Modulation of polyamine metabolism in Arabidopsis thaliana by salicylic acid.

Polyamines (PAs) play important roles in plant defense against pathogens, but the regulation of PA metabolism by hormone-mediated defense signaling pathways has not been studied in depth. In this study, the modulation of PA metabolism by salicylic acid (SA) was analyzed in Arabidopsis by combining the exogenous application of this hormone with PA biosynthesis and SA synthesis/signaling mutants. SA induced notable modifications of PA metabolism, mainly consisting in putrescine (Put) accumulation both in whole-plant extracts and apoplastic fluids. Put was accumulated at the expense of increased biosynthesis by ARGININE DECARBOXYLASE 2 and decreased oxidation by copper amine oxidase. Enhancement of Put levels by SA was independent of the regulatory protein NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1) and the signaling kinases MKK4 and MPK3, but depended on MPK6. However, plant infection by Pseudomonas syringae pv. tomato DC3000 elicited Put accumulation in an SA-dependent way. The present study demonstrates a clear connection between SA signaling and plant PA metabolism in Arabidopsis and contributes to understanding the mechanisms by which SA modulates PA levels during plant-pathogen interactions.

Botrydial confers Botrytis cinerea the ability to antagonize soil and phyllospheric bacteria.

The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillusamyloliquefaciens strain MEP218 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP218 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP218. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea.

Mechanisms of plant protection against two oxalate-producing fungal pathogens by oxalotrophic strains of Stenotrophomonas spp.

KEY MESSAGE: Oxalotrophic Stenotrophomonas isolated from tomato rhizosphere are able to protect plants against oxalate-producing pathogens by a combination of actions including induction of plant defence signalling callose deposition and the strengthening of plant cell walls and probably the degradation of oxalic acid. Oxalic acid plays a pivotal role in the virulence of the necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum. In this work, we isolated two oxalotrophic strains (OxA and OxB) belonging to the bacterial genus Stenotrophomonas from the rhizosphere of tomato plants. Both strains were capable to colonise endophytically Arabidopsis plants and protect them from the damage caused by high doses of oxalic acid. Furthermore, OxA and OxB protected Arabidopsis from S. sclerotiorum and B. cinerea infections. Bacterial inoculation induced the production of phenolic compounds and the expression of PR-1. Besides, both isolates exerted a protective effect against fungal pathogens in Arabidopsis mutants affected in the synthesis pathway of salicylic acid (sid2-2) and jasmonate perception (coi1). Callose deposition induced by OxA and OxB was required for protection against phytopathogens. Moreover, B. cinerea and S. sclerotiorum mycelial growth was reduced in culture media containing cell wall polysaccharides from leaves inoculated with each bacterial strain. These findings suggest that cell walls from Arabidopsis leaves colonised by these bacteria would be less susceptible to pathogen attack. Our results indicate that these oxalotrophic bacteria can protect plants against oxalate-producing pathogens by a combination of actions and show their potential for use as biological control agents against fungal diseases.

Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea.

Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70-80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.

Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

Novel components of leaf bacterial communities of field-grown tomato plants and their potential for plant growth promotion and biocontrol of tomato diseases.

This work aimed to characterize potentially endophytic culturable bacteria from leaves of cultivated tomato and analyze their potential for growth promotion and biocontrol of diseases caused by Botrytis cinerea and Pseudomonas syringae. Bacteria were obtained from inner tissues of surface-disinfected tomato leaves of field-grown plants. Analysis of 16S rRNA gene sequences identified bacterial isolates related to Exiguobacterium aurantiacum (isolates BT3 and MT8), Exiguobacterium spp. (isolate GT4), Staphylococcus xylosus (isolate BT5), Pantoea eucalypti (isolate NT6), Bacillus methylotrophicus (isolate MT3), Pseudomonas veronii (isolates BT4 and NT2), Pseudomonas rhodesiae (isolate BT2) and Pseudomonas cichorii (isolate NT3). After seed inoculation, BT2, BT4, MT3, MT8, NT2 and NT6 were re-isolated from leaf extracts. NT2, BT2, MT3 and NT6 inhibited growth of Botrytis cinerea and Pseudomonas syringae pv. tomato in vitro, produced antimicrobial compounds and reduced leaf damage caused by B. cinerea. Some of these isolates also promoted growth of tomato plants, produced siderophores, the auxin indole-3-acetic and solubilized inorganic phosphate. Thus, bacterial communities of leaves from field-grown tomato plants were found to harbor potentially endophytic culturable beneficial bacteria capable of antagonizing pathogenic microorganisms and promoting plant growth, which could be used as biological control agents and biofertilizers/biostimulators for promotion of tomato plant growth.

