation on plant growth and

development. Vast areas of tropical and subtropical countries in Latin America, Africa

and Asia are prone to very limited phosphorus availability (Sanchez and Uehara 1980)

and lack of economic resources in these nations have made use of phosphorus in the form

of conventional fertilizers almost impossible as a cost-effective means for improving

bean yields. Moreover, various natural and man-made processes like fixation of

phosphorus to iron and aluminum oxides, soil weathering and erosion, soil acidification

10

etc. make phosphorus unavailable for crops. Therefore, the most effective means to

improve phosphorus acquisition is selection of efficient genotypes which are tolerant to

phosphorus deficiency. Our work has focused on exploring various root architectural

traits of common bean that may improve phosphorus efficiency of this crop.

Phosphorus availability and root architecture of common bean

Common bean genotypes vary substantially in the acquisition of phosphorus,

thereby differing in their ability to adapt to low phosphorus soil situations (Lynch and

Beebe 1995; Yan et al. 1995a; 1995b). Spatial localization of roots is an important

determinant in acquisition of soil resources, especially in the case of heterogeneous

environments (Drew and Saker 1978; Fitter and Stickland 1991; Snapp et al. 1995). Root

architecture of common bean has been associated with increased yield of crops in

phosphorus stress conditions, with low phosphorus-adapted genotypes capable of

efficient topsoil foraging (Bonser et al. 1996; Liao et al. 2001; Lynch and Brown 2001;

Liao et al. 2004). Phosphorus, being relatively immobile in soil, is depleted from the

rhizosphere by root activity. In that situation the plant must continue to explore new soil

volumes to obtain adequate nutrition. Since phosphorus tends to be found at higher

concentrations in the upper soil layers in native soils (Enwezor and Moore 1966; Keter

and Ahn 1986; Pothuluri et al. 1986), phosphorus-efficient plants with shallower growth

angles can explore those layers and acquire immobile nutrients like phosphorus, as well

as minimize competition among and within root systems (Bonser et al. 1996; Ge 1999;

Liao et al. 2001; Lynch and Brown 2001; Rubio et al. 2001; Rubio et al. 2003). A

shallow root system in common bean is highly advantageous in low phosphorus soil

because it enhances increased phosphorus uptake efficiency and increases plant

productivity (Lynch 1995; Bonser et al. 1996; Liao et al. 2001; Lynch and Brown 2001;

Rubio et al. 2003).

Phosphorus availability is an important factor for regulation of root growth angle.

It has been found that low phosphorus availability can alter growth angle of basal roots

(BRGA), resulting in root architectural plasticity in response to phosphorus availability

(Bonser et al. 1996; Liao et al. 2001). In common bean, some genotypes exhibit

11

shallower or deeper basal roots in response to low phosphorus availability (Bonser et al.

1996; Liao et al. 2001). The effect of phosphorus on gravitropic sensitivity was specific

to phosphorus and could not be mimicked by other mineral deficiencies (Bonser et al.

1996). Besides growth angle, various other root traits like production of adventitious

roots (Miller et al. 2003), lateral root branching (Zhu and Lynch 2004), formation of

aerenchyma (Fan et al. 2003), and density and length of root hairs (Bates and Lynch

1996; Ma et al. 2001) are affected by low phosphorus availability. These traits may lead

to efficient more phosphorus uptake and adaptation to phosphorus-deficient soils (Bonser

et al. 1996; Liao et al. 2001; 2004).

OVERVIEW OF RESEARCH PROJECTS

My research work focuses on the regulation of growth angle of basal roots, which

together with the primary root constitute the root system of common bean. The

development of the common bean root system begins with the taproot, followed closely

by development of basal roots (eight to twelve in number) (Miller et al. 2003) from two

to three whorls (Fig. 1.1) at the root-shoot interface i.e. the region between lower part of

hypocotyls and upper part of primary root (Zobel 1986), adventitious roots emerging

from hypocotyls and lateral roots developing from each of the other root classes. The

taproot is generally the deepest of all these root types, with a GSA of 0°, and the

adventitious roots are the shallowest, growing approximately horizontally. The basal

roots vary in their growth angles, which could be regulated through their gravitropic

response. Just after emergence, the tip of each basal root maintains its own growth

trajectory which can be designated as the gravitropic set point angle (Digby and Firn

1995), thus providing the greatest opportunity for variation in overall root system depth,

and helping in efficient acquisition of resources from soil. Growth angle of basal roots

vary substantially among bean genotypes (Bonser et al. 1996; Liao et al. 2001) and also

within one individual plant.

In common bean, ethylene treatment modifies basal root angle. The ethylene

precursor 1-aminocyclopropane-1-carboxylic acid (ACC) makes basal roots shallower,

while the ethylene inhibitor aminoethoxyvinylglycine (AVG) makes basal roots deeper

12

(Zhang 2002). There is an overall correlation of ethylene production with basal root angle

in several shallow genotypes, but not in deep genotypes. These data suggest that genetic

variation in ethylene production or responsiveness could be related to basal root angle

(Zhang 2002). Ethylene has been shown to affect gravitropism of maize primary roots

(Lee et al. 1990), tomato shoots (Madlung et al. 1999), flower-stalks of snapdragon

(Philosoph-Hadas et al. 1998), and GSA of rye coleoptiles (Edelmann 2002). In addition,

ethylene has been shown to interact with low phosphorus availability in regulating

growth angle of basal roots, aerenchyma formation, root hair formation, maintenance of

primary root elongation, and lateral rooting (He et al. 1992; Lynch and Brown 1997;

Borch et al. 1999; Zhang 2002; Fan et al. 2003; Zhang et al. 2003).

Therefore, this thesis research aims to study regulation of growth angle of basal

roots in common bean from nutritional, hormonal and positional points of view. I

employed common bean genotypes contrasting in growth angle of basal roots and two

different populations of recombinant inbred lines (RILs). These RILs vary substantially

in growth angle of basal roots (BRGA), but share a common genetic background, making

them ideal tools for study of root traits.

In the second chapter, we investigate the basal root graviresponse in detail, with

special reference to the position of root origin, which I have shown to be a major factor

determining basal root shallowness. In addition, experiments were conducted to test the

hypothesis that ethylene is involved in genetic, positional and nutrition-induced variation

of BRGA.

We developed a sophisticated image-analysis program to analyze time-lapse

photographs of basal roots of common bean for kinematic studies. This approach was

used to measure basal root growth in response to low phosphorus and ethylene in space-

time coordinates. In the third chapter, I describe the algorithm of the image-analysis

program, and in the fourth chapter, I present results from the kinematic analysis. Study of

root kinematics has been employed previously by other researchers in investigating root

growth (Fraser et al. 1990; Liang et al. 1997; Ma et al. 2003; van der Weele et al. 2003)

but not plagiogravitropic growth or curvature.

13

In the fifth chapter, we investigate possible cross-talk between ethylene and auxin

in influencing growth angle of basal roots. Auxin sensitivity of plagiogravitropic roots

has not been examined. It is possible that, like lateral roots (Muday and Haworth 1994),

basal roots contain less auxin, therefore auxin concentrations that would inhibit primary

root elongation would enhance basal root growth. Therefore, we focus on exploring the

genotypic difference in the sensitivity of basal roots to exogenous auxin application.

The work presented in this thesis will hopefully aid in understanding basal root

architectural traits of common bean which will be beneficial for improving their

adaptation to limited resource environments. Basal roots together with the primary root

forms the scaffolding of the entire root system architecture of common bean. Therefore,

study of basal root architecture and growth will improve our knowledge that can be

applied in the development of bean genotypes that are productive even under low fertility

soil prevalent in developing nations.

14

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Yan X, Beebe SE, Lynch JP (1995a) Genetic variation for phosphorus efficiency of

common bean in contrasting soil types: II. Yield response. Crop Science 35,

1094-1099.

Yan X, Lynch JP, Beebe SE (1995b) Genetic variation for phosphorus efficiency of

common bean in contrasting soil types: I. Vegetative response. Crop Science 35,

1086-1093.

Young L, Evans M, Hertel R (1990) Correlations between gravitropic curvature and

auxin movement across gravistimulated roots of Zea mays. Plant Physiology 92,

792-796.

Zhang YJ (2002) 'Ethylene and phosphorus responses in plants.' PhD thesis,

(Pennsylvania state University, PA).

Zhang YJ, Lynch JP, Brown KM (2003) Ethylene and phosphorus availability have

interacting yet distinct effects on root hair development. Journal of Experimental

Botany 54, 2351-2361.

Zhu JM, Lynch JP (2004) The contribution of lateral rooting to phosphorus acquisition

efficiency in maize (Zea mays) seedlings. Functional Plant Biology 31, 949-958.

Zobel RW (1986) Rhizogenetics (root genetics) of vegetable crops. Hortscience 21, 956-

959.

23

Figure 1.1 A macroscopic view of a 2-d old germinated bean seedling showing basal roots emerging from the 3 distinct whorls. Whorls are designated as 1, 2, and 3 from theshoot-side to primary root-side.

CHAPTER 2: GENETIC, POSITIONAL, AND NUTRITIONAL REGULATION

OF ROOT PLAGIOTROPISM MODULATED BY ETHYLENE

Paramita Basu1, Yuan-Ji Zhang2, Jonathan P. Lynch1, 2 and Kathleen M. Brown1, 2

1Intercollege Program in Plant Physiology, The Pennsylvania State University Park, PA

16802, USA

2Department of Horticulture, The Pennsylvania State University Park, PA 16802, USA

25

ABSTRACT

Plagiogravitropic growth of roots strongly affects root architecture and topsoil

exploration, which is important for the acquisition of depth-dependent soil resources such

as phosphorus. Here we show that basal roots of Phaseolus vulgaris L. develop from 2-3

definable whorls at the root-shoot interface and exhibit position-dependent

plagiogravitropic growth. The whorl closest to the shoot produces the shallowest roots,

while lower whorls produce deeper roots. Genotypes vary in both the average growth

angles of roots within whorls and the range of growth angles, i.e. the difference between

the shallowest and deepest basal roots within a root system. Since ethylene has been

implicated in both gravitropic and edaphic stress responses, we studied the role of

ethylene and its interaction with phosphorus availability in regulating growth angles of

genotypes with shallow or deep basal roots. There is only a small correlation between

growth angle and ethylene production in the basal rooting zone, but ethylene sensitivity is

strongly correlated with growth angle. Basal roots emerging from the uppermost whorl

are more responsive to ethylene treatment, displaying shallower angles and inhibition of

growth. Ethylene sensitivity is greater for shallow than for deep genotypes and for plants

grown with low phosphorus compared to those supplied with high phosphorus. Ethylene

exposure increases the range of angles, though deep genotypes grown in low phosphorus

are less affected. Our results show that ethylene mediates regulation of growth angle by

position of origin, genotype, and phosphorus availability.

Key words: basal roots, ethylene, gravitropism, Phaseolus vulgaris, phosphorus, root

architecture

26

INTRODUCTION

A simplified system for studying gravity responses, gravistimulation of

orthogravitropic organs, has led to enormous advances in understanding the mechanisms

of gravity sensing and response in plants. However, few plant parts are actually

orthogravitropic, but instead grow at some other angle with respect to gravity (i.e. they

are plagiogravitropic). According to Firn & Digby (1997), every organ has a “gravitropic

set point angle” (GSA), a somewhat stable angle of growth that is controlled by

developmental and environmental factors, including gravity itself. Plagiogravitropism

and the regulation of the GSA are poorly understood, but these phenomena characterize

the most graviresponsive organs and have important ecological and agricultural

implications.

One important consequence of root plagiogravitropism is its influence on root

architecture and soil resource acquisition. For example, phosphorus, a relatively

immobile nutrient, is heterogeneously distributed in most soils, with greatest availability

in upper soil layers and decreasing availability with depth (Pothuluri et al. 1986). The

seedling roots of bean (basal roots) establish characteristic growth angles very early. The

initial growth trajectory determines the vertical distribution of root length in the soil,

including not only the basal root axes, but also the lateral roots that develop later (Liao et

al. 2001). Overall root system depth determines the efficiency of exploration for shallow

resources such as phosphorus (Lynch and Brown 2001) and deep resources such as water

(Ho et al. 2004; Ho et al. 2005). Comparative analysis of contrasting genotypes indicates

that shallowness of seedling roots is closely correlated with phosphorus efficiency in

bean and maize (Bonser et al. 1996; Liao et al. 2001; Liao et al. 2004; Zhu et al. 2005).

In this paper, we examine regulation of basal root angle in common bean

(Phaseolus vulgaris L.). Its root system consists of a primary root, a variable number

(eight to twelve) of basal roots (Miller et al. 2003) originating from the root-shoot

interface i.e. the region between lower part of hypocotyl and upper part of primary root

(Zobel 1986), adventitious roots emerging from the subterranean hypocotyl, and lateral

roots developing from each of the other root classes. The taproot reaches a length of 2-3

cm two days after seed imbibition, the basal roots emerge three days after imbibition, and

27

the adventitious roots develop after about twelve days. Basal roots, together with the

primary root, constitute the major scaffolding of the root system, since these root types

appear earliest. Growth angles of basal roots (BRGA) vary with genotype and result in

variation in distribution of total root length with depth (Bonser et al. 1996; Liao et al.

2001). In some genotypes, basal roots grow shallower with decreased phosphorus

availability (Bonser et al. 1996; Liao et al. 2001), indicating genetic variation for both

growth angle and for its plasticity in response to phosphorus availability.

Lynch and Brown (2001) mentions that ethylene, a plant hormone often

associated with stress responses, is likely to be important for basal root gravitropic

responses to low phosphorus availability. Ethylene is intimately involved with auxin in

the control of differential growth responses (Harper et al. 2000) and is known to

modulate gravitropic responses in roots and shoots (Abeles et al. 1992; Philosoph-Hadas

et al. 1996; Madlung et al. 1999; Edelmann 2002; Edelmann et al. 2002). Low

phosphorus availability increases ethylene production by roots of bean and tomato plants

(Borch et al. 1999; Lynch and Brown 2001). Preliminary evidence from our lab indicates

that ethylene treatment makes common bean basal roots shallower, while ethylene

inhibitors make them deeper (Zhang 2002).

We hypothesize that ethylene might be involved in genetic, positional and

nutrition-induced variation of growth angle of basal roots. To investigate this hypothesis,

we used common bean genotypes contrasting in BRGA and recombinant inbred lines

(RILs) generated from two different populations demonstrating contrasting root

architecture. These genotypes vary in BRGA yet share a common genetic background,

allowing us to evaluate the involvement of ethylene in regulating the growth angle of

basal roots.

METHODS

Common bean (Phaseolus vulgaris L.) genotypes G19833 and DOR364, with

contrasting responses to low phosphorus availability were used to generate a population

of F12 RILs (obtained from CIAT, Cali, Colombia). G19833 is a large, black-seeded

genotype from the Andean gene pool and has an indeterminate bush growth habit (Yan et

28

al. 1995a) while DOR364 is of Mesoamerican origin (Singh et al. 1991) and has an

indeterminate bush habit (Type II), erect stems and small seeds (Singh 1982). G19833 is

better adapted to phosphorus-limited conditions and has a shallower root system than

DOR364 (Lynch 1995; Bonser et al. 1996; Beebe et al. 1997; Liao et al. 2001). The two

parental genotypes and six RILs were selected for these experiments based on their

growth angle. RILs were selected that had shallow or deep basal roots, according to a

screening under low phosphorus availability (Liao et al. 2004). We also used the “L88”

RILs developed by Dr. Jim Kelly (Michigan State University) from a cross of B98311

and TLP19. B98311 is drought resistant Mesoamerican genotype from the MSU breeding

program and possesses a Type II growth habit and a deep vigorous primary root (Frahm

et al. 2004) and TLP19 was developed for tolerance to low phosphorus at the

International Center for Tropical Agriculture (CIAT, Cali, Colombia) and also possesses

a Type II growth habit. The RILs descending from the cross between these two parents

share a common genetic background, yet segregate for root architectural traits as well as

adaptation to abiotic stress.

Seeds were surface sterilized with 6% sodium hypochlorite for 5 min, rinsed

thoroughly with distilled water and scarified with a razor blade. Seeds were germinated at

28°C in darkness for 2 d in rolled germination paper (25.5 x 37.5 cm Anchor Paper Co.,

St. Paul, MN, USA) moistened with either low or high phosphorus nutrient solution,

which was composed of (in µM) 3000 KNO3, 2000 Ca(NO3)2, 250 MgSO4, 25 KCl, 12.5

H3BO3, 1 MnSO4, 1 ZnSO4, 0.25 CuSO4, 0.25 (NH4)6Mo7O24, and 25 Fe-Na-EDTA. For

high phosphorus solutions, 1000 µM NH4H2PO4 was added; for low phosphorus, 500 µM

(NH4)2SO4 was added. Germinated seeds with radicals approximately 2-3 cm long were

transferred to growth pouches consisting of a sheet of 30 x 24 cm blue germination paper

(Anchor Paper Co., St. Paul, MN, USA) inserted into a polyethylene bag of the same size

with evenly spaced (3 cm apart) holes for aeration. Pouches were open at the bottom to

allow direct contact with the nutrient solution containing high (1 mM) or low (0 mM)

phosphorus as described above. The pouches were stiffened by attaching perforated

plexiglass sheets to stabilize the root system. The pouches were suspended in nutrient

solution and covered with aluminum foil to prevent illumination of the roots. Root

29

systems were photographed after 2 d growth in pouches and basal root angles were

determined using Matlab 7.0TM (Mathworks Inc., Natick, MA, USA). Growth angles of

basal roots were measured relative to the vertical, i.e. larger angles indicate shallower

basal roots. The range of growth angles for each plant was calculated by subtracting the

minimum growth angle from the maximum growth angle exhibited by the basal roots of

an individual plant.

For ethylene measurement, fresh tissue bearing basal roots were harvested from 3

d old seedlings. The segments were separated into three basal root whorls with a razor

blade and enclosed individually in 9 ml vials capped with septa at 25°C. Ethylene was

sampled with a 1-cc syringe from the headspace of the vials 2 hr later and quantified by

gas chromatography (HP6890 gas chromatograph equipped with a flame ionization

detector and an activated alumina column, Hewlett-Packard Company, Wilmington, DE,

USA). In a preliminary experiment, we measured ethylene from the intact tissue (whole

segment of basal rooting zone) compared with tissues divided into three whorls. We

found that dividing the root tissue into separate whorls did not significantly affect

ethylene production when compared with the amount of ethylene measured from intact

tissue of the entire segment of rooting zone (data not shown). In addition, we measured

endogenous ethylene production from the tissue of the root-shoot interface separately

from the basal roots arising from each whorl and found that the average endogenous

ethylene production from the root-shoot interface tissue amounts to 11-12 nL L-1 (S.E. ±

0.3 to ± 0.8), whereas the ethylene production from the basal root of each whorl amounts

to 18 – 26 nL L-1 g -1 fresh weight (S.E. ± 0.6 to ± 1.1). This measurement shows that the

effect of tissue of the root-shoot junction was negligible in producing ethylene compared

to that of the basal roots.

In an initial study of ethylene sensitivity of basal roots, we treated the parent

genotypes, TLP19 and B98311 with ethylene immediately after transfer to growth

pouches containing 0 or 1 mM phosphorus. The seedlings in the pouches were placed in

air-sealed chambers (53 cm x 36 cm x 31 cm) containing air or ethylene (0.6 µL L-1) for 1

d at 25-26°C. Images of the root system were recorded with digital camera after 1 d of

ethylene treatment, and growth angles were measured between the vertical and the line

30

connecting the base of the basal root emergence with the root tip at 24 h. Ethylene

concentrations were monitored by gas chromatography. Moreover, to test the

involvement of ethylene in regulating BRGA, we treated the seedlings with ethylene

action inhibitor MCP (EthylBloc, Floralife Inc., Walterboro, SC, 0.43% 1-

methylcyclopropane). The seedlings were treated with MCP just after transplanting to the

pouch and the seedlings were kept in air–sealed growth chambers, 118 L in volume. MCP

gas was released by adding EthylBloc (4 mg EthylBloc per 0.08 ml buffer per liter air

space) in plastic weigh-boat placed inside the roof of the chamber and buffer added to it

via a syringe inserted through a rubber septum. The seedlings were treated for 24 h and

growth angles were measured from the digital images of the basal roots.

Phosphorus content was measured in tissue bearing basal roots harvested from 3 d

old seedlings of the deep and shallow genotypes of the DOR364 X G19833 RILs. Fresh

tissue containing basal roots was harvested, dried at 60°C and weighed. Dried samples

were ground, ashed at 500°C for 10 h and analyzed for phosphorus content

spectrophotometrically (Murphy and Riley 1962).

To study the effect of ethylene on BRGA, exogenous ethylene was applied to

germinated seedlings immediately after transfer to growth pouches containing low or

high phosphorus. Seedlings in pouches were exposed to air or concentrations of ethylene

ranging from 0.1 to 0.8 µL L-1 for up to 48 h at 25-26°C. Ethylene concentrations were

monitored by gas chromatography. The concentration of ethylene in control chambers

varied from 0.028 µl L-1 to 0.041 µl L-1. Digital images of the roots were taken after 24

and 48 h and the basal root growth angles were measured as the angle between the

vertical and the line connecting the root tip positions at 24 h and 48 h using Matlab 7.0TM

(Mathworks Inc., Natick, MA, USA). Root growth (increase in length between 24 and 48

h) was assessed from the same digital images. The experiments were repeated 3 times

with 2-3 plants per genotype per treatment each time. The slope of the ethylene dose-

response curve was estimated by the slope of the linear regression line fitted to BRGA vs.

ethylene concentration data for each genotype, each whorl position and each phosphorus

treatment, and was defined as the ethylene sensitivity.

31

For assessing the effect of exogenous ethylene on the range of BRGA, we

calculated the growth angles as the angle between the vertical axis and the line

connecting the root tip positions at 0 h and 48 h. From these measurements, the range of

growth angles was calculated by subtracting the minimum growth angle from the

maximum growth angle exhibited by the basal roots of each individual plant.

Ethylene concentration in the rhizosphere around bean plants was measured at the

Pennsylvania State University Horticulture Farm at Rock Springs, PA, USA. Plastic tubes

with screened openings and fitted with septa were placed in the soil within 15 cm of the

base of the bean plants growing in fertilized, irrigated soil (Typic Haplauf). Plant spacing

was 45 cm within rows. Accumulated ethylene in the tubes was collected with syringes at

24 h intervals for 3 d.

Where statistical analyses were appropriate, the data were analyzed by analysis of

variance (ANOVA) for the main effects (phosphorus, ethylene, genotype and whorl of

origin). Both ANOVA and calculations of ethylene response functions were performed

with SPSS (SPSS Graduate Pack, version 12, for Windows, SPSS Inc.).

RESULTS

Morphology of basal root production

Basal roots comprise a major part of the bean root system. These roots emerge

within 3 d of germination from distinct whorls at the root-shoot junction (Fig. 2.1 insert).

We designated the whorls bearing basal roots from top (closest to the shoot) to bottom as

1, 2, 3 successively. Whorl 1 typically bears fewer roots than the lower whorls (Table

2.1). The number of basal roots per whorl varies among genotypes. B98311, TLP19, and

G19833 each typically have three whorls of basal roots, but DOR364 typically has only

two whorls (Table 2.1). There was no significant effect of phosphorus on the number of

basal roots per whorl or the number of whorls.

Basal root angle depends on genotype and position of origin

We examined the growth angles of parents and selected RILs from the L88

population, derived from a cross of the phosphorus-efficient genotype TLP19 with the

32

drought tolerant genotype B98311. As expected, TLP19 has shallower basal roots, while

B98311 has deeper basal roots (Fig. 2.1). RILs 15 and 57 have shallower basal roots

compared to RILs 7 and 76 (Fig 2.1). The growth angle of basal roots of all genotypes

varied with position of origin (Fig. 2.1). Basal roots emerging from whorl 1 are

consistently shallower than those from whorl 3.

The effects of genotype and phosphorus on basal root angle were first tested on

selected RILs derived from the cross of the parent lines G19833 and DOR364 that

exhibited differences in growth angle in preliminary screening under low phosphorus

availability (Zhang 2002; Liao et al. 2004). Basal roots of most genotypes of the G19833

x DOR364 RIL population grew shallower under low phosphorus (data not shown). The

extent of the phosphorus effect (plasticity) varied with genotype, but shallow genotypes

as a group were not significantly more responsive to phosphorus treatment than deep

genotypes (data not shown). Genotype had a much greater effect on BRGA than

phosphorus treatment (F values from ANOVA were 283 and 11.7 respectively). Analysis

of regulation of growth angles of RILs in the G19833 X DOR364 population may be

complicated by the fact that G19833 differ DOR364 in the number of whorls, and the

RIL population shows segregation for this trait. The majority of experiments were

therefore performed using L88 genotypes.

