Functional Characterization Of Two MAPKKK Genes In Rice

Rice has been recognized as a model plant for researches of cereal species and one of the most important agronomical crops in the world. Mitogen-activated protein kinase (MAPK) cascades (MAPKKK-MAPKK-MAPK sequencial phosphorylation) have been suggested to be involved in various signaling pathways for stress responses and plant development. However, very few genes in the MAPK cascades have been characterized in rice. Here, a drought hypersensitive T-DNA mutant (dsm1) and an increased leaf angle T-DNA mutant (ila1) were identified in rice. Both the mutated genes (DSM1 and ILA1) encode Raf-like MAPK kinase kinase (MAPKKK) in rice. This study focused on the functional analysis of these two genes, and the main results are as follows.Two allelic dsm1 mutants were more sensitive than wild type plants to drought stress at both seedling and panicle development stages. The dsm1 mutants lost water more rapidly than wild-type plants under drought stress, which was in agreement with the increased drought sensitivity phenotype of the mutant plants. DSM1-RNA interference lines were also hypersensitive to drought stress. The predicted DSM1 protein belongs to a B3 subgroup of plant Raf-like MAPKKKs and was localized in the nucleus. Kinase assay demonstrated that MBP was strongly phosphorylated by the DSM1 kinase domain (KD) and that the DSM1 KD had autophosphorylation activity regardless of the presence or absence of MBP. By real-time PCR analysis, the DSM1 gene was induced by salt, drought, and abscisic acid, but not by cold. Microarray analysis revealed that two peroxidase (POX) genes, POX22.3 and POX8.1, were sharply down-regulated compared to wild type, suggesting that DSM1 may be involved in reactive oxygen species (ROS) signaling. Peroxidase activity, electrolyte leakage, chlorophyll content, and 3,3’-diaminobenzidine staining revealed that the dsm1 mutant was more sensitive to oxidative stress due to an increase in ROS damage caused by the reduced POX activity. Overexpression of DSM1 in rice increased the tolerance to dehydration stress at the seedling stage.Leaf angle of the ila1 mutant was greatly increased at tillering and heading stages compared with the wild type. Complementation experiment suggested that the increased leaf angle phenotype was caused by disruption of the ILA1 gene. Paraffin sections analysis of leaf lamina showed that the ILA1 gene is involved in sclerenchymatous cell development. ILA1 was a nuclear localized protein and specifically expressed in leaf lamina. Kinase assay demonstrated that MBP was strongly phosphorylated by the ILA1 and that the ILA1 also had autophosphorylation activity. Using a yeast two-hybrid screen, we identified six uncharacterized proteins belonging to the same family as ILA1 interacting proteins (IIPs). We also demonstrated that IIP2 and IIP4 physically interact with ILA1 by using BiFC and Co-IP. IIP2 and IIP4 were also nuclear localized proteins and showed transcription activity in yeast, suggesting that they may function as transcription factors.