Regulation Of Calcium On Activity And Function Of Plasma Membrane Proton Pump Of Soybean At Different Growth Stages Under Acid Rain Stress

Acid rain?AR_?is a worldwide environmental problem,leading to huge economic losses on agriculture and forestry.Hence,finding ways for improving resistance of plants to AR is significant to alleviate the negative effects caused by AR_.Calcium(Ca²⁺),as one of the essential nutrients and the second messenger of signal transduction in plants,has became a research focus on plant tolerance to abiotic stresses.In this paper,we study effects of exogenous Ca²⁺(Ca²⁺1/ 5 mM,Ca²⁺2/ 10 mM)on growth,chlorophyll content,root permeability,activity of plasma membrane proton pump,nutrient elements and relative expression of GmPHA1 at transcriptional level in soybean during three different growth stages exposed to AR_{AR1/ pH 4.5,AR2/ pH 3.5}by advanced technologies such as high performance liquid chromatograph,inductively coupled plasma and Real-time PCR.The main results are as follows:?1?Effects of exogenous Ca²⁺ on growth and resistance of soybean at three growth stages exposed to AR were investigated.The results show that AR₁ or AR₂ decreased the biomass and chlorophyll content,and increased root permeability of soybean,the degree of effects: AR₁<AR₂,seedling stage >filling stage >podding stage;AR₁ + Ca²⁺1 or AR₁ + Ca²⁺2 increased the biomass and chlorophyll content of soybean,as well as decreased the root permeability compared with those of AR₁ treatment group;The biomass and chlorophyll content of soybean treated with AR₂ + Ca²⁺1 or AR₂ + Ca²⁺2 were still lower than those of the control,but higher than those treated with AR₂,while the root permeability was still higher than the control,but significantly lower than that treated with AR₂.Additionally,the alleviated effects of AR₂ + Ca²⁺1 were better than those treated with AR₂ + Ca²⁺2.The above results illustrate that the application of Ca²⁺ can alleviate the inhibition of AR on soybean growth,promote chlorophyll synthesis and maintain the integrity of root plasma membrane,but the alleviated effects were limited by the intensity of AR_,and also depended on the concentration of exogenous Ca²⁺.The degree of alleviated effects on growth and resistance of soybean: seedling stage >filling stage >podding stage.After a 5-day recovery,the biomass,chlorophyll content and root permeability of soybean treated with AR₁ + Ca²⁺1 or AR₁ + Ca²⁺2 were close to the level of the control,showing that the alleviated effects of AR₁ + Ca²⁺1 or AR₁ + Ca²⁺2 were persistent,not temporary;The biomass and chlorophyll content of soybean treated with AR₂ + Ca²⁺1 or AR₂ + Ca²⁺2 treatment were still lower than those of the control,but higher than those of AR₂,and root permeability was lower than that of AR₂ treatment group,indicating that Ca²⁺ accelerated the recovery period of growth,chlorophyll content and root permeability of soybean,however,the recovery degree was limited by the acidity of AR_.?2?Effect of exogenous Ca²⁺ on activity of plasma membrane proton pump and nutrients contents in soybean of three growth stages exposed to AR were investigated.The results show that AR₁ increased the proton pump activity of soybean,the degree of the increase: seedling stage< filling stage< podding stage.However,AR₂ inhibited the proton pump activity.The degree of the inhibition: seedling stage >filling stage >podding stage;the contents of NO3-,P,K,Na,Mg and Zn decreased in both AR₁ and AR₂ treatment groups,the degree of the decrease: AR₁<AR₂.In the AR₁ + Ca²⁺1 treatment group,the activities of plasma membrane proton pump and calcium pump of soybean root were close to the level of the control,and lower than those of AR₁ treatment group.Besides,the contents of NO3-,P,Mg and Zn in the treatment was close to those in the control treatment,while the K content was still lower than that of the control,but significantly higher than that of AR₁ treatment group.In the AR₂ + Ca²⁺1 treatment group,the activities of plasma membrane proton pump and calcium pump of soybean root were lower than those of the control,but significantly higher than those of AR₂ treatment group.In addition,the contents of NO3-,P,Mg and Zn in this treatment were lower than those of the control,but higher than those of AR₂ treatment