Arabidopsis Root Development Regulation by the Endogenous Folate Precursor, Para-Aminobenzoic Acid, via Modulation of the Root Cell Cycle.

The continuous growth of roots depends on their ability to maintain a balanced ratio between cell production and cell differentiation at the tip. This process is regulated by the hormonal balance of cytokinin and auxin. However, other important regulators, such as plant folates, also play a regulatory role. In this study, we investigated the impact of the folate precursor para-aminobenzoic acid (PABA) on root development. Using pharmacological, genetic, and imaging approaches, we show that the growth of Arabidopsis thaliana roots is repressed by either supplementing the growth medium with PABA or overexpressing the PABA synthesis gene GAT-ADCS. This is associated with a smaller root meristem consisting of fewer cells. Conversely, reducing the levels of free root endogenous PABA results in longer roots with extended meristems. We provide evidence that PABA represses Arabidopsis root growth in a folate-independent manner and likely acts through two mechanisms: (i) the G2/M transition of cell division in the root apical meristem and (ii) promoting premature cell differentiation in the transition zone. These data collectively suggest that PABA plays a role in Arabidopsis root growth at the intersection between cell division and cell differentiation.

A novel role for the chloride intracellular channel protein Clic5 in ciliary function.

CLIC5 belongs to a family of ion channels with six members reported so far. In vertebrates, the CLIC5 gene encodes two different isoforms, CLIC5A and CLIC5B. In addition to its ion channel activity, there is evidence for further functions of CLIC5A, such as the remodeling of the actin cytoskeleton during the formation of a functional glomerulus in the vertebrate kidney. However, its specific role is still incompletely understood and a specific functional role for CLIC5B has not been described yet. Here we report our findings on the differential expression and functions of Clic5a and Clic5b during zebrafish kidney development. Whole-mount in situ hybridization studies revealed specific expression of clic5a in the eye and pronephric glomerulus, and clic5b is expressed in the gut, liver and the pronephric tubules. Clic5 immunostainings revealed that Clic5b is localized in the cilia. Whereas knockdown of Clic5a resulted in leakiness of the glomerular filtration barrier, Clic5b deficient embryos displayed defective ciliogenesis, leading to ciliopathy-associated phenotypes such as ventral body curvature, otolith deposition defects, altered left-right asymmetry and formation of hydrocephalus and pronephric cysts. In addition, Clic5 deficiency resulted in dysregulation of cilia-dependent Wnt signalling pathway components. Mechanistically, we identified a Clic5-dependent activation of the membrane-cytoskeletal linker proteins Ezrin/Radixin/Moesin (ERM) in the pronephric tubules of zebrafish. In conclusion, our in vivo data demonstrates a novel role for Clic5 in regulating essential ciliary functions and identified Clic5 as a positive regulator of ERM phosphorylation.

Formin-mediated bridging of cell wall, plasma membrane, and cytoskeleton in symbiotic infections of Medicago truncatula.

Legumes have maintained the ability to associate with rhizobia to sustain the nitrogen-fixing root nodule symbiosis (RNS). In Medicago truncatula, the Nod factor (NF)-dependent intracellular root colonization by Sinorhizobium meliloti initiates from young, growing root hairs. They form rhizobial traps by physically curling around the symbiont.1,2 Although alterations in root hair morphology like branching and swelling have been observed in other plants in response to drug treatments3 or genetic perturbations,4-6 full root hair curling represents a rather specific invention in legumes. The entrapment of the symbiont completes with its full enclosure in a structure called the "infection chamber" (IC),1,2,7,8 from which a tube-like membrane channel, the "infection thread" (IT), initiates.1,2,9 All steps of rhizobium-induced root hair alterations are aided by a tip-localized cytosolic calcium gradient,10,11 global actin re-arrangements, and dense subapical fine actin bundles that are required for the delivery of Golgi-derived vesicles to the root hair tip.7,12-14 Altered actin dynamics during early responses to NFs or rhizobia have mostly been shown in mutants that are affected in the actin-related SCAR/WAVE complex.15-18 Here, we identified a polarly localized SYMBIOTIC FORMIN 1 (SYFO1) to be required for NF-dependent alterations in membrane organization and symbiotic root hair responses. We demonstrate that SYFO1 mediates a continuum between the plasma membrane and the cell wall that is required for the onset of rhizobial infections.

