Ascorbate, plant hormones and their interactions during plant responses to biotic stress.

Plants can experience a variety of environmental stresses that significantly impact their fitness and survival. Additionally, biotic stress can harm agriculture, leading to reduced crop yields and economic losses worldwide. As a result, plants have developed defense strategies to combat potential invaders. These strategies involve regulating redox homeostasis. Several studies have documented the positive role of plant antioxidants, including Ascorbate (Asc), under biotic stress conditions. Asc is a multifaceted antioxidant that scavenges ROS, acts as a co-factor for different enzymes, regulates gene expression, and facilitates iron transport. However, little attention has been given to Asc and its transport, regulatory effects, interplay with phytohormones, and involvement in defense processes under biotic stress. Asc interacts with other components of the redox system and phytohormones to activate various defense responses that reduce the growth of plant pathogens and promote plant growth and development under biotic stress conditions. Scientific reports indicate that Asc can significantly contribute to plant resistance against biotic stress through mutual interactions with components of the redox and hormonal systems. This review focuses on the role of Asc in enhancing plant resistance against pathogens. Further research is necessary to gain a more comprehensive understanding of the molecular and cellular regulatory processes involved.

Identification of ascorbate- and salicylate-responsive miRNAs and verification of the spectral control of miR395 in Arabidopsis.

We assumed that miRNAs might regulate the physiological and biochemical processes in plants through their effects on the redox system and phytohormones. To check this hypothesis, the transcriptome profile of wild-type Arabidopsis and lines with decreased ascorbate (Asc), glutathione (GSH), or salicylate (Sal) levels were compared. GSH deficiency did not influence the miRNA expression, whereas lower levels of Asc and Sal reduced the accumulation of 9 and 44 miRNAs, respectively, but only four miRNAs were upregulated. Bioinformatics analysis revealed that their over-represented target genes are associated with the synthesis of nitrogen-containing and aromatic compounds, nucleic acids, and sulphate assimilation. Among them, the sulphate reduction-related miR395 - ATP-sulfurylase couple was selected to check the assumed modulating role of the light spectrum. A greater induction of the Asc- and Sal-responsive miR395 was observed under sulphur starvation in far-red light compared to white and blue light in wild-type and GSH-deficient Arabidopsis lines. Sal deficiency inhibited the induction of miR395 by sulphur starvation in blue light, whereas Asc deficiency greatly reduced it independently of the spectrum. Interestingly, sulphur starvation decreased only the level of ATP sulfurylase 4 among the miR395 target genes in far-red light. The expression level of ATP sulfurylase 3 was higher in far-red light than in blue light in wild-type and Asc-deficient lines. The results indicate the coordinated control of miRNAs by the redox and hormonal system since 11 miRNAs were affected by both Asc and Sal deficiency. This process can be modulated by light spectrum, as shown for miR395.

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.

Light-Dependent Control of Metabolism in Plants.

The energy of sunlight is converted into chemical energy during photosynthesis in plants [...].

Light-Dependent Regulatory Interactions between the Redox System and miRNAs and Their Biochemical and Physiological Effects in Plants.

Light intensity and spectrum play a major role in the regulation of the growth, development, and stress response of plants. Changes in the light conditions affect the formation of reactive oxygen species, the activity of the antioxidants, and, consequently, the redox environment in the plant tissues. Many metabolic processes, thus the biogenesis and function of miRNAs, are redox-responsive. The miRNAs, in turn, can modulate various components of the redox system, and this process is also associated with the alteration in the intensity and spectrum of the light. In this review, we would like to summarise the possible regulatory mechanisms by which the alterations in the light conditions can influence miRNAs in a redox-dependent manner. Daily and seasonal fluctuations in the intensity and spectral composition of the light can affect the expression of miRNAs, which can fine-tune the various physiological and biochemical processes due to their effect on their target genes. The interactions between the redox system and miRNAs may be modulated by light conditions, and the proposed function of this regulatory network and its effect on the various biochemical and physiological processes will be introduced in plants.

Organelle-specific localization of glutathione in plants grown under different light intensities and spectra.

Plant ascorbate and glutathione metabolism counteracts oxidative stress mediated, for example, by excess light. In this review, we discuss the properties of immunocytochemistry and transmission electron microscopy, redox-sensitive dyes or probes and bright-field microscopy, confocal microscopy or fluorescence microscopy for the visualization and quantification of glutathione at the cellular or subcellular level in plants and the quantification of glutathione from isolated organelles. In previous studies, we showed that subcellular ascorbate and glutathione levels in Arabidopsis are affected by high light stress. The use of light-emitting diodes (LEDs) is gaining increasing importance in growing indoor crops and ornamental plants. A combination of different LED types allows custom-made combinations of wavelengths and prevents damage related to high photon flux rates. In this review we provide an overview on how different light spectra and light intensities affect glutathione metabolism at the cellular and subcellular levels in plants. Findings obtained in our most recent study demonstrate that both light intensity and spectrum significantly affected glutathione metabolism in wheat at the transcriptional level and caused genotype-specific reactions in the investigated Arabidopsis lines.

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.

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.

Identification of a redox-dependent regulatory network of miRNAs and their targets in wheat.

Reactive oxygen species and antioxidants have an important role in the regulation of plant growth and development under both optimal and stress conditions. In this study, we investigate a possible redox control of miRNAs in wheat (Triticum aestivum ssp. aestivum). Treatment of seedlings with 10 mM H2O2 via the roots for 24 h resulted in decreased glutathione content, increased half-cell reduction potential of the glutathione disulphide/glutathione redox pair, and greater ascorbate peroxidase activity compared to the control plants. These changes were accompanied by alterations in the miRNA transcript profile, with 70 miRNAs being identified with at least 1.5-fold difference in their expression between control and treated (0, 3, 6 h) seedlings. Degradome sequencing identified 86 target genes of these miRNAs, and 6722 possible additional target genes were identified using bioinformatics tools. The H2O2-responsiveness of 1647 target genes over 24 h of treatment was also confirmed by transcriptome analysis, and they were mainly found to be related to the control of redox processes, transcription, and protein phosphorylation and degradation. In a time-course experiment (0-24 h of treatment) a correlation was found between the levels of glutathione, other antioxidants, and the transcript levels of the H2O2-responsive miRNAs and their target mRNAs. This relationship together with bioinformatics modelling of the regulatory network indicated glutathione-related redox control of miRNAs and their targets, which allows the adjustment of the metabolism to changing environmental conditions.

Redox regulation of free amino acid levels in Arabidopsis thaliana.

Abiotic stresses induce oxidative stress, which modifies the level of several metabolites including amino acids. The redox control of free amino acid profile was monitored in wild-type and ascorbate or glutathione deficient mutant Arabidopsis thaliana plants before and after hydroponic treatment with various redox agents. Both mutations and treatments modified the size and redox state of the ascorbate (AsA) and/or glutathione (GSH) pools. The total free amino acid content was increased by AsA, GSH and H2 O2 in all three genotypes and a very large (threefold) increase was observed in the GSH-deficient pad2-1 mutant after GSH treatment compared with the untreated wild-type plants. Addition of GSH reduced the ratio of amino acids belonging to the glutamate family on a large scale and increased the relative amount of non-proteinogenic amino acids. The latter change was because of the large increase in the content of alpha-aminoadipate, an inhibitor of glutamatic acid (Glu) transport. Most of the treatments increased the proline (Pro) content, which effect was due to the activation of genes involved in Pro synthesis. Although all studied redox compounds influenced the amount of free amino acids and a mostly positive, very close (r > 0.9) correlation exists between these parameters, a special regulatory role of GSH could be presumed due to its more powerful effect. This may originate from the thiol/disulphide conversion or (de)glutathionylation of enzymes participating in the amino acid metabolism.

Pleiotropic effect of chromosome 5A and the mvp mutation on the metabolite profile during cold acclimation and the vegetative/generative transition in wheat.

BACKGROUND: Wheat is the leading source of vegetable protein in the human diet, and metabolites are crucial for both plant development and human nutrition. The recent advances in metabolomics provided an opportunity to perform an untargeted metabolite analysis in this important crop. RESULTS: Wheat was characterised at the metabolite level during cold acclimation and transition from the vegetative to the generative phase. The relationship between these changes and chromosome 5A and the maintained vegetative phase (mvp) mutation was also investigated. Samples were taken from the shoots and crowns during four developmental stages: plants grown at 20/17°C, after cold treatment but still during the vegetative phase, at the double ridge and during spikelet formation. The levels of 47 compounds were identified by gas chromatography-mass spectrometry, of which 38 were annotated. The cold treatment, in general, increased the concentrations of osmolites but not in all lines and not equally in the shoots and crowns. The accumulation of proline was not associated with the vernalisation process or with frost tolerance. The mvp mutation and chromosome 5A substitutions altered the amounts of several metabolites compared to those of the Tm and CS, respectively, during each developmental stage. The Ch5A substitution resulted in more substantial changes at the metabolite level than did the Tsp5A substitution. While Ch5A mainly influenced the sugar concentrations, Tsp5A altered the level of tricarboxylic acid cycle intermediates during the vegetative/generative transition. A much higher trehalose, proline, glutamine, asparagine, and unidentified m/z 186 content was detected in crowns than in shoots that may contribute to the frost tolerance of crowns. CONCLUSIONS: Substantial influences of chromosome 5A and the mvp mutation on metabolism during four different developmental stages were demonstrated. The distinct and overlapping accumulation patterns of metabolites suggest the complex genetic regulation of metabolism in the shoots and crowns.

Central role of the flowering repressor ZCCT2 in the redox control of freezing tolerance and the initial development of flower primordia in wheat.

BACKGROUND: As both abiotic stress response and development are under redox control, it was hypothesised that the pharmacological modification of the redox environment would affect the initial development of flower primordia and freezing tolerance in wheat (Triticum aestivum L.). RESULTS: Pharmacologically induced redox changes were monitored in winter (T. ae. ssp. aestivum cv. Cheyenne, Ch) and spring (T. ae. ssp. spelta; Tsp) wheat genotypes grown after germination at 20/17°C for 9 d (chemical treatment: last 3 d), then at 5°C for 21 d (chemical treatment: first 4 d) and subsequently at 20/17°C for 21 d (recovery period). Thiols and their disulphide forms were measured and based on these data reduction potentials were calculated. In the freezing-tolerant Ch the chemical treatments generally increased both the amount of thiol disulphides and the reduction potential after 3 days at 20/17°C. In the freezing-sensitive Tsp a similar effect of the chemicals on these parameters was only observed after the continuation of the treatments for 4 days at 5°C. The applied chemicals slightly decreased root fresh weight and increased freezing tolerance in Ch, whereas they increased shoot fresh weight in Tsp after 4 days at 5°C. As shown after the 3-week recovery at 20/17°C, the initial development of flower primordia was accelerated in Tsp, whereas it was not affected by the treatments in Ch. The chemicals differently affected the expression of ZCCT2 and that of several other genes related to freezing tolerance and initial development of flower primordia in Ch and Tsp after 4 d at 5°C. CONCLUSIONS: Various redox-altering compounds and osmotica had differential effects on glutathione disulphide content and reduction potential, and consequently on the expression of the flowering repressor ZCCT2 in the winter wheat Ch and the spring wheat Tsp. We propose that the higher expression of ZCCT2 in Ch may be associated with activation of genes of cold acclimation and its lower expression in Tsp with the induction of genes accelerating initial development of flower primordia. In addition, ZCCT2 may be involved in the coordinated control of the two processes.

The expression of CBF genes at Fr-2 locus is associated with the level of frost tolerance in Bulgarian winter wheat cultivars.

The regulation of the majority of cold-regulated genes in plants is mediated by CBF (C-repeat binding factors) transcription factor family. Natural differences in frost tolerance (FT) of wheat have been mapped to the Fr-2 (Frost Resistance-2) locus on chromosome group 5 and are associated with variation in threshold induction temperatures and/or transcript levels of CBF genes. This study used real-time reverse-transcription polymerase chain reaction (qRT-PCR) to compare the relative expression levels of four T. aestivum CBF genes (TaCBF15.2, TaCBFA19, TaCBFA2 and TaCBFD21) in crown tissue of two Bulgarian hexaploid winter wheat cultivars (Milena and Russalka) with distinct levels of low-temperature (LT) tolerance but same vernalization requirement, and the spring cultivar Chinese Spring. The transcription profiles of the selected TaCBF genes showed that they are induced by cold treatment at 2 °C. Analysis of transcript abundance revealed that the four TaCBF genes were expressed at higher levels in the frost tolerant Milena than in the susceptible Russalka. Largest differences (fivefold and fourfold) in expression levels between both winter cultivars were observed in two of the analysed genes, TaCBF15.2 and TaCBFA19, respectively. The higher steady-state expression levels of TaCBF genes before the onset of the LT treatment in Milena, combined with stronger induction by cold treatment, suggest that these molecular responses to LT are associated with superior FT development capacity. The results expand our understanding of the molecular mechanisms underlying LT acclimation in Bulgarian wheat and can be used for development of functional markers for improvement of FT wheat-breeding programmes.

Different accumulation of free amino acids during short- and long-term osmotic stress in wheat.

The effect of wheat chromosome 5A on free amino acid accumulation induced by osmotic stress was compared in chromosome 5A substitution lines with different freezing tolerance. Treatment with 15% polyethylene glycol (PEG) resulted in greater total free amino acid content even after 3 days compared to the controls. The ratio of amino acids belonging to various amino acid families differed after 3-week treatment in the control and PEG-treated plants only in the case of the freezing-sensitive substitution line. There was a transient increase with a maximum after 3 days in the amounts of several amino acids, after which their concentrations exhibited a more gradual increase. During the first days of osmotic stress the Glu, Gln, Asp, Asn, Thr, Ser, Leu, and His concentrations were greater in the tolerant substitution line than in the sensitive one, while the opposite relationship was observed at the end of the PEG treatment. The coordinated changes in the levels of individual amino acids indicated that they are involved in both the short- and long-term responses to osmotic stress. The alterations differed in the two chromosome 5A substitution lines, depending on the stress tolerance of the chromosome donor genotype.

Light Intensity- and Spectrum-Dependent Redox Regulation of Plant Metabolism.

Both light intensity and spectrum (280-800 nm) affect photosynthesis and, consequently, the formation of reactive oxygen species (ROS) during photosynthetic electron transport. ROS, together with antioxidants, determine the redox environment in tissues and cells, which in turn has a major role in the adjustment of metabolism to changes in environmental conditions. This process is very important since there are great spatial (latitude, altitude) and temporal (daily, seasonal) changes in light conditions which are accompanied by fluctuations in temperature, water supply, and biotic stresses. The blue and red spectral regimens are decisive in the regulation of metabolism because of the absorption maximums of chlorophylls and the sensitivity of photoreceptors. Based on recent publications, photoreceptor-controlled transcription factors such as ELONGATED HYPOCOTYL5 (HY5) and changes in the cellular redox environment may have a major role in the coordinated fine-tuning of metabolic processes during changes in light conditions. This review gives an overview of the current knowledge of the light-associated redox control of basic metabolic pathways (carbon, nitrogen, amino acid, sulphur, lipid, and nucleic acid metabolism), secondary metabolism (terpenoids, flavonoids, and alkaloids), and related molecular mechanisms. Light condition-related reprogramming of metabolism is the basis for proper growth and development of plants; therefore, its better understanding can contribute to more efficient crop production in the future.

Transcriptome profiling of pepper leaves by RNA-Seq during an incompatible and a compatible pepper-tobamovirus interaction.

Upon virus infections, the rapid and comprehensive transcriptional reprogramming in host plant cells is critical to ward off virus attack. To uncover genes and defense pathways that are associated with virus resistance, we carried out the transcriptome-wide Illumina RNA-Seq analysis of pepper leaves harboring the L3 resistance gene at 4, 8, 24 and 48 h post-inoculation (hpi) with two tobamoviruses. Obuda pepper virus (ObPV) inoculation led to hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a systemic infection without visible symptoms (compatible interaction). ObPV induced robust changes in the pepper transcriptome, whereas PMMoV showed much weaker effects. ObPV markedly suppressed genes related to photosynthesis, carbon fixation and photorespiration. On the other hand, genes associated with energy producing pathways, immune receptors, signaling cascades, transcription factors, pathogenesis-related proteins, enzymes of terpenoid biosynthesis and ethylene metabolism as well as glutathione S-transferases were markedly activated by ObPV. Genes related to photosynthesis and carbon fixation were slightly suppressed also by PMMoV. However, PMMoV did not influence significantly the disease signaling and defense pathways. RNA-Seq results were validated by real-time qPCR for ten pepper genes. Our findings provide a deeper insight into defense mechanisms underlying tobamovirus resistance in pepper.

Regulation of gene expression by chromosome 5A during cold hardening in wheat.

Cold hardening is necessary to achieve the genetically determined maximum freezing tolerance and to reduce yield losses in winter cereals. The aim of the present study was to determine a set of genes with an important role in this process, by comparing of chromosome 5A substitution lines with different levels of freezing tolerance, since chromosome 5A is a major regulator of this trait. During 21 days of treatment at 2 degrees C, 303 genes were up-regulated, while 222 were down-regulated at most sampling points, and 156 at around half of them (out of the 10,297 unigenes studied). The freezing-tolerant substitution line exhibited 1.5 times as many differentially expressed genes than the sensitive one. The transcription of 78 genes (39 up-regulated) proved to be chromosome 5A-dependent. These genes encoded proteins involved in transcriptional regulation, defence processes and carbohydrate metabolism. Three of the chromosome 5A-related genes, coding for a cold-responsive, a Ca-binding and an embryo and meristem-related protein, were genetically mapped and characterized in further detail. The present experimental system was appropriate for the selection of chromosome 5A-related genes involved in short- and long-term cold acclimation in wheat. By modifying the expression of these genes it may be possible to improve freezing tolerance.

Differential effects of cold, osmotic stress and abscisic acid on polyamine accumulation in wheat.

The effects of cold, osmotic stress and abscisic acid (ABA) on polyamine accumulation were compared in the moderately freezing-sensitive wheat (Triticum aestivum L.) variety Chinese Spring (CS) and in two derived chromosome 5A substitution lines, CS(T. spelta 5A) and CS(Cheyenne 5A), exhibiting lower and higher levels of freezing tolerance, respectively. When compared with the other treatments, putrescine (Put) and spermidine (Spd) levels were much greater after cold treatment, spermine (Spm) following polyethylene glycol-induced (PEG) osmotic stress and Spm and cadaverine (Cad) after ABA treatment. During 3-week cold stress, the Put concentration, first exhibited a transient increase and decrease, and then gradually increased. These alterations may be due to changes in the expression of genes encoding the enzymes of Put synthesis. The Put content was higher in the freezing-tolerant chromosome 5A substitution line than in the sensitive one after 3 weeks of cold. In contrast to cold, ABA and PEG induced a continuous decrease in the Spd level when applied for a period of 3 weeks. The Spm content, which increased after PEG and ABA addition, was twice as high as that of Put during ABA treatment at most sampling points, but this difference was lower in the case of PEG. The Cad level, induced to a great extent by ABA, was much lower when compared with that of the other polyamines. The present experiments indicate that cold, osmotic stress and ABA have different effects on polyamines, and that some of these changes are affected by chromosome 5A and correlate with the level of stress tolerance.

Light intensity and spectrum affect metabolism of glutathione and amino acids at transcriptional level.

The effects of various light intensities and spectral compositions on glutathione and amino acid metabolism were compared in wheat. Increase of light intensity (low-normal-high) was accompanied by a simultaneous increase in the shoot fresh weight, photosynthetic activity and glutathione content. These parameters were also affected by the modification of the ratios of blue, red and far-red components (referred to as blue, pink and far-red lights) compared to normal white light. The photosynthetic activity and the glutathione content decreased to 50% and the percentage of glutathione disulfide (characterising the redox state of the tissues) in the total glutathione pool doubled in far-red light. The alterations in the level and redox state of the antioxidant glutathione resulted from the effect of light on its synthesis as it could be concluded from the changes in the transcription of the related genes. Modification of the light conditions also greatly affected both the amount and the ratio of free amino acids. The total free amino acid content was greatly induced by the increase of light intensity and was greatly reduced in pink light compared to the normal intensity white light. The concentrations of most amino acids were similarly affected by the light conditions as described for the total free amino acid content but Pro, Met, Thr, ornithine and cystathionine showed unique response to light. As observed for the amino acid levels, the expression of several genes involved in their metabolism also enhanced due to increased light intensity. Interestingly, the modification of the spectrum greatly inhibited the expression of most of these genes. Correlation analysis of the investigated parameters indicates that changes in the light conditions may affect growth through the adjustment of photosynthesis and the glutathione-dependent redox state of the tissues. This process modifies the metabolism of glutathione and amino acids at transcriptional level.