Changes in physiological activities and root exudation profile of two grapevine rootstocks reveal common and specific strategies for Fe acquisition

In several cultivation areas, grapevine can suffer from Fe chlorosis due to the calcareous and alkaline nature of soils. This plant species has been described to cope with Fe deficiency by activating Strategy I mechanisms, hence increasing root H+ extrusion and ferric-chelate reductase activity. The degree of tolerance exhibited by the rootstocks has been reported to depend on both reactions, but to date, little emphasis has been given to the role played by root exudate extrusion. We studied the behaviour of two hydroponically-grown, tolerant grapevine rootstocks (Ramsey and 140R) in response to Fe deficiency. Under these experimental conditions, the two varieties displayed differences in their ability to modulate morpho-physiological parameters, root acidification and ferric chelate reductase activity. The metabolic profiling of root exudates revealed common strategies for Fe acquisition, including ones targeted at reducing microbial competition for this micronutrient by limiting the exudation of amino acids and sugars and increasing instead that of Fe(III)-reducing compounds. Other modifications in exudate composition hint that the two rootstocks cope with Fe shortage via specific adjustments of their exudation patterns. Furthermore, the presence of 3-hydroxymugenic acid in these compounds suggests that the responses of grapevine to Fe availability are rather diverse and much more complex than those usually described for Strategy I plants

Accumulation and tolerance of cadmium in a non- metallicolous ecotype of Silene vulgaris Garcke (Moench)

En este estudio, se analizó el efecto de diferentes concentraciones de Cd2+ sobre un ecotipo de Silene vulgaris Garcke (Moench). La concentración de 60 μM de Cd2+ provocó una ligera inhibición del crecimiento de las plantas mientras que la concentración más alta (120 μM) redujo drásticamente la biomasa y la elongación de la raíz y los brotes. Además, se detectaron altos niveles de Cd2+ en las plantas, un coeficiente de bioacumulación elevado en las raí- ces y un bajo factor de translocación indicando que el ecotipo de S. vulgaris empleado en este estudio presenta una alta capacidad de acumulación de Cd2+ en las raíces y sería un buen candidato para la fitoestabilización, lo que contribuiría a reducir los niveles de Cd2+ en el suelo. Además, los resultados obtenidos indican que se debe tener precaución con el origen de esta planta, ya que podría representar una fuente adicional de Cd2+ en la dieta humana

Exploring the interplay between metabolic pathways and taxane production in elicited Taxus baccata cell suspensions

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.Agencia Estatal de Investigación | Ref. PID2020-113438RB-I00Agència de Gestió d’Ajuts Universitaris i de Recerca | Ref. 2021 SGR00693Ministerio de Ciencia, Innovación y Universidades | Ref. FPU18/00850Ministerio de Universidades | Ref. 33.50.460A.75

Neurofuzzy logic predicts a fine-tuning metabolic reprogramming on elicited Bryophyllum PCSCs guided by salicylic acid

Novel approaches to the characterization of medicinal plants as biofactories have lately increased in the field of biotechnology. In this work, a multifaceted approach based on plant tissue culture, metabolomics, and machine learning was applied to decipher and further characterize the biosynthesis of phenolic compounds by eliciting cell suspension cultures from medicinal plants belonging to the Bryophyllum subgenus. The application of untargeted metabolomics provided a total of 460 phenolic compounds. The biosynthesis of 164 of them was significantly modulated by elicitation. The application of neurofuzzy logic as a machine learning tool allowed for deciphering the critical factors involved in the response to elicitation, predicting their influence and interactions on plant cell growth and the biosynthesis of several polyphenols subfamilies. The results indicate that salicylic acid plays a definitive genotype-dependent role in the elicitation of Bryophyllum cell cultures, while methyl jasmonate was revealed as a secondary factor. The knowledge provided by this approach opens a wide perspective on the research of medicinal plants and facilitates their biotechnological exploitation as biofactories in the food, cosmetic and pharmaceutical fields.Xunta de Galicia | Ref. ED431E 2018/07Xunta de Galicia | Ref. ED431D2017/1

A Combined Phenotypic and Metabolomic Approach for Elucidating the Biostimulant Action of a Plant-Derived Protein Hydrolysate on Tomato Grown Under Limited Water Availability

