The upregulation of thiamine (vitamin B₁) biosynthesis in Arabidopsis thaliana seedlings under salt and osmotic stress conditions is mediated by abscisic acid at the early stages of this stress response

Vitamin B₁ (thiamine) is essential for the proper functioning of all living organisms as it plays important roles in carbohydrate catabolism, NADPH and ATP synthesis and in the formation of nucleic acids. The biologically active form of thiamine is its diphosphate analog (TDP) which functions as a cofactor for a number of critical enzymes (Figure 1A) including pyruvate decarboxylase (PDC), pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), branched-chain α-ketoacid dehydrogenase (BKDH), transketolase (TK), acetolactate synthase (AHAS) and 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) [1]. Plants can synthesize TDP de novo from simple precursors via biosynthetic pathways that bear the hallmarks of the systems utilized by both bacteria and yeast [2–5]. In this regard, the early stages of TDP biosynthesis include two parallel pathways. One is similar to the mechanism found in bacteria and leads from 5-aminoimidazole ribonucleotide (AIR) to the pyrimidine moiety of thiamine (4-amino-2-methyl-5-hydroxymethylpyrimidine monophosphate, HMP-P) and engages only a single enzyme, a product of the THIC gene that has been characterized in Arabidopsis thaliana [6]. In the second of the parallel pathways, the thiazole moiety of thiamine (4-methyl-5-(2-hydroxyethyl)-thiazole phosphate, HET-P) is synthesized from glycine, NAD⁺ and a sulfur donor protein, in a similar manner to the system utilized in yeast [7]. The major enzyme involved in the HET-P formation is encoded by the THI1 gene, which has now been characterized in both Arabidopsis and Zea mays [8, 9].

Two further thiamine biosynthetic steps include an additional phosphorylation of HMP-P to 4-amino-2-methyl-5-hydroxymethylpyrimidine diphosphate (HMP-PP) and the condensation of HMP-PP with HET-P to form thiamine monophosphate (TMP). These two reactions are catalyzed by a single protein, the product of the TH1 gene in Arabidopsis [10] or THI3 gene in Z. mays [11], which is thus a bifunctional enzyme with both HMP-P kinase and TMP synthase activities. TMP does not possess any recognized physiological function to date but constitutes a thiamine resource or transitional stage for further TDP synthesis. In the latter case, an unphosphorylated form of thiamine is required. As has been shown previously in Z. mays [12], TMP is dephosphorylated by a relatively unspecific phosphatase which can also use TDP as a substrate. All of the steps described above proceed in chloroplasts [13], but the process of thiamine conversion to TDP occurs in the cytosol and is catalyzed by thiamine pyrophosphokinase, encoded by the TPK genes [14]. To fulfill its biological function, TDP must be transported to chloroplasts or mitochondria but the required transporters have not yet been identified. The thiamine biosynthesis pathway in plants is precisely regulated through TDP-dependent riboswitch mechanisms on the THIC gene and in ancient plant taxa also the THI1 gene levels [15].

It has been well documented that thiamine biosynthesis is activated during plant adaptation responses to persistent abiotic stress conditions such as salting and flooding [16], cold, heat and drought [17, 18], and oxidative stress [19, 20]. However, it has not yet been determined how thiamine biosynthesis and activation responds to the above stimuli at the early stages of the corresponding stress responses (sensing). In our current study, we further characterize the role of thiamine biosynthesis upregulation in the early response to oxidative, osmotic and salt stress in plants. In the experiments, we analyze changes in the expression levels of all thiamine-biosynthetic genes and of genes encoding the major TDP-dependent enzymes such as TK, KGDH, PDH, PDC and DXPS in Arabidopsis seedlings. We also show for the first time that abscisic acid (ABA), but not salicylic acid (SA), is involved in the upregulation of thiamine biosynthesis in the plant response to abiotic stress.

Because their growth and development are affected by environmental fluctuations, plants require fast responses to these changes for the maintenance of internal homeostasis. Hence, a number of sensitive mechanisms have evolved in plants to allow rapid sensing of environmental cues and facilitate growth modifications for adaptation and survival [21, 22]. As stress has an impact on the carbon fixation and overall energy status of plants, most previous genetic, proteomic and metabolic studies of this area have dealt with changes in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and photosynthesis [23–25]. However, these earlier reports have presented data that was obtained under very different growth or stress conditions, and also over quite different time scales and using different measurement parameter. This makes it difficult to compare the results from different laboratories in a meaningful way.

