Central role of the flowering repressor ZCCT2 in the redox control of freezing tolerance and the initial development of flower primordia in wheat
© Gulyás et al.; licensee BioMed Central Ltd. 2014
Received: 5 November 2013
Accepted: 25 March 2014
Published: 7 April 2014
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.).
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.
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.
KeywordsGlutathione Redox state Initial development of flower primordia Freezing tolerance Wheat ZCCT2 gene
Throughout their life cycle plants are affected by various abiotic stresses, such as drought, extreme temperature, high salt concentration and cold, and these cause notable yield reductions in agriculture worldwide. The genetically determined level of freezing tolerance is achieved during cold acclimation, which is a relatively slow, adaptive response during autumn, when the temperature, day length and light intensity usually decrease gradually . Two main signalling pathways ensure the reprogramming of the plant metabolism in Arabidopsis during this process; one is dependent on abscisic acid (ABA), whereas the other is not . In the ABA-independent pathway the C-REPEAT BINDING FACTOR/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF/DREB1) plays a central role both in Arabidopsis and in crop species, including wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) . At least 11 different CBF gene-coding sequences were mapped at the Fr-2 locus of chromosome 5A in wheat, and CBF14 has been found to be one of the most effective ones in increasing freezing tolerance both in wheat and barley [4–6]. CBFs are characterized by a plant-specific APETALA2/ETHYLENE-RESPONSIVE ELEMENT BINDING domain (AP2/ERF) [7, 8], which interacts with the C-repeat elements present in the promoter region of their target genes. These are COLD-REGULATED (COR) genes making up the CBF regulon, the activation of which increases freezing tolerance. One of these genes, COR14b, is well characterized in barley and wheat [9, 10]. It is differentially expressed in freezing-sensitive and freezing-tolerant genotypes, and helps to protect the photosynthetic apparatus from photo-oxidative damage during exposure to high-intensity light at freezing temperatures.
The decreasing temperature during autumn also fulfils the vernalization requirement of winter cereals and ensures the correct timing of the vegetative/generative transition and the protection of freezing-sensitive flowers . In contrast, spring cereals do not require any cold treatment to induce flowering. Allelic differences in the main wheat VERNALIZATION genes VRN1, VRN2 and VRN3 determine the timing of the transition from vegetative to reproductive development. The MADS-box transcription factor VRN1 promotes flowering by inhibiting genes in the VRN2 locus [12, 13]. The VRN2 locus contains two genes, ZCCT1 and ZCCT2 (encoding ZINC-FINGER/CONSTANS, CONSTANS-LIKE, TOC1 domain transcription factors) that are both involved in flowering repression . VRN3 encodes a RAF kinase inhibitor-like protein that displays a high degree of sequence identity to Arabidopsis FLOWERING LOCUS T (FT) protein . The FT protein is a long-distance flowering signal that moves from the leaves to the apices through the phloem and promotes flowering . The interactions between these three genes and their possible effect on freezing tolerance have been recently reviewed [11, 16].
The coordinated regulation of vernalization and cold acclimation has been demonstrated in wheat, since VRN1 allelic variation influences the duration of the expression of low temperature-induced genes . In particular, mutations in the VRN1 promoter, resulting in high VRN1 transcript levels under both long and short days dampen the expression of the COR genes and lower freezing tolerance, especially under long-day conditions [16, 18]. In addition, maximum freezing tolerance usually coincides with vernalization saturation in barley . Thus, the hypothesis of VRN1 pleiotropy would explain the fact, long known to breeders, that winter-type genotypes of wheat and barley carrying a vernalization-sensitive (“winter”) allele at the VRN1 locus are more freezing-tolerant than spring-type cultivars. Another link between the regulation of vernalization and the stress response exists through the NUCLEAR FACTOR Y complex (NF-Y) consisting of A, B and C subunits. An interaction between NF-YB and ZCCT (VRN2) proteins has been detected in wheat , and NF-Y has also proved to be involved in tolerance to abiotic stress in Arabidopsis. The NF-Y complex may affect the stress response through its interaction with the bZIP proteins controlling ABA signalling, as shown in Arabidopsis.
