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Plant and Cell Physiology Advance Access originally published online on August 27, 2009
Plant and Cell Physiology 2009 50(10):1786-1800; doi:10.1093/pcp/pcp121
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Temporal Expression Patterns of Hormone Metabolism Genes during Imbibition of Arabidopsis thaliana Seeds: A Comparative Study on Dormant and Non-Dormant Accessions

Jeremy Preston1,5, Kiyoshi Tatematsu1,5,6, Yuri Kanno1, Tokunori Hobo2, Mitsuhiro Kimura3, Yusuke Jikumaru1, Ryoichi Yano1, Yuji Kamiya1 and Eiji Nambara1,3,4,*

1Growth Regulation Research Group, RIKEN Plant Science Center, 1-7-22, Suehiro-cho, Tsurumi, Yokohama, 230-0045 Japan
2Bioscience and Biotechnology Center, Nagoya University, Furocho, Chikusa, Nagoya, 464-8601 Japan
3Department of Cell & Systems Biology, University of Toronto, 25 Willcocks Street, Toronto, Ontario M5S 3B2, Canada
4The Centre for the Analysis of Genome Evolution and Function (CAGEF), University of Toronto, Toronto, Ontario M5S 3B2, Canada

*Corresponding author: E-mail, eiji.nambara{at}utoronto.ca; Fax, +1-416-978-5878.


   Abstract
Top
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Supplementary data
 Funding
 Acknowledgments
 References
 
Seed imbibition is a prerequisite for subsequent dormancy and germination control. Here, we investigated imbibition responses of Arabidopsis seeds by transcriptomic and hormone profile analyses using dormant [Cape Verde Islands (Cvi)] and non-dormant [Columbia (Col)] accessions. Once imbibed, seeds of both accessions swelled most up to 3 h, reflecting water uptake. Microarray analysis showed that in both accessions, seeds imbibed for 15 min, 30 min and 1 h were less active in gene expression than at 3 h. More than 2,000 genes were either up-regulated or down-regulated in seeds imbibed for 3 h. Some genes up-regulated at 3 h were already induced in seeds imbibed for 1 h, suggestive of genome reprogramming early after the onset of imbibition. Imbibition-induced genes in seeds imbibed for 3 h included those up-regulated in both Col and Cvi (common) or unique to either accession (accession specific). Up-regulated genes that were both common and Cvi-specific were over-represented for sugar metabolism and the pentose phosphate pathway, whereas Col-specific genes were over-represented for ribosomal protein genes. Quantification of plant hormones showed that ABA and salicylic acid (SA) contents were higher, but gibberellin A4 (GA4), N6-({Delta}2-isopentenyl)adenine (iP), jasmonic acid (JA), JA–isoleucine (JA-Ile) and IAA were lower in imbibed seeds of Cvi compared with Col. In addition, changes in IAA and JA were initiated before 1 h, whereas ABA and JA-Ile declined 3 h after the onset of imbibition. An increase in GA4 and iP appeared to be correlated temporally with the initiation of secondary water uptake, which marks the completion of germination.

Keywords: Arabidopsis - Gene expression - Hormone metabolism - Microarray - Seed imbibition

Abbreviations: Col, Columbia; Cvi, Cape Verde Islands; GA, ; 4, gibberellin A4; GO, gene ontology; IAM, indole-3-acetamide; IAOx, indole-3-acetaldoxime; iP, N6-({Delta}2-isopentenyl)adenine; IPA, indole-3-pyruvic acid; JA, jasmonic acid; LC-ESI-MS/MS, liquid chromatography–electron spray ionization–tandem mass spectrometry; NCED, 9-cis-epoxycarotenoid dioxyge-nase; RT–PCR, reverse transcription–PCR; SA, salicylic acid; SID2, SALICYLIC ACID INDUCTION-DEFICIENT 2; TAM, tryptamine; UGT, glucosyltransferase.


   Introduction
Top
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Supplementary data
 Funding
 Acknowledgments
 References
 
Dry seeds contain a limited amount of water. However, many can survive for periods of dry storage and then germinate following subsequent imbibition. There are three phases of water uptake: passive water uptake by the dry seeds (phase I), a phase where there is little water uptake (phase II) and water uptake related to the completion of germination and seedling growth (phase III) (Bewley 1997Go, Finch-Savage et al. 2006Go). The transition from phase II to III marks the completion of germination; it does not occur in dormant or dead seeds, for example. Current genomics approaches using Arabidopsis seeds have been largely focused on the transition from phase II to III (Ogawa et al. 2003Go, Nakabayashi et al. 2005Go, Cadman et al. 2006Go, Carrera et al. 2007Go, Finch-Savage et al. 2007Go, Carrera et al. 2008Go, Tatematsu et al. 2008Go; reviewed in Holdsworth et al. 2007Go). On the other hand, little work has been reported on the molecular changes occurring during phase I (Bewley 1997Go). An early event after the start of imbibition is resumption of energy metabolism, such as respiration (Morohashi and Shimokoriyama 1972Go, Hourmant and Pradet 1981Go, Botha et al. 1992Go), and the glycolytic and oxidative pentose phosphate pathways resume activity during phase I, as do the Kreb’s cycle enzymes. Since the 1960s, several groups have shown early resumption of protein and mRNA synthesis (Bewley and Black 1994Go). Rajjou et al. (2004Go, 2008Go) reported that, in the presence of {alpha}-amanitin, there is a resumption of protein synthesis using stored mRNAs as templates and these play a role during and/or after seed imbibition.

