Research On The Mechanism Of Spinach Seedlings Response Under Copper Stress And Regulatory Effect

Copper（Cu）is an indispensable trace element in plant growth and development,but its excessive content affected plant growth and human health.In recent years,due to the unreasonable use of Cu-containing pesticides and manure in agricultural production,garbage incineration,sewage irrigation,copper mining and smelting and other human behaviors,the content of heavy metal Cu in the soil has seriously exceeded the standard.Spinach（Spinacia oleracea L.）,a herb belonging to the Caryophyllales order,contains large amounts of vitamins,carotenoids,organic acids and alkaline minerals,as well as antioxidantsis,and is characterized by large leaf area,high relative growth rate,and high rate of heavy metal absorption.In this study,spinach seedlings were used as pot experimental materials.The changes of growth,antioxidant enzymes,gas exchange parameters,chlorophyll fluorescence parameters,absorption and transport of mineral elements,and cell ultrastructure of spinach seedlings were first studied under different treatments of Cu concentrations.Then,three different plant hormones（GA₃,IAA and NAA）were sprayed into the leaves of Cu-stressed seedlings,and the optimal concentration of plant hormones was explored to further reveal the regulatory effect of plant hormones on the mechanism of Cu-tolerance in spinach seedling.The main results were as follow:1.Effects of different concentrations Cu treatment on growth physiological indexes of spinach seedlingsIn this experiment,11 groups of Cu concentrations were set,and aimed at observation the changes of growth physiological indexes of spinach seedlings.The results showed that at the 100mg L^-1Cu concentration,the biomass increased significantly,and antioxidant enzymes（SOD,POD,CAT and APX）were no significant change,photosynthetic parameters（P_n、g_s、T_r、Y（Ⅱ）and q P）increased,N,K,Mg,Ca,Fe and Zn content in leaf increased,the root of Ca,Fe and Zn contents increased,the organelles structure were complete and clearly visible.It shows that the 100mg L^-1Cu concentration can promote plant growth,increase the photosynthetic rate,and promote the mineral elements absorption.However,at the Cu concentration(800-1000 mg L^-1Cu concentration),the biomass decreased,MDA content significantly increased,the antioxidant enzymes activities increased to the highest after different degrees of decline,the contents of chlorophyll a and b reduced,the P_n,g_sand T_rdecreased,and Fv/Fm,q P,NPQ and Y（Ⅱ）reduced,except for the ambiguous structure of nucleus and mitochondria,all other organelles were completely visible.It showed that higher Cu concentration affected the life activities of plants,but plants maintained a better state of physiological activities by regulating the antioxidant system,which showed that plants had a strong Cu tolerance.2.Changes of growth physiological parameters of spinach seedlings under high concentration Cu stressWhen adding 700mg kg^-1Cu concentration,spinach seedling growth decreased,MDA,O₂^-and H₂O₂contents increases,proline content,soluble protein content and antioxidant enzyme activity also increased,chlorophyll a and chlorophyll b,carotenoid content,photosynthetic parameters（P_n,g_sand T_r）,and chlorophyll fluorescence parameters（Fv/Fm,q P,NPQ,Y（Ⅱ））reduced.The Cu content in leaves and roots increased significantly,and the Cu content in leaves was significantly higher than that in roots.In the leaf and root,Mg and Ca contents decreases,and N,P,K,Fe and Zn contents increased.The chloroplast in leaf cells swelled,the nucleolus blurred,but the rest of the organelles were clearly visible,indicate that under high concentration of Cu stress,the leaves and roots absorbed a large amount of Cu²⁺and the Cu²⁺mainly accumulated in the leaf,leading to the intensification of oxidative stress response in plants,chlorophyll synthesis,gas exchange,some mineral element absorption and other physiological activities were affected,and some organelle structure changed.Even so,the plants can still actively increase the content of osmotic regulating substances,increase the activity of antioxidant enzymes,and resist the damage of high Cu concentration to plants.It can be seen that spinach seedlings have strong Cu tolerance.3.Regulation effect of adding different concentrations of GA₃on spinach seedlings under high concentration of Cu stressIn this experiment,different concentrations of GA₃solutions were applied to the leaves of 700mg kg^-1Cu stressed seedlings.After 7 days,plant samples were collected for measurement of indicators.The results show that the application of 3-5 mg L^-1GA₃significantly decreased the Cu content in the leaf,O₂^-and H₂O₂content decreased,oxidative damage significantly reduced,the content of soluble protein increased,the parameters of gas exchange and chlorophyll fluorescence increased,the activities of antioxidant enzymes increased,the absorption of mineral elements promoted,and the plant biomass increased.However,when the concentration of GA₃was higher than 40 mg L^-1,the oxidative damage in plants enhanced,the activity of antioxidant enzymes,photosynthetic parameters and chlorophyll fluorescence parameters decreased,and the biomass also decreased,indicating that the high concentration of GA₃intensified the damage of Cu stress and seriously affected the physiological function of spinach seedlings.Therefore,it can be concluded that the concentration of 3-5 mg L^-1GA₃has the best alleviating effect on the toxicity of spinach seedlings under Cu stress.4.Regulation effect of spraying IAA solution with different concentrations on spinach seedlings under high concentration of Cu stressIn this experiment,6 groups of IAA solutions with different concentrations were sprayed into the leaves of seedlings under Cu stress.After 7 days of treatment,the physiological parameters were measured.The results showed that low concentration of IAA(10-40mg L^-1)could increase the Cu content in the roots and decrease the Cu content in the leaves of seedlings under Cu stress.At this point,the MDA,O₂^-and H₂O₂content reduced,antioxidant enzyme（SOD,CAT）activity increased,chlorophyll a and chlorophyll b,carotenoids,P_nand g_s,and Fv/Fm,NPQ,q P,ETR,mineral element absorption increased,the damaged to leaf organelles also alleviated,showed that exogenous add this concentration of IAA can induce the synthesis of antioxidant enzymes and enhance photosynthesis,promote the mineral elements absorption,alleviate the stress injury of high concentration to the spinach seedlings,and the alleviating effect was most obvious when the concentration of IAA reached 60mg L^-1.At this time,Cu content in leaves and roots decreased to the lowest level,oxidative stress reaction in seedlings was the lowest,plant growth,photosynthesis and mineral element absorption all increased to the highest level.Therefore,compared with other IAA concentrations,60mg L^-1IAA had the most obvious effect on Cu stress injury relief.5.Regulation effect of exogenous spraying of different concentrations of NAA solution on spinach seedlings under high concentration of Cu stressUnder Cu stress,10μmol L^-1NAA solution showed the most obvious regulation effect when spraying 5 different concentrations of NAA solution to spinach seedling leaves.At this time,the seedling leaves of Cu content decreased significantly,the roots of Cu content significantly increased,the growth also significantly increased,MDA,O₂^-and H₂O₂content decreased to a minimum,antioxidant enzymes（SOD,POD,CAT and APX）and proline,soluble protein contents increased,the chlorophyll content and P_n,g_sand C_iincreased to the largest,q P and ETR also increased to the maximum,the root of the mineral elements contents enhanced.The chloroplast number increase,grana lamella neatly,cell wall,cell membrane,nucleus,mitochondria and vacuoles were clearly visible,the nucleus was clearly separated from the nuclear fluid.It showed that add 10μmol L^-1NAA solution can improve plant antioxidant capacity,increase the contents of osmotic regulation substances,reduce intracellular membranous peroxide damage,reduce the interference of chlorophyll synthesis,promote the photosynthesis,alleviate the Cu damage to the organelles,thus,the stress effect of high concentration on spinach seedlings was alleviated.