Yeast Elicitor Research Articles

Release and biological activity of diffusible signal compounds from elicited plant cells

Summary Treatment of cell suspension cultures of soybean, alfalfa, or tobacco with macromolecular elicitors resulted in the release of low molecular mass (500–2000 u (Da)) endogenous elicitors that could pass through a dialysis membrane into the surrounding culture medium. The active compounds induced phenylalanine ammonia-lyase (PAL) activity in cells of all three species, isoflavonoid phytoalexin accumulation in soybean and alfalfa cells, and an oxidative burst in soybean cells. Release of factors from soybean cells was inhibited by the RNA synthesis inhibitor actinomycin D, but not by diphenylene iodonium, an inhibitor of the oxidative burst. The activity of the factors released from tobacco cells in response to the proteinaceous elicitor cryptogein, or from alfalfa cells in response to yeast elicitor, was unaffected by pretreatment with sodium periodate or proteinase K. In contrast, treatment of the factor(s) from soybean cells with proteinase K or trypsin destroyed its ability to induce the phytoalexin glyceollin. Ion-exchange chromatography revealed that the active material from cell cultures of each species consisted of at least two components. One component released from soybean cells appears to be a small peptide.

Identification and Characterization of an S-Adenosyl-L-methionine:Δ24-Sterol-C-methyltransferase cDNA from Soybean

In plants, the dominant sterols are 24-alkyl sterols, which play multiple roles in plant growth and development, i.e. as membrane constituents and as precursors to steroid growth regulators such as brassinosteroids. The initial step in the conversion of the phytosterol intermediate cycloartenol to the 24-alkyl sterols is catalyzed by S-adenosyl-L-methionine: delta 24-sterol-C-methyl-transferase (SMT), a rate-limiting enzyme for phytosterol biosynthesis. A cDNA clone (SMT1) encoding soybean SMT was isolated from an etiolated hypocotyl cDNA library by immunoscreening using an anti-(plasma membrane) serum. The deduced amino acid sequence of the SMT1 cDNA contained three conserved regions found in S-adenosyl-L-methionine-dependent methyltransferases. The overall structure of the polypeptide encoded by the SMT1 cDNA is most similar to the predicted amino acid sequence of the yeast ERG6 gene, the putative SMT structural gene. The polypeptide encoded by the SMT1 cDNA was expressed as a fusion protein in Escherichia coli and shown to possess SMT activity. The growing soybean vegetative tissues had higher levels of SMT transcript than mature vegetative tissues. Young pods and immature seeds had very low levels of the SMT transcript. The SMT transcript was highly expressed in flowers. The expression of SMT transcript was suppressed in soybean cell suspension cultures treated with yeast elicitor. The transcriptional regulation of SMT in phytosterol biosynthesis is discussed.

Purification and Partial Characterization of Tomato Extensin Peroxidase.

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI,PII,EPIII, and EPIV) were resolved; only the latter two cross-linked tomato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were cross-linked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cells of tomato. A highly sensitive enzyme-linked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Lipoxygenases in Bryonia dioica Jacq. Tendrils and Cell Cultures

Lipoxygenases in differentiated and undifferentiated tissues of Bryonia dioica were characterized. The enzymes from tendrils and tissue cultures were purified. Leaf, stem and root tissue as well as cell cultures predominantly contain enzymes with acidic pH-optima while those from tendrils are most active at alkaline pH values around 8.5. The purified enzyme from tendrils consists of a major isoform with pI = 6.5 and minor constituents with pI = 6.7 and 7.3, and predominantly produces 13-hydroperoxylinolenic acid from α-linolenic acid while the preparation from cell cultures contains at least seven isoforms in the pI range from 6.3–6.7 and 7.3–7.5. The major reaction product formed from α-linolenic acid was 9-hydroperoxylinolenic acid. All B. dioica enzymes showed an apparent molecular mass of 96 kDa and crossreacted with an antiserum raised against the lipoxygenase from oil bodies of cucumber. The properties of the tendril lipoxygenase are typical for class-1 enzymes while those of the other tissues resemble the clasen enzymes. Lipoxygenase activity in cell cultures was induced by yeast elicitor and methyl jasmonate. The activity in tendrils was constant during all phases of tendril coiling. The results are discussed in the context of jasmonate biosynthesis in B. dioica tendrils and jasmonate signalling during the coiling process.

Phytoalexin Accumulation in Omithopus sativus as a Response to Elicitor Treatment

An isoflavanoid derivative has been isolated as a phytoalexin from intact plants and cell suspension cultures of Ornithopus sativus after treatment with CuSO4, yeast elicitor, or a spore suspension of Colletotrichum trifolii. Based on its physical and spectroscopic properties the substance was identified as glabridin. After induction, the transient in crease in the content of this phytoalex in was preceded by a transient increase in the activities of PAL and CHS.

Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures.

To deter pathogenic microorganisms and herbivores, plants have developed an inducible chemical defense system. It is known that the induced synthesis of low molecular weight compounds can be provoked by exposing cultured cells to fungal cell wall fragments. In this study we show that endogenous jasmonic acid and its methyl ester accumulate rapidly and transiently after treatment of plant cell suspension cultures of Rauvolfia canescens and Eschscholtzia californica with a yeast elicitor. Thirty-six plant species tested in cell suspension culture could be elicited with respect to the accumulation of secondary metabolites by exogenously supplied methyl jasmonate. Addition of methyl jasmonate initiates de novo transcription of genes, such as phenylalanine ammonia lyase, that are known to be involved in the chemical defense mechanisms of plants. These data demonstrate the integral role of jasmonic acid and its derivatives in the intracellular signal cascade that begins with interaction of an elicitor molecule with the plant cell surface and results, ultimately, in the accumulation of secondary compounds.

