Oscar A. Ruiz

Spermine accumulation under salt stress

Polyamines have long been recognized to be linked to stress situations, and it is generally accepted that they have protective characteristics. However, little is known about their physiological relevance in plants subjected to long-term salt stress. In order to precise their importance, two rice (Oryza sativa) cultivars differing in their salt tolerance were salinized for 7, 14 and 21 days. The activities of some of the enzymes involved in polyamine metabolism, free polyamines and proline contents were evaluated. Arginine decarboxylase and S-adenosyl-L-methionine decarboxylase activities were reduced in both cultivars as a consequence of salt treatment. However, spermidine synthase activity was reduced in the salt tolerant cultivar (var Giza) but not in the salt sensitive (var El Paso), while no polyamine oxidase activity was detected. During the salinization period, putrescine and spermidine levels decreased in both cultivars, although less dramatically in Giza. Simultaneously, spermine accumulations occur in both varieties, while proline accumulation was major in the sensitive one. However, spermine accumulation induced by treatment with spermidine synthase inhibitor cyclohexylamine, determined no reduction in leaf injury associated with salt stress in both cultivars. The data presented suggest that spermine accumulation is not a salt tolerance trait.

Polyamine oxidase activity contributes to sustain maize leaf elongation under saline stress

The possible involvement of apoplastic reactive oxygen species produced by the oxidation of free polyamines in the leaf growth of salinized maize has been studied here. Salt treatment increased the apoplastic spermine and spermidine levels, mainly in the leaf blade elongation zone. The total activity of polyamine oxidase was up to 20-fold higher than that of the copper-containing amine oxidase. Measurements of H(2)O(2), *O(2)(-), and HO* production in the presence or absence of the polyamine oxidase inhibitors 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane suggest that, in salinized plants, the oxidation of free apoplastic polyamines by polyamine oxidase by would be the main source of reactive oxygen species in the elongation zone of maize leaf blades. This effect is probably due to increased substrate availability. Incubation with 200 microM spermine doubled segment elongation, whereas the addition of 1,19-bis-(ethylamine)-5,10,15 triazanonadecane and 1,8-diamino-octane to 200 microM spermine attenuated and reversed the last effect, respectively. Similarly, the addition of MnCl(2) (an *O(2)(-) dismutating agent) or the HO* scavenger sodium benzoate along with spermine, annulled the elongating effect of the polyamine on the salinized segments. As a whole, the results obtained here demonstrated that, under salinity, polyamine oxidase activity provides a significant production of reactive oxygen species in the apoplast which contributes to 25-30% of the maize leaf blade elongation.

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.

Perturbation of spermine synthase Gene Expression and Transcript Profiling Provide New Insights on the Role of the Tetraamine Spermine in Arabidopsis Defense against Pseudomonas viridiflava

The role of the tetraamine spermine in plant defense against pathogens was investigated by using the Arabidopsis (Arabidopsis thaliana)-Pseudomonas viridiflava pathosystem. The effects of perturbations of plant spermine levels on susceptibility to bacterial infection were evaluated in transgenic plants (35S::spermine synthase [SPMS]) that overexpressed the SPMS gene and accumulated spermine, as well as in spms mutants with low spermine levels. The former exhibited higher resistance to P. viridiflava than wild-type plants, while the latter were more susceptible. Exogenous supply of spermine to wild-type plants also increased disease resistance. Increased resistance provided by spermine was partly counteracted by the polyamine oxidase inhibitor SL-11061, demonstrating that the protective effect of spermine partly depends on its oxidation. In addition, global changes in gene expression resulting from perturbations of spermine levels were analyzed by transcript profiling 35S::SPMS-9 and spms-2 plants. Overexpression of 602 genes was detected in 35S::SPMS-9 plants, while 312 genes were down-regulated, as compared to the wild type. In the spms-2 line, 211 and 158 genes were up- and down-regulated, respectively. Analysis of gene ontology term enrichment demonstrated that many genes overexpressed only in 35S::SPMS-9 participate in pathogen perception and defense responses. Notably, several families of disease resistance genes, transcription factors, kinases, and nucleotide- and DNA/RNA-binding proteins were overexpressed in this line. Thus, a number of spermine-responsive genes potentially involved in resistance to P. viridiflava were identified. The obtained results support the idea that spermine contributes to plant resistance to P. viridiflava.