Freezing tolerance and initial development of flower primordia, like many adaptive and developmental processes, are under redox control in plants . Unfavourable environmental conditions induce oxidative stress . Reactive oxygen species (ROS), such as superoxide radicals, hydrogen peroxide, hydroxyl radicals and singlet oxygen may accumulate to toxic levels, leading to serious injury or plant death because of redox imbalance . However, a moderate increase in the ROS level may activate various defence mechanisms through redox signalling pathways [26, 27]. The enzymatic and non-enzymatic compounds in the antioxidant system may be affected, including ascorbate and glutathione, which are the heart of the redox hub .
Alterations in ROS and antioxidant levels are not only induced by various environmental effects, but may also occur during the growth and development of plants. Tissue-, cell- and compartment-specific spatial and temporal variations in their levels are especially important. One of the most important antioxidants is glutathione [glutathione was used generically in this paper to indicate reduced glutathione (GSH) and glutathione disulphide (GSSG)], which is a multifunctional metabolite that interacts with several molecules through thiol-disulphide exchange and de-glutathionylation and also participates in detoxification, defence, metabolism, redox signalling and the regulation of transcription and protein activity [26, 29]. Changes in the amount and ratio of GSH and GSSG affect cellular reducing capacity and half-cell reduction potential, which can be used as stress markers [30, 31]. The biosynthesis of GSH was stimulated by low temperature in wheat, and this change was greater in freezing-tolerant genotypes than in sensitive ones . After 3 weeks of cold treatment there was a correlation between the H2O2, ascorbate and glutathione contents, the ascorbate/dehydroascorbate (ASA/DHA) and GSH/GSSG ratios, glutathione reduction potential and freezing tolerance in wheat . Besides their involvement in cold acclimation, ascorbate and glutathione are also involved in vernalization. The flowering time of ASA-deficient Arabidopsis mutants was shifted substantially . The overexpression of the first enzyme in glutathione biosynthesis led to earlier flowering and an increased GSSG level even at optimal growth temperature . A similar alteration was only observed in wild-type Arabidopsis at 4°C. Thus, it was suggested that an increase in GSSG content or changes in the reduction potential of glutathione partially mimicked seed vernalization treatment . Alterations in the GSSG content may influence flowering time through the OXIDATIVE STRESS2 (OXS2) transcription factor .
Based on the cited results it was hypothesized that changes in the redox potential of glutathione may affect freezing tolerance and the initial development of flower primordia in wheat. It could be predicted that the pharmacological modification of the redox state of glutathione and its precursors would modify the thiol-dependent redox potential in winter wheat genotypes even at optimum growth temperature and in spring wheat genotypes only at low temperature, since the latter usually activate the protective mechanisms after stronger environmental effects. This hypothesis was tested by comparing freezing tolerance and the initial development of flower primordia after the pharmacological modification of the glutathione redox state in one winter and one spring wheat genotype. The effect of redox changes on the expression of genes related to freezing tolerance and the initial development of flower primordia was studied.
