Testing the polar auxin transport model with a selective plasma membrane H+-ATPase inhibitor

Auxin is unique among plant hormones in that its function requires polarized transport across plant cells. A chemiosmotic model was proposed to explain how polar auxin transport is derived by the H⁺ gradient across the plasma membrane (PM) established by PM H⁺-adenosine triphosphatases (ATPases). However, a classical genetic approach by mutations in PM H⁺-ATPase members did not result in the ablation of polar auxin distribution, possibly due to functional redundancy in this gene family. To confirm the crucial role of PM H⁺-ATPases in the polar auxin transport model, we employed a chemical genetic approach. Through a chemical screen, we identified protonstatin-1 (PS-1), a selective small-molecule inhibitor of PM H⁺-ATPase activity that inhibits auxin transport. Assays with transgenic plants and yeast strains showed that the activity of PM H⁺-ATPases affects auxin uptake as well as acropetal and basipetal polar auxin transport. We propose that PS-1 can be used as a tool to interrogate the function of PM H⁺-ATPases. Our results support the chemiosmotic model in which PM H⁺-ATPase itself plays a fundamental role in polar auxin transport.     

水杨酸诱发的丹参悬浮培养细胞内H2O2迸发与其培养基碱化的关系

水杨酸(salicylic acid,SA)处理可诱导丹参悬浮培养细胞内H2O2产生及其培养基碱化。利用NADPH氧化酶抑制剂咪唑(imidazole,IMD)、H2O2淬灭剂二甲基硫脲(dimethylthiourea,DMTU)、质膜H+-ATPase抑制剂钒酸钠(Na3VO4)及激活剂壳梭孢菌素(fusicoccin,FC)处理丹参悬浮培养细胞,探讨SA诱导的H2O2迸发与培养基碱化之间的关系。结果表明,H2O2可促发培养基碱化,IMD和DMTU抑制SA诱发的培养基碱化,说明H2O2参与SA诱发的培养基碱化过程;SA抑制质膜H+-ATPase活性,Na3VO4引发培养基碱化并使H2O2迸发时间提前,FC处理逆转了SA诱导的培养基碱化及H2O2迸发,说明质膜H+-ATPase调控培养基pH值变化,培养基碱化促进了H2O2产生。因此,丹参悬浮培养细胞内H2O2水平与其培养基碱化程度之间相互关联、共同作用,协同响应SA的诱导。     

Phosphorylation-dephosphorylation of membrane proteins controls the microsomal H-ATPase activity of corn roots

The Mg²⁺-dependent H⁺-ATPase activity of a sealed microsomal vesicle fraction isolated from corn (Zea mays L.) roots appears to be controlled by a phosphorylation-dephosphorylation cycle. Phosphorylation of the microsomal fraction is carried out by a Ca²⁺/calmodulin (CaM)-stimulated process. The H⁺-ATPase activity decreases with increasing phosphorylation of the membranes and becomes only slightly uncoupled by ionophores and less inhibited by dicyclohexylcarbodiimide (DCCD), diethylstilbestrol (DES), NO₃⁻ and vanadate. The inhibitory effect of phosphorylation is greater on the NO₃⁻-sensitive H⁺-ATPase activity than on the vanadate-sensitive activity. Restoration of H⁺-ATPase activity is achieved by allowing the phosphorylated membranes to dephosphorylate either in the absence or presence of exogenous alkaline phosphatase. Moreover, the presence of fluphenazine during the Ca²⁺/CaM-stimulated treatment inhibits membrane phosphorylation and protects the H⁺-ATPase activity from inhibition.     

拟南芥AtJ3通过核质转运调控植物质膜H~+-ATPase活性与ABA响应

拟南芥AtJ3(Arabidopsis thaliana Dna J homolog 3)为一蛋白分子伴侣,在植物体内可通过与PKS5(SOS2-like protein kinase 5)蛋白激酶形成复合物来抑制PKS5的活性;同时AtJ3-PKS5复合物可对质膜上H~+-ATPase质子转运活性进行正向调节,并参与对外源ABA的响应。为揭示AtJ3-PKS5复合物参与质膜H~+-ATPase活性调节及对外源ABA响应中的作用,本研究以拟南芥AtJ3、PKS5不同突变体为材料,在盐及ABA共同处理下对AtJ3-PKS5复合物的功能及作用机制进行了探讨。结果显示,在2种因素共同处理下,AtJ3-PKS5复合物可同时对处理因素进行响应。即AtJ3-PKS5复合物可对质膜上H~+-ATPase质子转运活性进行调节,并使细胞内p H值发生变化,同时还可诱导ABI5下游ABA响应基因的表达;外源ABA可引起AtJ3从细胞核向细胞质的转运,从而增强了AtJ3对H~+-ATPase活性的调节。说明AtJ3-PKS5复合物在对H~+-ATPase活性调节及对外源ABA响应的交互代谢途径中起着关键调节子的作用。     

