The Plant Journal (2010) 61, 611–622
doi: 10.1111/j.1365-313X.2009.04088.x
Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds Jose´ M. Barrero1,†, Anthony A. Millar2,†, Jayne Griffiths1, Tomasz Czechowski3, Wolf R. Scheible3, Michael Udvardi3, John B. Reid4, John J. Ross4, John V. Jacobsen1 and Frank Gubler1,* 1 CSIRO Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia, 2 Australian National University, Canberra ACT 2601, Australia, 3 Max-Planck Institute of Molecular Plant Physiology, Am Mu¨hlenberg 1, 14476 Golm, Germany, and 4 School of Plant Science, University of Tasmania, Tasmania, Australia Received 13 August 2009; revised 14 October 2009; accepted 9 November 2009; published online 31 December 2009. * For correspondence (fax +612 6246 5000; e-mail
[email protected]). † These authors contributed equally to this work.
SUMMARY Seed dormancy is a very important trait that maximizes the survival of seed in nature, the control of which can have important repercussions on the yield of many crop species. We have used gene expression profiling to identify genes that are involved in dormancy regulation in Arabidopsis thaliana. RNA was isolated from imbibed dormant (D) and after-ripened (AR) ecotype C24 seeds, and then screened by quantitative RT-PCR (qRT-PCR) for differentially expressed transcription factors (TFs) and other regulatory genes. Out of 2207 genes screened, we have identified 39 that were differentially expressed during the first few hours of imbibition. After analyzing T-DNA insertion mutants for 22 of these genes, two displayed altered dormancy compared with the wild type. These mutants are affected in genes that encode a RING finger and an HDZip protein. The first, named DESPIERTO, is involved in ABA sensitivity during seed development, regulates the expression of ABI3, and produces a complete loss of dormancy when mutated. The second, the HDZip (ATHB20), is expressed during seed germination in the micropylar endosperm and in the root cap, and increases ABA sensitivity and seed dormancy when mutated. Keywords: seed dormancy, germination, ABA, transcription factor profiling, ATHB20.
INTRODUCTION The transition from dormancy to germination in seeds is a major control point that plants must transgress during their life cycle. This event is critical to the survival of the species, and is governed by a complex interaction of environmental and genetic factors (Finch-Savage and Leubner-Metzger, 2006; Finkelstein et al., 2008; Holdsworth et al., 2008a). Because of this complexity, the mechanisms governing dormancy acquisition during seed development and dormancy decay during after-ripening (a process that occurs during dry storage, through which dormancy is lost) are not fully understood, even though they have major consequences in both nature and agriculture (Gubler et al., 2005). Several approaches have been used to dissect the genetic pathways leading to dormancy acquisition, maintenance and decay (reviewed in Koornneef et al., 2002 and in Holdsworth et al., 2008b). Mutagenesis has yielded several ª 2009 CSIRO Journal compilation ª 2009 Blackwell Publishing Ltd
mutants that display a dormancy-related phenotype in several species. The study of these mutants has revealed the importance of factors like abscisic acid (ABA), gibberellins, light, temperature, nutrition and seed coat, and of processes like epigenetic regulation, RNA stability and protein degradation. However, the highly pleiotropic phenotypes of many of these mutants make it difficult to understand the roles of the affected genes in dormancy and germination. A number of global gene expression analyses have been performed in Arabidopsis and in barley, comparing dormant (D) with after-ripened (AR) seeds (Nakabayashi et al., 2005; Cadman et al., 2006; Finch-Savage et al., 2007; Carrera et al., 2008; Barrero et al., 2009). These studies highlight the importance of ABA- and stress-related genes in the D condition, and of genes related to protein degradation, 611
612 Jose M. Barrero et al. reserve mobilization and cell-wall modification in the AR condition. Despite the identification of numerous differentially expressed genes by array analyses, there is little direct evidence for their roles in dormancy. The interpretation of previous results is complex because the expression of genes related to dormancy release may overlap with the expression of genes involved in the germination process, especially if long imbibition times are used. This has the effect of masking the master genes that are directly related to dormancy release, and identifying many downstream genes that are not directly involved in dormancy control. Some reports have clearly shown the importance of early events during dormancy release, such as CYP707A2 induction, which reduces ABA levels in Arabidopsis seeds after 6 h of hydration (Millar et al., 2006), or the recently reported production of nitric oxide in the aleurone 3 h after imbibition (Liu et al., 2009). In this paper we report the use of an extended highthroughput quantitative RT-PCR (qRT-PCR) platform for comparing the expression of 2207 genes, including all Arabidopsis transcription factors (TFs) and other regulatory genes, between D and AR seeds during the first 45 min and 3 h of imbibition. This approach provides increased sensitivity to detect differences in the expression of dormancy-related TFs, compared with microarray experiments, allowing the identification of ‘master’ genes regulating dormancy. We identified 39 genes that are differentially expressed between D and AR seeds. Only two of them, a RING finger-encoding gene named DESPIERTO (DEP) and the HDZip gene ATHB20, displayed altered dormancy when mutated, indicating a high degree of gene redundancy and
the high robustness of the dormancy program. Using physiological assays and gene expression analyses we have characterized DEP and ATHB20, reaching the conclusion that these genes are involved in ABA sensitivity during seed development and germination, respectively. RESULTS Transcription factor profiling of D and AR seeds The Arabidopsis ecotype C24 produces dormant seeds that require a period of several months at room temperature to fully after-ripen (Koornneef et al., 2000; Millar et al., 2006). We used D (
