Isolation, functional characterization, and expression analysis of grapevine (Vitis vinifera L.) hexose transporters: differential roles in sink and source tissues

Three hexose transporters (VvHT3, VvHT4, and VvHT5) were cloned from Vitis vinifera L. and functionally characterized in the hexose transport-impaired Saccharomyces cerevislae mutant EBY.VW4000. Both VvHT4 and VvHT5 facilitated glucose uptake, with Kms of 137 mu M and 89 mu M, respectively. VvHT3 was not functional in the yeast system but a VvHT3:GFP (green fluorescent protein) fusion protein was targeted to the plasma membrane in plant cells. In young 'sink' leaves, transcript levels of all five VvHTs and a cell wall invertase (VvcwlNV) were low. In mature leaves, there were increased levels of VvHT1, VvHT3, VvHT5, and VvcwiNV transcripts, suggesting that mature leaves may have an increased capacity for apoplastic sucrose hydrolysis and hexose retrieval. In grape berries, VvHT1, VvHT2, and VvHT3 transcript levels were found to be significantly higher than those of VvHT4 and VvHT5. VvHT1 was most highly expressed early in berry development but decreased during the period of rapid sugar accumulation, while VvHT2 and VvHT3 expression remained high during this accumulation phase. VvcwiNV expression occurred throughout berry development but peaked just prior to veraison. It is clear that the machinery to transport the hexose molecules produced through the cleavage of sucrose, by cell wall invertase, is present in the berry. This agrees with the suggestion that hexose accumulation to high levels during the ripening phase occurs through an apoplastic pathway. Interestingly, there is no direct relationship between VvHT gene expression and hexose accumulation, which suggests either that transcription is not the main determinant of transport activity or that other transport pathways are also active.