Physiological And Molecular Mechanisms For Involvement Of Acid Phosphatases In Microbial Symbiosis And Phosphorus Utilization In Soybean

Acid red soil is a typical agricultural soil type in South China,which has strong phosphorus?P?fixation,thereby lead to high accumulative P in soils that crops hardly uptake.Therefore,it is urgent to improve crop P efficiency,and promote the utilization of accumulative P.Both symbiosis with beneficial microorganisms and secretion of acid phosphatases can improve P efficiency in crops,and promote utilization potentials of accumulative P in soils.Rhizobium and arbuscular mycorrhizal fungi?AMF?are important beneficial symbiotic bacteria.Purple acid phosphatase?PAP?is a kind of acid phosphatases which contribute significantly to P efficiency.In acid red soil,the reports were fewer about the effects of the inoculation with rhizobia on indigenous AMF,and P uptake and yield.At the molecular level,there are few reports about the possible function of nodule and mycorrhizal induced PAPs.On the basis of previous studies,in the present study,a monoculture/intercropping field experiment in acid red soils was carried out to study the effects of rhizobium inoculation on AM colonization,P uptake and yield of maize/soybean with two contrast soybean varieties with different P efficiency,and maize as plant materials under different P treatments and rhizobium inoculation conditions.At the same time,the soybean transgenic plants were employed to explore the involvement of noduleinduced purple acid phosphatase gene GmPAP21 in nodule P metabolism.In addition,a dual localization analysis system of mycorrhizal infection structures and GUS staining,and the protein heterologous expression systems were used to analysis the possible mechanism of involvement of mycorrhizal-induced purple acid phosphatase gene GmPAP33 in mycorrhizal symbiosis system.These results will help to explore the possible molecular and physiological mechanism of rhizobium and AMF involving crop P efficiency.The main results are as follows:1.Field experiment results showed that yield and biomass of soybean/maize did not reduced at the P application of 130 kg/hm² compared to the P application of 160 kg/hm²,and P application of 40 kg/hm² significantly increased the activities of rhizosphere acid phosphatases in both soybean and maize plants,suggesting that fertilizer P application can be reduced in the field.Soybean/maize intercropping system has obvious intercropping advantages,and intercropped soybean as a contributor promoted the growth of intercropped maize.2.The results from soil nutrients of root interaction zone in soybean/maize intercropping showed,rhizobium inoculation significantly increased the content of available P in the soils of root interaction zone under the P application of 160 kg/hm²,and significantly increased the content of available nitrogen?N?under the P application of 130 kg/hm².3.The number and dry weight of root nodules,and nitrogen and phosphorus contents in root nodules significantly increased with increasing P application and rhizobium inoculation compared with low P without rhizobium inoculation.However,rhizobium inoculation did not affect the contents of N/P and the activities of rhizosphere acid phosphatase in both soybean and maize plants.AM colonizations of soybean and maize were between 35%⁶0%.P application significantly influenced AM colonization of soybean,but had no effect on AM colonization of maize.4.Comparison of two soybean varieties with different P efficiency showed that yield,biomass,root surface area,and N and P contents in P-efficient soybean variety BX10 were significantly higher than P-inefficient soybean variety BD2 under the P applications of 130kg/hm² and 160 kg/hm² with different monoculture/intercropping mode and rhizobium inoculation treatment.N content and rhizosphere acid phosphatase activities of intercropped maize with BD2 was significantly higher than that with BX10 under the P application 130kg/hm² with rhizobium inoculation conditions.5.The results from GUS histochemical localization of the transgenic plants with nodule-induced purple acid phosphatase gene GmPAP21 promoter showed that N and P deficiencies had a significant effect on the expression localization of GmPAP21.Under non-inoculated conditions,GmPAP21 was induced by P starvation at LNLP and HNLP treatments compared with LNHP and HNHP treatments.GUS activity was mainly detected in the roots,leaves,stems,flowers,pods and seeds,and a strong signal was also detected in both root tips and whole root tissues.Conversely,transgenic lines in LNHP and HNHP displayed only slight GUS staining in stems,flowers,pods and seeds.Under rhizobium inoculation conditions,GUS driven by the GmPAP21 promoter was abundantly expressed in roots and nodules,followed by flowers,stems,leaves and seeds under LNLP conditions.GmPAP21 was also expressed in nodules under HNLP and LNHP conditions,but the staining was notably lower.No detectable GUS staining in nodules was observed in HNHP.6.Hydroponic experiments of the over-expressing GmPAP21 transgenic soybean plants showed that over-expressing GmPAP21 significantly reduced plant dry weight and contents of N and P,dry weight and N and P contents of root nodules compared with wild-type soybean plants under high P with rhizobium inoculation conditions.In contrast,there were no obvious differences between transgenic plants and wild-type plants under low P conditions.Combined with the results of GmPAP21 promoter analysis,these results indicate that GmPAP21 is a new P-starvation responsive purple acid phosphatase,which might involve in nodule P metabolism besides participating in P reutilization in plants.7.GUS histochemical localization of the transgenic plants with mycorrhizal-induced purple acid phosphatase gene GmPAP33 promoter was carried out using the dual localization analysis of mycorrhizal infection structure and GUS staining,and the results showed that GmPAP33 was mainly expressed in arbuscule-containing cortex cells.Subcellular localization of the transient expression in both tobacco leaf epidermal cells and Arabidopsis protoplast cells showed that GmPAP33 was mainly located in the plasma membrane.This indicates that GmPAP33 might be involved in P recycling and reutilization in mycorrhizal arbuscular membrane structures.8.Biochemistry functional analysis of GmPAP33 showed that the optimal pH of GmPAP33 was 4,the optimal temperature was 50?.The different cation or anion had a great influence on GmPAP33 enzyme activity.Bivalent cations Mg²⁺and Mn²⁺significantly stimulated enzyme activity of GmPAP33,while the other cations K⁺,Na⁺,Ba²⁺,Al³⁺and Zn²⁺,molybdate and EDTA inhibited enzyme activity of GmPAP33.GmPAP33 had wide substrate specificity.Substrate specificity analysis of GmPAP33 showed that the relative activities of the tested substrates were lower,indicating that phospholipids might be the optimum substrate for GmPAP33.Combining with the previous results,these results showed that GmPAP33 is a new mycorrhizal-induced purple acid phosphatase gene,which might be involved in hydrolysis and recycling of phospholipids in mycorrhizal arbuscular membrane structures.In summary,the reduction of P fertilizer application did not reduce the yield of soybean and maize.Soybean and maize intercropping system had intercropping advantage,which obviously improved P uptake and yield of maize.rhizobium inoculation improved nodule characteristics,but had no promotion to P uptake and yield of soybean and maize.This suggests that the potential of promoting P uptake by rhizobium inoculation yet need to explore in many ways in acid soils.GmPAP21 was involved in P metabolism of root nodules besides internal P reutilization in plants.Mycorrhizal induced GmPAP33 was possibly involved in hydrolysis and recycling of phospholipids in mycorrhizal arbuscular membrane structures.The results in this study provide a theoretical basis for increasing the utilization of accumulative P in soils and improving P efficiency in plants through beneficial microbial symbiosis.