A critical review on natural compounds interacting with the plant plasma membrane H+-ATPase and their potential as biologicals in agriculture

The plant plasma membrane (PM) H⁺-ATPase is an essential enzyme controlling plant growth and development. It is an important factor in response to abiotic and biotic stresses and is subject to tight regulation. We are in demand for new sustainable natural growth regulators and as a key enzyme for regulation of transport into the plant cell the PM H⁺-ATPase is a potential target for these. In this review, we have evaluated the known non-protein natural compounds with regulatory effects on the PM H⁺-ATPase, focusing on their mechanism of action and their potential as biologicals/growth regulators in plant production of future sustainable agriculture.     

Efficient iron plaque formation on tea(Camellia sinensis) roots contributes to acidic stress tolerance

Tea plants grow in acidic soil, but to date, their intrinsic mechanisms of acidic stress tolerance have not been elucidated. Here, we assessed the tea plant response to growth on NHt4 nutrient media having different p H and iron levels. When grown in standard NHt4 nutrient solution(iron insufficient, 0.35 mg Là1 Fe2t), tea roots exhibited significantly lower nitrogen accumulation, plasma membrane Ht-ATPase activity, and protein levels; net Htefflux was lower at pH 4.0 and 5.0 than at pH 6.0. Addition of30 mg Là1 Fe2t(iron sufficient, mimicking normal soil Fe2tconcentrations) to the NHt4 nutrient solution led to more efficient iron plaque formation on roots and increased root plasma membrane Ht-ATPase levels and activities at p H 4.0 eland 5.0, compared to the p H 6.0 condition. Furthermore,plants grown at pH 4.0 and 5.0, with sufficient iron,exhibited significantly higher nitrogen accumulation than those grown at pH 6.0. Together, these results support the hypothesis that efficient iron plaque formation, on tea roots, is important for acidic stress tolerance. Furthermore,our findings establish that efficient iron plaque formation is linked to increased levels and activities of the tea root plasma membrane Ht-ATPase, under low pH conditions.     

硫化氢对低温胁迫下甜樱桃柱头和子房线粒体功能的影响

为探索低温胁迫下外源硫化氢(H₂S)对甜樱桃花的柱头和子房线粒体功能的影响,本研究以甜樱桃品种‘早大果’花枝为试材,在-2 ℃低温下喷施0.05 mmol·L^-1硫氢化钠(NaHS,H₂S供体)和15 μmmol·L^-1 次牛磺酸(HT、H₂S清除剂),测定柱头和子房线粒体中活性氧、抗氧化酶和线粒体膜通透性转换孔(MPTP)开放程度、膜流动性、膜电位和细胞色素(Cyt c/a)比值变化。结果表明: 低温胁迫导致线粒体内过氧化氢(H₂O₂)和丙二醛(MDA)含量显著增加,线粒体MPTP明显增大,膜流动性降低,膜电位和线粒体Cyt c/a吸光度比值、膜H⁺-ATPase活性显著下降,线粒体结构受到损伤。低温胁迫下,外施0.05 mmol·L^-1 NaHS可显著降低低温胁迫下柱头和子房线粒体H₂O₂和MDA含量,在较长时间内维持较高的超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性,减小线粒体MPTP开放程度,增强线粒体膜流动性,提高线粒体膜电位、Cyt c/a值和膜H⁺-ATPase活性;NaHS清除剂HT则抵消NaHS对上述参数的影响。综上所述,外源H₂S可以提高低温胁迫下甜樱桃柱头和子房线粒体抗氧化酶活性,减少H₂O₂和MDA积累,提高膜H⁺-ATPase活性,稳定线粒体膜结构和功能,进而缓解低温胁迫对花器官的伤害。     

一氧化氮调节盐胁迫下小麦幼苗根部质膜H+-ATPase和焦磷酸酶活性提高耐盐性

采用外源一氧化氮(NO)供体硝普钠(SNP)研究了NO对盐胁迫下小麦(Triticum aestivum L.)幼苗耐盐性的影响.结果表明,0.1 mmol/L SNP处理显著缓解了1 50 mmol/L NaCl胁迫对小麦幼苗生长的抑制效应,包括水分丧失以及叶绿素降解,从而提高了小麦幼苗的耐盐性.进一步结合1 mg/mL血红蛋白处理则显著逆转了SNP诱导的上述效应;利用亚硝酸钠和铁氰化钾作为对照也证实了NO对小麦幼苗耐盐性的专一性调节作用,并可能与NO对小麦幼苗根部质膜H -ATPase和焦磷酸酶活性诱导有关.此外,尽管NO显著提高了盐胁迫下小麦幼苗根部细胞质膜H -ATPase和焦磷酸酶的ATP水解活性,但是对跨膜H 转运则没有明显影响.应用外源CaSO4和EGTA处理也证实,Ca2 可能在NO诱导的质膜H -ATPase和焦磷酸酶活性的提高过程中起信号作用.另外,分析盐胁迫下小麦幼苗根部Na 和K 含量的变化也发现,NO对Na 含量没有明显影响,但是却显著提高了K 水平和K /Na 比,这可能也是NO提高小麦幼苗耐盐性的原因之一.     

