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Low temperature (LT) negatively affects plant growth and development via the alteration of the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Among RNS, tyrosine nitration, the addition of an NO2 group to a tyrosine residue, can modulate reduced nicotinamide-dinucleotide phosphate (NADPH)-generating systems and, therefore, can alter the levels of NADPH, a key cofactor in cellular redox homeostasis. NADPH also acts as an indispensable electron donor within a wide range of enzymatic reactions, biosynthetic pathways, and detoxification processes, which could affect plant viability. To extend our knowledge about the regulation of this key cofactor by this nitric oxide (NO)-related post-translational modification, we analyzed the effect of tyrosine nitration on another NADPH-generating enzyme, the NADP-malic enzyme (NADP-ME), under LT stress. In Arabidopsis thaliana seedlings exposed to short-term LT (4 °C for 48 h), a 50% growth reduction accompanied by an increase in the content of superoxide, nitric oxide, and peroxynitrite, in addition to diminished cytosolic NADP-ME activity, were found. In vitro assays confirmed that peroxynitrite inhibits cytosolic NADP-ME2 activity due to tyrosine nitration. The mass spectrometric analysis of nitrated NADP-ME2 enabled us to determine that Tyr-73 was exclusively nitrated to 3-nitrotyrosine by peroxynitrite. The in silico analysis of the Arabidopsis NADP-ME2 protein sequence suggests that Tyr73 nitration could disrupt the interactions between the specific amino acids responsible for protein structure stability. In conclusion, the present data show that short-term LT stress affects the metabolism of ROS and RNS, which appears to negatively modulate the activity of cytosolic NADP-ME through the tyrosine nitration process.

Drought is considered one of the abiotic stresses with significant implications on plant productivity. Previously, we have shown that water deficit produces a differential nitro-oxidative stress in roots and leaves of Lotus japonicus L. plants. Using this model legume, we studied the nitro-oxidative stress in drought-stressed roots by complementary biochemical, cellular and proteomic approaches. Cellular analyses of root cross-sections by confocal laser scanning microscopy (CLSM) using specific fluorescent probes for superoxide radical (O.-), nitric oxide (NO-), peroxynitrite (ONOO) and S-nitrosothiols (SNOs) showed that drought stress causes a differential cellular localization of these reactive species. Mainly, O.- and ONOO- had a wide distribution in almost all root cell types (xylem, parenchyma, and peridermis), whereas NO and SNOs accumulated in cortical cells (peridermis). Liquid chromatography-electrospray/mass spectrometry (LC-ES/MS) analyses showed that the content of ascorbate, S-nitrosoglutaathione (GSNO), and reduced glutathione (GSH) in drought-stressed roots was drastically diminished. Nitroproteome analysis by two-dimensional gel electrophoresis and mass spectrometry allowed to identify 13 tyrosine-nitrated proteins such as methionine synthase, Hsp70, adenosyl-homocysteinase, peroxidase, alcohol dehydrogenases, glutamine synthetase, fructokinase, 1,3-beta-glucanase, chitinases, endochitinase, among others which are directly (24%) or indirectly (74%) related to plant defense. Taken together, these results indicate that drought-stressed roots have an active metabolism of reactive oxygen and nitrogen species (ROS and RNS) characterized by an increase of protein nitration and accumulation of NO and SNOs in cortical cells. The possibility of autophagy taking place in the stressed roots is also discussed.

Nitric oxide (NO) has emerged as an essential biological messenger in plant biology that usually transmits its bioactivity by post-translational modifications such as S-nitrosylation, the reversible addition of an NO group to a protein cysteine residue leading to S-nitrosothiols (SNOs). In recent years, SNOs have risen as key signalling molecules mainly involved in plant response to stress. Chief among SNOs is S-nitrosoglutathione (GSNO), generated by S-nitrosylation of the key antioxidant glutathione (GSH). GSNO is considered the major NO reservoir and a phloem mobile signal that confers to NO the capacity to be a long-distance signalling molecule. GSNO is able to regulate protein function and gene expression, resulting in a key role for GSNO in fundamental processes in plants, such as development and response to a wide range of environmental stresses. In addition, GSNO is also able to regulate the total SNO pool and, consequently, it could be considered the storage of NO in cells that may control NO signalling under basal and stress-related responses. Thus, GSNO function could be crucial during plant response to environmental stresses. Besides the importance of GSNO in plant biology, its mode of action has not been widely discussed in the literature. In this review, we will first discuss the GSNO turnover in cells and secondly the role of GSNO as a mediator of physiological and stress-related processes in plants, highlighting those aspects for which there is still some controversy.

