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.     

Localized auxin biosynthesis and postembryonic root development in Arabidopsis

Auxin is a phytohormone essential for plant development. Due to the high redundancy in auxin biosynthesis, the role of auxin biosynthesis in embryogenesis and seedling development, vascular and flower development, shade avoidance and ethylene response were revealed only recently. We previously reported that a vitamin B₆ biosynthesis mutant pdx1 exhibits a short-root phenotype with reduced meristematic zone and short mature cells. By reciprocal grafting, we now have found that the pdx1 short root is caused by a root locally generated signal. The mutant root tips are defective in callus induction and have reduced DR5::GUS activity, but maintain relatively normal auxin response. Genetic analysis indicates that pdx1 mutant could suppress the root hair and root growth phenotypes of the auxin overproduction mutant yucca on medium supplemented with tryptophan (Trp), suggesting that the conversion from Trp to auxin is impaired in pdx1 roots. Here we present data showing that pdx1 mutant is more tolerant to 5-methyl anthranilate, an analogue of the Trp biosynthetic intermediate anthranilate, demonstrating that pdx1 is also defective in the conversion from anthranilate to auxin precursor tryptophan. Our data suggest that locally synthesized auxin may play an important role in the postembryonic root growth.Key words: auxin synthesis, root, PLP, PDX1The plant hormone auxin modulates many aspects of growth and development including cell division and cell expansion, leaf initiation, root development, embryo and fruit development, pattern formation, tropism, apical dominance and vascular tissue differentiation.^1–3 Indole-3-acetic acid (IAA) is the major naturally occurring auxin. IAA can be synthesized in cotyledons, leaves and roots, with young developing leaves having the highest capacity.^4,5Auxin most often acts in tissues or cells remote from its synthetic sites, and thus depends on non-polar phloem transport as well as a highly regulated intercellular polar transport system for its distribution.²The importance of local auxin biosynthesis in plant growth and development has been masked by observations that impaired long-distance auxin transport can result in severe growth or developmental defects.^3,6 Furthermore, a few mutants with reduced free IAA contents display phenotypes similar to those caused by impaired long-distance auxin transport. These phenotypes include defective vascular tissues and flower development, short primary roots and reduced apical dominance, or impaired shade avoidance and ethylene response.^7–15 Since these phenotypes most often could not be rescued by exogenous auxin application, it is difficult to attribute such defects to altered local auxin biosynthesis. By complementing double, triple or quadruple mutants of four Arabidopsis shoot-abundant auxin biosynthesis YUCCA genes with specific YUCCA promoters driven bacterial auxin biosynthesis iaaM gene, Cheng et al. provided unambiguous evidence that auxin biosynthesis is indispensable for embryo, flower and vascular tissue development.^8,13 Importantly, it is clear that auxin synthesized by YUCCAs is not functionally interchangeable among different organs, supporting the notion that auxin synthesized by YUCCAs mainly functions locally or in a short range.^6,8,13The central role of auxin in root meristem patterning and maintenance is well documented,^1,2,16 but the source of such IAA is still unclear. When ¹⁴C-labeled IAA was applied to the five-day-old pea apical bud, the radioactivity could be detected in lateral root primordia but not the apical region of primary roots.¹⁷ Moreover, removal of the shoot only slightly affected elongation of the primary root, and localized application of auxin polar transport inhibitor naphthylphthalamic acid (NPA) at the primary root tip exerted more profound inhibitory effect on root elongation than at any other site.¹⁸ These results suggest that auxin generated near the root tip may play a more important role in primary root growth than that transported from the shoot. In line with this notion, Arabidopsis roots have been shown to harbor multiple auxin biosynthesis sites including root tips and the region upward from the tip.⁴Many steps of tryptophan synthesis and its conversion to auxin involve transamination reactions, which require the vitamin B₆ pyridoxal 5-phosphate (PLP) as a cofactor. We previously reported that the Arabidopsis mutant pdx1 that is defective in vitamin B₆ biosynthesis displays dramatically reduced primary root growth with smaller meristematic zone and shorter mature cortical cells.