final thesis 35

Quality of grain from crosses between Australian and tetraploid wheat

varieties.

Nomah Sangu

A thesis submitted in fulfilment of the requirement for the degree of

Master of Philosophy

Faculty of Science

The University of Sydney

November 2018

"

i

Statement of originality

This thesis is submitted to the University of Sydney in fulfilment of the requirement for

the degree of Master of Philosophy. The work presented in this thesis is to the best of

my knowledge and belief, original except as acknowledged in the text. The author

hereby declares that this thesis contains no material previously published or written by

another person unless stated otherwise. This work has not been submitted, in part or

full, for the purpose of obtaining any other degree.

Nomah Sangu

Signature:…………………………… Date: ……………………………….

ii

Acknowledgements

I would like to express my sincere gratitude to my advisor, Professor Les Copeland, for

his unwavering support and encouragement during the course of my studies. I would also

like to thank my associate supervisor Professor Richard Trethowan for his support.

I would like to extend my appreciation to the staff in the Faculty of Science at The

University of Sydney for their help whenever I needed it. I am grateful for the help from

Ms. Iona Gyorgy, Dr Sahar Aldehar, Dr Ali Khoddami, Dr Nicolas Daneri and Mr. Michael

Turner.

I would like to thank my parents, siblings and friends for their never ending support and

encouragement. My sincere gratitude also goes to my husband, Jonathan and my two

daughters, Shalom and Sheerah for their endless love, support, patience and

encouragement throughout my studies.

iii

Abstract

Wheat grain proteins and starch quality are commonly affected by abiotic stress

conditions. In order to reduce the effects of climate change, there is need to select heat

tolerant varieties usually grown in warmer regions. The University of Sydney’s Plant

Breeding Institute is working to improve heat tolerance through novel crosses between

commercial Australian wheat cultivars and the ancestral tetraploid, Emmer wheat

(Triticum dicoccum), which has good heat tolerance. Several potential lines have been

identified in the breeding programme, but there is need to determine their grain quality

attributes. The objective of this project was to study the effect of heat stress on the

quality attributes of heat stressed wheat grains grown in Narrabri, NSW in 2014. The

heat stressed wheat plants were sown 2 weeks after the normal planting date. The

average maximum daily temperature in the early stages of grain filling for the unstressed

grain was 22⁰ C, whereas that for the stressed grain was 29⁰ C. Thirteen out of 200

wheat lines were chosen, based on yield, 95% of Suntop and screenings < 30%. Protein

content and swelling index of glutenin was determined. SDS-PAGE was used to analyse

both the soluble and insoluble protein fractions. Proteomic analysis was used to

determine the identity of the proteins of interest. Total starch content, amylose content of

the starch and pasting properties of both flour and starch was measured. Heat stress

caused an increase in protein content and swelling index of glutenin. Qualitative

differences were observed when SDS-PAGE gels were run. The main proteins affected

by heat stress, as identified by proteomics, were serpins. Heat stress caused a

significant reduction in total starch content in most but not all of the genotypes, but no

significant changes were observed for amylose content. Heat stress resulted in a

significant difference between unstressed and heat stressed grain in pasting viscosity

(PV) and breakdown viscosity (BV) of flour, as well as trough viscosity (TV) and final

viscosity (FV) of starch. SDS-PAGE was used to analyse protein extracts, but no

differences were observed between parent and respective progeny for the soluble

protein, whereas qualitative changes in protein expression (presence or absence of

protein bands) were observed in grain from entry 3 and its progenies entries 68, 176,

177. Proteins were sent for proteomic analysis and were identified as serpins. Serpins

are termed soluble proteins, but their occurrence in the insoluble protein fraction maybe

due to the effect of heat stress which changes solubility or the ability to associate with the

gluten network. Therefore, serpins may be associated with improved heat tolerance.

iv

Since serpins require strong detergents for their extraction, they may be closely linked to

gluten-forming proteins and thus may have an effect on dough quality.

v

Contents

Statement of originality .............................................................................................................................................. i

Acknowledgements ................................................................................................................................................... ii

Abstract ...................................................................................................................................................................... iii

Chapter 1 Literature Review ........................................................................................................................................1

1.1 Introduction ..........................................................................................................................................................1

1.2 Origins of wheat ..................................................................................................................................................2

1.3 Characteristics of ancient wheats .....................................................................................................................3

1.4 Wheat grain structure .........................................................................................................................................4

1.5 Starch ...................................................................................................................................................................6

1.6 Wheat endosperm proteins ...............................................................................................................................9

1.7 Wheat quality assessment ..............................................................................................................................12

1.8 Influence of wheat physiology on grain yield and quality ............................................................................14

1.9 Effect of growth environment on starch properties ......................................................................................16

1.10 Effect of growth environment on protein properties ...................................................................................17

1.11 Analytical methods for grain proteins and starch .......................................................................................18

1.12 SDS-PAGE ......................................................................................................................................................18

1.13 Swelling index of glutenin ..............................................................................................................................21

1.14 Proteomics .......................................................................................................................................................22

1.15 Amylose content .............................................................................................................................................22

1.16 Rapid Visco Analysis (RVA) ..........................................................................................................................24

1.17 Objectives ........................................................................................................................................................27

Chapter 2 Materials and Methods .............................................................................................................................28

2.1 Grain selection ..................................................................................................................................................28

2.2 Growing conditions ...........................................................................................................................................28

2.3 Protein Extraction .............................................................................................................................................29

2.4 Preparation of protein reagent ........................................................................................................................30

2.5 Protein Assay ....................................................................................................................................................30

2.6 Swelling Index of Glutenin ...............................................................................................................................30

2.7 SDS-PAGE ........................................................................................................................................................31

2.8 Starch extraction ...............................................................................................................................................31

2.9 Total starch content ..........................................................................................................................................32

2.10 Amylose content .............................................................................................................................................32

2.11 Pasting characteristics ...................................................................................................................................32

2.12 Statistical Analysis ..........................................................................................................................................33

CHAPTER 3 RESULTS AND DISCUSSION .........................................................................................................34

3.1 Summary of mean values ................................................................................................................................34

3.2 Protein content .................................................................................................................................................35

3.3 Swelling index of glutenin ................................................................................................................................38

3.4 Total starch content ..........................................................................................................................................39

vi

3.5 Amylose content ...............................................................................................................................................40

3.6 Pasting properties of flour ................................................................................................................................42

3.7 Pasting properties of starch.............................................................................................................................43

3.8 Comparison of mean RVA profile values ......................................................................................................49

3.9 SDS-PAGE Gels ...............................................................................................................................................50

3.10 Proteomics .......................................................................................................................................................50

CHAPTER 4 Conclusions ..........................................................................................................................................55

References ...............................................................................................................................................................58

vii

List of figures

Figure 1.1 Anatomy of wheat grain. ………………………………………………………………………………...............................................5

Figure 1.2 Light microscopic view through the wheat aleurone layer. …………………………………….............. ……. …………… ..6

Figure 1.3 Double helixes packing configuration according to crystalline type. …………………............................................7

Figure 1.4 Granular and molecular structures of A (disk-shaped) and B-(spherical) granules of

starch.…………………………………………………………………………………………………………………………………………………………………………..8

Figure 1.5 Schematic diagram of amylopectin with a branch point at the 1,6 position. ……………..…................................9

Figure 1.6 Structural model for high molecular weight glutenin subunits. …………………….…...........................................11

Figure 1.7 Flow chart of assimilates for grain filling. .…………………………………………………..…….…......................................15

Figure 1.8 Polymerization of acrylamide.……………………………………………………………………………....…..................................19

Figure 1.9 Separation principle of electrophoresis.……………………………………………….......................................................20

Figure 1.10 Typical Rapid Visco Analyser pasting curve identifying characteristic features. ………………………………………26

Figure 3.1 RVA profiles for flour………………………………………………………………………………………………………………………………..44

Figure 3.1 RVA profiles for starch………………………………………………………………………………………………………………………….....47

Figure 4.2 SDS-PAGE true replicate gels for the insoluble protein fraction………………………………………………………………..51

Figure 4.3 Replicate gels for soluble protein fraction…………………………………………………………………………………………….....52

viii

List of tables

Table 1.1 Summary of the types and characteristics of wheat grain

prolamins.…………………………………………………………………………………………….…...……………………...…………………………..…...10

Table 1.2 Summary of desirable wheat quality attributes and end products.………………………………………………………..13

Table 2.1 Breeding lines selected for the study…………………......................................................................................29

Table 3.1 Table of mean values………………………………………………………………………………………………………………………....…35

Table 3.2 Values for Protein Content, SIG,Total Starch Content and Amylose Content……………………………………..…..37

Table 3.3 RVA values for flour………………………………………………………………………………………………………….…………….………45

Table 3.4 RVA profile values for starch……………………………………...……………….........................……………………………….…48

Table 3.5 RVA meav values………………………………...……………...........…...………………………………………………………..………….49

Table 4.1 Summary of proteomics results…...………………..................………………....…...…………………………………………..…54

1

Chapter 1 Literature Review

1.1 Introduction

Cereal grains are the foundation of the human food chain. Wheat is one of the major

food crops and has various applications both in food and non-food industries. Wheat

quality refers to the fitness of grain for a specific end-use, which can involve processing

into foods, feeds or industrial products. The most crucial determinants of grain quality are

proteins and starch, which constitute up to 9-20% and about 73% of cereal grains,

respectively (Gutierrez-Alamo et al., 2008). Between 2010 and 2050, world food demand

is expected to rise by at least 50% due to increasing population and the growing demand

for a "westernised" diet in developing countries (Tai et al., 2014).

Due to the need to feed the evergrowing world population, it is important to protect the

crops from climatic changes and this requires application of productive food security

measures (Yumurtaci, 2015). Climate change characterised by high temperatures, water

deficit and intense rainfall are jeopardising crop quality and yield. Elevated temperatures

will continue to threaten future sustainable crop production, such that world cereal

production is predicted to be reduced from the year 2030 (Yumurtaci, 2015). Some

climate change models predict that Africa and North America will have reduced crop

yields due to rising temperatures in the 2040s (Liu et al., 2013).

To mitigate the effects of climate change and to reduce losses due to heat stress, there

is need to select for heat tolerant varieties normally grown in warmer regions (Tai et al.,

2014). The University of Sydney's Plant Breeding Institute is developing new wheat lines

with superior drought and heat tolerance to boost production in more difficult climatic

conditions. One method being used to improve heat tolerance is through novel crosses

between commercial Australian wheat cultivars and the ancestral tetraploid, Emmer

wheat (Triticum dicoccum), which have desirable heat tolerance but lack modern

agronomic and yield attributes. Emmer wheat has drought and heat tolerance attributes

and is increasingly being used as a pool for desirable genes in wheat breeding

(Zaharieva et al., 2010; Trethowan and Mujeeb-Kazi, 2008).

2

Numerous promising new lines have been identified, but their grain quality attributes are

not known. Knowledge of the characteristics of the grain proteins and starch would be

beneficial to the end users such as plant breeders and the food industry.

1.2 Origins of wheat

About 50-70 million years ago marks the evolution of the family Poacea (grasses); the

subdivision Pooideae, including wheat, barley and oats, emerged about 20 million years

ago (Peng et al., 2011). The majority of the people on Earth derive a substantial part of

their nutrition from the grass family, which is composed of about 10,000 species

classified into 600 to 700 genera. Grasses belong to a group of plants called

monocotyledons, which includes all flowering plants with a single seed leaf (Kellog,

2001). The domestication of plants and animals played a pivotal role in the human

evolution. Use of primitive progenitors is an ideal way to crop advancement due to their

increased resistance to environmental stresses, and thus provides hope for future

breeding programs (Nevo, 2007; Peng et al,. 2011).

Wheat was first cultivated about 10,000 years ago under the “Neolithic Revolution”,

which was characterised by settled farming. Wheat has an incomparable diversity and

has secured a place in the culture and even in the religion of various communities

worldwide. The first domesticated forms of wheat were landraces chosen by farmers

from wild wheats. Domestication involved choosing genetic characteristics that

distinguished plants from their wild forms (Shewry, 2009). All Triticum species emanated

from the “Fertile Crescent” of the Near East, including the eastern parts of the

Mediterranean and south eastern areas of Turkey (Matsuoka, 2011; Luo et al,. 2007;

Shewry 2009; Heun et al,. 1997). The ancient wheats were diploid einkorn (genome AA)

and tetraploid emmer (genome AABB) wheats. Allotetraploid wild emmer wheat, Triticum

dicoccoides (TD) 2n = 28, the ancestor of cultivated wheats, has donated two genomes

to bread wheat and forms a pivotal role in wheat evolution and improvement (Nevo,

2014; Peng et al., 2011; Dvorak et al., 2012).

The genus Triticum is made up of six biological species of wheat at three ploidy levels.

These species are subdivided into three sections: Monococcum (has diploid species);

Dicoccoidea (contains tetrapliod species) and Triticum (comprises hexaploid species).

3

Triticum urartu, Triticum turgidum and Triticum aestivum are important to the wheat

lineage (Matsuoka, 2011; Dvorak et al., 2012). The main two types of cultivated wheats

are the hexaploid bread wheat, T. aestivum (2n = 6x = 42, AABBDD), which constitutes

about 95 percent of world wheat production and the tetraploid, hard or durum wheat, T.

turgidum (2n = 4x = 28, AABB) constituting the other 5 percent (Peng et al., 2011; Nevo,

2014). T. aestivum emanated from a hybridisation between cultivated tetraploid emmer,

T. dicoccum and the goat grass Aegilops tauschii (DD), but the resulting hexaploid was

spelt (Peng et al., 2011; Nevo, 2014; Blatter et al., 2004; Dvorak et al., 2012; Shewry,

2009).

Free-threshing cultivated wheat emerged as a result of mutations in three major genes

under the process of wheat evolution. These three main genes are Br, Tg and q, which in

their wild types are characterised by a brittle rachis, tenacious glume and the non-free

threshing character, respectively. A dominant mutant at the q locus transformed the

recessive mutations at the Tg (tenacious glume) locus and gave rise to these free forms

(Shewry, 2009; Peng et al., 2011; Dvorak et al., 2012; Blatter et al., 2004). Wheat grains

have unique characteristics which makes them suitable for various end uses.

1.3 Characteristics of ancient wheats

Emmer is a hulled type of wheat that grows naturally in the Near East and is still grown in

the Mediterranean Basin (Pagnotta et al., 2005). Emmer wheat is still a vital crop in

Ethiopia and is grown in marginal lands in Italy and India (Zaharieva et al., 2010;

Cazzato et al., 2013). Spelt wheat is also still an important grain crop in marginal areas

in south eastern Europe, mainly in Germany and Switzerland (Cazzatoet al., 2013). In

the recent past, due to the rising demand for more natural and organic produce, there

was a new interest in emmer wheat, owing to its beneficial attributes for conditions like

high blood cholesterol and allergies, as a rich genetic pool for wheat breeding, and for its

capability to thrive in low fertility soils (Pagnotta et al., 2005; Cazzato et al., 2013;

Pagnotta et al., 2008).

Emmer wheat is popular for its tolerance to fungal diseases like stem rust, which are

dominant in wet areas and it has also demonstrated to be resistant to high temperature

stress. Due to the high protein composition of its grain, emmer wheat has the capacity to

4

be used in the manufacture of various food products (Konvalina et al., 2012; Zaharieva

et al., 2010). Emmer wheat contains high amounts of resistant starch, fibre and

antioxidants (Pagnotta et al., 2008), and can be used to produce bread with a more

appealing flavour, taste and colour than that from bread wheat. It has a good quality for

pasta with reduced stickiness, adequate firmness and dark colour. In India, the majority

of traditional foods were observed to have a delightful taste and flavour when they were

made from emmer wheat rather than from durum or bread wheat (Zaharieva et al.,

2010).

1.4 Wheat grain structure

The wheat grain is defined as a caryopsis, which refers to a fruit where the ovary wall is

joined to the single seed. Starting from the centre to the exterior, the layers are starchy

endosperm, the aleurone layer, the seed coat (epidermis and testa), the pericarp, the

hypodermis and the epidermis (Antoine et al., 2003). Figure 1.1 shows the anatomy of a

wheat grain. The cereal grain is a complicated organ composed of numerous tissues

with various physiological functions. The endosperm constitutes the majority of the

cereal grain and is made up of starch-granule filled cells in a protein matrix.

Arabinoxylans and beta-glucans are the major constituents of the endosperm cell walls

(Jaaskelainen et al., 2013).

In wheat kernels, the aleurone is a monocellular layer that constitutes the exterior part of

the endosperm, and produces the enzyme alpha-amylase, which is secreted during

germination into the starchy endosperm and facilitates breakdown of starch into maltose

and glucose. The aleurone constitutes 7-9 % of the kernel by weight and 45-50 % of the

bran component and contains the greater proportion of the metabolically essential

enzymes. Figure 1.2 shows a microscopic view through the wheat aleurone layer. The

pericarp makes up 5 % of the kernel, and is composed of about 6 % protein, 2 % ash, 20

% cellulose and 0.5 % lipid (Faltermaier et al., 2014). The germ is also termed the

embryo of wheat and constitutes 2.5-3.5 % of the kernel. The germ consists of two main

components, the embryonic axis and the scutellum, a storage organ. Germ is made up of

25 % protein, 18 % carbohydrates, 16 % oil and 5 % ash (Faltermaier et al., 2014).

5

Cell walls consist of structural proteins which influence physical attributes of the wall.

These are grouped into proteins which are high in glycine, proline and hydroxyproline.

