Workshops	
  in	
  next-­‐genera1on	
  
science	
  at	
  UNC	
  Charlo7e	
  in	
  2014	
  
Workshop	
  3	
  -­‐	
  Tools	
...
Tomato	
  Pollen	
  RNA-­‐Seq	
  
Seeking	
  biological	
  significance	
  
	
  
Slides	
  by	
  Ivory	
  Clabaugh	
  Blakl...
What	
  do	
  we	
  already	
  know?	
  
What	
  CAN	
  we	
  	
  
learn	
  from	
  this	
  data?	
  
What	
  	
  
are	
  ...
Given	
  that	
  “impaired	
  pollen	
  development	
  under	
  high-­‐temperature	
  
condi1ons	
  has	
  been	
  implica...
!  Can't	
  use	
  these	
  data	
  to	
  find	
  out	
  what	
  makes	
  this	
  cul1var	
  more	
  
heat-­‐tolerant	
  th...
What	
  CAN	
  we	
  	
  
learn	
  from	
  this	
  data?	
  
!  	
  Effects	
  of	
  stress	
  on	
  gene	
  expression	
  ...
What	
  do	
  we	
  already	
  know?	
  
7	
  
What	
  do	
  we	
  already	
  know?	
  
Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009
doi:10.1093/...
Pollen	
  quality	
  
The	
  following	
  papers	
  use	
  this	
  graph	
  layout.	
  
And	
  they	
  show	
  similar	
  ...
•  Mild	
  chronic	
  heat	
  stress	
  reduces	
  	
  
sugar	
  content	
  in	
  some	
  cul1vars	
  	
  
but	
  not	
  H...
Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009
doi:10.1093/jxb/erp234 Advance Access publication 23 ...
What	
  do	
  we	
  already	
  know?	
  
Open access – Research article
THIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED
‘ET...
What	
  do	
  we	
  already	
  know?	
  
Firon 2012	
  
Acquired	
  thermo	
  tolerance	
  in	
  
pollen	
  may	
  be	
  u...
Exploring	
  the	
  Results	
  
Compare	
  to	
  microarray	
  (Frank	
  2009)	
  
Pathway	
  visualiza1on	
  with	
  Lyco...
Compared	
  to	
  Frank	
  2009	
  microarray	
  
•  Direct	
  comparison	
  made	
  difficult	
  by	
  lack	
  of	
  
mappi...
•  Frank	
  2009	
  microarray	
  study	
  showed	
  
this	
  gene	
  was	
  up-­‐regulated.	
  	
  
•  Only	
  weakly	
  ...
class	
  I	
  heat	
  shock	
  protein	
  3	
  	
  
Frank	
  et	
  al	
  show	
  this	
  gene	
  (LesAffx.10596.1.S1_at)	
 ...
Annota1on	
  improvements	
  
from	
  RNA-­‐Seq	
  
18	
  
Extra	
  Exon	
   19	
  
Under	
  Coun1ng	
  
20	
  
References	
  –	
  Firon 2006	
  
“The	
  prevalence	
  of	
  high	
  ambient	
  temperatures	
  in	
  a	
  
significant	
 ...
References	
  –	
  Firon 2012	
  
“Impaired	
  pollen	
  development	
  under	
  high-­‐temperature	
  
condi1ons	
  has	
...
References	
  –	
  Frank 2009	
  	
  
“Although	
  no	
  significant	
  differences	
  in	
  gene	
  expression	
  between	
...
Using	
  LycoCyc	
  to	
  visualiza1on	
  
gene	
  expression	
  changes	
  
wings	
  2014	
  
24	
  
LycoCyc	
  
•  Curated	
  database	
  of	
  metabolic	
  pathways,	
  
reac1ons,	
  enzymes,	
  and	
  genes	
  for	
  tom...
Consider	
  the	
  context...	
  
•  Most	
  bioinforma1cs	
  souware	
  projects	
  are	
  
funded	
  by	
  grants,	
  wh...
Recent	
  mee1ng	
  about	
  scien1fic	
  souware	
  
sustainability	
  
•  Ann	
  requests:	
  please	
  consider	
  these...
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	
 ...
Prac1ce:	
  Select	
  Upload	
  Data	
  from	
  File	
  
