Function Analysis Of Cysteine Proteinase Gene βVPE And γVPE In Arabidopsis Thaliana

Vacuolar processing enzyme(VPE)is a cysteine proteinase which is originally identified for maturation and activation of proteins in plants vacuolar.In plant,VPE regulate the PCD by vacuole-mediated cell death.In the angiosperm,PCD of the tapetum is a precondition for the development of pollen.Many transcription factors are involved in anther cell differentiation and tapetum development.The expression of βVPEwas decreased in spl/nzz and emsl/exsmutants,and is upregulated in tdfmutants.For these reasons,we speculated that βVPEmay play an essential role in flower development.The differentiation of TEs is a typical PCD of vascular plants.Vacuolar membranebreakdown is the crucial event in TE PCD.Previous studies showed that the absence of yVPE delayed vacuole degradation after leaves infected by TMV in the yvpe mutant.So,we speculated that yVPEmay play an essential role in stem development.Based on the above results,we researched the role of βVPE during anther development using βvpe mutant,and researched the role ofγVPE during stem development using yvpe mutant.The results are as follows:(1)Function analysis of βVPE in anther development.The result of Real-time PCR and GUS staining showed that βVPE was expressed specifically in the tapetum from stages 5-8 of anther developmentand transformed into the mature enzyme before stages 7-8.The result of paraffin section and transmission electron microscopeshowed that the degradation of tapetum cell wall was failed,the formation of tapetum and elaioplast was decreased significantly,and the development of some pollen was abnormal.The result of catalysis and Western bolt showed that the mature βVPE protein could induce the maturation of CEP1 in vitro and the maturation of CEP1,RD19A,and RD19C were seriously inhibited in βvpe mutants.These results indicate thatβVPE is indirectly involved in pollen development and tapetal cell degradation by participating in the maturation of cysteine proteases.(2)Function analysis of yVPE in stem development.The result of Real-time PCR and immunohistochemistry showed that the yVPE protein was expressed specifically in cambium cells and developingxylem during stem development.The result of paraffin section and transmission electron microscopeshowed that the degradation of fiber cells intracellular contents was delayed and the secondary fiber cell walls was thickened.The result of catalysis and Western bolt showed that the mature yVPE protein could induce the maturation of CEP1 in vitro and the maturation of CEP1 was seriously inhibited in yvpe mutants.Transcriptome analysis showed that the expression of 2,001 genes was significantly altered expression in the yvpe mutants,and most of them are important for secondary cell wall formation and PCD.These results demonstrate that γVPE is indirectly involved inxylem PCD by participating in the maturation of CEP1.In conclusion,PVPE and yVPE regulate the development of pollen and xylem respectively by activating the maturation of other proteases and participating in the PCD process of anther tapetum and stem xylem.PVPE and yVPE play an important role in regulating PCD of Arabidopsis thaliana.