The Molecular Mechanism Of KNR6 Regulating Kernel Number Per Row In Maize

Maize(Zea mays L.)grain yield is a complex quantitative trait which is consisted of several components including kernel number per row(KNR),kernel row number(KRN),ear length(EL)and ear weight(EW),each of traits is controlled by an array of genes with minor genetic effect.KERNEL NUMBER PER ROW6(KNR6)is the previously reported gene for regulating ear length and kernel number per row in our laboratory.However,the acting mechanism and genetic regulatory network of KNR6 in kernel number per row are little known.In this study,we'll uncover the molecular mechanism of KNR6 in the regulation of the ear-inflorescence development and in turn kernel number per row.The main results are as follows:1.KNR6 physically interacts with AGAP: Using immunoprecipitation mass spectrometry(IP-MS),we identified 135 proteins which are involved in biological processes including the RNA translation,biosynthesis and catabolism,ATP metabolism,and intracellular transport.The interaction between KNR6 and a maize Arf GTPase activating protein(AGAP)was in vitro confirmed by yeast two hybrid(Y2H),firefly luciferase complementation imaging assay(LCI-assay),bimolecular fluorescence complementation(Bi FC)and pull-down assay.And the co-expression assay showed the overlapped RNA enrichment domains of both KNR6 and AGAP in meristems of the ear-inflorescence,inferring an overlapped enrichment of the proteins translated from both genes,which provide a spatial probability for the protein interaction of both KNR6 and AGAP.2.KNR6 proteins have kinase activity and can phosphorylate AGAP: We expressed in vitro and purified KNR6 protein.The self-phosphorylation activity of KNR6 and its phosphorylation activity to myelin basic protein(MBP),a common substrate,were tested.The results showed that KNR6 phosphorylate itself and MBP.However,those KNR6 isoforms with mutation at the 74 th and 172 th amino acid residuals lost their kinase activity,indicating that the 74 th and 172 th amino acid residuals are required for kinase activity of KNR6.Additionally,by ?-[18O4] ATP labeling phosphorylation experiment,we identified 63 proteins including AGAP they are considered as the phosphorylation substrates of KNR6.These proteins participate in the DNA transcription,protein binding,intracellular transport and development processes.Furthermore,AGAP as one of the substrates could be phosphorylated by KNR6 in vitro.Moreover,two 14-3-3 proteins also interacted with KNR6 and could be phosphorylated by KNR6.The yeast three-hybrid assay showed taht 14-3-3,KNR6 and AGAP form heterotrimers in vitro,implicating that 14-3-3 might be a molecule cofactor participating in the regulatory pathway of the KNR6-AGAP.3.Pleiotropy of AGAP in vegetative and reproductive development: We created the AGAP gene-edited lines by CRISPR/Cas9.In the AGAP knockout lines(agapko),the deletion causes the premature termination of AGAP translation.Compared with the non-transgenic siblings(AGAPNT),the agapko1 lines showed a shorter the length of both inflorescence meristem(IM)and the mature ear,and less kernel number per row.The agapko2 showed significant phenotype changes with dwarf plant,twisted internodes and curved leaves.Specifically,the ear development was dramatically inhibited,leading in the failure of reproduction,and the tassels presented claw-like shap,showing the pleiotropy of AGAP in vegetative and reproductive traits.4.AGAP genetically interacts with KNR6 to synergistically regulate ear length and kernel number per row: We also created the KNR6 gene-edited lines by CRISPR/Cas9.Through phenotyping the traits of KNR6-edited lines,we found that KNR6 controls KNR,EL and IM length,but not the other traits.Sequentially,knr6ko1 were crossed to agapko1,and then four genotypes AGAP/KNR6,agapko1/KNR6,AGAP/knr6ko1 and agapko1/knr6ko1 were identified from the segregation population.Each of single mutants: agapko1/KNR6 and AGAP/knr6ko1,showed a shorter ear with less kernels than AGAP/KNR6,but each of single mutants had a longer ear with more kernels than the double mutant agapko1/knr6ko1,indicating that AGAP genetically interacted with KNR6 to regulate ear length and kernel number per row cooperatively.5.AGAP interacted with small G-proteins participating in vesicle trafficking:Subcellular localization showed that AGAP and two Arf GTPase1(ARF1)proteins were located at Golgi.LCI-assay and Bi FC assay indicated that AGAP interacts physically with two ARF1 proteins.Furthermore,transmission electron microscope(TEM)observation of Golgi apparatus in the cells of the agapko1 roots revealed AGAP mutation strongly affect the structure of Golgi,showing a thinner Golgi cisternae and circularized Golgi apparatus.The fluorescent dye FM4-64 is used to monitor endocytosis in plant,we found the FM4-64 labelled endocytosis is delay,and agglomeration of the BFA(brefeldin A)bodies is reduced in the agapko cells,indicating that AGAP participates in vesicle trafficking.We suggest that AGAP participate in vesicle trafficking at endocytosis and Golgi/ER network through interacting with small G-protein ARF1 members.In summary,we hypothesize that KNR6 phosphorylates and then activates AGAP,and phosphorylated AGAP proteins participate in the endocytosis through interacting small G-protein ARF1.It is well known that the vesicle trafficking in plant species is involved in the polar auxin transport by determining the intracellular distribution of auxin efflux and infflux carriers.Therefore,we suggest that KNR6-AGAP-ARF1 complex modulates the ear length and kernel number through participating in the regulation of the auxin-mediated inflorescence development..