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Treatment of tomato plants (Lycopersicon esculentum Mill.) with fusicoccin (FC), an activator of the plasma-membrane H+-ATPase which maintains an electrochemical gradient across the plasma membrane, resulted in a dose-dependent accumulation of transcripts for intra- and extracellular pathogenesis-related (PR) proteins. The accumulation of PR protein transcripts was paralleled by an increase in leaf salicylic acid (SA) content. Transcripts of PR proteins and SA started to accumulate 3 h after FC treatment. 2-Aminoindan-2-phosphonic acid, an inhibitor of SA synthesis, was used to assess the role of SA in FC-mediated induction of PR gene expression. 2-Aminoindan-2-phosphonic acid was found to suppress the accumulation of SA but not the induction of PR gene expression in response to FC treatment. Furthermore, in transgenic tobacco plants overexpressing a bacterial salicylate hydroxylase gene (nahG-tobacco), PR transcripts accumulated after FC treatment to levels similar to those observed in control tobacco plants. The data indicate a role for the proton gradient across the plasma membrane in the SA-independent induction of PR gene expression.

Chorismate synthase, the seventh enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate which is the last common precursor in the biosynthesis of numerous aromatic compounds in bacteria, fungi and plants. The enzyme has an absolute requirement for reduced FMN as a cofactor, although the 1,4-anti elimination of phosphate and the C(6proR)-hydrogen does not involve a net redox change. The role of the reduced FMN in catalysis has long been elusive. However, recent detailed kinetic and bioorganic approaches have fundamentally advanced our understanding of the mechanism of action, suggesting an initial electron transfer from tightly bound reduced flavin to the substrate, a process which results in C-O bond cleavage. Studies on chorismate synthases from bacteria, fungi and plants revealed that in these organisms the reduced FMN cofactor is made available in different ways to chorismate synthase: chorismate synthases in fungi--in contrast to those in bacteria and plants--carry a second enzymatic activity which enables them to reduce FMN at the expense of NADPH. Yet, as shown by the analysis of the corresponding genes, all chorismate synthases are derived from a common ancestor. However, several issues revolving around the origin of reduced FMN, as well as the possible regulation of the enzyme activity by means of the availability of reduced FMN, remain poorly understood. This review summarizes recent developments in the biochemical and genetic arena and identifies future aims in this field.

For a better understanding of the molecular and biochemical processes involved in orthophosphate (Pi) uptake at the root/soil interface, we cloned a Pi-transporter c DNA (LePT1) from a root air-specific cDNA library of tomato (Lycopersicon esculentum Mill.). The corresponding protein belongs to the growing family of ion transporters with twelve putative transmembrane domains. It is highly homologous to recently isolated Pi transporters from higher plants, yeast and fungi. When expressed in a Pi-uptake-deficient yeast mutant, the L. esculentum phosphate transporter 1 (LePT1) protein exhibits an apparent Km of 31 MicroM. The transporter is still active at submicromolar Pi concentrations and mediates highest Pi uptake at pH 5. The activity of LePT1 is dependent on the electrochemical membrane potential mediated by the yeast P-type H + - ATPase. Transcript levels of LePT1 in tomato seedlings are detectable in all vegetative organs under Pi-sufficient conditions, with highest concentrations in root hairs. In situ hybridization studies demonstrate cell-specific expression of LePT1 in the tomato root. The LePT1 mRNA is detectable in peripheral cell layers such as rhizodermal and root cap cells. Under Pi-deprivation condition, mRNA levels are also detectable in young stelar tissue. This work presents molecular and biochemical evidence for distinct root cells playing an important role in Pi acquisition at the root/soil interface.

Three plastidic chorismate synthase isozymes (CS1, CS2 and CS2 delta) of tomato were identified by isolation of the corresponding cDNAs. These three cDNAs are derived from only two genes (LeCS1 and LeCS2). This additional complexity results from differential splicing of the primary transcript of one of the genes (LeCS2) giving rise to two different transcripts (CS2 and CS2 delta transcripts). All three isozymes were individually expressed in Escherichia coli both as precursor proteins with N-terminal transit peptides and as mature proteins. Only the mature but not the precursor isozymes CS1 and CS2 were enzymatically active. The enzyme CS2 delta was unstable in E. coli. Both CS1 and CS2 were purified to near homogeneity and their enzymatic properties were analyzed. They differ substantially in their Km values for the substrate 5-enol-pyruvylshikimate 3-phosphate (11 and 80 microM for the mature forms of CS1 and CS2, respectively). The two isozymes appear to be active only as oligomers, and the potential physiological implications of this result are discussed.

