Some Properties of a Functional Reconstituted Plasmalemma H-ATPase Activated by Fusicoccin

Fusicoccin was shown to stimulate the ATP-driven, intravesicular acidification of liposomes reconstituted with crude fusicoccin receptors and the H⁺-translocating ATPase, both solubilized from maize (Zea mays L.) plasma membrane. The present paper reports optimal conditions for dual reconstitution and fusicoccin activation as well as the biochemical characterization of the effect of fusicoccin on this system. Fusicoccin stimulation of proton pumping was dependent on pH and fusicoccin concentration. Its specificity was demonstrated by the positive effect of two cotylenins that have a high affinity for fusicoccin receptors and by the negative response to 7,9-epideacetylfusicoccin, an inactive fusicoccin derivative. Kinetic measurements at different ATP concentrations showed that fusicoccin increases the V_max of the enzyme. Fusicoccin stimulation of maize H⁺-ATPase was also maintained when receptors from maize were substituted by those from spinach (Spinacia oleracea L.).     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.     

Nitric oxide enhances salt tolerance in maize seedlings through increasing activities of proton-pump and Na+/H+ antiport in the tonoplast

Nitric oxide (NO), an endogenous signaling molecule in animals and plants, mediates responses to abiotic and biotic stresses. Our previous work demonstrated that 100 μM sodium nitroprusside (SNP, an NO donor) treatment of maize seedlings increased K⁺ accumulation in roots, leaves and sheathes, while decreasing Na⁺ accumulation (Zhang et al. in J Plant Physiol Mol Biol 30:455–459, 2004b). Here we investigate how NO regulates Na⁺, K⁺ ion homeostasis in maize. Pre-treatment with 100 μM SNP for 2 days improved later growth of maize plants under 100 mM NaCl stress, as indicated by increased dry matter accumulation, increased chlorophyll content, and decreased membrane leakage from leaf cells. An NO scavenger, methylene blue (MB-1), blocked the effect of SNP. These results indicated that SNP-derived NO enhanced maize tolerance to salt stress. Further analysis showed that NaCl induced a transient increase in the NO level in maize leaves. Both NO and NaCl treatment stimulated vacuolar H⁺-ATPase and H⁺-PPase activities, resulting in increased H⁺-translocation and Na⁺/H⁺ exchange. NaCl-induced H⁺-ATPase and H⁺-PPase activities were diminished by MB-1. 1-Butanol, an inhibitor of phosphatidic acid (PA) production by phospholipase D (PLD), reduced NaCl- and NO-induced H⁺-ATPase activation. In contrast, applied PA stimulated H⁺-ATPase activity. These results suggest that NO acts as a signal molecule in the NaCl response by increasing the activities of vacuolar H⁺-ATPase and H⁺-PPase, which provide the driving force for Na⁺/H⁺ exchange. PLD and PA play an important role in this process.     

H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids

The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K⁺ uptake, suggesting a stimulation of the plasmalemma H⁺ pump. The evaluation of weak acid-induced H⁺ extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged form of the acid, partially masking the proton pump-mediated extrusion of H⁺. The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segments a significant increase in `real' H⁺ extrusion, roughly matching the increase in net K⁺ uptake. The presence of K⁺ significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K⁺ absorption. In maize root segments, the effects of fusicoccin and those of butyric acid on both K⁺ uptake and H⁺ extrusion are clearly synergistic, thus suggesting distinct modes of action. These results support the view that the activity of the plasmalemma H⁺ pump is regulated by the value of cytosolic pH.     

