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First published online July 31, 2009; 10.1104/pp.109.142018 Plant Physiology 151:641-654 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
CHOTTO1, a Putative Double APETALA2 Repeat Transcription Factor, Is Involved in Abscisic Acid-Mediated Repression of Gibberellin Biosynthesis during Seed Germination in Arabidopsis1,[W],[OA]RIKEN Plant Science Center, Yokohama, Kanagawa 230–0045, Japan (R.Y., Y. Kanno, Y.J., K.N., Y. Kamiya, E.N.); and Department of Cell and Systems Biology (E.N.), and Centre for the Analysis of Genome Evolution and Function (E.N.), University of Toronto, Toronto, Ontario, Canada M5S 3B2
The phytohormones abscisic acid (ABA) and gibberellins (GAs) are the primary signals that regulate seed dormancy and germination. In this study, we investigated the role of a double APETALA2 repeat transcription factor, CHOTTO1 (CHO1), in seed dormancy, germination, and phytohormone metabolism of Arabidopsis (Arabidopsis thaliana). Wild-type seeds were dormant when freshly harvested seeds were sown, and these seeds were released from dormancy after a particular period of dry storage (after-ripening). The cho1 mutant seeds germinated easily even in a shorter period of storage than wild-type seeds. The cho1 mutants showed reduced responsiveness to ABA, whereas transgenic plants constitutively expressing CHO1 (p35S::CHO1) showed an opposite phenotype. Notably, after-ripening reduced the ABA responsiveness of the wild type, cho1 mutants, and p35S::CHO1 lines. Hormone profiling demonstrated that after-ripening treatment decreased the levels of ABA and salicylic acid and increased GA4, jasmonic acid, and isopentenyl adenine when wild-type seeds were imbibed. Expression analysis showed that the transcript levels of genes for ABA and GA metabolism were altered in the wild type by after-ripening. Hormone profiling and expression analyses indicate that cho1 seeds, with a short period of storage, resembled fully after-ripened wild-type seeds. Genetic analysis showed that the cho1 mutation partially restored delayed seed germination and reduced GA biosynthesis activity in the ABA-overaccumulating cyp707a2-1 mutant background but did not restore seed germination in the GA-deficient ga1-3 mutant background. These results indicate that CHO1 acts downstream of ABA to repress GA biosynthesis during seed germination.
Because of the sessile nature of plants, seed dormancy and germination are tightly regulated by both internal and external cues. Seed dormancy inhibits precocious germination and germination of matured dry seeds (Bewley, 1997  ; Baskin and Baskin, 1998; Donohue, 2005; Fenner and Thompson, 2005). Seed dormancy confers either inhibition or delay of germination, thereby enabling the seed to germinate under proper conditions for plant survival. Seed dormancy status is initiated during seed maturation and is released by endogenous and environmental factors such as phytohormones, temperature, nutrients, and light (Finkelstein et al., 2008; Holdsworth et al., 2008). Precocious germination and deep seed dormancy after harvest is an agricultural and industrial problem causing huge economic losses; therefore, it is imperative to understand how to control seed dormancy and germination in each plant species (Gubler et al., 2005).
The phytohormones abscisic acid (ABA) and GAs are major endogenous factors that regulate seed dormancy and germination. ABA is essential for the induction and maintenance of seed dormancy and inhibits seed germination, while GA is required for the promotion of seed germination. It has been demonstrated that both biosynthesis and inactivation of ABA and GA are important for regulation of seed dormancy and germination (Nambara and Marion-Poll, 2005
Seed dormancy is released by subsequent dry storage of matured seeds, a process called after-ripening. The molecular mechanism of seed dormancy and after-ripening has been studied in detail (Bailly, 2004
In a previous report, we identified and characterized ABA-insensitive seed germination mutants, named chotto1 (cho1; Nambara et al., 2002
Seed Dormancy and Germination in cho1 Mutants and p35S::CHO1 Lines
Two cho1 mutant alleles, cho1-1 and cho1-3, were used to examine the role of CHO1 in seed dormancy and germination. We previously showed that the cho1-1 mutation causes an amino acid substitution in the second AP2 domain, whereas the cho1-3 mutation disrupts the CHO1 gene by a DNA insertion in the fifth exon (Yamagishi et al., 2009
We next investigated the effect of after-ripening on the ABA responsiveness during germination of wild-type and cho1-3 mutant seeds. Seeds were dry stored for 0, 1, 2, 4, and 8 weeks and imbibed without stratification. Germination frequencies in the presence of (+)-S-ABA of both wild-type and cho1-3 seeds were higher when seeds were dry stored for longer periods (Fig. 1B). Interestingly, wild-type and mutant seeds germinated fully after 2 weeks of dry storage, and the ABA responsiveness of both the wild type and mutants decreased further by prolonged dry storage for 8 weeks (Fig. 1B). The ABA responsiveness of cho1 mutants on germination was less than that in the wild type when seeds were sown after 1, 2, and 4 weeks of dry storage, as reported previously (Yamagishi et al., 2009 ). However, the ABA responsiveness of cho1 mutants was comparable to that of wild-type seeds when these seeds were after-ripened for 8 weeks. To further investigate the function of CHO1, we made transgenic plants that express CHO1 under the control of the cauliflower mosaic virus 35S promoter, designated as p35S::CHO1 lines. Quantitative reverse transcription (QRT)-PCR analysis indicated that the transcript levels of CHO1 in 24-h imbibed seeds were 4-fold higher in the p35S::CHO1 lines than in the wild type (Fig. 1C). Without applying exogenous ABA, the germination frequencies after 1 week of dry storage were restored to the wild-type level in two p35S::CHO1 lines (Fig. 1B). However, after 2 or more weeks of dry storage, p35S::CHO1 showed full germination frequencies similar to the wild type and cho1 mutants (Fig. 1B), suggesting that seed dormancy was normally released by after-ripening even in the p35S::CHO1 lines. However, p35S::CHO1 seeds showed higher responsiveness to exogenous ABA than wild-type and cho1-3 mutant seeds after a series of dry storage periods (Fig. 1B). Even after 8 weeks of dry storage, germination of p35S::CHO1 lines was effectively suppressed by 0.5 or 1.0 µM exogenous ABA, in which germination of the wild type and cho1-3 was permissive.