Maternal tissues are composed of the exterior pericarp, interior pericarp (cross wall and

tube cells), pigmented seed coat (testa), the nucellar epidermis and the nucellus. The cell

walls of external layers are thick, hydrophobic and made of cellulose, xylans, lignin (10-

12 %) and provide protection (Saulnier et al., 2012). Among these, the thickest is the

aleurone layer (up to 65µm), the outer pericarp is of an average thickness 15–30 µm and

the seed coat measures 5–8 µm. The outer pericarp and aleurone layer contain large

amounts of highly ferulolylated arabinoxylans (Barron et al., 2007). Aleurone cells

secrete enzymes to hydrolyse nutrients for the germinating embryo. The nucellar

epidermis is the maternal tissue encompasing the endosperm and embryo (Evers and

Millar, 2006). Wheat kernels have a mean length of about 8 mm and are about 35 mg in

weight (Faltermaier et al., 2014).

Figure 1.1 Figure 1.1 Anatomy of wheat grain. From Saulnier et al., 2012

6

Figure 1.2 Light microscopic view through the wheat aleurone layer. Left to right: endosperm, aleurone cells, hyaline layer, testa, and pericarp. From Buri et al., 2004.

1.5 Starch

Starch is a combination of two main d-glucan polymers, amylose, a mainly linear α(1-4)-

d-glucan, and amylopectin, a highly branched, linear α(1-4)-d-glucan with α(1-6) linkages

(Li et al., 2013; Zhang et al., 2013a; Waduge et al., 2014). Starch is a major component

of various foods and its characteristics and associations with other elements, especially

water and lipids, are of fascination in the food manufacturing world and for the human

diet. Starches are classified into crystalline types namely A and B polymorphs. The A-

type structures are closely packed together, and the water molecules occur in-between

the double helical structures, as shown in Figure 1.3, while the B forms have a more

open structure and the water molecules are situated at the centre of the six double

helical cavity (Corre et al., 2010). The A- and B-type granules show variations in terms of

structure, crystallinity, amylopectin branch chain length and disposition and physical

characteristics such as swelling and gelatinisation (Li et al., 2013).

7

Figure 1.3 Double helices packing configuration according to crystalline type. From Corre et al., 2010.

Wheat starch granules have a bimodial size distribution, and depending on their shape

and size, can be divided into A- and B-type. The A-type starch granules have a lenticular

or disk configuration, and have a mean diameter ranging from 10 to 48 µm whereas the

B-type granules are spherically or polygonally shaped with a diameter of not more than

10 µm. The A-type starch granules exhibit narrow perforations on the exterior and coiling

channels on the interior part of the granules (Li et al., 2013). Figure 1.4 shows the

proposed granular and molecular configurations of the A and B granules of wheat starch.

Starch granules are semi-crystalline, meaning they are made up of both crystalline and

amorphous structures (Vamadevan and Bertoft, 2015). The morphological structure of

starch granules is composed of alternating amorphous and semi-crystalline layers called

growth rings. The amorphous growth rings consist of amylopectin and amylose chains in

a fairly disordered configuration, while the semi-crystalline regions are made up of

amylopectin chains carrying alternating crystalline and amorphous layers of about 9-11

nm thick in a lamellar order. The proportion of long to short branch chains affects the

configuration of amylopectin chains, which in turn influences the arrangement and thus

the structure and dimensions of the starch granule (Salman et al., 2009). Figure 1.5

shows amylopectin, with a branching point situated at the 1,6 location.

8

The degree of polymerisation of amylose ranges between 900 and 3,300 glucosyl units

and its chains are made up of around 270-525 glucose residues. The degree of

polymerisation of amylopectin lies between 4,800 and 15,900, with a chain length of the

branches of 18-27 glucose units (Vamadevan and Bertoft, 2015).

Starch granule crystallinity ranges from 15 to 45 % (Perez and Bertoft, 2010; Jane,

2006). Their diameter varies between less than 1 µm to about 100 µm. Native starch

granules contain around 10 % moisture. Amylose and amylopectin constitute 98-99 % of

the dry mass of the starch granules, with the rest consisting of minute quantities of lipids

and minerals (Copeland et al., 2009). Amylose constitutes upto 25-30 % of most wheat

starches, whereas amylopectin is 70-75 % of wheat starch. Amylose readily retrogrades

to form firm gels and strong films, whereas amylopectin produces soft gels and weak

films. Amylose is produced by granular-bound starch synthase, whereas amylopectin is

produced by soluble starch synthase acting together with starch branching enzymes

(Vamadevan and Bertoft, 2015).

Figure 1.4 Granular and molecular structures of A (disk-shaped) and B- (spherical) granules of starch. Figure 1.4 Granular and molecular structures of A (disk-shaped) and B-(spherical) granules of starch.

From Jane, 2006.

9

Figure 1.5 Schematic diagram of amylopectin with a branch point at the 1,6 position. From Zobel, 1988.

1.6 Wheat endosperm proteins

Cereal grains are the source of more than 200 million tones of proteins for the

nourishment of humanity and livestock. Seed proteins can be classified into three

classes: storage proteins, metabolic proteins, and protective proteins (Shewry et al.,

2002). Prolamins are the main storage proteins of all cereals. The prolamins of wheat

are chiefly responsible for the distinctive viscosity and elasticity characteristics of

wheat gluten. Gluten is a tenacious viscoelastic matrix characteristic of wheat flour

that gives cohesiveness to dough. Prolamins are divided into three classes: high-level

sulphur (S-rich), low-level sulphur (S-poor) and high molecular weight (HMW)

prolamins (Shewry et al., 2002). Prolamins are synthesised at the endoplasmic

reticulum during seed maturation and stored in subcellular molecules of the

developing endosperm (Zhang et al., 2013b). Table 1.1 shows characterisation of

wheat grain prolamins.

Wheat proteins can be classified into four categories: albumins (dissolve in water),

globulins (dissolve in dilute salt solutions), gliadins (dissolve in 70 % ethanol) and

glutenins (dissolve in dilute acids and bases). Gluten proteins consist of two

10

components – the gliadins, which dissolve in alcohol and the glutenins, which do not

dissolve in alcohol (Barak et al., 2015; Ang et al., 2010).

Table 1.1 Summary of the types and characteristics of wheat grain prolamins (gluten proteins). From Shewry and Halford, 2002.

aC-type LMW subunits are essentially polymeric forms of α- and γ-gliadins and D-type LMW subunits polymeric ω -gliadins. The B-type

LMW subunits constitute a discrete group of S-rich prolamins. bCys is present in D-type LMW subunits, but not ω-gliadins.

The distinctive attributes of wheat grain are mainly attributed to the gluten-forming

storage proteins of its endosperm. Gluten proteins constitute some of the largest protein

molecules that exist. Glutenins fall into three categories depending on their

electrophoretic movement in SDS-PAGE, the A-, B- and C- regions of electrophoretic

gels. Group A (MW of 80,000-120,000 Da) are the high molecular weight glutenin

subunits. The B- (42,000 to 51,000 Da) and C- (30,000-40,000 Da) types are the low

molecular weight glutenin subunits (Gianibelli et al., 2001).

High molecular weight glutenin subunits (HMW-GS) contain non-recurring nitrogen and

carbon terminal regions, with a central recurring domain which bestows elasticity to

protein molecules. The HMW-GS carry a spiral central region, while the two terminal

sections are alpha helical. HMW-GS are responsible for dough elasticity and enable the

dough to trap gas bubbles released by yeast and thus allowing the dough to rise during

11

Figure 1.6 Structural model for high molecular weight glutenin subunits (Shewry et al., 1989).

baking (Anjum et al., 2007). The HMW-GS can be divided into x- and y-type, with

molecular weight ranging from 83,000 to 88,000 and 67,000 to 74,000, respectively

(Wieser, 2007). Figure 1.6 shows structural model for high molecular weight glutenin

subunits.

Gliadins constitute on average of half of the total prolamins of gluten (Ang et al., 2010;

Shewry et al., 1986). The α-, β- and γ-gliadins are monomeric proteins containing

intrachain disulphide bonds. The gliadins comprise a central part carrying recurring

sequences, with high levels of proline and glutamine, and two non-recurrent regions at

the N- and C-terminal ends (Barak et al., 2015). The molecular weight of α-gliadins

ranges from 30,000 to 34,000 Da, while that of the γ-type lies between 26,000 to 36,000

Da. The recurring regions of gliadins are composed of a combination of poly-L-proline ll

and beta-reverse turn structures, whereas the non-recurring regions consist of alpha-

helical structures (Ang et al.,2010). Gliadins exist as monomers and influence gluten

extensibility, while glutenins occur as high molecular weight polymers and responsible

for gluten elasticity and strength (Falcao-Rodrigues et al., 2005; Shewry et al., 1986).

Albumin and globulin proteins each contribute about 10% of the total composition of

flour. They are termed soluble proteins due to their ease of isolation from flour using

dilute salt solution. Albumins and globulins are mostly enzymes or proteins that play a

12

role in cell metabolic processes but can have an influence on flour protein content and

dough characteristics (Merlino et al., 2012; Merlino et al., 2009).

1.7 Wheat quality assessment

Wheat quality refers to the fitness of grain for a specific end-use (Varzakas et al., 2014).

Wheat quality assessment is usually based on the various physical, chemical,

biochemical and rheological characteristics. The visco-elastic characteristics of the

gluten proteins causes the distinct baking attributes of wheat (Marchetti et al., 2012;

Goesaert et al., 2005; Veraverbeke and Delcour, 2002; Ferrari et al., 2014; Khan et al.,

2012). One of the principal determinants of wheat quality is grain hardness, which is an

important part of wheat classification as it influences milling, baking and end-product

quality (Pasha et al., 2010; Bordes et al., 2008; Bettge and Morris, 2000). Grain texture

is usually determined by numerous environmental and physicochemical attributes such

as protein content of the kernel, size of the kernel and moisture content. In soft grained

wheat, there is an empty space surrounding the starch granules and this lack of

continuity offers a pathway for mechanical pressure, resulting in soft material which is

readily disrupted (Bettge and Morris, 2000).

Wholemeal flour is coarser in texture than white flour. In wholemeal flour processing,

bran and germ are retained, thereby making it higher in fibre and more nutritious than

white flour (Bordes et al., 2008). Gluten protein quality is influenced by the proportion of

monomeric gliadins to polymeric glutenins. There is need to reach an equilibrium

between dough viscosity and elasticity to attain high quality bread making (Goesaert et

al., 2005; Veraverbeke and Delcour, 2002). An important tool used for the evaluation of

wheat flour yield is the 1000 kernel weight, which is a measure of the size of the seed

and is the weight of 1,000 seeds (grams) (Khan et al., 2012). Two other critical

determinants in the fitness of wheat flour for specific end-uses are wheat flour colour and

brightness. Whiteness for steamed breads and salted noodles, and yellowness for

alkaline noodles, are examples. The occurrence of yellow pigments and their precursors,

and also bran, influences the brightness and colour of flour (Marti et al., 2013). Table 1.2

is a summary of desirable wheat quality attributes and end-products.

13

Table 1.2 Summary of desirable wheat quality attributes and end products.

End Product Desirable wheat quality attribute References

Course

textured flour

Hard Wheat Pasha et al., 2010

Bread Hard textured grain, high protein content,

good dough extensibility, high water

uptake, kneading tolerance

Hill, 2014; Bruckner et al.,

2001

Cakes,

cookies,

pastries

Soft wheat with low protein content, small

flour particle size

Hill, 2014; Moiraghi et al.,

2013; Moiraghi et al., 2011;

Pasha et al., 2010

Sweet

biscuits

Soft kernel texture, low protein content,

low level of vitreous kernel, low water

retention capacity

Duncan, 2000; Igrejas et al.,

2002

Pasta Hard textured grain, high protein content,

non-stickiness, low cooking loss

Kill and Turnbull, 2001;

Wrigley et al 2016;

Varzakas et al., 2014; Marti

et al., 2013

Chinese raw

noodles

Hard wheat with strong gluten, high

protein levels.

Hou, 2010; Habernicht et

al., 2002

Dry white

Chinese

noodles

Medium textured to hard textured wheat,

medium protein level, medium to high

gluten strength, low polyphenol activity,

reduced ash content

He et al., 2004; Habernicht

et al., 2002

Japanese

Udon

noodles

Soft textured wheat flour with low protein

content, increased starch viscosity,

increased flour swelling capacity

Hou, 2010; He et al., 2004;

Habernicht et al., 2002

Broiler feed Soft wheat Yegani and Korver, 2012

Pig feed High fibre content, high digestible energy Jha et al., 2011

14

The creation of an unbroken and strong matrix of solidified gluten proteins which traps

starch granules, reducing their expansion and solubilisation in cooking water, is the

major determinant of good pasta cooking quality (Varzakas et al., 2014; Marti et al.,

2013). Low level of noodle brightness and decreased colour durability over time

emanates from polyphenol oxidase in wheat flour (Habernicht et al., 2002). The ideal

quality soft textured wheat flour gives rise to cookies with a low thickness and soft

texture. The extent of cookie spread is largely dependent on the quantity of free water;

as the amount of water in the dough increases, there is an increase in the water uptake

by sugar, thereby reducing dough viscosity, and thus making dough easier to flow

(Moiraghi et al., 2011).

1.8 Influence of wheat physiology on grain yield and quality

Drought conditions cause a detrimental effect on wheat yields worldwide. The three

sources of carbon reserves for grains are photoassimilates from pre-anthesis re-

translocated to the grain; dry matter produced after anthesis and translocated directly to

the grain and photoassimilates produced post-anthesis and briefly stored before

retranslocation to the grain (Pheloung and Siddique, 1991; Blum et al., 1994; Latiri et al.,

2013; Inoue et al., 2004). The rate of grain maturation in cereals relies on the availability

of current photoassimilates and the ability of retranslocation of assimilates from the

vegetative parts to the grains. Fructans are non-digestible carbohydrates with numerous

nutritional characteristics including effects on mineral uptake (Blum et al., 1994).

Fructans are the main components of the water-soluble carbohydrates deposited in the

stem and contribute more than 80 % of the total water soluble carbohydrates. Water

soluble carbohydrates are sugars such as fructans, sucrose, glucose and fructose which

are deposited in the stem as reserves, from where they are later used as a reservoir for

remobilization to the developing grains (Inoue et al., 2004). Fructans mainly accumulate

in the lower stem internodes and are produced from sucrose when the photoassimilate

supply is more than the plant’s immediate demand (Li et al., 2013).

Both the vegetative and reproductive phases of wheat development are negatively

affected by high temperature stress. When plants are subjected to a particular extent of

15

heat stress, significant compensatory processes may occur such as regulation of water

or nutrient absorption. Plants develop a compensatory effect to satisfy the huge sink

demand, which includes a higher rate of retranslocation of assimilates produced before

anthesis, and this consequently produce a notable increase in yield (Ma et al., 2014). A

model shown in Figure 1.7 has been developed to demonstrate the various fluxes of

assimilates to the grains.

Figure 1.7 Flow chart of assimilates for grain filling. F1 (assimilates synthesised from leaves), F2

(assimilates from ears and awns), F3 (temporary storage of assimilates), F4s (remobilisation of assimilates

stored after anthesis), F4L (remobilisation of assimilates), D5 (demand of assimilates), F5 (supply of

assimilates to the grains), F6 (assimilates used by respiration). From Latiri et al., 2013.

The ideal growth temperature of wheat lies between 22 and 25 ͦC and desiccation occurs

when temperature rises above 35 ̊ C. Heat stress conditions cause a sharp decrease in

rate of photosynthesis post anthesis, thereby reducing the amount of current assimilates

translocated to the grain, but at the same time increasing the remobilisation of

assimilates from stem reserves. Photosynthates made before anthesis contribute

significantly to grain filling after exposure of wheat to long periods of heat stress (Inoue

et al., 2004; Pheloung and Siddique, 1991). After exposure to elevated temperatures, the

rate of photosynthetic activity of the flag leaf is decreased and remobilisation of

photoassimilates impeded causing a major reduction in grain weight owing to a decrease

in the grain filling period and grain development rate (Inoue et al., 2004). Photoassimilate

accumulation before anthesis may contribute from 10 % to more than half of the grain

16

yield based on environmental status, genotype and agronomical practices in wheat.

During the grain maturation period in wheat, nitrogen remobilisation to the grains is more

than nitrogen assimilation and contributes about 50 to 92 % of the nitrogen assimilated

into the grain at maturity (Pampana et al., 2007).

The susceptibility to water stress in wheat depends on the growth stage. Imposition of

water stress during the initial stages of grain filling has a profound effect on yield due to a

decline in endosperm cell number, and therefore sink volume for dry matter storage.

Exposure to water stress conditions results in a rapid reduction in grain water potential

and solute potential and these can hinder metabolic processes involved in grain filling.

Water stress conditions cause a substantial decline in rate of flag leaf photosynthetic

activity leading to early senescence of the flag leaf (Ahmadi and Baker, 2001).

Exposing wheat to water shortages at the initial grain filling stages causes a decrease in

grain weight and hence grain yield. Drought stress induces the untimely grain

dessication causing a significant reduction in grain sucrose and grain weight. Extreme

drought and excessive water supply results in starch content reduction. Under extreme

water stress conditions after anthesis, the proportion of amylopectin to amylose and

amylose content in the grain is greatly reduced (Chang-Xing et al., 2009).

1.9 Effect of growth environment on starch properties

Environmental conditions can influence wheat starch and protein quality (Liu et al.,

2011). Increased temperatures during the grain maturation period results in a decline in

the starch content of wheat grain (Nhan and Copeland, 2014; Tester et al., 1995). Two

crucial enzymes in the starch synthesis pathway, starch synthase and branching

enzyme, hold a pivotal role in inhibiting starch accumulation when temperatures rise

above 25⁰C during the grain filling period (Matsuki et al., 2003). Consequently,

amylopectin morphology is affected since both enzymes are involved in the extension

and branching of amylopectin molecules. Temperature stress also causes higher

gelatinisation enthalpy due to the higher stability of the crystallite configuration

emanating from the extended helices of the longer branch chains of amylopectin

(Matsuki et al., 2003). Gelatinisation enthalpy is the total amount of energy required to

cause starch to change from crystalline to gelatinisation.

17

Exposure of wheat to rising temperatures leads to a rise in the total amylose content of

wheat starch because amylopectin is more sensitive to high temperature stress than

amylose (Liu et al., 2011; Matsuki et al., 2003; Tester et al., 1995). Low water stress

after anthesis causes a substantial reduction in the proportion of B-type starch granules.

It also results in a reduction in lipid content of starches, as well as higher set back and

final viscosity which decreases the retrogradation of amylose when hot pastes are

cooled (Tester et al., 1995). Low water stress after anthesis limits the amount of

photoassimilation for grain maturation, but does not interfere with remobilisation of

carbon to the grain. The retranslocation of carbon reserves in the vegetative tissues and

their movement to the grain needs the commencement of whole plant senescence

(Bahrani et al., 2011).

1.10 Effect of growth environment on protein properties

Bread-making potential of wheat flour depends on grain protein content and constitution,

which are in turn affected by the genetic and environmental variation and their

interactions (Malik et al., 2013; Hasniza et al., 2014). The critical factors influencing

protein composition in mature wheat grain is wheat growing time, nitrogen accessibility

and temperature (Malik et al., 2013). Water availability and fertiliser application rate are

critical determinants influencing protein build up in wheat grain. When nitrogen is applied

late during plant development, a rise in the SDS-extractable protein in the grain is

observed, which correlates negatively with gluten strength. In general, polar forces such

as electrostatic and hydrogen bonding between protein molecules are weakened by

higher grain water content (Johansson et al., 2001). High temperature stress causes a

decline in the size of glutenin polymers, thereby diminishing the dough strength. Heat

stress during the grain maturation period leads to reduction of glutenin to gliadin ratio

(Zhu and Khan, 2001). Elevated temperatures can facilitate time to anthesis causing a

reduced grain filling period, thus resulting in a reduced grain yield (Asseng et al., 2011).

Elevated temperatures reduces the grain filling period and shortens time to physiological

maturity, causing a reduction in kernel weight. Rate of deposition of the various types of

protein subunits differs upon exposure to high temperatures (Hurkman et al., 2009). Heat

18

stress results in the creation of reactive oxygen species in the cytoplasm, as well as a

rise in the amount of stress/defense proteins and globulin storage proteins in the

endosperm of the fully developed grain (Hurkman et al., 2009). The build up of protein

components is most susceptible to water stress at late stages of the grain filling period.

Exposure to falling soil water potential after anthesis causes a decrease in the protein

components globulin, gliadin, glutenin and glutenin/gliadin ratio (Chang-Xing et al.,

2009). Wheat flour quality depends on protein and starch content, and there are different

analytical methods used to characterise these grain components.

1.11 Analytical methods for grain proteins and starch

1.12 SDS-PAGE

SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) is used to

separate and characterise biological molecules such as peptides, proteins, nucleotides

and nucleic acids. SDS can create hydrophobic bonding with nucleic acids and

nucleotides, thus conveying a negative charge on them and consequently making them

in polyacrylamide gel slabs in the presence of an electric field. SDS-PAGE separates

proteins depending on size and molecular weight, as well as comparing protein

composition of various samples, and number and size of polypeptide units (Bean and

Lookhart, 2000). Knowledge of the type of protein present in flour is important in order to

determine the end use of the flour. SDS-PAGE is especially important for the separation

of high molecular weight glutenin subunits, which are associated with bread quality

(Bean and Lookhart, 2000).

Addition of SDS, an anionic detergent, to protein mixtures breaks the disulphide bonds

and gives rise to production of highly negatively charged protein molecules (Kralova,

1999). In order to concentrate the protein sample into a sharp band, prior to entering the

fractionating gel, there is need to use a stacking gel. This will ultimately give more

distinct protein bands in the separating gel (Walker, 2002). Gradient gels are suitable to

separate proteins with a broad range of molecular weight values. SDS-PAGE involves

use of a discontinuous gel with a top stacking gel and a bottom resolving gel which have

dissimilar pH values and polyacrylamide concentrations (Brunelle and Green, 2014).

19

Wheat proteins are normally isolated in gels ranging from 6 to 17 percent, according to

the protein components being isolated (Bean and Lookhart, 2000).

There is a positive correlation between molecular weight and distance travelled by the

SDS-polypeptide complex in constant concentration gels (Bietz et al., 1992;Chiou et al.,

1999

Figure 1.8 Polymerization of acrylamide. From Walker, 2002.

Acrylamide chains are joined in a head-to-tail style forming extended chains and

sometimes every now and then a bis-acrylamide molecule is added into the extending

polymer (Walker, 2002), as shown in Figure 1.8 above. The pore size of polyacrylamide

gels is reduced as the total acrylamide concentration increases. The commonly used

gels have an acrylamide concentration of between 7 and 18 %, higher concentration gels

tend to change the protein structure, whereas gels with low concentration are unstable

(Jan-Christer, 2011). Radicals commence the process of polymerization, which occurs in

closed cassettes, in the absence of oxygen. Polymerization of vertical slab gels mainly

occurs between glass plates and lateral spacers which controls the thickness of the gel.

The speed of polymerization is determined by temperature, pH, oxygen and monomer

20

concentration (Jan-Christer, 2011). Figure 1.9 shows the separation principle of

electrophoresis.

Figure 1.9 Separation principle of electrophoresis. Proteins with different net charges and sizes migrate in an electric field in the presence of buffer with different migration velocities. The different proteins form discrete zones. From Bean and Lookhart, 2000.

Proteins exposed to some chemical changes and some very basic or acidic proteins are

unable to attach to SDS, thus producing unusually high molecular masses after SDS-

PAGE. This problem is avoided by increasing the acrylamide concentration (Chiou et al.,

1999). Bromophenol blue is the ionisable tracking dye which enables the electrophoresis

to be observed. It is essential to continue running electrophoresis until the dye runs off

the gel bottom so as to enable proteins to migrate along the whole gel, therefore

improving the band separation (Bietz et al., 1992).

Advantages of polyacrylamide gels are that they are replicable and are inert, and the thin

gels improve resolution due to good heat transmission. The excellent resolution by SDS-

21

PAGE enables the analysis of glutenin subunit composition (Bietz et al., 1992). SDS-

PAGE is also fairly cheap and convenient technique for analysing grain proteins.

However, it has a limitation in that it requires post-isolation staining to visualize the

bands and quantification can be challenging. Toxicity of acrylamide is avoided by using

precast gels (Bean and Lookhart, 2000).

1.13 Swelling index of glutenin

Different foods require dough with unique properties such as strength, extensibility and

mixing tolerance. There is need for predictive test methods which can be used to

separate grain depending on the dough-quality potential. Wheat grain buyers and millers

rely on the SIG test method to evaluate protein quality and there is potential for breeders

to use it for ideal dough strength potential (Uthayakuramaran et al., 2007). The swelling

index of glutenin test is an easier technique for measuring wheat processing quality, as

well as a criterion determining if gluten quality is strong or weak. The SIG test is now

commonly used to estimate the gluten strength and is an important parameter in

international trade specifications (Oikonomou et al., 2013). The SIG test is an essential

criterion in a breeding program for assessing the amount and quality of insoluble

glutenin. Many samples can be analysed in a limited amount of time, it is highly

reproducible and requires a small sample size (Wang and Kovacs 2002;

Uthayakuramaran et al., 2007).

The quantity of insoluble glutenin is indicated by these constitutional variants: relative

glutenin content (proportion of insoluble glutenin relative to total protein) and absolute

glutenin content (proportion of insoluble glutenin in flour), (Wang and Kovacs, 2002a). In

the SIG test, flour (40 mg) is mixed with deionized water and then left to swell in non-

reducing solvents (SDS, lactic acid or both), after which mixture is centrifuged at low

speed. The swelling time and mixing force in non-reducing solvents determine the

swelling potential of the glutenin. The SIG test must be carried out at a constant room

temperature (24⁰ C) so as to prevent variations in responses to temperature with

different cultivars (Wang and Kovacs 2002; Uthayakuramaran et al., 2007). The addition

of water improves the accuracy of the technique since flour readily suspends in water. If

1.2 mL of solvent is used for glutenin swelling, an ideal sample size is about 35–45 mg.

22

Glutenin swelling depends on the mixing strength, proportion of solvent-to-sample, and

the swelling time and temperature (Wang and Kovacs, 2002).

1.14 Proteomics Proteomics is an analytical method used to identify unknown proteins after separation by

electrophoresis or HPLC. First, the proteins are digested into peptides, whose mass is

estimated by a mass spectrometer. The peptide mass is compared to a database with

the theoretical mass of known protein sequence. The match with the highest score is

then concluded to be the identity of the protein (de Hoog and Mann, 2004).

Mass spectroscopy is a reliable technique for identification and quantification of proteins

(Bansal et al., 2016). A mass spectrometer is made up of an ion source (e.g., ESI –

electrospray ionization or MALDI - matrix assisted laser desorption ionization) which

ionises the protein sample and change them into gas; mass analyser (quadrupole or

TOF - time of flight), which separates the ions depending on their mass to charge (m/z)

ratios; a detector which records the amount of ions from the analyser; and a computer to

analyse the data and give rise to the mass spectra (Pedreschi et al., 2010).

The mass spectrometer provides data on the mass and intensity of the peptide peaks,

while the tandem mass spectra are merely for peptide identification (de Hoog and Mann,

2004). The protein in question is first broken down into smaller peptides usually through

the use of a trypsin enzyme. The trypsin enzyme works by distinctively cutting proteins

on the carboxyl-terminal side of arginine and lysine residues. The two soft ionization

techniques for mass spectrometry (ESI and MALDI) produce very little fragmentation and

usually whole ions are formed. The ions then enter the mass analysers such as TOF or

quadrupole. Quadrupole ion traps confine the ions in a powerful electromagnetic field

and continuously discharge them into the detector depending on their mass to charge

values. Ions which have a smaller mass/charge values will arrive at the detector first

(Pedreschi et al., 2010).

1.15 Amylose content

The proportion of amylose to amylopectin largely affects the end-use properties of

starches. Gelatinization, solubility, pasting properties and bread volume depend on

23

amylose content. Amylose content determination is thus an essential quality attribute for

the bulk of starch-based products (Mahmood et al., 2007; Zhu et al., 2008). Amylose

content is the principal attribute determining the capacity of the starch granule to absorb

water and expand. This is a crucial predictive tool to measure quality, eg, noodle quality.

For instance, in Japanese Udon noodles, the consistency of the cooked product is a

crucial characteristic which emanates from the ability of the starch granule to absorb

water, expand and maintain its structure (Wrigley et al., 2016).

The most popular method for amylose content determination of starch is iodine-based

colorimetry. The colorimetric method is not always very accurate due to the formation of

an association between iodine and macromolecules amylopectin chains, which absorb

light at a wavelength almost the same as that of the amylose-iodine compound. In

addition, the medium sized polymers have an effect on the iodine-binding process (Zhu

et al., 2008). Some degradation of the starch, however, can occur at the dispersion

temperature (100 ̊C) for starch, and after mixing with iodine, there is some change in

absorbance values, which needs a 15 min waiting period (Mahmood et al., 2007).

Starch-lipid complexes can also be created and this can affect the result. Therefore,

there is need to use water-alcohol solutions to extract lipids from the starch before the

analysis, (McGrance et al., 1998).

It is essential to completely dissolve the starch so as to obtain an accurate determination

of amylose content (McGrance et al., 1998). The starch solvents include perchloric acid,

hydrochloric acid, sodium hydroxide, potassium hydroxide and dimethyl sulphoxide. The

largest variation in the absorption of amylose and amylopectin in the presence of iodine

occurs at the optimum value for amylose absorption, which is about 620 nm based on

the botanical origin, hence this is the point for single-wavelength predictions. A second

wavelength is used at the peak of amylopectin absorption, 535-550 nm (Mahmood et al.,

2007.)

An alternative method for amylose content determination is the particular creation of

amylopectin complexes with the lectin concanavalin A (Con A). Con A particularly binds

with branched polysaccharides characterised by α-d-glucopyranosyl or α-d-

mannopyrannosyl units resulting in the creation of a precipitate in specified conditions of

24

pH, temperature and ionic strength (Gibson et al., 1997). This technique relies on the

precipitation of amylopectin by Con A in a solubilised, lipid-free starch sample, followed

by its removal by centrifugation. The supernatant contains amylose which is then

measured post its amylolytic hydrolysis to glucose by α-amylase and amyloglucosidase.

It is then presented as a percentage of the glucose produced by the amylolytic hydrolysis

of the total starch in a distinct aliquot of the dissolved sample (ie before the Con A

treatment) (Gibson et al., 1997; Yun et al., 1990).

Preparation of the Con A in solvent in concentrated form has the advantage of indefinite

storage without microbial contamination. It is essential to remove excess Con A after

precipitation with amylopectin. This is achieved by heat denaturation of protein in the

supernatant, followed by centrifugation (Yun et al., 1990). In this study, I chose the Con

A method because it is fast and accurate.

1.16 Rapid Visco Analysis (RVA)

The viscosity of starch pastes is determined for two reasons: (i) starch-paste viscosity is

an estimator of end-use functionality in the food manufacturing world and for other

industries (for example paper production), (ii) starch-paste viscosities is another option

for falling number measurements. The falling number test is an internationally

standardised method for sprout damage detection (Wrigley et al., 2016). The starch

paste peak viscosity values has become the most popular method of selecting for

improved quality for Japanese noodles in Australian wheat breeding programs (Crosbie,

1991). The general aims of rheological measurements are: (i) to determine the

mechanical characteristics of the starch paste (ii) to characterise and mimic the

behaviour of the paste during processing (Wrigley et al., 2016).

The minimum viscosity achieved is indicative of the stability of starch gel at high

temperatures. The final viscosity attained depicts the gel strength of products at low

temperatures (Wrigley et al., 2016). Wheat flour and water mixtures are essential

ingredients in the production of different food products (Lei et al., 2008). The RVA (Rapid

Visco Analyser) is an essential tool for determining the viscous characteristics of heated

starch and flour, and for determining the association between functionality and structural

attributes (Blazek and Copeland, 2008).

25

The RVA is a heating and cooling equipment designed for testing products high in starch

and similar substances which need tight control of temperature and shear (Nelles et al.,

2000). The aims of starch gelatinization (cooking) include enhancement of palatability of

starchy foods, change of the physical &/ or chemical functional properties of the starch.

Gelatinization is the disintegration of the molecular organization of starch granules when

heated above the gelatinization temperature in excess water (minimum of 14 water

molecules per single anhydrous glucose unit) (Nelles et al., 2000). Pasting involves

swelling of starch granules and loss of amylose, thereby causing a rapid increase in

viscosity. When starch granules are both heated and under shear forces, they are partly

disrupted and the viscosity is reduced due to the re-organization of the leaked amylose.

Further cooling causes re-alignment of polymers leading to the formation of a gel, which

is described as a “setback” or rise in viscosity (Doutch et al., 2012; Fujiwara et al., 2016).

Swelling tests are basic techniques which measure water absorption during the process

of starch gelatization. In wheat starches, amylopectin is responsible for water uptake,

swelling and pasting of starch molecules, while amylose and lipids seem to reverse

these activities. Viscoelasticity of starch pastes and gels emanates from the association

of amylose and amylopectin after they absorb water. When starch paste is cooled,

amylose chains are aggregated by hydrogen-bonding to form a gel (Blazek and

Copeland, 2008).

During activity of the RVA, starch slurry is heated from 50 to 95 ̊C in 4 to 8 minutes, the it

is held at 95 ̊C for 2-5 minutes prior to dropping the temperature to 50 ̊C, in a total

experimental time of 13 to 23 minutes. The optimum shear force is a fast (10 sec) stir at

960 rev/min, after which it is stirred at 160 rev/min throughout the duration of the heating

and cooling process (Nelles et al., 2000). Viscosity breakdown is caused by soluble

starch granules arranging themselves depending on the stirring direction and granular

disruption. Viscosity ultimately rises owing to the decline of energy in the system and

eventual hydrogen bonding between starch polymers (setback) during cooling (Nelles et

al., 2000). Several factors affect pasting properties include size of starch granules, starch

composition, processing ingredients and type of chemicals used pretreatment. The

cooling period after gelatinization enables the establishment of the extent of setback

26

(Zhou et al., 1998). Figure 1.10 shows the typical RVA pasting curve identifying

characteristic features.

Figure 1.10 Typical Rapid Visco Analyser pasting curve identifying characteristic features. From Zhou et al., 1998

Pasting temperature shows the lowest temperature needed to cook a specific sample.

Peak viscosity depicts the ability of the starch to bind water molecules, while final

viscosity indicates the capacity of starch to retrograde into a gelatinous paste following

cooling. The hold viscosity depicts the capacity of a starch paste to tolerate heat and

shear forces. Setback is associated with retrogradation of starch granules (Singh et al.,

2010). Acids, chelating agents like ethylenediaminetetra-acetic acid (EDTA) and heavy

metals such as mercuric chloride and silver nitrate are used to inhibit the action of α-

amylase (Batey et al., 1997). The RVA has the following advantages: small sample size,

capacity to establish temperature profiles and storage of computer data (Cozzolino etal.,

2013; Zhou et al., 1998). All these analytical methods are used to characterise wheat

grain proteins and starch.

27

1.17 Objectives

The wheat lines used in this study are new and have never been investigated for grain

quality. Hence, the objective of this project was to study the quality attributes of some of

the new stress-tolerant breeding lines by characterising the properties of proteins and

starch, in grain that was grown in Narrabri, NSW in 2014. Yield of the lines was not

diminished after exposure to heat stress. Only 13 lines performed well following heat

stress. Lines which performed well were lines whose yield was 95 % of Suntop and

screenings < 30%. Lines were selected from a very large database (over 200 entries)

based on yield, screenings and Thousand Kernel Weight (TKW). Heat stress was

applied by sowing the heat stressed plants two weeks after the normal planting date.

