María Pilar López-Gresa

Induction of p-Coumaroyldopamine and Feruloyldopamine, Two Novel Metabolites, in Tomato by the Bacterial Pathogen Pseudomonas syringae

Inoculation of tomato plants (Solanum lycopersicum cv. Rutgers) with Pseudomonas syringae pv. tomato led to the production of a hypersensitive-like response in this pathovar of tomato. Accumulation of hydroxycinnamic acid amides (HCAA) of tyramine (p-coumaroyltyramine and feruloyltyramine) and dopamine (p-coumaroyldopamine and feruloyldopamine) was detected after bacterial infection. Two of them, p-coumaroyldopamine and feruloyldopamine, are described for the first time. The accumulation of HCAA was preceded by an increment of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) gene expression. HCAA also accumulated in transgenic NahG tomato plants overexpressing a bacterial salicylic hydroxylase. However, treatment of plants with the ethylene biosynthesis inhibitor, aminoethoxyvinilglycine, led to a reduction in the accumulation of THT transcripts and HCAA. Together, the results suggest that pathogen-induced induction of ethylene is essential for HCAA synthesis, whereas salicylic acid is not required for this response. In addition, notable antibacterial and antioxidant activities were found for the new HCAA, thus indicating that they could play a role in the defense of tomato plants against bacterial infection.

The characterization of transgenic tomato overexpressing gibberellin 20-oxidase reveals induction of parthenocarpic fruit growth, higher yield, and alteration of the gibberellin biosynthetic pathway

Fruit-set and growth in tomato depend on the action of gibberellins (GAs). To evaluate the role of the GA biosynthetic enzyme GA 20-oxidase (GA20ox) in that process, the citrus gene CcGA20ox1 was overexpressed in tomato (Solanum lycopersicum L.) cv Micro-Tom. The transformed plants were taller, had non-serrated leaves, and some flowers displayed a protruding stigma due to a longer style, thus preventing self-pollination, similar to GA(3)-treated plants. Flowering was delayed compared with wild-type (WT) plants. Both yield and number of fruits per plant, some of them seedless, were higher in the transgenic plants. The Brix index value of fruit juice was also higher due to elevated citric acid content, but not glucose or fructose content. When emasculated, 14-30% of ovaries from transgenic flowers developed parthenocarpically, whereas no parthenocarpy was found in emasculated WT flowers. The presence of early-13-hydroxylation and non-13-hydroxylation GA pathways was demonstrated in the shoot and fruit of Micro-Tom, as well as in two tall tomato cultivars (Ailsa Craig and UC-82). The transgenic plants had altered GA profiles containing higher concentrations of GA(4), from the non-13-hydroxylation pathway, which is generally a minor active GA in tomato. The effect of GA(4) application in enhancing stem growth and parthenocarpic fruit development was proportional to dose, with the same activity as GA(1). The results support the contention that GA20ox overexpression diverts GA metabolism from the early-13-hydroxylation pathway to the non-13-hydroxylation pathway. This led to enhanced GA(4) synthesis and higher yield, although the increase in GA(4) content in the ovary was not sufficient to induce full parthenocarpy.

Diversity and relationships in key traits for functional and apparent quality in a collection of eggplant: fruit phenolics content, antioxidant activity, polyphenol oxidase activity, and browning.

Eggplant (Solanum melongena) varieties with increased levels of phenolics in the fruit present enhanced functional quality, but may display greater fruit flesh browning. We evaluated 18 eggplant accessions for fruit total phenolics content, chlorogenic acid content, DPPH scavenging activity, polyphenol oxidase (PPO) activity, liquid extract browning, and fruit flesh browning. For all the traits we found a high diversity, with differences among accessions of up to 3.36-fold for fruit flesh browning. Variation in total content in phenolics and in chlorogenic acid content accounted only for 18.9% and 6.0% in the variation in fruit flesh browning, and PPO activity was not significantly correlated with fruit flesh browning. Liquid extract browning was highly correlated with chlorogenic acid content (r = 0.852). Principal components analysis (PCA) identified four groups of accessions with different profiles for the traits studied. Results suggest that it is possible to develop new eggplant varieties with improved functional and apparent quality.

