The Involvement Of SRNA In Apple(Malus Domestica)Floral Transition

Apple is one of the most important fruit trees in the world.During flower induction,the transition from vegetative growth to reproductive growth in the meristem(hereafter,refered to floral transition)is critical to floral bud development and production.Explore the mechanism underline apple floral transition is therefore important to accelerate breeding for apple and other perennial woody fruit trees.sRNA is a kind of small non coding RNA,which control the gene network by regulating target genes and cover plant development,stress response,hormone pathways.Until now,the study of sRNA-mediated floral transition in plants are mainly focused on the role of miR156 and miR172,meanwhile,little is known about the role of siRNA in this process.In this study,we used vegetative and floral buds from apple trees as materials,sRNA sequencing and comprehensive bioinformatic analysis were performed;meanwhile,the key factors of miRNA biogenesis pathway,MdAGO1 and MdAGO10,were cloned and explored their functions in apple.The main results are as following:1 Through sRNA sequencing,we found except the conserved role of floral transition regulator miR156-miR172,a large number of sRNAs exemplified by 33 previously annotated miRNAs and 6 novel members displayed significant differential expression(DE)patterns in vegetative and floral stages.Notably,most of these DE-miRNAs in floral transition displayed opposite expression changes in previously reported phase transition in apple trees.Bioinformatic analysis suggested most of the DE-miRNAs target the transcripts involved in SPL gene regulation,GA pathway,stress responses,and auxin pathways,with further suggestion that there is an inherent link between physiological changes and developmental reprogramming during the floral transition.We also observed significant changes in 24 nucleotide(nt)siRNAs that are hallmarks for RNA-dependent DNA methylation pathway,suggestive of the correlation between elevated DNA methylation levels in floral bud and the floral transition.2 MdAGO1 and MdAGO1O were cloned from apple,they contain PAZ domain and PIWI domain which are conserved in AGO proteins.MdAGO1O displayed high similarity with AGO 10 from other plants,while MdAGO1 has specific insertion only in woody fruit trees such as apple and pear.Subcellular localization showed both MdAGO1 and MdAGO10 localized in nucleus.Then we performed functional complement analysis by over-expressing MdAGO1 and MdAGO10 in Arabidopsis agol-27 and pnh-2 mutant under the driven by Arabidopsis AGO1 and AGO 10 promoters.The expression MdAGO1 and MdAGO10 could successfully rescue the defects on phenotypes and miRNA levels in ago1-27 and pnh-2 respectively,and recovered to that of wild type.Notably,MdAGO1 could also complement the defect phenotype of pnh-2.We checked the background of complementary plants and finally confirmed the rescued phenotype came from MdAGO1 expression.Further analysis showed the reason may come from the specific insertion sequence at N terminal in apple AGO 1.