Arabidopsis putative MAP kinase kinase kinases Raf10 and Raf11 are positive regulators of seed dormancy and ABA response.

We isolated an Arabidopsis ABA-insensitive mutant, ais143, by activation tagging screen. T-DNA was found to be located in the coding region of a putative mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) gene, Raf10, thereby abolishing its expression in the mutant. ais143 exhibited reduced seed dormancy as well as reduced ABA sensitivity. The phenotypes were complemented by the wild-type Raf10 gene, and the overexpression (OX) of Raf10 resulted in delayed seed germination and enhanced ABA sensitivity. Raf10 has high sequence identity to another MAP3K, Raf11. Parallel analysis of Raf11 knockout (KO) and OX lines showed that their phenotypes were similar to those of Raf10 KO and OX lines. An ais143 raf11 double mutant exhibited stronger phenotypes than single mutants, indicating the functional redundancy between Raf10 and Raf11. Transcript analysis revealed that the expression of many ABA-associated genes, including the key regulatory genes ABI3 and ABI5, was altered in the Raf10 and Raf11 OX lines. Recombinant Raf10 and Raf11 proteins exhibited kinase activity, which was inhibited by the MAP3K inhibitor BAY 43-9006 but not by the MAP2K inhibitor U0126. Collectively, our data indicate that Raf10 and Raf11 kinases are important regulators of seed dormancy and ABA response and that they affect the expression of ABI3, ABI5 and other ABA-regulated genes.

Isolation and functional characterization of CE1 binding proteins.

BACKGROUND: Abscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. RESULTS: To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. CONCLUSIONS: Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions of other CEBFs remain to be determined. Our in vivo functional analysis of several CEBFs suggests that they are likely to be involved in ABA and/or sugar response. Together with previous results reported by others, our current data raise an interesting possibility that the coupling element CE1 may function not only as an ABRE but also as an element mediating biotic and abiotic stress responses.