Transgenic tomato plants overexpressing tyramine N-hydroxycinnamoyltransferase exhibit elevated hydroxycinnamic acid amide levels and enhanced resistance to Pseudomonas syringae.

Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-overexpressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection.

Native-Invasive Plants vs. Halophytes in Mediterranean Salt Marshes: Stress Tolerance Mechanisms in Two Related Species

Dittrichia viscosa is a Mediterranean ruderal species that over the last decades has expanded into new habitats, including coastal salt marshes, ecosystems that are per se fragile and threatened by human activities. To assess the potential risk that this native-invasive species represents for the genuine salt marsh vegetation, we compared its distribution with that of Inula crithmoides, a taxonomically related halophyte, in three salt marshes located in “La Albufera” Natural Park, near the city of Valencia (East Spain). The presence of D. viscosa was restricted to areas of low and moderate salinity, while I. crithmoides was also present in the most saline zones of the salt marshes. Analyses of the responses of the two species to salt and water stress treatments in controlled experiments revealed that both activate the same physiological stress tolerance mechanisms, based essentially on the transport of toxic ions to the leaves—where they are presumably compartmentalized in vacuoles—and the accumulation of specific osmolytes for osmotic adjustment. The two species differ in the efficiency of those mechanisms: salt-induced increases in Na+ and Cl− contents were higher in I. crithmoides than in D. viscosa, and the osmolytes (especially glycine betaine, but also arabinose, fructose and glucose) accumulated at higher levels in the former species. This explains the (slightly) higher stress tolerance of I. crithmoides, as compared to D. viscosa, established from growth inhibition measurements and their distribution in nature. The possible activation of K+ transport to the leaves under high salinity conditions may also contribute to salt tolerance in I. crithmoides. Oxidative stress level—estimated from malondialdehyde accumulation—was higher in the less tolerant D. viscosa, which consequently activated antioxidant responses as a defense mechanism against stress; these responses were weaker or absent in the more tolerant I. crithmoides. Based on these results, we concluded that although D. viscosa cannot directly compete with true halophytes in highly saline environments, it is nevertheless quite stress tolerant and therefore represents a threat for the vegetation located on the salt marshes borders, where several endemic and threatened species are found in the area of study.

Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs

Significance N6-methyladenosine (m6A) modification has been found to constitute an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. Although the influence of the m6A cellular machinery has been suspected to occur in the plant virus cycle, it has never been proved. Here we have identified a plant protein with m6A demethylase activity (atALKBH9B) and demonstrate that this protein removes m6A modification from RNA in vitro. Remarkably, we found that m6A abundance on the viral genome of alfalfa mosaic virus is influenced by atALKBH9B activity and regulates viral infection. This study extends the vast repertoire that plants exploit to control cytoplasmic-replicating RNA viruses. N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

Bacillus subtilis IAB/BS03 as a potential biological control agent

We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent.

Effects of Salt Stress on Three Ecologically Distinct Plantago Species.

Comparative studies on the responses to salt stress of taxonomically related taxa should help to elucidate relevant mechanisms of stress tolerance in plants. We have applied this strategy to three Plantago species adapted to different natural habitats, P. crassifolia and P. coronopus–both halophytes–and P. major, considered as salt-sensitive since it is never found in natural saline habitats. Growth inhibition measurements in controlled salt treatments indicated, however, that P. major is quite resistant to salt stress, although less than its halophytic congeners. The contents of monovalent ions and specific osmolytes were determined in plant leaves after four-week salt treatments. Salt-treated plants of the three taxa accumulated Na+ and Cl- in response to increasing external NaCl concentrations, to a lesser extent in P. major than in the halophytes; the latter species also showed higher ion contents in the non-stressed plants. In the halophytes, K+ concentration decreased at moderate salinity levels, to increase again under high salt conditions, whereas in P. major K+ contents were reduced only above 400 mM NaCl. Sorbitol contents augmented in all plants, roughly in parallel with increasing salinity, but the relative increments and the absolute values reached did not differ much in the three taxa. On the contrary, a strong (relative) accumulation of proline in response to high salt concentrations (600–800 mM NaCl) was observed in the halophytes, but not in P. major. These results indicate that the responses to salt stress triggered specifically in the halophytes, and therefore the most relevant for tolerance in the genus Plantago are: a higher efficiency in the transport of toxic ions to the leaves, the capacity to use inorganic ions as osmotica, even under low salinity conditions, and the activation, in response to very high salt concentrations, of proline accumulation and K+ transport to the leaves of the plants.

