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The screen identified a T-DNA line that was deficient in the accumulation of GA-Fl. By imaging NPF3-YFP in root tip cells where the vacuoles are small and by using chemical and genetic markers that stain the tonoplast and plasma membrane, we were able to rule out vacuolar localization ( seedlings with Brefeldin A (BFA), an inhibitor of ARF–GTP exchange factors, for 15min and imaged NPF3-YFP by confocal microscopy.

This treatment resulted in the depletion of NPF3-YFP from the plasma membrane and accumulation of the transporter in intracellular vesicles, indicating that NPF3 is rapidly targeted to the plasma membrane in a BFA-dependent manner ( seedlings with GA or paclobutrazol (paclo; an inhibitor of GA synthesis) with and without BFA did not show a significant difference compared with respective control treatments, implying that NPF3 subcellular localization or its BFA sensitivity are not directly affected by GA.

We could not detect any difference to the WT in response to nitrate for any of the tested lines (Supplementary Fig. However, overexpression of of 0.5m M (Supplementary Fig. Kinetic uptake assays of weak acids are, however, notoriously difficult to interpret.

The propensity of weak acids to diffuse across cellular membranes at acidic p H and the cumulative nature of the uptake assays makes it difficult to accurately estimate the apparent is likely to be an overestimation.

The screen was performed visually by testing 10 seedlings per T-DNA line using the confocal microscope.However, the identity of GA transporters involved in this process has remained elusive.Here we describe a search for GA transporters by screening for mutants defective in GA-Fl accumulation.Moreover, additional support for endodermis localization of NPF3 was obtained by analysing cell type-specific expression data (Supplementary Fig.3a) derived from microarray studies of RNA bound to ribosomes, which were immunoprecipitated by the use of epitope-tagged ribosomal protein from seedlings transcripts and translation are consistent with a role for NPF3 in GA-Fl accumulation in the root endodermis.

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