N-MYC DOWN-REGULATED-LIKE Proteins Regulate Meristem Initiation by Modulating Auxin Transport and MAX2 Expression

Background

N-MYC DOWN-REGULATED-LIKE (NDL) proteins interact with the Gβ subunit (AGB1) of the heterotrimeric G protein complex and play an important role in AGB1-dependent regulation of lateral root formation by affecting root auxin transport, auxin gradients and the steady-state levels of mRNA encoding the PIN-FORMED 2 and AUXIN 1 auxin transport facilitators. Auxin transport in aerial tissue follows different paths and utilizes different transporters than in roots; therefore, in the present study, we analyzed whether NDL proteins play an important role in AGB1-dependent, auxin-mediated meristem development.

Methodology/Principal Findings

Expression levels of NDL gene family members need to be tightly regulated, and altered expression (both over-expression and down-regulation) confers ectopic growth. Over-expression of NDL1 disrupts vegetative and reproductive organ development. Reduced expression of the NDL gene family members results in asymmetric leaf emergence, twinning of rosette leaves, defects in leaf formation, and abnormal silique distribution. Reduced expression of the NDL genes in the agb1-2 (null allele) mutant rescues some of the abnormal phenotypes, such as silique morphology, silique distribution, and peduncle angle, suggesting that proper levels of NDL proteins are maintained by AGB1. We found that all of these abnormal aerial phenotypes due to altered NDL expression were associated with increases in basipetal auxin transport, altered auxin maxima and altered MAX2 expression within the inflorescence stem.

Conclusion/Significance

NDL proteins, together with AGB1, act as positive regulators of meristem initiation and branching. AGB1 and NDL1 positively regulate basipetal inflorescence auxin transport and modulate MAX2 expression in shoots, which in turn regulates organ and lateral meristem formation by the establishment and maintenance of auxin gradients.     

ERECTA Family Genes Regulate Auxin Transport in the Shoot Apical Meristem and Forming Leaf Primordia

