Map-based Cloning And Functional Analysis Of PAA1,a Gene Responsible For Panicle Apical Abortion In Rice (Oryza Sativa L.)

Rice?Oryza sativa?is an important staple crop for more than half of the global population.Grain number per panicle is one of key components for determining rice grain yield.Large panicle with numerous spikelets is the major target of rice breeding programs worldwide.However,the phenomenon of spikelets abortion on the apical portion of panicle frequently occurs in rice breeding programs and results in the significant decrease in the panicle size and grain number per panicle,which finally reducing the grain yield.To date,the genetic and molecular mechanism underlying the spikelets abortion remains largely elusive.Here,we identified a new mutant,panicle apical abortion1?paa1?,and examined the phenotypic characterization and specific defects of paa1 panicles during the course of panicle development.We isolated the corresponding gene PAA1 responsible for panicle apical abortion by map-based cloning and further investigated its function to reveal the molecular mechanism of panicle apical abortion in rice.Results from our experiment are summarized as follows:1.Compared with the wild-type,the mutant paa1 showed no visible abnormalities during vegetative development stage.However,after heading,the paa1 mutant displayed smaller and slightly erect panicles with aborted spikelets on the apical portion of panicles,and then these aborted spikelets were disappeared at the stage of grain maturity,resulting in comprehensive reductions in the panicle length,grain number per panicle,1000-grain weight and the plant height.Consequently,the grain yield in paa1 was influenced.2.Detailed investigation to various developmental stages prior to heading indicated that the apical spikelets in paa1 exhibited apparent growth inhibition at later panicle development stage.Finally,these aberrant spikelets became shriveled.To further analyze the cellular alternations in paa1 mutant,we performed TEM and TUNEL assay,and the results showed that the organized cell contents of aborted spikelets are disappeared and exhibit DNA degradation.Furthermore,the paa1 degenerated spikelets were identified with the H₂O₂ burst,MDA increment and up-regulated expressions of OsVPE2 and OsVPE3,two indicators of PCD.These results indicated that apical spikelets degeneration of paa1 is caused by programmed cell death,likely induced by the accumulation of hydrogen peroxide.3.Through the method of map-based cloning,the PAA1 locus was narrowed to an 82-kb region between makers C1 and H-4 in the long arm of chromosome 2,which includes nine predicted open reading frames.DNA sequence comparison revealed a single nucleotide substitution of A?G occurred in the second intron of gene LOC_Os02g45160.The mutation would alter the splicing site,resulting in two abnormal transcripts.Moreover,two abnormal transcripts both lead to premature terminations of proteins translation in paa1 mutant.Analyses of genetic complementation and RNA interference verified that LOC_Os02g45160 is indeed the PAA1 gene.PAA1 encodes an aluminum-activated malate transporter and belongs to ALMT family in rice.The PAA1 protein consists of 488 amino acid residues and has seven potential transmembrane helices in the N-terminal of PAA1.PAA1 is specially localized to the plasma membrane and expressed in various tissues,including roots,stems,leaf blades,leaf sheathes and panicles.5.Heterologous expression of PAA1 in E.coli showed that PAA1 acts as a dicarboxylate transporter,facilitating the malate transport.Used two-electrode voltage clamp method,Xenopus oocytes expressing PAA1 could show significant malate current,including inward and outward current.Furthermore,addition of Al to the bathing solution had no significant effect on the inward current in oocytes expressing PAA1 and preloaded with malate,suggesting PAA1 is an aluminum-independent malate transporter.6.In rice seedlings,the exposure to malate significantly enhanced the expression level of PAA1 in wild-type seedlings and the malate accumulation in the shoots of paa1 was significantly reduced compared with that of wild-type rice.The malate content was determined in various organs at the reproductive stage,the result showed the paa1 mutant exhibited significantly reduced malate content in apical panicles compared with the wild-type,suggesting that mutation of PAA1 led to defective accumulation in panicle,and finally,resulted in the abnormal phenotype of panicle apical abortion in the paa1 mutant.7.The measurement of pyridine nucleotide contents showed that the levels of NAD⁺,NADP⁺and NADPH in aborted spikelets of paa1 are significantly reduced,in comparison with the apical spikelets of WT.Moreover,aborted spikelets of paa1 exhibited the disturbance of cellular redox balance,accompany with the decreased level of NADPH/NADP⁺ratio and increased level of NADH/NAD⁺ratio.These results indicated that the decrease of malate content in paa1 apical spikelets causes the disturbance of NAD?H?or NADP?H?-related cellular redox homeostasis,resulting in ROS burst and finally inducing cell death.