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 transketolase-overexpressing 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.
Highlights
The Calvin Benson (C3) cycle is the primary pathway of atmospheric CO2 uptake and fixation into organic molecules
This effect of TK on carbon allocation may be due to its central location in the C3 cycle where it catalyzes the reversible transfer of a two-carbon molecule to glyceraldehyde 3-phosphate (G3P) from sedoheptulose 7-phosphate, generating xylulose 5-phosphate (Xu5P) and ribose 5-phosphate; or fructose 6-phosphate to produce Xu5P and erythrose 4-phosphate
TK enzyme assays confirmed that these plants had increased TK activity compared with that of wild-type plants, the differences (1.76- to 2.5fold increase compared with wild-type plants) in TK activity were less pronounced than the differences in transcript abundance (Figure 2B; Supplemental Table 1)
Summary
The Calvin Benson (C3) cycle is the primary pathway of atmospheric CO2 uptake and fixation into organic molecules. Analysis of tobacco plants expressing an antisense construct for plastid TK demonstrated that a small reduction in the activity of this enzyme by 20 to 40% inhibited photosynthesis and significantly decreased the levels of aromatic amino acids and phenylpropanoids (Henkes et al, 2001) Comparison of these results with those from analysis of antisense SBPase and FBPaldolase plants showed that these changes are unique to TK and do not reflect a general reduction in the availability of carbon from the C3 cycle (Haake et al, 1998, 1999; Harrison et al, 1998). One hypothesis that this work has raised is that increasing TK activity in the plant may have the potential to increase carbon assimilation through the C3 cycle and to increase phenylpropanoid metabolism
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