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The occasion of The Company of Biologists' workshop ‘Cellular gateways: expanding the role of endocytosis in plant development’ on 22–25 April 2018, at Wiston House, an Elizabethan mansion in West Sussex, England, witnessed stimulating and lively discussions on the mechanism and functions of endocytosis in plant cells. The workshop was organized by Jenny Russinova, Daniël Van Damme (both VIB/University of Ghent, Belgium) and Takashi Ueda (National Institute for Basic Biology, Okazaki, Japan), and aimed to bridge the gap in knowledge about the endocytic machinery and its cargos in the plant field.

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.

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.

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.

In addition to the classical functions of flavonoids in the response to biotic/abiotic stress conditions, these phenolic compounds have been implicated in the modulation of various developmental processes. These findings suggest that flavonoids are more integral components of the plant signaling machinery than traditionally recognized. To understand how flux through the flavonoid pathway affects plant cellular processes, we used wild-type and chalcone isomerase mutant (transparent testa 5, tt5) seedlings grown under anthocyanin inductive conditions, in the presence or absence of the flavonoid intermediate naringenin, the product of the chalcone isomerase enzyme. Because flavonoid biosynthetic genes are expressed under anthocyanin inductive conditions regardless of whether anthocyanins are formed or not, this system provides an excellent opportunity to specifically investigate the molecular changes associated with increased flux through the flavonoid pathway. By assessing genome-wide mRNA accumulation changes in naringenin-treated and untreated tt5 and wild-type seedlings, we identified a flavonoid-responsive gene set associated with cellular trafficking, stress responses and cellular signaling. Jasmonate biosynthetic genes were highly represented among the signaling pathways induced by increased flux through the flavonoid pathway. In contrast to studies showing a role for flavonoids in the control of auxin transport, no effect on auxin-responsive genes was observed. Taken together, our data suggest that Arabidopsis can sense flavonoids as a signal for multiple fundamental cellular processes.

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.

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.

Anthocyanins are flavonoid pigments that accumulate in the large central vacuole of most plants. Inside the vacuole, anthocyanins can be found uniformly distributed or as part of sub-vacuolar pigment bodies, the Anthocyanic Vacuolar Inclusions (AVIs). Using Arabidopsis seedlings grown under anthocyanin-inductive conditions as a model to understand how AVIs are formed, we show here that the accumulation of AVIs strongly correlates with the formation of cyanidin 3-glucoside (C3G) and derivatives. Arabidopsis mutants that fail to glycosylate anthocyanidins at the 5-O position (5gt mutant) accumulate AVIs in almost every epidermal cell of the cotyledons, as compared to wild-type seedlings, where only a small fraction of the cells show AVIs. A similar phenomenon is observed when seedlings are treated with vanadate. Highlighting a role for autophagy in the formation of the AVIs, we show that various mutants that interfere with the autophagic process (atg mutants) display lower numbers of AVIs, in addition to a reduced accumulation of anthocyanins. Interestingly, vanadate increases the numbers of AVIs in the atg mutants, suggesting that several pathways might participate in AVI formation. Taken together, our results suggest novel mechanisms for the formation of sub-vacuolar compartments capable of accumulating anthocyanin pigments.

Plants produce a very large number of specialized compounds that must be transported from their site of synthesis to the sites of storage or disposal. Anthocyanin accumulation has provided a powerful system to elucidate the molecular and cellular mechanisms associated with the intracellular trafficking of phytochemicals. Benefiting from the unique fluorescent properties of anthocyanins, we show here that in Arabidopsis (Arabidopsis thaliana), one route for anthocyanin transport to the vacuole involves vesicle-like structures shared with components of the secretory pathway. By colocalizing the red fluorescence of the anthocyanins with green fluorescent protein markers of the endomembrane system in Arabidopsis seedlings, we show that anthocyanins are also sequestered to the endoplasmic reticulum and to endoplasmic reticulum-derived vesicle-like structures targeted directly to the protein storage vacuole in a Golgi-independent manner. Moreover, our results indicate that vacuolar accumulation of anthocyanins does not depend solely on glutathione S-transferase activity or ATP-dependent transport mechanisms. Indeed, we observed a dramatic increase of anthocyanin-filled subvacuolar structures, without a significant effect on total anthocyanin levels, when we inhibited glutathione S-transferase activity, or the ATP-dependent transporters with vanadate, a general ATPase inhibitor. Taken together, these results provide evidence for an alternative novel mechanism of vesicular transport and vacuolar sequestration of anthocyanins in Arabidopsis.

International audience; 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.

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.

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.

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 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.

The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes. In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R. Substitution of six amino acids in P1 with the C1 amino acids results in P1(*), whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively. We have used P1(*) in combination with various promoters to uncover two mechanisms for R function. On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1. This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent. The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor. A second function of R requires cis-regulatory elements in addition to the C1/P1 DNA-binding sites for R-enhanced transcription of a1. We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes. Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo.

Summary As we begin to unravel the molecular mechanisms of the regulation of anthocyanin biosynthesis, we must remember that evolution and selective had significant opportunities to explore new ways to color plants, flowers, and seeds. Model plant systems such as maize, petunia, and Arabidopsis will continue to provide the framework to understand how pigment accumulation is controlled. However, it is important to investigate how nature has exploited variations in these central prototypes to provide the amazing diversity found today in the type and distribution of anthocyanin pigments.

Background Natural products have numerous medicinal applications and play important roles in the biology of the organisms that accumulate them. Few methods are currently available for identifying proteins that bind to small molecules, therefore the discovery of cellular targets for natural products with pharmacological activity continues to pose a significant challenge in drug validation. Similarly, the identification of enzymes that participate in the biosynthesis or modification of natural products remains a formidable bottleneck for metabolic engineering. Flavonoids are one large group of natural products with a diverse number of functions in plants and in human health. The coupling of flavonoids to small ceramic and glass beads provides a first step in the development of high-throughput, solid-support base approaches to screen complex libraries to identify proteins that bind natural products. Results The utilization of small glass and ceramic beads as solid supports for the coupling of small molecules was explored. Initial characterization of the beads indicated uniform and high capacity loading of amino groups. Once the beads were deemed adequate for the linking of small molecules by the coupling of NHS-fluorescein followed by microscopy, chemical hydrolysis and fluorometry, the flavonoid naringenin was modified with 1,4-dibromobutane, followed by the attachment of aminopropyltriethoxysilane. After NMR structural confirmation, the resulting 7-(4-(3-(triethoxysilyl)propylamino)butoxy) naringenin was attached to the ceramic beads. Conclusion Our results demonstrate that ceramic and glass beads provide convenient solid supports for the efficient and facile coupling of small molecules. We succeeded in generating naringenin-coupled ceramic and glass beads. We also developed a convenient series of steps that can be applied for the solid-support coupling of other related flavonoids. The availability of solid-support coupled naringenin opens up new opportunities for the identification of flavonoid-binding proteins.

Background Little is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively.