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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.

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 host's 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.

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.

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.

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Phytopathogenic fungi responsible for post-harvest diseases on fruit and vegetables cause important economic losses. We have previously reported that harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol) is active against the causal agents of green and gray molds Penicillium digitatum and Botrytis cinerea, respectively. Here, antifungal activity of harmol was characterized in terms of pH dependency and conidial targets; also photodynamic effects of UVA irradiation on the antimicrobial action were evaluated. Harmol was able to inhibit the growth of both post-harvest fungal disease agents only in acidic conditions (pH 5), when it was found in its protonated form. Conidia treated with harmol exhibited membrane integrity loss, cell wall disruption, and cytoplasm disorganization. All these deleterious effects were more evident for B. cinerea in comparison to P. digitatum. When conidial suspensions were irradiated with UVA in the presence of harmol, antimicrobial activity against both pathogens was enhanced, compared to non-irradiated conditions. B. cinerea exhibited a high intracellular production of reactive oxygen species (ROS) when was incubated with harmol in irradiated and non-irradiated treatments. P. digitatum showed a significant increase in ROS accumulation only when treated with photoexcited harmol. The present work contributes to unravel the antifungal activity of harmol and its photoexcited counterpart against phytopathogenic conidia, focusing on ROS accumulation which could account for damage on different cellular targets.

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