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Reactive oxygen species are believed to perform multiple roles during plant defense and possibly as cellular signaling molecules. In animals, nitric oxide (NO) is an important redox-active signaling molecule. Here we show that infection of resistant, but not susceptible, tobacco with tobacco mosaic virus resulted in enhanced NO synthase (NOS) activity. Furthermore, administration of NO donors or recombinant mammalian NOS to tobacco plants or tobacco suspension cells triggered expression of the defense-related genes encoding pathogenesis-related 1 protein and phenylalanine ammonia lyase (PAL). These genes were also induced by cyclic GMP (cGMP) and cyclic ADP-ribose, two molecules that can serve as second messengers for NO signaling in mammals. Consistent with cGMP levels. Furthermore, NO-induced activation of PAL was blocked by 6-anilino-5,8-quinolinedione and 1H-(1,2,4)-oxadizole[4,3-alpha]quinoxalin-1-one, two inhibitors of guanylate cyclase. Although 6-anilino-5,8-quinolinedione fully blocked PAL activation, inhibition by 1H-(1,2,4)-oxadiozole[4,3-alpha]quinoxalin-1-one was not entirely complete, suggesting the existence of cGMP-independent, as well as cGMP-dependent, NO signaling. We conclude that several critical players of animal NO signaling are also operative in plants

We analyzed lignin content and composition in transgenic tobacco (Nicotiana tabacum) lines altered in the expression of the early phenylpropanoid biosynthetic enzymes L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase (C4H). The reduction of C4H activity by antisense expression or sense suppression resulted in reduced levels of Klason lignin, accompanied by a decreased syringyl/guaiacyl monomer ratio as determined by pyrolysis gas chromatography/mass spectrometry. Similar reduction of lignin levels by down-regulation of L-phenylalanine ammonia-lyase, the enzyme preceding C4H in the central phenylpropanoid pathway, did not result in a decreased syringyl/guaiacyl ratio. Rather, analysis of lignin methoxyl content and pyrolysis suggested an increased syringyl/guaiacyl ratio. One possible explanation of those results is that monolignol biosynthesis from L-phenylalanine might occur by more than one route, even at the early stages of the core phenylpropanoid pathway, prior to the formation of specific monolignol precursors

A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both L-phenylalanine (PAL activity) and L-tyrosine (tyrosine ammonialyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same pH and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine

The signaling pathways that allow plants to mount defenses against chewing insects are known to be complex. To investigate the role of jasmonate in wound signaling in Arabidopsis and to test whether parallel or redundant pathways exist for insect defense, we have studied a mutant (fad3-2fad7-2fad8) that is deficient in the jasmonate precursor linolenic acid. Mutant plants contained negligible levels of jasmonate and showed extremely high mortality (approximately 80%) from attack by larvae of a common saprophagous fungal gnat, Bradysia impatiens (Diptera: Sciaridae), even though neighboring wild-type plants were largely unaffected. Application of exogenous methyl jasmonate substantially protected the mutant plants and reduced mortality to approximately 12%. These experiments precisely define the role of jasmonate as being essential for the induction of biologically effective defense in this plant-insect interaction. The transcripts of three wound-responsive genes were shown not to he induced by wounding of mutant plants but the same transcripts could be induced by application of methyl jasmonate. By contrast, measurements of transcript levels for a gene encoding glutathione S-transferase demonstrated that wound induction of this gene is independent of jasmonate synthesis. These results indicate that the mutant will be a good genetic model for testing the practical effectiveness of candidate defense genes.

Nodulation and nitrogen fixation genes of Mesorhizobium loti are encoded on the chromosome of the bacterium. Nevertheless, there is strong evidence that these genes can be transferred from an inoculant strain to nonsymbiotic mesorhizobia in the field environment. Here we report that the chromosomal symbiotic element of M. loti strain ICMP3153 is transmissible in laboratory matings to at least three genomic species of nonsymbiotic mesorhizobia. The element is 500 kb in size, integrates into a phe-tRNA gene, and encodes an integrase of the phage P4 family just within its left end. The entire phe-tRNA gene is reconstructed at the left end of the element upon integration, whereas the 3' 17 nucleotides of the tRNA gene are present as a direct repeat at the right end. We termed the element a symbiosis island on the basis of its many similarities to pathogenicity islands. It may represent a class of genetic element that contributes to microbial evolution by acquisition

Hydroxyl radicals may be responsible for oxidative damage during drought or chilling stress. We have shown that the presence of mannitol in chloroplasts can protect plants against oxidative damage by hydroxyl radicals (B. Shen, R.G. Jensen, H.J. Bohnert [1997] Plant Physiol 113:1177-1183). Here we identify one of the target enzymes that may be protected by mannitol. Isolated thylakoids in the presence of physiological concentrations of Fe2+ generated hydroxyl radicals that were detected by the conversion of phenylalanine into tyrosine. The activity of phosphoribulokinase (PRK), a thiol-regulated enzyme of the Calvin cycle, was reduced by 65% in illuminated thylakoids producing hydroxyl radicals. Mannitol (125 mM) and sodium formate (15 mM), both hydroxyl radical scavengers, and catalase (3000 units mL-1) prevented loss of PRK activity. In contrast, superoxide dismutase (300 units mL-1) and glycine betaine (125 mM) were not effective in protecting PRK against oxidative inactivation. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity was not affected by hydroxyl radicals. We suggest that the stress-protective role of mannitol may be to shield susceptible thiol-regulated enzymes like PRK plus thioredoxin, ferredoxin, and glutathione from inactivation by hydroxyl radicals in plants

Arabidopsis ecotype Columbia (Col-0) seedlings, transformed with a phenylalanine ammonia-lyase 1 promoter (PAL1)beta-glucuronidase (GUS) reporter construct, were inoculated with virulent and avirulent isolates of Peronospora parasitica. The PAL1 promoter was constitutively active in the light in vascular tissue but was induced only in the vicinity of fungal structures in the incompatible interaction. A double-otaining procedure was developed to distinguish between GUS activity and fungal structures. The PAL1 promoter was activated in cells undergoing lignification in the incompatible interaction in response to the pathogen. Pretreatment of the seedlings with 2-aminoindan-2-phosphonic acid (AIP), a highly specific PAL inhibitor, made the plants completely susceptible. Lignification was suppressed after AIP treatment, and surprisingly, pathogen-induced PAL1 promoter activity could not be detected. Treatment of the seedlings with 2-hydroxyphenylaminosulphinyl acetic acid (1,1-dimethyl ester) (OH-PAS), a cinnamyl alcohol dehydrogenase inhibitor specific tor the lignification pathway, also caused a shift toward susceptibility, but the effect was not as pronounced as it was with AIP. Significantly, although OH-PAS suppressed pathogen-induced lignification, it did not suppress pathogen-induced PAL1 promoter activation. Salicylic acid (SA), supplied to AIP-treated plants, restored resistance and both pathogen-induced lignification and activation of the PAL1 promoter. Endogenous SA levels increased significantly in the incompatible but not in the compatible combination, and this increase was suppressed by AIP but not by OH-PAS. These results provide evidence of the central role of SA in genetically determined plant disease resistance and show that lignification per se, although providing a component of the resistance mechanism, is not the deciding factor between resistance and susceptibility.

In higher plants, sugars are required not only to sustain heterotrophic growth but also to regulate the expression of a variety of genes. Environmental stresses, such as pathogen infection and wounding, activate a cascade of defense responses and may also affect carbohydrate metabolism. In this study, the relationship between sugar- and stress-activated signal transduction pathways and the underlying regulatory mechanism was analyzed. Photoautotrophically growing suspension culture cells of Chenopodium rubrum were used as a model system to study the effects of the metabolic regulator D-glucose and of different stress-related stimuli on photosynthesis, sink metabolism, and defense response by analyzing the regulation of mRNAs for representative enzymes of these pathways. Glucose as well as the fungal elicitor chitosan, the phosphatase inhibitor endothall, and benzoic acid were shown to result in a coordinated regulatory mechanism. The mRNAs for phenylalanine ammonia-lyase, a key enzyme of defense response, and for the sink-specific extracellular invertase were induced. In contrast, the mRNA for the Calvin cycle enzyme ribulose bisphosphate carboxylase was repressed. This inverse regulatory pattern was also observed in experiments with wounded leaves of C. rubrum plants. The differential effect of the protein kinase inhibitor staurosporine on mRNA regulation demonstrates that the carbohydrate signal and the stress-related stimuli independently activate different intracellular signaling pathways that ultimately are integrated to coordinately regulate source and sink metabolism and activate defense responses. The various stimuli triggered the transient and rapid activation of protein kinases that phosphorylate the myelin basic protein. The involvement of phosphorylation in signal transduction is further supported by the effect of the protein kinase inhibitor staurosporine on mRNA levels

Systemic acquired resistance (SAR) has been reported to be associated with lesion-mimic mutants. Tobacco plants expressing vacuolar and apoplastic yeast-derived invertase (vaclnv and cwlnv, respectively) develop spontaneous necrotic lesions similar to hypersensitive responses caused by avirulent pathogens. Therefore, SAR and metabolic alterations leading to the activation of defense-related responses were studied in these plants. Defense-related gene transcripts, callose content, peroxidase activities, and levels of salicylic acid were found to be elevated. The defense reactions were accompanied by increased resistance toward potato virus Y and were measured as decreased viral spreading and reduced multiplication in systemic leaves of the transgenic plants. Interestingly, the accumulation of pathogenesis-related (PR) protein transcripts (PR-Q) and repression of photosynthetic gene transcripts (chlorophyll a/b binding protein) were inversely correlated and required the same threshold level of hexoses for induction and repression. Expression of a cytosolic yeast-derived invertase in transgenic tobacco plants with equally increased levels of sugars neither displayed SAR responses nor showed decreased levels of photosynthetic genes. It is suggested that hexose sensing in the secretory pathway is essential for mediating the activation of defense-related genes as well as repression of photosynthetic genes in vaclnv and cwlnv plants

Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes that include phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). In this work we demonstrate that PAL and CHS mRNAs accumulate in leaves of Arabidopsis thaliana (L.) Heynh. upon exposure to low temperature in a light-dependent manner. The regulation of the PAL1 gene expression by low temperature and light was examined by analyzing the expression of the beta-glucuronidase (uidA) reporter gene in transgenic Arabidopsis plants containing the uidA gene of Escherichia coli under the control of the PAL1 promoter. The results indicate that the accumulation of PAL1 mRNA is transcriptionally regulated. Histochemical staining for beta-glucuronidase activity showed that the PAL1 promoter is preferentially activated in photosynthetically active cells, paralleling anthocyanin accumulation. Moreover, we show that light may also be implicated in the regulation of the CHS gene in response to bacterial infiltration. Finally, using two transparent testa Arabidopsis mutants that are unable to accumulate anthocyanins, we demonstrate that these pigments are not required for successful development of freezing tolerance in this species.