Recent studies in animal systems have shown that NO can interact with fatty acids to generate nitro-fatty acids (NO-FAs). They are the product of the reaction between reactive nitrogen species and unsaturated fatty acids, and are considered novel mediators of cell signaling based mainly on a proven anti-inflammatory response. Although these signaling mediators have been described widely in animal systems, NO-FAs have scarcely been studied in plants. Preliminary data have revealed the endogenous presence of free and protein-adducted NO-FAs in extra-virgin olive oil (EVOO), which appear to be contributing to the cardiovascular benefits associated with the Mediterranean diet. Importantly, new findings have displayed the endogenous occurrence of nitro-linolenic acid (NO-Ln) in the model plant Arabidopsis thaliana and the modulation of NO-Ln levels throughout this plant's development. Furthermore, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO-Ln was involved in plant-defense response against different abiotic-stress conditions, mainly by inducing the chaperone network and supporting a conserved mechanism of action in both animal and plant defense processes. Thus, NO-Ln levels significantly rose under several abiotic-stress conditions, highlighting the strong signaling role of these molecules in the plant-protection mechanism. Finally, the potential of NO-Ln as a NO donor has recently been described both in vitro and in vivo. Jointly, this ability gives NO-Ln the potential to act as a signaling molecule by the direct release of NO, due to its capacity to induce different changes mediated by NO or NO-related molecules such as nitration and S-nitrosylation, or by the electrophilic capacity of these molecules through a nitroalkylation mechanism. Here, we describe the current state of the art regarding the advances performed in the field of NO-FAs in plants and their implication in plant physiology.

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Nitro-fatty acids (NO-FAs) are the product of the reaction between reactive nitrogen species derived of nitric oxide (NO) and unsaturated fatty acids. In animal systems, NO-FAs are considered novel signaling mediators of cell function based on a proven antiinflammatory response. Nevertheless, the interaction of NO with fatty acids in plant systems has scarcely been studied. Here, we examine the endogenous occurrence of nitro-linolenic acid (NO-Ln) in Arabidopsis and the modulation of NO-Ln levels throughout this plant’s development by mass spectrometry. The observed levels of this NO-FA at picomolar concentrations suggested its role as a signaling effector of cell function. In fact, a transcriptomic analysis by RNA-seq technology established a clear signaling role for this molecule, demonstrating that NO-Ln was involved in plant defense response against different abiotic-stress conditions, mainly by inducing heat shock proteins and supporting a conserved mechanism of action in both animal and plant defense processes. Bioinformatics analysis revealed that NO-Ln was also involved in the response to oxidative stress conditions, mainly depicted by HO, reactive oxygen species, and oxygen-containing compound responses, with a high induction of ascorbate peroxidase expression. Closely related to these results, NO-Ln levels significantly rose under several abiotic-stress conditions such as wounding or exposure to salinity, cadmium, and low temperature, thus validating the outcomes found by RNA-seq technology. Jointly, to our knowledge, these are the first results showing the endogenous presence of NO-Ln in Arabidopsis (Arabidopsis thaliana) and supporting the strong signaling role of these molecules in the defense mechanism against different abiotic-stress situations.

Nitric oxide (NO) is a biological messenger that orchestrates a plethora of plant functions, mainly through post-translational modifications (PTMs) such as S-nitrosylation or tyrosine nitration. In plants, hundreds of proteins have been identified as potential targets of these NO-PTMs under physiological and stress conditions indicating the relevance of NO in plant-signaling mechanisms. Among these NO protein targets, there are different antioxidant enzymes involved in the control of reactive oxygen species (ROS), such as H2O2, which is also a signal molecule. This highlights the close relationship between ROS/NO signaling pathways. The major plant antioxidant enzymes, including catalase, superoxide dismutases (SODs) peroxiredoxins (Prx) and all the enzymatic components of the ascorbate-glutathione (Asa-GSH) cycle, have been shown to be modulated to different degrees by NO-PTMs. This mini-review will update the recent knowledge concerning the interaction of NO with these antioxidant enzymes, with a special focus on the components of the Asa-GSH cycle and their physiological relevance.