Range of basal root growth angles

Since the difference in growth angles among the basal roots within a root system

varied among genotypes (Fig. 2.1), we calculated the range of growth angles, defined as

the difference between the shallowest and the deepest roots (see Methods). The range of

basal root growth angles varied with genotype, and was smaller for deep genotypes than

for shallow genotypes (Table 2.2). There was no significant effect of phosphorus

treatment on angle range, so the range data for high and low phosphorus treatments were

pooled together.

33

Effect of genotype, phosphorus treatment and position of origin on ethylene

production

To test the hypothesis that higher ethylene production results in shallower basal

root growth, we measured ethylene production rates in basal roots of different genotypes.

Since the growth angle of basal roots may be determined at a very early stage of

development, ethylene production was measured just as the basal roots were emerging

and the roots were 0.6 – 2.6 cm long. In both shallow and deep genotypes, the whorl 1

produced significantly more ethylene than the two lower whorls when ethylene

production was expressed on a fresh weight basis (Fig. 2.2A) or per basal root (Fig.

2.2B). Ethylene production was significantly higher in the uppermost whorl (P <0.001)

when ethylene production was expressed per g fresh weight or per basal root. Ethylene

production per basal root, but not per g fresh weight, was significantly less in deep than

shallow genotypes (P <0.05) and higher with low phosphorus treatment (P <0.001). There

was a weak positive correlation between ethylene production and growth angle of basal

roots (r2 = 0.234, P <0.001), which resulted from the higher ethylene production and

larger angles in whorl 1. Ethylene production was not correlated with genotypic and

phosphorus-related angle differences.

Basal root growth depends only on root position of origin

Roots from lower whorls elongated significantly faster (P <0.001) than those from

the upper whorl, regardless of P treatment and genotype (Fig. 2.3). Root elongation rate

exhibited a weak negative correlation with ethylene production (r2 = 0.123, P <0.001).

There was no significant effect of genotype or phosphorus treatment on root growth rates.

Similar results were obtained when the growth rate of basal roots was assessed between

24 and 48 h (data not shown). G19833, which has three whorls of basal roots, showed

growth patterns very similar to those in Fig. 2.3, with no significant effect of phosphorus

treatment (data not shown). DOR364, which has two whorls of basal roots, had growth

rates similar to whorls 2 and 3 in Fig. 2.3, and low phosphorus reduced growth rates by

about 20%, an effect not observed in L88 genotypes or G19833.

34

Ethylene treatment alters basal root growth angles, their range and root growth

Figure 2.4 shows the effect of MCP and exogenous ethylene (0.6 µL L-1) on the

growth angle of the parent genotypes of L88 population. Ethylene treatment significantly

(P <0.001) increased the shallowness of the genotypes (TLP19, B98311) in all whorls.

On the other hand, MCP made the basal roots significantly (P <0.001) deeper. We found

significant differences between control, MCP and ethylene treatments in regulating

growth angles. There were significant interactions between genotype and position of

origin (P <0.001) as well as between hormone (ethylene and MCP) treatments and

position of origin (P <0.001). However, phosphorus did not significantly affect BRGA in

this experiment (Table 2.4 in appendix).

We observed that neither genotype, phosphorus treatment, nor ethylene treatment

had a significant effect on the internal phosphorus content of tissue bearing basal roots

from 3-d old seedlings (mean P content = 6.15 mg/g dry weight), although seedlings

grown a few days longer in high phosphorus accumulate about 10% more phosphorus

than those grown without phosphorus (Bonser et al. 1996).

For a more detailed examination of the effects of ethylene on BRGA, the

seedlings of three shallow and three deep genotypes from the L88 population were

exposed to different ethylene concentrations to generate dose-response functions. An

example of ethylene dose-responses for the shallow parent (TLP19) grown in low

phosphorus nutrient solution is provided in Fig. 2.5. Ethylene sensitivity was defined as

the slope of the ethylene response function for each genotype, whorl and phosphorus

treatment. Ethylene sensitivity was greater in shallow genotypes compared to deep

genotypes, and the basal roots growing from the upper whorl were more responsive than

the basal roots of lower whorls (Fig. 2.6, Table 2.3). The basal roots were more

responsive to exogenous ethylene treatment when grown with low phosphorus compared

to high phosphorus (Fig. 2.6, Table 2.3). Ethylene sensitivity was well correlated with

growth angle in both low and high phosphorus availability (Fig. 2.7). Most basal roots

grown with low phosphorus were highly responsive to ethylene treatment, while in high

phosphorus, responsiveness increased with shallowness.

35

Growth of most basal roots was significantly reduced by the low concentrations of

ethylene used in this experiment (up to 0.8 µL L-1), but this effect depends on position of

origin (Table 2.3). The ethylene sensitivity of the growth response was calculated as the

slope of the dose-response function (Fig. 2.8). Basal roots from whorl 3 were

considerably less sensitive to ethylene than roots originating from the upper whorls.

There was a small but significant phosphorus x whorl interaction originating primarily

from the greater ethylene sensitivity of low-phosphorus roots from whorl 1 (Fig. 2.8).

Shallow genotypes were somewhat less sensitive to ethylene inhibition of growth than

deep genotypes. The growth rate of the basal roots showed a strong negative correlation

with growth angle (r2 = 0.51, P <0.001 for treatments shown in Fig. 2.8). When ethylene

treatments were excluded, the correlation was 0.30 (P <0.001).

In addition to reducing growth and increasing basal root angle, exogenous

ethylene treatment increased the range of growth angles of shallow genotypes under both

phosphorus treatments (Fig. 2.9). Phosphorus deficiency reduced the range of growth

angles for the deep genotype only at high ethylene concentrations (Fig. 2.9). Ethylene

treatment resulted in a larger increase in range of shallow genotypes compared to deep

genotypes (Fig. 2.9).

DISCUSSION

The angle of growth of basal roots is a primary determinant of the distribution of

roots with soil depth (Bonser et al. 1996; Ge et al. 2000; Liao et al. 2001). In this study,

we show that the position of root origin has more influence on BRGA than the previously

reported effects of genotype and phosphorus availability. Basal roots arise from an

approximately one-centimeter region at the root-shoot interface. They emerge from two

to three distinct whorls in this region, and there is genetic variation for whorl number and

basal root number (Fig. 2.1, Table 2.1). There are typically three to four basal roots per

whorl in the lower whorls and two to three basal roots in the upper whorl. The diversity

in root architecture of common bean is generated partly by the variation in basal root

number as well as by variation in growth angles of basal roots.

36

Neither the basal root number nor the whorl number was affected by phosphorus

availability, which is not surprising since basal roots emerge while seedling growth is still

dependent on cotyledonary reserves. It is possible that maternal nutrition affects these

variables, but this was not tested in this study. The seeds used in these experiments were

produced in fertilized fields.

The position of emergence of basal roots was the major determinant of basal root

shallowness. Within a root system, basal roots grew at increasingly deeper angles from

the upper to the lower whorls (Fig. 2.1). Genotypes varied in both the mean angle of

growth from each whorl and the range of basal root angles within root systems (Fig 1,

Fig. 2.9, Table 2.2). Thus, the distribution of basal roots within the soil volume would be

skewed to shallower or deeper soil layers by larger or smaller growth angles, and the

vertical distribution would be greater in genotypes with a larger range of angles (Table

2.2). A large range of BRGA could be useful in environments when both shallow

resources, such as phosphorus, and deep resources, such as water, are limiting (Ho et al.

2005).

Ethylene production did not explain variation in basal root angles. Ethylene

production was not correlated with BRGA, and there was only a weak negative

correlation between ethylene production and basal root growth rate in the concentration

range employed here. Neither ethylene production nor growth rates were related to

variation in growth angles among genotypes. Earlier experiments on ethylene production

from excised root tips from eight genotypes grown for six days likewise showed no

significant effect of phosphorus or genotype on ethylene production despite large

differences in growth angles (Zhang 2002).

Tissue sensitivity to ethylene appears to be far more important in determining the

BRGA than the amount of ethylene produced by the basal roots. The initial experiment

with exogenous ethylene showed significant effects of ethylene and position of origin on

two parent genotypes (Fig. 2.4). Detailed studies of L88 genotypes showed that ethylene

sensitivity (change in growth angle) was greater with low phosphorus availability, in

genotypes with shallower root systems, and in roots from upper whorls (Figs. 2.6, 2.7,

Table 2.3). Thus, there was a strong correlation between ethylene sensitivity and growth

37

angle (Fig. 2.7), which supports the hypothesis that growth angle may be partially

regulated by ethylene, and that differences in ethylene sensitivity may explain variation

in growth angle with whorl, genotype, and phosphorus availability.

Basal roots from whorls 1 and 2 responded to ethylene by reducing elongation

(Fig 2.8), a well-known root response to ethylene (Abeles et al. 1992). However, the

deepest, fastest growing roots, which emerged from whorl 3, were remarkably insensitive

to ethylene inhibition of growth (Fig 2.8). The high correlation between BRGA and the

root elongation rate suggests that these processes are linked. This link is probably

indirect, since low phosphorus plants did not show consistently higher ethylene

sensitivity for elongation, but did for angle, and the difference in ethylene sensitivity

between shallow and deep genotypes was larger for angle than for growth (Fig. 2.6, 2.8).

The concentration of ethylene in soil varies with biological, physical and

chemical processes like soil moisture, soil organic matter, soil texture, and soil

temperature (Arshad and Frankenberger 2002). Our measurement of ethylene

concentration in the agricultural field soil of Penn State Univeristy, central Pennsylvania

ranges from 45 to 60 nL L-1 in the root zone around bean plants. However, in some soils,

ethylene concentrations of up to 10 µL L-1 have been reported (Abeles et al. 1992) and

stress conditions (e.g. nutrient stress, water logging, flooding) may result in even higher

concentrations (Abeles et al. 1992). Ethylene concentration varies with soil depth

(Campbell and Moreau 1979) with highest levels in the top 10 cm. Soil ethylene could be

important in natural and agricultural ecosystems because even low concentrations in the

root zone could affect plant growth and development. Our ethylene sensitivity experiment

shows that basal roots grow shallower even at very low ethylene concentrations, 100-200

nL L-1, while higher concentrations have a larger effect on growth angle and also reduce

basal root elongation (Figs. 2.6-2.8). Therefore it is likely that ethylene in soil has an

important role in regulating root development, including growth angle.

This report provides evidence that ethylene plays a significant role in regulating

root architectural responses to low phosphorus availability. Both low phosphorus and

ethylene affect root traits likely to affect phosphorus acquisition and utilization, including

aerenchyma formation, basal root growth angle, lateral root density, and root hair

38

development (He et al. 1992; Lynch and Brown 1997; Borch et al. 1999; Fan et al. 2003;

Zhang et al. 2003). In several cases, ethylene and phosphorus interact in a manner that

suggests ethylene mediation of responses to low phosphorus availability. Ethylene action

was required for a subset of low-phosphorus-induced events leading to increased root hair

length and density in Arabidopsis, and ethylene had different effects at high and low

phosphorus availability (Zhang et al. 2003). Likewise, in Arabidopsis primary roots, the

ethylene action inhibitor 1-methylcyclopropene (MCP) increased cell elongation in the

growth zone of plants growth with high phosphorus but reduced it when phosphorus

availability was low (Ma et al. 2003). In common bean, an ethylene synthesis inhibitor

increased main root elongation and reduced lateral root density under high phosphorus

availability, but did the opposite under low phosphorus availability (Borch et al. 1999).

Roots of 5 week-old common bean plants subjected to phosphorus deficiency produced

twice as much ethylene per unit dry weight as roots supplied with adequate phosphorus

(Borch et al. 1999). We suggested that increased ethylene production and altered ethylene

sensitivity could play a significant role in root responses to phosphorus deficiency (Borch

et al. 1999). In the experiments with much younger plants reported here, we did not

observe a significant effect of phosphorus treatment on endogenous ethylene production

in the basal rooting zone, but basal roots of plants grown with low phosphorus

maintained growth rates equivalent to phosphorus-treated plants, and these plants did not

manifest reduced phosphorus content at this early stage. Despite this, plants grown with

low phosphorus availability were more responsive to increasing ethylene by producing

larger growth angle than plants grown with high phosphorus (Fig. 2.6). Thus, ethylene

perception may mediate the regulation of BRGA by phosphorus availability.

BRGA has important implications for resource acquisition. Results from

geometric modeling, growth studies in controlled environments, and field experiments

show that shallow-rooted genotypes are better adapted to low phosphorus availability

than deep-rooted genotypes (Bonser et al. 1996; Liao et al. 2001; Liao et al. 2004; Ho et

al. 2005; Zhu et al. 2005). Shallow basal roots not only increase topsoil exploration, but

produce less intraplant and interplant competition for phosphorus (Ge et al. 2000; Lynch

and Brown 2001; Rubio et al. 2001; Rubio et al. 2003). The results reported here show

39

that genotypic or low phosphorus-induced increases in ethylene sensitivity of basal roots

result in shallower roots. This would be beneficial for phosphorus acquisition by

increasing topsoil exploration and reducing overlap of the phosphorus depletion zones

(Ge et al. 2000; Lynch and Brown 2001). Since ethylene is normally present in soil,

alteration in BRGA would be a typical feature of field performance, with differential

responsiveness based on the genotype and position of origin, i.e. whorl. A second effect

of ethylene response in the field would be greater range of growth angle of basal roots

(Fig. 2.9). Under low phosphorus treatment, and especially in the presence of ethylene,

shallow genotypes produce more dispersed basal roots compared to deep genotypes,

which would facilitate efficient phosphorus acquisition from the topsoil. While basal

roots from the upper whorls would exploit upper soil horizons, basal roots from lower

whorls, which are less responsive to ethylene, would grow progressively deeper and

explore different soil domains. This has important implications for water acquisition,

which can pose a problem for shallow rooted genotypes (Ho et al. 2005). A greater range

of BRGA would increase the depth of soil exploration and therefore the acquisition of

heterogeneously distributed resources, including phosphorus and water (Ho et al. 2004).

Our results indicate that ethylene may be a modifier of root responses to nutrient

availability and that ethylene perception may be a central aspect of the response of basal

roots to low phosphorus availability (Lynch and Brown 1997). In addition, our study

shows that the position of emergence of basal roots plays a key role in determining the

direction of plagiogravitropic growth, and acts in concert with environmental cues such

as phosphorus and endogenous signals such as ethylene. The observed variation in basal

root growth angle within closely related genotypes under phosphorus stress and in

response to ethylene increases the scope for selection and breeding of crops with

improved adaptation to low soil phosphorus availability (Lynch 1998).

ACKNOWLEDGEMENT

The authors gratefully acknowledge support from US-AID Bean-Cowpea CRSP.

40

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Campbell RB, Moreau RA (1979) Ethylene in a compacted field soil and its effect on

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Ge Z, Rubio G, Lynch JP (2000) The importance of root gravitropism for inter-root

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starved or phosphate-starved roots of Zea mays L. during aerenchyma formation.


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Lynch JP (1995) Root architecture and plant productivity. Plant Physiology 109, 7-13.





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Fabaceae). Economic Botany 45, 379-396.

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Zhang YJ, Lynch JP, Brown KM (2003) Ethylene and phosphorus availability have

interacting yet distinct effects on root hair development. Journal of Experimental

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efficiency in maize (Zea mays). Functional Plant Biology 32, 749-762.

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44

Figure 2.1. Effect of genotype and position of origin on basal root angle of common bean. Insert shows a close up view of a young seedling (3 d after imbibition) showing three distinct whorls bearing emerging basal roots. All genotypes are from the L88 population. The growth angle of the basal roots was measured after 2 d growth in pouches. The bars show mean growth angles of basal roots emerging from each whorl of 10-12 plants per genotype, with data pooled over phosphorus treatments, ± SE.

45

Figure 2.2. Endogenous ethylene production per gram fresh weight (pooled over both phosphorus treatments together (A) and per basal root (separately for both phosphorus treatments) (B) by the segments of the root-shoot junction bearing basal roots. Segments were harvested 3 d after imbibition. Values shown are means of 8 plants from each of three shallow and three deep genotypes from the L88 population ± SE.

46

Figure 2.3. Growth rate of basal roots measured during the first 24 h growth in pouches. Values shown are means of 8 plants from each of three shallow and three deep genotypes from the L88 population (pooled over both phosphorus treatments together) ± SE. Growth rate is significantly affected only by whorl of origin (P <0.001).

47

Figure 2.4. Effect of MCP and 0.6 µl L-1 ethylene on basal root angle of parent genotypes of L88 populations. The plants were treated with either MCP or ethylene for 24 h immediately after transferring to the pouch. Values shown are means of 10-12 plants per genotype ± SE, with data pooled over both high and low phosphorus treatments.

48

Figure 2.5. Ethylene sensitivity of basal root angles for whorls 1, 2 and 3 of a shallow genotype (TLP19) grown in low phosphorus. The angle was measured for the growth occurring between 24 and 48 h. Values shown are means of basal roots of 5-7 plants per ethylene treatment ± SE.

49

Figure 2.6. Ethylene sensitivity of basal root growth angle as a function of genotype, whorl and phosphorus treatment (low and high P) in three shallow and three deep genotypes from the L88 population. Ethylene sensitivity was measured as the slope of the response functions as illustrated in Fig. 6. Statistical analysis corresponding to these data is shown in Table 3.

50

Figure 2.7. Correlation between ethylene sensitivity and growth angle of basal roots of six L88 genotypes grown in low (low P) and high (high P) phosphorus treatments. Angles on the X-axis are of control plants without ethylene.

51

Figure 2.8. Ethylene sensitivity of growth response of basal roots as a function of genotype, whorl and phosphorus treatment (low and high P) in three shallow and three deep genotypes from the L88 population. Growth was measured between 24 and 48 h. Ethylene sensitivity was calculated as the slope of the response curve (ethylene concentration vs. growth).

52

Figure 2.9. Effect of exogenous ethylene on the range of growth angles of three shallow and three deep genotypes from the L88 population grown in low (low P) or high (high P) phosphorus. Angles were measured for growth occurring between 0 and 48 h. The range of growth angles for each plant was calculated by subtracting the minimum angle from the maximum angle produced by the basal roots of each plant. Values shown are means of the range of growth angles of 4- 7 plants per genotype per ethylene treatment ± SE.

53

Table 2.1. Average number of basal roots per whorl in four parent genotypes. The numbers designate mean numbers of basal roots of 6-8 plants ± SE. The upper whorl is designated as whorl 1, while the lower whorl as whorl 3.

Number of basal roots per whorl

Genotype Whorl 1 Whorl 2 Whorl 3

B98311 2.5±0.2 2.7±0.1 3.5±0.1

TLP19 2.3±0.1 3.2±0.1 3.9±0.1

G19833 3.2±0.2 3.9±0.2 4.1±0.1

DOR364 3.1±0.2 3.9±0.1

Table 2.2 Range of growth angles of basal roots per plant in six genotypes (three deep and three shallow) from the L88 population. The three deep genotypes used for the experiment of growth angle measurement are B98311, RIL7 and RIL76, while the three shallow genotypes are TLP19, RIL15 and RIL57. N = 4-7 plants per genotype.

Genotypes Mean

angle

Standard

deviation

Range of

angles

Min. growth

angle

Max. growth

angle

Deep 41.7 14.0 39.3 21.3 60.6

Shallow 56.4 18.0 54.5 28.5 82.9

54

Table 2.3. ANOVA of growth angle and growth response of basal roots from contrasting genotypes (shallow and deep) of the L88 population as affected by exogenous ethylene treatment. The three deep genotypes used for the experiment of growth angle measurement are B98311, RIL7 and RIL76, while the three shallow genotypes are TLP19, RIL15 and RIL57.

Growth angle Growth rate

Effect DF F-value P-value F-value P-value

Genotype 1 701.9 <0.001 25.29 <0.001

Phosphorus 1 0.178 0.673 4.102 0.046

Ethylene 5 220.0 <0.001 118.1 <0.001

Whorl 2 2218 <0.001 730.5 <0.001

Genotype*Phosphorus 1 2.741 0.098 0.193 0.662

Genotype*Ethylene 5 2.620 0.023 0.642 0.718

Genotype*Whorl 2 64.83 <0.001 0.515 0.584

Phosphorus*Ethylene 5 11.34 <0.001 0.193 0.965

Phosphorus*Whorl 2 3.484 0.031 3.854 0.021

Ethylene*Whorl 10 4.957 <0.001 11.54 <0.001

55

CHAPTER 2 APPENDIX In an initial experiment, we investigated if the genotypic difference in growth

angles of basal roots varies with the basal root lengths. Therefore, we grew contrasting

(shallow and deep) genotypes for 2 d after germination of the seedlings in the growth

pouch containing low or high phosphorus nutrient solution. After 2 d growth in the

pouches, digital images were captured and growth angles of basal roots (BRGA) were

determined at a fixed radius of 2 cm from the base the emerging position of each basal

root (Fig. 10). We observed that even with a fixed root length, the shallow and deep

genotypes significantly (P < 0.001) differ from each other in BRGA.

0

20

40

60

80

100

whorl 1 whorl 2 whorl 3

Bas

al ro

ot a

ngle

(deg

ree

from

ver

tical

)

deep genotypeshallow genotype

Figure 2.10. Genotypic variation in basal root angle for shallow (RIL57) and deep (RIL7) genotypes of common bean genotypes grown in the pouch system for 2 days in low phosphorus. The bars show mean growth angles of basal roots emerging from each whorl of 7 plants per genotype ± SE. The growth angles were measured at a fixed radius of 2 cm from the base of the emerging position of each basal root.

56

Table 2.4. ANOVA of BRGA of two contrasting parent genotypes (TLP19 and B98311) as affected by genotype, phosphorus, ethylene/MCP treatment or whorls. BRGA

Source DF F-value P-valueGenotype 1 204.5482 <0.001Phosphorus 1 1.154849 0.283Ethylene/MCP 2 309.0635 <0.001Whorl 2 551.1713 <0.001Genotype * Phosphorus 1 0.357171 0.550Genotype * Ethylene/MCP 2 1.896532 0.151Genotype * Whorl 2 22.99848 <0.001Phosphorus* Ethylene/MCP 2 0.175863 0.839Phosphorus * Whorl 2 0.254257 0.776Ethylene/MCP* Whorl 4 18.11323 <0.001Genotype *Phosphorus * Ethylene/MCP 2 0.93561 0.393Genotype *Phosphorus* Whorl 2 1.197749 0.303Genotype * Ethylene/MCP * Whorl 4 1.619132 0.168Phosphorus * Ethylene/MCP * Whorl 4 0.513042 0.726Genotype *Phosphorus*Ethylene/MCP * Whorl 4 5.169682 <0.001

CHAPTER 3: KINEMATIC ANALYSIS OF ROOT GROWTH AND

GRAVITROPISM USING SEMI-AUTOMATED IMAGE

ANALYSIS

Paramita Basu1, Anupam Pal2, Jonathan P. Lynch1,3, Kathleen M. Brown1,3

1Intercollege Program in Plant Physiology, The Pennsylvania State University, University

Park, PA 16802 USA; 2Department of Mechanical Engineering, The Pennsylvania State University, University

Park, PA 16802 USA; 3Department of Horticulture, The Pennsylvania State University, University Park, PA

16802 USA.

58

ABSTRACT

We employed a new kinematic approach based on computer-aided image analysis

to measure root growth and curvature. Although computer-assisted kinematic analysis

has been applied to primary root growth of Arabidopsis, it has not been employed for

study of plagiogravitropic growth or for thicker-rooted species. The primary difficulty in

kinematic study of thicker rooted species like bean is that the epidermal cells are not

visible, resulting in images of roots devoid of any trackable patterns. Our objective was to

develop a way to study spatio-temporal patterns of growth of bean basal roots in a

reliable, semi-automated way while minimizing user interventions to allow large scale

experiments. Graphite particles sprinkled on the roots created random patterns that could

be followed by image analysis. Images of the growing roots were captured using a high

resolution digital camera at 5 minute intervals for 4-6 hours. Here we describe a newly

developed image-analysis program, KineRoot, based on MatlabTM 7.0, that can track the

displacement of the patterns created by the graphite particles over space and time using a

highest correlation search method. The tracking algorithm also took advantage of the

color difference between the root and the background for higher accuracy and reliability.

Following pattern tracking, the edges of the roots were determined automatically by an

‘edge detection’ algorithm which provided root diameter and root midline. Local growth

rate of the root was measured by projecting the tracked points on the midline. From the

shape of the root midline, root curvature was calculated. By combining curvature

measurement with root diameter, differential growth ratio between two sides of a bending

root was also calculated. The growth and curvature zones of the basal roots were

identified by analyzing the spatio-temporal tracking information of the patterns on the

root. Root growth velocity and diameter were measured as functions of distance from the

root tip and time. The new software was able to produce growth velocity data with high

degree of accuracy and consistency. The growth zone of a basal root may span 1-6 mm

from the tip and changes with time. Therefore, grouping data from a 4-6 h experiment in

time-averaged mean results in inaccurate estimates of growth zones of a root. The upper

side of the root grew 2-4% more than the lower side resulting in a downward bend. This

59

new approach of computer aided image analysis and measurement provides a new tool

for kinematic analysis for not only roots, but also other plant organs of tubular shape.

