Cytokinin oxidase/dehydrogenase inhibitors: progress towards agricultural practice.

Cytokinin oxidase/dehydrogenase (CKX) inhibitors reduce the degradation of cytokinins in plants and thereby may improve the efficiency of agriculture and plant tissue culture-based practices. Here, we report a synthesis and structure-activity relationship study of novel urea derivatives concerning their CKX inhibitory activity. The best compounds showed sub-nanomolar IC50 values with maize ZmCKX1, the lowest value yet documented. Other CKX isoforms of maize (Zea mays) and Arabidopsis were also inhibited very effectively. The binding mode of four compounds was characterized based on high-resolution crystal complex structures. Using the soil nematode Caenorhabditis elegans, and human skin fibroblasts, key CKX inhibitors with low toxicity were identified. These compounds enhanced the shoot regeneration of Lobelia, Drosera, and Plectranthus, as well as the growth of Arabidopsis and Brassica napus. At the same time, a key compound (namely 82), activated a cytokinin primary response gene ARR5:GUS and cytokinin sensor TCSv2:GUS, without activating the Arabidopsis cytokinin receptors AHK3 and AHK4. This strongly implies that the effect of compound 82 is due to the upregulation of cytokinin signalling. Overall, this work presents highly effective and easily prepared CKX inhibitors with a low risk of environmental toxicity for further investigation of their potential in agriculture and biotechnology.

Comprehensive LC-MS/MS Analysis of Nitrogen-Related Plant Metabolites.

We have developed and validated a novel LC-MS/MS method for simultaneously analyzing amino acids, biogenic amines, and their acetylated and methylated derivatives in plants. This method involves a one-step extraction of 2-5 mg of lyophilized plant material followed by fractionation of different biogenic amine forms and exploits an efficient combination of hydrophilic interaction chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry. This approach enables high-throughput processing of plant samples, significantly reducing the time needed for analysis and its cost. We also present a new synthetic route for deuterium-labelled polyamines. The LC-MS/MS method was rigorously validated by quantifying levels of nitrogen-related metabolites in seedlings of seven plant species including Arabidopsis, maize, and barley, all of which are commonly used model organisms in plant science research. Our results revealed substantial variations in the abundance of these metabolites between species, developmental stages, and growth conditions, particularly for the acetylated and methylated derivatives and the various polyamine fractions. However, the biological relevance of these plant metabolites is currently unclear. Overall, this work contributes significantly to the field of plant science by providing a powerful analytical tool and setting the stage for future investigations into the functions of these nitrogen-related metabolites in plants.

PGI1-mediated vascular oxidative pentose phosphate pathway modulates photosynthesis via long-distance cytokinin signaling.

In Arabidopsis, the plastidial isoform of phosphoglucose isomerase, PGI1, mediates growth and photosynthesis, likely due to its involvement in the vascular production of cytokinins (CK). To examine this hypothesis, we characterized pgi1-2 knockout plants impaired in PGI1 and pgi1-2 plants specifically expressing PGI1 in root tips and vascular tissues. Moreover, to investigate whether the phenotype of pgi1-2 plants is due to impairments in the plastidial oxidative pentose phosphate pathway (OPPP) or the glycolytic pathway, we characterized pgl3-1 plants with reduced OPPP and pfk4pfk5 knockout plants impaired in plastidial glycolysis. Compared with wild-type (WT) leaves, pgi1-2 leaves exhibited weaker expression of photosynthesis- and 2-C-methyl-D-erythritol 4-P (MEP) pathway-related proteins, and stronger expression of oxidative stress protection-related enzymes. Consistently, pgi1-2 leaves accumulated lower levels of chlorophyll, and higher levels of tocopherols, flavonols and anthocyanins than the WT. Vascular- and root tip-specific PGI1 expression countered the reduced photosynthesis, low MEP pathway-derived CK content, dwarf phenotype and the metabolic characteristics of pgi1-2 plants, reverting them to WT-like levels. Moreover, pgl3-1, but not pfk4pfk5 plants phenocopied pgi1-2. Histochemical analyses of plants expressing GUS under the control of promoter regions of genes encoding plastidial OPPP enzymes exhibited strong GUS activity in root tips and vascular tissues. Overall, our findings show that root tip and vascular PGI1-mediated plastidial OPPP activity affects photosynthesis and growth through mechanisms involving long-distance modulation of the leaf proteome by MEP pathway-derived CKs.

The crosstalk of far-red energy and signaling defines the regulation of photosynthesis, growth, and flowering in tomatoes.

This study investigated the effect of light intensity and signaling on the regulation of far-red (FR)-induced alteration in photosynthesis. The low (LL: 440 µmol m-2 s-1) and high (HL: 1135 µmol m-2 s-1) intensity of white light with or without FR (LLFR: 545 µmol m-2 s-1 including 115 µmol m-2 s-1; HLFR: 1254 µmol m-2 s-1 + 140 µmol m-2 s-1) was applied on the tomato cultivar (Solanum Lycopersicon cv. Moneymaker) and mutants of phytochrome A (phyA) and phytochrome B (phyB1, and phyB2). Both light intensity and FR affected plant morphological traits, leaf biomass, and flowering time. Irrespective of genotype, flowering was delayed by LLFR and accelerated by HLFR compared to the corresponding light intensity without FR. In LLFR, a reduced energy flux through the electron transfer chain along with a reduced energy dissipation per reaction center improved the maximum quantum yield of PSII, irrespective of genotype. HLFR increased net photosynthesis and gas exchange properties in a genotype-dependent manner. FR-dependent regulation of hormones was affected by light signaling. It appeared that PHYB affected the levels of abscisic acid and salicylic acid while PHYA took part in the regulation of CK in FR-exposed plants. Overall, light intensity and signaling of FR influenced plants' photosynthesis and growth by altering electron transport, gas exchange, and changes in the level of endogenous hormones.

Stomatal effects and ABA metabolism mediate differential regulation of leaf and flower cooling in tomato cultivars exposed to heat and drought stress.

Co-occurring heat and drought stresses challenge crop performance. Stomata open to promote evaporative cooling during heat stress, but close to retain water during drought stress, which resulted in complex stomatal regulation under combined heat and drought. We aimed to investigate stomatal regulation in leaves and flowers of perennial, indeterminate cultivars of tomatoes subjected to individual and combined heat and drought stress followed by a recovery period, measuring morphological, physiological, and biochemical factors involved in stomatal regulation. Under stress, stomata of leaves were predominantly affected by drought, with lower stomatal density and stomatal closing, resulting in significantly decreased photosynthesis and higher leaf temperature. Conversely, stomata in sepals seemed affected mainly by heat during stress. The differential patterns in stomatal regulation in leaves and flowers persisted into the recovery phase as contrasting patterns in stomatal density. We show that flower transpiration is regulated by temperature, but leaf transpiration is regulated by soil water availability during stress. Organ-specific patterns of stomatal development and abscisic acid metabolism mediated this phenomenon. Our results throw light on the dual role of stomata in heat and drought tolerance of vegetative and generative organs, and demonstrate the importance of considering flower surfaces in the phenotyping of stomatal reactions to stress.

Plant nucleoside N-ribohydrolases: riboside binding and role in nitrogen storage mobilization.

Cells save their energy during nitrogen starvation by selective autophagy of ribosomes and degradation of RNA to ribonucleotides and nucleosides. Nucleosides are hydrolyzed by nucleoside N-ribohydrolases (nucleosidases, NRHs). Subclass I of NRHs preferentially hydrolyzes the purine ribosides while subclass II is more active towards uridine and xanthosine. Here, we performed a crystallographic and kinetic study to shed light on nucleoside preferences among plant NRHs followed by in vivo metabolomic and phenotyping analyses to reveal the consequences of enhanced nucleoside breakdown. We report the crystal structure of Zea mays NRH2b (subclass II) and NRH3 (subclass I) in complexes with the substrate analog forodesine. Purine and pyrimidine catabolism are inseparable because nucleobase binding in the active site of ZmNRH is mediated via a water network and is thus unspecific. Dexamethasone-inducible ZmNRH overexpressor lines of Arabidopsis thaliana, as well as double nrh knockout lines of moss Physcomitrium patents, reveal a fine control of adenosine in contrast to other ribosides. ZmNRH overexpressor lines display an accelerated early vegetative phase including faster root and rosette growth upon nitrogen starvation or osmotic stress. Moreover, the lines enter the bolting and flowering phase much earlier. We observe changes in the pathways related to nitrogen-containing compounds such as ß-alanine and several polyamines, which allow plants to reprogram their metabolism to escape stress. Taken together, crop plant breeding targeting enhanced NRH-mediated nitrogen recycling could therefore be a strategy to enhance plant growth tolerance and productivity under adverse growth conditions.

Biochemical and structural basis of polyamine, lysine and ornithine acetylation catalyzed by spermine/spermidine N-acetyl transferase in moss and maize.

