Evidence for the involvement of cysteine proteases in the regulation of methyl jasmonate-induced cell death in grapevine

A new system to study programmed cell death (PCD) in plants is described. Grapevine (Vitis vinifera L., cv. Limberger) leaves and suspension cells were induced to undergo a form of cell death that mimics the hypersensitive response (HR) by treatment with a lipid-derived molecule, methyl jasmonate (MeJA). This chemical-induced cell death was accompanied by the characteristic features of apoptosis in animal and plant cells, such as typical changes in nuclear morphology, the fragmentation of the nucleus and protoplast collapse. Local and ectopic treatment of grapevine leaves with phenylmethylsulfonyl fluoride (PMSF), leupeptin, and especially with a specific inhibitor of cysteine proteases, E-64, inhibited MeJA-induced cysteine protease activity and blocked PCD triggered by 50 muM MeJA. These results indicate that proteolysis plays a crucial role in MeJA-induced apoptosis and that this type of PCD can be regulated by activity poised between the cysteine protease and the cysteine protease inhibitor.

A bestatin primes grapevine cells for augmented elicitation of the hypersensitive-like cell death and associated defense responses by methyl jasmonate

Localized treatment of grapevine (Vitis vinifera L. cv. Limberger) leaves with bestatin, an inhibitor of some aminopeptidases in plants and animals, augmented the sensitivity for methyl jasmonate-induced hypersensitive-like response. Enhanced resveratrol accumulation was associated with potentiated activation of genes encoding phenylalanine ammonia-lyase (PAL). The augmentation of PAL gene induction was proportional to the length of pretreatment with bestatin, indicating time-dependent printing of the cells. Exogenously supplied bestatin also potentiated other characteristic elicitor-induced short- and long-term defense responses in cell suspensions of grapevine including strong medium alkalinization and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Bestatin therefore appears to be exerting its effects close to the level of transcriptional control of defense-related genes, where it might inhibit a regulatory protease. Strikingly, the ability of bestatin to potentiate grapevine PAL gene elicitation and resveratrol accumulation, emphasizes an important role for defense response potentiation in acquired plant disease resistance.

New findings to the role of tunikamycin in grapevine: Disease defense responses

Exogenous methyl jasmonate (MeJA) is an effective trigger of cellular damage resulting in the development of limited necrotic lesions that mimic the hypersensitive reaction (HR) lesions associated with resistance to avirulent pathogens. Localized treatment of leaves of intact grapevines (Vitis vinifera L. cv. Limberger) or excised leaves with tunikamycin stimulates an agonist-dependent mechanism operating at an early step in the signal pathway for induction of MeJA-dependent UR-like response. With respect to tunikamycin, the fine control mechanism has shown to be both, concentration- and time-dependent. The same treatment also antagonized H2O2 accumulation from the MeJA-induced oxidative burst suggesting that this type of reactive oxygen intermediate plays a minor role in the induction of the HR in grapevine cells challenged by exogenous MeJA. Moreover, our results indicate that the activation of defense reactions of grapevine, at least in part, is dependent and sensitive to N-linked glycosylation.

Immunodetection of PR-1-like proteins in grapevine leaves infected with Oidium tuckeri and in elicited suspension cell cultures

Three pathogenesis-related (PR-1-like) proteins extractable at pH 8.0 were found to accumulate in grapevine leaves after fungal pathogen (Oidium tuckeri) infection. These proteins were called gPR-1 (grapevine pathogenesis-related) proteins. Estimated molecular masses in SDS-containing gels were: gPR-1a 15.5 kDa; gPR-1b 16.8 kDa; gPR-1c 17.7 kDa. Antiserum raised against tobacco PR-la reacted specifically with grapevine counterparts. Likewise, stimulation of gPR-1 protein accumulation was observed when a set of prototype elicitors was added to grapevine cell suspension cultures. Results with inducing elicitors also showed that the extracellular PR-1-like proteins represent the only isoforms of this prominent group of pathogenesis-related proteins found in grapevin

Methyl jasmonate induces a hypersensitive-like response of grapevine in the absence of avirulent pathogens

Methyl jasmonate (MeJA), a common plant secondary compound, when applied to the surface of grapevine leaves, caused the formation of lesions that mimic a typical hypersensitive response. Sustained exposure of grapevines to 50 µM MeJA provoked tissue damage, stimulated salicylic acid production, and expression of defense-related genes. Besides these local responses, after several days systemic expression of defense-related genes was induced as well, Thus, grapevine cells that perceived MeJA generated a cascade of events acting at local, short and long distances and causing the coordinated expression of specific defense responses with a timing and magnitude similar to the hypersensitive response against pathogens, MeJA represents a powerful tool to investigate the signals and their respective pathways involved in mechanism of induced disease resistance of grapevine.

