Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers

The necrotrophic pathogen Gibberella pulicaris infects potato tubers through wounds that contain fungitoxic secondary metabolites such as the phytoalexins rishitin and lubimin. In order to colonize tuber tissue, the fungus must possess a mechanism to tolerate potato defense compounds. In this paper, we show that a gene, Gpabc1, that codes an ATP-binding cassette (ABC) transporter is required for tolerance to these phytoalexins and for virulence on potato. The Gpabc1 gene, isolated in the course of a differential cDNA screen, shares high sequence homology with the ABC1 gene of Magnaporthe grisea. G. pulicaris mutants deficient in Gpabc1 were still able to metabolize rishitin but lost their tolerance to this phytoalexin as well as their virulence on potato. These results strongly suggest that the Gpabc1-encoded ABC transporter is necessary for tolerance of G. pulicaris to rishitin and that this tolerance is required for virulence on potato.     

Potato phytoalexin elicitors in Phytophthora infestans spore germination fluids

Media from germinating spores of Phytophthora infestans contain substances that elicit accumulation of the phytoalexin rishitin in potato tuber slices. Gel permeation chromatography of media extracts indicates the presence of several substancec. The active substances can be precipitated with ammonium sulfate, are heat labile and pronase-sensitive, which suggests that they are proteins.     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Biotransformation of potato stress metabolites rishitin,lubimin, and 15-dihydro lubimin by potato and soybean cell cultures

Potato callus and cell suspensions of potato and soybean were exogenously supplied with potato phytoalexin rishitin, much of which was converted by both species to an unknown tenatively identified as glutinosone. Exogenous lubimin was unaffected by the potato cell culture, but was transformed to 15-dihydro lubimin by the soybean cell suspensions. The stability of the exogenous lubimin may be ascribed to a second block in the rishitin pathway of the potato cell culture.Abbreviations MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - TMV 2,4-dichlorophenoxyacetic acid, 2,4-D; Tobacco Mosaic Virus - TLC thin layer chromatography - GC gas chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance     

An in vitro control mechanism for potato stress metabolite biosynthesis

Ethylene/oxygen (E/O₂) elevates sesquiterpenoid stress metabolite (SSM) levels in potato (Solanum tuberosum L.) tuber tissue which is reacting hypersensitively. To determine whether E/O₂ retards SSM turnover, a measured amount of rishitin was applied to tuber tissue which was then incubated in air or E/O₂, and rishitin disappearance was monitored. No difference in the rate of rishitin disappearance was detected between air and E/O₂ incubations. However, tissue treated with rishitin and incubated in E/O₂ accumulated intermediates of the katahdinone and phytuberin pathways. This was not the case in rishitin-air treatments. These results suggest the dual involvement of ethylene and SSM intermediates in the regulation of the biosynthesis of SSM, compounds which may serve as phytoalexins.     

Effects of Controlled Atmospheres on Production of Sesquiterpenoid Stress Metabolites by White Potato Tuber: Possible Involvement of Cyanide-resistant Respiration

Levels of katahdinone (solavetivone), lubimin, rishitin, and phytuberin, sesquiterpenoid stress metabolites of white potato (Solanum tuberosum), were monitored in tuber slices which were challenged with an extract of Phytophthora infestans and incubated under controlled atmospheres. A mixture of ethylene in air enhanced stress metabolite production. This enhancement was amplified by higher partial pressures of oxygen. Stress metabolite production was inhibited by salicylhydroxamic acid. These results suggest the involvement of cyanide-resistant respiration in the production of potato stress metabolites, compounds which may serve as phytoalexins.     

Detoxification of sesquiterpene phytoalexins byGibberella pulicaris (Fusarium sambucinum) and its importance for virulence on potato tubers

Summary Gibberella pulicaris (Fusarium sambucinum) is a major cause of dry-rot of stored potatoes (Solanum tuberosum) worldwide. The ability of field strains ofG. pulicaris to cause dry-rot is correlated with their ability to detoxify sesquiterpene phytoalexins produced by potato. All highly virulent field strains can detoxify the sesquiterpenes rishitin and lubimin. Meiotic recombinational analysis indicates that rishitin detoxification can be controlled at two or more loci. High virulence has been associated with one of these loci, designatedRiml. Detoxification of rishitin and lubimin comprises a complex pattern of reactions involving epoxidation, dehydrogenation, and cyclization. To date, seven lubimin metabolites and one rishitin metabolite have been characterized. Genes for rishitin and lubimin detoxification are being cloned fromG. pulicaris in order to more rigorously analyze the role and regulation of sesquiterpene metabolism in potato dry-rot. Our results indirectly support a role for sesquiterpene phytoalexins in resistance of potato tubers to dry-rot and may enhance research on alternative control strategies for this economically important potato disease.     

Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum

Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites. Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 g g^–1 dry wt) was the most predominant followed by lubimin (171 g g^–1 dry wt). The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures. The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites.     

