Inadequate Niacin Intake Disrupts Growth and Retinol Homeostasis Resulting in Higher Liver and Lower Serum Retinol Concentrations in Male Rats.

BACKGROUND: Niacin-derived nicotinamide adenine dinucleotide is an essential cofactor for many dehydrogenase enzymes involved in vitamin A (VA) metabolism. Several countries with high prevalence of VA deficiency rely on maize, a poor source of available niacin, as a dietary staple. OBJECTIVES: This study evaluated the interaction of dietary niacin on VA homeostasis using male Sprague-Dawley rats, aged 21 d (baseline body weight 88.3 ± 6.6 g). METHODS: After 1 wk of acclimation, baseline samples were collected (n = 4). Remaining rats (n = 54) were split into 9 groups to receive low tryptophan, VA-deficient feed with 3 different amounts of niacin (0, 15, or 30 mg/kg) and 3 different oral VA doses (50, 350, or 3500 nmol/d) in a 3 × 3 design. After 4 wk, the study was terminated. Serum, livers, and small intestine were analyzed for retinoids using high-performance liquid chromatography. Niacin and metabolites were evaluated with nuclear magnetic resonance. Plasma pyridoxal-P (PLP) was measured with high-performance liquid chromatography. RESULTS: Niacin intake correlated with serum retinol concentrations (r = 0.853, P < 0.001). For rats receiving the highest VA dose, liver retinol concentrations were lower in the 30-mg/kg niacin group (5.39 ± 0.27 µmol/g) than those in the 0-mg/kg and 15-mg/kg groups (9.18 ± 0.62 and 8.75 ± 0.07 µmol/g, respectively; P ≤ 0.05 for both). This phenomenon also occurred in the lower VA doses (P ≤ 0.05 for all). Growth and tissue weight at endline were associated with niacin intake (P ≤ 0.001 for all). Plasma PLP correlated with estimated niacin intake (r = 0.814, P < 0.001). CONCLUSIONS: Optimal niacin intake is associated with lower liver VA and higher serum retinol and plasma PLP concentrations. The extent to which vitamin B intake affects VA homeostasis requires further investigation to determine if the effects are maintained in humans.

The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway.

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 (th3) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 (DXS1), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

Protocol for a Case-Control Study to Investigate the Association of Pellagra With Isoniazid Exposure During Tuberculosis Preventive Treatment Scale-Up in Malawi.

Background: Pellagra is caused by niacin (vitamin B3) deficiency and manifested by a distinctive dermatitis. Isoniazid is critical for treating tuberculosis globally and is a component of most regimens to prevent tuberculosis. Isoniazid may contribute to pellagra by disrupting intracellular niacin synthesis. In 2017, Malawian clinicians recognized a high incidence of pellagra-like rashes after scale-up of isoniazid preventive treatment (IPT) to people living with HIV (PLHIV). This increase in pellagra incidence among PLHIV coincided with a seasonal period of sustained food insecurity in the region, which obscured epidemiological interpretations. Although isoniazid has been implicated as a secondary cause of pellagra for decades, no hypothesis-driven epidemiological study has assessed this relationship in a population exposed to isoniazid. We developed this case-control protocol to assess the association between large-scale isoniazid distribution and pellagra in Malawi. Methods: We measure the relative odds of having pellagra among isoniazid-exposed people compared to those without exposure while controlling for other pellagra risk factors. Secondary aims include measuring time from isoniazid initiation to onset of dermatitis, comparing niacin metabolites 1-methylnicotinamide (1-MN), and l-methyl-2-pyridone-5-carboxamide (2-PYR) in urine as a proxy for total body niacin status among subpopulations, and describing clinical outcomes after 30-days multi-B vitamin (containing 300 mg nicotinamide daily) therapy and isoniazid cessation (if exposed). We aim to enroll 197 participants with pellagra and 788 age- and sex-matched controls (1:4 ratio) presenting at three dermatology clinics. Four randomly selected community clinics within 3-25 km of designated dermatology clinics will refer persons with pellagra-like symptoms to one of the study enrollment sites for diagnosis. Trained study dermatologists will conduct a detailed exposure questionnaire and perform anthropometric measurements. A subset of enrollees will provide a casual urine specimen for niacin metabolites quantification and/or point-of-care isoniazid detection to confirm whether participants recently ingested isoniazid. We will use conditional logistic regression, matching age and sex, to estimate odds ratios for the primary study aim. Discussion: The results of this study will inform the programmatic scale-up of isoniazid-containing regimens to prevent tuberculosis.

Elevated plasma cystathionine is associated with increased risk of mortality among patients with suspected or established coronary heart disease.

BACKGROUND: Elevated circulating cystathionine levels are related to atherosclerotic cardiovascular disease, a leading cause of death globally. OBJECTIVE: We investigated whether plasma cystathionine was associated with mortality in patients with suspected or established coronary heart disease (CHD). METHODS: Data from 2 independent cohorts of patients with suspected stable angina pectoris (SAP) (3033 patients; median 10.7 y follow-up; 648 deaths) or acute myocardial infarction (AMI) (3670 patients; median 7.0 y follow-up; 758 deaths) were included. Hazard ratios with 95% CIs per SD increment of log-transformed cystathionine were calculated using Cox regression modeling. Endpoint data was obtained from a national health registry. RESULTS: Among patients with SAP, there was a positive association between plasma cystathionine and death (age- and sex-adjusted HRs [95% CI] per SD: 1.23 [1.14, 1.32], 1.29 [1.16, 1.44], and 1.17 [1.05, 1.29] for total, cardiovascular, and noncardiovascular mortality, respectively). Corresponding risk estimates were 1.28 (1.19, 1.37) for all-cause, 1.33 (1.22, 1.45) for cardiovascular, and 1.19 (1.06, 1.34) for noncardiovascular death among AMI patients. In both cohorts, estimates were slightly attenuated after multivariate adjustments for established CHD risk factors. Subgroup analyses showed that the relation between cystathionine and all-cause mortality in SAP patients was stronger among nonsmokers and those with lower plasma concentration of pyridoxal-5'-phosphate (P-interaction ≤ 0.01 for both). CONCLUSIONS: Elevated plasma cystathionine is associated with both cardiovascular and noncardiovascular mortality among patients with suspected or established CHD. The joint risk associations of high plasma cystathionine with lifestyle factors and impaired vitamin B-6 status on mortality need further investigation. This trial was registered at clinicaltrials.gov as NCT00354081 and NCT00266487.

Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Redesigning thiamin synthesis: Prospects and potential payoffs.

Thiamin is essential for plant growth but is short-lived in vivo and energetically very costly to produce - a combination that makes thiamin biosynthesis a prime target for improvement by redesign. Thiamin consists of thiazole and pyrimidine moieties. Its high biosynthetic cost stems from use of the suicide enzyme THI4 to form the thiazole and the near-suicide enzyme THIC to form the pyrimidine. These energetic costs lower biomass yield potential and are likely compounded by environmental stresses that destroy thiamin and hence increase the rate at which it must be made. The energy costs could be slashed by refactoring the thiamin biosynthesis pathway to eliminate the suicidal THI4 and THIC reactions. To substantiate this design concept, we first document the energetic costs of the THI4 and THIC steps in the pathway and explain how cutting these costs could substantially increase crop biomass and grain yields. We then show that a refactored pathway must produce thiamin itself rather than a stripped-down analog because the thiamin molecule cannot be simplified without losing biological activity. Lastly, we consider possible energy-efficient alternatives to the inefficient natural THI4- and THIC-mediated steps.

Plasma cystathionine and risk of acute myocardial infarction among patients with coronary heart disease: Results from two independent cohorts.

