ARF-GTPase as a Molecular Switch for Polar Auxin Transport Mediated by Vesicle Trafficking in Root Development

Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.¹ In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.² Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.³ AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.^1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.^5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.⁴ As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,⁷ and overexpression of OsAGAP interferes with localization of AUX1.¹ Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.¹ Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.¹ Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.⁸ The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.¹ So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10⁻⁷ M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.⁹ GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.⁶ Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.¹⁰ OsAGAP possesses an ARF GTPase-activating function in rice.¹¹ Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.¹Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.¹² An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.     

Evaluating the function of putative hormone transporters

Hormones typically serve as long distance signaling molecules. To reach their site of action, hormones need to be transported from the sites of synthesis. Many plant hormones are mobile, thus requiring specific transport systems for the export from their source cells as well as subsequent import into target cells. Hormone transport in general is still poorly understood. Auxin is probably the most intensively studied plant hormone concerning transport in the moment. To advance our understanding of hormone transport we need two principal data sets: information on the properties of the transport systems including substrate specificity and kinetics, and we need to identify candidate genes for the respective transporters. Physiological transport data can provide an important basis for identifying and characterizing candidate transporters and to define their in vivo role. A recent publication in Plant Physiology highlights how kinetic and specificity studies may help to identify cytokinin transporters.¹Key words: kinetin, zeatin, adenine, phytohormone, transportBy definition, hormones are compounds that interact at low concentrations with cellular receptors to modulate signal transduction pathways. A comparison of the chemical structures of animal and plant hormones suggests potential common origins. Peptide hormones are found in both kingdoms and share common processing mechanisms (e.g., TRH, vasopressin and kinins in animals; systemins, phytosulfokines, self incompatibility peptides in plants).^2,3 Steroid hormones derived from cholesterol such as testosterone, cortisol and calcitriol regulate development in mammals; the steroid hormone brassinolide is essential for plant development.⁴ Glutamate can serve as metabolite and signal in both plants and animals.^5,6 Finally, lipid and phospholipid-derived signaling compounds such as linoleic acid and arachidonic acid also function in both plants and animals; with phospholipid-derived prostaglandins and eicosanoids bearing similarities to the plant defense compound jasmonic acid.⁷Other signaling compounds present in animals have yet to be shown to function in plants, e.g., glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone or thyroid-stimulating hormone have been not been described to exist in plants.⁸ Compounds structurally similar to animal amine-derived hormones derived from tyrosine and tryptophan (such as catecholamines and thyroxine) are also present in plants, but appear to function primarily in herbivore defense.⁹The best characterized, and arguably most important plant hormones, bear little similarity to animal hormones and are mechanistically distinct. These include auxins, cytokinins, gibberellins, abscisic acid, ethylene and an apparent carotenoid-derivative, the MAX-dependent regulator of auxin signaling.^10,11 Arguably, the stress response compound salicylic acid, which functions in stress, wounding and defense responses could also be considered a plant hormone.¹²Hormonal signaling mechanisms can be categorized as autocrine (acting at the site of biosynthesis), paracrine (acting in adjacent or proximal cells), and endocrine (acting in cells distal to the site of production). In both, plants and animals, paracrine and endocrine hormone action is mediated and influenced by multiple long distance delivery systems. Hormones move primarily through the circulatory system in animals, but, in plants, are mobilized by transpiration and source-sink flows, which can be directed by chemisomotically-driven cellular uptake and efflux. However, the mechanisms driving uptake and efflux at the cellular level, as well as the proteins that mediate this movement, are surprisingly similar in plants and animals, despite the dissimilarities of plant and animal cell structure (central vacuoles, cell walls and H⁺ versus K⁺/Na²⁺ in/out gradients).Surprisingly little is known about plant hormone transport. Most hormones have autocrine activity, but in order to act at a distance or to even act on adjacent cells they must be transported across membranes. The existence of cellular export and import mechanisms are suggested by the presence of multiple hormones in the phloem sap^13,14 and the well documented polar long distance movement of auxin.