After-ripened seeds reduce the ABA level in response to imbibition more rapidly than freshly harvested seeds (Ali-Rachedi et al., 2004
Next, to investigate the effect of the cho1 mutation on the accumulation of other plant hormones, hormone profiling was performed in 36-h imbibed seeds using LC-ESI-MS/MS. Seeds were dry stored for 1 and 6 weeks before imbibition. Endogenous levels of GA4, indole-3-acetic acid (IAA), JA, trans-zeatin (tZ), iP, and SA were determined. Interestingly, after-ripening treatment caused significant changes in the levels of not only ABA but also GA4, JA, iP, and SA in imbibed wild-type seeds. GA4, JA, and iP levels were increased, but ABA and SA levels were decreased in imbibed seeds with after-ripening treatment for 6 weeks relative to those for 1 week (Table I ). Imbibed seeds of cho1-1 and cho1-3 contained lower levels of ABA and SA but higher levels of GA4, JA, and iP than wild-type seeds after 1 week of dry storage (Table I). GA4 levels were 2-fold higher in cho1 mutants than in the wild type, whereas ABA levels were 2-fold lower in cho1 mutants. However, after 6 weeks of dry storage, there was no significant difference in all of these hormone levels between the wild type and cho1 mutants.
Changes in the Expression Levels of ABA and GA Metabolism Genes in cho1 Mutants QRT-PCR analysis was performed in dry seeds and imbibed seeds treated with 1 week of dry storage to investigate the expression pattern of genes involved in biosynthesis and inactivation of ABA and GA. The transcript levels of ABA inactivation genes (CYP707A2 and CYP707A3) were up-regulated in cho1 mutants relative to the wild type, while ABA biosynthesis genes (ZEP and NCED9) were down-regulated in the mutants (Fig. 3A ). Up-regulation of CYP707A2 in cho1 mutants appears to be a precocious induction, with a peak at 6 h in contrast to the wild type, with a peak at 12 h, whereas the higher levels of the CYP707A3 mRNA were observed in cho1 mutants at 24 h after imbibition. On the other hand, expression of ZEP and NCED9 in cho1 mutants was down-regulated after 12 h of imbibition. Expression levels of NCED6 and CYP707A1 in cho1 mutants were comparable to those in the wild type (Fig. 3A). Among GA biosynthesis genes, the transcript levels of GA3ox1 and GA3ox2 were significantly increased after imbibition (Fig. 3B). The induction of these genes in cho1 mutants was approximately 10-fold more exaggerated in cho1 mutants than in the wild type after imbibition. By contrast, the transcript levels of GA20ox3, a GA biosynthesis gene, were lower in cho1 mutants than in the wild type, which is the opposite direction in terms of higher accumulation of GA4 in the cho1 mutant than in the wild type (Table I; Fig. 3B). There was no significant difference in the transcript levels of GA20ox1, GA2ox2, and GA2ox6 between the wild type and cho1 mutants (Fig. 3B).
Furthermore, we investigated transcript levels of genes involved in the regulation of germination and hormone metabolism and signaling: seed dormancy and germination (DOG1, FUSCA3 [FUS3], PHYTOCHROME-INTERACTING FACTOR-LIKE5 [PIL5], SPATULA [SPT]), ABA signaling (ABI3, ABI4, ABI5), ABA response (RD29B), and GA signaling (REPRESSOR OF GA1-3 [RGA], GA-INSENSITIVE [GAI], RGA-LIKE2 [RGL2], RGL3; Fig. 3C; Supplemental Fig. S1). Of these genes, expression patterns of FUS3, PIL5, and SPT were altered in the cho1 mutant (Fig. 3C), but the transcript levels of DOG1, ABI, and RGA/GAI genes were not remarkably altered in the mutant (Fig. 3C; Supplemental Fig. S1). The transcript levels of RD29B seem to correlate with the ABA level (Fig. 2; Supplemental Fig. S1). Expression of FUS3 was down-regulated in cho1 mutants relative to the wild type after 12 h of imbibition. PIL5 expression was found to be down-regulated more than 2-fold in cho1 mutants after 24 h of imbibition when compared with the wild type, while SPT expression was up-regulated in cho1 mutants after 24 h of imbibition.
