The Vacuolar Proton-Cation Exchanger EcNHX1 Generates pH Signals for the Expression of Secondary Metabolism in Eschscholzia californica.

Cell cultures of Eschscholzia californica react to a fungal elicitor by the overproduction of antimicrobial benzophenanthridine alkaloids. The signal cascade toward the expression of biosynthetic enzymes includes (1) the activation of phospholipase A2 at the plasma membrane, resulting in a peak of lysophosphatidylcholine, and (2) a subsequent, transient efflux of vacuolar protons, resulting in a peak of cytosolic H(+). This study demonstrates that one of the Na(+)/H(+) antiporters acting at the tonoplast of E. californica cells mediates this proton flux. Four antiporter-encoding genes were isolated and cloned from complementary DNA (EcNHX1-EcNHX4). RNA interference-based, simultaneous silencing of EcNHX1, EcNHX3, and EcNHX4 resulted in stable cell lines with largely diminished capacities of (1) sodium-dependent efflux of vacuolar protons and (2) elicitor-triggered overproduction of alkaloids. Each of the four EcNHX genes of E. californica reconstituted the lack of Na(+)-dependent H(+) efflux in a Δnhx null mutant of Saccharomyces cerevisiae. Only the yeast strain transformed with and expressing the EcNHX1 gene displayed Na(+)-dependent proton fluxes that were stimulated by lysophosphatidylcholine, thus giving rise to a net efflux of vacuolar H(+). This finding was supported by three-dimensional protein homology models that predict a plausible recognition site for lysophosphatidylcholine only in EcNHX1. We conclude that the EcNHX1 antiporter functions in the elicitor-initiated expression of alkaloid biosynthetic genes by recruiting the vacuolar proton pool for the signaling process.

"Self" and "non-self" in the control of phytoalexin biosynthesis: plant phospholipases A2 with alkaloid-specific molecular fingerprints.

The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the "self" plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate "self-made" from "foreign" alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins.

Alkaloid metabolism in thrips-Papaveraceae interaction: recognition and mutual response.

Frankliniella occidentalis (Pergande), the Western Flower Thrips (WFT), is a polyphagous and highly adaptable insect of the order Thysanoptera. It has a broad host range but is rarely found on Papaveraceae, which might be due to deterrent effects of alkaloids present in most species of this family. In order to test the adaptive potential of WFT, we investigated its interaction with two Papaveraceae offered as sole feeding source. We found that WFT are able to live and feed on leaves of Eschscholzia californica and Chelidonium majus. Both plants respond to thrips feeding by the enhanced production of benzophenanthridine alkaloids. Furthermore, cell cultures of E. californica react to water insoluble compounds prepared from adult thrips with enhanced alkaloid production. During feeding, WFT take up benzophenanthridine alkaloids from either plant and from an artificial feeding medium and convert them to their less toxic dihydroderivatives. This was shown in detail with sanguinarine, the most cytotoxic benzophenanthridine. A similar conversion is used in plants to prevent self-intoxication by their own toxins. We conclude that WFT causes a phytoalexin-like response in Papaveraceae, but is able to adapt to such host plants by detoxification of toxic alkaloids.

Assay of phospholipase A activity.

Phospholipases of the A type constitute a large family of esterases that catalyze the hydrolysis of the fatty acid ester bonds in phospholipids and thus generate lysophospholipids and fatty acids. Both products or their metabolites are important signal molecules in the cellular adaptation to stress, developmental processes and several diseases in plants and animals. The assay of PLA activity has been much promoted by the availability of phospholipid substrates with fluorophores at one or two fatty acids. The double labeled compounds display an increase of fluorescence due to the escape from intramolecular quenching or FRET. They thus allow the sensitive monitoring of PLA activity even without a separation of the hydrolysis products. This chapter is focused on the proper use of fluorescent (BODIPY) labelled substrates for assays of PLA activity in cells and subcellular fractions by fluorimetric analysis and classical or confocal microscopy.

Signal transfer in the plant plasma membrane: phospholipase A(2) is regulated via an inhibitory Gα protein and a cyclophilin.

The plasma membrane of the California poppy is known to harbour a PLA2 (phospholipase A2) that is associated with the Gα protein which facilitates its activation by a yeast glycoprotein, thereby eliciting the biosynthesis of phytoalexins. To understand the functional architecture of the protein complex, we titrated purified plasma membranes with the Gα protein (native or recombinant) and found that critical amounts of this subunit keep PLA2 in a low-activity state from which it is released either by elicitor plus GTP or by raising the Gα concentration, which probably causes oligomerization of Gα, as supported by FRET (fluorescence resonance energy transfer)-orientated fluorescence imaging and a semiquantitative split-ubiquitin assay. All effects of Gα were blocked by specific antibodies. A low-Gα mutant showed elevated PLA2 activity and lacked the GTP-dependent stimulation by elicitor, but regained this capability after pre-incubation with Gα. The inhibition by Gα and the GTP-dependent stimulation of PLA2 were diminished by inhibitors of peptidylprolyl cis-trans isomerases. A cyclophilin was identified by sequence in the plasma membrane and in immunoprecipitates with anti-Gα antibodies. We conclude that soluble and target-associated Gα interact at the plasma membrane to build complexes of varying architecture and signal amplification. Protein-folding activity is probably required to convey conformational transitions from Gα to its target PLA2.

Overproduction of alkaloid phytoalexins in California poppy cells is associated with the co-expression of biosynthetic and stress-protective enzymes.

Many plant cells respond to pathogens by the induction of phytoalexin biosynthesis, but the underlying changes of gene expression are often obscured by their close linkage to the complex rearrangements during pathogen defense, especially the hypersensitive cell death. In root-derived cell cultures of Eschscholzia californica, the overproduction of cytotoxic benzophenanthridine alkaloids can be triggered by a minimum of pathogen pressure that does not evoke hypersensitive reactions. Such conditions activate a signal chain that is initiated by a short contact to low concentrations of yeast glycoprotein elicitor and includes a transient acidification of the cytoplasm. In contrast, high elicitor concentrations signal via an increase of jasmonate and trigger hypersensitive cell death, preceded by a drastic decay of translatable mRNAs. The main changes in protein and mRNA patterns caused by either signal path were compared by 2D proteomic separation, MS/MS sequencing and mRNA-in vitro translation. The four proteins showing the highest overexpression were identical between cells that received low or high-elicitor treatment and overlapped with the three proteins most up-regulated by artificial pH shifts. They comprised one biosynthetic enzyme (norcoclaurine:SAM 4' O-methyl-transferase) plus a unique combination of stress-protective proteins: a heat shock protein (hsp 70); a peptidyl-prolyl-cis/trans isomerase (cyclophilin); and a glyceraldehyde-3-phosphate dehydrogenase. It appears that overproduction of the benzophenanthridine phytoalexins requires the up-regulation of a rate-limiting biosynthetic enzyme plus the coordinated expression of a specific set of protective enzymes and thus is managed like an oxidative stress.

Phytochemical and antimicrobial characterization of Macleaya cordata herb.

Macleaya cordata (plume poppy) is a source of bioactive compounds, mainly isoquinoline alkaloids which are used in phytopreparations with anti-inflammatory and antimicrobial activities. In this study, the alkaloids sanguinarine, chelerythrine, their dihydro derivatives, protopine and allocryptopine and phenolics, gallic, protocatechuic, p-hydroxybenzoic, m-hydroxybenzoic, gentisic, p-coumaric, caffeic, ferulic and sinapic acids were determined in extracts prepared from M. cordata aerial part, seeds, and seed capsules using HPLC with UV detection and/or LC/MS with electrospray ionization. The highest content of sanguinarine and chelerythrine was found in capsules. Protopine and allocryptopine were major alkaloids in leaves including footstalks. The seed oil contained dihydrosanguinarine, dihydrochelerythrine and twelve fatty acids of which linoleic, oleic, palmitic and stearic acids predominated. In addition, sanguinarine reductase, a key enzyme in sanguinarine/dihydrosanguinarine equilibrium in plants, was found for the first time, in the soluble proteins of leaves. Finally, extracts were tested for antimicrobial activity using the microdilution method on standard reference bacterial strains.