¹⁵ Brassinosteroid receptors have been demonstrated as integral plasma membrane proteins which receive the hormone signal from outside the cell.¹⁶ This suggests a need for the hormone to first move into the apoplasm after biosynthesis. However, until recently, only the cellular auxin transport mechanisms mediated by the AUX/LAX, PIN and AtABCB/PGP proteins has been well characterized (reviewed in ref. ¹⁷).The study of these transporters has benefited from the use of plant, yeast and animal expression systems to characterize the proteins involved. Analyses of auxin transport proteins have capitalized on earlier suppression cloning and radiotracer uptake studies used successfully to characterize ion and metabolite transporters in yeast.^18–21 In cases where yeast systems have proven intractable for analysis of auxin transport proteins, heterologous systems based on mammalian cell systems have proven to be highly effective for radiotracer uptake studies.^18–23 Xenopus oocyte expression has been successfully utilized to characterize the AUX/LAX family of auxin influx symporters.^24,25 Plant cell culture systems have also been used to characterize transport proteins. This can however be problematic when endogenous substrates are metabolized by the cells, as is the case with IAA in tobacco BY-2 and Arabidopsis cell cultures.¹⁹ It is also difficult to assess the function of plant proteins in undifferentiated cell cultures, which may differ from the native function in phloem or xylem parenchyma cells.A recent article describes the use of a heterologous expression system based on the fission yeast S. pombe to express and characterize the PIN1 auxin efflux protein after knock-out of the endogenous yeast PIN-like gene AEL1.²¹ Previously, PIN1 had only been functionally expressed in plant cell systems and was nonfunctional when expressed in baker''s yeast or mammalian cells.^19,22 This report suggests that PIN1, interacts synergistically with the AtABCB19/PGP19 auxin efflux transporter, but appears to also mediate auxin efflux on its own, consistent with the distant phylogenetic similarity of the auxin efflux transporter protein family to major facilitator proteins.Subsequent work in the Murphy lab has shown that S. pombe can be used for comparisons of all known auxin transporters in a single system in which all ABC transporters and a solitary AUX1-like gene had been knocked out (Yang and Murphy, unpublished). This system also allows for the more detailed analyses of substrate specificity, transport kinetics and coupling mechanisms (primary and secondary active transport, uniport, cotransport antiport) necessary for functional assignment of auxin transport proteins. This system may also provide an attractive alternative to baker''s yeast when functional expression of a plant protein in Saccharomyces cerevisiae proves unsuccessful.Similar efforts are required for characterizing the transport of all other plant hormones including cytokinin. Arabidopsis transporters mediating both trans-zeatin and adenine uptake had been identified using yeast as an expression system.²⁶ Recently, the Schulz and Frommer labs provided a reference data set for trans-zeatin uptake by characterizing radiolabeled trans-zeatin uptake in Arabidopsis cell cultures.¹ The data show that the uptake kinetics of trans-zeatin are multiphasic, indicating the presence of both low- and high-affinity transport systems. The protonophore CCCP is an effective inhibitor of cytokinin uptake, consistent with H⁺-mediated uptake. Other physiologically active cytokinins such as isopentenyladenine and benzylaminopurine are effective competitors of trans-zeatin uptake, whereas allantoin had no inhibitory effect. Adenine competes for zeatin uptake indicating that degradation products of cytokinin oxidases can be transported by the same systems. Comparison of adenine and trans-zeatin uptake in Arabidopsis seedlings reveals similar uptake kinetics. Kinetic properties as well as substrate specificity determined in cell cultures are compatible with the hypothesis that members of the plant-specific PUP transporter family may play a role in adenine transport to scavenge extracellular adenine. In addition, the findings are also compatible with the hypothesis that this class of transporters may be involved at least in low affinity (µM range) cytokinin uptake. PUPs are encoded by a large gene family of 21 members, so it is conceivable that other members of the family may be involved in high affinity transport. Systematic analyses of single knock outs in Arabidopsis and combinations thereof my help to shed more light on the role of PUPs in cytokinin transport.     

