Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC

Abstract

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3'-untranslated region of THIC. Strong evidence is provided for the involvement of an iron-sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.

Fig. 1.

Arabidopsis THIC characteristics. (A) (Upper) Exon–intron structure of Arabidopsis THIC indicating the location of the SAIL_793_H10 insertion. (Lower) PCR analysis indicating plant genotypes employing primer pairs specific for either WT or the T-DNA insertion in thiC, respectively. From left to right: molecular marker, WT, three lines heterozygous for thiC, and a homozygous line (L13). (B) Amino acid sequence alignment of THIC homologs identified in Arabidopsis thaliana (At), Oryza sativa (Os), Bacillus subtilis (Bs), and Escherichia coli (Ec). Amino acids identical in at least two of the sequences are shaded in red. The asterisk denotes the identified C(X)₂C(X)₄C motif. (C) (Left) Quantitative RT-PCR of THIC transcript in WT or thiC plants. The values indicated are the percentage mRNA levels relative to WT (value = 100). Error bars indicate the standard deviation of the experiment performed in triplicate. (Right) Western blot analysis of the same samples probed with a THIC antibody. L13 and L17 refer to lines 13 and 17, respectively. (D) thiC mutant phenotype. (Left) WT and progeny of selfed THIC/thiC plants grown in sterile culture lacking vitamin B1. Approximately one quarter of the population shows a pale green phenotype and does not develop beyond the cotyledon stage. (Right) Supplementation with vitamin B1 (0.5 μM) restores growth of thiC to that of WT. Ten-day-old seedlings grown under long day conditions. (E) (Left) Enlarged picture of two seedlings from D. (Right) Total chlorophyll content (Chl) of WT and thiC (L13) under the same conditions. Error bars indicate the standard deviation of three independent experiments.

Fig. 2.

Rescue of Arabidopsis thiC mutants. (A) (Left) Rescue of thiC by supplementation with thiamin after transfer to soil. Ten-day-old segregating thiC seedlings grown on medium lacking vitamin B1 (see Fig. 1D) were transferred to soil and watered with the indicated concentrations of thiamin (μM). Plants are 30 days after transfer to soil. (Right) Total vitamin B1 content of seeds of rescued plants. (B) Progeny of rescued thiC lines as described in A compared with segregating nonsupplemented thiC from a heterozygous population. The numbers indicate the concentration of thiamin (μM) supplied to the mother plants. Seedlings (10 days old) were grown in the absence of vitamin B1. As a control, a segregating thiC seedling from a heterozygous mother grown in the presence of vitamin B1 is shown. (C) Total vitamin B1 content of WT (circles), thiC progeny rescued with 100 μM (squares) and 1.5 μM (triangles) thiamin, respectively, grown in the absence of thiamin over the time period indicated. (D) Total chlorophyll content (Chl) of the same seedlings as in C. Error bars (some of which are sufficiently small that they fall within the symbols) indicate the standard deviation of three independent experiments. DAG, days after germination.

Fig. 3.

Subcellular localization of THIC in Arabidopsis. The N-terminal 90 amino acids of THIC were fused to either the N or C terminus of YFP to give N90THIC-YFP and YFP-N90THIC, respectively, and transiently expressed in isolated A. thaliana mesophyll protoplasts. Confocal laser scanning microscopy was used to monitor fluorescence.

Fig. 4.

Expression analysis of THIC as assessed by RT-PCR. Transcript abundance in the indicated tissues (A) and days after germination (DAG) in the absence of thiamin (B) grown under long day conditions (LD, 100 μmol photons m⁻² s⁻¹ white light for 16-h/8-h dark). (C) Samples of whole seedlings were analyzed 6 days after growing in either continuous light (LL) or LD, or continuous dark (DD). In addition, samples of 6-day-old etiolated seedlings were analyzed at the indicated times after transfer to light, as well as after a 24-h LD cycle. (D) Seedlings grown in the presence and absence of 0.5 μM thiamin under LD. In all cases, Actin2 mRNA serves as the control.

Fig. 5.

THIC is an iron–sulfur cluster protein. (A) RNA interference of CpnifS as evidenced by the chlorotic phenotype. Plants were grown on soil for 14 days, after which silencing was induced by spraying with 2% (vol/vol) ethanol every 4 days. The plants shown are 19 days after induction of CpnifS silencing. (B) (Upper) Transcript abundance of THIC in WT or CpnifS plants (as shown in A). WT-C, WT-E, L6-C, and L6-E refer to wild-type or CpnifS line 6 control or ethanol-treated plants, respectively. Actin2 mRNA serves as the control. (Lower) Western blot of the same samples probed with a THIC antibody in addition to the Ponceau-S stained membrane where RBCL indicates the large subunit of Rubisco and serves as a protein loading control. (C) Total thiamin content of the same samples described in B. (D) UV-visible spectrum of Arabidopsis THIC as isolated aerobically (black line), and 1 min after addition of 0.3 M sodium dithionite (red line).