Synergistic activation of plasma membrane H+– ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin

The regulation of the H⁺-ATPase of plasma membrane is a crucial point in the integration of transport processes at this membrane. In this work the regulation of H⁺-ATPase activity induced by changes in turgor pressure was investigated and compared with the stimulating effect of fusicoccin (FC). The exposure of cultured cells of Arabidopsis thaliana L. (ecotype Landsberg 310–14-2) to media containing mannitol (0. 15 or 0. 3 M ) or polyethylene glycol 6000 (PEG) (15. 6% or 22% w/v) resulted in a decrease in the turgor pressure of the cells and in a strong stimulation of H⁺ extrusion in the incubation medium. The osmotica-induced H⁺ extrusion was (1) inhibited by the inhibitor of plasma membrane H⁺-ATPase, erythrosin B (EB), (2) dependent on the external K⁺ concentration, (3) associated with a net K⁺ influx, and (4) lead to an increase of cellular malate content. These results show that the reduction of external osmotic potential stimulates the activity of plasma membrane H⁺-ATPase
The effect of mannitol was only partially inhibited by treatments with cycloheximide (CH) and cordycepin, which block protein and mRNA synthesis, respectively. All the effects of osmotica were qualitatively and quantitatively similar to those induced by 5 μ M FC. However, when FC and mannitol (or PEG) were fed together, their effects on H⁺ extrusion appeared synergistic, irrespective of whether FC was present at suboptimal or optimal concentrations. This behaviour suggests that the modes of action of FC and of the osmotica on H⁺-ATPase activity differ at least in some step(s)     

Patch-clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER-located auxin binding protein from maize (Zm-ERabp1), was investigated using the patch-clamp technique (whole-cell configuration). Following a lag-phase of 30–40 seconds, indole-3-acetic acid and 1-naphthylacetic acid induced an outwardly directed current of positive charge in a concentration-dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg²⁺-ATP in the patch-pipette and was abolished after addition of erythrosin B, an inhibitor of H⁺-ATPase, to the protoplasts indicating that the plasma membrane H⁺-ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm-ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm-ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm-ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H⁺-ATPase is activated, and (ii) that the activation of the H⁺-ATPase by such proteins is initiated from outside the plasma membrane.     

Enhanced H+/ATP coupling ratio of H+-ATPase and increased 14-3-3 protein content in plasma membrane of tomato cells upon osmotic shock

Modulation of proton extrusion and ATP-dependent H⁺ transport through the plasma membrane in relation to the presence of 14-3-3 proteins in this membrane in response to osmotic shock was studied in tomato ( Lycopersicon esculentum Mill. cv. Pera) cell cultures. In vivo H⁺ extrusion by cells was activated rapidly and significantly after adding 100 m M NaCl, 100 m M KCl, 50 m M Na₂SO₄, 1.6% sorbitol or 2 µ M fusicoccin to the medium. The increase in H⁺ extrusion by cells treated with 100 m M NaCl was correlated with an increase of H⁺ transport by the plasma membrane H⁺-ATPase (EC 3.6.1.35), but not with changes in ATP hydrolytic activity of this enzyme, suggesting an increased coupling ratio of the enzyme. Immunoblot experiments showed increased amounts of 14-3-3 proteins in plasma membrane fractions isolated from tomato cells treated with 100 m M NaCl as compared to control cells without changing the amount of plasma membrane H⁺-ATPase. Together, these data indicate that in tomato cells an osmotic shock could enhance coupling between ATP hydrolysis and proton transport at the plasma membrane through the formation of a membrane 14-3-3/H⁺-ATPase complex.     

Influence of ATPase activity on PPi dependent H-transport in tonoplast vesicles of Acer pseudoplatanus

Tonoplast H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H⁺-transports were also additive. The H⁺ -PPase inhibition did not change ATP-dependent H⁺-transport but H⁺-ATPase inhibition inhibited the PPi dependent H⁺-transport. Because H⁺-PPase was reported to transport H⁺ and K⁺ into the vacuole (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H⁺-ATPase activity could modify the H⁺/K⁺ stoichiometry for the benefit of K⁺-transport.     

