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.

What
CAN
we

learn
from
this
data?

5

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

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/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,
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,
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

Pollen
quality

The
following
papers
use
this
graph
layout.

And
they
show
similar
data.

What
do
we
already
know?

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”

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

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,
2
Department of Fruit Tree Sciences, Institute of Plant Sciences, The Volcani Center, Agricultural Research Organization, POB 6,
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

What
do
we
already
know?

‘ETHYLENE 2012’
1
2
Abstract
Background and
aims
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 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

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.

‘ETHYLENE 2012’
1
2
Abstract
Background and
aims
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 2012: pls024; doi:10.1093/aobpla/pls024, available online at www.aobplants.oxfordjournals.org & The Authors 2012 1
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

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)

49

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

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

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

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

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

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