•  Upload forLycoCyc.tsv	
  
•  Made	
  in	
  Differen1al	
  
Expr...
File	
  contains	
  log2FC	
  for	
  DE	
  genes	
  
•  No	
  header	
  
•  1st	
  column	
  lists	
  genes	
  
•  2nd	
  ...
Prac1ce:	
  Upload	
  forLycoCyc.tsv!
1.  Select	
  file	
  
Differen1alExpression/
results/forLycCyc.tsv	
  
2.  Enter	
  1...
Auer	
  upload,	
  Omics	
  Table	
  appears	
  
•  Omics	
  Control	
  Panel	
  shows	
  heat	
  map	
  legend,	
  
opaci...
Expression	
  results	
  overlaid	
  on	
  pathways	
  
•  Click-­‐drag	
  to	
  move	
  pathways	
  diagram	
  
•  Overla...
Prac1ce:	
  Zoom	
  to	
  hormones	
  
•  Click-­‐drag	
  to	
  move	
  pathways	
  diagram	
  
•  Note:	
  Overlay	
  col...
Prac1ce:	
  Click	
  line	
  to	
  see	
  reac1on	
  info	
  
•  Click	
  Keep	
  Open	
  to	
  keep	
  popup	
  in	
  vie...
More	
  op1ons	
  
•  Tip:	
  To	
  dismiss,	
  click	
  upper	
  right	
  corner	
  when	
  
cursor	
  is	
  looks	
  lik...
Prac1ce:	
  Click	
  Omics	
  to	
  see	
  barcharts	
  
40	
  
Prac1ce:	
  Go	
  to	
  pathway	
  page	
  
•  Click	
  pathway	
  name	
  to	
  open	
  pathway	
  page	
  in	
  a	
  
ne...
Prac1ce:	
  View	
  pathway	
  page	
  
•  Click	
  More	
  Detail	
  to	
  see	
  
structures,	
  enzyme	
  
names	
  
• ...
Prac1ce:	
  Overlay	
  fold-­‐change	
  
results	
  on	
  pathway	
  page	
  
•  Choose	
  Customize	
  or	
  Overlay	
  
...
Prac1ce:	
  Overlay	
  fold-­‐change	
  
results	
  on	
  pathway	
  page	
  
•  Choose	
  Customize	
  or	
  Overlay	
  
...
45	
  
•  Upload	
  Fold-­‐change	
  
file	
  
•  Enter	
  1	
  
•  Click	
  Apply	
  to	
  keep	
  
window	
  open	
  	
  ...
•  Reac1ons	
  lines	
  
with	
  DE	
  genes	
  
thicker,	
  color-­‐
coded	
  	
  
46	
  
Prac1ce:	
  
View	
  overlay	
  
•  Go	
  back	
  to	
  Cellular	
  
Overview	
  
•  Inves1gate	
  down-­‐regulated	
  
transporters	
  	
  
•  Or	
  pick	...
Comparing	
  tomato	
  and	
  
Arabidopsis	
  pollen	
  
wings	
  2014	
  
48	
  
Arabidopsis	
  Comparison	
  
•  See	
  folder	
  in	
  the	
  tomatopollen	
  repository	
  
–  ArabidopsisComparison	
  ...
Results	
  
•  See:	
  AtComparison.html	
  
•  Take-­‐home:	
  
– Pollen	
  from	
  tomato	
  and	
  Arabidopsis	
  have	...
Prac1ce:	
  Follow-­‐up	
  	
  
•  Pollen	
  experts:	
  Review	
  genes	
  that	
  are	
  
– highly	
  expressed	
  in	
 ...
See	
  files	
  in	
  results	
  folder	
  (1	
  of	
  2)	
  
•  atCompEnsembl.tsv	
  lists	
  
– average,	
  normalized	
 ...
See	
  files	
  in	
  results	
  folder	
  (2	
  of	
  2)	
  
•  atCompBoth.tsv	
  same	
  as	
  in	
  
atCompEnsembl.tsv	
...
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RNA-Seq data analysis at wings 2014 - Workshop 3 Biological Interpretation

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Slides from Workshop 3 of WiNGs Conference held at the UNC Charlotte City Center campus in May 2014.