A comparison of the sites of phytoalexin accumulation and of biosynthetic activity in potato tuber tissue inoculated with biotic elicitors

Aged discs cut from Kennebec potato tubers were inoculated with one of the following: an elicitor preparation from mycelia of Phytophthora infestans race 4, zoospores from either race 4 or race TY complex of this fungus, or sodium arachidonate. At 24 hr intervals after inoculation, four successive 0.5 mm thick layers of tissue were cut from the discs. This tissue was analysed for accumulated phytoalexins and also used to prepare cell-free enzyme systems for lubimin biosynthesis. In tissue treated with either the elicitor preparation or race 4 zoospores, levels of phytoalexin accumulation were highest in the first layer of tissue. Surprisingly, however, cell-free lubimin biosynthesis from [1-¹⁴C]isopentenyl pyrophosphate was also generally greater in preparations derived from the first 0.5 mm of tissue. Accumulation of phytoalexins in tissue inoculated with zoospores from race TY complex was very low, whereas cell-free biosynthetic activity was initially comparable to that seen in preparations from tissue treated with the elicitor preparation. By the end of the experimental period lower layers of tissue from discs treated with sodium arachidonate contained the highest levels of phytoalexins and yielded cell-free enzyme preparations with the greatest lubimin biosynthetic activity.     

Studies on the biosynthesis and metabolism of the phytoalexin lubimin and related compounds in Datura stramonium L.

Arachidonic acid, cellulase, CuSO₄, a sonicate of Phytophthora infestans mycelium and a spore suspension of Penicillium chrysogenum all elicited the formation of the sesquiterpenoid phytoalexins lubimin, 3-hydroxylubimin and rishitin in fruit cavities of Datura stramonium. 3-Hydroxylubimin was the predominant phytoalexin formed after treatment of the fruits with arachidonic acid, cellulase and the P. infestans preparation. Copper sulphate was a potent elicitor of lubimin but not 3-hydroxylubimin. The fungus P. chrysogenum metabolized lubimin and 3-hydroxylubimin to 15-dihydrolubimin and 3-hydroxy-15-dihydrolubimin respectively, both in fruit cavities inoculated with spores of this fungus and in pure culture. The 15-dihydrolubimin formed in the fruits by the fungus was further metabolized (by the fruits) to both isolubimin and 3-hydroxy-15-dihydrolubimin. The precursor-product relationships between all of the subject compounds was investigated by feeding experiments with ³H-labelled compounds. 2-Dehydro-[15-³H₁]lubimin was rapidly and efficiently incorporated into lubimin and may be the direct precursor of lubimin in planta. 3-Hydroxy[2-³H₁]lubimin was incorporated into the nor-eudesmane rishitin but 10-epi-3-hydroxy[2-³H₁]lubimin was not. An updated scheme for the biosynthesis and metabolism of lubimin and related compounds in infected tissues of solanaceous plants is presented.We thank Mr Vic Swetez for the provision of plant material, Mrs Margaret Huffee for technical assistance, Dr David Ewing for help with obtaining NMR spectra, and the Agricultural and Food Research Council for financial support.     

Novel study on the elicitation of hypersensitive response by polyunsaturated fatty acids in potato tuber.

A GC-MS procedure was carried out for the simultaneous and unequivocal quantitation of both potato phytoalexin (rishitin and lubimin) accumulation and the rate of disappearance of polyunsaturated fatty acids (PUFA) and some of their esters tested as possible elicitors. Potato 5-lipoxygenase and lipolytic acyl hydrolase play a key role in hypersensitive response (HR) induction. As expected, arachidonic acid, its hydrolysable esters, and eicosapentaenoic acid elicited much higher HR than the other PUFA tested, although the latter were equally affected by potato 5-lipoxygenase. Hydroxyl radicals appear to be actively involved in the browning process. The polyaminoacid poly-L-lysine did not show any eliciting activity.     

Effect of Mastoparan on Phospholipase A2 Activity in Potato Tubers Treated with Fungal Elicitor

Participation of phospholipase (PL) A₂ in signal trans-ductionhas been reported to elicitate a resistance reaction in potatocells by inoculation of an incompatible race of Phytophthorainfestans, the late blight fungus, or by treatment with fungalelicitor hyphal wall components (HWC). Mastoparan, a genericG protein activator, has been shown to activate PLA in a G protein-dependentmanner in animal cells. We analyzed the effects of mastoparanand the inactive analog Mas-17 on PLA₂ activity in potato tubers.In healthy potato tubers, the activation of PLA₂ by mastoparanwas detected in the soluble fraction, but not micro-somal fraction.However, in potato tubers treated with HWC, PLA₂ activity wasstimulated by mastoparan in both soluble and microsomal fractions.Pretreatment of the microsomal fraction with neomycin, a PLCinhibitor, and staurosporine, a protein kinase inhibitor, inhibitedthe mastoparan-induced activation of PLA₂. This suggested thatthe PLA₂ activation in potato tubers by mastoparan was mediatedby the PLC pathway and protein phospho-rylation. We also examinedthe accumulation of potato phytoalexin rishitin. Mastoparanstimulated rishitin accumulation induced by HWC, but did notinduce the accumulation. This indicated that mastoparan mightactivate the signal transduction pathway in the resistance reactionsinduced in potato tubers. (Received March 12, 1998; Accepted August 6, 1998)     

