Cytokinin is an important regulator of plant growth and development. In Arabidopsis thaliana, the two-component phosphorelay mediated through a family of histidine kinases and response regulators is recognized as the principal cytokinin signal transduction mechanism activating the complex transcriptional response to control various developmental processes. Here, we identified an alternative mode of cytokinin action that uses endocytic trafficking as a means to direct plant organogenesis. This activity occurs downstream of known cytokinin receptors but through a branch of the cytokinin signaling pathway that does not involve transcriptional regulation. We show that cytokinin regulates endocytic recycling of the auxin efflux carrier PINFORMED1 (PIN1) by redirecting it for lytic degradation in vacuoles. Stimulation of the lytic PIN1 degradation is not a default effect for general downregulation of proteins from plasma membranes, but a specific mechanism to rapidly modulate the auxin distribution in cytokinin-mediated developmental processes.

Cross-talk between plant cells and their surroundings requires tight regulation of information exchange at the plasma membrane (PM), which involves dynamic adjustments of PM protein localization and turnover to modulate signal perception and solute transport at the interface between cells and their surroundings. In animals and fungi, turnover of PM proteins is controlled by reversible ubiquitylation, which signals endocytosis and delivery to the cell's lytic compartment, and there is emerging evidence for related mechanisms in plants. Here, we describe the fate of Arabidopsis PIN2 protein, required for directional cellular efflux of the phytohormone auxin, and identify cis-and trans-acting mediators of PIN2 ubiquitylation. We demonstrate that ubiquitin acts as a principal signal for PM protein endocytosis in plants and reveal dynamic adjustments in PIN2 ubiquitylation coinciding with variations in vacuolar targeting and proteolytic turnover. We show that control of PIN2 proteolytic turnover via its ubiquitylation status is of significant importance for auxin distribution in root meristems and for environmentally controlled adaptations of root growth. Moreover, we provide experimental evidence indicating that PIN2 vacuolar sorting depends on modification specifically by lysine 63 -linked ubiquitin chains. Collectively, our results establish lysine 63 -linked PM cargo ubiquitylation as a regulator of polar auxin transport and adaptive growth responses in higher plants.P lants have evolved a repertoire of mechanisms for continuously adapting vital parameters in response to fluctuating environmental conditions. Sensing and responding to such variations depend to a large extent on the activity of plasma membrane (PM)-associated proteins that function in stimulus perception and solute transport. Specifically, adjustments in subcellular distribution of PM proteins are an efficient means to modulate their activity and involve continuous protein cycling between PM and endosomes as well as irreversible targeting for degradation in the lytic vacuole/lysosome (1).An evolutionary conserved machinery controls PM protein sorting for degradation, and, specifically in animals and fungi, it was demonstrated that PM protein fate is decisively influenced by their reversible ubiquitylation, triggering cargo endocytosis and delivery to the lytic compartment (2-4). Related mechanisms appear to be operative in plants (5-9) because, recently, ubiquitylation has been described for some plant PM proteins, linking nutrient transport and stimulus perception to endocytic protein turnover (6-9). Strikingly, different patterns of protein ubiquitylation have been observed, with mono-, di-, as well as polyubiquitylation implicated in regulating endocytic trafficking and degradation of distinct proteins (5). This resembles the situation in nonplant organisms, in which mono-and polyubiquitylation have been associated with cargo endocytosis (10).Vacuolar sorting was also demonstrated for PIN1-type auxin carrier proteins, which are instrumental for ...

The polarized transport of the phytohormone auxin [1], which is crucial for the regulation of different stages of plant development [2, 3], depends on the asymmetric plasma membrane distribution of the PIN-FORMED (PIN) auxin efflux carriers [4, 5]. The PIN polar localization results from clathrin-mediated endocytosis (CME) from the plasma membrane and subsequent polar recycling [6]. The Arabidopsis genome encodes two groups of dynamin-related proteins (DRPs) that show homology to mammalian dynamin-a protein required for fission of endocytic vesicles during CME [7, 8]. Here we show by coimmunoprecipitation (coIP), bimolecular fluorescence complementation (BiFC), and Förster resonance energy transfer (FRET) that members of the DRP1 group closely associate with PIN proteins at the cell plate. Localization and phenotypic analysis of novel drp1 mutants revealed a requirement for DRP1 function in correct PIN distribution and in auxin-mediated development. We propose that rapid and specific internalization of PIN proteins mediated by the DRP1 proteins and the associated CME machinery from the cell plate membranes during cytokinesis is an important mechanism for proper polar PIN positioning in interphase cells.

SUMMARYRemarkable progress in various techniques of in vivo fluorescence microscopy has brought an urgent need for reliable markers for tracking cellular structures and processes. The goal of this manuscript is to describe unexplored effects of the FM (Fei Mao) styryl dyes, which are widely used probes that label processes of endocytosis and vesicle trafficking in eukaryotic cells. Although there are few reports on the effect of styryl dyes on membrane fluidity and the activity of mammalian receptors, FM dyes have been considered as reliable tools for tracking of plant endocytosis. Using plasma membrane-localized transporters for the plant hormone auxin in tobacco BY-2 and Arabidopsis thaliana cell suspensions, we show that routinely used concentrations of FM 4-64 and FM 5-95 trigger transient re-localization of these proteins, and FM 1-43 affects their activity. The active process of re-localization is blocked neither by inhibitors of endocytosis nor by cytoskeletal drugs. It does not occur in A. thaliana roots and depends on the degree of hydrophobicity (lipophilicity) of a particular FM dye. Our results emphasize the need for circumspection during in vivo studies of membrane proteins performed using simultaneous labelling with FM dyes.

BackgroundAbundance and distribution of the plant hormone auxin play important roles in plant development. Besides other metabolic processes, various auxin carriers control the cellular level of active auxin and, hence, are major regulators of cellular auxin homeostasis. Despite the developmental importance of auxin transporters, a simple medium-to-high throughput approach to assess carrier activities is still missing. Here we show that carrier driven depletion of cellular auxin correlates with reduced nuclear auxin signaling in tobacco Bright Yellow-2 (BY-2) cell cultures.ResultsWe developed an easy to use transient single-cell-based system to detect carrier activity. We use the relative changes in signaling output of the auxin responsive promoter element DR5 to indirectly visualize auxin carrier activity. The feasibility of the transient approach was demonstrated by pharmacological and genetic interference with auxin signaling and transport. As a proof of concept, we provide visual evidence that the prominent auxin transport proteins PIN-FORMED (PIN)2 and PIN5 regulate cellular auxin homeostasis at the plasma membrane and endoplasmic reticulum (ER), respectively. Our data suggest that PIN2 and PIN5 have different sensitivities to the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Also the putative PIN-LIKES (PILS) auxin carrier activity at the ER is insensitive to NPA in our system, indicating that NPA blocks intercellular, but not intracellular auxin transport.ConclusionsThis single-cell-based system is a useful tool by which the activity of putative auxin carriers, such as PINs, PILS and WALLS ARE THIN1 (WAT1), can be indirectly visualized in a medium-to-high throughput manner. Moreover, our single cell system might be useful to investigate also other hormonal signaling pathways, such as cytokinin.

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Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation.