Study On Evaluation And Mechanism Of Cold Tolerance Of Cotton At Seedling Emergence Stage

Cotton（Gossypium hirsutum L.）is an important cash crop in China,and occupies an important position in the national economy.Cotton originating from tropical and subtropical regions is sensitive to low temperature.Low temperature in spring is one of the main environmental factors limiting cotton production in China.To investigate the response mechanism of cotton to low temperature at seedling emergence stage,a total of 200germplasms in the northern extra early maturing cotton region,the Yellow River basin cotton region,the Yangtze River basin cotton region,the northwest inland cotton region and foreign varieties were used as research materials,and three treatments of constant chilling（4℃）,diurnal variation of chilling（16℃/4℃）and suitable temperature（28℃/25℃）were set to investigate the genotypic differences of their cold tolerance traits in response to chilling stress,and to establish a cold tolerance evaluation model.We further investigated the physiological indicators such as antioxidant enzyme activity,phytohormone content and transcriptional expression level under chilling stress,and revealed the physiological and molecular mechanisms of cotton to response chilling stress at seedling emergence stage using materials with significant differences in response to chilling.The main findings were as follows.1.There were significant differences in the response of different genotypes of cotton to chilling at seedling emergence stage.Under chilling stress,phenotypic indicators such as seedling emergence rate and seedling length,as well as physiological indicators such as soluble sugar and free amino acid content,differed significantly among cotton germplasm.The variation coefficients of cold tolerance traits ranged from 5.31%to 98.41%;There is a certain correlation between the cold tolerance traits of the two low temperature treatments.By establishing a cold tolerance evaluation model of cotton at seedling emergence stage,Y=-4.10+0.58X4+0.40X14+0.32X1+0.22X5（r=0.96）,X4_{seedlings length},X1_{emergence rate},and X5_{dry weight} under CC stress,and X14_{germination rate} under DVC stress can be determined as evaluation parameters.2.There are both commonalities and differences in the regulatory mechanisms of different genotypes of cotton in response to constant chilling and diurnal variation of chilling stress at seedling emergence stage.A total of 584 significantly associated single nucleotide polymorphisms（SNPs）(-log₁₀（p）≥6.41)were identified,and 35 quantitative trait locus（QTL）occurred stably,of which 5 were associated to QTLs by constant chilling and diurnal variation of chilling stress,respectively,and 25 were co-associated;The accumulation of dry matter weight in seedling under both low chilling stresses was associated with the flavonoid biosynthesis process regulated by Gh＿A10G0500（Gh TT7）;phenotypes such as seedling emergence rate and seedling dry matter weight under constant chilling stress were associated with Gh＿D09G0189（Gh SAL1）SNP variant,which clearly indicates that the IP₃ signaling pathway through Gh SAL1 negatively regulates cold tolerance in cotton.3.Phenotypic indicators such as seedling emergence and hypocotyl length,as well as physiological indicators such as antioxidant enzyme activities and hormones such as trans-zeatin（t ZR）were significantly higher in cold-tolerant than cold-sensitive cotton varieties with constant chilling treatment;a total of 11705 differentially expressed genes（DEGs）were obtained from transcriptome sequencing,and circadian rhythm regulation pathways were significantly enriched,and the expression levels of the involved genes were significantly higher in cold-tolerant variety.The expression levels of the genes involved in the cold-tolerant varietiy were significantly up-regulated.It is hypothesized that cold-tolerant variety could regulate phytohormone content and reduce oxidative damage through the circadian pathway.Compared with the cold-sensitive variety XLZ84,the cold-tolerant variety XLZ16 showed significant increases in seedling emergence and hypocotyl length by 44.76%and 9.41%,and superoxide dismutase（SOD）,peroxidase（POD）and catalase（CAT）activities by 84.81%,105.50%and 17.42%after 4 d of constant chilling treatment.After 12 h of chilling treatment,the content of growth-promoting hormones such as growth hormone（IAA）and trans-zeatin（t ZR）increased significantly by 29.95%and 36.86%,while the content of anti-stress hormones such as vanilloid alcohol（TY）and cis-12-oxo-phytodienoic acid（cis-OPDA）decreased significantly by 49.97%and 35.27%.Principal coordinate analysis（PCo A）and orthogonal partial least squares discriminant analysis（OPLS-DA）further demonstrated the differences in the physiological mechanisms of different cold-tolerant cotton variety in response to chilling at seedling emergence stage.4.Phenotypic indexes such as hypocotyl length and elongation rate,as well as physiological indexes such as antioxidant enzyme activity and hormones such as IAA were significantly higher in cold-tolerant cotton variety than cold-sensitive variety under diurnal variation of chilling stress;transcriptome sequencing results indicated that cold-tolerant variety had higher respiratory metabolism intensity and antioxidant capacity under diurnal variation of chilling stress,thus resisting chilling damage.Compared with the cold-sensitive variety XLZ38,the cold-tolerant variety KN27-3 showed a 45.02%increase in hypocotyl length and 105.67%and 42.67%increase in SOD and POD activities after 7 d of diurnal variation of chilling treatment.After 54 h of treatment,IAA,t ZR and other growth-promoting hormones were higher in KN27-3,with 2.88-fold and 5.28-fold differences,while abscisic acid（ABA）（42 h of stress）was lower,with 4.08-fold differences.A total of 7535 DEGs were identified by transcriptome sequencing,and it was found that DEGs of enzymes related to the anabolic pathway,antioxidant enzymes such as CAT1 and glutathione peroxidase（GPX4）,and osmoregulators such as asparagine and GDP-mannose were more up-regulated in the cold-tolerant variety KN27-3.In summary,there were significant differences in response of different cotton varieties to low temperature during seedling emergence stage;Under low temperature stress,the antioxidant enzyme activity,hormone content promoting plant growth and other physiological indicators of cold tolerant varieties were significantly higher than those of cold sensitive varieties,and maintained a higher respiratory metabolic intensity and osmotic adjustment capacity.The expression of cotton CBF genes were low under low temperature stress which were not induced by stress.It is speculated that the cold tolerance of field cold tolerant cotton varieties in this study is regulated by ABA and CBF independent ways at seedling emergence stage,so as to achieve the balance of low temperature response and growth and development.