Equilibrative nucleoside transporter 1 (ENT1) is critical for pollen germination and vegetative growth in Arabidopsis

Abstract

ENT1 of Arabidopsis thaliana was the first member of the equilibrative nucleoside transporter (ENT) family to be identified in plants and characterized as a cellular, high-affinity nucleoside importer. Evidence is presented here for a tonoplast localization of ENT1 based on proteome data and Western blot analyses. Increased export of adenosine from reconstituted tonoplast preparations from 35S:ENT1 mutants compared with those from the wild type and ENT1-RNAi mutants support this view. Furthermore, increased vacuolar adenosine and vacuolar 2′3′-cAMP (an intermediate of RNA catabolism) contents in ENT1-RNAi mutants, but decreased contents of these metabolites in 35S:ENT1 over-expresser mutants, were observed. An up-regulation of the salvage pathway was detected in the latter mutants, leading to the conclusion that draining the vacuolar adenosine storage by ENT1 over-expression interferes with cellular nucleotide metabolism. As a consequence of the observed metabolic alterations 35S:ENT1 over-expresser mutants exhibited a smaller phenotypic appearance compared with wild-type plants. In addition, ENT1:RNAi mutants exhibited significantly lower in vitro germination of pollen and contained reduced internal and external ATP levels. This indicates that ENT1-mediated nucleosides, especially adenosine transport, is important for nucleotide metabolism, thus influencing growth and pollen germination.

Nucleotide Metabolism in Plants

“…24) mutants (Schroeder et al, 2018). Consistently, transgenic lines with increased vacuolar nucleoside export (Bernard et al, 2011) are smaller than the wild type.…”

“…The details of this process have not been investigated so far. The tonoplast membrane possesses a nucleoside exporter (equilibrative nucleoside transporter 1 [ENT1, At1g70330]; Bernard et al, 2011) for the release of nucleosides into the cytoplasm. For adenosine, the turnover of S-adenosyl Met (SAM), used for methylation reactions, is another important source (Figs.…”

3D Imaging of Tapetal Mitochondria Suggests the Importance of Mitochondrial Fission in Pollen Growth

“…Freshly opened flowers were collected from wild-type and elm1 mutant in pollen suspension buffer containing 50 mM of K 2 HPO 4 , and 1% Suc and 4% sorbitol at pH 5.8 (Bernard et al, 2011), and vortexed vigorously. One-hundred microliters of pollen suspension was used for intracellular ATP measurement and protein quantification.…”

Current Progress in Tonoplast Proteomics Reveals Insights into the Function of the Large Central Vacuole

“…For example, the characterization of the copper transporter COPT5 (Klaumann et al, 2011), the molybdate transporter MOT2 (Gasber et al, 2011), and the nucleoside transporter ENT1 (Bernard et al, 2011) was initiated by vacuolar proteome data.…”