This study will focus on the following protein analytical methods: Sodium dodecyl sulfate

polyacrylamide gel electrophoresis, (SDS-PAGE), total protein content, swelling index of

glutenin and proteomics. Protein was not determined by NIR due to miscommunication

with breeders. Protein analytical methods will provide information on the type of proteins

present in the wheat grains. Knowledge of the type and amount of protein present affect

the end use of flour. The starch analytical methods include RVA (Rapid visco analysis),

total starch content and amylose content. Starch and amylose content values are

important in determining the quality of wheat flour.

28

Chapter 2 Materials and Methods

2.1 Grain selection

Wheat was grown in Narrabri in 2014. Table 2.1 shows the breeding lines selected for

this project. Suntop (11) is the standard. 2-49/C/K(3) is the parent to lines 68, 176 and

177. Sokoll (2) is the parent to entries 36 and 52. PBW550 (6) is the parent to entries

126, 129, 134 and 190. According to information obtained from Professor Richard

Trethowan, Director of the breeding program, these hexaploid parents were each

crossed (as a female) with tetraploid emmer wheat (as a male) and the resulting F1

progeny then backcrossed to each hexaploid parent (by using the F1 as a female and

the parent as the male). Hexaploid like plants from the BC1F1 were then selected and

double haploids made (around 10 per plant). BC1F1 is the crossing of a hybrid with one

of its parents. The wheat lines were selected based on a combination of screenings,

yield and TKW (thousand kernel weight). Lines which had a yield of more than 95 % of

the standard Suntop after heat stressing were selected. Lines which had a screening

value of less than 30 % after heat stressing were also selected. Screenings are defined

as unmillable material passing through a 2mm slotted screen after 40 shakes in grams

as percentage of grain sample weight (%). Therefore, screenings of less than 30 percent

indicate that the greater portion of grain was well developed.

2.2 Growing conditions

Wheat was grown at the I.A. Watson Grains Research Centre at Narrabri (situated 512

km northwest of Sydney at latitude 30.3324° S, 149.7812° E and an elevation of 212 m

above sea level) and is located on the northwestern slopes of NSW. Heat stressing was

achieved by planting the heat stressed plants 2 weeks after the normal planting date.

Two weeks was chosen because this was a realistic delay in a practical situation. The

average maximum temperature recorded for the last 2 weeks of the grain filling period for

the unstressed plants was 33.5 ͦC, whereas that for the heat stressed plants was 37.5 ͦC.

Predominant soil type at the experimental site was a black [AE] Vertosol cracking clay

with high water retention. AE means an A horizon distinguished by the eluviation of clay,

iron, aluminium, or organic matter. Irrigation was applied two months after sowing the

heat stressed plants, as well as at 2 weeks before harvesting the unstressed wheat

29

grain. The average annual rainfall for the season was 210 mm and the average annual

irrigation was 75 mm.

Table 2.1 Breeding lines selected for the study

Breeding line Description PEDIGREE Yield % Screenings TKW

Suntop (11) Standard 100.0 40.98 27.13

Sokoll (2) Parent 103.7 27.5 25.56

36 Progeny SOKOLL/2/SOKOLL/35883 M500110 98.0 29.1 31.52

52 Progeny SOKOLL/2/SOKOLL/35888 M500132 114.2 22.58 30.4

PBW550 (6) Parent 118.4 29.29 28.74

129 Progeny PBW550/2/PBW550/18343 KC75 121.7 29.79 29.07




2-49/C/K (3) Parent 64.46 27.36 28.42

68 Progeny 2-49/C//KENNEDY/4/2-49/C/K/3/35883 99.5 23.65 31.74



2.3 Protein Extraction

Whole meal flour was milled from wheat grain using a coffee grinder and sieved to pass

through a 500 µm screen. Flour samples for each cultivar were fractionated sequentially

into soluble and insoluble proteins according to the method of Wilkes and Copeland,

(2008), as described briefly as follows. Wholemeal flour was measured (200 mg) and

proteins were extracted by sonication for 10 s at 10 W with a Misonix Micron Ultrasonic

cell disruptor. Sonication helps to loosen the association between particles. Ice was used

30

to cool and the process repeated. Centrifugation was done at 16,000 x g for 20 min at

4 ̊C and the supernatant set aside. 1.0 mL of Tris buffer was used to sonicate the pellet

again and centrifugation done. The soluble protein fraction is the combined

supernatants. 1.0 mL of solution that contain 7M urea, 2M thiourea, 2mM

Tributylphosphine, 1 % (w/v) ASB-14 (amidosulfobetaine-14), and 40 mM Tris base was

used to extract the pellet. Sonication was done for 10 sec at 10 W and centrifugation

done. This is termed insoluble proteins.

2.4 Preparation of protein reagent

50 mL of 95 % ethanol was used to solubilise Coomassie Brilliant Blue G-250 (100 mg),

after which 100 mL 85 % (w/v) phosphoric acid was mixed in. Water was topped to make

a total volume of 1 litre. The resulting solution contained 0.01 % (w/v) Coomassie

Brilliant Blue G-250, 4.7 % (w/v) ethanol, and 8.5 % (w/v) phosphoric acid.

2.5 Protein Assay

A procedure from Bradford, (1976), was used to estimate the amount of protein. The

Coomassie dye-binding protocol was used and bovine serum albumin utilised as the

standard. A volume of 0.1 mL of solution was transferred into 12 x 100 mm glass tubes.

These had approximately 10 to 100 µg protein. Tris buffer was used to top up the

solution in the test tube to 0.1 Ml. The test tubes had 5 mL of protein reagent added to

them, and a vortex mixer was used to mix. Between 2 min and 1 hour, absorbance was

estimated at a wavelength of 595 nm against a reagent blank. The reagent blank

consisted of 0.1 mL of the Tris buffer mixed with 5 mL of protein reagent.

2.6 Swelling Index of Glutenin

Swelling index of glutenin was measured according to Wang and Kovacs (2002a;

2002b), with some modifications as follows. A 1.5 mL microcentrifuge tube had whole

meal flour (40 mg), together with 0.6 mL of deionized water added and kept at 20 ̊C. A

single tube vortex mixer was used to thoroughly mix the solution for 5 sec. A

Thermomixer (Eppendorf, Germany) at 1,400 rpm was used to mix the solution for 20

min at 24 ⁰C. Vortexing was done at 10 min and 20min. Vortexing helps break lumps in

31

the mixture. 0.6 mL of 1.5 % SDS solution was mixed in and the tube vortexed for 5 sec.

Centrifugation was done at 5000 rpm for 5 min. A syringe was used to scoop away the

supernatant and foam. The swollen residue was weighed. SIG was calculated as follows:

mass of swollen residue/original sample mass (12% moisture).

2.7 SDS-PAGE

The soluble and insolube protein fractions were fractionated by SDS-PAGE following the

protocol by Laemmli, (1970), and as modified by Wilkes and Copeland, (2008). Between

8-15 µg were loaded in each well when running gels. Approximately 50 µg of protein was

added to 62.5 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 2% (w/v) SDS, and 5% (v/v) 2-

mercaptoethanol. The solution was heated in a boiling water bath at 95 ̊C for 5 min. A

10% resolving gel (0.375M Tris, pH 8.8) with a 4% stacking gel (0.125M Tris, pH 6.8)

was used in electrophoresis. A buffer which consisted of 25 mM Tris, 192 mM glycine,

0.1% SDS at pH 8.3 was used in electrophoresis at a constant current (35 mA) at 10 ̊C.

A fixing solution of 10% methanol and 7.5% acetic acid was used to fix gels for a

minimum of 1 hour. Colloidal dye (0.1% Coomassie Brilliant Blue, 8% ammonium sulfate,

0.8% phosphoric acid, and 20% methanol) was used to stain gels overnight. A destaining

solution with 1% acetic acid was used and gels scanned using a Bio-Rad GS-800

densitometer.

2.8 Starch extraction

Starch was extracted following the protocol of Matheson and Welsh (1990) as described

below. A total volume of 300 mL of 0.2 M ammonium hydroxide solution was used to

soak wheat grains (100 g) for 24 hr. Another 300 mL of 0.2 M ammonium hydroxide was

used to blend the grain for 30 sec. A 250 µm nylon mesh was used to filter and the

residue was blended again in 0.2 M ammonium hydroxide solution and filtration

repeated. Blending and filtration was done over and over until solution was clear.

Centrifugation (5000 g) of the filtrates was done for 10 min at room temperature and the

supernatant thrown away. The resulting pellet has two different layers: the bottom white

pellet layer, and the brown top layer. The tailing was separated from the prime starch

layer. A spatula was used to separate the bottom from the top brown layer and mixed

with 0.2 M ammonium hydroxide. Centrifugation (5000 g) was done for 10 min at room

32

temperature. The brown layer of starch was thrown away and the white starch was

mixed together with the other white starch layer from recovered from the first process.

Water was used to wash the precipitate and centrifugation done at 5000 g for 10 min. A

solution of 0.2 M acetic acid was used to blend the precipitate and a120 µm nylon mesh

used in filtration. Centrifugation (5000 g) of solution was done for 10 min at room

temperature. Water, ethanol, water and acetone was sequentially used to wash the

precipitate. The starch pellet was kept overnight in a fume hood to dry to about 14%

moisture. Starch was stored at room temperature.

2.9 Total starch content

Total starch content of flour was determined using a Megazyme Total Starch kit

according to the instructions provided with the kit.

2.10 Amylose content

Amylose content of both flour and starch was determined using an Amylose/Amylopectin

Megazyme kit according to the instructions provided with the kit.

2.11 Pasting characteristics

The Rapid Visco Analyser RVA-4 (Perten Instruments, Macquarie Park, Australia,) was

used to determine the pasting properties of both starch and flour based on the Standard

Method 1 (STD1) which comes with the instrument as described below. Deionized water,

22 mL was added to 2.5 g of flour (10 % moisture), as well as 0.1% silver nitrate. The

was used to heat samples from 50 ⁰C to 95 C̊ within 3 min, after which samples were

held at 95 ⁰C for 5 min. Temperatures were dropped to 50 ̊C and were maintained at 50

⁰C for 7 min. The mixing paddle had a revolution of 960rpm for 10 s, before dropping to

160 rpm for the rest of the procedure. Peak viscosity (PV), viscosity at trough (TV) and

final viscosity (FV) were measured and breakdown viscosity (BV, which is the difference

between PV and minimum viscosity, MV) and setback viscosity (SV, which is the

difference between FV and MV) were recorded using the Thermocline software. Pasting

time (Pt) and pasting temperature (PT) were measured as well.

33

2.12 Statistical Analysis

All analytical tests were done on duplicate flour or starch samples. Means and

standard deviations were calculated with Microsoft Excel 2007. Paired t-test using Excel

was used to determine significant differences between attributes at P < 0.05.

34

CHAPTER 3 RESULTS AND DISCUSSION

In the subsequent presentation of results, T1 is the unstressed grain, whereas T2 is the

heat stressed grain. T2 was planted 2 weeks after T1, so experienced longer, higher

temperatures during grain filling. Figure 3.1 shows the daily min-max temperature for the

growing to sowing period. The average maximum temperature recorded for the last 2

weeks of the grain filling period for the unstressed plants was 33.5 ⁰C, whereas that for

the heat stressed plants was 37.5 ⁰C. The number of days with temperatures of more

than 30 and 35⁰C for T1 was 4, whereas that for T2 was 9. Figure 3.1 is a graph showing

daily min-max temp during the sowing-harvest period. The limitation of this study is that

only one season data was recorded and results may change over many seasons. Suntop

(11) is the standard. 2-49/C/K (3) is the parent to lines 68, 176 and 177. Sokoll (2) is the

parent to lines 36 and 52. PBW550 (6) is the parent to lines 126, 129, 134 and 190.

Figure 3.1 Graph showing daily min-max temp during the sowing--harvest period

3.1 Summary of mean values

Table 3.1 of mean values demonstrates that heat stress caused a significant increase in

protein content and SIG, but caused a significant reduction in total starch content. The

0

5

10

15

20

25

30

35

40

45

0 1 2 3 4 5 6 7 8

tem

pe

ratu

re (⁰C

)

time (months)

min max

35

protein content, SIG and starch content values for the heat stressed and unstressed

grain were significantly different from each other. There was no significant change in

amylose content following heat stress.

Table 3.1 Table of mean values

Protein % SIG Starch % Amylose%

T1 12.9±1e 5.4±0.9j 68.0±4k 27.0±2q

T2 15.4±0.5d 6.2±0.7f 65.0±5s 26.0±3q

Values are means ± SD of duplicate determinations. These are true replicates. Means is average of 13

values. Values with different letters within a column are significantly different (p< 0.05). T1- Unstressed

grain. T2 – Heat stressed grain. SIG - swelling index of glutenin

3.2 Protein content

Heat stress caused a significant increase in protein content. The unstressed and the

heat stressed grain were pIanted 2 weeks apart. The average maximum temperature

recorded for the last 2 weeks of the grain filling period for the unstressed plants was

33.5 ⁰C, whereas that for the heat stressed plants was 37.5 ⁰C. The number of days to

maturity for the unstressed wheat grain ranged from 148 to 156, whereas those for the

heat stressed wheat grain lied between 103 and 110. The unstressed 2-49/C/K (3) had

the same number of days to maturity as the unstressed standard. The results from the

present study show that protein content values for the unstressed (T1) and heat

stressed (T2) varieties were significantly different (p<0.05) from each other, as shown in

table 3.2. Suntop, the standard, recorded the highest increase in protein content, while

entry 134, derived from entry PBW550, had the smallest increase in protein content.

Cunningham Kennedy, parent to entry 68, 176 and 177 recorded the second highest

rise (4.2%) in protein content following heat stressing.

Numerous studies have reported that heat and drought stress affect both quality and

quantity of wheat protein by increasing the amount of protein in grain. Growth and

maturation of wheat plants, as well as quality attributes, are affected by heat effects.

The grain filling period is the most affected. There are conflicting results on the effect of

heat stress on wheat quality, (Spiertz et al., 2006). Different opinions exist on whether

36

heat stress has beneficial or detrimental impact on wheat quality attributes. Blumenthal

et al., 1991 showed that exposing wheat plants to heat stress produced grain with

weaker dough attributes. However, Modelstad et al., 2011, observed a positive

correlation between gluten quality and an elevated temperature from heading to about

halfway in the grain filling period.

Results from this study are in agreement with those obtained by Liu et al., (2017), who

found that high temperature regime resulted in an increased protein concentration in the

grains. Wheat was exposed to different temperature treatments of 27/17⁰C, 31/21⁰C,

35/25⁰C, and 39/29⁰C at 3 and 6 days post anthesis, as well as 10 days post anthesis.

In a study by Chinnusamy et al., (2003), 32 wild and cultivated wheat cultivars were

grown in the field, with the heat stressed wheat sown 4 weeks after the normal planting

date. The heat stressed plants were exposed to temperatures above 35 ⁰C, and a rise in

grain protein content was observed. Similar results were also obtained by Li et al.,

(2013), who observed an increase in protein content after heat stress. A rise in protein

quantity may be attributed to increased rates of grain nitrogen deposition & to reduced

rates of carbohydrate biosynthesis under high temperature stress environment. The

relative rise in protein concentration following exposure to heat stress may be due to a

decrease in grain weight and grain diameter, (Liu et al., 2017). The reduction in grain

weight and diameter was due to the disruption of endosperm cell multiplication, decline

in quantity of amyloplasts, formation of disfigured starch granules and the inhibition of

starch granule biosynthesis. TGW was measured for both T1 and T2.

In another study by Stone et al., (1995), 75 wheat cultivars were exposed to a 3 day

period of elevated temperatures (40⁰ C) at either 10 or 30 days post anthesis under a

specified environmental conditions. This gave rise to a significant increase in grain

protein content. Labuschagne et al., (2016), who exposed 2 wheat varieties to heat

stress in pot trials in a greenhouse, as well as Li et al., (2013), who investigated effect of

heat stress under field conditions also observed a significant increase in flour protein

content following heat stressing. Viswanathan and Khanna-Chopra, (2001), exposed

wheat to heat stress in a field environment also observed a rise in nitrogen content in two

wheat varieties after heat stress exposure.

37

Table 3.2 Values for Protein Content, SIG (Swelling Index of Glutenin) , Total Starch Content and Amylose Content.

Values are means ± SD of duplicate determinations. Mean of 13 genotypes. Values with different letters within a

column are significantly different (p< 0.05) from each other. Letters are random. Flour moisture content values

ranged from 14 to 17%, whereas starch moisture content values lay between 10 and 14%. Protein and starch values

corrected to a common moisture basis.

