Hypersensitive cell death is an important defense reaction of plants to pathogen infection and is accompanied by lipid peroxidation processes. These may occur non-enzymatically by the action of reactive oxygen species or may be catalyzed by enzymes such as α-dioxygenases, lipoxygenases, or peroxidases. Correlative data showing increases in 9-lipoxygenase products in hyper-sensitively reacting cells have so far suggested that a large part of lipid peroxidation is mediated by a specific set of 9-lipoxygenases. To address the significance of 9-lipoxygenases for this type of pathogen response in potato, RNA interference constructs of a specific pathogen-induced potato 9-lipoxygenase were transferred to potato plants. Significantly reduced 9-lipoxygenase transcript levels were observed in transgenic plants after pathogen treatment. In addition, 9-lipoxygenase activity was hardly detectable, and levels of 9-lipoxygenase-derived oxylipins were reduced up to 12-fold after pathogen infection. In contrast to wild type plants, high levels of non-enzymatically as well as 13-lipoxygenase-derived oxylipins were present in 9-lipoxygenase-deficient plants. From this we conclude that during the normal hypersensitive response in potato, lipid peroxidation may occur as a controlled and directed process that is facilitated by the action of a specific 9-lipoxygenase. If 9-lipoxygenase-mediated formation of hydroperoxides is repressed, autoxidative lipid peroxidation processes and 13-lipoxygenase-mediated oxylipins synthesis become prominent. The unaltered timing and extent of necrosis formation suggests that the origin of lipid hydroperoxides does not influence pathogen-induced cell death in potato.

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with Km values of 50 and 10 μm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring inA. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg2+-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg2+-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.

Plants are continuously exposed to attack by potential phytopathogens. Disease prevention requires pathogen recognition and the induction of a multifaceted defense response. We are studying the non-host disease resistance response of parsley to the oomycete, Phytophthora sojae using a cell culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide sequence (Pep-13) present within an abundant cell wall transglutaminase. Following recognition of this elicitor molecule, parsley cells mount a defense response, which includes the generation of reactive oxygen species (ROS) and transcriptional activation of genes encoding pathogenesis-related (PR) proteins or enzymes involved in the synthesis of antimicrobial phytoalexins. Treatment of parsley cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked both Pep-13-induced phytoalexin production and the accumulation of transcripts encoding enzymes involved in their synthesis. In contrast, DPI treatment had no effect upon Pep-13-induced PRgene expression, suggesting the existence of an oxidative burst-independent mechanism for the transcriptional activation ofPR genes. The use of specific antibodies enabled the identification of three parsley mitogen-activated protein kinases (MAPKs) that are activated within the signal transduction pathway(s) triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast co-transfection system overexpressing wild-type and loss-of-function MAPK mutants, we show an essential role for post-translational phosphorylation and activation of MAPKs for oxidative burst-independentPR promoter activation.

The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn2+/Co2+-hypersensitive yet displays significantly enhanced Cd2+ and Ni2+ tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf− cells compared with wild-type cells in the presence of elevated Zn2+concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd2+ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.

Salutaridinol 7-O-acetyltransferase (EC 2.3.1.150) catalyzes the conversion of the phenanthrene alkaloid salutaridinol to salutaridinol-7-O-acetate, the immediate precursor of thebaine along the morphine biosynthetic pathway. We have isolated a cDNA clone that corresponds to the internal amino acid sequences of the native enzyme purified from a cell suspension culture of opium poppy Papaver somniferum. The recombinant enzyme acetylated the 7-hydroxyl moiety of salutaridinol in the presence of acetyl-CoA. The apparent K m value for salutaridinol was determined to be 9 μm and 54 μm for acetyl-CoA. The gene transcript was detected in extracts from Papaver orientale and Papaver bracteatum in addition to P. somniferum. Genomic DNA gel blot analysis indicated that there is likely a single copy of this gene in the P. somniferum genome. The amino acid sequence of salutaridinol 7-O-acetyltransferase is most similar (37% identity) to that of deacetylvindoline acetyltransferase ofCatharanthus roseus. Salutaridinol 7-O-acetyltransferase is the second enzyme specific to morphine biosynthesis for which we have isolated a cDNA. Taken together with the other cDNAs cloned encoding norcoclaurine 6-O-methyltransferase, (S)-N-methylcoclaurine 3′-hydroxylase, the cytochrome P-450 reductase, and codeinone reductase, significant progress has been made toward accumulating genes of this pathway to enable the end goal of a biotechnological production of morphinan alkaloids.