A collection of temporal gene expression patterns is useful to define a physiological phase. Hughes and Galau (1989Go, 1991Go) determined the temporal expression patterns of many genes in cotton seeds and defined phases during seed development. Similar expression analyses using many marker genes revealed that the transition from seed development to germination occurs gradually and such genes for late embryogenesis and for germination co-exist during the transition (Harada et al. 1988Go, Hoecker et al. 1995Go, Fernandez, 1997Go, Nambara et al. 2000Go). Recent advances in gene expression analysis using microarrays allow for large-scale analysis from a single piece of material, and time-course analysis using microarrays provides a series of marker genes during germination (Nakabayashi et al. 2005Go). Moreover, transcriptome analysis is powerful when it is combined with metabolite analysis (Hirai et al. 2004Go, Keurentjes et al. 2006Go, Hirano et al. 2008Go), and these techniques have been utilized for investigating seed physiology (Ruuska et al. 2002Go, Fait et al. 2006Go, Penfield et al. 2006Go).

Plant hormones play key roles in the regulation of various plant processes. ABA and gibberellins are implicated in the control of seed dormancy and germination (Nambara and Marion-Poll 2005Go, Yamaguchi 2008Go). These are also involved in the seed responses to environmental conditions, such as light (Oh et al. 2009Go), temperature (Yamauchi et al. 2004Go, Toh et al. 2009) and nutrients (Matakiadis et al. 2009Go). ABA content is high in dry seeds and declines during imbibition (phase I), whereas gibberellin content increased in germinating seeds during phase II. It is notable that, although little is known about the physiological responses during phase I, the reduction of ABA is a rapid response during phase I in both Columbia (Col) and Cape Verde Islands (Cvi) seeds (Ali-Rachedi et al. 2004Go, Kushiro et al. 2004Go, Okamoto et al. 2006Go, Millar et al. 2006Go). ABA 8'-hydroxylase (CYP707A) plays an essential role in this decline (Okamoto et al. 2006Go). ABA contents decline in both dormant seeds, such as C24 and Cvi (Ali-Rachedi et al. 2004Go, Millar et al. 2006Go), and non-dormant Col seeds imbibed in non-permissive conditions (Seo et al. 2006Go, Toh et al. 2008Go) during early imbibition, but increase thereafter. In addition, many plant hormones also influence seed dormancy and germination (Kucera et al. 2006). Metabolism of different hormones is mutually dependent, and a change in one eventually alters the contents of others. Because of the diverse chemical nature of plant hormones, it had been difficult to quantify them simultaneously in a single plant material, but recent advances in hormone analysis techniques have overcome this barrier (Chiwocha et al. 2005Go, Kojima et al. 2009Go). Because of the lack of physiological knowledge on the imbibition responses during phase I, a hormone metabolism study will provide the useful fingerprints of this phase.

We previously published a kinetic study of transcriptomes on Col dry seeds after 6, 12 and 24 h of imbibition, which showed that they changed with time. Notably, the transcriptomes of imbibed seeds were fully reprogrammed from dry seeds within 6 h after the onset of imbibition (Nakabayashi et al. 2005Go). The aim of this study is to collect and characterize the marker genes for the seed imbibition response and to define the timing of resumption of transcription after the start of imbibition. We performed gene expression analysis during early stages after seed imbibition to determine when obvious changes occurred. We used non-dormant Col and dormant Cvi accessions of Arabidopsis (Koornneef et al. 2004Go) to compare if gene expression patterns resemble each other or not. This analysis allows for the identification of genes induced in both accessions as well as those induced in an accession-specific manner. We also performed hormone profiling on imbibed seeds at different imbibition times to follow how hormone metabolism genes are regulated during and after imbibition.


   Results and Discussion
Top
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Supplementary data
 Funding
 Acknowledgments
 References
 
Seed imbibition of Col and Cvi accessions
Under our conditions, non-dormant Col seeds completed germination after 30–36 h of imbibition on moistened filter paper, whereas dormant Cvi seeds did not germinate even in favorable conditions unless treated with cold moistening, nitrate application or after-ripening.

Phase I is characterized by passive water uptake and can be monitored by seed swelling after the onset of imbibition. Dry seeds of Cvi are larger than those of Col (Col, length 0.381 ± 0.025 mm, width 0.194 ± 0.015 mm; Cvi, length 0.423 ± 0.026 mm, width 0.262 ± 0.020 mm). Once imbibed, Col seeds swelled more abruptly than Cvi seeds (Fig. 1A, B), but seeds of both accessions ceased swelling mostly by 3 h (Fig. 1). A second swelling, an indication of completion of germination, was observed only in Col seeds 30 h after the start of imbibition.


Figure 1
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  		Fig. 1 Measurement of seed size during and after the start of imbibition. Col (A) and Cvi (B) wild-type seeds were imbibed on moistened filter paper. Pictures of the seeds were taken at the indicated time points. Relative width is the ratio of imbibed seed width relative to dry seed width. The average relative width ± SD (n = 20) is shown. The same data for water uptake during the first 3 h are magnified in the insets.

 
Microarray analysis of imbibed seeds
Triplicate microarray experiments were performed on dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h of both accessions (Fig. 2). As shown in Fig. 2, both Col and Cvi seeds showed a similar change in transcriptomes.