Fusicoccin receptors. Evidence for endogenous ligand

The binding of fusicoccin to the microsomal preparations of maize roots in vitro is increased several-fold when segments of the tissue are washed for 2 h in distilled water before homogenization. Addition of freeze-dried wash solution to microsomal preparations of spinach leaves or fresh roots, washed roots, or coleoptiles of maize inhibited the binding of fusicoccin to particulate fractions. The freeze-dried material also blocked fusicoccin-promoted H⁺ extrusion from maize root segments. Roots may contain one or more water-soluble compounds competing with fusicoccin at the receptor level; such ligands might play a physiological role as modulators of the H⁺/K⁺ exchange system in higher plants.Abbreviation FC Fusicoccin     

Use of an extracellular,ion-selective,vibrating microelectrode system for the quantification of K+, H+, and Ca2+ fluxes in maize roots and maize suspension cells

An ion-selective vibrating-microelectrode system, which was originally used to measure extracellular Ca²⁺ gradients generated by Ca²⁺ currents, was used to study K⁺, H⁺ and Ca²⁺ transport in intact maize (Zea mays L.) roots and individual maize suspension cells. Comparisons were made between the vibrating ion-selective microelectrode, and a technique using stationary ion-selective microelectrodes to measure ionic gradients in the unstirred layer at the surface of plant roots. The vibrating-microelectrode system was shown to be a major improvement over stationary ion-selective microelectrodes, in terms of sensitivity and temporal resolution. With the vibrating ion microelectrode, it was easy to monitor K⁺ influxes into maize roots in a background K⁺ concentration of 10 mM or more, while stationary K⁺ electrodes were limited to measurements in a background K⁺ concentration of 0.3 mM or less. Also, with this system it was possible to conduct a detailed study of root Ca²⁺ transport, which was previously not possible because of the small fluxes involved. For example, we were able to investigate the effect of the excision of maize roots on Ca²⁺ influx. When an intact maize root was excised from the seedling at a position 3 cm from the site of measurement of Ca²⁺ transport, a rapid fourfold stimulation of Ca²⁺ influx was observed followed by dramatic oscillations in Ca²⁺ flux, oscillating between Ca²⁺ influx and efflux. These results clearly demonstrate that wound or perturbation responses of plant organs involve transient alterations in Ca²⁺ transport, which had previously been inferred by demonstrations of touch-induced changes in cytoplasmic calcium. The sensitivity of this system allows for the measurement of ion fluxes in individual plant cells. Using vibrating K⁺ and H⁺electrodes, it was possible to measure H⁺efflux and both K⁺ influx and efflux in individual maize suspension cells under different conditions. The availability of this technique will greatly improve our ability to study ion transport at the cellular level, in intact plant tissues and organs, and in specialized cells, such as root hairs or guard cells.Symbol X amplitude of vibration The authors would like to thank Richard Sanger for his invaluable work on the design and improvement of the ion-selective vibratingmicroelectrode system. The research presented here was supported in part by U.S. Department of Agriculture Competitive Grant No. 90-37261-5411 to Leon Kochian and William Lucas.     

Evidence for Cotransport of Nitrate and Protons in Maize Roots : II. Measurement of NO(3) and H Fluxes with Ion-Selective Microelectrodes

We report here on an investigation of net nitrate and proton fluxes in root cells of maize (Zea mays L.) seedlings grown without (noninduced) and with (induced) 0.1 millimolar nitrate. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, WJ Lucas [1987] Plant Physiol 84: 1177-1184) was utilized to quantify net ionic fluxes from the measurement of electrochemical potential gradients for NO₃⁻ and H⁺ within the unstirred layer at the root surface. The nitrate-inducibility, pH dependence, and concentration dependence of net NO₃⁻ uptake correlated quite closely with the electrical response of maize roots to nitrate under the same experimental conditions (as described in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289). Additionally, it was found that potential inhibitors of the plasmalemma H⁺-ATPase (vandate, diethylstilbestrol), which were shown to abolish the electrical response to NO₃⁻ (in PR McClure, LV Kochian, RM Spanswick, JE Shaff [1990] Plant Physiol 93: 281-289), dramatically inhibited NO₃⁻ absorption. These results strongly indicate that the NO₃⁻ electrical response is due to the operation of a NO₃⁻ transport system in the plasmalemma of maize root cells. Furthermore, the results from the H⁺-ATPase inhibitor studies indicate that the NO₃⁻ transport system is linked to the H⁺-ATPase, presumably as a NO₃⁻/H⁺ symport. This is further supported by the pH response of the NO₃⁻ transport system (inhibition at alkaline pH values) and the change in net H⁺ flux from a moderate efflux in the absence of NO₃⁻, to zero net H⁺ flux after exposing the maize root to exogenous nitrate. Although these results can be explained by other interpretations, the simplest model that fits both the electrical responses and the NO₃⁻/H⁺ flux data is a NO₃⁻/H⁺ symport with a NO₃⁻:H⁺ flux stoichiometry >1, whose operation results in the stimulation of the H⁺-ATPase due to the influx of protons through the cotransport system.     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

