ABSTRACT
We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021.
Subject(s)
Arabidopsis Proteins/analysis , Arabidopsis , Glucosyltransferases/analysis , Hypocotyl , Microscopy/methods , Arabidopsis/chemistry , Arabidopsis/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Glucosyltransferases/metabolism , Hypocotyl/chemistry , Hypocotyl/metabolismABSTRACT
Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gßγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gßγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Cell Membrane/metabolism , Glucosyltransferases/metabolism , Receptors, G-Protein-Coupled/metabolism , Cell Wall/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Membrane Proteins/metabolism , Multiprotein Complexes/metabolism , Mutation/genetics , Protein Binding , Seedlings/growth & development , Seedlings/ultrastructure , Signal Transduction , Stress, Physiological , trans-Golgi Network/metabolism , trans-Golgi Network/ultrastructureABSTRACT
All plant cells are surrounded by a cell wall that determines the directionality of cell growth and protects the cell against its environment. Plant cell walls are comprised primarily of polysaccharides and represent the largest sink for photosynthetically fixed carbon, both for individual plants and in the terrestrial biosphere as a whole. Cell wall synthesis is a highly sophisticated process, involving multiple enzymes and metabolic intermediates, intracellular trafficking of proteins and cell wall precursors, assembly of cell wall polymers into the extracellular matrix, remodeling of polymers and their interactions, and recycling of cell wall sugars. In this review we discuss how newly fixed carbon, in the form of UDP-glucose and other nucleotide sugars, contributes to the synthesis of cell wall polysaccharides, and how cell wall synthesis is influenced by the carbon status of the plant, with a focus on the model species Arabidopsis (Arabidopsis thaliana).
