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Tobacco (Nicotiana tabacum L.) plants were transformed with an antisense construct of the chloroplast triose phosphate/phosphate translocator (TPT). Three transformant lines of the T4 progeny, which showed a large decrease in the transcript level of the TPT were used for further biochemical and physiological characterisation. In all antisense lines tested, TPT transport activity was diminished by 50-70% compared with the wild type (WT). Despite this high reduction in the transport capacity, alpha TPT plants lacked any visible phenotype. Hexokinase and alpha-amylase activities were increased in alpha TPT plants compared with the WT, whereas activities of ribulose-1,5-bisphosphate carboxylase/oxygenase and ADP-glucose pyrophosphorylase (AGPase) were not affected. At the end of a 14-h light period, leaf starch contents in alpha TPT lines were similar to those of the WT and controls, indicating that a decrease in the TPT had no effect on starch accumulation. Sucrose contents were diminished by more than 50% in alpha TPT lines compared with control plants. The time course of starch accumulation revealed a transient increase in the starch content in a selected alpha TPT line after 6 h in the light, followed by a decrease towards the end of the light period. Labelling with 14C indicated that during the dark and light (late afternoon) periods starch is mobilised at higher rates in alpha TPT lines than in the controls. Glucose/fructose ratios at the end of the dark period were increased from 1.2 in control plants to 2 in alpha TPT lines indicating increased amylolytic starch degradation. Initial rates of [14C] glucose transport in isolated chloroplasts were increased by a factor of 2-3 in alpha TPT plants compared with the WT. Rates of CO2 assimilation were substantially diminished in the alpha TPT lines in high CO2 and low O2, but remained unaffected in ambient CO2. The rate of photosynthetic electron transport during the induction of photosynthesis in saturating CO2 exhibited pronounced oscillations only in WT and control plants. Oscillations were less pronounced in alpha TPT plants, indicating that phosphate limitation of photosynthesis is lowered in alpha TPT plants compared with the WT. It is proposed that photoassimilates are more readily directed into starch biosynthesis in alpha TPT plants. This is supported by determinations of 3-phosphoglycerate levels (an activator of AGPase) during the transition from dark to light in high CO2.

The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing the TPT gene isolated from the C4 plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C3 plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more 14CO2 in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in alphaTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as alpha-amylase or hexokinase were increased in alphaTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, alpha-amylase activity exhibited a pronounced diurnal variation in alphaTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 microl x l(-1)) and low O2 (2%) the rate of CO2 assimilation was decreased in the alphaTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type.

Transgenic tobacco (Nicotiana tabacum L.) plants with decreased and increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were used to study the control the TPT exerts on the flux of starch and sucrose biosynthesis, as well as CO2 assimilation, respiration and photosynthetic electron transport. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (alphaTPT) and tobacco lines overexpressing a TPT gene from Flaveria trinervia (FtTPT) were used. In ambient CO2, there was no or little effect of altered TPT transport activities on either rates of photosynthetic electron transport and/or CO2 assimilation. However, in elevated CO2 (1500 microl x 1(-1)) and low O2 (2%) the TPT exerted strong control on the rate of CO2 assimilation (control coefficient for the wild type; C(J(A))(TPT) = 0.30) in saturating light. Similarly, the incorporation of 14C into starch in high CO2 was increased in tobacco plants with decreased TPT activity, but was reduced in plants overexpressing the TPT from F. trinervia. Thus, the TPT exerted negative control on the rate of starch biosynthesis with a C(J(Starch))(TPT) = -0.19 in the wild type estimated from a hyperbolic curve fitted to the data points. This was less than the positive control strength on the rate of sucrose biosynthesis (C(J(Suc))(TPT) = 0.35 in the wild type). Theoretically, the positive control exerted on sucrose biosynthesis should be numerically identical to the negative control on starch biosynthesis unless additional metabolic pathways are affected. The rate of dark respiration showed some correlation with the TPT activity in that it increased in FtTPT overexpressors, but decreased in alphaTPT plants with an apparent control coefficient of C(J(Res))(TPT) = 0.24. If the control on sucrose biosynthesis is referred to as "gain of carbon" (positive control) and the control on starch biosynthesis as well as dark respiration as a "loss of carbon" (negative control) for sucrose biosynthesis and subsequent export, the sum of the control coefficients on dark respiration and starch biosynthesis would be numerically similar to the control coefficient on the rate of sucrose biosynthesis. There was also some control on the rate of photosynthetic electron transport, but only at high light and in elevated CO2 combined with low O2. The control coefficient for the rate of photosynthetic electron transport was C(J(ETR))(TPT) = 0.16 in the wild type. Control coefficients were also calculated for plants with elevated and lowered TPT activity. Furthermore, the extent to which starch degradation/glucose utilisation compensates for the lack of triose phosphate export was assessed. The TPT also exerted control on metabolite contents in air.

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