The Characteristics And Rhizosphere Effects In Alleviating Al-toxicity Of Pinus Massoniana Root Exudation In Acid-aluminum Environment

Aluminum?Al?usually exists in soil with the form of silicate and has little effect on the growth of crops.But the soil pH become below 5.0,the soluble phytotoxic species,such as Al³⁺,Al?OH?²⁺and Al?OH?₂⁺will be dissolved in a large amount,while for most plants,even the micromolar treatment under 10?M Al³⁺could inhibit root growth quickly.The total area of acid soil in China is about 2.03×10⁷ hm²,soil acidification,which leads to soil active aluminum stress,was considered to be an important factor of forest degradation.The effect of aluminum on plant growth is closely related to its chemical forms in soil solution.The high-activity aluminum such as Al³⁺,Al?OH?²⁺and Al?OH?₂⁺in soil are the most important factors causing aluminum damage in plants.But the root exudation could alter the form of aluminum in soil,change the soil microecological environment,and reduce the toxicity of soil aluminum to plants.Although a great deal of research has been done on aluminum toxicity and its mechanism of aluminum tolerance in plants,these studies are mainly focused on herbaceous plants,and whether woody plants have the same aluminum tolerance as herbaceous plants has not yet been determined.Pinus massoniana is a typical representative of woody plants in acid soil,and it is also the main tree species for afforestation and industrial raw materials in southern China,which plays an important role in forestry production.Recently,the phenomenon of large area growth decline of Pinus massoniana has appeared,and the study suggested,by Houtian Liu et al.,the acid-aluminum hazard was one of the main causes of the decline of Pinus massoniana.Therefore,how to alleviate the toxic effect of aluminum on Pinus massoniana has become a key problem for the sustainable development of Pinus massoniana industry in southern China.In order to investigate the aluminum tolerance of Pinus massoniana and the mechanism of Aluminum-toxic mitigation,different aluminum tolerant Pinus massoniana seedlings were used as experimental materials in this study.The exudation characteristics of roots and its effects of root exudation on aluminum morphology and soil micro-ecology in soil were studied by soil culture method,and the regulation and mechanism of organic acids,secreted by root system,on root system and root tip of Pinus massoniana were studied by hydroponic method.The regulation of exogenous organic acids on the content of aluminum in Pinus massoniana,the migration coefficient of aluminum and the content of apical aluminum were also discussed in this study.The aim of this study was to clarify the effect of root exudation on the microecology of aluminized soil and the ability of aluminum toxicity of Pinus massoniana,and to reveal the mechanism of aluminum tolerance of Pinus massoniana.The main results are as follows:1.The exudation characteristics of Pinus massoniana root system under aluminum acid environment were clarified.Aluminum-tolerant Pinus massoniana?FJ5?could secrete more organic acids than aluminum-sensitive?GD20?under aluminum stress,which may be one of the main mechanisms of aluminum tolerance.The ability of acid phosphatase in root exudation of Pinus massoniana was significantly increased under aluminum stress,and the ability of tolerance to aluminum was higher than that of aluminum sensitive type in root exudation of Pinus massoniana.It was suggested that exogenous Al³⁺could promote the secretion of acid phosphatase in roots,which may be one of the mechanisms of aluminum tolerance of Al-tolerant Pinus massoniana.Although the ability of acid total phenol in root exudation of Pinus massoniana and Al-sensitive type was significantly increased under aluminum stress,the Al-sensitive type was higher than the Al-tolerant,indicating that the mechanisms of aluminum tolerance of Pinus massoniana was not caused by exogenous Al³⁺to stimulate the secretion of total phenol in root system of Pinus massoniana.Although the content of protein and amino acids in root exudation of Al-tolerant Pinus massoniana was always higher than that of Al-sensitive type,the effect of exogenous Al³⁺on protein and amino acids in root exudation of FJ5 and GD20 was not obvious.The results showed that the change of protein and amino acid in root exudation of Pinus massoniana was also not one of the mechanisms.2.The regulation of root exudation of Pinus massoniana on the form of aluminum under the acid-aluminum environment was revealed.This transformation could reduce the content of active aluminum in the soil,thus reducing the toxicity of aluminum to plants,which may be one of the main reasons of the relief of aluminum stress by the root exudation of Pinus massoniana.The content of organic complex aluminum in GD20 soil was lower than that of FJ5,while the result of acid-soluble inorganic aluminum was exactly opposite,indicating that FJ5 root exudation were more likely to synthesize active aluminum ions in soil into stable organic complex aluminum.The contents of organic complex aluminum and aluminum humate in the soil treated by rhizosphere soil and exogenous root exudation of Pinus massoniana were higher than non-rhizosphere soil,while the contents of monomeric hydroxy aluminum[Al?OH?²⁺,Al?OH?²⁺],exchangeable aluminum[Al³⁺]and acid-soluble inorganic aluminum[Al?OH?