JXB Advance Access originally published online on October 21, 2008
Journal of Experimental Botany 2008 59(15):4133-4143; doi:10.1093/jxb/ern253
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RESEARCH PAPER |
Modulation of thiamine metabolism in Zea mays seedlings under conditions of abiotic stress
1Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland
2Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
* To whom correspondence should be addressed: E-mail: rapala{at}mol.uj.edu.pl
Received 7 August 2008; Revised 14 September 2008 Accepted 16 September 2008
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Abstract |
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The responses of plants to abiotic stress involve the up-regulation of numerous metabolic pathways, including several major routes that engage thiamine diphosphate (TDP)-dependent enzymes. This suggests that the metabolism of thiamine (vitamin B1) and its phosphate esters in plants may be modulated under various stress conditions. In the present study, Zea mays seedlings were used as a model system to analyse for any relation between the plant response to abiotic stress and the properties of thiamine biosynthesis and activation. Conditions of drought, high salt, and oxidative stress were induced by polyethylene glycol, sodium chloride, and hydrogen peroxide, respectively. The expected increases in the abscisic acid levels and in the activities of antioxidant enzymes including catalase, ascorbate peroxidase, and glutathione reductase were found under each stress condition. The total thiamine compound content in the maize seedling leaves increased under each stress condition applied, with the strongest effects on these levels observed under the oxidative stress treatment. This increase was also found to be associated with changes in the relative distribution of free thiamine, thiamine monophosphate (TMP), and TDP. Surprisingly, the activity of the thiamine synthesizing enzyme, TMP synthase, responded poorly to abiotic stress, in contrast to the significant enhancement found for the activities of the TDP synthesizing enzyme, thiamine pyrophosphokinase, and a number of the TDP/TMP phosphatases. Finally, a moderate increase in the activity of transketolase, one of the major TDP-dependent enzymes, was detectable under conditions of salt and oxidative stress. These findings suggest a role of thiamine metabolism in the plant response to environmental stress.
Key words: Abscisic acid, antioxidant enzymes, maize, oxidative stress, salt stress, thiamine phosphate synthase, thiamine pyrophosphokinase, transketolase, water stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Thiamine (vitamin B1) functions as a precursor of thiamine diphosphate (TDP) which serves as a coenzyme in a number of the major metabolic pathways, including acetyl-CoA synthesis, the tricarboxylic acid cycle, anaerobic ethanolic fermentation, the oxidative pentose phosphate pathway, the Calvin cycle, the branched–chain amino acid pathway, and plant pigment biosynthesis (Friedrich, 1987). Animals can only synthesize TDP from exogenous thiamine sources but bacteria, yeasts, and higher plants can do so from simple universal precursors (for reviews, see Begley et al., 1999; Nosaka, 2006; Roje, 2007). Thiamine biosynthetic pathways (Fig. 1) include independent syntheses of two substituted thiazole and pyrimidine compounds, 4-methyl-5-(2-hydroxyethyl)thiazole phosphate (HET-P) and 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate (HMP-PP), which are then coupled to form thiamine monophosphate (TMP). Recent evidence suggests that HET-P is synthesized by plants via a pathway similar to that in yeast, and involving cysteine, glycine, and probably NAD+ (Chatterjee et al., 2007). One of the later steps in thiazole ring formation is catalysed by an enzyme encoded by the Arabidopsis thi1 gene (Godoi et al., 2006) or its orthologues that have been identified in many plant species. In contrast, and with similarities to bacteria rather than yeast, the pyrimidine moiety (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate, HMP-P) seems to be formed in plants from 5-aminoimidazole ribonucleotide (AIR), with the involvement of a protein encoded by the thiC gene (Raschke et al., 2007). The last two steps in the TMP synthesis pathway involve the phosphorylation of HMP-P by HMP-P kinase (EC 2.7.4.7 [EC] ) and the condensation of HMP-PP with HET-P by TMP synthase (TMPS, EC 2.5.1.3 [EC] ). In plants, these two reactions are catalysed by a single two-domain protein, TH1 in Arabidopsis (Ajjawi et al., 2007b) and its orthologues in other plant species (Rapala-Kozik et al., 2007). It is generally accepted that plant TMP synthesis proceeds in plastids (Belanger et al., 1995). However, TDP can only be formed from free thiamine in the cytoplasm due to the catalytic action of thiamine pyrophosphokinase (TPK, EC 2.7.6.2 [EC] ) (Ajjawi et al., 2007a). Hence, the dephosphorylation of TMP, the export of de novo synthesized TMP or thiamine from chloroplasts to cytoplasm, and the import of TDP by chloroplasts and mitochondria are essential parts of the overall mechanism of TDP synthesis and interorganellar distribution.