Exocyst subunit Exo70B2 is linked to immune signaling and autophagy.

During the immune response, activation of the secretory pathway is key to mounting an effective response, while gauging its output is important to maintain cellular homeostasis. The Exo70 subunit of the exocyst functions as a spatiotemporal regulator by mediating numerous interactions with proteins and lipids. However, a molecular understanding of the exocyst regulation remains challenging. We show that, in Arabidopsis thaliana, Exo70B2 behaves as a bona fide exocyst subunit. Conversely, treatment with the salicylic acid (SA) defence hormone analog benzothiadiazole (BTH), or the immunogenic peptide flg22, induced Exo70B2 transport into the vacuole. We reveal that Exo70B2 interacts with AUTOPHAGY-RELATED PROTEIN 8 (ATG8) via two ATG8-interacting motives (AIMs) and its transport into the vacuole is dependent on autophagy. In line with its role in immunity, we discovered that Exo70B2 interacted with and was phosphorylated by the kinase MPK3. Mimicking phosphorylation had a dual impact on Exo70B2: first, by inhibiting localization at sites of active secretion, and second, it increased the interaction with ATG8. Phosphonull variants displayed higher effector-triggered immunity (ETI) and were hypersensitive to BTH, which induce secretion and autophagy. Our results suggest a molecular mechanism by which phosphorylation diverts Exo70B2 from the secretory into the autophagy pathway for its degradation, to dampen secretory activity.

Auxin biosynthesis and cellular efflux act together to regulate leaf vein patterning.

Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

The Medicago truncatula DREPP Protein Triggers Microtubule Fragmentation in Membrane Nanodomains during Symbiotic Infections.

The initiation of intracellular host cell colonization by symbiotic rhizobia in Medicago truncatula requires repolarization of root hairs, including the rearrangement of cytoskeletal filaments. The molecular players governing microtubule (MT) reorganization during rhizobial infections remain to be discovered. Here, we identified M. truncatula DEVELOPMENTALLY REGULATED PLASMA MEMBRANE POLYPEPTIDE (DREPP), a member of the MT binding DREPP/PCaP protein family, and investigated its functions during rhizobial infections. We show that rhizobial colonization of drepp mutant roots as well as transgenic roots overexpressing DREPP is impaired. DREPP relocalizes into symbiosis-specific membrane nanodomains in a stimulus-dependent manner. This subcellular segregation coincides with DREPP-dependent MT fragmentation and a partial loss of the ability to reorganize the MT cytoskeleton in response to rhizobia, which might rely on an interaction between DREPP and the MT-organizing protein SPIRAL2. Taken together, our results reveal that establishment of symbiotic associations in M. truncatula requires DREPP in order to regulate MT reorganization during initial root hair responses to rhizobia.

Settling for Less: Do Statoliths Modulate Gravity Perception?

Plants orientate their growth either towards (in roots) or away from (in shoots) the Earth's gravitational field. While we are now starting to understand the molecular architecture of these gravity response pathways, the gravity receptor remains elusive. This perspective looks at the biology of statoliths and suggests it is conceivable that their immediate environment may be tuned to modulate the strength of the gravity response. It then suggests how mutant screens could use this hypothesis to identify the gravity receptor.

The MKK7-MPK6 MAP Kinase Module Is a Regulator of Meristem Quiescence or Active Growth in Arabidopsis.

