| Grapes(Vitis L.),as one of the most important economic crops,are cultivated extensively throughout the world.The cold regions of northern China have gradually emerged as major grape-growing areas.However,low temperature stress,including freezing injury,severely limits the sustainable development of these regions.Recent studies show that melatonin(MEL)plays a pivotal role in plant resistance to abiotic stresses such as low temperature,drought and salinity,but the physiological responses and molecular mechanisms underlying MEL’s mitigation of cold stress in grapes remain elusive.In this study,physiological morphology,photosynthetic pigment content,membrane lipid peroxidation,antioxidant enzyme activity,ultrastructure and endogenous hormone levels of ’Cabernet Sauvignon’seedlings were assessed to determine the protective effects of MEL under simulated late frost stress.Subsequently,transcriptomic analysis of the dynamic response mechanisms of MEL to mitigate simulated late frost stress in ’Cabernet Sauvignon’ seedlings was performed to identify key genes involved in the grapevine response to MEL and simulated late frost.Finally,the HSFA6b gene was cloned,bioinformatically analysed,overexpressed and transformed in order to study its positive regulation of cold tolerance in transgenic vine calluses via the JA pathway.The results provide theoretical and practical insights into the involvement of MEL in grapevine cold tolerance.Key findings include:1.Optimal exogenous MEL concentration for mitigating simulated late frost stress in ’Cabernet Sauvignon’ seedlings was determined by subjecting control(Mock)and grape seedlings treated with varying MEL concentrations(50,100,150,200,and 300 μM)to simulated late frost conditions.Results indicated that exogenous MEL at different concentrations significantly alleviated grape leaf wilting,chlorophyll degradation,and malondialdehyde(MDA)accumulation while promoting soluble sugar accumulation,with 150 μM MEL proving most effective in mitigating simulated late frost stress.2.Physiological regulation of exogenous MEL on ’Cabernet Sauvignon’ seedlings under simulated late frost stress was assessed by pre-treating seedlings with 150 μM MEL.Compared to the Mock group,MEL-treated seedlings exhibited reduced leaf wilting and a 25%increase in green seedling recovery rate.Additionally,MEL-treated seedlings demonstrated lower hydrogen peroxide(H2O2)and superoxide anion(O2·-)accumulation and increased catalase(CAT),superoxide dismutase(SOD),and peroxidase(POD)activities by 8-fold,1.2-fold,and 1.9-fold,respectively.Ultrastructural observations revealed that MEL treatment maintained normal leaf cell and chloroplast structures,ensuring functional homeostasis.Hormone content analysis showed that MEL-treated seedlings had higher abscisic acid(ABA),indoleacetic acid(IAA),trans-zeatin riboside(tZR),and endogenous MEL levels compared to the Mock group,with endogenous MEL and ABA levels 23.88%and 14.57%higher,respectively.However,gibberellic acid(GA3)levels in MEL-treated seedlings were lower than in the Mock group.This suggests that exogenous MEL treatment triggers ’Cabernet Sauvignon’ seedlings’ reactive oxygen species clearance,photosynthetic system homeostasis,and endogenous hormone regulation under simulated late frost stress,thereby promoting normal growth.3.Low-temperature-responsive genes were identified using transcriptome sequencing analysis of grape leaves from Mock and MEL-treated seedlings under simulated late frost stress.Results revealed that,compared to the Mock group,the MEL-treated group contained 171 conserved low-temperatureresponsive genes and 776 exogenous MEL-specific induced expression genes.Notably,differentially expressed genes in the MEL-treated group were significantly enriched in pathways related to chloroplast complexes,carbohydrate transport,redox enzyme activities,and monoterpenoid biosynthesis.MEL treatment upregulated the expression of heat stress transcription factor VvHSFA6b,key JA signaling transduction gene VvOPR11,reductase gene VvFRO2,and starch and sucrose metabolism gene VvBGAL10.4.Functional validation of MEL-responsive key gene VvHSFA6b in conferring cold tolerance in grapes through the JA signaling pathway under simulated late frost stress was conducted.The open reading frame(ORF)of the VvHSFA6b gene is 1080 bp long,encoding 360 amino acids,and belongs to the HSF family with high conservation.Overexpression of the VvHSFA6b gene was performed in ’Cabernet Sauvignon’ callus tissue for gene function verification.Following low-temperature treatment,the fresh weight changes in transgenic grape callus(VvHSFA6b-OE)were significantly lower than those in untransformed grape callus(UT).Furthermore,the expression levels of low-temperature-responsive genes VvICE1s,VvCBFs,VvCOR413,and VvRD22,as well as JA content,were significantly higher in VvHSFA6b-OE grape callus compared to UT callus.Key genes involved in JA synthesis and signal transduction,such as VvAOC2,VvAOS2,VvLOX4,and VvOPR11,were significantly upregulated,while VvJAZ1 and VvJAZ9 were significantly downregulated.These findings suggest that the VvHSFA6b gene enhances cold tolerance in transgenic grape callus through the JA-ICE1-CBF pathway. |