In our current study, which for the first time evaluates the early response stage ("stress sensing") rather than the long-term adaptation to stress in plants, we compared (i) the expression levels of the genes involved in thiamine/TDP biosynthesis pathways; (ii) the expression levels of genes coding for the most important TDP-requiring enzymes; and (iii) the thiamine and its phosphate ester contents, in Arabidopsis seedlings treated with abiotic stressors including PQ, NaCl and SOR. The application of NaCl or SOR into the plant growth media during the first few hours of treatment is sensed by the plants as general water stress rather than as salt- or SOR-specific effects [26]. It is generally accepted, that stress-generated damage involves the most important pathways and hence an obvious need to upregulate the main biosynthetic processes, including the biosynthesis of TDP. Indeed, we observed in our present experiments that the expression of all TDP-biosynthesis genes had already been increased at 2 h after the application of two water-stress agents (Figure 2). Interestingly, these genes showed decreased expression under salt stress over a longer time scale of several days, suggesting an extensive ion penetration into plant tissues [26] that could possibly generate new stimuli and adaptation processes. However, in our SOR stress experiments, the most of the early observed changes, particularly those involving the THIC gene, were maintained over a long time scale. This may indicate that a hormonal signaling mechanism rather than water availability controls plant growth under these conditions [27]. Surprisingly, we did not observe any marked influence of PQ on the expression of TDP biosynthesis genes over a time scale of a few hours, which is in contrast to the findings of an earlier study of long-lasting PQ-generated oxidative stress [20].

Our observed increases in gene expression during initial stages of the plant stress responses may indicate that (i) the translated proteins are susceptible to damage under unfavorable conditions and an increase of transcription is therefore required to maintain the proper thiamine cellular levels; (ii) the TDP-dependent enzymes are destroyed and, parallel to their re-synthesis, a new requirement for their cofactors emerges; and/or (iii) thiamine production is necessary to overcome the consequences of stress or to develop new adaptation strategies. Our analysis of thiamine and its phosphate analogs in stressed plants (Figure 3) showed that the observed changes in the expression of thiamine biosynthetic genes correlated with the activity of the encoded enzymes. The increase in the thiamine and TDP contents during the early Arabidopsis response (sensing) to water stress is consistent with similar changes recently reported in Z. mays seedlings subjected to long-lasting osmotic, oxidative and salinity stress [19]. This suggests that during stress sensing and adaptation in plants, the biosynthesis of thiamine is tightly regulated. Moreover, this regulation does not seem to be specific to the type of stress and could benefit from two different mechanisms previously identified in plants. The first type of possible regulation occurs at the genetic level and can apply to the THIC gene promoter as it contains a TDP-dependent riboswitch [15]. The other regulation process can operate at the protein level, as was demonstrated in a study of Z. mays, where TMP synthase activity was strongly inhibited by an increased supply of HMP-PP, produced through the activity of the kinase domain of the same protein, THI3 [11].

To test the hypothesis that the effects of stress conditions on thiamine compound pools in plant tissues arise from the increased requirement for TDP from the TDP-dependent main catabolic pathways, we analyzed the stress-dependent changes in the expression of several genes encoding TDP-dependent enzymes (Figure 4). The mitochondrial production of acetyl CoA and the tricarboxylic acid cycle are assumed to be the first plant sensors of oxidative stress [23, 28, 29]. Our current analyses identified KGDH and PDH as the main source of stress perturbation. However, the upregulation of the expression of these two mitochondrial TDP-dependent enzymes can result not only from the increased requirement for mitochondrial potential restoration, respiration control, nitrogen metabolism or glutamate signaling [30] but also reflect the activation of a stress signaling response to a metabolic imbalance as a result of a KGDH side reaction resulting in reactive oxygen species production [29].

The TK enzyme, which operates in the chloroplastic Calvin-Benson cycle (CBC), is involved in carbon fixation [31] and its activity is a limiting factor for sucrose production and photosynthesis [32]. As it functions also in the pentose phosphate pathway, TK is additionally important for the generation of NADPH, cooperating with a variety of oxidant-scavenging systems [23, 33–35]. The early reported increase in TK transcripts in Arabidopsis agrees with the changes in TK activity observed in Z. mays under salt and oxidative stress [19] and supports the hypothesis that this enzyme is involved in plant stress protection. DXPS is another TDP-dependent enzyme which is involved in the production of powerful antioxidants, the carotenoids [36, 37]. The activation of carotenoid synthetic pathways has been observed during osmotic, salt and drought stress [38–40]. In our present study, however, no significant changes in the expression of DXPS-encoding gene were observed over 24 h of stress treatment, possibly because its regulation occurs at the posttranscriptional level [41].

The well characterized and essential roles of the phytohormones in a wide range of stress adaptive processes in plants [42, 43] prompted us to search for candidate regulators of the activation of thiamine biosynthesis and TDP-dependent metabolic processes during salt and osmotic stress sensing and in the later adaptation phase. Our focus on SA was based on previous reports of an activation of a cabbage THIJ-like gene product involved in HMP phosphorylation via the SA-dependent signaling pathway [44] and an involvement of SA in thiamine-induced plant resistance to pathogen attacks [45]. However, our treatments of Arabidopsis plants with exogenous SA did not cause any observable changes in expression of genes involved in thiamine biosynthesis (Figures 5 and 6).

