Map-Based Cloning And Functional Analysis Of Two Key Genes OsDAAT1 And FLO19 Involved In Rice Grain Chalkiness

Rice（Oryza sativa L.）is one of the most important cereal crop worldwide,feedingmore than half of the world’s population as the staple food.With increases in population and improvements in the standard of living,the demand for rice high yield and superior quality is increasing day by day.Rice grain quality is determined by many components such as appearance,processing,cooking and eating qualities.Grain chalkiness is the opaque portion in rice endosperm due to loosely packing of starch granules and protein bodies during grain filling.Grain chalkiness is an undesirable trait that not only affects the grain appearance,but also affects the cooking and eating properties,and milled rice quality and market value.Therefore,studying the molecular mechanism and regulatory network of chalkiness formation has important scientific value and production guidance significance for rice endosperm development and genetic improvement.Although numerous genes associated with grain chalkiness have been detected by various groups,the molecular mechanism for this trait is poorly understood.In the study,we identified two chalky mutants,named D-amino acid aminotransferase1（daat1）and floury endosperm 19（flo19）from the mutant library defective in rice endosperm development.Molecular cloning and transgenic complementation revealed that the Os DAAT1 allele encodes a D-amino acid aminotransferase and the FLO19 allele encodes a plastid-localized pyruvate dehydrogenase E1α1 subunit.The biological functions of Os DAAT1 and FLO19 were studied and the role of each gene in rice endosperm development and chalkiness formation was discussed,repectively.The results are summarized below.Part Ⅰ:Map-based cloning and functional analysis of Os DAAT1.1.The daat1 mutant was isolated from a tissue culture-derived population of the japonica rice cultivar Nipponbare.In contrast to the transparent endosperm observed in wild type seeds,the daat1 showed defective endosperm at maturity,with the translucent endosperm in the dorsal part of seeds,but floury in the abdomen.Both grain length and width of the daat1 mutant were markedly increased,with thickness reduced compared with wild type.Consistently,the daat1 mutant had a slower grain filling rate and reduced1000-grain weight.Further analyses found lower contents of amylose and crude protein in mutant kernels,but the contents of total starch and lipid were similar compared to the wild type.The daat1 mutant displayed lower plant height and slower growth throughout the entire growth period.Thus,Os DAAT1 plays an important role in endosperm development and plant growth.2.Cytological observation of early stage of endosperm development showed that the belly part of the daat1 mature grains was consisted of scattered and spherical starch granules（SGs）,in contrast to the tightly and polyhedral packed SGs in the back of the daat1 and the whole endosperm of wild type.Notably,in addition to defective SGs,the swelling and aberrant endoplasmic reticulum（ER）was observed in the daat1 endosperm cells through transmission electron microscopy（TEM）anaylsis.Therefore,it was suggested that the Os DAAT1 mutation may affect starch grains formation and ER structure in the endosperm cells of rice.3.To determine the genetic basis of the chalky grain phenotype of the daat1 mutant,we performed reciprocal crosses between daat1 and the wild type.As a result,all progeny displayed chalky phenotype when daat1 works as the female parent,but normal phenotype when wild type works as the female parent.Furthermore,when either type of F₁（daat1×WT and WT×daat1）was self-pollinated,all progeny displayed the normal phenotype,even for the 25%of the seeds that were homozygous for the daat1 mutation.Thus,it was suggested that Os DAAT1 functions in the maternal tissues for control of grain chalkiness formation in rice.4.To map the Os DAAT1 gene,we constructed the F₂ populations from a cross between daat1 and the indica cultivar N22.The Os DAAT1 locus was placed in a 48 kb region of the chromosome 3,which consists of four open reading frames（ORFs）.Genomic sequence analysis indicated that LOC＿Os03g24460 is the only gene that is different between the wild type and daat1,which harbours a 4931-bp deletion from the 93^st of forth exon to upstream region in daat1.And this deletion caused no expression of LOC＿Os03g24460 in the daat1 mutant.Furthermore,complementation test,CRISPR/Cas9 system and RNAi experiments confirmed that LOC＿Os03g24460 may be the candidate gene.5.Phylogenetic analysis showed that Os DAAT1 homologous proteins can be widely found in eukaryotes and prokaryotes and Os DAAT1 may descend from prokaryotes.Multiple sequence alignment analysis demonstrated that Os DAAT1 has high similarity to orthologs from monocots and eudicots.In addition,real-time PCR（RT-qPCR）and RNA fluorescent in situ hybridization experiments revealed that Os DAAT1 was expressed ubiquitously,with relatively higher expression level in the vegetative organs of rice,especially in the vascular tissues of the node and rachilla,than that in development endosperm.Subcellular localization and subcellular fractionation experiments indicate that Os DAAT1 is a cytoplasm-localized protein.6.In vitro enzyme assays demonstrated that Os DAAT1 exhibits wide-spectrum D-amino acid aminotransferase activity,which is responsible for the interconversion of different D-AAs.The D-Ala content of the daat1 seed,first leaf and first stem was significantly higher than that in wild type.Notably,both D-Ala of wild type were present at very low levels in the second stem but were present at very high levels in daat1.In addition,the D-Ala was significantly higher in F₁（daat1×WT）than that in F₁（WT×daat1）,which correspond well to the differences