The Secretion,Composition And Function Of Pollination Drop In Ginkgo Biloba L.

Pollination drops（PDs）,being involved in pollen capture,pollen germination and fertilization of gymnosperms,are the secretions produced by ovulesand are the essential components in pollination system.Previous studies have reported the secretion and function of PDs in gymnosperms.However,the chemical components of PDs and molecular mechanisms of secretion in gymnosperms are largely unknown.Specilally,although several studies have demonstrated that microRNAs（miRNAs）were also detectable outside cells,named extracellular miRNAs,whether extracellular miRNAs presented in plants are still unknown.In this study,by high-throughput sRNA sequencing and GC-MS technology,we identified a large amount of metabolites and miRNAs in PDs of G.biloba.In addition,we used the RNA-seq,sRNA sequencing and molecular biology to explore the molecular regulation mechanisms of PD secretion.The main results are as follows:（1）The composition of G.biloba PDs mainly contained 23 kinds of sugars,accounting for 45.70%of the total chemical components,such as monosaccharides（sucrose and glucose）and disaccharide（lactose,maltose and lyxose）.The second abundant component was organic acids that consisted of 18 kinds（15.94%）,including glucoheptonic acid,pyruvic acid,lactic acid and so on.The third component was alcohols（13;15.39%）,such as xylitol,sorbitol and inositol.In addition,19 secondary metabolites（11.94%）including flavonoid and terpenoid compounds were identified.These metabolites are mainly involved in galactose metabolism,starch and sucrose metabolism pathways.（2）The pollen was cultured by using a 200-fold dilution of PD.After 24 hours,the outer wall of pollen was removed,and some pollen stated to germinate within 48 hours,suggesting that PDs are indispensable for pollen germination and growth.According to the compositions and concentration of sugar in PDs,we further added fucose,maltose,sorbose,sucrose,glucose and fructose to the culture medium for pollen germination experiment.The results showed that,although the pollen can exhibit water swelling and starch granules accumulation,but don’t germinate into pollen tubes.Taken together,these results indicated that the germination of G.biloba pollen is difficult and takes a long time,whereas the involvement of PDs can promote pollen germination,being different from angiosperms and other gymnosperms.However,the specific components in PDs are necessary for pollen tube germination and growth still need further study.（3）While G.biloba pollen was cultured in conventional medium,the culture medium was polluted after 48 h.After adding chlorogenic acid,the culture medium was polluted after 96 h.However,the conventional mediums with the additive of lauric acid,lactic acid and maleic acid were almost not polluted during 108 h,showing their significant inhibitions on pollution of medium during pollen culture.The OD600 data analysis further confirmed that lactic acid,maleic acid and monoglyceride laurate have lower OD600 values,indicating that they have significant antibacterial functions.（4）We successfully constructed sRNA library and detected 45 known and 550 novel miRNAs in G.biloba PDs.Four of these known miRNAs expressed in PD were further validated by reverse transcriptase polymerase chain reaction（RT-PCR）and quantitative real-time PCR（qRT-PCR）.Potential target analysis of known and novel miRNAs indicated that some miRNAs are probably involved in carbohydrate metabolism,glycan biosynthesis and metabolism,hormone signaling,biosynthesis of secondary metabolites,lipid metabolism,and defense response,consistent with metabolomics results.Interestingly,we found that almost all known miRNAs identified from PDs also expressed in ovules through comparative analysis of miRNA expression.qRT-PCR analysis confirmed that some miRNAs are expressed in both PDs and ovules,and some miRNAs in PDs expressed higher than that in ovules,which implied the miRNAs of PDs may secret from ovules.（5）Through high-throughput sequencing of secretory ovlues and pollinated ovules,we identified a potential secretion related Gb₂3187 and starch metabolism associated genes including Gb₃2463,Gb₀5693 and Gb₂8387 that all showed high-regulated expression in secretory ovlues.sRNA analysis further confirmed that miR1083,miR858b and miR858a might be involved in the regulation of secretions,and 12 miRNAs such as miR319i,miR159f and miR166d-5p are involved in the starch metabolism pathway.（6）Gibberellin（GA）signaling and synthesis related genes Gb₁1704,Gb₀5787 and Gb₁6591 showed up-regulated expression in secretory ovules.In addition,jasmonic acid（JA）signaling negative regulating genes Gb₁7431 and Gb₁7432 showed down-regulated expression in secretory ovules,whereas JA synthesis genes Gb₃9942,Gb₀3931,Gb₁8696 were up-regulated expression.Exogenous spraying GA and JA experiments showed that 100μmol of methyl jasmonate and short-time gibberellin treatments can effectively promote the secretion of PDs,indicating that jasmonate and gibberellin may be involved in regulating the secretion of G.biloba PDs.（7）Phylogenetic tree analysis of the secretion related genes between G.biloba and other plant species displayed that Gb₁5814,Gb₁9348 and Gb₂3187 were most likely to involve in secretory process of G.biloba ovule.qRT-PCR results showed that the expression of three genes were up-regulated during ovule secretion.We further cloned these genes and constructed overexpression vectors.By transient transformation in the tobacco leaves,we found that Gb₁5814 and Gb₁9348 mainly located on the plasma membrane,while Gb₂3187 located on the plasma membrane and nucleus.The functions of these genes will be further studied and verified in the future.