Breeding Lines Protein % SIG Starch % Amylose %

Suntop_T1 10.4±0.1t 5.1±0.1d 68.0±0.2j 27.0±1d Suntop_T2 15±0.2s 6.8±0.1g 65.0±0.3n 26.0±1d C/Kennedy_T1 11.3±0.1d 3.9±0.2t 69.0±0.1q 27.0±1f

C/Kennedy_T2 15.5±0.1a 4.7±0.4g 65.0±0.1d 28.0±2f

68_T1 13.2±0.2q 5.1±0.1w 74.0±0.2t 30.0±1g

68_T2 14.8±0.2b 5.3±0.5g 67.0±0.1j 32.0±1g

176_T1 12.3±0.1s 4.3±0.1k 71.0±0.1o 30.0±1q

176_T2 14.6±0.1c 5.8±0.7p 73.0±0.2l 29.0±1q

177_T1 12.4±0.1e 4.0±0.1s 71.0±0.1k 28.0±1b

177_T2 15.1±0.2f 5.4±0.1j 65.0±0.1s 29.0±1b Sokoll_T1 13.6±0.1f 6.0±0.1c 68.0±0.3f 26.0±1v Sokoll_T2 15.5±0.1d 6.7±0.1p 65.0±0.1q 32.0±1v

36_T1 13.3±0.2r 6.0±0.1s 67.0±0.3u 29.0±1a

36_T2 15.2±0.1g 6.8±0.1j 69.0±0.1d 26.0±1a

52_T1 13.4±0.1b 6.2±0.1c 70.0±0.1f 30.0±1d

52_T2 15.6±0.1h 6.5±0.1b 71.0±0.2n 27.0±1d

PBW550_T1 13.4±0.1v 5.3±0.1t 67.0±0.1p 26.0±1v

PBW550_T2 16.2±0.3i 6.8±0.1d 60.0±0.1v 24.0±1v

126_T1 13.5±0.1k 5.7±0.1m 69.0±0.2s 24.0±1w

126_T2 15.8±0.1j 6.3±0.1r 67.0±0.1k 24.0±1w

129_T1 13.4±0.2y 6.0±0.1n 70.0±0.1a 26.0±1i

129_T2 16.1±0.1k 6.8±0.1v 69.0±0.2u 26.0±2i

134_T1 14.4±0.1f 6.4±0.3k 63.0±0.1f 24.0±1u

134_T2 15.7±0.1x 6.4±0.5t 58.0±0.1b 27.0±1u

190_T1 13.3±0.3e 6.2±0.1e 60.0±0.1f 27.0±1h

190_T2 15.2±0.2m 6.4±0.1u 58.0±0.1k 26.0±2h

38

3.3 Swelling index of glutenin

SIG is an essential predictor of the gluten strength. It is a simple, quick test that gives a

reasonable comparison between samples. Heat stressing caused a significant increase

in the swelling index of glutenin. There was a 0.2 to 1.7 rise in the swelling index of

glutenin when wheat was subjected to heat stressing. Suntop experienced the highest

rise in SIG, while entry 190 had the lowest increase. Results from the present study

contrast with results obtained by Li et al., (2013), who observed a decrease in the mean

swelling index of glutenin under heat stress. Findings from the present study showed a

significant rise in swelling index of glutenin for heat stressed wheat. This is in agreement

with the results of Guzman et al., (2016), who observed a significant rise in swelling

index of glutenin following wheat exposure to heat stress. The variation on the effect of

heat stress on the SIG between these studies may be due to the distinctive flactuations

in the quantity of polymeric proteins. Gluten protein synthesis occurs during the grain

filling phase and research has shown that high temperature stress conditions have an

effect on the frequency and time span of the biosynthesis of glutenins and gliadins, their

constitution and the arrangement of polymeric proteins, (Li et al., 2013).

Gluten strength is a major parameter for predicting the rheological and processing

characteristics of wheat. In a report by Li et al., (2015), fifteen common wheat and nine

durum wheat cultivars were evaluated among three environments: no stress, drought

stress and heat stress. They found that for durum wheat, SIG accurately predicted gluten

strength in zero stress and heat stress environment. This is consistent with results from

the present study, where there was a significant rise in SIG following heat stress.

Moldestad et al., (2011), investigated seventeen field trials with four different wheat

varieties over three years. Their aim was to study the effect of natural variation in

temperature during grain filling on wheat gluten quality. Their results were in agreement

with results from the present study. A positive correlation was observed between gluten

quality and an increased stress from heading to almost halfway in the grain maturation

phase. In a study by Hurkman et al., (2013), one wheat cultivar was planted in

greenhouses under medium and elevated temperature regimes. After exposure to heat

39

stress, the greater proportion of high molecular weight glutenin subunits increased.

Ratios of high molecular weight to low molecular weight glutenin subunits also increased.

Another study by Zhang et al., (2001) also observed a 2 to 4-fold increase in HMW-GS

concentration in wheat from 33 days after athesis to maturity. In the study, spring wheat

was exposed to water deficit and/or high temperature periods at spikelet initiation,

anthesis or both. At maturity, the HMW-GS concentration was highest in heat stressed

grains.

Majoul-Haddad et al., (2013), subjected winter bread wheat to four hours of heat stress

at 38 ̊C on four successive days during the grain filling period. Glutenin protein fractions

were significantly increased at eight days after anthesis following heat stress treatment.

In a report by Don et al., (2005), four wheat lines were grown in different temperature

conditions under greenhouse environments. Heat stress caused a reduction in

HMW/LMW ratio of glutenin macropolymer (GMP), but increased glutenin particle size.

Considering effect of heat stress on dough mixing characteristics, size of glutenin

particles is more important than GMP concentration for dough making time.

Physicochemical methods such as gluten index can be used to effectively predict gluten

strength. The timing, time span and intensity of heat stress have an effect on the grain

quality attributes (Nhan and Copeland, 2014). Drought and heat stress have an influence

on the rate and duration of the build-up and composition of glutenins and gliadins, their

constitution, and the size distribution of polymeric proteins, (Li et al., 2013; Liu et al.,

2017). The distribution of the monomeric and polymeric proteins are vital factors affecting

wheat flour quality, (Daniel and Triboi, 2012).

3.4 Total starch content

Results from the present study show that exposure to heat stress resulted in a significant

(p<0.0.5) reduction in total starch content in most of the genotypes except for entries 36

and 176. Entry 68 experienced the highest drop in total starch content after heat stress.

A study by Spiertz et al., (2006), found that heat shock caused a negative effect on

starch biosynthesis. Results from the present study are consistent with Liu et al., (2017),

who observed a reduction in total starch content of wheat after exposure to high

temperature at anthesis. Their study showed that exposure of wheat to high temperature

at anthesis had a more significant effect than high temperature stress applied at grain

40

filling stages. In the present study, high temperatures were experienced during the grain

filling period. A reduction in starch content due to heat stress may be attributed to

reduced amylopectin concentration. Reduced starch concentration following heat

stressing may be explained by the disruption of endosperm cell division, reduced

amyloplast numbers, deformed starch granules, and restriction of starch granule

biosynthesis (Liu et al., 2017).

A significant reduction in total starch content was also observed by Chinnusamy and

Khanna-Chopra, (2003), after 32 wild and cultivated wheat genotypes were exposed to

temperatures above 35 ͦC. This reduction may be due to decreased activity of soluble

starch synthase. Under heat stress conditions, the relative percentage of protein to

starch rises because of a reduction in starch synthesis. Similar results to the present

study were also obtained by Tester et al., (1994), who observed a significant reduction in

starch content following heat stressing.

Research has shown that on average 25% of the reduction in total starch may be due to

smaller A-type starch granules, which means reduced number of amyloplasts, and 75%

due to fewer A-type starch granules. Wheat was subjected to temperatures more than 30

⁰C after anthesis and a significant decline in total starch content was observed by Liu et

al., (2010). In a study of 4 wheat cultivars, Matsuki et al., (2014) found that raising

maturation temperatures of wheat (30 ⁰C) reduced the total starch content per kernel by

32-49 %.The quality of wheat flour products relies on the starch content due to

associations between starch and water during gelatinization and pasting.

3.5 Amylose content

Results from the present study showed no significant change in amylose content after

exposure of wheat to heat stress, as shown in table 3.2. There are contrasting reports

on amylose content of wheat following heat stress. The present results are in contrast

with Liu et al., (2017) who found that wheat subjected to heat stress after anthesis

resulted in a significant increase in amylose content. They observed a rise in amylose

content of starch with increasing temperature above 30 ͦC during the first 14 days after

anthesis. The increase in amylose content in this study was maybe due to the timing of

the heat stress, which was at 10 days after anthesis. The study by Liu et al., 2017

41

differs from the present study because heat stress was applied at 3, 6 and 10 days after

anthesis, whereas in the present study, heat stress was applied during the grain filling

period. Results from the present study are in agreement with the study by Li et al.,

(2013), who did not find a significant increase in amylose content after exposure to heat

stress. However, they found a significant rise in amylose content after wheat was

exposed to drought stress, but not heat stress. The present study found no change in

amylose content maybe due to the timing of the heat stress, and also maybe because

grain was grown over just one season.

A study on the effect of heat stress on hulled rice by Ahmed et al., (2014), observed a

significant decrease in amylose content of the grains. Amylose is synthesised by a

single enzyme called granule-bound starch synthase (GBSS). The alteration in the ratio

of amylose-amylopectin after exposure to elevated temperature stress is due to a

reduction in GBSS activity. This may also be attributed to the comparatively reduced

action of the enzymes which participate in amylopectin production in the endosperm

during maturation which will have an effect on amylose-amylopectin ratio (Ahmed et al.,

2014).

A report by Stone et al., (1995), observed a reduction of 1.5% (P<0.1) in amylose

content in 64 % of the cultivars after exposure to heat 30 days after anthesis. However,

some of the cultivars exhibited no significant response to amylose content after heat

stressing, which is in agreement with results from the present study. Only one variety

showed a significant rise in amylose content following heat stressing. Similar results

were also obtained by Tester et al., (1994), who observed a rise in total amylose

content with increasing growth temperatures.

A study done by Yu et al., (2015), showed a significant decline in amylose content after

wheat was subjected to heat stress. The results contradict those of Labuschagne et al.,

(2009), who found a significant increase in amylose content in one of the varieties

following heat stress. The variation in amylose content after heat stress may be

because the rate of total starch, amylose, and amylopectin accumulation is reduced

from the mid to late stages of grain maturation phase under heat stress conditions. Heat

stress conditions have an influence on starch biosynthesis by slowing down plant

42

senescence and/ or by reduction of the grain filling period. Research has shown that

heat stress conditions significantly reduces the activities of starch synthase and

granule-bound starch synthase, which ultimately changes the composition of protein

and total starch, (Yu et al., 2015). Some reports have shown that heat stress reduction

of total starch content was owing to the reduced amylopectin content, while amylose

content remains relatively constant. Results similar to those obtained in the present

study were observed by Liu et al., (2010), who found that amylose concentration

remained relatively constant after heat stressing. A study of 4 wheat cultivars by

Matsuki et al., (2003), observed that amylose content was not significantly affected by

raised maturation temperatures (30 ̊C), despite some variations in response between

cultivars.

3.6 Pasting properties of flour

RVA was performed for both flour and starch because if differences exist between the two,

it implies that, other components in flour, other than starch are contributing to these

differences. The peak viscosities for flour ranged from 1156 cP to 2169 cP, as shown in

table 3.3. The breakdown viscosities were between 94.5 cP and 848 cP. The highest final

viscosity was 2338 cP while the lowest was 1867cP. The RVA profiles are shown in figure

3.1 demonstrate that all the entries show similar behaviour. Results from the present study

showed a significant drop in PV, TV and BV, as shown in table 3.3 with later sowing. This is

in agreement with results obtained by Liu et al., (2011), who observed reduction in PV, BV,

SV and FV in two wheat cultivars after exposure to temperatures above 30 ͦC after anthesis.

Heat stress causes a change in the structure and decreases the quantity of type A starch

granules, and ultimately starch biosynthesis is hindered and starch content and starch

viscosity are reduced, (Liu et al., 2011).

In a study by Wang et al., (2017), flours and starches of 10 leading popular Chinese wheat

cultivars were investigated under normal and heat stress conditions during the grain-filling

period. They observed no significant differences in TV, FV and PT, but a significant change

in PV and BV, which is in agreement with the results obtained in the present study.

However, for SV, the present study did not observe a significant change, while results from

Wang et al., (2017), showed a significant change. Zhong et al., (2005), subjected four

43

cultivars of rice to two temperature treatments, 22 ⁰C and 32 ⁰C. Heat stressing caused a

significant decrease in SV, as well as a rise in BV and SV, and this contradicts with results

from the present study. No significant differences were observed for other pasting

parameters between the two temperature conditions.

3.7 Pasting properties of starch

The peak viscosity of starch ranged from 2242.5 cP to 3188 cP, as shown in Table 3.4.

Entry 11_T1 had the highest peak viscosity. The setback viscosity lied between 560 cP and

913 cP. The pasting time for starch ranged from 5.4 to 6.7. The pasting temperature ranged

from 69 to 85.6 ̊C. Figure 3.2 shows the pasting profiles of starch for all the entries. Results

from the present study demonstrate a significant increase in PV and TV in most of the

entries following heat stressing, except for entries 6, 11 and 126 where a decrease in PV

was observed. This is in agreement with results obtained by Yu et al., 2015, who observed

a significant increase in PV and TV for two wheat varieties following heat stressing. Yu et

al., (2015), also observed a significant drop in setback viscosity, but peak time and pasting

temperature were not affected by heat stress, and this is similar to what was observed in

the present study.

A rise in FV may be accredited to an increase in PV, which could possibly have emanated

from the exudation of an increased quantity of amylose into the free water and increased

extent of starch swelling, thus causing a more gelatinous system to form during cooling and

and cause a rise in the FV. Starch granules with a lower BV values indicate increased

firmness and are ideal as a component for food subjected to elevated temperatures and

shear forces, (Singh et al., 2010).

44

Figure 3.2 RVA profiles for flour. Entry 11 is the standard. Entry 3 is parent to entries 68, 176 and 177. Entry 6 is parent to 126, 129, 134 and 190. Entry 2 is parent to 36 and 42. T1 is flour from unstressed grain and T2 is flour from heat stressed

45

Table 3.3 RVA values for flour

These are means of duplicates. Values with the same letters within a column are significantly different (p< 0.05). PV is peak viscosity, TV is trough viscosity, BV is breakdown, FV is final viscosity, SV is setback viscosity. Pt is Pasting time and PT is Pasting temperature. Entry 11 is the standard. Entry 3 is parent to entries 68, 176 and 177. Entry 6 is parent to 126, 129, 134 and 190. Entry 2 is parent to 36, 42. T1 is flour from unstressed grain and T2 is flour from heat stressed grain

Breeding Lines

PV TV BV FV SV Pt PT

(11) Suntop_T1 2146.5±15x 1327.5±23u 819.0±14e 2140.0±22f 812.5±20j 5.0±1y 83.0±1e

(11) Suntop_T2 1391.0±24a 1243.0±15u 284.5±20y 1985.0±26f 878.5±14j 5.0q1±1y 86.0±0e

(3) C/Kennedy_T1 1899.0±18e 1219.0±16w 681.0±15d 2169.0±21v 951.0±22d 5.5f 86.0±1r

(3) C/Kennedy_T2 1492.5±13g 1106.0±17w 386.5±15q 1989.5±15v 883.5±24d 6.0±2f 88.0±1r

68_T1 1621.0±21n 1187.5±22q 433.5±25s 2166.0±12d 979.0±22s 5.0±1y 87.0±1v

68_T2 1483.0±20t 1163.0±16q 320.0±16k 2096.0±14d 933.0±5±13s 6.0±1y 87.0±2v

176_T1 2014.0±17e 1363.0±13z 651.0±19m 2271.0±22e 907.5±12j 6.0±2k 87.0±1w

176_T2 1769.0±14k 1325.0±16z 444.0±22c 2189.5±21e 865.0±24j 6.0±1k 88.0±1w

177_T1 1964.0±25p 1332.0±26c 632.5±12y 2218.5±25y 886.5±26t 6.0±1j 87.0±1c

177_T2 1650.0±18q 1282.5±14c 367.5±14v 2107.0±29y 824.5±15t 6.0±1j 88.0±1c

(2) Sokoll_T1 1303.0±15b 1066.0±13j 238.0±23w 2008.0±29b 943.0±17t 5.3±1t 87.0±1h

(2) Sokoll_T2 1079.0±14r 976.5±23j 102.5±14r 1854.0±22b 878.0±16t 5.0±1t 87.0±1h

36_T1 1589.0±24e 1199.0±19t 390.0±13w 2220.0±23e 1021.0±24q 5.0±1k 87.0±0s

36_T2 1415.0±22 1156.0±16 259.0±21c 2140.0±19e 984.0±23q 5.0±1k 71.0±1s

52_T1 1511.5±14 1131.0±12 380.5±12q 2126.0±15b 995.0±21e 5.0±1j 88.0±1g

52_T2 1416.0±14l 1134.0±16x 282.0±16fp 2088.0±15b 954.0±22e 5.0±1j 72.0±0g

(6) PBW550_T1 1861.0±24o 1088.0±24b 773.0±22k 1892.5±12q 804.5±13j 5.0±2e 87.0±1d

(6) PBW550_T2 1225.5±14l 848.0±13b 378.0±13b 1552.0±12q 704.0±12j 5.0±1e 88.0±d

126_T1 1810.5±22r 1064.5±19n 746.0±18l 1820.5±21z 756.0±25p 5.0±2h 86.0±1f

126_T2 1702.0±22e 1090.0±13n 612.5±12j 1881.0±22z 791.5±11p 5.0±1h 88.0±0f

129_T1 1917.5±22q 1099.0±19u 818.5±13x 1865.5±12c 766.5±22d 5.0±1d 86.3±g

129_T2 1662.0±23w 1065.5±18u 596.5±21p 1876.0±19c 810.5±24d 5.0±1d 88.0±0g

134_T1 1433.5±13f 932.5±17e 501.0±13e 1677.0±14g 744.5±16g 5.0±1q 89.0±1q

134_T2 1407.5±22d 922.0±15e 485.5±12e 1706.5±14g 784.5±12g 5.0±2q 88.0±1q

190_T1 1533.5±14e 977.5±17m 556.0±17v 1724.5±11y 747.0±11i 5.0±1e 88.0±0d

190_T2 1321.5±21y 887.0±12m 434.0±25f 1599.0±22y 712.0±31i 5.0±1e 87.0±0d

46

In a study by Lu et al., (2016), the influence of heat stress at 1–15 days post pollination,

as well as 16–30 post pollination on the starch physicochemical characteristics of four

waxy maize varieties was investigated. Lu et al., (2016), observed that heat stress did

not affect the pasting temperature in all varieties, and this is in agreement with results

from the present study. However, heat stress subjected at 1-15 DAP, caused a

significant reduction in trough and final viscosities. This is in contrast to results of the

present study, where heat stress resulted in a significant increase in TV and FV. Maize

starch exposed to high temperatures at 16-30 DAP, showed higher PV than those at 1-

15 DAP. The difference in these results might lie in the time of the exposure to heat

stress. In the study by Lu et al., 2016, heat stress was implemented at the early stages

of grain maturation phase, whereas in the present study heat was applied at the late

stages of grain filling. Early grain maturation stage is an important phase for division and

transformation of endosperm cells, and metabolic disruptions may occur to plants

exposed to heat stress at this stage. The difference in pasting may be due to other grain

constituents which can affect the pasting behaviour, (Lu et al., 2016).

