Interplay between secondary metabolites and plant hormones in silver nitrate-elicited Arabidopsis thaliana plants.

Plants produce a myriad of specialized compounds in response to threats such as pathogens or pests and different abiotic factors. The stress-related induction of specialized metabolites can be mimicked using silver nitrate (AgNO3) as an elicitor, which application in conservation agriculture has gained interest. In Arabidopsis thaliana, AgNO3 triggers the accumulation of indole glucosinolates (IGs) and the phytoalexin camalexin as well as pheylpropanoid-derived defensive metabolites such as coumaroylagmatins and scopoletin through a yet unknown mechanism. In this work, the role of jasmonic (JA) and salicylic acid (SA) signaling in the AgNO3-triggered specialized metabolite production was investigated. To attain this objective, AgNO3, MeJA and SA were applied to A. thaliana lines impaired in JA or SA signaling, or affected in the endogenous levels of IGs and AGs. Metabolomics data indicated that AgNO3 elicitation required an intact JA and SA signaling to elicit the metabolic response, although mutants impaired in hormone signaling retained certain capacity to induce specialized metabolites. In turn, plants overproducing or abolishing IGs production had also an altered hormonal signaling response, both in the accumulation of signaling molecules and the molecular response mechanisms (ORA59, PDF1.2, VSP2 and PR1 gene expression), which pointed out to a crosstalk between defense hormones and specialized metabolites. The present work provides evidence of a crosstalk mechanism between JA and SA underlying AgNO3 defense metabolite elicitation in A. thaliana. In this mechanism, IGs would act as retrograde feedback signals dampening the hormonal response; hence, expanding the signaling molecule concept.

Spermine deficiency shifts the balance between jasmonic acid and salicylic acid-mediated defence responses in Arabidopsis.

Polyamines are small aliphatic polycations present in all living organisms. In plants, the most abundant polyamines are putrescine (Put), spermidine (Spd) and spermine (Spm). Polyamine levels change in response to different pathogens, including Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). However, the regulation of polyamine metabolism and their specific contributions to defence are not fully understood. Here we report that stimulation of Put biosynthesis by Pst DC3000 is dependent on coronatine (COR) perception and jasmonic acid (JA) signalling, independently of salicylic acid (SA). Conversely, lack of Spm in spermine synthase (spms) mutant stimulated galactolipids and JA biosynthesis, and JA signalling under basal conditions and during Pst DC3000 infection, whereas compromised SA-pathway activation and defence outputs through SA-JA antagonism. The dampening of SA responses correlated with COR and Pst DC3000-inducible deregulation of ANAC019 expression and its key SA-metabolism gene targets. Spm deficiency also led to enhanced disease resistance to the necrotrophic fungal pathogen Botrytis cinerea and stimulated endoplasmic reticulum (ER) stress signalling in response to Pst DC3000. Overall, our findings provide evidence for the integration of polyamine metabolism in JA- and SA-mediated defence responses, as well as the participation of Spm in buffering ER stress during defence.

Morphological, physiological, and molecular scion traits are determinant for salt-stress tolerance of grafted citrus plants.

Introduction: Citrus productivity has been decreasing in the last decade in the Mediterranean basin as a consequence of climate change and the high levels of salinity found in the aquifers. Citrus varieties are cultivated grafted onto a rootstock, which has been reported as responsible for plant tolerance to adverse situations. However, other important factors for stress tolerance relying in the scion have been less studied. The aim of this study was to evaluate the effect of the grafted scion on citrus tolerance to salt stress. Methods: Four different citrus rootstock/scion combinations were subjected to salt stress for 30 days, using Carrizo citrange (CC) or Citrus macrophylla (CM) as rootstocks, and Navelina orange (NA) or Oronules mandarin (OR) as scions. CM-OR was the most tolerant combination, whereas CC-NA was the most sensitive one. Results and discussion: Our results support the idea that the rootstock plays an important role in salt stress tolerance, but scion is also crucial. Thus, photosynthesis and transpiration, processes regulated by abscisic acid and jasmonic acid, are determinant of plant performance. These photosynthetic parameters were not affected in plants of the salt-tolerant combination CM-OR, probably due to the lower intoxication with Cl- ions, allowing a better performance of the photosynthetic machinery under stress conditions. The different stomatal density of the two citrus scions used in this work (higher in the sensitive NA in comparison to the tolerant OR) also contributes to the different tolerance of the grafted plants to this adverse condition. Additionally, CsDTX35.1 and CsDTX35.2, genes codifying for Cl- tonoplast transporters, were exclusively overexpressed in plants of the salt-tolerant combination CM-OR, suggesting that these transporters involved in Cl- compartmentalization could be crucial for salt stress tolerance. It is concluded that to improve citrus tolerance to high salinity, it is important that scions have a versatile photosynthetic system, an adequate stomatal density, and a proper modulation of genes coding for Cl- transporters in the tonoplast.