JA is associated with the defense against necrotrophic pathogens and herbivorous insects [46] but its involvement in salt stress-dependent gene regulation has also been suggested [47]. In our current study, supplementation of growth medium with JA caused a slightly increased thiamine content in Arabidopsis seedlings, resulting from the activation of thiamine biosynthetic genes, mostly TH1 (Figures 5 and 6). Although the mechanism underlying thiamine biosynthesis regulation by JA requires further analysis, it is noteworthy that a cis-acting element T/GBOXATPIN2 (AACGTG) was identified in the TH1 promoter region [48] which is responsible for activating this gene in response to JA upon wounding [49].

ABA regulates nearly 10% of the protein-coding genes in plants [50] and commonly participates in the transcriptional regulation processes that operate under salt and osmotic stress conditions [51–54]. The upregulation of the THI1, THIC, TH1 and TPK genes and the 3.5-fold increase in the total thiamine levels in Arabidopsis seedlings under the influence of an external ABA supply that we observed in this study provide the first evidence for the involvement of ABA in thiamine biosynthesis under stress conditions (Figures 5, 6). These findings are also interpretable in the light of previous analyses of THI1and THIC promoters that showed the presence of an ABA-responsive element (ABRE) in the THI1 promoter [16] and putative stress-related elements in the THIC promoter, including ABRE and drought-response element [20]. In silico analysis of the TPK promoter has identified a dehydration and ABA-inducible expression element MYBATRD22 (CTAACCA) and of the TH1 promoter an ABRE - ABRELATERD1 (ACGTG) element.

Our current analysis of the expression of TDP-dependent enzymes in seedlings grown on ABA-containing medium showed a transcript accumulation for KGDH- and TK-encoding genes but no significant effects on PDH gene transcripts. These data are consistent with our previous observations of the increased ABA content and TK activity in Z. mays seedlings treated with NaCl and polyethylene glycol [19]. Taken together, these data are consistent with the well established hypothesis regarding the linkages between ABA and plant mitochondrial functionality [55–57].

Although the application of exogenous ABA has often been used to mimic water stress responses in plants [58], it is generally accepted that both ABA-dependent and ABA-independent processes are involved in these types of responses [59]. In an attempt to further validate the hypothesis that ABA is responsible for thiamine biosynthesis in the early response (sensing) of plants to water stress generated by salt or SOR, a mutant Arabidopsis plant (aba1) was employed in our present experiments. This mutant is defective in the production of zeaxanthin epoxidase, an enzyme that catalyzes two early steps in the generation of the epoxycarotenoid precursor as part of the ABA biosynthetic pathway [60]. In the analysis, aba1 showed different behaviors in response to salt and osmotic stress. Under salt stress, a decrease in the THI1 and THIC expression levels in the aba1 mutant plants was observed, confirming the regulation of these thiamine biosynthetic steps by ABA. The expression of TH1 and TPK was found to be maintained as in the wild type plant. In contrast, in the presence of SOR the activation of thiamine biosynthetic genes was found not to be markedly different from wild type plants, suggesting that some additional, e.g. post-transcriptional, regulatory processes for thiamine biosynthesis operate under these conditions, although the details remain to be clarified.

Our present findings suggest a contribution of ABA to the transcriptional regulation of the two first genes that function in thiamine biosynthesis (THI1 and THIC) in Arabidopsis seedlings subjected to salt and osmotic stress, particularly at the early stages of the stress response (i.e. stress sensing). However, other as yet unidentified endogenous mediators must also be involved and the relative importance of ABA becomes less significant over a longer time scale (i.e. during the plant adaptation to stress). Moreover, the TH1 and TPK genes appear to be regulated by endogenous factors other than ABA, but also by ABA when it is present in the growth medium.

On the basis of the data obtained in this study as well as the results of previous reports, a role for thiamine biosynthesis and TDP-dependent metabolic pathways in abiotic stress sensing and adaptation can be hypothesized (Figure 9). Briefly, abiotic stressors which appear in the plant environment begin to inactivate the major metabolic pathways, the crucial enzymes of which can act as stress sensing centers (i.e. KGDH, TK). The urgent requirement for the rapid reactivation of these factors generates the secondary processes or stimuli that initiate a further adaptation phase. The activation of TDP biosynthesis processes leads to thiamine/TDP overproduction, which supports defense responses such as NADPH production or carotenoid biosynthesis. TDP production and the subsequent activities of TDP-dependent enzymes come under ABA control but is not the sole regulator of these processes. However, thiamine compounds themselves can also act as a significant source of antioxidants [61, 62] or play a stress alarmone function as has been suggested in bacteria under carbon starvation conditions [63].

We here demonstrate for the first time that TDP biosynthesis processes are quickly activated during the early phase (sensing) of the Arabidopsis response to salt and osmotic stress. The produced TDP is incorporated into TDP-dependent enzymes that are up-regulated at the same time such as KGDH, PDH, TK, all of which are involved in the main metabolic pathways that respond to stress conditions in plants. ABA seems to be involved in the activation of genes encoding enzymes that function in thiamine and TDP biosynthesis as well as the TDP-dependent KGDH and TK genes. We propose a working model for the contribution of thiamine to the stress sensing and adaptation processes in plants.