Transcript
Page 1: RNA-Seq data analysis at wings 2014 - Workshop 3 Biological Interpretation

Workshops  in  next-­‐genera1on  science  at  UNC  Charlo7e  in  2014  

Workshop  3  -­‐  Tools  for  biological  interpreta1on    

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Tomato  Pollen  RNA-­‐Seq  

Seeking  biological  significance    

Slides  by  Ivory  Clabaugh  Blakley  

2  

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What  do  we  already  know?  

What  CAN  we    learn  from  this  data?  

What    are  we    

trying  to  learn?  

3  

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Given  that  “impaired  pollen  development  under  high-­‐temperature  condi1ons  has  been  implicated  in  reduced  yields  in  a  large  number  of  crop  systems”  (Firon  et  al  2012)  

What  biological  mechanisms  could  poten3ally  be  manipulated  by  plant  growers,    breeders  and/or  bio-­‐engineers  to  increase  pollen  heat  tolerance  in  tomato  and  other  crops  so  as  to  prevent  loss-­‐of-­‐yield  in  the  face  of  high  temperatures.  

What    are  we    

trying  to  learn?  

4  

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!  Can't  use  these  data  to  find  out  what  makes  this  cul1var  more  heat-­‐tolerant  than  other  cul1vars.    

!  We  CANNOT  comment  on  expression  differences  that  take  place  during  other  stages  in  developing  pollen.  

!  We  CANNOT  comment  on  expression  differences  that  take  place  in  the  anthers,  or  anywhere  else  in  the  plant.  

!  We  CANNOT  comment  on  structural  differences.  

!  We  CANNOT  compare  pollen  to  other  sample  types,  e.g.,  leaves  or  roots.  

What  CAN  we    learn  from  this  data?  

5  

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What  CAN  we    learn  from  this  data?  

!    Effects  of  stress  on  gene  expression  in  pollen      –  treatment  versus  control–  GO,    LycoCyc  

!    Rela1ve  expression  levels  between  genes  –  RPKM  

!    Gene  annota1on  completeness  &  accuracy    –  novel  genes,  splicing  events  –  IGB,  Cufflinks  

!  Differen1al  splicing  (if  there's  enough  data)    !    Types  of  genes  expressed  in  mature  tomato  pollen  

 –  compare  with  Arabidopsis  (2013  Plant  Phys)   6  

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What  do  we  already  know?  

7  

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What  do  we  already  know?  

Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009doi:10.1093/jxb/erp234 Advance Access publication 23 July, 2009This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Transcriptional profiling of maturing tomato (Solanumlycopersicum L.) microspores reveals the involvement of heatshock proteins, ROS scavengers, hormones, and sugars inthe heat stress response

Gil Frank1, Etan Pressman1, Ron Ophir2, Levia Althan1, Rachel Shaked1, Moshe Freedman1, Shmuel Shen1 and

Nurit Firon1,*

1 Department of Vegetable Research, Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, POB 6,Bet Dagan, 50250, Israel2 Department of Fruit Tree Sciences, Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, POB 6,Bet Dagan, 50250, Israel

Received 5 February 2009; Revised 25 June 2009; Accepted 26 June 2009

Abstract

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of thedeveloping pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, ispoorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) ofmicrospores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried outusing a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP)techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. Thedata obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than theclassical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers,sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use ofcDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomatoAffymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependentprotein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerantcultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes ascandidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of themolecular events underlying the HSR of maturing microspores of a crop plant, tomato.

Key words: cDNA-AFLP, gene expression, heat stress response, microarray, microspore maturation, tomato.

Introduction

Most crop plants are exposed to heat stress (HS) duringsome stage of their life cycle. HS, defined as the temper-atures above normal optimum, is expected to become a morefrequent and acute problem in the coming years (Sato et al.,2000). Exposure to HS reduces yield and decreases thequality of many crops, including vegetable crops (Kinet and

Peet, 1997; Wien, 1997; Boote et al., 2005). Peet et al. (1998)demonstrated in tomato that at daily mean temperatures of29 !C (32/26 !C day/night), fruit number, fruit weight perplant, and seed number per fruit were markedly decreasedcompared with at 25 !C. Plants also encounter high temper-ature damage during spring and autumn when grown in the

* To whom correspondence should be addressed. E-mail: [email protected]ª 2009 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

by guest on April 18, 2014

http://jxb.oxfordjournals.org/D

ownloaded from

Pollen grains of heat tolerant tomato cultivars retain higher carbohydrateconcentration under heat stress conditions

