Mechanisms Of Vacuolar Proton Pump In Tolerance Of Aluminum Toxicity In Arabidopsis Thaliana

Aluminum is a key factor for crop damage by acid soil.When human activity and plant root activity lead to a pH below 5.5,the aluminum in the soil is released from unsoluble aluminum compounds to form free aluminum with toxicity.In extracellular apart,aluminum can chelate cell wall to make cell wall elastic loss;Inside the cell,aluminum breaks down the structure of large molecules such as proteins and nucleic acids.To response to aluminum toxicity,plants have evolved both extracellular and intracellular detoxification mechanisms:the former mainly secreted organic acids,especially free aluminum outside malic acid chelation,while the latter incorporated.These two mechanisms require proton gradients formed by proton pumps in the plasma membrane and vacuolar membranes,respectively.The genome of the model plant Arabidopsis thaliana has 11 AHAs members,while the vacuolar membrane proton pump is divided into vacuolar proton ATP enzyme(V-ATPase)and vacuolar proton pyrophosphatase(PPase).VHA-a2 and VHA-a3 subunits of V-ATPase and AVP1 of v-ppase control the growth and development of plants and the stress response.However,the functional mechanism of these vacuolar proton pumps in response to external aluminum stress in plants remains largely unkown.This study used plant molecular genetics,plant morphology,fluorescence methods of chemistry,analytical chemistry,finding that VHA-a2 and VHA-a3 are key proton pumps to adapted to aluminum toxicity environment of acidification in A.thaliana,and revealing that A.thaliana can coordinate the excretion of intracellular organic acid and protons by suppressing VHA-a2 and VHA-a3 activity,so that detoxification mechanism of extracellular aluminum can be achieved by controlling plasma membrane proton pump and organic acid transporter by vacuolar proton pump.The main research contents are presented as follows1.VHA-A2 and VHA-A3 are key proton pumps in Arabidopsis thalianaFirstly,wild type(WT,Col-0)and vacuolar proton pump VHA-a2 and VHA-a3 double mutants(vha-a2 a3)in Colombian background were used to find that under acidic conditions,compared with WT,the double mutants showed the growth characteristics in stress tolerance,with longer root length and fresh weight,and were sensitive to other metals such as calcium,manganese and zinc.However,vha-a1 and avp1 of vha-a2 or vha-a3 monomutant with opposite golgi network did not show an resistance.Furthermore,the vha-a2 a3 avp1 mutant exhibited a similar aluminum growth phenotype to that of vha-a2 a3.It was found that vha-a2 a3 was only in acidic pH<5.4show resistance to aluminum.In addition,the specific Al3+ tolerance phenotype of vha-a2 a3 was further confirmed by the treatment of different types of aluminum ion compounds(AlCl3 and Al2(SO4)3)and different concentrations of aluminum ions.Through quantitative PCR and vacuolar proton pump activity analysis,it was found that aluminum treatment reduced the expression and protein activity of vha-a2 and vha-a3 genes.These results indicated that vha-a2 and vha-a3 functionally redundant regulation of A.thaliana in response to aluminum toxicity.2.Double mutation of vha-a2 and vha-a3 reduced absorption of aluminum in rootsThe roots of plants,especially the apices,are the targets of aluminum toxicity.The content and distribution of WT and vha-a2 a3 root tip aluminum were analyzed by using the aluminum ions staining reagent hematoxylin and fluorescent reagent morin(Aluminum Ionophore I)under the condition of no Al3+.The results showed that compared with WT,the root tip meristem of vha-a2 a3 had no significant distribution of sappanine staining,and the fluorescence intensity of mulberry was weak.Due to the specific distribution of sappanine and mulberry pigment combining with the aluminum in cell wall and cytoplasm,the cell wall and cell contents of vha-a2 a3 apical cell were lower.To further confirm the results,roots of WT and vha-a2 a3 treated by 300 ?M AlCl3 for 72 hours were detected using ICP-MS,respectively,and then aluminum content of vha-a2 a3 was found to be 60%of WT.3.The plasma membrane proton pump is involved in the aluminum resistance of the vha-a2 a3 mutantThrough the analysis of V-ATPase and V-PPase of A.thaliana and plasma membrane proton pump activity,it was found that V-ATPase and V-PPase activity were inhibited by Al3+ treatment,while plasma membrane proton pump activity was activated.Menwhile,qPCR was used to detect expression change in 8 PM H+-ATPase protein-coding genes(AHA1,AHA2,AHA3,AHA4,AHA7,AHA8,AHA10)between WT and vha-a2 a3 roots treated by 250 ?M Al3+ for 12 h.It was found that AHA1,AHA2 and AHA7 were up-regulated expression after Al3+ treatment.However,compared with WT,the expression levels of the three genes of vha-a2 a3 were further increased by 2,4 and 3 times,indicating that plasma membrane proton pumps,especially AHA2,may be involved in the aluminum tolerance of the vha-a2 a3 mutant.To confirm this,an analysis of the aha2 mutant plants showed that the mutant plants showed sensitivity to aluminum processing compared with WT,and the roots contained high levels of aluminum.To further confirm the role of activated plasma membrane proton pumps in the aluminum resistance of the vha-a2 a3 mutants,an external additive plasma membrane proton pump activator was used.fusicoccin(FC)and the inhibitor vanadate(VA),it was found that FC treatment lead to aluminum growth resistance similar to that of the vha-a2 a3 