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RNA-Seq data analysis at wings 2014 - Workshop 3 Biological Interpretation

  1. 1. Workshops  in  next-­‐genera1on   science  at  UNC  Charlo7e  in  2014   Workshop  3  -­‐  Tools  for  biological   interpreta1on     1  
  2. 2. Tomato  Pollen  RNA-­‐Seq   Seeking  biological  significance     Slides  by  Ivory  Clabaugh  Blakley   2  
  3. 3. What  do  we  already  know?   What  CAN  we     learn  from  this  data?   What     are  we     trying  to  learn?   3  
  4. 4. 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  
  5. 5. !  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  
  6. 6. 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  
  7. 7. What  do  we  already  know?   7  
  8. 8. What  do  we  already  know?   Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009 doi:10.1093/jxb/erp234 Advance Access publication 23 July, 2009 This 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 (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the 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, Israel 2 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 the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using 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. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical 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 of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular 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. byguestonApril18,2014http://jxb.oxfordjournals.org/Downloadedfrom Pollen grains of heat tolerant tomato cultivars retain higher carbohydrate concentration 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, Israel b 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 male gametophyte 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 and germinability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number and quality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitive cultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, and a parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starch accumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that the carbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest that heat-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 yield and impairs the quality of many crops, including vegetable crops. The prevalence of high ambient temperatures in a significant proportion of the tomato-growing areas of the world is one of the most crucial problems in tomato production. Chronic heat stress, even of a mild degree, has been shown to disrupt the normal development of the gametes and thereby fruit set. Levy et al. (1978) compared the effects of high temperatures on a susceptible and a tolerant tomato cultivar and found that heat stress affected mainly the pollen grains; it reduced their viability and the effect was more pronounced in the susceptible cultivar. Sato et al. (2000) found that among five tomato cultivars grown under mild high-temperature conditions (32 8C day and 26 8C night) only cv. FLA 7156 set fruits. They suggested that differences among cultivars in pollen release and germination under heat stress are the most crucial factors in determining fruit set. Porch and Jahn (2001) found that heat stress caused indehiscence of the anthers, reduced pollen viability and reduced yield in a heat-sensitive genotype of bean (Phaseolus vulgaris); the anthers and pollen of a heat-tolerant genotype were generally normal under the same conditions. Starch biosynthesis during the final phases of pollen maturation is critical in determining pollen quality not only because starch is a reserve source of energy for pollen germination but it may also serves as a checkpoint of pollen maturity. In dicots, such as tomato, starch accumulation peaks at 3 days before anthesis, while the mature pollen grains are considered starchless. In monocots (such as maize) starch accumulates during pollen maturation and the mature pollen grains contain starch. In several maize genetically controlled male-sterile mutants it was shown that pollen inviability was associated with starch-deficiency (Datta et al., 2002). www.elsevier.com/locate/scihorti Scientia Horticulturae 109 (2006) 212–217 * Corresponding author. Tel.: +972 3 9683470; fax: +972 3 9669642. E-mail addresses: pressman@agri.gov.il, pressman@volcani.gov.il (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 article THIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED ‘ETHYLENE 2012’ Ethylene is involved in maintaining tomato (Solanum lycopersicum) pollen quality under heat-stress conditions Nurit 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, Israel 2 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 (Solanum lycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024 Abstract Background and aims Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-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 pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling pathway and altering ethylene levels on tomato pollen functioning under heat stress. AoB PLANTS http://aobplants.oxfordjournals.org/AoB PLANTS http://aobplants.oxfordjournals.org/ Firon  2006   effect  of  heat  stress  on   pollen  carbohydrates   Frank,  2009   Microarray  study   Firon,  2012   Manipula1on  of  ethylene   pathway   8  
  9. 9. Pollen  quality   The  following  papers  use  this  graph  layout.   And  they  show  similar  data.   What  do  we  already  know?   9  
  10. 10. •  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 carbohydrate concentration 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, Israel b 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 male gametophyte 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 and germinability, caused reduced fruit set and markedly reduced the numbers of seeds per fruit. In the heat-tolerant cultivars, however, the number and quality of pollen grains, the number of fruits and the number of seeds per fruit were less affected by high temperatures. In all the heat-sensitive cultivars, the heat-stress conditions caused a marked reduction in starch concentration in the developing pollen grains at 3 days before anthesis, and a parallel decrease in the total soluble sugar concentration in the mature pollen, whereas in the four heat-tolerant cultivars tested, starch accumulation at 3 days before anthesis and soluble sugar concentration at anthesis were not affected by heat stress. These results indicate that the carbohydrate content of developing and mature tomato pollen grains may be an important factor in determining pollen quality, and suggest that heat-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 yield and impairs the quality of many crops, including vegetable crops. The prevalence of high ambient temperatures in a significant proportion of the tomato-growing areas of the world is one of the most crucial problems in tomato production. Chronic heat stress, even of a mild degree, has been shown to disrupt the normal development of the gametes and thereby fruit set. Levy et al. (1978) compared the effects of high temperatures on a susceptible and a tolerant tomato cultivar and found that heat stress affected mainly the pollen grains; it reduced their viability and the effect was more pronounced in the susceptible cultivar. Sato et al. (2000) found that among five tomato cultivars grown under mild high-temperature conditions (32 8C day and 26 8C night) only cv. FLA 7156 set fruits. They suggested that differences among cultivars in pollen release and germination under heat stress are the most crucial factors in determining fruit set. Porch and Jahn (2001) found that heat stress caused indehiscence of the anthers, reduced pollen viability and reduced yield in a heat-sensitive genotype of bean (Phaseolus vulgaris); the anthers and pollen of a heat-tolerant genotype were generally normal under the same conditions. Starch biosynthesis during the final phases of pollen maturation is critical in determining pollen quality not only because starch is a reserve source of energy for pollen germination but it may also serves as a checkpoint of pollen maturity. In dicots, such as tomato, starch accumulation peaks at 3 days before anthesis, while the mature pollen grains are considered starchless. In monocots (such as maize) starch accumulates during pollen maturation and the mature pollen grains contain starch. In several maize genetically controlled male-sterile mutants it was shown that pollen inviability was associated with starch-deficiency (Datta et al., 2002). www.elsevier.com/locate/scihorti Scientia Horticulturae 109 (2006) 212–217 * Corresponding author. Tel.: +972 3 9683470; fax: +972 3 9669642. E-mail addresses: pressman@agri.gov.il, pressman@volcani.gov.il (E. Pressman). 0304-4238/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2006.03.007 10  
  11. 11. Journal of Experimental Botany, Vol. 60, No. 13, pp. 3891–3908, 2009 doi:10.1093/jxb/erp234 Advance Access publication 23 July, 2009 This 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 (Solanum lycopersicum L.) microspores reveals the involvement of heat shock proteins, ROS scavengers, hormones, and sugars in the 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, Israel 2 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 the developing pollen grains. The high temperature response, especially at this most HS-sensitive stage of the plant, is poorly understood. To obtain an overview of molecular mechanisms underlying the HS response (HSR) of microspores, a detailed transcriptomic analysis of heat-stressed maturing tomato microspores was carried out using 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. The data obtained reveal the involvement of specific members of the small heat shock protein (HSP) gene family, HSP70 and HSP90, in addition to the HS transcription factors A2 (HSFA2) and HSFA3, as well as factors other than the classical 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 of cDNA-AFLP enabled the detection of genes representing pollen-specific functions that are missing from the tomato Affymetrix chip, such as those involved in vesicle-mediated transport and a pollen-specific, calcium-dependent protein kinase (CDPK2). For several genes, including LeHSFA2, LeHSP17.4-CII, as well as homologues of LeHSP90 and AtVAMP725, higher basal expression levels were detected in microspores of cv. Hazera 3042 (a heat-tolerant cultivar) compared with microspores of cv. Hazera 3017 (a heat-sensitive cultivar), marking these genes as candidates for taking part in microspore thermotolerance. This work provides a comprehensive analysis of the molecular 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) during some stage of their life cycle. HS, defined as the temper- atures above normal optimum, is expected to become a more frequent and acute problem in the coming years (Sato et al., 2000). Exposure to HS reduces yield and decreases the quality 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 of 29 °C (32/26 °C day/night), fruit number, fruit weight per plant, and seed number per fruit were markedly decreased compared 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: vcfiron@volcani.agri.gov.il 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. byguestonApril18,2014http://jxb.oxfordjournals.org/Downloadedfrom 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  
  12. 12. What  do  we  already  know?   Open access – Research article THIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED ‘ETHYLENE 2012’ Ethylene is involved in maintaining tomato (Solanum lycopersicum) pollen quality under heat-stress conditions Nurit 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, Israel 2 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 (Solanum lycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024 Abstract Background and aims Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-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 pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling 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 response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or 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 pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor 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 pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls. Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathwayor reducing ethylenelevels increased tomato pollen sensitivity to heatstress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality. * Corresponding author’s e-mail address: vcfiron@volcani.agri.gov.il Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-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.   12  