Changes in the amount and redox state of thiols
Effect of the compounds on fresh weight
Redox regulation of gene expression
Redox control of freezing tolerance
Effect of redox treatments on the initial development of flower primordia and H2O2accumulation in the shoot apices
Modification of the redox state of thiols
It was shown that the redox state of the thiols was modified by the addition of reductants, oxidants and osmotica to the nutrient solution in hydroponically grown wheat seedlings. The redox state of glutathione was affected not only by GSH and GSSG, but also by ASA, H2O2, NaCl and PEG, indicating that this modification was not a simple feed-back control of its synthesis or reduction by the substrate, but part of a more general redox control process. ASA and H2O2 may affect the redox state of glutathione through the ascorbate-glutathione cycle, whereas NaCl and PEG may influence it through the osmotic stress-induced accumulation of H2O2. Changes in the GSSG content and EGSH/GSSG value, which were closely correlated with each other (Additional file 6) were only observed at optimal growth temperature in the freezing-tolerant Ch but not in Tsp after treatment with the various compounds, leading to great differences in these parameters between the treated seedlings of the two genotypes. At 20/17°C the EGSH/GSSG value was generally increased significantly by the treatments in Ch compared to the control, whereas there was no significant change in Tsp. However, if the chemical treatments were combined with cold (5°C), the EGSH/GSSG value exhibited a similar general change in Tsp like the one observed for Ch at 20/17°C, whereas it was partly restored to the value detected before the cold treatment in Ch. These differences between the two genotypes may be due to the different levels of antioxidants before the treatments, as shown by the higher GSSG content and EGSH/GSSG value in Ch compared to Tsp, and result in the different expression of genes related to freezing tolerance and the initial development of flower primordia in the two genotypes. This is supported by the fact that a change (20 mV) in the EGSH/GSSG value similar to that observed for wheat in the present study dramatically decreased the seed viability of four plant species .
Besides gluthathione, the other two thiols, cysteine and hydroxymethylglutathione may also modify the cellular redox environment, and consequently the structure and activity of redox-responsive molecules . However, changes in the redox state of glutathione may have the greatest effect on the redox environment, since its concentration was 3- to 4-fold greater than that of hydroxymethylgluthione and 10-fold greater than that of cysteine. The importance of the maintenance of the appropriate glutathione redox state is also indicated by the contrasting effect of 1 mM and 2 mM GSH on the redox state of cysteine and glutathione. This difference may be explained by the GSH sensitivity of the key enzyme of cysteine synthesis, adenosine-5'-phosphosulfate reductase. Accordingly, we assume that it is not affected by the 1 mM GSH concentration, but may be severely inhibited by the 2 mM GSH concentration. Consequently, the amount of Cys which is the precursor of GSH, as well as the GSH concentration will be reduced by 2 mM GSH. The marked increase in CySS and GSSG may be explained by the severe inhibition of cysteine reductase and glutathione reductase by 2 mM GSH .
It should be mentioned that at 20/17°C the various compounds added only induced an increase in the concentration of the disulphide forms and half-cell reduction potential of glutathione and the two other thiols in Ch. At 5°C, however, the redox state of both GSH and cysteine was similar in the two wheat genotypes, but in Tsp the percentage of hmGSSG was only 1–2%, and the total level (reduced + disulphide forms) was decreased to 20–30% of the control value after the majority of the treatments. By contrast, the ratio of hmGSSG was increased (to 21–65%) by nearly all treatments at 5°C in Ch. Based on this difference between the winter and spring wheat genotypes, the hmGSH/hmGSSG couple may have a special role in the regulation of the redox-responsive molecules involved in cold acclimation and the initial development of flower primordia in Poaceae, where hmGSH is a homologue of GSH (the cysteine is replaced by a serine).
The influence of cold on redox changes described earlier  was intensified when combined with various chemical treatments in the present study, both in Ch and Tsp. Both the combined application of cold and various redox agents and cold treatment alone had a greater effect on the redox system in Ch than in Tsp, and there was a strong correlation between freezing tolerance and redox changes . The effect of exogenous GSH on tolerance to low temperature was also shown in tobacco . In addition, PEG-induced osmotic stress resulted in greater changes in the amount and redox state of glutathione in a tolerant wheat genotype than in a sensitive one .
