Spatial Distribution Of Soil Enzyme Activities Against Heavy Metal Stress Characterized By In Situ Zymography

The rapidly growing population,industrial progress and technical innovations have increased the concentration of heavy metals around the globe.Heavy metals can be hazardous to soil,plant and human health through the soil-crop-food chain.Heavy metals usually affect the growth and morphology of plants,disrupt the microbial metabolism and the biochemical reactions of the soil,as well as change the soil enzymes.Although soil enzyme activity is crucial for assessing heavy metals contaminated soils and soil fertility,the spatial distribution of enzyme activity in heavy metal-contaminated soils remains unclear.In this study,under the pot experiment,the spatial distribution of enzyme activities under heavy metal stress was analyzed by in situ zymography,and the response mechanism of soil-plant enzyme activities system to heavy metal stress was discussed,and the alleviation mechanism of rhizobium-alfalfa symbiosis under copper stress was revealed.The main results as follows:1.Under heavy metal stress,the growth of alfalfa was significantly inhibited,the biomass and the content of chlorophyll in leaf decreased significantly.Over-accumulation of Pb,Zn and Cd in root of alfalfa,and the transfer coefficient was less than 1.0,which indicates the benefits of phytostabilization of heavy metals.In extremely heavy pollution treatment,the malondialdehyde content（MDA）in alfalfa root was increased 34.7%,and was subjected to severe oxidative stress.Catalase activity（CAT）in alfalfa decreased with increasing heavy metal concentrations.In moderate pollution and extremely heavy pollution treatment,the soilβ-glucosidase activities increased by 34.4%and 33.9%,respectively.In addition,the correlation analysis showed that the catalase and saccharase were the most sensitive to the Pb,Zn and Cd pollutions in soil.2.The distribution of enzyme activities showed that both phosphatase andβ-glucosidase activities were associated with the roots and were rarely distributed throughout the soil.In addition,the total hotspot areas of enzyme activities were the highest in extremely heavy pollution soil.The hotspot areas of phosphatase were 3.4%,1.5%and 7.1%under none,moderate and extremely heavy pollution treatment,respectively,but increased from 0.1%to0.9%forβ-glucosidase with the increasing pollution levels.Compared with the traditional method of enzyme activities,zymography can directly and accurately reflect the distribution and extent of enzyme activity in heavy metals polluted soil.3.The rhizobium-alfalfa symbiosis can promote the growth of alfalfa,increase the biomass and the chlorophyll content of alfalfa,improve the contents of total nitrogen and total phosphorus,promote the uptake of copper in alfalfa,reduce the ability of copper transfer,and help to stabilize the metal.The biomass of shoot and root in the rhizobium inoculation treatments were increased by 9.5%-35.2%and 2.6%-33.3%,respectively.After rhizobium inoculation,the total nitrogen content in the root of alfalfa increased 5.7%-9.2%.At 600 and800 mg·kg^-1 Cu concentration,compared with the non-inoculated plants,the plants with rhizobium inoculation increased the total phosphorus content in root of plants by 6.35%and7.41%,respectively.The rhizobium inoculation could significantly increase copper content and the uptake in root and enhance metal extraction efficiency.4.With the increase of copper stress,the malondialdehyde content（MDA）increased gradually,while rhizobium inoculation significantly inhibited the production of MDA and alleviated the oxidative stress of plants.At 800 mg·kg^-1 Cu,the MDA content in shoot and root decreased by 17.8%and 24.6%,respectively.In addition,the rhizobium-alfalfa symbiosis could stimulate the antioxidant enzyme system,enhanced the activities of SOD,POD,CAT and APX,promoted the enzymatic reaction to consume oxygen free radical,improved the balance of plant cell,released the stress.5.Compared with the non-inoculated treatment,soil alkaline phosphatase andβ-glucosidase increased significantly in rhizobium-alfalfa symbiosis.In addition,at 0 and 800mg·kg^-1 Cu,rhizobium inoculation significantly increased the alkaline phosphatase activity in rhizosphere by 13.6%and 38.3%respectively,compared with the control treatment.For both control and rhizobium inoculation treatments,the distribution of enzyme activities showed that phosphatase activity was associated with the roots and it was high in the root tips.In addition,Compared with the non-inoculated treatments,rhizobium inoculation also increased the total hotspot areas of phosphatase under copper stress of 0 and 800 mg·kg^-1,which increased by 26.1%and 39.3%,respectively.In this paper,we visualised the spatial patterns of enzymatic hotspots in alfalfa rhizosphere,and elucidated the phosphatase response to rhizobium inoculation for the first time.