Two Genes In PLATZ Family Regulate Rice Inflorescence Development

Panicle architecture and grain shape are key factors for determining rice grain yield.Thus it is important to elucidate the molecular mechanism and genetic network underlying the regulation of the development of the two characters.Previous studies in this laboratory showed that OsPLATZ1 is involved in the regulation of panicle and grain development in rice.OsPLATZ2 shares high sequence-identities with OsPLATZ1,and their expression patterns are similar,implying that they probably exert similar function.PLATZs are a kind of DNA binding proteins dependent on zinc and act as transcription regulators.The only reported PLATZ member in rice is GL6/GS6(OsPLATZ2),a grain length gene,yet the underlying mechanism of which has not been fully clarified.Meanwhile,the functions and regulatory mechanisms of PLATZs in other plants species have also been rarely reported.This study was conducted to reveal the biological functions of OsPLATZ1 and OsPLATZ2,and to explore the regulatory pathways involving OsPLATZ1,through transcriptome sequencing analysis and protein-interaction assay.The main results of this study are as follows:(1)A phylogenetic tree of PLATZ family members was constructed by searching the protein sequences in public database,and it was found that there are 12,17 and 15 PLATZ members in the genomes of Arabidopsis,maize and rice,respectively.(2)Single and double mutants of OsPLATZ1/OsPLATZ2,and transgenic materials with over-expression of OsPLATZ1 or OsPLATZ2 were constructed for genetic analysis.The results showed that loss of single gene function of OsPLATZ1 or OsPLATZ2 leads to increased numbers of secondary branch,and reduced grain size and plant height.The phenotype of OsPLATZ1-over-expression transgenic plants varies with the expression levels of exogenous genes,showing generally decreased numbers of primary and secondary branchers and grains per panicle,yet increased grain size.The results proved that OsPLATZ1 and OsPLATZ2 are redundantly involved in the regulation of panicle branching and grain development in rice,as well as the control of plant height.(3)The subcellular localization of OsPLATZ1 and OsPLATZ2 was analyzed using the transient expression system with rice protoplasts,and the results showed that both OsPLATZ1 and OsPLATZ2 are localized in the nucleus.The results of RNA in situ hybridization assay showed that OsPLATZ1 is highly expressed in the primordia of primary branches,secondary branches and spikelets,and strong signals were also detected in lemma and palea primordia.This expression pattern is consistent with the phenotypes that the number of panicle branches increases and the grain size decreases in the loss-of-function mutant,while the number of panicle branches decreases and the grain size increases in the over-expression plants.The expression pattern of OsPLATZ1 is highly consistent with the reported expression pattern of OsPLATZ2,which further indicates that both OsPLATZ1 and OsPLATZ2 regulate panicle branching and grain shape formation.(4)In order to reveal the downstream pathways of OsPLATZ1 controlling panicle development,transcriptome sequencing analysis was conducted using young panicles of OsPLATZ1 mutant in this study.The results showed that the differentially expressed genes were significantly enriched in biological processes such as photosynthetic carbon fixation,amino acid and carbohydrate metabolism,indicating that OsPLATZ1 influences inflorescence branching and grain development by regulating photosynthesis and metabolic processes.(5)The protein interactions among OsPLATZ1/OsPLATZ2 and OsTFC1,GRFs,and SLR1 were further explored using the Y2 H system.It was found that full-length proteins of OsTFC1,OsGRF2,OsGRF5,OsGRF8 and OsGRF9 interact with both OsPLATZ1 and OsPLATZ2,while full-length proteins of OsGRF1,OsGRF3,GRF4,OsGRF7,OsGRF10,OsGRF12 and SLR1 interact only with OsPLATZ2.The results suggested that OsPLATZ1 and OsPLATZ2 may be involved in small RNA transcription,GRFs and SLR1 related pathways.In summary,this study proved that OsPLATZ1 and OsPLATZ2 negatively regulate inflorescence branching and positively regulate grain size in rice.The consistency was clarified between the spatiotemporal expression pattern of OsPLATZ1 and the mutant phenotype of osplatz1.Both OsPLATZ1 and OsPLATZ2 may be involved in RNA polymerase Ⅲ-mediated noncoding RNA transcription via the interactions with OsTFC1,and may regulate rice development through the interactions with multiple members of GRF family.This study reveals new functions of rice PLATZs,and provides clues to elucidate the molecular mechanism and genetic network involving PLATZs underlying the regulation of panicle and grain development.