Suspension-cultured Parsley Cells Research Articles

Two differentially regulated class II chitinases from parsley.

Two distinct cDNA clones, PcCHI1 and PcCHI2, with high sequence similarity to plant chitinases were isolated from parsley (Petroselinum crispum), expressed in Escherichia coli, and the encoded proteins functionally identified as endochitinases. Different expression patterns of the corresponding mRNAs and proteins in infected and uninfected parsley plants indicated distinct roles of the two isoforms in both pathogen defense and plant development. Infection of parsley leaf buds with Phytophthora sojae resulted in the rapid, transient and highly localized accumulation of PcCHI1 mRNA and protein around infection sites, whereas PcCHI2 mRNA and protein were systemically induced at later infection stages. Similar differences in the timing of induction were observed in elicitor-treated, suspension-cultured parsley cells. In uninfected plants, PcCHI1 mRNA was particularly abundant in the transmitting tract of healthy flowers, suggesting a role in the constitutive protection of susceptible transmitting tissue of the style against pathogen ingress and/or in the fertilization process, possibly by affecting pollen tube growth. Localization of PcCHI2 mRNA and protein in the parenchymatic collenchyme of young pedicels may indicate a function in the constitutive protection of this tissue. In addition to such distinct roles of PcCHI1 and PcCHI2 in preformed and induced pathogen defense, both chitinases may have endogenous regulatory functions in plant development.

Pretreatment of Parsley Suspension Cultures with Salicylic Acid Enhances Spontaneous and Elicited Production of H2O2.

Suspension-cultured cells of parsley (Petroselinum crispum L.) were used to study the regulation of extracellular H2O2. After resuspension, the washed cells regulated the H2O2 concentration spontaneously to a constant level that was greatly increased when the cultures were pretreated for 1 d with salicylic acid (SA). The H2O2 level was further increased on addition of a fungal elicitor preparation, macromolecular chitosan, the sterol-binding polyene macrolide amphotericin B, the G protein-activating peptide mastoparan, or La3+. In all cases, this induced H2O2 burst was also greatly enhanced in cell suspensions pretreated with SA. Both the spontaneous and the induced H2O2 production were decreased by the protein kinase inhibitor K-252a. It is suggested that production of extracellular H2O2 occurs by an endogenously controlled plasma membrane enzyme complex that requires continuous phosphorylation for function and whose activity is increased by pretreatment of the cells with SA. This system can also receive various external stimuli, including those resulting from binding of fungal elicitor. SA can induce acquired resistance against pathogens. The conditioning of the parsley suspension culture by SA represents, therefore, a model for the long-term regulation of apoplastic H2O2 concentration by this signal substance, as suggested previously for the wound hormone methyl jasmonate.

Oligopeptide elicitor-mediated defense gene activation in cultured parsley cells.

We have used suspension-cultured parsley cells (Petroselinum crispum) and an oligopeptide elicitor derived from a surface glycoprotein of the phytopathogenic fungus Phytophthora megasperma f.sp. glycinea to study the signaling pathway from elicitor recognition to defense gene activation. Immediately after specific binding of the elicitor by a receptor in the plasma membrane, large and transient increases in several inorganic ion fluxes (Ca2+, H+, K+, Cl-) and H2O2 formation are the first detectable plant cell responses. These are rapidly followed by transient changes in the phosphorylation status of various proteins and by the activation of numerous defense-related genes, concomitant with the inactivation of several other, non-defense-related genes. A great diversity of cis-acting elements and trans-acting factors appears to be involved in elicitor-mediated gene regulation, similar to the apparently complex nature of the signal transduced intracellularly. With few exceptions, all individual defense responses analyzed in fungus-infected parsley leaves have been found to be closely mimicked in elicitor-treated, cultured parsley cells, thus validating the use of the elicitor/cell culture system as a valuable model system for these types of study.

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.

Methyl jasmonate conditions parsley suspension cells for increased elicitation of phenylpropanoid defense responses

Pre-incubation of suspension-cultured parsley cells with methyl jasmonate greatly enhances their ability to respond to fungal elicitors by secretion of coumarin derivatives. The effect is most pronounced at relatively low elicitor concentration and also observed for the incorporation of esterified hydroxycinnamic acids and of “lignin-like” polymers into the cell wall. These three responses correspond to defense reactions induced locally when a fungal pathogen attacks plant cells. In contrast, the conditioning of parsley cells by the signal substance methyl jasmonate is reminiscent of the developmental nature of systemic acquired resistance and renders the cells more effective for the elicitor-induced local defense reactions.

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 Mg 2+, Ca 2+ and Co 2+. 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.

The protein kinase inhibitor, K-252a, decreases elicitor-induced Ca 2+ 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 Ca 2+ uptake, K + release and external alkalinization, and increases uptake of 45Ca 2+ 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.

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g(-1) of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g(-1) h(-1). Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with (2)H2O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.

Rapid Response of Suspension-cultured Parsley Cells to the Elicitor from Phytophthora megasperma var. sojae

Large and rapid increases in the activities of two enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase and 4-coumarate:CoA ligase, occurred in suspension-cultured parsley cells (Petroselinum hortense) treated with an elicitor preparation from Phytophthora megasperma var. sojae. Highest enzyme activities were obtained with an elicitor concentration similar to that required for maximal phenylalanine ammonialyase induction in cell suspension cultures of soybean, a natural host of the fungal pathogen.The changes in phenylalanine ammonia-lyase activity in parsley cells were caused by corresponding changes in the mRNA activity for this enzyme. Phenylalanine ammonia-lyase mRNA activity increased much faster and transiently reached a much higher level in elicitor-treated than in irradiated cell cultures. In contrast to irradiation, treatment of the cells with the elicitor did not induce the enzymes of the flavonoid glycoside pathway, as demonstrated for acetyl-CoA carboxylase and chalcone synthase. Induction of these enzymes by light was abolished by simultaneous application of the elicitor.

Comparison of acetyl-CoA carboxylases from parsley cell cultures and wheat germ

The activity of acetyl-CoA carboxylase of suspension-cultured cells of parsley ( Petroselinum hortense Hoffm.) is greatly stimulated by light soon after transferring cells to new culture medium. Parsley acetyl-CoA carboxylase has been purified from frozen cells by treatment of the crude protein extract with Dowex 1 × 2 and polyethyleneimine, precipitation with (NH 4) 2SO 4, chromatography on DEAE-cellulose and blue Sepharose CL-6B, and gel filtration on Sepharose 6B. A recovery of about 8% has been achieved with a 300-fold increase in specific activity. Wheat germ acetyl-CoA carboxylase has been purified 2180-fold by a similar procedure. The two carboxylases have the following characteristics: Molecular weights of 840,000 for the parsley carboxylase and 700,000 for the wheat germ carboxylase have been estimated from the elution volumes of a calibrated Sepharose 6B column. Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the carboxylases from parsley and wheat each are composed of one large subunit ( M r = 210,000 and 240,000, respectively) and possibly one smaller polypeptide component ( M r = 105,000 and 98,000, respectively). Avidin-binding experiments demonstrated that the 240,000 — M r component of wheat germ carboxylase is the biotin-containing subunit of this enzyme. No isoenzymes of the parsley carboxylase could be demonstrated.