Leaves are produced postembryonically at the flanks of the shoot apical meristem. Their initiation is induced by a positive feedback loop between auxin and its transporter PIN-FORMED1 (PIN1). The expression and polarity of PIN1 in the shoot apical meristem is thought to be regulated primarily by auxin concentration and flow. The formation of an auxin maximum in the L1 layer of the meristem is the first sign of leaf initiation and is promptly followed by auxin flow into the inner tissues, formation of the midvein, and appearance of the primordium bulge. The ERECTA family genes (ERfs) encode leucine-rich repeat receptor-like kinases, and in Arabidopsis (Arabidopsis thaliana), this gene family consists of ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERL2. Here, we show that ERfs regulate auxin transport during leaf initiation. The shoot apical meristem of the er erl1 erl2 triple mutant produces leaf primordia at a significantly reduced rate and with altered phyllotaxy. This phenotype is likely due to deficiencies in auxin transport in the shoot apex, as judged by altered expression of PIN1, the auxin reporter DR5rev::GFP, and the auxin-inducible genes MONOPTEROS, INDOLE-3-ACETIC ACID INDUCIBLE1 (IAA1), and IAA19. In er erl1 erl2, auxin presumably accumulates in the L1 layer of the meristem, unable to flow into the vasculature of a hypocotyl. Our data demonstrate that ERfs are essential for PIN1 expression in the forming midvein of future leaf primordia and in the vasculature of emerging leaves.Leaves are formed during postembryonic development by the shoot apical meristem (SAM), a dome-shaped organ with a stem cell reservoir at the top and with leaf initiation taking place slightly below in the peripheral zone. The initiation of leaf primordia depends on the establishment of auxin maxima at the site of initiation (Braybrook and Kuhlemeier, 2010). Auxin is polarly transported through the epidermal layer of the meristem to the incipient primordium initiation site (Heisler et al., 2005) and then moves inward, where it promotes the formation of a vascular strand (Scarpella et al., 2006; Bayer et al., 2009). The developing vascular tissue acts as an auxin sink, depleting auxin in the epidermal layer (Scarpella et al., 2006). PIN1, an auxin efflux protein, is a central player in the formation of auxin maxima and is involved in the transport of auxin in both the epidermis and the forming vascular strand during leaf initiation (Benková et al., 2003; Reinhardt et al., 2003). PIN1 is the earliest marker for midvein formation (Scarpella et al., 2006), which starts to form before a leaf primordium bulges out of the meristem. The mechanisms determining PIN1 expression and polar localization in the SAM are central to understanding leaf initiation. In the L1 layer of the SAM, PIN1 is polarly localized in the plasma membrane toward cells with higher auxin concentration (Jönsson et al., 2006; Smith et al., 2006). Formation of the vein is explained by the canalization hypothesis, in which high auxin flux reinforces PIN1 expression (Kramer, 2008). Of all plasma membrane-localized PIN family transporters, only PIN1 has been detected in the vegetative SAM and linked with the initiation of rosette leaves (Guenot et al., 2012). At the same time, rosette leaves are positioned nonrandomly in pin1 mutants, suggesting that additional PIN1-independent mechanisms also have a role in regulating leaf initiation (Guenot et al., 2012).Here, we investigate the role of ERECTA family receptor-like kinases during leaf initiation in Arabidopsis (Arabidopsis thaliana). Previously, ERECTA family genes (ERfs) have been shown to be involved in the regulation of epidermis development and of plant growth along the apical-basal/proximodistal axis in aboveground organs (Torii et al., 1996; Shpak et al., 2004, 2005). Triple erecta (er), erecta-like1 (erl1), and erl2 mutants (er erl1 erl2) form a rosette with small, round leaves that lack petiole elongation. During the reproductive stage, the main inflorescence stem exhibits striking elongation defects and reduced apical dominance. ER has been implicated in vascular development, with the er mutation causing radial expansion of xylem (Ragni et al., 2011) and premature differentiation of vascular bundles (Douglas and Riggs, 2005). Recently, the dwarfism of described mutants was attributed to the function of ERf genes in the phloem, where they perceive signals from the endodermis (Uchida et al., 2012a). In the epidermis, all three genes inhibit the initial decision of protodermal cells to become meristemoid mother cells (Shpak et al., 2005). In addition, ERL1 and to a lesser extent ERL2 are important for maintaining cell proliferative activity in stomata lineage cells and for preventing terminal differentiation of meristemoids into guard mother cells. The activity of ERf receptors in the epidermis is regulated by a different set of peptides than in the phloem. EPIDERMAL PATTERNING FACTOR1 (EPF1) and EPF2 are expressed in stomatal precursor cells. They inhibit the development of new stomata in the vicinity of a forming stoma (Hara et al., 2007, 2009; Hunt and Gray, 2009). EPF-LIKE9 (EPFL9)/stomagen is expressed in the mesophyll, and, in contrast, it promotes the development of stomata (Kondo et al., 2010; Sugano et al., 2010). EPFL4 and EPFL6/CHALLAH are expressed in the endodermis, and their perception by phloem-localized ERfs is critical for stem elongation (Uchida et al., 2012a).While ERfs are very strongly expressed in the vegetative SAM and in forming leaf primordia, only recently has it become clear that these genes are involved in the regulation of meristem size and leaf initiation (Uchida et al., 2012b, 2013). It was suggested that ERfs regulate stem cell homeostasis in the SAM via buffering its cytokinin responsiveness by an unknown mechanism (Uchida et al., 2013). Here, we further investigate the involvement of ERfs in the control of leaf initiation and phyllotaxy. Our data suggest that ERfs are essential for PIN1 expression in the vasculature of forming leaf primordia. Based on analysis of the DR5rev::GFP reporter, auxin may accumulate in the L1 layer of the SAM in the mutant but is not able to move into the vasculature, consistent with drastically reduced PIN1pro:PIN1-GFP expression there. These data suggest that the convergence of PIN1 expression in the inner tissues of the SAM during leaf initiation is a complex process involving intercellular communications enabled by ERfs. The importance of ERfs for efficient auxin transport is further supported by reduced phototropic response in the er erl1 erl2 mutant.     

