Proteomics Study Of The Response Of Rice Roots To Aluminum Stress And Functional Analysis Of OsTCTP

Rice (Oryza sativa L.) is not only an important crop worldwide, but also a model species of monoeotyledons for researches. With the advent of the completion of the rice genome sequencing and various omics technologies, the rice functional genomics research has entered a new phase. One of the major limitations to crop production worldwide on acid soils is the prevalence of soluble aluminum (Al) ions. Therefore, great efforts have been spent on the discovery of potential Al tolerance mechanisms and a number of studies suggest that over-expression of some important regulatory or functional genes in Al sensitive plants can significantly improve Al stress resistance.To investigate the Al tolerance mechanisms of rice, we used an iTRAQ (isobaric tags for relative and absolute quantitation) labeling, bioinformatics analysis and qPCR measurements to compare the root proteins of the Al-sensitive rice cultivar (7098) and the Al-tolerant rice cultivar (Nipponbare). We found that the root proteins were significantly affected by Al treatments. In total,106proteins were differentially expressed upon Al toxicity in sensitive and tolerant cultivars. They cover a wide range of molecular functions, including energy, stress and defense, protein turnover (synthesis, modification and degradation), metabolism (catalyst), signal transduction, transport and intracellular traffic, cell structure, cell growth/division and transcription. The amount of62proteins changed significantly in7098after Al treatments and76in Nipponbare, sharing32proteins. Our results showed that7098and Nipponbare respond to Al toxicity differentially at the protein level. KOBAS analysis showed that glycolysis/gluconeogenesis is the most significantly up-regulated biochemical process in response to excess Al. The mRNA levels of eight proteins mapped in the glycolysis/gluconeogenesis were further analyzed by qPCR and the expression levels of all the eight genes are higher in tolerant cultivar than in sensitive cultivar, suggesting that these compounds may promote Al tolerance by modulating the production of available energy.Furthermore, we also make a thoroughly functional analysis of the stress-responsive gene, OsTCTP (translationally controlled tumor protein). Firstly, we cloned the gene of OsTCTP from cv. Nipponbare and southern blot analysis revealed that OsTCTP is a single-copy gene with two differentially spliced transcripts and further study showed only one transcript can be translated into a mature protein. Secondly, T-DNA mutant analysis showed that OsTCTP gene is regulated at translational level. We also used western blotting to investigate the expression patterns of OsTCTP at protein level under normal and stress conditions. The results demonstrated that OsTCTP is constitutively expressed in all the tissues or organs in various tissues and organs investigated, especially in actively dividing and differentiating tissue types. The protein level of OsTCTP is upregulated by mercury, copper, hydrogen peroxide and ABA; but not by NaCl, PEG, SA, JA, NAA and mechanical damage. Lastly, we investigated its role in Hg tolerance through OsTCTP-OX and OsTCTP-RNAi transgenic rice plants. The results indicated that OsTCTP may improve the Hg tolerance by regulating the activities of SOD, CAT, APX and POD to scavenge Hg-induced ROS.