To investigate the effect of after-ripening on the expression of ABA and GA metabolism genes, QRT-PCR analysis was performed using seeds that had been dry stored for 0, 1, 4, and 9 weeks prior to imbibition. It has been reported that the transcript levels of ABA and GA metabolism genes, such as ZEP, NCED9, CYP707A2, and GA3ox1, are affected by after-ripening in Arabidopsis seeds (Cadman et al., 2006
Effects of cho1 Mutation in ABA-Overaccumulating cyp707a2 and GA-Deficient ga1 Mutants
The results described above indicate that the transcript levels of ABA and GA metabolism genes and levels of ABA and GA in imbibed seeds are affected by cho1 mutations. It is known that ABA regulates GA metabolism and vice versa (Toyomasu et al., 1994
Then, seed germination was analyzed in the ga1-3 mutant and the ga1-3 cho1-3 double mutant. Both ga1-3 and ga1-3 cho1-3 mutants did not germinate when 4-week-stored seeds were used for germination assays (Fig. 6A ). Exogenous application of GA4 recovered germination frequencies in both ga1-3 and ga1-3 cho1-3 mutants in a dose-dependent manner. There was no difference in the GA response curve between ga1-3 and ga1-3 cho1-3 mutants (Fig. 6A). We then analyzed the expression of GA3ox1 and GA3ox2. When compared with the wild type, GA3ox1 gene expression was transiently up-regulated after 6 h of imbibition in ga1-3 mutants, whereas GA3ox2 gene expression was significantly down-regulated in the ga1-3 mutants during the course of imbibition (Fig. 6B), consistent with the previous study showing that GA3ox1 and GA3ox2 gene expression is regulated by a negative feedback mechanism and a positive feedback mechanism, respectively, in imbibed seeds (Yamaguchi et al., 1998 ).
We next examined the effect of the cho1-3 mutation on the expression of hormone metabolism genes under GA-deficient conditions. The cho1-3 mutation further enhanced expression of GA3ox1, but not of GA3ox2, when the expression patterns in ga1-3 cho1-3 were compared with those in ga1-3 (Fig. 6B). On the other hand, expression of ABA metabolism genes (ZEP, NCED6, NCED9, CYP707A1-3) and ABA response genes (ABI5, RD29B) was compared between ga1-3 and ga1-3 cho1-3. There was no significant difference in the transcript levels of these genes between these two mutants in both dry seeds and imbibed seeds (Supplemental Fig. S3).
To examine the site of CHO1 expression, histochemical analysis of GUS expression was performed using pCHO1::GUS transgenic seeds. When 16-h imbibed seeds were stained, GUS staining was detected in the radicle of the embryo in pCHO1::GUS seeds (Fig. 7A ). The staining was intense at the tip of the radicle but absent in the endosperm or testa. When sections of the GUS-stained seeds were examined, pCHO1::GUS expression was observed in the radicle except for the provascular.
Next, the effect of after-ripening on the expression pattern of CHO1 in imbibed seeds was investigated using freshly harvested seeds and 4-week-stored seeds. In the wild type, CHO1 expression was moderately up-regulated in response to imbibition in freshly harvested seeds, while it was up-regulated after 6 h of imbibition in 4-week-stored wild-type seeds (Fig. 7B), suggesting that after-ripening resulted in precocious up-regulation of CHO1 expression after imbibition. Interestingly, this up-regulation of CHO1 expression was observed even in freshly harvested cho1-1 mutant seeds (Fig. 7B). In contrast, this was not observed in the cho1-3 mutation due to the disrupted CHO1 expression as a result of the DNA insertion mutation. CHO1 expression was induced in the cyp707a2-1 mutant at 6 h in a similar manner as in the wild type (Fig. 7C). The mRNA of CHO1 maintained a higher level at 24 h in cyp707a2-1 than in the wild type. This difference does not seem to be the direct effect of ABA overaccumulation, because exogenous ABA does not induce CHO1 expression at this time (Yamagishi et al., 2009 ). On the other hand, CHO1 expression was not altered by the ga1-3 mutation or by the application of GA (Fig. 7C).
It has been shown that CHO1 is a member of AP2-like subfamily, which is composed of at least 18 members in the Arabidopsis genome (Kim et al., 2006
CHO1 Plays a Role in ABA-Mediated Repression of Seed Germination after Imbibition of Dry Seeds
Seed dormancy is induced during seed maturation and is released by subsequent dry storage, a process called after-ripening. The dry storage increases the germination potential of seeds, enabling the seeds to germinate upon imbibition (Holdsworth et al., 2008
In this study, we characterized the effects of after-ripening in wild-type seeds for its germination potential (Fig. 1, A and B), ABA responsiveness (Fig. 1B), endogenous levels of ABA, GA4, iP, JA, and SA (Table I), and transcript levels of genes involved in ABA and GA metabolism (Fig. 4). Characterization of the cho1 mutants using these criteria demonstrates that the shortly stored cho1 seeds resembled fully after-ripened wild-type seeds. The cho1 mutants display the phenotype as if it is hypersensitive to after-ripening. A simple hypothesis to explain this is that this phenotype acquires proper induction of primary dormancy but has low ability to maintain seed dormancy after imbibition of dry seeds. Nondormant mutants might exhibit reduced dormancy even in freshly harvested seeds, and after-ripening hypersensitive mutants acquire primary dormancy in fresh seeds but are released by after-ripening relatively easily. We previously discussed that the genetic components for ABA sensitivity in seed development are similar, but distinct, from those after imbibition of dry seeds (Yamagishi et al., 2009
Loss of function of CHO1 reduced the ABA responsiveness, whereas its gain of function led to the opposite result (Fig. 1B). The ABA responsiveness of the wild type, cho1 mutants, and p35S::CHO1 lines decreased in a manner dependent on the period of dry storage. The reduction of ABA responsiveness continued to the 8-week storage period, even though all genotypes were fully germinated by 2 weeks of dry storage. This result indicates that after-ripening continued to reduce the ABA responsiveness even after the seed was completely released from primary dormancy, as is the case of after-ripening-dependent gene expression (Carrera et al., 2008
It has been reported that the reduction in ABA levels in imbibed seeds is exaggerated by after-ripening prior to imbibition (Ali-Rachedi et al., 2004
In addition, our comprehensive plant hormone measurement revealed that after-ripening treatment decreased the levels of ABA and SA and increased GA4, JA, and iP levels in imbibed wild-type seeds (Table I). It has been reported that SA and cytokinin are involved in the regulation of seed germination (Riefler et al., 2006
Our gene expression analysis showed that the transcript levels of genes involved in ABA and GA metabolism were altered in cho1 mutants (Fig. 3). When 1-week-stored cho1 seeds were imbibed, transcript levels of ABA biosynthesis genes (ZEP, NCED9) were down-regulated, whereas those of ABA inactivation genes (CYP707A2, CYP707A3) and GA biosynthesis genes (GA3ox1, GA3ox2) were up-regulated in cho1 mutants relative to the wild type (Fig. 3, A and B). Among these genes, the changes of GA3ox1 and GA3ox2 mRNA levels were remarkable and increased more than 10-fold in cho1 mutants when compared with the wild type. These expression patterns are correlated with changes in ABA and GA4 levels (Table I; Fig. 2). In addition, we found that expression patterns of the regulatory genes involved in ABA and GA metabolism were also altered in cho1 mutants (Fig. 3C). These include PIL5 and SPT genes, which are involved in the negative regulation of seed germination (Oh et al., 2004
Recent transcriptome analysis using dormant Cvi-0 has revealed that after-ripening affects the transcription of ABA and GA metabolism genes after imbibition (Cadman et al., 2006
Hormone metabolism is mutually regulated and constitutes a network to coordinate hormone balance (Seo et al., 2009
GA biosynthesis genes are expressed in hypocotyl and radicle of imbibed Arabidopsis seeds (Yamaguchi et al., 2001
In this study, our gel mobility shift assay demonstrated that the AP2 domains of CHO1 bind to the DNA element recognized by ANT in a specific manner (Fig. 8; Nole-Wilson and Krizek, 2000
Amino acid sequences of AP2 domains are highly conserved among AP2-like family members. The AP2 domains of CHO1 are most similar to those of PLT1 and PLT2 (both 94% identity) and ANT (86%). It has been demonstrated that overexpression of the ANT gene results in increased size in leaves, seeds, and flowers (Krizek, 1999
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) Columbia accession was used as the wild type in this study. cho1-1, cho1-3, and cyp707a2-1 mutants were reported previously (Kushiro et al., 2004
To compare seed dormancy and germination, freshly harvested seeds and dry-stored seeds were sown and imbibed on water-agarose at 23°C under continuous white light as described above. Radicle emergence was scored daily as seed germination for up to 7 d. For ABA- and GA-responsiveness tests, freshly harvested seeds and dry-stored seeds were sown on water-agarose containing (+)-S-ABA or GA4 and imbibed at 23°C under continuous white light. Radicle emergence was scored after 5 or 7 d. In each germination test, approximately 50 seeds were used, and two experiments were carried out using two independent seed lots.
