Abscisic acid (ABA) is a phytohormone that regulates many physiological functions, such as plant growth, development and stress responses. The MAPK cascade plays an important role in ABA signal transduction. Several MAPK and MAPKK molecules are reported to function in ABA signaling; however, there have been few studies related to the identification of MAPKKK upstream of MAPKK in ABA signaling. In this study, we show that an Arabidopsis MAPKKK, MAPKKK18 functions in ABA signaling. The expression of MAPKKK18 was induced by ABA treatment. Yeast two-hybrid analysis revealed that MAPKKKK18 interacted with MKK3, which interacted with C-group MAPK, MPK1/2/7. Immunoprecipitated kinase assay showed that the 3xFlag-tagged MAPKKK18, expressed in Arabidopsis plants, was activated when treated with ABA. These results indicate the possibility that the MAPK cascade is composed of MAPKKK18, MKK3 and MPK1/2/7 in ABA signaling. The transgenic plants overexpressing MAPKKK18 (35S:MAPKKK18) and its kinase negative mutant (35S:MAPKKK18 KN) were generated, and their growth was monitored. Compared with the WT plant, 35S:MAPKKK18 and 35S:MAPKKK18 KN showed smaller and bigger phenotypes, respectively. Senescence of the rosette leaves was promoted in 35S:MAPKKK18, but suppressed in 35S:MAPKKK18 KN. Furthermore, ABA-induced leaf senescence was accelerated in 35S:MAPKKK18. These results suggest that MAPKKK18 controls the plant growth by adjusting the timing of senescence via its protein kinase activity in ABA dependent manners.

The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca(2+) signaling occurred upstream of the MEKK1-MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.

a b s t r a c tThe MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.

Regulation of cell proliferation is crucial for establishing the shape of plant leaves. We have identified (), a loss-of-function mutant of which exhibits a narrowed- and rolled-leaf phenotype in rice. was found to be an ortholog of (), which positively regulates cell proliferation. The reduced leaf size of plants with enlarged cells and the increased size ofoverexpressing leaves with normal-sized cells indicate that is a positive regulator of leaf proliferation and that mutation triggers a compensation syndrome, as does Expression analysis revealed that is predominantly expressed on the epidermis of leaf primordia, which is different from the location of A protein movement assay demonstrated that MKB3 moves from anexpressing domain to a non-expressing domain, which is required for normal leaf development. Our results suggest that rice and have conserved functions and effects on leaf development. However, the expression pattern of and direction of protein movement are different between rice and, which might reflect differences in leaf primordia development in these two species.

Mitogen-activated protein kinase (MAPK) cascades play important roles in plant responses to various environmental stimuli, including high salt or drought levels. Arabidopsis MAP3Kδ4 with PAS (period circadian protein, arylhydrocarbon receptor nuclear translocator protein and single-minded protein) domain is one of the Raftype MAPKKKs whose function has not been identified to date. Previous studies have shown that the MAP3Kδ4 overexpressing transformant exhibits vigorous growth. In this study, RT-PCR analysis showed that MAP3Kδ4 transcripts are increased through stress treatments, such as high salt, osmosis, drought and cold and the plant hormone, abscisic acid (ABA). The precipitation of MAP3Kδ4 using its specific antibody showed that ABA treatment markedly induces the activity of this enzyme. Furthermore, the ABA-mediated inhibition of seed germination was relieved in transgenic Arabidopsis over-expressing MAP3Kδ4. These results suggested that MAP3Kδ4 plays an important role in ABA signalling. Transgenic Arabidopsis over-expressing MAP3Kδ4 also exhibited enhanced tolerance to salt stress. The results obtained in this study demonstrated that MAP3Kδ4 was active during ABA-related responses and is involved in both stress tolerance and increased biomass. Therefore, MAP3Kδ4 and its counterpart genes are important with respect to agricultural developments.