Ammonium enhances resistance to salinity stress in citrus plants

In this work, we demonstrate that NH4+ nutrition in citrange Carrizo plants acts as an inducer of resistance against salinity conditions. We investigated its mode of action and provide evidence that NH4+ confers resistance by priming abscisic acid and polyamines, and enhances H2O2 and proline basal content. Moreover, we observed reduced Cl− uptake as well as enhanced PHGPx expression after salt stress. Control and N-NH4+ plants showed optimal growth. However, N-NH4+ plants displayed greater dry weight and total lateral roots than control plants, but these differences were not observed for primary root length. Our results revealed that N-NH4+ treatment induces a similar phenotypical response to the recent stress-induced morphogenetic response (SIMRs). The hypothesis is that N-NH4+ treatment triggers mild chronic stress in citrange Carrizo plants, which might explain the SIMR observed. Moreover, we observed modulators of stress signaling, such as H2O2 in N-NH4+ plants, which could acts as an intermediary between stress and the development of the SIMR phenotype. This observation suggests that NH4+ treatments induce a mild stress condition that primes the citrange Carrizo defense response by stress imprinting and confers protection against subsequent salt stress

Absence of Cu–Zn superoxide dismutase BCSOD1 reduces Botrytiscinerea virulence in Arabidopsis and tomato plants, revealinginterplay among reactive oxygen species, callose and signallingpathways

Plants activate responses against pathogens, including the oxida-tive burst. Necrotrophic pathogens can produce reactive oxygenspecies (ROS) that benefit the colonization process. Previously, wehave demonstrated that tomato plants challenged with Botrytiscinerea accumulate ROS and callose, together with the inductionof genes involved in defence, signalling and oxidative metabolism.Here, we studied the infection phenotype of the Dbcs od1 strain inboth tomato and Arabidopsis plants. This mutant lacks bcsod1,which encodes Cu–Zn superoxide dismutase (SOD). This enzymecatalyses the conversion of superoxide ion (O–2) into hydrogen per-oxide (H2O2). ROS play a protective role and act as signals inplants. Dbcsod1 displayed reduced virulence compared with wild-type B05.10 in both species. Plants infected with Dbcsod1 accu-mulated less H2O2and more O–2than those infected with B05.10,which is associated with an increase in the defensive polymer cal-lose. This supports a major role of fungal SOD in H2O2productionduring the plant–pathogen interaction. The early induction of thecallose synthase gene PMR4 suggested that changes in ROSaltered plant defensive responses at the transcriptional level. Themetabolites and genes involved in signalling and in response tooxidative stress were differentially expressed on Dbcsod1 infec-tion, supporting the notion that plants perceive changes in ROSbalance and activate defence responses. A higher O2–/H2O2ratioseems to be beneficial for plant protection against this necrotroph.Our results highlight the relevance of callose and the oxylipin 12-oxo-phytodienoic acid (OPDA) in the response to changes in theoxidative environment, and clarify the mechanisms that underliethe responses to Botrytis in Arabidopsis and tomato plants.This work was supported by grants from the Spanish Ministry of Scienceand Innovation (AGL2010-22300-C03-01-02 and AGL2013-49023-C03-01-02), co-funded by the European Regional Development Funds (ERDF)and by Generalitat Valenciana Grupos de Excelencia PROMETEO/2012/066. J.L.-C. and O.C.-S. were recipients of research contracts from thePROMETEO/2012/066 grant. E.F.-C. was the recipient of research con-tracts from grant AGL2010-22300-C03-01. J.L.-C. is the recipient of aresearch contract from grant AGL2013-49023-C03-01

NH4+ protects tomato plants against Pseudomonas syringae by activation of systemic acquired acclimation