A report by Liu et al., (2011), exposed two wheat varieties to high temperatures (above

25 ⁰C) for 3 days at varying times post anthesis. The results observed were similar to

those from the present study since the SV and BV were significantly reduced following

heat stressing. High temperature stress significantly reduces starch accumulation and

concentration and consequently reduces starch viscosity, (Liu et al., 2011). High

temperature stress significantly reduces starch accumulation and concentration and

consequently reduces starch viscosity, (Liu et al., 2011). Similar results to the present

study were obtained by Panozzo and Eagles, (1998), who observed a significant rise

among cultivars for peak viscosity when seven wheat cultivars were grown in fifteen

environments with different temperatures for grain filling period, including temperatures

above 30 ͦC. Heat stress resulted in an increase in PV. Research by Wang et al., (2017),

investigated flours and starches of 10 leading popular Chinese wheat cultivars, under

normal and heat stress conditions during grain filling period. The obtained results go

hand in hand with results of the present study where a significant decrease in the

setback viscosity was recorded. However, the present study observed a significant

increase in most genotypes in PV, TV and FV similar to results by Wang et al., (2017).

47

Figure 3.3 RVA profiles for starch. Entry 3 is parent to entries 68, 176 and 177. Entry 6 is parent to 126, 129, 134 and 190.

Entry 2 is parent to 36 and 42. T1 is starch from unstressed grain and T2 is starch from heat stressed grain.

48

Table 3.4 RVA profile values for starch

Breeding Lines PV TV BV FV SV Pt PT

(11) Suntop_T1 3188.0±18e 2007.0±0.9e 1181.0±11g 2920.0±19k 913.0±24b 5.4±3t 69.0±2y

(11) Suntop_T2 2564.5±13a 1996.0±0.8q 569.0±13g 2875.0±14g 879.5±9u 6.1±6s 72.2±3f

(3) C/Kennedy_T1 2356.0±13g 1829.0±9x 527.0±8w 2503.0±8w 674.0±22k 6.0±1y 83.1±1r

(3) C/Kennedy_T2 2521.0±14p 2045.0±12w 435.0±12w 2879.0±13q 534.0±23g 6.0±1e 83.0±2y

68_T1 2990.0±15k 1670.0±19u 678.0±17n 2467.0±21l 789.0±11x 6.0±1p 83.0±1b

68_T2 3104.0±19m 1890.0±16k 534.0±18n 2897.0±21s 645.0±14r 6.0±2t 83.0±1i

176_T1 2526.0±15u 1671.0±13s 855.0±21d 2591.0±17g 920.0±18n 6.0±1v 80.0±1w

176_T2 2868.5±18u 2173.0±17c 695.5±22d 3043.5±15e 870. 5±14m 6.0±1n 83.0±1q

177_T1 3010.0±14p 2303.0±15r 707.0±12q 3456.0±20d 115.03±14r 6.0±1z 83.0±1d

177_T2 3149.0±25k 2491.0±18u 658.0±13q 3773.0±22p 128.2±16t 6.0±2h 83.0±2j

(2)Sokoll_T1 2242.5±9r 1754.0±12c 488.5±2g 2342.0±19q 588.0±18x 6.2±1s 83.0±3k

(2) Sokoll_T2 2540.0±8h 2137.0±09q 403.0±0.6g 2697.0±18z 560.0±9t 6.7±0d 84.0±1t

36_T1 2354.5±7j 1576.0±2b 779.0±0.8b 2280.0±14c 703.0±22b 5.6±1c 71.8±2j

36_T2 2477.0±7t 1856.0±7z 621.0±0.9b 2578.0±19p 721.0±11j 6.1±1c 74.6±3y

52_T1 2456.0±10h 1980.0±23m 786.0±9j 2345.0±11b 879.0±22s 6.1±1e 83.0±1r

52_T2 2567.0±13k 1879.0±22n 567.0±6j 2567.0±12w 678.0±21a 6.2±1g 82.0±1x

(6) PBW550_T1 3057.5±21q 2265.5±19k 792.0±17g 3227.5±21b 962.0±12w 6.0±1h 84.0±1a

(6) PBW550_T2 2878.0±22q 2175.0±16b 638.0±15g 2869.0±22t 1170.0±13d 6.0±2r 82.0±1z

126_T1 3099.0±13e 2538.5±17g 560.5±11w 3568.0±23r 1029.5±14c 6.0±1s 79.0±1n

126_T2 2969.0±15g 2499.5±13f 467.0±13w 3666.0±22d 1166.5±17h 6.0±1h 81.0±1d

129_T1 2345.0±20y 2456.0±14r 678.0±24e 2578.0±21b 1290.0±13e 6.0±2k 83.0±2r

129_T2 2567.0±23y 2256.0±10f 534.0±21e 2987.0±22b 1456.0±15e 6.0±1b 81.0±1c

134_T1 2345.0±13d 2678.0±13g 734.0±14z 3456.0±16h 987.0±21s 6.0±2s 84.0±1b

134_T2 2517.0±12d 2890.0±15b 567.0±16z 3678.0±1h 789.0±21c 6.0±3g 83.0±1v

190_T1 2890.0±22x 1980.0±11s 690.0±12f 2765.0±13e 145.06±23t 6.0±2y 82.0±2d

190_T2 3076.0±23x 2178.0±11a 498.0±22f 2978.0±15e 123.4±20t 6.0±1l 81.0±1q

Values are means ± SD of duplicates. Values with different letters within a column are significantly different (p< 0.05). PV is

peak viscosity, TV is trough viscosity, BV is breakdown, FV is final viscosity, SV is setback viscosity. Pt is pasting time

and PT is pasting temperature. Entry 11 is the standard. Entry 3 is parent to entries 68, 176 and 177. Entry 6 is parent to

126, 129, 134 and 190.

49

3.8 Comparison of mean RVA profile values

Table 3.5 shows that heat stress caused a significant drop in the PV of flour, whereas

there was no significant change in the PV of starch. This means that the heat stress is

having an effect on other components of flour, apart from starch, such as non-starch

polysaccharides. The table also shows that there was no change in FV for flour but a

significant increase for starch following heat stress. It is important to investigate the

other flour components which cause differences in pasting behaviour, such as non-

starch polysaccharides.

Table 3.5 RVA mean profile values

PV TV BV FV SV Pt PT

F_T1 1739±256k 1153±134g 586±187b 2023±203x 870±102a 5±0.3b 87±1.4j

F_T2 1463±196r 1099±150g 381±139m 1928±207x 846±207a 5±0.5b 85±6j

S_T1 2682±357f 2054±362n 727±174w 2808±468c 945±245z 6±0.2v 81±5p

S_T2 2754±258f 2190±295n 553±87d 3037±408s 918±305z 6±0.2v 81±4p

Values are means ± SD of duplicate determinations. Values with different letters within a column are significantly different

(p< 0.05). PV is peak viscosity, TV is trough viscosity, BV is breakdown, FV is final viscosity, SV is setback viscosity. F_T1

means flour from unstressed plants. F_T2 is flour from heat stressed plants. S_T1 is starch from unstressed plants. S_T2

is starch from heat stressed plants.

50

3.9 SDS-PAGE Gels

SDS-PAGE was used to analyse both the soluble and insoluble protein fractions so as

to determine effect of heat stress on expression of proteins between parents and

progeny. When the insoluble protein fractions were fractionated by SDS-PAGE,

qualitative changes in protein expression (presence or absence of protein bands) were

observed in grain from entries 68, 176, 177 and entry 2-49/C/K(3), which is a parent to

entries 68, 176 and 177. Qualitative differences in protein expression were observed for

proteins of MW of 43 and 50 kDa as shown in Figure 3.4. There are clear bands in

entries 68, 176 and 177 which are not expressed in entry 3. Boxes are used to mark

differences in protein bands. The gels shown in Figure 3.4 are true replicates, meaning

they are from separate protein extracts. However, when the soluble protein extracts

were analyzed using SDS-PAGE, no differences were observed between parent and

respective progeny, as shown in Figure 3.5.

3.10 Proteomics After qualitative differences were observed between parent, entry 3 and progenies,

entries 68,176 and 177, proteins were sent for proteomic analysis. Bands in boxes were

cut from the first gel image from entries 68, 176 and 177, as shown in figure 3.4 and

sent for proteomic analysis. The summary for the results is shown in table 3.6. Two

bands were cut from each of the entries. The upper band was of molecular weight of 50

kDa, while the lower band was of molecular weight of 45 kDa. Only the lower bands

gave a spectra classified as good. Spectra represent the relative abundance estimations

for the protein. The lower bands were identified as members of the serpin family of

proteins. Other classes of identified proteins include globulins and catalase.

51

Figure 3.4 SDS-PAGE true replicate gels for the insoluble protein fraction. True replicates. Qualitative differences between 3, 68, 176 and 177 are enclosed in the boxes. Bands from first gel image was sent for proteomics.

52

Serpins (serine proteinase inhibitors) are permanent inhibitors of serine proteinases of

the chymotrypsin family. The majority of serpins are indeed permanent inhibitors of

serine proteinases of the chymotrypsin family it has been popularly known for decades

now that some serpins restrict cysteine proteases and others have lost their capacity to

inhibit proteases and have changed their roles. Inhibitory serpins have eight to nine α-

helices (A – H), three β-sheets (A – C), and a reactive center loop (RCL); also known as

a reactive site loop, (RSL) with a distinct bait pattern for the serpin’s victim proteinase

(Roberts and Hejgaard, 2004). Serpins easily dissolve in salt solutions and constitute up

to 4% of the total proteins in the fully developed grain endosperm. Due to their

occurrence in dough liquor, serpins have an effect on grain quality attributes. Dough

liquor is the soluble fraction of dough. Moreover, they contain amino acid patterns in

their configuration which mimic the glutamine-rich recurring pattern present in prolamin

storage protein in the endosperm (Østergaard et al., 2000).

The protein scores ranged from 39 to 96. The score represents the probability of

theoretical detection for peptides under a distinct set of experimental conditions. The

Figure 3.5 Replicate gels of the soluble protein fraction

53

score is determined from the peptide’s sequence and the adjacent amino acids, and a

series of modifiable factors that represent the experimental conditions (Parker, 2001).

The higher the score the better. Serpins are soluble proteins, but the reason they occur

in insoluble protein fraction maybe due to the effect of heat stress, which may alter

solubility. Serpins in wheat, barley and rye grain occur in both ‘free’ and ‘bound’ forms,

and the latter is isolated only using a reducing agent. Almost half of the total serpin

found in the endosperm occur in the ‘bound’ form (Hejgaard, 1978). Serpins are a

member of the albumin group of proteins and are mostly water-soluble serine proteinase

inhibitors (Hasniza et al., 2014). Serpins are salt-soluble proteins and constitute about 4

% of the total protein in the fully developed ceral grain endosperm (Cane et al., 2008).

These proteins are related to quality. One attribute of serpins that might affect grain

quality attribute is the capacity to create complexes by disulfide bonding between

serpins and beta-amylase protein molecules which also occur in the grain endosperm

(Cane et al., 2008).

A study by Hasniza et al., (2014) obtained similar results with the present study for

proteomics. They investigated changes in proteins for wheat grown in different locations.

Peptide mass fingerprinting identified the proteins as mostly globulin and serpin

isoforms, together with triticin. They observed that the warmer 2009 season produced

grain with globulin and serpin isoforms, together with stress and heat cushioning

proteins. Serpins and globulins easily dissolve in water and salt solutions proteins and

would presumably been isolated in the soluble protein component. However, these

proteins were found in the insoluble protein component both in the present study and

the one by Hasniza et al., (2014), and these need powerful detergents and chaotropic

agents for their extraction. Soluble protein fraction was not sent for proteomic analysis.

This demonstrates that they may be intimately linked to gluten-forming proteins and

therefore may have an effect on dough quality. In a study by Majoul-Haddad et al.,

(2013), the effect of 4 hours of heat shock at 38 ⁰C, implemented on 4 successive days

during the grain filling stage of winter bread wheat was investigated. They observed that

none of the HMW-GS and only 13 storage proteins were affected by heat stress. A

triticin precursor was also observed to be abundant at maturity in heat stressed grain,

and this is consistent with results from the present study.

54

Table 3.6 Summary of proteomics results

Lane Band Spectra ID Accession No Score

68T1 Upper Beta amylase,partial [Triticum aestivum]

gi |32400764 62

68T1 Lower Good No significant human or wheat (SP or NCBI) hits

68T2 Upper No significant wheat (SP or NCBI) hits

68T2 Lower Good Serpin - ZIA OS=Triticum aestivum GN=WZCI PE=1 SV=1

SPZIA_WHEAT 45

176T1 Upper Cytochrome b6-f complex iron-sulfur subunit, chloroplastic OS=Triticum aestivum GN=petC PE=2 SV=1

UCRIA_WHEAT 39

176T1 Lower Good Serpin – ZIA OS=Triticum aestivum GN=WZCI PE=1 SV=1

SPZIA_WHEAT 54

176T2 Upper No significant human or wheat (SP or NCBI) hits

176T2 Lower Good Serpin – ZIA OS=Triticum aestivum GN=WZCI PE=1 SV=1 Catalase- 1 OS=Triticum aestivum GN=CATI PE=2 SV=1

SPZIA_WHEAT CATA1_WHEAT

96 40

177T1 Upper Poor Globulin – 3A [Triticum aestivum] gi |390979705 63 177T1 Lower Good Serpin – ZIA OS=Triticum

aestivum GN=WZCI PE=1 SV=1

SPZIA_WHEAT

72

177T2 Upper Poor Diphosphomevalonate decarboxylase [Triticum urartu]

gi |474368068 65

177T2 Lower Good Serpin – ZIA OS=Triticum aestivum GN=WZCI PE=1 SV=1 Catalase- 1 OS=Triticum aestivum GN=CATI PE=2 SV=1

SPZIA_WHEAT CATA1_WHEAT

60 41

The Lane represents the entry number. The Upper band are protein of molecular weight 50 kDa, while the lower band are protein of molecular weight of 44 kDa. The spectra represent the relative abundance estimations for the protein. Good spectra means protein is more abundant. The ID provides the name of the protein. The accession number is the unique identifier assigned to the protein by the database used to generate the report. The score represents the probability of theoretical detection for peptides under a distinct set of experimental conditions. The higher the score, the better.

55

CHAPTER 4 Conclusions

Heat stress caused a significant increase in protein content as well as the swelling index

of glutenin. Cunningham Kennedy (entry 3), the parent, experienced the highest

percentage rise in protein content, which was approximately twice as much as that for

the progenies, entries 68, 176 and 177. Genotypes respond differently to environmental

conditions. Knowledge of the type and amount of protein present in flour is important so

as model and predict the dough attributes associated with these proteins. Moreover, this

will benefit plant breeders for selecting germplasm for further breeding studies. Entries

176 and 177, which are progenies for entry 3, had approximately the same increase in

swelling index of glutenin. When the soluble protein extracts were analysed using SDS-

PAGE, no differences were observed between parent and respective progeny.

However, when the insoluble protein fractions were fractionated by SDS-PAGE,

qualitative changes in protein expression (presence or absence of protein bands) were

observed in grain from entries 68, 176, 177 and 2-49/C/K(3), which is a parent to entries

68, 176 and 177. Qualitative differences in protein expression were observed for

proteins of MW of 43 and 50 kDa. There were clear bands in entries 68, 176 and 177

which were not expressed in entry 3. The fortuitous finding of clear qualitative

differences between protein bands in SDS-PAGE gels may lead to the identification of a

protein associated with heat tolerance, by proteomics.

After qualitative differences were observed between parent, entry 3 and progenies,

entries 68, 176 and 177, proteins were sent for proteomic analysis. Bands were cut from

the second set of replicates from entries 68, 176 and 177, as shown in Figure 4.1.

Proteomic analysis identified the proteins as serpins and globulins. Serpins are soluble

proteins, but the reason they occur in insoluble protein fraction maybe due to the effect

of heat stress, which may alter solubility. Hasniza et al., (2014), also reported serpins in

the insoluble protein fraction identified by peptide mass fingerprinting. This is in

agreement with findings from the present study, where serpins occurred in the insoluble

56

protein fraction. These proteins could in some way be related to improved heat

tolerance. Moreover, research has shown that due to the fact that serpins occur in the

insoluble protein component, which requires powerful detergents for their extraction, it

demonstrates that they may be intimately linked to gluten-forming proteins and therefore

may have an effect on dough quality (Hasniza et al., 2014). The linkages may be

hydrogen bonds, disulphide or ionic bonds.

Exposure of wheat to heat stress resulted in a significant decrease in total starch

content, and no significant changes were observed for amylose content. The progenies,

entries 68 and 176 experienced a drop in total starch content which was twice as much

as that for the parent, entry 3. Heat stress caused a significant drop in peak viscosity of

flour for most genotypes, whereas heat stress did not cause any significant change in

peak viscosity for starch. RVA was performed for both flour and starch because if

differences exist between the two, it implies that other components in flour, other than

starch are responsible for these differences. There is need to explore the other

constituents which occur in flour, such as non-starch polysaccharides, which are

causing differences in pasting behaviour in the present study.

