Arabidopsis Raf-Like Kinase Raf10 Is a Regulatory Component of Core ABA Signaling.

Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABAregulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.

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.

AtERF15 is a positive regulator of ABA response.

KEY MESSAGE: The Arabidopsis AP2/ERF family transcription factor AtERF15 is nuclear-localized and positively regulates ABA and stress responses. Abscisic acid (ABA) is a major plant hormone that controls the expression of hundreds genes involved in various aspects of plant growth and development, such as seed development, germination, seedling growth and abiotic stress response. Several cis-elements mediating the ABA-regulated gene expression have been reported, and one of the regulatory elements is Coupling Element 1 (CE1). We previously isolated a group of AP2/ERF family proteins that bind CE1, but their functions are mostly unknown. In this study, we demonstrate that one of the CE1 binding factors (CEBFs), AtERF15, is involved in ABA response. To investigate the AtERF15 function, we generated its overexpression (OX) lines by expressing the AtERF15 coding region under the control of CaMV 35S promoter and analyzed their phenotypes. We found that the AtERF15 OX lines were hypersensitive to ABA at the germination stage. The ABA hypersensitivity was also observed in our root elongation assay of seedlings. Furthermore, the transgenic lines were hypersensitive to high salinity and high osmolarity at the seedling establishment stage, and the transgenic seedlings were drought-tolerant. We also determined the tissue-specific expression pattern and the subcellular localization of AtERF15. Our results indicate that it is highly expressed in roots and embryos and nuclear-localized. Collectively, our data suggest that AtERF15 is a positive regulator of ABA response.

ATHB17 is a positive regulator of abscisic acid response during early seedling growth.

We performed activation tagging screen to isolate abscisic acid (ABA) response mutants. One of the mutants, designated ahs10 (ABA-hypersensitive 10), exhibited ABA-hypersensitive phenotypes. TAIL-PCR analysis of the mutant revealed that T-DNA was inserted in the promoter region of the Arabidopsis gene, At2g01430, which encodes a homeodomain-leucine zipper protein ATHB17. Subsequent expression analysis indicated that ATHB17 was activated in ahs10. To recapitulate the mutant phenotypes, we prepared ATHB17 OX lines and investigated their phenotypes. The results showed that ATHB17 confers ABA-hypersensitivity and drought tolerance. On the contrary, ATHB17 knockout lines were ABA-insensitive and drought-sensitive, further demonstrating that ATHB17 is involved in ABA and water-stress responses. Interestingly, the ATHB17 effect on seedling growth in the presence of ABA was observed only during the postgermination seedling establishment stage, suggesting that it functions during a narrow developmental window of early seedling growth.

Arabidopsis zinc finger proteins AtC3H49/AtTZF3 and AtC3H20/AtTZF2 are involved in ABA and JA responses.

There are 68 CCCH zinc finger protein genes in the Arabidopsis genome. However, only a few of them have been characerized functionally. In this paper, we report the function of two Arabidopsis CCCH zinc finger proteins AtC3H49/AtTZF3 and AtC3H20/AtTZF2. To investigate their functions, we examined their expression patterns and analyzed their overexpression and knockout(KO)/RNA interference (RNAi) phenotypes. Both AtC3H49/AtTZF3 and AtC3H20/AtTZF2 genes were expressed in various vegetative tissues and in flowers, and their encoded proteins were localized in the cytoplasm. Overexpression of AtC3H49/AtTZF3 or AtC3H20/AtTZF2 conferred ABA hypersensitivity, reduced transpiration and enhanced drought tolerance. Their overexpression also altered the plant growth pattern. The transgenic plants grew slowly during the early stage of growth, but their growth rates were accelerated at later stages, and mature plants were larger than the wild-type plants. Moreover, the transgenic plants displayed delayed senescence and enhanced longevity. Subsequent experiments showed that jasmonic acid (JA)-induced senescence was also delayed. Microarray and quantitative reverse transcription-PCR analyses indicated that the expression of a number of genes involved in JA, ABA and biotic/abiotic stress responses was altered in the transgenic lines. Recombinant AtC3H49/AtTZF3 and AtC3H20/AtTZF2 proteins displayed RNase activity in vitro, suggesting that they may be involved in mRNA turnover process. The knockout/RNAi lines of AtC3H49/AtTZF3 and AtC3H20/AtTZF2 exhibited weak phenotypes, presumably because of their functional redundancy.

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.

AtNEK6 interacts with ARIA and is involved in ABA response during seed germination.

ARIA is an ARM repeat protein that is a positive regulator of ABA response. To identify ARIA-interacting proteins, we conducted yeast two-hybrid screening. One of the positive clones obtained from the screen encoded a protein kinase, AtNEK6, which belongs to the NIMA (Never In Mitosis, gene A)-related kinase family. We analyzed AtNEK6 over-expression (OX) and knockout (KO) lines to investigate its in vivo function. The AtNEK6 OX lines grew slowly, whereas the KO line germinated and grew faster than wild type plants. AtNEK6 also affected ABA and stress responses. During seed germination, AtNEK6 OX lines were hypersensitive to ABA and high osmolarity, whereas its KO line was partially insensitive to ABA and high osmolarity. Previously, AtNEK6 was shown to be involved in epidermal cell morphogenesis. Our results indicate that AtNEK6 is also involved in plant growth regulation and responses to ABA and high osmolarity during the seed germination stage.

DREB2C interacts with ABF2, a bZIP protein regulating abscisic acid-responsive gene expression, and its overexpression affects abscisic acid sensitivity.

ABF2 is a basic leucine zipper protein that regulates abscisic acid (ABA)-dependent stress-responsive gene expression. We carried out yeast two-hybrid screens to isolate genes encoding ABF2-interacting proteins in Arabidopsis (Arabidopsis thaliana). Analysis of the resulting positive clones revealed that two of them encode an AP2 domain protein, which is the same as AtERF48/DREB2C. This protein, which will be referred to as DREB2C, could bind C-repeat/dehydration response element in vitro and possesses transcriptional activity. To determine its function, we generated DREB2C overexpression lines and investigated their phenotypes. The transgenic plants were ABA hypersensitive during germination and seedling establishment stages, whereas primary root elongation of seedlings was ABA insensitive, suggesting developmental stage dependence of DREB2C function. The DREB2C overexpression lines also displayed altered stress response; whereas the plants were dehydration sensitive, they were freezing and heat tolerant. We further show that other AP2 domain proteins, DREB1A and DREB2A, interact with ABF2 and that other ABF family members, ABF3 and ABF4, interact with DREB2C. Previously, others demonstrated that ABF and DREB family members cooperate to activate the transcription of an ABA-responsive gene. Our result implies that the cooperation of the two classes of transcription factors may involve physical interaction.

An ARIA-interacting AP2 domain protein is a novel component of ABA signaling.

ADAP is an AP2-domain protein that interacts with ARIA, which, in turn, interacts with ABF2, a bZIP class transcription factor. ABF2 regulates various aspects of the abscisic acid (ABA) response by controlling the expression of a subset of ABA-responsive genes. Our expression analyses indicate that ADAP is expressed in roots, emerging young leaves, and flowers. We found that adap knockout mutant lines germinate more efficiently than wild-type plants and that the mutant seedlings grow faster. This suggests that ADAP is involved in the regulation of germination and seedling growth. Both germination and post-germination growth of the knockout mutants were partially insensitive to ABA, which indicates that ADAP is required for a full ABA response. The survival rates for mutants from which water was withheld were low compared with those for wild-type plants. The result shows that ADAP is necessary for the response to stress induced by water deprivation. Together, our data indicate that ADAP is a positive regulator of the ABA response and is also involved in regulating seedling growth. The role of ADAP is similar to that of ARIA, which is also a positive regulator of the ABA response. It appears that ADAP acts through the same ABA response pathway as ARIA.