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The adequate provision of TDP is essential for maintaining the optimal activities of all TDP-dependent enzymes. Hence, the organisms which can synthesize thiamine de novo would be expected to co-ordinate the rate of thiamine biosynthesis with the activities of the TDP-dependent metabolic pathways. Interestingly, TDP itself may take part in the regulation of thiamine biosynthesis by riboswitch binding (Bocobza et al., 2007) or through the activity of TDP-dependent transcription factors (Nosaka, 2006).
The basis for the present study was the assumption that a regulatory link between thiamine biosynthesis and thiamine-dependent pathways may be detectable in higher plants under conditions of abiotic or biotic stress. Several of the numerous metabolic pathways which respond to stress factors engage TDP-dependent enzymes and some universal examples include the accumulation of soluble sugars that have multiple regulatory roles (Couée et al., 2006), increased energy generation for the synthesis of numerous stress-related proteins or osmoprotectants (Zhu, 2002), and the up-regulation of NADPH-requiring antioxidant systems (Valderrama et al., 2006). However, only a few TDP-dependent enzymes have actually been reported to be up-regulated under stress conditions, the best studied example being pyruvate decarboxylase (EC 4.1.1.1 [EC] ) which directs glycolysis toward ethanolic fermentation (Drew, 1997). Pyruvate decarboxylase has been shown to be strongly induced in Arabidopsis and a few other plant species by anoxia and by treatment with low temperatures, mannitol, high salinity, abscisic acid (ABA), oxygen radical generation or wounding (Conley et al., 1999; Kürsteiner et al., 2003).
There is currently no direct experimental evidence for the up-regulation of thiamine/TDP biosynthesis in plants under abiotic or biotic stress other than a recent finding that the expression of the thiazole synthesizing gene thi1 in Arabidopsis is activated by flooding, high salinity, and sugar deprivation (Ribeiro et al., 2005). There are, however, other reported phenomena that suggest a role of thiamine in plant stress responses, although the exact molecular mechanisms remain to be elucidated. These include the observed accumulation of thiamine triphosphate and adenosine thiamine triphosphate, recently hypothesized to function as universal signalling molecules, in withering Arabidopsis (Makarchikov et al., 2003; Bettendorff et al., 2007) and the induction of systemic acquired resistance in Arabidopsis and several other plant species against some fungal and bacterial infections through the activation of pathogenesis-related genes (PR) by thiamine compounds (Ahn et al., 2005). On the other hand, a rice gene OsDR8 was shown to have a dual function in disease resistance induction and thiamine accumulation (Wang et al., 2006). This gene shows a high sequence similarity to the thiazole-synthesizing gene thi1 of Arabidopsis and its baker's yeast orthologue THI4 which, in addition, have been suggested to play a role, although as yet unclear, in DNA repair mechanisms (Machado et al., 1996, 1997).
In the present study, the effects of specific abiotic stress conditions on thiamine biosynthesis in Zea mays were evaluated. The maize seedlings were separately subjected to water, salt, and oxidative stress and changes in the ABA and antioxidant enzyme levels were evaluated together with the properties of the thiamine metabolism pathways, i.e. the concentration of thiamine and its phosphate esters, the activity of thiamine-synthesizing and activating enzymes and the activity of a major TDP-dependent enzyme, transketolase (TK, EC 2.2.1.1 [EC] ).
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Materials and methods |
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Plant materials and growth conditions
Seeds of Zea mays L. were surface-sterilized in 20% (v/v) commercial bleach for 20 min and then washed six times with sterile distilled water. The seeds were then germinated for 2 d and grown in sterilized soil for a further 8 d in a greenhouse at a temperature of between 24–26 °C, under a photosynthetically active light source with the photon flux density of 400 µmol m–2 s–1, and with the photoperiod of 14/10 h (day/night). The seeds were also watered daily. For a further two days, the seedlings were irrigated daily with solutions of either (i) polyethylene glycol 6000 (PEG) at 5%, 10%, and 20%, (ii) NaCl at 0.1, 0.2, and 0.4 M, or (iii) H2O2 at 10, 25, and 50 mM.
Extract preparation
Maize seedling leaves were homogenized using a mortar and pestle under liquid nitrogen. Soluble proteins were then extracted by homogenizing the leaf powder (5 g) in 30 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 0.15 M NaCl, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 mM EDTA, 1 mM dithiothreitol (DTT), and 1% polyvinylpyrrolidone. The homogenate was then centrifuged at 14 000 g for 30 min at 4 °C. The resulting supernatant was stored at –20 °C and used for the detection of thiamine and its phosphate esters, and for antioxidant enzyme assays. To determine the activity of enzymes involved in thiamine biosynthesis, the extracts were extensively dialysed against 50 mM phosphate buffer (pH 8.0) for 48 h at 4 °C. The protein concentration in the homogenates was determined by the method of Bradford (1976), using bovine serum albumin standards.
For ABA detection, the leaf powder was extracted for 24 h at 4 °C in the dark with a solution of 80% methanol in 20 mM bicarbonate buffer (pH 8.0). The mixture was then centrifuged at 5000 g for 15 min at 4 °C. The supernatant was vacuum-dried at <30 °C and stored at –20 °C until use in the ABA assay.
Quantification of thiamine and thiamine phosphate esters
The maize seedling extracts were treated with 12% trichloroacetic acid (TCA) (1:2 v/v) at 4 °C and centrifuged for 5 min at 5000 g. The protein pellet was discarded and TCA was removed from the supernatant by ether extraction. The thiamine, TMP, and TDP levels were quantified in the de-acidified supernatant by reverse-phase high pressure liquid chromatography (RP-HPLC) separation, with a post-column derivatization and fluorometric detection (Lee et al., 1991). Solvent A contained 15 mM ammonium citrate (pH 4.2) and solvent B consisted of 0.1 M formic acid containing 0.4% diethylamine (pH 3.2). A gradient elution (0–90% B in 16 min) at a flow rate of 1 ml min–1 was applied. The fluorogenic derivatization was performed using 0.0025% sodium hexacyanoferrate (III) in 2.25% NaOH, pumped through a Gilson Minipuls peristaltic pump with a flow rate of 0.8 ml min–1. The HPLC equipment used consisted of (i) a Shimadzu (Kyoto, Japan) model LC-9A HPLC pump with a Shimadzu FCV-9AL proportioning valve, (ii) a Knauer (Bad Homburg, Germany) model A0263 manual injector equipped with a 100 µl sample loop, (iii) a Merck cartridge LiChrosphere 100RP-18 (5 µm) column (250x4 mm) with a cartridge precolumn (4x4 mm), (iv) a Shimadzu model RF-535 fluorescence monitor set at 365 nm excitation wavelength and 430 nm emission wavelength, and (v) a Shimadzu Class-VP (version 4) software/hardware package for pump control and data acquisition and analysis.
Assays for enzymes involved in thiamine synthesis and utilization
The activity of TMPS was determined in a mixture containing 50 mM phosphate/borate buffer (pH 9.0), 60 µM HMP-PP (synthesized according to Brown, 1970), 60 µM HET-P (synthesized according to Leder, 1970), 10 mM MgCl2 and 200 µl of extracts in a final volume of 0.5 ml. After incubation of the assay mixture for 2 h at 37 °C, the reaction was stopped by the addition of HCl (to the final concentration of 0.2 M) and heating at 90 °C for 5 min. The precipitate was removed by centrifugation and the levels of TMP in the supernatant (after 3-fold dilution with mobile phase) were determined by isocratic RP-HPLC with post column derivatization and fluorescence detection. The mobile phase consisted of a mixture of 0.15 M ammonium citrate (pH 4.2) and 80% acetonitrile with 0.08% TFA (90:10 v/v). A flow rate of 1 ml min–1 was applied.
