Location: Plant, Soil and Nutrition Research
Project Number: 8062-21000-049-003-A
Project Type: Cooperative Agreement
Start Date: Sep 1, 2021
End Date: Aug 31, 2024
Objective:
Plants use complex physiological and biochemical mechanisms to adapt to environmental stresses throughout their development and growth continuously. Among these, the translocation of mineral nutrients throughout the plant and the regulation of the ion composition within the cells of the different tissues play a central role. The overall objective of this research is to elucidate the structural and functional characteristics of various membrane transport proteins known to mediate these processes. The specific objectives are to develop, improve and implement new methodologies to characterize the invitro transport activity of various families of plant membrane transporters. The correlation between the recorded invitro characteristics and other cellular and whole plant observations will provide insight into the functional roles of the transporter in the context of its in planta roles and determinants of important agronomic traits.
Approach:
Functional Analysis of Membrane Transporters: The existing electrophysiological platforms will be improved to allow for recording the activity of the targeted membrane transporters expressed in heterologous systems. Currently, plant transporters are expressed in frog eggs and their transport activity is characterized by measuring ion transport using a two-electrode voltage clamp approach. The goal is to set up a more flexible and efficient expression system using Human Embryonic Kidney (HEK293) cells. These cells are easily transformed (transiently and stably) and produce large amounts of recombinant protein (e.g., membrane transporters) while being amendable to functional analysis via electrophysiological approaches. These cell cultures are hardy and require low maintenance. Transport in HEK293 expressing cells will be assayed electrophysiologically in platforms utilizing solid supported lipid bilayers and a glass patch-clamp approach. These methodologies will allow an understanding of the nature of the substrates transported by these proteins. These novel expression systems and electrophysiological platforms will also be used to carry out the functional comparison of natural allelic variations of these transporters, with the goal of establishing the structural motifs that determine and underlie the functional characteristics of a given transporter. The collaborator will provide the various transporters, associated with Zn, Cu, Fe and heavy metal transport, and their allelic variants, as well as the cellular and whole plant ion profiles to perform the functional correlations.