Lotus tenuis x L. corniculatus interspecific hybridization as a means to breed bloat-safe pastures and gain insight into the genetic control of proanthocyanidin biosynthesis in legumes.

BACKGROUND: Proanthocyanidins (PAs) are secondary metabolites that strongly affect plant quality traits. The concentration and the structure of these metabolites influence the palatability and nutritional value of forage legumes. Hence, modulating PAs in the leaves of forage legumes is of paramount relevance for forage breeders worldwide. The lack of genetic variation in the leaf PA trait within the most important forage species and the difficulties in engineering this pathway via the ectopic expression of regulatory genes, prompted us to pursue alternative strategies to enhance this trait in forage legumes of agronomic interest. The Lotus genus includes forage species which accumulate PAs in edible organs and can thus be used as potential donor parents in breeding programs. RESULTS: We recovered a wild, diploid and PA-rich population of L. corniculatus and crossed with L. tenuis. The former grows in an alkaline-salty area in Spain while the latter is a diploid species, grown extensively in South American pastures, which does not accumulate PAs in the herbage. The resulting interspecific hybrids displayed several traits of outstanding agronomic relevance such as rhizome production, PA levels in edible tissues sufficient to prevent ruminal bloating (around 5 mg of PAs/g DW), biomass production similar to the cultivated parent and potential for adaptability to marginal lands. We show that PA levels correlate with expression levels of the R2R3MYB transcription factor TT2 and, in turn, with those of the key structural genes of the epicatechin and catechin biosynthetic pathways leading to PA biosynthesis. CONCLUSIONS: The L. tenuis x L. corniculatus hybrids, reported herein, represent the first example of the introgression of the PA trait in forage legumes to levels known to provide nutritional and health benefits to ruminants. Apart from PAs, the hybrids have additional traits which may prove useful to breed forage legumes with increased persistence and adaptability to marginal conditions. Finally, our study suggests the hybrids and their progeny are an invaluable tool to gain a leap forward in our understanding of the genetic control of PA biosynthesis and tolerance to stresses in legumes.

The communities of tomato (Solanum lycopersicum L.) leaf endophytic bacteria, analyzed by 16S-ribosomal RNA gene pyrosequencing.

Endophytic bacterial communities of tomato leaves were analyzed by 16S-rRNA gene pyrosequencing and compared to rhizosphere communities. Leaf endophytes mainly comprised five phyla, among which Proteobacteria was the most represented (90%), followed by Actinobacteria (1,5%), Planctomycetes (1,4%), Verrucomicrobia (1,1%), and Acidobacteria (0,5%). Gammaproteobacteria was the most abundant class of Proteobacteria (84%), while Alphaproteobacteria and Betaproteobacteria represented 12% and 4% of this phylum, respectively. Rarefaction curves for endophytic bacteria saturated at 80 OTUs, indicating a lower diversity as compared to rhizosphere samples (> 1700 OTUs). Hierarchical clustering also revealed that leaf endophytic communities strongly differed from rhizospheric ones. Some OTUs assigned to Bacillus, Stenotrophomonas, and Acinetobacter, as well as some unclassified Enterobacteriaceae were specific for the endophytic community, probably representing bacteria specialized in colonizing this niche. On the other hand, some OTUs detected in the leaf endophytic community were also present in the rhizosphere, probably representing soil bacteria that endophytically colonize leaves. As a whole, this study describes the composition of the endophytic bacterial communities of tomato leaves, identifying a variety of genera that could exert multiple effects on growth and health of tomato plants.

Thermospermine catabolism increases Arabidopsis thaliana resistance to Pseudomonas viridiflava.

This work investigated the roles of the tetraamine thermospermine (TSpm) by analysing its contribution to Arabidopsis basal defence against the biotrophic bacterium Pseudomonas viridiflava. The participation of polyamine oxidases (PAOs) in TSpm homeostasis and TSpm-mediated defence was also investigated. Exogenous supply of TSpm, as well as ectopic expression of the TSpm biosynthetic gene ACL5, increased Arabidopsis Col-0 resistance to P. viridiflava, while null acl5 mutants were less resistant than Col-0 plants. The above-mentioned increase in resistance was blocked by the PAO inhibitor SL-11061, thus demonstrating the participation of TSpm oxidation. Analysis of PAO genes expression in transgenic 35S::ACL5 and Col-0 plants supplied with TSpm suggests that PAO 1, 3, and 5 are the main PAOs involved in TSpm catabolism. In summary, TSpm exhibited the potential to perform defensive functions previously reported for its structural isomer Spm, and the relevance of these findings is discussed in the context of ACL5 expression and TSpm concentration in planta. Moreover, this work demonstrates that manipulation of TSpm metabolism modifies plant resistance to 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.

