Molecular Mechanism Of MATE Transporter DG1 Regulating Rice Grain Filling

Rice is the most important food crop in the world,and the staple food of more than half of the world’s population.The grain filling of rice directly determines yield and quality.As the global climate warms,extreme weather becomes more frequent.During the flowering and grain filling stage of rice,it is easy to encounter the damage of high temperature in summer,leading to reduced yield or even no harvest.Therefore,analyzing the molecular mechanism behind the effect of high temperature on rice grain filling can provide theoretical guidance for molecular design breeding to solve rice grain filling under high temperature and ensure yield and quality.In this study,a mutant dg1（defective grain-filling1）with defective grain filling was selected from the EMS mutant library of the backbone parent Shuhui 527（R527）of heavy panicle hybrid rice,and the phenotype analysis,gene mapping and function study were carried out.The main findings are as follows:1.Phenotypic analysis of the dg1 mutant found that the dg1 mutant mainly showed delayed grain filling and poor filling.The grain filling time of dg1 was extended 27 days,and there were24.68%of incompletely filled seeds in Wenjiang,Sichuan,resulting in a thousand-grain weight decreased 21.36%;histocytology analysis found that the programmed cell death of dg1endosperm cells and the filling rate of starch in endosperm were significantly slower than wild type;physiological index measurement showed that the content of sucrose,fructose and glucose in dg1 caryopsis was higher than wild type.The total starch content is lower than wild type.2.Genetic analysis shows that the mutant phenotype is controlled by a pair of recessive genes.Using Mut Map analysis,it is found that the dg1 phenotype is caused by the base variation from G to A at position 786 of the LOC＿Os03g12790 genome,which leads to the premature stop of protein translation.This gene encodes multidrug and toxic compound extrusion transporter,named DG1.Genetic complementation and Nipponbare gene knockout further confirmed that DG1 is the gene that controls the phenotype of the dg1 mutant.3.The two systems of Xenopus oocytes and rice protoplasts proved that DG1 has ABA efflux activity.4.Analysis of expression patterns found that DG1 was mainly expressed in nodes,leaves and roots;GUS staining showed that DG1 was mainly expressed in nodes,leaves,roots and rachilla;GUS stained tissue sections observed that DG1 was mainly expressed in leaf vascular bundles and roots central column;immunofluorescence analysis of DG1 is mainly expressed in the parenchyma cells between the enlarged vascular bundles and diffuse vascular bundles in the nodes,the phloem of the leaf vascular bundles and the central large vascular bundles of the rachilla.Using 35S::DG1^{c DNA}-e GFP positive plant root tips to subcellular localization of DG1found that it is located on the cell membrane.5.The results of ABA and ABA precursor determination found that the ABA content in caryopses 3 and 5 days after fertilization of dg1 was significantly lower than that of the wild type,while the content of stems and leaves was significantly higher than that of the wild type;on the other hand,The content of ABA precursor substances 9,-cis-neoxanthin and 9-cis-violoxanthin in leaves are 40 times and 10 times that in caryopsis and stems,respectively;phenotype,³H-ABA fedding and exogenous ABA staining have proved that DG1 mediates the long-distance transport of ABA.That is to say,the leaves are the main tissues and organs for ABA synthesis,and they are transported to the caryopsis through the transporter DG1 to meet the needs of grain filling.The middle nodes play a key role in the ABA transport process.6.DG1 mediates the long-distance transport of ABA,regulates genes related to starch synthesis,and promotes grain filling.Analysis of RNA-sequence data of wild type and dg1caryopsis 5 days after fertilization found that about 80%of the key starch synthesis genes in the mutant dg1 were significantly lower than wild type;the ABA-induced caryopsis experiment found that ABA can regulate the starch in wild type synthesize the expression of key genes NF-YB1,NF-YC12,Osb ZIP58,RPBF,b HLH144,Os AGPL3,Wx,Os BEIIb,Os ISA1 and Os AGPL2.so that the seeds can develop normally.7.DG1 gene is induced by temperature,the expression of temperature rise is higher,and the ABA transport efficiency is also higher.The phenotype of the dg1 mutant at mild temperature was significantly weaker than that of high temperature conditions.WT,dg1 and complementary lines were treated under controlled temperature.It was found that dg1 had no grain filling defect phenotype at an average daily temperature of about 24°C.A large number of incompletely filled seeds were produced at an average daily temperature of about 30°C;³H-ABA transport experiments proved that DG1 regulates the efficiency of ABA transport from leaves to caryopsis in response to temperature.8.The sequence and function of DG1 gene are conserved in grasses.Phylogenetic tree analysis found that DG1 orthologous genes have about 50%protein sequence similarity in angiosperms,and up to about 80%in gramineous plants;use a Mu mutant（mu1004348）of DG1 in maize.Phenotypic analysis revealed that zmdg1 exhibited similar to dg1 with delayed grain filling and kernel filling defects.In summary,this study revealed the long-distance ABA transport system and biological significance.That is,ABA is mainly synthesized in the leaves,loaded into the phloem,and then transported to the enlarged vascular bundles,and from the enlarged vascular bundles to the diffusion vascular bundles,and further enters the caryopsis through the rachilla vascular bundles,the expression levels of a series of key starch synthesis genes in caryopsis were regulated to promote grain filling.;in this process,the ABA efflux transporter DG1 plays a key role,and the expression of the gene DG1 encoding this protein is regulated by temperature,coordinating the transport efficiency of ABA from leaves to caryopsis,To ensure the normal development of seeds at various temperatures.