In recent years, the study of nitric oxide (NO) in plant systems has attracted the attention of many researchers. A growing number of investigations have shown the significance of NO as a signal molecule or as a molecule involved in the response against (a)biotic processes. NO can be responsible of the post-translational modifications (NO-PTM) of target proteins by mechanisms such as the nitration of tyrosine residues. The study of protein tyrosine nitration during development and under biotic and adverse environmental conditions has increased in the last decade; nevertheless, there is also an endogenous nitration which seems to have regulatory functions. Moreover, the advance in proteome techniques has enabled the identification of new nitrated proteins, showing the high variability among plant organs, development stage and species. Finally, it may be important to discern between a widespread protein nitration because of greater RNS content, and the specific nitration of key targets which could affect cell-signaling processes. In view of the above point, we present a mini-review that offers an update about the endogenous protein tyrosine nitration, during plant development and under several abiotic stress conditions.

Trichoderma harzianum Rifai is a well-known biological control agent (BCA) effective against a wide range of phytopathogens. Since colonization and persistence in the target niche is crucial for biocontrol effectiveness we aimed to: (i) shed light on the olive roots colonization process by T. harzianum CECT 2413, (ii) unravel the fate of its biomass upon application, and (iii) study the in planta interaction with the soil-borne pathogen Verticillium dahliae Kleb. Fluorescently-tagged derivatives of CECT 2413 and V. dahliae and confocal laser scanning microscopy were used. In vitro assays showed for the first time mycoparasitism of V. dahliae by T. harzianum, evidenced by events such as hyphal coiling. In planta assays revealed that CECT 2413 profusely colonized the rhizoplane of olive roots. Interestingly, biomass of the BCA was visualized mainly as chlamydospores. This observation was independent on the presence or absence of the pathogen. Evidence of inner colonization of olive roots by CECT 2413 was not obtained. These results suggest that CECT 2413 is not able to persist in a metabolically-active form when applied as a spore suspension. This may have strong implications in the way this BCA should be introduced and/or formulated to be effective against Verticillium wilt of olive.

Low temperature severely affects plant growth and development. To overcome this constraint, several plant species from regions having a cool season have evolved an adaptive response, called cold acclimation. We have studied this response in olive tree (Olea europaea L.) cv. Picual. Biochemical stress markers and cold-stress symptoms were detected after the first 24 h as sagging leaves. After 5 days, the plants were found to have completely recovered. Control and cold-stressed plants were sequenced by Illumina HiSeq 1000 paired-end technique. We also assembled a new olive transcriptome comprising 157,799 unigenes and found 6,309 unigenes differentially expressed in response to cold. Three types of response that led to cold acclimation were found: short-term transient response, early long-term response, and late long-term response. These subsets of unigenes were related to different biological processes. Early responses involved many cold-stress-responsive genes coding for, among many other things, C-repeat binding factor transcription factors, fatty acid desaturases, wax synthesis, and oligosaccharide metabolism. After long-term exposure to cold, a large proportion of gene down-regulation was found, including photosynthesis and plant growth genes. Up-regulated genes after long-term cold exposure were related to organelle fusion, nucleus organization, and DNA integration, including retrotransposons.