Minimum user interventions also make it a useful tool for analyzing a large amount of

high resolution image data for relatively longer times. Spatio-temporal analysis of root

growth shows that it is important to study growth rate simultaneously in space and time

to accurately characterize growth of basal roots of common beans.

60

INTRODUCTION

Plant growth is characterized by spatio-temporal variation in expansion of various

regions of the plant body. Therefore, in order to understand the mechanism behind plant

morphogenesis, we need to analyze the processes (cell division, expansion and

differentiation) involved in controlling the growth of the organs with respect to both time

and space. A number of researchers have investigated the changing growth zones by

employing ‘kinematic’ analysis. Kinematics is an aspect of dynamics which involves

study of physical motion (acceleration and growth velocity) without reference to the

forces resulting in the movement (Gandar 1983). This approach has been widely used in

determining the growth profile of elongating plant organs, such as root, stem, leaf, and

perianth, in which the spatial distribution of growth may or may not be “steady” i.e. time

independent.

Kinematic analysis has been employed to the study of primary root growth for a

long time (Goodwin and Stepka 1945; Erickson and Sax 1956) and has become more

established in the last couple of decades (Silk and Erickson 1979; Gandar 1980, 1983;

Beemster and Baskin 1998). Using a compound microscope, Goodwin and Stepka (1945)

measured cell division and the displacement of epidermal cells in Phleum roots over a

short period of time and thereby identified four regions of a growing root— the root cap,

the slowly growing meristematic zone, rapidly elongating region at the base of the

meristematic zone and zone of relatively slowly elongating cells. Later studies have

combined measurement of incremental organ growth and increase in cell length and cell

number to define the spatial distribution of rate of root elongation (Erickson and Sax

1956; Goodwin and Avers 1956; Bertaud et al. 1986; Ben-Haj-Salah and Tardieu 1995;

Beemster et al. 1996; Sacks et al. 1997). In addition, relative elemental growth rate,

describing the displacement of points along the growing organ at any one instant in time,

has also been analyzed for the two-dimensional growth of leaves (Erickson 1966).

Kinematic analysis has been widely applied to growth of both monocot as well as dicot

leaves (Bernstein et al. 1993; Ben-Haj-Salah and Tardieu 1995; Fiorani et al. 2000;

Tardieu et al. 2000), and to the growth of perianth in Lilium (Gould and Lord 1989).

61

Various researchers have also made use of this technique in studying the influence of

different types of environmental variations on spatial and temporal growth of different

organs e.g., effect of low water potential in maize root (Sharp et al. 1988; Fraser et al.

1990; Sharp et al. 2004), effect of temperature on maize primary root (Pahlavanian and

Silk 1988; Walter et al. 2002), supply of nitrogen on fescue leaves (Gastal and Nelson

1994), water stress on tall fescue leaves (Durand et al. 1995) and primary root of maize

(Liang et al. 1997; Sacks et al. 1997), effect of irradiance on maize roots (Muller et al.

1998), effect of salinity on sorghum leaves (Bernstein et al. 1993; 1995), and wheat

leaves (Hu et al. 2000). Kinematic analysis has also been employed to describe the

influence of biotic stress such as aphid infestation on elongation rate of alfalfa shoot

(Girousse et al. 2005). Recently, the effect of phosphorus deficiency has been studied on

the elongation rate of the primary root of Arabidopsis (Ma et al. 2003) and grass leaf

growth (Kakanova et al. 2006). Application of the kinematic approach in such diverse

studies shows the utility of the method in understanding the details of plant growth.

For kinematic analysis of motion of a plant organ at high spatio-temporal

resolution, researchers applied different methods in visualizing and analyzing the spatial

patterns of elongation of the segments of the organ (Erickson and Sax 1956; Gandar

1983). Scientists have marked growing zones of plant organs with various markers such

as ink, graphite particles, carbon-water mixture, needle holes etc. throughout the growing

zone and measured the displacement of the markers over time for analyzing spatio-

temporal variation in growth rate (Sharp et al. 1988; Gould and Lord 1989; Ben-Haj-

Salah and Tardieu 1995; Sacks et al. 1997; Beemster and Baskin 1998; Granier and

Tardieu 1998; Muller et al. 1998; Granier and Tardieu 1999; Hu et al. 2000). The

displacement of the above mentioned identifiable markers on the surfaces of the growing

organs were measured manually by a ruler or by a binocular microscope, or by taking

time-lapse photographs using still camera or video camera (Sharp et al. 1988; Gould and

Lord 1989; Bernstein et al. 1993; Ben-Haj-Salah and Tardieu 1995; Sacks et al. 1997;

Beemster and Baskin 1998; Granier and Tardieu 1998; Muller et al. 1998; Granier and

Tardieu 1999; Hu et al. 2000). More recently, instead of marking the growing organ,

another group of researchers measured spatio-temporal displacements of natural

62

landmarks such as vein structures on leaves (Schmundt et al. 1998) or computationally

discernible patterns on the roots (van der Weele et al. 2003). Then they applied various

methods of image analysis for quantification of growth. Schmundt et al. (1998) used

image sequence analysis for measurement of growth in leaves of Ricinus communis and

Nicotiana tabacum which has been termed as the ‘optical growth analysis’. This study

used visualization of leaf vein structures using infra-red light and then application of

computer-assisted image analysis software based on ‘structure-tensor’ approach (Jahne

1997) to obtain high resolution growth maps of leaves. The Schmundt et al. (1998) study

resulted in a picture of the actual growth rates and changes in growth rates over time of

the actively expanding leaves. Later the method by Schmundt et al., (1998) was modified

by Walter et al., (2002), who applied the automated technique of image sequence analysis

for detailed study of relative elemental growth rate distribution of growing maize primary

root influenced by variation in root temperature. Recently van der Weele et al., (2003)

introduced a new computer-assisted technique which involved the combination of two

methods, ‘structure tensor’ (Jahne 1997) and ‘robust matching’ algorithm (Black and

Anandan 1996) to measure the expansion profile of a growing root at high spatio-

temporal resolution. They captured digital images of the root of Arabidopsis at 5 or 10 s

intervals. Then nine images were selected for a stack where the ‘structure tensor’ method

was used to find a line of minimum variation in pixel intensity and define the moving and

nonmoving portions of the root. Then van der Weele et al., (2003) used the ‘robust

matching’ algorithm to statistically match the patterns on the root and obtain a velocity

profile of the root.

Growth rate of a plant organ can be analyzed by two different approaches:

Eulerian approach and Lagrangian approach. In the Eulerian approach, tissue growth is

measured as a function of a spatial coordinate, e.g. root growth rate as a function of

distance from the root tip, whereas in the Lagrangian approach, growth of a particular

region of the plant organ is followed over time, e.g. lengths of a particular group of cells

on a root as a function of time. The Eulerian approach aims at studying the distribution of

tissue expansion and rate of cell division along the axis of the growing organ. According

to this approach, position and size of a growing area of the organ are determined by the

63

spatial location of the organ from a fixed reference. On the other hand, the Lagrangian

description refers to the motion of the individual cells with respect to a reference which

contributes to the growth of a specific region (Gandar 1983; Fiorani et al. 2000).

Although the Eulerian approach has been primarily used to identify relative elongation

zones of the root at a fixed time and the Lagrangian approach has been used to

characterize growth of cells over space and time, both approaches can be used in spatio-

temporal frame of reference. The requirement is that one should be able to measure

growth over a relatively long period of time and over a sufficiently long region.

In most of the studies discussed above the primary objective was to characterize

the growth of the plant organ. However our goal for kinematic study is not only to

characterize local root growth, but also to identify and characterize the bending regions of

the basal roots in response to gravity. We aim to identify the growth and graviresponding

zones of the basal roots, to investigate the relation between these zones, and to determine

how these are affected by low phosphorus availability and ethylene treatment. Therefore

whereas one-dimensional kinematic study in the direction of growth is sufficient for

identifying and characterizing growing area of the roots, at least two-dimensional

kinematic study is essential for our purposes. In addition we aim to study basal root

growth and bending over a relatively long period of time (4-6 hours) to accommodate the

time scales associated with changes in growth angle of basal roots. Our measurement and

analysis are further complicated by the lack of details in the thick basal roots of 24-48 h

old common bean seedlings since the epidermal cells are invisible under the

magnification of microscope. The ‘structure tensor’ method used by a number of

researchers (Schmundt et al. 1998; van der Weele et al. 2003) calculates local root or leaf

growth velocity with high degree of confidence only if there are a large number of high

contrast patterns. In the absence of such patterns the ‘structure tensor’ method can only

produce a very sparse velocity field with low confidence. Therefore, in the present work,

we developed a novel semi-automated image processing system in Lagrangian frame of

reference to analyze the gravitropic growth of basal roots in common bean where we take

advantage of patterns not only at a pixel site, but also in its neighborhood. As a result, the

new approach can generate reliable root growth data even in regions where there are very

64

low contrast patterns or no patterns as long as the neighborhood is large enough to

include identifiable patterns. This approach is also particularly suitable for finding two-

dimensional growth velocity of the root for relatively longer times. Furthermore this new

software also automatically detects root edge generating the root midline for calculation

of root curvature, diameter and differential growth ratio between two sides of a bending

root.

In the following section we present the experimental protocol for growing the

bean seedlings, addition of marker particles and photographing the roots. Then we

describe the image analysis algorithm followed by actual measurements from the

analyzed images. We present representative results from a growing root using our new

approach. In chapter 4 we make use of this new kinematics method to address scientific

questions on graviresponse of plagiogravitropic roots.

METHODS

Experimental method

Common bean (Phaseolus vulgaris L.) genotype TLP19 developed for tolerance

to low phosphorus at the International Center for Tropical Agriculture (CIAT, Cali,

Colombia) was employed for this study. TLP19 has an indeterminate bush habit i.e. Type

II growth habit. TLP19 produces shallow basal roots, and within one plant we observed

shallow basal roots emerging from the top (closest to the shoot) whorls (whorl 1 and

whorl 2), with progressively deeper basal roots emerging from the lower whorl (whorl 3)

as shown in Fig. 3.1.








65


transferred to a sheet of 30 x 24 cm blue germination paper (Anchor Paper Co., St. Paul,

MN, USA) stiffened by attaching perforated plexiglass sheets to stabilize the root system.

The bottom of the blue paper with plexiglass was placed to allow direct contact with the

nutrient solution containing high (1 mM) or low (0 mM) phosphorus as described above.

The germination paper containing seedling was suspended in nutrient solution and

covered with aluminum foil to prevent illumination of the roots.

Graphite particles were sprinkled on the roots carefully without disturbing the

plants and these particles created patterns on the otherwise uniformly colored basal root

that could be followed in image analysis. Figure 3.2 shows a photo of the graphite

sprinkling procedure using a dropper fitted with a pipette tip. A small amount of graphite

powder was drawn into the pipette tip and then blown on the roots from close proximity.

During this procedure extra precaution was taken to not touch the roots and also not

change the orientation of the seedling with respect to the gravity. Images of root systems

were captured at fixed intervals (5 min) using a high resolution (6 Megapixel) digital

single lens reflex camera (Nikon D70s) fitted with 105 mm Nikkor micro lens 1 d after

the emergence of basal roots in pouches. Images were captured for 4-6 h. The captured

images had a resolution of 10-20 µm/pixel. Figure 3.3 shows a photo of the image

capturing setup. The seedling in the blue germination was placed in a water-sealed

plexiglass box maintained at 25-26 °C. Photographs of the seedlings were captured from

outside the plexiglass box. During time lapse photography of the growing roots, the blue

germination paper containing growing seedlings were kept inside the plexiglass box to

have uniform growth conditions e.g. temperature, humidity etc. in controls and treatments

like ethylene and MCP. For capturing image the camera was triggered at fixed intervals

of time by a laptop computer through a universal serial bus (USB) cable. Plants were

grown in complete darkness and photos were captured using the camera’s flash to

minimize light exposure of the roots. To avoid shadows from direct flash which interferes

with image analysis, light from the flashes was bounced off a sheet of white paper placed

on top of the plexiglass box. A ruler was attached to the supporting plexiglass sheet for

calibrating pixel dimensions into millimeters.

66

RESULTS

Image Analysis

To measure spatio-temporal changes in root growth we developed a sophisticated

image analysis software, titled KineRoot, using Matlab 7.0TM (Natick, MA). The software

features an easy-to-use graphical user interface which is shown in Fig. 3.4. It allows

loading of a large number of images (limited by the computer’s memory only) and then

playing the images as a movie at desired speeds and moving from one frame to another

with the click of a mouse button. Furthermore, by measuring the millimeter marks on the

ruler, KineRoot also allows easy spatial calibration of the images from pixels to

millimeters. Image analysis by KineRoot is divided into two basic steps.

Step 1 : Tracking of Selected Points on the Roots

From all the time sequence images loaded on to KineRoot, the user selects an image

as the reference which shows the root tip and the body of the basal root most clearly. In

the reference image the user selects 10-15 points along the basal root. The choice of the

points is arbitrary with the only requirement that they be chosen sequentially along the

root. Then the user identifies the point lying on the root tip. From these points, 25 points

are generated by interpolation using cubic splines (Press 1997). Starting from the images

immediately preceding and following the reference image, these 25 interpolated points

are then tracked in time in all other images sequentially by using as a reference the image

where the points have been tracked immediately before the current image. For tracking

the points, three methods, their variations and combinations are used:

(a) Highest Correlation Coefficient Search

After the user selects the points on the reference frame and the points are

interpolated, the user specifies a search radius of R pixels within which the new location

of a tracked point is searched in the current frame. The user also specifies a template

radius N which is used to calculate the correlation coefficient between the reference

image and the current image. Figure 3.5 schematically shows the pattern matching

67

algorithm using the highest correlation search method. For example, if the coordinate of a

selected point in the reference image (Fig. 5A) is 0 0( , )x y , then the new location ( *, *)x y

of this point in the current image (Fig. 5B) is searched within 0 0*x R x x R− ≤ ≤ +

and 0 0*y R y y R− ≤ ≤ + . Let the intensity of pixels within a (2 1) (2 1)N N+ × + box

surrounding the point 0 0( , )x y in the reference image be 0 ( , )I x y where

0 0 to x x N x N= − + and 0 0 to y y N y N= − + , and in the current image the intensity of

(2 1) (2 1)N N+ × + box of pixels centered around the point ( *, *)x y be *( , )I x y . The

correlation coefficient between these two boxes of pixels is

0 0

0 0

0 0 0

**2 2

* *

( , ) *( *, *)

( ( , ) ) ( *( , ) *)

N N

x N y N

x N y N y Nx N

o ox x N y y N x x N y y N

I x x y y I x x y yC

I x y I I x y I

=− =−

+ + ++

= − = − = − = −

+ + + +=

− −

∑ ∑

∑ ∑ ∑ ∑, (1)

where 0I and *I are average pixel intensities i.e.

0 0

0 0

0 02

1 ( , )(2 1)

x N y N

x x N y y NI I x y

N

+ +

= − = +

=+ ∑ ∑ (2)

**

* 2* *

1 *( , )(2 1)

y Nx N

x x N y y N

I I x yN

++

= − = −

=+ ∑ ∑ . (3)

For a point located at 0 0( , )x y in the reference frame, the correlation coefficient C is

calculated for all points ( *, *)x y located within 0 0*x R x x R− ≤ ≤ +

and 0 0*y R y y R− ≤ ≤ + in the current frame. The point where correlation coefficient C is

highest is chosen as the new location ( *, *)x y of the point 0 0( , )x y in the current image.

After all 25 points were tracked to the current image, the new locations of the points were

used as reference for the following image. This algorithm was followed until all 25

interpolated points were tracked in all images.

The amount of computation necessary to track a point depends on the search radius

R and pixel box size N. Minimizing both R and N improves the speed of tracking.

However, too small values of R and N compromise the accuracy of tracking. Therefore an

optimum choice of R and N is important for both computational efficiency and accuracy

68

of the method. The overall root growth data presented in Fig. 2.3 of Chapter 2 were used

to calculate maximum expected displacement 560 24(x ×∆ = x maximum growth/day ≈ 0.1

mm) of any marker point over five minutes. An optimal value for search radius

2 0.2 mmR x= ∆ = was used to ensure that even in the rare case of very high growth rate

the algorithm could track the marker points while avoiding unnecessary computations.

For estimating the optimum value of N, the average spacing of patterns on the last image

was used because as the root grows the gap between the graphite particles increase in the

growth zone and in the last image the gap between two graphite particles becomes

highest. In our measurements N = 0.25 mm produced the most optimum results.

Since all of the images were colored, we calculated the correlation coefficients Cr,

Cg and Cb for red, green and blue color intensities respectively using equation (1) where

I0 and I* were replaced by the appropriate color intensities. For searching the highest

correlation coefficient, in most of the analysis we used the average of Cr, Cg and Cb as an

estimate of the overall similarity between two boxes of pixels. However, the user could

also choose a search algorithm based on only one color, if necessary.

In order to make the algorithm efficient, the user had the choice of using the

velocity of the individual points to provide a better initial guess to the search algorithm.

When a point 0 0( , )x y moves to ( *, *)x y in time tδ the two-dimensional velocity of the

point is

0 0* *,x x y yu vt tδ δ− −

= = . (3)

Therefore to find the location ( ', ')x y in the following image we could limit our search

area within * ' *x u t R x x u t Rδ δ+ − ≤ ≤ + + and * ' *y v t R y y v t Rδ δ+ − ≤ ≤ + + under the

assumption that the change in velocity of a point is small within a span of three

consecutive images. This approach of using the velocity of the points to predict the new

location of the points helps the program track all points more accurately and efficiently.

(b) Highest Color Weighted Correlation Coefficient Search

The highest correlation search method described above matches boxes of pixels

irrespective of whether the pixels are on the root or on the germination paper behind it.

69

Although this method works in more than 70% of the kinematic studies, in the cases

where as the root grows and moves into an area of the germination paper where the

texture of the paper is very different from the reference image, the algorithm tends to

struggle to track the points accurately.

To overcome this problem we improved the algorithm by introducing a weighting

factor ( , )w x y based on color of the pixel into the calculation of correlation coefficient C.

With this new algorithm the user selects a small area of the image covering only the root

and then another area covering only the germination paper. Colors from each of these

areas are averaged and stored as root color ( , ,r rR G B ) and background color ( , ,b b bR G B )

where R, G and B are the intensities of red, green and blue respectively, and range

between 0 and 1. Figure 3.6 shows a schematic for calculating the weighting factor w. If

the difference of intensity of any color between the root and the background is less than

0.2, the weighting factor w is assigned a value of 1 (e.g. the blue color in Fig 3.6),

otherwise the weighting factor is calculated by linear interpolation for pixels which has

color intensity between that of the root and the background. If the color intensity is

outside the root-background color intensity range, w is assigned a value of 1 or 0

depending on proximity to the root color or background color respectively. Using the

weighting factor the color weighted correlation coefficient is defined as

0 0

0 0

0 0 0 0 0 0

**2 2

* *

( , ) *( *, *) ( , ) *( *, *)

( ( , ) ) ( *( , ) *)

N N

x N y Nc x N y N y Nx N

o ox x N y y N x x N y y N

I x x y y I x x y y w x x y y w x x y yC

I x y I I x y I

=− =−

+ + ++

= − = − = − = −

+ + + + + + + +=

− −

∑ ∑

∑ ∑ ∑ ∑ (4)

where w0 and w* are the weighting factors for pixels in the reference image and the

current image respectively. The color -based weighting factors reduce the importance of

the pixels from the background paper in calculating the correlation coefficients between

two boxes of pixels. As a result, even if the texture of the background paper changes

drastically the software is able to track the points on the root reliably. It should be noted

that in case of low contrast images where the intensity difference between the root and

background is less than 0.2 in all three colors the weighting factor becomes 1. As a result

70

the color weighted highest correlation search approach changes to highest correlation

search approach described in the previous section.

(c) Minimum Pixel Intensity Difference Search

Although the above two methods track the points reliably, they are computationally

expensive and relatively slow. Therefore, instead of calculating correlation coefficients,

for roots that do not bend an easier approach is to calculate the normalized root-mean-

square (R.M.S) difference in color intensity between two boxes of pixels

20 0 02

0 0 02

1 [ ( , ) *( * , * )](2 1)

1 ( , )(2 1)

N N

x N y NN N

x N y N

I x x y y I x x y yN

DI x x y y

N

=− =−

=− =−

+ + − + ++

=+ +

+

∑ ∑

∑ ∑, (5)

and find the point where D between the two boxes is minimum. As the root bends,

relative movement of the graphite particles within a box of pixels becomes large and two-

dimensional. Therefore when intensity difference between a subgroup of pixels within the

(2N+1)2 box is minimum, intensity difference in another subgroup becomes large. As a

result the software cannot determine the absolute minimum value of D and the tracking

method using minimum pixel intensity difference search approach becomes inconsistent.

This problem does not arise when the root grows along a straight line minimizing the

relative movement of graphite particles within the (2N+1)2 box of pixels.

Using tracking history

In addition to the three methods described above, we also employed a variation

where instead of using the previous image only as the reference, the user could include

more images including the one where the user first selected the points as reference which

we call ‘the history tracking method’. For example, if there are 50 images and the user

used the 35th image to select the points then, in the absence of history tracking, while

locating the points on the 34th image 35th image will be used as reference, for 33rd image

34th will be the reference and so on. However, using history tracking the user could also

use other images where points have already been tracked as a reference also, e.g. for the

71

22nd image the reference could be all of 23rd, 24th, 25th and the initial reference 35th

image. Therefore in this case I0 will be the 23rd image, I1 will be the 24th image, I2 will be

the 25th image and I3 will be the 35th. We calculate a weighted average of the correlation

coefficients putting higher weight on the I0 image and then followed by progressively

lesser weights on the images which are further away from the current image in time. So

the average correlation coefficient for history tracking is,

0

0

n

i ii

h n

ii

p CC

p

=

=

=∑

∑ (6)

where Ci is the coefficient of correlation between the current image I* and reference

image Ii, and pi’s are the weights on the each of the correlations such that 1i ip p +> . Then

we search for maximum average correlation coefficient Ch to locate the best match for the

points.

Using more than one method

In addition to the three tracking methods and the variation above, in the cases where

no one method succeeded in tracking the points, we employed a combination of two

methods. In that case, an average selection criterion was calculated from two methods.

For example, when we combined both the highest correlation coefficient method with the

difference method, we defined a new selection parameter

( (1 )) / 2P C D= + − (7)

and searched for the point where P is highest.

Each of the tracking methods described above have different computational loads.

Since our objective is to track the marker points reliably with minimum possible

computation, the methods are ranked and chosen according to their computational

efficiency in the following order, minimum pixel intensity difference search method,

highest correlation coefficient search method, highest color weighted correlation

coefficient search method, combination of difference and correlation search method and

correlation search with track history method. After tracking the marker points, the

72

algorithm for each method provides a confidence measure of marker tracking, and if the

confidence measure is too low the next tracking method with higher computational load

is chosen. For both the correlation coefficient search method and color weighted

correlation coefficient search methods the minimum correlation coefficient for tracking

all 25 points in all frames provides the confidence measure F = Cmin. For the minimum

pixel intensity difference search method, confidence measure is given by

max1F D= − where Dmax is minimum value of D. A threshold value of confidence measure

F = 0.8 was used before moving to the next method.

Step 2 : Automatic Edge Detection and Finding the Midline of the Roots

Since the selected points on the root can be located anywhere along the root

diameter, we must find the root midline and project these points on the midline to

estimate root growth. To identify the root midline we first need to find the edges of the

root. An ‘edge’ in an image is defined as a line at which the gradient of color intensity

has a local peak. However, quite often the edge in an image cannot be accurately marked

by maximizing derivative of the pixel intensities directly because of noise in the image or

blurriness at the edge. Over the years many methods have been developed for automatic

detection of edges from digital images (Prewitt 1970; Sobel 1978; Canny 1986). Among

these the most popular method is the edge detection algorithm by Canny (1986). For

detecting the edges of the roots we use a large portion of the Canny algorithm coupled

with customizations based on the specific nature of the root images. The Canny algorithm

has three steps out of which we use two steps, and replace the third step with a simpler

method by customizing for the specific feature of the basal root images.

(a) Noise smoothing and image gradient— Since an edge is identified by a sudden change

in color within a span of few pixels i.e. strong color gradient, we need to make sure that

the strongest color gradients of the image do not reflect either the noise in the image or

the dark graphite particles on the image. Therefore before detecting the edge of the root

we need to smooth noise by convolving the image with a Gaussian filter. A two-

dimensional Gaussian filter is defined as

73

2 2

222

1( , )2

x y

G x y e σσ πσ

+− = , (8)

and is shown in Fig. 3.7A. The parameter σ controls the spread of the filter contributing

to the blurriness of the filtered image which can be changed in KineRoot by changing

filter radius. The pixel intensity I(x, y) is convolved with this filter for noise smoothing.