Plant-derived protein hydrolysates (PHs) are an important category of biostimulants able to increase plant growth and crop yield especially under environmental stress conditions. PHs can be applied as foliar spray or soil drench. Foliar spray is generally applied to achieve a relatively short-term response, whereas soil drench is used when a long-term effect is desired. The aim of the study was to elucidate the biostimulant action of PH application method (foliar spray or substrate drench) on morpho-physiological traits and metabolic profile of tomato grown under limited water availability. An untreated control was also included. A high-throughput image-based phenotyping (HTP) approach was used to non-destructively monitor the crop response under limited water availability (40% of container capacity) in a controlled environment. Moreover, metabolic profile of leaves was determined at the end of the trial. Dry biomass of shoots at the end of the trial was significantly correlated with number of green pixels (R2 = 0.90) and projected shoot area, respectively. Both drench and foliar treatments had a positive impact on the digital biomass compared to control while the photosynthetic performance of the plants was slightly influenced by treatments. Overall drench application under limited water availability more positively influenced biomass accumulation and metabolic profile than foliar application. Significantly higher transpiration use efficiency was observed with PH-drench applications indicating better stomatal conductance. The mass-spectrometry based metabolomic analysis allowed the identification of distinct biochemical signatures in PH-treated plants. Metabolomic changes involved a wide and organized range of biochemical processes that included, among others, phytohormones (notably a decrease in cytokinins and an accumulation of salicylates) and lipids (including membrane lipids, sterols, and terpenes). From a general perspective, treated tomato plants exhibited an improved tolerance to reactive oxygen species (ROS)-mediated oxidative imbalance. Such capability to cope with oxidative stress might have resulted from a coordinated action of signaling compounds (salicylic acid and hydroxycinnamic amides), radical scavengers such as carotenoids and prenyl quinones, as well as a reduced biosynthesis of tetrapyrrole coproporphyrins