In vivo and in vitro effects of copper and cadmium on the plasma membrane H+-ATPase from cucumber (Cucumis sativus L.) and maize (Zea mays L.) roots

The plasmalemma vesicles isolated from cucumber and maize roots were used to study the effect of Cu²⁺ and Cd²⁺ on the hydrolytic and proton pumping activities of ATPase. In vivo application of metal ions to the plant growth solutions resulted in stimulation of the proton transport in maize. In cucumber roots the action of metals was not the same: cadmium stimulated the H⁺ transport through plasmalemma whereas Cu²⁺ almost completely inhibited it. Copper ions decreased the hydrolytic activity of H⁺-ATPase in cucumber, without any effect on this activity in membranes isolated from maize roots. The effect of cadmium on the hydrolytic activities was opposite: ATP-hydrolysis activity in plasmalemma was not altered in cucumber, whereas in maize its stimulation was observed. The amount of accumulated metals was not the main reason of different influence of metals on H⁺-ATPase activity in tested plants. In in vitro experiments Cu²⁺ inhibited H⁺ transport in the cucumber, to a higher degree than Cd²⁺ and both metals did not change this H⁺-ATPase activity of plasmalemma isolated from corn roots. Cu²⁺ added into the incubation medium reduced the hydrolytic activity of ATPase in the plasma membrane isolated from cucumber as well as from corn roots. Cd²⁺ diminished the hydrolytic activity of ATPase in cucumber, and no effect of Cd²⁺ in the plasmalemma isolated from corn roots was found. Our results indicated different in vitro and in vivo action of both metals on H⁺-ATPase and different response of this enzyme to Cu²⁺ and Cd²⁺ in maize and cucumber.     

Active extrusion of protons into deionized water by roots of intact maize plants

The investigations were focussed on the question as to whether roots of intact maize plants (Zea mays L. cv Blizzard) release protons into deionized H₂O. Plants in the six to seven leaf stage depressed the pH of deionized H₂O from 6 to about 4.8 during an experimental period of 4 hours. Only one-third of the protons released could be ascribed to the solvation of CO₂ in H₂O. The main counter anions released were Cl⁻, NO₃⁻, and SO₄²⁻. At low temperature (2°C), the H⁺ release was virtually blocked while a relatively high amount of K⁺ was released. The presence of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the external solution increased the H⁺ secretion significantly. Addition of vanadate to the outer medium inhibited the H⁺ release while fusicoccin had a stimulating effect. Substituting the nutrient solution of deionized H₂O resulted in a substantial increase of the membrane potential difference from −120 to −190 millivolts. The experimental results support the conclusion that the H⁺ release by roots of intact maize plants is an active process driven by a plasmalemmalocated ATPase. Since the net H⁺ release was not associated with a net uptake of K⁺, it is unlikely to originate from a K⁺/H⁺ antiport.     

