The Myo-inositol pathway does not contribute to ascorbic acid synthesis.

Ascorbic acid (AsA) biosynthesis in plants predominantly occurs via a pathway with d-mannose and l-galactose as intermediates. One alternative pathway for AsA synthesis, which is similar to the biosynthesis route in mammals, is controversially discussed for plants. Here, myo-inositol is cleaved to glucuronic acid and then converted via l-gulonate to AsA. In contrast to animals, plants have an effective recycling pathway for glucuronic acid, being a competitor for the metabolic rate. Recycling involves a phosphorylation at C1 by the enzyme glucuronokinase. Two previously described T-DNA insertion lines in the gene coding for glucuronokinase1 show wild type-like expression levels of the mRNA in our experiments and do not accumulate glucuronic acid in labelling experiments disproving that these lines are true knockouts. As suitable T-DNA insertion lines were not available, we generated frameshift mutations in the major expressed isoform glucuronokinase1 (At3g01640) to potentially redirect metabolites to AsA. However, radiotracer experiments with 3 H-myo-inositol revealed that the mutants in glucuronokinase1 accumulate only glucuronic acid and incorporate less metabolite into cell wall polymers. AsA was not labelled, suggesting that Arabidopsis cannot efficiently use glucuronic acid for AsA biosynthesis. All four mutants in glucuronokinase as well as the wild type have the same level of AsA in leaves.

Suppression of the ribosomal L2 gene reveals a novel mechanism for stress adaptation in soybean.

Pseudomonas syringae pv. glycinea bacteria or zoospores of the fungus Phytophthora sojae were used to trigger a hypersensitive reaction (HR) in cell cultures of soybean (Glycine max [L.] Merr. cv. Williams 82). During a screen for genes that show an altered expression as a response to dying neighbour cells we have identified a gene fragment that is specifically but transiently down-regulated in an HR. The corresponding cDNA codes for the ribosomal protein L2 (rpL2) of 80S ribosomes, which is essential for the peptidyl-transferase activity. Two gene copies of rpL2 exist in soybean and both genes are transcribed. The temporary down-regulation of the rpL2 genes is followed by a transient block in the synthesis of new proteins as visualised by pulse-labelling experiments using 35S-amino acids. The same basic phenomenon was also found after treatment of soybean cells with other stress-causing compounds such as elicitors or heavy metals. It is suggested that the transient block in protein synthesis allows a more rapid depletion of, for example, signal molecules with a short half-life time and thus leads to a faster adaptation of the cellular protein inventory to the new environmental conditions.

WY-14,643 and other agonists of the peroxisome proliferator-activated receptor reveal a new mode of action for salicylic acid in soybean disease resistance.

Inoculation of soybean (Glycine max [L.] Merr.) cell-suspension cultures with avirulent bacteria results in a salicylic acid (SA)-controlled programmed cell death (pcd). To unravel the nature of the SA-dependent step in pcd, a screening procedure for complementing compounds was performed. Diverse chemicals that are well known as activating ligands for orphan receptors in animals, particularly receptors of the PPAR (peroxisome proliferator-activated receptor) subfamily, were found to be active. These include the compounds WY-14643, flufenamic acid, LY-171883, tolbutamide, indomethacin and clofibrate. A new marker gene (DD-CA9) from soybean that is induced in the hypersensitive reaction by SA and by PPAR ligands was isolated by differential display, and showed homology to antifungal lectins. In plants, SA is also involved in a signal transduction pathway leading to systemic acquired resistance (SAR). The PPAR ligands which act on the pcd pathway for plant resistance induce a beta-1,3-glucanase gene in soybean at high concentrations but do not induce marker genes of the SAR pathway such as the PR-1 gene in tobacco or Arabidopsis. Thus SA seems to act on two independent plant defence pathways that can now be separately activated by synthetic compounds. We propose a model for the control of pcd by SA in soybean, in which SA induces the transcription of (novel) genes required for the final completion of the cell death program.

Defence gene expression in soybean is linked to the status of the cell death program.

