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.

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.

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 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.

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.

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.

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.

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.

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.

LED Lighting - Modification of Growth, Metabolism, Yield and Flour Composition in Wheat by Spectral Quality and Intensity.

The use of light-emitting diode (LED) technology for plant cultivation under controlled environmental conditions can result in significant reductions in energy consumption. However, there is still a lack of detailed information on the lighting conditions required for optimal growth of different plant species and the effects of light intensity and spectral composition on plant metabolism and nutritional quality. In the present study, wheat plants were grown under six regimens designed to compare the effects of LED and conventional fluorescent lights on growth and development, leaf photosynthesis, thiol and amino acid metabolism as well as grain yield and flour quality of wheat. Benefits of LED light sources over fluorescent lighting were manifested in both yield and quality of wheat. Elevated light intensities made possible with LEDs increased photosynthetic activity, the number of tillers, biomass and yield. At lower light intensities, blue, green and far-red light operated antagonistically during the stem elongation period. High photosynthetic activity was achieved when at least 50% of red light was applied during cultivation. A high proportion of blue light prolonged the juvenile phase, while the shortest flowering time was achieved when the blue to red ratio was around one. Blue and far-red light affected the glutathione- and proline-dependent redox environment in leaves. LEDs, especially in Blue, Pink and Red Low Light (RedLL) regimens improved flour quality by modifying starch and protein content, dough strength and extensibility as demonstrated by the ratios of high to low molecular weight glutenins, ratios of glutenins to gliadins and gluten spread values. These results clearly show that LEDs are efficient for experimental wheat cultivation, and make it possible to optimize the growth conditions and to manipulate metabolism, yield and quality through modification of light quality and quantity.

Experiences of radiological examinations of buildings in Hungary.

Natural radioisotopes occur everywhere in the environment, being a source of exposure to the general population. Everyone is continuously exposed to terrestrial and cosmic radiations both indoors and outdoors, which are the main contributors to external exposure of individuals. There were made many ambient dose rate and indoor gamma radiation and radon concentration measurements in Hungarian by different laboratories. The main goal of the present work is the summarisation and evaluation of the latest results of the Laboratory of National Public Health Center National Research Directorate for Radiobiology and Radiohygiene. The reviewed examinations were made between 1995 and 2016. The average ambient dose rate was 103 ± 17 nSv/h and the average indoor gamma dose rate was 155 ± 47 nSv/h based on the data of 382 and 581 sampling points, respectively. The average indoor radon concentration was 108 Bq/m3 with the median value of 75 Bq/m3 based on the data of 415 sampling points. We performed an additional analysis of the results of 233 personal surveyed buildings where sophisticated gamma radiation and/or indoor radon concentration measurements were made. We were also interested in has got any affect the presence of slag to the radiation levels of the buildings? We found that usually elevated radiation can be detected in houses which contain slag compared to buildings without slag. In addition we conclude that the recommended minimum duration of short-term radon measurement shall be at least three days even if it does by closed 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.

Transcript and hormone analyses reveal the involvement of ABA-signalling, hormone crosstalk and genotype-specific biological processes in cold-shock response in wheat.

The effect of one-day cold-shock on the transcriptome and phytohormones (auxin, cytokinins, abscisic, jasmonic and salicylic acids) was characterised in freezing-sensitive (Chinese Spring), highly freezing-tolerant (Cheyenne) and moderately freezing-tolerant (Chinese Spring substituted with Cheyenne's 5A chromosome) wheat genotypes. Altogether, 636 differentially expressed genes responding to cold-shock were identified. Defence genes encoding LEA proteins, dehydrins, chaperons and other temperature-stress responsive proteins were up-regulated in a genotype-independent manner. Abscisic acid was up-regulated by cold accompanied by adherent expression of its metabolic genes. Data revealed the involvement of particular routes within ABA-dependent signalling in response to cold-shock in the examined genotypes. Cold-shock affected gene expression along carbohydrate metabolic pathways. In photosynthesis, cold-shock changed the expression of a number of genes in the same way as it was previously reported for ABA. Overrepresentation analysis of the differentially expressed genes supported the ABA-signalling and carbohydrate metabolism results, and revealed some pronounced biological process GO categories associated with the cold-shock response of the genotypes. Protein network analysis indicated differences between the genotypes in the information flow along their signal perception and transduction, suggesting different biochemical and cellular strategies in their reaction to cold-shock.

The mvp2 mutation affects the generative transition through the modification of transcriptome pattern, salicylic acid and cytokinin metabolism in Triticum monococcum.

Wild type and mvp2 (maintained vegetative phase) deletion mutant T. monococcum plants incapable of flowering were compared in order to determine the effect of the deleted region of chromosome 5A on transcript profile and hormone metabolism. This region contains the vernalization1 (VRN1) gene, a major regulator of the vegetative/generative transition. Transcript profiling in the crowns of T. monococcum during the transition and the subsequent formation of flower primordia showed that 306 genes were affected by the mutation, 198 by the developmental phase and 14 by the interaction of these parameters. In addition, 546 genes were affected by two or three factors. The genes controlled by the deleted region encode transcription factors, antioxidants and enzymes of hormone, carbohydrate and amino acid metabolism. The observed changes in the expression of the gene encoding phenylalanine ammonia lyase (PAL) might indicate the effect of mvp2 mutation on the metabolism of salicylic acid, which was corroborated by the differences in 2-hydroxycinnamic acid and cinnamic acid contents in both of the leaves and crowns, and in the concentrations of salicylic acid and benzoic acid in crowns during the vegetative/generative transition. The amount and ratio of active cytokinins and their derivatives (ribosides, glucosides and phosphates) were affected by developmental changes as well as by mvp2 mutation, too.

Comparison of redox and gene expression changes during vegetative/generative transition in the crowns and leaves of chromosome 5A substitution lines of wheat under low-temperature condition.

The aim of our experiments was to investigate the effect of chromosome 5A on the thiol-dependent redox environment and on the transcription of cold- and vernalization-related genes during the vegetative/generative transition in crowns and leaves of wheat. Chinese Spring, a moderately freezing-tolerant variety, and its more and less tolerant substitution lines - [CS(Ch5A)] and [CS(Tsp5A)], respectively - with different combinations of vernalization alleles were compared. At low temperature, the amount of cystine and glutathione disulphide and the related redox potentials increased in the crowns but not in the leaves. In the crowns of the substitution lines, the concentration and redox state of thiols were different only at the vegetative and double ridge (start of the generative transition) stages. The expression of the vernalization-related VRN1 gene increased significantly during the transition both in the crowns and leaves. The transcription of the freezing tolerance-related CBF14, COR14b and COR39 genes markedly increased in both organs after 2 weeks at 4 °C when the seedlings were still in the vegetative stage. This increment was greater in CS(Ch5A) than in CS(Tsp5A). The Ch5A chromosome in CS genetic background enhanced the expression of CBF regulon even in the generative phase in crown that is the key organ for overwintering and freezing tolerance. At certain developmental stages, both the thiol and the transcript levels differed significantly in the two substitution lines.

Phytoremediation capacity of aquatic plants is associated with the degree of phytochelatin polymerization.

Phytochelatins (PCs) play important role in phytoremediation as heavy metal binding peptides. In the present study, the association between heavy metal removal capacity and phytochelatin synthesis was compared through the examination of three aquatic plants: Elodea canadensis, Salvinia natans and Lemna minor. In case of a Cd treatment, or a Cd treatment combined with Cu and Zn, the highest removal capacity was observed in L. minor. At the same time, E. canadensis showed the lowest removal capacity except for Zn. The heavy metal-induced (Cu+Zn+Cd) oxidative stress generated the highest ascorbate level in L. minor. Cd in itself or combined with the other two metals induced a 10-15-fold increase in the amount of ɣ-glutamylcysteine in L. minor while no or smaller changes were observed in the other two species. Correspondingly, the total PC content was 6-8-fold greater in L. minor. In addition, PCs with higher degree of polymerization were only observed in L. minor (PC4, PC6 and PC7) while PC2 and PC3 occurred in E. canadensis and S. natans only. The correlation analysis indicated that the higher phytoremediation capacity of L. minor was associated with the synthesis of PCs and their higher degree of polymerization.

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.