Niloufer G. Irani

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.

Clathrin-mediated endocytosis: the gateway into plant cells

Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, hormonal signaling and communication with the environment including nutrient delivery, toxin avoidance, and pathogen defense. The major endocytic mechanism in plants depends on the coat protein clathrin. It starts by clathrin-coated vesicle formation at the plasma membrane, where specific cargoes are recognized and packaged for internalization. Recently, genetic, biochemical and advanced microscopy studies provided initial insights into mechanisms and roles of clathrin-mediated endocytosis in plants. Here we summarize the present state of knowledge and compare mechanisms of clathrin-mediated endocytosis in plants with animal and yeast paradigms as well as review plant-specific regulations and roles of this process.

The clathrin adaptor complex AP-2 mediates endocytosis of brassinosteroid insensitive1 in Arabidopsis.

In mammals, clathrin-mediated endocytosis (CME) depends on the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2). Our work identifies the components of the Arabidopsis thaliana AP-2 and shows that the machinery of CME in plants is evolutionarily conserved. Our data reveal that AP-2 mediates the endocytosis of the brassinosteroid receptor BRI1. Clathrin-mediated endocytosis (CME) regulates many aspects of plant development, including hormone signaling and responses to environmental stresses. Despite the importance of this process, the machinery that regulates CME in plants is largely unknown. In mammals, the heterotetrameric ADAPTOR PROTEIN COMPLEX-2 (AP-2) is required for the formation of clathrin-coated vesicles at the plasma membrane (PM). Although the existence of AP-2 has been predicted in Arabidopsis thaliana, the biochemistry and functionality of the complex is still uncharacterized. Here, we identified all the subunits of the Arabidopsis AP-2 by tandem affinity purification and found that one of the large AP-2 subunits, AP2A1, localized at the PM and interacted with clathrin. Furthermore, endocytosis of the leucine-rich repeat receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1), was shown to depend on AP-2. Knockdown of the two Arabidopsis AP2A genes or overexpression of a dominant-negative version of the medium AP-2 subunit, AP2M, impaired BRI1 endocytosis and enhanced the brassinosteroid signaling. Our data reveal that the CME machinery in Arabidopsis is evolutionarily conserved and that AP-2 functions in receptor-mediated endocytosis.

Receptor endocytosis and signaling in plants

The emerging complexity of plant endocytic systems puts it on a par with their animal counterparts, reflecting an essential role in signal transduction. The endocytic machinery regulates the space and the time of signal transduction and processing in the cell. Plants possess numerous cell surface receptor-like kinases (RLKs) (more than 600 members in Arabidopsis thaliana and 1100 in rice), a trend attributed to their indeterminate mode of growth, the absence of cell migration, and the need for adaptation towards the environment. Thus, plants would require a robust and highly plastic endocytic system in order to integrate multiple signaling cues from neighboring cells as well as the environment. Although a comprehensive understanding of how plant endocytosis impacts signaling pathways is still lacking, experimental evidence suggests that both plant and animal endosomal systems extensively control signaling.

V-ATPase-activity in the TGN/EE is required for exocytosis and recycling in Arabidopsis

In plants, vacuolar H+-ATPase (V-ATPase) activity acidifies both the trans-Golgi network/early endosome (TGN/EE) and the vacuole. This dual V-ATPase function has impeded our understanding of how the pH homeostasis within the plant TGN/EE controls exo- and endocytosis. Here, we show that the weak V-ATPase mutant deetiolated3 (det3) displayed a pH increase in the TGN/EE, but not in the vacuole, strongly impairing secretion and recycling of the brassinosteroid receptor and the cellulose synthase complexes to the plasma membrane, in contrast to mutants lacking tonoplast-localized V-ATPase activity only. The brassinosteroid insensitivity and the cellulose deficiency defects in det3 were tightly correlated with reduced Golgi and TGN/EE motility. Thus, our results provide strong evidence that acidification of the TGN/EE, but not of the vacuole, is indispensable for functional secretion and recycling in plants.

PP2A Phosphatases: The “On-Off” Regulatory Switches of Brassinosteroid Signaling

Protein phosphatase 2A acts as both a positive and negative regulator of brassinosteroid signaling, depending on where in the pathway the phosphatase acts. Inactivation of ligand-bound plasma membrane receptors is crucial for the regulation of their signaling outputs. The internalization of activated receptors and their subsequent targeting for recycling or degradation is controlled by posttranslational modifications, of which phosphorylation and dephosphorylation play an important role. Recent work suggests that a similar mechanism acts on the brassinosteroid (BR) receptor BR INSENSITIVE 1 (BRI1) in Arabidopsis thaliana to switch off BR signaling. The degradation of BRI1 requires a protein phosphatase 2A (PP2A)–mediated dephosphorylation that is specified by methylation of the phosphatase by a leucine carboxylmethyltransferase on membranes. PP2A is also reported to act positively on BR signaling by targeting the transcription factor BRASSINAZOLE-RESISTANT 1 (BZR1), a component downstream of BRI1. Thus, PP2A proteins play a dual role in the regulation of the BR pathway to switch between inhibition and activation of the BR signaling, depending on their substrate specificity and localization.

