Studies On The Physiological Mechanisms Of Overexpressing Ub In Improving The High Temperature Tolerance In Tobacco

Organism produces new functional proteins in the cell cycle constantly. At the sametime, the invalid proteins should be degraded when they finished their functions. Asimportant as it’s synthesize, protein degradation also plays a key role in physiologicalmetabolism, growth and development of the organisms. Ubiquitin/26S proteasome pathway（UPP） is the most important selective proteolytic pathway so far. In this pathway, ubiquitin（Ub） is attached to proteins destined for degradation. The conjugated proteins are thenrecognized and degraded by the26S proteasome. Under stress conditions, cell wouldaccumulate a large number of abnormal or damaged proteins. In order to maintain normalcellular function, these damaged proteins should be removed in time. It is reported that theexpression of ubiquitin is increased under stress condition, and the degradation of short-lived regulatory proteins is accelerated.In our previous work, a monoubiquitin gene Ta-Ub2from wheat （Triticumaestivum）was cloned, a sense vector with Ta-Ub2was constructed and transformed into tobacco. Inthis study, the sense Ta-Ub2transgenic plants （T2-2, T2-11, T2-13） and WT （NC89） plantswere used to study the responses of transgenic plants to high temperature stress. Weinvestigated the expression of Ub proteins, membrane damage, accumulation and removingof reactive oxygen species （ROS）, photosynthesis and protein carbonylation, etc. The resultsshowed that overexpressing Ub enhanced high temperature tolerance in the transgenicseedlings. The main results were as follows:（1） Immumology analysis of Ub protein in sense transgenic plants indicated thatoverexpressing Ta-Ub2could not significantly alter the level of free ubiquitin pool intransgenic tobaccos, but polyubiquitin increased. After high temperature stress, theaccumulation of polyubiquitin decreased in transgenic plants, however, the accumulation ofconjugated ubiquitin increased in transgenic plants. （2） Under high temperature stress, overexpressing Ta-Ub2enhanced germination andseedlings growth of sense transgenic plants, released the growth inhibition resulted fromhigh temperature stress, suggesting the enhanced high temperature tolerance byoverexpressing Ub.（3） High temperature stress inhibited the photosynthesis of the tobacco plants. Comraredwith WT, photosynthesis indexes of sense Ta-Ub2transgenic plants were ameliorated, and.the decrease of the actual efficiency of PSII photochemistry （ФPSII） and the maximalefficiency of PSII photochemistry （Fv/Fm） were also alleviated under high temperaturecondition.（4） Under high temperature stress, the transgenic plants had a better protection of theproteins in thylakoid membranes than WT, especially a better protection of D1protein.（5） High temperature stress destroyed the membrane integrity and stability, and inducedreactive oxygen species （ROS） accumulation, and ultimately resulted in evident increase inmalondialdehyde （MDA） content and electrolyte leakage. Overexpressing Ta-Ub2decreased the peroxidant level and protected the membrane integrity and stability oftransgenic plants from high temperature stress.（6） High temperature stress caused the increase in superoxide radical （O₂^-） production andH₂O₂content in tobacco plants. The acculation of O₂^-and H2O2in WT was higher than thatin transgenic plants. Several main antioxidant enzymes such as SOD, CAT, POD and APXin sense Ta-Ub2transgenic plants maintained higher activity than WT under hightemperature stress, which can inhibit the increase of ROS production, MDA content andelectrolyte leakage caused by stress conditions.（7） Immumology analysis of carbonyls protein indicated that the contents of proteincarbonyls were increased by high temperature stress in both transgenic tobaccos and WT.The contents of protein carbonyls in tobaccos with overexpressing Ta-Ub2were less thanthat in WT. These suggested that overexpressing Ta-Ub2gene enhanced the antioxidantability of proteins. From all the results above, it was suggested that overexpressing the Ta-Ub2geneenhanced the tolerance of the transgenic plants to high temperature stress. These resultsproposed a new stratagem for enhancing the tolerance of plants to high temperature stresscondition.