  13. 13. 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 article THIS PAPER IS PART OF A SPECIAL ISSUE ENTITLED ‘ETHYLENE 2012’ Ethylene is involved in maintaining tomato (Solanum lycopersicum) pollen quality under heat-stress conditions Nurit 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, Israel 2 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 (Solanum lycopersicum) pollen quality under heat-stress conditions. AoB PLANTS 2012: pls024; doi:10.1093/aobpla/pls024 Abstract Background and aims Exposure to higher-than-optimal temperatures reduces crop yield and quality, mainly due to sensitivity of developing pollen grains. The mechanisms maintaining high pollen quality under heat-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 pollen heat-stress response and thermotolerance by assessing the effects of interfering with the ethylene signalling 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 response sensor (ERS)-like ethylene receptor—and the corresponding wild type were exposed to control or 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 pretreating cv. Micro-Tom tomato plants, prior to heat-stress exposure, with an ethylene releaser or inhibitor 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 pollen grains accumulated only 40 % of the sucrose level accumulated by the wild type. Pretreatment of tomato plants with an ethylene releaser increased pollen quality under heat stress, with an over 5-fold increase in the number of germinating pollen grains per flower. Pretreatment with an ethylene biosynthesis inhibitor reduced the number of germinating pollen grains following heat-stress exposure over 5-fold compared with non-treated controls. Conclusions Ethylene plays a significant role in tomato pollen thermotolerance. Interfering with the ethylene signalling pathwayor reducing ethylenelevels increased tomato pollen sensitivity to heatstress, whereas increasing ethylene levels prior to heat-stress exposure increased pollen quality. * Corresponding author’s e-mail address: vcfiron@volcani.agri.gov.il Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-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 13  
  14. 14. Exploring  the  Results   Compare  to  microarray  (Frank  2009)   Pathway  visualiza1on  with  LycoCyc   Gene  Ontology  enrichment  analysis   Novel  Gene  search   14  
  15. 15. 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  
  16. 16. •  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  
  17. 17. 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  
  18. 18. Annota1on  improvements   from  RNA-­‐Seq   18  
  19. 19. Extra  Exon   19  
  20. 20. Under  Coun1ng   20  
  21. 21. 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  
  22. 22. 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  
  23. 23. 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  
  24. 24. Using  LycoCyc  to  visualiza1on   gene  expression  changes   wings  2014   24  
  25. 25. 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  
  26. 26. 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  
  27. 27. Recent  mee1ng  about  scien1fic  souware   sustainability   •  Ann  requests:  please  consider  these  issues  when   you  review  proposals   27   h7p://arxiv.org/abs/1404.7414  
  28. 28. Prac1ce:  Go  to  SolGenomics.net   •  Select  Pathways   28  
  29. 29. Select  Solanum   lycopersicum   database   29  
  30. 30. Prac1ce:  Select   Cellular  Overview   30  
  31. 31. 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  
  32. 32. Prac1ce:  Select  Upload  Data  from  File   •  Upload forLycoCyc.tsv   •  Made  in  Differen1al   Expression  Markdown   (previous  workshop)     32  
  33. 33. 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  
  34. 34. 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  
  35. 35. Auer  upload,  Omics  Table  appears   •  Omics  Control  Panel  shows  heat  map  legend,   opacity  sevngs   – Tip:  move  Opacity  Controller  to  right   35  
  36. 36. Expression  results  overlaid  on  pathways   •  Click-­‐drag  to  move  pathways  diagram   •  Overlay  colors  indicate  up  or  down-­‐regulated   enzymes   36   hormones  
  37. 37. 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  
  38. 38. Prac1ce:  Click  line  to  see  reac1on  info   •  Click  Keep  Open  to  keep  popup  in  view,  new   op1ons  appear   38  
  39. 39. More  op1ons   •  Tip:  To  dismiss,  click  upper  right  corner  when   cursor  is  looks  like  a  hand     39  
  40. 40. Prac1ce:  Click  Omics  to  see  barcharts   40  
  41. 41. Prac1ce:  Go  to  pathway  page   •  Click  pathway  name  to  open  pathway  page  in  a   new  tab   41  
  42. 42. 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  
  43. 43. Prac1ce:  Overlay  fold-­‐change   results  on  pathway  page   •  Choose  Customize  or  Overlay   Omics  Data  on  Pathway   Diagram   43  
  44. 44. 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. 45. 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!
  46. 46. •  Reac1ons  lines   with  DE  genes   thicker,  color-­‐ coded     46   Prac1ce:   View  overlay  
  47. 47. •  Go  back  to  Cellular   Overview   •  Inves1gate  down-­‐regulated   transporters     •  Or  pick  another  reac1on/ pathway  to  inves1gate   47   Prac1ce:  Explore  other  reac1ons    
  48. 48. Comparing  tomato  and   Arabidopsis  pollen   wings  2014   48  
  49. 49. 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)   49  
  50. 50. 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     50  
  51. 51. 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?   51  
  52. 52. 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)   52  
  53. 53. 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)   53  

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