Functional Analysis Of Seed Specific Genes OsLTPL36 And OsEnF In Rice

Endosperm,the main component of rice seed,not only nourishes the embryo and support its development,but also provides calories and proteins for humans.The development processes of endosperm were composed of different phases including mitosis,endoreduplication,accumulation of nutrients,as well as the programmed cell death(PCD)of starch.Although many researches on the development of endosperm were conducted and some of the endosperm development related genes were identified,the molecular mechanism of endosperm development and the accumulation of nutrients,especially lipid,in endosperm,remain elusive.We selected 10 endosperm-specific genes annotated as expressed proteins or transcription factors for further analysis.T-DNA insertion mutations and reverse genetic strategy such as RNAi and over,expression method were carried out to create the transgenic rice plants,which were used to study the biological function and the mechanism of these genes.Here we report the functions of an ns-LTP,OsLTPL36 and a F-box protein,OsEnF.The main results are as follows:1.Analysis of the protein sequences of OsLTPL36 suggested the presence of an N-terminal hydrophobic signal peptide of 22 amino acid residues.The molecular model of OsLTPL36 shows that the molecular surface representation of OsLTPL36 model resembles a "hand" and can grip lipid tightly.Hence,it was concluded that this a-helix could impose a significant hydrophobic character to OsLTPL36.2.Transcriptional profiling and in situ hybridization analysis confirmed that the expression of OsLTPL36 was specifically high in the developing seed.It was strongly expressed in developing seed coat and endosperm aleurone cells.The expression pattern of OsLTPL36 suggested that it might play a role in seed development.3.No obvious morphological difference between OsLTPL36-RNAi lines and the wide type were observed.However,the seed setting rate and 1000-grain weight of OsLTPL36-RNAi lines were decreased in compared with the WT.We also observed that OsLTPL36-RNAi lines grains displayed higher chalkiness.Several major storage substances of the seeds analysis demonstrated that the seed total lipid and protein contents in three OsLTPL36-RNAi lines were lower than that in WT.Furthermore,the content of C16:0(palmitic acid),C18:0(stearic acid),C18:1(oleic acid),C18:2(linoleic acid)and C18:3(linolenic acid)were significantly reduced.The observation of starch morphology using scanning electron microscopy(SEM)showed that the packaging of starch granules in the endosperm of OsLTPL36-RNAi lines(i5-5,i8-3 and i23-2)were loose,round and smaller.The data demonstrated that OsLTPL36,was critical important not only for lipid metabolism but also for starch granules in the endosperm.4.Cytological results showed that the embryo development process was delayed in OsLTPL36-RNAi lines and the RNAi lines developed embryo with abnormal morphology.The results indicated that OsLTPL36 might be involved in embryo development.The seed germination rates of the OsLTPL36-RNAi lines were significantly declined.The germinated seeds of RNAi lines developed into abnormal seedling with no root or only two leaves.These results confirmed that stored lipids of seed played a vital role in seed germination and seedling growth.5.We identified OsEnF,a putative F-box protein,which showed specific expression in rice developing seed,with the highest expression level in seed of 7 DAP.pOsEnF:gfp and in situ hybridization analysis confirmed that OsEnF was exclusively expressed in developing seed.These results suggested that OsEnF might function in rice seed development.6.Genotype identification and phenotypic analysis of T-DNA insertion mutants of OsEnF shows the seed setting rate and 1000-grain weight were significantly reduced compared with the WT.In addition,we found that the grain length and width were decreased.From 5 DAP seed,the surface of seeds in osenf mutant became shrunken and white.Histological analysis showed that the embryo and endosperm development was abnormal and the number of endosperm cell was decreased.Endosperm cell cannot fully populated or irregularity aggregated results in a cavity formed in the ovary,endosperm cell filling capsules a contraction,eventually led to the shrinking of seed.The similar phenotype was observed in OsEnF-RNAi lines.Scanning electron microscopy(SEM)showed that the packaging of starch granules in the endosperm of WT and OsEnF-OX lines were polygonal and tightly packed.In contrast,the starch granules were loose,round and smaller in osenf and OsEnF-RNAi lines.The reduced expression of OsEnF resulted in structure defected of embryo and abnormal growth of seedling.OsEnF may be a important regulator for rice seed development,participate in the endosperm cell division and storage matter accumulation.7.Yeast-two hybrid assay confirmed the direct interaction of OsEnF with OSK1 and OSK20.We used the OsEnF in pGBKT7 vector as bait to screen cDNA library of rice developing seed in yeast.OsEnF interacted with OsFIE2 in vitro and the interaction was confirmed by Pull-down and Y2H experiment.In the cell-free degradation system,we also demonstrated that OsFIE2 could be degraded by host plant 26S proteasome system.Taken together,our results might suggest that OsEnF is involved in the 26S proteasome pathway by interacting with Oryza sativa Skp1 and that it targets the substrate OsFIE2 for degradation,controlling endosperm cell proliferation.