Stefan Hanstein

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.

CO2-Triggered Chloride Release from Guard Cells in Intact Fava Bean Leaves. Kinetics of the Onset of Stomatal Closure

The influence of CO2 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 CO2 concentration within the cuvette of 350 μL L−1, an increase of 250 μL L−1CO2 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 CO2 was kept constant (CO2 clamp). Without CO2 clamp, substomatal CO2 increased by 120 μL L−1 upon “light off.” The response to an increase in CO2 within the cuvette from 250 to 500 μL L−1 in dark-adapted leaves was equivalent to the response to an increase from 350 to 600 μL L−1 in the light. No Cl− efflux was triggered by 2-min CO2 pulses (150–800 μL L−1). After a switch from 350 μL L−1 to CO2-free cuvette air, the guard cells were less sensitive to a rise in CO2 and to light off, but the sensitivity to both stimuli partially recovered. Changes in CO2 also caused changes of the guard cell apoplastic voltage, which were generally faster than the observed Cl−responses, and which also promptly occurred when CO2 did not initiate Cl− efflux. The comparatively slow activation of Cl− efflux by CO2 indicates that an intermediate effector derived from CO2 has to accumulate to fully activate plasma membrane anion channels of guard cells.

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.

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.

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.

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.

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 enzymes 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.