The Arabidopsis thiamin‐deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B₁ biosynthesis pathway

Abstract

Thiamin diphosphate (TPP, vitamin B ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes.

On the nature of thiamine triphosphate in Arabidopsis

“…., 2015). By contrast, the pale green 1 (pale1) mutant, impaired in the TMP phosphatase, TH2 (At5g32470), is deficient in leaf TDP (Mimura et al, 2016, Hsieh et al, 2017. We examined the B 1 vitamer profile of these lines and could confirm that TDP was increased in THIC THI1 OE but decreased in pale1 ( Figure 3B).…”

Ascorbate and Thiamin: Metabolic Modulators in Plant Acclimation Responses

“…Phosphorylation of HMP-P to HMP-PP, and subsequently condensation of HMP-PP and HET-P, is done by the action of a bifunctional enzyme, thiamin monophosphate pyrophosphorylase (TH1), which eventually leads to the formation of thiamin monophosphate (TMP) [30,110]. TMP is dephosphorylated to thiamin by a haloacid dehalogenase (HAD) family phosphatase (TH2) [111], and subsequently pyrophosphorylated to thiamin pyrophosphate (TPP) by TPP kinases (TDPKs), which are located in the cytosol (Figure 3) [110,112].…”

“…In addition to its role in central metabolism, studies in different plant species have shown that alterations in the levels of thiamin vitamers result in smaller plants, chlorosis of the leaves, growth retardation, delayed flowering, fitness cost, and an influence on yield penalty [25,26,105,111,133,134]. Some of these phenotypes, such as chlorosis and the delayed flowering, have also been shown to be dependent on the light regime [25,32].…”

Extrachloroplastic PP7L Functions in Chloroplast Development and Abiotic Stress Tolerance

“…In screens for mutants with altered chloroplast biology at this stage, most studies have identified chloroplast-localized proteins involved in chloroplast development and showing a photosynthesis phenotype. Exceptions include PALE GREEN1 (Hsieh et al, 2017) and SCO3 (Albrecht et al, 2010), which are localized to mitochondria and peroxisomes, respectively. Interestingly, the cytoskeleton is altered in sco3-1, and microtubule inhibitors have similar effects on chloroplast biogenesis to sco3-1 (Albrecht et al, 2010), suggesting a role for the cytoskeleton and peroxisomes in chloroplast biogenesis.…”