UPLC-ESI-QTOF-MS assisted targeted metabolomics to study the enrichment of vinca alkaloids and related metabolites in Catharanthus roseus plants grown under controlled LED environment.

Enrichment of pharmaceutically important vinca alkaloids, vinblastine and vincristine, in the leaves of Madagascar periwinkle (Catharanthus roseus) plants through different pre- or postharvest treatments or cultivation conditions, e.g., exposing the plants to UV-irradiation, has been in focus for decades. Controlled LED environment in the visible light range offers the possibility of monitoring the changes in the concentration of metabolites in the vinca alkaloid-related pathway without involving UV-related abiotic stress. In the frame of our targeted metabolomics approach, 64 vinca alkaloids and metabolites were screened with the help of a UPLC-ESI-QTOF-MS instrumental setup from the leaf extracts of C. roseus plants grown in chambers under control (medium light), low light, and high blue / high red/ high far-red conditions. Out of the 14 metabolites that could be assigned either unambiguously with authentic standards or tentatively with high resolution mass spectrometry-based methods, all three dimer vinca alkaloids, that is, 3',4'-anhydrovinblastine, vinblastine and vincristine showed an at least nine-fold enrichment under high blue irradiation when compared with the control conditions: final concentrations of 961 mg kg-1 dry weight, 33.8 mg kg-1 dry weight, and 11.7 mg kg-1 dry weight could be achieved, respectively. As supported by multivariate statistical analysis, the key metabolites of the vinca alkaloid pathway were highly represented among the metabolites that were specifically stimulated by high blue light application.

Hierarchical contribution of Argonaute proteins to antiviral protection.

Antiviral RNAi is the main protective measure employed by plants in the fight against viruses. The main steps of this process have been clarified in recent years, primarily relying on the extensive genetic resources of Arabidopsis thaliana. Our knowledge of viral diseases of crops, however, is still limited, mainly due to the fact that A. thaliana is a non-host for many agriculturally important viruses. In contrast, Nicotiana benthamiana has an unparalleled susceptibility to viruses and, since it belongs to the Solanaceae family, it is considered an adequate system for modeling infectious diseases of crops such as tomatoes. We used a series of N. benthamiana mutants created by genome editing to analyze the RNAi response elicited by the emerging tomato pathogen, pepino mosaic virus (PepMV). We uncovered hierarchical roles of several Argonaute proteins (AGOs) in anti-PepMV defense, with the predominant contribution of AGO2. Interestingly, the anti-PepMV activities of AGO1A, AGO5, and AGO10 only become apparent when AGO2 is mutated. Taken together, our results prove that hierarchical actions of several AGOs are needed for the plant to build effective anti-PepMV resistance. The genetic resources created here will be valuable assets for analyzing RNAi responses triggered by other agriculturally important pathogenic viruses.

L-Aminoguanidine Induces Imbalance of ROS/RNS Homeostasis and Polyamine Catabolism of Tomato Roots after Short-Term Salt Exposure.

Polyamine (PA) catabolism mediated by amine oxidases is an important process involved in fine-tuning PA homeostasis and related mechanisms during salt stress. The significance of these amine oxidases in short-term responses to salt stress is, however, not well understood. In the present study, the effects of L-aminoguanidine (AG) on tomato roots treated with short-term salt stress induced by NaCl were studied. AG is usually used as a copper amine oxidase (CuAO or DAO) inhibitor. In our study, other alterations of PA catabolism, such as reduced polyamine oxidase (PAO), were also observed in AG-treated plants. Salt stress led to an increase in the reactive oxygen and nitrogen species in tomato root apices, evidenced by in situ fluorescent staining and an increase in free PA levels. Such alterations were alleviated by AG treatment, showing the possible antioxidant effect of AG in tomato roots exposed to salt stress. PA catabolic enzyme activities decreased, while the imbalance of hydrogen peroxide (H2O2), nitric oxide (NO), and hydrogen sulfide (H2S) concentrations displayed a dependence on stress intensity. These changes suggest that AG-mediated inhibition could dramatically rearrange PA catabolism and related reactive species backgrounds, especially the NO-related mechanisms. More studies are, however, needed to decipher the precise mode of action of AG in plants exposed to stress treatments.

Effect of ascorbate and hydrogen peroxide on hormone and metabolite levels during post-germination growth in wheat.

The modulation of hormone and metabolite levels by ascorbate (ASA) and hydrogen peroxide (H2 O2 ) was compared during post-germination growth in shoots of wheat. Treatment with ASA resulted in a greater reduction of growth than the addition of H2 O2 . ASA also had a larger effect on the redox state of the shoot tissues as shown by the higher ASA and glutathione (GSH) levels, lower glutathione disulfide (GSSG) content and GSSG/GSH ratio compared to the H2 O2 treatment. Apart from common responses (i.e., increase of cis-zeatin and its O-glucosides), the contents of several compounds related to cytokinin (CK) and abscisic acid (ABA) metabolism were greater after ASA application. These differences in the redox state and hormone metabolism following the two treatments may be responsible for their distinct influence on various metabolic pathways. Namely, the glycolysis and citrate cycle were inhibited by ASA and they were not affected by H2 O2 , while the amino acid metabolism was induced by ASA and repressed by H2 O2 based on the changes in the level of the related carbohydrates, organic and amino acids. The first two pathways produce reducing power, while the last one needs it; therefore ASA, as a reductant may suppress and induce them, respectively. H2 O2 as an oxidant had different effect, namely it did not alter glycolysis and citrate cycle, and inhibited the formation of amino acids.

The role of methyl salicylate in plant growth under stress conditions.

Methyl salicylate is a volatile compound, the synthesis of which takes place via the salicylic acid pathway in plants. Both compounds can be involved in the development of systemic acquired resistance and they play their role partly independently. Salicylic acid transport has an important role in long-distance signalling, but methyl salicylate has also been suggested as a phloem-based mobile signal, which can be demethylated to form salicylic acid, inducing the de-novo synthesis of salicylic acid in distal tissue. Despite the fact that salicylic acid has a protective role in abiotic stress responses and tolerance, very few investigations have been reported on the similar effects of methyl salicylate. In addition, as salicylic acid and methyl salicylate are often treated simply as the volatile and non-volatile forms of the same compound, and in several cases they also act in the same way, it is hard to highlight the differences in their mode of action. The main aim of the present review is to reveal the individual role and action mechanism of methyl salicylate in systemic acquired resistance, plant-plant communication and various stress conditions in fruits and plants.

Improvement of Drought Tolerance by Exogenous Spermidine in Germinating Wheat (Triticum aestivum L.) Plants Is Accompanied with Changes in Metabolite Composition.

Drought is one of the most important environmental factors reducing the yield and production of crops, including wheat. Polyamines are closely associated with plant stress tolerance. The present study investigated the mechanisms through seed germination with spermidine protecting wheat varieties from drought stress. In the first experiment, the effects of spermidine on the germination of wheat varieties, namely Rakhshan, Mihan, Sirvan and Pishgam, were investigated in three drought levels, namely 0, -2, and -4 MPa induced by polyethylene glycol 6000. Analysis of variance indicated that spermidine, drought stress and interaction between varieties and drought stress were significant for all traits, and with severity of stress, all traits significantly decreased. In the second experiment, detailed gene expression and non-targeted metabolomics analyses were carried out using the Rakhshan and Mihan varieties after germination, with or without spermidine treatment and/or drought stress. According to the biomass parameters, the Mihan variety showed relatively better growth compared to the other variety, but the Rakhshan one showed more pronounced responses at gene expression level to exogenous spermidine than the Mihan variety. Overall, these results showed that spermidine increased the drought tolerance of wheat at the germination stage, due to specific role of polyamine metabolism in the development of effective responses under drought stress.

Light Spectral Composition Modifies Polyamine Metabolism in Young Wheat Plants.

Although light-emitting diode (LED) technology has extended the research on targeted photomorphogenic, physiological, and biochemical responses in plants, there is not enough direct information about how light affects polyamine metabolism. In this study, the effect of three spectral compositions (referred to by their most typical characteristic: blue, red, and the combination of blue and red [pink] lights) on polyamine metabolism was compared to those obtained under white light conditions at the same light intensity. Although light quality induced pronounced differences in plant morphology, pigment contents, and the expression of polyamine metabolism-related genes, endogenous polyamine levels did not differ substantially. When exogenous polyamines were applied, their roborative effect were detected under all light conditions, but these beneficial changes were correlated with an increase in polyamine content and polyamine metabolism-related gene expression only under blue light. The effect of the polyamines on leaf gene expression under red light was the opposite, with a decreasing tendency. Results suggest that light quality may optimize plant growth through the adjustment of polyamine metabolism at the gene expression level. Polyamine treatments induced different strategies in fine-tuning of polyamine metabolism, which were induced for optimal plant growth and development under different spectral compositions.

Mutations in Rht-B1 Locus May Negatively Affect Frost Tolerance in Bread Wheat.

The wheat semi-dwarfing genes Rht (Reduced height) are widely distributed among the contemporary wheat varieties. These genes also exert pleiotropic effects on plant tolerance towards various abiotic stressors. In this work, frost tolerance was studied in three near-isogenic lines of the facultative variety 'April Bearded' (AB), carrying the wild type allele Rht-B1a (tall phenotype), and the mutant alleles Rht-B1b (semi-dwarf) and Rht-B1c (dwarf), and was further compared with the tolerance of a typical winter type variety, 'Mv Beres'. The level of freezing tolerance was decreasing in the order 'Mv Beres' > AB Rht-B1a > AB Rht-B1b > AB Rht-B1c. To explain the observed differences, cold acclimation-related processes were studied: the expression of six cold-related genes, the phenylpropanoid pathway, carbohydrates, amino acids, polyamines and compounds in the tricarboxylic acid cycle. To achieve this, a comprehensive approach was applied, involving targeted analyses and untargeted metabolomics screening with the help of gas chromatography/liquid chromatography­mass spectrometry setups. Several cold-related processes exhibited similar changes in these genotypes; indeed, the accumulation of eight putrescine and agmatine derivatives, 17 flavones and numerous oligosaccharides (max. degree of polymerization 18) was associated with the level of freezing tolerance in the 'April Bearded' lines. In summary, the mutant Rht alleles may further decrease the generally low frost tolerance of the Rht-B1a, and, based on the metabolomics study, the mechanisms of frost tolerance may differ for a typical winter variety and a facultative variety. Present results point to the complex nature of frost resistance.

Far-Red Light Coordinates the Diurnal Changes in the Transcripts Related to Nitrate Reduction, Glutathione Metabolism and Antioxidant Enzymes in Barley.

Spectral quality, intensity and period of light modify many regulatory and stress signaling pathways in plants. Both nitrate and sulfate assimilations must be synchronized with photosynthesis, which ensures energy and reductants for these pathways. However, photosynthesis is also a source of reactive oxygen species, whose levels are controlled by glutathione and other antioxidants. In this study, we investigated the effect of supplemental far-red (735 nm) and blue (450 nm) lights on the diurnal expression of the genes related to photoreceptors, the circadian clock, nitrate reduction, glutathione metabolism and various antioxidants in barley. The maximum expression of the investigated four photoreceptor and three clock-associated genes during the light period was followed by the peaking of the transcripts of the three redox-responsive transcription factors during the dark phase, while most of the nitrate and sulfate reduction, glutathione metabolism and antioxidant-enzyme-related genes exhibited high expression during light exposure in plants grown in light/dark cycles for two days. These oscillations changed or disappeared in constant white light during the subsequent two days. Supplemental far-red light induced the activation of most of the studied genes, while supplemental blue light did not affect or inhibited them during light/dark cycles. However, in constant light, several genes exhibited greater expression in blue light than in white and far-red lights. Based on a correlation analysis of the gene expression data, we propose a major role of far-red light in the coordinated transcriptional adjustment of nitrate reduction, glutathione metabolism and antioxidant enzymes to changes of the light spectrum.

Role of ethylene in ER stress and the unfolded protein response in tomato (Solanum lycopersicum L.) plants.

The unfolded protein response (UPR) plays a significant role in the maintenance of cellular homeostasis under endoplasmic reticulum (ER) stress, which is highly dependent on the regulation of defense-related phytohormones. In this study, the role of ethylene (ET) in ER stress and UPR was investigated in the leaves of intact tomato (Solanum lycopersicum) plants. Exogenous application of the ET precursor 1-aminocyclopropane-1-carboxylic acid not only resulted in higher ET emission from leaves but also increased the expression of the UPR marker gene SlBiP and the transcript levels of the ER stress sensor SlIRE1, as well as the levels of SlbZIP60, after 24 h in tomato leaves. Using ET receptor Never ripe (Nr) mutants, a significant role of ET in tunicamycin (Tm)-induced ER stress sensing and signaling was confirmed based on the changes in the expression levels of SlIRE1b and SlBiP. Furthermore, the analysis of other defense-related phytohormones showed that the Tm-induced ET can affect positively the levels of and response to salicylic acid. Additionally, it was found that nitric oxide production and lipid peroxidation, as well as the electrolyte leakage induced by Tm, is regulated by ET, whereas the levels of H2O2 and proteolytic activity seemed to be independent of ET under ER stress in the leaves of tomato plants.

Methylglyoxal induces stress signaling and promotes the germination of maize at low temperature.

Maize is sensitive to cold injury, especially during germination. Since cold causes oxidative stress, compounds that promote the accumulation of free radical forms, such as the reactive aldehyde (RA) methylglyoxal (MG), may be suitable to trigger a systemic defense response. In this study, maize seeds were soaked in MG solution for one night at room temperature, before germination test at 13°C. The exogenous MG enhanced the germination and photosynthetic performance of maize at low temperature. Transcriptome analysis, hormonal, and flavonoid profiling indicated MG-induced changes in photosystem antenna proteins, pigments, late embryogenesis abundant proteins, abscisic acid (ABA) derivatives, chaperons, and certain dihydroflavonols, members of the phenylpropanoid pathway. MG-response of the two maize cultivars (A654 and Cm174) were somewhat different, but we recorded higher endogenous hydrogen peroxide (H2 O2 ) and lower nitric oxide (NO) level in at least one of the treated genotypes. These secondary signal molecules may provoke some of the changes in the hormonal, metabolic and gene expression profile. Decreased auxin transport, but increased ABA degradation and cytokinin and jasmonic acid (JA) synthesis, as well as an altered carbohydrate metabolism and transport (amylases, invertases, and SWEET transporters) could have promoted germination of MG-pretreated seeds. While LEA accumulation could have protected against osmotic stress and catalase expression and production of many antioxidants, like para-hydroxybenzoic acid (p-HBA) and anthocyanins may have balanced the oxidative environment for maize germination. Our results showed that MG-pretreatment could be an effective way to promote cold germination and its effect was more pronounced in the originally cold-sensitive maize genotype.

Polyamine Metabolism under Different Light Regimes in Wheat.

Although the relationship between polyamines and photosynthesis has been investigated at several levels, the main aim of this experiment was to test light-intensity-dependent influence of polyamine metabolism with or without exogenous polyamines. First, the effect of the duration of the daily illumination, then the effects of different light intensities (50, 250, and 500 µmol m-2 s-1) on the polyamine metabolism at metabolite and gene expression levels were investigated. In the second experiment, polyamine treatments, namely putrescine, spermidine and spermine, were also applied. The different light quantities induced different changes in the polyamine metabolism. In the leaves, light distinctly induced the putrescine level and reduced the 1,3-diaminopropane content. Leaves and roots responded differently to the polyamine treatments. Polyamines improved photosynthesis under lower light conditions. Exogenous polyamine treatments influenced the polyamine metabolism differently under individual light regimes. The fine-tuning of the synthesis, back-conversion and terminal catabolism could be responsible for the observed different polyamine metabolism-modulating strategies, leading to successful adaptation to different light conditions.

Activation of Local and Systemic Defence Responses by Flg22 Is Dependent on Daytime and Ethylene in Intact Tomato Plants.

The first line of plant defence responses against pathogens can be induced by the bacterial flg22 and can be dependent on various external and internal factors. Here, we firstly studied the effects of daytime and ethylene (ET) using Never ripe (Nr) mutants in the local and systemic defence responses of intact tomato plants after flg22 treatments. Flg22 was applied in the afternoon and at night and rapid reactions were detected. The production of hydrogen peroxide and nitric oxide was induced by flg22 locally, while superoxide was induced systemically, in wild type plants in the light period, but all remained lower at night and in Nr leaves. Flg22 elevated, locally, the ET, jasmonic acid (JA) and salicylic acid (SA) levels in the light period; these levels did not change significantly at night. Expression of Pathogenesis-related 1 (PR1), Ethylene response factor 1 (ERF1) and Defensin (DEF) showed also daytime- and ET-dependent changes. Enhanced ERF1 and DEF expression and stomatal closure were also observable in systemic leaves of wild type plants in the light. These data demonstrate that early biotic signalling in flg22-treated leaves and distal ones is an ET-dependent process and it is also determined by the time of day and inhibited in the early night phase.

Unfinished story of polyamines: Role of conjugation, transport and light-related regulation in the polyamine metabolism in plants.

Polyamines play a fundamental role in the functioning of all cells. Their regulatory role in plant development, their function under stress conditions, and their metabolism have been well documented as regards both synthesis and catabolism in an increasing number of plant species. However, the majority of these studies concentrate on the levels of the most abundant polyamines, sometimes providing data on the enzyme activity or gene expression levels during polyamine synthesis, but generally making no mention of the fact that changes in the polyamine pool are very dynamic, and that other processes are also involved in the regulation of actual polyamine levels. Differences in the distribution of individual polyamines and their conjugation with other compounds were described some time ago, but these have been given little attention. In addition, the role of polyamine transporters in plants is only now being recognised. The present review highlights the importance of conjugated polyamines and also points out that investigations should not only deal with the polyamine metabolism itself, but should also cover other important questions, such as the relationship between light perception and the polyamine metabolism, or the involvement of polyamines in the circadian rhythm.

Comparison of Light Condition-Dependent Differences in the Accumulation and Subcellular Localization of Glutathione in Arabidopsis and Wheat.

This study aimed to clarify whether the light condition-dependent changes in the redox state and subcellular distribution of glutathione were similar in the dicotyledonous model plant Arabidopsis (wild-type, ascorbate- and glutathione-deficient mutants) and the monocotyledonous crop species wheat (Chinese Spring variety). With increasing light intensity, the amount of its reduced (GSH) and oxidized (GSSG) form and the GSSG/GSH ratio increased in the leaf extracts of both species including all genotypes, while far-red light increased these parameters only in wheat except for GSH in the GSH-deficient Arabidopsis mutant. Based on the expression changes of the glutathione metabolism-related genes, light intensity influences the size and redox state of the glutathione pool at the transcriptional level in wheat but not in Arabidopsis. In line with the results in leaf extracts, a similar inducing effect of both light intensity and far-red light was found on the total glutathione content at the subcellular level in wheat. In contrast to the leaf extracts, the inducing influence of light intensity on glutathione level was only found in the cell compartments of the GSH-deficient Arabidopsis mutant, and far-red light increased it in both mutants. The observed general and genotype-specific, light-dependent changes in the accumulation and subcellular distribution of glutathione participate in adjusting the redox-dependent metabolism to the actual environmental conditions.

The effects of putrescine pre-treatment on osmotic stress responses in drought-tolerant and drought-sensitive wheat seedlings.

Recent studies have demonstrated that exogenous polyamines have protective effects under various stress condition. A broader understanding of the role of the polyamine pool fine regulation and the alterations of polyamine-related physiological processes could be obtained by comparing the stress effects in different genotypes. In this study, the impact of pre-treatment with putrescine in response to osmotic stress was investigated in the drought-tolerant Katya and drought-sensitive Zora wheat (Triticum aestivum) cultivars. Photosynthetic performance, in vivo thermoluminescence emission from leaves, leaf temperature, polyamine and salicylic acid levels, contents of osmoprotectants, and activities of antioxidant enzymes in the leaves were investigated not only to reveal differences in the physiological processes associated to drought tolerance, but to highlight the modulating strategies of polyamine metabolism between a drought-tolerant and a drought-sensitive wheat genotype. Results showed that the tolerance of Katya under osmotic stress conditions was characterized by higher photosynthetic ability, stable charge separation across the thylakoid membrane in photosystem II, higher proline accumulation and antioxidant activity. Thermoluminescence also revealed differences between the two varieties - a downshift of the B band and an increase of the afterglow band under osmotic stress in Zora, providing original complementary information to leaf photosynthesis. Katya variety exhibited higher constitutive levels of the signaling molecules putrescine and salicylic acid compared to the sensitive Zora. However, responses to exogenous putrescine were more advantageous for the sensitive variety under PEG treatment, which may be in relation with the decreased catabolism of polyamines, suggesting the increased need for polyamine under stress conditions.

Acclimation of photosynthetic processes and metabolic responses to elevated temperatures in cereals.

The aim of the present work was to better understand the molecular mechanisms of heat acclimation processes in cereals. A large number of winter and spring wheat, barley and oat varieties were grown under either control conditions (22/20°C) or under a mild heat stress (30°C) that induce the acclimation processes. The temperature dependence of chlorophyll a fluorescence induction and gas exchange parameters showed that heat acclimation increased the thermotolerance of the photosynthetic apparatus, but these changes did not differ sharply in the winter-spring type cereals. Similarly, to wheat, elevated temperature also led to increasing transpiration rate and reduced water use efficiency in barley and oat plants. A non-targeted metabolomic analysis focusing on polar metabolites in two selected barley (winter type Mv Initium and spring type Conchita) and in two oat varieties (winter type Mv Hópehely and spring type Mv Pehely) revealed substantial differences between both the two species and between the acclimated and non-acclimated plants. Several compounds, including sugars, organic acids, amino acids and alcohols could be separated and detected. The expression level of the CYP707, HSP90, galactinol synthase, raffinose synthase and α-galactosidase genes showed genotype-dependent changes after 1 day; however, the CYP707 was the only one, which was still upregulated in at least some of the genotypes. Results suggest that heat acclimation itself does not require general induction of primary metabolites. However, induction of specific routes, e.g. the induction of the raffinose family oligosaccharides, especially the synthesis of galactinol, may also contribute the improved heat tolerance in cereals.

Addition of Aegilops biuncialis chromosomes 2M or 3M improves the salt tolerance of wheat in different way.

Aegilops biuncialis is a promising gene source to improve salt tolerance of wheat via interspecific hybridization. In the present work, the salt stress responses of wheat-Ae. biuncialis addition lines were investigated during germination and in young plants to identify which Aegilops chromosomes can improve the salt tolerance of wheat. After salt treatments, the Aegilops parent and the addition lines 2M, 3M and 3M.4BS showed higher germination potential, shoot and root growth, better CO2 assimilation capacity and less chlorophyll degradation than the wheat parent. The Aegilops parent accumulated less Na in the roots due to an up-regulation of SOS1, SOS2 and HVP1 genes, while it contained higher amount of proline, fructose, glucose, galactose, maltose and raffinose. In the leaves, lower Na level was accompanied by high amount of proline and increased expression of NHX2 gene. The enhanced accumulation of sugars and proline was also observed in the roots of 3M and 3M.4BS addition lines. Typical mechanism of 2M addition line was the sequestration of Na into the vacuole due to the increased expression of HVP1 in the roots and NHX2 in the leaves. These results suggest the Aegilops chromosomes 2M and 3M can improve salt tolerance of wheat in different way.

Salicylic Acid Signalling in Plants.

Ten articles published in the "Special Issue: Salicylic Acid Signalling in Plants" are summarized, in order to get a global picture about the mode of action of salicylic acid in plants, and about its interaction with other stress-signalling routes. Its ecological aspects and possible practical use are also discussed.

Molecular background of cadmium tolerance in Rht dwarf wheat mutant is related to a metabolic shift from proline and polyamine to phytochelatin synthesis.

Plant height is among the most important agronomic traits influencing crop yield. Wheat lines carrying Rht genes are important in plant breeding due to their both higher yield capacity and better tolerance to certain environmental stresses. However, the effects of dwarf-inducing genes on stress acclimation mechanisms are still poorly understood. Under the present conditions, cadmium stress induced different stress responses and defence mechanisms in the wild-type and dwarf mutant, and the mutant with the Rht-B1c allele exhibited higher tolerance. In the wild type after cadmium treatment, the abscisic acid synthesis increased in the leaves, which in turn might have induced the polyamine and proline metabolisms in the roots. However, in the mutant line, the slight increment in the leaf abscisic acid content accompanied by relatively high salicylic acid accumulation was not sufficient to induce such a great accumulation of proline and putrescine. Although changes in proline and polyamines, especially putrescine, showed similar patterns, the accumulation of these compounds was antagonistically related to the phytochelatin synthesis in the roots of the wild type after cadmium stress. In the dwarf genotype, a favourable metabolic shift from the synthesis of polyamine and proline to that of phytochelatin was responsible for the higher cadmium tolerance observed.