Changes in cytosolic Mg2+ levels can regulate the activity of the plasma membrane H+-ATPase in maize.

Plant PM (plasma membrane) H+-ATPase, a major consumer of cellular ATP, is driven by the MgATP complex which may dissociate at low cytosolic Mg2+ activity. We investigated whether hydrolytic activity of PM H+-ATPase is inhibited at ATP concentrations exceeding the Mg2+ concentration. Activity in isolated maize PMs was measured at pH 6.5 in the presence of 5 mM Mg2+ (high) or 2 mM Mg2+ (low), whereas K+ was applied at concentrations of 155 mM (high) or 55 mM (low). In all experiments, with membrane vesicles either from roots or leaves, the enzyme activity decreased in the presence of Mg2+-free ATP. At inhibitory ATP concentrations, the activity was not influenced by the K+ concentration. The activity was restored after increasing the Mg2+ concentration. ATP inhibition also occurred at pH 7.5. Kinetic modelling shows that Mg2+-free ATP acted as a competitive inhibitor with a Ki in the range of the Km. Ki decreased by 75% at low K+ concentration. Ki was one order of magnitude lower at pH 7.5 compared with pH 6.5. The observed inhibition is consistent with a concept in which down-regulation of the cytosolic Mg2+ activity is involved in (phyto)hormonal stress responses.

Hot-Water Hemicellulose Extraction from Fruit Processing Residues.

Hemicelluloses are an abundant biopolymer resource with interesting properties for applications in coatings and composite materials. The objective of this investigation was to identify variables of industrially relevant extraction processes that increase the purity of hemicelluloses extracted from fruit residues. Our main finding is that extraction with subcritical water, followed by precipitation with alcohol, can be adjusted to yield products with a purity of at least 90%. Purity was determined based on the total concentration of glucose, galactose, xylose, arabinose, and mannose after hydrolysis with sulfuric acid. In the first experimental design (DoE methodology), the effects of extraction temperature (95-155 °C) and time (20-100 min) on yield and purity were studied. A clear trade-off between yield and purity was observed at high temperatures, indicating the selective removal of impurities. In the second experimental design, the influence of extract pH and alcohol concentration on yield and purity was investigated for the raw extract and a concentrate of this extract with 1/6 of the original volume. The concentrate was obtained by ultrafiltration through ceramic hollow-fiber membranes. The highest purity of 96% was achieved with the concentrate after precipitating with 70% alcohol. Key factors for the resource efficiency of the overall process are addressed. It is concluded that extraction with subcritical water and ultrafiltration are promising technologies for producing hemicelluloses from fruit residues for material applications.

Silicon-mediated growth promotion in maize (Zea mays L.) occurs via a mechanism that does not involve activation of the plasma membrane H+-ATPase.

Silicon (Si)-mediated growth promotion of various grasses is well documented. In the present study, Si-induced changes in maize shoot growth and its underlying mechanisms were studied. Maize plants were grown with various concentrations of Si (0-3 mM) in the nutrient solution. Silicon nutrition improved plant expansion growth. Silicon-supplied maize plants (0.8 and 1.2 mM) showed higher plant height and leaf area compared to no-Si amended plants. It was assumed that Si-induced expansion growth was due to positive Si effects on plasma membrane (PM) H+-ATPase. In this context, western blot analysis revealed an increase in PM H+-ATPase abundance by 77% under Si nutrition. However, in vitro measurements of enzyme activities showed no significant effect on apoplast pH, proton pumping, passive H+ efflux and enzyme kinetics such as Km, Vmax, and activation energy. Further, these results were confirmed by in vivo ratiometric analysis of apoplastic pH, which showed non-significant changes upon Si supply. In contrast, 1 mM Si altered the relative transcripts of specific PM H+-ATPase isoforms. Silicon application resulted in a significant decrease of MHA3, and this decrease in transcription seems to be compensated by an increased concentration of H+-ATPase protein. From these results, it can be concluded that changes in cell wall composition and PM H+-ATPase may be responsible for Si-mediated growth improvement in maize.

Structure-Function Relationships of Antimicrobial Peptides and Proteins with Respect to Contact Molecules on Pathogen Surfaces.

