Exogenous Melatonin Induced Physiological Mechanism Enhances Drought Tolerance In Tartary Buckwheat

Drought stress is one of the major obstacles for agricultural production worldwide.Meanwhile,drought stress often occurs in both natural and agricultural ecosystems.Evidence showed that drought stress had an antagonistic effect on plant growth and development,photosynthetic activities,antioxidant activities,and alteration of molecular functions.However,plants themselves suffer from phytotoxicity under elevated levels of drought stress because of limiting antioxidant and detoxification capacity.Tartary buckwheat is one of the nutritious minor cereals and is grown in high-cold mountainous areas of arid and semi-arid zones where drought is a common phenomenon,potentially reducing the growth and yield.Therefore,improving drought resistance in crops is a pre-requisite for achieving economic benefits.Hence,hormonal applications under such conditions may provide additional power to strengthen the plant defensive system in order to combat the stress generated by xenobiotics.The multifunctional signaling molecules melatonin（N-acetyl-5-methoxytryptamine）is widely dispersed in different parts of plants and is responsible for many physiological responses to hostile environmental conditions.Therefore,the present experiment was conducted to understand the stress responses of the Tartary buckwheat plant towards drought as well as to elucidate the possible protective mechanism of melatonin in drought stress mitigation through a series of experiments.The salient results are summarized as follows:1.The main purpose of this experiment was to investigate the drought stress effects on seed germination,physiology,and biological mechanisms,for screening the most resistant Tartary buckwheat genotypes.Seeds from 14 cultivars were treated with polyethylene glycol（PEG）,which induced four stress conditions（0%,10%,20%and 30%）to evaluate germination percentage and seedling growth parameters.The results showed significant differences in germination percentage,relative water content（RWC）,and all growth parameters among the cultivars after exposing to drought stress.In addition,on the basis of stress tolerance index,three tolerant（Qian Ku,Xi Nong 9943 and Xi Nong 9940）,and one susceptible（Qian Ku-4）genotypes were identified for carrying out further investigation through physiological and biochemical mechanisms against PEG-induced drought stress at germination stage.The results of the study showed that the levels of antioxidant activities significantly increased by superoxide dismutase（SOD）,peroxidase（POD）,catalase（CAT）and ascorbate peroxidase（APX）enzymes in the plumule tissues of the tested four cultivars as the water potential reached the highest level.However,the activities of secondary metabolites like phenylalanine ammonialyase（PAL）,polyphenol oxidase（PPO）,phenolics,flavonoids,and total antioxidant capacity were increased abruptly under severe stress conditions in tolerant genotypes as compared to the sensitive genotypes.A high variation of drought responses was identified among genotypes,which made it possible to screen drought tolerant cultivar（Xi Nong 9943）at the germination stage.These findings would help to choose a suitable parent for future breeding programs.2.This study examined for a better understanding of tolerance mechanisms under severe drought at seedling stage.The experiment was conducted with four cultivars,including two tolerant（Xi Nong 9943 and Xi Nong 9940）,one susceptible（Qian Ku-4）,and one check（Xi Qiao-2）cultivars,and four water regimes such as T₀-80%field capacity（FC）used as no drought stress,T₁-60%FC,T₂-40%FC and T₃-20%FC,respectively.Drought stress declined RWC,stomatal properties,chlorophyll（Chl）pigments;besides,its increased MDA contents,hydrogen peroxide（H₂O₂）and O₂^·-of all Tartary buckwheat genotypes.In addition,the tolerant genotypes accumulated higher osmotic solutes like proline and soluble protein and lower ROS and MDA content than the sensitive genotypes under drought conditions.The antioxidant activity such as SOD,POD,CAT and APX were enhanced with the increasing stress levels.However,the activities of secondary metabolites like phenolics,flavonoids,PAL and PPO,and total antioxidant capacity were sharply increased in tolerant variety compared to the susceptible variety under drought.Higher accumulation of osmolyte solute and improvement of physiological and biochemical trials and less oxidative damage through improving antioxidant defense system are the mechanisms of drought tolerance in selected Tartary buckwheat genotype,Xi Nong 9943 followed by Xi Nong 9940.3.In this part of study,we evaluated the influence of melatonin supplementation on plant morphology and different physiological activities in the Xi Nong 9943 variety of Tartary buckwheat under drought stress.The experiment was designed with the following six treatments-（i）control（80%FC）（Cont）,（ii）control and 100μM melatonin（Cont+Mel100）（iii）drought（20%FC）（D）,（iv）drought and 50μM melatonin（D+Mel50）,（v）drought and 100μM melatonin（D+Mel100）,and（vi）drought and 200μM melatonin（D+Mel200）.Drought stress decreased plant growth and biomass production compared to the control.Drought（D）also decreased Chl a,b,and the Fv/Fm ratio by 54%,70%,and 8%,respectively,which was associated with the disorganized stomatal properties.Under drought（D）treatment,H₂O₂,O₂^·-,and MDA contents increased by 2.30,2.43,and 2.22-folds,respectively,which caused oxidative stress.In contrast,proline and soluble sugar content were increased by 84%and 39%,respectively.However,exogenous melatonin（100μM）could improve plant growth by preventing ROS-induced oxidative damage by increasing photosynthesis,enzymatic antioxidants（SOD,POD,CAT and APX）,secondary metabolites like PAL,phenolics,and flavonoids,total antioxidant scavenging,and maintaining RWC and osmoregulation substances under water stress.Therefore,our study suggested that exogenous melatonin could accelerate drought resistance by enhancing photosynthesis and antioxidant defense in Tartary buckwheat plants.4.This study was also designed with three treatments according to our previous study results,such as i)control（80%FC）（Ctr）,（ii）drought（20%FC）（D）,and（iii）drought and100μM melatonin（DM）,respectively,to reveal and further clarify the potential role of exogenous melatonin on physiological and molecular changes in the Xi Nong 9943 variety of Tartary buckwheat plants against drought stress by evaluating transcriptome analysis.This observation aims to provide a better understanding of how the exogenous melatonin regulates the differential gene expressions in plants,including photosynthesis,carotenoid biosynthesis,secondary metabolites related genes and transcriptional factor in response to drought stress.Exogenous melatonin significantly alleviated carotenoid and chlorophyll depletion and ROS accumulation by improving enzymatic and non-enzymatic antioxidants under drought stress.By the transcriptome analysis,4329,3265 and 3753 differential expressed genes（DEGs）were identified in the comparisons of D vs.Ctr,DM vs.Ctr,and DM vs.D,respectively.Among them,843 DEGs were commonly expressed in all compressions and associated with main photosynthesis,carotenoid and secondary metabolites biosynthesis-related genes.Moreover,transcript families such as b HLH,WRKY,MYB-related,B3,NAC and FAR1 related gene expression were calculated more in all comparisons.The q PCR and RNA-sequence analysis results suggested that exogenous melatonin is closely associated with drought stress moderator by boosted antioxidant defense mechanisms through regulating the genes involved in significant DEGs changes,photosynthesis,carotenoid and secondary metabolites biosynthesis,and transcriptional factor.These findings indicated that melatonin could improve drought stress tolerance directly or indirectly by protecting enhanced ROS accumulation in Tartary Buckwheat.Our results helped to understand the role of melatonin in drought stress and provide a new perspective regarding the roles of melatonin in Tartary buckwheat plants against abiotic stress.In addition,findings of the current study would be helpful for future breeding programs to develop new cultivar and fulfill the food security as well as food industrial demand.Moreover,it clarifies the process of developing new approaches in plant cultivation,and provides guidance to industrial agriculture in drought condition.