Histological and microarray analysis of the direct effect of water shortage alone or combined with...

"The definitive version is available at www.blackwell-synergy.com" doi: 10.1111/j.1399-3054.2010.01394.x.

Szucs, Attila, Katalin Jäger, Manuela E Jurca, Attila Fábián, Sándor Bottka, Agnes Zvara, Beáta Barnabás, andAttila Fehér. 2010. “Histological and microarray analysis of the direct effect of water shortage alone or combined with heat on early grain development in wheat (Triticum aestivum).” Physiologia plantarum 140 (2) (October): 174-88. doi:10.1111/j.1399-3054.2010.01394.x.

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Histological and microarray analysis of the direct effect of water shortage alone or combined

with heat on early grain development in wheat (Triticum aestivum L.)

Attila Szűcs1,2, Katalin Jäger2, Manuela E. Jurca1, Attila Fábián2, Sándor Bottka1, Ágnes Zvara3, Beáta

Barnabás2, Attila Fehér1* 1 Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, Temesvári

krt. 62, H-6726, Szeged, Hungary 2 Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik u. 2, H-2462,

Martonvásár, Hungary 3 Laboratory of Functional Genomics, Biological Research Center, Hungarian Academy of Sciences,

Temesvári krt. 62, H-6726

Key-words: cereal, environmental stress, gene expression, genomics, seed development

* author for correspondance: Prof. Attila Fehér, Institute of Plant Biology, BRC, HAS, 6701 Szeged,

P.O.Box 521., Hungary. [email protected]



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Abstract

Based on the in silico analysis of the representation of expressed sequence tags (ESTs) in wheat grain-

related cDNA libraries, a specific 15k oligonucleotide microarray has been developed in order to

monitor environmental stress-dependent gene expression changes in the wheat caryopses. Using this

array, the effect of water withdrawal, with and without additional heat stress, has been investigated

during the first five days of kernel development on two wheat cultivars differing in their drought

sensitivity. Water shortage affected (more than 2-fold change) the expression of only 0.5% of the

investigated genes. A parallel heat treatment increased the ratio of responding genes to 5-7% due to

the temperature stress and/or the increased water deficit due to enhanced evaporation. It could be

established that the two cultivars, differing in their long-term adaptation capabilities to drought,

responded to the short and direct stress treatments on the same way. In response to the combined

drought and heat treatment, the coordinately altered expression of genes coding for storage proteins,

enzymes involved in sugar/starch metabolism, histone proteins, heat shock proteins, proteases,

tonoplast aquaporins as well as several transcription factors has been observed. These gene expression

changes were in agreement with histological data that demonstrated the accelerated development of

the embryo as well as the endosperm.

Introduction

Wheat, planted on around 2.1x108 hectares and harvested at approximately 6x108 tons per year

(http://faostat.fao.org/), is one of the most important cereals on the world. Although during the last 40

years the size of the harvested area did not change by more than 10%, the wheat yield was tripled due

to the introduction of modern agricultural techniques and improved cultivars. However, a continuously

rising demand for wheat consumption, especially in the developing region of the world, is evident as

indicated by the price of wheat that doubled in the last decade (http://stats.oecd.org/). At the same

time, there is an unfavorable change in the climate with increased average temperature and lowered

water availability in many wheat-growing areas. Therefore, yield increase is still in the forefront of

wheat breeding efforts but with an emphasis on the increased stability of grain production under

various environmental conditions.

Modern molecular techniques may provide useful tools to speed up the breeding process providing

deep insights into the developmental and molecular pathways affecting final yield. As grain

development, the primary determinant of wheat yield, is concerned, it can be divided into three phases.

The first phase (1-14 days after pollination; 1-14 DAP) starts with the event of double fertilization.

The zygote divides, and the embryo partially differentiates by the end of this period. The nucleus of

the fertilized central cell divides rapidly without cytokinesis resulting in a coenocyte with



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approximately 2.000 nuclei by 72 hours after fertilization (Chojecki et al. 1986). Cellularization takes

place during the subsequent 24 hours. The endosperm cells further divide, expand and differentiate

that determine the number of endosperm cells of the mature grain (up to 300.000 cells; Evers and

Millar 2002). Because there is a high correlation between the number of endosperm cells and the total

weight of the seed, the final yield is largely compromised if this developmental period is subjected to

unfavorable conditions.

During the grain filling period (14-28 DAP) nutrient reserves, starch and gluten proteins, are deposited

into the endosperm cells, which stop dividing. Obviously, this period also directly affects the yield, as

grain size and quality are strongly dependent on reserve accumulation.

The last stage starts at around 28 DAP when the deposition of storage molecules is ending and the

seed starts to desiccate. At 42 DAP the grains are physiologically fully matured.

Of course, there is a considerable variability in the above time scale depending on the cultivar. In

addition to the genotype, environmental conditions have also a significant impact on grain

development. Grain filling often coincides with dry and warm summer periods in many wheat-

growing areas. Therefore, the stress sensitivity of this special developmental phase is considered to be

crucial and is in the focus of research and breeding efforts (Barnabás et al. 2008, Blum 1998, Blum

2005, Boyer and Westgate 2004, Dupont and Altenbach 2003, Gibson and Paulsen 1994, Nicolas et al.