¹⁹ In the current investigation, we found that the root tips of pdx1 have reduced cell division capability and reduced DR5::GUS activity, although the induction of this reporter gene by exogenous auxin was not changed. Reciprocal grafting indicates that the short-root phenotype of pdx1 is caused by a root local rather than shoot generated factor(s). Importantly, pdx1 suppresses yucca mutant, an auxin overproducer, in root hair proliferation although it fails to suppress the hypocotyl elongation phenotype.²⁰ Our work thus demonstrated that pdx1 has impaired root local auxin biosynthesis from tryptophan. To test whether the synthesis of tryptophan is also affected in pdx1 mutant, we planted pdx1 together with wild-type seeds on Murashige and Skoog (MS) medium supplemented with 5-mehtyl-anthranilate (5-MA), an analogue of the Trp biosynthetic intermediate anthranilate.²¹ Although pdx1 seedlings grew poorly under the control conditions, the growth of wild-type seedlings was more inhibited than that of the pdx1 seedlings on 10 µM 5-MA media (Fig. 1A–D). Compared with the elongated primary root on MS, wild-type seedlings showed very limited root growth on 5-MA (Fig. 1E). The relatively increased tolerance to 5-MA of pdx1 thus indicates that the pdx1 mutant may be defective in Trp biosynthesis, although amino acid analysis of the bulked seedlings did not find clear changes in Trp levels in the mutants (our unpublished data).Open in a separate windowFigure 1The pdx1 mutant seedlings are relatively less sensitive to toxic 5-methyl anthranilate (5-MA). (A and C) Five-day-old seedlings of the wild type (Col-0) (A) or pdx1 (C) on MS medium. (B and D) Five-day-old seedlings of the wild type (B) or pdx1 (D) on MS medium supplemented with 10 µM 5-MA. (E) Eight-day-old seedlings of the wild type or pdx1 on MS medium without or with 10 µM 5-MA supplement. Sterilized seeds were planted directly on the indicated medium and after two days of cold treatment, the plates were incubated under continuous light at 22–24°C before taking pictures.We reported that PDX1 is required for tolerance to oxidative stresses in Arabidopsis.¹⁹ Interestingly, redox homeostasis appears to play a critical role in Arabidopsis root development. The glutathione-deficient mutant root meristemless1 (rml1) and the vitamin C-deficient mutant vitamin C1 (vtc1) both have similar stunted roots.^22,23 Nonetheless, pdx1 is not rescued by either glutathione or vitamin C¹⁹ suggesting that the pdx1 short-root phenotype may not be resulted from a general reduction of antioxidative capacity. Interestingly, ascorbate oxidase is found to be highly expressed in the maize root quiescent center.²⁴ This enzyme can oxidatively decarboxylate auxin in vitro, suggesting that the quiescent center may be a site for metabolizing auxin to control its homeostasis.²⁵ It is therefore likely that the reduced auxin level in pdx1 root tips could be partially caused by increased auxin catabolism resulted from reduced vitamin B₆ level. We thus conducted experiments to test this possibility. A quiescent center-specific promoter WOX5 driven bacterial auxin biosynthetic gene iaaH²⁶ was introduced into pdx1 mutant. The transgenic seeds were planted on media supplemented with different concentrations of indoleacetamide (IAM), the substrate of iaaH protein. Although promotion of lateral root growth was observed at higher IAM concentrations, which indicates increased tryptophan-independent auxin production from the transgene, no change in root elongation was observed between pdx1 with or without the WOX5::iaaH transgene at any concentration of IAM tested (data not shown), suggesting that the pdx1 short-root phenotype may not be due to increased auxin catabolism.Taken together, in addition to auxin transport; temporally, spatially or developmentally coordinated local auxin biosynthesis defines the plant growth and its response to environmental changes.^8,14,15     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

Creating drought- and salt-tolerant cotton by overexpressing a vacuolar pyrophosphatase gene

Increased expression of an Arabidopsis vacuolar pyrophosphatase gene, AVP1, leads to increased drought and salt tolerance in transgenic plants, which has been demonstrated in laboratory and field conditions. The molecular mechanism of AVP1-mediated drought resistance is likely due to increased proton pump activity of the vacuolar pyrophosphatase, which generates a higher proton electrochemical gradient across the vacuolar membrane, leading to lower water potential in the plant vacuole and higher secondary transporter activities that prevent ion accumulation to toxic levels in the cytoplasm. Additionally, overexpression of AVP1 appears to stimulate auxin polar transport, which in turn stimulates root development. The larger root system allows AVP1-overexpressing plants to absorb water more efficiently under drought and saline conditions, resulting in stress tolerance and increased yields. Multi-year field-trial data indicate that overexpression of AVP1 in cotton leads to at least 20% more fiber yield than wild-type control plants in dry-land conditions, which highlights the potential use of AVP1 in improving drought tolerance in crops in arid and semiarid areas of the world.Key words: drought tolerance, proton pump, salt tolerance, transgenic cotton, vacuolar membraneDrought and salinity are major environmental factors that limit agricultural productivity in most parts of the world.