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 liquid chromatography (HILIC), reversed phase (RP) chromatography with pre-column derivatization, and tandem mass spectrometry (MS). 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-labeled 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 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.

• Keywords
• Acetylated amino acids, LC-MS/MS, acetylated biogenic amines, amino acids, biogenic amines, methylated amino acids, plant metabolism,
• MeSH
• Arabidopsis metabolism   growth & development   MeSH
• Chromatography, Liquid MeSH
• Nitrogen * metabolism   MeSH
• Hordeum metabolism   growth & development   MeSH
• Liquid Chromatography-Mass Spectrometry MeSH
• Zea mays metabolism   growth & development   MeSH
• Polyamines metabolism   analysis   MeSH
• Plants metabolism   MeSH
• Tandem Mass Spectrometry * methods   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitrogen * MeSH
• Polyamines MeSH

In the context of global climate change and the increasing need to study plant response to drought, there is a demand for easily, rapidly, and remotely measurable parameters that sensitively reflect leaf water status. Parameters with this potential include those derived from leaf spectral reflectance (R) and chlorophyll fluorescence. As each of these methods probes completely different leaf characteristics, their sensitivity to water loss may differ in different plant species and/or under different circumstances, making it difficult to choose the most appropriate method for estimating water status in a given situation. Here, we present a simple comparative analysis to facilitate this choice for leaf-level measurements. Using desiccation of tobacco (Nicotiana tabacum L. cv. Samsun) and barley (Hordeum vulgare L. cv. Bojos) leaves as a model case, we measured parameters of spectral R and chlorophyll fluorescence and then evaluated and compared their applicability by means of introduced coefficients (coefficient of reliability, sensitivity, and inaccuracy). This comparison showed that, in our case, chlorophyll fluorescence was more reliable and universal than spectral R. Nevertheless, it is most appropriate to use both methods simultaneously, as the specific ranking of their parameters according to the coefficient of reliability may indicate a specific scenario of changes in desiccating leaves.

• Publication type
• Journal Article MeSH

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.

• Keywords
• abiotic stress, biostimulant, growth, plant phenotyping,
• Publication type
• Journal Article MeSH

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, β-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.

• Keywords
• chlorophyll fluorescence, elevated CO2, gas exchange, grain yield, heat stress, targeted metabolomic analysis, wheat,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Pisum sativum, abiotic stress, developmental plasticity, drought, oleic acid, phloem,
• MeSH
• Biological Transport MeSH
• Nitrogen metabolism   MeSH
• Phloem anatomy & histology   genetics   physiology   MeSH
• Adaptation, Physiological MeSH
• Stress, Physiological MeSH
• Genotype MeSH
• Pisum sativum anatomy & histology   genetics   physiology   MeSH
• Oleic Acid metabolism   MeSH
• Plant Leaves anatomy & histology   genetics   physiology   MeSH
• Droughts MeSH
• Plant Exudates MeSH
• Carbon metabolism   MeSH
• Water physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Nitrogen MeSH
• Oleic Acid MeSH
• Plant Exudates MeSH
• Carbon MeSH
• Water MeSH

Chloroplasts, the sites of photosynthesis in higher plants, have evolved several means to tolerate short episodes of drought stress through biosynthesis of diverse metabolites essential for plant function, but these become ineffective when the duration of the stress is prolonged. Cyanobacteria are the closest bacterial homologs of plastids with two photosystems to perform photosynthesis and to evolve oxygen as a byproduct. The presence of Flv genes encoding flavodiiron proteins has been shown to enhance stress tolerance in cyanobacteria. In an attempt to support the growth of plants exposed to drought, the Synechocystis genes Flv1 and Flv3 were expressed in barley with their products being targeted to the chloroplasts. The heterologous expression of both Flv1 and Flv3 accelerated days to heading, increased biomass, promoted the number of spikes and grains per plant, and improved the total grain weight per plant of transgenic lines exposed to drought. Improved growth correlated with enhanced availability of soluble sugars, a higher turnover of amino acids and the accumulation of lower levels of proline in the leaf. Flv1 and Flv3 maintained the energy status of the leaves in the stressed plants by converting sucrose to glucose and fructose, immediate precursors for energy production to support plant growth under drought. The results suggest that sugars and amino acids play a fundamental role in the maintenance of the energy status and metabolic activity to ensure growth and survival under stress conditions, that is, water limitation in this particular case. Engineering chloroplasts by Flv genes into the plant genome, therefore, has the potential to improve plant productivity wherever drought stress represents a significant production constraint.

• Keywords
• Hordeum vulgare L., biomass, metabolites, photosynthesis, plastid biotechnology, yield,
• Publication type
• Journal Article MeSH