Abstract:
Salvaging of nucleobases from storage tissues plays an important role in providing nucleobases to cells which are unable to synthesize sufficient amounts for their needs, e.g. during early phases of seedling development, when cells divide rapidly. Cellular uptake systems for pyrimidines have been described so far, but in higher eukaryotes, transporters of thymine and uracil have not been characterized. AtUPS1 and PvUPS1 were recently identified as transporters of allantoin in Arabidopsis and French bean, respectively. However, in contrast to tropical legumes, Arabidopsis mainly uses amino acids for long distance transport. Allantoin transport has not been described in Brassicaceae. Thus, the physiological substrates of UPS transporters in Arabidopsis may be compounds structurally related to allantoin.
The Arabidopsis genome encodes five UPS transporters. For a better understanding of the role of members of the AtUPS family, different members were characterized in this work by (i) detailed investigation of substrate specificities when expressed in Xenopus oocytes (AtUPS1, 2), (ii) analysis of transport mechanism and regulation (AtUPS1, 2, 5), (iii) analysis of expression pattern in plants by promoter-GUS studies and RT-PCR, and (iv) investigation of transgenic plants deficient or lower in expression of AtUPS members due to T-DNA insertions or PTGS (post transcriptional gene silencing) (AtUPS1, 2, 5).
AtUPS1 and AtUPS2 have broad substrate specificities covering cyclic purine degradation products as well as pyrimidines. Moreover, they both mediated high-affinity transport of uracil (~ 6 µM). AtUPS1 and AtUPS2 seemed to transport uncharged substrates by H+-cotransport. With the exception of AtUPS2, a nucleotide-binding domain (Walker A motif) is predicted in the UPS sequences. It might have a role in the regulation of transport mediated by AtUPS proteins. In addition, two splicing variants of AtUPS5 were cloned and analyzed (AtUPS5 and a shorter variant, AtUPS5s). AtUPS5 also mediated the transport of allantoin, uracil and other heterocycles. AtUPS5s might be involved in the regulation of transport mediated by AtUPS5 by interaction or complex formation with UPS5, since coexpression of both proteins in yeast decreased the transport efficiency of AtUPS5 significantly.
Moreover, the expression patterns of members of the AtUPS family suggest functions in nucleobase salvaging, for example the high expression of AtUPS1 during germination and the early stages of seedling development. AtUPS4 might play a role in uracil supply to pollen. AtUPS2 and AtUPS5 might serve the long-range transport of nucleobases and possibly the transport involved in cellular turnover. In addition, expression of members of the AtUPS family was elevated in plants grown under conditions that can lead to nucleotide deficiency (nitrogen deficiency, salt stress). Arabidopsis ups2 and ups5 T-DNA insertion mutants and ups1 lines, in which transcript levels were reduced by PTGS, were more tolerant to (5-fluorouracil) 5-FU, compared to the wild type. The results suggest that in Arabidopsis UPS transporters are the main transporters of uracil and potentially other nucleobases, whereas during evolution legumes may have taken advantage of the low selectivity of UPS proteins for the long distance transport of allantoin.
Nevertheless, for a detailed understanding of the transport of nucleobases and derivatives in plants, knowledge about their cellular and subcellular concentrations under dynamic conditions of living plant cells is necessary. Therefore, as a starting point for the imaging of nucleobases in plant cells, a FRET-based sensor for the purine hypoxanthine was developed and tested in vitro.