Host-Pathogen Interactions: XV. Fungal Glucans Which Elicit Phytoalexin Accumulation in Soybean Also Elicit the Accumulation of Phytoalexins in Other Plants

A β-glucan isolated from the mycelial walls of Phytophthora megasperma var. sojae and a glucan purified from yeast extract stimulate the accumulation of phytoalexins in red kidney bean, Phaseolus vulgaris, and stimulate the accumulation of the phytoalexin, rishitin, in potato tubers, Solanum tuberosum. These glucans have previously been shown to be potent elicitors of glyceollin accumulation in soybean, Glycine max.

Treatment of kidney bean cotyledons with the glucan elicitors resulted in the accumulation of at least five fungistatic compounds. These compounds migrate during thin layer chromatography identically to the fungistatic compounds which accumulate in kidney beans which have been inoculated with Colletotrichum lindemuthianum, a fungal pathogen of kidney beans.

Potatoes accumulate as much as 29 micrograms of rishitin per gram fresh weight following exposure to the glucan from Phytophthora megasperma var. sojae and as much as 19.5 micrograms of rishitin per gram fresh weight following exposure to yeast glucan. Potatoes accumulated 28 micrograms of rishitin per gram fresh weight following inoculation with live Phytophthora megasperma var. sojae.

     

Metal Selectivity of Exocytosis in α-Toxin-Permeabilized Bovine Chromaffin Cells

Abstract: Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca²⁺, Ba²⁺, Sr²⁺, Pb²⁺, La³⁺, Cd²⁺, Co²⁺, Tb³⁺, Mn²⁺, and Zn²⁺ on the release of norepinephrine (NE) from staphylococcal α-toxin-permeabilized bovine chromaffin cells. Pb²⁺, La³⁺, Cd²⁺, Sr²⁺, and Ba²⁺ activated NE secretion accompanied by the release of intragranular dopamine β-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺ was nonadditive with Ca²⁺, indicating a common site of action. In contrast, the Ba²⁺-evoked NE release was additive with Ca²⁺ and the Ca²⁺ agonists Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺, suggesting that Ba²⁺ activates secretion at a site distinct from the Ca²⁺ receptor. In distinction to the NE release evoked by Pb²⁺, La³⁺, Cd²⁺, and Ba²⁺, the Sr²⁺-evoked NE release was associated with a significant elevation of Ca²⁺ concentration in the medium and abolished by Ca²⁺ chelation. This indicates that the secretagogue effect of Sr²⁺ was indirect and secondary to the displacement of bound Ca²⁺. Co²⁺ and Mn²⁺ inhibited the NE release evoked by Ca²⁺, Sr²⁺, Pb²⁺, La³⁺, and Cd²⁺ but had no effect on the Ba²⁺-dependent secretion. Tb³⁺ and Zn²⁺ were without effect on exocytosis.     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.     

Ca 2+ transport activity in mitochondria from some plant tissues

Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca²⁺. Additions of Ca²⁺ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca²⁺ was largely accumulated. Accumulation of Ca²⁺ required phosphate and, in most cases, was stimulated by Mg²⁺ and ADP or ATP. Ca²⁺ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca²⁺ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca²⁺ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca²⁺ binding sites sensitive to uncouplers, La³⁺, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca²⁺, probably due to lack of a specific Ca²⁺ carrier.     

Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans

In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd²⁺ is strongly inhibited by Ca²⁺ ions; furthermore, Ca²⁺ reduces the net accumulation of Cd²⁺ by worms. Both Ca²⁺ and a variety of calcium uptake inhibitors (nifedipine, La³⁺, verapamil) depress the dose response of the transgene to Cd²⁺. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd²⁺ treated worms, but has a much larger effect in the case of Mn²⁺, reflecting its much greater affinity for this ion.     

Stimulation of Potassium Uptake by Abscisic Acid in Potato Tuber Tissues. Relation with H+ and Ca2+ Fluxes

Previous results with potato tuber discs showed that a treatment with abscisic acid stimulated K⁺ uptake. In this investigation, we determine the relationship between increased K' uptake and H⁺extrusion, and Ca²⁺ fluxes by treating tissues with specific Ca²⁺ channel blocker (La³⁺), calmodulin (CaM) inhibitors (chlorpromazine and W₇), and with Ca²⁺ ionophore (A23187). K⁺ uptake increased with increasing external pH whether tissues were treated with ABA or not. Treatment of tissues with La³⁺ inhibited K⁺ uptake, whereas CaM inhibitors have no effect. By contrast ABA and A23187 produced a synergistic effect, suggesting that ABA may act in part, on K⁺ uptake, like a Ca²⁺ agonist, in accord with Huddart's hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.