La metabolómica como herramienta para la evaluación fisiológica y nutricional en citricultura

La metabolómica tiene como objetivo el análisis de todos los metabolitos de bajo peso molecular presentes en un organismo, tejido o tipo celular concreto en un estadio de desarrollo dado y bajo unas condiciones ambientales particulares, proporcionando así una descripción detallada del fenotipo bioquímico. En este trabajo se analizaron, mediante cromatografía líquida en fase reversa acoplada a espectrometría de masas (RPLC/ESI-QTOF-MS) los perfiles de metabolitos secundarios de tres patrones de cítricos: citrange Carrizo, citrumelo CPB4475 y mandarino Cleopatra cultivados en condiciones óptimas en tres localizaciones distintas. Los metabolitos se emplearon como marcadores para clasificar las muestras atendiendo al genotipo y a la localización mediante análisis clúster jerárquico (HCA) seguido de análisis discriminante basado en mínimos cuadrados parciales (PLS-DA), que permite la identificación de las variables importantes en dicha clasificación. Este análisis reveló una influencia importante del genotipo incluso por encima del ambiente, aunque éste también influyó en la composición metabólica de forma significativa. En conclusión, esta tecnología puede ser empleada para investigar variaciones sutiles en el metabolismo debidas a alteraciones genéticas o cambios en el ambiente, a modo de técnica de evaluación fisiológica.Este estudio fue financiado por el Ministerio de Educación y Ciencia y la Universitat Jaume 1a través de los proyectos AGL201 0-22195-C03-01, P11B2012-06 Y P11B2013-2

Effects of cadmium on gas exchange and phytohormone contents in citrus

The effect of increased Cd2+ concentrations in the watering solution on citrus physiology was studied by using two citrus genotypes, Cleopatra mandarin and Carrizo citrange. Cadmium content in roots and leaves was tested together with measurements of leaf damage, gas exchange parameters, and hormonal contents. Citrus roots efficiently retained Cd2+ avoiding its translocation to the shoots and Cleopatra mandarin translocated less Cd2+ than Carrizo. With increasing Cd2+ concentration all gas exchange parameters were decreased more in Carrizo than in Cleopatra mandarin. Cd-induced increases in abscisic acid and salicylic acid contents were observed in leaves but not in roots of both genotype

Root Involvement in Plant Responses to Adverse Environmental Conditions

limate change is altering the environment in which plants grow and survive. An increase in worldwide Earth surface temperatures has been already observed, together with an increase in the intensity of other abiotic stress conditions such as water deficit, high salinity, heavy metal intoxication, etc., generating harmful conditions that destabilize agricultural systems. Stress conditions deeply affect physiological, metabolic and morphological traits of plant roots, essential organs for plant survival as they provide physical anchorage to the soil, water and nutrient uptake, mechanisms for stress avoidance, specific signals to the aerial part and to the biome in the soil, etc. However, most of the work performed until now has been mainly focused on aerial organs and tissues. In this review, we summarize the current knowledge about the effects of different abiotic stress conditions on root molecular and physiological responses. First, we revise the methods used to study these responses (omics and phenotyping techniques). Then, we will outline how environmental stress conditions trigger various signals in roots for allowing plant cells to sense and activate the adaptative responses. Later, we discuss on some of the main regulatory mechanisms controlling root adaptation to stress conditions, the interplay between hormonal regulatory pathways and the global changes on gene expression and protein homeostasis. We will present recent advances on how the root system integrates all these signals to generate different physiological responses, including changes in morphology, long distance signaling and root exudation. Finally, we will discuss the new prospects and challenges in this field

La actividad fotosintética como indicador de la tolerancia de los cítricos al estrés provocado por la inundación del sustrato