Estudio metabolómico y genómico de cultivos celulares de zanahoria

El objetivo principal de este trabajo de investigación fue caracterizar los metabolitos secundarios producidos en dos líneas celulares de zanahoria. Para ello, uno de los objetivos concretos fue la caracterización de la producción de compuestos de naturaleza isoprenoide y compuestos fenólicos en suspensiones celulares de una línea celular verde de Dacus carota en condiciones control y en condiciones de elicitación con ciclodextrinas, jasmonato de metilo, hexenol y β-glucano separadamente o en combinación. De la misma manera, se caracterizó la producción de isoprenoides y compuestos fenólicos en suspensiones celulares de una línea celular naranja de D. carota en condiciones control y en condiciones de elicitación con ciclodextrinas y jasmonato de metilo. Asimismo, se estudio la producción de fitosteroles y carotenoides y la expresión de genes implicados en su ruta biosintética en suspensiones celulares de una línea celular naranja de D. carota en presencia de inhibidores de la ruta de biosíntesis de carotenoides y fitosteroles. Los resultados derivados de este estudio muestran que la línea celular de zanahoria verde biosintetizó de manera constitutiva los siguientes isoprenoides: carotenoides, α-tocoferol, clorofilas a y b y fitosteroles. El principal isoprenoide encontrado en la línea verde fue la luteína, una xantofila amarilla. La caracterización de la línea celular de zanahoria verde mostró que los fitoesteroles y compuestos fenólicos se acumularon principalmente en el medio extracelular (15100 μg/L y 477,5 μg/L, respectivamente) en presencia de ciclodextrinas. A diferencia de los compuestos mencionados anteriormente, el β-caroteno (1138,1 μg/L), la luteína (25949,5 μg/L), el α-tocoferol (8063,8 μg/L) y la clorofila a (1625,1 μg/L) y b (9958,3 μg/L) se acumularon principalmente dentro de las células. Por lo tanto, los ciclodextrinas fueron capaces de inducir la ruta biosintética del mevalonato, aumentando la biosíntesis de fitoesteroles y compuestos fenólicos, y acumulándolos fuera de las células. Sin embargo, en ausencia de ciclodextrinas, las células de la zanahoria acumularon principalmente carotenoides a través de la ruta biosintética del 4-fosfato de metileritritol. Por lo tanto, el uso de ciclodextrinas permitiría la acumulación extracelular de fitoesteroles y compuestos fenólicos incrementando el flujo de carbono hacia la ruta citosólica del mevalonato y hacia la ruta de biosíntesis fenilpropanoide. Además, β-glucano fue capaz de inducir la acumulación intracelular de α-tocoferol en la línea de células de zanahoria verde. Por otro lado, la línea celular de zanahoria naranja biosintetizó de manera constitutiva carotenoides y fitoesteroles. Los principales isoprenoides acumulados en esta línea celular fueron β-caroteno y luteína. El análisis de esta línea naranja en condiciones de elicitación (50 mM ciclodextrinas) mostró que los fitoesteroles se acumulaban principalmente en el medio extracelular. Sin embargo, el β-caroteno y la luteína se acumularon esencialmente dentro de las células. El tratamiento combinado de ciclodextrinas y jasmonato de metilo mejoró la acumulación extracelular de compuestos fenólicos. Por lo tanto, la línea celular de zanahoria naranja bajo condición de elicitación proporciona un sistema biotecnológico eficiente para producir compuestos bioactivos. La adición de Terbinafina y Diflufenican que inhiben las rutas biosintéticas de fitoesteroles y carotenoides respectivamente, provocó una alta acumulación de escualeno y fitoeno. Estos compuestos bioactivos no suelen acumularse y, debido a esto, la línea celular de zanahoria naranja tratada con estos inhibidores es una fuente alternativa para producirlos. Los niveles de expresión más altos del gen escualeno sintasa se encontraron después de 24h de tratamiento con el inhibidor terbinafina. Esto puede explicar la alta acumulación intracelular de escualeno sin efecto negativo sobre los niveles de fitosteroles. Además, las ciclodextrinas no aumentaron los niveles de expresión de escualeno sintasa. Este hecho puso de manifiesto que los ciclodextrinas no funcionan como moléculas inductoras en la biosíntesis de los fitoesteroles, sino que son capaces de extraerlos de las membranas celulares. Por último, los niveles de expresión del gen fitoeno desaturasa 1 sugirieron que la acumulación del contenido total de carotenoides podría estar vinculada a la expresión de este gen. Sin embargo, el tratamiento con diflufenican disminuyó los niveles de expresión de fitoeno sintasa 1 y fitoeno desaturasa, lo que sugiere que la alta acumulación de fitoeno no se correlacionó con un alto nivel de expresión de estos genes. The main objective was to characterize secondary metabolites produced in two carrot cell lines. Thus, one of the specific objectives was to characterize the production of isoprenoid and phenolic compounds in cell suspensions of a Daucus carota green cell line under control conditions and under elicitation conditions with cyclodextrins, methyl jasmonate, Hexenol and β-glucan separately or in combination. Moreover, the production of isoprenoids and phenolic compounds was characterized in cell suspensions of orange D. carota cell line under control conditions and under conditions of elicitation with cyclodextrins and methyl jasmonate. It was also studied the production of phytosterols and carotenoids and the expression of genes involved in their biosynthetic pathway in cell suspensions of orange D. carota cell line in the presence of inhibitors of the biosynthesis pathway of carotenoids and phytosterols. The results derived from this study refer to the green carrot cell line constitutively biosynthesized the following isoprenoids: carotenoids, α-tocopherol, chlorophyll a and b, and phytosterols. The main isoprenoid found in this green carrot line was lutein, a yellow xanthophyll. The characterization of the green carrot cell line showed that phytosterols and phenolic compounds wew mainly accumulated in the extracellular medium (15100 μg/L and 477.5 μg/L, respectively) in the presence of cyclodextrins. Unlike the aforementioned compounds, β-carotene (1138.1 μg/L), lutein (25949.5 μg/L), α-tocopherol (8063.8 μg/L) and chlorophyll 1625.1 μg/L) and b (9958.3 μg / L) were mainly accumulated within the cells. Therefore, cyclodextrins were able to induce the biosynthetic pathway of mevalonate, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulating them outside the cells. However, in the absence of cyclodextrins, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate biosynthetic pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of phytosterols and phenolic compounds by increasing the flow of carbon towards the mevalonate cytosolic pathway and towards the phenylpropanoid pathway. In addition, β-glucan was able to induce the intracellular accumulation of α-tocopherol in the green carrot cell line. On the other hand, the orange carrot cell line constitutively biosynthesized carotenoids and phytosterols. The major isoprenoids accumulated in this cell line were β-carotene and lutein. Analysis of this orange line under elicitation conditions (50 mM cyclodextrins) showed that phytosterols accumulated mainly in the extracellular medium. However, β-carotene and lutein were essentially accumulated within the cells. Combined treatment of cyclodextrins and methyl jasmonate improved the extracellular accumulation of phenolic compounds. Therefore, the orange carrot cell line under elicitation condition provides an efficient biotechnological system to produce bioactive compounds. The addition of Terbinafine and diflufenican, which inhibited the biosynthetic pathways of phytosterols and carotenoids respectively, caused a high accumulation of squalene and phytoene. These bioactive compounds are not usually accumulated and, because of this, the orange carrot cell line treated with these inhibitors is an alternative source to produce them. The highest expression levels of the square gene were found after 24h of treatment with the inhibitor terbinafine. This fact may explain the high intracellular accumulation of squalene with no negative effect on phytosterol levels. In addition, the cyclodextrins did not increase the expression levels of squalene synthase. This fact revealed that cyclodextrins do not function as induction molecules in the biosynthesis of phytosterols, but are capable of extracting them from cell membranes. Finally, gene expression levels of squalene synthase suggest that the accumulation of total carotene content could be linked to the expression of this gene. However, treatment with diflufenican decreased expression levels of phytoene synthase 1 and phytoene desaturase, suggesting that high phytoene accumulation did not correlate with a high level of expression of these genes

