Metal Sensing by the IRT1 Transporter-Receptor Orchestrates Its Own Degradation and Plant Metal Nutrition.

Plant roots forage the soil for iron, the concentration of which can be dramatically lower than those needed for growth. Soil iron uptake uses the broad metal spectrum IRT1 transporter that also transports zinc, manganese, cobalt, and cadmium. Sophisticated iron-dependent transcriptional regulatory mechanisms allow plants to tightly control the abundance of IRT1, ensuring optimal absorption of iron. Here, we uncover that IRT1 acts as a transporter and receptor (transceptor), directly sensing excess of its non-iron metal substrates in the cytoplasm, to regulate its own degradation. Direct metal binding to a histidine-rich stretch in IRT1 triggers its phosphorylation by the CIPK23 kinase and facilitates the subsequent recruitment of the IDF1 E3 ligase. CIPK23-driven phosphorylation and IDF1-mediated lysine-63 polyubiquitination are jointly required for efficient endosomal sorting and vacuolar degradation of IRT1. Thus, IRT1 directly senses elevated non-iron metal concentrations and integrates multiple substrate-dependent regulations to optimize iron uptake and protect plants from highly reactive metals.

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 BRASSINOSTEROID INSENSITIVE1 (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. Loss of Desi3a leads to hypersensitivity to BRs, indicating that Desi3a acts as a negative regulator of BR signaling. Besides, 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 to enhanced 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.

A toggle switch in plant nitrate uptake.

In plants, the uptake of nitrate from the soil is a critical process controlled by complex regulatory networks that target nitrate transporters in the roots. In this issue, Ho et al. (2009) show that phosphorylation of the CHL1 nitrate transporter allows the plant root to sense and respond to different nitrate concentrations in the soil.

Differential metal sensing and metal-dependent degradation of the broad spectrum root metal transporter IRT1.

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, which 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. A short-term exposure to cadmium decreased the cell surface levels of IRT1 through endocytosis and degradation, but with a lower efficiency than observed for other IRT1 metal substrates. We demonstrated that IRT1 endocytosis in response to cadmium is mediated through the direct binding of cadmium to histidine residues within the regulatory loop of IRT1. However, we revealed that the affinity of the metal sensing motif is much lower for cadmium compared to other metal substrates of IRT1. Finally, we proved that cadmium-induced IRT1 degradation takes place through ubiquitin-mediated endocytosis driven by the UBC35/36 E2 ubiquitin-conjugating enzymes and the IDF1 E3 ubiquitin ligase. Altogether, this work sheds light on the mechanisms of cadmium-mediated downregulation of IRT1 and provides an additional molecular basis for cadmium accumulation and toxicity in plants.

Advanced Cataloging of Lysine-63 Polyubiquitin Networks by Genomic, Interactome, and Sensor-Based Proteomic Analyses.

The lack of resolution when studying the many different ubiquitin chain types found in eukaryotic cells has been a major hurdle to our understanding of their specific roles. We currently have very little insight into the cellular and physiological functions of Lys-63 (K63)-linked ubiquitin chains, although they are the second most abundant forms of ubiquitin in plant cells. To overcome this problem, we developed several large-scale approaches to characterize (1) the E2-E3 ubiquitination machinery driving K63-linked ubiquitin chain formation and (2) K63 polyubiquitination targets to provide a comprehensive picture of K63 polyubiquitin networks in Arabidopsis (Arabidopsis thaliana). Our work identified the ubiquitin-conjugating enzymes (E2s) UBC35/36 as the major drivers of K63 polyubiquitin chain formation and highlights the major role of these proteins in plant growth and development. Interactome approaches allowed us to identify many proteins that interact with the K63 polyubiquitination-dedicated E2s UBC35/36 and their cognate E2 variants, including more than a dozen E3 ligases and their putative targets. In parallel, we improved the in vivo detection of proteins decorated with K63-linked ubiquitin chains by sensor-based proteomics, yielding important insights into the roles of K63 polyubiquitination in plant cells. This work strongly increases our understanding of K63 polyubiquitination networks and functions in plants.

Endocytosis of BRASSINOSTEROID INSENSITIVE1 Is Partly Driven by a Canonical Tyr-Based Motif.

Clathrin-mediated endocytosis (CME) and its core endocytic machinery are evolutionarily conserved across all eukaryotes. In mammals, the heterotetrameric adaptor protein complex-2 (AP-2) sorts plasma membrane (PM) cargoes into vesicles via the recognition of motifs based on Tyr or di-Leu in their cytoplasmic tails. However, in plants, very little is known about how PM proteins are sorted for CME and whether similar motifs are required. In Arabidopsis (Arabidopsis thaliana), the brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) undergoes endocytosis, which depends on clathrin and AP-2. Here, we demonstrate that BRI1 binds directly to the medium AP-2 subunit (AP2M). The cytoplasmic domain of BRI1 contains five putative canonical surface-exposed Tyr-based endocytic motifs. The Tyr-to-Phe substitution in Y898KAI reduced BRI1 internalization without affecting its kinase activity. Consistently, plants carrying the BRI1Y898F mutation were hypersensitive to BRs. Our study demonstrates that AP-2-dependent internalization of PM proteins via the recognition of functional Tyr motifs also operates in plants.

