Rice On The Cell Membrane Non-specific Lipid Transfer Protein Receptor

Nonspecific lipid transfer protein (nsLTP) is a class of low molecular weight, basic proteins, consisting of fourα-helices fold stabilized by four intramolecular disulfide bonds which provides a hydrophobic cavity for lodging a large variety of lipids and hydrophobic molecules. So far, nsLTP was proposed to promote the spreading of systemic acquired resistance signal in arabidopsis, and induce the adhesion of the pollen tube to an artificial stylar matrix in vitro, suggesting a receptor-like molecule likely exists in the cell membranes. In this study, using green fluorescence protein (GFP) as a visual marker, rice nsLTP144 was shown to be mainly located in the cell wall but partly associated with the cell membranes. The fluorescence signal on the cell membranes was not uniform, which suggests nsLTP144 could bind to the protein molecule(s) in the cell membranes. To test this hypothesis, recombinant protein Trx-nsLTP144 /Trx-nsLTP110 was labeled with iodine-125 and incubated with rice plasma membranes to determine if the protein can bind to rice cell membranes. The binding assay indicated that the binding was specific, saturable and could be competed by the unlabeled protein, suggesting that there are specific binding sites on rice plasma membranes. Protease treatment of the plasma membranes could abolish the binding but NaIO₄ and glycosidase could not, indicating that nsLTP144 bound to plasma membranes protein(s) without carbohydrate moiety. Using the homobifunctional cross-linking regent bis(sulfosuccinimidyl)suberate (BS³) and rice plasma membranes incubated with ¹²⁵I-Trx-nsLTP144, we identified, after SDS-polyacrylamide gel electrophoresis and autoradiography, a putative protein receptor on the rice plasma membranes with the molecular mass around 60 kDa. NsLTP144 can not trigger extracelluar alkalization in arabidopsis, but can abolish the extracellular alkalization effect of phytopathogen elicitor cryptogein, suggesting that cryptogein and nsLTP144 may bind to the same membrane protein(s). In vitro pull-down assay showed that nsLTP144 interacted with OsCaM1, a possible extracellular calmodulin, implying that nsLTP144 and OsCaM1 could function in the same signal transduction pathway. These results shed light on revealing the roles of nsLTP in vivo and make it promising to finally characterize the plasma membranes receptor of nsLTP. The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE). Ten protein spots with strikingly higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry (MS) analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.