Figure 3.7B shows a close up image of a basal root and Fig. 3.7C shows the convolved

image

( , ) ( , ) ( , )

( , ) ( , ).i j

I x y I x y G d d

I x i y j G i jξ η

ξ η ξ η η ξ= − −

= − −

∫ ∫∑∑

(9)

As a result of convolution with the Gaussian filter, the image gets blurred which allows

better calculation of the color gradient of the image. Figure 3.7D shows the magnitude of

the color gradient ( ) ( )22I Ix y∂ ∂∂ ∂

+

of the image averaged for red, green and blue colors.

The darker streaks in the image which represent higher color gradient show the edges in

the image.

(b) Edge enhancement— The image color gradient presented in Fig. 3.7D shows that

although the gradient identifies the edges, the peak gradient corresponding to the edge

spreads over more than one pixel width resulting in a smudged edge. To identify the true

edge in the image, the Canny edge detector identifies the local maxima along the edge

and suppresses all other high gradient values in image as shown in Fig. 3.7E. For detailed

algorithm of the process of non-maxima suppression please refer to Canny (1986).

(c) Edge finding— Although the Canny edge detection algorithm has one more step in

which the edge points are linked together to generate the final edge, we apply an easier

approach knowing that the roots are elongated objects and the edges can be found if we

move perpendicular to the lines joining the tracked points. However there might be

another root near the edge which can be picked erroneously by the computer. To prevent

this error, the user measures the approximate root diameter, which is then used as the

search radius for finding the root edge from the non-maxima suppressed image gradient

(Fig. 3.7E). Figure 3.7F shows the final edge detected image. The two edges of the root

74

are outlined by the green and the yellow lines. By taking the average of the two edges we

can also identify the root midline.

Since the root edge is determined by the magnitude of color gradient in the image, it

is important to use the maximum available contrast between the root and the background.

For our experiments the background germination paper is blue whereas the root color is

light gray. When we compared the individual red, green and blue colors between the root

and the background we found that the instead of averaging all three colors, the red color

produces the highest contrast whereas the blue color has the least contrast between the

root and the background. Therefore, for edge detection, best results were obtained using

the intensity of red color of the pixels. But for tracking the points we used the average

correlation coefficient calculated from all three colors.

Measurements

Once the root midline is found, we project the tracked points on the midline (i.e.

drop perpendicular on the midline) and measure the distance Sp of the projected points

from the root tip along the midline of the root as shown in Fig. 3.8A. For our subsequent

measurements we use Sp to compute root growth velocity and relative elongation rate. In

addition we also directly measure the root diameter D at any point along the root length.

Figure 8B shows the schematic of the space time mapping of marker points where

distance of the marker points from the root tip is along the vertical axis and time is on the

horizontal axis. Note here that since we use the root tip as our spatial reference, it is held

fixed in the ‘root length map’ plot. The region where the distance between consecutive

marker points changes more rapidly over time than other areas along the root identifies

the growth zone as shown in Fig. 3.8B.

Knowing the distance of the tracked points from root tip allows us to calculate root

growth velocity as a function of distance from the root tip and time. If a point p is located

at Sp distance from the root tip at time t, and after δt time it moves to Sp+δSp distance

from the root tip as shown in Fig. 3.9 then the growth velocity of the point p

( , ) pp p p

SU U S t

tδδ

= = . (10)

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As the root grows, the marker points move away from the root tip and the points further

away from the root tip move away faster than the point closer to the root tip due to the

cumulative velocity of all points lying between the root tip and the marker point. As a

result the root tip velocity increases with distance from the root tip. But once the marker

points are outside the growth zone, the growth velocity becomes constant. This

description of root growth velocity following individual marker points is a Lagrangian

description. In the Eulerian description, we describe root growth velocity as a function of

distance from the root tip s and time t (Gandar 1983)

( , )u u s t= . (11)

The relative elongation rate describes the rate of relative growth of a small segment of the

root of length over a short time as illustrated in Fig. 3.10 where a root segment of length l

grows to l+δl over time δt. Therefore relative elongation rate

lrl tδδ

= . (12)

Relative elongation rate r(s,t) can also be calculated by taking derivative of the root

growth velocity u(s,t) with respect to distance from the root tip s (Taiz and Zeiger 1998).

Since we are also interested in bending of the roots, one of the important

parameters to calculate from image analysis is the root curvature. Curvature of a curve is

the reciprocal of radius of curvature i.e. the radius of a circle that matches the curve at a

point (x, y) and is given by

( )

2

2

3/ 221

d ydx

dydx

κ = +

(13)

where y=y(x) is the equation which describes the root midline. To calculate the root

diameter d at distance s from the root tip, a line locally perpendicular to the root midline

is drawn. The distance between the two points of intersection of the two edges with this

perpendicular line is the root diameter at distance s from the root tip. As a root responds

to gravity and bends toward gravity, one side of the root grows more than the other side.

Therefore the ratio of local growth rate along the two edges of the root can be used to

characterize graviresponse of a root. Following Silk and Erickson (1978), the differential

76

growth ratio of two arcs of length δsu and δsl on the upper and lower edges of an element

of bending root can be calculated as

22

u

l

s ds d

δ κδ κ

+=

−. (14)

Example measurements

In this section we present representative measurements from one basal root to

demonstrate the performance of the software and the kind of results obtainable from it.

Figure 3.11 shows an example of marker point tracking and automatic edge detection

using a montage of 8 selected images of basal roots at 45 min intervals from an image

sequence of 72 images originally captured at 5 min intervals. The 2 d old seedling with

emerging basal roots was grown in growth pouch in high phosphorus nutrient solution

(see methods). The images were captured beginning 36 h after the emergence of the basal

roots. The red dots are the marker points selected by the user at 120 min and tracked in

other frames by KineRoot using highest correlation search method. Note that after the

user selected the marker points they were interpolated to generate a total of 25 points

which were tracked in all frames. To avoid crowding of the points, here we only show

the points selected by the user. After the points were tracked, automatic edge detection

identified the edges of the root which are outlined using yellow and green lines. The

average of the root edge lines generates the root midline which is shown by the bold

white line. The root tip is identified by the cyan asterisk symbol. The marker points were

projected on the midline to calculate distance Sp from the root tip along the midline.

As the root grows, the marker points move away from each other (Fig. 3.11). The

region where the points move away from each other faster than other regions identifies

the growth zone of the root. This qualitative assessment of root growth zone is more

prominent in the root length map plot in Fig. 3.12 where the distance of the marker points

from the root tip is on the vertical axis and time is on the horizontal axis. The top most

diagonal line starting at 3.5 mm at time 0 min and ending at 7 mm at 355 min shows an

overall growth of 3.5 mm during 355 min of the selected root segment. The points

located between 0.8-2.2 mm from the root tip at the initial time tend to separate more

77

compared to points in other regions of the root which identifies the growth zone of the

root.

Figure 3.13A shows the measured growth velocity as a function of distance from

the root tip. The gray dots in Fig. 3.13A show the growth velocity of all 25 marker points

from 72 images taken over a period of 6 h at 5 min intervals. The superimposed bold line

is the mean of growth velocity after grouping the data in bins of 0.5 mm. The raw data

from KineRoot forms a well clustered group showing the robustness of the algorithm. As

anticipated, the growth velocity at the root tip starts from 0 and continues to increase

through the growth zone and becomes nearly constant after about 5.5 mm from the root

tip. Figure 3.13B show the mean relative elongation rate as a function of distance from

the root tip. The error bars are standard deviation bars. The mean data shows that the

growth zone spans up to 6 mm from the root tip. However the error bars on relative

elongation rate plot are relatively large which show temporal variation in the elongation

rate rather than scatter in the data.

In Fig. 3.14 we show a color isocontour plot of relative elongation rate as a

function of both distance from the root tip and time which depicts temporal variation in

relative elongation rate. Distance from the root tip is along the vertical axis and time is

along the horizontal axis. Color shows the rate of relative elongation with reds, oranges

and yellows showing high values, and light and dark blues showing low rates of

elongation. The length of the growth zone increases with time from approximately 1.5

mm (1-2.5 mm from root tip) at 60 min to 4 mm (1-5 mm from root tip) at 350 min. The

apical boundary of the growth zone remains almost constant at 1 mm from the root tip.

But the distal end of the growth zone expands lengthening the growth zone. In addition,

the rate of elongation also increases with time as shown by the large red region beyond

270 min compared to mostly green elongation zone before that.

Detection of root edge allows us to also measure root diameter in space time

coordinates. Figure 3.15 shows the time averaged root diameter as a function of distance

from the root tip. Clearly the diameter of the root near the tip is minimum and reaches a

nearly constant magnitude at about 1.5 mm from the root tip. The small error bars in Fig.

78

3.15 show that as the root grows in length by about 3.5 mm over 6 h, the root diameter

remains nearly constant during this period.

Since our interest in kinematics of basal roots of common bean is not only in root

growth but also in graviresponse, we show in Fig. 3.16A the mean curvature of the root

midline measured from KineRoot. Although curvature is very low for this particular root,

Fig. 3.16A shows two regions of bending— the apical bending zone spanning 1-3.5 mm

from the root tip and the distal bending zone spanning 3.5-5.5 mm from the root tip.

Using equation (14) we also calculate the differential growth ratio between two edges of

the root which is shown in Fig. 3.16B. Since the root for this example study bends very

little over 6 h (Fig. 3.11), the differential growth ratio is also relative small. The upper

edge of the root has grown 2-4% more than the lower edge.

DISCUSSION

This chapter presents a new approach in kinematics using a semi-automated

image analysis software, KineRoot, for the study of growth and graviresponse of basal

roots of common bean. Since the basal root of a 36 h old common bean seedling is

devoid of any patterns for spatio-temporal tracking and size of the roots are such that the

epidermal cells are also not visible in the usual microscopic magnification, we sprinkled

graphite particles to add patterns to the root for tracking by KineRoot. Although use of

ink or graphite particles as marker points has been used before (Erickson and Sax 1956;

Sacks et al. 1997; Beemster and Baskin 1998; Muller et al. 1998), addition of marker

points has been a tedious procedure because clearly visible marker had to be added very

carefully for tracking because touching can damage and/or change root growth. However

in KineRoot the computer matches patterns within boxes of pixels surrounding a marker

point through correlations, so there is no need for any particular type or placement of

markers on the roots, and any point on the root can be used as a marker point even if

there is no graphite particle exactly at that point, as long as there are some uniquely

identifiable color patterns around the roots. As a result KineRoot is more suitable for

kinematic study of large number of roots with minimum user interventions. Furthermore,

the method of pattern matching allows us to track the marker points on the roots for

79

extended periods even if they deviate from a straight trajectory and bend. This kinematic

study was limited to 4-6 h because the basal root tends to respond to gravity within this

period of time. But if necessary, even longer studies can also be performed using

KineRoot with the only limitation being the computing power.

Graphite particles added to the root for creating trackable patterns separate with

time in the growth zone, as shown in Fig. 3.17. The existing algorithms based on

‘structure tensor’ method (Schmundt et al. 1998; van der Weele et al. 2003) search for a

path of minimum pixel intensity difference in a stack of 7-9 images to generate the

velocity field of the plant organ. Therefore, in any portion of the plant organ where there

are very few patterns this method cannot generate velocity with sufficient confidence,

and as a result, produce a velocity field which is very sparse. In case of a growing root, it

is the zone of interest, the growth zone which becomes less populated with patterns with

time and the structure tensor method generates very few high confidence velocity

measurements there. But KineRoot not only matches patterns at a pixel site, but also

from its neighboring sites. As a result even when the patterns separate away within the

growth zone, KineRoot can track marker points with high confidence based on patterns

in its neighboring pixels.

As shown in Fig. 3.11 and Fig. 3.13 KineRoot automatically tracks the marker

points and detects edges of the roots generating reliable growth data. The growth velocity

data generated by KineRoot follow the description of root growth found in the literature

(Taiz and Zeiger 1998). The growth zone of roots can be divided into two main regions,

the meristem (zone of cell division) and zone of elongation. As the cells divide, they

successively pass through the elongation zone and then in the maturation zone growth

ceases as cells become mature with differentiated characteristics (Dolan et al. 1993; Taiz

and Zeiger 1998). Rate of elongation of the root is regulated by the combined function of

cell production in the meristem and cumulative cell expansion in both meristem and

growth zone (Beemster and Baskin 1998). But for analysis of growth in thicker roots of

common bean, it is difficult to measure the cell production in the meristem while the root

is growing. Our analysis of relative elongation rate (Fig. 3.13B) shows that the rate of

relative elongation is not zero close to the root tip which gives an estimate that cell

80

production in the meristem contributes to the cell displacement from the meristem into

the growth zone.

Color isocontour plots show that relative elongation rate varies as a function of

both space and time in Fig. 3.14. This type of representation of bivariate data allows easy

identification of spatio-temporal patterns of growth of the basal roots. The spatio-

temporal isocontour of relative elongation rate also explains the large standard deviations

in Fig. 3.13B. Since the length of the growth zone as well as rate of elongation change

with time, grouping data from the entire duration of the experiment introduces

variability, resulting in large standard deviation in mean relative elongation rate (Fig.

3.13B).

Identification of the root edge allows us to not only locate the root midline but

also measurement of root diameter. It is interesting to note that root diameter for this

example study remained nearly constant during the nearly 6 h study where as root length

grew by 3.5 mm (Fig. 3.15). The root midline was used to estimate the curvature of the

root as it grows (Fig. 3.16A). The measurement of root curvature is very important for

the study of graviresponse. When combined with root diameter, root curvature can also

be used to calculate differential growth ratio between two sides of a bending root,

because a root can only bend if one side grows more than the other side. In this case since

the bending of the root is minimal, the differential growth ratio is also minimal with the

upper edge growing 2-4% more than the lower edge of the root.

Our approach of nearly automatic image analysis and measurement using colored

images provides a new tool for application of kinematic techniques to the analysis of

spatio-temporal growth of plant organs over long time spans as long as there are

discernible patterns in the images for tracking on the organ. In the following chapter we

show more detailed results and analysis of growth and graviresponse of basal roots of

common been under various treatments using this new software.

81

ACKNOWLEDGEMENT

We would like to thank Dr. Anupam Pal for developing the kinematic program and also

helping in analyzing and discussing the results from kinematic studies.

82

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Beemster GTS, Masle J, Williamson RE, Farquhar GD (1996) Effects of soil resistance to

root penetration on leaf expansion in wheat (Triticum aestivum L.): kinematic

analysis of leaf elongation. Journal of Experimental Botany 47, 1663-1678.

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86

Figure 3.1. Photo showing the root system of a 2 d old common bean plant (TLP19) in the growth pouch. The plant shows both shallow and deep basal roots growing from the root-shoot interface.

87

Figure 3.2. Photo showing sprinkling of graphite particles on the basal roots of a 1 day old bean seedling using dropper fitted with a pipette tip.

88

Figure 3.3. Photo of the experimental setup showing the position of the camera and two flash units to capture high resolution photos of the basal root of a bean seedling. Bean seedling in the polyethylene pouch was placed inside an air-tight plexiglass box and maintained at temperatures between 25-26°C. Time lapse photography was driven by a laptop computer connected to the camera by a USB cable. Photos were captured using two flashes and light from the flashes was bounced off white papers placed on the top of the plexiglass box.

89

Figure 3.4. Screenshot of the graphical user interface of the image analysis software ‘KineRoot’.

90

Figure 3.5. Schematic showing pattern matching algorithm by finding the highest correlation coefficient between two boxes of pixels. The yellowish projections in the blue background represent the growing root whereas the black spots show the markers due to graphite particles, and (A) shows the reference image and (B) shows the current image. The red circle in (A) is being tracked (B). We choose all pixels within the red square in (A) and correlate it with the cyan boxes in (B). When the dotted cyan box is centered around the green circle in (B), the correlation with (A) is low because of mismatch of the graphite markers, whereas when the solid cyan box placed centered around the red circle, correlation coefficient with the red box in (A) reaches its maximum value identifying the new location of the point in the current image. Note, that there is no requirement for the points to be on a graphite particle for tracking.

A

B

A

B

(x0, y0)

(x*, y*)

91

Figure 3.6. Schematic showing the weights for calculating color-weighted correlation coefficients based on color of the pixel and sampled colors of the root (Rr, Gr, Br) and the background (Rb, Gb, Bb). The red , green and blue lines show the weighting factors for the corresponding colors. If the difference in color intensity between the root and the background is less than 0.2, weighting factor is assigned a value of 1, otherwise weighting factor is w is calculated by linear interpolation for a pixel whose color intensity lies between that of the root and the background. If the color intensity of a pixel is outside this range, a value of 1 or 0 is assigned based on the proximity to the root color or background color respectively.

92

Figure 3.7. Steps of automatic edge detection: (A) two dimensional Gaussian filter, (B) close up image of a basal root, (C) basal root image after noise smoothing by convolution with the Gaussian filter, (D) magnitude of the gradient of the smoothed image showing blurry edges, (E) edge enhanced by non-maxima suppression, (F) detected edges of the root shown by green and yellow lines and the centerline shown by thick white line.

93

Fig. 3.8. (A) Schematic showing projection of tracked points on the root centerline. Distance of the projected tracked points from the root tip Sp is measured along the root centerline. From the detected root edge we also measure the root diameter D as a function of distance from the root tip and time. (B) Schematic showing the spatio-temporal trajectory of the tracked points. The region where the gap between the points increases rapidly with time, identifies the growth zone.

A

Growth zone

Sp

time

B

94

Fig. 3.9. Schematic illustrating the calculation of root growth velocity with respect to the root tip. If a marker point located at Sp distance from the root tip at time t moves to Sp+ δSp distance from the root tip over time interval δt, the growth velocity of the point is given by Up= δSp/δi.

Root tip reference

S2

0

1

2 3

4 5

6

S4 S2 + δS2

S4 + δS4

Time t Time t+δt

95

Fig. 3.10. Schematic showing the growth of a small segment of the root from an initial length l to l+δl over a short period of time δt. Therefore the relative elongation rate is

defined as the fractional change in length per unit time, lrl tδδ

= .

l

l lδ+

time t

time t tδ+

96

Figure 3.11. Montage of 8 images of a basal root at 45 min intervals from a sequence of 72 images originally captured at 5 min intervals. The green and yellow lines show the edges detected by KineRoot and the bold white line shows the root midline. The red dots show the tracked points.

97

Fig. 3.12. Root length map showing the growth of the root by plotting distance of the marker points from the root tip along the root midline at 5 min time intervals.

98

Figure 3.13. (A) Root growth velocity plotted as a function of distance from the root tip. The gray dots show the growth velocity of 25 tracked points in 72 frames. The bold line shows the average growth velocity after grouping the data in bins of 0.5mm. The error bars are standard deviation bars. (B) Mean relative elongation rate plotted against distance from the root tip with standard deviation error bars.

A

B

99

Figure 3.14. Colored isocontours of rate of relative elongation plotted as a function of distance from the root tip and time. Reds, oranges and yellows show high rate of elongation whereas light and dark blues show low/zero rate of elongation.

100

Figure 3.15. Mean root diameter plotted as a function of distance from the root tip. The error bars show standard error.

101

Figure 3.16. (A) Mean root curvature and (B) differential growth ratio between the upper and lower sides of the root plotted as a function of distance from the root tip. Positive curvature and differential growth ratio greater than 1 indicate downward bending and vice-versa. The error bars show standard error.

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Fig. 3.17. Two images of the root at (A) the initial time and (B) after 6 h showing the spreading of the marker points due to growth.

CHAPTER 4: GROWTH AND CURVATURE OF BASAL ROOTS OF COMMON

BEAN (PHASEOLUS VULGARIS L.) ANALYZED USING

KINEMATIC APPROACH

Paramita Basu1, Anupam Pal2, Jonathan P. Lynch1, 3, Kathleen M. Brown1, 3

1Intercollege Program in Plant Physiology, Penn State University, University Park, PA

16802 USA; 2Department of Mechanical Engineering, Penn State University, University Park, PA

16802 USA; 3Department of Horticulture, Penn State University, University Park, PA 16802 USA.

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ABSTRACT

Using a newly developed image-analysis program KineRoot, we measured root

growth and curvature of basal roots of common bean using a kinematic approach.

Although computer-assisted kinematic analysis has been applied to primary root growth

of Arabidopsis, it has not been employed for study of plagiogravitropic growth or for

thicker-rooted species. Use of KineRoot permits study of spatio-temporal patterns of

growth of bean basal roots in a reliable, semi-automated way while minimizing user

interventions to allow large scale experiments. We identify and measure the local patterns

of root growth and graviresponding zones of the basal roots, investigate the velocity

profiles within these zones and determine how these zones are affected by low

phosphorus availability and ethylene treatment. We observe that basal roots accelerate

growth rate of the upper whorls at the cost of lower growth rate in lower whorls in

response to low phosphorus availability. Apart from root growth, one of the most

important aspects of this study is to characterize the bending of the basal roots which

leads to graviresponse and reflects shallowness or deepness of basal roots. Root curvature

results from differential growth between upper and lower edges of the root, and the

direction of this curvature varied over time, producing a waving motion. Therefore study

of spatio temporal patterns of differential growth ratio of a growing root allows

identification and measurement of root bending zones and bending amount. Our results

show that ethylene and MCP treatments do not alter local root curvature, but alter the

span and duration of the bending of the root upward or downward which causes altered

response to gravity, thereby producing shallow and deep roots respectively. The results

from this study show new aspects of plagiogravitropic response of basal roots which have

not been observed before.

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INTRODUCTION

Root architecture, i.e. the three-dimensional spatial configuration of roots, varies

greatly with genotype and environment, influencing plant adaptability and productivity.

In common bean, basal roots emerging from the root-shoot interface, together with the

primary root, form the scaffold of the bean root system. The growth angle of the basal

roots is a major determinant of topsoil exploration, and therefore efficient soil resource

acquisition, especially in environments with phosphorus limitation (Bonser et al. 1996;

Liao et al. 2001). Genotypes vary in basal root growth angle, and some genotypes

respond to phosphorus availability by becoming shallower or deeper (Bonser et al. 1996;

Liao et al. 2001). The position of origin (whorl) is a major determinant of growth angle,

with roots arising from upper whorls growing more shallow than those from lower whorls

(Chapter 2). Growth dynamics responsible for basal root growth and response to gravity

are the subject of this chapter.

Despite intense research on root gravitropism, the detailed mechanism behind the

perception and response to gravity is not yet clearly elucidated. The process of

gravisensing occurs by the perception of gravity stimulus in the root cap statocytes (Sack

1991), followed by graviresponse leading to the growth response in the elongation zones

of the root (Baluska and Hasenstein 1997). The Cholodny-Went theory has been

established as the framework for studying root gravitropism according to which

downward curvature of roots in response to gravity is induced by asymmetric

redistribution of auxin within the elongation zone with accumulation of higher auxin

content along the lower flank of the bending root. Since the optimal concentration of

auxin necessary for root growth is much lower than that for shoot growth (Eliasson et al.

1989), higher auxin content would be inhibitory to root growth i.e. roots are more

sensitive to auxin than shoots. This indicates that increased auxin content in the lower

side of a bending root would result in localized growth inhibition, thereby leading to

downward curvature.

When a vertical root is gravistimulated i.e. the root is reoriented to a certain angle

from the vertical, root curvature is initiated and the root starts to bend until the root tip is

aligned towards the gravitational vector. By using computer-based video digitizing

106

system for tracking marker beads along the surface of maize roots, Ishikawa et al. (1991)

observed that a group of cells termed distal elongation zone (DEZ), lying between the

root apical meristem and the central elongation zone (CEZ), exhibit enhanced elongation

rate in a gravistimulated root. The curvature which initiates in the DEZ is the result of

inhibition of elongation in DEZ and CEZ on the lower flank as well as acceleration of

cell elongation in the DEZ on the upper flank of the gravistimulated root (Mullen et al.

1998a; Wolverton 2002). When the bending root comes to a vertical position, the growth

resumes to the symmetrical pattern (Evans and Ishikawa 1997). However, in vertically

growing roots, the maximum rate of cell elongation occurs in the CEZ with a growth rate

three times more than in the DEZ (Ishikawa and Evans 1993).

To analyze the mechanism of root growth and graviresponse, it is important to

identify the regions of the root where growth and bending take place, and also measure

the rate/amount of growth/curvature in space-time coordinates under different treatments.

The method by which this is done is called the ‘kinematic’ analysis. Kinematics is an

aspect of dynamics which involves the study of physical motion (acceleration and growth

velocity) without reference to the forces resulting in the movement (Gandar 1983).

Kinematic analysis has been used by a number of researchers in investigating the growth

zones of an elongating root. This approach has been employed in the study of primary

root growth for a long time (Goodwin and Stepka 1945; Erickson and Sax 1956) and has

become more established in studying both temporal and spatial distribution of a growing

root in the last couple of decades (Erickson and Sax 1956; Goodwin and Avers 1956; Silk

and Erickson 1979; Gandar 1980, 1983; Pahlavanian and Silk 1988; Beemster et al. 1996;

Sacks et al. 1997; Beemster and Baskin 1998; Walter et al. 2002; Ma et al. 2003; van der

Weele et al. 2003). Besides root, the growth profiles of elongating leaf, stem, perianth

etc. have also been analyzed by using kinematic approach (Gould and Lord 1989;

Bernstein et al. 1993; Ben-Haj-Salah and Tardieu 1995; Fiorani et al. 2000; Tardieu et al.