Genetic Diversity and Host Range of Rhizobia Nodulating Lotus tenuis in Typical Soils of the Salado River Basin (Argentina)

ABSTRACT A total of 103 root nodule isolates were used to estimate the diversity of bacteria nodulating Lotus tenuis in typical soils of the Salado River Basin. A high level of genetic diversity was revealed by repetitive extragenic palindromic PCR, and 77 isolates with unique genomic fingerprints were further differentiated into two clusters, clusters A and B, after 16S rRNA restriction fragment length polymorphism analysis. Cluster A strains appeared to be related to the genus Mesorhizobium, whereas cluster B was related to the genus Rhizobium. 16S rRNA sequence and phylogenetic analysis further supported the distribution of most of the symbiotic isolates in either Rhizobium or Mesorhizobium: the only exception was isolate BA135, whose 16S rRNA gene was closely related to the 16S rRNA gene of the genus Aminobacter. Most Mesorhizobium-like isolates were closely related to Mesorhizobium amorphae, Mesorhizobium mediterraneum, Mesorhizobium tianshanense, or the broad-host-range strain NZP2037, but surprisingly few isolates grouped with Mesorhizobium loti type strain NZP2213. Rhizobium-like strains were related to Rhizobium gallicum, Rhizobium etli, or Rhizobium tropici, for which Phaseolus vulgaris is a common host. However, no nodC or nifH genes could be amplified from the L. tenuis isolates, suggesting that they have rather divergent symbiosis genes. In contrast, nodC genes from the Mesorhizobium and Aminobacter strains were closely related to nodC genes from narrow-host-range M. loti strains. Likewise, nifH gene sequences were very highly conserved among the Argentinian isolates and reference Lotus rhizobia. The high levels of conservation of the nodC and nifH genes suggest that there was a common origin of the symbiosis genes in narrow-host-range Lotus symbionts, supporting the hypothesis that both intrageneric horizontal gene transfer and intergeneric horizontal gene transfer are important mechanisms for the spread of symbiotic capacity in the Salado River Basin.

Effect of salt stress on the regulation of maize (Zea mays L.) genes involved in polyamine biosynthesis

A cDNA for spermidine synthase (SPDS), which converts putrescine to the higher polyamine spermidine using decarboxylated S-adenosylmethionine as a cofactor, was isolated from Zea mays leaves (Zmspds2A). Comparison of the deduced amino acid sequence revealed a high homology (81.9%) with Oryza sativa SPDS2. RT-PCR analyses showed that Zmspds2A was equally expressed in leaves, stem and roots. In contrast, transcripts of other genes related to polyamine biosynthesis (Zmodc, adc and samdc) showed tissue-specific regulation. The effect of salt stress on the expression of all these genes in maize leaves exposed to NaCl solutions of different concentrations was analysed. Our results showed that only Zmodc and Zmspds2A were up-regulated by salt stress; whereas the other two genes were barely affected by this treatment. In addition to Zmspds2A, a second transcript encoding a maize spermidine synthase (Zmspds2B) that also became up-regulated by salt stress, was identified. Comparison of partial cDNA sequences of transcripts Zmspds2A and Zmspds2B with the corresponding genomic DNA region revealed the existence of alternative splicing mechanism, opening a new aspect in plant polyamine biosynthesis modulation under abiotic stress.

