Identification Of The Function Of OsPLATZ,a Rice Member Of Zinc-dependent DNA-binding Protein Family

Plant height and panicle architecture are important agronomic traits and closely related to crop yield.Therefore,it is significant to reveal their regulation mechanism and network.In this study,a gene encoding rice Plant A/T-rich sequence-and Zinc-binding protein,OsPLATZ,was identified.Functional knockout mutants of OsPLATZ displayed various variations,such as shorter plant height,larger panicles,smaller grain size,and lower seed germination rate.The pleiotropism indicates that OsPLATZ plays important roles in the development of rice plant height and panicle traits.It was reported that The PLATZ superfamily is a novel type of plant-specific zinc-dependent DNA binding protein that non-specifically binds to A/T-rich sequences and inhibits transcription.In 2001,the first member of this family was reported to be found in peas.There are six PLATZ homologues in rice,yet their functions have not been reported.Moreover,functions of members in this superfamily are also rarely reported in other plants.In this study,the biological function of OsPLATZ was revealed by transgenic overexpression and CRISPR/Cas9 mediated gene editing.The expression pattern of this gene was analyzed by Quantitative RT-PCR?qRT-PCR?and in situ hybridization.The subcellular localization of OsPLATZ was studied using protoplast transient expression system.Furthermore,yeast two-hybrid?Y2H?was used to screen interacting-factors of OsPLATZ.The main results are as follows:?1?A gene editing method based on CRISPR/Cas9 system was used to perform functional knockout of OsPLATZ.Genotyping,phenotyping and co-segregation analysis were carried out with the obtained T₀ and T₁ transgenic plants and the F₃ hybrids descended from a cross between the knockout mutant osplatz and wild type.The results showed that the mutants display multiple defects throughout the whole lifetime from seed germination to maturation,such as lower seed germination rate,shorter plant height,shorter leaves,smaller grain size and increased numbers of secondary branches and spikelets in heterozygous individuals.These results indicated that OsPLATZ has pleiotropic effects on rice growth and development.?2?The results of qRT-PCR experiments showed that the expression level of OsPLATZ was high and specific at the panicle formation stage.Further,the expression signals were detected specifically in primordia of both primary and secondary branches by in situ hybridization.The expression pattern is consistent with the panicle phenotypes of the knockout mutants,further demonstrating that OsPLATZ is involved in regulating the panicle development in rice.?3?As for the dwarf phenotype in the knockout mutant osplatz,the result of co-segregation analysis with F₃ plants showed that it is a semi-dominant trait.?4?Two over-expression vectors ofOsPLATZwere constructeddrivenby Ubi promoters with and without 3×FLAG-tag,respectively.Both constructs were used to transform wild type rice and multiple independent transformants were obtained,creating materials for further study of biological functions and molecular mechanism of OsPLATZ.?5?The result of subcellular localization analysis indicated that OsPLATZ is localized in the nucleus.Combining this result and the pleiotropism,it is reasonable to infer that OsPLATZ is probably a transcription regulator.?6?OsPLATZ was used as a bait to screen the cDNA library of rice young panicles by Y2H assay,and nine candidate OsPLATZ interacting-factors were obtained.Further verification showed that OsPLATZ interacts with the protein encoded by Os09g0416500gene.In summary,this study preliminarily revealed the biological functions and expression pattern of OsPLATZ and identified an interacting factor of OsPLATZ,laiding a foundation for further elucidation of the molecular mechanism of OsPLATZ involved in the regulation of rice growth and development,as well as providing new resources for further understanding the functions of PLATZ superfamily.