Actinidic Acid,a New Triterpene Phytoalexin from Unripe Kiwi Fruit

Seven phytoalexins (1-7), including a new compound, were isolated from the peel of unripe kiwi fruit (Actinidia deliciosa cv. Golden King) that had been wounded and inoculated with Colletotrichum musae. The new phytoalexin (1) was identified as 2α,3β,23-trihydroxy-12,20(30)-ursadien-28-oic acid, and named actinidic acid. Phytoalexins 2-6 are known triterpenes but have not previously been described as phytoalexins. Phytoalexin 7 is the same triterpene as the phytoalexin of nectarine fruit.     

Changes in the content and biosynthesis of phytoalexins in banana fruit

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-(13)C] and [2-(13)C]cinnamic acids, and [2-(13)C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4'-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

Host-Pathogen Interactions : XXV. Endopolygalacturonic Acid Lyase from Erwinia carotovora Elicits Phytoalexin Accumulation by Releasing Plant Cell Wall Fragments

Heat-labile elicitors of phytoalexin accumulation in soybeans (Glycine max L. Merr. cv Wayne) were detected in culture filtrates of Erwinia carotovora grown on a defined medium containing citrus pectin as the sole carbon source. The heat-labile elicitors were highly purified by cation-exchange chromatography on a CM-Sephadex (C-50) column, followed by agarose-affinity chromatography on a Bio-Gel A-0.5m gel filtration column. The heat-labile elicitor activity co-purified with two α-1,4-endopolygalacturonic acid lyases (EC 4·2·2·2). Endopolygalacturonic acid lyase activity appeared to be necessary for elicitor activity because heat-inactivated enzyme preparations did not elicit phytoalexins. The purified endopolygalacturonic acid lyases elicited pterocarpan phytoalexins at microbial-inhibitory concentrations in the soybean-cotyledon bioassay when applied at a concentration of 55 nanograms per milliliter (1 × 10⁻⁹ molar). One of these lyases released heat-stable elicitors from soybean cell walls, citrus pectin, and sodium polypectate. The heat-stable elicitor-active material solubilized from soybean cell walls by the lyase was composed of at least 90% (w/v) uronosyl residues. These results demonstrate that endopolygalacturonic acid lyase elicits phytoalexin accumulation by releasing fragments from pectic polysaccharides in plant cell walls.     

Inhibition of Biosynthesis of Polyisoprenol Sugars in Chick Embryo Microsomes by Tunicamycin

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-¹³C] and [2-¹³C]cinnamic acids, and [2-¹³C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4′-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

The role of Ca2+ in elicitation of phytoalexin synthesis in cell culture of onion (Allium cepa L.)

Summary Treatment of Allium cepa L. cellsuspension cultures with a biotic elicitor derived from the fungus Botrytis cinerea, resulted in phytoalexin synthesis. Two phytoalexins, 5-octylcyclopenta-1,3-dione and 5-hexyl-cyclopenta-1,3-dione, were accumulated in cultured onion cells. Removal of extracellular Ca²⁺ by the calcium chelator ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid abolished the elicitor-mediated phytoalexin synthesis. The calcium channel blockers, verapamil and 8-N,N-(dimethylamino)octyl-3,4,5-trimethoxybenzoate caused similar effects, whereas the addition of the Ca²⁺ ionophore A23187 enhanced the accumulation of phytoalexins in the absence of the elicitor. Increase in the cytoplasmic Ca²⁺ concentration in elicitor-treated onion cells was observed as monitored by the fluorescent calcium indicator indo-1. These observations suggest that Ca²⁺ acts as a second messenger in the regulation of phytoalexin synthesis in cultured onion cells.Abbreviations A23187 4-bromo-calcium ionophore - cAMP adenosine 3,5-cyclic monophosphate - [Ca²⁺]_cyt cytoplasmic Ca²⁺ concentration - EGTA ethylene glycol bis(b-aminoethyl ether) N,N-tetraacetic acid - EtOH ethanol - Et₂O diethyl ether - fr.wt fresh weight - HR hypersensitive response - PIPES piperazine N,N-bis-(2-ethanesulfonic acid) - TMB-8 [8-N,N-(dimethylamino)] octyl-3,4,5-trimethoxy-benzoate - Tsl tsibulin     

The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.     

