Molecular Mechanism Of Tapetal Development Regulated By Cysteine Proteases In Brassica Napus

Rapeseed oil is one of the major edible oil and heterosis utilization is considered to be the most efficient way to increase yield and improve quality.Male sterility is the most efficient way to use heterosis.As previous study showed that the only difference between the near isogenic lines 7365AB(Bnams3ms3ms4bms4b/ Bna Ms3ms3ms4bms4b)lies in the Bn Ms3 locus which is responsible for the fertility of 7365 B.In Bnms3 mutant,the expression of some cysteine proteases associated with tapetal degradation was affected.The degradation of tapetal is considered to be a programmed cell death(PCD)event.The major papain-like cysteine proteases(PLCPs)are crucial executors during proper timing of tapetal PCD.In this study,PLCPs were utilized to demonstrate the function of tapetal in anther development in Brassica napus.The results were described as follows.1.Differential expression of four PLCPs in the male fertile and male sterile linesThirty-one cysteine proteases have been investigated by RT-PCR in the 7365 A mutant and the wild-type 7365 B in B.napus.The results showed that Bn CP1(AT1G06260)and Bna C.CP20.1(AT1G29090)could only be detected in the wild type but not in the 7365 A mutant;however,Bn CP13(AT5G45890)and Bn CP21(AT3G49340)only expressed in the 7365 A mutant but not in the wild type;other cysteine proteases showed no differences between the wild type and 7365 A mutant.2.Expression of at CP13 and at CP20 prior to the tetrad stage impair anther developmentAs Arabidopsis and B.napus are both members of Brassicacea,they share high sequence identity in their open reading frames(ORFs)and have similar gene functions.There are multiple copies in B.napus,so the functions of the four cysteine proteases in Arabidopsis were firstly investigated.To identify the function of these four genes during anther development,the T-DNA insertion mutants in Arabidopsis were investigated.The mutants displayed no visibly altered phenotype during vegetative and reproductive growth compared with wild-type plants.Meanwhile,the expression of four cysteine proteases with the double 35 S promoter displayed no visibly altered phenotype during vegetative and reproductive growth compared with wild-type plants.In the wild-type tapetum,PCD begins at tetrad stage but RT-PCR showed these genes were not expressed until the uninucleate stage.Then,the anther specific promoter Bn A9 was employed to control the expression of these four cysteine proteases.The results demonstrated that the expression of CP13 and CP20 with the A9 promoter exhibited defects in pollen development.So,we explored the functions of Bn CP20 and Bn CP13 in our study.3 Function analysis of Bna C.CP20.1 and Bna C.CP13.4.Bna C.CP20.1and Bna C.CP13.4 were cloned from the flower bud of 7365 B plants and 7365 A plants in B.napus,respectively.The predictive protein analysis indicated that they all shared the typical motif of cysteine proteinases,such as: EX3RX3FX2NX3IX3 N,GCNGG motif,conserved residue Gln and three conserved catalytic residues(Cys-HisAsn).The expression pattern of Bna C.CP20.1 displayed that it merely expressed in anther tapetal cells and pollen grains from the uninucleate stage until mature pollen grains formation during anther development in 7365B;and Bna C.CP13.4 only expressed in anther tapetal cells from the miscrospores release from the tetrad stage unit mature pollen grains stage in 7365 A.Moreover,we also investigated the expression level of Bna C.CP20.1 and Bna C.CP13.4 in other three male-sterile materials: such as S45 A mutant in B.napus,hau CMS mutant and D.berthautii CMS mutant in B.juncea.q RTPCR analysis confirmed that Bna C.CP20.1 was expressed only at late stages during anther development in the wild-type plants,but not in the male sterile lines;and Bna C.CP13.4 was expressed only in the male sterile lines during late stages of anther development,but not in the wild-type.Cross-sections of anthers from the transgenic plants and wild-type plants were used to examine the pollen development process.The results displayed that the tapetal cells of male sterile kept on enlarging and vacuolating,the release of microspores was blocked and most microspores couldn‘t be released from tetrad,the tapetum and microspores degradation together,resulting in male sterility.The following results of cytochemical staining for callose wall in the wild type and the transgenic plants with aniline blue demonstrated that it was the degradation defect of callose around tetrad that caused the microspores could not be separated normally.Scanning electron microscopy and transmission electron microscopy illustrated that the development of pollen exine was abnormal and the exine structure was defective on the transgenic microspores.TUNEL assay showed that tapetal PCD occurred earlier in transgenic lines than the wild type.These results suggested that tapetal dysfunction in transformants and premature tapetal PCD might have adverse consequences during anther development,such as loss secretory function in tapetum.These findings suggest that timely expression of cysteine proteases is necessary for tapetal degeneration and pollen development.