Research On The Function Of Viviparous1-Regulated Aleurone Development In Rice

Rice is an important crop,the endosperm of which is the main edible part containing starch endosperm and aleurone layer.The rice endosperm in the filling stage includes three parts:aleurone layer,subaleurone layer and starch endosperm from outside to inside.The aleurone layer is rich in fatty acids,vitamins,and mineral elements,and thus it has been proposed to improve the nutritional quality of grains by increasing the thickness of the aleurone layer.Little is known about the molecular mechanism of rice aleurone layer development,which restricts the application of this strategy in the improvement of rice nutritional quality.VP1(Viviparous1)is a member of the B3 transcription factor gene family,which was involved in seed dormancy.This study found that rice OsVP1 expressed mainly in the aleurone layer and embryo of the grain,and not only participates in the regulation of rice dormancy,but also affects the development of aleurone layer.The results showed that the osvp1 mutant had a thicker aleurone layer tissue compared with the wild type.In addition,this study also analyzed the biological functions of PHS-B3a and PHS-B3b,the other two B3 transcription factors in rice.It was found that mutation of these genes could induced pre-harvest sprouting in high temperature and humidity environment.The main findings of this study are summarized below:1.Expression pattern of OsVP1:we investigated the expression of OsVP1 in different tissues and in different days after fertilization(DAF)through quantitative real-time PCR(qRT-PCR).,The results showed that the expression of OsVP1 was almost undetectable in vegetative organs.OsVP1 is mainly expressed in the developing seeds,starting from 10 DAF,and continues to,but expressed in the later stage of seeds.GUS staining results of pOsVP1::GUS transgenic plant seeds showed that OsVP1 had the highest expression in the dorsal aleurone layer since 10DAF by using the OsVP1 promoter to drive GUS tag expression.2.The osvp1 mutants were successfully generated by CRISPR/Cas9 technology,and the phenotypic analysis of which was found that the osvp1 mutant showed pre-harvest sprouting phenomenon compared with wild type.In addition,the dorsal aleurone layer of the osvp1 mutant was significantly thickened.Further analysis of p 13 01 Ubi-OsVP 1-eGFP overexpression transgenic materials showed that the thickness of the dorsal aleurone layer of OsVP1 overexpression seeds was significantly thinner compared with wild type,indicating that OsVP1 could participate in the regulation of the development of rice aleurone layer.3.The results of the aleurone layer resin section showed that the difference in the thickness of the aleurone layer between the wild type and the mutant was mainly in the dorsal of the grain,in which the wild type only had 3-5 layers of cells,while the mutant had 4-7 layers of cells.There was no difference in the thickness of the ventral aleurone layer.Transmission electron microscopy showed that the mutant had a thicker aleurone layer with higher level of storage proteins than the wild type,suggesting that OsVP1 may be involved in the differentiation of the aleurone.4.In order to have a better understanding of the mechanism by how OsVP1 regulating the development of rice aleurone layer,transcriptome sequencing was performed on the aleurone layer of 10DAF wild type and mutants.Compared with the wild type,a total of 2479 differentially expressed genes were detected in the two mutants,of which 1422 were upregulated and 1057 were down-regulated.GO enrichment analysis showed that the relevant genes such as aleurone grain membrane and ubiquitin ligase complex were significantly enriched.KEGG analysis found that these differentially expressed genes were mainly involved in some important metabolic pathways such as photosynthesis,plant signal transduction,glutathione metabolism,glycolysis,and starch sucrose metabolism.Among them,multiple auxin-related genes were differentially expressed in wild-type and osvp1 mutants.In addition,the expression of the aleurone layer-specific expression gene OsNF-YB1 in osvp1 mutants was decreased.5.RT-PCR results showed that by comparing the expression of key regulatory genes for aleurone layer development in wild type and osvp1,we found that the expression of TWINKLE,OsDEK1 and OsROS1a in osvp1 mutants was significantly down-regulated.As to whether OsVP1 could participate in the development of the aleurone layer by regulating these genes was still a question to be studied.6.Our previous studyfound that the PRC2 gene OsFIEl is also involved in the regulation of the aleurone layer,and the aleurone layer of osfie1 mutant is significantly thicker than that of the wild type.Here,we found that the thickness of the aleurone layer of the osfiel osvp1 double mutant was significantly increased compared with that of the single mutants.Considering that OsFIEl is dependent on gibberellin(GA)for regulating aleurone layer thickness,we also use GA to treat the wild-type and osvp1 mutants plants at heading stage.The results showed that there was no significant difference in aleurone thickness after treatment,suggesting that OsVP1 and OsFIE1 may not in the same pathway for regulating the development of aleurone layer.In addition,yeast two hybrid and luciferase complementation experiments also showed there was no interaction between the two proteins.7.In addition,we used CRISPR/Cas9 technology to knock out two B3 transcription factor,which designated as PHS-B3a and PHS-B3b,respectively.The phenotypic analysis results found that their mutations can cause pre-harvest sprouting in high temperature and humid conditions.