Functional Analysis Of Reactive Oxygen Species Metabolism Related Genes In Upland Cotton Under Salt Stress

Cotton?Gossypium spp.?is a widely cultivated polyploid economic crop.Various stresses,such as salt stress,can directly lead to a considerable negative impact on cotton growth and development and lint yield.Reactive oxygen species?ROS?are natural byproduct of the active and reactive oxygen metabolites.As one type of signaling molecules,ROS are widely involved in regulating different biological processes.Maintaining a basal level of ROS is essential for proper cellular ROS signaling,which may be important for the stress tolerance of plants.And,ROS levels that are too low are cytostatic for cells,whereas ROS levels that are too high are cytotoxic.Thus,it is very important to study the metabolic process of ROS for improving plant stress tolerance.In cotton,some studies added evidence of the role of individual ROS metabolism-related genes in response to abiotic/biotic stresses.However,no information on ROS metabolism network and its related regulatory mechanism under salt stress is currently available at genome-wide level in cotton.In this study,we systematically identified three ROS-related gene family,including respiratory burst oxidase homologs?rboh?,superoxide dismutase?SOD?and catalase?CAT?,in the genome of upland cotton,and analyzed their expression patterns during developmental stages and under various stresses.And,we predicted the putative molecular regulatory mechanisms of the three gene family at the transcriptional and posttranscriptional level.In addition,we obtained a predicted targeting relationship between ghr-miR414c and GhFSD1gene and validate their biological functions under salt stress.The main results were as follows:1.Genome-wide characterization and expression analysis of the SOD gene family.There were 18 members in upland cotton SOD gene family,encoding Cu/Zn-SOD,Mn-SOD and Fe-SOD.These GhSOD genes distributed at different densities across 12 of the 26chromosomes.Based on the phylogenetic relationships of the SOD gene family from upland cotton and the nine-flagship species considered pivotal references for understanding plant evolution,we found that the genes encoding Mn-and Fe-SODs were older than the genes encoding Cu/Zn-SODs because they were thought to have been generated from the same ancestral enzyme,while the Cu/Zn-SODs evolved separately and did not possess such a similarity.Meanwhile,Cu/Zn-SODs separated from the family and occupied the dominant position during plant evolution to respond to the various abiotic and biotic stresses.The expression pattern of 18 GhSOD genes were tested in different tissues/organs and developmental stages,and different abiotic stresses and abscisic acid,which indicated that the SOD gene family possessed temporal and spatial specificity expression specificity and might play important roles in reactive oxygen species scavenging caused by various stresses in upland cotton.In addition,miRNA-mediated posttranscriptional regulation were predicted and analyzed.We found that 20 ghr-miRNAs targeted 14 GhSODs at 33 prediction sites,and most of the targeted sites located in the CDS regions.Among these putative targeting relationships,the expression of GhFSD1 gene was regulated by salt stress,and that the CDS of GhFSD1 harbored a potential ghr-miR414c targeting site.2.ghr-miR414c affected salt tolerance of cotton by regulating ROS metabolism under salinity stress.We cloned the primary transcript of ghr-miR414c and the full-length ORF of GhFSD1 from upland cotton.And,the expression profiles of ghr-miR414c and GhFSD1under salinity stress were monitored,and the results showed that salt stress induced expression of GhFSD1 and obviously increased SOD enzyme activities and H₂O₂concentrations,while ghr-miR414c showed expression patterns opposite to those of GhFSD1.The miRNA ghr-miR414c targeted the coding sequence region of GhFSD1,and the target relationship between them was experimentally confirmed by transient transfections,quantitative analysis and 5'RLM-RACE in tobacco.Ectopic expression of GhFSD1 in Arabidopsis conferred salinity stress tolerance by improving primary root growth and biomass,whereas Arabidopsis constitutively expressing ghr-miR414c showed hypersensitivity to salinity stress?100 and 150?mM NaCl treatment?.Silencing GhFSD1 by VIGS in cotton caused an excessive hypersensitive phenotype to salinity stress,whereas overexpressing miR414c decreased the expression of GhFSD1 and increased sensitivity to salinity stress,yielding a phenotype similar to that of GhFSD1-silenced cotton.Based on our data,we hypothesized that,without salt stress,the transcript level of GhFSD1 was regulated by ghr-miR414c via post-transcriptional gene silencing to maintain the ROS levels for normal metabolism in cotton cells.When cotton plants were exposed to salt stress,the in-planta production of ROS was induced and the expression of ghr-miR414c was reduced.The down-regulated expression of ghr-miR414c dampened down the inhibition of GhFSD1,leading to the increased GhFSD1 expression level and elevated SOD activity.As a result,the enhanced total SOD enzyme activity contributed to scavenge the excessive salt-induced ROS in plant cells,which alleviated cell membrane injury in response to salt stress.3.Comprehensive analysis of the rboh gene family.In the study,26 putative Ghrboh genes were identified and characterized,which were phylogenetically classified into six subfamilies and distributed at different densities across 18 of the 26 chromosomes or scaffolds.The results of synteny and collinearity analysis revealed that the common and lineage-specific WGD or polyploidy events and segmental duplications,which generated duplicate copies of plant rboh genes and were widespread throughout plant history,were the major factor responsible for the Ghrboh gene family expansion.Expression profiles of the gene family were analyzed and detected in different tissues/organs and developmental stages and different abiotic stresses,which indicated that the Ghrboh genes exhibited temporal and spatial specificity expression specificity and might play important roles in cotton development and stress tolerance through modulating NADPH oxidase-dependent ROS induction and other signaling pathways.4.Genome-wide characterization and bioinformatics analysis of the CAT gene family.In the present study,we identified 7 CAT genes in the genome of upland cotton for the first time at the genome-scale level.The results of the phylogenetic and synteny analysis showed that the GhCAT genes were divided into two groups,and WGD or polyploidy events contributed to the expansion of the Gossypium CAT gene family.Expression patterns analysis showed that the GhCAT gene family possessed temporal and spatial specificity and was induced by the hot,cold,drought,salt atresses and Verticillium dahliae infection.In addition,we found that the GhCAT gene family,which might be regulated by alternative splicing events,played roles in cotton development and stress tolerance through modulating the ROS metabolism.Taken together,our study helps lay the foundation for further exploring the roles of ROS-related genes in response to stress in upland cotton,broadens our insight into the mechanism of plant stress resistance,provides a theoretical basis for potential applications towards the genetic improvement of cotton.