Cloning And Functional Analysis Of The PAY1 Gene Controlling Plant Architecture And Grain Yield Of Rice (Oryza Sativa L.)

Plant architecture, usually referred to as the three-dimensional organization of the aerial part of a plant, is mainly determined by factors including branching (tillering) pattern, plant height, leaf shape and arrangement, and inflorescence morphology. Plant architecture is a complex of the important agronomic traits that determine grain yield. During the process of crop domestication and improvement, desirable plant architecture was the main selection direction for obtaining high-yielding varieties. Therefore, understanding the mechanism underlying plant architecture will facilitate the breeding of crop varieties with high-yield potential.In this study, a mutant PAY1 (PLANT ARCHITECTURE AND YIELD 1) which had a more compact plant architecture was characterized from the ethyl methane sulphonate (EMS)-mutagenized population of YIL55, an introgression line which derived from the progenies between wild rice and cultivated rice and showed short plant height, more tillers, larger tiller angle, less grains and low yield. Compared with YIL55, the PAY] mutant exhibited greater plant height, lower tiller number, smaller tiller angle, thicker stems, larger panicles, more grains per panicle and higher grain yield. The PAY1 gene was isolated through map-based cloning and its molecular function had also been analyzed.Genetic analysis showed that the changed penotype of the PAY1 mutant was controlled by a single dominant gene. Using an F2 population derived from the cross between the PAY1 mutant and Nipponbare, PAY1 was mapped on the long arm of chromosome 8 and further delimited within a 51-kb region. Comparing the genomic sequence of the mapping region (-51-kb) between the YIL55 and PAY1, only a single nucleotide change, g to a, at position+1244 in exon 4 of LOC_Os08g31470 was found, which resulted in a single amino acid substitution from Glutamine to Arginine in the PAY1 mutant.To verify whether the altered penotype of the PAY1 mutant was caused by a single nucleotide change of the LOC_Os08g31470 gene, an over-expression (OE) vector driven by the Ubiquitin promoter was generated and introduced it into YIL55. All the transgenic lines displayed the PAY1-like phenotype. In contrast, a pRNAi construct was introduced into the PAY1 mutant, and 10 independent RNAi transgenic lines showed plant architectures very close to YIL55. These results strongly confirmed that LOC_Os08g31470 is PAY1.Sequence comparison between genomic DNA and cDNA revealed that the PAY1 gene was composed of five exons and four introns that encoded a protein with 590 amino acids. Sequence analysis indicated that the PAYI cDNA is 2172-bp long, with an ORF of 1773-bp, a 172-bp 5’-untranslated region (UTR), and a 227-bp 3’-UTR.Transient expression of the construct p35S::PAY1-GFP showed that GFP-PAY1 localized to the nucleolus, indicating that PAY1 was a nuclear protein. The transcriptional activation assay showed that expression of the payl and BD (GAL4 binding domain) fusion protein in yeast resulted in a weak reporter gene expression while the PAY1 and BD fusion protein in yeast abolishes the weak auto-activation, implying that the payl has weak transcriptional activity in yeast. The transcriptional activity of PAY1 was analyzed using a dual luciferase reporter (DLR) assay system in Arabidopsis protoplasts. The result showed that payl significantly reduced the relative luciferase activity compared with the GAL4 BD negative control while the PAY1 slightly reduced the relative luciferase activity. Taken together, these results demonstrated that payl is a nuclear transcription factor functioned as transcriptional repressors and the point mutation in PAY1 abolished its transcription repression function.RT-PCR and GUS stain showed that PAY1 is ubiquitously in the examined rice organs, but especially highly expressed in leaves. RNA in situ hybridization revealed that PAY1 was predominantly expressed in the leaf primordia, young leaves, shoot apical meristem, tiller buds, the primordia of primary and secondary branches and the developing spikelets. Futher experiments showed that the mutation of PAY1 let to defective uptake capacity of IAA and basipetal PAT activity, and the reduced basipetal IAA transport activity led to the altered endogenous IAA distribution in the PAY1 mutant plant, which maybe the reason of the changed plant architecture of the PAYl mutant.The PAY1 allele was introgressed into indica cultivar Teqing (TQ) and 9311, respectively, using marker-assisted background selection. Compared with TQ plant and 9311 paint, both TQ-PAY1-NIL and 9311-RAY1-NIL plants showed taller height, less tiller number, thicker clums, larger panicles, and increased grain yield per plant, indicating that PAY1 is an important dominant regulator of plant architecture and grain yield in rice, and findings concerning PAY1 will be of value for breeding high-yielding rice.To further analyze the role of PAY1 in the regulatory network of rice plant architecture, the relationship between PAY1 and the PROG1(PROSTRATE GROWTH 1) gene was studied. The results showed that the expression level of PROG1 was significantly inhibited in the PAY1 mutant, which indicated that the interaction between PAY1 and PROG1 may exist. However, current results could not confirm that there is a direct interaction between PAY1 and PROG1, and further studies are still needed.