Boron Uptake Assay in Xenopus laevis Oocytes

Background

Boron is essential for plant growth but is toxic when it is accumulated. Boron has structural functions in the cell wall through cross-linking of pectic polysaccharides at rhamnogalacturonan II regions (Funakawa and Miwa, 2015). In solution, B exists primarily as boric acid at physiological pH [B(OH)₃ + H₂O ⇆ B(OH)₄^- + H⁺ (pKa = 9.24)]. Boric acid is a small neutral molecule and thus shows significant passive diffusion across biological membranes (Dordas et al., 2000). In addition, plants utilize boric acid channels belonging to major intrinsic protein (MIP, aquaporin) family and borate exporters (BOR family) to maintain B homeostasis (Takano et al., 2008).

Initially, maize PIP1 was expressed in Xenopus leavis oocytes and shown to facilitate boric acid uptake by 30% over the water-injected oocytes (Dordas et al., 2000). Then, Arabidopsis NIP5;1 was shown to facilitate boric acid uptake five to nine-fold over the water-injected oocytes (Takano et al., 2006). Together with the finding that Arabidopsis mutants lacking NIP5;1 activity showed lower B uptake into roots and severe growth defects under low B conditions, NIP5;1 was identified as a boric acid channel. NIP5;1 homologs, NIP6;1 and NIP7;1 from Arabidopsis, Lsi1 (NIP2;1) from rice, NIP2;1 from barley, and TLS1 (NIP3;1) from maize were also shown to facilitate boric acid uptake using Xenopus oocytes (Tanaka et al., 2008; Li et al., 2011, Mitani-Ueno et al., 2011; Schnurbusch et al., 2010; Durbak et al., 2014). Importantly, OsLsi1 transported boric acid, silicic acid, arsenite, and water, and AtNIP5;1 transported boric acid, arsenite, and water, but not silicic acid in Xenopus oocytes (Mitani-Ueno et al., 2011). Xenopus oocytes were also used for characterization of barley Bot1, a borate exporter for high-B tolerance, by electrophysiology (Nagarajan et al., 2015).

As another heterologous expression system, yeast Saccharomyces cerevisiae has been used for transport studies of B. In this system, Arabidopsis BOR1 and yeast ScBOR1 decreased B concentrations in the cells, and thus were characterized as borate exporters (Takano et al., 2002; Takano et al., 2007). In contrast, expression of AtNIP5;1 and its rice ortholog OsNIP3;1 increased boric acid uptake (Hanaoka et al., 2014). As a convenient but indirect assay, survival assay has been employed using Saccharomyces cerevisiae. Expression of BOR1 homologs conferred tolerance to toxic B conditions (Nozawa et al., 2006; Miwa et al., 2007; Sutton et al., 2007; Cañon et al., 2013; Hayes et al., 2015) and expression of NtXIP1;1, AtNIP4;1, HvPIP1;3 and HvPIP1;4 increased sensitivity (Fitzpatrick and Reid 2009; Bienert et al., 2011; Di Giorgio et al., 2016).

Nevertheless, Xenopus oocytes expression system is advantageous for ‘quantitative’ transport studies (Miller and Zhou, 2000; Yesilirmak and Sayers, 2009). It has apparently low intrinsic transport activity for many substrates. It tends to produce comparable levels of membrane proteins in the plasma membrane dependent on the amount of injected cRNA. Recently we utilized these merits for analysis of AtNIP5;1 variants. AtNIP5;1 is localized on the soil-side plasma membrane domain of the outermost root cell layers of roots (Takano et al., 2010). We found that the phosphorylated state of conserved threonine residues in ‘TPX’ repeat in N-terminal region of AtNIP5;1 is associated with the polar localization of NIP5;1 (Wang et al., 2017). Using the AtNIP5;1 variant in which conserved threonines were substituted to alanine, we showed that the polar localization is important for efficient B translocation in plant roots. Importantly, the direct boric acid uptake assay in X. laevis oocytes clarified that the threonine residues are not involved in the boric acid uptake activity of NIP5;1 channel (Wang et al., 2017).

Here, we describe the protocol of boric acid transport assay using Xenopus oocytes (Figure 1) and inductively coupled plasma-mass spectrometry (ICP-MS), which is applicable for various other metalloids and metals.