Stress tolerance mechanisms in Juncus: responses to salinity and drought in three Juncus species adapted to different natural environments

Comparative studies on the responses to salinity and drought were carried out in three Juncus species, two halophytes (Juncus maritimus Lam. and Juncus acutus L.) and one more salt-sensitive (Juncus articulatus L.). Salt tolerance in Juncus depends on the inhibition of transport of toxic ions to the aerial part. In the three taxa studied Na+ and Cl- accumulated to the same extent in the roots of salt treated plants; however, ion contents were lower in the shoots and correlated with the relative salt sensitivity of the species, with the lowest levels measured in the halophytes. Activation of K+ transport at high salt concentration could also contribute to salt tolerance in the halophytes. Maintenance of cellular osmotic balance is mostly based on the accumulation of sucrose in the three species. Yet, neither the relative salt-induced increase in sugar content nor the absolute concentrations reached can explain the observed differences in salt tolerance. In contrast, proline increased significantly in the presence of salt only in the salt-tolerant J. maritimus and J. acutus, but not in J. articulatus. Similar patterns of osmolyte accumulation were observed in response to water stress, supporting a functional role of proline in stress tolerance mechanisms in Juncus.

Unraveling Salt Tolerance Mechanisms in Halophytes: A Comparative Study on Four Mediterranean Limonium Species with Different Geographic Distribution Patterns

We have performed an extensive study on the responses to salt stress in four related Limonium halophytes with different geographic distribution patterns, during seed germination and early vegetative growth. The aims of the work were twofold: to establish the basis for the different chorology of these species, and to identify relevant mechanisms of salt tolerance dependent on the control of ion transport and osmolyte accumulation. Seeds were germinated in vitro, in the presence of increasing NaCl concentrations, and subjected to “recovery of germination” tests; germination percentages and velocity were determined to establish the relative tolerance and competitiveness of the four Limonium taxa. Salt treatments were also applied to young plants, by 1-month irrigation with NaCl up to 800 mM; then, growth parameters, levels of monovalent and divalent ions (in roots and leaves), and leaf contents of photosynthetic pigments and common osmolytes were determined in control and stressed plants of the four species. Seed germination is the most salt-sensitive developmental phase in Limonium. The different germination behavior of the investigated species appears to be responsible for their geographical range size: L. narbonense and L. virgatum, widespread throughout the Mediterranean, are the most tolerant and the most competitive at higher soil salinities; the endemic L. santapolense and L. girardianum are the most sensitive and more competitive only at lower salinities. During early vegetative growth, all taxa showed a strong tolerance to salt stress, although slightly higher in L. virgatum and L. santapolense. Salt tolerance is based on the efficient transport of Na+ and Cl− to the leaves and on the accumulation of fructose and proline for osmotic adjustment. Despite some species-specific quantitative differences, the accumulation patterns of the different ions were similar in all species, not explaining differences in tolerance, except for the apparent activation of K+ transport to the leaves at high external salinity, observed only in the most tolerant L. narbonense and L. virgatum. This specific response may be therefore relevant for salt tolerance in Limonium. The ecological implications of these results, which can contribute to a more efficient management of salt marshes conservation/regeneration programs, are also discussed.