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ARS Home » Northeast Area » Ithaca, New York » Robert W. Holley Center for Agriculture & Health » Plant, Soil and Nutrition Research » Research » Publications at this Location » Publication #378512

Research Project: Genetic and Genomic Characterization of Crop Resistance to Soil-based Abiotic Stresses

Location: Plant, Soil and Nutrition Research

Title: Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids

Author
item STUEBLER, MARTIN - Cornell University
item MANZER, ZACHARY - Cornell University
item LIU, HAN-YUAN - Cornell University
item MILLER, JULIA - Cornell University
item RICHTER, ANNETT - Boyce Thompson Institute
item KRISHNAN, SRINIVASAN - Boyce Thompson Institute
item SELIVANOVITCH, EKATERINA - Cornell University
item BANUNA, BARITUZIGA - Cornell University
item JANDER, GEORG - Boyce Thompson Institute
item REIMHULT, ERIK - University Of Natural Resources & Applied Life Sciences - Austria
item ZIPFEL, WARREN - Cornell University
item ROEDER, ADRIENNE - Cornell University
item Pineros, Miguel
item DANIEL, SUSAN - Cornell University

Submitted to: ACS Applied Materials and Interfaces
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/21/2024
Publication Date: 4/9/2024
Citation: Stuebler, M., Manzer, Z., Liu, H., Miller, J., Richter, A., Krishnan, S., Selivanovitch, E., Banuna, B., Jander, G., Reimhult, E., Zipfel, W.R., Roeder, A., Pineros, M., Daniel, S. 2024. Plant Membrane-On-A-Chip: A Platform for Studying Plant Membrane Proteins and Lipids. ACS Applied Materials and Interfaces. 16(16). https://doi.org/10.1021/acsami.3c18562.
DOI: https://doi.org/10.1021/acsami.3c18562

Interpretive Summary: Transport proteins embed within the cellar membrane mediate the transport of ions and nutrients entering and leaving the cell. Therefore, they play key roles in regulating many essential biological and environmental plant responses. Understanding processes occurring at the biomembrane cellular level requires developing new techniques that allow the recreation of the complex microenvironment occurring at these molecular scales. In this study, we report on the development of a methodology that allows for the self-assembly of native plant cell membranes derived from the surface of plant cells, along both its membrane proteins and lipid environment, .thereby creating a biomimetic plant membrane on a supporting glass surface (a “chip”). This “chip” is compatible with several microscopy, biophysical and analytical tools commonly used by scientists to understand biological phenomena occurring at this interface. We demonstrate the utility and generality of this technology by characterizing the diffusion and orientation of proteins from three different plant species using this platform. This new methodology has multiple immediate applications in studies characterizing cellular phenome, such as for example, protein-protein interactions, lipid-proteins interactions, and measurement of transport across the membrane and other similar applications where recreating and preserving the complex membrane environment is essential.

Technical Abstract: The plant cell plasma membrane is a semi-permeable barrier that serves two important functions: (1) transport of essential molecules in and out of the cell, and (2) sensory transduction of environmental stimuli and developmental signals. The plasma membrane is a two-dimensional, fluid mosaic material composed of primarily lipids and proteins. Considerable progress has been made over the past two decades to understand plant membrane proteins from a genetic perspective, but complementary advances in tools for physiological, electrophysiological, and biochemical characterization of plant membrane proteins have been limited. This work describes the development of Plant Membrane-on-a-Chip, a new cell-free platform consisting of a supported proteo-lipid membrane derived from the plant plasma membrane (PM) that captures the essential features of the membrane and is compatible with state-of-the-art microscopy and biophysical characterization tools. Three model plant species (Arabidopsis thaliana, Nicotiana benthamiana, and Zea mays) were successfully used to form planar plant membranes through the straightforward production of membrane vesicles from protoplasts. Transgenic membrane proteins expressed in the plant cells are directly incorporated with defined orientation and their surrounding lipids into supported membranes without using detergents or reconstitution processes. Supported membranes can be integrated with biosensors and other techniques for the investigation of the biophysical properties of their constituents. Our plant-based supported membranes, therefore, provide a novel tool to isolate and study membrane properties, transport phenomena, biophysical processes, and the protein-lipid interactions that drive them. We illustrate single-molecule tracking of plant membrane proteins using this platform and characterization of their diffusion behavior and orientation.