Auxin acts independently of DELLA proteins in regulating gibberellin levels

Shoot elongation is a vital process for plant development and productivity, in both ecological and economic contexts. Auxin and bioactive gibberellins (GAs), such as GA₁, play critical roles in the control of elongation,^1–3 along with environmental and endogenous factors, including other hormones such as the brassinosteroids.^4,5 The effect of auxins, such as indole-3-acetic acid (IAA), is at least in part mediated by its effect on GA metabolism,⁶ since auxin upregulates biosynthesis genes such as GA 3-oxidase and GA 20-oxidase and downregulates GA catabolism genes such as GA 2-oxidases, leading to elevated levels of bioactive GA₁.⁷ In our recent paper,¹ we have provided evidence that this action of IAA is largely independent of DELLA proteins, the negative regulators of GA action,^8,9 since the auxin effects are still present in the DELLA-deficient la cry-s genotype of pea. This was a crucial issue to resolve, since like auxin, the DELLAs also promote GA₁ synthesis and inhibit its deactivation. DELLAs are deactivated by GA, and thereby mediate a feedback system by which bioactive GA regulates its own level.¹⁰ However, our recent results,¹ in themselves, do not show the generality of the auxin-GA relationship across species and phylogenetic groups or across different tissue types and responses. Further, they do not touch on the ecological benefits of the auxin-GA interaction. These issues are discussed below as well as the need for the development of suitable experimental systems to allow this process to be examined.Key words: auxin, gibberellins, DELLA proteins, interactions, elongation     

Arabidopsis FAB1A/B is possibly involved in the recycling of auxin transporters

Fab1/PIKfyve produces Phosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) from Phosphatidylinositol-3-phosphate (PtdIns 3-P), and is involved not only in vacuole/lysosome homeostasis, but also in transporting various proteins to the vacuole or recycling proteins on the plasma membrane (PM) through the use of endosomes in a variety of eukaryotic cells. We previously demonstrated that Arabidopsis FAB1A/B functions as PtdIns-3,5-kinase in both Arabidopsis and fission yeast and plays a key role in vacuolar acidification and endocytosis. Although the conditional FAB1A/B knockdown mutant revealed an auxin-resistant phenotype to a membrane-impermeable auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), the mutant did not exhibit this phenotype to a membrane-permeable artificial auxin, naphthalene 1-acetic acid (NAA). The difference in the sensitivities to 2,4-D and NAA is similar to those of the auxin-resistant mutants such as aux1. Taken together, these results suggest that impairment of the function of Arabidopsis FAB1A/B might cause a defect in the membrane recycling capabilities of the auxin transporters and inhibit proper auxin transport into the cells in Arabidopsis.Key words: auxin signaling, auxin transporter, recycling of plasma membrane proteinsPhosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) exists on the external membrane of multi-vesicular bodies (MVBs) at very low levels in eukaryotic cells,^1,2 and plays key roles in endomembrane homeostasis including endocytosis, vacuole/lysosome formation and vacuolar acidification.^1,3 PtdIns (3,5) P₂ deficiency causes an enlarged vacuolar structure in yeast and mammalian cells.^4,5 FAB1 forms a protein complex with its regulatory molecules, and synthesizes PtdIns (3,5) P₂ from PtdIns 3P.^6–9 In Arabidopsis, there are four Fab1/PIKfyve orthologs (FAB1A, FAB1B, FAB1C and FAB1D) in the genome, and the double homozygous mutant of FAB1A and FAB1B exhibited the male gametophyte lethal phenotype.¹⁰ Previously, we reported that conditional loss-of-function and gain-of-function mutants of FAB1A/B impair endomembrane homeostasis and reveal various developmental phenotypes.¹¹ Interestingly, lateral root formation by exogenous auxin, which is known as a typical auxin-responsive phenotype, was largely impaired when FAB1A/B expression was conditionally downregulated or upregulated. From these results, we speculated that the defect in the endocytosis process in fab1a/b mutants might inhibit the precise recycling process of auxin transporters on the PM, thereby inhibiting proper auxin transport into the plant cells.¹¹ In this report, we tested this hypothesis to assess the sensitivity on auxin-dependent lateral root formation to a membrane permeable auxin, NAA, in the fab1a/b knockdown mutant.     

