Preliminary Study On The Physiological,biochemical And Molecular Mechanism Of Response To Low Temperature Stress In Empurpled Mutant HS2(Gossypium Hirsutum L.)

China is the largest cotton producer and consumer country in the world,cotton planting and related industries play an important role in our country’s national economy.Low temperature stress is one of the most serious disasters that restrict crop growth and development and affect agricultural production.In recent years,Xinjiang,the largest cotton-producing region in China,has been more frequent due to early-spring and latefrosts,even "low spring cold" invasion,which have seriously affected the yield and quality of cotton in Xinjiang region.Therefore,the research on the cold-resistant mechanism of cotton and the selection of cold-resistant varieties have become an urgent task to ensure and promote the stable development of cotton industry in China.The upland cotton empurpled mutant(HS2)is a leaf color mutant that exhibits strong cold resistance under low temperature conditions and outdoor in the falling and winter.In this paper,during the seed germination and seedling stages,we used the methods of molecular biology,physiology and biochemistry to comprehensively analyze the differences in plant cell morphology,tissue staining,physiology and biochemistry,and the expression levels of genes related to cold stress in HS2 and C312 plants under low temperature stress.We preliminarily discussed the regulation mechanism of response to cold stress in HS2,which will provid some new understanding of cold resistance mechanism and cotton breeding of cold resistance.The results were listed as follows:(1)By analyzing the anthocyanin content of C312 and HS2,we know that the anthocyanin content in each tissue and organ of HS2 is higher than that of C312.In addition,HS2 is more sensitive to light,and the anthocyanin content of HS2 under light conditions is significantly higher than that of indoor culture.We speculate that the change in leaf color of the mutant is related to the accumulation of anthocyanins in the body.(2)By observing the phenotypes of C312 and HS2 under low temperature stress during the seed germination and seedling stages,we found that HS2 exhibited strong cold resistance at both stages.Observed by histochemical staining,it is found that there is a big difference in the degree of cell membrane damage under low temperature stress between C312 and HS2,and it is positively correlated with the content of MDA.We speculate that the cell membrane is one of the main parts of cold damage.It can be seen that HS2 has better cell membrane integrity at low temperatures.It may be that the dynamic balance of active oxygen in the body is relatively stable,so it has a stronger protective effect on cell membranes.(3)Through to the physiological and biochemical analysis of C312 and HS2 at seed germination stage and seedling stage under low temperature stress,the activities of peroxidase(POD)and catalase(CAT),glutathione and soluble protein content in root of HS2 at seed germination stage were significantly increased compared with that of C312.In addition,the contents of anthocyanin,glutathione and soluble protein in leaves of HS2 increased significantly compared with that of C312 at seedling stage.These results indicate that HS2 can better adapt to low temperature stress than C312.The accumulation of soluble protein,glutathione and anthocyanin and the increase of antioxidant enzyme activity in the body may play an important role in its strong low temperature tolerance.(4)By analyzing the expression levels of cold resistance-related genes in leaves of C312 and HS2 at the seedling stage under low temperature stress,the expression of Gh COR47 and Gh SOD2 genes in HS2 leaves is significantly higher than that of C312 at each stage of low temperature stress;the expression of Gh CBF1 and Gh CBF3 genes in HS2 leaves is significantly higher than those without cold treatment under low temperature stress;the expression of Gh MYB113,Gh ANS,Gh GST and Gh UFGT genes in HS2 leaves is significantly higher than that of C312 at each stage of low temperature stress.Low temperature stress can induce the expression of cold resistance-related genes such as Gh CBF1,Gh CBF3,Gh COR47,and the expression of anthocyanin metabolismrelated genes such as Gh MYB113,Gh ANS,Gh GST and Gh UFGT in HS2 leaves.We speculate that the increased expression of these genes may promote the accumulation of antioxidant enzymes,anthocyanins and cold-resistant proteins in plants,maintain the balance of endogenous reactive oxygen species and the structure of cell membranes,thereby enhancing the ability of HS2 to resist low temperature stress.Through analysis of the phenotypic observation,detection of physiological and biochemical indicators and expression level of related genes in C312 and HS2 under low temperature stress,we found that HS2 has obvious cold resistance compared with C312.Compared with C312 under low temperature stress,HS2 has enhanced antioxidant enzyme activity,increased antioxidant and soluble protein content,and significantly increased the expression level of related cold-resistance genes.We speculate that the changes in these biochemical indicators and gene expression levels enable HS2 to keep the endogenous balance of reactive oxygen species in the body under cold stress.The homeostasis of reactive oxygen species enhances the tolerance of HS2 under cold stress,reduces cell membrane damage,and enhances its own cold resistance.