Development of Fe-deficiency responses in cucumber (Cucumis sativus L.) roots: involvement of plasma membrane H(+)-ATPase activity

One of the mechanisms through which some strategy I plants respond to Fe-deficiency is an enhanced acidification of the rhizosphere due to proton extrusion. It was previously demonstrated that under Fe-deficiency, a strong increase in the H(+)-ATPase activity of plasma membrane (PM) vesicles isolated from cucumber roots occurred. This result was confirmed in the present work and supported by measurement of ATP-dependent proton pumping in inside-out plasma membrane vesicles. There was also an attempt to clarify the regulatory mechanism(s) which lead to the activation of the H(+)-ATPase under Fe-deficiency conditions. Plasma membrane proteins from Fe-deficient roots submitted to immunoblotting using polyclonal antibodies showed an increased level in the 100 kDa polypeptide. When the plasma membrane proteins were treated with trypsin a 90 kDa band appeared. This effect was accompanied by an increase in the enzyme activity, both in the Fe-deficient and in the Fe-sufficient extracts. These results suggest that the increase in the plasma membrane H(+)-ATPase activity seen under Fe-deficiency is due, at least in part, to an increased steady-state level of the 100 kDa polypeptide.     

Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a major role in the activation of ion and nutrient transport and has been suggested to be involved in several physiological processes, such as cell expansion and salt tolerance. Its activity is regulated by a C-terminal autoinhibitory domain that can be displaced by phosphorylation and the binding of regulatory 14-3-3 proteins, resulting in an activated enzyme. To better understand the physiological consequence of this activation, we have analyzed transgenic tobacco (Nicotiana tabacum) plants expressing either wild-type plasma membrane H(+)-ATPase4 (wtPMA4) or a PMA4 mutant lacking the autoinhibitory domain (DeltaPMA4), generating a constitutively activated enzyme. Plants showing 4-fold higher expression of wtPMA4 than untransformed plants did not display any unusual phenotype and their leaf and root external acidification rates were not modified, while their in vitro H(+)-ATPase activity was markedly increased. This indicates that, in vivo, H(+)-ATPase overexpression is compensated by down-regulation of H(+)-ATPase activity. In contrast, plants that expressed DeltaPMA4 were characterized by a lower apoplastic and external root pH, abnormal leaf inclination, and twisted stems, suggesting alterations in cell expansion. This was confirmed by in vitro leaf extension and curling assays. These data therefore strongly support a direct role of H(+)-ATPase in plant development. The DeltaPMA4 plants also displayed increased salt tolerance during germination and seedling growth, supporting the hypothesis that H(+)-ATPase is involved in salt tolerance.     

Lysophosphatidylcholine specifically stimulates plasma membrane H(+)-ATPase from corn roots

The plasma membrane H(+)-ATPase activity from corn seedling roots is shown to be stimulated 3- to 4-fold by the addition of lysophosphatidylcholine (lysoPC). This effect clearly differs from that of other detergents by both the magnitude and the absence of inhibition at higher concentrations. LysoPC decreases the apparent Km for MgATP, increases Vmax of the ATPase reaction but does not change its pH optimum. On the contrary, the acid phosphatase activity associated with plasma membranes is not influenced by lysoPC. A lysoPC stimulation is also demonstrated for the solubilized preparation of the H(+)-ATPase. It is assumed that lysoPC stimulation of the plant plasma membrane H(+)-ATPase is not only due to permeabilization of the vesicles for MgATP, but also to direct action on the enzyme.     

Abscisic acid and 14-3-3 proteins control K channel activity in barley embryonic root

Germination of seeds proceeds in general in two phases, an initial imbibition phase and a subsequent growth phase. In grasses like barley, the latter phase is evident as the emergence of the embryonic root (radicle). The hormone abscisic acid (ABA) inhibits germination because it prevents the embryo from entering and completing the growth phase. Genetic and physiological studies have identified many steps in the ABA signal transduction cascade, but how it prevents radicle elongation is still not clear. For elongation growth to proceed, uptake of osmotically active substances (mainly K(+)) is essential. Therefore, we have addressed the question of how the activity of K(+) permeable ion channels in the plasma membrane of radicle cells is regulated under conditions of slow (+ABA) and rapid germination (+fusicoccin). We found that ABA arrests radicle growth, inhibits net K(+) uptake and reduces the activity of K(+) (in) channels as measured with the patch-clamp technique. In contrast, fusicoccin (FC), a well-known stimulator of germination, stimulates radicle growth, net K(+) uptake and reduces the activity of K(+) (out) channels. Both types of channels are under the control of 14-3-3 proteins, known as integral components of signal transduction pathways and instrumental in FC action. Intriguingly, 14-3-3 affected both channels in an opposite fashion: whereas K(+) (in) channel activity was fully dependent upon 14-3-3 proteins, K(+) (out) channel activity was reduced by 14-3-3 proteins by 60%. Together with previous data showing that 14-3-3 proteins control the activity of the plasma membrane H(+)-ATPase, this makes 14-3-3 a prime candidate for molecular master regulator of the cellular osmo-pump. Regulation of the osmo-pump activity by ABA and FC is an important mechanism in controlling the growth of the embryonic root during seed germination.     

Phosphorylation of Thr-948 at the C terminus of the plasma membrane H(+)-ATPase creates a binding site for the regulatory 14-3-3 protein

The plant plasma membrane H(+)-ATPase is activated by the binding of 14-3-3 protein to the C-terminal region of the enzyme, thus forming an H(+)-ATPase-14-3-3 complex that can be stabilized by the fungal toxin fusicoccin. A novel 14-3-3 binding motif, QQXYpT(948)V, at the C terminus of the H(+)-ATPase is identified and characterized, and the protein kinase activity in the plasma membrane fraction that phosphorylates this threonine residue in the H(+)-ATPase is identified. A synthetic peptide that corresponds to the C-terminal 16 amino acids of the H(+)-ATPase and that is phosphorylated on Thr-948 prevents the in vitro activation of the H(+)-ATPase that is obtained in the presence of recombinant 14-3-3 and fusicoccin. Furthermore, binding of 14-3-3 to the H(+)-ATPase in the absence of fusicoccin is absolutely dependent on the phosphorylation of Thr-948, whereas binding of 14-3-3 in the presence of fusicoccin occurs independently of phosphorylation but still involves the C-terminal motif YTV. Finally, by complementing yeast that lacks its endogenous H(+)-ATPase with wild-type and mutant forms of the Nicotiana plumbaginifolia H(+)-ATPase isoform PMA2, we provide physiological evidence for the importance of the phosphothreonine motif in 14-3-3 binding and, hence, in the activation of the H(+)-ATPase in vivo. Indeed, replacing Thr-948 in the plant H(+)-ATPase with alanine is lethal because this mutant fails to functionally replace the yeast H(+)-ATPase. Considering the importance of the motif QQXYpTV for 14-3-3 binding and yeast growth, this motif should be of vital importance for regulating H(+)-ATPase activity in the plant and thus for plant growth.     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

Function of transport H+-ATPases in plant cell plasma and vacuolar membranes of maize under salt stress conditions and effect of adaptogenic preparations

Participations of electrogenic H+-pumps of plasma and vacuolar membranes represented by E1-E2 and V-type H+-ATPases in plant cell adaptation to salt stress conditions has been studied by determination of their transport activities. Experiments were carried out on corn seedlings exposed during 1 or 10 days at 0.1 M NaCl. Preparations Methyure and Ivine were used by seed soaking at 10(-7) M. Plasma and vacuolar membrane fractions were isolated from corn seedling roots. In variants without NaCl a hydrolytical activity of plasma membrane H+-ATPase was increased with seedling age and its transport one was changed insignificantly, wherease the response of the weaker vacuolar H+-ATPase was opposite. NaCl exposition decreased hydrolytical activities of both H+-ATPases and increased their transport ones. These results demonstrated amplification of H+-pumps function especially represented by vacuolar H+-ATPase. Both preparations, Methyure mainly, caused a further increase of transport activity which was more expressed in NaCl variants. Obtained results showed the important role of these H+-pumps in plant adaptation under salt stress conditions realized by energetical maintenance of the secondary active Na+/H+ -antiporters which remove Na+ from cytoplasm.     