Comparative functional genomics of salt stress in related model and cultivated plants identifies and overcomes limitations to translational genomics.

One of the objectives of plant translational genomics is to use knowledge and genes discovered in model species to improve crops. However, the value of translational genomics to plant breeding, especially for complex traits like abiotic stress tolerance, remains uncertain. Using comparative genomics (ionomics, transcriptomics and metabolomics) we analyzed the responses to salinity of three model and three cultivated species of the legume genus Lotus. At physiological and ionomic levels, models responded to salinity in a similar way to crop species, and changes in the concentration of shoot Cl(-) correlated well with tolerance. Metabolic changes were partially conserved, but divergence was observed amongst the genotypes. Transcriptome analysis showed that about 60% of expressed genes were responsive to salt treatment in one or more species, but less than 1% was responsive in all. Therefore, genotype-specific transcriptional and metabolic changes overshadowed conserved responses to salinity and represent an impediment to simple translational genomics. However, 'triangulation' from multiple genotypes enabled the identification of conserved and tolerant-specific responses that may provide durable tolerance across species.

Comparative ionomics and metabolomics in extremophile and glycophytic Lotus species under salt stress challenge the metabolic pre-adaptation hypothesis.

The legume genus Lotus includes glycophytic forage crops and other species adapted to extreme environments, such as saline soils. Understanding salt tolerance mechanisms will contribute to the discovery of new traits which may enhance the breeding efforts towards improved performance of legumes in marginal agricultural environments. Here, we used a combination of ionomic and gas chromatography-mass spectrometry (GC-MS)-based metabolite profilings of complete shoots (pooling leaves, petioles and stems) to compare the extremophile Lotus creticus, adapted to highly saline coastal regions, and two cultivated glycophytic grassland forage species, Lotus corniculatus and Lotus tenuis. L. creticus exhibited better survival after exposure to long-term lethal salinity and was more efficient at excluding Cl⁻ from the shoots than the glycophytes. In contrast, Na+ levels were higher in the extremophile under both control and salt stress, a trait often observed in halophytes. Ionomics demonstrated a differential rearrangement of shoot nutrient levels in the extremophile upon salt exposure. Metabolite profiling showed that responses to NaCl in L. creticus shoots were globally similar to those of the glycophytes, providing little evidence for metabolic pre-adaptation to salinity. This study is the first comparing salt acclimation responses between extremophile and non-extremophile legumes, and challenges the generalization of the metabolic salt pre-adaptation hypothesis.

In vitro and in vivo inhibition of plant polyamine oxidase activity by polyamine analogues.

Polyamine oxidase from Avena sativa L. cv. Cristal seedlings was purified to homogeneity using a simple four-step purification protocol including an infiltration washing technique. The enzyme had a high affinity for spermidine and spermine (K(m) approximately 5.5 and 1.2 microM, respectively), and also oxidized norspermidine (K(m) approximately 64.0 microM). Natural and synthetic diamines, cyclohexylamine, the putrescine analogue 1-aminooxy-3-aminopropane, and several polyamine analogues had inhibitory effects on polyamine oxidase activity and none were substrates. No inhibitory effect was observed on spermidine oxidation when the reaction product 1,3-diaminopropane was added. By contrast, 1-aminooxy-3-aminopropane showed mixed inhibition kinetics and a K(i) value of 0.113 mM. In addition, in vitro enzymatic activity assays showed that the oligoamine [3,8,13,18,23,28,33,38,43,48-deca-aza-(trans-25)-pentacontene], the tetramine 1,14-bis-[ethylamino]-5,10-diazatetradecane, and the pentamine 1,19-bis-[ethylamino]-5,10,15-triazanonadecane, displayed potent competitive inhibitory activities against polyamine oxidase with K(i) values of 5.8, 110.0 and 7.6 nM, respectively, where cyclohexylamine was a weak competitive inhibitor with a K(i) value of 0.5 mM. These analogues did not inhibit mycelial growth of the fungus Sclerotinia sclerotiorum (Lib.) De Bary and the bacterium Pseudomonas viridiflava (Burkholder) Dowson in vitro. On the contrary, with concentrations similar to those used for polyamine analogues, guazatine (a well-known fungicide and at the same time, a polyamine oxidase inhibitor) inhibited ( approximately 85%) S. sclerotiorum mycelial growth on Czapek-Dox medium. Finally, the analogue 1,19-bis-ethylamino-5,10,15-triazanonadecane inhibited polyamine oxidase activity observed in segments of maize leaves in vivo. The results obtained provide insights into research on the influence of polyamine oxidase activity on plant biotic and abiotic stresses.