1984, Saini and Westgate 1999, Yang and Zhang 2006). However, at least in certain regions such as

in Central Europe, hot and/or dry episodes are more and more frequent during the earlier phases of

wheat development (Bogataj and Sušnik 2007, Olesen et al. 2007). Therefore, the biological effect of

short but extreme hot and dry weather periods needs to be investigated during various developmental

phases of wheat to gain sufficient amount of data to establish cultivation/breeding/molecular strategies

to diminish the effects of these adverse conditions on the final yield.

Herewith the histological and molecular analysis on the consequences of heat and/or water limitation

during the earliest phase of wheat grain development (first five days after anthesis) is reported to give

some insights into the stress sensitivity of this hardly investigated developmental stage. For this

purpose, a custom oligonucleotide microarray representing almost 15.000 genes expressed during

wheat grain development has been developed to allow easy and affordable monitoring of gene

expression changes in developing wheat caryopses.



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Materials and methods

Plant material

Drought-tolerant Plainsman V and drought-sensitive Cappelle Desprez winter wheat genotypes were

used in the experiment. After germination 60 seedlings of each genotype were vernalized for 7 weeks

at 4 oC, then transplanted into a sand-soil-peat (1:3:1) mixture (2 kg/pot) and grown in a phytotron

using the T1 spring climatic programme (Tischner et al. 1997) for 9 weeks, until the beginning of

flowering. During this period the initial max/min temperature (12.5/5.5 oC) was increased to 23/14 °C.

The plants were emasculated prior to flowering. At the time of anthesis the spikes were hand-

pollinated and subjected to stress treatments starting at one hour after pollination.

Stress treatments and sample collection

Stress treatments applied for 5 days during early seed development involved either total water

withholding alone (hereafter treatment D) or total water withholding in parallel with heat stress at

34/24 °C (hereafter treatment DH). Prior to and immediately following the DH treatment the plants

were kept at a transitional temperature of 28 °C during the dawn and dusk phases of the plant growth

programme, when illumination was set to 1/3 of the total light intensity. The volumetric water content

of the soil (VWC) during treatments was measured with a HH2 moisture meter equipped with an SM

200 moisture sensor (Delta T Devices Ltd., Cambridge, UK).

The main spikes of the plants were used for sample collection. In each treatment kernels were

collected from six spikelets located in the middle of each of three spikes 1, 2, 3, 4 and 5 days after

pollination. The kernels located on one side of each spikelet were used for histological studies and

those on the other side for gene expression studies.

Histological investigations

For light microscopy, kernels were fixed for 4 h at room temperature in primary fixative containing

4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.05 M Na-cacodylate buffer (CB), pH

7.2. After fixation, the samples were washed in CB and dehydrated through a gradient series of

ethanol, infiltrated with UnicrylTM resin (SPI Supplies Inc. West Chester, PA) according to the

manufacturer’s instructions and UV-polymerized for 10 days at -20ºC. Semithin sections (0.5–1 μm)

were cut using an Ultracut E microtome (Reichert-Jung GmbH, Heidelberg, Germany). The periodic

acid-Schiff reaction and 0.25 % (w/v) Coomassie Brilliant Blue R-250 were used to stain the sections

for polysaccharides and proteins, respectively. Sections of the wheat kernels were examined with an

Olympus BX51 (Olympus, Tokyo, Japan) light microscope. Micrographs were taken using a Canon

G6 camera, and CellP software (Olympus Life Science Europa GmbH, Hamburg, Germany) was used



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for image processing and analysis. The data were statistically analyzed with ANOVA (SPSS for

Windows, 10.0).

Planning and construction of seed-specific 15K wheat oligonucleotide array

In order to identify a gene set preferentially expressed during wheat seed development, the “Wheat

Transcript assembly 2” dataset from TIGR (The Institute for Genomic Research;

http://plantta.jcvi.org/cgi-bin/plantta_release.pl, updated July 2007) has been analysed. 156 out of the

451 EST libraries represented in the database were found to be related to seed development (from egg

cells and zygotes to mature grains). EST contig or singleton sequences having at least one EST

occurrences in any seed specific cDNA library have been used for further analysis. Using the PICKY

software (http://www.complex.iastate.edu/download/Picky/), 358621 50-60-mer oligos have been

designed for the 121343 sequences in two steps (the software accepts only 65536 input sequences in

one run). After removing the duplicated oligo sequences, we analyzed the cross-hybridization among

the oligo sequences and the assembled contigs and EST sequences by the BLASTN software

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The oligos were first filtered to keep ony those which had low

potential for cross hybridization to other contigs (length of alignment*identity [%]/length of oligo was

less than 90%) but had perfect match to their target contig (length of alignment*identity [%]/length of

oligo was 100%). Furthermore, only probes targeting genes having at least 2 ESTs from grain

development-related cDNA sources have been retained. All of the oligos (14.966) were connected to

the wheat EST contig databases: TIGR Transcript Assembly 2, KEGG (Kyoto Encyclopedia of Genes

and Genomes; http://www.genome.jp/kegg/) and DFCI Gene Indices (Dana Farber Cancer Institute;

http://compbio.dfci.harvard.edu/tgi/tgipage.html) based on sequence identity (at least 97%) and all of

the relevant information was inserted into a relational database. The annotation file with raw and

normalized data was uploaded to the NCBI GEO database (Accession number: GSE18205;

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE18205).