N. Firon a, R. Shaked a, M.M. Peet b, D.M Pharr b, E. Zamski c,K. Rosenfeld a, L. Althan a, E. Pressman a,*

a Department of Vegetable Crops, ARO, The Volcani Center, Bet Dagan, Israelb Department of Horticultural Science, NCSU, Raleigh, NC, USA

c Institute of Plant Sciences and Genetics, Faculty of Agriculture, Rehovot 76100, Israel

Received 9 May 2005; received in revised form 13 March 2006; accepted 15 March 2006

Abstract

Exposure to high temperatures (heat stress) causes reduced yield in tomatoes (Lycopersicon esculentum), mainly by affecting malegametophyte development. Two experiments were conducted where several tomato cultivars were grown under heat stress, in growth chambers(day/night temperatures of 31/25 8C) or in greenhouses (day/night temperatures of 32/26 8C), or under control (day/night temperatures of 28/22 8C) conditions. In heat-sensitive cultivars, heat stress caused a reduction in the number of pollen grains, impaired their viability andgerminability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number andquality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitivecultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, anda parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starchaccumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that thecarbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest thatheat-tolerant cultivars have a mechanism for maintaining the appropriate carbohydrate content under heat stress.# 2006 Elsevier B.V. All rights reserved.

Keywords: Lycopersicon esculentum; Cultivars; Heat stress; Heat tolerance; Pollen quality; Starch; Sugars; Tomato

1. Introduction

Exposure to higher than optimal temperatures reduces yieldand impairs the quality of many crops, including vegetablecrops. The prevalence of high ambient temperatures in asignificant proportion of the tomato-growing areas of the worldis one of the most crucial problems in tomato production.Chronic heat stress, even of a mild degree, has been shown todisrupt the normal development of the gametes and therebyfruit set. Levy et al. (1978) compared the effects of hightemperatures on a susceptible and a tolerant tomato cultivar andfound that heat stress affected mainly the pollen grains; itreduced their viability and the effect was more pronounced inthe susceptible cultivar. Sato et al. (2000) found that among fivetomato cultivars grown under mild high-temperature conditions

(32 8C day and 26 8C night) only cv. FLA 7156 set fruits. Theysuggested that differences among cultivars in pollen release andgermination under heat stress are the most crucial factors indetermining fruit set. Porch and Jahn (2001) found that heatstress caused indehiscence of the anthers, reduced pollenviability and reduced yield in a heat-sensitive genotype of bean(Phaseolus vulgaris); the anthers and pollen of a heat-tolerantgenotype were generally normal under the same conditions.

Starch biosynthesis during the final phases of pollenmaturation is critical in determining pollen quality not onlybecause starch is a reserve source of energy for pollengermination but it may also serves as a checkpoint of pollenmaturity. In dicots, such as tomato, starch accumulation peaksat 3 days before anthesis, while the mature pollen grains areconsidered starchless. In monocots (such as maize) starchaccumulates during pollen maturation and the mature pollengrains contain starch. In several maize genetically controlledmale-sterile mutants it was shown that pollen inviability wasassociated with starch-deficiency (Datta et al., 2002).

www.elsevier.com/locate/scihortiScientia Horticulturae 109 (2006) 212–217

* Corresponding author. Tel.: +972 3 9683470; fax: +972 3 9669642.E-mail addresses: [email protected], [email protected]

(E. Pressman).

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.scienta.2006.03.007

Open access – Research articleTHIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED‘ETHYLENE 2012’

Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditionsNurit Firon1*, Etan Pressman1, Shimon Meir2, Reham Khoury1 and Leviah Altahan1

1 Department of Vegetable Research, Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel2 Postharvest and Food Sciences, Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel

Received: 11 June 2012; Returned for revision: 17 July 2012; Accepted: 14 August 2012; Published: 23 August 2012

Citation details: Firon N, Pressman E, Meir S, Khoury R, Altahan L. 2012. Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024

Abstract

Background andaims

Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due tosensitivity of developing pollen grains. The mechanisms maintaining high pollen quality underheat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene in-volvement in the pollen heat-stress response. Here we elucidated ethylene’s involvement in pollenheat-stress response and thermotolerance by assessing the effects of interfering with the ethylenesignalling pathway and altering ethylene levels on tomato pollen functioning under heat stress.

Methodology Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene responsesensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to controlor heat-stress growing conditions, and pollen quality was determined. Starch and carbohy-drates were measured in isolated pollen grains from these plants. The effect of pretreatingcv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser orinhibitor of ethylene biosynthesis on pollen quality was assessed.

Principal results Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant re-duction in the total number of pollen grains, reduction in the number of viable pollen and ele-vation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollengrains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatmentof tomato plants with an ethylene releaser increased pollen quality under heat stress, with anover 5-fold increase in the number of germinating pollen grains per flower. Pretreatment withan ethylene biosynthesis inhibitor reduced the number of germinating pollen grains followingheat-stress exposure over 5-fold compared with non-treated controls.

Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylenesignalling pathwayor reducing ethylene levels increased tomato pollen sensitivity to heat stress,whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.

* Corresponding author’s e-mail address: [email protected]

Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original work is properly cited.

AoB PLANTS http://aobplants.oxfordjournals.org/AoB PLANTS http://aobplants.oxfordjournals.org/

AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024, available online at www.aobplants.oxfordjournals.org & The Authors 2012 1

Firon  2006  effect  of  heat  stress  on  pollen  carbohydrates  

Frank,  2009  Microarray  study  

Firon,  2012  Manipula1on  of  ethylene  

pathway  

8  

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Pollen  quality  

The  following  papers  use  this  graph  layout.  And  they  show  similar  data.  

What  do  we  already  know?  

9  

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•  Mild  chronic  heat  stress  reduces    sugar  content  in  some  cul1vars    but  not  Hazera  3042.  

•  Reduces  pollen  starch  in    Hazera  3042  –  Only  when  applied  to  early  stages  –  A-­‐5  but  not  A-­‐3  or  Anthesis  ("A  minus  5  days")  

–  A-­‐5  is  most  heat-­‐sensi1ve  stage  of  pollen  development  

•  Reduces  pollen  grain  count,  pollen  viability  in  Hazera  3042,  but  effects  on  Hazera  3017  are  more  severe  –  Hazera  3042  is  “heat  tolerant”  –  Hazera  3017  is  “heat  sensi1ve”  

What  do  we  already  know?  

Firon 2006   Pollen grains of heat tolerant tomato cultivars retain higher carbohydrateconcentration under heat stress conditions

N. Firon a, R. Shaked a, M.M. Peet b, D.M Pharr b, E. Zamski c,K. Rosenfeld a, L. Althan a, E. Pressman a,*

a Department of Vegetable Crops, ARO, The Volcani Center, Bet Dagan, Israelb Department of Horticultural Science, NCSU, Raleigh, NC, USA

c Institute of Plant Sciences and Genetics, Faculty of Agriculture, Rehovot 76100, Israel

Received 9 May 2005; received in revised form 13 March 2006; accepted 15 March 2006

Abstract

Exposure to high temperatures (heat stress) causes reduced yield in tomatoes (Lycopersicon esculentum), mainly by affecting malegametophyte development. Two experiments were conducted where several tomato cultivars were grown under heat stress, in growth chambers(day/night temperatures of 31/25 8C) or in greenhouses (day/night temperatures of 32/26 8C), or under control (day/night temperatures of 28/22 8C) conditions. In heat-sensitive cultivars, heat stress caused a reduction in the number of pollen grains, impaired their viability andgerminability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number andquality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitivecultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, anda parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starchaccumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that thecarbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest thatheat-tolerant cultivars have a mechanism for maintaining the appropriate carbohydrate content under heat stress.# 2006 Elsevier B.V. All rights reserved.

Keywords: Lycopersicon esculentum; Cultivars; Heat stress; Heat tolerance; Pollen quality; Starch; Sugars; Tomato

1. Introduction

Exposure to higher than optimal temperatures reduces yieldand impairs the quality of many crops, including vegetablecrops. The prevalence of high ambient temperatures in asignificant proportion of the tomato-growing areas of the worldis one of the most crucial problems in tomato production.Chronic heat stress, even of a mild degree, has been shown todisrupt the normal development of the gametes and therebyfruit set. Levy et al. (1978) compared the effects of hightemperatures on a susceptible and a tolerant tomato cultivar andfound that heat stress affected mainly the pollen grains; itreduced their viability and the effect was more pronounced inthe susceptible cultivar. Sato et al. (2000) found that among fivetomato cultivars grown under mild high-temperature conditions

(32 8C day and 26 8C night) only cv. FLA 7156 set fruits. Theysuggested that differences among cultivars in pollen release andgermination under heat stress are the most crucial factors indetermining fruit set. Porch and Jahn (2001) found that heatstress caused indehiscence of the anthers, reduced pollenviability and reduced yield in a heat-sensitive genotype of bean(Phaseolus vulgaris); the anthers and pollen of a heat-tolerantgenotype were generally normal under the same conditions.