Irrepressible,truncated Auxin Response Factors: Natural roles and applications in dissecting auxin gene regulation pathways

The molecularly well-characterized auxin signal transduction pathway involves two evolutionarily conserved families interacting through their C-terminal domains III and IV: the Auxin Response Factors (ARFs) and their repressors the Aux/IAAs, to control auxin-responsive genes, among them genes involved in auxin transport.^1,2 We have developed a new genetic tool to study ARF function. Using MONOPTEROS (MP)/ARF5, we have generated a truncated version of MP (MPΔ),³ which has lost the target domains for repression by Aux/IAA proteins. Besides exploring genetic interactions between MP and Aux/IAAs, we used this construct to trace MP’s role in vascular patterning, a previously characterized auxin dependent process.^4,5 Here we summarize examples of naturally occurring truncated ARFs and summarize potential applications of truncated ARFs as analytical tools.     

Where and how does phototropin transduce light signals in the cell?

Light plays pivotal roles as an important environmental signal in plant growth and development. In Arabidopsis, phototropin 1 (phot1) and 2 (phot2) are the photoreceptors that mediate phototropism, chloroplast relocation, stomatal opening and leaf flattening, in response to blue light. However, little is known about how phototropins transduce the signals after the light is perceived. Changes induced by blue light in terms of intracellular localization patterns of phot2 in Arabidopsis were examined. Phot2 distributed uniformly in the plasma membrane under dark conditions. Upon irradiation with blue light, some of the phot2 associated with the Golgi apparatus. It was also shown that the kinase domain, but not the photosensory domain, is required for a plasma membrane and Golgi localization. Furthermore a kinase fragment, lacking the photosensory domain, constitutively triggered physiological responses in planta. Thus, the plasma membrane and the Golgi apparatus appear to be the most likely sites for the initial step of phot2 signal transduction. The Golgi apparatus facilitates vesicle trafficking and delivery of membrane proteins to the required locations in the cell. Therefore, this study implicates the regulation of vesicle trafficking by the Golgi apparatus as a mechanism by which phot2 elicits its cellular responses.Key words: Golgi apparatus, kinase, light signal transduction, photoreceptor, phototropin, vesicle traffickingA range of physiological responses in plants is brought about by blue (390–500 nm) and ultraviolet-A (320–390 nm) light. Phototropin, one of major classes of blue light photoreceptors in plants, mediates responses such as phototropism, chloroplast relocation, light-induced stomatal opening and leaf flattening.^1–6 The dicotyledon Arabidopsis, possesses two phototropins, termed phot1 and phot2, which have both overlapping and distinct functions.^5,7 Phototropins consist of two functional domains, a N-terminal photosensory domain, containing two LOV (Light, Oxygen, Voltage) domains (LOV1 and LOV2) and a flavin-mononucleotide (FMN) chromophore and a regulatory serine/threonine kinase domain at the C-terminus.⁸To understand the mechanism of phototropin signal transduction, we expressed phot2 derivatives with translationally-fused green fluorescent protein (GFP) in a phot1phot2 double mutant in a wild type background in Arabidopsis.^9,10 Phototropin is a membrane- associated protein lacking a membrane spanning domain.⁸ Phot1 fused to GFP (P1G) is mainly localized to the plasma membrane, regardless of the light conditions.⁶ This property was retained when phot2 was fused to GFP (P2G).⁹ A part of P2G associates with punctate structures in the cytoplasm in response to blue light. The punctate P2G colocalized with KAM1ΔC:mRFP, a Golgi marker, we therefore conclude that phot2 associated with the Golgi apparatus in a blue light-dependent manner.⁹ This association was observed even in the presence of brefeldin A (BFA), an inhibitor of the vesicle trafficking.⁹To determine which domain of phot2 is responsible for the Golgi association, fragments of phot2 were fused to GFP and expressed in protoplasts.⁹ The N-terminal fragment fused to GFP (P2NG) was distributed uniformly in the cytoplasm. By contrast, the C-terminal fragment fused to GFP (P2CG) localized to both plasma membrane and punctate structures. The latter was shown to be the Golgi apparatus with the aid of the Golgi marker, KAM1ΔC:mRFP.⁹ These observations were corroborated from data using transgenic plants.