Extraction and purification of ABA, GA4, IAA, tZ, iP, SA, and JA were performed by solid-phase extraction as described (Dobrev and Kamínek, 2002
Extraction and purification of ABA were performed as described by Saika et al. (2007) For simultaneous measurement of ABA, GA4, IAA, tZ, iP, SA, and JA, 50 mg of frozen seed samples was mixed with 500 µL of 80% (v/v) methanol containing 1% (v/v) acetic acid and internal standards (D6-ABA, D2-GA4, D5-tZ, D6-iP, D2-IAA, D6-SA, D2-JA), mashed up with a TissueLyser (Qiagen), and then extracted at –30°C overnight. Samples were centrifuged at 14,000g for 10 min at 4°C, and the pellet was washed with 80% (v/v) methanol containing 1% (v/v) acetic acid. Combined supernatant extracts were evaporated to obtain extracts in water containing 1% acetic acid and applied to preequilibrated Oasis HLB column cartridges (Waters). After washing with 1 mL of water containing 1% (v/v) acetic acid, all hormones were eluted with 1 mL of 80% (v/v) methanol containing 1% (v/v) acetic acid. The eluting materials were evaporated to obtain extract in water containing 1% (v/v) acetic acid and applied to preequilibrated Oasis MCX column cartridges (Waters). After washing the MCX cartridges with 1 mL of water containing 1% (v/v) acetic acid, the acidic and neutral fraction that contained ABA, GA4, IAA, SA, and JA was eluted with 2 mL of methanol. Two hundred microliters of the acidic and neutral fraction was transferred, evaporated, and reconstituted with water for SA analysis. The MCX cartridges were further washed with 1 mL of water containing 5% (v/v) aqueous ammonia, and the basic fraction that contained tZ and iP was eluted with 2 mL of 60% (v/v) methanol containing 5% (v/v) aqueous ammonia. Acidic and neutral fractions were further applied to preequilibrated Oasis WAX column cartridges (Waters). After washing the WAX cartridges with 2 mL of methanol and water containing 1% (v/v) acetic acid, the acidic fractions that contained ABA, GA4, IAA, and JA were eluted with 2 mL of methanol containing 1% (v/v) acetic acid. Each eluting fraction was evaporated, reconstituted with water containing 1% (v/v) acetic acid, and injected into a LC-ESI-MS/MS apparatus (Agilent 6410; Agilent) equipped with a ZORBAX Eclipse XDB-C18 column (Agilent). The amount of each hormone was determined by spectrometer software (MassHunter version B.01.02). The LC conditions and MS parameters are listed in Supplemental Tables S1 and S2.
Total RNA preparation from seeds, first-strand cDNA synthesis, and QRT-PCR were performed as described previously (Okamoto et al., 2006
To construct p35S::CHO1 plasmid, a 1.7-kb cDNA fragment of CHO1 was amplified from a wild-type cDNA library by PCR using the following primers: 5'-CACCCCTATTCCCAAAGATGAAGAACAATAAC-3' and 5'-TCATTCCAACCCAAAAACCGGTG-3' (an underline indicates the sequence for the directional cloning into pENTR/D-TOPO vector), and cloned into pENTR/D-TOPO vector (Invitrogen). After the sequence was verified, the cloned cDNA was converted into pGWB2 binary vector, just downstream of the cauliflower mosaic virus 35S promoter and upstream of the nopaline synthase transcription terminator by LR clonase reaction according to the manufacturer's protocol (Invitrogen). To construct pCHO1::GUS plasmid, a 2.2-kb fragment of genomic DNA that contains a putative promoter region upstream of the ATG start codon of CHO1 was amplified from wild-type genomic DNA by PCR using the following primers: 5'-CACCCGTGTGCATGGTAGCTGACACGTG-3' and 5'-ATTGTTCTTCATCTTTGGGAATAGG-3', and cloned into pENTR/D-TOPO vector. After the sequence was verified, the cloned genomic DNA was converted to pGWB3 binary vector, just upstream of the GUS reporter gene by LR clonase reaction.
The constructed vectors were introduced into the Agrobacterium tumefaciens strain GV3101 by electroporation and transformed into Arabidopsis by the floral dipping method (Clough and Bent, 1998
GUS staining was performed as described previously (Yamaguchi et al., 2001
The gel mobility shift assay was performed as described in previous reports (Fukazawa et al., 2000
Nucleotide sequences of the double-stranded BS15 oligonucleotides used for the gel mobility shift assays were according to the report of Nole-Wilson and Krizek (2000)
The following materials are available in the online version of this article.
We thank Drs. George Stamatiou and Danielle Vidaurre for critical reading of the manuscript. We also thank Drs. Shinjiro Yamaguchi, Kiyoshi Tatematsu, Jutaro Fukazawa, and Masanori Okamoto for their helpful discussion and suggestions and Ms. Sachiyo Harada for her technical assistance. Received May 24, 2009; accepted July 23, 2009; published July 31, 2009.
