Divergent Transport Mechanisms for Pyrimidine Nucleosides in Petunia Pollen

Abstract

Peunia Aybrida pollen exhibits divergent transport m ms for pymidine nocleosides. Uridine and cytidine show all the properties of being actively trsported, a nucleoside tramport mechaism not hitherto reported in plant cells. Contrasting with this, thymidine trnsport has the properties of a nonactive, camrier-mediated system. Reo for these different mechanisms are considered to lie in the hig demand for uridine and cytidine, obined perhaps from stylar tissue, for the biosynthetic reactin of the pollen tube, while thymidine demand is lower due to the absence of DNA replication in germinating Petuia polle.Gene expression and RNA synthesis in pollen can be followed by incorporation of labeled uridine into RNA (13,19), while pollen DNA repair is monitored by labeled tymidine incorporation into DNA (8-10). In addition it has been reported that the pool of these and other nucleic acid precursors increase in the style of Petunia hybrida after pollination (20), and since pollen and pollen tube interact closely with stylar tissue in Petunia (17), then the style could be a good source of nucleic acid and nucleotide precursors for the biosynthetic reactions of the elongating pollen tube. There is a need therefore to understand the uptake of these precursors by pollen since the incorporation of nucleoside precursors in other biological systems is limited by the rate of entry into the cell (1,3,16,19). Pollen cultures also provide us with a convenient system for studying the mechanism ofpyrimidine nucleoside transport in plant cells, for comparison with other cells. The mechanism ofpyrimidine nucleoside transport varies according to the organism, having been shown to be active in certain bacterial cells (4,14,15) and nonactive, carriermediated in animal cells (11,17,18,22 MATERIAIS AND METHODSPetunia hybrida pollen, clone W166H, was germinated using a method described previously (8, 23). After 2 h at a RH of 100%, pollen was germinated in a liquid medium containing 10% sucrose/0.01% boric acid (pH 5.5), 5 mg pollen/ml. Germination achieved was 75%. Transport, measured over the first 30 min, was initiated by addition of sterile aqueous 6-[3H]uridine (2 uCi/ml, 24 Ci/mmol) or 6-[3H]thymidine (2 jCi/ml, 23.8 Ci/ mmol) or 2-['4C]cytidine (0.1 Ci/ml, 27.2 mCi/mmol) at different carrier nucleoside concentrations. After 30 min transport, the pollen was washed three times with ice-cold medium containing 1 mm of the corresponding nucleoside. The amount of nucleoside transported was calculated from the TCA-soluble radioactivity. Examination of this acid-soluble fraction by paper electrophoresis as described previously (9) showed that all the radioactivity was present as pyrimidine nucleoside'or nucleotide. Efflux was measured by resuspending washed cells, already subjected to 30 min transport, in transport medium containing the agent to be tested. After 30 min, radioactivity was determined in pollen and medium. All radioactivity was determined by liquid scintillation counting in a Packard Tricarb 460 CD liquid scintillation sy...

Purine Nucleoside Transport in Petunia Pollen Is an Active, Carrier-Mediated System Not Sensitive to Nitrobenzylthioinosine and Not Renewed during Pollen Tube Growth

“…The properties of the nucleoside transport system described here and elsewhere (11,12) pool is quite large and the chances then of the label entering DNA being repaired is extremely low compared to the label from thymidine (4). Another aspect of DNA repair which arises from the present investigation is that relating to changes in nucleoside transport during pollen germination.…”

“…Sulfydryl-binding reagents are known to be effective inhibitors of nucleoside uptake in Escherichia coli (18) and animal cells (25). We showed that these reagents are potent inhibitors of pyrimidine nucleoside transport in Petunia pollen (11,12), and as shown in Table II, NEM and PAO are also effective inhibitors of pollen purine nucleoside uptake. Similarly the metal-complexing reagents EDTA and EGTA are effective inhibitors of both purine nucleoside (Table II) (Table IV) at concentrations normally used for animal cells (25).…”

“…Before culturing, the pollen was placed in a chamber with 100% humidity for 2 h, then quickly transferred to an Erlenmeyer flask containing a sterile solution of 10% sucrose in 0.01% boric acid (pH 5.5) for culture at 25°C in a shaking water bath (5). Pollen inhibitors DCCD' and NBD-Cl, and the proton translocators CCCP and DNP (11,12) were added to the pollen cultures during transport determinations. As shown in Table II, these compounds inhibited purine nucleoside transport very effectively, and did so with an equally effective inhibition of ATP buildup in the pollen.…”

“…Pollen from Petunia hybrida L. clone W166H was collected from plants grown under glasshouse conditions and stored at -1 5°C before use as described previously (11,12) Radiochemicals were purchased from Amersham Radiochemicals, England, and other chemicals from Sigma.…”

Functional Characterization and Expression Analysis of a Gene, OsENT2, Encoding an Equilibrative Nucleoside Transporter in Rice Suggest a Function in Cytokinin Transport

“…Pollen cells of petunia (Petunia hybrida) are able to uptake exogenous nucleosides via both an active transport system and a facilitated diffusion (Kamboj and Jackson, 1984, 1985, 1987. Cotyledons separated from castor bean (Ricinus communis) efficiently import endosperm-derived secretion products, including purine and pyrimidine bases, nucleosides, and AMP, but not ATP ( Kombrink and Beevers, 1983 H]transzeatin riboside (tZR) were supplied to soybean (Glycine max) explants, they were incorporated and immediately metabolized into the corresponding nucleotides (Singh et al, 1988).…”