The redox state can be modified not only by various pharmacological compounds , but also by the overexpression or inhibition of the related enzymes. Thus, the increased expression of a gene encoding an enzyme with both glutathione S-transferase and glutathione reductase activities affected the amount of glutathione and its redox state in tobacco . Changes in the activity of these and other enzymes may lead to the oxidation of GSH and indirectly to that of other compounds involved in the ascorbate-glutathione cycle, and to changes in the cellular redox potential . Similar redox changes were described in mutants deficient in ascorbate and glutathione or in the enzymes involved in the reduction of their oxidised forms, leading to an increase in the cytosolic redox potential compared to wild-type plants . Similarly to the pharmacological modification of the redox state, the use of hypomorphic mutants or RNAi transgenic lines would also allow the cellular redox environment to be modified gradually, thus facilitating the study and promoting the understanding of its regulatory role.
Although monitoring the endogenous redox changes induced by various environmental effects makes it possible to clarify their role in growth, development and the stress response, the pharmacological modification of the levels of various redox components is an important tool to obtain additional information about their participation in these processes, as shown in the case of chilling in maize .
Redox control of freezing tolerance
The importance of endogenous redox changes during cold acclimation and their correlation with freezing tolerance was shown in wheat seedlings . The exogenous application of redox compounds and osmotica induced a great increase in oxidized thiols and simultaneously increased freezing tolerance in the winter wheat genotype Ch, but not in the spring genotype Tsp. Comparing the effect of the various compounds tested, it can be concluded that, rather than having specific effects, the individual compounds have a similar influence on the ascorbate-glutathione cycle and on the redox potential of the GSH/GSSG couple, resulting in an improvement in freezing tolerance. The increase in the amount of GSSG could be important in this process, since the higher tolerance of transgenic tobacco seedlings to salt and chilling stress was also related to the elevated GSSG concentrations . Changes in the amount and ratio of GSH and GSSG may influence the metabolism through the thiol/disulphide conversion or the (de)glutathionylation of proteins, which modifies their activity. Changes in the Cys/CySS and hmGSH/hmGSSG ratios may have a similar effect on proteins and subsequently on freezing tolerance as shown by the different effects of 0.5 mM and 1 mM GSSG on the ratio of disulphide forms and subsequently on freezing tolerance in Tsp. The redox potential of glutathione showed a moderate correlation with freezing tolerance (r2: 0.64) in Ch (winter wheat), whereas there was no correlation in Tsp (spring wheat) (r2: 0.08), indicating that the redox changes induced by the various treatments tested only improved freezing tolerance in the winter genotype.
Redox control of the initial development of flower primordia
In contrast to the improvement of freezing tolerance in Ch, a different adaptive strategy was observed in the spring genotype Tsp after the various treatments, involving an accelerated growth of the shoots and roots and a quicker initial development of the flower primordia. The changes observed after the combined application of cold and the various compounds were accompanied by increased GSSG content, which was also involved in the initiation of flowering in Arabidopsis. The importance of the fine regulation of GSSG content is also indicated by the stronger effect of its higher concentration on the initial development of flower primordia in Tsp in the present experiment. The redox changes depending on the redox state of glutathione may be important developmental signals affecting the whole metabolism and, consequently, the growth and development of plants. As in wheat, the involvement of ASA in controlling the initial development of flower primordia was also shown in Arabidopsis, where flowering was delayed in ASA-deficient mutants under long-day conditions . Whereas in an earlier study the developmental stage of the flower primordia did not correlate with the endogenous level of various antioxidants during the 3-week cold hardening , the exogenous application of redox agents accelerated the initial development of flower primordia during the recovery period after growth at low temperature. This contradiction can be explained by the different redox processes occurring during cold treatment and the subsequent recovery, or by the more drastic effect of exogenous redox compounds. The concentrations and oxidation levels of ascorbate and glutathione may affect the flowering time via the control of H2O2 levels through the ascorbate-glutathione cycle. This assumption is confirmed by the present findings, since the effect of exogenous H2O2 on the initial development of flower primordia was similar to that of GSH and ASA. In addition, a correlation was found between ascorbate peroxidase activity, H2O2 level and flowering time when an ascorbate peroxidase-deficient mutant was compared to wild-type and overexpressing Arabidopsis plants . The mutants, which had the highest H2O2 content, flowered first and the transgenic plants with the lowest H2O2 content last. Osmotica may also induce H2O2 accumulation and subsequently to stress-induced early flowering . The importance of H2O2 in the control of flowering at the gene expression level was shown by transcriptome analysis in Arabidopsis, where H2O2 increased the expression of a CONSTANS-LIKE protein . The genetic basis of stress-induced early flowering was recently described in plants  and the results were used to elaborate a model for the redox regulation of flowering .