Lipoxygenases are key enzymes in the synthesis of oxylipins and play an important role in the response of plants to wounding and pathogen attack. In cultured potato cells treated with elicitor from Phytophthora infestans, the causal agent of late blight disease, transcripts encoding a linoleate 9-lipoxygenase and a linoleate 13-lipoxygenase accumulate. However, lipoxygenase activity assays and oxylipin profiling revealed only increased 9-lipoxygenase activity and formation of products derived therefrom, such as 9-hydroxy octadecadienoic acid and colneleic acid. Furthermore, the 9-lipoxygenase products 9(S),10(S),11(R)-trihydroxy-12(Z)-octadecenoic and 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid were identified as novel, elicitor-inducible oxylipins in potato, suggesting a role of these compounds in the defense response against pathogen attack. Neither 13-lipoxygenase activity nor 13-lipoxygenase products were detected in higher amounts in potato cells after elicitation. Thus, formation of products by the 9-lipoxygenase pathway, including the enzymes hydroperoxide reductase, divinyl ether synthase, and epoxy alcohol synthase, is preferentially stimulated in cultured potato cells in response to treatment with P. infestanselicitor. Moreover, elicitor-induced accumulation of desaturase transcripts and increased phospholipase A2 activity after elicitor treatment suggest that substrates for the lipoxygenase pathway might be provided by de novo synthesis and subsequent release from lipids of the endomembrane system.

Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves.

Using synthetic inhibitors, it has been shown that the ectopeptidase dipeptidyl peptidase IV (DP IV) (CD26) plays an important role in the activation and proliferation of T lymphocytes. The human immunodeficiency virus-1 Tat protein, as well as the N-terminal nonapeptide Tat(1–9) and other peptides containing the N-terminal sequence XXP, also inhibit DP IV and therefore T cell activation. Studying the effect of amino acid exchanges in the N-terminal three positions of the Tat(1–9) sequence, we found that tryptophan in position 2 strongly improves DP IV inhibition. NMR spectroscopy and molecular modeling show that the effect of Trp2-Tat(1–9) could not be explained by significant alterations in the backbone structure and suggest that tryptophan enters favorable interactions with DP IV. Data base searches revealed the thromboxane A2 receptor (TXA2-R) as a membrane protein extracellularly exposing N-terminal MWP. TXA2-R is expressed within the immune system on antigen-presenting cells, namely monocytes. The N-terminal nonapeptide of TXA2-R, TXA2-R(1–9), inhibits DP IV and DNA synthesis and IL-2 production of tetanus toxoid-stimulated peripheral blood mononuclear cells. Moreover, TXA2-R(1–9) induces the production of the immunosuppressive cytokine transforming growth factor-β1. These data suggest that the N-terminal part of TXA2-R is an endogenous inhibitory ligand of DP IV and may modulate T cell activation via DP IV/CD26 inhibition.

Hydroxycinnamoyl-CoA:tyramineN-(hydroxycinnamoyl)transferase (THT; EC 2.3.1.110) catalyzes the transfer of hydroxycinnamic acids from the respective CoA esters to tyramine and other amines in the formation ofN-(hydroxycinnamoyl)amines. Expression of THT is induced byPhytophthora infestans, the causative agent of late blight disease in potato. The amino acid sequences of nine endopeptidase LysC-liberated peptides from purified potato THT were determined. Using degenerate primers, a THT-specific fragment was obtained by reverse transcription-polymerase chain reaction, and THT cDNA clones were isolated from a library constructed from RNA of elicitor-treated potato cells. The open reading frame encoding a protein of 248 amino acids was expressed in Escherichia coli. Recombinant THT exhibited a broad substrate specificity, similar to that of native potato THT, accepting cinnamoyl-, 4-coumaroyl-, caffeoyl-, feruloyl- and sinapoyl-CoA as acyl donors and tyramine, octopamine, and noradrenalin as acceptors tested. Elicitor-induced THT transcript accumulation in cultured potato cells peaked 5 h after initiation of treatment, whereas enzyme activity was highest from 5 to 30 h after elicitation. In soil-grown potato plants, THT mRNA was most abundant in roots. Genomic Southern analyses indicate that, in potato, THT is encoded by a multigene family.