Biology subjects

Vert, G.

Publications and source records attributed to Vert, G..

8 recordsLinked to original sources

bioRxiv2026

TurboID-based proteomic profiling reveals proxitome of the IRT1 metal transporter and new insight into metal uptake regulation in plants

IRT1 is the major root iron transporter responsible for broad spectrum metal absorption in Arabidopsis root epidermal cells. Non-iron metal substrates of IRT1 were recently shown to regulate IRT1 cell surface levels by endocytosis and vacuolar degradation. TurboID-based proximity labeling was recently developed to detect protein:protein interactions and thus shed light on the intricate regulation of proteins in living cells. Although TurboID-based proximity labeling technology has been successfully established in mammals, its application in plant systems remains limited and inexistent for highly hydrophobic multispann transmembrane proteins. Here, we established TurboID for proximity labeling of IRT1 and identified 494 IRT1-specific proximal proteins, including the previously reported FYVE1 IRT1 interacting protein. To showcase the biological relevance of identified IRT1 proximal proteins, we characterized further the NHX5 Na+(K+)/H+ antiporter and the RGLG2 E3 ubiquitin ligase. We validated both IRT1 proximal proteins as IRT1 partners using several orthogonal assays. We also highlight the contribution of NHX-type antiporters and RGLG-type E3 ligases in plants responses to non-iron metal nutrition and IRT1 endocytosis. Overall, our work showcases the power of TurboID to identify new interacting proteins for plant transporters, expanding the application of this technology to proteins notoriously difficult to work with.

plant biology↗

bioRxiv2025

Broad application of plant protein tagging with ALFA tag for nanobody-based imaging and biochemical approaches

Epitope tags are broadly used for detecting, modifying or purifying proteins of interest, but their breadth of application is often rather poor. Recently, the rationally designed ALFA tag and its ALFA Nanobody complements the repertoire of epitope tags and emerged as a highly versatile epitope tag characterized in various animal models and that outperforms existing tags. Here we evaluated the ALFA tag/ALFA Nanobody technology in plants and showcase its application for in planta protein detection across multiple compartments or cellular structures, protein:protein interaction studies, protein purification, induced-proximity approaches, and super-resolution microcopy. Most importantly, we shed light on the edge of ALFA tagging technology for proteins difficult to tag for topological or functionals reasons. We provide the proof of concept of ALFA tag technology for the detection and functional analysis of the Arabidopsis IRT1 iron transporter. Overall, such versatile and validated toolbox of ALFA tag and ALFA Nanobody applications will serve as a resource of great interest for functional studies in plants.

plant biology↗

bioRxiv2024

Metal sensing properties of the disordered loop from the Arabidopsis metal transceptor IRT1

The plant IRT1 iron transporter is a plasma membrane protein that takes up iron in root upon iron-limited conditions. Besides its primary metal substrate iron, IRT1 also transports other divalent metals that overaccumulate in plants when soil iron is low and IRT1 is highly expressed. We previously reported that the intracellular regulatory loop between transmembrane helices TM4 and TM5, comprising IRT1 residues from 144 to 185, is involved in the post-translational regulation of IRT1 by its non-iron metal substrates. Upon excess of zinc, IRT1 (144-185) undergoes phosphorylation by the CIPK23 kinase followed by its ubiquitination by IDF1 to target IRT1 for vacuolar degradation. This zinc-dependent downregulation of IRT1 requires the presence of four histidine (H) residues in IRT1 loop, that directly bind zinc. However, how selective metal binding is achieved and how this allows downstream regulation to take place is largely unknown. Here, we characterized the metal binding properties and structure of IRT1 loop to better understand the molecular basis of non-iron metal sensing and signaling. Using a combination of circular dichroism and NMR, we demonstrate that zinc and manganese bind to IRT1 loop with nanomolar range affinity, and that metal binding does not trigger structuration of the loop. We prove that zinc and manganese binding is mediated by the four H residues and identify aspartic acid (D) residue D173 as helping in metal coordination and participating to metal sensing and metal-dependent degradation of IRT1 in plants. Altogether, our data provide further evidence of how the regulatory loop of IRT1 senses high cytosolic divalent metal concentrations to regulate metal uptake in plants.

plant biology↗

bioRxiv2024

Direct non-transcriptional link between brassinosteroid perception and cortical microtubule reorientation drives hypocotyl growth