Experimental toolbox for quantitative evaluation of clathrin-mediated endocytosis in the plant model Arabidopsis.

Clathrin-mediated endocytosis (CME) is a crucial cellular process implicated in many aspects of plant growth, development, intra- and intercellular signaling, nutrient uptake and pathogen defense. Despite these significant roles, little is known about the precise molecular details of how CME functions in planta To facilitate the direct quantitative study of plant CME, we review current routinely used methods and present refined, standardized quantitative imaging protocols that allow the detailed characterization of CME at multiple scales in plant tissues. These protocols include: (1) an efficient electron microscopy protocol for the imaging of Arabidopsis CME vesicles in situ, thus providing a method for the detailed characterization of the ultrastructure of clathrin-coated vesicles; (2) a detailed protocol and analysis for quantitative live-cell fluorescence microscopy to precisely examine the temporal interplay of endocytosis components during single CME events; (3) a semi-automated analysis to allow the quantitative characterization of global internalization of cargos in whole plant tissues; and (4) an overview and validation of useful genetic and pharmacological tools to interrogate the molecular mechanisms and function of CME in intact plant samples.This article has an associated First Person interview with the first author of the paper.

Birth, life and death of the Arabidopsis IRT1 iron transporter: the role of close friends and foes.

MAIN CONCLUSION: IRT1 intracellular dynamics and function are finely controlled through protein-protein interactions. In plants, iron uptake from the soil is tightly regulated to allow optimal growth and development. Iron acquisition in Arabidopsis root epidermal cells requires the IRT1 transporter, which also mediates the entry of non-iron metals. In this mini-review, we describe how protein-protein interactions regulate IRT1 intracellular dynamics and IRT1-mediated metal uptake to maintain iron homeostasis. Recent interactomic data provided interesting clues on IRT1 secretion and the putative involvement of COPI- and COPII-mediated pathways. Once delivered to the plasma membrane, IRT1 can interact with other components of the iron uptake machinery to form an iron acquisition complex that likely optimizes iron entrance in root epidermal cells. Then, IRT1 may be internalized from the plasma membrane. In the past decade, IRT1 endocytosis emerged as an essential mechanism to control IRT1 subcellular localization and thus to tune iron uptake. Interestingly, IRT1 endocytosis and degradation are regulated by its non-iron metal substrates in an ubiquitin-dependent manner, which requires a set of interacting-proteins including kinases, E3 ubiquitin ligases and ESCRT complex subunits. This mechanism is essential to avoid non-iron metal overload in Arabidopsis when the iron is scarce.

Endocytosis in plants: Peculiarities and roles in the regulated trafficking of plant metal transporters.

The removal of transmembrane proteins from the plasma membrane via endocytosis has emerged as powerful strategy in the regulation of receptor signalling and molecule transport. In the last decade, IRON-REGULATED TRANSPORTER1 (IRT1) has been established as one of the key plant model proteins for studying endomembrane trafficking. The use of IRT1 and additional other metal transporters has uncovered novel factors involved in plant endocytosis and facilitated a better understanding of the role of endocytosis in the fine balancing of plant metal homoeostasis. In this review, we outline the specifics of plant endocytosis compared to what is known in yeast and mammals, and based on several examples, we demonstrate how studying metal transport has contributed to extending our knowledge of endocytic trafficking by shedding light on novel regulatory mechanisms and factors.

Regulation of Root Nutrient Transporters by CIPK23: 'One Kinase to Rule Them All'.

Protein kinases constitute essential regulatory components in the majority of cellular processes in eukaryotic cells. The CBL-INTERACTING PROTEIN KINASE (CIPK) family of plant protein kinases functions in calcium (Ca2+)-related signaling pathways and is therefore involved in the response to a wide variety of signals in plants. By covalently linking phosphate groups to their target proteins, CIPKs regulate the activity of downstream targets, their localization, their stability and their ability to interact with other proteins. In Arabidopsis, the CIPK23 kinase has emerged as a major hub driving root responses to diverse environmental stresses, including drought, salinity and nutrient imbalances, such as potassium, nitrate and iron deficiencies, as well as ammonium, magnesium and non-iron metal toxicities. This review will chiefly report on the prominent roles of CIPK23 in the regulation of plant nutrient transporters and on the underlying molecular mechanisms. We will also discuss the different scenarios explaining how a single promiscuous kinase, such as CIPK23, may convey specific responses to a myriad of signals.