The GO (gene ontology) terms, and the Affymetrix Wheat GeneChipTM identifiers, were also included,

where they were available. Putative tissue (pericarp, embryo and endosperm) locations of the

transcripts were assigned using literature data (Drea et al. 2005, Wan et al. 2008). The custom

microarrays have been produced through Agilent’s eArray system

(https://earray.chem.agilent.com/earray/).

RNA isolation

Total cellular RNA was purified from isolated, snap-frozen, developing seeds (3 and 5-DAP) using the

RNAzol reagent (Sigma, St-Louis, USA). To avoid genomic DNA contamination, each RNA



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preparation was treated with RNAse-free DNAse according to the manufacturer’s instructions (Sigma,

St-Louis, USA). RNA was then quantified and checked for integrity by an Agilent 2100 Bioanalyser.

Probe preparation, microarray hybridization and data analysis

1 µg total RNA was used to synthesize Cyc3 and Cyc5 labeled aRNA using AMBION (Austin, USA)

Amino Allyl MessageAmpTM II aRNA Amplification Kit. The hybridization and washing was

performed as recommended by Agilent. Image analysis and normalization was done by the Agilent

Feature Extraction 10.1 software using default GE2-v5_10_Apr08 protocol (Linear and Lowess). Two

replicates were used for both condition and one of them was dye-swapped to minimize dye

incorporation errors. Transcriptome data analysis was carried out by Agilent GeneSpring 10.1

software. Those entities where the background subtracted raw signal were always lower than 75 were

removed (15%) from further examination. Using 2way ANOVA 4567 oligos were found to produce

reproducible signals at p < 0.05 applying the Benjamini Hochberg false discovery rate (Benjamini and

Hochberg 1995) and the p value computation was asymptotic. 1697 out of the 4567 probes were

found to detect at least 2-fold signal intensity difference in response to any of the treatments as

compared to the controls. This dataset of 1697 probes was used for further analysis. In hierarchical

clustering the similarity measure was Euclidean and the linkage rule was Centroid.

Real-time quantitative PCR

Real-time quantitative RT-PCR was assayed on an ABI Prism 7700 sequence detection system and the

accumulation of PCR products was measured in real time as the increase in SYBR Green fluorescence

using ROX (glycine conjugate of 5-carboxy-X-rhodamine, succinimidyl ester) as a passive reference

dye according to the manufacturer’s (Applied Biosystems) instruction. Primers are listed as a

supplementary material (see Appendix S1 in Supporting information). Two transcripts, which showed

consistent expression in every sample in the microarray experiment (TA85550, TA64564), have been

chosen as internal controls for data normalization (geNorm method, Vandesompele et al. 2002). Real

time PCR reactions were carried out in triplicate and the results were analyzed using the comparative

CT method based on the advice of the manufacturer (Applied Biosystems) and averaged according to

the geNorm method (Vandesompele et al. 2002).

Results

Effect of water shortage and heat stress on early seed development



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Wheat plants have been subjected to two kind of stress treatments during the first five days of grain

development. Water withdrawal during this period resulted in 52-56% decrease of volumetric water

content of the soil as compared to the controls. When this treatment was combined with heat (34/24 °C

day/night) in addition to the elevated temperature, a further decrease of the soil water content (down to

24% of the control’s value) was also manifested due to increased evaporation (Table 1). The effect of

heat stress alone was not tested as under field conditions extreme heat in most of the cases coincides

with water limitation at the time of wheat seed development.

In order to assess the effect of a short period of heat and/or drought stress on early seed development,

microscopic sections were prepared from kernels of wheat plants subjected to heat and/or water

shortage during their first 5 days of development (1-5 DAP) as above.

As the size of the endosperm and the number and size of endosperm cells were considered, significant

difference was observed between the investigated cultivars (Plainsman V and Cappelle Desprez) at 5

DAP (Table 2) indicating a genotypic difference in endosperm development. Despite of it, the

endosperm of both cultivars responded on the same way to the stress treatments (Table 2, Figs 1 and

2). Drought (treatment D) did not result in significant differences neither in the endosperm area, cell

number or cell size nor in starch and protein accumulation (based on histological staining) in any of

the cultivars as compared to the untreated controls. In contrast, under drought and heat conditions

(treatment DH), the endosperm was considerably enlarged at the end of the 5-day period and w as full

of starch granules and proteinaceous substances (Table 2, Figs 1 and 2). Interestingly, under this

condition the difference between the endosperm size of the two cultivars disappeared (Table 1)

indicating either a more pronounced acceleration of endosperm development of the Cappelle Desprez

genotype in response to treatment DH or the existence of a limit of endosperm size that was reached

by both cultivars under DH conditions at 5 DAP and could not be exceeded even in the Plainsman V

cultivar.

