Functional Analysis Of Alfalfa Γ-tocopherol Methyltransferase(Γ-TMT)Gene In Response To Abiotic Stresses

Tocopherols as a group of lipid-soluble compounds are essential for animal nutrition and well known as powerful antioxidants.Tocopherols have a good effect on improving livestock immunity and reproductive performance.Adding tocopherol to feed can prevent meat apoilage,increase meat stability and prolong the shelf life,allowing for the distribution of meat or processed meat products beyond market borders.In addition,tocopherols are involved in maintaining the dynamic homeostasis of reactive oxygen species(ROS)in the plants by cleaning oxygen free radicals,thereby improving the resistance to abiotic stresses.Therefore,to explore and study the functions of crucial genes in the tocopherol biosynthetic pathway have great potential application for breeding new resistant germplasms,improving the nutritional composition of forage varieties,and improving the supply and quality of livestock products.Among the four tocopherol isforms,α-tocopherol catalyzed by the y-tocopherol methyltransferase(y-TMT)is the predominant form in plant leaf tissue and has the strongest biological activity.Our previous study showed that the alfalfa γ-TMT gene(MsTMT)responsed to various abiotic stresses,such as drought and salt.However,whether overexpression of MsTMT confers increased drought and salinity tolerance to alfalfa and the underlying mechanisms are still unclear.Here,we produced transgenic Arabidopsis and alfalfa plants by overexpressing MsTMT under the control of 35S promoter.And then,the biosynthesis of α-tocopherol and the stress resistance at different developmental stage of these transgenic plants were studied.The main results are as follows:(1)Overexpression of MsTMT gene in Arabidopsis resulted in the specific accumulation of α-tocopherol in seeds.In addition,the germination of transgenic seeds was delayed under salt stress,which was associated with the dynamic homeostasis of ABA and GA by influencing the ABA and GA biosynthesis genes.(2)Overexpression of MsTMT gene promoted the mucilage secretion and also changed the metal ions exchange sites and braching structure of mucilage.These changes may lead to the alteration in calcium crosslinks and hydrophilicity for transgenic Arabidopsis seeds,thereby further affecting the seed’s perception of external salinity stress.(3)MsTMT gene plays distinct roles in the response to salt stress during germination and seedling stage of transgenic Arabidopsis,indicating that this gene is essential for the ability of plants to adapt to the adverse environment.(4)Overexpression of MsTMT gene significantly promoted the metabolic flow of tocopherol biosynthesis in alfalfa and was instruental in directing pathway flux towardsα-tocopherol.High Performance Liquid Chromatography(HPLC)analysis showed that the contents of α-tocopherol and total tocopherols were both increased in transgenic plant leaves.(5)Stomatal development of transgenic alfalfa leaves was significantly inhibited,which may be caused by repressing the transcription level of SPCHLESS gene.The reduced stomatal density led to a sharp decrease in stomatal conductance and transpiration efficiency,which in turn improved the water use efficiency of transgenic plants.(6)The drought resistance of transgenic alfalfa was significantly enhanced with less oxidative damage and more osmotic substances,such as free proline and soluble sugar.RNA-seq and qRT-PCR analysis suggest that MsTMT gene is involved in both ABA-dependent and ABA-independent pathways in the drought response.