KRN4 Control Maize Kernel Row Number By Cis-regulating UB3 Expression In Maize

Rice(Oryza sativa L.)and maize(Zea mays L.)are monocotyledons of graminaceae.Their branches are composed of tillers produced from axillary meristems(AMs)and inflorescence branches produced from branch meristems(BMs).The mechanism of AMs and BMs development in grasses is similar,and genes regulating branching are always orthologs.unbranched3(UB3)is a SBP-box gene,responsible for maize kernel row number(KRN)variation and yield traits by regulating the rate of lateral primordia initiation,but how UB3 negatively regulates axillary meristems is still unclear.KRN4 is a quantitative trait locus(QTL)for KRN by regulating the expression of UB3,located in ~60kb downstream of UB3 transcription start site(TSS),how KRN4 regulates UB3 expression distantly is remain unknown.To uncover the effects of UB3 in modulating grass branches,genetic transformation was performed in both rice and maize.In addition,the hypothesis of KRN4 regulating UB3 expression in distance is established.The main finds are:1.Overexpressed UB3 repressed calli and branches development in maize and rice respectively: UB3 overexpressed in maize calli,the differentiation and regeneration of maize calli were repressed significantly,and the growth of seedlings was compromised;UB3 overexpressed in rice,plant growth including plant height,tiller number and panicle branches number were decreased;while UB3 expressed at an appropriate level in rice,the number of panicle branches was higher,while tiller number and plant height were slightly reduced.2.UB3 regulated rice branches developmentthrough cytokinin signal pathway: RNA-seq was performed using the shoot apicals(SAs)and young panicle(YPs)RNA of UB3 overexpressed transgenic lines,different expression genes(DEGs)on cytokinin pathway were detected.Cytokinin level of seedlings of transgenic lines was lower than that in non-transgenic plants.Combined RNA-seq data and Ideal Plant Architecture1(IPA1)Chromatin Immunoprecipitation quantitative PCR(Ch IP-seq)data,two genes(LONELY GUY1(LOG1)and LOC_OS04G44354)on cytokinin biosynthesis and cytokinin oxidase/dehydrogenase2(Os CKX2)on cytokinin degradation were found.Thus,overexpression of exogenous UB3 in rice dramatically suppressed tillering and panicle branching as a result of a greater decrease in the amount of active cytokinin.By contrast,moderate expression of UB3 suppressed tillering slightly,but promoted panicle branching by cooperating with SPL genes(SPL7,SPL14,SPL17),resulting in a larger grain number per panicle in rice.3.The UB3 network in regulating maize KRN: RNAs isolated from immature ear of ub3::mum plants and wild type plants were used for RNA-seq,DEGs showed 17 DEGs associated with cytokinin biosynthesis were upregulated,and 3 DEGs associated with cytokinin degradation were downregulated.Therefore,UB3 negatively regulating cytokinin level might exhit in maize inflorescence development.In addition,genes on CLAVATA-WUSCHEL feedback loop,including THICK TASSEL DWARF1(td1),FASCIATED EAR2(fea2),COMPACT PLANT2(ct2)and FASCIATED EAR3(fea3),and other classic genes like FON2-LIKE CLE PROTEIN1(Zm FCP1),WUSCHEL1(Zm WUS1)and CLAVATA3(Zm CLV3)were also detected.EMSAs showed that UB3 protein could directly target the promoter regions of Zm FCP1 and Zm WUS1,indicating that expressions of both Zm FCP1 and Zm WUS1 might be regulated by UB3 directly.4.KRN4 enhance UB3 expression in cis: KRN4 is a 3.1 kb intergenic region responsible for KRN,located in ~60kb downstream of UB3.The mu-TE inserted into the KRN4 region destroyed the structure of KRN4,so the expression level of UB3 in the immature ear of krn-mum1 dropped at least one times,and both the KRN and primary tassel branches number(PTBN)increased significantly.UB3 expression level in the ear with KRN4 allele is much higher than that with KRN4 NX allele which contains a 1.2-Kb insertion.Transient assay in maize protoplast showed KRN4 or KRN4 NX is able to enhance the firefly luciferase(luc)gene expression compared to minimal 35 S,and the enhancer activity of KRN4 is much higher than that of KRN4 NX.In addition,KRN4 and KRN4 NX can also enhance expression of the luciferase droved by p UB3.5.KRN4 binding proteins analysis: OCS Element Binding Factors1(OBF1)and OCS Element Binding Factors4(OBF4)were identified through yeast one hybrid(Y1H)and EMSAs with E1 segment.Transient assay in maize protoplast showed OBF1 and OBF4 proteins could bind on E1 segment and enhance luc gene expression.6.UB2 binds and positively regulating UB3 expression: UB2 is the orthologous genes of UB3 in maize,and enhance the ear and tassel phenotype in the ub3 and ub2 double mutant.In immature ear of UB2 overexpressed transgenic lines,the expression level of UB3 increased dramatically;in ub2::mum in which UB2 expression level was reduced,UB3 expression level decreased significantly.Thus UB2 promotes UB3 expression in maize immature ear.Ch IP-q PCR using immature ears of UB2 overexpressed transgenic lines was performed,DNA fragments at KRN4-P4 and p UB3-P sites were significant enriched in the two biological repeats compared with the control.The luciferase complementation image(LCI)assays showed UB2 was able to interact with OBF1 and OBF4.7.Summary of KRN4 regulating cis-regulating UB3 expression modulating KRN: UB2 protein mediated the chromatin loops formation between KRN4 and UB3 promoter so that KRN4 close to UB3 promoter spatially;Then KRN4 binding proteins OBF1 and OBF4 were recruited to form transcription complex with UB2 protein;Under the cooperation with RNA polymerase II(RNAP II),transcription complex target on UB3 promoter,activate and enhance the UB3 expression.Combined the network of UB3 regulating branches in rice and the RNA-seq data in maize immature ear,we speculated that UB3 is able to regulate the genes on the cytokinin pathway to response KRN variation;in addition,UB3 could target on the promoter region of FCP1 and WUS1 on the CLAVATA-WUSCHEL feedback loop to regulate the inflorescence meristem size.