Hydrogen emission from nodulated soybeans [Glycine max (L.) Merr.] and consequences for the productivity of a subsequent maize (Zea mays L.) crop

Hydrogen (H₂) is a by-product of the symbiotic nitrogen fixation (N₂ fixation) between legumes and root-nodule bacteria (rhizobia). Some rhizobial strains have an uptake hydrogenase enzyme (commonly referred to as Hup⁺) that recycles H₂ within the nodules. Other rhizobia, described as Hup^?, do not have the enzyme and the H₂ produced diffuses from the nodules into the soil where it is consumed by microorganisms. The effect of this phenomenon on the soil biota and on the soil itself, and consequent stimulation of plant growth, has been demonstrated previously. Soybeans [Glycine max (L.) Merr.] cv. Leichhardt, inoculated with either a Hup⁺ strain (CB1809) or one of two Hup^? strains (USDA442 or USDA16) of Bradyrhizobium japonicum and uninoculated soybeans, plus a non-legume control [capsicum (Capsicum annuum L.)] were grown in the field at Ayr, North Queensland, Australia. The objectives were to examine (1) relationships between N₂ fixation and H₂ emission, and (2) the influence H₂-induced changes in soil might have during the legume phase and/or on the performance of a following crop. Strains CB1809 and USDA442 were highly effective in N₂ fixation (“good” fixers); USDA16 was partly effective (“poor” fixer). The soil had a large but non-uniformly distributed naturalised population of B. japonicum and most uninoculated control plants formed nodules that fixed some N₂. These naturalised strains were classified as “poor fixers” of N₂ and were Hup⁺. H₂ emissions from nodules were assessed for all treatments when the soybean crop was 62 days old. Other parameters of symbiotic N₂ fixation and plant productivity were measured when the crop was 62 and 96 days old and at crop maturity. Immediately after final harvest, the land was sown to a crop of maize (Zea mays L.) in order to determine the consequences of H₂ emission from the soybean crop on maize growth. It was estimated that soybeans inoculated with USDA442, the highly effective Hup^– strain of B. japonicum, fixed 117 kg shoot N/ha (or about 195 kg total N/ha if the fixed N associated with roots and nodules was taken into account), and contributed about 215,000 l H₂ gas per hectare to the ecosystem over the life of the crop. The volume of H₂ evolved from soybeans nodulated by the Hup⁺ strain CB1809 was only 6% of that emitted by the USDA442 treatment, but there was no indication that soybean inoculated with USDA442 benefited from the additional H₂ input. The shoot biomass, grain yield, and amounts of N fixed (105 kg shoot N/ha, 175 kg total N/ha) by the CB1809 treatment were little less than for USDA442 plants. Three days after the soybean crop was harvested, the plots were over-sown with maize along the same row lines in which the soybeans had grown. This procedure exposed the maize roots to whatever influence soybean H₂ emission might have had on the soil and/or the soil microflora immediately surrounding soybean nodules. The evidence for a positive effect of soybean H₂ emission on maize production was equivocal. While the consistent differences between those pre-treatments that emitted H₂ and those that did not indicated a trend, only one difference (out of the 12 parameters of maize productivity that were measured) was statistically significant at P?<?0.05. The findings need substantiation by further investigation.     

H Efflux and Hexose Transport under Imposed Energy Status in Maize Root Tips

The relationship between changes in H⁺ flux and sugar transport in maize Zea mays L. DEA root tips have been investigated using two methods for controlling the cellular nucleotide level: (a) incubation in the presence of a glucose analog, the 2-deoxyglucose, which decreased the ATP level to less than 15% of its initial value within 60 minutes without changing the ADP and AMP levels; (b) an hypoxic treatment which also decreased the ATP level but with a concomitant rise in ADP and AMP. In both cases the rate of hexose transport was not modified until ATP had dropped to 70% of its initial value; then it decreased with the cellular ATP level. The residual uptake rate at very low ATP concentrations still represented 50% of the maximum rate with the dGlc treatment but only the diffusion rate in anoxia. H⁺ efflux was abolished in anoxia but not by the 2-deoxyglucose treatment, in spite of a lower cellular ATP concentration. Our results are consistent with an inhibition of H⁺-ATPase activity in anoxia by the high levels of cellular ADP and AMP, and provide in vivo evidence that sugar uptake is dependent upon the proton motive force rather than cellular ATP concentration. The absence of stimulation of H⁺ extrusion by ferricyanide in either normoxic or hypoxic conditions suggests that a redox system does not appear to contribute to H⁺ secretion under the conditions of this investigation.     