Starch biosynthesis during the final phases of pollenmaturation is critical in determining pollen quality not onlybecause starch is a reserve source of energy for pollengermination but it may also serves as a checkpoint of pollenmaturity. In dicots, such as tomato, starch accumulation peaksat 3 days before anthesis, while the mature pollen grains areconsidered starchless. In monocots (such as maize) starchaccumulates during pollen maturation and the mature pollengrains contain starch. In several maize genetically controlledmale-sterile mutants it was shown that pollen inviability wasassociated with starch-deficiency (Datta et al., 2002).

www.elsevier.com/locate/scihortiScientia Horticulturae 109 (2006) 212–217

* Corresponding author. Tel.: +972 3 9683470; fax: +972 3 9669642.E-mail addresses: [email protected], [email protected]

(E. Pressman).

0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.scienta.2006.03.007

10  

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Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009doi:10.1093/jxb/erp234 Advance Access publication 23 July, 2009This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Transcriptional profiling of maturing tomato (Solanumlycopersicum L.) microspores reveals the involvement of heatshock proteins, ROS scavengers, hormones, and sugars inthe heat stress response

Gil Frank1, Etan Pressman1, Ron Ophir2, Levia Althan1, Rachel Shaked1, Moshe Freedman1, Shmuel Shen1 and

Nurit Firon1,*

1 Department of Vegetable Research, Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, POB 6,Bet Dagan, 50250, Israel2 Department of Fruit Tree Sciences, Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, POB 6,Bet Dagan, 50250, Israel

Received 5 February 2009; Revised 25 June 2009; Accepted 26 June 2009

Abstract

Above-optimal temperatures reduce yield in tomato largely because of the high heat stress (HS) sensitivity of thedeveloping pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, ispoorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) ofmicrospores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried outusing a combination of Affymetrix Tomato Genome Array and cDNA-amplified fragment length polymorphism (AFLP)techniques. The results were corroborated by reverse transcription-PCR (RT-PCR) and immunoblot analyses. Thedata obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than theclassical HS-responsive genes. The results also indicate HS regulation of reactive oxygen species (ROS) scavengers,sugars, plant hormones, and regulatory genes that were previously implicated in other types of stress. The use ofcDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomatoAffymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependentprotein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerantcultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes ascandidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of themolecular events underlying the HSR of maturing microspores of a crop plant, tomato.

Key words: cDNA-AFLP, gene expression, heat stress response, microarray, microspore maturation, tomato.

Introduction

Most crop plants are exposed to heat stress (HS) duringsome stage of their life cycle. HS, defined as the temper-atures above normal optimum, is expected to become a morefrequent and acute problem in the coming years (Sato et al.,2000). Exposure to HS reduces yield and decreases thequality of many crops, including vegetable crops (Kinet and

Peet, 1997; Wien, 1997; Boote et al., 2005). Peet et al. (1998)demonstrated in tomato that at daily mean temperatures of29 !C (32/26 !C day/night), fruit number, fruit weight perplant, and seed number per fruit were markedly decreasedcompared with at 25 !C. Plants also encounter high temper-ature damage during spring and autumn when grown in the

* To whom correspondence should be addressed. E-mail: [email protected]ª 2009 The Author(s).

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

by guest on April 18, 2014

http://jxb.oxfordjournals.org/D

ownloaded from

What  do  we  already  know?  

Frank 2009 Microarray study  •  STHS  –  short  term  heat  stress,  44°C,  ho7er  than  the  MCHS  

(mild  chronic  heat  stress)  •  Compared  heat-­‐sensi1ve,  heat-­‐tolerant  cul1vars,  but  

observed  no  difference  observed  in  heat  responses    

• Only  104  genes  up-­‐regulated  by  heat,  none  down-­‐regulated  • Up-­‐regulated  genes  included  –  Heat  Shock  Proteins  –  Hormones  –  ethylene  –  JA  –  Reac1ve  oxygen  species  scavengers  –  Carbohydrate  biosynthesis  –  Stress  responses  

11  

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What  do  we  already  know?  

