Functional Characterization of the Arabidopsis Abscisic Acid Transporters NPF4.5 and NPF4.6 in Xenopus Oocytes

Abstract

Few proteins have been characterized as abscisic acid transporters. Several of them are NRT1/PRT Family (NPF) transporters which have been characterized in yeast using reporter systems. Because several members of the NPF4 subfamily members were identified in yeast as ABA transporters, here, we screened for ABA transport activity the seven members of the NPF4 subfamily in Xenopus oocytes using cRNA injection and ³H-ABA accumulation. The ABA transport capacities of NPF4.2, NPF4.5, NPF4.6, and NPF4.7 were confirmed. The transport properties of NPF4.5 and NPF4.6 were studied in more detail. Both ABA transporter activities are pH-dependent and slightly pH-dependent apparent Km around 500 μM. There is no competitive inhibition of the ABA-analogs pyrabactin and quinabactin on ABA accumulation demonstrating a different selectivity compared to the ABA receptors. Functional expression of these ABA transporters in Xenopus oocyte is an opportunity to start structure-function studies and also to identify partner proteins of these hormone transporters.

Keywords: Km; abscisic acid; hormone; pH; transport.

Figure 1

Screen for ABA transport activity of Arabidopsis NPF4 subfamily members in Xenopus oocytes. Control (noninjected) and NPF4-injected oocytes were bathed in 1 μM ³H-ABA (pH = 6.0), and ³H accumulation in oocytes was quantified after 20 min. Values are mean +/− SEM (n = 5–12 oocytes, biological replicates). ***P < 0.001, **0.001 < P < 0.005, two-sided t-test after comparison with control oocytes.

Figure 2

pH-dependent ³H accumulation in NPF4.5 and NPF4.6 in Xenopus oocytes. Control (noninjected, black circles), NPF4.5 (green circles) and NPF4.6 (red circles) injected oocytes were bathed in 1 μM ³H-ABA (pH = 5.0 to 7.5), and ³H accumulation in oocytes was quantified after 20 min. Values are mean +/− SEM (n = 6–20 oocytes, biological replicates). ***P < 0.001, **0.001 < P < 0.005, two-sided t-test after comparison with control oocytes.

Figure 3

Effect of external ABA concentration on ³H accumulation in NPF4.5 and NPF4.6-expressing oocytes. ³H-accumulation in NPF4.5 (A) and NPF4.6 (B) expressing oocytes depending on the wide external ABA concentrations range (0–5 μM ³H-ABA). The solid lines are least-squares Michaelian fits. Data are mean +/− SE (n = 6–10 oocytes, biological replicates).

Figure 4

Effect of pyrabactin and quinabactin on ³H accumulation in NPF4.5 and NPF4.6 in Xenopus oocytes. Control (noninjected), NPF4.5 and NPF4.6 injected oocytes were bathed in 1 μM ³H-ABA (pH = 6.0), and ³H accumulation in oocytes was quantified after 20 min in the presence or in the absence of 0.5 or 5 μM of pyrabactin or quinabactin. Values are mean +/− SEM (n = 9–11 oocytes, biological replicates). The pyrabactin and quinabactin treatment has no significant effect on ³H accumulation (two-sided t-test).