2000; Girousse et al. 2005; Kakanova et al. 2006). Application of the kinematic approach

in such diverse studies shows the utility of the method in understanding the details of

plant growth.

107

To investigate the mechanism behind the longitudinal growth of a root, most

studies depend on the spatial analysis of tissue expansion rate of the elongating zone (Silk

1992) which allows accurate analysis of local events resulting in root elongation. The

expansion growth of a root accelerates and decelerates within a zone of few millimeters

in length. Organ growth is determined by both cell expansion as well as cell division

leading to cell production throughout the growth zone at a given time (Beemster and

Baskin 1998). For quantifying root growth, Walter et al., (2002) applied the tensor

method of image sequence analysis based on intensity gradients where they obtained

velocities at relatively few pixels, resulting in extensive interpolation. Recently the

RootflowRT method (van der Weele et al. 2003) has been developed for measuring the

expansion profile of root elongation at high spatio-temporal resolution by combining the

tensor method with a robust matching algorithm for attaining confident measurements

from more than 50% of pixels. Using these techniques for the quantification of expansion

rates of roots, the growth zone of root can be divided into two distinct zones, an apical

region with steadily increasing velocity and a subapical zone with steeply increasing

velocity with an abrupt transition zone in between (van der Weele et al. 2003). Although

these image sequence analysis methods are elegant in their applications for analysis of

growth in roots, shoots and leaves, they heavily depend on visible natural patterns on the

plant organ. For thicker rooted species like bean where the epidermal cells are not visible

under normal magnification and resolution of microscope, lack of natural patterns on the

root poses a big challenge in estimating local root growth and curvature using these

methods. Although graphite particles add visible markers on the roots, the traditional way

of carefully placing the markers and then tracking them in space-time is very tedious and

does not permit study of a large number of roots. As the roots grow, the graphite particles

also separate in the growth zone, reducing the trackable patterns. Therefore if the tensor

structure method is applied to the kinematic study of bean basal roots sprinkled with

graphite particles, it will be able to calculate root velocity at very few pixels in the

growth zone even when coupled with the robust matching algorithm because of severe

lack of patterns. Therefore a new image analysis method was developed where patterns

from neighboring pixels were used to automatically track marker points resulting in

108

velocity measurements even in locations where there are no visible patterns. Chapter 3

describes the image analysis method in detail.

We employed this non-invasive method of kinematic analysis to measure basal

root growth and curvature in response to gravity. Our objectives were to (1) identify the

growth and graviresponding zones of the basal roots, (2) investigate the velocity profiles

within these zones, (3) find the relationship between the growth and the graviresponding

zones, (4) study the time evolution of these zones, and (5) determine how these are

affected by low phosphorus availability and ethylene treatment.

MATERIALS AND METHODS

Plant Culture

Common bean (Phaseolus vulgaris L.) genotype TLP19 developed for tolerance


Colombia) was employed for this study. TLP19 has an indeterminate bush habit i.e. Type

II growth habit. TLP19 produces shallow basal roots and within one plant, we observed

shallow basal roots emerging from the top (closest to the shoot) whorls (whorl 1 and

whorl 2), with progressively deeper basal roots emerging from the lower whorl (whorl 3).









transferred to a sheet of 30 x 24 cm blue germination paper (Anchor Paper Co., St. Paul,

MN, USA) stiffened by attaching perforated plexiglass sheets to stabilize the root system.

The bottom of the blue paper with plexiglass was placed to allow direct contact with the

nutrient solution containing high (1 mM) or low (0 mM) phosphorus as described above.

109

The germination paper containing seedling was suspended in nutrient solution and

covered with aluminum foil to prevent illumination of the roots.

Treatment with ethylene and inhibitors of ethylene action

For ethylene treatment the growth pouch containing the bean seedling in low or

high phosphorus nutrient solution was placed inside a water-sealed plexiglass chamber

(37 L in volume). The seedling was treated with 0. 6 ul L-1 ethylene gas 36 h after the

emergence of the basal roots in the growth pouch and continued for 6-8 h of basal root

growth during which the basal root tends to respond to gravity. It should be noted that to

maintain uniform growth conditions e.g. temperature, humidity etc. the controls were also

placed inside the plexiglass box during time lapse photography.

The ethylene action inhibitor, MCP (EthylBloc, Floralife Inc., Walterboro, SC,

0.43% 1-methylcyclopropene) was used to test the role of ethylene in high or low

phosphorus availability. The plants were kept inside the similar water sealed plexiglass

chamber (37 L in volume). MCP gas was released through the reaction of EthylBloc

powder added to a plastic weighing plate inside the chamber and water added to the plate

by a syringe inserted through a rubber stopper. The ratio of EthylBloc powder to water

was calculated to be 4 mg EthylBloc per 0.08 ml water per liter air space.

Imaging procedure

Graphite particles were sprinkled on the roots carefully without disturbing the

plants and these particles created patterns that could be followed in image analysis on the

otherwise uniformly colored basal root (Chapter 3). During this procedure extra

precaution was taken not to touch the roots and also not to change the orientation of the

seedling with respect to the gravity because touching the root or changing their

orientation with respect to gravity may damage/change their natural behavior. We

checked the effect of adding graphite particles on the length of basal roots and compared

with the controls (Fig. 4.8. in Appendix). Images of root systems were captured at fixed

intervals (5 min) using a high resolution (6 Megapixel) digital single lens reflex camera

(Nikon D70s) fitted with 105 mm Nikkor micro lens. Images were captured for 4-6 h.

110

The captured images had a resolution of 10-20 µm/pixel. Figure 3.3 (Chapter 3) shows a

photo of the image capturing setup. The seedling in the growth pouch was placed in an

airtight plexiglass box maintained at 25-26 °C. Photographs of the seedlings were

captured from outside the plexiglass box. The camera was triggered at fixed intervals of

time by a laptop computer through a universal serial bus (USB) cable. Plants were grown

in complete darkness and photos were captured using the camera’s flash to minimize

light exposure of the roots. To avoid shadows from direct flash which interfere with

image analysis, light from the flashes was bounced off the white paper placed on the top

of the plexiglass box. A ruler was attached to the supporting plexiglass sheet for

calibrating pixel dimensions into millimeters.

Measurements

Using a new semi-automated image analysis software, KineRoot (see Chapter 3

for details), marker points are tracked on the root in time. Initially the user selects 10-15

marker points along the body of the root which are then spatially interpolated to generate

a total of 25 marker points. KineRoot automatically tracks the position of these marker

points in all of the images by matching patterns surrounding the marker points. After the

marker points are tracked in all images, the edges of the root are identified by KineRoot

using an edge detection algorithm. From the detected edges of the root, the midline is

calculated by averaging the two edges which is used as the longitudinal axis for

measurement of local root growth velocity. The marker points are projected on the root

midline, and distance of the projected marker points from the root tip along the midline

are measured. Local root velocities relative to the root tip are then calculated as,

( , ) s s s sv s tt t

δ δδ δ

+ −= = (1)

where a marker point at distance s from the root tip moves to s+δs distance from the root

tip over time δt with velocity v(s, t). Spatial derivative of root growth velocity gives the

relative elongation rate of the root

( , ) ve s ts

δδ

= . (2)

111

To calculate a meaningful derivative of growth velocity, a smoothed curve was fitted to

the growth velocity data following the overlapping polynomial smoothing procedure used

by Beemster and Baskin (1998) at every time step.

After determining the root midline, the curvature of the root κ is calculated as the

reciprocal of the radius of curvature R of the root midline at point (x, y),

( )

2

2

3/ 22

1

1

d ydx

dydx

Rκ = =

+

. (3)

It should be noted here that (x, y) is the coordinate of a point on the root midline in the

fixed two-dimensional Cartesian frame of reference attached to the plexiglass sheet

holding the germination paper. Therefore (x, y) coordinates of all points lying on the root

midline identify the shape and location of the midline. On the other hand s is the

coordinate of a point on the root midline in the moving one dimensional curvilinear frame

of reference attached to the root tip. Coordinate s of any point on the root gives the

distance from root tip along the root midline for calculation of growth but does not

provide the shape of the root. Therefore s cannot be used to calculate the curvature.

To calculate the root diameter d at distance s from the root tip, a line locally

perpendicular to the root midline is drawn. The distance between the two points of

intersection of the two edges with this perpendicular line is the root diameter at distance s

from the root tip. As a root responds to gravity and bends toward gravity, one side of the

root grows more than the other side. Therefore the ratio of local growth rate along the

two edges of the root can be used to characterize graviresponse of a root. Following Silk

and Erickson (1978), the differential growth ratio of two arcs of length δsu and δsl on the

upper and lower edges of an element of bending root can be calculated as

22

u

l

s ds d

δ κδ κ

+=

−. (4)

112

RESULTS

Time history of root growth rate

Figure 4.1 shows the overall basal root growth rate as a function of time in high

and low phosphorus nutrient solutions for control, ethylene and MCP treatments during

the kinematic study. Data from tracking of the root tip were grouped in bins of 1 h, and

averages and standard errors for each bin were calculated, and plotted in Fig. 4.1. During

the time of the study, rate of root growth for most treatments tends to remain nearly

constant for basal roots emerging from whorl 1. However, in phosphorus deficient

nutrient solutions, roots from whorl 1 of controls had a low rate of growth to begin with

and then increased rapidly. Under low P treatment, ethylene inhibited growth rate in

whorl 1 (orange line), whereas MCP enhanced growth rate (magenta line), which then

drops after a certain period. In phosphorus sufficient conditions, control, ethylene and

MCP treatment all show very similar time course of root growth rate for whorl 1. In low

P, growth rate of roots emerging from whorl 3 in controls was less than all other

treatments, whereas application of MCP in low P (magenta line) increased growth rate

compared to other treatments in whorl 3. While MCP treatment increased growth rate in

low P roots from whorl 3, ethylene treatment reduced growth rate under low P treatment.

Root growth velocity

Although overall growth rate of the entire root in Fig. 4.1 indicates the effects of

different treatments on basal root elongation, for understanding the details of root growth

it is important to identify the growth zone and measure the local patterns of growth.

Toward this end Fig. 4.2 shows the velocity profiles of bean basal roots as functions of

distance from the root tip. In the frame of reference fixed with the root tip, any point on

the root moves away from the root tip as the tissue in between grows. Therefore, the

further the point is from the root tip, the higher the velocity. To calculate the average

velocity profiles, data from smoothed velocity profiles of individual pairs of images were

grouped in bins of 0.5 mm. Each of the curves represents data collected from 8-13 roots

obtained from 3-5 plants per treatment for 4-6 hours at 5 min intervals. Although

temporal variations in growth velocity were observed in a few roots, there was no

113

consistent temporal pattern in growth velocity that could be extracted. Therefore the

curves in Fig. 4.2 not only show average of multiple roots, but also averages over time.

Figure 4.2 shows that in whorl 1, growth velocity tends to increase up to about 6 mm

from the root tip, then slows down for next 2.5 mm followed by another increment before

reaching a constant value by about 13.5 mm from the root tip in all but ethylene treatment

under low P. Up to about 4 mm from the root tip, all treatments show similar growth

velocity and then separate into two groups— a higher velocity group comprised of

controls and MCP treatments in low P and a lower velocity group consisting of the rest.

At 9 mm from the root tip, the groups merge and the second velocity increment begins.

This second elongation zone is rather high for controls under low P (blue line) and can

therefore explain the high overall growth rate for controls in low P in whorl 1. Under

ethylene treatment in low P (orange line), root growth velocity is reduced and the second

elongation zone does not exist.

Figure 4.2 also shows growth velocity averages from roots in whorl 3 where the

notable difference with whorl 1 is that this second elongation zone of the root does not

appear in any of the treatments. Furthermore, growth velocity at the end of the elongation

zone (>14 mm) is higher in whorl 3 compared to whorl 1 for all treatments except

controls in low P (blue line). Similar to Fig. 4.1, growth is enhanced in whorl 1 for

controls under low P and this enhanced growth is compensated in whorl 3. Application

of MCP strongly increases growth velocity in low P but only slightly increased it in high

P (Fig. 4.2). Ethylene increases growth velocity in low P but reduces it in high P.

Relative elongation rate

The spatial derivative of root growth velocity with respect to distance from the

root tip in Fig. 4.2 is the relative elongation rate, which is shown in Fig. 4.3 for whorls 1

and 3 under different treatments. The relative elongation rate quantifies the local

expansion rate of tissue along the body of the basal roots. As anticipated from Fig. 4.2,

roots in whorl 1 show a bimodal growth zone in all but ethylene in low P treatments

(orange line). The peak closer to the root tip spans about 6-7 mm with a consistent

relative elongation rate for all treatments, but the second peak is highly sensitive to

114

different treatments, and varies in both magnitude and span. But in whorl 3 except for

controls in high P (red line), none of the other treatments generated a detectable bimodal

relative elongation peak. However, this unimodal relative elongation zone of the roots of

whorl 3 does not make the growth zone shorter compared to whorl 1, rather it joins the

two growth zones. In both whorls 1 and 3, the peak elongation zone is located at about

2.5-3 mm from the root tip. While in whorl 1 rate of relative elongation drops rapidly and

then increases to form the second peak, in whorl 3 rate of relative elongation

monotonically declines.

To understand the bimodal shape of the relative elongation zone in whorl 1, it is

important to examine the data not only as a function of distance from the root tip, but also

as a function of time. To illustrate the spatio-temporal variations in relative elongation

rate, Fig. 4.4 shows, as an example, color isocontours of average relative elongation rate

of the roots of whorl 1 in controls under high P and low P. The reds, oranges and yellows

show relatively high rate of relative elongation whereas the blues and greens show

relatively low values. The relative elongation zone in low P is spread out between 2-13

mm with clearly distinguishable peaks. In high P the peak elongation zone near the root

tip maintains a consistent magnitude before breaking up in to two peaks after 120 min.

But the second peak which is observed between 6-11 mm in Fig. 4.3 does not correspond

to this yellow-orange band between 3.5-6 mm in Fig. 4.4. Rather it is the average of the

high relative elongation rates discontinuously spread between 6 and 11 mm from the root

tip in Fig. 4.4. Furthermore, presence of large amount of yellows and reds in low P

indicate higher relative elongation rates in low P compared to high P (Fig. 4.4). Both

isocontour plots show that relative elongation rate has underlying time dependence, apart

from the variability caused by root to root variations— relative elongation rate is low at

the beginning and then increases over time.

Root curvature and differential growth

Apart from root growth, one of the most important aspects of this study is to

characterize the bending of the basal roots which leads to graviresponse. In many of the

studies we observed that as the root grows, it develops a wavy motion with the root

115

periodically bending upward and downward. Figure 4.5 shows an example of this wave

motion of the root. 16 images of a root from whorl 1 captured at 20 min intervals were

digitally processed by Adobe Photoshop 7.0 TM (Adobe Systems Inc, San Jose, CA) and

superimposed in Fig. 4.5. The trajectory of the root tip is shown by red dots on the tip and

joining the red lines.

When a root bends downward, root growth is higher along the upper edge of the

root compared to the lower edge, and vice versa. Therefore, for downward bending,

differential growth ratio δsu/δsl > 1 and for upward bending δsu/δsl < 1. Measurements of

curvature and root diameter (results not shown) allow calculation of the differential

growth ratio between the upper and lower edges of the roots using equation (4). Two

examples of measured differential growth ratios are shown in Fig. 4.6 where spatio-

temporal changes in differential growth ratio are plotted as color isocontours for two

control roots—one grown in high P and the other in low P. The inserts show three

snapshots of the growing roots. The timings of the images with respect to the isocontour

plots are identified by magenta arrows (Fig. 4.6). In both high and low P treatments, the

left and right side images show that the root is bending downward resulting in differential

growth ratio greater than 1 (red-yellow colors), while the middle images show a subtle

bend upward resulting in differential growth ratio less than 1.0 (blue colors). In this

example, differential growth is nearly uniform along the length of the root in high P as

shown by mostly vertical color bands, whereas the direction of curvature of the root

changes along the body of the root in low P, as colors change in the vertical direction.

Both isocontour plots show periodic temporal changes in differential growth ratio

above and below 1 indicating wavy motion of the root. By applying frequency analysis

using Fourier transform of the differential growth ratio as a function of time at fixed

distances from the root tip, we identified the time periods of the wavy motion of roots

under different treatments which are listed in Table 4.1. Interestingly, application of MCP

tends to reduce the waviness of root growth and no dominant frequency of waviness

could be identified. Ethylene increased the time periods of upward-downward bending

for whorl 1 in both high and low P treatments compared to controls. But in the whorl 3,

compared to controls, ethylene only affected waviness under low P treatment.

116

The spatio-temporal oscillatory changes in differential growth ratio around 1

indicate that averaging differential growth ratio either in space or time will even out the

variations associated with upward or downward bending. Therefore from the isocontour

plots, the spatio-temporal areas where the root is bending upward vs. downward are

identified by setting threshold differential growth ratios of 0.99 for upward bending and

1.01 for downward bending. The solid black lines on the isocontour plots in Fig. 4.6

identify the downward bending regions (δsu/δsl > 1.01) and the areas enclosed by the

dotted black line show the spatio-temporal area of upward bending (δsu/δsl < 0.99).

Differential growth ratio 0.99 ≤ δsu/δsl ≤ 1.01 indicates a straight growing root with

negligible curvature. From each kinematic study of bean basal roots, we measured the

percentage of spatio-temporal areas of upward and downward bending of the roots, and

also calculated the average differential growth ratios during upward and downward

bending. Figure 4.7A shows the percentage of spatio-temporal area for upward and

downward bending of roots emerging from whorl 1 under ethylene and MCP treatments

relative to controls, whereas Fig. 4.7B shows the average differential growth ratios during

upward and downward bending for controls, ethylene and MCP. The dominant bending

pattern of the roots is toward gravity (empty bars) with only 8-10% spatio-temporal areas

occupied by upward bending in controls whereas downward bending is 50% of the

spatio-temporal area (data not shown). Under high phosphorus conditions, the spatio-

temporal area of upward bending increases more than 56% compared to controls,

whereas in low P the increase in upward bending is 25%. On the other hand application

of ethylene causes a reduction in spatio-temporal area for downward bending by 3-5%.

MCP, on the other hand, reduces spatio-temporal area for upward bending by 64% in

high P and 41% for low P while increasing downward bending areas by about 4-5%

compared to controls. Although the spatio-temporal areas of upward vs. downward

bending are influenced by different treatments, the average differential growth ratio

during either upward or downward curvature remain constant irrespective of treatments

as shown in Fig. 4.7B, albeit with high root to root variability.

117

DISCUSSION

Using a novel image analysis system, this kinematic study shows local patterns of

root growth and bending of basal roots for common bean. We grew plants under

controlled environment with the specific objective of studying root growth and

graviresponse in different phosphorus treatments and hormonal stimuli like ethylene. Our

earlier studies show that ethylene application influences both basal root growth and

gravitropic angle (Chapter 2). In this kinematic study, the ethylene action inhibitor MCP

was applied to explore the role of ethylene in regulating plagiogravitropic growth locally.

The results from this study show new aspects of plagiogravitropic response of

basal roots which have not been observed before. One of the most interesting

observations from this study is the response of the basal roots in controls under

phosphorus deficient conditions. Figures 4.1 and 4.2 show that in low P treatment, plants

respond by accelerating growth of the roots in upper whorls (blue lines). Our earlier

measurements (Fig. 2.1 in Chapter 2) indicate that basal roots from whorl 1 are

significantly shallower than those of whorl 3. Therefore by enhancing growth of roots

from whorl 1, plants try to acquire limited nutrients like phosphorus more efficiently

from the top soil. This elevated growth rate of the roots in whorl 1 is compensated by a

reduction in growth rate in whorl 3 (Figs. 4.1 and 4.2). This is consistent with the earlier

observations in the field study of increased total basal root length of shallow roots in the

upper soil horizon compared to deep roots (Liao et al. 2001; Liao et al. 2004).

A close look at the rate of relative elongation of roots from whorl 1 in Fig. 4.3

reveals that this additional growth rate of basal roots in low P is a result of enhanced

elongation in the second elongation zone which is apical to the root maturation zone. But

in whorl 3, the second elongation zone does not exist. In addition, the first elongation

zone is reduced compared to other treatments. This phenomenon of accelerated growth of

controls in low P is visualized better when studied in spatio-temporal coordinates using

color isocontour in Fig. 4.4. Clearly, in low P the elongation zone is larger and also the

rate of elongation is higher in whorl 1 which might account for adaptive behavior of basal

roots from different whorls under phosphorus deficient conditions.

118

Application of ethylene inhibits root growth in both high and low P conditions

for whorl 1 and whorl 3 (Fig. 4.2) which is consistent with our findings presented in Fig.

2.8 of Chapter 2. The reduction in growth under ethylene treatment in low P is a result of

nonexistence of the second elongation zone even in whorl 1 (Fig. 4.3). In high P

conditions, ethylene causes the second elongation zone to move further from the root tip

and makes this second elongation process very infrequent (data not shown) resulting in

an overall reduction in root growth. Although we notice similar behavior in whorl 3

under ethylene treatment, the deviations from controls due to ethylene are relatively less.

On the other hand, application of the ethylene action inhibitor MCP causes the root

growth rate to increase in low P but root growth rate remains similar to controls in high P

(Fig. 4.1), a result which is contradictory to what we observed in other studies (Fig. 5.17

in Appendix of Chapter 5). This inconsistency could be due the fact that to get good

quality trackable images, imaging had to be started immediately after the application of

MCP with no time for pre-treatment which might have caused delayed activation of MCP

effects.

Together with prior growth angle, root curvature determines shallowness or

deepness of basal roots. Root curvature results from differential growth between upper

and lower edges of the root. Therefore, study of spatio temporal patterns of differential

growth ratio of a growing root allows identification and measurement of root bending

zones and degree of bending. Earlier works have shown that as the root grows it follows a

wavy path (Simmons et al. 1995; Shabala and Newman 1997; Mullen et al. 1998b; Buer

et al. 2000; Buer et al. 2003). Similar observations are made in this study also—an

example is shown in Fig. 4.5. However, not every image clearly displayed this kind of

wavy motion. But measurement of differential growth ratios in kinematic studies show

subtle wavy motions of the roots, even when it may be difficult to identify them directly

on the images, as shown by the changes in the color in isocontour plots of differential

growth ratio in Fig. 4.6. Through systematic frequency analysis using Fourier transforms,

the periodicity of the waviness of root growth was measured for different treatments and

tabulated in Table 4.1. In agreement with Buer et al. (2003), the periodicity

119

measurements show that application of ethylene alters the waviness of root motion. But

surprisingly, MCP nearly eliminates waviness of root motion.

The waviness of root growth also makes it difficult to average differential growth

ratios over space or time (Fig. 4.6) for further quantification of root bending, because

alternating high and low values when averaged together flatten the profiles of differential

growth ratio between the upper and lower edges of the root. Therefore from each

isocontour plot, upward and downward bending spatio-temporal areas were identified

(Fig. 4.6). If differential growth was higher than 1%, root growth was identified as a

curved growth; otherwise root growth was categorized as a straight. Therefore,

differential root growth ratio greater than 1.01 was identified as downward bending and

0.99 or lower was identified as upward bending. These thresholds were then used to

calculate the space-time areas of upward and downward bending (Fig. 4.6). Fig. 4.7A

shows a 56% increase in the spatio-temporal area for upward bending due to ethylene

whereas application of MCP reduces upward bending by up to 64% compared to controls.

Not surprisingly, the basal roots from whorl 1 respond to gravity and bend mostly toward

gravity and occasionally upward. However even the subtle upward movement of the

basal roots over a long period of time and the longer length of the root can alter root

architecture. In agreement with our earlier results (Figs. 2.4 and 2.6 in Chapter 2), Fig.

4.7A shows that ethylene increases the percentage of time and root length contributing to

upward bending and reduces the spatio-temporal area for downward bending resulting in

shallower roots, while maintaining similar curvatures as controls (Fig. 4.7B). On the

other hand, MCP makes the roots deeper by reducing the time and length of the roots

contributing to upward bending and increasing the time and length of the roots

contributing to downward bending—a result similar to our earlier observations (Fig. 2.4).

Although effects of ethylene or MCP on root architecture in Chapter 2 show the overall

behavior, these new results explain the dynamics associated with graviresponse. In a

seemingly surprising discovery we find that local root curvature remains unaffected by

ethylene or MCP treatments, but the spatio-temporal duration associated with upward vs.

downward motion of the root is affected which causes the root to respond to gravity

differently under different treatments. Since differential growth ratio remains unaltered,

120

local elongation rates along upper and lower edges of the roots under ethylene/MCP

treatments are equally affected in each edge.