Soybean cell cultures (cv. Williams 82) respond to Pseudomonas syringae bacteria expressing the avirulence gene AvrA with a hypersensitive reaction, a programmed cell death (PCD) of plant cells to pathogen attack. This PCD is under control of salicylic acid (SA) via an unknown mechanism. In the presence of low concentrations of SA, the cells undergo a very rapid cell death, which needs only half of the time required for the normal hypersensitive reaction (HR). Northern blot studies for defence-related genes show that the expression of many of these genes is tightly linked to the status of the cell death program rather than to pathogen-derived elicitors. Thus the expression is much faster in the SA-accelerated PCD than in the normal hypersensitive reaction. In contrast, other pathogen-responsive genes are induced independently of the speed of PCD, indicating a divergent signalling mechanism. The production of reactive oxygen species during the oxidative burst of bacteria-inoculated soybean cells is slightly enhanced in the presence of SA but occurs at the same time as in untreated cells, suggesting that SA exhibits the control of the PCD downstream of the oxidative burst. Consistent with these findings a HR-specific marker gene is neither directly induced by H2O2 or SA. However, this gene shows a high expression in the regular HR and is induced much faster in the SA-accelerated PCD.

Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ-specific expression patterns.

The expression pattern of the single-copy gene UDP-glucose dehydrogenase (Ugd) was analysed in transgenic Arabidopsis plants by promoter:GUS and GFP fusions, Western blots, activity assays and histochemical activity staining. The enzyme oxidizes UDP-glucose to UDP-glucuronic acid and thus directs carbohydrates irreversibly into a cell wall-specific pool of nucleotide sugars. UDP-glucuronic acid is the central intermediate in the interconversion pathway to other nucleotide sugars, including the UDP-derivatives of arabinose, xylose, apiose and galacturonic acid which account for half the biomass of a typical Arabidopsis leaf cell wall. These activated sugars are needed as substrates for the biosynthesis of matrix polysaccharide polymers. In plants up to 5 days old the Ugd gene is strongly expressed in young roots, but very little in hypocotyls. Older plants show a more uniform expression pattern with a preference for the vascular system. A complex expression pattern was observed in flowers with high activity in the stamen, stigma and nectaries. Meristems in the leaf axil of rosette and inflorescence leaves exhibit a high level of activity of the Ugd gene. Although many of the growing tissues show high activity levels of the Ugd gene, others such as the hypocotyl and the cotyledons of young seedlings do not. Instead these tissues efficiently incorporate 3H-inositol into their cell walls. This indicates the biosynthesis of UDP-glucuronic acid through an alternative pathway via the oxidation of inositol to glucuronic acid and subsequent activation to the nucleotide sugar. The data strongly suggest two alternative pathways for matrix polysaccharide precursors with spatial and developmental regulation.

Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea.

Soybean (Glycine max [L.] Merr.) cell suspension cultures (cv. Williams 82) inoculated with the pathogenic bacteria Pseudomonas syringae pv. glycinea respond with a hypersensitive reaction (HR) when the bacteria express the avirulence gene avrA. A mRNA differential display was established for this system to allow the identification of genes induced during the HR. Six PCR-fragments (DD1-DD6) from the differential display analysis were identified, which are induced during the HR. Database searches revealed that the fragment DD1 encodes chalcone isomerase and DD2 was identified as ubiquitin. The fragment DD3 shares significant homology to the signalling molecule 14-3-3. The partial DD4 product is homologous to the enhancer of rudimentary from Drosophila and an uncharacterized homologue of it from Arabidopsis. The fragment DD5 is similar to glucose-6-phosphate dehydrogenase which provides NADPH to the cell. The PCR-product DD6 seems to be a new leucine-rich-repeat disease resistance gene from soybean, which is significantly induced during the HR. All of the identified genes are clearly induced during a HR in infected plants of the same cultivar, indicating that results from the cell culture model system can be transferred to intact plants. These studies show that complex mRNA differential display is a powerful tool to identify new induced gene in plant-pathogen interactions.

Characterization and cloning of cutinase from Ascochyta rabiei.

Ascochyta rabiei, the causal agent of Ascochyta blight on chickpea plants, secretes a cutinase in the culture filtrate when it is induced by cutin or hydroxylated fatty acids. This cutinase is the main esterase in the culture fluids. The enzyme was purified to homogeneity by three successive chromatographic steps. It showed an apparent molecular weight of 22 kD in SDS-PAGE and cleaved ester bonds of 3H-labelled cutin or p-nitrophenylbutyrate with maximal activities around pH 8. As a serine esterase, cutinase is strongly inhibited by organophosphorous compounds and the most effective inhibitor 2,3,5-trichloropyridine-6-(O-methyl-O-n-butyl)-phosphateester++ + (MAT 9564) shows a Ki value of 0.8 nM. The cutinase gene was cloned from a genomic cosmid library by screening with two oligonucleotides directed against cutinase consensus peptides. The gene was subcloned to a 1.7 Kb SaII/HindIII-insert and sequenced. The cutinase gene codes for a 223 amino acid protein with strong homology to other fungal cutinase sequences. The purified cutinase is encoded by a single copy gene.

