Physiological Basis And Molecular Mechanism Of Lotus Flower Bud Abortion

Lotus(Nelumbo nucifera)is one of the most famous perennial aquatic flowers in China.It’s an ornamental and edible plant that is widely cultivated.During the growing season,the lotus produces flower buds at each node and most of them were aborted because of unfavorable environmental changes,such as leaf shading by other leaves and rainy weather.Adverse environmental conditions will reduce reproductive success,slow seed,and fruit development,and even threatens survival.This different strategy could help lotus to terminate flowering with low reproductive success and minimize energy waste,yet the mechanism remains unclear.In this work,we conducted an integrated analysis of morphological and cytological observations,endogenous phytohormone assay,high throughput sequencing,and transgenics validations to uncover the underlying regulatory mechanism.The main findings are as follows:1.The research first observed the morphology of lotus flower buds and found that most lotus buds were aborted under the unfavorable growth conditions.It was proved by histochemical staining that compared with the normal flower buds,the aborting flower buds stained by Diaminobenzidine(DAB),Nitrotetrazolium Blue chloride(NBT),and Evans Blue,showed more dark yellow,indicating that reactive oxygen species(ROS)was produced during the aborting process in the lotus flower buds.Subsequently,TdT-mediated dUTP-biotin nick end labeling(TUNEL)test found,aborted flower buds were stained with fluorescein isothiocyanate(FITC)and 4’,6-dimidino-2-phenylindole(DAPI),the nucleus emits green fluorescence,which indicates that programmed cell death(PCD)has occurred in the aborting lotus flower buds.These indicate PCD may be an important reason of lotus flower bud abortion.2.Hormones play important roles in the PCD process.To verify the hormone change in aborting lotus flower buds,High-Performance Liquid Chromatography(HPLC)method was used to determine the hormone content between aborting flower buds and normal flower buds.The results showed the changes in hormones are determined by auxin(IAA)and ABA(Abscisic acid),especially the ABA achieved 136.67ng/g in the well-developed flower buds.Among them,abscisic acid content decreased by nearly 50%.Therefore we speculated that ABA may reduce the ratio of flower buds by exogenous application.To further verify the function of ABA in the PCD of lotus flower buds,through exogenous application different concentrations of ABA and its inhibitor—sodium tungstate(Tungstate),we found with adding ABA,the abortion rate of flower buds reduced,and the best concentration is 80 μM,which could significantly increase the amount of flower about 65%,verified ABA plays a positive role in reducing the abortion rate.3.The experiment further analyzed the changes of gene expression in aborted flower buds through high-throughput sequencing,results showed there are 345 genes with significant expression differences in the “plant hormone signal transduction”(ko04075)pathway,among them there are 17 auxins response proteins and 19 gibberellin carrier proteins were significantly up-regulated.5 ABA synthesis-related genes were significantly down-regulated,especially the 9-cis-epoxycarotenoid dioxygenase(NCED)decreased by up to 8 times.At the same time,autophagy-related proteins autophagy-related proteins 1(ATG1),autophagy-related proteins 13(ATG13),autophagy-related proteins 11(ATG11),autophagy-related proteins 101(ATG101),PCD related genes,programmed cell death protein 4(PDCD4),(programmed cell death protein 5,PDCD5),phenylalanine aminolyase(PAL)are also showed a significant increase.In the meantime,the expression of SNF1-related protein kinase 1(SnRK1)coordinated cell growth,played the role of an energy sensor,and induced PCD,showing a significant up-regulation trend,these indicate ABA plays an important role in the regulatory pathway of flower bud abortion.4.To verify the effect of NnSnRK1 on lotus flower bud abortion and research its relationship with ABA.We constructed NnSnRK1-interference vectors and NnSnRK1-overexpression vectors used IL-60 gene expression systems to verify the functions of NnSnRK1.Results showed,under the NnSnRK1-overexpression infection,the protein kinase activity and expression of SnRK1 protein kinase increased significantly,and the amount of flower decreased,rather under the NnSnRK1-interference infection,the protein kinase activity and expression of SnRK1 decreased significantly,the flower: leaf ratio increased by 40%.Moreover,it also indicates the flower bud abortion was inhibited,lotus showed strong flowering ability.Further experiments verified when lotus infected with NnSnRK1-interference plasmid were treated with ABA,NnSnRK1 expression level and protein kinase activity were significantly reduced,and the amount of flower was also significantly increased compared with NnSnRK1-interference,which can be reversed by Tungstate treatment.In summary,this study reveals a potential new way that adjusts the timing of flowering by controlling the abortion of lotus flower buds.NnSnRK1 play an important role in the flower development.By regulating the abortion of lotus flower buds,it participates in the signal pathway of ABA regulation as a negative regulator.It plays an important role in coordinating the relationship between lotus vegetative growth/stress response and reproduction growth.