Identification and analysis of Arabidopsis thiamine pyrophosphate transporters

Thiamine pyrophosphate (TPP) serves as a cofactor in universal metabolic pathways, including glycolysis, the pentose phosphate pathway (PPP) and the tricarboxylic acid cycle; moreover, it is essential for the proper functioning of all organisms. Recently, several steps of the plant thiamine biosynthetic pathway have been characterised, and a mechanism of feedback regulation for thiamine biosynthesis via riboswitching has been unravelled. In plants, thiamine is made in the chloroplasts and then transferred to the cytosol to generate the active form of thiamine, TPP. The mitochondria and chloroplasts must import TPP from the cytosol because both organelles contain TPP-dependent enzymes. In Arabidopsis, two members of the mitochondrial carrier family (MCF), AtTpc1 and AtTpc2, export TPP to the mitochondria, but the chloroplast TPP carrier is still unknown. This project aims to identify thiamine chloroplast transporter(s) and investigate mitochondrial thiamine transporters in Arabidopsis. The approaches used to achieve the aims of this project include phylogenetic analysis, mutant analysis, polymerase chain reaction techniques and gene construct techniques. AtTpc1 and AtTpc2 have a limited effect on plant growth under normal conditions, at least in terms of stem length and secondary stem length. Moreover, phylogenetic analysis showed that AtFolt1 (Arabidopsis folate transporter) and CTT (AT3G51870) are putative chloroplast TPP transporters. The inhibition of AtFolt1 by about 50% and CTT by about 80% was associated with reduced TPP levels in leaves. Thus, AtFolt1 and CTT may be chloroplast TPP transporters.