Andrés Gárriz

Apoplastic Polyamine Oxidation Plays Different Roles in Local Responses of Tobacco to Infection by the Necrotrophic Fungus Sclerotinia sclerotiorum and the Biotrophic Bacterium Pseudomonas viridiflava

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decarboxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast. Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis due to infection by S. sclerotiorum. Specific inhibition of diamine and PA oxidases attenuated the PA-induced enhancement of leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PA oxidase inhibitor. It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting pathogenesis strategies. This response affects the pathogens ability to colonize host tissues and results are detrimental for plant defense against necrotrophic pathogens that feed on necrotic tissue; on the contrary, this response plays a beneficial role in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to significant changes in host susceptibility to different kinds of pathogens.

Ecological and agronomic importance of the plant genus Lotus. Its application in grassland sustainability and the amelioration of constrained and contaminated soils

The genus Lotus comprises around 100 annual and perennial species with worldwide distribution. The relevance of Lotus japonicus as a model plant has been recently demonstrated in numerous studies. In addition, some of the Lotus species show a great potential for adaptation to a number of abiotic stresses. Therefore, they are relevant components of grassland ecosystems in environmentally constrained areas of several South American countries and Australia, where they are used for livestock production. Also, the fact that the roots of these species form rhizobial and mycorrhizal associations makes the annual L. japonicus a suitable model plant for legumes, particularly in studies directed to recognize the mechanisms intervening in the tolerance to abiotic factors in the field, where these interactions occur. These properties justify the increased utilization of some Lotus species as a strategy for dunes revegetation and reclamation of heavy metal-contaminated or burned soils in Europe.

The Sesquiterpene Botrydial Produced by Botrytis cinerea Induces the Hypersensitive Response on Plant Tissues and Its Action Is Modulated by Salicylic Acid and Jasmonic Acid Signaling

Botrytis cinerea, as a necrotrophic fungus, kills host tissues and feeds on the remains. This fungus is able to induce the hypersensitive response (HR) on its hosts, thus taking advantage on the hosts defense machinery for generating necrotic tissues. However, the identity of HR effectors produced by B. cinerea is not clear. The aim of this work was to determine whether botrydial, a phytotoxic sesquiterpene produced by B. cinerea, is able to induce the HR on plant hosts, using Arabidopsis thaliana as a model. Botrydial induced the expression of the HR marker HSR3, callose deposition, and the accumulation of reactive oxygen species and phenolic compounds. Botrydial also induced the expression of PR1 and PDF1.2, two pathogenesis-related proteins involved in defense responses regulated by salicylic acid (SA) and jasmonic acid (JA), respectively. A. thaliana and tobacco plants defective in SA signaling were more resistant to botrydial than wild-type plants, as opposed to A. thaliana plants defective in JA signaling, which were more sensitive. It can be concluded that botrydial induces the HR on its hosts and its effects are modulated by host signaling pathways mediated by SA and JA.

Physiological and molecular implications of plant polyamine metabolism during biotic interactions

During ontogeny, plants interact with a wide variety of microorganisms. The association with mutualistic microbes results in benefits for the plant. By contrast, pathogens may cause a remarkable impairment of plant growth and development. Both types of plant–microbe interactions provoke notable changes in the polyamine (PA) metabolism of the host and/or the microbe, being each interaction a complex and dynamic process. It has been well documented that the levels of free and conjugated PAs undergo profound changes in plant tissues during the interaction with microorganisms. In general, this is correlated with a precise and coordinated regulation of PA biosynthetic and catabolic enzymes. Interestingly, some evidence suggests that the relative importance of these metabolic pathways may depend on the nature of the microorganism, a concept that stems from the fact that these amines mediate the activation of plant defense mechanisms. This effect is mediated mostly through PA oxidation, even though part of the response is activated by non-oxidized PAs. In the last years, a great deal of effort has been devoted to profile plant gene expression following microorganism recognition. In addition, the phenotypes of transgenic and mutant plants in PA metabolism genes have been assessed. In this review, we integrate the current knowledge on this field and analyze the possible roles of these amines during the interaction of plants with microbes.

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. α-Difluoromethylornithine (DFMO) and α-difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.

Defense responses in two ecotypes of Lotus japonicus against non-pathogenic Pseudomonas syringae.

