Identification Of Kaempferol Glycosides And Its Effect On Pollen Tube Growth And Seed Number In Malus

Malus plants are an important cultivated fruit plants.The flavonoids in Malus not only affect the color of flowers and fruits,but also participate in regulating plant growth and development and resistance to stress.In this study,based on isolated and identified three kaempferol glycosides,different flower color genotypes of Malus were used to analyze the relationship between the content of flavonoids in flower and seed number,and synthesis competition and regulatory mechanism among different flavonoid compounds,and the mechanism of seed number affected by kaempferol glycosides.This research further enriched the biological functions of flavonoids metabolism in Malus.The main results are as follows:1. The content of kaempferol glycosides negatively correlated with the content of anthocyanin in different tissues of flower and positively correlated with seed number in Malus.Three kaempferol glycosides were isolated and purified from petals of Malus by various chromatographic means.After using liquid chromatography-mass spectrometry?LC-MS?and nuclear magnetic resonance?NMR?,the three compounds were identified as kaempferol 3-O-?-xylopyranoside,kaempferol 3-O-?-arabinofuranoside and kaempferol3-O-?-rhamnopyranoside.By analyzing different tissues of 14 genotypes of Malus,it was found that kaempferol glycosides were mainly accumulated in flowers and extremely low in leaves and fruits.In addition,white flower genotypes accumulated relatively more kaempferol glycosides and less anthocyanin in petals,pistil and stamens,while,red flower genotypes accumulated relatively more anthocyanin and less kaempferol glycosides.After correlation analysis between seed number and the content of anthocyanin and kaempferol glycosides respectively in flower,it was found that a negative correlation was showed between seed number and anthocyanin content,the correlation coefficient was?=-0.868,and a positive correlation was showed between seed number and kaempferol glycosides content,the correlation coefficient was?=0.642.After overexpressed Md MYB10 in‘Royal Gala'?Malus domestica?,the seed number decreased significantly.2. The synthesis competition between kaempferol glycosides and anthocyanin was regulated by F3'HII?flavonoid 3'-hydroxylase?and FLS?flavonol synthase?in flower of Malus.By analyzing the expression levels of key genes of the flavonoid pathway F3'HI,F3'HII,FLS,DFR?dihydroflavonol 4-reductase?,ANS?anthocyanidin synthase?and UFGT?UDP-glycose:flavonoid 3-O-glycosyltransferase?in flower of three different development stages,leaves and fruits of Malus,it was found that,at the early development stage of flower,the expression level of F3'HII is relatively low and the expression level of FLS is relatively high in the white flower genotypes when compared with the red flower genotypes.At the later development stages of flower,there is no significant difference in genes expression between the two types of genotypes.In addition,there was no significant difference in the expression levels of F3'HII and FLS in leaves and fruits between the two genotypes.Through comparative analysis of F3'HII and FLS expressions in different tissue,it was found that the expression level of F3'HII in flowers was significantly lower than that in leaves and fruits,while FLS was the opposite.After overexpressed Md MYB10 in‘Royal Gala',the expression level of Md F3'HII was significantly increased and the expression level of Md FLS was significantly decreased,and more anthocyanin and less kaempferol glycosides were accumulated in the transgenic flower.As the Md F3'HII RNAi in GL3apple,the content of kaempferol in the transgenic leaves was significantly increased,while the expression of Md FLS was not affected.Overexpression of Md F3'HII in Arabidopsis wild type?Col-0?and F3'H mutant?tt7?both increased the quercetin/kaempferol ratio in leaf and inflorescence.These results indicate that the synthesis competition between kaempferol glycosides and anthocyanin was regulated by F3'HII and FLS in flower of Malus.3. The seed number of Malus affected by kaempferol glycosides through regulating the pollen tube growth.After artificially pollinating pollen of‘Granny Smith'?M.domestica?,a comparative analysis of the pollen tube growth in the styles between wild-type‘Royal Gala'and overexpressing Md MYB10 transgenic lines revealed that the growth rate of pollen tubes in Md MYB10 overexpressing styles was significantly slower than growth rate in wild type.After external application of different flavonoid compounds to the pollen of different genotypes of Malus in pollen growth medium for 4 h,it was found that quercetin glycosides and anthocyanin had little effect on the growth of pollen tubes,while,kaempferol glycosides,especially kaempferol-3-rhamnoside,significantly promoted pollen tube growth.Through transcriptome and quantitative expression analysis,we found that the genes expression of pollen tube growth of apple was regulated by kaempferol-3-rhamnoside,such as:genes of heat shock proteins,genes involved in small GTPase and MAPK signaling,genes involved in microtubule movement and so on.The application of kaempferol-3-rhamnoside enhanced the accumulation of Ca²⁺at the tip of pollen tubes.However,with the application of EGTA simutaneously,the accumulation of Ca²⁺disappeared and the growth of pollen tubes were inhibited.These results indicate that kaempferol-3-rhamnoside might regulate the growth of pollen tubes through the above genes and Ca²⁺.By applying kaempferol-3-rhamnoside on the pistil stigma of M.‘Adams'and M.‘Raidant',belonging to the red flower genotypes,which significantly increased seed numbers.After overexpressed Md F3'HII in the F3'H mutant?tt7?of Arabidopsis,its seed number returned to the wild type level.These results indicate that the seed number of Malus affected by kaempferol glycosides through regulating the pollen tube growth.