The abundance of transcripts specific for several tomato (Lycopersicon esculentum L.) genes encoding enzymes of the prechorismate pathway was analyzed in different organs of mature plants utilizing a dot-blot assay which was developed for this purpose. The transcript levels were determined for two 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15), one shikimate kinase (EC 2.7.1.71), one 5-enolpyruvylshikimate 3-phosphate synthase (EC 2.5.1.19), and two chorismate synthase (EC 4.6.1.4) genes in leaves, cotyledons, stems, roots, and flowers. These organ-specific expression patterns were compared with that of the phenylalanine ammonia-lyase (EC 4.3.1.5) gene family of tomato.

The re-formation of vacuoles in miniprotoplasts (evacuolated mesophyll protoplasts) of tobacco was investigated under different conditions. When a constant osmolarity was maintained, increasing the concentration of NaCl in the medium enhanced the regeneration of vacuoles compared to the control (0.5 M mannitol used as osmoticum). An enhanced growth rate of miniprotoplasts could also be observed under low-osmolarity conditions, by substitution of NaCl for KCl or NaNO3, or with different effectors (glycinebetaine and methyljasmonate). Using the polymerase chain reaction, one cDNA fragment of the B-subunit of the vacuolar ATPase and two fragments of the tonoplast-bound pyrophosphatase (PPase) of tobacco were cloned. Southern blot analyses indicates that for both proteins more than one gene is present in tobacco. During the regeneration of vacuoles the transcript level of the PPase increased earlier than that of the B-subunit of the vacuolar ATPase under all conditions tested (0.5 M mannitol, 0.3 M mannitol, and 0.25 M NaCl, respectively). Under salt-stress conditions (0.25 M NaCl used as osmoticum), the expression level of both proton pumps is enhanced compared to the control. This increase is not specifically due to salt stress but generally to an increased growth rate of the vacuole, since under low-osmolarity conditions the expression of the vacuolar pumps is enhanced, too.

The fibres of the green-lint mutant (Lg) of cotton (Gossypium hirsutum L.) are suberized and contain a large proportion of wax. The unidentified components of the wax were separated into a colourless fluorescent fraction and a yellow pigmented fraction. Using ultraviolet spectroscopy and nuclear-magneticresonance ((1)H-NMR) spectroscopy, esterified trans-caffeic acid was identified as the only phenolic component in the colourless fraction. This fraction was further purified and was shown to contain caffeic acid esterified to fatty acids (mainly ω-hydroxy fatty acids), and glycerol in molar ratios of 4∶5∶5. When 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (EC 4. 3. 1. 5.) was added to ovules cultured in vitro, at the beginning of secondary wall formation, the fibres remained white and the colourless caffeic-acid derivative and the yellow compounds could no longer be detected by ultraviolet spectroscopy. Fibres grown in the presence of AIP were also examined in the electron microscope. Secondary cell walls were present in the treated fibres, but the electron-opaque suberin layers were replaced by apparently empty spaces. This result indicates that cinnamic-acid derivatives are covalently linked to suberin and have a structural role within the polymer or are involved in anchoring the polymer to the cellulosic secondary wall. Purified cell walls of green cotton fibres contained about 1% (of the dry weight) of bound glycerol, 0.9% of the glycerol being extractable with the wax fraction and 0.1% remaining in the cell-wall residue. The corresponding values for white fibres were 0.03% (total), 0.02% (wax), and 0.01% (cell-wall residue). Fibres synthesizing their secondary walls in the presence of AIP contained about normal amounts of bound glycerol in the wax fraction, but glycerol accumulation in the cell-wall residue was inhibited by about 95%. These observations indicate that glycerol is an important constituent of cotton-fibre suberin. Considerable amounts of bound glycerol could also be determined in exhaustively extracted cell walls of the cork layer of potato periderm (1.2%) and smaller amounts in the outer epidermal cell wall of Agave americana L. leaf (0.1%) indicating that the presence of glycerol in suberins and possibly also in cutins may be more widespread than previously thought.

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.

The light-induced increase in the activity of hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (CQT) in hypocotyls of etiolated buckwheat seedlings is not suppressed by aminooxy acetic acid, an inhibitor of cinnamic acid formation in this tissue. Incubation of hypocotyls in 2 mmol l(-1) trans-cinnamate in the dark does not increase CQT activity. Thus, trans-cinnamate does not appear to mediate the effect of light on CQT in buckwheat hypocotyls.