As the size of the embryo is considered, it was not significantly increased as a consequence of

treatment D in comparison to the untreated control neither in the drought sensitive nor in the tolerant

genotype (Figs 1-3). However, the embryos in both Cappelle Desprez and Plainsman V seeds were

extremely large, 8- and 12-fold larger, respectively, in DH-treated grains as compared to the controls

(Figs 1-3) at 5 DAP. The enlarged embryos contained many large vacuolated cells (Fig. 1 I, 2 I) that

could not be observed in the embryos developed under control or water-limited conditions. It was thus

established that both cultivars responded to the two treatments in a similar way: as compared to the

control, treatment D had no effect on the kernels, but endosperm and embryo development as well as

storage material accumulation were speeded up or enhanced by treatment DH.



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In order to obtain a more detailed view of the stress-induced changes, the gene expression pattern of

the seeds was also investigated using a custom wheat grain microarray.

Microarray analysis of general gene expression changes in wheat seeds exposed to heat and/or

drought during the first five days after pollination

For gene expression analysis, fluorescently labeled cDNA probes were prepared from control and

treated (D or DH) Plainsman V and Cappelle Desprez caryopses isolated at 5 DAP. These probes were

hybridized to custom Agilent microarrays carrying oligonucleotides representing 14.966 wheat genes

preferentially expressed during various phases of seed development, as determined by in silico gene

expression analysis based on the frequency of ESTs in various cDNA libraries (see Materials and

methods).

85% percent of the oligonucleotides synthesized onto the microarray exhibited detectable fluorescent

signal following hybridization with at least one of the above cDNA probes, validating the success of

the in silico approach to select seed-expressed transcripts for the microarray.

Evaluation of the hybridization data revealed that the combined effect of heat and drought (treatment

DH) was much significant on the gene expression profile of the cultivars than that of the water

withholding alone (treatment D) (Fig. 4; see Appendix S2 in Supporting information for the detailed

list of genes with at least 2-fold change in their expression). Treatment D had a statistically significant

effect (see Materials and methods) on the expression of only 71 genes in Plainsman V and 69 genes in

Cappelle Desprez. In contrast, 783 genes of Cappelle Desprez and 990 genes of Plainsman V,

representing 5 and 7% of the investigated genes, respectively, responded to the combined treatment

(DH). The overlaps between theses subsets were very high: 88% (369 out of 418) of up-regulated and

86% (315 out of 365) of down regulated genes changed in both cultivars on the same way. There was

no any gene up regulated in one of the cultivars and down regulated in the other. The number of genes

responding to any of the treatments with a significantly altered (>=2) expression level in any of the

cultivars is summarized in Table 3.

Stress-affected expression of genes in the various grain tissues

We have compared the expression of genes representing various tissues of the developing wheat

caryopses. Unfortunately, the number of wheat sequences annotated to various tissues is still very

limited, but nevertheless those that could be clearly assigned to tissue-types (179 probes) showed that

while the expression of genes associated with the maternal pericarp was hardly affected, most of the

endosperm- and embryo-associated genes were considerably up regulated in response to treatment DH

but not during treatment D (Fig. 5).



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Specific changes in the gene expression pattern of wheat seeds subjected to the combined effect

of drought and heat during the first five days after pollination

Detailed investigations revealed that similarly annotated genes often responded on the same way to the

combined effect of drought and heat (Fig. 6, see also Appendix S3 in Supporting information for more

details).

One of the most obvious groups of coordinately expressed genes was of those coding for storage

proteins. Most of these genes represented by 23 probes on the array were strongly up-regulated in

response to the DH, but not in the D, treatment in both investigated cultivars (Fig. 6 A). These data are

in a good agreement with the increased level of protein staining in the endosperm of DH-treated seeds

as well as with the preferential up-regulation of endosperm-related transcripts (Figs 1-2, 5).

Genes involved in starch metabolism (11 probes) were more differentially regulated (Fig. 6 B) but the

expression of many of them was also considerably increased (the beta-amylase gene represented by

TA61328_4565 exhibited the highest increase among all genes tested), while some of the genes,

especially those implicated in sugar transport processes, were down-regulated (for detailed data, see

Appendix S3 in Supporting information).

In agreement with the treatment, several heat shock protein genes (9 probes) were also up-regulated in

the treated seeds (in response to D and DH as well), but few of them proved to be unresponsive

indicating that they may have different expression kinetics or altered functions (Fig. 6 C).

It could also be recognized that most of the histone genes (21 probes) were down-regulated in the

treated seeds as compared to controls (Fig. 6 D).

There was an interesting pattern observed in the case of genes coding for proteases and aquaporins

(Fig. 6 E-G). While subtilisin-like serine proteinase genes (6 probes, Fig. 6 E) were characteristically

down-regulated, two genes coding for aspartic proteases (Fig. 6 F) were strongly up-regulated in the

DH-treated seeds, while other types were unaffected (data not shown). Aquaporin genes also exhibited

a differential expression response depending on their cellular localization. Tonoplast-intrinsic proteins

(3 probes) were up-regulated in both cultivars (Fig. 6 G) while there were no significant changes in the

expression of plasma membrane integral aquaporins genes (4 probes, data not shown).