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.

Ornithine and arginine decarboxylase activities and effect of some polyamine biosynthesis inhibitors on Gigaspora rosea germinating spores.

The pathways for putrescine biosynthesis and the effects of polyamine biosynthesis inhibitors on the germination and hyphal development of Gigaspora rosea spores were investigated. Incubation of spores with different radioactive substrates demonstrated that both arginine and ornithine decarboxylase pathways participate in putrescine biosynthesis in G. rosea. Spermidine and spermine were the most abundant polyamines in this fungus. The putrescine biosynthesis inhibitors alpha-difluoromethylarginine and alpha-difluoromethylornithine, as well as the spermidine synthase inhibitor cyclohexylamine, slightly decreased polyamine levels. However, only the latter interfered with spore germination. The consequences of the use of putrescine biosynthesis inhibitors for the control of plant pathogenic fungi on the viability of G. rosea spores in soil are discussed.

Polyamine metabolism during the germination of Sclerotinia sclerotiorum ascospores and its relation with host infection.

• Polyamine biosynthesis inhibitors were used to study polyamine metabolism during the germination of Sclerotinia sclerotiorum ascospores, and to evaluate the potential of polyamine biosynthesis inhibition for the control of ascospore-borne diseases in plants. • The effects of inhibitors on ascospore germination, free polyamine levels, ornithine decarboxylase activity and development of disease symptoms on tobacco (Nicotiana tabacum) leaf discs inoculated with ascospores were determined. • α-Difluoromethylornithine inhibited ornithine decarboxylase and decreased free spermidine levels, but had no effect on ascospore germination. Both, the spermidine synthase inhibitor cyclohexylamine and the S-adenosyl-methionine decarboxylase inhibitor methylglyoxal bis-[guanyl hydrazone] decreased free spermidine levels, but only the latter inhibited ascospore germination, at concentrations of 5 mm or higher. Lesion development on leaf discs was reduced by cyclohexylamine and methylglyoxal bis-[guanyl hydrazone], but not by α-difluoromethylornithine. In the absence of inhibitors, dormant ascospores contained higher polyamine levels than mycelium. • Ascospore germination did not depend on ornithine decarboxylase activity and inhibitors of this enzyme will probably have a limited potential for the control of ascospore-borne plant diseases. On the contrary, spermidine synthase and S-adenosyl-methionine decarboxylase could be more suitable targets for fungicidal action. The relative insensitivity of ascospore germination to polyamine biosynthesis inhibitors may be caused by their high polyamine content.

The putrescine analogue 1-aminooxy-3-aminopropane perturbs polyamine metabolism in the phytopathogenic fungus Sclerotinia sclerotiorum.

The effects of the putrescine analogue 1-aminooxy-3-aminopropane on fungal polyamine metabolism were evaluated using Sclerotinia sclerotiorum as an experimental model. The compound inhibited ornithine decarboxylase, spermidine synthase, and S -adenosyl-methionine decarboxylase in mycelial extracts. Addition of 1-aminooxy-3-aminopropane at 1 mM to the culture medium did not reduce mycelial growth and caused a 29% decrease in free spermidine and a two-fold increase in free spermine. When added 4.5 h before the determination of ornithine decarboxylase, 1-aminooxy-3-aminopropane reduced in vivo activity of this enzyme by 40-50%. When added 48 h before the determination, 1-aminooxy-3-aminopropane at 0.01 and 0.1 mM caused a slight increase of in vivo ornithine decarboxylase activity, while it had no effect at 1 mM. Comparison of the action of 1-aminooxy-3-aminopropane with that of other inhibitors of polyamine biosynthesis suggested that its effects on in vivo ornithine decarboxylase activity resulted from a balance between direct inhibition of enzyme activity and indirect stimulation of enzyme synthesis and/or activity mediated by the decrease in spermidine levels, which in turn was due to inhibition of spermidine synthase and S -adenosyl-methionine decarboxylase. The potential of 1-aminooxy-3-aminopropane as a tool for studies on fungal polyamine metabolism and for the control of plant diseases of fungal origin is discussed.