Response Of Antioxidant System In Rice Seedlings To Exogenous Ca²⁺ And Acid Rain

Acid rain?AR?is a global environmental problem.The economic losses caused by acid rain pollution in agroforestry are immeasurable.Exploring effective ways to reduce acid rain damage to plants is imminent.Calcium ion(Ca²⁺)acts as the second messenger within the cell,triggering many important physiological and biochemical processes,and thus affecting the response and adaptation of plants to the external environment.In this paper,rice seedlings were treated with Ca²⁺and acid rain for 5 days?stress period?and recovered under control conditions for 5 days?recovery period?.To investigate the mechanism of exogenous Ca²⁺?5m M?and acid rain?pH 3.5,pH 2.5?on the growth,reactive active oxygen?ROS?content,and malondialdehyde?MDA?content,membrane permeability?MP?,antioxidant enzyme activity,antioxidant content,antioxidant isoenzyme pattern,and gene expression rice seedling leaves and roots during the stress and recovery stages.It provides new direction to find effective ways to control and reduce disasters to alleviate the damage of acid rain on plant growth.The results of the experiment are as follows:?1?The effect of exogenous Ca²⁺and acid rain on the growth of rice seedlings under acid rain stress is known as:Acid rain?pH 3.5,pH 2.5?increased the level of reactive oxygen species[superoxide anion?O₂?^-?,hydrogen peroxide?H₂O₂?]and membrane lipid peroxidation?MDA,plasma membrane permeability?of rice seedlings,thus inhibited the growth of the above-ground and below-ground rice seedlings.The degree of inhibition increased with the increase of acid rain intensity,the largest decline in growth index is leaf area and the inhibition of leaves was greater than that of roots.The addition of exogenous Ca²⁺reduced the content of reactive oxygen species?O₂?^-,H₂O₂?,reduced membrane lipid peroxidation,and reduced the inhibition of acid rain?pH 3.5,pH 2.5?stress on rice seedling growth,the effect on roots was better than that of leaves.During the recovery period,the growth recovery of rice seedlings supplemented with exogenous Ca²⁺was higher than that of rice seedlings treated with single acid rain.?2?The effect of exogenous Ca²⁺and acid rain on antioxidant enzyme activity and antioxidant content of rice under acid rain stress can be seen as follows:During stress period,the activities of superoxide dismutase?SOD?,catalase?CAT?,peroxidase?POD?,aseorbateperoxidase?APX?and dehydroascorbate reductase?DHAR?in leaves and roots of rice seedlings were increased under acid rain?pH 3.5?stress,and the activity of glutathione reductase?GR?and the ratios of ascorbic acid/dehydroascorbic acid?ASA/DHA?and glutathione/glutathiol?GSH/GSSG?were decreased.Acid rain?pH 2.5?increased the activity of SOD,CAT and DHAR in rice seedlings,and decreased POD,APX,GR activity and ASA/DHA,GSH/GSSG ratio.The activities of POD,APX,GR in rice seedlings under high-strength acid rain?pH 2.5?stress were reduced,and the ability to scavenge reactive oxygen species was reduced.It is one of the reasons that acid rain has a different effect on growth by acid rain intensity.Exogenous Ca²⁺could increase the activity of antioxidant enzymes in rice seedlings,promote the conversion of antioxidants from oxidized to reduced,and increase the ratio of ASA/DHA and GSH/GSSG.The ROS content is negatively correlated with APX,GR,ASA and GSH.The increase of APX and GR activity and the decrease of reducing antioxidant?ASA and GSH?content can reduce ROS content.During the recovery period,exogenous Ca²⁺increased antioxidant enzyme activity and antioxidant content in rice seedlings that still had active oxygen accumulation.Antioxidant enzyme activity and antioxidant content of rice seedlings were lower than that of roots,and the ability of leaves to remove active oxygen was not as good as that of roots,this is one of the reasons why roots are more resistant to acids than leaves.?3?The effect of exogenous Ca²⁺and acid rain on the isozyme patterns and gene expression of rice antioxidant enzymes under acid rain stress can be seen as follows:During the stress period,the antioxidant enzyme bands of rice seedlings were thickened under acid rain?pH 3.5,pH 2.5?,but the changes in brightness were not obvious;gene expression was upregulated.The addition of exogenous Ca²⁺caused antioxidase isoenzyme bands of rice seedlings thicker and brighter,and the up-regulation of gene expression was higher than that of the single acid rain treatment group.Acid rain can only increase the total enzyme activity by increasing the isozyme synthesis amount.Acid rain increases total enzyme activity by increasing isozyme synthesis,Adding exogenous Ca²⁺can not only synthesize more enzymes,but also activate inactive enzymes,thereby increasing the total activity of antioxidant enzymes.After principal component analysis,the relative expression levels of Cu/Zn-SOD1and Cu/Zn-SOD2,Cu/Zn-SOD3,CAT1,CAT3,POD1,APX1,APX2,APX4,DHAR1,GR1 and GR2 in rice seedlings under acid rain stress were significantly increased.The negative correlation between APX4,GR1 and GR2 and MDA content was the strongest.During the recovery period,rice seedlings with exogenous Ca²⁺had greater increase in enzyme content,enzyme activity,and relative gene expression compared with single acid rain-stressed rice seedlings,and had stronger self-recovery ability.In summary,exogenous 5 mM Ca²⁺increased antioxidant enzyme synthesis and activates non-expressed active antioxidant enzymes by increaing antioxidase gene expression?particularly APX4,GR1 and GR2?,simultaneously promoted the conversion of the oxidized antioxidant to the reduced form,thereby increased antioxidant enzyme activity?especially APX and GR?and the ratio of reduced/oxidized antioxidant,thereby increased rice seedlings'ability to scavenge reactive oxygen species,and reduce the degree of oxidative damage.This is one of the reasons why Ca²⁺alleviates the inhibition of acid rain on the growth of rice seedlings,thereby increasing the acid resistance of rice seedlings.The addition of exogenous5 mM Ca²⁺had better effect on rice seedling roots than leaves.Exogenous Ca²⁺can not only alleviate the acid rain damage to rice seedlings,but also increase the self-repairing ability of rice seedlings after stress relief,and promote self-repair of rice seedlings.This study can provide a theoretical basis for reducing acid rain damage or reducing agricultural economic losses.