Recently we have shown that cultured cells of the higher plant Corydalis sempervirens Pers., adapted to growth in the presence of high concentrations of the herbicide glyphosate, a potent specific inhibitor of the shikimate pathway enzyme 5-enolpyruvylshikimic acid 3-phosphate (EPSP) synthase (EC 2.5.1.19, 3-phosphoshikimate 1-carboxyvinyltransferase) oversynthesize the EPSP synthase protein (Smart et al., 1985, J. Biol. Chem. 260, 16338-16346). We now report that the EPSP synthase protein can be detected in cells of the adapted as well as of the non-adapted strain by the use of protein A-colloidal gold immunocytochemistry. The overproduced EPSP synthase in the glyphosate-adapted cells is located exclusively in the plastid and we find no evidence for the existence of extra-plastidic EPSP synthase in either strain.

Fluctuations in the activity of phenylalanine ammonia-lyase (PAL) in tissues of buckwheat seedlings were monitored by an intact cell assay and compared with results obtained with the conventional in vitro assay. Good correlation between measurements with the different assays was found for PAL activity changes during 1) growth of seedlings in the dark, 2) illumination of seedlings, and 3) incubation of hypocotyl segments. When leaf disks of buckwheat, strawberry, or sunflower were allowed to float on either water or a sugar solution, PAL activities determined with the intact cell assay increased irrespective of the presence of sugar, while PAL activities determined in cell homogenates increased only in disks floating on a sugar solution. Possible explanations for these discrepancies are discussed.

A procedure is described which permits the estimation of the relative activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5.) in intact plant cells, exemplified by buckwheat hypocotyls. Hypocotyl segments are incubated at pH 5.5 with L-[3-(3)H]phenylalanine. N(3)HH2, which is liberated from phenylalanine by the action of phenylalanine ammonia-lyase, equilibrates with tissue water to yield (3)HOH, which is recovered by sublimation. Participation of phenylalanine transaminase in the reactions leading to (3)HOH formation is excluded, and it is conclusively shown that (3)HOH is formed intracellularly and not by enzymatic activity leaking out of wounded tissue.

Homogenates of hypocotyls of light-grown mung-bean (Vigna radiata (L.) Wilczek) seedlings catalyzed the formation of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) from the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl-coenzyme A. Apparent Km values for ACC and malonyl-CoA were found to be 0.17 mM and 0.25 mM, respectively. Free coenzyme A was an uncompetitive inhibitor with respect to malonyl-CoA (apparent Ki=0.3 mM). Only malonyl-CoA served as an effective acyl donor in the reaction. The D-enantiomers of unpolar amino acids inhibited the malonylation of ACC. Inhibition by D-phenylalanine was competitive with respect to ACC (apparent Ki=1.2 mM). D-Phenylalanine and D-alanine were malonylated by the preparation, and their malonylation was inhibited by ACC. When hypocotyl segments were administered ACC in the presence of certain unpolar D-amino acids, the malonylation of ACC was inhibited while the production of ethylene was enhanced. Thus, a close-relationship appears to exist between the malonylation of ACC and D-amino acids. The cis- as well as the trans-diastereoisomers of 2-methyl- or 2-ethyl-substituted ACC were potent inhibitors of the malonyltransferase. Treatment of hypocotyl segments with indole-3-acetic acid or CdCl2 greatly increased their content of ACC and MACC, as well as their release of ethylene, but had little, or no, effect on their extractable ACC-malonylating activity.

Both enantiomers of α-aminooxy-β-phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%.

Previous reports on the incorporation of [(14)C]adenine into adenosine-3':5'-cyclic monophosphate (cyclic AMP) in oat (Avena sativa L.) and maize (Zea mays L.) coleoptile sections, chick-pea (Cicer arietinum L.) embryos and barley (Hordeum vulgare L.) aleurone layers were reexamined. Separation of labelled nucleotides on DEAE-Sephadex A 25 showed that a peak of (14)C activity, previously considered to be cyclic AMP, is not identical with this compound. Attempts to detect the cyclic nucleotide by means of a highly specific protein-kinase assay in various plant tissues (Nicotiana tabacum L., tissue culture; Catharanthus roseus Don., tissue culture; Lycopersicon esculentum Mill, seedlings; Nicotiana tabacum, pith parenchyma; Avena sativa, coleoptiles) failed even though up to 100 g of plant material was extracted and a number of control experiments were carried out to insure that cyclic AMP, if present in the extracts, could be measured.

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33-40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments (3)H is stereospecifically released from the pro-3S-position of L-[2,3-(3)H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, (3)H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.