Expression of genes and poteins involving salt-tolerance mechanisms in rice Oryza sativa L. analyzed by cDNA-AFLP and proteomics

Abstract

Plants respond to environmental stress with a number of physiological and biochemical changes. High salinity is one of the most important stresses severely limiting growth and development of rice (Oryza sativa L.), and thus reducing its crop productivity. The metabolic pathways for salt stress responses are likely to be very complicated. In this study, molecular markers associated with salt stress of O. sativa were identified. Differential expression profiles of transcripts in salt-tolerant (Homjan) and salt-susceptible (Pathumthani 1) varieties under a time course of salt stress treatment were examined by RNA arbitrary-primed (RAP)-PCR. Subsequently, salt-tolerant (Pokkali) and salt-susceptible (IR29) varieties were used for complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) analysis. Several specific/differential expression transcripts were observed. One transcripts from RAP-PCR and seven transcripts from cDNA-AFLP were cloned and sequenced. A RAP-PCR fragment significantly match protein kinase domain containing protein while three cDNA-AFLP fragments significantly similar to lipin-N terminal conserve region (Lpin), ATP synthase subunit C (ATPsubc) and cytochrome P450 monooxygenase (CYP450), hypothetical protein₂₄₀ , hypothetical protein₁₈₆, hypothetical protein₂₀₆ and hypothetical protein₁₇₁, respectively. The expression levels of three known transcripts and that of hypothetical protein₁₇₁ (HTP₁₇₁) (from cDNA-AFLP) upon salt stress treatment in both Pokkali and IR29 varieties were examined by quantitative real-time PCR and semi-quantitative RT-PCR, respectively. Hypothetical protein217 (HTP217) were used for internal standard. Proteomic studies based on one-dimensional gel electrophoresis and LC-MS/MS of proteins in leaves of Pokkali and IR29 were carried out. A total of 206 differential expressed proteins significantly matched previously deposited sequences in the NCBI database. Of these, 113 proteins matched those having known functions and 93 proteins significantly similar to hypothetical proteins. In total, 43, 24 and 46 proteins were found in Pokkali, IR29 or both varieties, respectively. These proteins could be categorized to 13 functional categories including those, for example, in cellular metabolism, oxidation reduction, RNA processing, structural protein, photosynthesis, cell rescue and defence, transport and binding proteins etc. Cysteine protease (CTP) was down–regulated during salt stress. This expression of a transcript encoding this protein was further examined by semi-quantitative RT PCR. Semi-quantitative RT-PCR revealed that the expression level of hypothetical protein₁₇₁ was not significantly different in leaves of IR29 (P > 0.05). In contrast, that of this gene in Pokkali was significantly up-regulated at 3 to 12 hpt (P < 0.05). CTP₂₀₁ was significantly down-regulated at 3 to 24 hpt in IR29 but that in Pokkali was significantly higher than control at 6 and 12 hpt (P < 0.05). Quantitative real-time PCR indicated that the expression of lipin N-terminal conserved region family protein in IR29 at 6 hpt was significantly lower than that at 24 hpt (P < 0.05). The expression levels of this gene in Pokkali at 0 and 3 hpt were significantly lower than that at 24 hpt (P < 0.05). ATP synthase subunit C family protein in leaves of IR29 at 3 hpt was significantly greater than that of the control (P < 0.05) but its expression in Pokkali was comparable during the stress treatment (P > 0.05). Cytochrome p450 monooxygenase was less abundantly expressed at 6 hpt than that at 24 hpt in IR29 (P < 0.05). In Pokkali, its expression level at 12 hpt was significantly higher than that of the control (P < 0.05).