Identification And Functional Analysis Of Proteins Related To The Cold Acclimation In Grapevine Buds

Grape is one of the fruit crops planted worldwide.China is one of the centers of origin for the Vitis species and has multiplex germplasms.Temperature is one of the major determinants for the growth and development of grapes.It limits thermal niches occupation of organisms;thereby determining their geographic distribution.In China,the most producing areas experience cold winters,of which the main grown cultivars are susceptible to extreme low temperatures.The vines have to be covered in soil so as to survive the harsh winter season which is laborious and expensive.Cold hardiness has been a major breeding goal in regions subject to severe low winter temperatures.Through a phenomenon known as cold acclimation?CA?,plants have developed an adaptation strategy so as to withstand unfavorable extreme low temperatures.This process includes several changes including accumulation of protective proteins.A well-known Chinese wild species Amur grape?V.amurensis?is popularly known for its cold hardiness compared with the widely cultivated grapevine?V.vinifera?.Understanding the mechanisms involved in cold stress tolerance is essential for the breeding of grape cultivars with excellent freezing tolerance and ultimately for other agricultural crops.Here,we investigated the effect of CA on the proteomes of V.amurensis cv.Zuoshan-1 and V.vinifera cv.Jingzaojing buds to explore the cold-tolerance mechanisms used by these species in field conditions.Furthermore,protein Snakin-2 was selected for functional analysis as the candidate protein in response to low temperature.The main research results are as follows:1.The buds of V.amurensis cv.Zuoshan-1 and V.vinifera cv.Jingzaojing were sampled in vineyard of the Institute of Botany,Chinese Academy of Sciences at two-time points:late fall(October 20^th,2016)and early winter(December 2^nd,2016).Differential thermal analysis indicated that gradual changes in temperature caused the improvement of cold hardiness especially in V.amurensis.Thus,proteomic analysis was conducted for these samples to explore the different responses of buds during CA.2.A total of 472 and 713 differentially abundant proteins?DAPs?were identified between the two-time points in V.amurensis and V.vinifera,respectively.The two species shared 235 DAPs,which were mainly involved in the protein chaperone and metabolic pathways,particularly carbohydrate metabolism.These DAPs represent the general responses to CA in Vitis species.V.amurensis contained less unique DAPs?237?than V.vinifera?478?.A functional category analysis indicated that the phenylpropanoid biosynthesis pathway was enriched in V.amurensis.Contrasting patterns were observed between the two species for phenylalanine ammonia-lyase,cinnamoyl-CoA reductase 1,and shikimate O-hydroxycinnamoyl transferase,which accumulated in V.amurensis but decreased in V.vinifera.These findings provide new insights into the mechanisms by which cold hardiness is achieved in V.amurensis buds.3.The qRT-PCR analysis of the 12 genes encoding selected DAPs revealed a positive correlation between transcriptional and proteomic expression levels.However,the expression levels of the detected genes changed more dramatically than their corresponding proteomic levels.Genes such as those encoding remorin and ubiquinol showed an obvious accumulation at the transcription level in V.vinifera,but not at the protein level.This variation illustrates the importance of the combination of the transcriptomics and proteomics data analysis as both approaches can complement each other to minimize analytical errors.4.A gibberellin-regulated protein Snakin-2 showed an abrupt increase in V.vinifera?fold-change of 30.646?during CA.Hence it was selected for further functional analysis as the candidate cold-responsive protein.Comparative quantitative expression analysis of Snk-2 revealed that low temperature induces contrasting spatio-temporal expression patterns amongst the leaf,stem and roots in V.amurensis and V.vinifera.Generally,Snk-2 was differentially accumulated across all the tissues in both species.While stem had more increased expression in V.amurensis,leaf tissue had the highest expression levels in V.vinifera.Root tissue had significantly differential accumulation in both species.Further works still needed to discover the expression of Snakin-2 in different tissues during CA and its functional analysis in response to low temperature.