The expression profile of transcription factors represented on the microarray were also investigated

(Fig. 6 H). Both up- and down-regulated transcription factors could be identified in drought plus heat-

treated samples while water limitation alone did not significantly changed the expression of any of

these factors during the investigated period.

Validation of the microarray data by RT-QPCR



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Fourteen selected genes with various expression characteristics (unchanged, up- or down-regulated)

were chosen for an independent analysis of their relative transcript abundance in the various samples.

As shown on Fig. 7, the RT-QPCR experiments has validated the gene expression changes revealed by

the microarray data (R2 = 0.675 for 56 data points). Differences could only be observed in the

magnitude of the relative gene expression, especially in the case of those genes where the control

expression values were close to the background level in the microarray experiment. It is supposed that

the largely different range of sensitivity of the two methods, microarray hybridization and RT-QPCR,

explain these differences. The different normalization method and the specificity of the used probes

(hybridizing and PCR priming oligonucleotides), might also affected the sensitivity of detection

differentially, especially taking into account the complex allohexaploid genome of wheat and the

potential sequence differences among the alleles. Microarray and RT-QPCR techniques have been

shown to have different discriminative power as the homeologous and paralogous sequences are

considered (Poole et al. 2007).

Discussion

Using a custom wheat microarray to investigate seed-specific gene expression changes

Wheat has a huge and complex allohexaploid genome (~16000 Mb) with a high proportion (~80%) of

repetitive sequences that until now prevented the determination of the whole genome sequence of this

agronomically important species. This obviously causes difficulties in wheat functional genomics

despite recent progress in the enrichment of molecular genetic and physical maps of wheat

chromosomes as well as in the sequencing of isolated chromosomes and various cDNA libraries

(Gupta et al. 2008). Based on this latter, the number of wheat expressed sequence tags (ESTs) and

EST-based transcript assemblies (TAs) is continuously increasing in the databases (e.g. Houde et al.

2006, Wilson et al. 2004). In the absence of sufficient genome sequence data, these EST and TA

sequences representing the wheat transcriptome have been used as a basis to develop wheat

oligonucleotide microarrays for large-scale gene expression profiling studies (e.g. Clarke and Rahman

2005, Crismani et al. 2006, Golkari et al. 2007, Kawaura et al. 2006, Laudencia-Chingcuanco et al.

2006, Laudencia-Chingcuanco et al. 2007a, Wan et al. 2008, Wan et al. 2009, Winfield et al. 2009).

Although these sequences at present may represent only the half of the wheat genome (Wan et al.

2008), they have the advantage that they already carry information on their expression site/level,

namely their occurrence and frequency in various cDNA libraries (Houde et al. 2006, Wilson et al.

2004). Therefore, in addition to plan microarrays covering the most possible transcripts, specialized

arrays representing only subsets of genes active in specific tissues/treatments can be produced based

on the in silico analysis of EST data. Here we reported the planning and use of a custom wheat



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microarray that allowed us to investigate the expression of almost 15.000 transcripts selected on their

expression in the wheat grain from fertilization till maturation. As 85% of the oligonucleotides gave

signals well above the background if probed with labeled young wheat grain-derived cDNAs, the in

silico selection strategy can be considered as successful. In comparison, among the 61.000 probesets

of Affymetrix wheat microarray 33.000 showed significant binding to transcripts from wheat grains at

different developmental stages (Wan et al. 2008). Of these 14.500 proved to be regulated on a

developmental manner (Wan et al. 2008).

The combination of the custom microarray planning approach with the eArray system of Agilent

(https://earray.chem.agilent.com/earray/) provided for us an economical alternative of using

commercially available “whole genome” wheat arrays. In addition, the system is very flexible and can

be improved from experiment to experiment via the replacement of any of the oligonucleotides based

on the newly available sequence or gene expression data.

Comparison of drought sensitive and tolerant wheat cultivars

Two winter wheat cultivars previously characterized to differ in their drought tolerance (Guóth et al.

2008) have been chosen for the experiments. It was found that these two genotypes (Cappelle Desprez

as drought sensitive and Plainsman V as drought tolerant) hardly differed in their gene expression

responses. The absence of the differential response of these two genotypes is probably due to the fact

that water withdrawal for the relatively short (5 day) period hardly had immediate effects on early seed

development, including gene expression. Therefore the differences of the cultivars resulting in long-

term drought adaptation at the whole plant level (Guóth et al. 2008) could not be manifested at the

level of the developing grain.

Stress sensitivity of early grain development in wheat

Wheat yield is primarily dependent on grain number and weight. The dry matter content of a grain is

directly determined by the number of endosperm cells and the accumulated nutrients per cell

(Chojecki et al. 1986). Considering these two components, the increase in endosperm cell number (by

cell divisions) dominates the first half of grain development (app. 0-14 DAP) while the so called grain

filling period takes place subsequently (app. 14-28 DAP). The realization of the final yield of cereal

crops is equally dependent on the success of both phases. Although endosperm cell division and

reserve accumulation both can be seriously affected by adverse conditions, grain filling has been

extensively studied but less attention has been paid to the period directly following anthesis (for

review, Barnabas et al. 2008). Here we attempted to investigate, using histological and molecular



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approaches, the earliest period of seed development (1-5 DAP) in two different cultivars (drought

sensitive and tolerant) under various conditions (water shortage with or without parallel heat).