Phosphorylation-dependent interaction between plant plasma membrane H(+)-ATPase and 14-3-3 proteins

The H(+)-ATPase is a key enzyme for the establishment and maintenance of plasma membrane potential and energization of secondary active transport in the plant cell. The phytotoxin fusicoccin induces H(+)-ATPase activation by promoting the association of 14-3-3 proteins. It is still unclear whether 14-3-3 proteins can represent natural regulators of the proton pump, and factors regulating 14-3-3 binding to the H(+)-ATPase under physiological conditions are unknown as well. In the present study in vivo and in vitro evidence is provided that 14-3-3 proteins can associate with the H(+)-ATPase from maize roots also in a fusicoccin-independent manner and that the interaction depends on the phosphorylation status of the proton pump. Furthermore, results indicate that phosphorylation of H(+)-ATPase influences also the fusicoccin-dependent interaction of 14-3-3 proteins. Finally, a protein phosphatase 2A able to impair the interaction between H(+)-ATPase and 14-3-3 proteins was identified and partially purified from maize root.     

外源多胺对低氧胁迫下黄瓜幼苗根系生长及H+-ATP酶和H+-焦磷酸酶活性的影响

采用营养液水培方式,研究了根际低氧胁迫下外源多胺对黄瓜幼苗植株根系生长,内源多胺含量与质膜H -ATP酶、液泡膜H -ATP酶和焦磷酸酶活性的影响.结果表明,根际低氧胁迫显著抑制黄瓜幼苗根系的生长,外源Put(腐胺)和Spd(亚精胺)可缓解低氧胁迫对根系的生长抑制,多胺主要以Spd的形式发挥促进性的生理作用,Put通过转化为Spd发挥作用;低氧胁迫下黄瓜根系内源多胺含量略有提高,外源多胺处理可增加内源多胺的含量;低氧胁迫下外源Put和Spd处理后质膜H -ATP酶活性显著提高,外源多胺对黄瓜根系液胞膜H -ATP酶和H -焦磷酸酶活性没有明显影响,说明低氧胁迫下外源多胺主要通过提高质膜H -ATP酶活性而发挥生理作用.     

Cosuppression of a plasma membrane H(+)-ATPase isoform impairs sucrose translocation, stomatal opening, plant growth, and male fertility

The plasma membrane H(+)-ATPase builds up a pH and potential gradient across the plasma membrane, thus activating a series of secondary ion and metabolite transporters. pma4 (for plasma membrane H(+)-ATPase 4), the most widely expressed H(+)-ATPase isogene in Nicotiana plumbaginifolia, was overexpressed in tobacco. Plants that overexpressed PMA4 showed no major changes in plant growth under normal conditions. However, two transformants were identified by their stunted growth, slow leaf initiation, delayed stem bolting and flowering, and male sterility. Protein gel blot analysis showed that expression of the endogenous and transgenic pma4 was cosuppressed. Cosuppression was developmentally regulated because PMA4 was still present in developing leaves but was not detected in mature leaves. The glucose and fructose content increased threefold, whereas the sucrose content remained unchanged. The rate of sucrose exudation from mature leaves was reduced threefold and the sugar content of apical buds was reduced twofold, suggesting failure of sucrose loading and translocation to the sink tissues. Cosuppression of PMA4 also affected the guard cells, stomatal opening, and photosynthesis in mature leaves. These results show that a single H(+)-ATPase isoform plays a major role in several transport-dependent physiological processes.     

Effect of Seed Freezing and Storage Conditions on Plasma Membrane Properties of Norway Spruce (Piceo abies Karst.) Seedlings

Norway spruce (Picea abies Karst.) seeds were frozen and stored for 15 months at + 3, ? 25, ? 75 or ? 196°C. After storage, seeds were germinated for 9?14 days to determine viability and plasma membrane protein composition, H⁺-ATPase activity and fluidity. The results indicate no significant differences in viability of seed 14 days after germination. Biochemical analyses revealed increased plasma membrane fluidity in 9-day-old Norway spruce seedlings raised from seeds pretreated at ? 75 °C. and changes in the temperature profile of membrane fluidity in seedlings after pre-treatment of seeds at ? 25 °C. On the other hand, the same treatments did not result in changes in plasma membrane protein content, protein composition or ATPase activity. There was also no difference in plasma membrane H⁺-ATPase activity assayed in the presence of different ATP hydrolysis inhibitors. Based on the presented results, and other experimental data, we suggest that during early seedling growth, adaptation of seeds to ? 25 and ? 75°C freezing and/or storage temperature results in stability of the plasma membrane protein function and composition and increased fluidity or changes in the temperature-dependent fluidity profile of these membranes.     

