Identification And Functional Analysis Of Male Sterility Gene OsPDT1 In Rice (Oryza Sativa L. Ssp. Indica)

Rice is both a model plant for crop research and a globally important food crop.In recent years,more and more genes were cloned and analyzed in rice.As one of the most interesting fields,male sterility research has also obtained a serious of achievements.In male sterility rice,the mutant always could not produce fertility pollen grains in the anthers at maturation stage.Based on recent classification of anther developmental stages,the anthers were classified into 14 developmental stage.From stage 1 to 7,the cells of flower tissue formed,including microspore mother cells,anther wall cells and connective vascular tissue.From stage 8 of anther development,the tapetal cells initiate the programmed cell death（PCD）,which provides nutritions for microspores development.In this study,we identified a male sterility mutant Oryza sativa pectin defective tapetum 1（ospdt1）,which was involved in the PCD of tapetal cells and extine formation.The results as follows:1 Gene mapping and phenotype analysis of OsPDT11.1 OsPDT1 involved in the fertility development of pollen grains in riceCompared with wild type,ospdt1 mutant produced no filled grains.We found the ospdt1mutant failed to produce viable pollen grains at maturation stage.Transverse sections of anthers were performed,and no obvious defects were detected at metaphase of anther development in ospdt1 anthers.At late stage of anther development,we found the tapetum were highly vacuolated and pollen grains were aborted in ospdt1 anthers.Transmission electron microscopy were performed at stage 10 to 11,and the results showed that the exine of pollen wall was defective,which caused aborted microspores.At stage 11,the tapetal cells expanded abnormally and exhibited plasmolysis,which indicate that the degradation of tapetum was abnormal.1.2 OsPDT1 involved in the PCD of tapetal cellsAs the abnormal degradation of tepetum in above detection,we performed TUNEL assay to explore the PCD of tapetal cells.We found the PCD of tapetal cells was earlier in ospdt1 anthers,which appeared at stage 8a.We suggest the earlier PCD in the tapetal cells caused complete male sterility of the ospdt1 mutant.1.3 OsPDT1 encodes a galacturonosyltransferaseMap-based cloning was performed and OsPDT1 was mapped to chromosome 9 to a 35kb region.Sequencing of candidate genes within this region revealed a transversion from“T”to“A”in the ninth exon of Os09g0531900.We performed a functional complementation assay to confirm that Os09g0531900 was OsPDT1,which encodes a galacturonosyltransferase.We detected the intracellular localization of OsPDT1,but no observable signals were detected with transient expression of OsPDT1-GFP or OsPDT1_1-260aa-GFP.Transient expression of OsPDT1_1-141aa-GFP in the protoplasts resulted in fluorescent signals in the Golgi apparatus.We investigated the spatiotemporal expression pattern of OsPDT1,and the results of RT-q PCR indicated that OsPDT1was expressed in the root,stem,leaf,sheath,and spikelet.In situ RNA hybridization revealed more detailed information on OsPDT1 expression in specific tissues,and found OsPDT1 was predominantly expressed in the pollen mother cells,microspores and tapetal cells in the anther sac.2 Function analysis of OsPDT12.1 Pectin content in ospdt1 anther sac was not dramatically influencedAs homologous to GAUT1,OsPDT1 may participate in the synthesis of homogalacturonan（HG）,the most abundant pectic polysaccharides.We quantified the pectin in spikelets by means of carbazole colorimetry and conducted immunofluorescence using JIM5 and JIM7 antibodies to analyse the distribution of HG in the anther locule,however,no significant difference was observed.The cell wall contains the most abundant pectin content,our results conclude that the mutation of OsPDT1 did not dramatically affect the HG in the cell walls.Thus,we focused on the intracellular pectin.2.2 The intracellular distribution of Osi WAK1-EYFP was affected in ospdt1To explore the defects exhibited by the ospdt1 mutant,we investigated whether the Oryza sativa indica Wall-associated Kinase 1（Osi WAK1）works as a tool for intracellular pectin reasearch.We found the EXD of Osi WAK1 bound to polygalacturonide in vitro,and Osi WAK1-EYFP was localized in the secretory vesicle compartment（SVC）of wild type protoplasts.The Osi WAK1 fusion protein lacking the EXD（DEL-EYFP）barely accumulated in the SVC of wild type protoplasts.In the ospdt1 protoplasts,the SVC which containing Osi WAK1-EYFP was dramatically decreased.In the Osi WAK1-EYFP overexpressing line（WEO）,we found that the fluorescence signals of Osi WAK1-EYFP accumulated in the ball-like structures and cell periphery,however,the Osi WAK1-EYFP overexpressing line in the ospdt1 background（ospdt1/WEO）showed that fluorescence signals mainly accumulated around the cell periphery,and with rare cases,signals can be detected in the ball-like structures.We suggest the intracellular pectin,which may be defective in ospdt1 cytoplasm,affected the intracellular distribution of Osi WAK1-EYFP.Further research indicated that the localization of SVC could help Osi WAK1-EYFP to transport to the cell periphery.2.3 The OsiWAK1 is involved in PCD regulation of tapetal cellsThe Osi WAK1 RNA interference lines were explored,we found them were sterility and exhibited earlier PCD signals.We further investigated the fertility of WEO and found it exhibited severe sterility,in addition,the WEO anthers contained tetrad pollen grains at maturation stage.We further explored the PCD of tapetal cells,and found it was delayed in WEO anthers.2.4 OsPDT1 participates in the PCD of tapetal cells,at least in part,by affecting the function of Osi WAK1We detected the anther development of the ospdt1/WEO.The ospdt1/WEO exhibiteds similar phenotypes to WEO but not ospdt1.We investigated the PCD of tapetal cells in the ospdt1/WEO,and found the PCD signals were nromal in ospdt1/WEO at stage 8a.In summary,we consider the mutation of OsPDT1 might affect the intracellular pectin,which in turn influenced the inracellular distribution and function of Osi WAK1,and caused abnormal PCD of tepetum and absolute male sterility.