Lotus japonicus is a model legume broadly used to study many important processes as nitrogen fixing nodule formation and adaptation to salt stress. However, no studies on the defense responses occurring in this species against invading microorganisms have been carried out at the present. Understanding how this model plant protects itself against pathogens will certainly help to develop more tolerant cultivars in economically important Lotus species as well as in other legumes. In order to uncover the most important defense mechanisms activated upon bacterial attack, we explored in this work the main responses occurring in the phenotypically contrasting ecotypes MG-20 and Gifu B-129 of L. japonicus after inoculation with Pseudomonas syringae DC3000 pv. tomato. Our analysis demonstrated that this bacterial strain is unable to cause disease in these accessions, even though the defense mechanisms triggered in these ecotypes might differ. Thus, disease tolerance in MG-20 was characterized by bacterial multiplication, chlorosis and desiccation at the infiltrated tissues. In turn, Gifu B-129 plants did not show any symptom at all and were completely successful in restricting bacterial growth. We performed a microarray based analysis of these responses and determined the regulation of several genes that could play important roles in plant defense. Interestingly, we were also able to identify a set of defense genes with a relative high expression in Gifu B-129 plants under non-stress conditions, what could explain its higher tolerance. The participation of these genes in plant defense is discussed. Our results position the L. japonicus-P. syringae interaction as a interesting model to study defense mechanisms in legume species.

Response to long-term NaHCO3-derived alkalinity in model Lotus japonicus Ecotypes Gifu B-129 and Miyakojima MG-20: transcriptomic profiling and physiological characterization.

The current knowledge regarding transcriptomic changes induced by alkalinity on plants is scarce and limited to studies where plants were subjected to the alkaline salt for periods not longer than 48 h, so there is no information available regarding the regulation of genes involved in the generation of a new homeostatic cellular condition after long-term alkaline stress. Lotus japonicus is a model legume broadly used to study many important physiological processes including biotic interactions and biotic and abiotic stresses. In the present study, we characterized phenotipically the response to alkaline stress of the most widely used L. japonicus ecotypes, Gifu B-129 and MG-20, and analyzed global transcriptome of plants subjected to 10 mM NaHCO3 during 21 days, by using the Affymetrix Lotus japonicus GeneChip®. Plant growth assessment, gas exchange parameters, chlorophyll a fluorescence transient (OJIP) analysis and metal accumulation supported the notion that MG-20 plants displayed a higher tolerance level to alkaline stress than Gifu B-129. Overall, 407 and 459 probe sets were regulated in MG-20 and Gifu B-129, respectively. The number of probe sets differentially expressed in roots was higher than that of shoots, regardless the ecotype. Gifu B-129 and MG-20 also differed in their regulation of genes that could play important roles in the generation of a new Fe/Zn homeostatic cellular condition, synthesis of plant compounds involved in stress response, protein-degradation, damage repair and root senescence, as well as in glycolysis, gluconeogenesis and TCA. In addition, there were differences between both ecotypes in the expression patterns of putative transcription factors that could determine distinct arrangements of flavonoid and isoflavonoid compounds. Our results provided a set of selected, differentially expressed genes deserving further investigation and suggested that the L. japonicus ecotypes could constitute a useful model to search for common and distinct tolerance mechanisms to long-term alkaline stress response in plants.

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. Alpha-difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.

Polyamine Metabolism Responses to Biotic and Abiotic Stress

Plants have developed different strategies to cope with the environmental stresses they face during their life cycle. The responses triggered under these conditions are usually characterized by significant modifications in the metabolism of polyamines such as putrescine, spermidine, and spermine. Several works have demonstrated that a fine-tuned regulation of the enzymes involved in the biosynthesis and catabolism of polyamines leads to the increment in the concentration of these compounds. Polyamines exert different effects that could help plants to deal with stressful conditions. For instance, they interact with negatively charged macromolecules and regulate their functions, they may act as compatible osmolytes, or present antimicrobial activity against plant pathogens. In addition, they have also been proven to act as regulators of gene expression during the elicitation of stress responses. In this chapter, we reviewed the information available till date in relation to the roles played by polyamines in the responses of plants during biotic and abiotic stress.

Phenotypic and Genotypic Characterization of Mutant Plants in Polyamine Metabolism Genes During Pathogenic Interactions

Plants respond to pathogen attack by modifying defense gene expression and inducing the production of myriad proteins and metabolites. Among these responses, polyamine (PA) levels suffer remarkable modifications. Evidences demonstrate that plants make use of the polyamine biosynthetic pathway and the oxidative catabolism of these compounds in order to mount adequate defenses against pathogens. In Arabidopsis thaliana, putrescine is synthesized exclusively through the arginine decarboxylase (ADC) pathway, this enzyme exists as two isoforms named ADC1 and ADC2. Even though both isoforms participate in the response to pathogen attack, the mechanisms modulating ADC activity are not completely understood. Therefore, studies to clarify their roles are necessary. In this chapter, we describe the methods that can be applied for the study of plant-pathogen interactions using Arabidopsis adc mutant plants.