Map-Based Cloning And Functional Analysis Of Two Key Genes GPA6 And GPA8 Involved In Glutelin Trafficking In Rice Endosperm

Large amounts of storage proteins are accumulated in rice seeds and its content are closely related to rice quality.Therefore,it is of great significance to study the mechanism of storage protein synthesis,transport and deposition.The content of glutelin in rice storage protein is the highest,about 80%.The glutelin precursor is first synthesized in the endoplasmic reticulum,then transported to protein storage vacuole(PSV)by dense vesicle(DV)and precursor-accumulating vesicle(PAC),and finally processed by vacuolar processing enzymes into mature acidic and basic subunits that are deposited in protein bodyII(PBII).Although the trafficking mechanism of glutelin has made some progresses,its network pathway is still unclear.When the processes of synthesis,transportation and deposition of gluten are defected,large amounts of glutelin precursors will be accumulated in the seeds,so 57H mutants are ideal material to study the mechanism.In this study,two new 57H mutants named gpa8 and gpa6 were screened from the rice MNU and 60Co mutagenesis library.Enlarged DV,bent Golgi,and PMB structure were appeared in gpa8 mutant.Map-based cloning confirmed that GPA8 encoded the E-subunit of V-ATPase(OsVHA-E1)and its function was studied.It was found that V-ATPase regulated the budding and trafficking processes of DV by maintaining the pH homeostasis of trans-Golgi network(TGN),which further enriched the network regulatory pathways of glutelin synthesis,transportation and deposition.Enlarged DV and PMB structure were also appeared in gpa6 mutant.Map-based cloning confirmed that GPA6 encoded a Na+/H+antiporter(OsNHX5).The main research results are as follows:(1)Compared with wild-type ninggengl,gpa8 seeds have a opaque appearance,increased glutelin precursor,decreased 1000-grain weight and amylose content and no significant change in the total protein content.gpa8 mutant leaves showed lesion-mimics and early senescence from the seedling stage,which was more serious in the mature stage.(2)Through semi-thin sections and immunofluorescence analyses,it was found that PBII in the mutant of gpa8 decreased significantly and formed PMB structure.Tem observation confirmed that Golgi was severely bent and the numbers of Golgi were increased in gpa8 mutant.DV was significantly enlarged and aggregated around Golgi in gpa8 mutant.Therefore,the budding and trafficking of DV were both defected in gpa8 mutant.(3)The gpa8 mutant was hybridized with an indica variety N22 to generate a F2 segregation population for cloning the gene.It was found that the gene Os01g0659200 was missing two bases of GT at the beginning of the fifth intron,which resulted in the non-clipping of intron and the early termination of shift code.GPA8 encoded the E-subunit of V-ATPase,which contained an E-subunit domain.Phylogenetic tree analysis showed that this gene was conserved and its homologous genes were widespread in other eukaryotes.Tissue expression analysis revealed that the gene was constitutively expressed.The root tips of OsVHA-El-GFP fused complementary transgenic lines were observed and OsVHA-E1 was mainly localized on tonoplast.Most of the subunits of V-ATPase were isolated by immunoprecipation-mass spectrometry analysis of OsVHA-El-GFP fused complementary transgenic lines,indicating that OsVHA-El played a biological role in the formation of complex with other subunits in vivo.The V-ATPase enzyme activity of the mutant was significantly lower than that of the wild type.(4)Previous studies have shown that V-ATPase could regulate the pH homeostasis of some intracellular organelles,such as TGN and vacuole.It was found that the pH of TGN was significantly increased in gpa8 mutant,which was consistent with the previously observed cytological phenotype of post-Golgi trafficking passway.To a certain extent,this indicated that the pH of TGN regulated by V-ATPase was necessary for post-Golgi trafficking of glutelin precursor and had an important regulatory effect on the budding and trafficking of DV.We found that the pH of vacuole was also increased significantly,which may lead to premature leaf senescence.(5)Previous experimental results indicated that OsVHA-E1 was involved in the transport of rice glutelin precursors to protein storage vacuoles.In order to further analyze the biological function of this gene,we wanted to know whether this protein participated in the post-Golgi trafficking of other proteins,so we selected two marker proteins(vacuolar iron transporter 1:VIT1;secretory carrer membrane protein1:SCAMP1)to observe their subcellular localization in the protoplast.VIT1 and SCAMP1 were not correctly localized in the mutant,indicating that V-ATPase was essential for the post-Golgi transport pathway.(6)The gpa6 mutant seed was opaque in appearance and the glutelin precursor was significantly increased.Cytological experiments have shown that DV-mediated glutelin precursor transport in the mutant is defected,and DV fuses with the cell membrane to form complex abnormal structure PMBs,resulting in a decrease in PBII.Using the map-based cloning method,the target gene OsNHX5 was isolated and expressed constitutively.OsNHX5 was localized to Golgi,TGN and PVC and is capable of regulating the pH of three compartments to further regulate DV transport.