¹⁰ Hence the C-terminal kinase domain, but not the N-terminal photo-sensory domain, is essential for the association of phot2 with the plasma membrane and the Golgi apparatus.The Golgi network is a key player in vesicle trafficking, to and from ER, vacuoles, trans-Golgi network, endosome and the plasma membrane.¹¹ Membrane spanning proteins are delivered and recycled through the Golgi apparatus. Among the membrane spanning proteins that are especially interesting, with respect to phototropin function, are auxin carriers such as PIN proteins. Phototropic curvature, which is under the control of phototropin, is believed to be caused by an uneven distribution of auxin.¹² The intracellular distribution of PIN proteins is maintained and regulated by vesicle trafficking.¹³ Indeed, factors such as ADP-ribosylation factor1 (ARF1) and guanine-nucleotide exchange factors (GEFs), which are involved in vesicle trafficking, are indispensable for the proper distribution of PIN proteins.^14–17 It is intriguing that a light stimulus alters the distribution pattern of PIN proteins.¹⁸ Hence, a fascinating possibility arises that phot2 alters the intracellular distribution of PIN proteins by regulating vesicle trafficking at the level of the Golgi apparatus.Phototropins are members of the subfamily VIII of AGC kinases.¹⁹ Interestingly, PINOID, another member of the subfamily, is localized at the cell periphery and regulates the apical-basal polar distribution of PIN proteins.^20–22 Accordingly, overexpression of PINOID disturbs the auxin distribution in transgenic plants.^23,24 The kinase fragment of phototropin exhibits constitutive kinase activity in vitro.²⁵ Interestingly, the auxin distribution is disturbed in plants expressing P2CG, as is the case with PINOID.¹⁰ Hence, both PINOID and phot2 might alter the PIN protein distribution in the cell through a common mechanism, in response to distinct stimuli.To date, no authentic substrate has been described for any of the AGC VIII kinases.¹⁹ Considering the localization pattern of phototropins, the substrates are most likely to reside in the plasma membrane and/or the Golgi apparatus. NPH3, RPT2 and PKS1 are downstream factors for phototropic responses,^26–28 all associating with the plasma membrane. Although they interact preferentially with the N-terminal rather than the C-terminal domain of phot1,^26,29 it is also possible that the C-terminal kinase domain interacts transiently with these factors leading to their phosphorylation. However, at present the molecular functions of NPH3, RPT2 and PKS1 remain unclear and await future investigation.Although both phot1 and phot2 are localized to the plasma membrane, punctate structures are yet to be described for P1G. Instead, a part of phot1-GFP is released from the plasma membrane to the cytosol in response to a light stimulus.⁶ We recently reexamined the intracellular localization of P1G. A specific network-like structure in the cytoplasm in addition to intense plasma membrane staining was observed (Fig. 1). A similar pattern was observed for P2G although it is less clear.⁹ Hence, both phot1 and phot2 might be associating with a structure in the cytoplasm that has yet to be described, and which might be another site of phototropin signaling in the cell.Open in a separate windowFigure 1A light-induced network-like distribution pattern of P1G in the cytoplasm. The P1G seedlings grown under dark conditions⁶ were incubated in MS solution (diluted 50%) without (upper panels) or with (lower panels) 100 µM BFA. The cells were inspected with a confocal laser scanning microscope. Images taken before (left) or after (right) blue light illumination at 48 µmol m⁻² sec⁻¹ are shown. Bar = 10 µm.P2CG elicits some phototropin responses without a light stimulus.¹⁰ That is, chloroplasts were in the avoidance position and stomata opened without a blue light stimulus in the P2CG overexpressing plants. It is a fascinating possibility that phototropin elicits those responses through the regulation of vesicle trafficking, although other possibilities exist. Stomata open as the result of phosphorylation of the plasma membrane H⁺-ATPase³⁰ and it is unlikely that the vesicle trafficking is directly involved in this regulatory process. It is possible to conjecture that vesicle trafficking affects chloroplast positioning but how this would work remains to be determined. Overall how a single photoreceptor such as phototoropin might regulate diverse physiological responses awaits future study.     