Figure 2
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  		Fig. 2 Changes in transcript abundance after the start of imbibition of Col (A) and Cvi (B) seeds. From top to bottom: seeds imbibed for 15 min, seeds imbibed for 30 min, seeds imbibed for 1 h and seeds imbibed for 3 h vs. dry seed. The x-axis indicates log (fold change) and the y-axis indicates log (P-value). Bold lines indicate the cut-off values; P-value = 0.02, and fold change = 1.8.

 
Initiation of change in transcriptomes after the start of imbibition
Down-regulated genes. During the first hour after the start of imbibition, most mRNAs remained at the same levels, but those of one set of genes declined in seeds imbibed for 15 min (Table 1). Eighty-two Col genes and 32 Cvi genes were down-regulated >1.8-fold (P < 0.02) in seeds imbibed for 15 min relative to the dry state, and 23 genes were shared. This indicates that the down-regulation of these genes is common for Col and Cvi. In addition, down-regulation was not transient, so mRNA abundance of these genes was also low in seeds imbibed for 30 min and 1 h (Fig. 2). In seeds imbibed for 3 h, many genes were down-regulated (Fig. 2). Commonality of early down-regulation in Col and Cvi seeds indicates that this is a selective process and not a random mRNA degradation.


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  		Table 1 Number of imbibition-regulated genes

 
Publicly available expression data show that the 23 genes down-regulated in seeds imbibed for 15 min are highly co-regulated developmentally. Co-expression of these genes is visualized by a tool available at the BAR website (http://bbc.botany.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi; Toufighi et al. 2005Go). These genes are highly expressed similarly in mature pollen and stamens (Supplementary Fig. S1). Those highly over-represented included genes for polysaccharide metabolism (P = 1.18e-6) and cell wall modification (P = 3.68e-4).

Up-regulated genes. Seeds imbibed for 15 min, 30 min and 1 h showed similar transcript profiles to the dry seeds (Fig. 2). In both Col and Cvi, only limited numbers of genes showed an increase in mRNA abundance of a cut-off value of 1.8-fold (P < 0.02) (Table 1). On the other hand, many genes showed remarkable changes in mRNA abundance in seeds imbibed for 3 h (Fig. 2). There were 1,290 up-regulated genes in Col and 801 up-regulated genes in Cvi whose transcripts increased >1.8-fold (P < 0.02).

The up-regulation in seeds imbibed for 15 and 30 min was small and transient, and was not maintained at the subsequent time points, and these mRNAs were in low abundance (Supplementary Table S1). In contrast, although up-regulated genes in seeds imbibed for 1 h were also few, these were different from those in seeds imbibed for 15 and 30 min. We found four Col genes and five Cvi genes whose mRNA abundance was significantly increased in seeds imbibed for 1 h relative to those of dry seeds (Table 1, Fig. 3). Changes in mRNA abundance of some genes up-regulated at 1 h were notable and not transient, and these genes were further up-regulated in seeds imbibed for 3 h (Fig. 3). The mRNA abundance of AT4G35985 and AT5G59820 in Col increased 7.8- and 3.4-fold, respectively, in seeds imbibed for 1 h, and 104- and 30-fold, respectively, in seeds imbibed for 3 h (Fig. 3A). The mRNA abundance of AT3G19680 in Cvi increased 2.6-fold in seeds imbibed for 1 h and 85-fold in seeds imbibed for 3 h (Fig. 3B). Also, some other genes up-regulated at 3 h were induced in seeds imbibed for 1 h when different seed batches were used (data not shown). These data indicate that the remarkable changes in expression of a large number of genes in seeds imbibed for 3 h were initiated at 1 h after the onset of imbibition. It is also notable that many of the genes up-regulated at 3 h showed high mRNA abundance in seeds imbibed for 6 h, but not in seeds imbibed for 12 h, when compared with previously obtained microarray data (Nakabayashi et al. 2005Go).


Figure 3
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  		Fig. 3 The changes in mRNA abundance of genes up-regulated at 1 h in Col (A) and Cvi (B). The x-axis and y-axis show time after the start of imbibition (hours) and mRNA abundance obtained from triplicate microarray data, respectively. A solid line indicates a gene whose mRNA abundance is up-regulated at both 1 and 3 h, and a dotted line shows a gene whose mRNA abundance is temporarily induced at 1 h, but not at 3 h. A bar indicates the standard deviation. 0 h indicates dry seeds.

 
Genes induced in seeds imbibed for 1 h. Both Col and Cvi seeds showed remarkable changes in the transcriptome at 3 h, but some of this strong induction was already initiated at 1 h. Other genes also showed statistically significant changes in mRNA abundance in seeds imbibed for 15 min, 30 min and 1 h, but these changes were small and transient (i.e. they could not be seen at the following time point). Therefore, it is unclear if these genes contribute to the physiological responses during imbibition. In contrast, remarkable up-regulation of some of genes at 1 h indicates that transcriptional activity was enhanced at this time point.

Both of two early imbibition-responsive genes in Col are related to the stress responses. AT4G35985 is annotated as a senescence/dehydration-associated chloroplast protein that is structurally related to EARLY RESPONSIVE TO DEHYDRATION 7 (ERD7) (Kiyosue et al. 1994Go). ERD7 is also induced under high light stress (Kimura et al. 2003Go). Another early imbibition-responsive gene in Col is AT5G59820, which encodes ZAT12, a stress-inducible zinc finger protein essential for signaling of reactive oxygen species under stress conditions (Kazuoka et al. 2000Go, Davletova et al. 2005Go). On the other hand, one early imbibition-responsive gene in Cvi, AT3G19680, is annotated as a hypothetical protein. Interestingly, public microarray data indicate that expression of these genes responds rapidly to various biotic and abiotic stresses (Toufighi et al. 2005Go).

Gene ontology of genes up-regulated at 3 h. Biased gene ontology (GO) categories of these genes are useful to characterize their expression during phase I. At present, it remains unclear if these biased GO categories reflect the cause or result of physiological similarity and differences between Col and Cvi seeds. Nonetheless, these are potentially useful fingerprints of these seeds. Of genes up-regulated at 3 h in Col and Cvi seeds, 480 genes are shared (Fig. 4A), which is less than the shared genes in genes down-regulated at 3 h (Fig. 4B). These genes appeared to be regulated by seed imbibition independent of dormancy and germination. The GO categories of these genes were analyzed on the MIPS web site (http://mips.gsf.de/proj/funcatDB/search_main_frame.html), and were highly over-represented with respect to metabolism genes (P = 4.52e-7), in particular primary metabolism genes related to carbohydrates and the aspartate family (Fig. 4C). In addition, pentose phosphate pathway genes were enriched (P = 1.16e-4). This pathway is essential for the production of NADPH and nucleotide-sugars. Common genes were also highly over-represented for those related to the mitochondrial matrix, cellular defense and transport of micromolecules (Fig. 4D, Supplementary Table S2). Interestingly, enriched GO categories also included genes for leaf differentiation, suggesting that at least part of the imbibition response is not unique. This is in contrast to the down-regulated genes that are highly seed specific.


Figure 4
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  		Fig. 4 Genes up-regulated in seeds imbibed for 3 h. Venn diagrams of genes up-regulated at 3 h (A) and genes down-regulated at 3 h (B). The cut-off value is 1.8-fold (P < 0.02) relative to the mRNA abundance in the corresponding dry seeds. (C) Classification of the gene functions of genes up-regulated at 3 h. Genes were classified into 13 groups as described on the right. From left to right; whole genome (Genome), genes up-regulated at 3 h common to Col and Cvi (Common), Col-specific genes (Col) and Cvi-specific genes (Cvi). An asterisk indicates the biased GO categories. (D) Genes up-regulated at 3 h belonged to the biased GO category. Red and blue colors indicate higher and lower expression, respectively. The color scale with an average signal intensity is shown at the bottom. From left to right, relative mRNA abundance in dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h taken from microarray data.

 
Among the 801 up-regulated Cvi genes, induction of 321 was Cvi-specific. The biased GO categories of the Cvi-specific up-regulated genes resembled those induced in common in Col and Cvi seeds. A GO category (01.05.02 sugar, glucoside, polyol and carboxylate metabolism) was over-represented most significantly in both common (P = 5.14e-07) and Cvi-specific genes (P = 5.08e-07). Both gene sets included a distinct set of genes related to cellulose biosynthesis (Fig. 4C). Also, both common and Cvi-specific genes were enriched for those related to the pentose phosphate pathway and to mitochondrial functions (Fig. 4C). Respiration and energy production might be essential for both dormant and non-dormant seeds. It is also reasonable that dormant Cvi seeds activate additional sets of genes related to these processes due to the long heterotrophic period. Cadman et al. (2006Go) reported a core gene set whose expression is associated with seed dormancy (442 genes) and with germination (779 genes) in Cvi seeds. Notably, the 321 Cvi-specific genes reported here shared only one gene (At1g12290) with the 442 dormancy-associated genes, but there were 40 genes in common with the 779 germination-associated core genes. This indicates that the Cvi-specific up-regulation of these genes is not uniquely associated with maintenance of seed dormancy.

Among the 1,290 up-regulated genes in Col seeds imbibed for 3 h, induction of 810 genes was Col-specific. These genes showed biased GO categories different from those up-regulated in common or that were Cvi-specific (Supplementary Table S2). The GO category over-represented most exclusively was ribosome biogenesis (P = 1.05e-28), mostly ribosomal protein genes, which were previously characterized germination-associated genes in Col and Cvi (Cadman et al. 2006Go, Tatematsu et al. 2008Go). Also, Col-specific genes up-regulated at 3 h included AtTCP14 that encodes a basic helix–loop–helix (bHLH) transcription factor whose expression is associated with ribosomal protein genes in germinating seeds (Tatematsu et al. 2008Go). The present work shows that co-induction of these ribosomal protein genes is initiated early in imbibed Col seeds and their mRNA abundance remains high until 24 h after the onset of imbibition. The early induction of ribosomal protein genes is unique to other germination-associated genes, such as AtGA3ox genes, which are induced latter in germinating seeds (Ogawa et al. 2003Go).

Quantification of plant hormones
Quantification of several hormones was conducted on imbibed seeds. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was used to quantify ABA, gibberellin A4 (GA4), jasmonic acid (JA), JA–isoleucine conjugate (JA-Ile), salicylic acid (SA), IAA and N6-({Delta}2-isopentenyl)adenine (iP). Col and Cvi seeds displayed differences in their contents (Supplementary Fig. S2). Changes in plant hormones were compared with those of mRNAs transcribed from hormone metabolism genes. Two types of comparative analyses of hormones and transcripts of hormone metabolism genes were conducted: (i) kinetics of each accession; and (ii) differences between the two accessions.