³⁺]were lower than non-rhizosphere soil,indicating that the root exudation of Pinus massoniana could make active aluminum transforme into the form of humate aluminum and organic complex aluminum.The content of organic complex aluminum in GD20 soil was lower than that of FJ5,while the content of acid-soluble inorganic aluminum was higher than that of FJ5,which indicated that root exudation of FJ5 were more likely to synthesize into stable organic complexation aluminum with the active aluminum ions in the soil,which indicated that FJ5 root exudation was more capable of alleviating soil aluminum toxicity.3.The ability of root exudation of Pinus massoniana to regulate the microecology of aluminum-acid soil was determined.The PLAF content of soil microbes,such as soil bacteria,Gram-positive bacteria,Gram-negative bacteria,fungi,AM fungi,actinomycetes and protists,was deseased under A1³⁺stress.The root exudation of Pinus massoniana was beneficial to the growth and propagation of soil microorganisms,The content of microbial PLAF in the rhizosphere soil and the soil treated by exogenous root exudation were significantly higher than that of non-rhizosphere soil,the order was rhizosphere soil>soil treated by exogenous root exudation>non-rhizosphere soil,The content of the microbial PLAF in FJ5 rhizosphere soil and the soil treated by root exudation were higher than that of GD20;The activities of urease,phosphatase,invertase,polyphenol oxidase,dehydrogenase and hydrogen peroxide decreased significantly in Pinus massoniana soil under aluminum stress,but the decrease was the largest in non-rhizosphere soil and the least in rhizosphere.The root exudation could increase soil enzyme activity under Al³⁺stress.The order of soil enzyme activity was rhizosphere soil>soil treated by exogenous root exudation>non-rhizosphere soil.And the basic respiration rate of soil was significantly reduced under Al³⁺stress.The root exudation of Pinus massoniana could make the microbe more active in soil environment and release more CO₂,and the basic respiration rate of rhizosphere soil and the soil treated by exogenous root exudation were also significantly higher than that of non-rhizosphere soil.4.The effect of organic acids on the root system of Pinus massoniana was clarified.The growth of roots was inhibited,root activity was significantly decreased,and the injury of cell membrane was also significantly increased in two species of Pinus massoniana under Al³⁺stress.This stress could stimulate the stress response of Pinus massoniana and increase the content of osmotic regulators such as soluble sugar and soluble protein,and the activities of antioxidant enzymes such as POD,SOD and CAT increased significantly.The inhibitory effect of Al³⁺on Al-sensitive type was stronger than that on Al-resistant.The suitable concentration of organic acid could alleviate the inhibition of Al³⁺stress on the root growth of Pinus massoniana and enhance the root activity.The content of osmotic regulators,such as soluble sugar,soluble protein and Pro,and MDA gradually became normal.The activities of antioxidant enzymes,such as POD,SOD and CAT,continued to increase.The optimum alleviating concentration of oxalic acid,citric acid and malic acid respectively were0.2mmol·L^-1,0.02mmol·L^-1,and 0.01mmol·L^-1.5.The regulation mechanism of organic acids on the root tip of Pinus massoniana was revealed.The total sugar in cellulose I of the cell wall of two Pinus massoniana under Al³⁺stress was significantly accumulated,while the total sugar in cellulose II did not change significantly,indicating that the main binding site of aluminum in the root tip of Pinus massoniana may be in the hemicellulose I of the cell wall.There was no significant difference in total sugar content in the root tip cell wall hemicellulose of two species Pinus massoniana,which indicated that it may not be the dominant factor of aluminum tolerance in Pinus massoniana.The accumulation of uronic acid in the cell wall of root tip of GD20 was significant under Al³⁺stress,but no significant change in FJ5,indicating that the uronic acid and aluminum chelate in hemicellulose of root tip were the expression of Al-sensitive type Pinus massoniana rather than Al-resistance.The exogenous organic acids could reduce the combination of pectin and hemicellulose with aluminum in the cell wall of GD20 root,and the effect of oxalic acid was better than that of citric acid on sensitive GD20.Al³⁺stress could increase the contents of H₂O₂ and MDA in the root tip of Pinus massoniana,which was toxic to Pinus massoniana.And this stress also stimulated the stress response of Pinus massoniana to make the activities of POD,APX and GR in the root tip increased,then eliminated too much peroxide to maintain the balance of the system.IDH and GO may be the main metabolic enzymes affecting aluminum tolerance of Pinus massoniana.And the organic acids could effectively reduce the contents of H₂O₂ and MDA in root tip,and reduce the activities of antioxidant enzymes and related metabolic enzymes produced by Al³⁺stress,thus restoring the balance system of Pinus massoniana system.6.The regulation of organic acids on aluminum absorption and transport of Pinus massoniana was clarified.Aluminum stress could increase aluminum content in root tip and plant of Pinus massoniana,and the content of aluminum in all parts of GD20 was higher than that in FJ5.It indicated that the aluminum repellent ability of plants was also a kind of mechanism of aluminum tolerance of Pinus massoniana.The aluminum migration coefficient of FJ5 was lower than GD20,and the lower aluminum migration coefficient may be the main reason for its aluminum tolerance of FJ5.The suitable concentration of exogenous organic acids could reduce the aluminum content and the aluminum transport coefficient of Pinus massoniana,so as to alleviate aluminum toxicity.The optimum alleviating concentration of oxalic acid,citric acid and malic acid respectively were0.2mmol·L^-1,0.02mmol·L^-1,and 0.01mmol·L^-1,and the mitigation effect to GD20 was better than FJ5.Based on the results of the above aspects,the study has the following conclusions:?1?The aluminum stress could inhibit the root growth of Pinus massoniana,reduce the root activity and break the balance of physiological metabolism of root system.?2?The secretion of organic acids and acid phosphatase in roots,induced by aluminum,was the main mechanism of aluminum tolerance of Pinus massoniana.?3?The uronic acid and aluminum chelation in hemicellulose of root tip were the expression of Al-sensitive type Pinus massoniana rather than Al-resistance.?4?The root exudation changed the form of active aluminum in rhizosphere soil,which was one of the main reasons to alleviate aluminum stress of Pinus massoniana root exudation.?5?The root exudation of Pinus massoniana under aluminum stress could alleviate the toxcity of aluminum by changing the soil microecological characteristics.?6?The main mechanisms to alleviate aluminum toxicity of Pinus massoniana were that organic acid could effectively regulate the changes of root physiological characteristics,reduce the absorption of aluminum from roots and decrease the migration coefficient of aluminum in Pinus massoniana.