Efficient strategies for controlled release of nanoencapsulated phytohormones to improve plant stress tolerance.

Climate change due to different human activities is causing adverse environmental conditions and uncontrolled extreme weather events. These harsh conditions are directly affecting the crop areas, and consequently, their yield (both in quantity and quality) is often impaired. It is essential to seek new advanced technologies to allow plants to tolerate environmental stresses and maintain their normal growth and development. Treatments performed with exogenous phytohormones stand out because they mitigate the negative effects of stress and promote the growth rate of plants. However, the technical limitations in field application, the putative side effects, and the difficulty in determining the correct dose, limit their widespread use. Nanoencapsulated systems have attracted attention because they allow a controlled delivery of active compounds and for their protection with eco-friendly shell biomaterials. Encapsulation is in continuous evolution due to the development and improvement of new techniques economically affordable and environmentally friendly, as well as new biomaterials with high affinity to carry and coat bioactive compounds. Despite their potential as an efficient alternative to phytohormone treatments, encapsulation systems remain relatively unexplored to date. This review aims to emphasize the potential of phytohormone treatments as a means of enhancing plant stress tolerance, with a specific focus on the benefits that can be gained through the improved exogenous application of these treatments using encapsulation techniques. Moreover, the main encapsulation techniques, shell materials and recent work on plants treated with encapsulated phytohormones have been compiled.

Appraisal of Abelmoschus esculentus L. Response to Aluminum and Barium Stress.

Trace metal element (TME) pollution is a major threat to plants, animals and humans. Agricultural products contaminated with metals may pose health risks for people; therefore, international standards have been established by the FAO/WHO to ensure food safety as well as the possibility of crop production in contaminated soils. This study aimed to assess the accumulating potential of aluminum and barium in the roots, shoots and fruits of Abelmoschus esculentus L., and their effect on growth and mineral nutrition. The content of proline and some secondary metabolites was also evaluated. After treating okra plants with aluminum/barium (0, 100, 200, 400 and 600 µM) for 45 days, the results showed that Al stimulated the dry biomass production, whereas Ba negatively affected the growth and the fructification yield. The okra plants retained both elements and exhibited a preferential accumulation in the roots following the sequence: roots > shoots > fruits, which is interesting for phytostabilization purposes. Al or Ba exposure induced a decline in mineral uptake (K, Ca, Mg, Zn and Fe), especially in roots and shoots. In order to cope with the stress conditions, the okra plants enhanced their proline and total phenol amounts, offering better adaptability to stress.

Encapsulation Reduces the Deleterious Effects of Salicylic Acid Treatments on Root Growth and Gravitropic Response.

The role of salicylic acid (SA) on plant responses to biotic and abiotic stresses is well documented. However, the mechanism by which exogenous SA protects plants and its interactions with other phytohormones remains elusive. SA effect, both free and encapsulated (using silica and chitosan capsules), on Arabidopsis thaliana development was studied. The effect of SA on roots and rosettes was analysed, determining plant morphological characteristics and hormone endogenous levels. Free SA treatment affected length, growth rate, gravitropic response of roots and rosette size in a dose-dependent manner. This damage was due to the increase of root endogenous SA concentration that led to a reduction in auxin levels. The encapsulation process reduced the deleterious effects of free SA on root and rosette growth and in the gravitropic response. Encapsulation allowed for a controlled release of the SA, reducing the amount of hormone available and the uptake by the plant, mitigating the deleterious effects of the free SA treatment. Although both capsules are suitable as SA carrier matrices, slightly better results were found with chitosan. Encapsulation appears as an attractive technology to deliver phytohormones when crops are cultivated under adverse conditions. Moreover, it can be a good tool to perform basic experiments on phytohormone interactions.

Climate change-associated multifactorial stress combination: A present challenge for our ecosystems.

