Heinrich Kauss

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.

Proteolytic activation and stimulation by Ca2+ of glucan synthase from soybean cells

In homogenates from suspension‐cultured soybean cells, 1,3‐β‐d‐glucan synthase activity is increased through preincubation with trypsin or due to action of an endogenous enzyme which presumably is a protease as it is inhibited by soybean trypsin inhibitor. The 1,3‐β‐d‐glucan synthase in untreated membrane preparations is also reversibly stimulated by Ca2+. This Ca2+‐dependence is lost on proteolytic activation. Regardless as to whether the enzyme was rendered active by preincubation with trypsin or by the presence of Ca2+, it is inhibited by calmidazolium, trifluoperazine and polymyxin B, suggesting that the activation by Ca2+ is not mediated by calmodulin.

Chitosan-elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures

In suspension cultured cells of parsley (Petroselinum crispum), chitosan elicited a rapid deposition of the 1,3-ß-glucan callose on the cell wall and a slower formation of coumarins. With cells remaining in conditioned growth medium, fully N-deacetylated chitosans and partially N-acetylated chitosans were about equally active, the potency increased with the degree of polymerization up to several thousand and addition of reduced glutathione increased the sensitivity of the cells. These results indicate common initial events in the induction of callose and coumarin synthesis although two fully independent metabolic pathways are involved. When the cells were suspended in fresh growth medium, less chitosan was required, and fully N-deacetylated chitosan became the best callose elicitor.

Physiological changes in suspension-cultured soybean cells elicited by treatment with chitosan

Abstract Addition of soluble chitosan to suspension-cultured Glycine max cells inhibited the rate of increase in cell fresh weight, increased their glyceollin content and altered their cell wall composition. The amounts of wall-bound phenolic compounds and callose increased and the walls became more resistant to degradation by fungal enzymes. Poly-L-lysine caused the same effects as chitosan, whereas the monomers L-lysine and D-glucosamine were not active. All the reactions observed could be partly suppressed by Na-polygalacturonate.

Pretreatment of Parsley (Petroselinum crispum L.) Suspension Cultures with Methyl Jasmonate Enhances Elicitation of Activated Oxygen Species

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to demonstrate an influence of jasmonic acid methyl ester (JAME) on the elicitation of activated oxygen species. Preincubation of the cell cultures for 1 d with JAME greatly enhanced the subsequent induction by an elicitor preparation from cell walls of Phytophtora megasperma f. sp. glycinea (Pmg elicitor) and by the polycation chitosan. Shorter preincubation times with JAME were less efficient, and the effect was saturated at about 5 [mu]M JAME. Treatment of the crude Pmg elicitor with trypsin abolished induction of activated oxygen species, an effect similar to that seen with elicitation of coumarin secretion. These results suggest that JAME conditioned the parsley suspension cells in a time-dependent manner to become more responsive to elicitation, reminiscent of developmental effects caused by JAME in whole plants. It is interesting that pretreatment of the parsley cultures with 2,6-dichloroisonicotinic and 5-chlorosalicylic acid only slightly enhanced the elicitation of activated oxygen species, whereas these substances greatly enhanced the elicitation of coumarin secretion. Therefore, these presumed inducers of systemic acquired resistance exhibit a specificity different from JAME.

Carbohydrate-binding proteins from plant cell walls and their possible involvement in extension growth

Various plant tissues have been shown to contain proteins and glycoproteins which can bind carbohydrates with a high affinity and structural specifity. These lectins (or phytohemagglutinins) have recently become subject of growing interest in many laboratories, since they represent good tools for the study of cell surface carbohydrate groups. However, their physiological role in plants is completely unknown [ 1,2] . It has become clear during the past years that plant cell walls contain glycoproteins besides pectin, hemicelluloses and cellulose [3,4]. Although a great portion of the glycoprotein is regarded as covalently linked to the carbohydrates, some protein can be isolated from the walls by means of salt solutions. It has been considered that this fraction is representative of a precursor of the covalently bound glycoprotein; however the evidence obtained from pulse-chase experiments is not fully conclusive [S] After feeding [’ 4 C] proline to growing mung bean hypocotyl segments up to half of the radioactivity incorporated as proline and hydroxyproline into the wall proteins can be extracted with chaotropic agents [6] . This result suggested that the non-covalently bound glycoprolein constitutes a much greater portion of cell wall protein than had previously been tllc.ught. Tllis paper presents results to show that noncovalently bound protein fractions from growing as well as non-growing mung bean hypocotyl cell walls exhibit lectin properties. As the binding potential is strongly diminished towards more acidic pfl-values, it is suggested that the cell wall lectins are involved in plant cell extension growth.