The Mycorrhiza-and Trichoderma-Mediated Elicitation of Secondary Metabolism and Modulation of Phytohormone Profile in Tomato Plants

Arbuscular mycorrhiza and Trichoderma are well-known beneficial fungi whose plant growth promotion and defense elicitation effects are known. However, the molecular and biochemical processes underlying the beneficial effects of these priming microorganisms have not been fully elucidated yet. On this basis, the present work aimed to use metabolomics to dissect comprehensively the modulation of secondary metabolism induced by mycorrhiza and Trichoderma, using tomato as a model plant. To this aim, either mycorrhiza or Trichoderma were applied to tomato roots at transplanting using a commercial formulation and then harvested once the mutualistic relationship was well established. Shoots were analyzed using an MS-based untargeted metabolomics approach, and differential metabolites identified by multivariate statistics were subjected to pathway analysis. Together with promoting plant growth, the treatments induced a broad molecular reprogramming with the phenylpropanoid biosynthetic pathway (including defense phenolics like coumarins and glycosylated anthocyanins) being strongly elicited. An accumulation of auxins, cytokinins, and jasmonate (especially after treatment with Trichoderma) could be observed concerning phytohormone profiles. Overall, the broad and distinctive effects triggered by mycorrhiza and Trichoderma in tomato secondary metabolism supported both plant growth promotion and immunity

Accumulation and tolerance of cadmium in a non- metallicolous ecotype of Silene vulgaris Garcke (Moench)

Acumulación y tolerancia a cadmio en un ecotipo no metalífero de Silene vulgaris Garcke (Moench) En este estudio, se analizó el efecto de diferentes concentraciones de Cd 2+ sobre un ecotipo de Silene vulgaris Garcke (Moench). La concentración de 60 μM de Cd 2+ provocó una ligera inhibición del crecimiento de las plantas mientras que la concentración más alta (120 μM) redujo drásticamente la biomasa y la elongación de la raíz y los brotes. Además, se detectaron altos niveles de Cd 2+ en las plantas, un coeficiente de bioacumulación elevado en las raí-ces y un bajo factor de translocación indicando que el ecotipo de S. vulgaris empleado en este estudio presenta una alta capacidad de acumulación de Cd 2+ en las raíces y sería un buen candidato para la fitoestabilización, lo que contribuiría a reducir los niveles de Cd 2+ en el suelo. Además, los resultados obtenidos indican que se debe tener precaución con el origen de esta planta, ya que podría representar una fuente adicional de Cd 2+ en la dieta humana. Palabras clave: Metal pesado, Fitoestabilización, Caryophyllaceae. Abstract In this study, a pot experiment was developed using a non-metallicolous ecotype of Silene vulgaris Garcke (Moench) exposed to 0, 60 and 120 µM Cd 2+ for 13 days. The dose of 60 µM Cd 2+ had little effect on the growth of S. vulgaris plants, whereas the highest dose produced a drastic reduction in biomass, and root and shoot elongation. The high internal Cd 2+ concentration together with the high bioaccumulation coefficient in roots and the low translocation factor indicated that this ecotype could be a good candidate for the phytostabilisation of Cd 2+ -contaminated soils. In view of the widespread use of this plant in popular medicine and the cuisine of Mediterranean countries, the results obtained also suggest that caution needs to be taken concerning its origin since it could represent an additional source of Cd 2+ in the human diet

