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

Abstract

To investigate the effect of increased plastid transketolase on photosynthetic capacity and growth, tobacco (Nicotiana tabacum) plants with increased levels of transketolase protein were produced. This was achieved using a cassette composed of a full-length Arabidopsis thaliana transketolase cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed a major and unexpected effect of plastid transketolase overexpression as the transgenic tobacco plants exhibited a slow-growth phenotype and chlorotic phenotype. These phenotypes were complemented by germinating the seeds of transketolase-overexpressing lines in media containing either thiamine pyrophosphate or thiamine. Thiamine levels in the seeds and cotyledons were lower in transketolase-overexpressing lines than in wild-type plants. When transketolaseoverexpressing plants were supplemented with thiamine or thiamine pyrophosphate throughout the life cycle, they grew normally and the seed produced from these plants generated plants that did not have a growth or chlorotic phenotype. Our results reveal the crucial importance of the level of transketolase activity to provide the precursor for synthesis of intermediates and to enable plants to produce thiamine and thiamine pyrophosphate for growth and development. The mechanism determining transketolase protein levels remains to be elucidated, but the data presented provide evidence that this may contribute to the complex regulatory mechanisms maintaining thiamine homeostasis in plants.

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

“…6C and 6D), similar to previously observed in tobacco lines overexpressing TK (Khozaei et al, 2015). This may be due to a disruption of export of carbon from the CBB cycle, mainly erythrose-4-phosphate (E4P), a main intermediate in the synthesis of aromatic amino acids, phenylpropanoids synthesis, and isoprenoid biosynthesis (Khozaei et al, 2015). Cells grown under this condition showed about 32% decreased in vivo oxygen evolution rate (Fig.…”

“…Potential limiting steps in the CBB cycle in plants have been intensively studied using both experimental (antisense plants and overexpressing plants) and simulation (steadystate model and dynamic model) approaches (Farquhar et al, 1980;Khozaei et al, 2015;Raines, 2011;Zhu et al, 2007). However, there is only very limited experimental information in cyanobacteria.…”

“…TK exports carbon resources to the shikimate acid pathway, phenylpropanoid metabolism, and thiamine biosynthesis. Even minor changes (increased or reduced) in the TK activity in tobacco had dramatic effects on the photosynthetic carbon fixation (Henkes et al, 2001;Khozaei et al, 2015). These data demonstrated that TK is an important enzyme in the regulation of carbon fixation and shows the possibility to increase photosynthetic carbon fixation by increasing the level of TK.…”

Does Abiotic Stress Cause Functional B Vitamin Deficiency in Plants?

“…There are as yet no cases of full-blown rerouting in response to vitamin deficiency, although two studies provide indirect evidence for redirection of fluxes that may be, at least in part, active responses. In the first study, tobacco leaves overexpressing transketolase, a thiamin diphosphate-dependent enzyme, became moderately thiamin diphosphate-deficient and showed reduced isoprenoid synthesis via the thiamin diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS); this reduction was associated with lower DXS transcript levels (Khozaei et al, 2015). In the second study, a drastic, rapidonset tetrahydrofolate deficiency in Arabidopsis cells treated with antifolate drugs led to initial depletion of Met and S-adenosyl-Met pools, reflecting reduction in the tetrahydrofolate-dependent flux of one-carbon units to Met as one-carbon fluxes were adaptively reprioritized in favor of nucleotides (Loizeau et al, 2008).…”

Long-Distance Transport of Thiamine (Vitamin B1) Is Concomitant with That of Polyamines

“…S1), as a necessary cofactor for key metabolic enzymes involved in glycolysis and the citric acid cycle (Fitzpatrick and Thore, 2014). In plants, it is also necessary for the Calvin cycle, the biochemical route of carbon fixation (Khozaei et al, 2015). Therefore, thiamine is vital for cell energy supply in all organisms.…”