AtTHIC, a gene involved in thiamine biosynthesis in Arabidopsis thaliana

Abstract

Thiamine (vitamin B1) is an essential compound for organisms. It contains a pyrimidine ring structure and a thiazole ring structure. These two moieties of thiamine are synthesized independently and then coupled together. Here we report the molecular characterization of AtTHIC, which is involved in thiamine biosynthesis in Arabidopsis. AtTHIC is similar to Escherichia coli ThiC, which is involved in pyrimidine biosynthesis in prokaryotes. Heterologous expression of AtTHIC could functionally complement the thiC knock-out mutant of E. coli. Downregulation of AtTHIC expression by T-DNA insertion at its promoter region resulted in a drastic reduction of thiamine content in plants and the knock-down mutant thic1 showed albino (white leaves) and lethal phenotypes under the normal culture conditions. The thic1 mutant could be rescued by supplementation of thiamine and its defect functions could be complemented by expression of AtTHIC cDNA. Transient expression analysis revealed that the AtTHIC protein targets plastids and chloroplasts. AtTHIC was strongly expressed in leaves, flowers and siliques and the transcription of AtTHIC was downregulated by extrinsic thiamine. In conclusion, AtTHIC is a gene involved in pyrimidine synthesis in the thiamine biosynthesis pathway of Arabidopsis, and our results provide some new clues for elucidating the pathway of thiamine biosynthesis in plants.

Overexpression of Plastid Transketolase in Tobacco Results in a Thiamine Auxotrophic Phenotype

“…Tobacco mutants in thiamine biosynthesis exhibited a 50% reduction in chlorophyll pigments that was also fully reversible by the addition of exogenous thiamine (McHale et al, 1988). A similar result was also obtained in the Arabidopsis ThiC insertion mutants that were blocked in the biosynthesis of the pyrimidine moiety of the thiamine molecule, producing albino plants that exhibited slow growth, even though starch and sucrose levels accumulated up to 40 and 50% more, respectively, than wild-type plants (Raschke et al, 2007;Kong et al, 2008).…”

Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins

“…This could be due to positional eVects. It has long been known that chromatin state, Xanking sequences, methylation and transgene orientation at the insertion site can result in signiWcant diVerences in expression levels between multiple transgenic lines in animals (Clark et al 1994;Feng et al 2001;Wang et al 2010) and plants (Holtorf et al 1995;Guerineau et al 2003;Petsch et al 2005;Jones et al 2008;Kong et al 2008) even when transformed with identical genetic constructs. Furthermore, it is possible that there are additional regulatory elements upstream of the AGB1 promoter region used in this study (1 kb).…”

Orchestration of Thiamin Biosynthesis and Central Metabolism by Combined Action of the Thiamin Pyrophosphate Riboswitch and the Circadian Clock in Arabidopsis

“…This was achieved by generating two THIC expression cassettes, containing the promoter, the gene, and the 39 region (containing the TPP riboswitch) of THIC, with or without an A-to-G point mutation in the TPP riboswitch (i.e., A515G, relative to the stop codon) that reduces its activity (Sudarsan et al, 2005; see Supplemental Figure 3A online). These cassettes were introduced independently into the background of an Arabidopsis mutant in which a T-DNA insertion abolishes THIC expression (Kong et al, 2008). Interestingly, while plants harboring the native TPP riboswitch (thereafter referred as control plants) did not display any particular phenotype under short-day conditions (i.e., 10 h/14 h, day/night), those harboring the deficient TPP riboswitch exhibited chlorosis, growth retardation, and delayed flowering (observed in 10 independent transformed lines; Figure 3A; see Supplemental Figure 3B online).…”

“…In Arabidopsis thaliana, three enzymes participate together in the synthesize of thiamin monophosphate (TMP), namely, TH1 (Ajjawi et al, 2007a), THI1 (Belanger et al, 1995;Machado et al, 1996), and the TPP riboswitch-regulated THIC (Bocobza et al, 2007;Raschke et al, 2007;Wachter et al, 2007;Kong et al, 2008). Unlike in prokaryotes where TMP can be directly converted into TPP (Begley et al, 1999), in Arabidopsis, TMP is subsequently dephosphorylated into thiamin (Komeda et al, 1988), which is then pyrophosphorylated into TPP by the thiamin pyrophosphokinases (TPKs), namely, TPK1 and TPK2 (Ajjawi et al, 2007b).…”