The TPK assay mixture (0.5 ml) contained 50 mM phosphate/borate buffer (pH 9.0), 50 µM thiamine, 25 mM MgCl2, 20 mM ATP, and 200 µl of protein sample. After incubation of the assay mixture for 2 h at 37 °C, the reaction was stopped by acidification and heating at 90 °C for 5 min. The samples were centrifuged at 10 000 g for 10 min, diluted three times with 0.1 M potassium phosphate and analysed for TDP content by RP-HPLC with post column derivatization and fluorimetric detection as described above for the determination of thiamine and its phosphate esters.
For the assay of TDP/TMP acid phosphatase (EC 3.1.3.1 [EC] ) (TPPH), the products of TDP hydrolysis were determined by RP-HPLC in the same chromatographic system as that described above for the determination of TPK activity. The assay mixture (0.5 ml) contained 50 mM acetate (pH 5.0), 100 µM TDP, and 200 µl of protein sample. After incubation of the assay mixture for 30 min at 37 °C, the reaction was stopped and subjected to HPLC analysis.
The TK activity was determined spectrophotometrically by coupling its reaction with triosephosphate isomerase and glycerol-3-phosphate dehydrogenase (Masri et al., 1988). The assay mixture (final volume 200 µl) contained 67 µl of 0.05 M phosphate buffer, 4 µl of 30 mM MgCl2, 4 µl of 10 mM TDP, 10 µl of 5 mM NADH, 0.5 µl of glycerol-3-phosphate dehydrogenase–triosephosphate isomerase mix (2 mg ml–1), and 100 µl of leaf extract. After preincubation at 37 °C for 10 min, the pentose phosphate mixture (10 µl) was added. The decrease in the absorbance of NADH at 340 nm was recorded at 37 °C using a Power WaveX-Select microplate reader (Bio-Tek Instruments, Winooski, VT). The enzyme activity was expressed in terms of the amounts of NADH that were oxidized under these conditions.
Assays for antioxidant enzymes
Catalase (EC 1.11.1.6
[EC]
) (CAT) activity was determined by monitoring the H2O2 decomposition at 240 nm for 3 min (Aebi, 1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 5 mM H2O2 and 25 µl of enzyme extract in a 1 ml final volume. CAT activity was expressed as µmol H2O2 decomposed min–1 mg–1 protein.
The activity of glutathione reductase (EC 1.6.4.2 [EC] ) (GR) was determined in a 1 ml mixture containing 50 mM potassium phosphate buffer (pH 8.0), 0.1 mM Na2EDTA, 3 mM oxidized glutathione, and 100 µl of enzyme extract. The reaction was initiated by the addition of NADPH (0.15 mM) and the rate of the NADPH oxidation was monitored at 340 nm for 10 min (Jablonski and Anderson, 1978). Enzyme activity was expressed as nmol NADPH oxidized min–1 mg–1 protein.
Ascorbate peroxidase (EC 1.11.1.11 [EC] ) (APX) activity was determined by following the decrease in the absorbance at 290 nm for 3 min in a 1 ml mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H2O2, 0.1 mM EDTA, and 100 µl of enzyme extract. The activity of APX was expressed as the nmol ascorbate oxidized min–1 mg–1 protein (Nakano and Asada, 1981).
Assay for abscisic acid
Lyophilized extracts from 0.5 g of fresh maize seedling leaves were dissolved in TBS buffer (25 mM TRIS-HCl, 100 mM NaCl, 100 mM MgCl2, 3 mM NaN3, pH 7.5). Aliquots from this solution were then analysed for ABA levels using an indirect enzyme linked immunosorbent assay (ELISA) from a commercial kit (Phytodetect) according to the manufacturer's instructions.
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Results |
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In the present study, two sets of parameters were simultaneously evaluated in Zea mays seedlings subjected to abiotic stress: (i) typical hallmarks of the plant response to various types of stress, and (ii) the status of the thiamine biosynthesis and activation pathways. The first set included the ABA levels and the activities of three antioxidant enzymes, CAT, APX and GR. Thiamine metabolism was characterized by (i) the levels of total thiamine and each of the individual thiamine compounds, (ii) the activity of the enzymes TMPS, TPK, and TDP/TMP dephosphorylating phosphatase (TPPH) that control the pools of thiamine compounds, and (iii) the activity of a TDP-dependent enzyme, TK.
Water stress
As expected, the levels of ABA in maize seedlings increased sharply under conditions of water stress (drought) induced by treatment with PEG (Fig. 2). Of the three antioxidant enzymes tested in the present analyses, only APX was found not to respond to the drought-stress conditions.
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The total thiamine content in the seedling leaves was also observed to increase under conditions of water stress (by up to 55% in the presence of 20% PEG; Fig. 3A). In addition, the free thiamine to TDP ratio appeared to increase due to a drop in the TDP levels by up to 30% following treatment with 20% PEG. By contrast, the levels of TMP were relatively constant within the range of PEG concentrations used. The activities of the enzymes that control the endogenous pools of thiamine compounds also increased in a PEG concentration-dependent manner (Fig. 3B) in the order: TPPH >TPK >TMPS. At the highest PEG concentration, the activities of TPPH and TPK increased to 240% and 180% of the control levels, respectively. On the other hand, the apparent activation of TMPS under conditions of 5–20% PEG was not statistically significant. Moreover, equivocal results were obtained for TK activity (Fig. 3B) which seemed to increase significantly (by up to 140%) in the 5–10% PEG treated seedlings, but to return to control levels thereafter.
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Salt stress
Under the influence of NaCl at a concentration range of 0.1–0.2 M, increases in the ABA levels and antioxidant enzyme activities could be observed in the maize seedling leaves (Fig. 4). At yet higher salt concentration (0.4 M) these parameters remained relatively constant or even dropped slightly. In contrast to conditions of water stress, salt stress significantly induced the activity of APX. The effects of high salt on the activities of the other antioxidant enzymes were weaker than those of PEG-induced drought stress.
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The effects of salt stress upon the total thiamine pool and the distribution of thiamine compounds very closely matched those observed under water stress conditions (Fig. 5A). In addition, and similar to drought conditions, the activities of TPK and TPPH progressively increased by 80–90% (Fig. 5B). Once again, the smallest changes were observed for TMPS activity. Interestingly, a statistically significant enhancement of TK activity by c. 50% was found to be induced by salt stress, detectable for salt concentrations of 0.2 M and higher (Fig. 5B).
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Oxidative stress
Oxidative stress was induced by treating the maize seedlings with hydrogen peroxide at concentrations ranging from 10–50 mM. The determination of the ABA levels was influenced by artefacts as the measured values were lower than those obtained for the untreated seedlings (Fig. 6). Nevertheless, these determined levels increased nearly 2-fold between 10 mM and 50 mM H2O2. Of the antioxidant enzymes tested, CAT and APX were found to be strongly activated in a H2O2 concentration-dependent manner. The effects of H2O2 on GR activity were the lowest among the three types of abiotic stress analysed.
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The effects of H2O2 on the total thiamine pool and upon the distribution of thiamine compounds in maize seedling leaves were qualitatively similar to those observed for water and salt stress (Fig. 7A). However, the increase in total thiamine was much higher under oxidative stress conditions, and showed nearly a 2-fold up-regulation at the 50 mM H2O2 exposure level. Moreover, the free thiamine levels reached a 3-fold increase compared with normal tissue. Exclusive also to this type of abiotic stress, a regular increase (of up to 200%) in the TMP levels was observed at higher H2O2 concentrations. Surprisingly, however, TPPH was relatively insensitive to H2O2 treatment (Fig. 7B). The TPK activity increased 2-fold, which is the strongest response of this enzyme among the different abiotic stress conditions studied here. A clear 2-fold TMPS activity gain was also observed but only at the highest H2O2 concentration. Finally, and similar to the response to salt stress, the H2O2 treatment at 25 mM enhanced TK activity by nearly 70% (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The hypothesis that the de novo biosynthesis of thiamine and its further conversion (phosphorylation/dephosphorylation) in higher plants is up-regulated during the plant responses or adaptations to abiotic or biotic stress is relatively novel. Only one plant gene family, comprising orthologues of the yeast THI4 gene that control a single step in the complex thiamine biosynthetic pathways, has been shown to be up-regulated under conditions of abiotic stress (Ribeiro et al., 2005) and to play specialized roles against DNA damage or pathogen attack (Machado et al., 1996, 1997; Wang et al., 2006). Intuitively, a primary reason for an increase in the thiamine synthesis rate would be to supply TDP for the enzymes that are critically involved in several key reactions in the metabolic network and which must be reconfigured to deal with stress factors and to support adaptive responses. At least two major TDP-dependent pathways, the oxidative pentose phosphate pathway (Couée et al., 2006; Baxter et al., 2007) and the ethanolic fermentation (Drew, 1997; Conley et al., 1999; Kürsteiner et al., 2003) have been reported in numerous studies to be accelerated or induced under different abiotic stress conditions. In addition, pyruvate decarboxylase, the TDP-dependent enzyme involved in ethanolic fermentation, has been shown unequivocally to be up-regulated under specific stress conditions.
In the present study, the effects of abiotic stress on plant thiamine metabolism have been evaluated further. The model system for this study consisted of 8-d-old maize seedlings treated with PEG, NaCl or H2O2, which are agents commonly used to induce water, salt, and oxidative stress, respectively. Because of the relatively long stress duration time, the reported phenomena are assumed to represent an adaptation rather than an immediate reaction to the particular stress conditions. A set of control parameters, including the level of the universal hormone, ABA, and the activities of antioxidant enzymes such as CAT, GR, and APX was first evaluated (Figs 2, 4, 6) and confirmed that the response of the maize seedlings to these specific stress conditions displayed characteristics similar to those previously reported for the same plant model (Jiang and Zhang, 2002; Azevedo Neto et al., 2005).
Thiamine metabolism is dependent upon pools of individual thiamine compounds (free thiamine, TMP, and TDP) the sum of which is the total thiamine level. In the absence of any uptake of exogenous thiamine, as is the case in the plant model used for the present study, these pools are determined by the activity of the biosynthetic pathways that generate TMP, the activity of TPK that activates thiamine to TDP and the numerous phosphatases capable of dephosphorylating TDP and/or TMP. The finding that the total thiamine pool increases in the maize seedling leaves under stress conditions is a novel observation and for the first time clearly suggests a link between thiamine metabolism and abiotic stress in plants. Interestingly, this general increase in the thiamine content is associated with a moderate drop in the TDP levels and a high gain of free thiamine. Significantly, this result is unlikely to be just an artefact caused by the mobilization of numerous non-specific phosphatases following leaf tissue homogenization. If this has been the case, the TMP levels should also have dropped but they have remained stable except under oxidative stress where the TMP pool seemed to increase. It appears therefore that upon abiotic stress, the maize seedling does not appreciably increase the pool of TDP for use by the major TDP-dependent enzymes but, instead, accumulates reserves of free thiamine for their further activation, i.e. pyrophosphorylation by TPK, in response to increased metabolic demands. It is noteworthy also that the drop in the steady-state TDP levels, albeit moderate, may be important as TDP is probably the major regulatory factor for thiamine biosynthesis (Nosaka, 2006). The mechanisms underlying the repression of plant thiamine biosynthesis by thiamine compounds, probably exclusively by TDP, are not yet fully understood, but recently, a TDP-dependent riboswitch has been identified in the 3'-untranslated region of the pyrimidine-synthesizing gene of many flowering plants thiC (Bocobza et al., 2007; Raschke et al., 2007). One could speculate that virtually all new TDP molecules synthesized in the maize seedling in response to abiotic stress are quickly bound to TDP-requiring apoenzymes to maintain a low level of free TDP, the only form of this molecule that can exert regulatory effects.
In the present study also, the activities of three enzyme systems that control the pools of thiamine compounds in plant tissues were assessed. Of the major thiamine biosynthetic enzymes that are now known, an activity assay is only available for TMPS, which is the THI3 protein of maize (Rapala-Kozik et al., 2007). Inconsistent with the above-mentioned increase in the total thiamine pool, the activity of TMPS only slightly increased under all of the stress conditions tested except for the highest concentration of hydrogen peroxide, where a 2-fold activity gain was observed. In the recent study (Rapala-Kozik et al., 2007), an allosteric regulation of maize TMPS by ATP was suggested. This enzyme may also be inducible, as its activity increases during maize seed germination and seedling growth (Go
da et al., 2004). However, those two findings do not definitively show that TMPS is the major regulatory molecule of the entire thiamine biosynthesis pathway in plants as other biosynthetic step(s) may be rate-limiting and thus be target(s) of critical regulatory mechanisms. Indeed, abiotic stress-related mechanisms were recently suggested for the regulation of thi1 gene through the ABA-responsive element (Ribeiro et al., 2005), and the THIC iron–sulfur cluster protein which may be redox-activated by the thioredoxin–ferredoxin system (Raschke et al., 2007). Hence, the activity of TMPS may remain constant even when the overall rate of thiamine biosynthesis changes.