Based on the present experiment, GSH-dependent redox changes inhibit ZCCT2 transcription to a greater extent in Tsp than in Ch (Figure 6B). From the negative correlation between ZCCT2 and VRN1 transcript levels in Tsp, it can be supposed that the decrease in ZCCT2 expression may be associated with the increased expression of VRN1 in the present experiment. The repression of ZCCT2 (present in the VRN2 locus) by VRN1 was reported in a recent study . ZCCT2 may control VRN1 transcript levels through its interaction with NF-YB in a regulatory loop, in which NF-YB may have a positive effect on VRN1 expression  (Additional file 6). As a result of this regulation possibility, VRN1 expression was much greater in Tsp after the majority of the treatments compared to the control. VRN1 might have a positive effect on OXS2 and FKF1, which are positive regulators of flowering. According to our hypothesis this led to an accelerated initial development of shoot apices, shown by the more developed flower primordia of seedlings treated with redox compounds and osmotica compared with the control. Although correlation analysis did not reveal any relationship between the expression of ZCCT2, OXS2 and FKF1 in wheat (Additional file 6), ZCCT2 may activate OXS2 and FKF1 through NF-YB and VRN1. Interestingly, the effect of redox changes (EGSH/GSSG) on OXS2 expression may be mediated by ABA based on correlations between EGSH/GSSG value and NCED1 transcript levels (Additional file 6). This assumption is supported by the results obtained in Arabidopsis, where ABA and OXS2 were found to have an effect on drought-induced early flowering under long-day growth conditions . Besides having a stimulating effect on the initial development of flower primordia, the increased VRN1 expression in Tsp may also be responsible for the decrease in freezing tolerance, because of the inhibition of cold-responsive genes [16, 17]. The coordinated regulation of flowering and tolerance to low temperature was also described in Arabidopsis. The redox control of the initial development of flower primordia was shown not only in Tsp but also in Ch, where the low expression of OXS2 and FKF1 (which are closely correlated with each other) may be associated with the higher ZCCT2 transcript level, as indicated by the negative correlation between ZCCT2 and the other two genes (Additional file 6). Consequently, the initial development of flower primordia was delayed. The effect of ABA on flowering was also indicated by the close correlations between NCED1, OXS2 and VRN1 in Ch.
The application of redox-altering compounds (reductants, oxidants and osmotica) differentially affected the GSSG content and the EGSH/GSSG values, and consequently the expression of the flowering repressor ZCCT2, in the two genotypes. The much greater expression of ZCCT2 in Ch compared with Tsp after the various treatments was associated with the much lower expression of VRN1, the major regulator of the initial development of flower primordia, and with greater expression of genes increasing freezing tolerance. However, the much smaller ZCCT2 transcription (due to its strong repression by the various compounds tested) in Tsp compared to Ch was associated with much greater VRN1 expression and much lower transcript levels of the genes related to freezing tolerance. Based on the correlation between the expression of genes related to the initial development of flower primordia and cold acclimation improving freezing tolerance, a model was constructed to illustrate the coordinated control of the two processes. The effect of the various redox-altering compounds is mediated by alterations in GSSG concentrations and the EGSH/GSSG value in the proposed model, in which ZCCT2 has a central regulatory role.
