Molecular Mechanism And Applications Of Salt-solution Overcoming Self-incompatibility In Brassica Napus L.

Heterotic utilization of SI is employed as an important strategy for the hybrid seeds production of Brassica napus.The two-line hybrid seeds production based on salt-solution incapacitating SI has been elegantly exploited in B.napus,which makes it considerable to uncover the molecular mechanism involved in this phenomenon.In the present study,we revealed the molecular mechanism of salt-solution overcoming SI by many different technologies.The main findings of our study are as follow:1.Breakdown of SI through salt-solution depends on ionic and osmotic stress in B.napusOur study found that salt-solution with a concentration of 650-1450 mM could effectively break the SI and set many seeds.NaCl,KCl,CaCl₂ and Na₂SO₄ could overcome SI in different degrees,the osmotic stress agent mannitol(DM)can also break the SI but the effect is less than the salt-solution,and(NH₄)₂SO₄,as a protein denaturan,cannot destroy the SI.These findings indicated that breaking of SI through salt-solution is due to its interaction between ionic and osmotic stress.2.Salt-solution treatment has no significantly effect on SI-related genesBy employing qPCR analysis,we detected the expression of SI-related genes in the stigmas after different pollination treatments.The results showed the genes had no significant changes.In the following proteomics data,we identified some SI-related proteins,such as SRK,SLG,THL2,and GLO1,but none of them changed significantly.3.Salt stress-related genes are involved in salt-solution overcoming SIThe expression of related genes in salt stress pathway was analyzed by qPCR.It was found that the genes involved in Ca²⁺regulation(CBLs and CIPKs)and K⁺transport had significant changes in salt-solution overcoming self-incompatibility.It was speculated that they might be involved in salt-solution overcoming SI signaling pathway.The RNA-silencing plants were created by RNAi technology.We found that knock down expression of BnSOS2,BnMPK6 and BnPLD1 could significantly reduce the seed setting of compatible pollination and incompatible pollination after salt-solution treatment.It was speculated that BnSOS2,BnMPK6 and BnPLD1 were involved in salt-solution overcoming SI.BnMPK6 can interact with BnARC1,demonstrating that BnMPK6 may be ubiquitinated by ARC1 to degrade,which leads to the occurrence of SI.Salt-solution treatment can promote the occurrence of self-compatibility by inducing the expression of BnMPK6.4.iTRAQ analysis of salt-solution overcoming the SI of B.napusProteome(iTRAQ)analysis of the B.napus stigmas of un-pollinated(UP),pollinated with compatible pollen(PC),pollinated with incompatible pollen(PI),and pollinated with incompatible pollen after salt-solution treatment(NA),we detected 389,189 and 307differentially accumulated proteins(DAPs)in PC/UP,PI/UP and NA/UP,respectively.The functional annotation and analysis of DAPs implied that salt treatment-overcoming SI in B.napus was likely conferred by at least five different physiological mechanisms:1)the use of Ca²⁺as signal molecule;2)loosening of the cell wall to allow pollen tube penetration;3)synthesis of compatibility factor protein species for pollen tube growth;4)depolymerization of microtubule networks to facilitate pollen tube movement;5)inhibition of protein degradation pathways to restrain the SI response.5.Aspartyl protease(BnAP)and ribosomal large subunit(BnRPLS)are positive regulator gene of SIBased on the expression analysis of mRNAs and proteins,we successfully screened two functional candidate genes.(1)Overexpression of the aspartyl protease gene in B.napus'Westar'can reduce the the number of seeds to a certain extent,and weaken the effect of salt-solutin on SI,which demonstrated that BnAP is a positive regulator genes of SI.BnAP interacts with the positive regulatory proteins BnSRK,BnARC1 and BnMLPKf1 of SI,and also interacts with the negative regulatory proteins BnEXO70A1 and BnPLD1 of SI.BnAP is a protease that degrades proteins.It is speculated that it is activated by the upstream positive regulatory genes of SI,and then degrades the BnARC1 ubiquitin-labeled SC proteins,ultimately promoting the occurrence of SI.(2)Down-regulation of BnRPLS gene expression in B.napus‘Westar'by RNAi technology can completely break the self-incompatibility,indicating that BnRPLS is a positive regulator of SI.qPCR analysis revealed that the down-regulated expression of BnRPLS significantly inhibited the expression of the SI positive regulatory genes BnSRK and BnARC1,and significantly promoted the expression of the negative regulatory genes BnTHL1/2,BnEXO70A1 and BnGLO1.Protein interaction technology demonstrated that BnRPLS interacts with BnARC1 and also interacts with the BnEXO70A1 and BnGLO1.BnRPLS is a ribosomal protein involved in protein synthesis,which may promote the synthesis of BnSRK and BnARC1 proteins,resulting in down-regulation of BnEXO70A1and BnGLO1 proteins,thereby promoting the occurrence of SI.