Calcium-dependent phosphorylation regulates the plasma-membrane H+-ATPase activity of maize (Zea mays L.) roots

Phosphorylation/dephosphorylation of the plasma-membrane H⁺-ATPase (EC 3.6.1.35) could act as a regulatory mechanism to control its activity. In this work, a plasmalemma-enriched fraction from maize roots and a partially purified H⁺-ATPase were used to investigate the effects of Ca²⁺ and calmodulin on the H⁺-ATPase activity and on its phosphorylation status. Both the hydrolytic and the proton-pumping activities were reduced approximately 50% by micromolar Ca²⁺ concentrations while calmodulin did not show any effect either alone or in the presence of Ca²⁺. The lack of effect of calmodulin antagonists indicated that calmodulin was not involved in this response. The addition of staurosporine, a kinase inhibitor, abolished the inhibitory effect of Ca²⁺. Phosphorylation of plasma membrane and partially purified H⁺-ATPase showed the same behavior. In the presence of Ca²⁺ a polypeptide of 100 kDa was phosphorylated. This polypeptide cross-reacted with antibodies raised against the H⁺-ATPase of maize roots. The autoradiogram of the immunodetected protein clearly showed that this polypeptide, which corresponds to the H⁺-ATPase, was phosphorylated. Additional clear evidence comes from the immunoprecipitation experiments: the data obtained show that the H⁺-ATPase activity is indeed influenced by its state of phosphorylation. Received: 19 October 1998 / Accepted: 23 February 1999     

Recent Molecular Approaches to the Physiology of the Plasma Membrane Proton Pump

During the last 3 years, genes for plasma membrane H⁺-ATPases from fungi, protozoa and plants have been isolated. Sequence similarities indicate that H⁺-ATPases constitute a separate group with the family of ATPases with phosphorylated intermediates. Yeast is a convenient model system to approach the physiology of H⁺-ATPases by recombinant DNA methodologies. A mutational analysis of yeast H⁺-ATPase has demonstrated that the enzyme is essential and rate-limiting for growth. Intracellular pH homeostasis is one of the crucial functions of H⁺-ATPase. In addition, there are indications for the direct energization of some essential transport system. The regulation of ATPase activity is probably mediated by an interaction between the active site and an inhibitory domain at the carboxyl-terminus.     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

Factors associated with the instability of nitrate-insensitive proton transport by maize root microsomes

Proton transport catalyzed by the nitrate-insensitive, vanadate-sensitive H⁺-ATPase in microsomes from maize (Zea mays L.) roots washed with 0.25 molar KI decreased as a function of time at 0 to 4°C. The rate of proton transport was approximately one-half of that by freshly isolated microsomes after 6 to 18 hours of cold storage. The decrease in proton transport coincided with losses in membrane phosphatidylcholine and was not associated with a change in vanadate-sensitive ATP hydrolysis. A technique based on a protocol developed for the reconstitution of Neurospora crassa plasma membrane H⁺-ATPase (DS Perlin, K Kasamo, RJ Brooker, CW Slayman 1984 J Biol Chem 259: 7884-7892) was employed to restore proton transport activity to maize microsomes. These results indicated that the decline in proton transport by maize root membranes during cold storage was not due to degradation of the protein moiety of the H⁺-ATPase, but was due to the loss of phospholipids.     

N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N′(4-dimethylamino-α-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H⁺-ATPase in microsomal preparations; however, the plasma membrane-type H⁺-ATPase was unaffected. The H⁺-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H⁺-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H⁺-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.     