Polyamines such as spermidine and spermine are essential regulators of cell growth, differentiation, maintenance of ion balance and abiotic stress tolerance. Their levels are controlled by the spermidine/spermine N1 -acetyltransferase (SSAT) via acetylation to promote either their degradation or export outside the cell as shown in mammals. Plant genomes contain at least one gene coding for SSAT (also named NATA for N-AcetylTransferase Activity). Combining kinetics, HPLC-MS and crystallography, we show that three plant SSATs, one from the lower plant moss Physcomitrium patens and two from the higher plant Zea mays, acetylate various aliphatic polyamines and two amino acids lysine (Lys) and ornithine (Orn). Thus, plant SSATs exhibit a broad substrate specificity, unlike more specific human SSATs (hSSATs) as hSSAT1 targets polyamines, whereas hSSAT2 acetylates Lys and thiaLys. The crystal structures of two PpSSAT ternary complexes, one with Lys and CoA, the other with acetyl-CoA and polyethylene glycol (mimicking spermine), reveal a different binding mode for polyamine versus amino acid substrates accompanied by structural rearrangements of both the coenzyme and the enzyme. Two arginine residues, unique among plant SSATs, hold the carboxyl group of amino acid substrates. The most abundant acetylated compound accumulated in moss was N6 -acetyl-Lys, whereas N5 -acetyl-Orn, known to be toxic for aphids, was found in maize. Both plant species contain very low levels of acetylated polyamines. The present study provides a detailed biochemical and structural basis of plant SSAT enzymes that can acetylate a wide range of substrates and likely play various roles in planta.

Glucose-6-P/phosphate translocator2 mediates the phosphoglucose-isomerase1-independent response to microbial volatiles.

In Arabidopsis (Arabidopsis thaliana), the plastidial isoform of phosphoglucose isomerase (PGI1) mediates photosynthesis, metabolism, and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Microbial volatile compounds (VCs) with molecular masses of <45 Da promote photosynthesis, growth, and starch overaccumulation in leaves through PGI1-independent mechanisms. Exposure to these compounds in leaves enhances the levels of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (GPT2) transcripts. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized the responses of wild-type (WT), GPT2-null gpt2-1, PGI1-null pgi1-2, and pgi1-2gpt2-1 plants to small fungal VCs. In addition, we characterized the responses of pgi1-2gpt2-1 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. Fungal VCs promoted increases in growth, starch content, and photosynthesis in WT and gpt2-1 plants. These changes were substantially weaker in VC-exposed pgi1-2gpt2-1 plants but reverted to WT levels with vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves and showed that knocking out GPT2 reduced the expression of photosynthesis-related proteins in pgi1-2 plants. Histochemical analyses of GUS activity in plants expressing GPT2-GUS under the control of the GPT2 promoter showed that GPT2 is mainly expressed in root tips and vascular tissues around hydathodes. Overall, the data indicated that the PGI1-independent response to microbial VCs involves resetting of the photosynthesis-related proteome in leaves through long-distance GPT2 action.

Addressing the contribution of small molecule-based biostimulants to the biofortification of maize in a water restriction scenario.

Biostimulants have become an asset for agriculture since they are a greener alternative to traditionally used plant protection products. Also, they have gained the farmers' acceptance due to their effect on enhancing the plant's natural defense system against abiotic stresses. Besides commercially available complex products, small molecule-based biostimulants are useful for industry and research. Among them, polyamines (PAs) are well-studied natural compounds that can elicit numerous positive responses in drought-stressed plants. However, the studies are merely focused on the vegetative development of the plant. Therefore, we aimed to evaluate how drenching with putrescine (Put) and spermidine (Spd) modified the maize production and the yield quality parameters. First, a dosage optimization was performed, and then the best PA concentrations were applied by drenching the maize plants grown under well-watered (WW) conditions or water deficit (WD). Different mechanisms of action were observed for Put and Spd regarding maize production, including when both PAs similarly improved the water balance of the plants. The application of Put enhanced the quality and quantity of the yield under WW and Spd under WD. Regarding the nutritional quality of the grains, both PAs increased the carbohydrates content, whereas the contribution to the protein content changed by the interaction between compound and growth conditions. The mineral content of the grains was also greatly affected by the water condition and the PA application, with the most relevant results observed when Spd was applied, ending with flour richer in Zn, Cu, and Ca minerals that are considered important for human health. We showed that the exogenous PA application could be a highly efficient biofortification approach. Our findings open a new exciting use to be studied deep in the biostimulant research.

