THIAMIN REQUIRING2 is involved in thiamin diphosphate biosynthesis and homeostasis

Abstract

SUMMARY The THIAMIN REQUIRING2 (TH2) protein comprising a mitochondrial targeting peptide followed by a transcription enhancement A and a haloacid dehalogenase domain is a thiamin monophosphate (TMP) phosphatase in the vitamin B1 biosynthetic pathway. The Arabidopsis th2‐3 T‐DNA insertion mutant was chlorotic and deficient in thiamin diphosphate (TDP). Complementation assays confirmed that haloacid dehalogenase domain alone was sufficient to rescue the th2‐3 mutant. In pTH2:TH2‐GFP/th2‐3 complemented plants, the TH2‐GFP was localized to the cytosol, mitochondrion, and nucleus, indicating that the vitamin B1 biosynthetic pathway extended across multi‐subcellular compartments. Engineered TH2‐GFP localized to the cytosol, mitochondrion, nucleus, and chloroplast, could complement the th2 mutant. Together, these results highlight the importance of intracellular TMP and thiamin trafficking in vitamin B1 biosynthesis. In an attempt to enhance the production of thiamin, we created various constructs to overexpress TH2‐GFP in the cytosol, mitochondrion, chloroplast, and nucleus. Unexpectedly, overexpressing TH2‐GFP resulted in an increase rather than a decrease in TMP. While studies on th2 mutants support TH2 as a TMP phosphatase, analyses of TH2‐GFP overexpression lines implicating TH2 may also function as a TDP phosphatase in planta. We propose a working model that the TMP/TDP phosphatase activity of TH2 connects TMP, thiamin, and TDP into a metabolic cycle. The TMP phosphatase activity of TH2 is required for TDP biosynthesis, and the TDP phosphatase activity of TH2 may modulate TDP homeostasis in Arabidopsis.

Harnessing enzyme cofactors and plant metabolism: an essential partnership

“…Besides thiamin, thiamin monophosphate (TMP) and thiamin diphosphate (TDP) are the two other major forms of vitamers among the six currently known thiamin vitamers (Fitzpatrick & Chapman, 2020). Although TDP is considered to be the bioactive form, the possibility that thiamin and TMP also have biological functions can not yet be excluded (Hsieh et al., 2022). The pathways that plants use to synthesize B1 vitamers are distributed among plastids, cytosol, and mitochondria (Hsieh et al., 2022; Joshi et al., 2021; Mimura et al., 2016).…”

“…Although TDP is considered to be the bioactive form, the possibility that thiamin and TMP also have biological functions can not yet be excluded (Hsieh et al., 2022). The pathways that plants use to synthesize B1 vitamers are distributed among plastids, cytosol, and mitochondria (Hsieh et al., 2022; Joshi et al., 2021; Mimura et al., 2016). Because other studies have yielded in‐depth descriptions of thiamin biosynthesis pathways in different organisms (Hsieh et al., 2022), we will only briefly describe the general aspects of thiamin biosynthesis in plants.…”

“…The pathways that plants use to synthesize B1 vitamers are distributed among plastids, cytosol, and mitochondria (Hsieh et al., 2022; Joshi et al., 2021; Mimura et al., 2016). Because other studies have yielded in‐depth descriptions of thiamin biosynthesis pathways in different organisms (Hsieh et al., 2022), we will only briefly describe the general aspects of thiamin biosynthesis in plants. Regarding subcellular distribution, two members of the mitochondrial carrier family (MCF) were identified as being responsible for mediating TDP import into the mitochondria in both maize ( Zea mays ) and Arabidopsis (Frelin et al., 2012) (Table 2).…”

“…Despite relatively low amounts of TDP being sufficient for plant growth, previous studies have shown that only minor reductions (in the range of nM) are able to affect growth. Indeed, null mutations that abolish thiamin synthesis are fatal (Hsieh et al., 2022), whereas plants with reduced TDP levels are chlorotic and smaller and may display delayed flowering (Bocobza et al., 2013). Thus, the deficiency is expected to affect respiration and photosynthesis.…”

Ectopic expression of a bacterial thiamin monophosphate kinase enhances vitamin B1 biosynthesis in plants

“…In contrast, TMP is dephosphorylated to thiamin, which is then pyrophosphorylated to form TDP in plants (Figure 1a). Arabidopsis th2 mutants impaired in TMP phosphatase have reduced thiamin and TDP levels and a chlorotic phenotype (Hsieh et al, 2017(Hsieh et al, , 2022Mimura et al, 2016). We previously showed that feeding thiamin could rescue the th2 mutants (Hsieh et al, 2017(Hsieh et al, , 2022.…”