| Summary: | 博士 === 中興大學 === 生命科學系所 === 95 === The halophyte Mesembryanthemum crystallinum L. (ice plant) is an inducible CAM and a model plant for studying salt-tolerant mechanisms in higher plants. It contains specialized epidermal bladder cells (EBCs) which rapidly expend under salt stress. The major functions of EBCs are maintaining ion homeostasis and water storage. During plant development, increase in vacuolar pH was observed in EBCs. In addition, light-induced rapid change in vacuolar pH was found in juvenile stage of ice plant indicating EBCs are metabolic active cells. A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) is highly expressed in EBCs. It has high homology to yeast VPS4 (vacuolar protein sorting 4) and Arabidopsis AAA-type ATPase (ATPase associated with a variety of cellular activities). Immunofluorescence labeling showed mcSKD1 protein was located in cytoplasm around ER/Golgi network. Yeast two-hybrid screen was performed to identify mcSKD1-interacting proteins. Using a library constructed from Arabidopsis, catalase 3 and AGP21 (arabinogalactan protein 21) were identified. Using a library constructed from salt-treated ice plant roots, mcSNF1 (sucrose non-fermenting 1) and mcCPN1 (copine 1) were further characterized. Yeast SNF1 is a ser/thr kinase that plays an important role in carbon metabolism and stress signaling. The SNF1 protein identified in ice plant contained an extra UBA (ubiquitin associated) domain. Sequence analysis of mcCPN1 showed it contains a VWA domain for membrane trafficking and a RING domain for protein ubiquitination. The accumulation of mcSNF1 and mcCPN1 was both induced by salt stress. Co-immunoprecipitation experiment showed mcSKD1 interacted with both mcSNF1 and mcCPN1 in vitro. In vivo pull-down assay showed a 3-fold increase in the association between mcSKD1 and mcSNF1 under salt stress. Microsomal fractionation and immunolabeling experiments showed salt induced rapid changes in cellular localization of mcSKD1, mcSNF1, and mcCPN1. When the cytoskeleton was disrupted, the distribution of mcSKD1 and mcSNF1 was altered, as seen by abnormal aggregation around plasma membrane. The result suggested that mcSKD1 is trafficking along the cytoskeleton. Arabidopsis snf1 and copine 1 mutants showed more salt sensitive than the wild type and aggregation of atSKD1 inside mutant cells. The results suggested that mcSNF1 and mcCPN1 function together with mcSKD1 in ubiquitin-related protein trafficking and stress signal transduction in order to maintain normal growth under high salinity.
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