The Plant Journal (2013) 74, 815–828
doi: 10.1111/tpj.12167
Combining association mapping and transcriptomics identify HD2B histone deacetylase as a genetic factor associated with seed dormancy in Arabidopsis thaliana Ryoichi Yano1,*, Yumiko Takebayashi1, Eiji Nambara1,2, Yuji Kamiya1 and Mitsunori Seo1 RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan, and 2 Department of Cell and Systems Biology, and Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto, Ontario, M5S 3B2, Canada
1
Received 27 November 2012; revised 20 February 2013; accepted 27 February 2013; published online 7 March 2013. *For correspondence (e-mail
[email protected]).
SUMMARY Seed dormancy is an important adaptive trait that enables germination at the proper time, thereby ensuring plant survival after germination. In Arabidopsis, considerable variation exists in the degree of seed dormancy among wild-type accessions (ecotypes). In this paper, we identify a plant-specific HD2 histone deacetylase gene, HD2B (At5g22650), as a genetic factor associated with seed dormancy. First, genomewide association mapping of 113 accessions was used to identify single nucleotide polymorphisms that possibly explain natural variation for seed dormancy. Integration of genome-wide association mapping and transcriptome analysis during cold-induced dormancy cycling identified HD2B as the most plausible candidate gene, and quantitative RT-PCR analysis demonstrated that HD2B expression was up-regulated by cold and after-ripening (dry storage of mature seed), treatments that are known to break seed dormancy. Interestingly, quantitative RT-PCR analysis in 106 accessions revealed that the expression of HD2B in imbibed seeds was significantly suppressed in most of the dormant accessions compared with less-dormant accessions, suggesting that suppression of HD2B expression may be important to maintain seed dormancy in dormant accessions. In addition, transgenic seeds of a dormant Cvi-0 accession that carried a 2.5 kb genomic DNA fragment of HD2B cloned from a less-dormant Col-0 accession (ColHD2B/Cvi-0) exhibited reduced seed dormancy accompanied by enhanced expression of HD2B when after-ripened or cold-imbibed. Endogenous levels of gibberellin were found to be increased in the imbibed seeds of after-ripened ColHD2B/Cvi-0 compared with wild-type Cvi-0. These results suggest that HD2B plays a role in seed dormancy and/or germinability in Arabidopsis thaliana. Keywords: seed dormancy, germination, histone deacetylase, gibberellin, genome-wide association, transcriptomics, Arabidopsis thaliana.
INTRODUCTION Despite their sessile nature, plants have the ability to choose their habitats through precise germination responses to environmental factors (Donohue, 2005). Germination at the right time is the first requirement for successful growth and survival, because it influences subsequent life-history traits . In this sense, seed dormancy, which arrests seed growth and development even under conditions favorable to germination, is thought to be an important adaptive trait of plants (Bewley, 1997; Finch-Savage and Leubner-Metzger, 2006; Holdsworth et al., 2008). After-ripening (AR) refers to a dry storage period, which enhances dormancy release. Additional factors, such as low temperatures (Penfield et al., 2005; Kendall © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd
et al., 2011) and nitrate (Alboresi et al., 2005; Matakiadis et al., 2009), may affect seed dormancy during seed maturation, dry storage and after imbibition. Because seed dormancy is one of the most important plant traits that affect crop yields and their industrial use , an understanding of the complex molecular mechanisms of seed dormancy is highly desirable (Gubler et al., 2005). Increasing lines of evidence have demonstrated that the phytohormones abscisic acid (ABA) and gibberellin (GA) play essential roles in environmental regulation of seed dormancy and germination (Nambara and Marion-Poll, 2005; Finkelstein et al., 2008; Yamaguchi, 2008). ABA is essential to induce and maintain dormancy, while GA is 815
816 Ryoichi Yano et al. required for germination. Dormancy-affecting factors, such as AR, low temperatures and nitrate, are known to affect the expression of genes involved in ABA catabolism and GA biosynthesis, such as CYP707A2 and gibberellin 3boxidase (GA3ox) (Cadman et al., 2006; Finch-Savage et al., 2007; Carrera et al., 2008). Expression of GA3ox genes is also changed in Arabidopsis mutants with altered primary seed dormancy (Penfield et al., 2005) or AR responses (Yano et al., 2009). On the other hand, genetic studies on reduced dormancy (rdo) mutants in Arabidopsis have indicated an ABA-independent mechanism of seed dormancy (Peeters et al., 2002). RDO4/HISTONE MONOUBIQUITINATION 1 (RDO4/HUB1) encodes a C3HC4 RING finger protein that appears to be involved in histone H2B monoubiquitination (Liu et al., 2007). More recently, RDO2 was shown to encode a transcription elongation factor, TFIIS (Liu et al., 2011). RDO4/HUB1 and RDO2 are thought to interact with the RNA polymerase II-associated factor 1 complex (PAF1C), suggesting the important role of transcription in seed dormancy. There is natural variation in the degree of seed dormancy and germinability among wild-type accessions (ecotypes) or cultivars of Arabidopsis and crop species (Alonso-Blanco et al., 2003; Fujino et al., 2008; Bentsink et al., 2010; Sugimoto et al., 2010). The most popular approach to identify genes for natural variation is quantitative trait locus (QTL) studies. In Arabidopsis, QTL studies using recombinant inbred lines (RILs) have identified DELAY OF GERMINATION 1 (DOG1) as a gene contributing to natural variation in