Single point mutations in various domains of a plant plasma membrane H(+)-ATPase expressed in Saccharomyces cerevisiae increase H(+)-pumping and permit yeast growth at low pH.

In plants, the proton pump-ATPase (H(+)-ATPase) of the plasma membrane is encoded by a multigene family. The PMA2 (plasma membrane H(+)-ATPase) isoform from Nicotiana plumbaginifolia was previously shown to be capable of functionally replacing the yeast H(+)-ATPase, provided that the external pH was kept above pH 5.5. In this study, we used a positive selection to isolate 19 single point mutations of PMA2 which permit the growth of yeast cells at pH 4.0. Thirteen mutations were restricted to the C-terminus region, but another six mutations were found in four other regions of the enzyme. Kinetic studies determined on nine mutated PMA2 compared with the wild-type PMA2 revealed an activated enzyme characterized by an alkaline shift of the optimum pH and a slightly higher specific ATPase activity. However, the most striking difference was a 2- to 3-fold increase of H(+)-pumping in both reconstituted vesicles and intact cells. These results indicate that point mutations in various domains of the plant H(+)-ATPase improve the coupling between H(+)-pumping and ATP hydrolysis, resulting in better growth at low pH. Moreover, the yeast cells expressing the mutated PMA2 showed a marked reduction in the frequency of internal membrane proliferation seen with the strain expressing the wild-type PMA2, indicating a relationship between H(+)-ATPase activity and perturbations of the secretory pathway.     

汞对萝卜种子发芽及幼苗某些生理特性的影响

研究了重金属汞离子(Hg^2 )对萝卜种子发芽及幼苗某些生理特性的影响。结果表明,随着汞离子浓度的增加和处理时间的延长,萝卜种子的发芽率和发芽指数以及幼苗的生长势和生长量均明显下降。幼苗子叶叶绿素a、b的含量下降。幼苗根系、茎叶过氧化物酶的活性则明显升高。     

Plasma Membrane H+-ATPase in Maize Roots Induced for NO3- Uptake

Plasma membrane H+-ATPase was studied in maize (Zea mays L.) roots induced for NO3- uptake. Membrane vesicles were isolated by means of Suc density gradient from roots exposed for 24 h either to 1.5 mM NO3- or 1.5 mM SO4-. The two populations of vesicles had similar composition as shown by diagnostic inhibitors of membrane-associated ATPases. However, both ATP-dependent intravesicular H+ accumulation and ATP hydrolysis were considerably enhanced (60-100%) in vesicles isolated from NO3--induced roots. Km for Mg:ATP and pH dependency were not influenced by NO3- treatment of the roots. ATP hydrolysis in plasma membrane vesicles for both control and NO3--induced roots was not affected by 10 to 150 mM NO3- or Cl-. On the other hand, kinetics of NO3-- or Cl--stimulated ATP-dependent intravesicular H+ accumulation were modified in plasma membrane vesicles isolated from NO3-- induced roots. Immunoassays carried out with polyclonal antibodies against plasma membrane H+-ATPase revealed an increased steady-state level of the enzyme in plasma membrane vesicles isolated from NO3--induced roots. Results are consistent with the idea of an involvement of plasma membrane H+-ATPase in the overall response of roots to NO3-.     