A LOV Protein Modulates the Physiological Attributes of Xanthomonas axonopodis pv. citri Relevant for Host Plant Colonization

Recent studies have demonstrated that an appropriate light environment is required for the establishment of efficient vegetal resistance responses in several plant-pathogen interactions. The photoreceptors implicated in such responses are mainly those belonging to the phytochrome family. Data obtained from bacterial genome sequences revealed the presence of photosensory proteins of the BLUF (Blue Light sensing Using FAD), LOV (Light, Oxygen, Voltage) and phytochrome families with no known functions. Xanthomonas axonopodis pv. citri is a Gram-negative bacterium responsible for citrus canker. The in silico analysis of the X. axonopodis pv. citri genome sequence revealed the presence of a gene encoding a putative LOV photoreceptor, in addition to two genes encoding BLUF proteins. This suggests that blue light sensing could play a role in X. axonopodis pv. citri physiology. We obtained the recombinant Xac-LOV protein by expression in Escherichia coli and performed a spectroscopic analysis of the purified protein, which demonstrated that it has a canonical LOV photochemistry. We also constructed a mutant strain of X. axonopodis pv. citri lacking the LOV protein and found that the loss of this protein altered bacterial motility, exopolysaccharide production and biofilm formation. Moreover, we observed that the adhesion of the mutant strain to abiotic and biotic surfaces was significantly diminished compared to the wild-type. Finally, inoculation of orange (Citrus sinensis) leaves with the mutant strain of X. axonopodis pv. citri resulted in marked differences in the development of symptoms in plant tissues relative to the wild-type, suggesting a role for the Xac-LOV protein in the pathogenic process. Altogether, these results suggest the novel involvement of a photosensory system in the regulation of physiological attributes of a phytopathogenic bacterium. A functional blue light receptor in Xanthomonas spp. has been described for the first time, showing an important role in virulence during citrus canker disease.

New insights into the role of spermine in Arabidopsis thaliana under long-term salt stress ☆

Polyamines (putrescine, spermidine and spermine) are traditionally implicated in the response of plants to environmental cues. Free spermine accumulation has been suggested as a particular feature of long-term salt stress, and in the model plant Arabidopsis thaliana the spermine synthase gene (AtSPMS) has been reported as inducible by abscisic acid (ABA) and acute salt stress treatments. With the aim to unravel the physiological role of free spermine during salinity, we analyzed polyamine metabolism in A. thaliana salt-hypersensitive sos mutants (salt overlay sensitive; sos1-1, sos2-1 and sos3-1), and studied the salt stress tolerance of the mutants in spermine and thermospermine synthesis (acl5-1, spms-1 and acl5-1/spms-1). Results presented here indicate that induction in polyamine metabolism is a SOS-independent response to salinity and is globally over-induced in a sensitive background. In addition, under long-term salinity, the mutants in the synthesis of spermine and thermospermine (acl5-1, spms-1 and double acl5-1/spms-1) accumulated more Na(+) and performed worst than WT in survival experiments. Therefore, support is given to a role for these higher polyamines in salt tolerance mechanisms.

Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress

Polyamines have been globally associated to plant responses to abiotic stress. Particularly, putrescine has been related to a better response to cold and dehydration stresses. It is known that this polyamine is involved in cold tolerance, since Arabidopsis thaliana plants mutated in the key enzyme responsible for putrescine synthesis (arginine decarboxilase, ADC; EC 4.1.1.19) are more sensitive than the wild type to this stress. Although it is speculated that the over-expression of ADC genes may confer tolerance, this is hampered by pleiotropic effects arising from the constitutive expression of enzymes from the polyamine metabolism. Here, we present our work using A. thaliana transgenic plants harboring the ADC gene from oat under the control of a stress-inducible promoter (pRD29A) instead of a constitutive promoter. The transgenic lines presented in this work were more resistant to both cold and dehydration stresses, associated with a concomitant increment in endogenous putrescine levels under stress. Furthermore, the increment in putrescine upon cold treatment correlated with the induction of known stress-responsive genes, and suggested that putrescine may be directly or indirectly involved in ABA metabolism and gene expression.