Plant material and treatments
A freezing-sensitive, spring habit Triticum aestivum ssp. spelta (Tsp) accession and the freezing-tolerant, winter habit Triticum ae. ssp. aestivum cv. Cheyenne (Ch) wheat cultivar were studied. Following germination in Petri dishes (1 d 25°C, 3 d 5°C, 2 d 25°C), seedlings were grown on half-strength modified Hoagland solution with a photoperiod of 16 h, at 260 μmol m-2 s-1, 20/17°C and 70/75% RH in a growth chamber (Conviron PGV-15; Controlled Env., Ltd., Winnipeg, Canada) . Twenty seedlings were cultivated on 500 ml nutrient solution in plastic pots. The solution was changed every week and at the beginning and end of the chemical treatments. After 6 days of growth, various reductants (1 and 2 mM GSH and ASA), oxidants (0.5 and 1 mM GSSG, 2 mM H2O2) or osmotica (15% PEG, 100 mM NaCl) were added to the nutrient solution as a pre-treatment, in order to observe their influence on the initial development of flower primordia and cold acclimation. GSH, GSSG, ASA and H2O2 were chosen due to their involvement in the ascorbate-glutathione cycle, to see what changes they induced in the thiol content and redox potential and how these alterations influenced the other parameters investigated, whereas NaCl and PEG were included to determine the effect of the oxidative stress induced by osmotica. The concentrations of the various compounds were determined in preliminary experiments using a dilution series. To compare their effect on the redox environment at temperatures of 20/17°C and 5°C, they were also added to the nutrient solution during the first four days of cold treatment. The 3-week cold hardening was followed by 3 weeks of recovery at 20/17°C. Samples were collected for biochemical analysis and the fresh weight of shoots and roots was measured after 3 (Additional file 4) and 7 days (Figure 4) of treatment with the various compounds. There were 3 independent experiments each with 3 parallel samples.
Determination of freezing tolerance
Freezing tolerance was estimated at the end of the 3-week cold hardening period by freezing 1 cm leaf segments (covered with aluminium foil and placed in sand in glass tubes) at -11, -13 or -15°C for 1 h. The temperature was decreased to freezing temperatures gradually (2°C for 6 h, -2°C for 15 h, then 2°C decrease every 2 h). The leaf segments were kept at 2°C for 2 h after freezing, then placed in vials containing 10 ml ultrapure water (Milli-Q 50 water purification system) and shaken overnight at room temperature. Membrane injury was determined by measuring the electrolyte leakage with a conductometer, then all the samples were boiled to destroy the cell membranes and the conductivity was determined again. Relative electrolyte leakage was characterised as the ratio of the first and the second values . High values of electrolyte leakage indicate severe damage to the cell membranes and high freezing sensitivity. The data are shown in Figure 7.
Determination of thiols
The plant material was ground with liquid nitrogen in a mortar, after which 1 ml of 0.1 M HCl was added to 200 mg plant sample. Total thiol content was determined after reduction with dithiothreitol and derivatisation with monobromobimane . For the detection of oxidised thiols, the reduced thiols were blocked with N-ethylmaleimide, and next the excess of N-ethylmaleimide was removed with toluene . Oxidised thiols were reduced and derivatised as described for total thiols. The samples were analysed after the separation of cysteine, γ-glutamylcysteine (γEC), hydroxymethylglutathione (hmGSH, a homologue of GSH in Poaceae) and GSH by reverse-phase HPLC (Waters, Milford, MA, USA) using a W474 scanning fluorescence detector (Waters). The amount of reduced thiols was calculated as the difference between the amounts of total and oxidised thiols. The half-cell reduction potential of the thiol redox couples was calculated using the Nernst equation . Data referring to Cys, hmGSH and GSH after 3 d and 7 d treatment, are shown in Additional files 1, 2, 3 and Figures 1, 2, and 3.
