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.

Effect of the Winter Wheat Cheyenne 5A Substituted Chromosome on Dynamics of Abscisic Acid and Cytokinins in Freezing-Sensitive Chinese Spring Genetic Background.

The effect of short- and long-term cold treatment on the abscisic acid (ABA) and cytokinin (CK) metabolism, and their main biosynthesis- and signaling-related genes were investigated in freezing-sensitive and freezing-tolerant wheat genotypes. Varieties Cheyenne and Chinese Spring substituted with the 5A Cheyenne chromosome, which represented freezing-tolerant genotypes, were compared with the freezing-sensitive Chinese Spring. Hormone levels and gene expression data indicated that the short- and long-term cold treatments are associated with specific regulation of the accumulation of cold-protective proteins and phytohormone levels, as well as the expression profiles of the hormone-related genes. The significant differences were observed between the genotypes, and between their leaf and crown tissues, too. The level of dehydrins, including WCS120 protein, and expression of WCS120 gene were considerably higher in the freezing-tolerant genotypes after 21 days of cold treatment. Expression of Cor14b and CBF14, cold-responsive regulator genes, was increased by cold treatment in all genotypes, to higher extent in freezing-tolerant genotypes. Cluster analysis revealed that the tolerant genotypes had a similar response to cold treatment, regarding expression of the ABA and CK metabolic genes, as well as hormone levels in leaves. As far as hormone levels in crowns are concerned, however, the strongly freezing-tolerant Cheyenne variety clustered separately from the Chinese Spring and the substitution line, which were more similar to each other after both 1 and 21 days of cold treatment than to Cheyenne. Based on these results we concluded that the 5A chromosome of wheat might have both a direct and an indirect impact on the phytohormone-dependent cold-induced freezing tolerance. Based on the gene expression data, novel genetic markers could be developed, which may be used to determine the freezing tolerance level in a wide range of wheat varieties.

Circadian and Light Regulated Expression of CBFs and their Upstream Signalling Genes in Barley.

CBF (C-repeat binding factor) transcription factors show high expression levels in response to cold; moreover, they play a key regulatory role in cold acclimation processes. Recently, however, more and more information has led to the conclusion that, apart from cold, light-including its spectra-also has a crucial role in regulating CBF expression. Earlier, studies established that the expression patterns of some of these regulatory genes follow circadian rhythms. To understand more of this complex acclimation process, we studied the expression patterns of the signal transducing pathways, including signal perception, the circadian clock and phospholipid signalling pathways, upstream of the CBF gene regulatory hub. To exclude the confounding effect of cold, experiments were carried out at 22 °C. Our results show that the expression of genes implicated in the phospholipid signalling pathway follow a circadian rhythm. We demonstrated that, from among the tested CBF genes expressed in Hordeumvulgare (Hv) under our conditions, only the members of the HvCBF4-phylogenetic subgroup showed a circadian pattern. We found that the HvCBF4-subgroup genes were expressed late in the afternoon or early in the night. We also determined the expression changes under supplemental far-red illumination and established that the transcript accumulation had appeared four hours earlier and more intensely in several cases. Based on our results, we propose a model to illustrate the effect of the circadian clock and the quality of the light on the elements of signalling pathways upstream of the HvCBFs, thus integrating the complex regulation of the early cellular responses, which finally lead to an elevated abiotic stress tolerance.

Light and Temperature Signalling at the Level of CBF14 Gene Expression in Wheat and Barley.

The wheat and barley CBF14 genes have been newly defined as key components of the light quality-dependent regulation of the freezing tolerance by the integration of phytochrome-mediated light and temperature signals. To further investigate the wavelength dependence of light-induced CBF14 expression in cereals, we carried out a detailed study using monochromatic light treatments at an inductive and a non-inductive temperature. Transcript levels of CBF14 gene in winter wheat Cheyenne, winter einkorn G3116 and winter barley Nure genotypes were monitored. We demonstrated that (1) CBF14 is most effectively induced by blue light and (2) provide evidence that this induction does not arise from light-controlled CRY gene expression. (3) We demonstrate that temperature shifts induce CBF14 transcription independent of the light conditions and that (4) the effect of temperature and light treatments are additive. Based on these data, it can be assumed that temperature and light signals are relayed to the level of CBF14 expression via separate signalling routes.

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.

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.

Light-quality and temperature-dependent CBF14 gene expression modulates freezing tolerance in cereals.

UNLABELLED: C-repeat binding factor 14 (CBF14) is a plant transcription factor that regulates a set of cold-induced genes, contributing to enhanced frost tolerance during cold acclimation. Many CBF genes are induced by cool temperatures and regulated by day length and light quality, which affect the amount of accumulated freezing tolerance. Here we show that a low red to far-red ratio in white light enhances CBF14 expression and increases frost tolerance at 15°C in winter Triticum aesitivum and Hordeum vulgare genotypes, but not in T. monococcum (einkorn), which has a relatively low freezing tolerance. Low red to far-red ratio enhances the expression of PHYA in all three species, but induces PHYB expression only in einkorn. Based on our results, a model is proposed to illustrate the supposed positive effect of phytochrome A and the negative influence of phytochrome B on the enhancement of freezing tolerance in cereals in response to spectral changes of incident light. KEY WORDS: CBF-regulon, barley, cereals, cold acclimation, freezing tolerance, light regulation, low red/far-red ratio, phytochrome, wheat.

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.

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.