Map-Based Cloning And Functional Analysis Of SGD1 Gene Involved In Tocopherols Biosynthesis In Rice

Rice is one of the most important food crops,feeding more than half of the world population.Plant height is a major factor related to grain productivity.It is important for rice breeding to investigate the molecular mechanism underlying plant height development.Vitamin E(tocopherol and tocotrienols)are essential components of the human diet.Tocopherols perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radicals and protecting polyunsaturated fatty acids from lipid peroxidation.Because of these and other activities,dietary tocopherols are thought to play an important role in improving immune function and in limiting the incidence and progression of several degenerative human diseases including certain types of cancer,cataracts,neurological disorders,and cardiovascular disease Vitamin E is assumed to play important roles as antioxidants in plants.In contrast to well documented roles in animals the functions of Vitamin E in plants are much less understood.In this study,we investigated the functions of tocopherols in rice through tocopherols deficient mutant sgd1-1 and sd1-2.This provided a basis to clarify the functions of tocopherols in plants.The main results are as follows:(1)sgd1-1 and sgd1-2 showed dn-type dwarfism.Both mutants had reduced grain size and 1000-grain weight relative to wild type.The panicles of both mutants were significantly smaller.The secondary panicle branches and average number of spikelets per panicle were also significantly decreased in mutants.Histological observations showed that reduced cell size was the main reason for dwarfing and small grain size in the sgdl mutants.(2)Map-based cloning and complementation analysis confirmed that sgdl phenotypes were caused by loss-of-function mutations of the SGD1 gene.SGD1 encodes a homogentisate phytyltransferase(HPT)that catalyzes the key step in tocopherol biosynthesis.HPLC results confirmed that tocopherols were lacking in sgdl-1 and sgd1-2.SGD1 has its highest expression levels in leaves.Phylogenetic analysis and alignmemnt indicated that SGD1 has a high level of conservation with monocot HPT homologs.(3)Tocopherols are assumed to play important roles as antioxidants in plants.However,the levels of LOOH and MDA were not elevated in sgdl-2 compared to Nipponbare.Histochemical staining showed no significant ROS activity in gd1-2.Since tocopherol deficiency did not lead to oxidative damage we concluded that tocopherols are not major antioxidants in rice grown under normal conditions.(4)The sgd1-2 mutant was hypersensitive to cold stress,but not drought and salt stress.Free proline and soluble sugars were accumulated in the sgd1-2 mutant under norma1 conditions.These indicated a constitutive cold stress response was induced in sgd1-2 mutant.Our results suggest that constitutive cold stress response induced by tocopherol deficiency causes retarded plant development.(5)Both a-amylase activity assays and treatment with various concentrations of GA3 indicated that sgd1-2 is a GA-related mutant.The bioactive GA content was similar between wild type and sgdl-2.SLR1 protein was accumulated in the sgdl-2 mutant caused attributed to increased SLR1 gene transcript levels,indicating that the dwarf phenotype in sgd1-2 was also associated with SLR1 protein accumulation.