Open access – Research articleTHIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED‘ETHYLENE 2012’

Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditionsNurit Firon1*, Etan Pressman1, Shimon Meir2, Reham Khoury1 and Leviah Altahan1

1 Department of Vegetable Research, Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel2 Postharvest and Food Sciences, Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel

Received: 11 June 2012; Returned for revision: 17 July 2012; Accepted: 14 August 2012; Published: 23 August 2012

Citation details: Firon N, Pressman E, Meir S, Khoury R, Altahan L. 2012. Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024

Abstract

Background andaims

Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due tosensitivity of developing pollen grains. The mechanisms maintaining high pollen quality underheat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene in-volvement in the pollen heat-stress response. Here we elucidated ethylene’s involvement in pollenheat-stress response and thermotolerance by assessing the effects of interfering with the ethylenesignalling pathway and altering ethylene levels on tomato pollen functioning under heat stress.

Methodology Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene responsesensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to controlor heat-stress growing conditions, and pollen quality was determined. Starch and carbohy-drates were measured in isolated pollen grains from these plants. The effect of pretreatingcv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser orinhibitor of ethylene biosynthesis on pollen quality was assessed.

Principal results Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant re-duction in the total number of pollen grains, reduction in the number of viable pollen and ele-vation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollengrains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatmentof tomato plants with an ethylene releaser increased pollen quality under heat stress, with anover 5-fold increase in the number of germinating pollen grains per flower. Pretreatment withan ethylene biosynthesis inhibitor reduced the number of germinating pollen grains followingheat-stress exposure over 5-fold compared with non-treated controls.

Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylenesignalling pathwayor reducing ethylene levels increased tomato pollen sensitivity to heat stress,whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.

* Corresponding author’s e-mail address: [email protected]

Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original work is properly cited.

AoB PLANTS http://aobplants.oxfordjournals.org/AoB PLANTS http://aobplants.oxfordjournals.org/

AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024, available online at www.aobplants.oxfordjournals.org & The Authors 2012 1

Firon 2012 Ethylene study  •  Ethylene  receptor  mutant    (Never  ripe)  phenotype  –  pollen  more  sensi1ve  to  mild  chronic  heat  stress  

–  reduced  sucrose  in  mature  pollen.  

•  Applica1on  of  ethylene  releaser  prior  to  HS  increased  pollen  thermotolerance.  

•  Ethylene-­‐biosynthesis  inhibitor    reduced  basal  as  well  as    acquired  thermotolerance.  

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What  do  we  already  know?  

Firon 2012  Acquired  thermo  tolerance  in  pollen  may  be  used  for  the  iden8fica8on  of  molecular  mechanisms  in  heat  tolerance,  by  employing  next-­‐genera8on  sequencing  methods  at  the  pollen  cDNA  level.  

Heat  acclima1on  here  was  1  hour  at  32°C.      Treatment  in  current  study  was  32°C/26°C  day/night.  

Open access – Research articleTHIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED‘ETHYLENE 2012’

Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditionsNurit Firon1*, Etan Pressman1, Shimon Meir2, Reham Khoury1 and Leviah Altahan1

1 Department of Vegetable Research, Institute of Plant Sciences, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel2 Postharvest and Food Sciences, Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center,Bet Dagan 50250, Israel

Received: 11 June 2012; Returned for revision: 17 July 2012; Accepted: 14 August 2012; Published: 23 August 2012

Citation details: Firon N, Pressman E, Meir S, Khoury R, Altahan L. 2012. Ethylene is involved in maintaining tomato (Solanumlycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024

Abstract

Background andaims

Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due tosensitivity of developing pollen grains. The mechanisms maintaining high pollen quality underheat-stress conditions are poorly understood. Our recently published data indicate high heat-stress-induced expression of ethylene-responsive genes in tomato pollen, indicating ethylene in-volvement in the pollen heat-stress response. Here we elucidated ethylene’s involvement in pollenheat-stress response and thermotolerance by assessing the effects of interfering with the ethylenesignalling pathway and altering ethylene levels on tomato pollen functioning under heat stress.

Methodology Plants of the ethylene-insensitive mutant Never ripe (Nr)—defective in an ethylene responsesensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to controlor heat-stress growing conditions, and pollen quality was determined. Starch and carbohy-drates were measured in isolated pollen grains from these plants. The effect of pretreatingcv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser orinhibitor of ethylene biosynthesis on pollen quality was assessed.

Principal results Never ripe pollen grains exhibited higher heat-stress sensitivity, manifested by a significant re-duction in the total number of pollen grains, reduction in the number of viable pollen and ele-vation of the number of non-viable pollen, compared with wild-type plants. Mature Nr pollengrains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatmentof tomato plants with an ethylene releaser increased pollen quality under heat stress, with anover 5-fold increase in the number of germinating pollen grains per flower. Pretreatment withan ethylene biosynthesis inhibitor reduced the number of germinating pollen grains followingheat-stress exposure over 5-fold compared with non-treated controls.

Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylenesignalling pathwayor reducing ethylene levels increased tomato pollen sensitivity to heat stress,whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality.

* Corresponding author’s e-mail address: [email protected]

Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use,distribution, and reproduction in any medium, provided the original work is properly cited.

AoB PLANTS http://aobplants.oxfordjournals.org/AoB PLANTS http://aobplants.oxfordjournals.org/

AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024, available online at www.aobplants.oxfordjournals.org & The Authors 2012 1

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Exploring  the  Results  

Compare  to  microarray  (Frank  2009)  

Pathway  visualiza1on  with  LycoCyc  

Gene  Ontology  enrichment  analysis  

Novel  Gene  search  

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Compared  to  Frank  2009  microarray  

•  Direct  comparison  made  difficult  by  lack  of  mapping  between  gene  ids,  probe  set  ids.  

•  Only  6  of  104  up-­‐reg  genes  were  on  our  DE  list,  and  they  were  down-­‐regulated    

•  Interpreta1on:  The  treatments  triggered  very  different  responses.    – Mild  chronic  heat  stress  over  many  weeks  is  very  different  than  short-­‐term,  severe  heat  stress.  

•  Developmental  stages  were  not  consistent  between  studies.  

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•  Frank  2009  microarray  study  showed  this  gene  was  up-­‐regulated.    

•  Only  weakly  expressed  in  our  study.  

Cytosolic  class  II  small  heat  shock  protein  LeHSP17.4    

16  Primer  sequences  Frank  2008  used  in  RT-­‐PCR  

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class  I  heat  shock  protein  3    

Frank  et  al  show  this  gene  (LesAffx.10596.1.S1_at)  as  being  up  by  140  fold.  In  our  data  there  is  very  li7le  representa1on  (Solyc09g015020.1).  This  is  a  small  gene  (465bp)  and  the  size  selec1on  step  of  the  library  prep  may  have  eliminated  most  fragments  from  this  gene.      This  gene  is  en1rely  overlapped  by  another  gene,  so  even  the  reads  that  did  align  here,  will  not  be  counted  by  featureCounts.  

evidence  of  SNP  

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Annota1on  improvements  from  RNA-­‐Seq  

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Extra  Exon   19  

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Under  Coun1ng  

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References  –  Firon 2006  “The  prevalence  of  high  ambient  temperatures  in  a  significant  propor1on  of  the  tomato-­‐growing  areas  of  the  world  is  one  of  the  most  crucial  problems  in  tomato  produc1on.”    Firon,  N.,  Shaked,  R.,  Peet,  M.  M.,  Pharr,  D.  M.,  Zamski,  E.,  Rosenfeld,  K.,  et  al.  (2006).  Pollen  grains  of  heat  tolerant  tomato  cul1vars  retain  higher  carbohydrate  concentra1on  under  heat  stress  condi1ons.  Scien1a  Hor1culturae,  109(3),  212–217.  doi:10.1016/j.scienta.2006.03.007    

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References  –  Firon 2012  “Impaired  pollen  development  under  high-­‐temperature  condi1ons  has  been  implicated  in  reduced  yields  in  a  large  number  of  crop  systems  (Stone  2001;  Firon  et  al.  2006;  Prasad  et  al.  2006;  Mukesh  et  al.  2007).  In  tomato,  developing  pollen  grains  are  highly  sensi1ve  to  HS  (Pressman  et  al.  2002,  2006;  Firon  et  al.  2006).”    Firon,  N.,  Pressman,  E.,  Meir,  S.,  Khoury,  R.,  &  Altahan,  L.  (2012).  Ethylene  is  involved  in  maintaining  tomato  (Solanum  lycopersicum)  pollen  quality  under  heat-­‐stress  condi1ons.  AoB  Plants,  2012,  pls024.  doi:10.1093/aobpla/pls024  and  references  therein.    

