Analysis Of Metabolomics And Study Of The Functional Network Responding To NaCl Stress In Cotton

The utilization of 7% of land and 33% of irrigated land worldwide are soil salinization seriously affected.As a pioneer crop for repairing saline-alkali soil,cotton plays an important role in the utilization and improvement of saline-alkali land.Metabolomics can detect various metabolites of cotton under stress,and discover the metabolic regulation laws of cotton adapting to stress through the analysis of qualitative and quantitative.Therefore,it has great significance that conducting metabolomics analysis and analyzing the salt tolerance regulatory network of cotton under salt stress.In this study,salt stress-treated cotton was selected and used as the material to analyze the regulatory network of cotton through morphological,physiological,metabolomic and transcriptomic analysis under salt stress,in order to decipher the molecular regulatory mechanism of cotton in response to salt stress.The main research findings were as follows:(1)Salt stress led to the activation of antioxidant response system and metabolic regulation in cotton.The morphological and physiological changes of four cotton materials were analyzed under salt stress,and the results showed that salt stress caused cotton leaves to wilt due to water loss,and the total flavonoid and cysteine content of the leaves significantly increased.The DAB and DPBA staining results of cotton indicated that salt stress led to the accumulation of ROS and flavonols.(2)Widely targeted metabolomic analysis was conducted on cotton leaves at different time periods of salt stress.A total of 741 metabolites were identified,including flavonoids,phenolic acid,lipid,amino acid,organic acid,alkaloids,nucleotide,and saccharides alcohol compounds.Through the signal intensity of mass spectrometry,the abundance of flavonoids,lignans,and coumarins was significantly increased,while the abundance of terpenoids and vitamins was decreased.Compared with the 0 h,a total of 96 differential metabolites(56 up-regulated and 40 down-regulated)were identified after 3 h treatment,162 differential metabolites(77 up-regulated and 85 down-regulated)were identified after 24 h of salt stress,and 268 differential metabolites(130 up-regulated and 138 down-regulated)were identified after 48 h of salt stress.The flavonols,turnose,L-cysteine,and isopentenyl-7-N-glucosides were found continuously up-regulated during three time periods,which were candidated biomarkers of cotton under salt stress.(3)Joint analysis was conducted on differential metabolites(DMs)and differentially expressed genes(DEGs)under salt stress.The co-enrichment pathways of differential metabolites and differentially expressed genes mainly involved metabolic pathways such as flavonoids biosynthesis and amino acids biosynthesis.DMs and DEGs with positive regulatory relationships were also enriched in flavonoids pathway.K-means clustering analysis found that DEGs that continuously up-regulated were mainly enriched in the flavonoid biosynthesis and amino acid metabolism pathway.The differential metabolites with continuously increasing content mainly belonged to flavonoids and phenolic acids.The joint analysis results indicated that the flavonoid biosynthesis pathway played an important role in the response of cotton to salt stress.(4)Analysis of cotton flavonol synthase FLS gene family members.FLS is a key enzyme for synthesizing flavonols such as quercetin and kaempferol.Family member identification and expression pattern analysis of the gene encoding this enzyme were carried out.The results showed that 9,7,4,and 4family members were identified in Gossypium hirsutum,Gossypium barbadense,Gossypium raimondii,and Gossypium arboreum,respectively.The GhFLS promoter contained plant hormone responsive elements and abiotic stress responsive elements.The q RT-PCR experiments found that GhFLS1 was significantly up-regulated in various time periods of salt stress.(5)GhFLS1 plays a key role in cotton response to salt stress.Compared with wild-type Arabidopsis,the seed germination percentage and root length of the transgenic GhFLS1 gene Arabidopsis plant were significantly increased under salt stress.After applying quercetin,it can alleviate the damage of Arabidopsis and cotton under salt stress.The GhFLS1 gene was silenced using VIGS technology and subjected to salt stress.Compared with the control p YL156,the GhFLS1 gene silenced strain showed more severe symptoms under salt stress.The total flavonoid content in transgenic cotton with GhFLS1 was significantly increased,and the transpiration rate and net photosynthetic rate were also significantly increased.The accumulation of ROS in transgenic cotton leaves was significantly decreased under salt stress,which indicated that the salt tolerance of transgenic GhFLS1 cotton had significantly improved.The Luc result showed that GhFLS1 was regulated by the GhMYB26,and participated in flavonoid metabolism.Based on the above research results,cotton initiated antioxidant response and metabolic regulation to adapt to salt stress.The GhFLS1 gene and quercetin in the flavonoid synthesis pathway jointly participated in the regulation of cotton salt tolerance,and overexpression of this gene can improve the salt tolerance of cotton seedlings.This study lays a molecular foundation for further understanding the molecular regulatory network and salt tolerance signaling pathways response to salt stress in cotton.