1. Investigación

For the first time in the literature, a relationship between the root application of melatonin and the greater capacity for resistance against Psedomonas syringae DC3000 in tomato plants has been established. Root delivered melatonin (100 μM), induced systemic resistance against pathogen reducing disease incidence by 51%. Mechanisms of action used by melatonin were assessing through different physiological, metabolic, and genetic markers. As a physiological marker, photosynthetic efficiency was studied, with a TARGAS 1 portable photosynthesis system. Metabolic markers were analysed on leaf powder collected 1 week after the pathogen challenge. These markers analysed were grouped into those related to the scavenging of Reactive Oxygen Species (ROS) and oxidative stress (ascorbate peroxidase (APX) activity, hydrogen peroxide (H2O2) concentration, malondialdehyde (MDA) concentration, and proline concentration) and those related to defence mechanisms (ß-1,3-glucanase and chitinase). Genetic markers were studied on leaf powder collected 6 h and 10 h after pathogen challenge. For this, the differential expression of the genes PR1, PR2 and PR3 was studied. Upon pathogen challenge, melatonin reverted the negative effects of the pathogen in net photosynthesis rate achieving similar values to healthy plants. Melatonin reduced oxidative stress, according to lower MDA (29%) and H2O2 (46%), improving ROS scavenging potential by enhancing APX activity (83%) and proline concentration (44%). Melatonin simultaneously triggered the salicylic acid (SA)-mediated pathway and the jasmonic acid/ethylene (JA/ET)-mediated pathway as the enzymatic activities ß-1,3-glucanase (Pathogenesis-Related protein 2; PR2; 103%) and chitinase activitiy (Pathogenesis-Related protein 3; PR3; 44%), markers of the first and second pathways respectively, were enhanced. This enhanced activity was consistent with enhanced expression of genes encoding PR2 and PR3. Results obtained indicate that melatonin, a natural plant compound, could be used in tomato cultivation as an economical and ecofriendly chemical agent against biotic stress.

We evaluated the ability of metabolic elicitors extracted from Pseudomonas fluorescens N21.4 to induce systemic resistance (ISR) in Arabidopsis thaliana against the pathogen Pseudomonas syringae DC3000. Metabolic elicitors were obtained from bacteria free culture medium with n-hexane, ethyl acetate and n-butanol in three consecutive extractions. Each extract showed plant protection activity. The n-hexane fraction was the most effective and was used to study the signal transduction pathways involved by evaluating expression of marker genes of the salicylic acid (SA) signalling pathway (NPR1, PR1, ICS and PR2) and the jasmonic acid/ethylene (JA/ET) signalling pathway (PDF1, MYC2, LOX2 and PR3). In addition, the level of oxidative stress was tested by determining the activity of enzymes related to the ascorbate-glutathione cycle. N-hexane extracts stimulated both pathways based on overexpression of ICS, PR1, PR2, PDF1 and LOX2 genes. In addition, activity of the pathogenesis-related proteins glucanase (PR2) and chitinase (PR3), lipoxygenase and polyphenol oxidase was enhanced together with an increased capacity to remove reactive oxygen species (ROS). This was associated with less oxidative stress as indicated by a decrease in malondialdehyde (MDA), suggesting a causative link between defensive metabolism against P. syringae and ROS scavenging.

In the present work, for the first time in the literature, the relationship between the degree of air pollution, the physiological state of the plants and the allergenic capacity of the pollen they produce has been studied. The physiological state of Lolium perenne plants growing in two cities with a high degree of traffic, but with different levels of air pollution, Madrid and Ciudad Real, have been explored. The photosynthetic efficiency of the plants through the emission of fluorescence of PSII, the degree of oxidative stress (enzymatic activities related to the ascorbate-glutathione cycle), the redox state (reduced and oxidized forms of ascorbate and glutathione) and the concentration of malondialdehyde have been evaluated. During the development period of the plants, Madrid had higher levels of NO2 and SO2 than Ciudad Real. The greater degree of air pollution suffered by Madrid plants was reflected on a lower photosynthetic efficiency and a greater degree of oxidative stress. In addition, NADPH oxidase activity and H2O2 levels in pollen from Madrid were significantly higher, suggesting a likely higher allergenic capacity of this pollen associated to a higher air pollution.

Physiological, metabolic, and genetic changes produced by two plant growth promoting rhizobacteria (PGPR) Pseudomonas sp. (internal code of the laboratory: N 5.12 and N 21.24) inoculated in tomato plants subjected to moderate water stress (10% polyethylene glycol-6000; PEG) were studied. Photosynthesis efficiency, photosynthetic pigments, compatible osmolytes, reactive oxygen species (ROS) scavenging enzymes activities, oxidative stress level and expression of genes related to abscisic acid synthesis (ABA; 9-cis-epoxycarotenoid dioxygenase NCDE1 gene), proline synthesis (Pyrroline-5-carboxylate synthase P5CS gene), and plasma membrane ATPase (PM ATPase gene) were measured. Photosynthetic efficiency was compromised by PEG, but bacterial-inoculated plants reversed the effects: while N5.12 increased carbon fixation (37.5%) maintaining transpiration, N21.24 increased both (14.2% and 31%), negatively affecting stomatal closure, despite the enhanced expression of NCDE1 and plasma membrane ATPase genes, evidencing the activation of different adaptive mechanisms. Among all parameters evaluated, photosynthetic pigments and antioxidant enzymes guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) responded differently to both strains. N 5.12 increased photosynthetic pigments (70% chlorophyll a, 69% chlorophyll b, and 65% carotenoids), proline (33%), glycine betaine (4.3%), and phenolic compounds (21.5%) to a greater extent, thereby decreasing oxidative stress (12.5% in Malondialdehyde, MDA). Both bacteria have highly beneficial effects on tomato plants subjected to moderate water stress, improving their physiological state. The use of these bacteria in agricultural production systems could reduce the amount of water for agricultural irrigation without having a negative impact on food production.