聚乙二醇（PEG）浸种对茎瘤芥老化种子活力及其幼苗生长的影响

茎瘤芥品种‘永安小叶’老化种子经聚乙二醇（PEG）浸种后,其种子活力和幼苗生长与PEG浓度有一定的相关性,不同浓度PEG浸种后的茎瘤芥种子膜透性均降低,种子发芽率、发芽势、发芽指数和活力指数增大,幼苗根干重、茎干重和根长也均增大,幼苗叶片中丙二醛（MDA）含量降低。幼苗生长与种子活力的变化趋势一致,PEG浓度小于30%时呈增大趋势,大于30%时呈减小趋势,但均大于不做PEG处理的;幼苗叶片中MDA含量与种子膜透性变化趋势一致,PEG浓度小于25%时,呈降低趋势,大于25%时则呈升高趋势,但这些指标均低于不做PEG处理的。     

Molecular characterization of proteins in protein-body membrane that disappear most rapidly during transformation of protein bodies into vacuoles

During the post-germination growth of seeds, protein bodies fuse with one another and are converted to a central vacuole. To investigate this transition, protein-body membranes from dry seeds of pumpkin (Cucurbita sp.) were prepared and their protein components characterized. Five major proteins (designated MP23, MP27, MP28, MP32 and MP73) were detected in the protein-body membranes. A cDNA clone encoding both MP27 and MP32 has been isolated. The deduced precursor polypeptide was composed of a hydrophobic signal sequence, MP27 and MP32, in that order. A putative site of cleavage between MP27 and MP32 was located on the COOH-terminal side of asparagine 278, an indication that the post-translational cleavage may occur by the action of a vacuolar processing enzyme that converts proprotein precursors of seed proteins into the mature forms. Immunoelectron microscopic analysis showed that MP27 and MP32 were associated with protein-body membrane of dry pumpkin seeds. Among the five membrane proteins, MP27 and MP32 disappeared most rapidly during seedling growth. The degradation of MP27 and MP32 starts just after seed germination and proceeds in parallel with the transformation of the protein bodies into a vacuole.     

Seed vigour-related DNA marker in rice shows homology with acetyl CoA carboxylase gene

Germination potential, i.e. vigour manifested at sensu stricto germination, was studied in 22 rice varieties to distinguish between the genetic control for low vigour (lv) at fresh harvest and lv after ageing of seeds. For identifying seed vigour-associated DNA marker(s), correlative studies between (1) physiological parameters viz. germination of (a) unaged seeds under optimum and cold stressed condition, (b) aged seeds under optimal condition and (2) growth-associated biochemical parameters viz. alcohol dehydrogenase (ADH) and esterase (EST) activity during early germination of fresh seeds and antioxidant potential in dry embryos of fresh and aged (1 year) seeds were undertaken. ADH and EST activity as well as antioxidant potential showed positive correlation with speed of embryo emergence and early seedling growth of seeds. The reduction of antioxidant potential in aged seeds also appeared proportional to germination performance. Random amplified polymorphic DNA analysis between the varieties was correlated with the above-mentioned vigour parameters, using multiple regression analysis. This led to the identification of four bands associated with the vigour trait at 99% significance level; of these, the ~ 900-bp DNA fragment (named as HVAC 19), through basic local alignment search tool nucleotides analysis, revealed a sequence homology with acetyl CoA carboxylase (ACCase) gene of wheat at 94% similarity level.     

Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth

Fatty acid desaturases play important role in plant responses to abiotic stresses. However, their exact function in plant resistance to salt stress is unknown. In this work, we provide the evidence that FAD2, an endoplasmic reticulum localized ω-6 desaturase, is required for salt tolerance in Arabidopsis. Using vacuolar and plasma membrane vesicles prepared from the leaves of wild-type (Col-0) and the loss-of-function Arabidopsis mutant, fad2, which lacks the functional FAD2, we examined the fatty acid composition and Na+-dependent H+ movements of the isolated vesicles. We observed that, when compared to Col-0, the level of vacuolar and plasma membrane polyunsaturation was lower, and the Na+/H+ exchange activity was reduced in vacuolar and plasma membrane vesicles isolated from fad2 mutant. Consistent with the reduced Na+/H+ exchange activity, fad2 accumulated more Na+ in the cytoplasm of root cells, and was more sensitive to salt stress during seed germination and early seedling growth, as indicated by CoroNa-Green staining, net Na+ efflux and salt tolerance analyses. Our results suggest that FAD2 mediated high-level vacuolar and plasma membrane fatty acid desaturation is essential for the proper function of membrane attached Na+/H+ exchangers, and thereby to maintain a low cytosolic Na+ concentration for salt tolerance during seed germination and early seedling growth in Arabidopsis.