OsGLP3-7 positively regulates rice immune response by activating hydrogen peroxide,jasmonic acid,and phytoalexin metabolic pathways

Although germin-like proteins (GLPs) have been demonstrated to participate in plant biotic stress responses, their specific functions in rice disease resistance are still largely unknown. Here, we report the identification and characterization of OsGLP3-7, a member of the GLP family in rice. Expression of OsGLP3-7 was significantly induced by pathogen infection, jasmonic acid (JA) treatment, and hydrogen peroxide (H₂O₂) treatment. OsGLP3-7 was highly expressed in leaves and sublocalized in the cytoplasm. Overexpression of OsGLP3-7 increased plant resistance to leaf blast, panicle blast, and bacterial blight, whereas disease resistance in OsGLP3-7 RNAi silenced plants was remarkably compromised, suggesting this gene is a positive regulator of disease resistance in rice. Further analysis showed that OsGLP3-7 has superoxide dismutase (SOD) activity and can influence the accumulation of H₂O₂ in transgenic plants. Many genes involved in JA and phytoalexin biosynthesis were strongly induced, accompanied with elevated levels of JA and phytoalexins in OsGLP3-7-overexpressing plants, while expression of these genes was significantly suppressed and the levels of JA and phytoalexins were reduced in OsGLP3-7 RNAi plants compared with control plants, both before and after pathogen inoculation. Moreover, we showed that OsGLP3-7-dependent phytoalexin accumulation may, at least partially, be attributed to the elevated JA levels observed after pathogen infection. Taken together, our results indicate that OsGLP3-7 positively regulates rice disease resistance by activating JA and phytoalexin metabolic pathways, thus providing novel insights into the disease resistance mechanisms conferred by GLPs in rice.     

Methionine-induced phytoalexin production in rice leaves

The application of methionine on wounded rice leaves induced the production of rice phytoalexins, sakuranetin and momilactone A. This induction resulted from stimulation of phenylalanine ammonia-lyase and naringenin 7-O-methyltransferase activity. Jasmonic acid, ethylene, and active oxygen species are important as signal transducers in disease resistance mechanisms. However, although the endogenous level of jasmonic acid rapidly increased in reaction to wound, methionine treatment could not induced endogenous JA production. Ethylene induced the production of the flavonoid phytoalexin, sakuranetin, but did not induce the production of a terpenoid phytoalexin, momilactone A. On the other hand, a free radical scavenger, Tiron, counteracted the induction of both sakuranetin and momilactone A production in methionine-treated leaves. Active oxygen species may be important in methionine-induced production of phytoalexins.     

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl₂-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

A new isoflavan phytoalexin from leaflets of Lotus hispidus

A new isoflavonoid phytoalexin isolated from the fungus-inoculated leaflets of Lotus hispidus (hairy birdsfoot trefoil) has been identified as 5,4′-dimethoxy-7,2′-dihydroxyisoflavan (5-methoxyvestitol). Three known isoflavans (demethylvestitol, vestitol and sativan) are also produced by L. hispidus. The synthesis of 5-, 6- and 8-methoxyvestitol is described. Preparation of the pterocarpan analogues of 6- and 8-methoxyvestitol has allowed the structures of two additional legume phytoalexins to be unequivocally confirmed.     

Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.     

Phytoalexin induction in the sapwood of plants of the Maloideae (Rosaceae): Biphenyls or dibenzofurans

Following fungal inoculation or natural infection, five biphenyl phytoalexins (aucuparin and its 2′ and 4′ oxygenated derivatives) were induced variously in the sapwood of Aronia, Chaenomeles, Eriobotrya, Malus(three spp.) and of Sorbus aucuparia. By contrast, 14 dibenzofuran phytoalexins were induced variously in sapwood of Cotoneaster (7 spp.), Crateagus, Cydonia, Mespilus, Photinia, Pseudocydonia, Pyracantha, Pyrus and two Sorbus spp. (S. chamaemespilum and S. domestica). These were five cotonefurans, three eriobofurans, five pyrufurans and a 2,3,4,7,8-pentaoxygenated dibenzofuran trimethyl ether. No plant has yet been found to produce both types of phytoalexin, although o-hydroxybiphenyls are theoretically precursors of the dibenzofurans. The ability to synthesize either biphenyls or dibenzofurans appears to be genus-specific, except in the case of Sorbus. In 18 of the 38 species tested, these phytoalexins were accompanied by constitutive antifungal phenolics, most of which appeared to be released from bound (glycosidic) forms during the infection process. These were identified variously as hydroquinone, p-hydroxyacetophenone, acetovanillone, 5,7-dihydroxychromone, chrysin, sakuranetin and naringenin. Woody members of the subfamilies Prunoideae and Spiraeoideae failed to yield any phytoalexins on induction, but did contain constitutive antifungal compounds. The limited frequency of the phytoalexin response within the family as a whole is considered in relation to the accumulation of constitutive antifungal agents in these plants.     