These results are interesting because there were clear qualitative (presence or absence

of protein bands) differences in protein expression due to heat stress. There was also

evidence for the presence of serpins in the insoluble protein fraction. There is room to

further investigate why the serpin proteins occur in the insoluble protein component. It is

important to explore why serpins are implicated in quality. Due to their abundance in the

endosperm protein, and owing to their occurrence in the liquid state of bread dough,

serpins have the capacity to affect grain quality attributes. Moreover, they have amino

acid series units in their configuration which mimic the high level glutamine recurrent

pattern which occur in the prolamin storage proteins in the grain endosperm (Cane et

al., 2008).

57

In summary, the present study has shown that heat stress caused a significant rise in

protein content and the swelling index of glutenin. There was a reduction in total starch

content, but no significant change was observed for amylose content and pasting

properties following heat stress. Heat stress also affected insoluble proteins. Serpins

occurred in the heat stressed progeny, but were not evident in the parents.

58

References

Ahmadi, A., Baker, D., 2001. The effect of water stress on grain filling processes in

wheat. Journal of Agricultural Science 136, 257–269.

Ahmed, N., Tetlow, I.J., Nawaz, S., Iqbal, A., Mubin, M., Nawazul Rehman, M.S.,

Butt, A., Lightfoot, D.A., Maekawa, M., 2015. Effect of high temperature on grain

filling period, yield, amylose content and activity of starch biosynthesis enzymes in

endosperm of basmati rice. Journal of the Science of Food and Agriculture 95, 2237-

2243.

Amerah, A., 2015. Interactions between wheat characteristics and feed enzyme

supplementation in broiler diets. Animal Feed Science and Technology 199, 1–9.

Ang, S., Kogulanathan, J., Morris, G., Kok, M., Shewry, P.R., Tatham, A., Adams, G.,

Rowe, A., Harding, S.,2010. Structure and heterogeneity of gliadin: a hydrodynamic

evaluation. European Biophysics Journal 39, 255–261.

Anjum, F., Khan, M., Din, A., Saeed, M., Pasha, I., Arshad, M., 2007. Wheat

gluten: High molecular weight glutenin subunits, structure, genetics, and relation to

dough elasticity. Journal of Food Science 72(3), 56–63.

Antoine, C., Peyrone, S., Mabille, F., Lapierre, C., Bouchet, B., Abecassis, J.,

Rouau, X., 2003. Individual contribution of grain outer layers and their cell wall

structure to the mechanical properties of wheat bran. Journal of Agricultural and

Food Chemistry 51(7), 2026–2033.

Ashraf, M., 2010. Inducing drought tolerance in plants: Recent advances.

Biotechnology Advances 28, 169–183.

59

Asseng, S., Foster, I., Turner, C., 2011. The impact of temperature variability on

wheat yields. Global Change Biology 17, 997–1012.

Bahrani, A., Abad, H., Sarvestani, Z., Moafpourian, G., Band, A., 2011.

Remobilization of dry matter in wheat: effects of nitrogen application and post-

anthesis water deficit during grain filling. New Zealand Journal of Crop and

Horticultural Science 39(4), 279–293.

Bansal, M., Sharma, M., Kanwar, P., Goyal, A., 2016. Recent advances in

proteomics of cereals. Biotechnology and Genetic Engineering Reviews 32, 1–17.

Barak, S., Mudgil, D., Khatkar, B., 2015. Biochemical and functional properties of

wheat gliadins: A review. Critical Reviews in Food Science and Nutrition 55(3),

357–368.

Barron, C., Surget, A., Rouau, X., 2007. Relative amounts of tissues in mature

wheat (Triticum aestivum l.) grain and their carbohydrate and phenolic acid

composition. Relative amounts of tissues in mature wheat (Triticum aestivum L.)

grain and their carbohydrate and phenolic acid composition. Journal of Cereal

Science 45, 88–96.

Batey, I.L., Curtin, B.M., Moore, S.A., 1997. Optimization of Rapid-Visco Analyser

Test Conditions for Predicting Asian Noodle Quality. Cereal Chemistry 74(4), 497–

501.

Bean S., Lookhart, G., 2000. Electrophoresis of cereal storage proteins. Journal of

Chromatography 881, 23–36.

Bedo, Z., Vida, G., Lang, L., Karsai, I., 1998. Breeding for breadmaking quality using

old Hungarian wheat varieties. Euphytica 100, 179–182.

60

Bettge, A., Morris, C., 2000. Relationships among grain hardness, pentosan fractions,

and end-use quality of wheat. Cereal Chemistry 77(2), 241–247.

Bietz, J.A., 1992. Review. Electrophoresis and chromatography of wheat proteins:

available methods, and procedures for statistical evaluation of the data. Journal of

Chromatography 624, 53-80.

Blatter, R.H.E., Jacomet, S., Schlumbaum, A., 2004. About the origin of European

spelt (Triticum spelta l.): allelic differentiation of the hmw glutenin b1-1 and a1-2

subunit genes. Theoretical and Applied Genetics 108, 360–367.

Blazek J., Copeland, L., 2008. Pasting and swelling properties of wheat flour and

starch in relation to amylose content. Carbohydrate Polymers 71, 380–387.

Blum, A., 1998. Improving wheat grain filling under stress by stem reserve

mobilisation. Euphytica 100, 77–83.

Blum, A., Sinmena, B., Mayer, J., Golan, G., Shpiler, L., 1994. Stem reserve

mobilisation supports wheat-grain filling under heat stress. Australian Journal of

Plant Physiology 21, 771–81.

Blumenthal, C.S., Batey, I.L., Bekes, F., Wrigley. C.W., Barlow, E.W.R., 1991.

Seasonal changes in wheat-grain quality associated with high temperature during

grain filling. Australian Journal of Agricultural Research 42, 21-30.

Bordes, J., Branlard, G., Oury, F., Charmet, G., Balfourier, F., 2008. Agronomic

characteristics, grain quality and flour rheology of 372 bread wheats in a

worldwide core collection. Journal of Cereal Science 48, 569–579.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analytical

Biochemistry 72, 248-254.

61

Bruckner, P., Habernicht, D., Carlson, G., Wichman, D., Talbert, L., 2001.

Comparative bread quality of white flour and whole grain flour for hard red spring

and winter wheat. Crop Science 41, 1917–1920.

Brunelle, F., Green, F., 2014. One-dimensional SDS-polyacrylamide gel

electrophoresis (1D-SDS-PAGE). Methods in Enzymology 541, 151–159.

Buri, R., von Reding, W., Gavin, M.H., 2014. Description and characterization of

wheat aleurone. Cereal Foods World 49(5), 274–282.

Cane, K., Sharp, P.J., Eagles, H.A., Eastwood, R.F., Hollamby, G.J., Kuchel, H., Lu,

M., Martin, P.J., 2008. The effects on grain quality traits of a grain serpin protein and

the VPM1 segment in southern Australian wheat breeding. Australian Journal of

Agricultural Research 59, 883–890.

Cazzato, E., Tufarelli, V., Laudadio, V., Stellacci, A., Selvaggi, M., Leoni, B.,

Troccoli, C., 2013. Forage yield and quality of emmer (Triticum dicoccum) and

spelt (Triticum spelta l.) as affected by harvest period and nitrogen fertilization.

Acta Agricultural Scandinavica, Section B - Soil and Plant Science 63(7), 571–

578.

Chang-Xing, C., Ming-Rong, H., Zhen-Lin, W., Yue-Fu, W., Qi L., 2009. Effects of

different water availability at post-anthesis stage on grain nutrition and quality in

strong-gluten winter wheat. Comptes Rendus Biologies 332, 759–764.

Chinnusamy, V., Khanna-Chopra, R., 2003. Effect of heat stress on grain starch

content in diploid, tetraploid and hexaploid wheat species. Journal of Agronomy &

Crop Science 189, 242—249.

62

Ciaffi, M., Tozzi, L., Borghi, B., Corbellini, M., Lafiandra, D., 1996. Effect of Heat

Shock During Grain Filling on the Gluten Protein Composition of Bread Wheat.

Journal of Cereal Science 24, 91–100.

Copeland, L., Blazek, J., Salman, H., Tang, M., 2009. Form and functionality of

starch. Food Hydrocolloids 23, 1527–1534.

Corre, D.L.,Bras, J., Dufresne, A., 2010. Starch nanoparticles: A review.

Biomacromolecules 11, 1139–1153.

Crosbie, G.B., Chiu, P.C., Ross, A.S., 1991. Shortened temperature program for

application with a Rapid Visco Analyser in prediction of noodle quality in wheat.

Cereal Chemistry 79(4), 596-599.

Daniel, C., Triboi, E., 2002. Changes in wheat protein aggregation during grain

development: Effects of temperature and water stress. European Journal of

Agronomy 16, 1-12.

Day, I., Augustin, M., Batey, I., Wrigley, C., 2006. Wheat-gluten uses and industry

needs. Trends in Food Science and Technology 17(8), 82–90.

DePauw, R., Knox, R., Singh, A., Fox, S., Humphreys, D., Hucle, D., 2012.

Developing standardized methods for breeding preharvest sprouting resistant

wheat, challenges and successes in Canadian wheat. Euphytica 188, 7–14.

Don, C., Lookhart, G., Naeem, H., MacRichie, F., Hamer, R.J., 2005. Heat stress and

genotype affect the glutenin particles of the glutenin macropolymer-gel fraction.

Journal of Cereal Science 42, 69-80.

Duncan, M., 2000. Technology of Biscuits, Crakers and Cookies. Woodhead

Publishing, Third Edition.

63

Dupont, F., Hurkman, W., Vensel, W., Tanaka, C., Kothari, K., Chung, O.,

Altenbach, S. 2006. Protein accumulation and composition in wheat grains: Effects

of mineral nutrients and high temperature. European Journal of Agronomy 25, 96–

107.

Dvorak, J., Deal, K.R., Luo, M., You, F.M., von Borstel, K., Dehghani, H., 2012.

The origin of spelt and free-threshing hexaploid wheat. Journal of Heredity 103(3),

426–441.

Evers, T., Millar, S., 2006. Cereal grain structure and development: Some

implications for quality. Journal of Cereal Science 36, 261–284.

Falcao-Rodrigues, M., Moldao-Martins, M., da Costa, M.B., 2005. Thermal

properties of gluten proteins of two soft wheat varieties. Food Chemistry 93, 459–

465.

Faltermaier, A., Waters, D., Becker, T., Arendt, E., Gastl, M., 2014. Common

wheat (Triticum aestivum l.) and its use as a brewing cereal. A review. Journal of

the Institute of Brewing 120, 1–15.

Ferrari, M., Clerici, M., Chang, Y., 2014. A comparative study among methods

used for wheat flour analysis and for measurements of gluten properties using the

wheat gluten quality analyser. Food Science and Technology 34(2), 235–242.

Fujiwara, N., Hall, C., Carlson, R., 2016. A comparison of Rapid Visco Analyzer

starch pasting curves and test baking for under- and overdosed dry baked mixes.

Cereal Foods World 61(1), 29-37.

Gianibelli, M., Larroque, O., MacRitchie, F., Wrigley, C., 2001. Biochemical,

genetic, and molecular characterization of wheat glutenin and its component

subunits. Cereal Chemistry 78(6), 635–646.

64

Gibson, T.S., Solah, V.A., McCleary, B.V., 1997. A procedure to measure amylose in

cereal starches and flours with Concanavalin A. Journal of Cereal Science 25, 111–

119.

Goesaert, H., Brijs, K., Veraverbeke, W.S., Courtin, C.M., Gebruers, K.,Delcour,

J.A., 2005. Wheat flour constituents: how they impact bread quality, and how to

impact their functionality. Trends in Food Science and Technology 16, 12–30.

Guzman, C., Mondal, S., Govindan, V., Autrique, J.E., Posadas-Romano, G.,

Cervantes, F., Crossa, J., Vargas, M., Singh, R.P., Pena, R.J., 2016. Use of rapid

tests to predict quality traits of CIMMYT bread wheat genotypes grown under different

environments. Food Science and Technology 69, 327-333.

Habernicht, D.K., J. E. Berg, J.E., Carlson, G.R., Wichman, D.M., Kushnak, G.D.,

Kephart, K.D., Martin, J.M., Bruckner, P.L., 2002. Pan bread and raw Chinese

noodle qualities in hard winter wheat genotypes grown in water-limited

environments. Crop Science 42(5), 1396–1403.

Hasniza, N., Copeland, L., Wilkes M., 2014. Globulin expression in grain of

Australian hard wheat cultivars is affected by growth environment. Cereal

Chemistry 91(2), 159–169.

He, Z., Zhang, J.Y., Quail, K., Pena, R., 2004. Pan bread and dry white Chinese

noodle quality in Chinese winter wheats. Euphytica 139, 257–267.

Hejgaard, J., 1978. “Free” and “bound” β-amylases during malting of barley.

Characterisation by two-dimensional immunoelectrophoresis. Journal of the Institute

of Brewing 84, 43-46.

Heun, M., Schafer-Pregl, R., Klawan, D., Castagna, R., Accerbi, M., Borghi, B.,

Salamini, F., 1997. Site of einkorn wheat domestication identified by DNA

fingerprinting. Science 278, 312–314.

65

Hill, V., 2014. A Kaizen Approach to Food Safety. Quality Management in the

Value Chain from Wheat to Bread. Springer International Publishing, Switzerland.

Hou, G.G., 2010. Asian Noodles. Science, Technology and Processing. A John

Wiley & Sons, Inc, Hoboken, New Jersey.

Hurkman, W., Vensel, W., Tanaka, C., Whitehand, L., Altenbach, S., 2009. Effect

of high temperature on albumin and globulin accumulation in the endosperm

proteome of the developing wheat grain. Journal of Cereal Science 49, 12–23.

Hurkman, W.J., Tanaka, C.K., Vensel, W.H., Thilmony, R., 2013. Comparative

proteomic analysis of the effect of temperature and fertilizer on gliadin and

glutenin accumulation in the developing endosperm and flour from Triticum

aestivum L. Cv. Butte 86. Proteome Science 11(8), 1-15.

Igrejas, G., Guedes-Pinto, H., Carnide, V., Clement, J., Branlard, G., 2002.

Genetical, biochemical and technological parameters associated with biscuit

quality. ii. prediction using storage proteins and quality characteristics in a soft

wheat population. Journal of Cereal Science 38, 187–197.

Inoue, T., Inanaga, S., Sugimoto, Y., Siddig, Y., 2004. Contribution of pre-

anthesis assimilates and current photosynthesis to grain yield, and their

relationships to drought resistance in wheat cultivars grown under different soil

moisture. Photosynthetica 42(1), 99–104.

Jaaskelainen, A., Holopainen-Mantila, U., Tamminen, T., Vuorinen, T., 2013.

Endosperm and aleurone cell structure in barley and wheat as studied by optical

and raman microscopy. Journal of Cereal Science 57, 543–550.

Jan-Christer, J., 2011. Protein Purification: Principles, High Resolution Methods, and

Applications, Third Edition.

66

Jane, J., 2006. Current understanding on starch granule structures. Journal of

Applied Glycoscience 53, 205–213.

Jha, R., Overend, D., Simmins, P., Hickling, D., Zijlstra, R., 2011. Chemical

characteristics, feed processing quality, growth performance and energy

digestibility among wheat classes in pelleted diets fed to weaned pigs. Animal

Feed Science and Technology 170, 78–90.

Johansson, E., Prieto-Linde, M.L., Jonsson, J., 2001. Effects of wheat cultivar and

nitrogen application on storage protein composition and breadmaking quality.

Cereal Chemistry 78(1), 19–25.

Kellog, E., 2001. Evolutionary history of the grasses. Plant Physiology 125, 1198–

1205.

Khan, M., Anjum, F., Pasha, I., Shabbir, M., Hussain, S., Nadeem, M., 2012.

Application of enzyme-linked immunosorbent assay for the assessment of spring

wheat quality. Food and Agricultural Immunology 23(1), 1–15.

Khan, M., Bukhari, A., Dar, Z., Rizvi, S., 2013. Status and strategies in breeding for

rust resistance in wheat. Agricultural Sciences 4, 292–301.

Kill, R.C., Turnbull, K., 2001. Pasta & Semolina Technology. Blackwell Science

Limited.

Konvalina, P., Capouchova, I., Stehno, Z., 2012. Agronomically important traits of

emmer wheat. Plant Soil and Environment 58(8), 341–346.

Kralova, B., 1999. Electrophoretic methods for the isolation and characterization of

enzymes. Analytica Chimica Acta 383, 109–117.

67

Kumar, S., Kumari, P., Kumar, U., Grover, M., Singh, A., Singh, R., Sengar, R.,

2013. Molecular approaches for designing heat tolerant wheat. Journal of Plant

Biochemistry and Biotechnology 22(4), 359–371.

Labuschagne, M.T., Elago, O., Koen, E., 2009. Influence of extreme temperatures

during grain filling on protein fractions, and its relationship to some quality

characteristics in bread, biscuits, and durum wheat. Cereal Chemistry 86(1), 61-66.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head

bacteriophage T4. Nature 227, 680-685.

Latiri, K., Lhomme, J., Lawlor, D., 2013. Grain filling of durum wheat through

assimilate remobilisation under semi-arid conditions. Experimental Agriculture

49(2), 197–211.

Ledward, D., 1995. Effects of elevated growth temperature and carbon dioxide levels

on some physicochemical properties of wheat starch. Journal of Cereal Science 22,

63–71.

Lei, F., Ji-chun, T., Cai-ling, S., Chun, L., 2008. RVA and farinograph properties

study on blends of resistant starch and wheat flour. Carbohydrate Polymers 7(7),

812–822.

