Cloning and characterization of a cytosolic isoform of triosephosphate isomerase developmentally regulated in potato leaves

A full-length cDNA encoding a triosephosphate isomerase (TPI) was cloned from the wild potato Solanum chacoense. Sequence analysis showed that the cDNA has high levels of homology to sequences coding for cytoplasmic TPIs (cTPIs) and lower homology to plastidic TPI (pTPI) sequences. A specific polyclonal antiserum was generated against recombinant (6 x His) epitope-tagged cTPI expressed in Escherichia coli. In Solanum tuberosum this immune-serum recognized a single TPI polypeptide present in crude leaf extracts while an antigenic signal was not evident in purified chloroplasts. Immunoblot analysis demonstrated that cTPI was distributed in all vegetative and reproductive tissues. TPI activity was followed over a 3 months time course encompassing the whole development of potato plants. Activity expressed on a fresh weight basis was significantly higher in expanding leaves compared to mature and senescing leaves during the course of the experiment. Consistent with this, there was a gradient of TPI activity and cTPI protein along the shoot axis with the highest levels found in the youngest tissues. Analysis of TPI isoforms profiles by anion exchange chromatography demonstrated that (i) photosynthetic and non-photosynthetic tissues express 2 TPI isoforms and (ii) cTPI always represents the bulk of extractable TPI activity. Of all tissues surveyed, expanding leaves had the highest cTPI to pTPI ratio. The results are discussed in relation to the hypothesis that leaf cTPI plays a role in growing tissues, where glycolysis and respiration fulfill a key function in production of energy and C skeletons for biosynthetic purposes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.