The Complex Metabolomics Crosstalk Triggered by Four Molecular Elicitors in Tomato

The elicitation of plant secondary metabolism may offer interesting opportunities in the framework of sustainable approaches in plant science and in terms of their ability to prime resistance to biotic and abiotic stressors. The broad metabolic reprogramming triggered by different molecular elicitors, namely salicylate (SA), polyamines (PAs), and chitosan, was comprehensively investigated using a metabolomics approach and the tomato (Solanum lycopersicum L.) as the model crop. Six different treatments were compared: a negative control (no treatments), a second negative control treated with 1 M acetic acid (the reference for chitosan, since chitosan was solubilized in acetic acid), and four molecular elicitors, 1 mM 2,1,3-benzothiadiazole (BTH, a positive control), 10 mg/mL chitosan, 0.01 mM SA, and a 0.1 mM PA (putrescine, spermidine, and spermine). All treatments determined a slight increase in biomass, in particular following PA treatment. A broad reprogramming of secondary metabolism could be observed, including membrane lipid remodeling, phenylpropanoid antioxidants, and phytohormone crosstalk. Overall, our results suggest that PAs, SA, and BTH shared a systemic acquired resistance (SAR)-related response, whereas chitosan induced a more distinct induced systemic resistance (ISR)-like jasmonate-related response. These results pave the way towards the possible use of elicitors as a sustainable tool in plant science and agriculture by increasing crop resilience to biotic and abiotic stressors without detrimental effects on plant biomass

Acumulación y tolerancia a cadmio en un ecotipo no metalífero de Silene vulgaris Garcke (Moench)

En este estudio, se analizó el efecto de diferentes concentraciones de Cd2+ sobre un ecotipo de Silene vulgaris Garcke (Moench). La concentración de 60 μM de Cd2+ provocó una ligera inhibición del crecimiento de las plantas mientras que la concentración más alta (120 μM) redujo drásticamente la biomasa y la elongación de la raíz y los brotes. Además, se detectaron altos niveles de Cd2+ en las plantas, un coeficiente de bioacumulación elevado en las raices y un bajo factor de translocación indicando que el ecotipo de S. vulgaris empleado en este estudio presenta una alta capacidad de acumulación de Cd2+ en las raíces y sería un buen candidato para la fitoestabilización, lo que contribuiría a reducir los niveles de Cd2+ en el suelo. Además, los resultados obtenidos indican que se debe tener precaución con el origen de esta planta, ya que podría representar una fuente adicional de Cd2+ en la dieta humana.ABSTRACT: In this study, a pot experiment was developed using a non-metallicolous ecotype of Silene vulgaris Garcke (Moench) exposed to 0, 60 and 120 µM Cd2+ for 13 days. The dose of 60 µM Cd2+ had little effect on the growth of S. vulgaris plants, whereas the highest dose produced a drastic reduction in biomass, and root and shoot elongation. The high internal Cd2+ concentration together with the high bioaccumulation coefficient in roots and the low translocation factor indicated that this ecotype could be a good candidate for the phytostabilisation of Cd2+-contaminated soils. In view of the widespread use of this plant in popular medicine and the cuisine of Mediterranean countries, the results obtained also suggest that caution needs to be taken concerning its origin since it could represent an additional source of Cd2+ in the human diet