Brassinosteroids (BRs) are steroid-type phytohormones that are essential for plant growth, development and adaptation to environmental stresses. BRs promote cell wall remodelling through extensive reprogramming of gene expression, thus impacting on cell elongation and hypocotyl growth. The existence of non-genomic responses to BRs is however still unclear. Here we identified and characterized a new family of BRI1-interacting proteins identified by yeast two hybrid named MBAPs. We confirmed using several complementary approaches that MBAPs are genuine BRI1 partners in plant cells. We demonstrated that MBAPs are localized to cortical microtubules (CMTs) underlying the plasma membrane using a short conserved helix and where they contact with and are phosphorylated by BRI1. Combinations of mbap loss-of-function mutants showed hypersensitivity to BRs and establish MBAPs as negative regulators of BR responses. Surprisingly, mbap mutants showed unaffected typical downstream BR signaling readouts and BR target gene expression. Rather, mbap mutants displayed disordered CMT network and enhanced CMT reorganization upon BR perception. Altogether, our work shed light on the direct non-transcriptional connection between BR perception at the cell surface and CMT organization in the control of hypocotyl elongation.

plant biology↗

bioRxiv2022

Ubiquitination steers SRF3 plasma membrane nano-organization to specify signaling outputs

Organisms cope with myriads of competing and conflicting environmental signals. These signals are often perceived by cell surface receptor kinases to mount appropriate adaptive responses. However, it is not well understood by which mechanism single receptor kinases can transduce different signals. The plant receptor kinase SRF3 transduces low iron and bacteria-derived signals. We found that upon these signals, ubiquitinated SRF3 is recognized by clathrin-mediated endocytosis for vacuolar targeting. Live super resolution microscopy revealed that cell surface SRF3 is present in a fast diffusible fraction, which is sustained by ubiquitination, and that non-ubiquitinated SRF3 is present in immobile nanodomains. Ubiquitination-mediated degradation of SRF3 is required for signaling only under low iron but not upon flg22 perception. Flg22-triggered SRF3 phosphorylation leads to SRF3 accumulation in the immobile fraction in which degradation is restricted, thereby preventing low iron signaling. We therefore propose that ubiquitination-dependent plasma membrane nano-organization of SRF3 specifies its signal transduction pathways.

plant biology↗

bioRxiv2022

Cadmium-induced endocytosis of the broad spectrum root metal transporter of Arabidopsis

Iron is an essential micronutrient for plant growth and development. Under low iron conditions, Arabidopsis plants take up soil iron using the root iron transporter IRT1. In addition to iron, IRT1 also transports others divalent metals including cadmium that consequently accumulates into plant tissues and enters the food chain. IRT1 expression was shown to be regulated at the transcriptional and post-translational levels by its essential metal substrates to maximize iron uptake while limiting the accumulation of zinc, manganese or cobalt. Here, we characterized the regulation of IRT1 by cadmium and uncovered a cadmium-mediated downregulation of IRT1 protein by endocytosis. A short term exposure to cadmium indeed decreased the celle surface levels of IRT1 through endocytosis and degradation. This is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. Moreover, we demonstrated that cadmium-induced IRT1 degradation uses ubiquitin-mediated endocytosis driven by the IDF1 E3 ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and offers a unique opportunity to boost plant cadmium uptake in phytoremediation/phytoextraction strategies.

plant biology↗

bioRxiv2022

SUMO/deSUMOylation of the BRI1 brassinosteroid receptor modulates plant growth responses to temperature

Brassinosteroids (BRs) are a class of steroid molecules perceived at the cell surface that act as plant hormones. The BR receptor BRI1 offers a model to understand receptor-mediated signaling in plants and the role of post-translational modifications. Here we identify SUMOylation as a new modification, targeting BRI1 to regulate its activity. BRI1 is SUMOylated in planta on two lysine residues and the levels of BRI1-SUMO conjugates are controlled by the Desi3a SUMO protease. We demonstrate that BRI1 is deSUMOylated at elevated temperature by Desi3a, leading to increased BRI1 interaction with the negative regulator of BR signaling BIK1 and enhancing BRI1 endocytosis. Loss of Desi3a or BIK1 results in increased response to temperature elevation, indicating that BRI1 deSUMOylation acts as a safety mechanism necessary to keep temperature responses in check. Altogether, our work establishes BRI1 deSUMOylation as a molecular crosstalk mechanism between temperature and BR signaling, allowing plants to translate environmental inputs into growth response. SIGNIFICANCE STATEMENTThe brassinosteroid (BR) receptor BRI1 provides a paradigm for understanding receptor-mediated signaling in plants and contribution of post-translational modifications. Here, we show that BRI carries SUMO modifications in planta on two intracellular lysine residues and that temperature elevation triggers BRI1 deSUMOylation mediated by the Desi3a SUMO protease. Importantly, BRI1 deSUMOylation leads to downregulation of BR signaling via increased BRI1 interaction with the BIK1 negative regulator and increased BRI1 endocytosis. Loss of BRI1 deSUMOylation in desi3a mutants boosts plant responses to heat, indicating that BRI1 deSUMOylation acts as a brake to keep temperature responses in check. Our study uncovers a new post-translational modification targeting BRI1 and sheds light on its functional outcome for environmentally-controlled plant growth.

plant biology↗

bioRxiv2021

The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remain unknown. We identified a receptor kinase, SRF3 coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthase activity. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domain which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

plant biology↗