To understand the results from this study, it is important to distinguish the

difference between local measurements in space and time with global measurements such

as total root length or angle over a longer period of time. For example, Fig. 4.1 shows

growth rate as a function of time, therefore time integration of each curve in Fig. 4.1, i.e.

the area under each curve, provides total root growth over the study period only,

irrespective of the initial length of the roots. A root can grow fast for a short period of

time and then can slow down resulting in the same overall growth compared to another

root which grows at a steady pace. Therefore, while overall root growth rates presented in

Fig. 4.1 regulate total root length, this is not the only determinant because these results

are based on a relatively short time of 4-6 h, and further changes in growth rate over time

may significantly alter the total growth of the roots.

Basal root growth angle studies in pouch experiments in Chapter 2 show that

there is no statistically significant difference in behavior of basal roots of different whorls

under low and high P. But Figs. 4.1 and 4.2 show that locally root growth rate is higher in

whorl 1 and lower in whorl 3 under low P. This raises the question that if the local pattern

of supposedly plastic behavior of roots can be seen in kinematic studies, then why it is

not observed in overall root growth and growth angle studies in the growth pouch? There

could be several reasons behind this seemingly contradictory behavior. In kinematic

studies root growth was analyzed over a relatively short period of time (4-6 h) during

which the basal roots exhibited a tendency to adapt to low P conditions by enhancing

growth rate of roots from whorl 1. But in the long run this behavior is not visible. The

germination paper which supplies nutrients to the seedling absorbs nutrient solution, and

through capillary action distributes nutrients uniformly to all the roots. So even if there is

phosphorus deficiency in the nutrient solution, all the basal roots, irrespective of

shallowness, get the same amount of phosphorus. But in the field under phosphorus

depleted conditions, more phosphorus is available in the topsoil compared to the subsoil.

This difference between field condition and growth pouch in terms of nutrient gradient

might account for the changed behavior of the basal roots in the long run in laboratory

121

experiments, although initially they show adaptive behavior in kinematic studies. In

addition, the growth pouches are 24 cm wide. Therefore as the basal roots move near the

edge of the paper they are forced to bend resulting in changes in growth angle.

Using detailed kinematic analysis this paper makes surprising discoveries which

could not be studied using other approaches. Firstly, in response to low phosphorus

treatments plants accelerate growth rate of the upper whorls at the cost of lower growth

rate in lower whorls. Ethylene and MCP do not alter local root curvature, but alter the

span and duration of the bending of the root upward or downward and thereby produce

shallow and deep roots respectively.

ACKNOWLEDGEMENT

We would like to thank Dr. Anupam Pal for developing the kinematic program and also

helping in analyzing and discussing the results from kinematic studies.

122

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126

Fig. 4.1. Time course of basal root growth rate from whorl 1 and whorl 3 grown in high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.

127

Fig. 4.2. Spatial profiles of growth velocity of basal roots as a function of distance from the root tip of whorl 1 and whorl 3 grown on high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.

128

Fig. 4.3. Spatial profiles of relative elongation rate (or strain rate) versus distance from the root tip for the basal roots of whorl 1 and whorl 3 grown on high (1 mM) and low (0 mM) phosphorus nutrient solutions. The ethylene and MCP treated roots were exposed to 0.6 µl/L ethylene gas and 1 µl/L MCP gas respectively 36 h after the emergence of the basal roots. Data from 8-13 basal roots of 3-5 plant per treatment for 4-6 hours at 5 min intervals were grouped in bins of 0.5 mm. The values shown are means of each of the bins ±SE.

129

Fig. 4.4. Color isocontour plot of relative elongation rate of basal roots emerging from whorl 1 in controls under high P and low P conditions showing the spatio-temporal variation in relative elongation rate. The colors work as a separate axis apart from the horizontal or vertical axes, and show space time locations of high (red, orange, yellow) and low (blue, green) magnitudes of relative elongation rate. The data for each plot were collected from 8 roots of 4 plants, and grouped in space-time bins of 0.5 mm x 5 min and averaged within each space-time bin.

130

Fig. 4.5. Superimposed time lapse photos of a growing basal root shot at 20 min intervals showing the wavy motion of the tip (red dots) during a 5 h period. To make all 16 images of the root visible, we only show the edges of the roots after digitally processing each image in Adobe Photoshop 7.0TM (Adobe Systems Inc, San Jose, CA).

131

Fig. 4.6. Examples of spatio-temporal color isocontour plot of differential growth ratio between upper and lower edges of a basal root of whorl 1 in a control plant under low P and high P conditions. The red and yellow colors show differential growth ratio > 1, i.e. bending downward while blue shows differential growth ratio < 1, i.e. bending upward. The inserts show photographs of the root at specific times identified by the dotted magenta arrows. The spatio-temporal regions enclosed by solid black lines identify space-time locations of downward bending roots with differential growth ratio δsu/δsl > 1.01, and the area enclosed within the dotted line identifies upward bending root with differential growth ratio δsu/δsl < 0.99.

132

Fig. 4.7. Spatio-temporal comparison of bending of the basal roots emerging from whorl 1. (A) Percentage of spatio-temporal area (see Fig. 4.6) during upward (δsu/δsl < 0.99) and downward (δsu/δsl > 1.01) bending of the roots under different treatments compared to controls. (B) Average differential growth ratio during upward and downward bending of the roots. Data show mean of 8-13 basal roots of 3-5 plant per treatment ±SE.

133

Table 4.1. Periodicity of the wavy motion of the bean basal roots for different treatments identified by frequency analysis using Fourier transform of the differential growth ratio as a function of time at fixed distances from the root tip. MCP treatment resulted in elimination of detectable periodicity. Numbers show mean ± SE.

Treatment Whorl 1 Whorl 3

High P 120±11 min 180±3 min

Low P 105±5 min 108±12 min

High P + Ethylene 180±16 min 180±15 min

Low P +Ethylene 210±10 min 140±23 min

High P +MCP - -

Low P +MCP - -

134

CHAPTER 4 APPENDIX

0

1.5

3

4.5

whorl 1 whorl 2 whorl 3

Leng

th o

f bas

al ro

ots

(cm

)

control day 1control day 2before graphite day 1after graphite day 2

Figure 4.8. Graphite does not affect root growth. The graphite particles were sprinkled carefully on the basal roots which were used for the time lapse photography required for kinematic study. N= 4-5 plants ± SE.

CHAPTER 5: HORMONAL REGULATION OF GRAVITROPIC GROWTH OF

BASAL ROOTS – A CROSS-TALK BETWEEN ETHYLENE AND

AUXIN

Paramita Basu1, Jurgen Engelberth2, Jonathan P. Lynch1, 3, Kathleen M. Brown1, 3

1Intercollege Program in Plant Physiology, Penn State University, University Park, PA

16802 USA; 2Department of Entomology, Penn State University, University Park, PA 16802 USA. 3Department of Horticulture, Penn State University, University Park, PA 16802 USA.

136

ABSTRACT

Gravitropic growth of roots determines root architecture, which is essential for

efficient acquisition of soil resources. Auxin and ethylene are potential regulators of the

graviresponse of roots. Basal roots of common bean exhibit plagiogravitropic growth

which varies over time. We hypothesize that ethylene modulates the auxin effect on root

growth and plagiogravitropic curvature of basal roots. Parental genotypes and

recombinant inbred lines of common bean with contrasting basal root traits were

employed for this study. Lower whorls of basal roots had higher free auxin content and

were more sensitive to auxin inhibition of basal root growth compared to upper whorls.

However, transport of auxin from root-shoot junction using 3H-IAA shows more

transport of radiolabeled auxin to upper whorls than lower whorls. Ethylene did not affect

transport of 3H-IAA from the hypocotyls to the roots, but increased free IAA content in

the basal roots. Both ethylene and auxin make the basal roots shallower. Our results show

that auxin concentration in controls is near optimal. Application of aminovinylglycine

(AVG) or 1-methylcyclopropene (MCP) together with exogenous IAA increase root

growth and reduce shallowness in phosphorus sufficient conditions. However, AVG and

MCP do not reverse IAA-inhibition of growth in low phosphorus. These results point to a

phosphorus dependent interaction between ethylene and auxin in regulation of

elongation, but a phosphorus-independent interaction for control of growth angle.

137

INTRODUCTION

Root architecture, the three-dimensional distribution of a root system, is defined

in part by growth angles. Each and every plant organ has distinct and specific response to

gravity, which usually results in plagiogravitropic growth i.e. growth at an angle other

than 0o relative to the gravity vector. This stable angle was referred to as gravitropic set-

point angle (GSA) by Firn and Digby (1997). According to Firn and Digby, the growth of

most plant organs occurs at a stable angle determined by various factors, including

gravity itself. The basal roots of common bean are secondary roots arising from the root-

shoot interface which together with the primary root determine the scaffolding of the

bean root system. They exhibit plagiogravitropic growth which varies over time and also

in response to gravity. Growth angle of basal roots (BRGA) have been associated with

genotypic differences (Chapter 2) in acquisition of limited and immobile nutrients like

phosphorus (P) and adaptation to low-P soils (Bonser et al. 1996; Liao et al. 2001; 2004).

In addition, low phosphorus availability dramatically alters the BRGA in some genotypes

(Bonser et al. 1996; Liao et al. 2001).

Low phosphorus availability is the major constraint for plant productivity in

many terrestrial ecosystems. The growth angle of roots has important implications for

acquisition of soil resources. In common bean shallow-rooted genotypes are better

adapted to low phosphorus availability than deep rooted genotypes (Bonser et al. 1996;

Liao et al. 2001; Liao et al. 2004; Ho et al. 2005). Shallow basal roots not only increase

topsoil exploration but also produce less intraplant and interplant competition for limited

and immobile nutrients like phosphorus (Ge et al. 2000; Lynch and Brown 2001; Rubio

et al. 2001; Rubio et al. 2003).

Our previous work (Chapter 2) has shown that phosphorus stress increases the

ethylene sensitivity of basal roots, making the basal roots shallower. In addition, our

work has also shown that position of emergence of basal roots has important implications

since basal roots from upper whorls explore the upper soil horizon by becoming

shallower, while roots from lower whorls, which are less responsive to ethylene, maintain

a deeper growth angle and would explore different soil domains. Moreover, we have

shown that shallow genotypes produce a greater range of BRGAs than deep genotypes

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(Chapter 2) in the presence of ethylene, which would enhance topsoil foraging (Lynch

and Brown 2001).

Although variation in growth angles of basal roots has been observed, very little

is known about how the growth angles are regulated. While it seems certain that response

to gravity plays a major role in regulating BRGA, hormonal signals such as ethylene and

auxin most likely play a crucial role in determining genotypic differences. The

involvement of auxin in regulating curvature has been postulated by the Cholodny-Went

theory, which states that laterally redistributed auxin in a gravistimulated organ results in

a differential auxin gradient, promoting differential cell elongation on the opposite flanks

of the stimulated organ and cause downward bending (Blancaflor and Masson 2003). In

addition to its role in regulating gravitropic bending, auxin has another potential role in

controlling root elongation. Although low concentration of auxin applied to the nutrient

solution may stimulate root growth, higher concentration of exogenous auxin reduces

root elongation (Eliasson et al. 1989). Therefore, concentrations of auxin which promote

the growth of shoots inhibit growth of roots i.e. roots are more sensitive to auxin than

shoots.

Two types of auxin transport within roots have been reported: 1) a fast and non-

directional auxin transport in the phloem (Ljung et al. 2001) and 2) a slow, directional

polar transport. Non-polar transport of auxin through phloem occurs in both the basipetal

and the acropetal directions (5-20 cm/hr) coupled with transport of assimilates (sugar)

and inactive auxin conjugates (Baker 2000; Friml and Palme 2002), whereas polar

transport is specific for the cell-to cell movement (5-20 mm/hr) of free active auxin in a

directional manner. In roots, polar auxin transport occurs in two directions: acropetally

from the base of the root to the root apex through the central cylinder and basipetally

away from the root apex through the outermost epidermal and cortical cell layers (Lomax

et al. 1995; Rashotte et al. 2000). Basipetal auxin transport is required for root

gravitropism (Rashotte et al. 2000). In addition to polar transport, lateral auxin movement

across roots is stimulated by a change in gravity and this lateral transport drives

differential gravitropic growth (Muday 2001). Although the two types of transport could

be linked directly or indirectly (Cambridge and Morris 1996), the distinction between the

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contribution of non-polar and polar auxin transport in generating the auxin pool in

different tissues has not yet been clearly elucidated.

Besides auxin, another potential modulator of root gravitropism is ethylene

(Abeles et al. 1992). Although there is evidence that ethylene mediates gravitropic

responses in roots, shoots and cut-flower stems (Chadwick and Burg 1967; Wheeler and

Salisbury 1980; Lee et al. 1990; Philosoph-Hadas et al. 1996; Kiss et al. 1999; Madlung

et al. 1999; Edelmann 2002; Chang et al. 2004), other research shows that ethylene has

no effect on gravitropic response of plant organs (Harrison and Pickard 1986; Woltering

1991). Evidence suggests that production of an ethylene gradient across a gravistimulated

organ is associated with the manifestation of gravitropic bending (Philosoph-Hadas et al.

1996). However, the role of the ethylene gradient in the signal transduction mechanism

leading to the gravitropic response is still controversial (Madlung et al. 1999; Friedman et

al. 2005; Woltering et al. 2005). Although ethylene applied at low concentrations

promotes gravicurvature, continuous application at higher concentration proved to be

inhibitory in gravistimulated shoot and inflorescence (Madlung et al. 1999; Lu et al.

2002).

The importance of auxin in gravitropism, as well as the close interaction between

ethylene and auxin in various developmental processes including root development, has

already been illustrated by various authors. Extensive studies regarding the physiological

interaction between auxin (IAA) and ethylene have established that at least two kinds of

interactions might exist (Rahman et al. 2001). A well-established auxin-ethylene

interaction is that the application of exogenous auxin stimulates ethylene production

(Chadwick and Burg 1967) and the second potential interaction is that ethylene inhibits

polar and lateral auxin transport (Burg and Burg 1967; Suttle 1988). Ethylene treatment

of pea hypocoyls reduced the amount of auxin transport up to 95% (Burg and Burg 1967;

Ruegger et al. 1997). In roots, by reducing acropetal auxin transport, ethylene could

cause auxin depletion in the root apex, thereby reducing root elongation. A second

possibility is that ethylene retards polar auxin transport from the root tip to the elongation

zone, resulting in an insufficient auxin pool in the elongation zone and reducing root

elongation (Casson and Lindsey 2003). In addition, it has been shown in citrus leaves that

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ethylene treatment reduces endogenous IAA levels by increasing conjugation of IAA

(Riov et al. 1982) and that the increased auxin conjugation lowers movement of auxin

through the tissue. Increased IAA catabolism is another mechanism by which ethylene

reduces active IAA content (Sagee et al. 1990). Ethylene may affect auxin redistribution

(Lee et al. 1990) or rate of auxin transport (Burg and Burg 1967) or synthesis of auxin in

the root tip (Stepanova et al. 2005). Although the are many reports of ethylene inhibition

of auxin transport, other evidence shows that ethylene stimulates auxin transport (Morgan

and Gausman 1966). Recent work by Madlung et al. (1999) suggested that exogenous

ethylene induces a signal which either stimulates asymmetric redistribution of auxin or

alters auxin sensitivity of the cells of a gravistimulated organ, thereby regulating

graviresponse.

In this report, we investigate the possible role of cross-talk between auxin and

ethylene in regulating growth angle of basal roots (BRGA) as well as root growth. Since

ethylene regulates plagiogravitropic growth of basal roots at an early stage (Chapter 2), it

is possible that ethylene modulates auxin movement, thereby affecting the auxin gradient

needed for graviresponse. However, effects on auxin movement alone might not account

for the complexity of gravity-induced changes in growth rate and curvature patterns

during plagiogravitropic growth. There could be an interaction of auxin redistribution and

time-dependent change in auxin sensitivity as suggested by Ishikawa et al. (1991), if

auxin mediates the gravitropic response as proposed by Cholodny-Went theory.

Therefore we specifically hypothesize that ethylene modulates the auxin response during

root growth and plagiogravitropic curvature of basal roots. This could be possible either

by modification of auxin transport in the growing roots or by altering the sensitivity of

basal roots to auxin. We tested this hypothesis in plagiogravitropic basal roots of

common bean demonstrating genetic and nutrition-induced variation in basal root growth

angles.

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METHODS

Plant material

Two parent genotypes, B98311 and TLP19, were crossed by Dr. Jim Kelly at

Michigan State University to produce a population of recombinant inbred lines (RILs),

the L88 population consisting of 81 lines. The RILs descending from the cross between

these two parents share a common genetic background, yet segregate for root

architectural traits as well as adaptation to abiotic stress. In addition, they possess

commercial quality of black bean seeds. B98311 is drought-resistant Mesoamerican

genotype from the MSU breeding program and possesses a Type II growth habit and a

deep vigorous primary root (Frahm et al. 2004) and TLP19 was developed for tolerance


Colombia) and also possesses a Type II growth habit. Preliminary experiments showed

that TLP19 produces shallower basal roots both under low and high phosphorus

conditions. In addition to the parent genotypes, we used four contrasting RILs (two

shallow and two deep) in our experiments, selected based on basal root growth angles

assessed in preliminary experiments.

Comparison of growth angle of genotypes




St. Paul, MN, USA) and moistened with either low or high phosphorus nutrient solution,





transferred to growth pouches consisting of a sheet of 30 x 24 cm blue germination paper

(Anchor Paper Co., St. Paul, MN, USA) inserted into a polyethylene bag of the same size

with evenly spaced (3 cm apart) holes for aeration. Pouches were open at the bottom to

allow direct contact with the nutrient solution containing high (1 mM) or low (0 mM)

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phosphorus (P) as described above. The pouches were stiffened by attaching perforated

plexiglass sheets to stabilize the root system. The pouches were suspended in nutrient

solution and covered with aluminum foil to prevent illumination of the roots. Root

systems were photographed after 2 d growth in pouches and basal root angles were

determined using Matlab 7.0TM (Mathworks Inc., Natick, MA, USA). Growth angles of

basal roots were measured relative to the vertical, i.e. larger angles indicate shallower

basal roots.

Treatment with auxin and NPA

For experiments with auxin treatment, we conducted preliminary experiments to

determine a suitable method for application of indole-3-acetic acid (IAA) to the

seedlings. Lanolin paste has been the most widely used method for application of IAA,

however, in our case; this method was unsuitable because the preparation and application

procedures were laborious and the lanolin paste did not adhere well to the vertical

seedling. Therefore, we applied IAA in solution directly to the root-shoot interface of the

seedling growing in the pouch without disturbing the seedling. A small plastic ring (cut

from pipette tips) was attached around the root-shoot junction just above the basal root

emergence zone. The bottom of the ring was sealed with a small piece of blue

germination paper to prevent the leakage of solution added to the ring. Experiments were

conducted to determine the effect of IAA concentration on the growth angle and the

growth rate of basal roots. Solutions of IAA (0 – 40 nmol in 20 µl) were applied to the

growing seedlings twice: the first application was done immediately after the transfer of

the seedling to the pouch and the second was done 24 h after the transfer of the seedling

i.e. one day after the basal roots emerged. The roots were photographed after 24 and 48 h

and the basal root growth angles were measured as the angle between the vertical and the

line connecting the root tip positions at 24 h and 48 h using Matlab 7.0TM (Mathworks

Inc., Natick, MA, USA). Root growth (increase in length between 24 and 48 h) was

assessed from the same digital images. The experiments were repeated three times with

2-3 plants per genotype per treatment each time. The slope of the auxin dose-response

curve was estimated by the slope of the linear regression line fitted to BRGA vs. auxin

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concentration data for each genotype, each whorl position and each P-treatment, and was

defined as the auxin sensitivity.

In a separate experiment, NPA (1-N-naphthylphthalamic acid), an auxin transport

inhibitor, was applied to the seedlings to determine the dose required to examine the

effect of auxin at below-endogenous level on the growth angle and growth rate of basal

roots. Different concentrations of NPA (0 – 20 nmol) were applied in solution in the

similar way as described above.

Measurement of ethylene production

We measured endogenous ethylene production from the basal roots of the auxin

treated (30 nmol) seedlings compared with control plants. For ethylene measurement,

fresh tissue containing the basal roots was harvested 48 h after transfer of the seedlings to

the pouch. The segments were separated into three basal root whorls with a razor blade

and enclosed individually in 9 ml vials capped with septa at 25°. Ethylene was sampled

with a 1-ml syringe from the headspace of the vials 2 h later and quantified by gas

chromatography (HP6890 gas chromatograph) equipped with a flame ionization detector

and an activated alumina column, Hewlett-Packard Company, Wilmington, DE, USA).

Treatment with ethylene inhibitors

In order to assess the possible role of ethylene-auxin interaction on regulation of

growth angle, we treated the seedlings with the inhibitor of ethylene biosynthesis AVG

(aminoethoxyvinylglycine), or the ethylene action inhibitor MCP (EthylBloc, Floralife

Inc., Walterboro, SC 0.43% 1-methylcyclopropene), sometimes in combination with

IAA. For experiments with MCP, seedlings were grown as previously described in either

low or high phosphorus nutrient solution. After transferring to the growth pouches, the

seedlings were treated with 30 nmol IAA and kept inside air-tight growth chambers (118

liter plastic boxes). EthylBloc was added to a plastic weighing plate placed inside the

growth chambers and buffer was added to the plate via a syringe inserted through a

rubber stopper on the top of the chamber. The seedlings were treated for 24 h and 48 h

with MCP released through the reaction of EthylBloc powder with buffer. The basal roots

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were photographed at 24h and 48 h and the growth angles and root growth were

measured as previously described. The experiment involved 4 plants per genotype per

treatment. The ratio of EthylBloc powder to buffer was 4 mg EthylBloc per 0.08 ml

buffer per liter air space and the actual amount of EthylBloc powder was calculated based

on the volume of the growth chamber, yielding 1 µl L-1 MCP gas inside the chamber. For

AVG experiments, 60 µM AVG and 30 nmol IAA were added to the ring around the

root-shoot junction of the seedlings grown in either low or high phosphorus nutrient

solution.

Quantification of endogenous auxin

In a separate set of experiments, we quantified the amount of endogenous auxin

present in the basal roots. For this experiment, the seedlings of RILs 57 and 7 were

treated with 30 nmol IAA (determined from dose response experiment) or 15 nmol NPA

at the root-shoot interface or with 0.6 ul L-1 ethylene in low phosphorus nutrient solution.

In another set of experiment, the seedlings of RIL57 were treated with 0.6 ul L-1 ethylene

in either low or high phosphorus nutrient solution. The plants were treated with the

specified hormone after the transfer of the seedlings to the pouch and the second

application was done at 24 h after the transfer of the seedlings. The basal roots were

harvested and frozen in liquid nitrogen and then stored in -80°C for analysis of

endogenous free IAA by GC-MS/MS with methanol chemical ionization (Trace GC 2000

attached to a GCQ mass spectrometer, Thermo Finningan, San Jose, CA) as described by

Schmelz et al. (2003) and Engelberth et al. (2004) for analysis of multiple hormones

from a very small amount of tissue. For the analysis of auxin, basal rooting zones were

separated into three basal root whorls with a razor blade and put inside screw-cap vials,

each containing approximately 150 mg – 200 mg basal roots. Since the basal roots of

each plant were very small at the time of harvest, we combined basal roots from of 10-12

plants for each sample to be analyzed. The root samples were transferred to the screw-cap

FastPrep tubes (Qbiogene, Carlsbad, CA) containing Zirmil beads (Mountainside, NJ)

and the endogenous IAA was quantified from the root samples and compared with

[2H5]IAA as internal standards.

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Auxin transport analysis

Auxin transport was assessed using radioactive auxin, 5-3H-IAA (25 Ci/mmol)

purchased from American Radiolabeled Chemicals (St. Louis, MO). The stock solution

was made by diluting 5-3H-IAA (0.00004 M) with (1.5 mM) cold IAA to make a total

volume of 3 ml. 20 µl of the stock solution was placed in a plastic ring around the root-

shoot interface of a bean seedling (described above) with a pipette. Root segments were

harvested 24 h after application of 3H-IAA to evaluate the transport of labeled IAA to the

basal root segments. The basal roots of whorls 1 and 3, primary roots, the root-shoot

interface tissue segment where the label was applied, and hypocotyls were soaked

separately in vials containing 10 ml of Biosafe II, biodegradable and non-flammable,

scintillation fluid. Vials containing the samples were vortexed and incubated for 48 h at

room temperature, and counts of radioactivity from these samples were determined using

the scintillation counter (1500 Tri-carb Packard, Downers Grove, IL) for 2 min. In

another experiment, the radioactive seedlings in the pouch were treated with ethylene

inside a water-tight plexiglass chamber after application of 3H-IAA. The basal roots were

harvested after 24 h as described above.