Biostimulants as an Alternative to Improve the Wine Quality from Vitis vinifera (cv. Tempranillo) in La Rioja.

The application of biostimulants appears to be an environmentally friendly, innovative, and sustainable agronomical tool to mitigate the negative effects induced by adverse climatology in traditional grape-growing regions such as La Rioja (Spain). However, their mechanism of action in grapevines is still unclear. We evaluated how commercial substances (two from Ascophyllum nodosum extraction and one amino acids-based biostimulant) and the non-proteinogenic amino acid ß-aminobutyric acid (BABA) affect the quality and quantity of musts and grapes in Vitis vinifera L. cv. Tempranillo from a semi-arid region of La Rioja during two seasons. We hypothesized an enhancement in organic metabolites in berries and leaves in response to these treatments, changing the organoleptic characteristics of the final products. The treatments altered the primary metabolites such as carbohydrates, organic acids (AcOrg), and free amino acids, first in the leaves as the effect of the foliar application and second in grapes and musts. As the main result, the biostimulant efficiency depended on the climatology and vineyard location to improve the final yield. Whereas biostimulant application enhanced the yield in 2018 (less dry year), it did not help production in 2019 (dry year). BABA was the most efficient biostimulant, enhancing plant production. Regarding yield quality, the biostimulant application improved the musts mainly by enhancing the fumaric acid content and by reducing carbohydrates, except in BABA-treated plants, where they were accumulated. These results corroborate biostimulants as an exciting approach in wine production, especially for improving wine quality.

Presence and future of plant phenotyping approaches in biostimulant research and development.

Commercial interest in biostimulants as a tool for sustainable green economics and agriculture concepts is on a steep rise, being followed by increasing demand to employ efficient scientific methods to develop new products and understand their mechanisms of action. Biostimulants represent a highly diverse group of agents derived from various natural sources. Regardless of their nutrition content and composition, they are classified by their ability to improve crop performance through enhanced nutrient use efficiency, abiotic stress tolerance, and quality of crops. Numerous reports have described modern, non-invasive sensor-based phenotyping methods in plant research. This review focuses on applying phenotyping approaches in biostimulant research and development, and maps the evolution of interaction of these two intensively growing domains. How phenotyping served to identify new biostimulants, the description of their biological activity, and the mechanism/mode of action are summarized. Special attention is dedicated to the indoor high-throughput methods using model plants suitable for biostimulant screening and developmental pipelines, and high-precision approaches used to determine biostimulant activity. The need for a complex method of testing biostimulants as multicomponent products through integrating other -omic approaches followed by advanced statistical/mathematical tools is emphasized.

Priming with Small Molecule-Based Biostimulants to Improve Abiotic Stress Tolerance in Arabidopsis thaliana.

Biostimulants became a hotspot in the fight to alleviate the consequences of abiotic stresses in crops. Due to their complex nature, it is challenging to obtain stable and reproducible final products and more challenging to define their mechanism of action. As an alternative, small molecule-based biostimulants, such as polyamines have promoted plant growth and improved stress tolerance. However, profound research about their mechanisms of action is still missing. To go further, we tested the effect of putrescine (Put) and its precursor ornithine (Orn) and degradation product 1,3-diaminopropane (DAP) at two different concentrations (0.1 and 1 mM) as a seed priming on in vitro Arabidopsis seedlings grown under optimal growth conditions, osmotic or salt stress. None of the primings affected the growth of the seedlings in optimal conditions but altered the metabolism of the plants. Under stress conditions, almost all primed plants grew better and improved their greenness. Only Orn-primed plants showed different plant responses. Interestingly, the metabolic analysis revealed the implication of the N- acetylornithine and Orn and polyamine conjugation as the leading player regulating growth and development under control and stress conditions. We corroborated polyamines as very powerful small molecule-based biostimulants to alleviate the adverse abiotic stress effects.

Elevated CO2 Improves the Physiology but Not the Final Yield in Spring Wheat Genotypes Subjected to Heat and Drought Stress During Anthesis.