Dynamic Control of the High-Affinity Iron Uptake Complex in Root Epidermal Cells.

In plants, iron uptake from the soil is tightly regulated to ensure optimal growth and development. Iron absorption in Arabidopsis root epidermal cells requires the IRT1 transporter that also allows the entry of certain non-iron metals, such as Zn, Mn, and Co. Recent work demonstrated that IRT1 endocytosis and degradation are controlled by IRT1 non-iron metal substrates in a ubiquitin-dependent manner. To better understand how metal uptake is regulated, we identified IRT1-interacting proteins in Arabidopsis roots by mass spectrometry and established an interactome of IRT1. Interestingly, the AHA2 proton pump and the FRO2 reductase, both of which work in concert with IRT1 in the acidification-reduction-transport strategy of iron uptake, were part of this interactome. We confirmed that IRT1, FRO2, and AHA2 associate through co-immunopurification and split-ubiquitin analyses, and uncovered that they form tripartite direct interactions. We characterized the dynamics of the iron uptake complex and showed that FRO2 and AHA2 ubiquitination is independent of the non-iron metal substrates transported by IRT1. In addition, FRO2 and AHA2 are not largely endocytosed in response to non-iron metal excess, unlike IRT1. Indeed, we provide evidence that the phosphorylation of IRT1 in response to high levels of non-iron metals likely triggers dissociation of the complex. Overall, we propose that a dedicated iron-acquisition protein complex exists at the cell surface of Arabidopsis root epidermal cells to optimize iron uptake.

The many facets of protein ubiquitination and degradation in plant root iron-deficiency responses.

Organisms need to deal with the absolute requirement for metals and also their possible toxicity. This is achieved through an intricate network of signaling pathways that are integrated to ultimately fine-tune iron uptake and metabolism. The mechanisms by which plants cope with iron limitation and the associated genomic responses are well characterized. On top of this transcriptional cascade is another level of regulation involving the post-translational protein modification and degradation. The ubiquitination and/or degradation of several transcription factors in the iron-deficiency signaling pathways and metal transporters has recently come to light. In this review we discuss the mechanisms and possible roles of protein modification and turnover in the regulation of root iron-deficiency responses. We also highlight the tight coupling between metal sensing by E3 ubiquitin ligases or bifunctional transporters and protein degradation.

Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors.

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.

The bifunctional transporter-receptor IRT1 at the heart of metal sensing and signalling.

Transporters are at the centre of regulatory modules allowing optimal assimilation, distribution or efflux of substrate molecules. The IRT1 root metal transporter represents a textbook example in which detailed regulatory networks have been shown to integrate several endogenous and exogenous cues at various levels to regulate its expression and to fine tune iron uptake. Here, we summarise recent advances in the dissection of the transcriptional and posttranslational control of IRT1 by its various metals substrates and discuss the emerging role of IRT1 in the direct sensing of non-iron metals flowing through IRT1 to drive its degradation. We propose that transporters that also act as receptors are likely to be a common theme in the regulation of nutrient transport by sensing local nutrient concentrations.

Proteasome-independent functions of lysine-63 polyubiquitination in plants.

Contents Summary 995 I. Introduction 995 II. The plant Ub machinery 996 III. From Ub to Ub linkage types in plants 997 IV. Increasing analytical resolution for K63 polyUb in plants 998 V. How to build K63 polyUb chains? 998 VI. Cellular roles of K63 polyUb in plants 999 VII. Physiological roles of K63 polyUb in plants 1004 VIII. Future perspectives: towards the next level of the Ub code 1006 Acknowledgements 1006 References 1007 SUMMARY: Ubiquitination is a post-translational modification essential for the regulation of eukaryotic proteins, having an impact on protein fate, function, localization or activity. What originally appeared to be a simple system to regulate protein turnover by the 26S proteasome is now known to be the most intricate regulatory process cells have evolved. Ubiquitin can be arranged in countless chain assemblies, triggering various cellular outcomes. Polyubiquitin chains using lysine-63 from ubiquitin represent the second most abundant type of ubiquitin modification. Recent studies have exposed their common function in proteasome-independent functions in non-plant model organisms. The existence of lysine-63 polyubiquitination in plants is, however, only just emerging. In this review, we discuss the recent advances on the characterization of ubiquitin chains and the molecular mechanisms driving the formation of lysine-63-linked ubiquitin modifications. We provide an overview of the roles associated with lysine-63 polyubiquitination in plant cells in the light of what is known in non-plant models. Finally, we review the crucial roles of lysine-63 polyubiquitin-dependent processes in plant growth, development and responses to environmental conditions.

