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SUMMARY: Plants and bacteria have distinct pathways to synthesize the bioactive vitamin B1 thiamin diphosphate (TDP). In plants, thiamin monophosphate (TMP) synthesized in the TDP biosynthetic pathway is first converted to thiamin by a phosphatase, which is then pyrophosphorylated to TDP. In contrast, bacteria use a TMP kinase encoded by ThiL to phosphorylate TMP to TDP directly. The Arabidopsis THIAMIN REQUIRING2 (TH2)‐encoded phosphatase is involved in TDP biosynthesis. The chlorotic th2 mutants have high TMP and low thiamin and TDP. Ectopic expression of Escherichia coli ThiL and ThiL‐GFP rescued the th2‐3 mutant, suggesting that the bacterial TMP kinase could directly convert TMP into TDP in Arabidopsis. These results provide direct evidence that the chlorotic phenotype of th2‐3 is caused by TDP rather than thiamin deficiency. Transgenic Arabidopsis harboring engineered ThiL‐GFP targeting to the cytosol, chloroplast, mitochondrion, or nucleus accumulated higher TDP than the wild type (WT). Ectopic expression of E. coli ThiL driven by the UBIQUITIN (UBI) promoter or an endosperm‐specific GLUTELIN1 (GT1) promoter also enhanced TDP biosynthesis in rice. The pUBI:ThiL transgenic rice accumulated more TDP and total vitamin B1 in the leaves, and the pGT1:ThiL transgenic lines had higher TDP and total vitamin B1 in the seeds than the WT. Total vitamin B1 only increased by approximately 25–30% in the polished and unpolished seeds of the pGT1:ThiL transgenic rice compared to the WT. Nevertheless, these results suggest that genetic engineering of a bacterial vitamin B1 biosynthetic gene downstream of TMP can enhance vitamin B1 production in rice. Significance Statement: Introducing a bacterial gene encoding thiamin monophosphate kinase into Arabidopsis and rice creates an additional route for thiamin diphosphate biosynthesis and increases vitamin B1 contents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]