Heat and drought events often occur concurrently as a consequence of climate change and have a severe impact on crop growth and yield. Besides, the accumulative increase in the atmospheric CO2 level is expected to be doubled by the end of this century. It is essential to understand the consequences of climate change combined with the CO2 levels on relevant crops such as wheat. This study evaluated the physiology and metabolite changes and grain yield in heat-sensitive (SF29) and heat-tolerant (LM20) wheat genotypes under individual heat stress or combined with drought applied during anthesis at ambient (aCO2) and elevated CO2 (eCO2) levels. Both genotypes enhanced similarly the WUE under combined stresses at eCO2. However, this increase was due to different stress responses, whereas eCO2 improved the tolerance in heat-sensitive SF29 by enhancing the gas exchange parameters, and the accumulation of compatible solutes included glucose, fructose, ß-alanine, and GABA to keep water balance; the heat-tolerant LM20 improved the accumulation of phosphate and sulfate and reduced the lysine metabolism and other metabolites including N-acetylornithine. These changes did not help the plants to improve the final yield under combined stresses at eCO2. Under non-stress conditions, eCO2 improved the yield of both genotypes. However, the response differed among genotypes, most probably as a consequence of the eCO2-induced changes in glucose and fructose at anthesis. Whereas the less-productive genotype LM20 reduced the glucose and fructose and increased the grain dimension as the effect of the eCO2 application, the most productive genotype SF29 increased the two carbohydrate contents and ended with higher weight in the spikes. Altogether, these findings showed that the eCO2 improves the tolerance to combined heat and drought stress but not the yield in spring wheat under stress conditions through different mechanisms. However, under non-stress conditions, it could improve mainly the yield to the less-productive genotypes. Altogether, the results demonstrated that more studies focused on the combination of abiotic stress are needed to understand better the spring wheat responses that help the identification of genotypes more resilient and productive under these conditions for future climate conditions.

Optimizing growing conditions for hydroponic farming of selected medicinal and aromatic plants.

This work presents a pipeline for optimizing semi-hydroponic growth conditions and analyzes the impact on the growth and metabolism in three Mentha species (M. arvensis, M. x piperita, or M. spicata) and three Ocimum basilicum genotypes (́Chládek cervená́, ́Litrá, or ́Mánes). The plant growth and the content- of nitrogen-containing compounds, phenolics, and terpenoids were determined under different nitrate concentrations and salt stress. Different responses were observed among genotypes for both species. ́Chládek cervená́ had the best growth under low nitrate, with lower histamine and higher flavonoid levels. Mentha x piperita was the best mint species performing under low nitrate and salt stress. Altogether, we demonstrate that a combination of phenomics and metabolomics is ideal to identify the optimal growth conditions for these plants and the chemical markers associated with these conditions. Besides, we showed that both primary and secondary metabolites can be good markers for classifying both species.

Enhanced Yield of Pepper Plants Promoted by Soil Application of Volatiles From Cell-Free Fungal Culture Filtrates Is Associated With Activation of the Beneficial Soil Microbiota.

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants' responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota.

Seed Priming With Protein Hydrolysates Improves Arabidopsis Growth and Stress Tolerance to Abiotic Stresses.

The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 µl ml-1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.

Phenolic Compounds and Biological Activity of Selected Mentha Species.

Mentha species are widely used as food, medicine, spices, and flavoring agents. Thus, chemical composition is an important parameter for assessing the quality of mints. In general, the contents of menthol, menthone, eucalyptol, and limonene comprise one of the major parameters for assessing the quality of commercially important mints. Building further on the phytochemical characterization of the quality of Mentha species, this work was focused on the composition of phenolic compounds in methanolic extracts. Thirteen Mentha species were grown under the same environmental conditions, and their methanolic extracts were subjected to the LC-MS/MS (liquid chromatography-tandem mass spectrometry) profiling of phenolics and the testing their biological activities, i.e., antioxidant and tyrosinase inhibition activities, which are important features for the cosmetic industry. The total phenolic content (TPC) ranged from 14.81 ± 1.09 mg GAE (gallic acid equivalents)/g for Mentha cervina to 58.93. ± 8.39 mg GAE/g for Mentha suaveolens. The antioxidant activity of examined Mentha related with the content of the phenolic compounds and ranged from 22.79 ± 1.85 to 106.04 ± 3.26 mg TE (Trolox equivalents)/g for M. cervina and Mentha x villosa, respectively. Additionally, Mentha pulegium (123.89 ± 5.64 mg KAE (kojic acid equivalents)/g) and Mentha x piperita (102.82 ± 15.16 mg KAE/g) showed a strong inhibition of the enzyme tyrosinase, which is related to skin hyperpigmentation. The most abundant compound in all samples was rosmarinic acid, ranging from 1363.38 ± 8323 to 2557.08 ± 64.21 µg/g. In general, the levels of phenolic acids in all examined mint extracts did not significantly differ. On the contrary, the levels of flavonoids varied within the species, especially in the case of hesperidin (from 0.73 ± 0.02 to 109. 39 ± 2.01 µg/g), luteolin (from 1.84 ± 0.11 to 31.03 ± 0.16 µg/g), and kaempferol (from 1.30 ± 0.17 to 33.68 ± 0.81 µg/g). Overall results indicated that all examined mints possess significant amounts of phenolic compounds that are responsible for antioxidant activity and, to some extent, for tyrosinase inhibition activity. Phenolics also proved to be adequate compounds, together with terpenoids, for the characterization of Mentha sp. Additionally, citrus-scented Mentha x villosa could be selected as a good candidate for the food and pharmaceutical industry, especially due its chemical composition and easy cultivation, even in winter continental conditions.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

