Nontoxic and toxic oligopeptides with D-amino acids and unusual residues in Microcystis aeruginosa PCC 7806

Toxic and nontoxic peptides were isolated from the cyanobacterium Microcystis aeruginosa PCC 7806 by a procedure including extraction of cells with water-saturated 1-butanol, chromatography of the extract on silica gel plates and high performance liquid chromatography (HPLC) on Partisil-5. The toxin was shown to be only a minor constituent, being negatively charged and thus separable by electrophoresis, within the HPLC-purified fraction. It contained erythro-β-methyl-D-Asp, D-Glu, D-Ala, L-Leu, and L-Arg known to be part of the Microcystis peptide-toxin with M_r 994. The major part of the HPLC-purified fraction was assigned, however, to a nontoxic peptide with a M_r of 956. Partial hydrolysis studies of the nontoxic peptide(s) revealed amino acid sequences composed of D-Glu, N-methyl-Phe, and 3,4-dehydro-Pro, aside from the common L-amino acids. Cyclic linkage in the nontoxic peptide(s) appears likely.     

S-adenosyl-L-methionine:trans-caffeoyl-coenzyme A 3-O-methyltransferase from elicitor-treated parsley cell suspension cultures

An S-adenosyl-L-methionine:caffeoyl-CoA 3-O-methyltransferase was purified 82-fold from elicitor-induced parsley cell suspension cultures by ammonium sulfate fractionation, anionic exchange and hydrophobic interaction chromatographies, and chromatofocusing. The enzyme has an apparent pI of 5.7 and a molecular weight of approx 48,000 determined by gel filtration chromatography. Maximal activity was observed at pH 7.5 in 50 mM phosphate or Tris-HCl buffers and the additional presence of 0.5 M NaCl. The methyltransferase activity was dependent on Mg2+, whereas EDTA, Mn2+, and Ca2+ inhibited the reaction. The partially purified enzyme efficiently catalyzed the methylation of caffeoyl-CoA, but also accepted with low affinity various other caffeic esters as substrates. Dark-grown parsley cells contained considerable methyltransferase activity which was nevertheless increased approx threefold within 12 h following the addition of a crude fungal elicitor to the cell suspensions. We propose that the O-methyltransferase activity is an important component in the rapid resistance response of the cells, which depends on the formation of cell wall-bound ferulic polymers.     

Elicitor-Inducible Caffeoyl-Coenzyme A 3-O-Methyltransferase from Petroselinum crispum Cell Suspensions : Purification, Partial Sequence, and Antigenicity

Physiological regulation of nodule gas permeability has a central role in the response of legumes to such diverse factors as drought, defoliation, and soil nitrate. A new method for quantifying nodule respiration and O₂ permeability, based on noninvasive spectrophotometry of leghemoglobin, was evaluated using intact, attached nodules of Lotus corniculatus. First, the relationship between nodule respiration (O₂ consumption) rate and internal O₂ concentration was determined from the rate of decrease in fractional oxygenation of leghemoglobin (FOL) under N₂. The rate of increase of FOL under 100% O₂ was then used to calculate nodule O₂ permeability, after correcting for respiration. Inactivation of nitrogenase by exposure to 100% O₂ for 15 minutes led to decreases in both permeability and O₂-saturated respiration (V_max), but the brief (<15 seconds) exposures to 100% O₂ required by the assay itself had little effect on either parameter. A gradual increase in external O₂ concentration from 20 to 40% resulted in a reversible decrease in permeability, but no change in V_max. The new method is likely to be useful for research on nodule physiology and might also be applicable to agronomic research and crop improvement programs.     

Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.     

Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics

The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic acquired resistance, one can hypothesize that fungal pathogens might be recognized more readily and effectively.     

N-malonyltransferases from peanut

Three distinct N-malonyltransferases were purified from peanut seedlings, accepting either anthranilic acid, D-tryptophan, or 3,4-dichloroaniline, respectively, as a substrate. Partially purified malonyl-CoA:D-tryptophan malonyltransferase also catalyzed the formation of the corresponding malonic acid conjugate when 1-aminocyclopropane-1-carboxylic acid was employed as a substrate. These N-malonyltransferases were clearly distinguished from several O-malonyltransferase activities also present in the same seedlings. N-Malonic acid conjugates have been previously isolated from peanut either as a natural constituent or after feeding with xenobiotics. By analogy to the results reported with cultured parsley cells, multiple malonyltransferases in peanut may have a role in vacuolar transport. Crude extracts of young peanut seedlings were incapable of hydrolyzing the respective N-malonic acid conjugates. However, dialyzed extracts of older plants released malonic acid from malonyl-1-aminocyclopropane-1-carboxylic acid but not from malonyl-3,4-dichloroaniline, suggesting that some N-malonic acid conjugates may be metabolized in plants which are approaching senescence.     

Rapid effect of an elicitor on uptake and intracellular distribution of phosphate in cultured parsley cells

Cell suspension cultures of parsley (Petroselinum hortense) grown in synthetic medium take up most of the inorganic phosphate supplied with the medium within the initial 5 days after transfer. Nuclear magnetic resonance spectra of intact parsley cells from this growth stage revealed that approximately half of the phosphate was located within the vacuoles, whereas after 7 days of growth phosphate content of the vacuoles was relatively low. At both times, addition of an elicitor preparation from Alternaria carthami, which is not toxic to the cells, led to a temporary increase of vacuolar phosphate at the expense of cytoplasmic phosphate, even when excess phosphate was added to the medium. The rapid decrease of cytoplasmic phosphate might play a role in the redirection of phenylpropanoid metabolism reported for elicitor-treated parsley cells.     

Role of external carbonic anhydrase in light-dependent alkalization byFucus serratus L. andLaminaria saccharina (L.) Lamour. (Phaeophyta)

It has been proposed that many marine macroalgae are able to utilize HCO ₃ ^– for photosynthesis and growth, and that energy-dependent ion pumping is involved in this process. We have therefore studied the light-dependent alkalization of the surrounding medium by two species of marine macroscopic brown algae,Fucus serratus L. andLaminaria saccharina (L.) Lamour. with the aim of investigating the role of extracellular carbonic anhydrase (EC 4.2.1.1.) in the assimilation of inorganic carbon from the seawater medium. In particular, the influence of membrane-impermeable or slowly permeable carbonic-anhydrase inhibitors on the rate of alkalization of the seawater has been investigated. Inhibition of the alkalization rate occurred in both species at an alkaline pH (pH 8.0) but no inhibition was observed at an acidic pH (pH 6.0). The alkalization was found to be light-dependent and inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and, thus, correlated with photosynthesis. Alkalization by macroalgae has previously been shown to be proportional to inorganiccarbon uptake. We suggest that alkalization of the medium at alkaline pH in both of the species examined is mainly the consequence of an extracellular reaction. The reaction is catalyzed by extracellular carbonic anhydrase which converts HCO ₃ ^– to OH^– and CO₂; CO₂ is then taken up through the plasmalemma. However, we do not exclude the involvement of other mechanisms of inorganic-carbon uptake.Abbreviations AZ acetazolamide - CA carbonic anhydrase - CAext extracellular carbonic anhydrase - C_i inorganic carbon - DBS dextran-bound sulfonamide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PPFD photosynthetic photon flux density This study was carried out with financial support by SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Trygger's Fund for Scientific Research (Sweden), SJFR (Swedish Council for Forestry and Agricultural Research) and CICYT (Spain). Z. Ramazanov is an invited professor of Ministerio de Educación y Ciencia, Spain.