Competence for Elicitation of H2O2 in Hypocotyls of Cucumber Is Induced by Breaching the Cuticle and Is Enhanced by Salicylic Acid

To study H2O2 production, the epidermal surfaces of hypocotyl segments from etiolated seedlings of cucumber (Cucumis sativus L.) were gently abraded. Freshly abraded segments were not constitutively competent for rapid H2O2 elicitation. This capacity developed subsequent to abrasion in a time-dependent process that was greatly enhanced in segments exhibiting an acquired resistance to penetration of their epidermal cell walls by Colletotrichum lagenarium, because of root pretreatment of the respective seedlings with 2,6-dichloroisonicotinic acid. When this compound or salicylic acid was applied to abraded segments, it also greatly enhanced the induction of competence for H2O2 elicitation. This process was fully inhibited by 5 [mu]M cycloheximide or 200 [mu]M puromycin, suggesting a requirement for translational protein synthesis. Both a crude elicitor preparation and a partially purified oligoglucan mixture from Phytophthora sojae also induced, in addition to H2O2 production, a refractory state, which explains the transient nature of H2O2 elicitation. Taken together, these results suggest that the cucumber hypocotyl epidermis becomes conditioned for competence to produce H2O2 in response to elicitors by a stimulus resulting from breaching the cuticle and/or cutting segments. This conditioning process is associated with protein synthesis and is greatly enhanced when substances able to induce systemic acquired resistance are present in the tissue.

The protein kinase inhibitor, K-252a, decreases elicitor-induced Ca2+ uptake and K+ release, and increases coumarin synthesis in parsley cells

An elicitor preparation from fungal cell walls known to induce coumarin synthesis in suspension‐cultured parsley cells also elicits a rapid and transient Ca2+ uptake, K+ release and external alkalinization, and increases uptake of 45Ca2+ into the cells. The latter three responses were inhibited by the protein kinase inhibitor K‐252a at 0.2 μM. Elicitor‐induced coumarin synthesis, a process which requires gene activation, was greatly enhanced by K‐252a. These results suggest that protein phosphorylation might be involved in the initial steps of signal transduction as well as in the long‐term induction of coumarin synthesis.

Biosynthesis of ferulic acid esters of plant cell wall polysaccharides in endomembranes from parsley cells

A microsomal preparation from suspension‐cultured parsley cells is able to transfer ferulic acid from the respective CoA thioester to endogenous acceptors. The reaction is not enhanced by digitonin but stimulated by Mg2+, Ca2+ and Co2+. Spermine can partly replace divalent ions. Solubility properties and degradation by polysaccharide hydrolases suggest that the products are polymeric cell wall carbohydrates. Sucrose density gradient centrifugation revealed that the most active vesicle fraction is distinct from plasma membranes but does also not peak with IDPase. It is suggested that a subfraction of the Golgi‐apparatus is the source of enzyme and acceptors.

Activation by polyamines, polycations, and ruthenium red of the Ca2+-dependent glucan synthase from soybean cells

The 1,3‐β‐D‐glucan synthase in microsomal preparations from suspension‐cultured soy bean cells requires C2+ for activity. In the absence of Ca2+ the enzyme can also be activated by poly‐L‐Lys, poly‐L‐Orn and ruthenium red. Under these conditions it is either not or only slightly inhibited by La3+ and shows increased affinity for UDP‐glucose. Spermine, when present alone at 57 μM, effects relatively little activation but cooperates with 5 μM Ca2+ to greatly enhance the affinity of the enzyme towards UDP‐glucose, suggesting that both types of activation may occur at the same enzyme.