1 This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (grant no. 19570051) and by a Natural Sciences and Engineering Research Council of Canada Discovery Grant (to E.N.). 
2 Present address: Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, Cologne 50829, Germany. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Eiji Nambara (eiji.nambara{at}utoronto.ca).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.142018 * Corresponding author; e-mail eiji.nambara{at}utoronto.ca.
Ali-Rachedi S, Bouinot D, Wagner MH, Bonnet M, Sotta B, Grappin P, Jullien M (2004) Changes in endogenous abscisic acid levels during dormancy release and maintenance of mature seeds: studies with the Cape Verde Islands ecotype, the dormant model of Arabidopsis thaliana. Planta 219: 479–488[Web of Science][Medline] Alonso-Blanco C, Bentsink L, Hanhart CJ, Blankestijn-de Vries H, Koornneef M (2003) Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana. Genetics 164: 711–729 Bailly C (2004) Active oxygen species and antioxidants in seed biology. Seed Sci Res 14: 93–107[CrossRef] Baskin CC, Baskin JM (1998) Seeds: Ecology, Biogeography, and Evolution of Dormancy and Germination. Academic Press, San Diego Bentsink L, Jowett J, Hanhart CJ, Koornneef M (2006) Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis. Proc Natl Acad Sci USA 103: 17042–17047 Bewley JD (1997) Seed germination and dormancy. Plant Cell 9: 1055–1066[CrossRef][Web of Science][Medline] Cadman CS, Toorop PE, Hilhorst HW, Finch-Savage WE (2006) Gene expression profiles of Arabidopsis Cvi seeds during dormancy cycling indicate a common underlying dormancy control mechanism. Plant J 46: 805–822[CrossRef][Web of Science][Medline] Carrera E, Holman T, Medhurst A, Dietrich D, Footitt S, Theodoulou FL, Holdsworth MJ (2008) Seed after-ripening is a discrete developmental pathway associated with specific gene networks in Arabidopsis. Plant J 53: 214–224[CrossRef][Web of Science][Medline] Cernac A, Andre C, Hoffmann-Benning S, Benning C (2006) WRI1 is required for seed germination and seedling establishment. Plant Physiol 141: 745–757 Chandra A, Anand A, Dubey A (2007) Effect of salicylic acid on morphological and biochemical attributes in cowpea. J Environ Biol 28: 193–196[Web of Science][Medline] Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium mediated transformation of Arabidopsis thaliana. Plant J 16: 735–743[CrossRef][Web of Science][Medline] Curaba J, Moritz T, Blervaque R, Parcy F, Raz V, Herzog M, Vachon G (2004) AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis. Plant Physiol 136: 3660–3669 Derkx MPM, Karssen CM (1993) Effects of light and temperature on seed dormancy and gibberellin-stimulated germination in Arabidopsis thaliana: studies with gibberellin-deficient and -insensitive mutants. Physiol Plant 89: 360–368[CrossRef] Dobrev PI, Kamínek M (2002) Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J Chromatogr A 950: 21–29[CrossRef][Web of Science][Medline] Donohue K (2005) Seeds and seasons: interpreting germination timing in the field. Seed Sci Res 15: 175–187[CrossRef] Fenner M, Thompson K (2005) The Ecology of Seeds. Cambridge University Press, Cambridge, UK Finch-Savage WE, Cadman CS, Toorop PE, Lynn JR, Hilhorst HW (2007) Seed dormancy release in Arabidopsis Cvi by dry after-ripening, low temperature, nitrate and light shows common quantitative patterns of gene expression directed by environmentally specific sensing. Plant J 51: 60–78[CrossRef][Web of Science][Medline] Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy and the control of germination. New Phytol 171: 501–523[CrossRef][Web of Science][Medline] Finkelstein RR, Reeves W, Ariizumi T, Steber C (2008) Molecular aspects of seed dormancy. Annu Rev Plant Biol 59: 387–415[CrossRef][Medline] Frey A, Audran C, Marin E, Sotta B, Marion-Poll A (1999) Engineering seed dormancy by the modification of zeaxanthin epoxidase gene expression. Plant Mol Biol 39: 1267–1274[CrossRef][Web of Science][Medline] Fukazawa J, Sakai T, Ishida S, Yamaguchi I, Kamiya Y, Takahashi Y (2000) Repression of shoot growth, a bZIP transcriptional activator, regulates cell elongation by controlling the level of gibberellins. Plant Cell 12: 901–915 Galinha C, Hofhuis H, Luijten M, Willemsen V, Blilou I, Heidstra R, Scheres B (2007) PLETHORA proteins as dose-dependent master regulators of Arabidopsis root development. Nature 449: 1053–1057[CrossRef][Medline] Gazzarrini S, Tsuchiya Y, Lumba S, Okamoto