| Starch and storage protein are the main components of rice endosperm.The total content of starch and storage protein accounts for more than 90% of the total mass of rice endosperm.Their content and morphology directly determine the yield and quality of rice.Storage protein is synthesized in the rough endoplasmic reticulum and bud off to form protein body I(PB I)or transported to the protein storage vacuole then processing to form protein body II(PB II).Starch is synthesized in endosperm cells.The main biosynthetic pathway of starch and storage proteins have been widely reported,but the regulatory mechanism of storage protein and starch biosynthesis remained largely unclear.In addition,it is reported that high temperature can shorten the rice grain-filling process,lead to insufficient storage material accumulation and change of composition and structure of starch with reduced rice production and quality.However,the molecular mechanism of how high temperature affecting rice endosperm quality remained still unclear.Therefore,it is of great significance to discover the inner molecular mechanisms underlying how b ZIP transcript factors respond to high temperature and how they regulate grain starch and storage protein biosynthesis in rice.Here,a stable opaque mutant,opaque3(o3),was screened from the EMS mutated library of ‘Zhonghua 11’.Then OPAQUE3 was cloned by Mut Map method,and the genetic and regulatory mechanism were researched in detail.The main results were as follows:(1)We identified a rice mutant,o3,with opaque endosperm,decreased grain filling rate,significantly decreased contents of total starch,amylose and total protein in mature grains,significantly increased content of lipid,significantly decreased 1000-grain weight,and yield per plant.The grain glutelin and prolamin contents were significantly decreased,and the 57-k Da glutelin precursor was over-accumulated accompanied by decreased basic and acid subunits.Besides,protein folding and processing chaperones such as Os BIP1 and PDIL1-1 were significantly accumulated in the ER of o3 endosperm.(2)Transmission electron microscopy of the developing endosperm cells showed that the structure of o3 ER was abnormal,which was more curved and shortened than that in the wild type.A large number of storage protein precursors were accumulated in ER and ER-derived vesicles.Some PB II and PB I were fused,and some PB-II were broken into pieces.But the Golgi apparatus developed normally in the o3 mutant.These results indicated that O3 was involved in the regulation of protein processing and secretion in the ER.(3)Analysis by scanning electron microscopy,semi-thin section and transmission electron microscopy assay showed that there were many abnormal compound starch grains in o3 development endosperm cells,some amyloplasts disintegrated in advance,and a large number of single starch granules existed in o3 endosperm cells.The analysis of starch physicochemical properties showed that the short chain of amylopectin(DP 6～12)in o3 increased,and the long chain(DP 13～24)decreased.The starch viscosity and the initial gelatinization temperature of o3 decreased.Which showed that O3 regulates the formation of compound starch grains in endosperm cells.(4)We cloned O3 gene by combining the method of marker mapping and Mut Map.The wild type O3 can complement the o3 mutant phenotype.And the endosperm phenotype of the knockdown mutants were similar to that of the o3 mutant,and the grains were opaque with abnormal starch compound grains,decreased content of starch and protein,1000-grain weight.The 57-k Da precursor of glutelin accumulates in large quantities.Besides,overexpression of O3 in the wild type did not change the plants morphology and grain appearance.(5)O3 encodes a transmembrane b ZIP transcription factor(Osb ZIP60)which is highly expressed in developing grains.Subcellular localization showed that O3 was located in both the ER and the nucleus.O3 was mostly induced to the nucleus when ER stress happened.This indicated that O3 could respond to ER stress and participate in the regulation of protein processing and secretion in ER.(6)RNA-seq analysis showed that the differentially expressed genes between o3 and WT were mainly classified as ER protein processing and secretion,starch and sucrose metabolism,glycolipid metabolism.Ch IP-seq,Ch IP-q PCR,EMSA and LUC experiments showed that O3 could directly bind to the p UPRE-II-motif of the promoter of ER chaperone genes Os BIP1 and PDIL1-1 and activate their expression.The point mutation of O3 have decreased the activation.The osbip1 and pdil1-1 mutants in ZJ100 background showed similar opaque phenotypes to o3,and the moderate overexpression of Os BIP1 in the o3 mutant could partially restore the mutant phenotype to the wild-type.This result proved that O3 was involved in the regulation of ER protein processing and secretion by directly regulating Os BIP1 and PDIL1-1 in the ER.(7)Ch IP-seq,EMSA and Y1 H showed that O3 could directly bind to the O2-box in the promoters of the genes related to endosperm storage protein biosynthesis,Os Glu A2,Prol14,Glb to regulate their expression and participate in the synthesis of storage protein.O3 can also directly bind to the promoter GCN4-box of starch synthesis-related genes AGPL2,GBSSI,SBE1 and ISA2 to regulate their expression and participate in endosperm starch biosynthesis.(8)O3 was induced to high expression level under heat stress.The mature grains of o3 exhibited extreme floury under high temperature environment(35°C,12 h light/28°C,12 h dark).However,the wild type showed a little change.Compared with that in normal temperature,the genes related to ER stress,protein processing,such as Osb ZIP50,BIPs,PDILs,Calnexin,Os Fes1 C and Os Ero1 were significantly up-regulated in o3 under high temperature.Osb ZIP50 was significantly cleaved in o3 mutant,which was more obvious under high temperature.Which may activate the expression of BIPs,PDILs,and other related genes together with other transcription factors to help alleviate ER stress under high temperature in o3.These results show that O3 simultaneously regulates the biosynthesis of endosperm storage proteins and starch,and participates in maintaining ER homeostasis under high temperature to ensure the normal development of rice endosperm. |
