Regulation Mechanism Of GA₃ Treatments On Water And Development Of Grape(V.vinifera L.)berry

Gibberellin(GA₃)as one of the six main plant hormones,which is also a main growth regulator in the production of fruit trees,and it plays an indispensable role in plant growth,development and production applications.In the daily management of table grapes,the growth regulator containing GA₃as the main ingredient is used to treat the fruit to seedless or increase the size of the fruit in order to promote the value of the grape products.The previous research of our research group found that the water parameters of the fruits were significantly changed after GA₃treatment,but there are few study reported the the relationship between water and fruit growth and development after GA₃ treatment.Based on this,the?Red Globe‘table grape was used as the test material,and different concentrations of GA₃(T1:40 mg/L;T2:70mg/L)were used for dip the clusters,water as the control(CK).Treatment time is first swelling stage of grape(horizonal diameter 8-10mm).Samples were taken every day within one week and every 15 days until grapes were mature after treatments(based on 2018-2019 two-year repeated experiment).Using RNA sequencing(mRNA,nc RNA-seq),gene cloning techniques were used to explore the regulation mechanism of water on the growth and development of the fruit after GA₃treatment.The main results are as follows:1.The water and endogenous hormones of different concentrations of GA₃ will affect the growth and development of the fruit;the main water parameters in the fruit:free water content,water potential,osmotic potential,and turgor pressure are all significantly increased.The result is that the feedback is obtained soon(0-1d)after the treatment which is worth noting,and the change trend of the water parameters also changed with the double"S"growth curve of the grape.Compared with CK,although the change trend is similar,however,the main water parameters always remain at a significantly higher level,1 day to the maturity stage after treatment.In terms of endogenous hormone levels,the intervention of exogenous GA₃significantly increased the level of auxin(IAA),gibberellin(GA₃)and zeatin(ZT).Interestingly,the change trends of endogenous hormones and water parameters maintained similar results.The change trend of ABA is opposite to the growth-promoting hormone(IAA,GA₃,ZT).Combined with the two years datas of 2018 and 2019,using correlation and cluster analysis,showed that after different concentrations(T1,T2)GA₃ treated the fruit clusters,the cell length and size,the vertical and horizontal diameters,the single fruit weight,volume and cluster tightness of the fruit phenotype all have a significant increased impact.2.Comprehensive consideration of the change trends of water and endogenous hormones in fruits treated with different concentrations of GA₃,the fruits with high concentration(T2)were selected for 0 d(2 h)after treatment for transcriptome(mRNA and nc RNA)sequencing analysis,and a total of 436 differentially expressed genes(DEGs)were obtained at the mRNA level,350 were significantly up-regulated and 86 were significantly down-regulated.The GO classification annotation shows that the biological processes with the largest number of differentially expressed genes involve in"Stimulus response","Biological regulation"and"Transport activity";the KEGG enrichment classification shows that differentially expressed genes mainly involve in"Plant hormone signal transduction","Flavone biosynthesis","Phenylalanine metabolism"and other related metabolic pathways are enriched items.In order to further understand the biological pathways of grape berries in response to exogenous GA₃,the expression patterns of water transport and hormone-related genes were mapped and analyzed,the VIT?12s0028g01810 gene related to water transport(Biological Process:water transport GO:0006833)and gibberellin-related VIT?08s0007g5030 gene(Biological Process:gibberellin GO:0009739)was used as the two candidate genes for subsequent transformation experiments,and 21 DEGs were randomly selected for qRT-PCR verification,it was found that their expression trend remained similar to the expression level of the transcriptome data.3.The candidate water transport related VIT?12s0028g01810(VvGSK-3)gene and the gibberellin-related VIT?08s0007g5030(VvRAX2)gene were functionally identified and analyzed by constructing two overexpression vectors p CAMBIA1300-VvGSK-3-GFP and p CAMBIA1300-VvRAX2-GFP,the vector construction method is homologous recombination.Transformed them into the onion epidermis and tobacco leaves for subcellular localization,it was found that both VvGSK-3 and VvRAX2 were localized in the nucleus,which was consistent with the results of bioinformatics predictions.In order to further verify the biological functions of the two genes,transferred them into grape callus(cv.Monastrell)and tomato(Micro-Tom),it was found that the endogenous GA₃ content in VvGSK-3 and VvRAX2transgenic gene callus was significantly increased,and the content was sorted to VvRAX2>VvGSK-3>WT(wild type).The water parameters and fruit phenotype of VvGSK-3 and VvRAX2 transgenic tomato fruits were determined,it was found that the total water content,water potential,single fruit weight,vertical/horizontal diameter,and single fruit volume in the transgenic fruit were significantly higher than those of WT.The overall order of tomato size is:VvRAX2>VvGSK-3>WT.Combining the physiological and molecular results of the grape treated with exogenous GA₃,we speculate that water and endogenous hormones jointly regulate the phenotypic differences of grape fruits,thereby affecting their growth and development,and there may be mutual regulation or promotion between water and endogenous hormones.The relationship needs further deepening and systematic research.4.Through the non-coding RNA(nc RNA)sequencing analysis of the fruits treated with high concentration GA₃(T2),it was found that a total of 286 mirRNAs were identified,of which 14 were differentially expressed,9 were significantly up-regulated,and 5 were significantly down-regulated;2146 LncRNAs,79 were differentially expressed,46 were significantly up-regulated,33 were significantly down-regulated;CircRNA was 2583,and 12 were differentially expressed,6 were significantly up-regulated,and 6 were significantly down-regulated.From the GO classification and KEGG enrichment of the differentially expressed coding(mRNA)and non-coding RNA target genes,they mainly enriched in"hormone signal transduction","water transport","phenylpropane biosynthesis","carbon metabolism"and"growth"pathways,indicating that exogenous GA₃ has a direct impact on the growth and development of grape berries.With the targeted mirRNA of differentially expressed genes(DEmRNAs)as the center,the mRNA,LncRNA and LncRNA and CircRNA regulatory network(CeRNA mechanism)indicated that different RNAs can target a variety of RNAs,and there is a relationship of mutual regulation and influence between them.