Structure and mechanism of sanguinarine reductase, an enzyme of alkaloid detoxification.

Sanguinarine reductase is a plant enzyme that prevents the cytotoxic effects of benzophenanthridine alkaloids, which are the main phytoalexins of Papaveraceae. The enzyme catalyzes the reduction of sanguinarine, the most toxic benzophenanthridine, which re-enters the cytoplasm after its primary accumulation in the cell wall region has reached a threshold concentration. We present the sequence of the gene and protein of sanguinarine reductase isolated from cell cultures of Eschscholzia californica. High sequence similarities indicate that the enzyme evolved from a plant-specific branch of the ubiquitous Rossmann fold NAD(P)H/NAD(P)(+) binding reductases, with NADP-dependent epimerases or hydroxysteroid reductases as the most likely ancestors. Based on the x-ray structure of a close homolog, a three-dimensional model of the spatial conformation and catalytic site of sanguinarine reductase was established and used for in silico screening of known three-dimensional structures. Surprisingly, the enzyme shares high structural similarity with enzymes of human and bacterial origin, which have similar functions as the plant homologs but bear little amino acid sequence similarity. Using site-directed mutagenesis, a series of recombinant enzymes was generated and assayed to reveal the impact of individual amino acids and peptides in the catalytic process. It appears that relatively few innovations were required to generate this selective catalyst for alkaloid detoxication, notably an insertion of 13 amino acids and the generation of a novel catalytic triad of Cys-Asp-His were sufficient.

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation.

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A(2) of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A(2) and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A(2) activities.

Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica.

Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

Sanguinarine reductase, a key enzyme of benzophenanthridine detoxification.

Cultured cells of Eschscholzia californica respond to a yeast glycoprotein elicitor by producing benzophenanthridine alkaloids, which are excreted into the cell wall and the outer medium. These compounds, preferentially sanguinarine, are efficient phytoalexins because of their ability to intercalate double-stranded DNA (dsDNA), penetrate membranes and inhibit various enzymes containing SH-groups. Externally added sanguinarine is rapidly taken up by intact cells and converted to dihydrosanguinarine, which is substituted intracellularly according to the biosynthetic route. A 29.5 kDa soluble enzyme that catalyses the reduction of sanguinarine and chelerythrine by either NADPH or NADH has been isolated and purified to homogeneity. Benzophenanthridines that accumulate in the outer medium, mainly 10-OH-chelerythrine, chelirubine and macarpine, are converted by the isolated enzyme and by intact cells at much slower rates than sanguinarine. The cellular capacity of uptake and conversion of sanguinarine largely surpasses the rate of alkaloid production. We conclude that the sanguinarine produced by intact cells, after excretion and binding to cell wall elements, is rapidly reabsorbed and reduced to the less toxic dihydrosanguinarine, which then undergoes further biosynthetic reactions. This recycling process would allow the presence of the toxic phytoalexin at the cellular surface without taking the risk of injuring the producing cell.

The Galpha protein controls a pH-dependent signal path to the induction of phytoalexin biosynthesis in Eschscholzia californica.

The function of a Galpha protein in the elicitation of phytoalexin (benzophenanthridine) biosynthesis was characterized in cultured cells of California poppy (Eschscholzia californica). Both the decrease of Galpha content via antisense transformation and the expression of recombinant anti-Galpha single-chain antibodies strongly impaired the induction of alkaloid biosynthesis by low elicitor concentrations. All transgenic cell types were deficient in two elicitor-triggered early signal events: activation of phospholipase A2 (PLA2) and efflux of vacuolar protons. The lacking H+ efflux could be restored (1) by adding lysophosphatidylcholine (LPC), a product of PLA2 activity, to vacuoles in situ and (2) by exposing intact cells to isotonic, near-neutral HEPES buffers. The latter treatment induced alkaloid biosynthesis in the absence of elicitor and in Galpha-deficient cells. We conclude that Galpha mediates the stimulation of PLA2 by low elicitor concentrations and that the resulting peak of LPC initiates a transient efflux of vacuolar protons. In this way, an acidic peak of the cytoplasmic pH is generated that causes the expression of enzymes of phytoalexin production independent of the hypersensitive response.

