The Arabidopsis GNOM ARF-GEF mediates endosomal recycling,auxin transport,and auxin-dependent plant growth

Exchange factors for ARF GTPases (ARF-GEFs) regulate vesicle trafficking in a variety of organisms. The Arabidopsis protein GNOM is a brefeldin A (BFA) sensitive ARF-GEF that is required for the proper polar localization of PIN1, a candidate transporter of the plant hormone auxin. Mutations in GNOM lead to developmental defects that resemble those caused by interfering with auxin transport. Both PIN1 localization and auxin transport are also sensitive to BFA. In this paper, we show that GNOM localizes to endosomes and is required for their structural integrity. We engineered a BFA-resistant version of GNOM. In plants harboring this fully functional GNOM variant, PIN1 localization and auxin transport are no longer sensitive to BFA, while trafficking of other proteins is still affected by the drug. Our results demonstrate that GNOM is required for the recycling of auxin transport components and suggest that ARF-GEFs regulate specific endosomal trafficking pathways.     

Gravitropism of Arabidopsis thaliana Roots Requires the Polarization of PIN2 toward the Root Tip in Meristematic Cortical Cells

In the root, the transport of auxin from the tip to the elongation zone, referred to here as shootward, governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the PIN-FORMED auxin efflux carrier PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized toward the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor GNOM and by the regulatory A1 subunit of type 2-A protein phosphatase (PP2AA1) but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism. These results demonstrate that auxin flow in the root cortex is important for optimal gravitropic response.     

Coordination of apical and basal embryo development revealed by tissue-specific GNOM functions

Flowering-plant embryogenesis generates the basic body organization, including the apical and basal stem cell niches, i.e. shoot and root meristems, the major tissue layers and the cotyledon(s). gnom mutant embryos fail to initiate the root meristem at the early-globular stage and the cotyledon primordia at the late globular/transition stage. Tissue-specific GNOM expression in the gnom mutant embryo revealed that both apical and basal embryo organization depend on GNOM provascular expression and a functioning apical-basal auxin flux: GNOM provascular expression in gnom mutant background resulted in non-cell-autonomous reconstitution of apical and basal tissues which could be linked to changes in auxin responses in those tissues, stressing the importance of apical-basal auxin flow for overall embryo organization. Although reconstitution of apical-basal auxin flux in gnom results in the formation of single cotyledons (monocots), only additional GNOM epidermal expression is able to induce wild-type apical patterning. We conclude that provascular expression of GNOM is vital for both apical and basal tissue organization, and that epidermal GNOM expression is required for radial-to-bilateral symmetry transition of the embryo. We propose GNOM-dependent auxin sinks as a means to generate auxin gradients across tissues.     

Role of the GNOM gene in Arabidopsis apical-basal patterning – From mutant phenotype to cellular mechanism of protein action

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.     

PIN Auxin Efflux Carrier Polarity Is Regulated by PINOID Kinase-Mediated Recruitment into GNOM-Independent Trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development.     

Flavonoids act as negative regulators of auxin transport in vivo in arabidopsis

Polar transport of the plant hormone auxin controls many aspects of plant growth and development. A number of synthetic compounds have been shown to block the process of auxin transport by inhibition of the auxin efflux carrier complex. These synthetic auxin transport inhibitors may act by mimicking endogenous molecules. Flavonoids, a class of secondary plant metabolic compounds, have been suggested to be auxin transport inhibitors based on their in vitro activity. The hypothesis that flavonoids regulate auxin transport in vivo was tested in Arabidopsis by comparing wild-type (WT) and transparent testa (tt4) plants with a mutation in the gene encoding the first enzyme in flavonoid biosynthesis, chalcone synthase. In a comparison between tt4 and WT plants, phenotypic differences were observed, including three times as many secondary inflorescence stems, reduced plant height, decreased stem diameter, and increased secondary root development. Growth of WT Arabidopsis plants on naringenin, a biosynthetic precursor to those flavonoids with auxin transport inhibitor activity in vitro, leads to a reduction in root growth and gravitropism, similar to the effects of synthetic auxin transport inhibitors. Analyses of auxin transport in the inflorescence and hypocotyl of independent tt4 alleles indicate that auxin transport is elevated in plants with a tt4 mutation. In hypocotyls of tt4, this elevated transport is reversed when flavonoids are synthesized by growth of plants on the flavonoid precursor, naringenin. These results are consistent with a role for flavonoids as endogenous regulators of auxin transport.     

Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx, vesicle trafficking and root development

Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants.     

Vectorial information for Arabidopsis planar polarity is mediated by combined AUX1, EIN2, and GNOM activity

Cell polarity is commonly coordinated within the plane of a single tissue layer (planar polarity), and hair positioning has been exploited as a simple marker for planar polarization of animal epithelia . The root epidermis of the plant Arabidopsis similarly reveals planar polarity of hair localization close to root tip-oriented (basal) ends of hair-forming cells . Hair position is directed toward a concentration maximum of the hormone auxin in the root tip , but mechanisms driving this plant-specific planar polarity remain elusive. Here, we report that combinatorial action of the auxin influx carrier AUX1, ETHYLENE-INSENSITIVE2 (EIN2) , and GNOM genes mediates the vector for coordinate hair positioning. In aux1;ein2;gnom eb triple mutant roots, hairs display axial (apical or basal) instead of coordinate polar (basal) position, and recruitment of Rho-of-Plant (ROP) GTPases to the hair initiation site reveals the same polar-to-axial switch. The auxin concentration gradient is virtually abolished in aux1;ein2;gnom eb roots, where locally applied auxin can coordinate hair positioning. Moreover, auxin overproduction in sectors of wild-type roots enhances planar ROP and hair polarity over long and short distances. Hence, auxin may provide vectorial information for planar polarity that requires combinatorial AUX1, EIN2, and GNOM activity upstream of ROP positioning.     

