Effects Of Exogenous Melatonin On Morphological Structure And Physiological Traits Of Cotton Seedlings Under Salt Stress

Salt damage is one of the main abiotic stresses that restrict crop growth and yield quality.As an important economic crop in the world,cotton is susceptible to salt damage during its seedling stage.As a new type of plant hormone,melatonin plays an important role in plant resistance.At present,the effects of melatonin on plant are mainly concentrated on seed germination and anther fertility,while the mechanism of melatonin on cotton seedlings is rarely reported.Therefore,in this study,the upland cotton(Gossypium hirsutum L.)variety "Guoxin No.9" was used as the experimental material,and the hydroponic experiment was carried out in an artificial climate room.Different treatments of salt and melatonin were set to study the morphology,structure and physiology characteristics changes of cotton seedlings.The regulation effect of melatonin on the development of cotton seedlings under salt stress was investigated,and the salt resistance mechanism of melatonin was clarified.The main results of the study are showed as follows:1.Exogenous melatonin promoted the growth and development of cotton seedlings under salt stress.Salt stress significantly reduced the plant height,stem thickness,leaf area and biomass of cotton seedlings.After applying different concentrations of melatonin,the growth of cotton seedlings was promoted in different degrees,of which 200 μM melatonin promoting effect is the most significant.2.Exogenous melatonin improved the photosynthetic performance of the functional leaves of the main stem of cotton seedlings,and enhanced the photosynthetic capacity.Relative chlorophyll content(SPAD value),photosynthetic gas exchange parameters(net photosynthetic rate,transpiration rate,stomatal conductance,intercellular CO2 concentration)and chlorophyll fluorescence parameters(PSII maximum photochemical efficiency,the actual photochemical quantum yield and apparent electron transfer efficiency)of the functional leaves of the main stem of cotton seedlings under salt stress decreased,while the non-photochemical quenching coefficient increased.After applying different concentrations of melatonin,the photosynthetic characteristics of cotton seedlings were promoted in different degrees,and 200 μM melatonin effectively improved photosynthesis.3.Exogenous melatonin reduced the reactive oxygen species content of cotton seedlings under salt stress.Under salt stress,the content of reactive oxygen species(H2O2,O2-)and malondialdehyde increased significantly.Histochemical staining(DAB,NBT staining)of the leaves showed that the H2O2(brown dots)and O2-·(blue)areas in cotton leaves were all over the leaves;fluorescence detection of H2O2(green fluorescence)and O2-·(red fluorescence)in guard cells showed that the active oxygen content increased significantly,but the melatonin treatment significantly reduced the active oxygen content of the seedlings and reduced the accumulation of active oxygen.4.Exogenous melatonin improved the antioxidant capitaty,osmotic regulation substance,and the expression levels of antioxidant enzyme genes and ion transporter genes in cotton seedlings under salt stress.Salt stress led to a significant increase in the activity of antioxidant systems(SOD,POD,CAT,APX)and the content of reducing substances(AsA,GSH);the proline content of organic osmotic regulators increased,and the content of soluble sugar and soluble protein decreased;the content of inorganic osmotic regulator Na+and Cl-increased,while the content of K+ decreased.After applying melatonin,the activities of SOD,POD,CAT,APX,reducing substances(AsA,GSH),organic osmotic regulators,K+ content and K+/Na+ratio increased significantly,but the content of Na+and Cl-were significantly reduced;and the expression of antioxidant enzyme synthesis genes(GhSOD,GhPOD,GhCAT,GhAPX)and ion transport genes(GhNHX1,GhSOS1,GhAKT1)were significantly increased,of which 200 μM melatonin had the most obvious regulatory effect.5.Exogenous melatonin increased the content of endogenous hormone and gene expression in cotton seedlings under salt stress.Under salt stress,the content of abscisic acid(ABA),IAA and zeatin nucleoside(ZR)increased,the content of GA decreased.After applying melatonin,by down-regulating the expression of ABA synthesis genes(GhNCED2,GhNCED9)and up-regulating the expression of metabolic genes(GhCYP707A2,GhCYP707A4)to reduce ABA content;by up-regulating the expression of GA synthesis genes(GhGA2ox1,GhGA2ox2,GhGA20ox1,GhGA20ox2)and up-regulating the expression of auxin synthesis gene(GhYUC6)to increase auxin content.6.Exogenous melatonin improved the anatomical structure of cotton seedling stems and leaves under salt stress,and promoted stomata opening.Under salt stress,the thickness of cotton leaf,palisade tissue and sponge tissue,xylem and phloem area were increased significantly;the mesophyll cell structure was destroyed,the chloroplast length became smaller,the width increased,and the grana lamellar structure was disordered;the mitochondria were slightly swollen;the length,width and opening of stomata decreased significantly,while the number of stomata increased;the thickness of the pith in the stem,xylem thickness and phloem thickness decreased significantly.After melatonin treatment of seedlings,the structure of its leaves and stems had been significantly improved,and the diameter of the pith in the stem and the thickness of the xylem and phloem had been increased,the stomatal length and width of pore diameter were increased,and the number of stomata was decreased;the chloroplast shape was regular,the grana lamellar structure was complete,and the mitochondria structure did not change significantly.7.Correlation and principal component analysis were carried out on 21 representative indicators.It is found that the total dry weight(TDW)was significantly positively correlated with Pn,SPAD,Fv/Fm,IAA,stomata opening(SA),stem phloem thickness(SPT),stem xylem thickness(SXT),and TDW had a significantly negatively correlated with the number of stomata(SN),palisade tissue thickness(PT),Na+,NPQ,H2O2,O2-·,and sponge tissue thickness(ST).TDW,Pn,SPAD,Fv/Fm,SA,IAA were significantly negatively correlated with SN,PT,Na+,NPQ,ST,H2O2,O2-·.The 21 traits were grouped into two principal components,PT,Na+,NPQ,H2O2,O2-·,SPT,TDW,Pn,SPAD,IAA,SXT,Fv/Fm had a greater contribution to the first principal component,LAX,LAP,SOD,and Pro contributed more to the second principal component,explaining 90.44%of the total variation of 21 traits.In summary,melatonin reduced the permeability of the membrane system by improving the antioxidant system,increasing the pore size,and increasing the osmotic adjustment substances;by increasing the content of IAA,GA,ZR and reducing the content of ABA;and by regulating the expression of antioxidant enzymes and ion transport genes,the structure of stem and leaf;the photosynthetic capacity and salt resistance of cotton seedlings were improved.