Mammalian circadian rhythms are generated by a feedback loop in which BMAL1 and CLOCK, players of the positive limb, activate transcription of the cryptochrome and period genes, components of the negative limb. Bmal1 and Per transcription cycles display nearly opposite phases and are thus governed by different mechanisms. Here, we identify the orphan nuclear receptor REV-ERBalpha as the major regulator of cyclic Bmal1 transcription. Circadian Rev-erbalpha expression is controlled by components of the general feedback loop. Thus, REV-ERBalpha constitutes a molecular link through which components of the negative limb drive antiphasic expression of components of the positive limb. While REV-ERBalpha influences the period length and affects the phase-shifting properties of the clock, it is not required for circadian rhythm generation.

Seed dormancy is a trait of considerable adaptive significance because it maximizes seedling survival by preventing premature germination under unfavorable conditions. Understanding how seeds break dormancy and initiate growth is also of great agricultural and biotechnological interest. Abscisic acid (ABA) plays primary regulatory roles in the initiation and maintenance of seed dormancy. Here we report that the basic leucine zipper transcription factor ABI5 confers an enhanced response to exogenous ABA during germination, and seedling establishment, as well as subsequent vegetative growth. These responses correlate with total ABI5 levels. We show that ABI5 expression defines a narrow developmental window following germination, during which plants monitor the environmental osmotic status before initiating vegetative growth. ABI5 is necessary to maintain germinated embryos in a quiescent state thereby protecting plants from drought. As expected for a key player in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability, and activity are highly regulated by ABA during germination and early seedling growth.A bscisic acid (ABA) is a phytohormone regulating the initiation and maintenance of seed dormancy. It also plays an essential role in a plant's response to stress, particularly water deprivation, notably by regulating stomatal aperture (1). So far, ABA-insensitive screens have been widely used to identify molecular genetic components of the ABA signal transduction pathway (2, 3). In these screens, mutagenized Arabidopsis seeds were exposed to ABA concentrations that inhibit germination of wild-type (WT) seeds, and putative mutants that were able to germinate were isolated (2, 3). These screens have allowed the identification of several ABI (ABA-insensitive) genes (4-10) and recent studies have established that ABI1 and ABI3 are key players in vegetative and embryonic ABA responses, respectively (1, 11).Nonetheless, few reports have clarified the physiological role of ABA and mechanisms of action triggered by ABA during germination and early seedling growth. We were led to address these issues by the recent cloning and analysis of ABI5 by two independent groups (4, 5). The abi5 mutation is recessive and ABI5 encodes a putative transcription factor of the basic leucine zipper (bZIP) family (4, 5). The bZIP region of ABI5 shows extensive homology to previously characterized plant (bZIP) transcription factors capable of activating reporter genes containing ABA-responsive DNA elements (ABREs) (12)(13)(14). ABI5 also binds to ABREs in vitro (unpublished results) and dry seeds of abi5 show reduced transcript levels of ABA-responsive and ABRE-containing late embryonic genes such as AtEm1 and AtEm6 (4, 5). Together with the ABA insensitivity of abi5 mutants, these results show that ABI5 is the first bZIP plant factor found to be required in vivo to signal ABA-elicited responses.In the present work, we found that ABA regulates ABI5 accumulation and activity during a limited developmental window. On re...

SummaryThe development of a germinating embryo into an autotrophic seedling is arrested under conditions of water deficit. This ABA-mediated developmental checkpoint requires the bZIP transcription factor ABI5. Here, we used abi3-1, which is also unable to execute this checkpoint, to investigate the relative role of ABI3 and ABI5 in this process. In wild-type Arabidopsis plants, ABI3 expression and activity parallel those described for ABI5 following stratification. During this process, transcript levels of late embryogenesis genes such as AtEm1 and AtEm6 are also re-induced, which might be responsible for the acquired osmotic tolerance in germinated embryos whose growth is arrested. ABI5 expression is greatly reduced in abi3-1 mutants, which has low AtEm1 or AtEm6 expression. Cross complementation experiments showed that 35S-ABI5 could complement abi3-1, whereas 35S-ABI3 cannot complement abi5-4. These results indicate that ABI5 acts downstream of ABI3 to reactivate late embryogenesis programmes and to arrest growth of germinating embryos. Although ABI5 is consistently located in the nucleus, chromosomal immunoprecipitation (ChIP) experiments revealed that ABA increases ABI5 occupancy on the AtEm6 promoter.

Seed germination is antagonistically controlled by the phytohormones gibberellic acid (GA) and abscisic acid (ABA). GA promotes seed germination by enhancing the proteasome-mediated destruction of RGL2 (for RGA-LIKE2), a key DELLA factor repressing germination. By contrast, ABA blocks germination by inducing ABI5 (for ABA-INSENSITIVE5), a basic domain/leucine zipper transcription factor repressing germination. Decreased GA synthesis leads to an increase in endogenous ABA levels through a stabilized RGL2, a process that may involve XERICO, a RING-H2 zinc finger factor promoting ABA synthesis. In turn, increased endogenous ABA synthesis is necessary to elevate not only ABI5 RNA and protein levels but also, critically, those of RGL2. Increased ABI5 protein is ultimately responsible for preventing seed germination when GA levels are reduced. However, overexpression of ABI5 was not sufficient to repress germination, as ABI5 activity requires phosphorylation. The endogenous ABI5 phosphorylation and inhibition of germination could be recapitulated by the addition of a SnRK2 protein kinase to the ABI5 overexpression line. In sleepy1 mutant seeds, RGL2 overaccumulates; germination of these seeds can occur under conditions that produce low ABI5 expression. These data support the notion that ABI5 acts as the final common repressor of germination in response to changes in ABA and GA levels.

Several Arabidopsis mutants have been isolated that either bypass this ABA-mediated growth arrest or show an enhanced response to ABA (for review, see Finkelstein et al. 2002). The latter category includes mutations in ERA1, which encodes a subunit of a farnesyl transferase (Cutler et al. 1996), and ABH1, which encodes a mRNA cap binding protein (Hugouvieux et al. 2001). Mutants bypassing the ABA-mediated growth inhibition were designated abi1, abi2, abi3, abi4, abi5, abi8 (for ABAinsensitive), cho1, and cho2 (Koornneef et al. 1984;Finkelstein 1994;Lopez-Molina and Chua 2000;Finkelstein et al. 2002;Nambara et al. 2002). The ABI8, CHO1, and CHO2 genes have not yet been cloned Nambara et al. 2002). ABI1 and ABI2 encode two homologous serine/threonine phosphatases of class 2C (Leung et al. 1994(Leung et al. , 1997Meyer et al. 1994;Rodriguez et al. 1998), and both abi1 and abi2 have identical Gly-