Biological activity of the elicitor released from mycelium of a grapevine isolate of the necrotrophic fungus Botrytis cinerea

To obtain primary insight into the pathway(s) by which defense responses in grapevine (Vitis vinifera L.) are induced, suspension cultures of grapevine cells were treated with an elicitor released from the mycelium of the necrotrophic fungal pathogen Botrytis cinerea (PERs et FRIES). It induced a typical array of defense responses, including cell death accompanied by the production of H2O2 from the oxidative burst and accumulation of diverse groups of pathogenesis-related (PR) proteins and key enzymes of the general phenylpropanoid pathway, comprising phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI). Nuclear run-off experiments demonstrated that the fungal elicitor caused rapid transcriptional activation of genes encoding diverse defense-related products followed by a massive salicylic acid production.

Early cadmium-induced effects on reactive oxygen species production, cell viability and membrane electrical potential in grapevine roots

Cadmium (Cd) is one of the most worldwide concerned metal pollutants. It is able to induce reactive oxygen species production through indirect mechanisms causing oxidative stress. Vitis vinifera roots were treated with 100 μM Cd for 0-180 min or 20-100 μM Cd for 24 h. Fluorescence confocal microscopy showed elevated hydrogen peroxide and superoxide levels in the apical root segments. Two phases (after 30 min and 24 h) of the superoxide raised levels were observed. This was accompanied by the decrease in root cell viability. Cd in concentrations between 0.005-10 mM induced significant, but different changes in membrane electrical potential (EM) of the root epidermal cells. The low concentrations of Cd (0.005-0.01 mM) caused transient EM hyperpolarization followed by depolarization, whereas by higher concentrations (0.05-5.0 mM) EM was depolarized. In any case, the depolarization or hyperpolarization were only transient up to 5 mM Cd concentration indicating that the plasma membrane function was not irreversibly destroyed. Hyperpolarization of EM induced by fusicoccin (FC) was completely suppressed only in the presence of 10 mM Cd pointing to the inhibition of H+-ATPase. The results suggest that the Cd interactions, depending on cellular development, result in activation of a complex of various mechanisms such as peroxide and hydrogen peroxide production, which in turn may be a more probable reason for the root cell responses to Cd toxicity than the transient EM changes

Dissimilar responses of membrane potential (EM), permeability properties and respiration to cadmium and nickel in maize root cells

The short-term treatment with Cd2+ and Ni2+ triggered transient depolarization of transplasma membrane potential (EM) in the outer cortical root cells of two maize cultivars (cv. Premia and cv. Blitz), however, both metals changed the EM in a quantitatively different way. The magnitude and duration of EM depolarization were concentration dependent and were greater in the metal susceptible cv. Blitz. The highest EM depolarization was recorded with simultaneous application of Cd2+ + Ni2+ in both maize cultivars. The EM depolarization induced by Cd2+ or Cd2+ + Ni2+ but not Ni2+ alone was accompanied with a tremendous increase of membrane conductivity, but it was not accompanied with the effect of heavy metals (HM) on respiration. Simultaneous application of fusiccocin (FC) with Cd2+ or Cd2+ + Ni2+ during the EM depolarization, inability of FC to stop the depolarization by FC-enhanced proton extrusion and rapid restoration of EM, suggested a transient inhibition of the plasma membrane H+-ATPase by these toxic metals. Our data support the opinion that differences in the effects of the studied ions were not the result of their direct action on PM, but rather of their different influence on intracellular processes within root cells

Vector coherent state representations, induced representations, and geometric quantization: I. Scalar coherent state representations

Coherent state theory is shown to reproduce three categories of representations of the spectrum generating algebra for an algebraic model: (i) classical realizations which are the starting point for geometric quantization; (ii) induced unitary representations corresponding to prequantization; and (iii) irreducible unitary representations obtained in geometric quantization by choice of a polarization. These representations establish an intimate relation between coherent state theory and geometric quantization in the context of induced representations.Comment: 29 pages, part 1 of two papers, published versio

Testing the Master Constraint Programme for Loop Quantum Gravity III. SL(2,R) Models

This is the third paper in our series of five in which we test the Master Constraint Programme for solving the Hamiltonian constraint in Loop Quantum Gravity. In this work we analyze models which, despite the fact that the phase space is finite dimensional, are much more complicated than in the second paper: These are systems with an SL(2,\Rl) gauge symmetry and the complications arise because non -- compact semisimple Lie groups are not amenable (have no finite translation invariant measure). This leads to severe obstacles in the refined algebraic quantization programme (group averaging) and we see a trace of that in the fact that the spectrum of the Master Constraint does not contain the point zero. However, the minimum of the spectrum is of order $\hbar^2$ which can be interpreted as a normal ordering constant arising from first class constraints (while second class systems lead to $\hbar$ normal ordering constants). The physical Hilbert space can then be be obtained after subtracting this normal ordering correction.Comment: 33 pages, no figure