The Antimicrobial peptides (e.g. defensins, hevein-like molecules and food-protecting peptides like nisin) are able to interact specifically with contact structures on pathogen surfaces. Besides protein receptors, important recognition points for such contacts are provided by pathogen glycan chains or surface lipids. Therefore, structural data concerning surface exposed glycans and lipids are of the highest clinical interest since these recognition functions play a key role when optimising anti-infection therapies. Approaches in nanomedicine and nanopharmacology in which various biophysical techniques such as NMR (Nuclear Magnetic Resonance), AFM (Atomic Force Microscopy), SPR (Surface Plasmon Resonance) and X-ray crystallography can be combined with biochemical and cell-biological methods will lead to improved antimicrobial peptides by this rational drug design approach. Such a strategy is extremely well suited to support clinical studies focussing on an effective fight against multiresistant pathogens. The data sets which are described here can be considered as universal for the design of various antimicrobial drugs against certain pathogens (bacteria, viruses and fungi) which cause severe diseases in humans and animals. Furthermore, these insights are also helpful for progressing developments in the field of food conservation and food preservation. A detailed analysis of the structure-function relationships between antimicrobial peptides and contact molecules on pathogen surfaces at the sub-molecular level will lead to a higher degree of specificity of antimicrobial peptides.

Apoplastic pH signaling in barley leaves attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei.

To investigate apoplastic responses of barley (Hordeum vulgare L.) to the barley powdery mildew fungus Blumeria graminis f. sp. hordei, noninvasive microprobe techniques were employed. H(+)- and Ca(2+)-selective microprobes were inserted into open stomata of barley leaves inoculated with Blumeria graminis f. sp. hordei race A6 conidia. Resistance gene-mediated responses of barley genotype Ingrid (susceptible parent line) and the near-isogenic resistant Ingrid backcross lines (I-mlo5, I-Mla12, and I-Mlg) were continuously monitored from 20 min to 4 days after inoculation. The main events were categorized as short-term responses around 2 h after inoculation (hai), intermediate responses around 8 and 12 hai, and long-term responses starting between 21 and 24 hai. Short-term responses were rapid transient decreases of apoplastic H(+)- and Ca2+ activities that lasted minutes only. Kinetics were similar for all genotypes tested, and thus, these short-term responses were attributed as nonspecific first encounters of fungal surface material with the host plasma membrane. This is supported by the observation that a microinjected chitin oligomer (GlcNAc)8 yielded similar apoplastic alkalinization. Intermediate responses are trains of H+ (increase) spikes that, being different in susceptible Ingrid and penetration-resistant I-mlo5 (or I-Mlg), were interpreted as accompanying specific events of papillae formation. Long-term events were massive slow and long-lasting alkalinizations up to two pH units above control. Since these latter changes were only observed with near-isogenic hypersensitive reaction (HR)-mounting genotypes I-Mla12 and I-Mlg but not with I-mlo5 or, to a smaller extent, with susceptible Ingrid, both lacking significant rates of HR, they were rated as cell death specific. It is concluded that apoplastic pH changes are important indicators of host-pathogen interactions that correlate with both the different stages of fungal development and the different types of host defense response.

Nanoinfusion: an integrating tool to study elicitor perception and signal transduction in intact leaves.

• To study elicitor effects in intact leaves of Hordeum vulgare cv. Ingrid, chitin fragments were delivered to substomatal cavities with a micropipette. Responses were monitored by a calibrated reference microelectrode and a pH-sensitive microelectrode, simply positioned below neighbouring open stomata in the air-filled space of the substomatal cavities. • Flooding of a leaf spot of approx. 600 × 300 µm with physiological aqueous solutions caused an immediate transient polarization of the extracellular solution in the order of -25 ± 12 mV. Immediately after the pipette solution was consumed, the extracellular solution was again polarized, still before the cavities dried out. In dry cavities, the extracellular equilibrium potential was -34 ± 10 mV. On flooding, the extracellular pH rose to 5.7 ± 0.3 after approx. 12 ± 7 min and returned to a stable level of 5.2 ± 0.3 after 30 min. • Sequential infusion on the same leaf spot, first with elicitor-free solution, then with the same solution containing 25 µmN-acetyl-chitooctaose, into the still-flooded cavities yielded an elicitor-specific pH maximum between 6 and 7 approx. 10 min after flooding. A pronounced pH maximum > 7 occurred between 40 and 240 min after flooding in wild-type plants. • The use of elicitor nanoinfusion for the integrated development of resistance inducers in cereals, wine and poplar is discussed.

Diferulic acids in the cell wall may contribute to the suppression of shoot growth in the first phase of salt stress in maize.