Characterization of a Diffusible Signal Capable of Inducing Defense Gene Expression in Tobacco.

Treatment of tobacco (Nicotiana tabacum) cell-suspension cultures with cryptogein, an elicitin protein from Phytophthora cryptogea, resulted in the release of a factor(s) that diffused through a 1000-D cutoff dialysis membrane and was capable of inducing sesquiterpene cyclase enzyme activity (a key phytoalexin biosynthetic enzyme in solanaceous plants) when added to fresh cell-suspension cultures. The diffusible factor(s) was released from cells over a 20-h period and induced a more rapid induction of cyclase enzyme activity than did direct treatment of the cultures with pure elicitin protein. The diffusible factor also induced a more rapid accumulation of transcripts encoding for sesquiterpene cyclase, acidic and basic chitinase, and hsr203 (a putative hypersensitive response gene) than did elicitin treatment. The diffusible factor(s) was resistant to protease, pectinase, Dnase, and RNase treatments, was not extractable into organic solvents, and was not immunoprecipitable when challenged with polyclonal antibodies prepared against elicitin protein. The diffusible factor(s) could not induce the release of more factor, suggesting that it was a terminal signal. These results are consistent with the notion that cells directly challenged or stimulated by pathogen-derived elicitors release diffusible secondary signal molecules that orchestrate the induction of complementary defense responses in neighboring cells.

Salicylic Acid Is Needed in Hypersensitive Cell Death in Soybean but Does Not Act as a Catalase Inhibitor.

The function of salicylic acid (SA) in hypersensitive cell death was studied in a soybean (Glycine max)-Pseudomonas syringae pv glycinea system. The infection of cell cultures with bacteria leads to a hypersensitive reaction (HR), which is dependent on an appropriate avirulence gene and on low concentrations of SA. The requirement for SA is essential for a process shortly before the onset of the HR-caused cell death 5 to 6 h after infection with bacteria. SA given 10 to 12 h after infection or preincubation cannot rescue the completion of the cell death program. SA does not inhibit catalase or ascorbate peroxidase in soybean. In addition, the in vivo capacity of the cell culture for the rapid metabolism of H2O2 is not altered by SA. This clearly shows that SA is needed for the HR-caused cell death for a reaction downstream of the oxidative burst. Lipid peroxides accumulate during the HR, but the loss of membrane control precedes the generation of lipid peroxides. The accumulation of lipid peroxides in the HR can be prevented by lipid antioxidants. Nevertheless, cell death kinetics remain unaltered in the presence of antioxidants. It is concluded that lipid peroxides are a consequence of cell death, but not the primary cause of it.

Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase.

Hemicellulose is a major component of primary plant cell walls. Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose. The enzyme controlling the biosynthesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22), was cloned from soybean (Glycine max [L.] Merr.) by an antibody screening procedure. This enzyme is surprisingly homologous to the bovine sequence, which is the only other eukaryotic UDP-glucose dehydrogenase sequence known. The characteristic motifs of the enzyme, the catalytic center, a NAD-binding site, and two proline residues for main chain bends, are conserved within the prokaryotic and eukaryotic sequences. The soybean full-length cDNA clone encodes a protein of 480 amino acids with a predicted size of 52.9 kD. The enzyme is highly expressed in young roots, but lower expression levels were observed in expanding tissues of the epicotyl and in young leaves. The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors.

Function of the oxidative burst in hypersensitive disease resistance.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. Recent findings indicate that H2O2 from this oxidative burst plays a central role in the orchestration of the hypersensitive response: (i) as the substrate driving the cross-linking of cell wall structural proteins to slow microbial ingress prior to the deployment of transcription-dependent defenses and to trap pathogens in cells destined to undergo hypersensitive cell death, (ii) as a local threshold trigger of this programmed death in challenged cells, and (iii) as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. These findings provide the basis for an integrated model for the orchestration of the localized hypersensitive resistance response to attack by an avirulent pathogen.

Function of Oxidative Cross-Linking of Cell Wall Structural Proteins in Plant Disease Resistance.

