Using Xenopus laevis Oocytes to Functionally Characterize Plant Transporters

Abstract

Functionally characterizing plant membrane transport proteins is challenging. Typically, heterologous systems are used to study them. Immature eggs (oocytes) of the South African clawed frog Xenopus laevis are considered an ideal expression system for such studies. These large oocytes have a low number of endogenous transport systems in their plasma membranes and highly express foreign mRNA; the oocyte plasma membrane is the default destination of integral membrane proteins that lack recognized organellar sorting signals. These features facilitate almost background‐free characterization of putative plant membrane transporters. Here we describe how to isolate Xenopus laevis oocytes, prepare capped sense RNA (cRNA) of the maize boron importer TASSEL‐LESS1 (TLS1) as an example, microinject the cRNA into the isolated oocytes, and functionally assess the boron import capabilities of TLS1 in an oocyte swelling assay. These protocols can be easily adapted to study other plant and non‐plant transporters with putative import function. © 2019 by John Wiley & Sons, Inc.

Assessment of a 18F-Phenylboronic Acid Radiotracer for Imaging Boron in Maize

“…The slight hypersensitivity of Zmtls1 and Zmrte to PBA suggests that PBA likely is transported passively. Direct transport of PBA by TLS1 or RTE cannot be excluded and will need to be assessed in the future by, for example, oocyte transport assays [50]. The reported role of PBA in interfering with cell wall crosslinking could be a cause of the enhanced sensitivity of Zmtls1 to PBA, as Zmtls1 was shown to have a reduced RG-II-B crosslinking, increased RG-II monomers, and reduced cellular B content [24], which tempt the following speculations: (1) Less PBA is needed to occupy the remaining B-binding sites; and (2) the reduced cellular B content in Zmtls1 cannot successfully compete with PBA.…”

Molecular characteristics and physiological roles of Na+–K+–Cl− cotransporter 2

“…As will be shown later, it still played an important role in the progress attained. In the oocyte of X. laevis , moreover, foreign proteins commonly adopt their native functional characteristics and achieve high levels of activity (Long, O'Neill, & Cheeseman, 2018; Pike, Matthes, McSteen, & Gassmann, 2019; Wagner, Friedrich, Setiawan, Lang, & Broer, 2000).…”

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

“…This enables whole-cell current recording through only channelrhodopsin without the presence of contaminating currents from endogenous channels. Oocytes have been shown to efficiently express foreign transport proteins in large numbers with the correct subcellular localization, structure, organization, and post-translational modifications, thus preserving the protein's native functional characteristics (34,35). Expression of functional ChRs has been demonstrated in a variety of different cell types including human neurons, human embryonic kidney cells, Drosophila flies, Caenorhabditis elegans nematodes, and others, all with vastly different membrane compositions.…”