M, McCourt P (2004) The transcription factor FUSCA3 controls developmental timing in Arabidopsis through the hormones gibberellin and abscisic acid. Dev Cell 7: 373–385[CrossRef][Web of Science][Medline] Gubler F, Millar AA, Jacobsen JV (2005) Dormancy release, ABA and pre-harvest sprouting. Curr Opin Plant Biol 8: 183–187[CrossRef][Web of Science][Medline] Holdsworth MJ, Bentsink L, Soppe WJ (2008) Molecular networks regulating Arabidopsis seed maturation, after-ripening, dormancy and germination. New Phytol 179: 33–54[CrossRef][Web of Science][Medline] Kim S, Soltis PS, Wall K, Soltis DE (2006) Phylogeny and domain evolution in the APETALA2-like gene family. Mol Biol Evol 23: 107–120 Koornneef M, Jorna ML, Brinkhorst-van der Swan DLC, Karssen CM (1982) The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 61: 385–393[Web of Science] Koornneef M, van der Veen JH (1980) Induction and analysis of gibberellin-sensitive mutants in Arabidopsis thaliana (L.) Heynh. Theor Appl Genet 58: 257–263[CrossRef][Web of Science] Krizek BA (1999) Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs. Dev Genet 25: 224–236[CrossRef][Web of Science][Medline] Kushiro T, Okamoto M, Nakabayashi K, Yamagishi K, Kitamura S, Asami T, Hirai N, Koshiba T, Kamiya Y, Nambara E (2004) The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism. EMBO J 23: 1647–1656[CrossRef][Web of Science][Medline] Lefebvre V, North H, Frey A, Sotta B, Seo M, Okamoto M, Nambara E, Marion-Poll A (2006) Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy. Plant J 45: 309–319[CrossRef][Web of Science][Medline] Léon-Kloosterziel KM, Gil MA, Ruijs GJ, Jacobsen SE, Olszewski NE, Schwartz SH, Zeevaart JA, Koornneef M (1996) Isolation and characterization of abscisic acid-deficient Arabidopsis mutants at two new loci. Plant J 10: 655–661[CrossRef][Web of Science][Medline] Lopez-Molina L, Mongrand S, Chua NH (2001) A postgermination developmental arrest checkpoint is mediated by abscisic acid and requires the ABI5 transcription factor in Arabidopsis. Proc Natl Acad Sci USA 98: 4782–4787 Marin E, Nussaume L, Quesada A, Gonneau M, Sotta B, Hugueney P, Frey A, Marion-Poll A (1996) Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana. EMBO J 15: 2331–2342[Web of Science][Medline] Millar AA, Jacobsen JV, Ross JJ, Helliwell CA, Poole AT, Scofield G, Reid JB, Gubler F (2006) Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase. Plant J 45: 942–954[Web of Science][Medline] Mitchum MG, Yamaguchi S, Hanada A, Kuwahara A, Yoshioka Y, Kato T, Tabata S, Kamiya Y, Sun TP (2006) Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development. Plant J 45: 804–818[CrossRef][Web of Science][Medline] Mizukami Y, Fischer RL (2000) Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proc Natl Acad Sci USA 97: 942–947 Nambara E, Hayama R, Tsuchiya Y, Nishimura M, Kawaide H, Kamiya Y, Naito S (2000) The role of ABI3 and FUS3 loci in Arabidopsis thaliana on phase transition from late embryo development to germination. Dev Biol 220: 412–423[CrossRef][Web of Science][Medline] Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56: 165–185[CrossRef][Medline] Nambara E, Suzuki M, Abrams S, McCarty DR, Kamiya Y, McCourt P (2002) A screen for genes that function in abscisic acid signaling in Arabidopsis thaliana. Genetics 161: 1247–1255 Nole-Wilson S, Krizek BA (2000) DNA binding properties of the Arabidopsis floral development protein AINTEGUMENTA. Nucleic Acids Res 28: 4076–4082 Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, Yamaguchi S (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination. Plant Cell 15: 1591–1604 Oh E, Kim J, Park E, Kim JI, Kang C, Choi G (2004) PIL5, a phytochrome-interacting basic helix-loop-helix protein, is a key negative regulator of seed germination in Arabidopsis thaliana. Plant Cell 16: 3045–3058 Oh E, Yamaguchi S, Kamiya Y, Bae G, Chung WI, Choi G (2006) Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis. Plant J 47: 124–139[CrossRef][Web of Science][Medline] Okamoto M, Kuwahara A, Seo M, Kushiro T, Asami T, Hirai N, Kamiya Y, Koshiba T, Nambara E (2006) CYP707A1 and CYP707A2, which encode abscisic acid 8'-hydroxylases, are indispensable for proper control of seed dormancy and germination in Arabidopsis. Plant Physiol 141: 97–107 Penfield S, Josse EM, Kannangara R, Gilday AD, Halliday KJ, Graham IA (2005) Cold and light control seed germination through the bHLH transcription factor SPATULA. Curr Biol 15: 1998–2006[CrossRef][Web