Molecular Mechanism Of Different Exogenous Carbon Regulating Growth Of Grape Plantlets In Vitro

Carbon is an important component of plant cell,and as energy supply for plant growth.Exogenous carbon is converted into sugar in plants and synthesized into other substances.Sugar can also be used as an endogenous signal to regulate plant growth,development and respond to environmental changes.Exogenous carbon is related to the growth and development of grape plantlets in vitro.In order to study the molecular mechanism of the effect of exogenous carbon on the growth of grape plantlets in vitro,the experiment was cultured grape plantlets in vitro with exogenous carbon dioxide and sucrose.Combined with the physiological changes of grape plantlets,the differentially expressed genes and proteins were analyzed by RNA-seq and iTRAQ.The objective of this work was to build available genomic and proteomic resource to investigate the molecular mechanisms of exogenous carbon regulating plant growth and development.Grape?Vitis vinifera L.cv.‘Pinot Noir'?plantlets cultured with no sucrose,with 2%sucrose,with eCO₂(1000?mol·mol^-1)and with both 2%sucrose and eCO₂ were designated as S0,S1,C0 and Cs,respectively.The main results are as follows:1.Different exogenous carbon had different effects on grape plantlets in vitro.The eCO₂could reduce stomatal conductance and transpiration rate of grape plantlets in vitro.The eCO₂can promote light convert into chemical energy,through increase the relative electron transport rate?ETR?,photochemical quenching coefficient?qP?and net photosynthetic rate of leaves,but reducing non-photochemical quenching?NPQ?.The eCO₂ promotes leaf area and biomass increase of grape plantlets in vitro.The changes of soluble protein,soluble sugar and chlorophyll in the leaves may be affected by the sugar content in the medium,not related to eCO₂.After treatment with exogenous carbon,the GA and ZR of grape plantlets in vitro were correlated with the treatment of exogenous carbon,and there was no significant change in IAA and ABA content.2.Sugar is not only important for plant growth and development,but also for plant signal transduction,especially in plant hormone response.Through the transcriptomic and proteomic analysis,with pooled data for Cs,C0 and S1 compared with S0,70 differentially expressed genes?DEGs?and 65 differentially expressed proteins?DEPs?were identified.Based on biological functions and physiological characteristics,we identified 8 DEGs and 2 DEPs related to ethylene signaling process.Amongst the DEGs we focussed on ERF TFs,including ERF5?LOC100244353,LOC100247763,LOC100254616 and LOC100261260?,ERF105?LOC100249507 and LOC100259725?,ERF2?LOC100254640?and CTr?CTr7?.Also,there were 2 DEPs related to ethylene metabolism,such as S-adenosylmethionine synthase 5?SAM synthase 5;XP₀02280106.1?and 1-aminocyclopropane-1-carboxylic acid oxidase 2?ACC oxidase 2;NP₀01267871.1?were also identified.The transcriptome and proteome results suggested that exogenous carbon inhibits ethylene biosynthesis through ACC oxidase 2.Additionally,CTr7 and ERF5,which were up-regulated,are related to the ethylene signaling pathway.We speculate that exogenous carbon regulates plant growth through ethylene signaling pathways,but which inhibit ethylene biosynthesis.3.Transcriptomic and proteomic profiles associated with photosynthesis and growth in leaves of grape plantlets in vitro at different CO₂ concentration were analyzed.A total of 1814genes?465 up-regulated and 1349 down-regulated?and 172 proteins?80 up-regulated and 97down-regulated?were significantly differentially expressed in eCO₂ compared to S1.Photosynthesis-antenna,photosynthesis and metabolism pathways were enriched based on GO and KEGG.Simultaneously,9,6 and 48 proteins were involved in the three pathways,respectively.The leaf area,plantlet height,qP,?PSII and ETR increased under eCO₂,whereas Fv/Fm and NPQ decreased.Changes of these physiological indexes are related to the function of DEPs.After combined analysis of proteomic and transcriptomic,the results make clear that eCO₂ have different effects on gene transcription and translation.RbcS was not correlated with its mRNA level,suggesting that the change in the amount of RbcS is regulated at their transcript levels by eCO₂.However,Rca was negatively correlated with its mRNA level,it is suggested that the change in the amount of its corresponding protein is regulated at their translation levels by eCO₂.Transcriptomic,proteomic and physiological analysis were used to evaluate eCO₂ effects on photosynthesis.The eCO₂ triggered the RbcS and Rca up-regulated,thus promoting photosynthesis and then advancing transformation of grape plantlets from heterotrophic to autotrophic.This research will helpful to understand the influence of eCO₂ on plant growth and promote reveal the mechanism of plant transformation from heterotrophic to autotrophic.4.Transcriptomic and proteomic profiles associated with secondary metabolism and growth in leaves of grape plantlets in vitro at different CO₂ concentration were analyzed.The expression of flavonoid metabolism-related genes and proteins in plant leaves is up-regulated,while the stilbene synthase gene is down-regulated,and KEGG enrichment to flavonoid metabolic pathways is significantly changed.Among them,CHS?F3H?FAOMT and flavonoid-3,5'-hydroxylase genes related to flavonoid metabolic pathway were abundantly expressed,stilbene synthase was down-regulated and CHS was up-regulated in proteome.ECO₂ may promote the transfer of phenylpropane metabolism to flavonoid and anthocyanin pathways in plant secondary metabolism,and inhibit the synthesis of stilbene compounds in plant.5.According to the previous research results,the two genes including ERF5 and glucose-related GDP-L-galactose phosphorylase were selected for cloning and transient expression vector construction.We use bioinformatics analysis and predict the structure,physicochemical properties and subcellular localization of the two genes.The ERF5 and GDP-L-galactose phosphorylase gene fragments were cloned and identified by sequencing.The P-1300-GFP-ERF and P-1300-GFP-G transient expression vectors were constructed,and the subcellular localization of onion epidermal cells and tobacco were performed.Leaf explant transformation,the results indicate that the location and prediction of the two genes in the cell are consistent.