Trichoderma harzianum CECT24l3 has been demonstrated to be an effective biocontrol agent (BCA) ofVerticillíum wilt ( Verticiilium dahliae K leb.) of olive (VWO) under control conditions. Previous works also sho\·ved in vitro mycoparasitism of V dahliae by strain CECT2413 and its ability to promote olivc growth and extemal!intemal colonization in tomato plants. The prcsent study aimed to evaluate the colonization capability of olive roots (var. Picual) by CECT2413 during a prolonged period oftime (> 100 days). A gfp-labelled derivative of CECT2413 and confocai laser scanning microscopy (CLSM) were used. CLSM image1y showed that CECT2413-gfp profusely colonized olive roots during the fírst three days after inoculation (DAI). Mycelium and abundant asexual reproductive structures (conidiophore-like) were clearly visible over the root surface. From this time point, tluorescent fm1gal structures resembling chlamydospores were observed, while a progressive loss of mycel ium became apparent. T'he majority of CECT2413-gfp biomass during this period (from 7-14 DAf) corresponded to conidia and chlamydospores found over the epidermis and root hairs. Colonization preference of specitic root si tes was not evident and conidia, chlamydospores and mycelium were found all over the examined roots. Long-term observations revealed the presence of chlamydospores and conidia on the rhizoplane but lack ofmycelium. !n conclusion, strain CECT2413 is an excellent colonizer of olive roots which helps to understand is dfectiveness asan efficient BCA against VWO.

Verticillium wilt of olive tree (Olea europaea L.) (VWO), ca u sed by the soil-borne fungus Verticillium dahliae Kleb., is considered one ofthe most devastatíng diseases affecting this crop in many areas. Preventive measures su eh as the treatment of the root systems at the nursery stage with microbial antagonists emerges as a promising altemative. Species ofthe soil-borne fungus Trichoderma ha ve been widely described as biocontrol agents (BCAs) of severa[ phytopathogenic fungi by means of different mechanisms. In this study we aimed to evaluate the potential of T harzianum CECT 2413 as effective BCA of VWO. Strain CECT 2413 was shown to in vitro overgrow both the bighly-virulent, defoliating (D) and the midly-virulent, non-defoliating (ND) V. dah!iae pathotypes infecting olive. Inhibition of V dahliae growth in cHituring media where strain CECT 2413 had previously grown showed that this ability might be dueto excretion of secondary metabolites or cell wall degrading enzymes. Wben a suspension of conidia of T harzianum CECT 2413 were applied by irrigation to roots of nursery-propagated, three-month-old olives (cv. Picual) prior to the treatment with a D isolate ofthe pathogen, disease symptoms were significantly reduced compared to that observcd in control plants. M oreo ver, increase in the number and size of lea ves in plants treated \vith Trichoderrna was also reported. Biofonnulations ofthis BCA, alone or in combination with other Trichoderma spp. strains, have been recently patented and licensed.

La verticilosis del olivo (Olea europaea L.) (VO), causada por el hongo Verticillium dahliae Kleb., está considerada como una de las enfermedades más devastadoras de este cultivo, particularmente en la cuenca mediterránea. En la actualidad, esta enfermedad sólo puede ser neutralizada de manera efectiva mediante una estrategia de control integrado, incluyendo la lucha biológica. En este sentido, numerosas especies del hongo Trichoderma han sido descritas como agentes de control biológico (ACB) de hongos fitopatógenos. El objeto de este estudio ha consistido en la evaluación del potencial de la cepa T. harzianum CECT 2413 como ACB efectivo frente a la VO. La cepa CECT 2413 demostró ser capaz de crecer in vitro sobre aislados representativos de los patotipos defoliante (D) y no defoliante (ND) de V. dahliae que infectan al olivo. Además, la observación mediante microscopía láser confocal (CLSM) del enfrentamiento directo de ambos hongos marcados con eyfp (V. dahliae) y gfp (T. harzianum) reveló que el micoparasitismo es uno de los mecanismos implicados en este antagonismo. Cuando se aplicó mediante irrigación una suspensión de conidias de la cepa CECT 2413 a plantones de olivo de 3 meses de edad (cv. Picual) antes de la infección con un aislado D de V. dahliae, se observó una reducción significativa de los síntomas de la enfermedad frente a los observados en plantas no tratadas con Trichoderma. Finalmente, mediante observación de la interacción gfp-Trichoderma-raíz por CLSM, se constató que la cepa CECT 2413 es capaz de colonizar y permanecer en la rizosfera de los olivos durante un periodo prolongado de tiempo. En resumen, T. harzianum CECT 2413 ha demostrado ser un colonizador eficiente de raíces de olivo y un ACB efectivo frente al patotipo D de V. dahliae.