In the first phase of salt stress the elongation growth of maize shoots is severely affected. The fixation of shape at the end of the elongation phase in Poaceae leaves has frequently been attributed to the formation of phenolic cross-links in the cell wall. In the present work it was investigated whether this process is accelerated under salt stress in different maize hybrids. Plants were grown in nutrient solution in a growth chamber. Reduction of shoot fresh mass was 50% for two hybrids which have recently been developed for improved salt resistance (SR 03, SR 12) and 60% for their parental genotype (Pioneer 3906). For SR 12 and Pioneer 3906, the upper three leaves were divided into elongated and elongating tissue and cell walls were isolated from which phenolic substances and neutral sugars were determined. Furthermore, for the newly developed hybrids the activity of phenolic peroxidase in the cell wall was analysed in apoplastic washing fluids and after sequential extraction of cell-wall material with CaCl2 and LiCl. The concentration of ferulic acid, the predominant phenolic cross-linker in the grass cell wall, was about 5mgg(-1) dry cell wall in elongating and in elongated tissue. The concentration of diferulic acids (DFA) was 2-3mgg(-1) dry cell wall in both tissues. Salt stress increased the concentration of ferulic acid (FA) and DFA in the parental genotype Pioneer 3906, but not in SR 12. Both genotypes showed an increase in arabinose, which is the molecule at which FA and DFA are coupled to interlocking arabinoxylan polymers. In SR 12, the activity of phenolic peroxidase was not influenced by salt stress. However, in SR 03 salt stress clearly increased the phenolic peroxidase activity. Results are consistent with the hypothesis that accelerated oxidative fixation of shape contributes to growth suppression in the first phase of salt stress in a genotype-specific manner.

Modulatory ATP binding to the E2 state of maize plasma membrane H+-ATPase indicated by the kinetics of vanadate inhibition.

P-type ATPases, as major consumers of cellular ATP in eukaryotic cells, are characterized by the formation of a phosphorylated enzyme intermediate (E2P), a process that is allosterically coupled to translocation of cations against an electrochemical gradient. The catalytic cycle comprises binding of Mg-ATP at the nucleotide-binding domain, phosphorylation of the E1 state (E1), conformational transition to the E2P state, and dephosphorylation through the actuator domain and re-establishment of the E1 state. Recently, it has been suggested that, for several P-type ATPases, Mg-ATP binds to the phosphorylated enzyme, thereby accelerating the transition to the E1 state, before then becoming the enzyme's catalytic substrate. Here, we provide evidence supporting this viewpoint. We employed kinetic models based on steady-state kinetics in the presence and absence of the reversible inhibitor orthovanadate. Vanadate is generally considered to be a conformational probe that specifically binds to the E2 state, arresting the enzyme in a state analogous to the E2P state. Hydrolytic H(+) -ATPase activities were measured in inside-out plasma membrane vesicles isolated from roots and shoots of maize plants. For root enzymes, kinetic models of vanadate inhibition that allow simultaneous binding of Mg-ATP and vanadate to the same enzyme state were most plausible. For shoot enzymes, application of the competitive inhibitor Mg-free ATP attenuated vanadate inhibition, which is consistent with a model in which either Mg-free ATP or Mg-ATP is bound to the enzyme when vanadate binds. Therefore, data from roots and shoots indicate that binding of ATP species before transition to the E1 state plays an important role in the catalytic cycle of plant plasma membrane H(+) -ATPase.

In vitro effect of different Na+/K+ ratios on plasma membrane H+ -ATPase activity in maize and sugar beet shoot.

Plant growth is impaired primarily by osmotic stress in the first phase of salt stress, whereas Na+ toxicity affects the plant growth mainly in the second phase. Salinity leads to increased Na+/K+ ratio and thus displacement of K+ by Na+ in the plant cell. Relatively higher cytosolic Na+ concentrations may have an effect on the activity of plasma membrane (PM) H+ -ATPase. A decreased PM-H+ -ATPase activity could increase the apoplastic pH. This process could limit the cell-wall extensibility and thus reduce growth according to the acid growth theory. To compare the effect of Na+ on PM H+ -ATPase activity in salt-sensitive maize (Zea mays L.) and salt-resistant sugar beet (Beta vulgaris L.) shoot, PM vesicles were isolated from growing shoots of both species and ATPase activity was determined by assaying the P(i) released by hydrolysis of ATP. The H+ pumping activity was measured as the quenching of acridine-orange absorbance. An increased Na+/K+ ratio decreased the PM H+ -ATPase activity in vesicles of maize as well as of sugar beet shoots. Nevertheless, the detrimental effect of increased Na+/K+ ratio was more severe in salt-sensitive maize compared to salt-resistant sugar beet. At 25 mM Na+ concentration, hydrolytic activity was not affected in sugar beet. However, a significant decrease in hydrolytic activity was observed in maize at pH 7. In maize and sugar beet, reduction in active H+ flux was 20% and 5% at 25 mM Na+ concentration in the assay, respectively. The active H+ flux was decreased to 80% and 60%, when 100 mM K+ were substituted by 100mM Na+. We conclude that PM H+ -ATPases of salt-resistant sugar beet and maize shoot are sensitive to higher concentration of Na+. However, sugar beet PM-H+ -ATPases are relatively efficient and may have constitutive resistance against lower concentration (25 mM) of Na+ as compared to that of salt-sensitive maize.