Our data indicate that water withdrawal (resulting in 54% decrease of volumetric water content of the

soil) during the first five days of their development had only a moderate immediate effect on the wheat

grains. It is likely that the short period of water limitation exerted its primary effects on vegetative

tissues. However, the induction of stress responses in the seed by this treatment is indicated by the

elevated expression of heat shock proteins known to be responsive not only to heat but drought stress

as well (e.g. Kiyosue et al. 1994, Rizhsky et al. 2004). The data indicate that this level of drought

stress could be mostly alleviated by the plant resulting in the undisturbed development of the seed, at

least in a short term. However, it was also observed that the same conditions could significantly affect

the yield parameters indicating a long term effect on grain development (K. Jäger, A. Fábián and B.

Barnabás, in preparation).

In contrast, drastic changes could be observed in the seeds developed at a high temperature in addition

to the water limitation. Considering the histological studies, the remarkably increased size of the

embryo and the faster accumulation of proteins and starch could be clearly observed in response to the

DH treatment. Plant responses evoked by drought and heat stresses may interfere at several levels

resulting in a unique way to affect plant development. The combination of these stresses resulted in

gene expression changes that were qualitatively and quantitatively different from the sum of the

changes caused by the two individual treatments alone in vegetative tissues of Arabidopsis and

tobacco (Rizhsky et al. 2002, Rizhsky et al. 2004). In agreement, the combination of heat and drought

reduce the duration of wheat grain fill more than either treatment alone (Altenbach et al. 2003, Dupont

and Altenbach 2003).

It has also to be emphasized in this respect that heat not only served as a direct stress factor but

through enhanced evaporation it reduced the volumetric water content of the soil by further 22% under

the DH conditions in addition to the 54% decrease caused by water withdrawal alone. Thereforee, in

the presented experiments the direct or indirect effects of heat could not be separated . However, heat

has been shown by others to have immediate effect on the wheat seed accelerating its development

considerably (Altenbach et al. 2002, Altenbach et al. 2003, Dupont and Altenbach 2003, Nicolas et al.

1984).

These observations and the fact that drought and heat frequently coincide under field conditions

emphasize the importance of the further investigation of plant responses to this combined treatment in

controlled environments as well as in the field.

Gene expression changes in the developing (5 DAP) wheat grain in response to drought and heat



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One of the most remarkable changes observed in the relative gene expression pattern in response to the

DH treatment was the significantly increased transcript level of storage protein genes (Fig. 6). In

agreement with the accelerated accumulation of the storage proteins, the expression of genes coding

for tonoplast-intrinsic proteins (TIPs) that are channel proteins associated with storage protein

vacuoles in the grain (Forrest and Bhave 2008, Maurel 2007) have also been up-regulated in drought

and heat stressed seeds.

Temperature has already been shown to influence the temporal expression of gluten protein genes

during grain filling (Altenbach et al. 2002). The transcripts of these genes could be detected earlier

under high temperature regimes but the period of their expression was altogether shorter in accordance

with the accelerated development of the grains under these conditions (Altenbach et al. 2003, Dupont

and Altenbach 2003). Our microarray data also support the earlier co-ordinated accumulation of

storage protein genes in response to the combined drought and heat treatment. It has also been

previously observed that heat stress did not uncouple the coordinated expression of members of gluten

gene families, despite the fact that several but not all gliadin genes have heat shock consensus

elements in their promoter regions (Blumenthal et al. 1990).

Heat shock proteins themselves are known to be present in the cereal endosperm and to accumulate in

response to heat shock (Hurkman et al. 1998, Skylas et al. 2002). Hurkman and co-workers (Hurkman

et al. 1998) found, however, that the response of the Hsp70 gene is different if the seeds are subjected

to a short or a prolonged high temperature treatment. In the latter case, the gene expression was

dependent on the developmental state of the grain. The differential increase in the expression of the

various heat shock proteins in our experiment may also be related to the interaction of environmental

and developmental cues.

Another characteristic gene expression change observed in our studies was related to histone genes.

Most of them were down regulated in the DH samples. Drought and heat is known to reduce storage

capacity of the cereal grains by decreasing the number of endosperm cells (Nicolas et al. 1984,

Nicolas et al. 1985; for review, Saini and Westgate 1999). Drought was considered to reduce primarily

the cell division rate while heat resulted in the reduction of the cell division period and their combined

effect decreased the endosperm cell number the most seriously in wheat (by 52-60%; Nicolas et al.

1984). We could not observe the negative effect of drought treatment either on the reduction in

embryo and endosperm size and endosperm cell number, or in cell cycle/histone gene expression.

However, we started the treatment right after pollination in contrast to Nicolas and co-workers

(Nicolas et al. 1984) who started this treatment 7 days earlier, at a more sensitive developmental stage.

The co-ordinated decrease of cell cycle/histone gene expression in response to the combined drought

plus heat treatment is in agreement with their observations. Moreover, we could also observe a



14

significant acceleration of embryo growth in the DH seeds. It is supposed that the extensive cell

division period of the embryo has been accomplished in these seeds by 5 DAP that also contributed to

decreased cell cycle gene activity in comparison to controls.