BACKGROUND: Cystathionine is a thio-ether and a metabolite formed from homocysteine during transsulfuration. Elevated plasma cystathionine levels are reported in patients with cardiovascular disease; however prospective relationships with acute myocardial infarction (AMI) are unknown. We investigated associations between plasma cystathionine and AMI among patients with suspected and/or verified coronary heart disease (CHD). METHODS: Subjects from two independent cohort studies, the Western Norway Coronary Angiography Cohort (WECAC) (3033 patients with stable angina pectoris; 263 events within 4.8 years of median follow-up) and the Norwegian Vitamin Trial (NORVIT) (3670 patients with AMI; 683 events within 3.2 years of median follow-up) were included. RESULTS: In both cohorts, plasma cystathionine was associated with several traditional CHD risk factors (P < 0.001). Comparing the cystathionine quartile 4 to 1, age and gender adjusted hazard ratios (95% confidence intervals) for AMI were 2.08 (1.43-3.03) and 1.41 (1.12-1.76) in WECAC and NORVIT, respectively. Additional adjustment for traditional risk factors slightly attenuated the risk estimates, which were generally stronger in both cohorts among non-smokers, patients with higher age, and lower BMI or PLP status (P-interaction ≤ 0.04). Risk associations also tended to be stronger in patients not treated with B-vitamins. Additionally, in a subset of 80 WECAC patients, plasma cystathionine associated strongly negatively with glutathione, an important antioxidant and positively with lanthionine, a marker of H2S production (P < 0.001). CONCLUSIONS: Plasma cystathionine is associated with increased risk of AMI among patients with either suspected or verified coronary heart disease, and is possibly related to altered redox homeostasis.

Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function.

NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggest caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by yjeF The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX dehydratase activity and NAD(P)HX levels, showing that the mutation had little impact on NAD(P)HX repair in vivo However, these cells showed metabolome changes, particularly in amino acids, which exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had reduced levels of 'free' (i.e. weakly bound or unbound) pyridoxal 5'-phosphate. These results provide circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6.

Suboptimal Vitamin B Intakes of Zambian Preschool Children: Evaluation of 24-Hour Dietary Recalls.

BACKGROUND: Hidden hunger affects individuals who chronically consume an inadequate intake of at least 1 micronutrient and is associated with low dietary diversity. Little data are available on dietary intake or status assessment of B vitamins among preschool children in Zambia. OBJECTIVES: The aim of this study was to assess 24-hour dietary recall records obtained from Zambian children aged 3 to 7 years for B vitamin intake in relation to adequacy and change over time in the same community. METHODS: Twenty-four-hour dietary recalls were collected from 2 studies that were 2 years apart in the same district of Zambia. Data were retrospectively analyzed for B vitamin intake, that is, biotin, vitamin B12, folate, niacin, pantothenic acid, vitamin B6, riboflavin, and thiamin. The estimated average requirement (EAR) cut point method was used to assess inadequacy prevalence for EARs established by the Institute of Medicine in the United States. RESULTS: For all B vitamins, mean values were below the EARs established for children 4 to 8 years old. Relative to the EAR, children had the highest intakes of vitamin B6 with inadequacies of 77.9% and 60.1% in 2010 and 2012, respectively. The highest prevalence of inadequate intake was associated with folate, where ≥95% of the children had intakes below the EAR in both studies. CONCLUSIONS: All median vitamin B intakes were inadequate among these young children in rural Zambia. Future researchers and policy makers may need to consider B vitamin status in resource-poor areas of the country.

A plastidial pantoate transporter with a potential role in pantothenate synthesis.

The pantothenate (vitamin B5) synthesis pathway in plants is not fully defined because the subcellular site of its ketopantoate → pantoate reduction step is unclear. However, the pathway is known to be split between cytosol, mitochondria, and potentially plastids, and inferred to involve mitochondrial or plastidial transport of ketopantoate or pantoate. No proteins that mediate these transport steps have been identified. Comparative genomic and transcriptomic analyses identified Arabidopsis thaliana BASS1 (At1g78560) and its maize (Zea mays) ortholog as candidates for such a transport role. BASS1 proteins belong to the bile acid : sodium symporter family and share similarity with the Salmonella enterica PanS pantoate/ketopantoate transporter and with predicted bacterial transporters whose genes cluster on the chromosome with pantothenate synthesis genes. Furthermore, Arabidopsis BASS1 is co-expressed with genes related to metabolism of coenzyme A, the cofactor derived from pantothenate. Expression of Arabidopsis or maize BASS1 promoted the growth of a S. enterica panB panS mutant strain when pantoate, but not ketopantoate, was supplied, and increased the rate of [3H]pantoate uptake. Subcellular localization of green fluorescent protein fusions in Nicotiana tabacum BY-2 cells demonstrated that Arabidopsis BASS1 is targeted solely to the plastid inner envelope. Two independent Arabidopsis BASS1 knockout mutants accumulated pantoate ∼10-fold in leaves and had smaller seeds. Taken together, these data indicate that BASS1 is a physiologically significant plastidial pantoate transporter and that the pantoate reduction step in pantothenate biosynthesis could be at least partly localized in plastids.