Hydrolytic and pumping activity of H+-ATPase from leaves of sugar beet (Beta vulgaris L.) as affected by salt stress.

Cell wall extensibility plays an important role in plant growth. According to the acid-growth theory, lower apoplastic pH allows extension growth by affecting cell wall extensibility. A lowered apoplastic pH is presumed to activate wall-loosening enzymes that control plant growth. Plasma membrane (PM) H(+)-ATPases play a major role in the apoplastic acidification by H(+) transport from cytosol to the apoplast. A salt-induced decrease in H(+)-pumping activity of plasma membrane H(+)-ATPases in salt-sensitive maize plants has previously been found. This led us to formulate the hypothesis that salt-resistant plant species such as sugar beet (Beta vulgaris L.) may have a mechanism to eliminate the effect of higher salt concentrations on plasma membrane H(+)-ATPase activity. In the present study, sugar beet plants were grown in 1mM NaCl (control) or 150 mM NaCl in hydroponics. H(+)-ATPase hydrolytic and pumping activities were measured in plasma membrane vesicles isolated from sugar beet shoots. We found that plasma membrane H(+)-ATPase hydrolytic and pumping activities were not affected by application of 150 mM NaCl. Moreover, apoplastic pH was also not affected under salt stress. However, a decrease in plant growth was observed. We assume that growth reduction was not due to a decrease in PM-H(+)-ATPase activity, but that other factors may be responsible for growth inhibition of sugar beet plants under salt stress.

CO(2)-triggered chloride release from guard cells in intact fava bean leaves. Kinetics of the onset of stomatal closure.

The influence of CO(2) on Cl(-) release from guard cells was investigated within the intact leaf by monitoring the Cl(-) activity in the apoplastic fluid of guard cells with a Cl(-)-sensitive microelectrode. In illuminated leaves adapted to a CO(2) concentration within the cuvette of 350 microL L(-1), an increase of 250 microL L(-1) CO(2) triggered a transient rise in the apoplastic Cl(-) activity from 3 to 14 mM within 10 min. This Cl(-) response was similar to the Cl(-) efflux evoked by turning off the light, when the substomatal CO(2) was kept constant (CO(2) clamp). Without CO(2) clamp, substomatal CO(2) increased by 120 microL L(-1) upon "light off." The response to an increase in CO(2) within the cuvette from 250 to 500 microL L(-1) in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 microL L(-1) in the light. No Cl(-) efflux was triggered by 2-min CO(2) pulses (150-800 microL L(-1)). After a switch from 350 microL L(-1) to CO(2)-free cuvette air, the guard cells were less sensitive to a rise in CO(2) and to light off, but the sensitivity to both stimuli partially recovered. Changes in CO(2) also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl(-) responses, and which also promptly occurred when CO(2) did not initiate Cl(-) efflux. The comparatively slow activation of Cl(-) efflux by CO(2) indicates that an intermediate effector derived from CO(2) has to accumulate to fully activate plasma membrane anion channels of guard cells.

The apoplastic pH of the substomatal cavity of Vicia faba leaves and its regulation responding to different stress factors.

The apoplastic pH of the substomatal cavity is an essential determinant of stomatal movement. In detached leaves of Vicia faba substomatal apoplastic pH and its dependence on external (stress) factors was investigated using a non-invasive approach: pH-microsensors were inserted into open stomata, and upon contact with the apoplastic fluid, pH was measured continuously, as apoplastic pH was challenged by changed conditions of light, atmosphere (NH(3), CO(2)), and xylem sap (abscisic acid, cyanide, fusicoccin, pH, inorganic salts). Apoplastic pH proved extremely sensitive to infiltration and local flooding, which rapidly increased the apoplastic pH by more than 1.5 pH units. Recovery from infiltration took several hours, during which light effects on the apoplastic pH were strongly impeded. This indicates that pH tests carried out under such conditions may not be representative of the undisturbed leaf. NH(3), flushed across the stomata, yielded a rapid apoplastic alkalinization from which an apoplastic buffer capacity of 2-3 mM per pH unit was calculated. Fusicoccin, fed into the xylem sap acidified the apoplast, whereas cyanide alkalized it, thus underscoring the importance of the plasma membrane H(+) pump for apoplastic pH regulation. To address the question to what extent pH was a drought signal, the effect of iso-osmotic pH changes, fed into the xylem through the petiole were tested. It is demonstrated that the apoplastic response remained below 0.1 pH per pH unit imposed, regardless of the buffer capacity. An increase in the osmolarity of the bath solution (harbouring the cut petiole) using KCl, NaCl, CaCl(2) or sorbitol alkalized the substomatal apoplast. It is suggested that pH may only act as drought signal when accompanied by elevated osmolarity.

CO2 provides an intermediate link in the red light response of guard cells.

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.