seed dormancy in Arabidopsis (Bentsink et al., 2006). DOG1 functions in a genetic pathway that is independent of ABA (Nakabayashi et al., 2012), and its expression in imbibed seeds is down-regulated by AR (Bentsink et al., 2006). In rice, qLTG301, a major QTL responsible for low-temperature germinability, has also been cloned through a QTL approach using the Hayamasari and Italica Livorno strains (Fujino et al., 2008). In addition, Sdr4, a major QTL responsible for seed dormancy and domestication, has been cloned through a QTL approach using the japonica and indica cultivars (Sugimoto et al., 2010). In Arabidopsis, FLOWERING LOCUS C (FLC), a master regulator of reproductive phase transition, has also been reported to be involved in natural variation for low-temperature germinability (Chiang et al., 2009). FLC is considered to determine seasonal habitat choice in nature by controlling both seed germination and flowering time. Although QTL studies are promising, genome-wide association (GWA) mapping has recently emerged as a more comprehensive and robust approach to study natural variation (Nordborg and Weigel, 2008; Ingvarsson and Street, 2011). In Arabidopsis, GWA mapping has identified a number of single nucleotide polymorphisms (SNPs) that may explain the variation in 107 phenotypes, including seed dormancy and germination (Atwell et al., 2010). GWA
mapping has been shown to be useful to identify environmentally sensitive genetic loci underlying local adaptation in flowering time (Li et al., 2010). However, because GWA mapping uses a huge number of SNPs as explanatory variables, distinguishing true-positive SNPs from false-positive ones is a challenge (Tabangin et al., 2009). None of the candidate genes identified by GWA mapping has been biologically shown to play a role in seed dormancy or germination. In this study, we identified a HD2 histone deacetylase (HDAC) gene, HD2B (At5g22650), as a genetic factor associated with seed dormancy and germination through a combined approach of GWA mapping and transcriptome analyses. HD2-type HDAC was initially identified in maize as a plant-specific HDAC (Lusser, 1997). Some Arabidopsis homologs of HD2 HDACs have been reported to regulate seed germination, the salt stress response and stomatal closure in the Col-0 ecotype (Sridha and Wu, 2006; Colville et al., 2011; Luo et al., 2012a). However, little was known about the role of HD2B in seed dormancy. Our results indicate that expression of HD2B is affected by cold and AR, and was down-regulated in most dormant accessions compared with less-dormant accessions. In addition, we showed that enhanced HD2B expression was associated with reduced seed dormancy and increased endogenous GA accumulation in the dormant Cvi-0 background. Our results indicated that expression of HD2B is associated with seed dormancy and/or germinability in at least some accessions in Arabidopsis. RESULTS GWA mapping of seed dormancy using seeds afterripened for two months It is known that seeds of the Cvi-0 accession, a dormant Arabidopsis accession, require much longer AR to reduce seed dormancy compared with less-dormant accessions, such as Col-0 and Ler, when matured at 23°C (AlonsoBlanco et al., 2003; Ali-Rachedi et al., 2004; Preston et al., 2009). To extensively investigate natural variation for seed dormancy and perform GWA mapping, we analyzed germination in 117 accessions using seeds after-ripened for 2 months. Of our 117 accessions, 108 originated from European countries, while the others originated from other regions of the world (Figure 1a and Data S1). When freshly harvested seeds were after-ripened for 2 months, then imbibed on water/agarose at 23°C in the light, germination frequencies differed substantially among the 117 accessions after 3 and 7 days of imbibition (Figure 1b). Thirtytwo accessions, including Col-0 and Ler-1, germinated at rates higher than 90% after 7 days imbibition, but 27 accessions, including Cvi-0, germinated at rates lower than 10%, indicating that the former accessions had almost completely lost seed dormancy during AR but that the latter
© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd, The Plant Journal, (2013), 74, 815–828
HD2B is associated with seed dormancy 817
(a)
the highest (R2 = 0.215; Figure S1a,b). These SNPs were located on the HD2B gene (At5g22650), and were detected as significantly associated SNPs whichever phenotype parameters were used for GWA mapping (Figure 2b–d; arrows). We also detected strongly associated SNPs around the middle region of chromosome 3 (Figure 2b–d; inverted T symbols), where the DOG6 QTL has been previously reported (Bentsink et al., 2010). In addition, SNPs within 500 kb of DOG1 were detected as significantly associated when arcsine- or logit-transformed parameters were used for EMMAX calculation (Figure 2c,d; arrowheads).
(b)
Integration of GWA mapping and transcriptome analyses identified HD2B as the most plausible candidate associated with seed dormancy
Figure 1. Natural variation for seed dormancy in Arabidopsis seeds that were after-ripened for 2 months. (a) Geographic origins of the 117 Arabidopsis accessions used in this study. (b) Natural variation for seed dormancy in the 117 accessions. Seeds were after-ripened for 2 months and then imbibed at 23°C in the light. Germination frequencies were analyzed after 3 or 7 days imbibition. Error bars represent the SD of four biological replicates. Accessions with germination frequencies less than 10% or more than 90% after 7 days imbibition were designated as dormant or less-dormant, respectively.
retained dormancy after 2 months of AR. We designated accessions with germination >90% as less-dormant and those with germination