group.These results show that the exogenous Ca²⁺ could accelerate the hydrolysis of ATP and benefit to the absorption of NO3-,P,K,Mg and Zn by regulating the activity of plasma membrane proton pump,but the regulation degree of Ca²⁺ was limited by strong acid.The alleviating of the exogenous Ca²⁺: podding stage > filling stage > seedling stage.The variation of Fe,Mn,Cu and Mo elements were different,mainly attributed by the differences in growth rhythm and resistance of soybean at different growth stages.After 5-days recovery,the activity of proton pump in AR₁ treatment group was still higher than that of the control,while the activity of proton pump in AR₂ treatment group was lower.However,the proton pump activity in soybean treated with AR₁ + Ca²⁺1 had no difference from that of the control.Besides,the contents of NO3-,P,K,and Zn were close to those of the control.Those results illustrate that the exogenous Ca²⁺ could maintain the normal absorption of NO3-,P,K,and Zn by regulating the activity of plasma membrane proton pump,benefiting to the recovery of growth of soybean exposed to AR at pH 4.5.In AR₂ + Ca²⁺1 treatment group,the activity of proton pump was still lower than that of the control,but significantly higher than that of AR₂ treatment group.The contents of NO3-,P,K,Na,Mg and Zn were also higher than those treated with AR₂.Those indicated that exogenous Ca²⁺ accelerated recovery process of the activity of plasma membrane proton pump in AR₂ + Ca²⁺1 treatment group,which is benifical to the absorption of NO3-,P,K,Na,Mg and Zn,and the recovery of growth,chlorophyll content and stability of root membrane.However,the recovery degree of soybean in AR₂ + Ca²⁺1 treatment group was limited by strong acid.?3?Effect of exogenous Ca²⁺ on relative expression of GmPHA1 at transcriptional level of soybean at three growth stages exposed to AR was investigated.AR₁ increased the relative expression of GmPHA1?seedling stage 119%,podding stage 186%,filling stage 153%?,while AR₂ decreased the relative expression of GmPHA1?seedling stage 81%,podding stage 67%,filling stage 71%?.In AR₁+Ca²⁺1 treatment group,the relative expression of GmPHA1 was close to the level of the control,benefiting to maintain the activity of plasma membrane proton pump maintained at the normal level;The relative expression of GmPHA1 in soybean root of AR_<sup>2+Ca²⁺1 treatment group was higher than that of AR₂ treatment group increased,the rising range: podding stage >filling stage >seedling stage,being consistent with the variety regularity of the activity of plasma membrane proton pump.Those results indicate that the regulation of Ca²⁺ on the transcriptional level of GmPHA1 is one of the mechanisms that regulate the activity of proton pump under AR stress.After a 5-day recovery,the relative expression of GmPHA1 at transcriptional level of soybean in AR₁ or AR₂ treatment group was still higher than that of the control whereas the activity of plasma membrane proton pump was still lower than that of the control,indicating that the plasma membrane proton pump is also probably affected by other factors.The relative expression of GmPHA1 at transcriptional level in soybean treated with AR₁ + Ca²⁺1 was higher than that of the control,but lower than that treated with AR₁?seedling stage 125%,podding stage 206%,filling stage 123%?,benefiting to maintain the activity of proton pump at the normal level;For the AR₂ + Ca²⁺1 treatment group,the expression of GmPHA1 was higher than that treated with AR₂,and the plasma membrane proton pump activity is also higher than that treated with AR₂,indicating that Ca²⁺ regulated the GmPHA1 at transcription level to accelerate the recovery process of plasma membrane proton pump treated with AR₂ + Ca²⁺1 during the recovery period.In conclusion,an optimum concentration of exogenous Ca²⁺ can alleviate the inhibition of acid rain on soybean growth,promote the synthesis of chlorophyll and stability of root plasma membrane of soybean exposed to AR stress,and maintain the normal absorption of nutrient by regulating activities of soybean root plasma membrane proton pump to improve plant resistance to AR stress,but the alleviated degree of Ca²⁺ was limited by the strong AR_,and the regulation effect of Ca²⁺ on seedling stage of soybean is the best.