¹ Climate change will likely make many places worse in terms of water availability and soil salinization,² which will have negative impacts on food production in world agriculture. Yet, the demand for more food will continue to rise because of the growing world population that may reach 9 billon people by 2050.³ Therefore, the primary challenge we face during this century is the production of more food under the constraints of limited water and fertilizer on marginal soils.Many genes that respond to abiotic stresses have been identified in the model plant Arabidopsis,⁴ and some of them were shown to play important roles in protecting plants under abiotic stress conditions.⁵ The Arabidopsis vacuolar pyrophosphatase gene AVP1 appears to be one of the most promising genes that may be used to improve drought- and salt-tolerance in crops.⁶ Roberto Gaxiola''s group first demonstrated that overexpression of AVP1 could lead to significantly improved drought- and salt-tolerance in transgenic Arabidopsis plants.⁷ Later when this gene was introduced into tomato⁸ and rice,⁹ similar tolerance phenotypes were observed. Overexpression of AVP1 in cotton, not only improved drought- and salt-tolerance in greenhouse conditions, but also increased fiber yield in dryland field conditions.⁶ AVP1-expressing cotton plants produced larger root systems and bigger shoot biomass than controls when grown under hydroponic conditions in the presence of up to 200 mM NaCl.⁶ In the greenhouse, AVP1-expressing cotton plants also produced more root and shoot biomass than controls when grown under saline conditions or reduced irrigation.⁶ The increased yield by AVP1-expressing cotton plants is due to more bolls produced, which in turn is due to larger shoot system that AVP1-expressing cotton plants develop under saline or drought conditions.⁶The larger root systems of AVP1-expressing cotton plants under saline and water-deficit conditions allow transgenic plants access to more of the soil profile and available soil water resulting in increased biomass production and yield. Li et al. showed that the larger root systems of AVP1-overexpressing Arabidopsis is caused by increased auxin polar transport in the root, which stimulates root development in AVP1-overexpressing Arabidopsis plants.¹⁰ Furthermore, a recent comparative study of transgenic Arabidopsis lines that produce enlarged leaves showed that auxin levels were increased by 50% in AVP1-overexpressing plants.¹¹ To test if altered auxin level is responsible for the observed larger root systems in AVP1-expressing cotton plants, we germinated wild-type and AVP1-expressing cotton plants in the absence or presence of the auxin polar transport inhibitor Naphthylphthalamic acid (NPA). Both wild-type and AVP1-expressing cotton plants developed robust lateral root systems in the absence of NPA (Fig. 1A). The presence of 50 µM NPA resulted in nearly complete inhibition of lateral root development in wild-type plants, while lateral root development in AVP1-expressing plants was reduced, it was significantly greater than wild-type (Fig. 1B). These data indicate that AVP1-overexpression could overcome the inhibitory effects of NPA on root development in AVP1-expressing cotton plants, suggesting that either increased auxin transport or higher auxin concentration in the root systems of AVP1-expressing cotton plants is responsible for the observed larger root systems, and eventually for the increased boll numbers and fiber yields under dryland field conditions.Open in a separate windowFigure 1Root development of wild-type and AVP1-expressing cotton plants in the absence and presence of auxin transport inhibitor NPA. (A) Phenotype of cotton roots after 10 days of growth in the absence of NPA. WT, Wild-type; 1, 5, 9, three independent AVP1-overexpressing cotton lines. (B) Phenotype of cotton roots after 10 days of growth in the presence of 50 µm NPA.Many genes that may play important roles under water-deficit conditions have been tested in laboratory conditions,^4,5 but very few have been tested vigorously in field conditions. A bacterial cold shock protein gene was shown to improve drought tolerance in maize based on multi-year and multi-place field trial experiments,¹² and it appears that this gene will likely gain approval for commercial release and become the first genetically engineered product that demonstrates improved drought tolerance in a major crop in the U.S. Another example of increased drought tolerance supported by multiple field trial experiments is through downregulation of farnesylation in transgenic canola plants.¹³ Downregulation of farnesyltransferase by antisense or RNAi techniques in transgenic canola leads to increased sensitivity to abscisic acid, consequently resulting in smaller guard cell aperture under drought conditions. These transgenic canola plants lose less water through transpiration and are more drought resistant. Data from more than 5 years of field studies in Canada consistently proved that this approach can indeed increase drought tolerance in transgenic canola. Our study with AVP1-expressing cotton over the last several years in field conditions is another example that genetic engineering approach can be an efficient tool in generating drought-tolerant crops. AVP1-expressing cotton plants can establish a larger shoot mass in dryland conditions (Fig. 2), which results in increased boll numbers and fiber production. Our approach is likely applicable to other major crops as well.Open in a separate windowFigure 2Wild-type and AVP1-expressing cotton plants grown in the dryland field condition. Plants were planted in the middle of may 2009 and the picture was taken in the middle of July 2009 at the USDA experimental Farm in Lubbock, Texas.     

Auxin distribution and lenticel formation in determinate nodule of Lotus japonicus

Legumes can establish a symbiosis with rhizobia and form root nodules that function as an apparatus for nitrogen fixation. Nodule development is regulated by several phytohormones including auxin. Although accumulation of auxin is necessary to initiate the nodulation of indeterminate nodules, the functions of auxin on the nodulation of determinate nodules have been less characterized. In this study, the functions of auxin in nodule development in Lotus japonicus have been demonstrated using an auxin responsive promoter and auxin inhibitors. We found that the lenticel formation on the nodule surface was sensitive to the auxin defect. Further analysis indicated that failure in the development of the vascular bundle of the determinate nodule, which was regulated by auxin, was the cause of the disappearance of lenticels.Key words: auxin, lenticel, Lotus japonicus, nodulation, symbiotic nitrogen fixationLegumes (Fabaceae) constitute the third largest plant family with around 700 genera and 20,000 species.¹ Legume plants form root nodules through symbiosis with a soil microbe called rhizobia. This plant-microbe symbiosis in nodules mediates an harmonized exchange of chemical signals between host plants and rhizobia.² Nodules are biologically divided into two different groups, i.e., indeterminate nodules and determinate nodules. Indeterminate nodules, represented by Trifolium repens (white clover) and Medicago truncatula, are initiated from the inner cortex to form a persistent nodule meristem, which allows continuous growth, and leads to the formation of elongated nodules, whereas in determinate legumes, nodules are mostly developed from outer cortical cells and form spherical nodules.³Auxin is one of the most important regulators for nodule development. Since the possible involvement of auxin in nodule formation was first reported by Thimann,⁴ auxin distribution during nodulation has been studied in particular with indeterminate nodules.⁵ However, little is known about auxin involvement in determinate nodule formation. To evaluate auxin functions in the determinate nodulation of legume plants, we performed an auxin-responsive promoter analysis in detail. Using GH3:GUS transformed Lotus japonicus (a kind gift from Dr. Herman P. Spaink, Leiden State University, Netherlands),⁶ we detected auxin signals throughout the nodulation process, e.g., at the basal and front part of the nodule primordia, circumjacent to the infection zone of the young developing nodules (Fig. 1), and at the nodule vascular bundle in mature nodules. We also investigated the effect of several auxin inhibitors, including newly synthesized auxin antagonist PEO-IAA (kindly provided by Dr. Hayashi, Okayama University of Science, Japan),⁷ on the nodulation of L. japonicus, and revealed that auxin was required for forming a nodule vascular bundle and lenticels (Fig. 2).⁸Open in a separate windowFigure 1GH3:GUS expression in determinate nodule at 6 dpi. (A) GUS staining was observed in the central cylinder of the root vascular bundle and in the nodule. (B) Cross section of (A). GUS expression was observed around the infection zone of the nodule. Bars = 100 µm.Open in a separate windowFigure 2The effect of auxin inhibitor on nodule surface. (A) Typical mature nodule of L. japonicus at 21 dpi. Lenticels are pointed out by yellow arrowheads. (B) The treatment of auxin inhibitor (NPA 100 µM) inhibited lenticel formation on the nodule surface. Bars = 500 µm.In indeterminate legumes, auxin is accumulated at the site of rhizobia inoculation.⁹ This is caused by the inhibition of polar auxin transport by accumulation of flavonoids around the infection site, which are known as regulators of auxin transport. When flavonoid biosynthesis is reduced by the gene silencing of chalcone synthase, which catalyzes the first step of flavonoid synthesis, M. truncatula was unable to inhibit polar auxin transport and resulted in reduced nodule number.^10,11 A similar phenotype was observed when the auxin transporter gene was silenced.¹² In addition, treatment of polar auxin transport inhibitors such as NPA and TIBA induce pseudonodule formation,⁹ suggesting that auxin accumulation is required for nodulation of indeterminate legumes. In contrast, the treatment of polar auxin transport inhibitors in determinate nodules did not induce a nodule-like structure, suggesting a different function of auxin between indeterminate and determinate nodules. It is, however, of interest to investigate the involvement of flavonoids in determinate nodule formation, because several genes in the flavonoid biosynthesis pathway are upregulated at 2 dpi (days post inoculation) in L. japonicus.¹³Lenticels regulate gas permeability of nodules.¹⁴ Under low oxygen or water-logged conditions, they develop more extensively, whereas they collapse, or develop very little during insufficient water conditions, or under high oxygen pressure.^14,15 Because lenticel development on the nodule surface is accompanied with the nodule vascular bundle, growth regulators supplied from the vascular system likely facilitate lenticel development.¹⁵ Our data suggests that auxin is necessary to form the nodule vascular bundle, and in fact, auxin itself is one of the candidates of growth substances that control lenticel formation. It is necessary to analyze mutants, which lack in lenticel formation, but can form a nodule vascular bundle, for clarification of further mechanisms of lenticel development.     

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.     

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     

Multiple signaling pathways control nitrogen-mediated root elongation in maize

Response of root system architecture to nutrient availability is an essential way for plants to adapt to soil environments. Nitrogen can affect root development either as a result of changes in the external concentration, or through changes in the internal nutrient status of the plant. Low soil N stimulates root elongation in maize. Recent evidence suggests that plant hormones auxin and cytokinin, as well as NO signaling pathway, are involved in the regulation of root elongation by low nitrogen nutrition.Key words: nitrogen, root growth, auxin, cytokinin, NONitrogen acquisition is determined by N demand for plant growth. At low N stress, N demand for maximum plant growth rate is not matched by plant N uptake. To acquire adequate N, plants may increase root length density to explore a larger soil volume and/or increase N uptake activity. High root density is also an important root trait for competition with soil microorganisms.¹ Since nitrate is a highly mobile, non-adsorbing ion, theoretic analysis predicts that its uptake is not limited by transport through soil, and a small root system is sufficient for nitrate acquisition.^2–4 In field conditions, however, genotypes that are efficient in N acquisition generally had a larger root system and higher root length density.^5,6 Under conditions of insufficient N supply, N mass flow to roots may not be adequate to meet the N demand for plant growth. Even in N-sufficient soils, various soil constraints (low water content, etc) may reduce the N mass flow rate. In these cases, large root size and high density will be very important for the utilization of the spatially distributed N, especially newly mineralized N, and the competition for organic N with soil microorganisms.^7,8The development of lateral roots in Arabidopsis in response to nitrate supply has been widely studied.⁹ Less attention has been paid to primary root growth in response to N, possibly because root elongtion is insensitive to increased N supply in Arabidopsis.^10,11 In maize, however, root elongation was sigificantly promoted by suboptimal N supply, and inhibited by overdose supply of N (Fig. 1).