Linking protein kinase CK2 and auxin transport

Studies performed in different organisms have highlighted the importance of protein kinase CK2 in cell growth and cell viability. However, the plant signaling pathways in which CK2 is involved are largely unknown. We have reported that a dominant-negative mutant of CK2 in Arabidopsis thaliana shows phenotypic traits that are typically linked to alterations in auxin-dependent processes. We demonstrated that auxin transport is, indeed, impaired in these mutant plants, and that this correlates with misexpression and mislocalization of PIN efflux transporters and of PINOID. Our data establishes a link between CK2 activity and the regulation of auxin homeostasis in plants, strongly suggesting that CK2 might be required at multiple points of the pathways regulating auxin fluxes.Key words: protein kinase CK2, root development, auxin, PIN, PINOIDThe plant hormone auxin plays critical roles in plant growth and development.¹ The most abundant natural auxin is the indol-3-acetic acid (IAA), which is synthesized in young apical tissues and then transported to the growing zones of the stem and root. The major route for long distance IAA movement is via the vascular tissue, but, additionally, a slower transport via cell-to-cell (called polar transport) is critical to generate auxin gradients within tissues. Formation of correct auxin gradients is thought to be essential for many plant developmental processes.² In recent years, the IAA transporters have been identified, establishing the molecular basis to understand how auxin transport is regulated. In particular, the identification of the family of plasma-resident PIN proteins, the members of which function as IAA efflux carriers, and the knowledge of their polar localization in the plasma membrane (PM), contributed to generate models predicting the direction of IAA fluxes.^3,4The factors that govern PIN targeting to a particular membrane domain are still not understood. It is known that PIN proteins constitutively undergo cycles of exocytosis and endocytosis to and from the PM, using distinct sorting and recycling endosome trafficking pathways.^5–7 Phosphorylation/dephosphorylation by the Ser/Thr kinase PINOID (PID) and the protein phosphatase 2A, respectively, controls PIN proteins apical/basal localization at the PM, via the GNOM-mediated vesicle trafficking system.⁸ Interestingly, PID is a member of the plant AGC kinases, and, as it happens with its mammals AGC counterparts, is activated by a membrane-associated 3-phosphoinositide-dependent kinase (PDK1).⁹ Moreover, a functional similarity between PIN polar localization in response to auxin and glucose receptor (GLUT4) asymmetrical distribution in response to insulin, has been pointed out.¹⁰ In both cases, cargo proteins (GLUT4 and PIN, respectively) are transported from endosomal vesicles to PM and the process is mediated by PDK1-activated AGC kinases.Protein kinase CK2 is a Ser/Thr kinase evolutionary conserved in eukaryotes, which plays key roles in cell survival, cell division and other cellular processes. A loss-of-function mutant of CK2 in Arabidopsis, obtained by overexpression of a CK2α-inactive subunit, confirmed the essential role of this protein kinase for plant viability.¹¹ Moreover, CK2mut plants showed a dramatic decrease of lateral root formation, inhibition of root growth and overproliferation of root hairs. We have further demonstrated that auxin transport is impaired in this plants, which is concomitant with missexpression of most of the PM-resident PIN proteins, and of PID.