In addition to endosperm cell number, the number and size of the forming starch granules are the main

determinant of grain fill and final yield. The starch accumulation period was shown to be shifted

earlier and was shortened in time in wheat grains developed under high temperature conditions (for

review, Dupont and Altenbach 2003). In addition, the ratio of the larger A-type versus smaller B-type

starch granules was increased (Hurkman et al. 2003). These changes were found to be associated with

the decreased transcription and activity of key enzymes of starch metabolism and a consequently

reduced rate of the conversion of sucrose to starch in the grains (Hurkman et al. 2003, Keeling et al.

1993; for review, Dupont and Altenbach 2003). Limitation of sucrose availability for starch synthesis

was not observed (Bhullar and Jenner 1986, Nicolas et al. 1984). In contrast, drought has been shown

to remobilize stem carbohydrate reserves to the grains (for review, Yang and Zhang 2006). Water

limitation also results in a compressed grain filling period, but the rate of sucrose-to-starch conversion

and the activity of its key enzymes were shown to be affected by drought only under specific

conditions (Yang and Zhang 2006; for review, Yang et al. 2004a). Heat and drought together

drastically shortens the grain filling, and therefore the starch accumulation, period (for review, Dupont

and Altenbach 2003).

The genes coding for proteins involved in sugar/starch metabolism exhibited a differential response

towards the stress (DH) treatment in the experiments reported in the present paper. The up-regulated

expression of some of these genes was likely be associated with the earlier start of the grain filling

period under the DH conditions, similarly as observed for storage protein genes (see above). One of

the -amylase genes (TA61328_4565) was among the genes that showed the highest increase in

transcript levels of all genes investigated under these conditions. This may also indicate a stress

response in addition to developmental regulation. β-amylase induction and the resultant maltose

accumulation have been hypothesized to function as a compatible-solute stabilizing factor in response

to acute temperature stress (Kaplan and Guy 2004, Kaplan et al. 2006). Fructan, as a source of

osmotically active sugars, is also implicated in various abiotic stress responses (Livingston et al. 2009)

that may similarly explain the observed up-regulated expression of fructan exohydrolase genes in the

wheat grain under DH stress. Fructan exohydrolases otherwise have been shown to be predominantly

expressed in the straw (Van Riet et al. 2006), and to play important roles in carbohydrate reserve

mobilization in response to drought stress in wheat (Wardlaw and Willenbrink 2000, Willenbrink et al.

1998, Yang et al. 2004b).



15

The relative expression of starch branching enzymes was also increased in response to the stress

treatments that maybe in relation with the accumulation of A-type starch granules the ratio of which

increases in response to heat (Hurkman et al. 2003). Starch branching enzymes have previously been

shown to be preferentially associated with A-type starch granules (Peng et al. 2000).

The differential response of proteinase-coding genes to DH stress in the wheat grain might also be

associated with the accelerated development of the grain. Early after anthesis the wheat grain exhibits

almost exclusively serine protease activity that declines during further development with a parallel

augmentation of the activity of aspartic proteases (Dominguez and Cejudo 1996). Therefore it can be

well hypothesized that the compressed developmental time line of DH-treated grains (Dupont and

Altenbach 2003) results in earlier decline of serine and earlier increase in aspartic protease

transcription as compared to control seeds. Proteolytic enzymes are involved in various developmental

processes in plants (Schaller 2004). Aspartic proteases have a role in the seeds in storage protein

processing (Otegui et al. 2006); therefore their highly up-regulated expression in DH-treated seeds

might be directly related to the earlier and more intensive synthesis of storage proteins (see above).

The physiological role of serine proteinases during the early phase of seed development is not known

(Antao and Malcata 2005)

The presented results indicate that the expression pattern of hundreds of genes was altered in the grain

of both investigated wheat cultivars in response to the combined effect of drought and heat stress. This

co-ordinated rearrangement of gene activities was likely governed by transcription factors. 22

transcripts annotated to code for transcription factors were significantly (>=2-fold) up- or down-

regulated in DH-treated versus control grains. The highest up-regulation was observed for an AP2-

domain transcription factor the members of which family are known to respond to various abiotic

stresses including drought (Kizis et al. 2001, Song et al. 2005). Zinc finger transcription factors are

also known for their role in stress responses in plants (Sakamoto et al. 2004). MADS-box transcription

factors are expressed at high level during the early grain development phase of wheat (Laudencia-

Chingcuanco et al. 2007b), and probably that is why they show a declined expression level in the DH

samples with accelerated development.

As the tissue-specific localization of the responding genes is concerned, it could be established that

those genes expressed predominantly in the embryo or the endosperm were highly responsive and in

average their expression was increased at the fifth DAP. It is in agreement with the histological studies

showing the increased size and higher protein and starch content of embryos and endosperms in grains

developed under the DH conditions (Figs 1-3). In contrast, the genes expressed in the pericarp were

largely unresponsive to the DH treatment (Table 3).



