Unlocking protein-protein interactions in plants: A comprehensive review of established and emerging techniques.

Protein-protein interactions orchestrate plant development and serve as crucial elements for cellular and environmental communication. Understanding these interactions offers a gateway to unravel complex protein networks that will allow a better understanding of nature. Methods for the characterization of protein-protein interactions have been around for a long time, yet the complexity of some of these interactions fuels the development of new techniques that provide a better understanding of the underlying dynamics. In many cases, the application of these techniques is limited by the nature of the available sample. While some methods require an in vivo set up, others solely depend on protein sequences to study protein-protein interactions via an in silico set up. The vast amount of techniques available to date calls for a way to select the appropriate tools for the study of specific interactions. Here, we classify widely spread tools and new emerging techniques for the characterization of protein-protein interactions based on sample requirements while providing insights into the information that they can potentially deliver. We provide a comprehensive overview of commonly used techniques and elaborate on the most recent developments, showcasing their implementation in plant research.

A precise balance of TETRASPANIN1/TORNADO2 activity is required for vascular proliferation and ground tissue patterning in Arabidopsis.

The molecular mechanisms guiding oriented cell divisions in the root vascular tissues of Arabidopsis thaliana are still poorly characterised. By overlapping bulk and single-cell transcriptomic datasets, we unveiled TETRASPANIN1 (TET1) as a putative regulator in this process. TET1 is expressed in root vascular cells, and loss-of-function mutants contain fewer vascular cell files. We further generated and characterised a CRISPR deletion mutant and showed, unlike previously described mutants, that the full knock out is additionally missing endodermal cells in a stochastic way. Finally, we show that HA-tagged versions of TET1 are functional in contrast to fluorescent TET1 translational fusions. Immunostaining using HA-TET1 lines complementing the mutant phenotype suggested a dual plasma membrane and intracellular localisation in the root vasculature and a polar membrane localisation in the young cortex, endodermal and initial cells. Taken together, we show that TET1 is involved in both vascular proliferation and ground tissue patterning. Our initial results pave the way for future work to decipher its precise mode of action.

Structural and Evolutionary Aspects of Plant Endocytosis.

Endocytosis is an essential eukaryotic process that maintains the homeostasis of the plasma membrane proteome by vesicle-mediated internalization. Its predominant mode of operation utilizes the polymerization of the scaffold protein clathrin forming a coat around the vesicle; therefore, it is termed clathrin-mediated endocytosis (CME). Throughout evolution, the machinery that mediates CME is marked by losses, multiplications, and innovations. CME employs a limited number of conserved structural domains and folds, whose assembly and connections are species dependent. In plants, many of the domains are grouped into an ancient multimeric complex, the TPLATE complex, which occupies a central position as an interaction hub for the endocytic machinery. In this review, we provide an overview of the current knowledge regarding the structural aspects of plant CME, and we draw comparisons to other model systems. To do so, we have taken advantage of recent developments with respect to artificial intelligence-based protein structure prediction.

Chromatin attachment to the nuclear matrix represses hypocotyl elongation in Arabidopsis thaliana.

The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs (SAURs) in Arabidopsis thaliana. The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.

Biomolecular condensation orchestrates clathrin-mediated endocytosis in plants.

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

Guidelines for naming and studying plasma membrane domains in plants.

Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.