NH4 + nutrition provokes mild toxicity by enhancing H2O2 accumulation, which acts as a signal activating systemic acquired acclimation (SAA). Until now, induced resistance mechanisms in response to an abiotic stimulus and related to SAA were only reported for exposure to a subsequent abiotic stress. Herein, the first evidence is provided that this acclimation to an abiotic stimulus induces resistance to later pathogen infection, since NH4 + nutrition (N-NH4 +)-induced resistance (NH4 +-IR) against Pseudomonas syringae pv tomato DC3000 (Pst) in tomato plants was demonstrated. N-NH4 + plants displayed basal H2O2, abscisic acid (ABA), and putrescine (Put) accumulation. H2O2 accumulation acted as a signal to induce ABA-dependent signalling pathways required to prevent NH4 + toxicity. This acclimatory event provoked an increase in resistance against later pathogen infection. N-NH4 + plants displayed basal stomatal closure produced by H2O2 derived from enhanced CuAO and rboh1 activity that may reduce the entry of bacteria into the mesophyll, diminishing the disease symptoms as well as strongly inducing the oxidative burst upon Pst infection, favouring NH4 +-IR. Experiments with inhibitors of Put accumulation and the ABA-deficient mutant flacca demonstrated that Put and ABA downstream signalling pathways are required to complete NH4 +-IR. The metabolic profile revealed that infected N-NH4 + plants showed greater ferulic acid accumulation compared with control plants. Although classical salicylic acid (SA)-dependent responses against biotrophic pathogens were not found, the important role of Put in the resistance of tomato against Pst was demonstrated. Moreover, this work revealed the cross-talk between abiotic stress acclimation (NH4 + nutrition) and resistance to subsequent Pst infection.This work was supported by grants from the Spanish Ministry of Science and Innovation (AGL2013-49023-C3-2-R). The authors are grateful to the Serveis Centrals d’Instrumentació Científica (SCIC) from Universitat Jaume I (UJI, Castellón, Spain)

Putrescine Biosynthesis Inhibition in Tomato by DFMA and DFMO Treatment

This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4 + as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH4 + as the sole N source presented an increase of putrescine content in leaves (Fernández-Crespo et al., 2015). To assess the importance of putrescine accumulation, DL-α-(Difluoromethyl)arginine (DFMA) and DL-α-(Difluoromethyl)ornithine (DFMO), inhibitors of putrescine synthesis, were used as irreversible inhibitors of ADC and ODC enzymes, respectively (Fallon and Phillips, 1988), with the purpose of reducing cellular putrescine accumulation induced by NH4 + nutrition. The inhibitor solution containing 2 mM DFMA and 5 mM DFMO was applied directly to each pot during the week prior to sample collection. Putrescine content was reduced by 35.3% in tomato plants grown with NH4 +.The work was supported by a grant from the Spanish Ministry of Science and Innovation (AGL2013- 49023-C-2-R

The Histone Marks Signature in Exonic and Intronic Regions Is Relevant in Early Response of Tomato Genes to Botrytis cinerea and in miRNA Regulation

Research into the relationship between epigenetic regulation and resistance to biotic stresses provides alternatives for plant protection and crop improvement. To unravel the mechanisms underlying tomato responses to Botrytis cinerea, we performed a chromatin immunoprecipitation (ChIP) analysis showing the increase in H3K9ac mark along the early induced genes SlyDES, SlyDOX1, and SlyLoxD encoding oxylipin-pathway enzymes, and SlyWRKY75 coding for a transcriptional regulator of hormonal signaling. This histone mark showed a more distinct distribution than the previously studied H3K4me3. The RNAPol-ChIP analysis reflected the actual gene transcription associated with increased histone modifications. A different pattern of marks in the oxylipin-related genes against P. syringae supported a pathogen-specific profile, while no significant differences occurred in SlyWRKY75. The epigenetic regulation of SlyWRKY75 by the intron-binding miR1127-3p was supported by the presence of SlyWRKY75 pre-mRNA in control plants. Interestingly, mRNA was found to be accumulated in response to B. cinerea and P. syringae, while reduction in miRNA only occurred against B. cinerea. The intronic region presented a similar pattern of marks than the rest of the gene in both pathosystems, except for H3K4me3 in the miRNA binding site upon B. cinerea. We located the gene encoding Sly-miR1127-3p, which presented reduced H3K4me3 on its promoter against B. cinerea

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Response of tomato-pseudomonas pathosystem to mild heat stress

Higher plants suffer from mild heat stress when temperatures increase by 5 ◦C above optimum growth temperatures. This produces changes at the cellular and metabolic levels, allowing plants to adapt to heat conditions. This study investigated an increase of 5 ◦C above the optimum growth temperature (26 ◦C) of tomato plants in the tomato—Pseudomonas syringae pv. tomato pathosystem. A temperature increase above 26 ◦C affects plant development, the defensive pathways activated against Pseudomonas syringae pv. tomato strain DC3000 (PstDC3000), and the bacterial growth and virulence machinery. The results demonstrated that tomato plants were able to acclimate to mild heat stress, showing no symptoms of damage. Moreover, plants subjected to a 5 ◦C increase (T31 ◦C plants) showed higher basal levels of metabolites such as proline and putrescine, which probably act as compatible osmolytes. This demonstrates their importance as key components of thermotolerance. When grown under mild heat stress, plants were less susceptible to PstDC3000 and showed increased accumulation of abscisic acid, jasmonic acid-isoleucine, and spermine. In addition, the temperature increase negatively affected the infectivity of PstDC3000. Inhibition of the genes responsible for quorum sensing establishment and synthesis of flagellin and coronatine was observed in bacteria extracted from T31 ◦C plants. Analysis of the genes involved in the synthesis of the type III secretion system indicates the important role of this system in bacterial growth under these conditions. As the known resistance mechanisms involved in the defense against PstDC3000 were not activated, the changes in its virulence mechanisms under high temperatures may explain the lower infection observed in the T31 ◦C plants

