Arabidopsis leaf plasma membrane proteome using a gel free method: Focus on receptor-like kinases

Abstract The hydrophobic proteins of plant plasma membrane still remain largely unknown. For example in the Arabidopsis genome, receptor-like kinases (RLKs) are plasma membrane proteins, functioning as the primary receptors in the signaling of stress conditions, hormones and the presence of pathogens form a diverse family of over 610 genes. A limited number of these proteins have appeard in protein profiles. The detection of these proteins and thus the determination of their dynamics and tissue specificity, is technically challenging due to their low abundance and association to a lipid membrane. To identify new putative membrane proteins especially receptor systems, we used a gel free proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis leaf a highly purified plasma membrane fraction with the aqueous two-phase partitioning technique. By separating the proteins in the plasma membrane fraction with ion exchange and reverse phase chromatography and analyzing the resulting fractions on a MALDI-TOF mass spectrometer, over 900 proteins were detected. The plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, onethird displaying at least four trans-membrane segments. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic and metabolism. Of these proteins, 304 were annotated as membrane proteins, 69 were RLKs, distributed among the different receptor families in proportions reflecting the distribution in the genome. Of the RLKs that were identified, most are reported for the first time at the protein level and will constitute interesting targets for further functional studies

Genome-wide analysis of potassium channel genes in rice: expression of the osakt and oskat genes under salt stress

Potassium (K+), as a vital element, is involved in regulating important cellular processes such as enzyme activity, cell turgor, and nutrient movement in plant cells, which affects plant growth and production. Potassium channels are involved in the transport and release of potassium in plant cells. In the current study, three OsKAT genes and two OsAKT genes, along with 11 nonredundant putative potassium channel genes in the rice genome, were characterized based on their physiochemical properties, protein structure, evolution, duplication, in silico gene expression, and protein–protein interactions. In addition, the expression patterns of OsAKTs and OsKATs were studied in root and shoot tissues under salt stress using real-time PCR in three rice cultivars. K+ channel genes were found to have diverse functions and structures, and OsKATs showed high genetic divergence from other K+ channel genes. Furthermore, the Ka/Ks ratios of duplicated gene pairs from the K+ channel gene family in rice suggested that these genes underwent purifying selection. Among the studied K+ channel proteins, OsKAT1 and OsAKT1 were identified as proteins with high potential N-glycosylation and phosphorylation sites, and LEU, VAL, SER, PRO, HIS, GLY, LYS, TYR, CYC, and ARG amino acids were predicted as the binding residues in the ligand-binding sites of K+ channel proteins. Regarding the coexpression network and KEGG ontology results, several metabolic pathways, including sugar metabolism, purine metabolism, carbon metabolism, glycerophospholipid metabolism, monoterpenoid biosynthesis, and folate biosynthesis, were recognized in the coexpression network of K+ channel proteins. Based on the available RNA-seq data, the K+ channel genes showed differential expression levels in rice tissues in response to biotic and abiotic stresses. In addition, the real-time PCR results revealed that OsAKTs and OsKATs are induced by salt stress in root and shoot tissues of rice cultivars, and OsKAT1 was identified as a key gene involved in the rice response to salt stress. In the present study, we found that the repression of OsAKTs, OsKAT2, and OsKAT2 in roots was related to salinity tolerance in rice. Our findings provide valuable insights for further structural and functional assays of K+ channel genes in rice