A versatile Multisite Gateway-compatible promoter and transgenic line collection for cell type-specific functional genomics in Arabidopsis.

Multicellular organisms are composed of many cell types that acquire their specific fate through a precisely controlled pattern of gene expression in time and space dictated in part by cell type-specific promoter activity. Understanding the contribution of highly specialized cell types in the development of a whole organism requires the ability to isolate or analyze different cell types separately. We have characterized and validated a large collection of root cell type-specific promoters and have generated cell type-specific marker lines. These benchmarked promoters can be readily used to evaluate cell type-specific complementation of mutant phenotypes, or to knockdown gene expression using targeted expression of artificial miRNA. We also generated vectors and characterized transgenic lines for cell type-specific induction of gene expression and cell type-specific isolation of nuclei for RNA and chromatin profiling. Vectors and seeds from transgenic Arabidopsis plants will be freely available, and will promote rapid progress in cell type-specific functional genomics. We demonstrate the power of this promoter set for analysis of complex biological processes by investigating the contribution of root cell types in the IRT1-dependent root iron uptake. Our findings revealed the complex spatial expression pattern of IRT1 in both root epidermis and phloem companion cells and the requirement for IRT1 to be expressed in both cell types for proper iron homeostasis.

The dynamics of plant plasma membrane proteins: PINs and beyond.

Plants are permanently situated in a fixed location and thus are well adapted to sense and respond to environmental stimuli and developmental cues. At the cellular level, several of these responses require delicate adjustments that affect the activity and steady-state levels of plasma membrane proteins. These adjustments involve both vesicular transport to the plasma membrane and protein internalization via endocytic sorting. A substantial part of our current knowledge of plant plasma membrane protein sorting is based on studies of PIN-FORMED (PIN) auxin transport proteins, which are found at distinct plasma membrane domains and have been implicated in directional efflux of the plant hormone auxin. Here, we discuss the mechanisms involved in establishing such polar protein distributions, focusing on PINs and other key plant plasma membrane proteins, and we highlight the pathways that allow for dynamic adjustments in protein distribution and turnover, which together constitute a versatile framework that underlies the remarkable capabilities of plants to adjust growth and development in their ever-changing environment.

Unraveling K63 Polyubiquitination Networks by Sensor-Based Proteomics.

The polybiquitination of proteins can take on different topologies depending on the residue from ubiquitin involved in the chain formation. Although the role of lysine-48 (K48) polyubiquitination in proteasome-mediated degradation is fairly well characterized, much less is understood about the other types of ubiquitin chains and proteasome-independent functions. To overcome this, we developed a K63 polyubiquitin-specific sensor-based approach to track and isolate K63 polyubiquitinated proteins in plants. Proteins carrying K63 polyubiquitin chains were found to be enriched in diverse membrane compartments as well as in nuclear foci. Using liquid chromatography-tandem mass spectrometry, we identified over 100 proteins from Arabidopsis (Arabidopsis thaliana) that are modified with K63 polyubiquitin chains. The K63 ubiquitinome contains critical factors involved in a wide variety of biological processes, including transport, metabolism, protein trafficking, and protein translation. Comparison of the proteins found in this study with previously published nonresolutive ubiquitinomes identified about 70 proteins as ubiquitinated and specifically modified with K63-linked chains. To extend our knowledge about K63 polyubiquitination, we compared the K63 ubiquitinome with K63 ubiquitination networks based on the Arabidopsis interactome. Altogether, this work increases our resolution of the cellular and biological roles associated with this poorly characterized posttranslational modification and provides a unique insight into the networks of K63 polyubiquitination in plants.

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis.

In plants, the controlled absorption of soil nutrients by root epidermal cells is critical for growth and development. IRON-REGULATED TRANSPORTER 1 (IRT1) is the main root transporter taking up iron from the soil and is also the main entry route in plants for potentially toxic metals such as manganese, zinc, cobalt, and cadmium. Previous work demonstrated that the IRT1 protein localizes to early endosomes/trans-Golgi network (EE/TGN) and is constitutively endocytosed through a monoubiquitin- and clathrin-dependent mechanism. Here, we show that the availability of secondary non-iron metal substrates of IRT1 (Zn, Mn, and Co) controls the localization of IRT1 between the outer polar domain of the plasma membrane and EE/TGN in root epidermal cells. We also identify FYVE1, a phosphatidylinositol-3-phosphate-binding protein recruited to late endosomes, as an important regulator of IRT1-dependent metal transport and metal homeostasis in plants. FYVE1 controls IRT1 recycling to the plasma membrane and impacts the polar delivery of this transporter to the outer plasma membrane domain. This work establishes a functional link between the dynamics and the lateral polarity of IRT1 and the transport of its substrates, and identifies a molecular mechanism driving polar localization of a cell surface protein in plants.