¹² In addition, PIN proteins accumulated in endosomal vesicles and auxin gradients were disturbed, both in roots and shoots of CK2mut plants. In particular, root columella cells were depleted of auxin, although the maximum at the quiescent center was unchanged. Starch granule staining with lugol revealed that columella cells retained their fate, although their organization and/or cell shape were clearly affected (Fig. 1).Open in a separate windowFigure 1Lugol-stained starch granules in uninduced (−Dex) and Dex-induced (+Dex) CK2mut roots. In the central part of the figure, a sketch of the main morphogenetic characteristics of mutant roots (right plantlet) as compared to wild-type roots (left plantlet) is shown. Note the shorter roots, wavy phenotype, absence of lateral roots and overproliferation of root hairs in mutant plants.Our results strongly suggest that CK2 is a regulator of auxin-dependent responses, most likely by participating in the regulation of auxin transport. Strikingly, depletion of CK2 activity inhibits some auxin-dependent physiological responses whereas it enhances others. For instance, whereas shoot phototropism was completely absent, root gravitropism was enhanced.¹² Figure 2 shows a time-course of DR5rev::GFP-derived signal after changing the gravity vector, in mutant and control Arabidopsis roots. The progressive auxin translocation to the lower side of the root after gravistimulation is more rapid and sustained in mutant than in control roots, which is likely responsible for the enhanced response to gravity found in mutant roots. Based on these results, we postulate that CK2 might act at different points of the auxin-induced regulatory pathway. As far as is known, the core module that regulates auxin transport is constituted by the protein kinase PID and a protein of the NPH3-domain family. NPH3-containing proteins play important roles in phototropic and gravitropic responses, and regulate polarity and endocytosis of PIN proteins.¹³ As has been proposed by other authors, the participation of one AGC kinase and one NPH3-like protein upstream of an ARF factor might be a common theme in response to different stimulus that are signaled by auxin.¹⁴ We propose that one of the functions of CK2 is the regulation of the activity of core proteins (Fig. 3). Mammalian AGC kinases are well known substrates of CK2 and CK2-dependent phosphorylation is critical for a full display of their activity. The PID and the NPH3-containing protein sequences contain numerous acidic-based motifs that are predicted CK2 phosphorylation sites. Moreover, according to Arabidopsis phosphoproteome databases, several members of the NPH3-containing protein family are predicted to be phosphorylated.¹⁵ In addition, we do not discard the possibility that other proteins involved in PIN transport might also be regulated by CK2-dependent phosphorylation. Experiments are in progress in our laboratory to assess the regulatory role of CK2 in auxin transport.Open in a separate windowFigure 2Time course of auxin relocation during root gravitropic response, as visualized by DR5rev::GFP fluorescence. Root pictures were taken at the indicated times after changing the direction of the gravity vector. Translocation of auxin to the lower part of the root is more rapid in Dex-induced CK2mut plants. Arrows indicate asymmetrical DR5::GFP fluorescence.Open in a separate windowFigure 3Proposed model for the role of CK2 in regulating auxin transport. The core module that regulates auxin transport (shown here as a black box) is constituted by the protein kinase PID and a protein of the NPH3-domain family. PID regulates apical-basal targeting of PIN proteins, by phosphorylating conserved Ser residues present in PIN hydrophilic loops.¹⁶ On the other hand, the family of NPH3-containing proteins regulates polarity and endocytosis of PIN proteins.¹³ There is also a functional similarity between the intracellular transport of PIN proteins and that of the glucose receptor (GLUT4),¹⁰ two processes that are signaled by AGC kinases. We propose that CK2 might be a regulator of the activity of the core proteins, by phosphorylating either the AGC kinase and/or the NPH3-containing protein. Mammalian CK2 is a known regulator of the activity of AGC kinases and other proteins participating in signaling pathways, such as in the Wnt/β-catenin signaling pathway.¹⁷