Intracellular pH signals in the induction of secondary pathways--the case of Eschscholzia californica.

Transient peaks of the cytoplasmic pH are essential elements in a number of signal cascades that activate environmental responses or developmental processes in plant cells but little is known about the mechanisms of their generation. In many plant cells, elicitation of the hypersensitive response is preceded by a perturbation of the ionic balance at the plasma membrane including the inhibition of the proton pump and the influx of H+ from the apoplast. A basically different mechanism of cytoplasmic acidification that is fed by vacuolar protons has been discovered in cell suspensions of the California Poppy (Eschscholzia californica). These cells react to a yeast glycoprotein elicitor with the overproduction of benzophenanthridine alkaloids. Low elicitor concentrations trigger the biosynthesis of these phytoalexins without invoking elements of the hypersensitive response. Accumulated data support the existence of a signal path that includes the following steps: Links between the above events that connect them within a distinct signal path are substantiated by the phenotypes of transformed cell lines that either display lowered Galpha levels due to antisense transformation or express Galpha-binding antibodies in the cytoplasm. All of these cell lines lack the elicitor-activation of PLA2 and of vacuolar proton fluxes and show an impaired phytoalexin response to low elicitor concentrations. High elicitor concentrations trigger alkaloid biosynthesis via an increase of jasmonate at a pH-independent signal path.

Selective desensitization of jasmonate- and pH-dependent signaling in the induction of benzophenanthridine biosynthesis in cells of Eschscholzia californica.

The biosynthesis of benzophenanthridine alkaloids, phytoalexins of Eschscholzia californica, in cultured cells can be induced by a glycoprotein preparation from yeast, methyljasmonate, artificial acidification with permeant acids, or mild osmotic stress. Each of these stimuli strongly attenuated the subsequent response to the same stimulus (homologous desensitization). Elicitor contact and artificial acidification mutually desensitized the cells for either signal. In contrast, elicitor-treated cells maintained their responsiveness to methyljasmonate or hyperosmolarity (sorbitol). Elicitor concentrations that nearly saturated the alkaloid response did not cause a detectable increase of jasmonate content. Transient acidification of the cytoplasm is a necessary step of signaling by low elicitor concentrations but was not detectable after jasmonate treatment. Seen together, the data indicate the existence of a jasmonate-dependent and jasmonate-independent (Delta pH controlled) signal pathway towards the expression of benzophenanthridine biosynthesis. Selective desensitization allows either stimulus to activate a distinct share of the biosynthetic capacity of the cell and limits the accumulation of toxic defense metabolites.

Elicitor-activated phospholipase A(2) generates lysophosphatidylcholines that mobilize the vacuolar H(+) pool for pH signaling via the activation of Na(+)-dependent proton fluxes.

The elicitation of phytoalexin biosynthesis in cultured cells of California poppy involves a shift of cytoplasmic pH via the transient efflux of vacuolar protons. Intracellular effectors of vacuolar proton transport were identified by a novel in situ approach based on the selective permeabilization of the plasma membrane for molecules of < or = 10 kD. Subsequent fluorescence imaging of the vacuolar pH correctly reported experimental changes of activity of the tonoplast proton transporters. Lysophosphatidylcholine (LPC) caused a transient increase of the vacuolar pH by increasing the Na(+) sensitivity of a Na(+)-dependent proton efflux that was inhibited by amiloride. In intact cells, yeast elicitor activated phospholipase A(2), as demonstrated by the formation of LPC from fluorescent substrate analogs, and caused a transient increase of endogenous LPC, as determined by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry. It is suggested that LPC generated by phospholipase A(2) at the plasma membrane transduces the elicitor-triggered signal into the activation of a tonoplast H(+)/Na(+) antiporter.

Uric acid is a genuine metabolite of Penicillium cyclopium and stimulates the expression of alkaloid biosynthesis in this fungus.

On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor L-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor L-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.