In contrast to TMPS, the activity of TPK in the maize seedlings consistently increased under each of the abiotic stress conditions studied. This novel finding is consistent with the reported induction of this enzyme during seed germination and seedling growth (Goda et al., 2004). In contrast to the major thiamine biosynthetic genes, the repression of TPK synthesis by TDP is incomplete and a moderate activity of the enzyme is expressed constitutively (Nosaka, 2006). The up-regulation of TPK in response to abiotic stress is easily interpretable in terms of increased demands for TDP from TDP-dependent enzymes.
The activity gain of TPPH in the maize seedlings under abiotic stress was highest for water stress, comparable to that of TPK in salt-stress conditions, but the most marginal of the enzymatic changes observed under oxidative stress. This activity is probably attributable to multiple acid phosphatases of broad substrate specificity. The concerted increase observed for both phosphatase and TPK activities may ensure a quick equilibration between the free thiamine reservoir and free TDP pool and maintain the latter at a low level that will limit the extent of thiamine biosynthesis repression.
Among the major TDP-dependent enzymes, TK appeared to be most suitable to test for any up-regulation under abiotic stress conditions, whereby an increase in thiamine/TDP synthesis might be driven. The TK-catalysed interconversions of various sugar phosphates are part of the Calvin cycle and the oxidative pentose phosphate pathway and are also a source of important metabolites. These include ribose-5-phosphate which acts as a precursor of AIR which is the substrate for the biosynthesis of pyrimidine moiety (HMP-P) of thiamine molecule (Roje, 2007). In many plant species, TK operates both in plastids and in the cytoplasm but even in plants with an exclusively chloroplastic TK or incomplete cytoplasmic pentose phosphate pathway the sugar-phosphate pools in both compartments are exchangeable through effective transporters located in the organellar membrane (Kruger and von Schaewen, 2003). The pentose phosphate pathway produces NADPH which feeds a variety of ROS-scavenging systems, including the GR-catalysed generation of one of the most powerful low molecular mass antioxidants, reduced glutathione. NADPH is also required to regenerate ascorbate from monodehydroascorbate, the product of the hydrogen peroxide-scavenging APX-catalysed reaction (Arora et al., 2002). Therefore, the activation of the oxidative pentose phosphate pathway in plant cells is considered to be a major metabolic defence mechanism against oxidants (Couée et al., 2006; Valderrama et al., 2006; Baxter et al., 2007). Albeit not a regulatory enzyme, TK does adjust its expression in response to changes within the entire pathway to guarantee a balanced flow of all intermediates (Henkes et al., 2001). However, the stress-induced up-regulation of this enzyme has only been reported previously for a very specific case of re-hydration of the desiccation-tolerant plant Craterostigma plantagineum (Bernacchia et al., 1995). The present observation of a significant gain of TK activity in maize seedlings under salt and oxidative stress conditions is the first evidence that this TDP-dependent enzyme may play a more universal role in the plant response to abiotic stress.
The present results can be briefly discussed in the more general context of the metabolic network adjustments that occur in response to abiotic stress (Fig. 8). For all three types of stress studied herein, the generation of ROS increases above normal levels in all plant cell compartments (Zhu, 2002). In chloroplasts, the major source of excessive ROS is the photosynthetic electron chain which becomes overreduced due to the inhibition of photosynthetic carbon fixation associated with the inactivation of Calvin cycle enzymes (Holaday et al., 1992). A recovery of the latter, TK-dependent pathway will occur during the subsequent adaptation phase (Strand et al., 1999). To scavenge ROS, several chloroplast-localized antioxidant systems are up-regulated, some of which are NADPH-dependent, such as the Asada–Halliwell pathway (the glutathione–ascorbate cycle) (Arora et al., 2002). The major source of NADPH for chloroplast antioxidant defence is probably generated via photosynthesis rather than by the oxidative pentose phosphate pathway. Another important class of antioxidants, the carotenoids, is produced in chloroplasts by a mevalonate-independent pathway of isoprenoid synthesis which engages a TDP-dependent enzyme, 1-deoxy-D-xylulose 5-phosphate synthase (EC 4.1.3.37 [EC] ) (Lange et al., 1998). Unfortunately, there have been no reports of any possible up-regulation of this enzyme or indeed the entire pathway under abiotic stress.