Low temperature severely affects plant growth and development. Several plant species have evolved an adaptive response, named cold acclimation. In order to study this response in olive tree (Olea europaea), we maintained twomonths- old vegetative propagated olive trees cv. Picual at low temperature (14h at 10ºC with light, 10h at 4ºC at the dark) for 15 days. Control plants were maintained in field growing conditions. Cold stress symptoms were observed after the first 24h as sagging leaf and biochemical stress markers. After 5 days the plants were observed completely recovered. We extracted RNA from leaves of three control plants and the same number from 24h and 10 days cold stressed plants. We pooled RNA and made two cDNA libraries for sequencing. We used Illumina HiSeq 1000 sequencer.

Las nuevas técnicas de secuenciación masiva conocidas como NGS (del inglés “Next Generation Sequencing”) permiten realizar estudios donde se analicen la totalidad de los genes de un organismo, el genoma completo o cientos de miles de marcadores simultáneamente. El análisis de la expresión de los genes al nivel de ARNm se conoce como transcriptómica y suele dar una imagen bastante completa de cómo se modifica la expresión génica en función de la edad, del tejido o de cambios ambientales. El olivo es una planta con capacidad de adaptación al frio y responde a una bajada de las temperaturas con cambios en la expresión génica que le permiten soportar situaciones de bajas temperaturas sin sufrir daños. La aplicación del análisis transcriptómico basado en NGS nos ha permitido definir conjuntos de genes que responden de diversa manera para adaptarse a la nueva situación de estrés por frio. Encontramos genes de expresión temprana que se inducen rápidamente y suponen una primera respuesta de la planta al frio. Bastantes de estos genes vuelven al poco tiempo a su nivel de expresión basal aunque persista la situación de frio. Otros en cambio continúan sobre-expresados mientras se mantiene la situación de bajas temperaturas. Por último encontramos genes que se inducen de forma tardía y que junto con el grupo anterior parecen ser los responsables de la adaptación a bajas temperaturas, mientras que los de expresión temprana y transitoria podrían tener un efecto de respuesta de choque ante la llegada brusca de las bajas temperaturas.

Cepa de Trichoderma útil para el tratamiento y/o prevención de infecciones provocadas por hongos pertenecientes al género Verticillium. La invención se refiere al uso de la cepa de hongos Trichoderma atroviride IMI 206040 para la prevención y/o tratamiento de infecciones de plantas y/o suelos provocadas por hongos fitopatógenos pertenecientes al género Verticillium, preferiblemente por los patotipos defoliante o no defoliante de Verticillium dahliae, agente causante de la verticilosis del olivo. Además, la invención propone el uso de esta cepa de hongos en combinación con otros hongos del género Trichoderma.

The use of indigenous bacterial root endophytes with biocontrol activity against soil-borne phytopathogens is an environmentally-friendly and ecologically-efficient action within an integrated disease management framework. The earliest steps of olive root colonization by Pseudomonas fluorescens PICF7 and Pseudomonas putida PICP2, effective biocontrol agents (BCAs) against Verticillium wilt of olive (Olea europaea L.) caused by the fungus Verticillium dahliae Kleb., are here described. A gnotobiotic study system using in vitro propagated olive plants, differential fluorescent-protein tagging of bacteria, and confocal laser scanning microscopy analysis have been successfully used to examine olive roots–Pseudomonas spp. interactions at the single-cell level. In vivo simultaneous visualization of PICF7 and PICP2 cells on/in root tissues enabled to discard competition between the two bacterial strains during root colonization. Results demonstrated that both BCAs are able to endophytically colonized olive root tissues. Moreover, results suggest a pivotal role of root hairs in root colonization by both biocontrol Pseudomonas spp. However, colonization of root hairs appeared to be a highly specific event, and only a very low number of root hairs were effectively colonized by introduced bacteria. Strains PICF7 and PICP2 can simultaneously colonize the same root hair, demonstrating that early colonization of a given root hair by one strain did not hinder subsequent attachment and penetration by the other. Since many environmental factors can affect the number, anatomy, development, and physiology of root hairs, colonization competence and biocontrol effectiveness of BCAs may be greatly influenced by root hair’s fitness. Finally, the in vitro study system here reported has shown to be a suitable tool to investigate colonization processes of woody plant roots by microorganisms with biocontrol potential.