Statistical analysis

ANOVA and calculations of response functions of IAA, NPA, AVG and MCP

were performed in SPSS (SPSS graduate pack, version 12, for Windows, SPSS Inc.).

Each experiment consisted of 4 or 6 contrasting genotypes and 2 phosphorus levels. In

some cases, where phosphorus effect was insignificant, the data were pooled over both

phosphorus treatments. Genotype, whorl position and application of hormone or inhibitor

effects were tested at P < 0.05.

RESULTS

Basal root angle depends on genotype and position of origin

Basal roots emerge within 3 d of germination from distinct whorls at the root-

shoot junction (Fig. 5.1 inset). All the genotypes typically develop three whorls of basal

146

roots and we designate the whorls bearing basal roots from top (closest to the shoot) to

bottom as 1, 2, 3 successively. We examined the growth angles of parents and selected

RILs from the L88 population, derived from a cross of the phosphorus-efficient genotype

TLP19 with the drought tolerant genotype B98311. As expected, TLP19 has shallower

basal roots, while B98311 has deeper basal roots (Fig. 5.1). RILs 15 and 57 have

shallower basal roots compared to RILs 7 and 76 (Fig 5.1). The growth angle of basal

roots of all genotypes varies with position of origin (Fig. 5.1). Basal roots emerging from

whorl 1 are consistently shallower than those from whorl 3.

Treatment with auxin alters basal root growth angle and root growth

To determine the effect of exogenous auxin application on BRGA and root

growth, the seedlings of three shallow (TLP19, RIL57 and RIL15) and three deep

(B98311, RIL7 and RIL76) genotypes from the L88 population were exposed to different

auxin concentrations to generate dose-response functions. Two examples of auxin dose-

responses on BRGA and basal root growth for a deep parent (B98311 and a shallow

parent (TLP19) grown in low phosphorus nutrient solution are provided in Fig. 5.2.

Treatment with higher IAA concentration increases BRGA i.e. makes the basal roots

shallower. Auxin sensitivity was defined as the slope of the auxin response function for

each genotype, whorl and phosphorus treatment. Auxin sensitivity was greater in shallow

genotypes compared to deep genotypes, and the basal roots of the upper whorl were more

responsive to auxin treatment than those of lower whorls (Fig. 5.3, Table 1). Basal roots

grown with different phosphorus concentrations are equally responsive to auxin treatment

(Fig. 5.4).

In addition to increasing BRGA, auxin treatment affects growth of basal roots by

reducing root growth with increasing IAA concentrations (up to 40 nmol), but this effect

depends on position of origin of basal roots and genotype (Table 5.1). Basal roots

emerging from whorl 2 are more sensitive to auxin inhibition of root growth (Fig. 5.5)

than those of whorls 1 and 3 especially for deep genotypes.

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Ethylene production from auxin treated seedlings

To test the hypothesis that application of auxin results in increased ethylene

production, endogenous ethylene rates were measured in basal roots of two contrasting

(shallow genotype - RIL57 and deep genotype - RIL7) genotypes grown with low or high

phosphorus nutrient solution (see methods). In both shallow and deep genotypes treated

with IAA, basal roots of all three whorls produced significantly more ethylene than those

of the control seedlings when ethylene production is expressed on a fresh weight basis or

per basal root (Fig. 5.6; Table 5.3 in Appendix). There is a significant phosphorus x IAA

interaction originating primarily from greater effect of IAA on ethylene production in

low-phosphorus roots (Table 5.3 in Appendix). Moreover, the shallow genotypes grown

with low phosphorus were more responsive to IAA application, producing significantly

(P = 0.013) more endogenous ethylene, expressed per basal root, than deep genotypes

with IAA application and low phosphorus treatment.

Effect of NPA on growth angle and growth of basal roots

Table 5.2 shows the effect of NPA (15 nmol) on shallow (TLP19 and RIL57) and

deep (B98311 and RIL7) genotypes pooled over low and high phosphorus treatments.

Basal roots responded to increasing NPA concentrations by becoming shallower and

shorter, particularly the upper two whorls. With higher NPA concentration (20 nmol and

above), basal root growth was significantly inhibited, and in most cases the basal roots

became agravitropic (data not shown). NPA had little effect on BRGA of deep genotypes

with only 2 to 3 degrees increment, whereas NPA had a stronger effect on BRGA of

shallow genotypes especially in the upper two whorls, making them 10 to 11 degrees

shallower. Besides effects on BRGA, NPA treatment had stronger inhibitory effects on

basal root growth in shallow genotypes in the upper 2 whorls, reducing root growth by 7

to 13%. In deep genotypes, although root growth was inhibited by NPA, growth

inhibition was relatively less ranging from 5 to 10%. There are significant genotype x

NPA treatment, and genotype x whorl interactions for BRGA (Table 5.4 in Appendix),

but there is no significant interaction of NPA with other treatments for root growth.

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Phosphorus treatment had no significant effect on NPA response for BRGA or root

growth (Table 5.4 in Appendix).

Influence of ethylene inhibitors on BRGA and basal root growth

One of the goals of these experiments is to ask whether blocking ethylene action

or synthesis alters IAA regulation of BRGA and basal root growth, i.e. is the auxin effect

on BRGA and root growth due to auxin stimulation of ethylene synthesis? An initial

experiment showed that 60 µM AVG inhibited ethylene production from the basal roots

by 80% (data not shown). Figure 5.7 shows that the treatment with AVG (60 µM) plus

IAA (30 nmol) makes basal roots significantly deeper (P < 0.001) compared to IAA

alone, and shallow genotypes are significantly (P < 0.001) more responsive to the

treatment compared to the deep genotypes. Since phosphorus treatment has no significant

effect, the data are pooled over both low and high phosphorus treatments.

Basal root growth is inhibited by AVG treatment of plants in low phosphorus

nutrient solution. Inhibition of ethylene synthesis by AVG increases root elongation

under high phosphorus but reduces it under low phosphorus significantly compared to

IAA (P < 0.001) in all the whorls (Fig. 5.8, Table 5.5 in Appendix).

To verify the effect of AVG on BRGA and basal root growth, we treated the

seedlings with ethylene action inhibitor MCP plus IAA. The effect of MCP is similar to

that of AVG (Figs. 5.9, 5.10). Both AVG and MCP reverse the auxin effect on BRGA by

making basal roots deeper in both shallow and deep genotypes (Figs. 5.7 and 5.9).

However, the auxin inhibition of basal root growth is reversed by AVG and MCP only in

high phosphorus treatment (Figs. 5.8 and 5.10).

Free IAA concentrations are increased by ethylene

Free IAA levels were quantified by gas-chromatography-mass spectroscopy

(Schmelz et al. 2003; Engelberth et al. 2004) and the results are shown in Figs. 5.11 and

5.12. Figure 5.11 shows endogenous free IAA content, expressed as per basal root, for

roots of a deep (RIL7) and a shallow (RIL57) genotype grown in low phosphorus nutrient

solution. Our results indicate that the free IAA concentration, per basal root or per gram

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fresh weight, is always higher in the basal roots of whorl 3 than whorl 1 of both deep and

shallow genotypes (Fig. 5.11; Table 5.6 in Appendix). Exogenous IAA application to the

basal roots slightly, but not significantly, increases free endogenous IAA content, while

NPA treatment also did not significantly affect IAA content (Fig 5.11). On the other hand

exogenous ethylene treatment of the seedlings significantly increases free IAA

concentration in the basal roots of both whorls 1 and 3 compared to only NPA treatment

of both shallow and deep genotypes (Fig. 5.11). We also examine the effect of

phosphorus nutrition on the free IAA content in the basal roots of a shallow (RIL57)

genotype (Fig. 5.12). Phosphorus has no significant effect on free IAA content, but again

ethylene significantly increases it (Fig. 5.12; Tables 5.7-5.8 in Appendix). Free IAA

content per basal root number is more consistent among the various treatments compared

to per gram fresh weights of the tissue, probably due to its relation to root tips (Tables

5.6-5.8 in Appendix).

Basal Root Growth Rate vs. free IAA content

To study the relationship between basal root growth rate and free IAA content

within the root, data from free IAA measurements for controls, 15 nmol NPA treatment

and 30 nmol IAA treatment were combined with measured growth rates under these

treatments. Figure 5.13 shows the growth rate vs. free IAA content for whorls 1 and 3 of

two genotypes— a shallow (RIL57) and a deep (RIL7). In each of the four line segments

in Fig. 5.13 the left, middle and right points correspond to NPA treated roots, controls

and IAA treated roots respectively. Although we did not observe significant reduction in

IAA content by either NPA treatment or increase in free IAA content by exogenous IAA

treatment in the basal roots, there is a trend which shows that both NPA and IAA

treatments reduce basal root growth rate compared to controls. Furthermore, whorl 1

which contains less free IAA than whorl 3, grows nearly 50% less than whorl 3 for both

shallow and deep genotypes. But the genotypic difference in relationship between growth

rate and free IAA relationship is not clear from Fig. 5.13.

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3H IAA transport

The effect of ethylene on polar auxin transport from the hypocotyl to the basal

roots is shown as movement of radioactive IAA (Figure 5.14). Significantly more IAA is

transported to the basal roots of whorl 1 compared to whorl 3, when treated with 3H-AA

alone or in combination with exogenous ethylene application to the seedlings. When

compared with roots of whorl 3, the counts are significantly higher in whorl 1, which

indicates higher amount of radioactivity in basal roots of whorl 1 with both treatments

than those of whorl 3. However, ethylene had no significant effect on auxin transport

from the hypocotyl to the root system (Fig.5.14).

DISCUSSION

The overall objective of this work was to investigate the potential interaction of

auxin and ethylene in regulating plagiogravitropic growth. Auxin transport is required for

various developmental processes including root elongation, and gravity response (Muday

and Haworth 1994). Although basipetal auxin transport was shown to be a key regulator

of graviresponse in primary root of Arabidopsis (Rashotte et al. 2000), the acropetal

transport stream could also play a role by contributing auxin to the root tip and therefore

to the auxin pool engaged in basipetal transport. It has been shown that both acropetal

and basipetal auxin transport streams are involved in the production of lateral roots (Reed

et al. 1998). Basal roots of common bean are secondary roots and they resemble

adventitious roots in that they arise from tissue with shoot anatomy. Like adventitious

and lateral roots, they appear in a tetrarch pattern (Fig. 5.15 in Appendix). Since auxin

and other hormonal signals are channeled to the basal roots, it could be possible that the

growth and developmental processes of these roots are controlled by both auxin transport

polarities.

To understand the mechanism underlying the growth and graviresponse of bean

basal roots we show in Fig. 5.13 that the free auxin content in controls of both shallow

and deep genotypes is near optimum for root growth, although we know from the

literature that auxin level may be sub- or supraoptimal for growth. Even if no significant

variation in free IAA content due to application of NPA or exogenous IAA was found,

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the overall trend in Fig. 5.13 tends to indicate that any alteration of free auxin content by

application of NPA or exogenous IAA might cause inhibition of root growth.

Furthermore, free auxin content in whorl 3 is higher than that of whorl 1 even when auxin

content is expressed as per gram fresh weight (data not shown). These results also

indicate that auxin sensitivity of basal root growth rate is different in whorls 1 and 3.

Therefore, growth response to free auxin content curves of two whorls cannot be

combined in one curve and must be studied separately. When ethylene is applied to the

basal roots, it also increases free auxin content (Fig. 5.11A). Therefore, it can be

anticipated that similar to IAA treatment, ethylene will also inhibit basal root growth rate.

That is exactly what is shown by the negative growth response to ethylene treatment in

Fig. 2.8 in Chapter 2.

When compared to Fig. 5.11, a seemingly contradictory picture appears in Fig.

5.14 where more counts imply higher IAA in whorl 1 compared to 3. But Fig. 5.11 and

5.14 show two completely different phenomena— Fig. 5.11 shows free IAA content in

the basal roots whereas Fig. 5.14 shows transport of 3H-IAA from the root-shoot interface

to the basal roots. 3H-IAA may have been conjugated or metabolized or root tips may be

contributing significantly to IAA content resulting in the lack of correlation between 3H-

IAA and free IAA in our results. The chemiosmotic model of polar auxin transport

suggests that about half of the auxin movement from apoplast to the cell takes place

through passive diffusion following a concentration gradient (Taiz and Zeiger 1998)

while efflux of auxin from cytosol to apoplast is through PIN proteins. Therefore one of

the rate limiting factors of auxin transport is the auxin gradient. Since 3H-IAA is applied

at the root-shoot junction, the auxin gradient is highest along the hypocotyls-primary root

axis resulting in high auxin transport both to the primary root and the hypocotyls (Fig.

5.14). As a result, both hypocotyls and primary root show high counts of 3H-IAA. For

lateral transport of auxin to the basal roots, again auxin concentration gradient plays an

important role. Due to proximity to the location of 3H-IAA application, auxin gradient

along whorl 1 is anticipated to be higher than that of whorl 3 resulting in increased auxin

transport to whorl 1.

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Another interesting observation from the free IAA analysis is that there is no

difference in free IAA content due to phosphorus treatments (Fig. 5.12). This is

consistent with the fact that phosphorus treatment has a negligible effect on root growth

as well as graviresponse (Fig. 2.1, and Fig. 2.3 of Chapter 2). Free IAA content is

significantly higher in whorl 3 than in whorl 1 (Fig. 5.11) and also basal root growth rate

is higher in whorl 3 compared to whorl 1 (Fig. 5.8, 5.13).

Apart from growth response, one of our most important objectives for this study

was to understand the mechanism of graviresponse of the basal roots. Auxin dose-

response experiments using parent and recombinant inbred lines of common bean with

contrasting basal root traits show that auxin treatment increases BRGA, making basal

roots shallower (Table 5.1, Figs. 5.2-5.4). Shallow genotypes are more sensitive to auxin

treatment than deep genotypes and basal roots from upper whorls are more responsive

than lower whorls. These responses are very similar to those observed with exogenous

ethylene treatment (Chapter 2). To determine whether auxin-induced ethylene production

is responsible for changes in basal root growth angle and reduced root growth, we

examined the effects of exogenous auxin on ethylene production in the basal roots (Fig.

5.6) and the effects of ethylene inhibitors on auxin responses (Fig. 5.7-5.10). Generally,

auxin response could be attributed to ethylene production. However, there were

exceptions, e.g. endogenous ethylene production is much less in high phosphorus

treatment but the plants still respond equally well to IAA application in terms of

shallowness of basal roots (Figs. 5.2, 5.3). Earlier we have shown that there is no

correlation between ethylene production and BRGA (Chapter 2). However, we observed

a correlation between auxin-induced ethylene production and BRGA of shallow and deep

genotypes treated with 30 nmol IAA in both low P and high P treatments (Fig 5.16 in

Appendix). Our results with AVG or MCP application show that AVG or MCP

counteracts the IAA effect on BRGA i.e. both AVG and MCP make basal roots deeper.

Therefore, it seems likely that auxin-induced ethylene production is only one of several

factors affecting growth angles.

Another possibility is that hormonal effects on growth indirectly result in altered

growth angle. However, auxin inhibition of growth does not correlate with auxin effect

153

on BRGA. Although BRGA response to exogenous IAA application declines from whorl

1 to whorl 3 (Fig. 5.3), the growth reduction response of whorl 2 is greater than that of

whorls 1 and 3 (Fig. 5.5). Previous researchers have found that high concentrations of

auxin did not abolish the response of primary roots to gravity (Ishikawa and Evans 1993;

Muday and Haworth 1994). The lack of correlation between root growth inhibition and

shallowness (caused by less differential growth) suggests that the mechanism involved in

the differential growth and root elongation are not regulated in the same manner

(Madlung et al. 1999).

Previous works suggest that ethylene interacts with auxin transport in various

developmental processes like root elongation, and root hair development (Rahman et al.

2001; 2002). In order to explore a more detailed role of ethylene in gravitropic curvature,

we examined the effects of auxin in the presence of ethylene action and ethylene

synthesis inhibitor, MCP and AVG in regulating root shallowness and root elongation

(Figs. 5.7-5.10). Our results with application of AVG and MCP to intact roots showed

that blocking ethylene (even with addition of IAA) results in deeper basal roots (Figs.

5.7, 5.9). Moreover, AVG and MCP applications prevent IAA inhibition of root

elongation in high phosphorus (Figs. 5.8, 5.10). Moreover, as applying IAA or NPA to

the basal roots results in reduction of root growth, the concentration of IAA must be

optimal for growth within sensitivity of each root. Addition of IAA drives auxin

concentration to supraoptimal level resulting in inhibition of root growth. In high P

treatment, application of AVG or MCP with IAA causes auxin concentration to return

back to optimal level resulting in near normal root growth. But in low P, the interaction

pathway between auxin and ethylene seems to be unaffected. Therefore, application of

AVG or MCP plus IAA causes the root growth to be similar to IAA alone in low P. This

result points to a possible alteration of ethylene-auxin interaction under phosphorus

stress.

Alternatively we could consider the effect of auxin and ethylene in high and low

phosphorus treatments separately. When basal roots under high P treatment are treated

with ethylene inhibitors plus IAA, two possible situations might arise. One possibility is

that application of ethylene inhibitors may reduce free IAA content as exogenous

154

ethylene application increases free IAA content (Fig. 5.12), although we did not check

this possibility. The second alternative situation could be reduction of ethylene

production by AVG or action by MCP so that ethylene-induced inhibition of root growth

is reduced. But our results show that in high P conditions, exogenous IAA treatment does

not increase endogenous ethylene production in most cases (Fig. 5.6) and therefore, we

cannot expect that IAA induced ethylene is the cause of IAA-induced growth inhibition.

So, in high phosphorus treatment, perhaps it is the reduction of free IAA which results in

increasing root growth with ethylene inhibitors AVG (Fig. 5.8) or MCP (Fig. 5.10). This

possibility is consistent with the fact that exogenous IAA application has such a little

effect on free IAA, so that the effect of ethylene could overcome it easily (comparing

Figs. 5.11 A and B). In addition, this idea also supports that increasing auxin content in

basal roots under high P treatment results in supraoptimal auxin concentrations for root

growth.

On the other hand, in low P conditions, IAA treatment does increase endogenous

ethylene production (Fig. 5.6), however, application of MCP or AVG cannot reverse the

auxin-inhibition of root growth. (Figs. 5.8, 5.10). In addition, ethylene production is

higher in low P (Fig. 2.2 in Chapter 2) and sensitivity of basal root growth to ethylene is

similar in both high and low P treatments (Fig. 2.8 in Chapter 2). These results suggest

that AVG or MCP would have more effects on ethylene rather than auxin in low P

conditions. Also, when we treated the basal roots with MCP for 24 h immediately after

transferring the seedlings to the pouch, we observed significant reduction in root growth

(about 50% reduction) by MCP under low P compared to controls (Fig.5.17 in

Appendix). Our results are consistent with the results of Ma et al., (2003) on Arabidopsis

primary root growth that ethylene is more important for maintaining elongation of root

under low phosphorus than high phosphorus conditions. There is no evidence to show

any difference in auxin sensitivity or auxin content between P treatment (Figs. 5.5, 5.12)

and therefore root growth dependence on auxin under low P treatment should be similar

to that of high P treated plants.

The most common mechanism of the interaction between ethylene and auxin is

that ethylene reduces polar auxin transport (Burg and Burg 1966; Morgan and Gausman

155

1966; Beyer 1973). However, to date we do not know of any work which shows that

ethylene might increase free auxin content. Although previous works (Beyer and Morgan

1970; Riov et al. 1982) show that ethylene treatment reduces endogenous IAA level by

increasing conjugation of IAA, one can possibly speculate that ethylene treatment may

reduce auxin conjugation which would possibly increase in the production of free IAA

content. Auxin-peptide conjugates consisting of 80% of the total IAA pool in mature

Phaseolus seeds, are believed to be the major source of free IAA content of young

seedlings where synthesis of free IAA has been detected within 2 days of germination

(Bialek and Cohen 1992; Bialek et al. 1992). As known from literature, ethylene

regulates endogenous IAA level in plants. There is a evidence which shows that kinetin-

induced ethylene production markedly suppresses the conversion of indole-acetic acid

into auxin conjugates in the kinetin-treated hypocotyl segments (Lau and Yang 1973).

Alternatively ethylene might reduce IAA catabolism or increase synthesis of IAA

resulting in increased free IAA content. In addition, the role of ethylene in regulating

polar auxin transport in the root cap indicates that ethylene may influence the auxin sink

in the root, thereby regulating the localized shortage or surplus of IAA content (Ponce et

al. 2005).

While more research is necessary to fully explain the role of interaction of auxin

and ethylene in regulating plagiogravitropic growth of common bean basal roots, this

study has potential benefits. Our results show a phosphorus-dependent interaction

between ethylene and auxin in regulation of root elongation, but a phosphorus-

independent interaction for control of growth angle. Observations in this study provide

further understanding of various root traits controlled by endogenous hormonal signals

like ethylene and auxin, as we know that different root classes vary substantially in their

responses to different hormones. Root systems modify their growth and development by

altering their sensitivity and response to different hormones. Finally this work forms the

basis of further understanding of key molecular and biochemical components involved in

hormonal regulation of root growth which can be manipulated for regulating root

architecture in various environments.

156

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Figure 5.1. Effect of genotype and position of origin on basal root growth angle of common bean genotypes of the L88 population. The growth angle of the basal roots was measured after 2 d growth in pouches. The bars show mean growth angles of basal roots emerging from each whorl of 10-12 plants per genotype, with data pooled over phosphorus treatments, ± SE. Inset shows a close up view of a young seedling (1 d after transplanting to the growth pouch) showing distinct whorls with emerging basal roots.

163

Figure 5.2. Auxin sensitivity of growth angles and growth rate of basal roots for whorls 1, 2, 3 of a deep (B98311) and a shallow (TLP19) genotype grown in low phosphorus. The angle and growth were measured for the root growth occurring between 24 and 48 h. Values shown are means of 4-5 plants per auxin treatment ± SE.

164

Figure 5.3. Auxin sensitivity of growth angle of basal roots as a function of genotype and whorl position in three shallow (TLP19, RIL57 and RIL15) and three deep (B98311, RIL7 and RIL76) genotypes (pooled over both phosphorus treatments). Auxin sensitivity was calculated as the slope of the response curve (auxin concentration vs. growth angle of basal roots).

165

high Py = 0.0686x + 0.2292

R2 = 0.5239

low Py = 0.0751x - 0.162

R2 = 0.8577

0

1.5

3

4.5

6

20 35 50 65 80

Growth angle (without auxin)

Aux

in s

ensi

tivity

Figure 5.4. Correlation between auxin sensitivity and growth angle of basal roots of six L88 genotypes (deep genotypes B98311, RIL7, RIL76 and shallow genotypes TLP19, RIL57, RIL15) grown in low P and high P. The symbols show values for each genotype and for each whorl position. Growth angles on X-axis designate control plants without auxin treatment.

166

Figure 5.5. Auxin sensitivity of growth response of basal roots as a function of genotype and whorl in three shallow (TLP19, RIL57 and RIL15) and three deep (B98311, RIL7 and RIL76) genotypes (pooled over both phosphorus treatments together). Root growth was measured between 24 and 48 h. Auxin sensitivity was calculated as the slope of the response curve (auxin concentration vs. growth).

167

0

25

50

75

100

whorl 1 whorl 2 whorl 3 whorl 1 whorl 2 whorl 3

shallow genotype deep genotype

Ethy

lene

pro

duct

ion

(nl/h

/g F

W) control Low P

IAA Low Pcontrol High PIAA High P

Figure 5.6. Endogenous ethylene production per gram fresh weight by the segments of the root-shoot junction bearing basal roots of a deep (RIL7) and a shallow (RIL57) genotype treated with 30 nmol IAA in either low P or high P nutrient solution. Segments were harvested 48 h after transplanting. Values shown are means of 4-7 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.

168

0

25

50

75

100


Deep genotypes Shallow genotypesBas

al ro

ot a

ngle

(deg

rees

from

ver

tical

) control IAA AVG+IAA

Figure 5.7. Combined effect of AVG (60 µM) and IAA (30 nmol) on the growth angle of basal roots of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes (pooled over both phosphorus treatments). AVG prevents the increase in root shallowness caused by IAA (P <0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.

169

Figure 5.8. Combined effect of AVG (60 uM) and IAA (30 nmol) on the basal root growth of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes grown in low P or high P nutrient solution. AVG reverses the IAA-inhibition of growth only for plants grown with high phosphorus (P < 0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment per phosphorus treatment. Bars indicate standard errors.

170

0

25

50

75

100


Deep genotypes Shallow genotypesBas

al ro

ot a

ngle

(deg

rees

fro

m v

ertic

al)

control IAA MCP +IAA

Figure 5.9. Combined effect of MCP and IAA on the growth angle of basal roots of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes (pooled over both phosphorus treatments). MCP prevents the increase in root shallowness caused by IAA (P <0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment and phosphorus treatment. Bars indicate standard errors.