不同生境两种生态型芦苇叶片质膜H+-ATPase的比较

利用两相法化纯化质膜微囊,研究了分布西北沙地区的两种生态型芦苇(Phragmites communis trih.)水生芦苇和重度盐化草甸芦苇,分别简称为水芒和盐芦)叶片质膜H - ATPase的部分性质.结果显示,与水芦相比,盐芦质膜H -ATPase的ATP水解活性升高,Km值由1.27mmol\l降至Vmax没有显著差异.并且该酶活性对温度的敏感必玫PH谱型也发生了变化.以对硝基苯磷酸盐为底物,低浓度时盐芦的的质膜H -ATPase水解活性有差异.钡酸盐抑制实验表明,两种生态的质膜H -ATPase磷酸-酶区的催化性质不同.胰酶对质膜H -ATPase活性的活化谱型也存在差异,说明该酶C末端的结构或性质发生了变化.此外,与水芦相比,盐芦质膜H -ATPase的质子泵活性的耦联程度也升高了.以上结果明,当芦苇从水生环境向盐渍环境过渡时,质膜H -ATPase的催化性质发生了变化,这些变化可能是由酶结构的修饰和不同的同工酬酶谱引起的.H -ATPase催化性质的变化可能是对盐渍生境的适应性反应.     

Changes in plasmalemma K+Mg2+ -ATPase dephosphorylating activity and H+ transport in relation to seasonal growth and freezing tolerance of Festuca pratensis Huds

Changes in plasmalemma K⁺Mg²⁺-ATPase dephosphorylating activity and H⁺ transport were examined in freezing-tolerant and non-tolerant genotypes of the perennial grass species Festuca pratensis Huds. Enzyme activity and ΔμH⁺ were measured in plasmalemma fractions isolated from basal nodes and roots. Three types of experiments were undertaken: (i) a field experiment, utilizing the seasonal growth and cessation cycle of a perennial plant; (ii) a cold acclimation experiment in hydroponics; and (iii) an instant freezing test. A specific fluctuation in K⁺Mg²⁺-ATPase activity was found throughout the seasonal growth of the plants (i). The K⁺Mg²⁺-ATPase activity peaks for both the basal node and the root plasmalemma were determined early in the spring before the renewal of growth. The lowest activity values in roots occurred at the time approaching flowering, and in basal nodes at the transition into the growth cessation. The K⁺Mg²⁺-ATPase activity was approximately 50% lower in the basal node plasmalemma of freezing-tolerant plants than of non-tolerant ones, when assessed at the optimal growth stage in hydroponics. In hydroponics (ii) and in the freezing test (iii), temperature stress was followed by a more pronounced change in the level of K⁺Mg²⁺-ATPase activity than in that of H⁺ transport, and this change was more clearly differentiated in the basal node plasmalemma of contrasting genotypes than in the roots. Stress response was manifested differently in freezing-tolerant and non-tolerant plants at cold acclimation (4–2 °C) and at freezing (−8 °C) temperatures. Proton transport regulation via coupled changes in the hydrolysed ATP/transported proton ratio, as an attribute of freezing-tolerant plants, is discussed.     

Plasma membrane ATPase activity during pepino (Solanum muricatum) ripening

Plasma membrane-enriched samples were extracted from pepino fruit (cv. El Camino) by phase partitioning. H⁺-ATPase (EC 3.6.1.35) activity in these samples increased during late fruit development (immediately before the onset of ripening) and western blotting confirmed there was an increase in enzyme abundance at this time. H⁺-ATPase activity decreased during early ripening and then increased again in the final phase of ripening. Immunolocalisation showed the plasma membrane H⁺-ATPase was most abundant in the outer cell layers of the fruit, which are considered to have a major role in determining fruit texture. Fruit softening was not accelerated by harvest and there was no stimulation of H⁺-ATPase activity by harvest. An in vitro tensile test using fruit rings showed tissue softening proceeded faster at low apoplastic pH (4.5) than at pH 6.5; and tissue buffered at pH 6.5 softened less than unbuffered rings. Erythrosin B, an inhibitor of the plasma membrane H⁺-ATPase, also retarded softening in vitro. These data suggest that plasma membrane H⁺-ATPase activity may contribute to the onset of pepino softening through a reduction in apoplastic pH.     

磷饥饿对番茄幼苗H+分泌及Pi吸收的影响

磷饥饿条件下番茄幼苗的H⁺分泌速率明显提高。质膜质子泵专一性抑制剂钒酸盐能显著抑制番茄幼苗的H⁺分泌,也能显著抑制其Pi吸收。此结果表明,磷饥饿时番茄幼苗Pi吸收速率的变化与H⁺分泌速率的变化之间可能具有一定的相关性,并进一步暗示质膜H⁺-ATPase可能参与其中。本文结果还表明,Pi/H⁺的准量关系约为1:1。