Characterization of a rice gene family encoding type-A diterpene cyclases

We have previously isolated and characterized the rice (Oryza sativa) cDNAs, OsCyc1/OsCPS4, OsCyc2/OsCPS2, OsKS4, OsDTC1/OsKS7, OsDTC2/OsKS8 and OsKS10, which encode cyclases that are responsible for diterpene phytoalexin biosynthesis. Among the other members of this gene family, OsCPS1 and OsKS1 have been suggested as being responsible for gibberellin biosynthesis, OsKSL11 has recently been shown to encode stemodene synthase, and the functions of the three other diterpene cyclase genes in the rice genome, OsKS3, OsKS5 and OsKS6, have not yet been determined. In this study, we show that recombinant OsKS5 and OsKS6 expressed in E. coli converted ent-copalyl diphosphate into ent-pimara-8(14),15-diene and ent-kaur-15-ene, respectively. Neither product is a hydrocarbon precursor required in the biosynthesis of either gibberellins or phytoalexins. OsKS3 may be a pseudogene from which the translated product is a truncated enzyme. These results suggest that the diterpene cyclase genes responsible for gibberellin and phytoalexin biosynthesis are not functionally redundant.     

Accumulation of phytoalexins and isoflavone glucosides in a resistant and a susceptible cultivar of Cicer arietinum during infection with Ascochyta rabiei

After infection with spores of a virulent strain of Ascochyta rabiei the chickpea (Cicer arietinum) cultivars ILC 1929 (susceptible) and ILC 3279 (resistant) were compared with regard to pterocarpan phytoalexin and isoflavone accumulation. Quantitative HPLC analyses of total extracts of aerial parts were used to measure the induced formation of the phytoalexins medicarpin and maackiain and the accumulation of the constitutive isoflavones biochanin A and formononetin together with their, 7-0-glucosides and their 7-0-glucoside-6″-0-malonates. The two cultivars showed no significant difference in the level of isoflavones and isoflavone conjugates. On the other hand, the resistant cultivar ILC 3279 rapidly accumulated large amounts of both, phytoalexins (20–26 nmole g^?1 fr.w.) whereas cultivar ILC 1929 only produced very small amounts (5 nmole g^?1 fr.w.) of medicarpin. The data are discussed with regard to isoflavonoid metabolism and the significance of induced and constitutive levels of phytoalexins and isoflavones in resistance of chickpea towards A. rabiei.     

Circumstantial evidence for phytoalexin involvement in the resistance of peanuts to Aspergillus flavus

Three stilbene phytoalexins, elicited by slicing and incubating imbibed peanut kernels under aerobic conditions, inhibited spore germination and hyphal extension of Aspergillus flavus with ED50 values in the range 4.9-12.8 micrograms ml-1. Phytoalexin yield was dependent on cultivar, conditions and duration of incubation after slicing, and crop history. The yield of phytoalexin from ten cultivars studied, after slicing and incubating at 25 degrees C for 24 h, ranged from 28 to 935 micrograms per g fresh weight and was negatively correlated with dry kernel colonization by A. flavus [r = -0.868 when plotted as 1n (phytoalexin concn) against 1n (percentage peanut colonization)]. When the incubation period was extended to 96 h there was no such correlation. Reduced phytoalexin yields were obtained when sliced kernels of one cultivar studied were incubated in water or at 37 degrees C, and no phytoalexin was obtained when the slices were incubated under nitrogen gas or frozen before aerobic incubation. Drought stress during pod development in four cultivars studied reduced phytoalexin yields of sliced kernels incubated at 25 degrees C for 24 h by 17-65% compared with non-stressed controls.     

Host-Pathogen Interactions : XVIII. ISOLATION AND BIOLOGICAL ACTIVITY OF GLYCINOL, A PTEROCARPAN PHYTOALEXIN SYNTHESIZED BY SOYBEANS

A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,11aS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCl₂-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonas phaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucumerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol.