Carmen Castresana

Suppression of beta-1,3-glucanase transgene expression in homozygous plants.

A chimeric construct containing the Nicotiana plumbaginifolia beta‐1,3‐glucanase gn1 gene was introduced into Nicotiana tabacum SR1 to produce high levels of the enzyme constitutively. We determined that the GN1 protein represents a basic beta‐1,3‐glucanase isoform which accumulates into the vacuoles of the transgenic plants. Analysis of the progeny of the transgenic plant with the highest levels of gn1 expression revealed an unexpected phenomenon of gene suppression. Plants hemizygous for the T‐DNA locus contained high levels of gn1 mRNA and exhibited a 14‐fold higher beta‐1,3‐glucanase activity than untransformed plants. However, the expression of gn1 was completely suppressed in the homozygous plants: no corresponding mRNA or protein could be detected. This suppression mechanism occurs at a post‐transcriptional level and is under developmental control. In addition, by generating haploid plants we found that this silencing phenomenon is not dependent on allelic interaction between T‐DNA copies present at the same locus of homologous chromosomes, but rather is correlated with the transgene dose in the plant genome. We postulate that high doses of GN1 protein relative to the level(s) of other still unknown plant products could trigger the cellular processes directed to suppress gn1 expression.

Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens

Plant oxylipins are a large family of metabolites derived from polyunsaturated fatty acids. The characterization of mutants or transgenic plants affected in the biosynthesis or perception of oxylipins has recently emphasized the role of the so-called oxylipin pathway in plant defense against pests and pathogens. In this context, presumed functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death. However, the precise contribution of individual oxylipins to plant defense remains essentially unknown. To get a better insight into the biological activities of oxylipins, in vitro growth inhibition assays were used to investigate the direct antimicrobial activities of 43 natural oxylipins against a set of 13 plant pathogenic microorganisms including bacteria, oomycetes, and fungi. This study showed unequivocally that most oxylipins are able to impair growth of some plant microbial pathogens, with only two out of 43 oxylipins being completely inactive against all the tested organisms, and 26 oxylipins showing inhibitory activity toward at least three different microbes. Six oxylipins strongly inhibited mycelial growth and spore germination of eukaryotic microbes, including compounds that had not previously been ascribed an antimicrobial activity, such as 13-keto-9(Z),11(E),15(Z)-octadecatrienoic acid and 12-oxo-10,15(Z)-phytodienoic acid. Interestingly, this first large-scale comparative assessment of the antimicrobial effects of oxylipins reveals that regulators of plant defense responses are also the most active oxylipins against eukaryotic microorganisms, suggesting that such oxylipins might contribute to plant defense through their effects both on the plant and on pathogens, possibly through related mechanisms.

Oxylipins Produced by the 9-Lipoxygenase Pathway in Arabidopsis Regulate Lateral Root Development and Defense Responses through a Specific Signaling Cascade

Arabidopsis thaliana seedling growth with pure oxylipins resulted in root waving, loss of root apical dominance, and decreased root elongation. 9-Hydroxyoctadecatrienoic acid (9-HOT) was a potent inducer of root waving. Studies with noxy2 (for nonresponding to oxylipins2), a new 9-HOT–insensitive mutant, and coronatine insensitive1-1 (jasmonate-insensitive) revealed at least three signaling cascades mediating the oxylipin actions. Treatment with 9-HOT resulted in a reduction in lateral roots and an increase in stage V primordia. Roots showed strong 9-lipoxygenase (9-LOX) activity, and root primordia expressed 9-LOX genes. These results, along with findings that noxy2 and mutants with defective 9-LOX activity showed increased numbers of lateral roots, suggest that 9-HOT, or a closely related 9-LOX product, is an endogenous modulator of lateral root formation. Histochemical and molecular analyses revealed that 9-HOT activated events common to development and defense responses. A subset of 9-HOT–responding root genes was also induced in leaves after 9-HOT treatment or pathogen inoculation. The results that noxy2 displayed altered root development, enhanced susceptibility to Pseudomonas, and reduced the activation of 9-HOT–responding genes are consistent with mechanistic links among these processes. The nature of the changes detected suggests that oxylipins from the 9-LOX pathway function in cell wall modifications required for lateral root development and pathogen arrest.

PIOX, a New Pathogen-Induced Oxygenase with Homology to Animal Cyclooxygenase

Changes in gene expression induced in tobacco leaves by the harpin HrpN protein elicitor were examined, and a new cDNA, piox (for pathogen-induced oxygenase), with homology to genes encoding cyclooxygenase or prostaglandin endoperoxide synthase (PGHS), was identified. In addition to the amino acid identity determined, the protein encoded by piox is predicted to have a structural core similar to that of ovine PGHS-1. Moreover, studies of protein functionality demonstrate that the PIOX recombinant protein possesses at least one of the two enzymatic activities of PGHSs, that of catalyzing the oxygenation of polyunsaturated fatty acids. piox transcripts accumulated after protein elicitor treatment or inoculation with bacteria. Expression of piox was induced in tissues responding to inoculation with both incompatible and compatible bacteria, but RNA and protein accumulation differed for both types of interactions. We show that expression of piox is rapidly induced in response to various cellular signals mediating plant responses to pathogen infection and that activation of piox expression is most likely related to the oxidative burst that takes place during the cell death processes examined. Cyclooxygenase catalyzes the first committed step in the formation of prostaglandins and thromboxanes, which are lipid-derived signal molecules that mediate many cellular processes, including the immune response in vertebrates. The finding of tobacco PIOX suggests that more similarities than hitherto expected will be found between the lipid-based responses for plant and animal systems.

ALPHA -OXIDATION OF FATTY ACIDS IN HIGHER PLANTS: IDENTIFICATION OF A PATHOGEN-INDUCIBLE OXYGENASE (PIOX) AS AN ALPHA -DIOXYGENASE AND BIOSYNTHESIS OF 2-HYDROPEROXYLINOLENIC ACID

A pathogen-inducible oxygenase in tobacco leaves and a homologous enzyme from Arabidopsiswere recently characterized (Sanz, A., Moreno, J. I., and Castresana, C. (1998) Plant Cell 10, 1523–1537). Linolenic acid incubated at 23 °C with preparations containing the recombinant enzymes underwent α-oxidation with the formation of a chain-shortened aldehyde, i.e., 8(Z),11(Z),14(Z)-heptadecatrienal (83%), an α-hydroxy acid, 2(R)-hydroxy-9(Z),12(Z),15(Z)-octadecatrienoic acid (15%), and a chain-shortened fatty acid, 8(Z),11(Z),14(Z)-heptadecatrienoic acid (2%). When incubations were performed at 0 °C, 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was obtained as the main product. An intermediary role of 2(R)-hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid in α-oxidation was demonstrated by re-incubation experiments, in which the hydroperoxide was converted into the same α-oxidation products as those formed from linolenic acid. 2(R)-Hydroperoxy-9(Z),12(Z),15(Z)-octadecatrienoic acid was chemically unstable and had a half-life time in buffer of about 30 min at 23 °C. Extracts of cells expressing the recombinant oxygenases accelerated breakdown of the hydroperoxide (half-life time, about 3 min at 23 °C), however, this was not attributable to the recombinant enzymes since the same rate of hydroperoxide degradation was observed in the presence of control cells not expressing the enzymes. No significant discrimination between enantiomers was observed in the degradation of 2(R,S)-hydroperoxy-9(Z)-octadecenoic acid in the presence of recombinant oxygenases. A previously studied system for α-oxidation in cucumber was re-examined using the newly developed techniques and was found to catalyze the same conversions as those observed with the recombinant enzymes, i.e. enzymatic α-dioxygenation of fatty acids into 2(R)-hydroperoxides and a first order, non-stereoselective degradation of hydroperoxides into α-oxidation products. It was concluded that the recombinant enzymes from tobacco and Arabidopsis were both α-dioxygenases, and that members of this new class of enzymes catalyze the first step of α-oxidation in plant tissue.

An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.

In Arabidopsis the stomatal defense response, a feature of the innate immunity in plants, involves oxylipin-mediated mechanisms that are independent of the phytohormone abscisic acid.

Controlling hormone signaling is a plant and pathogen challenge for growth and survival

Plants and pathogens have continuously confronted each other during evolution in a battle for growth and survival. New advances in the field have provided fascinating insights into the mechanisms that have co-evolved to gain a competitive advantage in this battle. When plants encounter an invading pathogen, not only responses signaled by defense hormones are activated to restrict pathogen invasion, but also the modulation of additional hormone pathways is required to serve other purposes, which are equally important for plant survival, such as re-allocation of resources, control of cell death, regulation of water stress, and modification of plant architecture. Notably, pathogens can counteract both types of responses as a strategy to enhance virulence. Pathogens regulate production and signaling responses of plant hormones during infection, and also produce phytohormones themselves to modulate plant responses. These results indicate that hormone signaling is a relevant component in plant-pathogen interactions, and that the ability to dictate hormonal directionality is critical to the outcome of an interaction.

Diversity of the Enzymatic Activity in the Lipoxygenase Gene Family of Arabidopsis thaliana

Lipoxygenases (LOX) catalyze the oxygenation of polyunsaturated fatty acids, the first step in the biosynthesis of a large group of biologically active fatty acid metabolites collectively named oxylipins. In the present study we report the characterization of the enzymatic activity of the six lipoxygenases found in the genome of the model plant Arabidopsis thaliana. Recombinant expressed AtLOX-1 and AtLOX-5 had comparable oxygenase activity with either linoleic acid or linolenic acid. AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 displayed a selective oxygenation of linolenic acid. Analyses by high-performance liquid chromatography and gas chromatography-mass spectrometry demonstrated that AtLOX-1 and AtLOX-5 are 9S-lipoxygenases, and AtLOX-2, AtLOX-3, AtLOX-4 and AtLOX-6 are 13S-lipoxygenases. None of the enzymes had dual positional specificity. The determined activities correlated with that predicted by their phylogenetic relationship to other biochemically-characterized plant lipoxygenases.

Emerging Complexity in Reactive Oxygen Species Production and Signaling during the Response of Plants to Pathogens

Plants have evolved a complex immune system to perceive microbial pathogens and respond by producing defense compounds preventing infection. Defense hormones such as salicylic acid (SA), jasmonates, and ethylene are key signals regulating the production of antimicrobial defenses. Moreover, other

Activation of the Fatty Acid α-Dioxygenase Pathway during Bacterial Infection of Tobacco Leaves FORMATION OF OXYLIPINS PROTECTING AGAINST CELL DEATH

A pathogen-induced oxygenase showing homology to prostaglandin endoperoxide synthases-1 and -2 was recently characterized by in vitro experiments as a fatty acid α-dioxygenase catalyzing formation of unstable 2(R)-hydroperoxy fatty acids. To study the activity of this enzyme under in vivo conditions and to elucidate the fate of enzymatically produced 2-hydroperoxides, leaves of tobacco were analyzed for the presence of α-dioxygenase-generated compounds as well as for lipoxygenase (LOX) products and free fatty acids. Low basal levels of 2-hydroxylinolenic acid (0.4 nmol/g leaves fresh weight) and 8,11,14-heptadecatrienoic acid (0.1 nmol/g) could be demonstrated. These levels increased strongly upon infection with the bacterium Pseudomonas syringae pv syringae (548 and 47 nmol/g, respectively). Transgenic tobacco plants overexpressing α-dioxygenase were developed, and incompatible infection of such plants led to a dramatic elevation of 2-hydroxylinolenic acid (1778 nmol/g) and 8,11,14-heptadecatrienoic acid (86 nmol/g), whereas the levels of LOX products were strongly decreased. Further analysis of oxylipins in infected leaves revealed the presence of a number of 2-hydroxy fatty acids differing with respect to chain length and degree of unsaturation as well as two new doubly oxygenated oxylipins identified as 2(R),9(S)-dihydroxy-10(E),12(Z),15(Z)-octadecatrienoic acid and 2(R),9(S)-dihydroxy-10(E),12(Z)-octadecadienoic acid. α-Dioxygenase-generated 2-hydroxylinolenic acid, and to a lesser extent lipoxygenase-generated 9-hydroxyoctadecatrienoic acid, exerted a tissue-protective effect in bacterially infected tobacco leaves.