| Self-incompatibility is a genetic mechanism evolved in flowering plants to prevent inbreeding and promote outcrossing to maintain genetic diversity,which is one of the main ways to utilize heterosis in Brassica napus L.Therefore,it’s of great significance to study the mechanism of self-incompatibility in B.napus.Here,self-compatibility Westar and self-incompatibility W-3 were used as research materials,and by experimental techniques including ultra-high performance liquid chromatoc-tandem mass spectrometry(UPLCMS/MS),semi-in vitro pollination,sequence analysis and genetic analysis revealed the metabolic characteristics of unpollinated(UP),self-compatibility(SC)and selfincompatibility(SI)pollinated stigmas in B.napus,unearthed the candidate factor Ascorbate Peroxidase 1(APX1),and analyzed the role of Bna.APX1 in pollen-stigma interaction.The main research results are as follows:1.Metabolic characteristics of UP,SC and SI pollinated stigmasA total of 578 metabolites were detected in the pollinated stigmas of UP,SC and SI,mainly flavonoids(21.63%),organic acids(10.38%),amino acids(13.84%),nucleotides(8.65%)and lipids(8.30%).The metabolic characteristics of UP,SC and SI pollinated stigmas were significantly different,the total contribution value of principal component analysis(PCA)was 53.64%.Compared with UP,a total of 85 metabolites were differentially enriched after compatibility pollination(67 up-and 18 down-regulated),and120 metabolites were differentially enriched after incompatibility pollination(93 up-and27 down-regulated).These differentially enriched metabolites were mainly concentrated in flavonoid biosynthesis,lipids metabolism and secondary metabolite biosynthesis pathway.There were 22 differentially enriched metabolites between SC and SI pollination stigmas(SI vs SC: 18 up-and 4 down-regulated),the up-regulated metabolites including 8lysophatidylcholines with different molecular weight,O-phosphocholine,L-histidine,Ltyramine,L-ascorbate,2-hydroxybutanoic acid,N-formylmethionine,7-methylxanthine,creatine,2-hydroxybutanoic acid and E-3,4,5’-trihydroxy-3’-glucopyranosylstilbene,the down-regulated metabolites including threose,pinocembrin,amentoflavone,and tricin5-Orutinoside.2.Bna.APX1 promotes pollen hydration and germination by removing ROSIn plants,L-ascorbate(L-As A)relies on ascorbate peroxidase(APX)scavenging reactive oxygen species(ROS).Metabolome results showed that the L-As A content was significantly accumulated in SI pollination stigma,which was 2.33 times of that in SC pollination stigma.Correspondingly,the stigma ROS level detection and APXs activity detection results showed that the stigma ROS level was creased and APXs activity decreased to 0.68 times after SI pollination;the stigma ROS level decreased and APXs activity increased to 1.74 times after SC pollination.Inhibition of stigma ROS production can break SI by Diphenyleneiodonium(DPI),treatment stigma with APXs inhibitor ρ-Aminophenol,stigma ROS increased and pollen germination was inhibited after SC pollination.The available transcriptome results show that the transcription level of Bna.APX1 was the highest among the 8 genes that encoding APX protein in B.napus stigma.Genetic analysis showed that,compared with the wild type,the stigma ROS level of Bna.apx1 mutants were significantly increased after SC pollination,and the pollen hydration and germination were delayed;the unpollinated stigma ROS level of Bna.APX1-OE overexpressed lines were significantly decreased,and further decreased after SC pollination,the pollen hydration and germination were ahead.The pollen tube elongation and seed set in the mutants and overexpressed lines of Bna.APX1 were not affected after SC pollination.In addition,at the early stage after SI pollination(30 min before),the ROS level in stigma of overexpressed lines Bna.APX1-OE were significantly lower than that of the wild type,and SI pollen hydration rate and germination number were significantly higher than that of the wild type.However,the pollen tube could not pass through the stigma of Bna.APX1-OE,and the SI seed setting characteristics were not affected in Bna.APX1-OE lines.Above results indicate that Bna.APX1 promotes pollen hydration and germination by removing ROS,and it’s a compatibility factor in pollen-stigma interaction.In conclusion,in this study,the stigma metabolism characteristics of B.napus after self-(in)compatibility pollination were revealed by UPLC-MS/MS,which laid a foundation for the mining of pollen-stigma interaction factors in B.napus,and preliminarily revealed the function of Bna.APX1 in pollen-stigma interaction by analyzing the stigmatic metabolic characteristics after compatibility and incompatibility pollination,which contributing to the understanding of ROS mediated pollen-stigma interaction. |
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