Purification and characterization of S-adenosyl- l-methionine: Caffeic acid 3- O-methyltransferase from suspension cultures of alfalfa ( Medicago sativa L.)

Caffeic acid O-methyltransferase (COMT) is one of a group of proteins present in alfalfa cell cultures which can be photoaffinity labeled with S-adenosyl- l-[ methyl- 3H]methionine. The enzyme was purified to homogeneity from elicitor-treated suspension cultures and shown to exist as an active monomer of subunit M r 41,000. COMT could be separated into two forms on the basis of their isoelectric points and relative affinities for S-adenosylmethionine and S-adenosylhomocysteine. Both forms had equal affinities for caffeic acid, were highly specific for the 3-hydroxyl group of substituted cinnamic acids, and exhibited negligible activity toward flavonoid substrates. An antiserum raised against COMT from aspen immunoprecipitated alfalfa COMT activity. Peptide mapping studies indicated that the two forms of COMT and an isoflavone O-methyltransferase from alfalfa are closely related proteins. The extractable activity of COMT doubled over a 48-h period following exposure of alfalfa cell suspensions to a yeast elicitor preparation, and this was associated with a small change in the relative proportions of the two forms of the enzyme.

Elicitor‐induced hydroxycinnamoyl‐CoA:tyramine hydroxycinnamoyltransferase in plant cell suspension cultures

Treatment of Nicotiana glutinosa L. cell suspension cultures with chitosan results in the co‐induction of phenylalanine ammonia lyase, 4‐eoumarate:CoA ligase, tyrosine decarboxylase and tyramine hydroxycinnamoyltransferase, all involved in the biosynthesis of hydroxycinnamoyltyramines. The highest enzyme activities were observed around 12 h after addition of 0.8 to 1 mg chitosan per g fresh weight of cells. No hydroxycinnamoyltyramines could be detected by TLC or HPLC of extracts made from non‐treated or elicited cells. [14C]‐Tyramine was incorporated into insoluble polymeric material at a higher rate in elicitor‐treated than in non‐treated cells of N. glutinosa. Tyramine hydroxycinnamoyltransferase could be induced in suspension cultured cells of Eschscholtzia califortnca Cham, but not in cells of Phaseolus vulgaris L. or Catharanthus roseus (L.) G. Don by addition of a yeast elicitor to the growth medium.

Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis

The chemical synthesis of [6- 3H]protopine is described. Microsomal preparations from Eschscholtzia californica cell suspension cultures challenged with a crude elicitor preparation from yeast, catalyse the hydroxylation of [6- 3H]protopine with the concomitant formation of [11- 3H]dihydrosanguinarine and HO 3H. This reaction served as an assay for the enzyme. The hydroxylase reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. Cytochrome c, inhibitors such as prochloraz and ketoconazole and carbon monoxide inhibit the hydroxylation reaction, suggesting that the enzyme is a cytochrome P-450 linked monooxygenase. The hydroxylase was induced by the yeast elicitor ca eight-fold, 20 hr after challenging the plant cell culture with the yeast carbohydrate, having no effect on the cell dry weight of the culture. The hydroxylase is specifically present in different plant species that produce benzo[c]phenanthridine alkaloids in culture and also respond to induction by the elicitor.

Elicitation of Canthin-6-one Alkaloid Accumulation in Cell Suspension Cultures of Ailanthus altissima (Mill.) Swingle

Cell suspension cultures of Ailanthus altissima (Simaroubaceae) were cultivated under illumination and in the dark. Under influence of a yeast elicitor and cell wall preparations of various Phytophthora species, respectively, alkaloid formation was stimulated. Canthin-6-one and 1- methoxycanthin-6-one accumulation could be increased up to a 125-fold and 2.5-fold, respectively within 96 h by elicitors in dark grown cultures. In response to elicitors canthin-6-one is accumulated in the cells and the growth medium.

Dynamics of benzophenanthridine alkaloid production in suspension cultures of Eschscholtzia californica after treatment with a yeast elicitor

A number of benzophenanthridine alkaloids are induced in suspension cultures of Eschscholtzia californica after treatment with an elicitor prepared from yeast extract. The formation of the alkaloids sanguinarine, chelerythrine and macarpine has been studied in relation to; elicitor concentration, incubation time after elicitation, and culture age. A significant portion of these alkaloids is released into the medium. Sanguinarine and chelerythrine reach maximum levels a few hours after the time of elicitation. Thereafter, their levels decline and the amount of macarpine increases. Viability of elicited cells, as determined by their subsequent growth, is not significantly reduced. There is a good correlation between induced tyrosine decarboxylase activity and alkaloid formation.

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.

Host-Pathogen Interactions

An elicitor of glyceollin accumulation in soybeans (Glycine max L.) has been isolated from a commercially available extract of brewers' yeast. Yeast is not a known pathogen of plants. The elicitor was isolated by precipitation in 80% (v/v) ethanol followed by column chromatography on DEAE-cellulose, sulfopropyl-Sephadex, and concanavalin A-Sepharose. Compositional and structural analysis showed the elicitor to be a glucan containing terminal, 3-, 6-, and 3,6-linked glucosyl residues. The yeast elicitor stimulates the accumulation of glyceollin in the cotyledons and hypocotyls of soybeans when as little as 15 nanograms or 100 nanograms of the elicitor is applied to the respective tissues. The yeast elicitor is very similar in both structure and absolute elicitor activity to an elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, a pathogen of soybeans. These and other results of this laboratory suggest that plants are able to respond to the presence of a wide range of fungi by recognizing, as foreign to the plant, structural polysaccharides of the mycelial walls of the fungi.