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Quantification of Callose Deposition in Plant Leaves

Callose is an amorphous homopolymer, composed of β-1, 3-glucan, which is widespread in higher plants. Callose is involved in multiple aspects of plant growth and development. It is synthetized in plants at the cell plate during cytokinesis, in several stages during pollen development and is deposited at plasmodesmata to regulate the cell-to-cell movement of molecules. Moreover, it is produced in response to multiple biotic and abiotic stresses (Chen and Kim, 2009). Callose is considered to act as a physical barrier by strengthening the plant cell well to slow pathogen infection and to contribute to the plant’s innate immunity. Thus the callose staining method is useful to quantify activity of plant immunity. In addition, this staining can be used to visualize structures in plant tissue, where the callose may be implied whether during the development of plants or response against pathogen infection. This method is based on the use of methyl blue which reacts with (13)--glucans to give a brilliant yellow fluorescence in UV light. Moreover, calcofluor stains chitin present in fungal cell membranes and also binds to cellulose at locations where the cuticle is damaged.Authors thank Universitat Jaume I and the National R&D Plan (AGL2010-22300-C03-02, Spain for funding support

Implementation of a project-based learning to the coordination of subjects in the Agrifood and Rural Engineering Bachelor

The Bachelor’s Degree in Agrifood and Rural Engineering at Universitat Jaume I of Castelló has implemented in the second academic year a multidisciplinary project using a Project-Based Learning as the teaching method. Its final purpose is the acquisition of skills that should help the students to cope with their future career. This teaching-learning system has been used for three consecutive years since the degree was firstly implemented. Once a particular farm is assigned, the students are organized in groups and must fulfill their assigned tasks in a collaborative manner with the final goal of developing a project on that farm including viable improvements of the exploitation, taking into account the issues related to the different subjects involved. This work presents the results obtained along the three years, analyzed from two different points of view: student satisfaction and learning outcomes. Besides, the proposals for improvement of the weaknesses identified during the process are presented. The results show that the used method has promoted the acquisition of the competences proposed. Moreover the multidisciplinary approach has led to better results in the student performance than those obtained by students enrolled in an unidisciplinary project. Although improvement actions have solved some of the problems detected, there are still some weaknesses, mainly related to team working and tutorials that should be addressed in the future.El grado de Ingeniería Agroalimentaria y del Medio Rural de la Universitat Jaume I de Castelló viene aplicando en su segundo curso un proyecto multidisciplinar usando el Aprendizaje Basado en Proyectos como recurso docente en el que se pretende que los estudiantes adquieran competencias que les ayuden a enfrentarse a su futuro profesional. Este sistema de enseñanza-aprendizaje se ha llevado a cabo durante los tres cursos que está implantado el grado. Una vez asignado un tipo de explotación agrícola concreta y utilizando las herramientas del trabajo en equipo, los estudiantes deben ser capaces de desarrollar un proyecto sobre la explotación con propuestas de mejora que sean factibles y que abarquen aspectos relacionados con las diferentes disciplinas implicadas, aplicando los conocimientos adquiridos en éstas. El presente trabajo incluye los resultados obtenidos durante los tres años del proyecto desde dos puntos de vista importantes: la satisfacción del estudiante y los resultados de aprendizaje. Además se presentan las propuestas de mejora aplicadas en cada curso como respuesta a las debilidades detectadas durante el proceso. Los resultados demuestran que el método utilizado ha favorecido la adquisición de las competencias propuestas. Además, el enfoque multidisciplinar ha propiciado mejores resultados que los alcanzados por los estudiantes que realizaron trabajos unidisciplinares. Por otro lado, aunque las acciones de mejora han permitido solventar algunos de los problemas detectados, siguen persistiendo carencias, sobre todo a nivel de trabajo en equipo y de tutorización, que se proponen como mejoras para el futuro