of Science][Medline] Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokinin receptor mutants reveal functions in shoot growth, leaf senescence, seed size, germination, root development, and cytokinin metabolism. Plant Cell 18: 40–54 Saika H, Okamoto M, Miyoshi K, Kushiro T, Shinoda S, Jikumaru Y, Fujimoto M, Arikawa T, Takahashi H, Ando M, et al (2007) Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice. Plant Cell Physiol 48: 287–298 Sawada Y, Aoki M, Nakaminami K, Mitsuhashi W, Tatematsu K, Kushiro T, Koshiba T, Kamiya Y, Inoue Y, Nambara E, et al (2008) Phytochrome- and gibberellin-mediated regulation of abscisic acid metabolism during germination of photoblastic lettuce seeds. Plant Physiol 146: 1386–1396 Seo M, Hanada A, Kuwahara A, Endo A, Okamoto M, Yamauchi Y, North H, Marion-Poll A, Sun TP, Koshiba T, et al (2006) Regulation of hormone metabolism in Arabidopsis seeds: phytochrome regulation of abscisic acid metabolism and abscisic acid regulation of gibberellin metabolism. Plant J 48: 354–366[CrossRef][Web of Science][Medline] Seo M, Nambara E, Choi G, Yamaguchi S (2009) Interaction of light and hormone signals in germinating seeds. Plant Mol Biol 69: 463–472[CrossRef][Web of Science][Medline] Sun TP, Kamiya Y (1994) The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis. Plant Cell 6: 1509–1518[Abstract] Tan BC, Schwartz SH, Zeevaart JA, McCarty DR (1997) Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA 94: 12235–12240 Toh S, Imamura A, Watanabe A, Nakabayashi K, Okamoto M, Jikumaru Y, Hanada A, Aso Y, Ishiyama K, Tamura N, et al (2008) High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds. Plant Physiol 146: 1368–1385 Toyomasu T, Yamane H, Murofushi N, Inoue Y (1994) Effects of exogenously applied gibberellin and red light on the endogenous levels of abscisic acid in photoblastic lettuce seeds. Plant Cell Physiol 35: 127–129 Tyler L, Thomas SG, Hu J, Dill A, Alonso JM, Ecker JR, Sun TP (2004) Della proteins and gibberellin-regulated seed germination and floral development in Arabidopsis. Plant Physiol 135: 1008–1019 Yamagishi K, Tatematsu K, Yano R, Preston J, Kitamura S, Takahashi H, McCourt P, Kamiya Y, Nambara E (2009) CHOTTO1, a double AP2 domain protein of Arabidopsis thaliana, regulates germination and seedling growth under excess supply of glucose and nitrate. Plant Cell Physiol 50: 330–340 Yamaguchi S (2008) Gibberellin metabolism and its regulation. Annu Rev Plant Biol 59: 225–251[CrossRef][Medline] Yamaguchi S, Kamiya Y, Sun T (2001) Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination. Plant J 28: 443–453[CrossRef][Web of Science][Medline] Yamaguchi S, Smith MW, Brown RG, Kamiya Y, Sun T (1998) Phytochrome regulation and differential expression of gibberellin 3β-hydroxylase genes in germinating Arabidopsis seeds. Plant Cell 10: 2115–2126 Yamauchi Y, Ogawa M, Kuwahara A, Hanada A, Kamiya Y, Yamaguchi S (2004) Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds. Plant Cell 16: 367–378 Yamauchi Y, Takeda-Kamiya N, Hanada A, Ogawa M, Kuwahara A, Seo M, Kamiya Y, Yamaguchi S (2007) Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds. Plant Cell Physiol 48: 555–561
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ABSTRACT
RESULTS
2-isopentenyl)adenine (iP) after imbibition in the wild type; (2) after-ripening affects the transcript levels of genes involved in ABA and GA metabolism beyond seed dormancy release in the wild type; and (3) the pattern of phytohormone levels and transcript levels indicate that shortly stored cho1 mutant seeds mimic fully after-ripened wild-type seeds. Genetic analysis using cyp707a2-1 and ga1-3 mutants further suggested that CHO1 acts downstream of ABA to repress GA biosynthesis during seed imbibition. The possible role of CHO1 in the regulation of seed dormancy, germination, and phytohormone metabolism is discussed. 
-32P]dCTP and the Klenow fragment of DNA polymerase I (Takara). The binding reaction was performed by incubating 1 µg of purified CHO1-AP2 or CHO1-AP2m protein with 100 fmol of labeled probe in 20 µL of binding buffer (50 ng µL–1 poly[dI-dC], 20 mM Tris-HCl, pH 7.5, 3 mM MgCl2, 50 mM KCl, 1 mM EDTA, 10% [v/v] glycerol, 0.3 mg mL–1 bovine serum albumin, and 1 mM dithiothreitol) at 4°C for 30 min. Excess amounts of nonlabeled DNA were used as competitors. The reaction mixtures were separated on a 4% polyacrylamide gel in 1x Tris-borate/EDTA buffer at 4°C and analyzed with BAS-2500 imaging analyzer (Fujifilm).