16

It has to be mentioned, however, that the number of wheat genes that can be clearly annotated to

specific grain tissues is low (179 of them is represented on our array). Nevertheless, the same

phenomenon was observed, at later development stages, by Wan et al. (Wan et al. 2008) using a

microarray covering 55,000 wheat transcripts among which 896 could be associated with the same

tissues (pericarp, embryo, endosperm). This phenomenon is likely associated with the fact that drought

and heat accelerates grain development and the embryo and endosperm are the main places of these

developmental events.

Conclusions

In silico gene expression analysis-based custom microarray production can serve as an

affordable means to reveal organ/tissue specific gene expression changes in wheat. This approach was

successfully used to monitor gene expression in wheat grains at 5 DAP in response to environmental

conditions. Water withdrawal during the first 5 days of kernel development had only a limited direct

effect on the development of the embryo and the endosperm in agreement with the limited changes

observed in gene expression. However, high temperature applied in parallel, also increasing the water

shortage due to enhanced evaporation, could substantially accelerate seed development that was well

manifested both at the histological and gene expression levels. Considering the present and forecasted

climate changes, more emphasis should be given to the effect of high temperature with parallel water

limitation on cereal development in addition to the intensive research efforts on drought tolerance.

Moreover, the sensitivity of early grain development towards immediate stress factors also needs more

attention.

Acknowledgements

The presented work was supported by the grants GVOP-3.1.1-2004-05-0522/3.0; ‘Búzakalász’ NKFP

4-064/04; OTKA K-68064; EU-FP7-REGPOT 2007-1 AGRISAFE 203288 and by the DEAK Ltd.

(GOP-1.1.2-07/1-2008-00079).



17

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Supporting information

Additional Supporting information may be found in the online version of the article:

Appendix S1 Sequences of used QRT-PCR primers

Appendix S2 List of transcripts with significantly (change >=2) altered gene expression

Appendix S3 Co-ordinated expression of genes falling into certain functional categories (as heatmaps)



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Figure legends

Figure 1. Light micrographs of five-days-old control (A-C), drought-stressed (D-F) and drought-and-

heat-stressed (G-I) Cappelle Desprez kernels (A,D,G), endosperm cells (B,E,H) and embryos (C,F,I).

Sagittal sections were taken at the largest cross-sectional embryo area. Red staining: carbohydrates;

blue staining: proteins; e: embryo; en: endosperm; mp: maternal pericarp; s: starch granules. Bars

represent 1 mm (A,D,G); 30 μm (B,C,E); 120 μm (F,H,I).

Figure 2. Light micrographs of five-days-old control (A-C), drought-stressed (D-F) and drought-and-

heat-stressed (G-I) Plainsman V kernels (A,D,G), endosperm cells (B,E,H) and embryos (C,F,I).

Sagittal sections were taken at the largest cross-sectional embryo area. Red staining: carbohydrates;

blue staining: proteins; e: embryo; en: endosperm; mp: maternal pericarp; s: starch granules. Bars

represent 1 mm (A,D,G); 30 μm (B,C,E); 120 μm (F,H,I).

Figure 3. Embryo size in control and treated Cappelle Desprez (A) and Plainsman V (B) kernels

between the 1st and 5th days of development. D: water withdrawal; DH: combined water shortage and

heat (34/24 °C) treatment.

Figure 4. Hierarchical clustering of the overall gene expression pattern of the grains of the two

cultivars (CA- Cappelle Desprez; PA – Plainsman V) developed under drought (D) or drought and

heat (DH) conditions. Relative gene expression changes are shown in comparison to grains developed

under control conditions. Blue color means decreased, red color increased relative expression. The

intensity of the colors is proportional to the gene expression change.

Figure 5. Averaged relative expression of genes annotated to be predominantly expressed in various

regions (endosperm, pericarp, embryo,) of the grain in stressed (D drought; DH drought and heat)

grains of two wheat cultivars (CA- Cappelle Desprez; PA – Plainsman V). The mean and the range of

relative expression values (from lowest to highest) are shown as log2 of fold-change in the

hybridization intensity of probesets hybridized to cDNAs of stressed grains as compared to those of

untreated controls. Number of annotated genes involved in the analysis is shown in parentheses.

Figure 6. Averaged relative expression of genes falling into selected functional classes (A, storage

proteins; B, starch metabolism; C, heat shock proteins; D, histones; E, serine proteases; F, aspartic

proteases; G, tonoplast intrinsic proteins; F, transcription factors), in the two wheat cultivars (Cap -



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Cappelle Desprez; Pla – Plainsman V), under the two stress conditions (D drought; DH drought and

heat). The mean and the range of relative expression values (from lowest to highest) are shown as log2

of fold-change in the hybridization intensity of probesets hybridized to cDNAs of stressed grains as

compared to those of untreated controls. Number of annotated genes involved in the analysis is shown

in parentheses.

Figure 7. Validation of the microarray data by RT-QPCR. The relative expression of fourteen genes in

four cDNA samples was compared. Relative expression value (treated/untreated fold-change as log2)

of a given transcript (identified here with a Probe ID) in a given cDNA sample is shown as a data

point on the graph. MA – data from microarray, PCR – data from RT-QPCR. The points are linked

for better visibility. R2 as a correlation coefficient has been calculated by regression analysis.



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Fig7

Histological and microarray analysis of the direct effect of water shortage alone or combined with...

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