Drought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem-mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem-mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Exogenous application of ZnO nanoparticles and ZnSO4 distinctly influence the metabolic response in Phaseolus vulgaris L.

Nanomaterials-mediated contamination (including the highly reactive metal oxides ZnO nanoparticles) is becoming one of the most concerning issues worldwide. In this study, the toxic effects of two chemical species of Zn (ZnO nanoparticles and bulk ZnSO4) were investigated in bean plants, following either foliar or soil application, at concentrations from 250 to 2000 mg L-1 using biochemical assays, proteomics and metabolomics. The accumulation of Zn in plant tissues depended on the application type, zinc chemical form and concentration, in turn triggering distinctive morphological, physiological, and redox responses. Bean plants were more sensitive to the foliar than to the soil application, and high concentrations of ZnO NP and bulk ZnSO4 determined the highest plant growth inhibition and stress symptoms. However, low dosages of ZnSO4 induced a slight plant growth promotion and better physiological and antioxidative response. Low concentration of Zn leaded to increased activity of stress-related proteins and secondary metabolites with antioxidant capacity, while increasing concentration reached the exhausted phase of the plant stress response, reducing the antioxidant defense system. Such high concentrations increased lipids peroxidation, protein degradation and membranes integrity. Oxidative damage occurred at high concentrations of both chemical species of Zn. Foliar spraying impaired photosynthetic efficiency, while soil applications (especially ZnSO4) elicited antioxidant metabolites and proteins, and impaired chloroplast-related proteins involved in the electron transport chain and ATP production. Taken together, the results highlighted distinctive and nanoparticles-related toxic effects of ZnO in bean, compared to ionic forms of Zn.

Proteostatic Regulation of MEP and Shikimate Pathways by Redox-Activated Photosynthesis Signaling in Plants Exposed to Small Fungal Volatiles.

Microorganisms produce volatile compounds (VCs) with molecular masses of less than 300 Da that promote plant growth and photosynthesis. Recently, we have shown that small VCs of less than 45 Da other than CO2 are major determinants of plant responses to fungal volatile emissions. However, the regulatory mechanisms involved in the plants' responses to small microbial VCs remain unclear. In Arabidopsis thaliana plants exposed to small fungal VCs, growth promotion is accompanied by reduction of the thiol redox of Calvin-Benson cycle (CBC) enzymes and changes in the levels of shikimate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway-related compounds. We hypothesized that plants' responses to small microbial VCs involve post-translational modulation of enzymes of the MEP and shikimate pathways via mechanisms involving redox-activated photosynthesis signaling. To test this hypothesis, we compared the responses of wild-type (WT) plants and a cfbp1 mutant defective in a redox-regulated isoform of the CBC enzyme fructose-1,6-bisphosphatase to small VCs emitted by the fungal phytopathogen Alternaria alternata. Fungal VC-promoted growth and photosynthesis, as well as metabolic and proteomic changes, were substantially weaker in cfbp1 plants than in WT plants. In WT plants, but not in cfbp1 plants, small fungal VCs reduced the levels of both transcripts and proteins of the stromal Clp protease system and enhanced those of plastidial chaperonins and co-chaperonins. Consistently, small fungal VCs promoted the accumulation of putative Clp protease clients including MEP and shikimate pathway enzymes. clpr1-2 and clpc1 mutants with disrupted plastidial protein homeostasis responded weakly to small fungal VCs, strongly indicating that plant responses to microbial volatile emissions require a finely regulated plastidial protein quality control system. Our findings provide strong evidence that plant responses to fungal VCs involve chloroplast-to-nucleus retrograde signaling of redox-activated photosynthesis leading to proteostatic regulation of the MEP and shikimate pathways.