The Specification of Geometric Edges by a Plant Rab GTPase Is an Essential Cell-Patterning Principle During Organogenesis in Arabidopsis

Summary Plant organogenesis requires control over division planes and anisotropic cell wall growth, which each require spatial patterning of cells. Polyhedral plant cells can display complex patterning in which individual faces are established as biochemically distinct domains by endomembrane trafficking. We now show that, during organogenesis, the Arabidopsis endomembrane system specifies an important additional cellular spatial domain: the geometric edges. Previously unidentified membrane vesicles lying immediately beneath the plasma membrane at cell edges were revealed through localization of RAB-A5c, a plant GTPase of the Rab family of membrane-trafficking regulators. Specific inhibition of RAB-A5c activity grossly perturbed cell geometry in developing lateral organs by interfering independently with growth anisotropy and cytokinesis without disrupting default membrane trafficking. The initial loss of normal cell geometry can be explained by a failure to maintain wall stiffness specifically at geometric edges. RAB-A5c thus meets a requirement to specify this cellular spatial domain during organogenesis.

Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana.

A subset of genes in Arabidopsis thaliana is known to be up-regulated in response to a wide range of different environmental stress factors. However, not all of these genes are characterized as yet with respect to their functions. In this study, we used transgenic knockout, overexpression and reporter gene approaches to try to elucidate the biological roles of five unknown multiple-stress responsive genes in Arabidopsis. The selected genes have the following locus identifiers: At1g18740, At1g74450, At4g27652, At4g29780 and At5g12010. Firstly, T-DNA insertion knockout lines were identified for each locus and screened for altered phenotypes. None of the lines were found to be visually different from wildtype Col-0. Secondly, 35S-driven overexpression lines were generated for each open reading frame. Analysis of these transgenic lines showed altered phenotypes for lines overexpressing the At1g74450 ORF. Plants overexpressing the multiple-stress responsive gene At1g74450 are stunted in height and have reduced male fertility. Alexander staining of anthers from flowers at developmental stage 12–13 showed either an absence or a reduction in viable pollen compared to wildtype Col-0 and At1g74450 knockout lines. Interestingly, the effects of stress on crop productivity are most severe at developmental stages such as male gametophyte development. However, the molecular factors and regulatory networks underlying environmental stress-induced male gametophytic alterations are still largely unknown. Our results indicate that the At1g74450 gene provides a potential link between multiple environmental stresses, plant height and pollen development. In addition, ruthenium red staining analysis showed that At1g74450 may affect the composition of the inner seed coat mucilage layer. Finally, C-terminal GFP fusion proteins for At1g74450 were shown to localise to the cytosol.

In Vivo Imaging of Brassinosteroid Endocytosis in Arabidopsis

Increasing evidence shows the involvement of endocytosis in specific signaling outputs in plants. To better understand the interplay between endocytosis and signaling in plant systems, more ligand-receptor pairs need to be identified and characterized. Crucial for the advancement of this research is also the development of imaging techniques that allow the visualization of endosome-associated signaling events at a high spatiotemporal resolution. This requires the establishment of tools to track ligands and their receptors by fluorescence microscopy in living cells. The brassinosteroid (BR) signaling pathway has been among the first systems to be characterized with respect to its connection with endocytic trafficking, owning to the fact that a fluorescent version of BR, Alexa Fluor 647-castasterone (AFCS) has been generated. AFCS and the fluorescently tagged BR receptor, BR INSENSITIVE1 (BRI1) have been used for the specific detection of BRI1-AFCS endocytosis and for the delineation of their endocytic route as being clathrin-mediated. AFCS was successfully applied in functional studies in which pharmacological rerouting of the BRI1-BR complex was shown to have an impact on signaling. Here we provide a method for the visualization of endocytosis of plant receptors in living cells. The method was used to track endocytosis of BRI1-BR complexes in Arabidopsis epidermal root meristem cells by using fluorescent BRs. Pulse-chase experiments combined with quantitative confocal microscopy were used to determine the internalization rates of BRs. This method is well suited to measure the internalization of other plant receptors if fluorescent ligands are available.

Synthetic Protocol for AFCS: A Biologically Active Fluorescent Castasterone Analog Conjugated to an Alexa Fluor 647 Dye

Synthetic derivatization of hormonally active brassinosteroids (BRs) can provide useful small molecule tools to probe BR signaling pathways, such as fluorescent analogs. However, most biologically active BRs are not suitable for direct chemical conjugation techniques because their derivatization typically requires extensive synthetic work and chemistry expertise. Here, we describe an operationally simple, two-step procedure to prepare and purify an Alexa Fluor 647-castasterone (AFCS) from commercially available materials. The reported strategy is also amenable to the introduction of various other amine-based labeling groups.