Heavy Metal Tolerance Of Spirodela Polyrrhiza And The Underlying Mechanisms Mediated By Environmental Microorganisms Cooperating With Plant Growth Regulators

Heavy metal contamination that caused by anthropogenic activities is a widespread and serious environmental problem.It not only seriously affects plant growth and development,and then reduces crop yield and quality,but also threats to human health through the food chain.Up to date,some exogenous plant growth regulators or bacteria have been reported could improve the tolerance of plant to kinds of heavy metals,but the cooperated effects of bacteria and growth regulators on enhancing heavy metal tolerance in plants are still not clear;moreover,the interactions that occurred between plant growth regulators and bacteria,as well as their cooperations to the improvement of heavy metal tolerance are usually neglected in these studies.In this study,we found the bacteria could cooperate with some plant growth regulators to improve the tolerance of heavy metals to Spirodela polyrrhiza and revealed the underlying mechanism by using comparative transcriptomic analysis.The main results were obtained as follows.1.Based on non-sterile S.polyrrhiza,the effects of Cu,Cd and Pb on the growth and metabolism of duckweed were studied.The results showed that（1）With the increases of the concentration of Cu,Cd and Pb,the contents of photosynthetic pigment and soluble protein gradually decreased.When the concentration of Cu,Cd and Pb reached the highest levels,the photosynthetic pigment decreased by 59.27%-77.61%,and the soluble protein content decreased by 51.78%-68.73%;（2）Cell membrane stability was breaked,and the malondialdehyde（MDA）content finally increased by 48.78%-91.79%when grown under 20μM Cu,7.5μM Cd,or 50μM Pb;（3）superoxide dismutase（SOD）activity decreased by 6.95%-15.79%and catalase（CAT）activity decreased by 22.09%-72.94%;peroxidase（POD）activity increased by 102.31%-345.73%when grow under 30μM Cu,7.5μM Cd and 50μM Pb,while POD activity increased by 89.39%-227.32%when grown under 20μM Cu,7.5μM Cd and 50μM Pb;as the continuous increased of concentration of heavy metals,the POD and APX activities were observed to decrease.2.Based on non-sterile S.polyrrhiza,the effects of plant growth regulators,including salicylic acid（SA）,gibberellin（GA）,abscisic acid（ABA）and paclobutrazol（PP333）,on the growth and metabolism of S.polyrrhiza when responding to Cd（7.5μM）stress were investigated.Under Cd stress,the contents of photosynthetic pigments and soluble protein content significantly decreased,the contents of MDA,O₂^-,hydrogen peroxide（H₂O₂）and proline（Pro）that accumulated in cells significantly increased,the antioxidant enzymes including SOD and CAT activities decreased while the POD and APX activities all increased,and glutathione（GSH）and ascorbic acid（As A）increased.With exogenous application of SA,GA,ABA or PP333 to the cultivation of S.polyrrhiza,the photosynthetic pigment increased from 34.41%to 89.65%and the soluble protein content increased from 31.78%to 80.07%;at the same time,the concentrations of MDA,O₂^-,GSH and As A were found to have significant decreases,CAT activity increased,and POD activity decreased.In addition,ABA and PP333increased the content of Pro,and ABA further increased APX activity.According to the above results,SA,GA,ABA and PP333 could reduce the damage of Cd to S.polyrrhiza by controlling the dynamic balance of reactive oxygen species（ROS）and reduced the excessive accumulation of O₂^-in fronds to directly alleviate the oxidative stress of plants,inhibiting membrane lipid peroxidation,increasing the content of soluble protein,and maintaining the level of antioxidant;PP333 and ABA could mediate the cadmium tolerance of S.polyrrhiza through increasing the content of Pro,and ABA may also be involved in the mitigation of Cd-induced oxidative stress by enhancing the antioxidant system.3.Based on sterile S.polyrrhiza,the effects of environmental microorganisms cooperating with salicylic acid on the Cd toleranceof S.polyrrhiza were studied.As results showed,the cooperation between native environmental bacterial communities and salicylic acid（SA）increased the chlorophyll content of S.polyrrhiza and increased the relative abundance of Pseudomonadaceae,with value increased from 12.05%to 32.47%.With the cooperation of Pseudomonas sp.MB-1 with SA,the contents of MDA,POD,APX,GSH,As A and Pro that induced by the presence of Cd were significantly reduced.In comparison with the growth that application of SA or Pseudomonas sp.MB-1,the combination of Pseudomonas sp.MB-1 with SA contributed to a significant improvement,with the chlorophyll content increased by 70.90%.Moreover,the combination of SA and Pseudomonas sp.MB-1 also increased the flavonoid content of S.polyrrhiza,which level increase by 29.65%.In addition,the combination of Pseudomonas sp.MB-1 with GA,ABA and PP333 contributed to the chlorophyll content increases by 53.21%,12.50%and 30.38%,respectively.4.The underlying mechanism of the cooperation of Pseudomonas sp.MB-1 with SA to enhance the Cd tolerance of S.polyrrhiza was studied by using comparative transcriptomic analysis.As results showed,with the application of Pseudomonas sp.MB-1 and SA,the expression levels of genes related to flavonoids synthesis（CHS3,CHI,ANS,ANR and LAR）,photosynthesis pathway and chlorophyll synthesis（CHLH,CRD1 and POR）were up-regulated,while the expressions of chlorophyll degradation related genes（PPD,PAO,SGR and NOL）were down-regulated.In accordance with physiological studies,it was found that the cooperation could increase the intracellular flavonoid content of S.polyrrhiza.In conclusion,the cooperation between Pseudomonas sp.MB-1 with SA can directly remove reactive oxygen species by increasing the flavonoid content of S.polyrrhiza,which was mainly responsible for the maintaince of chlorophyll content,enhancing photosynthesis and then the Cd tolerance.