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In the cytoplasm, ROS may partially inhibit the enzymes involved in early glycolytic steps resulting in a re-routing of the main carbohydrate-metabolic flux from glycolysis to the pentose phosphate pathway (Ralser et al., 2007). The activation of the latter pathway is induced by the up-regulation of the regulatory enzymes involved in the oxidative steps (Couée et al., 2006; Valderrama et al., 2006), but also by some non-regulatory enzymes such as TK, as shown in this study. The up-regulated pentose phosphate pathway produces more NADPH, which is recycled via numerous antioxidant systems, such as the ascorbate-glutathione cycle, to restore the cytoplasmic redox equilibrium quickly (Valderrama et al., 2006). The main glycolytic pathway may also be directed to cytoplasmic ethanolic fermentation due the inhibition of the mitochondrial conversion of pyruvate to acetyl-CoA (see below). The up-regulation of the ethanolic fermentation TDP-dependent enzyme, pyruvate decarboxylase, under different types of abiotic stress has been well characterized (Drew, 1997; Kürsteiner et al., 2003). The entire glycolytic fermentation process allows for a quick generation of energy until the normal, much more effective aerobic respiration is recovered (Kürsteiner et al., 2003).
The mitochondrial production of acetyl-CoA and the tricarboxylic acid cycle ceases during the first response to abiotic stress (Sweetlove et al., 2002; Taylor et al., 2004; Baxter et al., 2007), partly due to the oxidative inactivation of lipoic-acid dependent components of pyruvate dehydrogenase and 
-ketoglutarate dehydrogenase complexes. These multienzyme complexes also contain TDP-dependent components. Both pathways are restored during the adaptation phase (Taylor et al., 2004).
The up-regulated TDP-requiring enzymes, which include cytoplasmic TK, cytoplasmic pyruvate decarboxylase and, possibly, also chloroplastic 1-deoxy-D-xylulose 5-phosphate synthase and, in addition, in the adaptation phase, chloroplastic TK, mitochondrial pyruvate dehydrogenase, and mitochondrial -ketoglutarate dehydrogenase drain the free TDP pools in chloroplasts, mitochondria, and the cytoplasm which are easily exchangeable via specialized transporters in the organellar membranes. This may be one factor that contributes to the acceleration of chloroplastic thiamine biosynthesis pathways which may also be activated by other mechanisms such as ABA or ROS signalling. In the cytoplasm, the up-regulated TPK restores the free TDP pool and, together with up-regulated phosphatases, allows for its quick equilibration with the free thiamine reservoir and maintains TDP at a level suitable for both feeding TDP-dependent enzymes and controlling thiamine biosynthesis.
In conclusion, the current work shows for the first time that the known effects of the plant response to abiotic stress correlate with the parameters that characterize plant thiamine metabolism. The major findings presented in stressed plant tissue are the increase of total thiamine content, the gain of the thiamine activating enzyme, TPK, and the induction of a representative TDP-dependent enzyme, TK.
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Acknowledgements |
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This work was supported in parts by grant 2P04C 017 27 from the Ministry of Science and Higher Education, Poland (to MR-K).
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Abbreviations |
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ABA, abscisic acid; AIR, 5-aminoimidazole ribonucleotide; APX, ascorbate peroxidase; CAT, catalase; DTT, dithiothreitol; ELISA, enzyme linked immunosorbent assay; GR, glutathione reductase; HET-P, 4-methyl-5-(2-hydroxyethyl)thiazole phosphate; HMP-P, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate; HMP-PP, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate; PEG, polyethylene glycol; PMSF, phenylmethylsulphonyl fluoride; ROS, reactive oxygen species; RP-HPLC, reverse-phase high pressure liquid chromatography; TCA, trichloroacetic acid; TDP, thiamine diphosphate; TK, transketolase; TMP, thiamine monophosphate; TMPS, thiamine monophosphate synthase; TPK, thiamine pyrophosphokinase; TPPH, TDP/TMP acid phosphatase.
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