Study On Effect Of Melatonin On Salt Tolerance Of Cotton And Function Of GhSNAT34 Gene

As a cash crop,cotton is often threatened by abiotic stress during its growth and development.The effects of abiotic stress can be alleviated by exogenous melatonin（MT）administration and genetic transformation of MT synthesis gene to increase MT concentration.Studies have reported that MT is involved in plant response to salt stress,but the molecular mechanism of MT regulating cotton response to salt stress is still unclear.In the present study,we used Zhong 9807 in G.hirsutum as a material,the molecular functions of melatonin in regulating salt tolerance in cotton were investigated from both endogenous and exogenous directions using plant physiology,bioinformatics,molecular biology,and genomics.The main findings are as follows:1 Cotton seedlings at the three leaf stage were pretreated with different concentrations of melatonin（0,20,50,100,200μM）,and 0μm was used as a control to treat the cotton seedlings with 100 m M/L concentration of Na Cl solution.At four days of salt stress treatment,20μM melatonin pretreated seedlings exhibited only partial wilting and did not die,although cotyledons were all wilted.To explore whether the endogenous melatonin content was related to cotton salt tolerance,we determined the endogenous melatonin levels in three leaf stage seedlings of two cotton species（Zhong9807,GK50）,and found that the melatonin contents in Zhong 9807 roots and leaves with better salt tolerance were higher than GK50.We speculated that the salt tolerance of cotton may have some relationship with the endogenous melatonin content.Meanwhile,to explore whether the endogenous melatonin content in cotton is involved in cotton’s defense against salt stress,we treated three leaf stage cotton seedlings with salt and measured the endogenous melatonin content 12 h later,with the clear water treatment as the control,and the results showed that the salt treatment induced the elevation of endogenous melatonin content.In conclusion,endogenous melatonin in cotton is involved in the defense of cotton against salt stress,and there is a relationship with cotton salt tolerance.2 We utilized three treatments:CK（control）,S（100 m M Na Cl）,and M-S（20μM melatonin+100 m M Na Cl）to investigate the protective effects of exogenous melatonin on cotton under salt stress,and the mechanism by which exogenous melatonin protects cotton from salt stress was explored by RNA-seq technical analysis.There were 786 significantly differentially expressed genes（DEGs）（356 upregulated and 430 downregulated）between the S and M-S treatments.Melatonin pretreatment altered the expression of genes related to redox.Furthermore,we found that AP2,WRKY,NAC transcription factor family members,which are induced by melatonin,may play important roles in salt tolerance,and melatonin orchestrates other phytohormones in the defense network of cotton against salt stress to induce signaling molecules through phosphatidylinositol signaling system related genes in response to salt stress.Taking our results together,we delineate the molecular role of melatonin in improving salt tolerance in cotton through a complex regulatory network to improve the tolerance of cotton to salt stress by regulating various mechanisms,such as the antioxidant system,phytohormones,transcription factors,phospholipid metabolism,and signaling molecules.3 We found a putative serotonin N-acetyl transferase（SNAT）gene GH＿D02G1113,through the integration of upland cotton raw SNAT structure characteristics,the phylogenetic relationships of genes family members,gene structure and expression patterns and abiotic stress factors,based on whole genome sequences of upland cotton SNAT gene family system is analyzed.Phylogenetic analysis showed that a total of 63SNAT genes were identified in the whole genome of upland cotton,which could be divided into 5 subclasses（subclass I-V）.Most SNAT members in each group had similar motif composition and gene structure characteristics.More than half of Gh SNAT members contained Gibberellin-responsive components and Me JA-responsive components.GA and Me JA may be important signals to regulate the Gh SNAT family and participate in the abiotic stress response.We used published RNA-seq data to investigate the expression pattern of Gh SNAT gene under cold,heat,salt and PEG stress.Under different stress conditions,the expression level of Gh SNAT gene changed,suggesting that Gh SNAT members were involved in the regulation of abiotic stress.4 We used virus-induced gene silencing technique to silence melatonin biosynthetic gene Gh SNAT34,which significantly reduced the content of endogenous melatonin in the silenced plants,reduced the reactive oxygen species scavenging system and Ca²⁺signal transduction,and resulted in decreased salt tolerance.Exogenous supplementation of appropriate amount of melatonin could alleviate the effect of salt stress on silenced plants.These results indicated that cotton Gh SNAT34plays an important role in regulating endogenous melatonin level,and participates in salt stress response by regulating endogenous melatonin content in cotton.