Pollen-specific Gene SKU5-SIMILAR 13 Regulates The Growth Of Pollen Tubes In Arabidopsis Thaliana

The growth of pollen tube in pistil is one of the key steps to ensure the completion of sexual reproduction in angiosperms,which is the basis for the improvement of crop yield.Despite its importance during pollination,little is known about the mechanisms that mediate pollen tube growth in female tissues.In this study,we investigated the functions of SKU5-Similar family 13(SKS13)in Arabidopsis regulating pollen tube growth in the transmitting tract during pollination using molecular biology,cell biology,and genetics.The main results are as follows:SKS13 is the highest and specifically expressed gene among the Arabidopsis 19 SKS genes in pollen.We found that the female gametophyte transmission efficiencies of sks13 mutants were normal,while the male gametophyte transmission efficiencies were significantly decreased,indicating that SKS13 is involved in maintaining the function of male gametophytes in Arabidopsis.The viability,morphology,and germination rate of the sks13 mutant pollen showed no obviously differences compared to those of the wild type.However,the length of pollen tubes in sks13 mutants was significantly shorter than that of the wild type in vitro,and the rate of pollen tube burst was significantly higher.The amount of pectin was reduced,and the amount of cellulose was increased at the apical region in the cell wall of the mutant pollen tube,which might cause the rupture of the growing pollen tube in vitro.The sks13 pollen tubes showed retarded growth in the transmitting tract,resulting in the decreased transmission efficiencies of male gametophyte in sks13 mutants,which result in shorter siliques and reduced seeds in siliques.It has been predicted that the SKS13 protein contains a signal peptide at its N-terminus.To study the subcellular localization of SKS13,two subcellular localization vectors were constructed,in which the GFP sequence was fused to the end of the signal peptide sequence(GFP-SKS13)or the C-terminus of SKS13(SKS13-GFP),respectively.These vectors were separately introduced into the sks13-1 mutant.The results showed that when GFP-SKS13 was expressed,SKS13 was localized inside the pollen tube,while when SKS13-GFP was expressed,it was localized in the cytoplasm and the apical cell wall region of the pollen tube.However,the fertility of the mutant was restored only after SKS13-GFP was introduced.Therefore,the correct localization of SKS13 is in the cytoplasm and the apical cell wall region of the pollen tube.Transcriptomics analysis revealed that the genes in sks13 mutant were significantly downregulated related to JA synthesis,metabolism,and signal transduction.The level of JA in the pollen of sks13-1 mutant was significantly reduced.Inhibition of JA biosynthesis in the wildtype pollen tubes resulted in shortened pollen tubes and disturbed the distribution of pectin in the pollen tube wall,which was similar to the phenotype of sks13 mutants.These results suggest that SKS13 plays an important role in JA synthesis and modifies the cell wall of the pollen tube by mediating the biosynthesis of JA.JINGUBANG(JGB)is a protein containing the WD-40 domain.Previous studies have shown that JGB can inhibit pollen germination and maintain the growth of pollen tubes in the transmitting tract by inhibiting JA biosynthesis.In this study,we found that the pollen germination rate in vitro and the distribution of pectin and cellulose in the pollen tube cell wall of the jgb sks13 mutants were similar to those of the jgb mutant,suggesting that SKS13 and JGB act separately to regulate the biosynthesis of JA during pollen tube growth.JGB inhibits,while SKS13 promotes the biosynthetic pathway.The expression level of SKS14 in pollen is second only to SKS13 in the SKS family.The transmission efficiencies of the male and the female gametes in sks14 mutant were normal.There was no significant difference between sks14 mutant and the wild type in the growth of pollen tubes in the transmitting tract,while the pollen tube of the sks13 sks14 mutants was significantly slower than that of the sks13 mutants,indicating that SKS13 and SKS14 play redundant roles in mediating the growth of pollen tubes in the transmitting tract.These results suggest that SKS13 functions in pollen tube growth in the transmitting tract by mediating the biosynthesis of JA that modifies the cell wall of the pollen tube.