Eva Zažímalová

ABP1 Mediates Auxin Inhibition of Clathrin-Dependent Endocytosis in Arabidopsis

Spatial distribution of the plant hormone auxin regulates multiple aspects of plant development. These self-regulating auxin gradients are established by the action of PIN auxin transporters, whose activity is regulated by their constitutive cycling between the plasma membrane and endosomes. Here, we show that auxin signaling by the auxin receptor AUXIN-BINDING PROTEIN 1 (ABP1) inhibits the clathrin-mediated internalization of PIN proteins. ABP1 acts as a positive factor in clathrin recruitment to the plasma membrane, thereby promoting endocytosis. Auxin binding to ABP1 interferes with this action and leads to the inhibition of clathrin-mediated endocytosis. Our study demonstrates that ABP1 mediates a nontranscriptional auxin signaling that regulates the evolutionarily conserved process of clathrin-mediated endocytosis and suggests that this signaling may be essential for the developmentally important feedback of auxin on its own transport.

Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

Cytokinin regulates root meristem activity via modulation of the polar auxin transport

Plant development is governed by signaling molecules called phytohormones. Typically, in certain developmental processes more than 1 hormone is implicated and, thus, coordination of their overlapping activities is crucial for correct plant development. However, molecular mechanisms underlying the hormonal crosstalk are only poorly understood. Multiple hormones including cytokinin and auxin have been implicated in the regulation of root development. Here we dissect the roles of cytokinin in modulating growth of the primary root. We show that cytokinin effect on root elongation occurs through ethylene signaling whereas cytokinin effect on the root meristem size involves ethylene-independent modulation of transport-dependent asymmetric auxin distribution. Exogenous or endogenous modification of cytokinin levels and cytokinin signaling lead to specific changes in transcription of several auxin efflux carrier genes from the PIN family having a direct impact on auxin efflux from cultured cells and on auxin distribution in the root apex. We propose a novel model for cytokinin action in regulating root growth: Cytokinin influences cell-to-cell auxin transport by modification of expression of several auxin transport components and thus modulates auxin distribution important for regulation of activity and size of the root meristem.

The PIN-FORMED (PIN) protein family of auxin transporters

SummaryThe PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.

Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes

Many aspects of plant development, including patterning and tropisms, are largely dependent on the asymmetric distribution of the plant signaling molecule auxin. Auxin transport inhibitors (ATIs), which interfere with directional auxin transport, have been essential tools in formulating this concept. However, despite the use of ATIs in plant research for many decades, the mechanism of ATI action has remained largely elusive. Using real-time live-cell microscopy, we show here that prominent ATIs such as 2,3,5-triiodobenzoic acid (TIBA) and 2-(1-pyrenoyl) benzoic acid (PBA) inhibit vesicle trafficking in plant, yeast, and mammalian cells. Effects on micropinocytosis, rab5-labeled endosomal motility at the periphery of HeLa cells and on fibroblast mobility indicate that ATIs influence actin cytoskeleton. Visualization of actin cytoskeleton dynamics in plants, yeast, and mammalian cells show that ATIs stabilize actin. Conversely, stabilizing actin by chemical or genetic means interferes with endocytosis, vesicle motility, auxin transport, and plant development, including auxin transport-dependent processes. Our results show that a class of ATIs act as actin stabilizers and advocate that actin-dependent trafficking of auxin transport components participates in the mechanism of auxin transport. These studies also provide an example of how the common eukaryotic process of actin-based vesicle motility can fulfill a plant-specific physiological role.

Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development

The signalling molecule auxin controls plant morphogenesis via its activity gradients, which are produced by intercellular auxin transport. Cellular auxin efflux is the rate-limiting step in this process and depends on PIN and phosphoglycoprotein (PGP) auxin transporters. Mutual roles for these proteins in auxin transport are unclear, as is the significance of their interactions for plant development. Here, we have analysed the importance of the functional interaction between PIN- and PGP-dependent auxin transport in development. We show by analysis of inducible overexpression lines that PINs and PGPs define distinct auxin transport mechanisms: both mediate auxin efflux but they play diverse developmental roles. Components of both systems are expressed during embryogenesis, organogenesis and tropisms, and they interact genetically in both synergistic and antagonistic fashions. A concerted action of PIN- and PGP-dependent efflux systems is required for asymmetric auxin distribution during these processes. We propose a model in which PGP-mediated efflux controls auxin levels in auxin channel-forming cells and, thus, auxin availability for PIN-dependent vectorial auxin movement.

A novel putative auxin carrier family regulates intracellular auxin homeostasis in plants

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program. The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) and cellular auxin transport. We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.

Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux

Postembryonic de novo organogenesis represents an important competence evolved in plants that allows their physiological and developmental adaptation to changing environmental conditions. The phytohormones auxin and cytokinin (CK) are important regulators of the developmental fate of pluripotent plant cells. However, the molecular nature of their interaction(s) in control of plant organogenesis is largely unknown. Here, we show that CK modulates auxin-induced organogenesis (AIO) via regulation of the efflux-dependent intercellular auxin distribution. We used the hypocotyl explants-based in vitro system to study the mechanism underlying de novo organogenesis. We show that auxin, but not CK, is capable of triggering organogenesis in hypocotyl explants. The AIO is accompanied by endogenous CK production and tissue-specific activation of CK signaling. CK affects differential auxin distribution, and the CK-mediated modulation of organogenesis is simulated by inhibition of polar auxin transport. CK reduces auxin efflux from cultured tobacco cells and regulates expression of auxin efflux carriers from the PIN family in hypocotyl explants. Moreover, endogenous CK levels influence PIN transcription and are necessary to maintain intercellular auxin distribution in planta. Based on these findings, we propose a model in which auxin acts as a trigger of the organogenic processes, whose output is modulated by the endogenously produced CKs. We propose that an important mechanism of this CK action is its effect on auxin distribution via regulation of expression of auxin efflux carriers.

The auxin influx carriers AUX1 and LAX3 are involved in auxin-ethylene interactions during apical hook development in Arabidopsis thaliana seedlings.

Dark-grown dicotyledonous seedlings form a hook-like structure at the top of the hypocotyl, which is controlled by the hormones auxin and ethylene. Hook formation is dependent on an auxin signal gradient, whereas hook exaggeration is part of the triple response provoked by ethylene in dark-grown Arabidopsis seedlings. Several other hormones and light are also known to be involved in hook development, but the molecular mechanisms that lead to the initial installation of an auxin gradient are still poorly understood. In this study, we aimed to unravel the cross-talk between auxin and ethylene in the apical hook. Auxin measurements, the expression pattern of the auxin reporter DR5::GUS and the localization of auxin biosynthesis enzymes and influx carriers collectively indicate the necessity for auxin biosynthesis and efficient auxin translocation from the cotyledons and meristem into the hypocotyl in order to support proper hook development. Auxin accumulation in the meristem and cotyledons and in the hypocotyl is increased ∼2-fold upon treatment with ethylene. In addition, a strong ethylene signal leads to enhanced auxin biosynthesis at the inner side of the hook. Finally, mutant analysis demonstrates that the auxin influx carrier LAX3 is indispensable for proper hook formation, whereas the auxin influx carrier AUX1 is involved in the hook exaggeration phenotype induced by ethylene.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis

Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.