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References  –  Frank 2009    “Although  no  significant  differences  in  gene  expression  between  the  cul1vars  were  detected  by  the  Tomato  Affymetrix  Genome  Array  hybridiza1ons,  higher  expression  levels  of  HSFA2  and  LeHSP17.4-­‐CII  genes  were  detected  by  semi-­‐quan1ta1ve  RT-­‐PCR  analyses  in  non-­‐stressed  (‘control’)  microspores  of  cv.  Hazera  3042  (the  heat-­‐tolerant  cul1var)  versus  microspores  of  cv.  Hazera  3017  (the  heat-­‐  sensi1ve  cul1var)  (Fig.  3A).  These  results  may  point  to  a  poten1al  benefit  for  microspores  that  exhibit  higher  basal  expression  levels  of  ‘protec1ve’  genes,  such  as  HSP  genes,  prior  to  exposure  of  plants  to  HS.”    Frank,  G.,  Pressman,  E.,  Ophir,  R.,  Althan,  L.,  Shaked,  R.,  Freedman,  M.,  et  al.  (2009).  Transcrip1onal  profiling  of  maturing  tomato  (Solanum  lycopersicum  L.)  microspores  reveals  the  involvement  of  heat  shock  proteins,  ROS  scavengers,  hormones,  and  sugars  in  the  heat  stress  response.  Journal  of  Experimental  Botany,  60(13),  3891–3908.  doi:10.1093/jxb/erp234   23  

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Using  LycoCyc  to  visualiza1on  gene  expression  changes  

wings  2014  

24  

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LycoCyc  

•  Curated  database  of  metabolic  pathways,  reac1ons,  enzymes,  and  genes  for  tomato  

•  Developed  by  Lukas  Mueller's  group  at  Cornell  

•  Uses  same  souware  as  PlantCyc,  AraCyc  – Has  many  features,  but  is  fragile.    

•  Prac3ce:  Form  teams  of  three  people  for  this  part  of  the  workshop  to  avoid  overloading  the  system    

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Consider  the  context...  

•  Most  bioinforma1cs  souware  projects  are  funded  by  grants,  which  means...  – Students,  postdocs,  &  professors  write  the  code  

•  We  can't  easily  match  the  robustness  or  user-­‐friendliness  of  commercial  projects  

•  Please  be  pa3ent  and  alert  when  using  souware  from  academic  projects  –  it  may  be  a  li7le  buggy,  a  li7le  quirky,  but  the  content  will  likely  be  very  high  quality  

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Recent  mee1ng  about  scien1fic  souware  sustainability  

•  Ann  requests:  please  consider  these  issues  when  you  review  proposals   27  

h7p://arxiv.org/abs/1404.7414  

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Prac1ce:  Go  to  SolGenomics.net  

•  Select  Pathways  

28  

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Select  Solanum  lycopersicum  database  

29  

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Prac1ce:  Select  Cellular  Overview  

30  

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Shows  annotated  tomato  metabolic  pathways  

•  Shapes  are  metabolites  •  Gray  panels  are  groups  of  related  pathways  •  Blue  &  gray  lines  are  to  reac1ons  •  Blue  lines  are  reac1ons  annotated    w/  a  gene   31  

hormones  

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Prac1ce:  Select  Upload  Data  from  File  

•  Upload forLycoCyc.tsv  •  Made  in  Differen1al  Expression  Markdown  (previous  workshop)  

 

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File  contains  log2FC  for  DE  genes  

•  No  header  •  1st  column  lists  genes  

•  2nd  column  lists  log2  fold-­‐changes  – Posi1ve:  up  in  treatment  

– Nega1ve:  down  in  treatment  

33  

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Prac1ce:  Upload  forLycoCyc.tsv!

1.  Select  file  Differen1alExpression/results/forLycCyc.tsv  

2.  Enter  1  in  Data  column(s)  to  use  

3.  Click  Submit  

1  

2  

34  

3  

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Auer  upload,  Omics  Table  appears  

•  Omics  Control  Panel  shows  heat  map  legend,  opacity  sevngs  – Tip:  move  Opacity  Controller  to  right  

35  

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Expression  results  overlaid  on  pathways  

•  Click-­‐drag  to  move  pathways  diagram  •  Overlay  colors  indicate  up  or  down-­‐regulated  enzymes  

36  

hormones  

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Prac1ce:  Zoom  to  hormones  

•  Click-­‐drag  to  move  pathways  diagram  •  Note:  Overlay  colors  indicate  up  or  down-­‐regulated  enzymes  

37  

what  you  see  auer  two  zoom  clicks  

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Prac1ce:  Click  line  to  see  reac1on  info  

•  Click  Keep  Open  to  keep  popup  in  view,  new  op1ons  appear  

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More  op1ons  

•  Tip:  To  dismiss,  click  upper  right  corner  when  cursor  is  looks  like  a  hand    

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Prac1ce:  Click  Omics  to  see  barcharts  