^12,13 Until recently less is known about the underlying physiological mechansms. It is well documented that cytokinin is a root-to-shoot signal communicating N availability in addition to nitrate itself.¹⁴ Exogenous cytokinin application suppresses the elongation of primary roots.¹⁵ Recent work in Arabidopsis overexpressing cytokinin synthase (IPT) demonstrate that long-term CK overproduction inhibited primary root elongation by reducing quantitative parameters of primary root meristem.¹⁶ By comparing two maize inbred lines whose root elongation had a differential response to low N stress, it was found that the change of cytokinin content in roots was closely related to low-N induced root elongation.¹³ In the N-sensitive genotype 478, cytokinin (Zeatin + Zeatin riboside) content was significantly lower at low N condition. While in N-insensitive genotype Wu312, cytokinin content was hardly affected at various N supplies. Higher N supply shortened the distance from root apex to the first visible lateral roots, a phenomenen similar to that caused by exogenous cytokinins. Furthermore, exogenous cytokinin 6-benzylaminopurine (6-BA) completely reversed the stimulatory effect of low nitrate on root elongation. All the data suggests that the inhibitory effect of high concentration of nitrate on root elongation is, at least in part, mediated by increased cytokinin level in roots.Open in a separate windowFigure 1Root elongation is inhibited at high nitrate supply.Auxin regulates many cellular responses crucial for plant development. Auxin plays a key role in establishing and elaborating patterns in root meristems.^17,18 Root elongation of Arabidopsis is enhanced by exogenous auxin at low concentrations, but is inhibited at high concentrations.¹⁹ In an earlier report, a high external nitrate supply (8 mM) did cause a 70% decrease in the auxin concentration of the root in soybean.²⁰ In maize, inhibition of root growth by high nitrate was found closely related to the reduction of IAA levels in roots and exogenous NAA and IAA restored primary root growth in high nitrate concentrations.²¹ Interesting, it was found that auxin concentrations in phloem exudates were reduced by a greater nitrate supply, suggesting that shoot-to-root auxin transport may be inhibited by high N supply. Considering the antagonism between auxin and cytokinin.²² it was possible that, by increasing the cytokinin level and decreasing the auxin level, high nitrate supply may have negative influences on root apex activity so that root apical dominance is weakened and, therefore, root elongation is suppressed and lateral roots grow closer to the root apex.Nitric oxide (NO) is emerging as an important messenger molecule associated with many biochemical and physiological processes in plants. The involvement of NO in IAA-induced adventitious root development has also been reported.²³ Given that nitrate is a substrate for NR-catalysed NO production, and root development and growth are closely related to NO, it is expected that NO may play a role in nitrate-dependent root growth. Surprisingly, endogenous levels of NO in the root apices of maize seedlings grown in high nitrate solution were much lower than those in apices grown in low nitrate. The nitrate-induced inhibition of root elongation in maize was markedly reversed by treatments of the roots with a NO donor (SNP) and IAA.²⁴ These data suggest that the arrest of root elongation by high levels of external nitrate concentrations may result from an alteration of endogenous NO levels in root apical cells. NR mediated NO production is unlikely to be involved in the nitrate-dependent NO production and root elongation because NR activity is lower at low N supply. A NO synthase (NOS) inhibitor reduced root elongation in maize plants grown in the low-nitrate medium, suggest that NOS activity may be inhibited in plants grown in high-nitrate solution, thus leading to a reduction of the endogenous NO levels.Taken together, high nitrogen supply increases cytokinin level, but decreases auxin and NO levels in roots of maize. Besides, it was well documented ethylene has a negative effect on root elongation of various plants.^25–27 Exogenous supply of cytokinin increase ethylene production (Stenlid 1982; Bertell et al., 1990). Recently, it was demonstrated in Arabidopsis that auxin transport from the root apex via the lateral root cap is required for ethylene-mediated inhibition of root growth.²⁸ Therefore, a complex multiple siganlling pathways may be involved in N-mediated root elongation (Fig. 2). Further study is required to understand how these pathways interact with each other to reduce root elongation in response to high nitrate supply.Open in a separate windowFigure 2A simplified model explaining nitogen-mediated root elongation in maize.