JA metabolism. JA is synthesized from {alpha}-linolenic acid released from plastid membranes, and the steps occur in peroxisomes using enzymes for fatty acid β-oxidation (Fig. 5A; Wasternack 2007Go). The amounts of JA and JA-Ile were markedly different between Col and Cvi dry and imbibed seeds (Fig. 5B). Col dry seeds, in contrast to Cvi, were high in JA and JA-Ile. In Col, a decline in JA was observed after 1 h of imbibition, suggesting that the initiation of this reduction does not require de novo transcription. On the other hand, JA-Ile contents remained high up to at least 3 h (Fig. 5B) and gradually decreased thereafter (Supplementary Fig. S2). Transcripts of JA biosynthesis genes, AtLOX6 and ORP3, were higher in Col than in Cvi (Fig. 5C). An Arabidopsis jar1 mutant, which is defective in the conversion of JA to JA conjugates, showed an ABA-hypersensitive phenotype in seeds (Staswijk et al. 1992Go). It is possible that reduction of JA-Ile in Col seeds is an accession-specific mechanism to regulate seed germination.


Figure 5
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  		Fig. 5 JA and JA-Ile contents and changes in the abundance of mRNAs transcribed from their metabolism genes. (A) JA metabolic pathway. 13-HPOT, 13S-hydroperoxy-(9Z,11E,15Z)-octadecatrienoic acid; 12,13-EOT, 9Z,11E,15Z,13S,12R)-12,13-epoxy-9,11,15-octadecatrienoic acid; cis-(+)-ODPA, cis-(+)-12-oxo-phytodienoic acid; OPC-8:0, 3-oxo-2-(2 [Z]-pentenyl)-cyclopentane-1-octanoic acid; OPC-6:0, 3-oxo-2-(2 [Z]-pentenyl)-cyclopropane-1-hexanoic acid; JA, jasmonic acid; JA-ME, JA methyl ester; JA-Ile, JA–isoleucine conjugate. (B) JA and JA-Ile contents of Col (open circle) and Cvi (filled circle) seeds. Triplicate experiments were performed on independent seed batches. An average with SD is shown. (C) Changes in mRNA abundance of JA metabolism genes in Col and Cvi seeds. Red and blue colors indicate higher and lower expression, respectively. The color scale with an average signal intensity is shown at the bottom. From left to right, relative mRNA abundance in dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h taken from microarray data.

 
SA metabolism. SA is synthesized from chorismate via either isochorismate or phenylalanine (Fig. 6A; Shah 2003Go). Rajjou et al. (2006Go) reported that SA enhances germination potential when Arabidopsis seeds are imbibed in salt-rich media. SA present in dry seeds decreased gradually following imbibition (Fig. 6B) and was undetectable at 24 h after imbibition in the seeds of both accessions Cvi and Col. Both accessions showed a similar changes in SA after imbibition, but the former contained more (approximately 2-fold). A decline in SA was evident at 3 h, which is earlier than the reduction in ABA.


Figure 6
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  		Fig. 6 SA content and changes in the abundance of mRNAs transcribed from SA metabolism genes. (A) SA metabolic pathway. (B) SA contents of Col (open circle) and Cvi (filled circle) seeds. Triplicate experiments were performed on independent seed batches. An average with SD is shown. (C) Changes in mRNA abundance of SA metabolism genes in Col and Cvi seeds. Red and blue colors indicate higher and lower expression, respectively. The color scale with an average signal intensity is shown at the bottom. From left to right, relative mRNA abundance in dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h taken from microarray data.

 
SALICYLIC ACID INDUCTION-DEFICIENT 2 (SID2), which encodes a plastid-localized isochorismate synthase (Strawn et al. 2007Go), is one of the down-regulated genes in both Col and Cvi seeds 3 h after imbibition (Fig. 6C). Consistent with a high amount of SA in Cvi, SID2 transcripts were higher than in Col dry and imbibed seeds. A large amount of SA is present in plants as conjugates with glucose, and several glucosyltransferases (UGTs) are proposed to affect this conjugation (Lim et al. 2002Go, Dean and Delaney 2008Go). Of these, UGT74F2 produced more transcripts in Cvi than in Col seeds after 3 h (Fig. 6C) and their abundance in the former appears to be related to the high amount of SA in this accession. The transcript abundances of SID2 and UGT74F2 appear to complement each other in the Cvi seeds, in that the one involved in SA biosynthesis declines as the one involved in SA conjugation increases. This may be the consequence of co-regulation.

Auxin metabolism. Auxins play a pivotal role in plant growth and development (Woodward and Bartel 2005Go), and are implicated in the regulation of seed germination in Arabidopsis (Brady et al. 2003Go, Liu et al. 2007Go). IAA, the major naturally occurring auxin, is synthesized de novo via multiple pathways from either tryptophan (Trp) or indole derivatives (Fig. 7A; Sugawara et al. 2009Go). The Trp-dependent pathways include the tryptamine (TAM) pathway, the indole- 3-pyruvic acid (IPA) pathway, the indole-3-acetamide (IAM) pathway and the indole-3-acetaldoxime (IAOx) pathway. Hormone measurement showed that dry seeds of Col contained 2-fold more IAA than the Cvi dry seeds. Upon imbibition, IAA content in Col seeds was increased rapidly at 1 h, maintained its level at 2 h and declined gradually (Fig. 7B, Supplementary Fig. S2). In contrast, Cvi seeds maintained IAA content until 3 h (Fig. 7B), and declined thereafter (Supplementary Fig. S2).