in the distribution of D-AAs between wild type and daat1.Furthermore,fluorescent D-Ala analogue（HADA）tracer experiments showed that D-AAs could be transported from stem to seed in rice.Together,these findings suggest that Os DAAT1 acts as a regulator in maternal tissues,and the regulation of D-AAs metabolism in maternal tissues by Os DAAT1 clearly influences grain chalkiness in rice.7.RT-qPCR analysis revealed that the Os DAAT1 mutation could up-regulate the expression of genes involved in endoplasmic reticulum（ER）stress response.Interestingly,exogenous D-AAs treatment activates the ER stress response genes transcription in developing endosperm,as well as seedlings in rice.These results suggest that Os DAAT1-mediated D-AAs homeostasis is critical for maintaining ER homeostasis.Transcriptome analysis showed that carbohydrate and amino acids metabolism was significantly affected in the developing seeds of daat1 mutant.Overall,Os DAAT1 plays an important role in the formation of grain chalkiness via regulating maternal D-AAs homeostasis associated with ER stress response.Part Ⅱ:Map-based cloning and functional analysis of FLO19.1.We performed a mutant screen of an EMS-treated rice cultivar Diangengyou1population and isolated a flo19 mutant which produced seeds with an opaque inner endosperm,but translucent in the peripheral region.The flo19 mutant had a slower grain filling rate and reduced 1000-grain weight.Consistently,grain length,thickness and width were markedly reduced in the mutant.Further analyses found lower contents of total starch and amylose in mutant kernels;crude protein content was also lower,but lipid contents were higher compared to the wild type.The flo19 mutant displayed lower plant height and slower growth throughout the entire growth period.Thus,FLO19 plays an important role in endosperm development and plant growth.2.In the central region of the wild type endosperm most amyloplasts were well developed and filled with densely packed,polyhedral and sharp-edged compound granules,whereas two types of abnormal amyloplasts were readily observed in the flo19mutant.One type contained irregular single starch granules that weakly stained with iodine.The other type had scattered starch granules,some of which were well stained with iodine.Compared with the wild type,the expression levels of most genes related to starch synthesis were down-regulated in the flo19 mutant,as well as the activity of ADP-glucose pyrophosphorylase（AGPase）.Therefore,the flo19 mutation caused defects in amyloplast development and starch biosynthesis during rice endosperm development.3.To identify the underlying gene,the flo19 mutant was crossed with an indica variety IR36 and then self-pollinated to generate an F₂ mapping population.The FLO19 locus was placed in an 80.5 kb region of the chromosome 4,which region consists of 12 ORFs.Sequence analysis showed that the 11^th ORF（LOC＿Os04g02900）in flo19 had a single nucleotide substitution of C to T in the second exon,leading to the transition of Alanine to Valine.Further,complementation test and CRISPR/Cas9 experiments confirmed that LOC＿Os04g02900 is the gene responsible for the flo19 phenotype.Bioinformatics analysis showed that LOC＿Os04g02900 was predicted to encode a plastid pyruvate dehydrogenase complex E1α1 subunit（pt PDC-E1-α1）subunit in rice.4.Phylogenetic tree analysis showed that three genes potentially encoded plastid PDC-E1-αandβsubunits;among them one gene encoded a pt PDC-E1-α1 and two encoded pt PDC-E1-β1 andβ2.Multiple sequence alignment revealed that pt PDC-E1-α1proteins shared high sequence conservation and that the mutation site was highly conserved in monocots and eudicots.Moreover,RT-qPCR revealed that pt PDC-E1-α1,pt PDC-E1-β1 and pt PDC-E1-β2 were widely expressed throughout the plant,although they displayed distinct patterns.Subcellular Localization experiments indicate that all the three pt PDC-E1 subunits were localized to the chloroplasts in the same pattern.Furthermore,strong GFP fluorescence was observed in the amyloplasts of the FLO19-GFP transgenic rice pericarp cells.Therefore,FLO19 is a plastid-localized protein.5.Yeast two-hybrid（Y2H）,bimolecular fluorescence complementation（BiFC）,coimmunoprecipitation（Co-IP）and immunoprecipitation-mass spectrometry（IP-MS）experiments showed that pt PDC-E1-α1 is a member of pt PDC complex,and there are interactions amongα1,β1 andβ2 of rice PDC-E1.The FLO19 mutation resulted in significantly decreased activity of pt PDC and lower monogalactosyldiacylglycerol（MGDG）and digalactosyldiacylglycerol（DGDG）required for amyloplast development,leading to abnormal SGs and defective starch biosynthesis in developing rice endosperm.6.FLO19 overexpression significantly increased seed size and weight,but did not affect other important agronomic traits,such as panicle length,tiller number and seed setting rate.Association studies based the 3K rice genome panel revealed a significant association between a 4 bp in the FLO19 promoter and grain length（P=1.1e-08）.Further analysis indicated that the haplotype,named pFLO19^L,with the 4 bp was positively associated with longer grain length,whereas haplotype pFLO19^S with the 4 bp deletion was associated with short grain length.Notably,most xian/indica（XI）and AUS cultivars contained pFLO19^L,while only 11.3%geng/japonica（GJ）cultivars carrying pFLO19^S,especially temperate japonica.A transient expression assay and realtime-PCR（RT-qPCR）showed that the pFLO19^L more effectively enhanced the expression of the reporter gene than pFLO19^S.Taken together,a variation in the promoter sequence of FLO19 affects its expression,which in turn controls seed development.This provides a new idea for clarifying the mechanism underlying starch biosynthesis,and implies a potential application of FLO19 for improving rice yield and quality.