40  

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Prac1ce:  Go  to  pathway  page  

•  Click  pathway  name  to  open  pathway  page  in  a  new  tab  

41  

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Prac1ce:  View  pathway  page  

•  Click  More  Detail  to  see  structures,  enzyme  names  

•  Click  twice  for  even  more  detail    

•  Scroll  down  for  curator's  notes  

42  

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Prac1ce:  Overlay  fold-­‐change  results  on  pathway  page  

•  Choose  Customize  or  Overlay  Omics  Data  on  Pathway  Diagram  

43  

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Prac1ce:  Overlay  fold-­‐change  results  on  pathway  page  

•  Choose  Customize  or  Overlay  Omics  Data  on  Pathway  Diagram  

44  

•  New  window  with  Customiza3on  Op3ons  opens    

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45  

•  Upload  Fold-­‐change  file  

•  Enter  1  •  Click  Apply  to  keep  window  open    – Clicking  OK  closes  window  

–  If  you  close  the  window,  you  can't  change  appearance    w/o  re-­‐uploading  

Prac1ce:  Upload  forLycoCyc.tsv!

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•  Reac1ons  lines  with  DE  genes  thicker,  color-­‐coded    

46  

Prac1ce:  View  overlay  

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•  Go  back  to  Cellular  Overview  

•  Inves1gate  down-­‐regulated  transporters    

•  Or  pick  another  reac1on/pathway  to  inves1gate  

47  

Prac1ce:  Explore  other  reac1ons    

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Comparing  tomato  and  Arabidopsis  pollen  

wings  2014  

48  

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Arabidopsis  Comparison  

•  See  folder  in  the  tomatopollen  repository  – ArabidopsisComparison  

•  Matched  tomato  with  Arabidopsis  genes  –  Two  methods  for  the  matching  

•  BLAST  best  matches  against  TAIR10  proteins  (Ann)  •  Mapping  downloaded  from  Ensembl  BioMart  (Gad  Miller)  

•  Compared  tomato  pollen  gene  expression  normalized  counts  (FPKM)  to  Arabidopsis    –  pollen  RPKM    –  rose7es  RPKM  (from  21-­‐day  old  plants)  

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Results  

•  See:  AtComparison.html  •  Take-­‐home:  – Pollen  from  tomato  and  Arabidopsis  have  roughly  similar  expression  profiles  

– Same  categories  of  genes  are  highly-­‐expressed  in  both,  including  many  that  were  up-­‐regulated  by  heat  in  the  tomato  RNA-­‐Seq  experiment  

– Excep1on:  Many  "unknown"  genes  highly  expressed  in  tomato    

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Prac1ce:  Follow-­‐up    

•  Pollen  experts:  Review  genes  that  are  – highly  expressed  in  both  tomato  and  Arabidopsis  pollen  

– up-­‐  or  down-­‐regulated  by  mild  chronic  heat  stress  in  tomato    

•  Look  up  "unknown"  genes  in  IGB  and  CNTRL-­‐click  gene  model  to  run  a  BLASTX  or  BLASTP  search  – Are  these  genes  found  in  other  plant  species?  If  yes,  how  closely  related  are  they  to  tomato?  

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See  files  in  results  folder  (1  of  2)  

•  atCompEnsembl.tsv  lists  – average,  normalized  counts  for  annotated  tomato  genes  in  treatment  and  control  (ave.cn,  ave.tr)  

– normalized  counts  for  Arabidopsis  genes  in  pollen  (pollen)  and  rose7es  (Ave.seedling)  

– Arabidopsis  homologs  according  to  Ensembl  BioMart  (or  NA  if  not  available)  

– differen1ally  expressed  or  not,  True  or  False  (de)  

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See  files  in  results  folder  (2  of  2)  

•  atCompBoth.tsv  same  as  in  atCompEnsembl.tsv    but  only  lists  genes  where  Ann  and  Gad's  homolog  matching  methods  agreed    

•  forAraCyc.tsv  data  file  that  can  be  loaded  into  the  AraCyc  Omics  viewer  tool    –  average,  normalized  counts  for  annotated  tomato  genes  in  treatment  and  control  (ave.cn,  ave.tr)  

–  normalized  counts  for  Arabidopsis  genes  in  pollen  (pollen)  and  rose7es  (Ave.seedling)  

–  Arabidopsis  homologs  according  to  Ensembl  BioMart  (or  NA  if  not  available)  

–  differen1ally  expressed  or  not,  True  or  False  (de)  

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