Figure 7
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  		Fig. 7 IAA content and changes in the abundance of mRNAs transcribed from IAA metabolism genes. (A) IAA metabolic pathway. (B) IAA content of Col (open circle) and Cvi (filled circle) seeds. Triplicate experiments were performed on independent seed batches. An average with SD is shown. (C) Changes in mRNA abundance of IAA metabolism genes in Col and Cvi seeds. Red and blue colors indicate higher and lower expression, respectively. The color scale with an average signal intensity is shown at the bottom. From left to right, relative mRNA abundance in dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h taken from microarray data.

 
A family of flavin monooxygenases encoded by YUCCA genes catalyzes the conversion of TAM to N-hydroxyl-TAM, and plays a regulatory role in IAA biosynthesis (Zhao et al. 2001Go, Cheng et al. 2006Go). Recent reports demonstrate that a member of the TAA1/SAV3/WEI8 family catalyzes a committed step in the IPA pathway and plays an essential role in plant development, shade avoidance and auxin–ethylene cross-talk (Stepanova et al. 2008Go, Tao et al. 2008Go). CYP79B2/B3 catalyzes a committed step in the IAOx pathway (Zhao et al. 2002Go). This pathway appears to be specific to the Brassicaceae, playing a prominent role in IAA biosynthesis in Arabidopsis (Sugawara et al. 2009Go). In addition, IAA is supplied from IAA conjugates, such as amino acids and sugars. In bean, amide-conjugated IAAs accumulate during seed maturation and these storage forms become a major source of IAA during germination (Bialek and Cohen 1992Go, Bialek et al. 1992Go). IAA–amino acid conjugates, such as IAA-Ala, IAA-Leu and IAA-Phe, are postulated to be a source of IAA during germination and seedling growth in Arabidopsis (Rampey et al. 2004Go). The Arabidopsis genome contains seven genes for IAA–amino acid conjugate hydrolases (LeClere et al. 2002Go), and four of them (ILR1, ILL1, ILL2 and IAR3) encode enzymes that cleave IAA–amino acid conjugates (Davies et al. 1999Go, LeClere et al. 2002Go). Of the four functional hydrolases, expression of only ILR1 could be monitored by the ATH1 genome array. ILR1 transcripts were abundantly present in seeds of both Col and Cvi. Consistent with the higher amount of IAA, ILR transcripts were > 2-fold more abundant in Col than in Cvi (Fig. 7C). In contrast, most of the genes that encoded enzymes for de novo IAA biosynthesis did not show a significant difference in transcript abundance except for a member of the TAA1 C-S lyases, At4g24670, CYP79B2 (At4g39950) and nitrilase3 (At3g44320). The difference in abundance of these transcripts correlates with the amount of IAA in Col and Cvi seeds, suggesting that they are involved in IAA biosynthesis. In contrast, the abundance of the transcripts of 11 YUCCA genes was relatively low in all seeds.

ABA metabolism. ABA is synthesized from xanthophylls via oxidative cleavage with some modifications and is inactivated by several catabolic pathways (Fig. 8A). ABA content was maintained for 3 h after the onset of imbibition and decreased by 6 h in both Col and Cvi, although its reduction was less in the latter (Fig. 8B). Initiation of a decrease in ABA was temporally correlated with the induction of CYP707A2, which is one of the up-regulated genes common to Col and Cvi (Fig. 8C), and this is correlated with the decline in ABA in both Col and Cvi. The greater induction of this gene in Col than in Cvi may be related to the more prominent reduction of ABA in the former accession. Moreover, Matakiadis et al. (2009Go) reported that enhanced induction of CYP707A2 by nitrate accelerates the decline in ABA at 6 h after the start of imbibition. Taken together, these findings suggest that the loss of ABA is related to an increase in newly synthesized CYP707A2 protein after imbibition. In addition, UGT75B1 was also up-regulated in Col and Cvi seeds (Fig. 8C). UGT75B1 catalyzes glucose conjugation to ABA in vitro (Lim et al. 2005Go), and its overexpression enhances the production of ABA-glucose ester (ABA-GE), but does not alter the ABA contents of Arabidopsis seedlings (Priest et al. 2006Go). It is of interest to test if the ABA conjugation pathway plays a regulatory role in imbibition response in seeds. The 9-cis-epoxycarotenoid dioxygenase9 (NCED9) gene was up-regulated in a Col-specific manner (Fig. 8C). NCED9 is a primary member of the NCEDs in seeds and is responsible for seed dormancy and thermoinhibition of germination (Lefebvre et al. 2006Go, Toh et al. 2008Go). However, Col-specific induction of this gene in the early stages following imbibition appears to contradict the ABA loss that occurs more rapidly in Col than in Cvi, suggesting that induction of NCED9 is not a regulatory process for ABA content during the early stages of imbibition.


Figure 8
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  		Fig. 8 ABA content of seeds and changes in the abundance of mRNAs transcribed from ABA metabolism genes. (A) ABA metabolic pathway. AB ald, abscisic aldehyde; ABA-GE, ABA glucose ester; PA, phaseic acid. (B) ABA contents of Col (open circle) and Cvi (filled circle) seeds. Triplicate experiments were performed on independent seed batches. An average with SD is shown. (C) Changes in mRNA abundance of ABA metabolism genes in Col and Cvi seeds. Red and blue colors indicate higher and lower expression, respectively. The color scale with an average signal intensity is shown at the bottom. From left to right, relative mRNA abundance in dry seeds and seeds imbibed for 15 min, 30 min, 1 h and 3 h taken from microarray data.

