Characteristics of triose phosphate/phosphate translocator from wheat and its role in the distribution of assimilates

In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point for partitioning of photosynthate between source and sink. Studies on the characteristic of TPT and its regulation on the distribution of assimilates are critical for improving the utilization rate of photosynthetic assimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat ( Triticum aestivum L. cv. Jing 411) leaves. Analysis of SDS-PAGE and, labeling with an irreversible specific inhibitor, [H-3](2)-DIDS (4, 4'-diisothiocyano-2, 2'-stilbenedisulfonate, DIDS) demonstrated that wheat TPT was a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the total membrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquely distributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles and mitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties of TPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetone phosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P, therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was strongly inhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulation of starch in chloroplasts, therefore the TPT protein of wheat controls the export-of TP out of chloroplasts into cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out of chloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat has much high transport efficiency, which plays an important role in the regulation of the distribution of assimilates in wheat chloroplasts.