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

1

Tomato Pollen RNA-‐Seq

Seeking biological significance

Slides by Ivory Clabaugh Blakley

2

What do we already know?

What CAN we learn from this data?

What are we

trying to learn?

3

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

!  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.


5


!   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


7


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

Pollen quality

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


9

•  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”


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

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


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







Abstract

Background andaims









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.

12


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.






Abstract

Background andaims









13

Exploring the Results

Compare to microarray (Frank 2009)

Pathway visualiza1on with LycoCyc

Gene Ontology enrichment analysis

Novel Gene search

14

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.

15

•  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

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

17

Annota1on improvements from RNA-‐Seq

18

Extra Exon 19

Under Coun1ng

20

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

21

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.

22

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

Using LycoCyc to visualiza1on gene expression changes

wings 2014

24

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

25

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

26

Recent mee1ng about scien1fic souware sustainability

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

h7p://arxiv.org/abs/1404.7414

Prac1ce: Go to SolGenomics.net

•  Select Pathways

28

Select Solanum lycopersicum database

29

Prac1ce: Select Cellular Overview

30

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

Prac1ce: Select Upload Data from File

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

32

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

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

Auer upload, Omics Table appears

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

35

Expression results overlaid on pathways

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

36

hormones

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

Prac1ce: Click line to see reac1on info

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

38

More op1ons

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

39

Prac1ce: Click Omics to see barcharts

40

Prac1ce: Go to pathway page

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

41

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

Prac1ce: Overlay fold-‐change results on pathway page

•  Choose Customize or Overlay Omics Data on Pathway Diagram

43

Prac1ce: Overlay fold-‐change results on pathway page

•  Choose Customize or Overlay Omics Data on Pathway Diagram

44

•  New window with Customiza3on Op3ons opens

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!

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

46

Prac1ce: View overlay

•  Go back to Cellular Overview

•  Inves1gate down-‐regulated transporters

•  Or pick another reac1on/pathway to inves1gate

47

Prac1ce: Explore other reac1ons

Comparing tomato and Arabidopsis pollen

wings 2014

48

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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