Auxin transport: ABC proteins join the club

The isolation of potential auxin carriers from Arabidopsis thaliana marks a breakthrough in the characterization of elements involved in auxin delivery. Current models suggest that asymmetrical localization of auxin uptake and efflux carriers within the plasma membrane control the establishment of auxin gradients via facilitated transport. Now, the analysis of mutants defective in Arabidopsis ABC proteins indicates that primary active transport might participate in the control of auxin homeostasis as well.     

Auxin Transport Inhibitors: III. Chemical Requirements of a Class of Auxin Transport Inhibitors

The structural requirements of a proposed class of auxin transport inhibitors have been shown to be very similar to those required to inhibit the cress (Lepidium sativum) root geotropic response. A 2-carboxyphenyl group separated by a conjugated system of atoms from a second aromatic ring appears to be necessary for a molecule to have high activity.     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

Effect of Ethylene on Auxin Transport

Ethylene was found to have no influence on auxin transport in hypocotyls of Helianthus annuus and Phaseolus vulgaris; coleoptiles of Zea mays; petiole sections of Gossypium hirsutum, Phaseolus vulgaris, and Coleus blumei. In the experiments described here, the tissues were treated with ethylene only during the 3 hours of polar transport. This short treatment is in contrast to the methods of others who found an effect of ethylene on auxin transport when plants grown in ethylene are used as experimental tissues.     

The Rate of Transport of Natural Auxin in Woody Shoots

A method is described for the estimation of the rate of movementand the quantity transported of the natural growth hormone instandard isolated segments of apple shoots. During controlledstorage diffusible auxin is collected, and later by dividingthe standard length of stem into small sections the locationof the auxin front is determined, from which the rate of transportis deduced. Temperature markedly affects both rate of transportand amount of auxin transported (cf. van der Weij, 1932), amaximum occurring at 27–30° C.; followed by a rapidfall to zero. The total diffusible auxin in a given length ofstem is not affected by storage temperatures below 30° C.but falls to zero at 42° C. The rate of transport and amounttransported are proportional to the oxygen tension over therange 0 to 5 per cent. O₂, and there is some evidence for destructionof auxin in tensions below 2 per cent.     

PIN-Dependent Auxin Transport: Action,Regulation, and Evolution

Auxin participates in a multitude of developmental processes, as well as responses to environmental cues. Compared with other plant hormones, auxin exhibits a unique property, as it undergoes directional, cell-to-cell transport facilitated by plasma membrane-localized transport proteins. Among them, a prominent role has been ascribed to the PIN family of auxin efflux facilitators. PIN proteins direct polar auxin transport on account of their asymmetric subcellular localizations. In this review, we provide an overview of the multiple developmental roles of PIN proteins, including the atypical endoplasmic reticulum-localized members of the family, and look at the family from an evolutionary perspective. Next, we cover the cell biological and molecular aspects of PIN function, in particular the establishment of their polar subcellular localization. Hormonal and environmental inputs into the regulation of PIN action are summarized as well.     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: Auxin Dose-Response Curves and Effects of Inhibitors of Polar Auxin Transport