Li, Y., Wu, Y., Hernandez-Espinosa, N., Pena, R.J., 2013. The influence of drought

and heat stress on the expression of end-use quality parameters of common wheat.

Journal of Cereal Science 57, 73-78.

Li, H., Cai, J., Jiang, D., Liu, F., Dai, T., Cao, W., 2013. Carbohydrates

accumulation and remobilization in wheat plants as influenced by combined

waterlogging and shading stress during grain filling. Journal of Agronomy and

Crop Science 199, 38–48.

68

Li, W., Shan, Y., Xiao, X., Luo, Q., Zheng, J., Ouyang, S., Zhang, G., 2013.

Physicochemical properties of a- and b-starch granules isolated from hard red and

soft red winter wheat. Journal of Agricultural and Food Chemistry 61, 6477–6484.

Li, Y.F., Wu, Y., Hernandez-Espinosa, N., Pena, R.J., 2015. Comparing Small-

Scale Testing Methods for Predicting Wheat Gluten Strength Across

Environments. Cereal Chemistry 92(3), 231–235.

Liu, L., Ikeda, T.M., Branlard, G., Peña, R.J., Rogers, W.J., Lerner, S.E., Kolman,

M.A., Xia, X., Wang, L., Ma, W., Appels, R., Yoshida, H., Wang, A., Yan, Y., He,

Z., 2010. Comparison of low molecular weight glutenin subunits identified by SDS-

PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat. Plant Biology 10, 124.

Liu, P., Guo, W., Jiang, Z., Pu, P., Feng, C., Zhu, X., Peng, Y., Kuang, A., Little,

C., 2011. Effects of high temperature after anthesis on starch granules in grains of

wheat (Triticum aestivum l.). Journal of Agricultural Science 149, 159–169.

Liu, J., Folberth, C., Yang, H., Rockstrom, J., Abbaspour, K., Zehnder, A.J.B.,

2013. A global and spatially explicit assessment of climate change impacts on

crop production and consumptive water use. Public Library of Science 8(2), 1-8.

Liu, L., Ma, J., Tian, L., Wang, S., Tang, L., Cao, W., Zhu, Y., 2017. Effect of

postanthesis high temperature on grain quality formation for wheat. Agronomy

Journal 109(5), 1970-1980.

Lu, D., Yang, H., Shen, X., Lu, W., 2016. Effects of high temperature during grain

filling on physicochemical properties of waxy maize starch. Journal of Integrative

Agriculture 15(2), 309–316.

Luo, C., Branlard, G., Griffin, W., McNeil, D., 2000. The effect of nitrogen and

sulphur fertilisation and their interaction with genotype on wheat glutenins and

quality parameters. Journal of Cereal Science 31, 185–194.

69

Luo, M.C., Yang, Z.L., You, F.M., Kawahara, T., Waines, J.G., Dvorak, J., 2007.

The structure of wild and domesticated emmer wheat populations, gene flow

between them, and the site of emmer domestication. Theoretical and Applied

Genetics 114, 947-959.

Ma, J., Huang, G., Yang, D., Chai, Q., 2014. Dry matter remobilization and

compensatory effects in various internodes of spring wheat under water stress.

Crop Science 54, 331–339.

Mahmood, T., Turner, M.A., Stoddard, F.L., 2007. Comparison of methods for

colorimetric amylose determination in cereal grains. Starch/Stärke 59, 357–365.

Majoul-Haddad, T., Bancel, E., Martre, P., Triboi E., Branlard, G., 2013. Effect of

short heat shocks applied during grain development on wheat (Triticum aestivum L.)

grain proteome. Journal of Cereal Science 57, 486-495.

Malik, A., Kuktaite, R., Johansson, E., 2013. Combined effect of genetic and

environmental factors on the accumulation of proteins in the wheat grain and their

relationship to bread-making quality. Journal of Cereal Science 57, 170–174.

Marchetti, L., Cardos, M., Campana, L., Ferrero, C., 2012. Effect of glutens of

different quality on dough characteristics and breadmaking performance. Food

Science and Technology 46, 224–231.

Marti, A., Seetharaman, K., Pagani, M., 2013. Rheological approaches suitable for

investigating starch and protein properties related to cooking quality of durum

wheat pasta. Journal of Food Quality 36, 133–135.

70

Matheson, N.K., Welsh, L.A., 1990. Estimation and fractionation of the essentially

un-branched (amylose) and branched (amylopectin) components of starches with

Concanavalin A. Carbohydrate Research 180, 301-313.

Matsuki, J., Yasui, T., Kohyama, K., Sasaki, T., 2003. Effects of environmental

temperature on structure and gelatinization properties of wheat starch. Cereal

Chemistry 80(4), 476–480.

Matsuoka, Y., 2011. Evolution of polyploid Triticum wheats under cultivation: The

role of domestication, natural hybridization and allopolyploid speciation in their

diversification. Plant and Cell Physiology 52(5), 750–764, 2011.

McGrance, S.J., Cornell, H.J., Rix, C.J., 1998. A simple and rapid colorimetric method

for the determination of amylose in starch products. Starch/Stärke 50(4), 158–163.

McIntosh, R., 1998. Breeding wheat for resistance to biotic stresses. Euphytica 100,

19–34.

Merlino, M., Leroy, P., Chambon, C., Branlard, G., 2009. Mapping and proteomic

analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum

aestivum l.). Theoretical and Applied Genetics 118, 1321–1337.

Merlino, M., Bousbata, S., Svensson, B., Branlard, G., 2012. Proteomic and

genetic analysis of wheat endosperm albumins and globulins using deletion lines

of cultivar Chinese spring. Theoretical and Applied Genetics 13, 12-17.

Moiraghi, M., Vanzetti, L., Bainotti, C., Helguera, M., Len, A., Prez, G., 2011.

Relationship between soft wheat flour physicochemical composition and cookie-

making performance. Cereal Chemistry 88(2), 130–136.

71

Moiraghi, M., de la Hera, D., Perez, G., Gomez, M., 2013. Effect of wheat flour

characteristics on sponge cake quality. Journal of the Science of Food and

Agriculture 93, 542–549.

Nelles, D., Dewar, J., Bason, M., Taylor, J., 2000. Maize starch biphasic pasting

curves. Journal of Cereal Science 31, 287–294.

Nevo, E., 2007. Evolution of wild wheat and barley and crop improvement: Studies

at the institute of evolution. Israel Journal of Plant Sciences 55(3-4), 251–262.

Nevo, E., 2014. Evolution of wild emmer wheat and crop improvement. Journal of

Systematics and Evolution 52(6), 673–696.

Nhan, M., Copeland, L., 2014. Effects of growing environment on properties of

starch from five Australian wheat varieties. Cereal Chemistry 91(6), 587–594.

Oh-Ishi, M., Maeda, T., 2002. Separation techniques for high-molecular-mass

proteins. Journal of Chromatography B 771, 49–66.

Oikonomou, G., Teixeira, A.G.V., Foditsch, C., Bicalho, M.L., Machado, V.S., Bicalho,

R.C., 2013. Fecal Microbial Diversity in Pre-Weaned Dairy Calves as Described by

Pyrosequencing of Metagenomic 16S rDNA. Associations

of Faecalibacterium Species with Health and Growth. Public Library of Sciences 8(4),

1-10.

Ostergaard, S., Olsson, L., Johnston, M., Nielsen, J., 2000. Increasing galactose

consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL

gene regulatory network. Nature Biotechnology 18, 1283-1286.

Pagnotta, M., Mondini, I., Atallah, M., 2005. Morphological and molecular

characterization of Italian emmer wheat accessions. Euphytica 146, 29–37.

72

Pagnotta, M., Mondini, L., Codianni, P., Fares, C., 2008. Agronomical, quality, and

molecular characterization of twenty Italian emmer wheat (Triticum dicoccum)

accessions. Genetic Resources and Crop Evolution 12, 1–12.

Pampana, S., Mariotti, M., Ercoli, L., Masoni, A., 2007. Remobilization of dry

matter, nitrogen and phosphorus in durum wheat as affected by genotype and

environment. Italian Journal of Agronomy 3, 303–314.

Panozzo, J.F., Eagles, H.A., 1998. Cultivar and environmental effects on quality

characters in wheat. I. Starch. Australian Journal of Agricultural Research 49, 757–

66.

Pasha, I., Anjum, F., Morris, C., 2010. Grain hardness: A major determinant of

wheat quality. Food Science and Technology International 16(6), 0511–12.

Pavlova, A.S., Dyudeeva, E.S., Kupryushkin, M.S., Amirkhanov, N.V., Pyshnyi, D.V.,

Pyshnaya, I.A., 2018. SDS-PAGE procedure: Application for characterisation of new

entirely uncharged nucleic acids analogs. Electrophoresis 39, 670-674.

Pedreschi, R., Hertog, M., Lilley, K.S., Nicolai, B., 2010. Proteomics for the food

industry: Opportunities and challenges. Critical Reviews in Food Science and

Nutrition 50, 680–692.

Peng, J., Sun, D., Nevo, E., 2011. Wild emmer wheat, Triticum dicoccoides,

occupies a pivotal position in wheat domestication process. Australian Journal of

Crop Science 5(9), 1127–1143.

Pe’rez, S., Bertoft, E., 2010. The molecular structures of starch components and

their contribution to the architecture of starch granules: A comprehensive review.

Starch/Starke 62, 389-420.

73

Pheloung, P., Siddique, K., 1991. Contribution of stem dry matter to grain yield in

wheat cultivars. Australian Journal of Plant Physiology 18, 53–64.

Roberts, T.R., Hejgaard, J., 2004. Serpins in plants and green algae. Functional and

Integrative Genomics 8, 1–27.

Salman, H., Blazek, J., Rubio-Lopez, A., Gilbert, E., Hanley, T., Copeland, L.,

2009. Structure-function relationships in a and b granules from wheat starches of

similar amylose content. Carbohydrate Polymers 75, 420–427.

Sanchez-Garcia, M., Royo, C., Aparicio, N., Martin-Sanchez, J., Alvaro, A., 2013.

Genetic improvement of bread wheat yield and associated traits in Spain during

the 20th century. Journal of Agricultural Science 151, 105–118.

Saulnier, L., Guillon, F., Chateigner-Boutin, A., 2012. Cell wall deposition and

metabolism in wheat grain. Journal of Cereal Science 56, 91–108.

Shewry, P., Tatham, A., Forde, J., Kreis, M., Miflin, B., 1986. The classification and

nomenclature of wheat gluten proteins: A reassessment. Journal of Cereal

Science 4, 97–106.

Shewry, P.R., Halford, N., Tatham, A., 1992. High molecular weight subunits of

wheat glutenin. Journal of Cereal Science 15, 105–120.

Shewry, P.R., Halford, N., Belton, P., Tatham, A., 2002. The structure and

properties of gluten: an elastic protein from wheat grain. Philosophical

Transactions of the Royal Society of London B 357, 133–142.

Shewry, P.R., 2009. Wheat. Journal of Experimental Botany 60(6), 1537-1553.

74

Singh, S., Gupta, A.K., Gupta, S.K., Kaur, N., 2010. Effect of sowing time on protein

quality and starch pasting characteristics in wheat (Triticum aestivum L.) genotypes

grown under irrigated and rain-fed conditions. Food Chemistry 122, 559-565.

Stone, P.J., Nicolas, M.E., 1998. The effect of duration of heat stress during grain

filling on two wheat varieties differing in heat tolerance: grain growth and fractional

protein accumulation. Australian Journal of Plant Physiology 25, 13-20.

Tai, A.P., Val Martin, M., Heald, C.L., 2014. Threat to future global food security

from climate change and ozone air pollution. Nature Climate Change 4, 817-821.

Tester, R.F, Morrison, W.R., Ellis, R.H., Piggott, J.R., Batts, G.R., Wheeler, T.R.,

Morisson, J.I.L.,Hadley, P., Ledward, D.A, 1995. Effects of elevated growth

temperature and carbon dioxide levels on some physicochemical properties of

wheat starch. Journal of Cereal Science 22, 63–71.

Tester, R.F, Morrison, W.R., Ellis, R.H., Piggott, J.R., Batts, G.R., Wheeler, T.R.,

Morisson, J.I.L., Hadley, P., Zhu, J., Khan, K., 2001. Effects of genotype and

environment on glutenin polymers and breadmaking quality. Cereal Chemistry 78(2),

125–130.

Trethowan, R.M., Mujeeb-Kazi, A., 2008. Novel germplasm resources for improving

environmental stress tolerance of hexaploidy wheat. Crop Science 48, 1255-1265.

Vamadevan, V., Bertoft, E., 2015. Structure-function relationships of starch

components. Starch/Starke 67, 55–68.

Varzakas, T., Kozub, N., Xynias, I.N., 2014. Quality determination of wheat:

genetic determination, biochemical markers, seed storage proteins in bread and

durum wheat germplasm. Journal of the Science of Food and Agriculture 94,

2819–2829.

75

Veraverbeke, W., Delcour, J., 2002. Wheat protein composition and properties of

wheat glutenin in relation to breadmaking functionality. Critical Reviews in Food

Science and Nutrition 42(3), 179–208.

Verma, S., Yunus, M., Sethi, S., 1998. Breeding for yield and quality in durum wheat.

Euphytica 100, 15–18.

Viswanathan, C., Khanna-Chopra, R., 2001. Effect of heat stress on grain growth,

starch synthesis and protein synthesis in grains of wheat (Triticum aestivum L.)

Varieties Differing in Grain Weight Stability. Journal of Agronomy & Crop Science

186, 1-7.

Waduge, R., Kalinga, D., Bertoft, E., Seetharaman, K., 2014. Molecular structure

and organization of starch granules from developing wheat endosperm. Cereal

Chemistry 91(6), 578–586.

Walker, J.M., 2002. SDS Polyacrylamide gel electrophoresis of Proteins. From:

The Protein Protocols Handbook, 2nd Edition Edited by: J. M. Walker Humana Press

Inc., Totowa, NJ.

Wang, C., Kovacs, M.I.P., 2002. Swelling index of glutenin test. I. Method and

comparison with sedimentation, gel-protein, and insoluble glutenin tests. Cereal

Chemistry 79(2), 183–189.

Wang, C., Kovacs, M.I.P., 2002a. Swelling index of glutenin test. II. Application in

prediction of dough properties and end-use quality. Cereal Chemistry 79(2), 190–196.

Wang, S., Li, T., Miao, Y., Zhang, Y., He, Z., Wang, S., 2017. Effects of heat stress

and cultivar on the functional properties of starch in Chinese wheat. Cereal Chemistry

94(3), 443–450.

Wieser, H., 2007. Chemistry of gluten proteins. Food Microbiology 24, 115–119.

76

Wilkes, M., Copeland, L., 2008. Storage of wheat grains at elevated temperatures

increases solubilization of glutenin subunits. Cereal Chemistry 85(3), 335-338.

Wrigley, C., Batey, I., Miskelly, D., 2016. Cereal Grains: Assessing and Managing

Quality. Elsevier Science & Technology. Second Edition, Woodhead Publishing.

Yegani, M., Korver, D., 2012. Review: Prediction of variation in energetic value of

wheat for poultry. Canadian Journal of Animal Science 92, 261–273.

Yu, X., Li, B., Wang, L., Chen, X., Wang, W., Gu, Y., Wang, Z., Xiong, F., 2015. Effect

of drought stress on the development of endosperm starch granules and the

composition and physicochemical properties of starches from soft and hard wheat.

Journal of the Science of Food and Agriculture 96, 2746–2754.

Yumurtaci, A., 2015. Utilization of wild relatives of wheat, barley, maize and oat in

developing abiotic and biotic stress tolerant new varieties. Emirates Journal of Food

and Agriculture 27(1), 1-23.

Yun, S., Matheson, N.K., 1990. Estimation of amylose content of starches after

precipitation of amylopectin by Concanavalin-A. Starch/Starke 42(8), 302-305.

Zaharieva, M., Ayana, N., Hakimi, A., Misra, S., Monneveux, P., 2010. Cultivated

emmer wheat (Triticum dicoccum), an old crop with promising future: a review.

Genetic Resources and Crop Evolution 57, 937–962.

Zeng, J., Gao, H., Li, G., 2013. Functional properties of wheat starch with different

particle size distribution. Journal of the Science of Food and Agriculture 94, 57–62.

Zhang, P., He, Z., Zhang, Y., Xia, X., Chen, D., Zhang, y., 2008. Association between

% SDS-unextractable polymeric protein (% UPP) and end-use quality in Chinese

bread wheat cultivars. Cereal Chemistry 85(5) 696-700.

77

Zhang, B., Li, X., Liu, J., Xie, F., Chen, L., 2013a. Supramolecular structure of a- and

b-type granules of wheat starch. Food Hydrocolloids 31, 68–73.

Zhang, W., Sangtong, V., Peterson, J., Scott, M., Messing, J, 2013b. Divergent

properties of prolamins in wheat and maize. Planta 237, 1465–1473.

Zhong, L.J., Cheng, F.M., Wen, X., Sun, Z.X., Zhang, G.P., 2005. The deterioration of

eating and cooking quality caused by high temperature during grain filling in early-

season Indica rice cultivars. Journal of Agronomy & Crop Science 191, 218-225.

Zhou, M., Robards, K., Glennie-Holmes, M., Helliwell, S., 1998. Review. Structure

and Pasting Properties of Oat Starch. Cereal Chemistry 75(3), 273–281.

Zhu, J., Khan, K., 2001. Effects of genotype and environment on glutenin polymers

and breadmaking quality. Cereal Chemistry 78(2), 125–130.

Zhu, T., Jackson, D.S., Wehling, R.L., Geera, B., 2008. Comparison of amylose

determination methods and the development of a dual wavelength iodine binding

technique. Cereal Chemistry 85(1), 51-58.

Zobel, H., 1988. Molecules to granules: A comprehensive starch review.

Starch/Starke 40(2), 23-31.

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