Humans negatively influence Earth ecosystems and biodiversity causing global warming, climate change as well as man-made pollution. Recently, the number of different stress factors have increased, and when impacting simultaneously, the multiple stress conditions cause dramatic declines in plant and ecosystem health. Although much is known about how plants and ecosystems are affected by each individual stress, recent research efforts have diverted into how these biological systems respond to several of these stress conditions applied together. Studies of such "multifactorial stress combination" concept have reported a severe decrease in plant survival and microbiome biodiversity along the increasing number of factors in a consistent directional trend. In addition, these results are in concert with studies about how ecosystems and microbiota are affected by natural conditions imposed by climate change. Therefore, all this evidence should serve as an important warning in order to decrease pollutants, create strategies to deal with global warming, and increase the tolerance of plants to multiple stressful factors in combination. Here we review recent studies focused on the impact of abiotic stresses on plants, agrosystems and different ecosystems including forests and microecosystems. In addition, different strategies to mitigate the impact of climate change in ecosystems are discussed.

Improvement of salicylic acid biological effect through its encapsulation with silica or chitosan.

Attacks of necrotrophic and biotrophic fungi affect a large number of crops worldwide and are difficult to control with fungicides due to their genetic plasticity. Encapsulation technology is a good alternative for controlling fungal diseases. In this work, encapsulated samples of salicylic acid (SA) with silica (Si:SA) or chitosan (Ch:SA) at three different ratios were prepared by spray drying, and morphological and physicochemical characterised. Therefore, size distribution, specific surface area, thermal stability, encapsulation efficiency, and in-vitro SA release were determined. Biological activity of encapsulated samples were tested against different fungi of agricultural interest at various concentrations (0-1000 µM). Treatments prepared with the lowest ratios for both capsules, were found to have the best antifungal effect in an in vitro system, inhibiting the mycelial growth of Alternaria alternata, Botrytis cinerea, Fusarium oxysporum and Geotrichum candidum. Similarly, treatments with the lowest ratios of both encapsulated samples reduced free SA toxicity on Arabidopsis thaliana seeds. In this system, plants treated with capsules had higher root and rosette development than those treated with free SA. In conclusion, a product with a great potential in agriculture that shows high antifungal capacity and low toxicity for plants have been developed through a controlled and industrially viable process.

Assessing of growth, antioxidant enzymes, and phytohormone regulation in Cucurbita pepo under cadmium stress.

One of the major problems worldwide is soil pollution by trace metal elements, which limits plant productivity and threatens human health. In this work, we have studied the effect of different concentrations of cadmium on Cucurbita pepo plants, evaluating different physiological and biochemical parameters: hormone signaling, metabolite concentration (malondialdehyde and hydrogen peroxide) and, in addition, the antioxidant enzyme activities of catalase and superoxide dismutase were evaluated. The production of biomass decreased under the Cd-stress. The results showed that C. pepo accumulates higher amounts of Cd2+ in roots than in shoots and fruits. Cd2+ differently affected the content of endogenous phytohormones. Furthermore, data suggest an essential involvement of roots in the regulation of tolerance to trace elements. As a result, indole acetic acid content increased in roots of treated plants, indicating that this phytohormone can stimulate root promotion and growth under Cd-stress. Similarly, salicylic acid content in roots and shoots increased in response to Cd2+, as well as abscisic acid levels in roots and fruits. In roots, the rambling accumulation pattern observed for jasmonic acid and salicylic acid suggests the lack of a specific regulation role against trace element toxicity. The activity of catalase and superoxide dismutase decreased, disrupted by the metal stress. However, the proline, malondialdehyde and hydrogen peroxide content significantly increased in Cd2+in all the analyzed tissues of the stressed plants. All these data suggest that C. pepo plants are equipped with an effective antioxidant mechanism against oxidative stress induced by cadmium up to a concentration of 500 µM.

Particle film technology modulates xanthophyll cycle and photochemical dynamics of grapevines grown in the Douro Valley.

Field-grown grapevines are often exposed to multiple environmental stresses, which challenges wine-growers to develop sustainable measures to sustain vine growth, yield, and quality. Under field conditions this task is demanding, due to differences in the magnitudes of stresses and associated plant responses. In this study we explored the hypothesis that kaolin-particle film application improves grapevine photoprotection through the regulation of xanthophyll cycle genes, limiting the thermal dissipation of excess energy under harsh environmental conditions. Hence, we selected two grapevine varieties, Touriga-Nacional (TN) and Touriga-Franca (TF), grown in the Douro Demarcated Region, and evaluated changes in light dissipation mechanisms, xanthophyll cycle components, and the expression of xanthophyll cycle genes during the 2017 summer season. The results showed that, from veraison to ripening, kaolin triggered the up-regulation of violaxanthin de-epoxidase (VvVDE1) and zeaxanthin epoxidase (VvZEP1) genes, indicating optimised regulation of the xanthophyll cycle. Kaolin treatment also decreased chlorophyll (Chla, Chlb, Chl(a+b)) and carotenoid (Car) accumulation under increasing summer stress conditions in both varieties and lowered the non-photochemical quenching (NPQ) of grapevines on ripening, suggesting a long-term response to summer stress. In addition, kaolin-treated grapevines showed increased Chla/Chlb and lower Chl(a+b)/Car ratios, displaying some features of high light adapted leaves. Overall, this study suggests that kaolin application enabled grapevines to benefit from fluctuating periods of summer stress by managing chlorophyll and carotenoid content and limiting down-regulation of both photochemistry and photoinhibition processes. Under Mediterranean field conditions, kaolin application can be considered an efficient method of minimising summer stress impact on grapevines.

Root exudates: from plant to rhizosphere and beyond.

KEY MESSAGE: This article describes the composition of root exudates, how these metabolites are released to the rhizosphere and their importance in the recruitment of beneficial microbiota that alleviate plant stress. Metabolites secreted to the rhizosphere by roots are involved in several processes. By modulating the composition of the root exudates, plants can modify soil properties to adapt and ensure their survival under adverse conditions. They use several strategies such as (1) changing soil pH to solubilize nutrients into assimilable forms, (2) chelating toxic compounds, (3) attracting beneficial microbiota, or (4) releasing toxic substances for pathogens, etc. In this work, the composition of root exudates as well as the different mechanisms of root exudation have been reviewed. Existing methodologies to collect root exudates, indicating their advantages and disadvantages, are also described. Factors affecting root exudation have been exposed, including physical, chemical, and biological agents which can produce qualitative and quantitative changes in exudate composition. Finally, since root exudates play an important role in the recruitment of mycorrhizal fungi and plant growth-promoting rhizobacteria (PGPR), the mechanisms of interaction between plants and the beneficial microbiota have been highlighted.

Hormonal and metabolic responses of Mexican lime plants to CTV infection.

Plant viral infections alter gene expression and metabolism in infected host. To study the molecular responses of Mexican lime to CTV infection, an analysis of plant metabolome in response to infection with severe (T318) or mild (T385) isolates of CTV was performed. Healthy plants and those infected with any of the two virus strains showed different metabolite profiles, at different stages of new sprout development. Proline content increased in plants infected with CTV, proportionally to the virulence of the virus strain. Abscisic acid content decreased after virus infection whereas jasmonic and salicylic acid levels increased. CTV infection had an impact on plant secondary metabolism, by stimulating the synthesis of different metabolites such as l-methylhistidine, phenylpropanoid derivatives. These metabolites are common responses of different organisms, including higher mammals, to viral diseases, and its presence in this system points to the existence of universal responses to virus infection among different kingdoms.

Root exudates from citrus plants subjected to abiotic stress conditions have a positive effect on rhizobacteria.

Plants are constantly releasing root exudates to the rhizosphere. These compounds are responsible for different (positive or negative) interactions with other organisms, including plants, fungi or bacteria. In this work, the effect of root exudates obtained from in vitro cultured citrus plants on two rhizobacteria (Pseudomonas putida KT2440 and Novosphingobium sp. HR1a) was evaluated. Root exudates were obtained from two citrus genotypes differing in their sensitivity to salt and heat stress and differentially affected the growth of both rhizobacteria. Root exudates from salt-stressed plants of C. macrophylla (salt tolerant) induced an increase in bacterial growth higher than that obtained from Carrizo citrange exudates (salt sensitive). Root exudates from heat-stressed plants also had a positive effect on bacterial growth, which was more evident in the heat-sensitive C. macrophylla. These results reveal that the growth of these rhizobacteria can be modulated through citrus root exudates and can change depending on both the stress conditions as well as the genotype. Biosensors P. putida KT2442 (pMIS5) and Novosphingobium sp. HR1a (pPAH) were used to test the presence of proline and salicylates in root exudates by measuring ß-galactosidase activity. This activity increased in the presence of root exudates obtained from stressed plants to a higher extent in the case of exudates obtained from the genotype resistant to each particular stress, indicating that those root exudates contain larger quantities of proline and salicylates, as it has been described previously. Our data reveals that both P. putida KT2442 (pMIS5) and Novosphingobium sp. HR1a (pPAH), could be used as biosensors of plant stress.

Salt stress alleviation in citrus plants by plant growth-promoting rhizobacteria Pseudomonas putida and Novosphingobium sp.