Blue‐light‐induced PIN3 polarization for root negative phototropic response in Arabidopsis

Root negative phototropism is an important response in plants. Although blue light is known to mediate this response, the cellular and molecular mechanisms underlying root negative phototropism remain unclear. Here, we report that the auxin efflux carrier PIN‐FORMED (PIN) 3 is involved in asymmetric auxin distribution and root negative phototropism. Unilateral blue‐light illumination polarized PIN3 to the outer lateral membrane of columella cells at the illuminated root side, and increased auxin activity at the illuminated side of roots, where auxin promotes growth and causes roots bending away from the light source. Furthermore, root negative phototropic response and blue‐light‐induced PIN3 polarization were modulated by a brefeldin A‐sensitive, GNOM‐dependent, trafficking pathway and by phot1‐regulated PINOID (PID)/PROTEIN PHOSPHATASE 2A (PP2A) activity. Our results indicate that blue‐light‐induced PIN3 polarization is needed for asymmetric auxin distribution during root negative phototropic response.     

Hormonal regulation of lateral root development in Arabidopsis modulated by MIZ1 and requirement of GNOM activity for MIZ1 function

Plant organ development is important for adaptation to a changing environment. Genetic and physiological studies have revealed that plant hormones play key roles in lateral root formation. In this study, we show that MIZU-KUSSEI1 (MIZ1), which was identified originally as a regulator of hydrotropism, functions as a novel regulator of hormonally mediated lateral root development. Overexpression of MIZ1 (MIZ1OE) in roots resulted in a reduced number of lateral roots being formed; however, this defect could be recovered with the application of auxin. Indole-3-acetic acid quantification analyses showed that free indole-3-acetic acid levels decreased in MIZ1OE roots, which indicates that alteration of auxin level is critical for the inhibition of lateral root formation in MIZ1OE plants. In addition, MIZ1 negatively regulates cytokinin sensitivity on root development. Application of cytokinin strongly induced the localization of MIZ1-green fluorescent protein to lateral root primordia, which suggests that the inhibition of lateral root development by MIZ1 occurs downstream of cytokinin signaling. Surprisingly, miz2, a weak allele of gnom, suppressed developmental defects in MIZ1OE plants. Taken together, these results suggest that MIZ1 plays a role in lateral root development by maintaining auxin levels and that its function requires GNOM activity. These data provide a molecular framework for auxin-dependent organ development in Arabidopsis (Arabidopsis thaliana).     

Vesicular cycling mechanisms that control auxin transport polarity

The polar transport of auxin controls many important plant growth and developmental processes. The polarity of auxin movement has long been suggested to be mediated by asymmetric distribution of auxin transport proteins, yet, until recently, little was known about the mechanisms that establish protein asymmetry in auxin-transporting cells. Now, a recent paper provides significant insight into the mechanism by which the GNOM protein controls the cycling of an auxin efflux carrier protein, PIN1, between the endosome and the plasma membrane. The dynamic movement of auxin transport proteins between internal compartments and the plasma membrane suggests mechanisms for alterations in auxin transport polarity in response to changing developmental or environmental regulation.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane association. Our results suggest a general model of large ARF-GEF function in which regulated changes in protein conformation control membrane association of the exchange factor and, thus, activation of ARFs.     

Calcium Could Be Involved in Auxin-Regulated Maintenance of the Quiescent Center in the <Emphasis Type="Italic">Arabidopsis</Emphasis> Root

Ca²⁺ transduces hormone and environmental signals to the Ca²⁺ sensors and relays them to the downstream target effectors in cells. During the post-embryonic developmental process, auxin plays a critical role in maintaining the mitotically inactive status of the quiescent center (QC) and the root growth and development that follows. In this report, we demonstrate that Ca²⁺ plays an important role in the maintenance of the QC, probably by regulating PIN1-mediated auxin transport. Perturbation of the intracellular Ca²⁺ levels with chemicals that modify the Ca²⁺ level decreases the endogenous auxin level and the size of the auxin maximum in the root tip and, at the same time, activates QC cell division and expansion. This decreased level of auxin is almost completely restored to the control level by the treatment of exogenous auxin. Interestingly, treatment with Ca²⁺ level modifying chemicals significantly decreased the PIN1 expression in the root vasculature. Taken together, we suggest that balancing Ca²⁺ homeostasis is one of contributing factors in establishing the proper auxin maximum in the root tip and maintaining the QC identity.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

Cytokinin-mediated cell cycling arrest of pericycle founder cells in lateral root initiation of Arabidopsis

In Arabidopsis, lateral root formation is a post-embryonic developmental event, which is regulated by hormones and environmental signals. In this study, via analyzing the expression of cyclin genes during lateral root (LR) formation, we report that cytokinins (CTKs) inhibit the initiation of LR through blocking the pericycle founder cells cycling at the G(2) to M transition phase, while the promotion by CTK of LR elongation is due to the stimulation of the G(1) to S transition. No significant difference was detected in the inhibitory effect of CTK on LR formation between wild-type plants and mutants defective in auxin response or transport. In addition, exogenously applied auxin at different concentrations could not rescue the CTK-mediated inhibition of LR initiation. Our data suggest that CTK and auxin might control LR initiation through two separate signaling pathways in Arabidopsis. The CTK-mediated repression of LR initiation is transmitted through the two-component signal system and mediated by the receptor CRE1.     

Genetic and Chemical Reductions in Protein Phosphatase Activity Alter Auxin Transport, Gravity Response, and Lateral Root Growth

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.