Soil waterlogging is a seasonal phenomenon associated to the incidence of heavy rains in the mediterranean basin. The main effect of substrate flooding is the reduction in the availability of O2 in the root environment which is directly related to a decrease in the plant ability for water absorption and the reduction in gas exchange parameters. In general, citrus are considered sensitive to root waterlogging although significant differences have been described to this respect among genotypes. In this sense, Cleopatra mandarin is considered highly sensitive whereas other genotypes such as Swingle citrumelo or Carrizo citrange are classified as more tolerant. In the present work, the relationship of different photosynthetic parameters such as gas exchange and chlorophyll fluorescence with tolerance to continuous flooding was assessed in the three above-mentioned citrus genotypes. Cleopatra mandarin revealed as very sensitive, citrumelo as moderately tolerant and Carrizo citrange as very tolerant. This gradation was correlated with the ability to keep elevated photosynthetic activity and maintaining it stable for longer time. These results suggest that photosynthetic activity could be used as indicator of tolerance to root waterlogging in citrusEn la cuenca mediterránea, el encharcamiento del suelo es un fenómeno estacional asociado a la incidencia de lluvias torrenciales. El principal efecto de la inundación del sustrato es la reducción en la disponibilidad de O2 en el entorno radicular, lo cual se traduce en una reducción de la capacidad de absorción de agua de las plantas y en un descenso del intercambio gaseoso. Los cítricos se consideran, en general, sensibles a la asfixia radicular aunque se han descrito notables diferencias entre genotipos. En este sentido, se considera al mandarino Cleopatra altamente sensible mientras que otros patrones como citrumelo Swingle o citrange Carrizo están clasificados como más tolerantes. En el presente trabajo, se evaluó la relación de distintos parámetros fotosintéticos, tales como el intercambio gaseoso y la fluorescencia de clorofilas, con la tolerancia al encharcamiento en los tres genotipos mencionados. Se observó una gradación en la tolerancia al encharcamiento continuado, siendo mandarino Cleopatra muy sensible, citrumelo moderadamente tolerante y citrange Carrizo muy tolerante. Esta gradación se correlacionó con la capacidad de mantener una elevada actividad fotosintética estable durante más tiempo. Estos resultados sugieren que la actividad fotosintética podría ser utilizada como indicador de la tolerancia al encharcamiento radicular en cítrico

Photosynthetic and antioxidant responses of Mexican lime (Citrus aurantifolia) plants to Citrus tristeza virus infection

The effect of Citrus tristeza virus (CTV) infection on photosynthetic activity and antioxidant metabolism was analysedin plants of the highly susceptible citrus genotype Mexican lime (Citrus aurantifolia). Two virus isolates differing intheir virulence (the severe T318 and the mild T385) were used in the experiments. CTV infection caused a reduction inphotosynthetic capacity in infected plants. This limitation was mainly due to a reduction in the carboxylative efficiencywhereas the limitation of CO2diffusion through the stoma had lower impact. The virus did not damage the antennaeand did not reduce the efficiency of light harvesting complexes. Oxidative damage occurred in infected plants, as evi-denced by the increase in malondialdehyde levels. Indeed, CTV infection caused an increase in ascorbate peroxidaseactivity in new shoots developed in infected plants during the 2 years of the experiment. Data suggest that the H2O2removal machinery was not damaged as a result of stress but the defence mechanism was overwhelmed with time dueto the continuing pressure of biotic stressUniversitat Jaume I (SPAIN) P1IB2012-06 P1IB2013-23 Spanish Ministerio de Economia y Competitividad (MINECO) AGL2010-22195-C03-0

Water Stress Responses of Tomato Mutants Impaired in Hormone Biosynthesis Reveal Abscisic Acid, Jasmonic Acid and Salicylic Acid Interactions

To investigate the putative crosstalk between JA and ABA in Solanum lycopersicum plants in response to drought, suppressor of prosystemin-mediated responses2 (spr2, JA-deficient) and flacca (flc, ABA-deficient) mutants together with the naphthalene/salicylate hydroxylase (NahG) transgenic (SA-deficient) line were used. Hormone profiling and gene expression of key enzymes in ABA, JA and SA biosynthesis were analyzed during early stages of drought. ABA accumulation was comparable in spr2 and wild type (WT) plants whereas expression of 9-cis-epoxycarotenoid dioxygenase 1 (NCED1) and NCED2 was different, implying a compensation mechanism between NCED genes and an organ-specific regulation of NCED1 expression. JA levels and 12-oxo-phytodienoic acid reductase 3 (OPR3) expression in flc plants suggest that ABA regulates the induction of the OPR3 gene in roots. By contrast, ABA treatment to flc plants leads to a reduction of JA and SA contents. Furthermore, different pattern of SA accumulation (and expression of isochorismate synthase and phenylalanine ammonia lyase 1) was observed between WT seedlings and mutants, suggesting that SA plays an important role on the early response of tomato plants to drought and also that JA and ABA modulate its biosynthesis. Finally, hormone profiling in spr2 and NahG plants indicate a crosstalk between JA and SA that could enhance tolerance of tomato to water stress

Effect of cadmium and calcium treatments on phytochelatin and glutathione levels in citrus plants

Industry residues, phosphate fertilisers and wastewater as a source of irrigation have considerably increased levels of heavy metals in the soil, mainly cadmium (Cd2+). To test the effects of a calcium (Ca2+) treatment on Cd2+ accumulation and plant tolerance to this heavy metal, plants of two citrus genotypes, Cleopatra mandarin (CM) and Carrizo citrange (CC), were watered with increasing concentrations of Cd2+, and phytochelatin (PC) and glutathione (GSH) content were measured. Both genotypes were able to synthesise PCs in response to heavy metal intoxication, although CM seems to be a better Cd2+ excluder than CC. However, data indicate that CC plants had a higher capacity for regenerating GSH than CM plants. In this context, the effects of Ca2+ treatment on Cd2+ accumulation, plant survival and PC, GSH and oxidised glutathione (GSSG) content were assessed. Data indicate that treatment with Ca2+ had two positive effects on citrus physiology: it reduced Cd+2 uptake into roots and also increased GSH content (even in the absence of Cd2+). Overall, the data indicate that although Cd2+ exclusion is a powerful mechanism to avoid heavy metal build-up into photosynthetic organs, the capacity to maintain optimum GSH levels to feed PC biosynthesis could also be an important factor in stress tolerance

A novel in vitro tissue culture approach to study salt stress responses in citrus

In citrus, a major crop throughout the world, growth and yield are seriously affected by salinity. Different approaches, including agronomical, physiological and molecular methods, have been used to address this problem. In this work, an in vitro experimental system has been developed to study the toxic effect of NaCl on three citrus genotypes, avoiding the ion filter that represents the root system. To carry out the experiments, shoots were obtained from nodal segments of Cleopatra mandarin, Carrizo citrange and citrumelo CPB4475 plants growing in a greenhouse. Shoots were cultured in control or NaCl-supplemented media. After testing several salt concentrations, 60 mM NaCl was selected as saline treatment. Shoots accumulated similar levels of chloride when cultured without roots and exhibited similar leaf damage. No increases in malondialdehyde levels were observed in any genotype (as a measure of oxidative stress). Similar patterns of hormonal signalling (in terms of abscisic acid and salicylic acid contents) were exhibited in the three genotypes, despite their different tolerance under field conditions. All data together indicate that, without root system, all genotypes had the same behaviour under salt stress. The in vitro culture system has been proved as a useful tool to study biochemical processes involved in the response of citrus to salt stres

Biotechnological approaches to study plant responses to stress

Multiple biotic and abiotic environmental stress factors affect negatively various aspects of plant growth, development, and crop productivity. Plants, as sessile organisms, have developed, in the course of their evolution, efficient strategies of response to avoid, tolerate, or adapt to different types of stress situations. The diverse stress factors that plants have to face often activate similar cell signaling pathways and cellular responses, such as the production of stress proteins, upregulation of the antioxidant machinery, and accumulation of compatible solutes. Over the last few decades advances in plant physiology, genetics, and molecular biology have greatly improved our understanding of plant responses to abiotic stress conditions. In this paper, recent progresses on systematic analyses of plant responses to stress including genomics, proteomics, metabolomics, and transgenic-based approaches are summarized

Utilización del cultivo de tejidos vegetales in vitro para el estudio de la respuesta de los cítricos al estrés salino

Este trabajo describe la puesta a punto de un sistema in vitro para el estudio de la toxicidad del NaCl en tres genotipos de cítricos, evitando el filtro de iones que constituye la raíz. Los cultivos se establecieron a partir de plantas de mandarino Cleopatra, citrange Carrizo y citrumelo CPB4475 cultivadas en invernadero. Se ensayaron diferentes concentraciones de NaCl y se seleccionó 60 mM como tratamiento salino para los diferentes experimentos. Los brotes de todos los genotipos estudiados acumularon concentraciones similares de iones cloruro cuando se cultivaron desprovistos del sistema radicular y mostraron los mismos daños foliares. No se observó incremento en la concentración de malondialdehido (como indicador del daño oxidativo) en ninguno de los genotipos y todos ellos mostraron patrones similares de señalización hormonal, con independencia de su tolerancia o sensibilidad cuando se cultivan en campo. El sistema in vitro descrito se postula como una herramienta útil para el estudio de los procesos bioquímicos implicados en la respuesta de los cítricos al estrés salino.In this work, an in vitro experimental system has been developed to study the toxic effect of NaCl on three citrus genotypes, avoiding the ion filter that represents the root system. Cultures were established from greenhouse growing plants of Cleopatra mandarin, Carrizo citrange and citrumelo CPB4475. Several salt concentrations were tested and 60 mM NaCl was selected as saline treatment. Shoots from all studied genotypes accumulated similar levels of chloride when cultured without roots and exhibited similar leaf damage. No increases in malondialdehyde levels (as a measure of oxidative stress) were observed in any genotype and similar patterns of hormonal signalling were exhibited in the three genotypes, despite their different tolerance under field conditions. The in vitro culture system has been proved as a useful tool to study biochemical processes involved in the response of citrus to salt stress.Este estudio fue financiado por el Ministerio de Ciencia e Innovación y la Fundació Bancaixa/Universitat Jaume I a través de la concesión de los proyectos AGL2010-22195-C03-01/AGR y P1 1B2009-01, respectivament