| Watermelon(Citrullus Lanatus)is originated from Africa and is a typical thermophilic crop.In recent years,the cultivation area for off-season watermelon production such as sunlight greenhouses and early spring greenhouses has significantly been increased in China.However,watermelon often face the problem of cold stress that seriously affect the growth and development of seedlings,and ultimately yield and quality.It has been proved that rootstock grafting is an effective technique to improve cold tolerance of watermelon.In this study,we use “Qingyanzhen No.1”(Cucurbita maxima × Cucurbita moschata)a cold-tolerant cultivar as rootstock,and “97103” a cold-sensitive inbred line as scion.Several aspects including physiological reactions and proteomics of grafted seedlings under cold stress were studied to provide theoretical basis for the application of grafting in watermelon production.The main results were as follows:1.The seedlings of 14 watermelon rootstock genotypes were exposed to low temperature to study the effects on some physiological and biochemical indicators.Principal component analysis,subordinate function analysis,and clustering analysis were used to comprehensively evaluate the cold tolerance of watermelon rootstocks.The results showed that there were significant differences in 12 parameters among 14 watermelon rootstocks.Twelve physiological indexes of rootstock were converted into five independent comprehensive indexes by principal component analysis.According to the D value of comprehensive cold resistance,14 rootstock cultivars were divided into four categories by subordinate function analysis and clustering analysis.Black-seeded pumpkin was a cold-resistant variety,whereas,Qingyanzhen No.1,Jingxinzhen No.2,and Qiangli No.1 were relative cold-resistant varieties.Eight rootstock varieties such as Longzhen No.1,Genlishen,Shenlitiemuzhen,Yongzhen No.1,Qianggen,Xizhen No.1,Bijiu and Jingxinzhen No.1,were medium cold-resistant varieties.Ansheng and Fengzhen No.1 were weak cold-resistant varieties.The comprehensive evaluation results are basically consistent with the observation results of morphological characteristics.2.Physiological response of watermelon seedlings grafted onto pumpkin(Qingyanzhen No.1)rootstock was studied under different low temperature treatments at 11 ℃,9 ℃,7 ℃,and 5 ℃ for 1 d,3 d,5 d,and 7 d respectively and the own-root plants were used as control.The physiological mechanism of watermelon seedlings under different temperature stress was discussed.The results indicated that with the decrease of temperature and the increase in treatment time period,the cold injury of own-root and grafted watermelon seedligns was gradually increased,especially the treatment at 7 ℃ and 5 ℃ showed the most significant effect.Grafted watermelon seedlings can tolerate the low temperature stress at 7 ℃ for 5 d and 5 ℃ for 3 d,respectively.However,the own-root watermelon seedlings can tolerate the low temperature stress only at 7 ℃ for 3 d and 5 ℃ for 1 d,respectively.With the increase in treatment time period,the chlorophyll content of watermelon seedlings decreased under cold stress.Relative electrolyte leakage and malondialdehyde content increased significantly.The content of soluble sugar and proline increased rapidly and then slowdowns.The CAT and SOD activities of watermelon seedling leaves showed an “up-down-up” trend under 11 ℃ treatment,which showed a trend of first decrease and then increase at 9 ℃,7 ℃ and 5 ℃.Under different degrees of low temperature stress,POD activity decreased first and then increased.Compared with own-root seedlings,grafted seedlings maintained more chlorophyll and osmoregulation substance content and higher antioxidant enzyme activity at all same treatment levels.3.Rootstock grafting can improve the cold tolerance of watermelon.However,the molecular mechanisms underlying this process remain unknown.Herein,we used an isobaric tag for relative and absolute quantification(iTRAQ)-based quantitative proteomics approach for the comparative analysis of protein abundances in self-grafted(SG)and pumpkin rootstock-grafted(RG)watermelon seedlings in response to cold stress.A total of 4,796 distinct proteins were identified,and 752 proteins were substantially differentially accumulated in grafted watermelon seedling leaves after 48 h cold stress.Based on bioinformatics analysis,the cold tolerance of RG watermelon seedlings might be related to more energy produced through photosynthesis,carbon metabolism,and oxidative phosphorylation,compared with that of SG watermelon seedlings.RG watermelon seedlings could cope with cold stress by improving the scavenging capacity of ROS and arginine biosynthesis.Posttranscriptional regulation and protein homeostasis also play important roles for grafted watermelon seedlings to adapt to cold stress.Several protein kinases involved in signal transduction may act as positive regulators in RG watermelon seedling leaves suffering from cold stress.In addition,iTRAQ data were confirmed to be reliable by the assays of physiological indicators and relative transcript levels of eight genes.Taken together,we speculated that the mechanism of improved cold toleranece of RG watermelon seedlings through the following strategies:(1)14-3-3 protein and protein kinases involeved in signal transduction may act as positive regulators in RG in response to cold stress;(2)arginine synthesis,posttranscriptional regulation,and protein homeostasis such as ribosome,purine metabolism,mRNA surveillance,and posttranslational modification also play important roles in grafted watermelon seedlings to adapt to cold stress;(3)more efficient antioxidant enzyme activity and the considerable accumulation of osmoregulation substances can improve ROS scavenging capacity;(4)stronger photosynthesis,more stable carbon metabolism and higher oxidative phosphorylation can generate more energy to cope with cold stress.In short,we analyzed the response mechanism of grafted watermelon seedlings under cold stress through physiological and proteomics results,providing new clues for further research of cold tolerance mechanism of RG watermelon seedlings. |
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