Biogenesis of Hydrogen Sulfide and Thioethers by Cystathionine Beta-Synthase.

AIMS: The transsulfuration pathway enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase are thought to be the major source of hydrogen sulfide (H2S). In this study, we assessed the role of CBS in H2S biogenesis. RESULTS: We show that despite discouraging enzyme kinetics of alternative H2S-producing reactions utilizing cysteine compared with the canonical condensation of serine and homocysteine, our simulations of substrate competitions at biologically relevant conditions suggest that cysteine is able to partially compete with serine on CBS, thus leading to generation of appreciable amounts of H2S. The leading H2S-producing reaction is condensation of cysteine with homocysteine, while cysteine desulfuration plays a dominant role when cysteine is more abundant than serine and homocysteine is limited. We found that the serine-to-cysteine ratio is the main determinant of CBS H2S productivity. Abundance of cysteine over serine, for example, in plasma, allowed for up to 43% of CBS activity being responsible for H2S production, while excess of serine typical for intracellular levels effectively limited such activity to less than 1.5%. CBS also produced lanthionine from serine and cysteine and a third of lanthionine coming from condensation of two cysteines contributed to the H2S pool. INNOVATION: Our study characterizes the H2S-producing potential of CBS under biologically relevant conditions and highlights the serine-to-cysteine ratio as the main determinant of H2S production by CBS in vivo. CONCLUSION: Our data clarify the function of CBS in H2S biogenesis and the role of thioethers as surrogate H2S markers. Antioxid. Redox Signal. 28, 311-323.

Metabolomic Evaluation of the Consequences of Plasma Cystathionine Elevation in Adults with Stable Angina Pectoris.

Background: An elevated circulating cystathionine concentration, which arises in part from insufficiencies of vitamin B-6, B-12, or folate, has been shown to be associated with cardiovascular disease (CVD) risk. Hydrogen sulfide (H2S) is a gasotransmitter involved in vasodilation, neuromodulation, and inflammation. Most endogenously produced H2S is formed by pyridoxal phosphate (PLP)-dependent enzymes by noncanonical reactions of the transsulfuration pathway that yield H2S concurrently form lanthionine and homolanthionine. Thus, plasma lanthionine and homolanthionine concentrations can provide relative information about H2S production in vivo.Objective: To determine the metabolic consequences of an elevated plasma cystathionine concentration in adults with stable angina pectoris (SAP), we conducted both targeted and untargeted metabolomic analyses.Methods: We conducted NMR and LC-mass spectrometry (MS) metabolomic analyses on a subset of 80 plasma samples from the Western Norway Coronary Angiography Cohort and selected, based on plasma cystathionine concentrations, a group with high cystathionine concentrations [1.32 ± 0.60 µmol/L (mean ± SD); n = 40] and a group with low cystathionine concentrations [0.137 ± 0.011 µmol/L (mean ± SD); n = 40]. Targeted and untargeted metabolomic analyses were performed and assessed with the use of Student's t tests corrected for multiple testing. Overall differences between the cystathionine groups were assessed by untargeted NMR and LC-MS metabolomic methods and evaluated by partial least squares discriminant analysis (PLS-DA) with significant discriminating metabolites identified with 99% confidence.Results: Subjects with high cystathionine concentrations had 75% higher plasma lanthionine concentrations (0.12 ± 0.044 µmol/L) than subjects with low cystathionine concentrations [0.032 ± 0.013 µmol/L (P < 0.001)]. Although plasma homolanthionine concentrations were notably higher than lanthionine concentrations, they were not different between the groups (P = 0.47). PLS-DA results showed that a high plasma cystathionine concentration in SAP was associated with higher glucose, branched-chain amino acids, and phenylalanine concentrations, lower kidney function, and lower glutathione and plasma PLP concentrations due to greater catabolism. The high-cystathionine group had a greater proportion of subjects in the postprandial state.Conclusion: These data suggest that metabolic perturbations consistent with higher CVD risk exist in SAP patients with elevated plasma cystathionine concentrations.