KEY MESSAGE: This work reveals the protective role of two rhizobacteria, Pseudomonas putida and Novosphingobium sp., on citrus plants subjected to salt stress conditions. Detrimental salt stress effects on crops are likely to increase due to climate change reducing the quality of irrigation water. Plant growth-promoting rhizobacteria (PGPRs) can mitigate stress-induced damage in plants cultivated under high salinity conditions. In this work, Citrus macrophylla (alemow) plants inoculated with the rhizobacteria Pseudomonas putida KT2440 or Novosphingobium sp. HR1a were subjected to salt stress for 30 days. Results showed that in absence of salt stress, Novosphingobium sp. HR1a induced a decrease of transpiration (E) and stomatal conductance (gs). Both rhizobacteria reduced salt stress-induced damage. Levels of abscisic acid (ABA) and salicylic acid (SA) were lower in inoculated plants under salt stress conditions. Similarly, under stress conditions maximum efficiency of photosystem II (Fv/Fm) in inoculated plants decreased to a lower extent than in non-inoculated ones. In stressed plants, Novosphingobium sp. HR1a also induced leaf accumulation of 3-indole acetic acid (IAA) and a delay in the decrease of quantum yield (ΦPSII). P. putida KT2440 inhibited root chloride and proline accumulation in response to salt stress. Although both bacterial species had beneficial effects on salt-stressed citrus plants, Novosphingobium sp. HR1a induced a better plant performance. Therefore, both strains could be candidates to be used as PGPRs in programs of inoculation for citrus protection against salt stress.

Citrus plants exude proline and phytohormones under abiotic stress conditions.

KEY MESSAGE: This article describes the root exudation of proline and phytohormones in citrus and their involvement in salt- and heat-stress responses. Plants are constantly releasing several compounds to the rhizosphere through their roots, including primary and secondary metabolites. Root exudation can be affected by growth conditions, including pH, nutrient availability, soil salinity, or temperature. In vitro-cultured plants of two citrus genotypes with contrasting tolerance to salt- and heat-stress conditions were used as plant material. Proline and phytohormone contents in root exudates from plants subjected to salt or high-temperature conditions were evaluated. In addition, tissue damage and lipid peroxidation together with endogenous levels of chloride, proline, and phytohormones were determined in roots and shoots. Proline was released in larger quantities to the rhizosphere when plants were subjected to salt or heat stress. In each stress condition, the concentration of this amino acid was higher in the exudates obtained from plants tolerant to this particular stress condition. On the other hand, root exudation of phytohormones salicylic acid, indole acetic acid, abscisic acid, and jasmonic acid generally increased under both adverse conditions. Results confirm a phytohormone exudation in citrus plants, which had not been described previously and can have an important role in the rhizosphere communication. Moreover, stress conditions and the different tolerance of each genotype to the particular stress significantly modify the exudation pattern both quantitatively and qualitatively.

A fast and precise method to identify indolic glucosinolates and camalexin in plants by combining mass spectrometric and biological information.

In this manuscript, a fast and accurate identification and quantitation by mass spectrometry of indolic glucosinolates and camalexin involved in defense in Arabidopsis thaliana are described. Two elicitation systems, inoculation with Botrytis cinerea and treatment with AgNO(3), were used in Col-0 wild-type and mutant genotypes impaired in the biosynthesis of the selected metabolites. Identification of analytes was carried out by nontargeted LC/ESI-QTOF-MS profiling. Confirmation of indolic glucosinolates and camalexin was achieved by their absence in the cyp79B2/B3 and pad3 mutants as well as their respective fragmentation upon collision-induced dissociation. Camalexin accumulation was induced only after AgNO(3) treatment, whereas all indolic glucosinolates were constitutively present. Inoculation with Botrytis did not influence camalexin concentration but caused most aliphatic and indolic glucosinolates contents to decrease. Only the pen 3.1 mutant showed increased indolic glucosinolate levels after Botrytis or AgNO(3) treatments. In addition, profiles of secondary metabolite in nontreated Col-0 and mutant plants were analyzed by means of partial least squares coupled to discriminant analysis (PLS-DA), and differences in the basal levels of indolic glucosinolates and tryptophan between cyp79B2/B3 plants and the rest of genotypes, including Col-0, were found. This probably has to be taken into consideration when comparing stress responses of Col-0 and cyp79B2/B3. The use of mutants carrying alterations in biosynthetic pathways is proposed as a useful strategy to identify secondary metabolites.