171

Figure 5.10. Combined effect of MCP and IAA on the basal root growth of deep (B98311 and RIL7) and shallow (TLP19 and RIL57) genotypes grown in low P or high P nutrient solution. MCP reverses the IAA-inhibition of growth only for plants grown with high phosphorus (P < 0.001). Values shown are means of the growth angles of 4 plants per genotype per hormone treatment per phosphorus treatment. Bars indicate standard errors.

172

Figure 5.11. Free IAA in common bean basal roots of seedlings of a deep genotype (RIL7) and a shallow genotype (RIL57) grown in low phosphorus nutrient solution for exogenous ethylene (A), exogenous IAA (B), and exogenous NPA (C) applications. Values shown are the mean of three samples each containing 12 to 20 basal roots ± SE.

0.0

0.2

0.3

0.5

0.6

whorl 1 whorl 3 whorl 1 whorl 3

Deep genotype Shallow gentoypeEndo

geno

us fr

ee IA

A c

onte

nt (n

g/ba

sal r

oot)

control ethyleneA

0.0

0.2

0.3

0.5

0.6



geno

us fr

ee IA

A c

onte

nt (n

g/ba

sal r

oot)

control NPAC

0.0

0.2

0.3

0.5

0.6



geno

us fr

ee IA

A c

onte

nt (n

g/ba

sal r

oot)

control IAAB

173

0.0

0.1

0.2

0.3

0.4

0.5


Low P High P

Endo

geno

us fr

ee IA

A c

onte

nt (n

g/ba

sal r

oot)

control

ethylene

Figure 5.12. Free IAA in common bean basal roots of seedlings of a shallow genotype (RIL57) grown in low (low P) and high (high P) phosphorus nutrient solution. Values shown are the mean of three samples each containing 12 to 22 basal roots ± SE.

174

Figure 5.13. Basal root growth rate vs. free IAA content per basal root plotted for whorls 1 and 3 of a shallow (RIL57) and a deep (RIL7) genotype. For each of the four line segments the left most symbols were for NPA treated basal roots, the middle points were for controls and the right most were for IAA treated basal roots.

175

0

15000

30000

45000

60000

75000

hypocotyl appliedzone

whorl 1 whorl 3 primaryroot

3-H

IAA

tran

spor

t (cp

m)

IAAethylene + IAA

Figure 5.14. Auxin transport activity in roots of common bean seedlings of a shallow genotype (RIL57). Amount of radioactive IAA transported to the basal roots of different whorls, primary roots, hypocotyls and applied zone( root-shoot junction) after application of 3H-IAA for 24 h. Individual segments were immersed in scintillation cocktail for 48 h. Values show means ± SE of 4 seedlings per treatment.

176

Figure 5.15. Anatomical sections of basal root emergence zone of a parent genotype TLP19. Figures A-C show the zone from where basal roots emerge. Figures D-E show the longitudinal view of the root-shoot interface from where the basal roots develop from 3 distinct whorls (Fig. D) and 1 whorl (Fig. E). Figure F shows the transverse section of the region just below the basal root emergence zone.

Table 5.1. ANOVA of growth angle and growth response of basal roots from contrasting genotypes –shallow (TLP19, RIL57, RIL15) and deep (B98311, RIL7, RIL76) as affected by exogenous auxin treatment.

Growth angle Growth rate


Genotype 1 1752.39 <0.001 4.23 0.039

Phosphorus 1 23.51 <0.001 0.15 0.696

Auxin 4 260.82 <0.001 340.27 <0.001

Whorl 2 3756.14 <0.001 722.75 <0.001

Genotype*Phosphorus 1 0.59 0.442 0.06 0.796

Genotype*Auxin 4 5.88 <0.001 20.75 <0.001

Genotype*Whorl 2 434.87 <0.001 5.58 0.003

Phosphorus*Auxin 4 0.05 0.994 0.11 0.977


Auxin*Whorl 8 10.63 <0.001 20.99 <0.001

178

Table 5.2. Effect of NPA treatment on the basal root growth angle (BRGA) and growth rate of two shallow (TLP19 and RIL57) and two deep (B98311 and RIL7) genotypes (pooled over both phosphorus treatments). Values shown are means of the growth angles of 4 plants per genotype per phosphorus treatment ± SE.

NPA treatment 0 nmol 15 nmol 0 nmol 15nmol

Genotype Whorl BRGA (degrees) Growth rate (cm/day)

Deep 1 51±1.13 54±0.85 1.05±0.04 0.98±0.05

Deep 2 42±1.26 44±1.05 1.22±0.05 1.09±0.03

Deep 3 32±1.16 35±0.98 1.79±0.03 1.70±0.04

Shallow 1 72±1.06 83±1.35 0.90±0.06 0.83±0.05

Shallow 2 60±1.10 70±1.21 1.07±0.06 0.93±0.07

Shallow 3 40±1.12 43±1.19 1.72±0.06 1.68±0.07

179

CHAPTER 5 APPENDIX

Figure 5.16. Correlation between endogenous ethylene production and growth angle of a deep genotype – RIL7 (A) and a shallow genotype-RIL57 (B) treated with 30 nmol exogenous IAA concentration in both low P and high P treatments.

Low Py = 0.7128x + 23.267

R2 = 0.3729, P = 0.011

High Py = 0.2549x + 35.727

R2 = 0.2102, P = 0.085

0

25

50

75

100

125

30 45 60 75 90Growth angle (degrees from vertical)

Ethy

lene

pro

duct

ion

(ng/

h/g

FW) A

High Py = 0.2233x + 49.846

R2 = 0.3694, P = 0.003

Low Py = 0.3951x + 35.849

R2 = 0.3948, P = 0.016

0

25

50

75

100

125

30 45 60 75 90 105Growth angle (degrees from vertical)

Ethy

lene

pro

duct

ion

(ng/

h/g

FW)

B

180

0

1

2

3

4


Deep genotype Shallow genotype

Gro

wth

rate

(cm

/day

)

low P low P + MCPhigh P high P+MCP

Figure 5.17. Effect of MCP on growth of basal roots of a deep genotype (B98311) and a shallow genotype (TLP19) in both low P and high P treatments for 24 h immediately after transferring to the growth pouch. Each bar shows mean of basal roots of 5-7 plants ±SE.

181

Table 5.3. ANOVA of endogenous ethylene production as affected by genotype, phosphorus, IAA treatment or whorls.

C2H4 (µL/h/g FW) C2H4 (µL/h/basal root)

Source DF F-value P-value F-value P-valueGenotype 1 14.295 0.000 34.033 0.000Phosphorus 1 7.922 0.006 2.297 0.133IAA application 1 68.011 0.000 20.218 0.000Whorl 2 18.298 0.000 1.589 0.209Genotype*Phosphorus 1 20.653 0.000 1.454 0.231Genotype*IAA application 1 0.014 0.905 0.548 0.461Genotype*Whorl 2 0.589 0.557 0.190 0.827Phosphorus*IAA application 1 41.257 0.000 9.358 0.003Phosphorus*Whorl 2 1.487 0.231 0.558 0.574IAA application*Whorl 2 0.778 0.462 0.324 0.724Genotype*Phosphorus*IAA application 1 0.633 0.428 7.665 0.007Genotype*Phosphorus*Whorl 2 0.710 0.494 1.351 0.264Genotype*IAA application*Whorl 2 0.778 0.462 0.851 0.430Phosphorus*IAA application*Whorl 2 0.450 0.639 0.238 0.788Genotype*Phosphorus*IAA application*Whorl 2 1.298 0.278 3.970 0.022 Table 5.4. ANOVA of BRGA and basal root growth as affected by genotype, phosphorus, NPA treatment (0, 10, 15 nmol) or whorls.

BRGA Root growth

Source DF F-value P-value F-value P-valueGenotype 1 952.949 0.000 16.737 0.000Phosphorus 1 1.089 0.297 0.022 0.881NPA treatment 2 31.666 0.000 3.972 0.019Whorl 2 882.378 0.000 365.004 0.000Genotype*Phosphorus 1 0.377 0.539 0.001 0.974Genotype* NPA treatment 2 6.525 0.002 0.026 0.975Genotype* Whorl 2 102.310 0.000 1.847 0.158Phosphorus* NPA treatment 2 0.275 0.760 0.010 0.990Phosphorus* Whorl 2 0.330 0.719 0.011 0.989NPA treatment* Whorl 4 1.571 0.180 0.231 0.921Genotype* Phosphorus * NPA treatment 2 0.148 0.863 0.005 0.995Genotype * Phosphorus * Whorl 2 0.026 0.974 0.024 0.976Genotype* NPA treatment* Whorl 4 2.056 0.085 0.105 0.981Phosphorus * NPA treatment * Whorl 4 0.097 0.983 0.029 0.998Genotype * Phopshorus* NPA* Whorl 4 0.032 0.998 0.012 1.000

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Table 5.5. ANOVA of basal root growth as affected by genotype, treatment of AVG+IAA and IAA or whorls.

Source DF F-value P-value

Low P (control, IAA, AVG+IAA)Genotype 1 0.626 0.429Hormone treatment 2 69.492 <0.001Whorl 2 124.855 <0.001Genotype* Hormone treatment 2 4.047 0.018Genotype * Whorl 2 0.209 0.812Hormone treatment* Whorl 4 4.965 <0.001Genotype * Hormone treatment* Whorl 4 0.721 0.578

Low P (control, AVG+IAA)Genotype 1 1.963 0.162Hormone treatment 1 1.161 0.282Whorl 2 108.529 <0.001Genotype* Hormone treatment 1 0.223 0.637Genotype * Whorl 2 0.503 0.605Hormone treatment* Whorl 2 0.107 0.899Genotype * Hormone treatment* Whorl 2 0.250 0.779

High P (control, IAA, AVG+IAA)Genotype 1 0.288 0.592Hormone treatment 2 73.459 <0.001Whorl 2 118.688 <0.001Genotype* Hormone treatment 2 1.824 0.163Genotype * Whorl 2 0.043 0.958Hormone treatment* Whorl 4 4.490 0.001Genotype * Hormone treatment* Whorl 4 0.635 0.638

High P (control, AVG+IAA)Genotype 1 1.963 0.162Hormone treatment 1 1.161 0.282Whorl 2 108.529 <0.001Genotype* Hormone treatment 1 0.223 0.637Genotype * Whorl 2 0.503 0.605Hormone treatment* Whorl 2 0.107 0.899Genotype * Hormone treatment* Whorl 2 0.250 0.779

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Table 5.6. Free IAA in common bean basal roots of seedlings of a deep genotype (RIL7) and a shallow genotype (RIL57) grown in low phosphorus nutrient solution. The reported values are the mean and S.E. of three samples each containing 117 to 280 mg of basal root tissue.

Free IAA (ng/g FW)

Treatments Deep genotype Shallow genotype

Whorl 1 Whorl 3 Whorl 1 Whorl 3

Control 26.2±6.0 28.0±0.5 25.8±3.1 27.9±5.4

IAA (30 nmol) 25.4±1.0 28.9±1.9 25.9±2.9 28.8±1.1

NPA (15 nmol) 23.2±1.9 27.7±1.6 21.6±1.3 26.4±6.1

C2H4 (0.6 µl L-1) 27.1±0.9 31.2±1.2 27.3±2.1 30.0±4.1

Table 5.7. Free IAA in common bean basal roots of seedlings of a shallow genotype (RIL57) grown in low (Low P) and high (High P) phosphorus nutrient solution. The reported values are the mean and S.E. of three samples each containing 117 to 240 mg of basal root tissue.

Free IAA (ng/g FW)

Treatments Low P High P

Whorl 1 Whorl 3 Whorl 1 Whorl 3

Control 25.8±3.1 27.9±5.4 25.4±1.3 27.7±2.5

C2H4 (0.6 µl L-1) 27.3±1.2 30.0±4.1 27.8±2.0 30.9±0.2

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Table 5.8. ANOVA of endogenous free IAA from a shallow genotype (RIL57) as affected by ethylene treatment, phosphorus or whorls (1 and 3).

Free IAA (ng/g FW) Free IAA (ng/basal root)


Phosphorus 1 0.009 0.926 0.172 0.684

Ethylene 1 1.229 0.284 20.519 <0.001

Whorl 1 1.523 0.235 24.765 <0.001

Phosphorus*Hormone 1 0.058 0.812 0.370 0.552


Hormone*Whorl 1 0.033 0.858 0.033 0.858

CHAPTER 6: SUMMARY OF THE WORK

The overall objective of the research work presented here was to study the root

architecture of common bean plants, with special focus on the basal root growth angle in

response to gravity in concert with various environmental cues like phosphorus and

endogenous hormonal signals such as ethylene and auxin. Basal roots of common bean

together with the primary root form the scaffolding of the entire root system. Basal roots

have been considered as the specialized lateral roots (Zobel 1991) developing from root-

shoot interface. However, we found that the basal roots emerge from root-shoot junction,

the anatomy of which displays typical shoot anatomy (Figs. 5.15 A-C), while the region

just below the basal root emergence zone displays root anatomy (Figs. 5.15 D-F).

Therefore, basal roots resemble adventitious roots although they appear in 4 xylem files

(tetrarch) like lateral roots.

Gravitropism does not necessarily mean vertical upward growth of shoots and

vertical downward growth of roots. While various reports exist on the root gravitropism,

they focus mainly on primary roots of Arabidopsis, maize, rice etc. Basal roots of

common bean exhibit plagiogravitropic growth i.e. grow at a predetermined set-point

angle other than 0° or 180° with respect to gravity. However, this angle of growth

changes with time. The growth angle of basal roots is a primary determinant of the roots

with soil depth which impacts phosphorus acquisition efficiency (Bonser et al. 1996; Ge

et al. 2000; Liao et al. 2001). Common bean genotypes vary substantially in the growth

angle of basal roots (Liao et al. 2004) and by altering their growth angles, the plants are

better adapted to nutrient limited environment like low phosphorus availability (Bonser et

al. 1996; Liao et al. 2001; Ho et al. 2005). Shallow basal roots not only increase topsoil

exploration, but also produce less intraplant and interplant competition for phosphorus

which are beneficial under conditions of non-uniform availability of phosphorus in soil

(Ge et al. 2000; Lynch and Brown 2001).

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The overall theme of this thesis research is to address the regulation of

plagiogravitropic growth of basal roots by genotypic, physiological and environmental

variations. A detailed study of the genetic and physiological basis of the basal root traits

which account for efficient phosphorus acquisition would increase the scope for selection

and breeding of crops with improved adaptation to low phosphorus availability (Lynch

1998). Although it may seem that the shallowness of the basal roots is correlated with

inhibition of basal root growth, we observed that even with fixed root length there is a

significant difference in the BRGA of shallow and deep genotypes (Appendix Fig. 2.10).

With a view to attain our objective, we first investigated the role of ethylene in

modulating the growth angle in interaction with phosphorus availability. Secondly, we

focused on the more detailed study of localized growth and curvature of basal roots by

kinematic approach using semi-automated image analysis software. We also measured

root growth velocity and diameter as functions of distance from the root tip and time. In

addition, the kinematic method was employed in investigating how the elongation and

curvature of basal roots are affected by phosphorus treatments, and application of

exogenous ethylene and ethylene action inhibitor, 1-methylcyclopropene (MCP). The

final project presented in this thesis is based on the subject of exploring the cross-talk

between auxin and ethylene in regulation of plagiogravitropic growth in response to low

phosphorus availability.

The secondary roots of other plant species are gravitropic (Yamashita et al. 1997;

Kiss et al. 2002; Mullen and Hangarter 2003). Similarly basal roots of common bean

genotypes are also gravitropic (Walk 2005) and the genotypes differ in the

graviresponsiveness. Moreover, phosphorus and ethylene were hypothesized to regulate

growth angle of basal roots leading to the production of shallow root system (Bonser et

al. 1996; Zhang 2002). The work presented in chapter 2 is the continuation of our

hypothesis that ethylene might play a role in regulating genetic, positional and nutrient

induced variation of growth angle of basal roots. The results of this project indicate that

ethylene might act as a modulator of root responses to nutrient availability. In addition,

ethylene perception may be an important factor in the response of basal roots to low

phosphorus availability (Lynch and Brown 1997). Moreover our study shows that

187

position of emergence of basal roots from root-shoot junction plays a key role in

determining the non-vertical orientation of basal roots. This study has important

implications where basal roots from upper whorls would explore upper soil horizon by

becoming shallower, while roots from lower whorls less responsive to ethylene maintain

deeper growth angle and would explore different soil domains. This dimorphic root

architecture would be beneficial in acquisition of limited nutrients like phosphorus and

water from soil minimizing the competition among the roots of an individual plant.

Chapter 3 of my dissertation work describes the kinematic approach using semi-

automated computer-aided image analysis program, KineRoot, used for measuring

localized growth of basal roots and curvature, while chapter 4 focuses on the results and

conclusions made from the experimental work using that technique. We developed a

semi-automated way to study the spatio-temporal patterns of root growth of bean in a

reliable way while reducing user interventions to allow large scale experiments. In this

project we studied the plagiogravitropic growth of thicker rooted species like common

bean. The primary difficulty in studying bean is that epidermal cells are invisible

resulting in images of roots devoid of any trackable patterns. Basal roots were sprinkled

with graphite particles randomly, while the KineRoot program was used to track the

displacement of the patterns of the graphite particles over space and time using a number

of algorithms from the digital images taken by time lapse photography over a period of 4-

6 h. The tracking algorithm also took advantage of the color difference between the root

and the background for higher accuracy and reliability. The new software enables us to

measure the local root growth, diameter, and root midline which was used in calculating

root curvature. In addition, the program was able to produce growth velocity data with a

high degree of accuracy and consistency. Spatio-temporal study of root growth is

beneficial for characterizing the root growth accurately.

Chapter 4 of the thesis aims to study the growth and curvature of basal roots of

common bean using the image-analysis program KineRoot. We identified and measured

the local patterns of root growth and graviresponding zones of the basal roots,

investigated the velocity profiles within these zones and determine how these zones are

affected by low phosphorus availability and ethylene treatment. We observed that basal

188

roots accelerate growth rate of the upper whorls at the cost of lower growth rate in lower

whorls in response to low phosphorus availability. Apart from root growth, one of the

most important aspects of this study was to characterize the bending of the basal roots

which leads to graviresponse and reflects shallowness or deepness of basal roots. Root

curvature results from differential growth between upper and lower edges of the root.

Therefore study of spatio temporal patterns of differential growth ratio of a growing root

allows identification and measurement of root bending zones and bending amount. Our

results show that ethylene and MCP treatments do not alter local root curvature, but alters

the span and duration of the bending of the root upward or downward which causes the

root to respond to gravity differently under different treatments and thereby produce

shallow and deep roots respectively. The results from this study show new aspects of

plagiogravitropic response of basal roots which has not been observed before.

Chapter 5 of the thesis focuses on the potential interaction of auxin and ethylene

in regulating the plagiogravitropic curvature and growth of basal roots. Our results

support the hypothesis that auxin-ethylene interaction regulates growth angles which are

also dependent on phosphorus availability. For this project we measured free auxin

content endogenously present in the basal roots and observed both higher auxin content

and higher sensitivity to auxin for root growth in basal roots of lower whorls than upper

whorls. In addition, we showed that more radio-labeled IAA transported to upper whorls

compared to lower whorls. Measurement of growth angles show that both ethylene and

auxin make the basal roots shallower. Our results show that auxin concentration in

controls is near optimal. Application of aminoethoxyvinylglycine (AVG) or MCP

together with exogenous IAA increases root growth and reduces shallowness in

phosphorus sufficient conditions. However, AVG and MCP do not reverse IAA-

inhibition of growth in low phosphorus. These results point to a phosphorus dependent

interaction between ethylene and auxin in regulation of elongation, but a phosphorus-

independent interaction for control of growth angle. In addition, our results show that

under low P treatment, ethylene inhibitors like AVG or MCP may have more effects on

ethylene rather than auxin resulting in root growth inhibition, whereas in high P treatment

ethylene inhibitors might affect the free IAA content resulting in increased root growth.

189

However, we did not check the second possibility. It would be worthwhile if this

possibility can be checked to have better understanding of the role of ethylene-auxin

interaction in controlling root elongation under different phosphorus availability.

However, there are several areas of future work which can be pursued to have a

more detailed study of the effects of auxin-ethylene interaction on plagiogravitropic

growth of basal roots. First of all, longitudinal sectioning of basal roots might be done to

analyze the auxin redistribution in the graviresponding basal roots which would be

beneficial for verifying the role of auxin gradient in regulating the graviresponse of basal

roots. Moreover, analysis of conjugated auxin in addition to free IAA content could be

carried out to estimate total IAA content which is contributed by both free IAA and

conjugated auxin inside the basal roots. While this report focuses on the hormonal effect

on growth and BRGA of basal roots at a very early stage, future study could explore the

effects of auxin and ethylene at a later stage of root growth and curvature. Greenhouse

experiments would be essential for the study of older bean basal roots since the basal

roots grown in pouch always become deeper after 5-6 d due to lack of space in the pouch,

resulting in the absence of difference of BRGA between shallow and deep genotypes.

My thesis research on plagiogravitropic growth of basal roots of common bean

identifies a new aspect of basal root growth in terms of position of origin i.e. whorls. It

shows that apart from all other factors such as genotypic variations, hormones,

phosphorus availability etc., the whorl from which basal roots emerge is one of the most

important determinants of root depth. Roots also vary in their responses to gravity based

on whorl position. Root depth is strongly responsive to exogenous ethylene, but weakly

correlated with endogenous ethylene production. Furthermore our kinematic study

indicates adaptive behavior of basal roots under phosphorus stress. This is the first time

kinematics has been used to analyze plagiogravitropic growth. We show that the change

in basal root depth is not a result of changes in root curvature; rather it is a result of

changes in time and span of upward vs. downward bending of the roots. We also show

that auxin makes basal roots shallower but auxin response is regulated by ethylene which

changes auxin sensitivity and auxin content of the basal roots.

190

REFERENCES








Kiss JZ, Miller KM, Ogden LA, Roth KK (2002) Phototropism and gravitropism in

lateral roots of Arabidopsis. Plant and Cell Physiology 43, 35-43.












Mullen JL, Hangarter RP (2003) Genetic analysis of the gravitropic set-point angle in

lateral roots of Arabidopsis. Space Life Sciences: Gravity-Related Processes in

Plants 31, 2229-2236.

Walk TC (2005) 'Variation in root architecture of common bean and effects on

phosphorus acquisition.' PhD thesis, (Pennsylvania state University, PA).

Yamashita M, Takyu T, Saba T (1997) Gravitropic reaction in the growth of tea roots.

Japanese Journal of Crop Science 66, 472-478.

Zhang YJ (2002) 'Ethylene and phosphorus responses in plants.' PhD thesis,

(Pennsylvania State University, PA).

191

Zobel R (1991) Root growth and development. In 'The Rhizosphere and Plant Growth.'

(Eds Keister DCregan P) pp. 61-71. (Kluwer: Dordrecht, The Netherlands).

VITA

PARAMITA BASU

EDUCATION

• PhD Plant Physiology, Pennsylvania State University, 2006 • M.Sc. Botany, Calcutta University, Kolkata, India, 1997 • B.Sc Botany, Calcutta University, Kolkata, India, 1994

AWARDS AND HONORS

• Thomas Walter Memorial Scholarship, Penn State University 2002-2005. • 3rd place in Twentieth Annual Graduate Exhibition, Penn State University 2005,

University Park, PA, USA. • Graduate Student Travel awards, College of Agricultural Sciences, Penn State

University, 2005, University Park, PA, USA. TEACHING EXPERIENCE

• Lecturer in Vidyasagar College for Women, Calcutta University, India 1997-1999. • Teaching Assistant for Conservation Biology, Penn State University, Fall 2001. • Teaching Assistant for Plant Ecology, Penn State University, Fall 2002. • Teaching Assistant for Plant Nutrition Lab, Penn State University, Spring 2004. • Teaching Assistant for Post Harvest Physiology, Penn State University, Spring 2005 SELECTED JORUNAL PUBLICATIONS • Paramita Basu, Yuan-Ji Zhang, Jonathan P. Lynch, and Kathleen M. Brown.

Genetic, positional and nutritional regulation of root plagiogravitropism modulated by ethylene. To be submitted to Functional Plant Biology.

• Paramita Basu, Anupam Pal, Jonathan P. Lynch, and Kathleen M. Brown. Kinematic analysis of root growth and gravitropism using semi-automated image analysis. In Preparation.

• Paramita Basu, Anupam Pal, Jonathan P. Lynch, and Kathleen M. Brown. Growth and curvature of basal roots of common bean (Phaseolus vulgaris L.) analyzed using kinematic approach. In Preparation.

• Paramita Basu, Jurgen Engelberth, Jonathan P. Lynch, and Kathleen M. Brown. Hormonal regulation gravitropic growth of basal roots – a cross-talk between ethylene and auxin. In Preparation.

OTHER SKILLS AND INTERESTS

• Microsoft Office (Word, Excel, Power Point) • Matlab • Adobe Photoshop • Adobe Illustrator • BLAST • Statview • SPSS • EndNote

genetic, physiological and environmental regulation …

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