Astronaut ophthalmic syndrome.

During and after missions on the International Space Station, some astronauts experience ophthalmic changes, including choroidal folds, optic disc edema, cotton-wool spots, globe flattening, and refraction changes. Astronauts with ophthalmic issues had significantly higher plasma concentrations of metabolites that are associated with the 1-carbon metabolic pathway than those without ophthalmic issues. We hypothesized that genetic differences might explain the metabolite differences. Indeed, genetics and B vitamin status were significant predictors of ophthalmic issues. We now have developed a hypothesis regarding the mechanisms that link 1-carbon pathway genetics and the condition that we suggest calling, "astronaut ophthalmic syndrome." We maintain that this condition is genetically predisposed and is associated with endothelial dysfunction that is induced by oxidative stress. Subsequent edema can hinder cerebrospinal fluid efflux and can lead to locally increased pressures in the subarachnoid space within the orbit, which impinges on the optic nerve and/or eye in affected individuals. Confirming this hypothesis will help characterize the genetics of 1-carbon pathway metabolism, homocysteine, oxidative stress, endothelial dysfunction, and cardiovascular and potentially other diseases.-Zwart, S. R., Gibson, C. R., Gregory, J. F., Mader, T. H., Stover, P. J., Zeisel, S. H., Smith, S. M. Astronaut ophthalmic syndrome.

Methylenetetrahydrofolate Dehydrogenase 1 Polymorphisms Modify the Associations of Plasma Glycine and Serine With Risk of Acute Myocardial Infarction in Patients With Stable Angina Pectoris in WENBIT (Western Norway B Vitamin Intervention Trial).

BACKGROUND: Serine and glycine interconversion and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1)-mediated 1-carbon transfer are the major sources of methyl groups for 1-carbon metabolism. Recently, plasma glycine and a common polymorphism in MTHFD1 have been associated with risk of acute myocardial infarction (AMI). It is, therefore, of interest to explore if these 2 pathways interact in relation to AMI. METHODS AND RESULTS: A total of 2571 participants in the WENBIT (Western Norway B Vitamin Intervention Trial) undergoing coronary angiography for stable angina pectoris were studied. Associations of plasma serine and glycine concentrations with risk of AMI across 2 common and functional MTHFD1 polymorphisms (rs2236225 and rs1076991) were explored in Cox regression models. During a median follow-up of 4.7 years, 212 patients (8.2%) experienced an AMI. In age- and sex-adjusted analyses, plasma glycine (P<0.01), but not serine (P=0.52), showed an overall association with AMI. However, interactions of MTHFD1 rs2236225 polymorphism with both plasma serine and glycine were observed (Pinteraction=0.03 for both). Low plasma serine and glycine were associated with an increased risk of AMI among patients carrying the rs2236225 minor A allele. Similarly, low plasma glycine showed stronger risk relationship with AMI in the rs1076991 CC genotype carriers but weaker associations in patients carrying the minor T allele (Pinteraction=0.02). CONCLUSIONS: Our results showed that 2 common and functional polymorphisms in the MTHFD1 gene modulate the risk associations of plasma serine and glycine with AMI. These findings emphasize the possible role of the MTHFD1 in regulating serine and glycine metabolism in relation to atherosclerotic complications. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT00354081.

Does Abiotic Stress Cause Functional B Vitamin Deficiency in Plants?

B vitamins are the precursors of essential metabolic cofactors but are prone to destruction under stress conditions. It is therefore a priori reasonable that stressed plants suffer B vitamin deficiencies and that certain stress symptoms are metabolic knock-on effects of these deficiencies. Given the logic of these arguments, and the existence of data to support them, it is a shock to realize that the roles of B vitamins in plant abiotic stress have had minimal attention in the literature (100-fold less than hormones) and continue to be overlooked. In this article, we therefore aim to explain the connections among B vitamins, enzyme cofactors, and stress conditions in plants. We first outline the chemistry and biochemistry of B vitamins and explore the concept of vitamin deficiency with the help of information from mammals. We then summarize classical and recent evidence for stress-induced vitamin deficiencies and for plant responses that counter these deficiencies. Lastly, we consider potential implications for agriculture.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.