2种玉米幼苗耐旱性生理机制研究

以白种皮(白玉米)和黄种皮(黄玉米)2个玉米栽培品种为材料,在水培条件下进行聚乙二醇(PEG-6000)模拟干旱胁迫处理,分析玉米叶片抗旱性相关生理特性和质膜H+-ATP酶活性的变化,探讨2种玉米幼苗耐旱性生理机制。结果表明:(1)在2%、5%、10%PEG-6000处理条件下,随处理浓度和时间的增加,2种玉米幼苗植株失水率上升,叶片蒸腾速率降低,气孔传导率下降;在所有相同处理条件下,白玉米植株失水率明显小于黄玉米,而叶片蒸腾速率和气孔传导率下降幅度明显大于黄玉米,即白玉米的耐旱性比黄玉米强。(2)在相同浓度PEG-6000处理下,白玉米叶片可溶性蛋白、可溶性糖含量、游离脯氨酸含量均高于黄玉米,它在干旱胁迫下的渗透调节能力强于黄玉米。(3)在抗氧化酶体系中,随着PEG-6000胁迫浓度的升高,2种玉米叶片CAT活性呈下降趋势,但白玉米CAT活性在2%和5%PEG-6000胁迫下均显著高于黄玉米,其叶片中H2O2含量显著低于黄玉米。(4)随着PEG-6000胁迫浓度的升高,白玉米叶片质膜H+-ATPase磷酸化水平及其与14-3-3蛋白的结合受到的抑制作用比黄玉米强,白玉米叶片质膜H+-ATPase活性比黄玉米叶片低,叶片气孔开度小于黄玉米,叶片蒸腾速率和气孔传导率均低于黄玉米,这可能是白玉米耐旱性强于黄玉米的一个重要机制。     

Indolacetic and humic acids induce lateral root development through a concerted plasmalemma and tonoplast H<Superscript>+</Superscript> pumps activation

Increasing evidences have indicated that humic substances can induce plant growth and productivity by functioning as an environmental source of auxinic activity. Here we comparatively evaluate the effects of indole-3-acetic acid (IAA) and humic acids (HA) isolated from two different soils (Inseptsol and Ultisol) and two different organic residues (vermicompost and sewage sludge) on root development and on activities of plasmalemma and tonoplast H⁺ pumps from maize roots. The data show that HA isolated from these different sources as well as low IAA concentrations (10⁻¹⁰ and 10⁻¹⁵ M) improve root growth through a markedly proliferation of lateral roots along with a differential activation not only of the plasmalemma but also of vacuolar H⁺-ATPases and H⁺-pyrophosphatase. Further, the vacuolar H⁺-ATPase had a peak of stimulation in a range from 10⁻⁸ to 10⁻¹⁰ M IAA, whereas the H⁺-pyrophosphatase was sensitive to a much broader range of IAA concentrations from 10⁻³ to 10⁻¹⁵ M. It is proposed a complementary view of the acid growth mechanism in which a concerted activation of the plasmalemma and tonoplast H⁺ pumps plays a key role in the root cell expansion process driven by environment-derived molecules endowed with auxinic activity, such as that of humic substances.     

Effect of Exogenous Putrescine, Spermidine, and Spermine on K Uptake and H Extrusion through Plasmamembrane in Maize Root Segments

The action of exogenous polyamines (putrescine, spermidine, and spermine) on `washing' and fusicoccin-stimulated K⁺ uptake and H⁺ extrusion through the plasmamembrane in maize (Zea mays L., hybrid line Plenus S 516) root apical segments was studied. The results showed that polyamines inhibit the washing-stimulated K⁺ influx and H⁺ extrusion without interfering with K⁺ uptake and H⁺ extrusion stimulated by fusicoccin. Spermidine appeared to be the most effective in inhibiting K⁺ uptake and H⁺ extrusion while putrescine showed a smaller inhibiting action with respect to the others. The analysis of kinetic constants indicated that the polyamines behave as competitive inhibitors with respect to K⁺.