mutant,while VA eliminated the aluminum resistance of the vha-a2 a3 mutant.These results indicated that A.thaliana inhibits vacuolar proton pump V-ATPase to further activate plasma membrane proton pump and responds to the aluminum stress in the environment.The plasma membrane proton pump discharges the protons in the cell and establishes the proton gradient on both sides of the plasma membrane.To analyze the effect of activated plasma membrane proton pump on proton emission,the acidification of the extractoplasmic body was observed under a confocal laser microscope using the extraplasmic plasmon fluorescence stain HPTS.Under normal conditions,the fluorescence intensity of extracellular of vha-a2 a3 was weaker than that of WT.Under the condition of aluminum treatment,the fluorescence of the mutant basically disappeared,indicating that the extracellular pH of vha-a2 a3 was lower than that of WT.It was found that vha-a2 a3 plants reduced more significantly the pH of the culture solution than WT.However,further the lowering pH of the culture condition did not promote the aluminum resistance of the WT and vha-a2 a3 plants.It can be seen that the proton efflux of the plasma membrane is a necessary condition for the resistance of the vha-a2 a3 mutant to aluminium.4.Excretion of organic acid contribute to the resistance of vha-a2 a3 mutant to aluminumUnder the condition of aluminum stress,the proton excretion of plasma membrane leads to the secretion of organic acids,especially malate and citrate..To investigate the possible role of organic acid secretion,the effects of aluminum treatment on the expression of malate transporter ALMT1 and citrate transporter MATE,as well as the secretion of malate and citrate.were analyzed.The results of q-PCR indicated that the expression of ALMT1 and MA TE in WT root was significantly up regulated by aluminum treatment,and the mutation of vha-a2 a3 further promoted the expression of these two genes.High performance liquid chromatography(HPLC)analysis showed that aluminum treatment WT roots,there were increased secretion content of 15.68 times malate and 8.42 times citrate.Compared with WT,while the secretion of malate and citrate.of vha-a2 a3 was further increased to 182%%and 118%.However,the malate and citrate in the root of vha-a2 a3 are similar to and lower than WT.Exogenous addition of malate and citrate resulted in the WT showing an aluminum resistant growth phenotype similar to that of vha-a2 a3.These results established the key role of malate and citrate secretion in the aluminum resistance of vha-a2 a3.malate and citrate.Plasma membrane proton pump inhibitor VA eliminated the secretion of malate and citrate promoted by aluminum treatment of WT and vha-a2 a3 roots.FC could not improve the aluminum resistance of almtl mutant,suggesting that the secretion of these organic acids depends on activation of plasma membrane proton pump.Furthermore,the organoacid transporters drug inhibitors NIF and A-9-C inhibit the aluminum resistance of vha-a2 a3,while the vacuolar proton pump inhibitor ConA can promote the secretion of WT malate and citrate and reduce the content of root aluminum,but has no effect on the almt1 mutant.These results showed that the secretion of al-induced organic acids was dependent on the decrease of vacuolar V-ATPase activity and activation of plasma membrane proton pump activity.5.The calcium signal of the vha-a2 a3 mutant was reduced by aluminum stressCalcium signaling is a component of plant in response to aluminum stress.ICP-MS analysis showed that aluminum treatment significantly reduced the calcium content of vha-a2 a3 root compared with WT.Under the condition of aluminum deficiency,compared with expressing calcium ion fluorescence vector YC3.6 in WT plants,the roots of vha-a2 a3 showed relatively weak fluorescence.After aluminum treatment,the calcium fluorescence of WT and vha-a2 a3 background was inhibited,and the calcium fluorescence intensity of the latter decreased faster than that of the former,indicating that calcium signal was positively correlated with the expressions of vha-a2 and vha-a3 under aluminum stress.In order to analyze whether the expression of aluminum inhibiting vha-a2 and vha-a3 was the result of calcium attenuation,we used the vacuolar calcium absorber transporters CAX1 and caxlcax3,and found that the mutant is sensitivity to aluminum toxicity.However,vacuolar calcium receptor CBL2 and CBL3 mutant cbl2 cbl3 showed the same aluminum response as vha-a2 a3,indicating that CBL2 and CBL3 were involved in the process of regulating the activity of aluminum inhibiting VHA-A2 and VHA-a3.In summary,plant cells use the vacuolar proton pump to store the metabolic organic acids in the vacuole,and under the aluminum stress,reduces the activity of the vacuolar proton pump,leading to the following results:1.Reduction of organic acids when it entried into vacuoles,which increase the content of organic acids in the cytoplasm and provide sufficient substrate for the transport of organic acids in the plasma membrane.2.Activate the plasma membrane proton pump,promote the proton gradient measured in the plasma membrane,and provide impetus for the efflux of organic acid anions;3.The activity of organic acid transporters was also activated.Under the condition of sufficient substrate and polarization of plasma membrane proton gradient,organic acid was efficiently transported to the extracellular,thus extracellular detoxification of aluminum is achieved and adapted to the aluminum stress.