Cloning And Functional Analysis Of ZmRPC2 Regulating Kernel Development In Maize

The improvement of maize yield per unit area is the most effective measure to meet the demand of maize yield in China.Since the kernel weight is an important factor controlling yield per unit area,it is of great theoretical and practical significance to clone the genes that control maize kernel shape and kernel weight and reveal the molecular mechanisms that regulate kernel development.In this study,we cloned ZmRPC2 which plays a role in maize kernel development by BSA-seq and map-based cloning,using a small kernel mutant named smk7 obtained by EMS mutagenesis.The mechanism of ZmRPC2 controlling maize kernel development was clarified.The main results are as follows:1.Phenotypic characterization and genetic analysis of smk7: The development of embryo and endosperm of smk7 was more slowly than that of WT,the length,width,thickness and 100-kernel weight of smk7 mature kernel were dramatically reduced by10.07 %,11.70 %,19.38 % and 30.05 %,respectively.Cytological observations revealed that in the smk7 kernels,the cells in basal endosperm transfer layer(BETL)lacked cell wall ingrowths,and the starch granules(SG)and protein bodies(PB)in the central starch endosperm(CSE)accumulated slowly.However,the mature kernels of smk7 were still vitreous endosperm.Genetic analysis showed that the smk7 mutant is a monogenic recessive mutation.2.Positional cloning of ZmSmk7: BSA-seq was performed and the smk7 gene was mapped to an 80-Mb interval on chromosome 6.By map-based cloning using 4300 mutant kernels,we cloned the smk7 gene Zm00001d035960 which encodes the second subunit of RNA polymerase Ⅲ(RNAPⅢ),and then it was named ZmRPC2.Sequencing analysis revealed that a SNP at the splicing site on the 36 th exon of ZmRPC2 in smk7 resulted in 26 bp intron retention in the 36 th intron,leading to frameshift and premature termination of the protein.3.Functional verification of ZmRPC2: The candidate gene ZmRPC2 was edited by the CRISPR/Cas9 gene editing system,and three independent ZmRPC2 knock-out transgenic lines were generated.Three allelic mutants showed kernel development defective phenotype,and these alleles cannot complement smk7,which confirmed that ZmRPC2 is the mutated gene in smk7.4.The molecular mechanism of ZmRPC2 regulating kernel development in maize:Yeast two-hybrid screening and bimolecular luciferase complementation experiment revealed that ZmRPC2 could interact with the fourth subunit of RNAPⅢ(RPC53),the11 th subunit of RNAPⅢ(AC40),and the subunit of transcription initiation factor TFⅢC(TFC1)and the regulatory factor of RNAPⅢ(FL3)to control the transcription of small RNA.When it was defective,the expression of 5s r RNA and t RNAs was reduced by more than two-fold relative to that in WT endosperm and a lot of genes related to DNA replication and cell cycle were differentially expressed,which leads to the cell proliferation and endoreduplication are blocked in endosperm,resulting in reduced endosperm cell number and smaller kernel in smk7.5.ZmRPC2 genetically interacts with FL3: In order to revealing the relationship between ZmRPC2 and FL3,which have the same function of controlling the transcription activity of RNAPⅢ,smk7 and fl3 were crossed to constructed a double mutant.We found that the expression level of ZmRPC2 was increased by 112.76 % in fl3 endosperm,which compensated for kernel development defects of fl3,so the size of fl3 kernels was not significantly different from that of the WT,indicating that ZmRPC2 has a compensatory effect on FL3;meanwhile,the effect of both smk7 and fl3 on cell proliferation of endosperm and 100-kernel weight was cumulative,leading to smaller endosperm size and kernel weight of smk7;fl3 relative to smk7,these results indicated that FL3 and ZmRPC2 act on the same pathway to affecting the transcription activity of RNAPⅢ,controling cell proliferation of endosperm and development of kernel in maize.In fl3 endosperm,a large number of genes related to protein synthesis,transport,processing,modification and decomposition were down-regulated,the synthesis of zeins was decreased and non-zeins was increased,and mature kernels showed soft endosperm.Meanwhile,in smk7 endosperm,the expression levels of these genes did not change,nor did the synthesis of zein and non-zein,so the mature kernels presented vitreous endosperm.These results indicated that ZmRPC2 can not regulate the synthesis of zeins,and that FL3 controls the synthesis of zeins achieved through other pathways.