Plant growth flexibly adapts to environmental conditions. Growth initiation itself may be conditional to a suitable environment, while the most common response of plants to adverse conditions is growth inhibition. Most of our understanding about environmental growth inhibition comes from studies on various plant hormones, while less is known about the signaling mechanisms involved. The mitogen-activated protein kinase (MAPK) cascades are central signal transduction pathways in all eukaryotes and their roles in plant stress responses is well-established, while increasing evidence points to their involvement in hormonal and developmental processes. Here we show that the MKK7-MPK6 module is a suppressor of meristem activity using genetic approaches. Shoot apical meristem activation during light-induced de-etiolation is accelerated in mpk6 and mkk7 seedlings, whereas constitutive or induced overexpression of MKK7 results in meristem defects or collapse, both in the shoot and the root apical meristems. These results underscore the role of stress-activated MAPK signaling in regulating growth responses at the whole plant level, which may be an important regulatory mechanism underlying the environmental plasticity of plant development.

Root Gravitropism Is Regulated by a Crosstalk between para-Aminobenzoic Acid, Ethylene, and Auxin.

Plants respond to gravitational force through directional growth along the gravity vector. Although auxin is the central component of the root graviresponse, it works in concert with other plant hormones. Here, we show that the folate precursor para-aminobenzoic acid (PABA) is a key modulator of the auxin-ethylene interplay during root gravitropism in Arabidopsis (Arabidopsis thaliana). In gravistimulated roots, PABA promotes an asymmetric auxin response, which causes the asymmetric growth responsible for root curvature. This activity requires the auxin response transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signaling, indicating that PABA activity requires both auxin and ethylene pathways. Similar to ethylene, exogenous PABA reverses the agravitropic root growth of the auxin transport mutant pin-formed2 (pin2) and the auxin biosynthetic double mutant with loss of function of weak ethylene insensitive (wei) genes, wei8wei2, but not the pin2wei8wei2 triple mutant. This finding suggests that PABA regulates the ethylene-dependent reciprocal compensation between auxin transport and biosynthesis. Furthermore, manipulation of endogenous free PABA levels by modulating the expression of the gene encoding its glucosylation enzyme, UDP-GLYCOSYL TRANSFERASE75B1, impacts the root graviresponse, suggesting that endogenous free PABA levels may play a crucial role in modulating the auxin-ethylene cross talk necessary for root gravitropism.

Characterization of auxin transporter PIN6 plasma membrane targeting reveals a function for PIN6 in plant bolting.

Auxin gradients are sustained by series of influx and efflux carriers whose subcellular localization is sensitive to both exogenous and endogenous factors. Recently the localization of the Arabidopsis thaliana auxin efflux carrier PIN-FORMED (PIN) 6 was reported to be tissue-specific and regulated through unknown mechanisms. Here, we used genetic, molecular and pharmacological approaches to characterize the molecular mechanism(s) controlling the subcellular localization of PIN6. PIN6 localizes to endomembrane domains in tissues with low PIN6 expression levels such as roots, but localizes at the plasma membrane (PM) in tissues with increased PIN6 expression such as the inflorescence stem and nectary glands. We provide evidence that this dual localization is controlled by PIN6 phosphorylation and demonstrate that PIN6 is phosphorylated by mitogen-activated protein kinases (MAPKs) MPK4 and MPK6. The analysis of transgenic plants expressing PIN6 at PM or in endomembrane domains reveals that PIN6 subcellular localization is critical for Arabidopsis inflorescence stem elongation post-flowering (bolting). In line with a role for PIN6 in plant bolting, inflorescence stems elongate faster in pin6 mutant plants than in wild-type plants. We propose that PIN6 subcellular localization is under the control of developmental signals acting on tissue-specific determinants controlling PIN6-expression levels and PIN6 phosphorylation.

Converging Light, Energy and Hormonal Signaling Control Meristem Activity, Leaf Initiation, and Growth.