Elicitation of soybean cells causes a rapid insolubilization of two cell wall structural proteins, p33 and p100. Likewise, a short elicitation of 30 min rendered cell walls more refractory to enzyme digestion as assayed by the yield of protoplasts released. This effect could be ascribed to protein cross-linking because of its insensitivity to inhibitors of transcription (actinomycin D) and translation (cycloheximide) and its induction by exogenous H2O2. Moreover, the induced loss of protoplasts could be prevented by preincubation with DTT, which also blocks peroxidase-mediated oxidative cross-linking. The operation of protein insolubilization in plant defense was also demonstrated by its occurrence in the incompatible interaction but not in the compatible interaction between soybean and Pseudomonas syringae pv glycinea. Likewise, protein insolubilization was observed in bean during non-host hypersensitive resistance to the tobacco pathogen P. s. pv tabaci mediated by the hypersensitive resistance and pathogenicity (Hrp) gene cluster. Our data strongly suggest that rapid protein insolubilization leads to a strengthened cell wall, and this mechanism functions as a rapid defense in the initial stages of the hypersensitive response prior to deployment of transcription-dependent defenses.

H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. We report here that H2O2 from this oxidative burst not only drives the cross-linking of cell wall structural proteins, but also functions as a local trigger of programmed death in challenged cells and as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. Thus, H2O2 from the oxidative burst plays a key role in the orchestration of a localized hypersensitive response during the expression of plant disease resistance.

Medicarpin and maackiain 3-O-glucoside-6'-O-malonate conjugates are constitutive compounds in chickpea (Cicer arietinum L.) cell cultures.

The pterocarpan phytoalexin conjugates medicarpin 3-O-glucoside-6'-O-malonate and maackiain 3-O-glucoside-6'-O-malonate were isolated from cell suspension cultures of chickpea (Cicer arietinum L.) cultivar ILC 3279 and structurally elucidated. Both pterocarpan conjugates are constitutive metabolites of the chickpea cell cultures. Upon application of an elicitor from yeast to the cell cultures a substantial increase in the level of the phytoalexin aglycones medicarpin and maackiain was observed although a delayed but significantly higher rise of the conjugates also occurred. The significance of the pterocarpan conjugates for phytoalexin production is discussed.

A new inexpensive customized plaque for choroidal melanoma iodine-125 plaque therapy.

The authors have developed a new inexpensive precious metal alloy plaque for use in customized iodine-125 plaque therapy. Each plaque is formed from two flat circular gold/palladium foils which are used in dental crown work. Using a simple manual mechanism, the two forms are stamped over a customized acrylic die shaped to the dimensions of the tumor base plus a 2-mm margin. Completed plaques consist of a back wall, a 2-mm side wall, and a 1.5-mm wide lip with holes for suture placement. Advantages include: simple construction from inexpensive components, customized shape, and iodine seeds that are readily visible on plane radiographs.

Response of epidermoid and non-epidermoid cancers of the head and neck to fast neutron irradiation: the Fermilab experience.

One hundred and six patients with locally advanced cancers of the head and neck were treated with neutrons at the Fermilab Neutron Therapy Facility. Of these, 44 patients were previously untreated, 33 were recurrent following attempted surgery and 29 patients had previously received a full course of radiation therapy with conventional radiation. Results were analyzed to study the influence of stage, previous management, site of origin and tumor histology on local control of the disease. The most significant factor determining the outcome in this series of patients is the histological type. For epidermoid carcinoma, long term local control was achieved in 17/35 patients (49%) in the previously unirradiated group. With non-epidermoid tumors (adenocarcinoma, cylindroma, muco-epidermoid carcinoma), the local control rate was 28/39 (72%). Disease-free survival analysis also shows a survival advantage in non-epidermoid lesions treated with neutrons. It is concluded that neutron beam therapy may probably be the treatment of choice for non-resectable or recurrent non-epidermoid cancers of the head and neck and requires a clinical trial to establish this observation.

Physiological effects of hyperthermia: response of capillary blood flow and structure to local tumor heating.

Specific capillary blood flow and small-vessel pathology in animal tumors (rhabdomyosarcoma BA-1112) of WAG/Rij rats were examined following local tumor heating to 40-44.5 degrees C. Blood flow in tumors heated to 40-41 degrees C for 40 minutes was reduced initially by about 50%, but returned to near preheating values within 72 hours, consistent with the histopathological observations indicating small-vessel dilation and temporary congestion. The application of hyperthermia greater than 43 degrees C (for 40 minutes) resulted in the virtual elimination of capillary blood flow, consistent with pathological findings of widespread vessel rupture and hemorrhage in this temperature range.