Transport of ¹⁴C-photosynthate in decapitated stems of Phaseolusvulgaris explants was dependent on the concentration of indole-3-aceticacid (IAA) applied to the cut surfaces of the stem stumps. Thephysiological age of the stem influenced the nature of the transportresponse to IAA with stems that had ceased elongation exhibitinga more pronounced response with a distinct optimum. Increasednutrient status of the explants had little influence on theshape of the IAA dose-response curve but increased, by two ordersof magnitude, the IAA concentration that elicited the optimalresponse. Applications of the inhibitor of polar auxin transport,1-(2-carboxyphenyl)-3-phenylpropane-1, 3-dione (CPD), affectedIAA-promoted transport of ¹⁴C-photosynthates. At sub-optimalIAA concentrations, CPD inhibited transport, whereas at supra-optimalIAA concentrations, ¹⁴C-photosynthate transport was marginallystimulated by CPD. Treatment with CPD resulted in a significantreduction in stem levels of [¹⁴C]IAA below the site of inhibitorapplication, while above this point, levels of [¹⁴C]1AA remainedunaltered. The divergent responses of auxin-promoted transportto CPD treatment are most consistent with a remote action ofIAA on photosynthate transport in the decapitated stems. Key words: Auxin, photosynthate, transport     

Aryl-Substituted alpha-Aminooxycarboxylic Acids: A New Class of Auxin Transport Inhibitors

Certain herbicidal aminooxyisovalerate analogs were noted in whole plant phytotoxicity bioassays to cause disoriented roots. Since this symptom is often characteristic of interference with the transport of the plant hormone auxin, the ability of several of these compounds to compete for the N-1-naphthylphthalamic acid (NPA) binding site in corn (Zea mays L.) coleoptile membranes was measured. Significant NPA binding activity was found, expecially for the 2,4-dichlorophenyl analog. Application of structure-activity principles from traditional auxin transport inhibitors to this new class of molecules led to the synthesis of the naphthyl analogue. This molecule was extremely active in competing for NPA binding and in eliciting whole plant growth regulator effects. Possible relationships between these molecules and the mode of auxin transport are discussed.     

Auxin Transport in Tendril Segments of Passiflora caerulea

The movement of auxin through tendril segments of Passiflora caerulca L. has been investigated using IAA-2-¹⁴C. It has been shown that (1) flux of IAA through the segments is strongly polarized basipetally: (2) the amount of ¹⁴C recovered in the basal receiver blocks increases linearly within a transport period of 6 h; (3) velocity of basipetal transport is 14.5 mm h^?1; (4) at least 70% of the radioactivity in the receiver blocks is confined to the IAA molecule: approximately 55% of ¹⁴C from methanolic extracts of the segments is IAA: (5) at low temperatures (2–4°C) the basipetal transport is abolished; (6) white light promotes basipetal transport, and this effect is abolished in a CO₂-free atmosphere; (7) no difference could be detected in ¹⁴C content between dorsal and ventral halves of tendril segments nor among individual dorsal and ventral receiver blocks.     

棉花li突变体生长素极性运输的减弱

陆地棉(Gossypium hirsutum L.)li突变体叶片卷曲,植株扭曲,种子表皮毛明显偏短。通过扫描电子显微镜(SEM)观察发现,li突变体的纤维发育在起始期与野生型植株并无明显差异,但在伸长期开始后,如开花后3d(3 day post anthesis,DPA),纤维伸长受阻;li突变体茎的形成层和韧皮部分化发育不完全,生长素由顶端向基部的极性运输能力下降,仅为野生型植株的大约三分之一。推测棉花li突变体包括纤维发育不良在内的多效性异常表型,与其生长素极性运输能力的下降有关。     

Overexpression of the Auxin Binding PROTEIN1 Modulates PIN-Dependent Auxin Transport in Tobacco Cells

Background

Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive.

Methodology/Principal Findings

Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations.

Conclusions/Significance

The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.     