 
In summary, time-course expression analysis identified genes that were up-regulated or down-regulated during and after imbibition. Only a small change in transcriptome was observed within 1 h after the start of imbibition, but there were many changes by 3 h in both Col and Cvi seeds. The mRNA abundance is determined by both synthesis (transcription) and degradation of mRNA. Therefore, the maintenance of mRNA abundance of the majority of genes during the first 1 h of imbibition may be a consequence of the balance between its synthesis and degradation. Alternatively, it may be due to a combination of inactive transcription and protection of stored mRNA from nucleases. We identified 10,821 Col genes and 11,457 Cvi genes whose mRNA abundance in dry seeds was called ‘Presence’ or ‘Marginal’ by MAS software in all triplicate experiments. Most of the genes (>99%) showed no significant differences in abundance of their transcripts even at 1 h after imbibition (cut-off 1.8-fold, P < 0.02). The high similarity of transcriptomes in dry seeds and in seeds imbibed for 15 min, 30 min and 1 h suggests that there is very little expression of most genes at these time points, and those that are still accumulated within 1 h are the same messages as those that are present in dry seeds. More active resumption of transcription and of RNA degradation is initiated after 1 h. Changes in mRNA abundance from a large number of genes in seeds imbibed for 3 h support this contention.

Imbibition-inducible genes in seeds imbibed for 3 h were either similar or accession specific in Col and Cvi seeds. Expression of both common and accession-specific genes was activated simultaneously at this time point. The induction of these genes was possibly independent of whether the seeds will germinate or not, but nevertheless was initiated by imbibition. Also, subsets of genes were induced in an accession-specific manner. The biased GO categories indicate that Cvi-specific expressed genes included those enriched for sugar metabolism and the pentose phosphate pathway, which are similar to those up-regulated in common in Col and Cvi. On the other hand, Col-specific genes were highly over-represented for ribosomal protein genes. These ribosomal protein genes are over-represented in Col seeds imbibed for 24 h, just before the completion of germination (Tatematsu et al. 2008Go). These genes also are co-induced in Cvi seeds imbibed for 24 h under conditions permissive for germination (Cadman et al. 2006Go). It is interesting that these ribosomal protein genes were induced in Col seeds imbibed for 3 h, which is many hours before the completion of germination. Loss of function of AtTCP14, a regulator of ribosomal protein genes during germination, led to delayed germination (Tatematsu et al. 2008Go), suggesting that co- regulation of these ribosomal protein genes is involved in germination.

To date, we had little knowledge on the physiological responses during phase I. The ABA decline is a known event that occurs during this period. The present study demonstrates that, in addition to ABA, the contents of JA, JA-Ile, SA and IAA are changed during phase I. Among the various hormones, changes in IAA were rapid and were evident even in seeds imbibed for 1 h (Fig. 7B) and preceded the resumption of transcription, suggesting their increase is regulated either by enzymes or by translated mRNAs stored in the dry seeds. IAA production during germination may occur via hydrolysis of IAA conjugates, and mRNA abundance of ILR1 seems to be correlated with IAA contents (Fig. 7B, C). The increase in IAA was transient and decreased in seeds imbibed for 3 h, suggesting that the increase may be an early response to imbibition. Other changes in JA, JA-Ile, SA and ABA were their reduction. It is notable that even though these changes occur early during imbibition, the timing of the initiation of these reductions is different. The reduction of JA was observed at 1 h, suggesting that it occurs before the resumption of transcription. On the other hand, the reduction of ABA was not seen at 3 h, and this timing is correlated with the resumption of transcription and with the induction of CYP707A2. These differences might depend on de novo transcription after the start of imbibition.

It is also notable that their contents vary between Col and Cvi. The most remarkable difference is the content of JA and JA-Ile. We could not detect JA and JA-Ile in Cvi dry seeds, although a significant amount of them were accumulated in Col dry seeds. On the other hand, the SA content in Cvi was double than in Col seeds in both dry and imbibed seeds. JA and SA are involved in defense responses, thus it is possible that Col and Cvi seeds activate defense responses using different hormones.


   Materials and Methods
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 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Supplementary data
 Funding
 Acknowledgments
 References
 
Plant materials and growth conditions
Arabidopsis thaliana (L.) Heynh seeds of Col and Cvi accessions were used. Plants were grown as previously described (Kushiro et al. 2004Go), and seeds were harvested and stored at room temperature. Seeds stored for 2 months were used for all experiments. For imbibition, dry seeds were directly sown on filter paper moistened with distilled water in a 10 cm diameter Petri plate and incubated at 22°C in continuous light as previously described (Nakabayashi et al. 2005Go).

Measurement of imbibed seed size
Dry seeds were first sown on dry filter paper, and photographed using an MZ-FLIII stereomicroscope (Leica Microsystems, Wetzlar, Germany) with a digital camera (DC300; Leica Microsystems). Then water was applied to the filter paper, and seeds were placed at 22°C under continuous white light. The length and width of 20 photographed seeds were measured by the software ImageJ (http://rsb.info.nih.gov/ij/). Duplicate experiments were performed and similar results were obtained.

RNA isolation and microarray analysis
Three biologically independent seed batches of Col and Cvi were used for microarray analyses. Procedures for RNA isolation and microarray analysis have been described previously (Nakabayashi et al. 2005Go). Differentially expressed genes were identified by statistical analysis implemented in the LIMMA package of Bioconductor (Smyth 2005Go). Genes with at least a 1.8-fold higher transcript abundance than dry seeds and a P-value of <0.02 were considered to be genes with significantly higher transcript abundance in seeds imbibed for 15 min, 30 min and 1 h. Advanced data analyses were performed using Microsoft Excel and TIGR MeV (Saeed et al. 2003Go; http://www.jcvi.org/cms/research/software/#c622). The heatmaps for the expression patterns of metabolism genes related to gibberellins, ethylene, cytokinins and brassinosteroids are shown in Supplementary Fig. S3.

Measurement of plant hormones
Extraction and purification of ABA, GA4, IAA, iP, SA, JA and JA-Ile were performed by solid-phase extraction. Stable isotope-labeled compounds used as internal standards were: D6-ABA (Icon Isotopes, Summit, NJ, USA); D2-GA4, D6-iP (Olchemim Ltd, Olomouc, Czech Republic); D2-IAA, D6-SA (Sigma-Aldrich, Oakville, ON, Canada); and D2-JA (Tokyo Kasei, Tokyo, Japan). [13C6]JA-Ile was synthesized with [13C6]Ile (Cambridge Isotope Laboratories, Andover, MA, USA) as described, and used as internal standard (Jikumaru et al. 2004Go).

For simultaneous measurements of ABA, GA4, IAA, iP, SA, JA and JA-Ile, 50 mg of frozen seed samples were mixed with 500 ml of 80% (v/v) methanol containing 1% (v/v) acetic acid and internal standards (D6-ABA, D2-GA4, D6-iP, D2-IAA, D6-SA, D2-JA, [13C6]JA-Ile), mashed with a TissueLyser (QIAGEN), and then extracted at –30°C overnight. Samples were centrifuged at 14,000xg for 10 min at 4°C, and the pellet washed with 80% (v/v) methanol containing 1% (v/v) acetic acid. Combined supernatant extracts were evaporated to obtain extracts in water containing 1% (v/v) acetic acid, and applied to a pre-equilibrated Oasis HLB column cartridge (30 mg, 1 ml, Waters). After washing with 1 ml of water containing 1% (v/v) acetic acid, all hormones were eluted with 2 ml of 80% (v/v) methanol containing 1% (v/v) acetic acid. The eluting materials were evaporated to obtain extracts in water containing 1% (v/v) acetic acid, and applied to a pre-equilibrated Oasis MCX column cartridge (30 mg, 1 ml, Waters). After washing the MCX cartridges with 1 ml of water containing 1% (v/v) acetic acid, the acidic fraction and a neutral fraction that contained ABA, GA4, IAA, SA, JA, JA-Ile and SA was eluted with 2 ml of methanol. A 200 µl aliquot of this fraction was transferred, evaporated, and reconstituted with water containing 1% (v/v) acetic acid for SA analysis. The MCX cartridges were further washed with 1 ml of water containing 5% (v/v) aqueous ammonia, and a basic fraction that contained iP was eluted with 2 ml of 60% (v/v) methanol containing 5% (v/v) aqueous ammonia. This fraction was applied to a pre-equilibrated Sep-Pak column (100 mg, 1 ml, Waters). After washing with 1 ml of chloroform containing 1% (v/v) triethylamine, a basic fraction that contained cytokinins was eluted with methanol : chloroform : triethylamine = 20 : 79: 1 (by vol.). This fraction was transferred, evaporated, and reconstituted with water containing 1% (v/v) acetic acid for cytokinin analysis. Acidic and neutral fractions were further applied to a pre-equilibrated Oasis WAX column cartridge (30 mg, 1 ml, Waters). After washing the WAX cartridges with 1 ml of water containing 1% (v/v) acetic acid and 2 ml of methanol, an acidic fraction that contained ABA, GA4, IAA, JA and JA-Ile was eluted with 2 ml of 80% (v/v) methanol containing 1% (v/v) acetic acid for analysis of ABA, GA4, IAA, JA and JA-Ile. All fractions containing targeted hormones were reconstituted with distilled water and injected into an LC-ESI-MS/MS (Agilent 6410, Agilent) equipped with a ZORBAX Eclipse XDB-C18 column (Agilent). The amount of each hormone was determined using spectrometer software (MassHunterTM v. B. 01. 02). The LC conditions and MS parameters are listed in Supplementary Tables S3 and S4.

Data deposition
Microarray data in this study are available on the NASCArrays website (http://affy.arabidopsis.info/). The reference number is NASCARRAYS-499.


   Supplementary data
Top
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Supplementary data
Funding
 Acknowledgments
 References
 
Supplementary data are available at PCP online.


   Funding
Top
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Supplementary data
 Funding
Acknowledgments
 References
 
Japanese Society for the Promotion of Science [Grant-in-Aid for Scientific Research (C) No. 19570051 to E.N.].


   Acknowledgments
Top
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Supplementary data
 Funding
 Acknowledgments
References
 
The authors acknowledge Professor Derek Bewley (University of Guelph) for critical reading of this manu- script and Dr. Hiroyuki Kasahara (RIKEN Plant Science Center) for comments and suggestions. The authors also thank Dr. Tom Downey and Ms. Sachiyo Harada for supporting microarray data analysis and technical assistance, respectively.


   Footnotes
 
5These authors contributed equally to this work. Back

6Present address: Laboratory of Plant Organ Development, National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki, Aichi, 444–8585 Japan Back


   References
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 Materials and Methods
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 Funding
 Acknowledgments
 References
 
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(Received August 13, 2009; Accepted August 20, 2009)
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