Morphology of shoot apices
Preliminary experiments showed that the shoot apices did not develop during the 3-week cold hardening period, therefore the initial development of flower primordia was monitored at the end of the 3-week recovery period, when the apices were isolated from the crowns of the seedlings under a Zeiss Stemi 2000-C stereomicroscope (Carl Zeiss Mikroskopie, Jena, Germany). The photos were taken with a Camedia digital camera using standardized exposure times and sensor settings. The photos of the apices are shown in Figure 8 and in Additional file 5. The developmental stages of the apices were determined based on the scale of Gardner et al. , which takes into account the appearance of new structures. The scale between 0 and 8 corresponds to the following developmental stages: 0 – vegetative apex, 1 – early elongation of the apex, 2 – elongation with single ridge, 3 – double ridge indicating the vegetative/generative transition, 4 – enlargement of spikelet primordia, 5 – empty glume primordia, 6 – lemma glume primordia, 7 – floret and anther primordia, 8 – terminal spike.
Detection of peroxides
H2O2 was visualized in the shoot apex by staining with 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dissolved in 0.1 M Na-K-phosphate buffer (pH 8.0) for 30 min . An Olympos BX 51 microscope (Olympos Optical Co. Ltd., Tokyo, Japan) fitted with a Camedia digital camera was used to study the stained shoot apices. The distribution of H2O2 in the apices is shown in Figure 8 and in Additional file 5.
Gene expression studies
Total RNA was extracted with TRI Reagent (Sigma) according to the manufacturer’s instructions and the samples were treated with DNase I enzyme (Promega). Reverse transcription was performed using M-MLV Reverse Transcriptase and Oligo(dT) 15 primer (Promega) according to the manufacturer’s instructions. The expression level of the target genes was determined with real-time RT-PCR using a CFX96 thermocycler (Bio-Rad), with primers as detailed in Additional file 7[6, 47, 53–55]. The samples originated from 3 independent experiments each with 3 repetitions. The relative quantities of the individual transcripts were calculated with the ∆∆Ct method , using the housekeeping gene encoding a protein similar to phosphoglucanate dehydrogenase (unigene identifier: Ta307930) for normalization . The gene expression value was set to 1 in control Ch plants and all other data were given as values relative to this in both genotypes in order to allow the two genotypes to be compared. The expression data are shown in Figures 5 and 6.
Data from three independent experiments were evaluated, and standard deviations are indicated on the figures. The statistical analysis was done using two-component (treatments, genotypes) analysis of variance (SPSS program). Significant differences were calculated with the t-test. The correlation analysis was done according to Guilford .
C-repeat binding transcription factor 14
Triticum ae. ssp. aestivum cv. Cheyenne
Reduction potential of cysteine
Reduction potential of hydroxymethyl-glutathione
Reduction potential of glutathione
FLAVIN-BINDING KELCH-REPEAT-BOX1 protein
Nuclear factor YB
Ascorbate peroxidase (stroma)
Triticum aestivum ssp. spelta
Major vernalization protein
ZINC-FINGER/CONSTANS, CONSTANS-LIKE, TOC1 domain flowering repressor protein.
The authors wish to thank A. Horváth and M. Fehér for their help in plant cultivation and treatment. Thanks are due to R. Boussicut, F. Taulemesse and V. Allard (INRA, UMR 1095 GDEC, France) for providing the VRN1 and ZCCT2 primer sequences, Maria Secenji (Biological Research Centre, Szeged, Hungary) for the sAPX1 primer sequences, Balázs Kalapos (Agricultural Institute, Martonvásár, Hungary) for the NCED1 primer sequences, Brend Zechmann (Karl-Franzens University, Graz, Austria), Attila Vágújfalvi, Balázs Tóth and Róbert Dóczi (Agricultural Institute, Martonvásár, Hungary) for the critical reading of the manuscript. This work was funded by the European Union (FP7-KBBE-2011-5, 289842 – ADAPTAWHEAT), by the Hungarian Research Technology and Innovation Fund (EU BONUS 12-1-2012-0024), the Hungarian Scientific Research Fund (OTKA K83642, CNK80781) and the Hungarian National Development Agency (TÁMOP-4.2.2.B-10/1-2010-0025).
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