Truffles Regulate Plant Root Morphogenesis via the Production of Auxin and Ethylene

Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction, i.e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle Tuber melanopsorum induce alterations in root morphology of the host Cistus incanus and the nonhost Arabidopsis (Arabidopsis thaliana; i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and IAA fully mimicked the root morphology induced by the mycelium for both host and nonhost plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene-insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungus-induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5∷GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffles synthesize ethylene from l-methionine probably through the α-keto-γ-(methylthio)butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions.Ectomycorrhizal symbioses are mutualistic interactions between filamentous fungi and plant roots. Truffles, which are ascomycete fungi renown for their aromatic fruiting bodies, form ectomycorrhizas (ECM) in temperate climates predominantly with trees (i.e. hazel [Corylus avellana], oaks [Quercus spp.]).In soil, microorganisms communicate with plants by exchanging chemical signals throughout the rhizosphere. Depending on the nature of the interaction, these molecules can be either volatiles or solutes (dissolved solids). For example, rhizobacteria induce growth promotion in Arabidopsis (Arabidopsis thaliana) through the action of the volatile compound 2,3-butanediol (Ryu et al., 2003). The sesquiterpene volatile (E)-β-caryophyllene is produced by maize (Zea mays) roots fed upon by arthropods, and serves as attractant to natural enemies of the insects (Rasmann et al., 2005). Nonvolatile signal molecules can also dissolve in water and diffuse in the soil. Indeed the nitrogen-fixing bacteria Rhizobium secrete a nodulation factor that induces changes in root morphology of legumes (Dénarié and Cullimore, 1993; Heidstra and Bisseling, 1996). Mycelium branching is induced in the mycorrhizal fungus Gigaspora margarita by 5-deoxy-strigol, a strigolactone exuded from the roots of the legume host Lotus japonicus (Akiyama et al., 2005).Also ectomycorrhizal fungi engage in a molecular dialogue with plants and produce chemical signals that modulate plant root/ECM morphogenesis. The indole alkaloid hypaphorine produced by ectomycorrhizal fungus Pisolithus tinctorius inhibits root hair elongation in the host Eucalyptus globules and the nonhost Arabidopsis (Béguiristain et al.,1995; Reboutier et al., 2002). Hormones, mainly indole-3-acetic acid (IAA), have also been often implicated in symbiotic interactions (Barker and Tagu, 2000; Martin et al., 2001; Sirrenberg et al., 2007; Contreras-Cornejo et al., 2009). Most studies involving hormones have focused at a late stage of interaction, when ECMs were developing or already formed (for review, see Barker and Tagu, 2000). Indeed IAA-overproducing mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum form significantly more mycorrhizas with the host Pinus pinaster than the wild type (Gay et al., 1994). Similarly applying exogenous IAA to the ectomycorrhizal system Piloderma croceum/Quercus robur also resulted in a more intense ECM colonization of the host compared to controls with no additional IAA (Herrmann et al., 2004).Truffles form ECM with a variety of hosts such as oaks, hazels, but also some shrubs (i.e. Cistus). Under laboratory conditions, the establishment of the symbiotic phase takes 2 to 3 months (Sisti et al., 1998; Miozzi et al., 2005). Strains of the same truffle species vary in their capacity to colonize a single host (Giomaro et al., 2000). Volatile organic compounds have been implicated in the signaling between truffle and plants. Splivallo et al. (2007) discussed the phytotoxic activity of fruiting body volatiles and Menotta et al. (2004) highlighted their potential role as mycorrhization signals. Fruiting bodies volatiles shortened primary roots of plants (Splivallo et al., 2007) while the effect of mycelial metabolites has not been addressed.Our aim here was to investigate how truffle mycelia modify plant root architecture. We focus our effort on an early stage of interaction (10 d) to explain changes in root morphology prior to ECM formation and highlight the action of diffusible signals on the host plant Cistus incanus and the nonhost Arabidopsis. Using Arabidopsis mutants, we did not aim to investigate the mechanism behind the IAA/ethylene cross talk but rather to show that the fungal metabolites are perceived in planta through both auxin and ethylene signaling pathways. We illustrate how truffle metabolites modify the auxin response of the root meristem using the auxin reporter line DR5∷GFP. We further demonstrate that both hormones are produced by truffles at concentrations that fully explain the root phenotypic responses of the host and nonhost plants. Last, we elucidate ethylene biosynthesis in truffles.