The development of leaf primordia is subject to light control of meristematic activity. Light regulates the expression of thousands of genes with roles in cell proliferation, organ development, and differentiation of photosynthetic cells. Previous work has highlighted roles for hormone homeostasis and the energy-dependent Target of Rapamycin (TOR) kinase in meristematic activity, yet a picture of how these two regulatory mechanisms depend on light perception and interact with each other has yet to emerge. Their relevance beyond leaf initiation also is unclear. Here, we report the discovery that the dark-arrested meristematic region of Arabidopsis (Arabidopsis thaliana) experiences a local energy deprivation state and confirm previous findings that the PIN1 auxin transporter is diffusely localized in the dark. Light triggers a rapid removal of the starvation state and the establishment of PIN1 polar membrane localization consistent with auxin export, both preceding the induction of cell cycle- and cytoplasmic growth-associated genes. We demonstrate that shoot meristematic activity can occur in the dark through the manipulation of auxin and cytokinin activity as well as through the activation of energy signaling, both targets of photomorphogenesis action, but the organ developmental outcomes differ: while TOR-dependent energy signals alone stimulate cell proliferation, the development of a normal leaf lamina requires photomorphogenesis-like hormonal responses. We further show that energy signaling adjusts the extent of cell cycle activity and growth of young leaves non-cellautonomously to available photosynthates and leads to organs constituted of a greater number of cells developing under higher irradiance. This makes energy signaling perhaps the most important biomass growth determinant under natural, unstressed conditions.

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.

Maternal control of PIN1 is required for female gametophyte development in Arabidopsis.

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.

Root gravitropism and root hair development constitute coupled developmental responses regulated by auxin homeostasis in the Arabidopsis root apex.

Active polar transport establishes directional auxin flow and the generation of local auxin gradients implicated in plant responses and development. Auxin modulates gravitropism at the root tip and root hair morphogenesis at the differentiation zone. Genetic and biochemical analyses provide evidence for defective basipetal auxin transport in trh1 roots. The trh1, pin2, axr2 and aux1 mutants, and transgenic plants overexpressing PIN1, all showing impaired gravity response and root hair development, revealed ectopic PIN1 localization. The auxin antagonist hypaphorine blocked root hair elongation and caused moderate agravitropic root growth, also leading to PIN1 mislocalization. These results suggest that auxin imbalance leads to proximal and distal developmental defects in Arabidopsis root apex, associated with agravitropic root growth and root hair phenotype, respectively, providing evidence that these two auxin-regulated processes are coupled. Cell-specific subcellular localization of TRH1-YFP in stele and epidermis supports TRH1 engagement in auxin transport, and hence impaired function in trh1 causes dual defects of auxin imbalance. The interplay between intrinsic cues determining root epidermal cell fate through the TTG/GL2 pathway and environmental cues including abiotic stresses modulates root hair morphogenesis. As a consequence of auxin imbalance in Arabidopsis root apex, ectopic PIN1 mislocalization could be a risk aversion mechanism to trigger root developmental responses ensuring root growth plasticity.

NO VEIN facilitates auxin-mediated development in Arabidopsis.

Local, efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. The auxin-efflux pattern is regulated by dynamic expression and asymmetric subcellular localization of PIN auxin-efflux proteins during plant organogenesis. Thus, the question of how the expression and subcellular localization of PIN proteins are controlled goes to the heart of plant development. It has been shown that PIN expression and polarity are established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means such as cell-fate determination. We found that the Arabidopsis NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem-cell maintenance in the root meristem, and cotyledon outgrowth and separation. NOV function underlies cell-fate decisions associated with auxin gradients and maxima, thereby establishing cell-type-specific PIN expression and polarity. We propose that NOV mediates cell acquisition of the competence to undergo auxin-dependent coordinated cell specification and patterning, thereby educing context-dependent auxin-mediated developmental responses.

Lateral root stimulation in the early interaction between Arabidopsis thaliana and the ectomycorrhizal fungus Laccaria bicolor: is fungal auxin the trigger?

Lateral root (LR) stimulation during early signal exchange between plant roots and ectomycorrhizal (ECM) fungi has recently been shown to be achieved by modulation of auxin gradients. We suggested that this modulation could occur through altered polar auxin transport (PAT) and through activation of auxin signalling pathways in the root. However, it remains unclear, which fungal molecules alter auxin pathways inside the plant partner. It has been suggested in previous studies that auxin released by the fungus could trigger observed plant responses during early signal exchange and later on during root colonization. Here we focus on the early interaction and we provide evidence for an alternative mechanism. Indeed, LR stimulation by the fungus in A. thaliana followed a totally different timing than with exogenously applied auxin. Furthermore, experimental conditions that excluded the exchange of soluble molecules while allowing exchange of volatile(s) between the plant and the fungus were sufficient for LR induction, therefore questioning the role of secreted fungal auxin. These data suggest that volatiles released by the fungus and sensed by the plant may act upstream of altered auxin signalling in the plant.

NO VEIN mediates auxin-dependent specification and patterning in the Arabidopsis embryo, shoot, and root.

Local efflux-dependent auxin gradients and maxima mediate organ and tissue development in plants. Auxin efflux is regulated by dynamic expression and subcellular localization of the PIN auxin-efflux proteins, which appears to be established not only through a self-organizing auxin-mediated polarization mechanism, but also through other means, such as cell fate determination and auxin-independent mechanisms. Here, we show that the Arabidopsis thaliana NO VEIN (NOV) gene, encoding a novel, plant-specific nuclear factor, is required for leaf vascular development, cellular patterning and stem cell maintenance in the root meristem, as well as for cotyledon outgrowth and separation. nov mutations affect many aspects of auxin-dependent development without directly affecting auxin perception. NOV is required for provascular PIN1 expression and region-specific expression of PIN7 in leaf primordia, cell type-specific expression of PIN3, PIN4, and PIN7 in the root, and PIN2 polarity in the root cortex. NOV is specifically expressed in developing embryos, leaf primordia, and shoot and root apical meristems. Our data suggest that NOV function underlies cell fate decisions associated with auxin gradients and maxima, thus establishing cell type-specific PIN expression and polarity. We propose that NOV mediates the acquisition of competence to undergo auxin-dependent coordinated cell specification and patterning, thereby eliciting context-dependent auxin-mediated developmental responses.

Mechanical induction of lateral root initiation in Arabidopsis thaliana.

Lateral roots are initiated postembryonically in response to environmental cues, enabling plants to explore efficiently their underground environment. However, the mechanisms by which the environment determines the position of lateral root formation are unknown. In this study, we demonstrate that in Arabidopsis thaliana lateral root initiation can be induced mechanically by either gravitropic curvature or by the transient bending of a root by hand. The plant hormone auxin accumulates at the site of lateral root induction before a primordium starts to form. Here we describe a subcellular relocalization of PIN1, an auxin transport protein, in a single protoxylem cell in response to gravitropic curvature. This relocalization precedes auxin-dependent gene transcription at the site of a new primordium. Auxin-dependent nuclear signaling is necessary for lateral root formation; arf7/19 double knock-out mutants normally form no lateral roots but do so upon bending when the root tip is removed. Signaling through arf7/19 can therefore be bypassed by root bending. These data support a model in which a root-tip-derived signal acts on downstream signaling molecules that specify lateral root identity.

The indole alkaloids brucine, yohimbine, and hypaphorine are indole-3-acetic acid-specific competitors which do not alter auxin transport.

The indole alkaloids brucine and yohimbine, just like hypaphorine, counteract indole-3-acetic acid (IAA) activity in seedling roots, root hairs and shoots, but do not appear to alter auxin transport in roots or in cultured cells. In roots, the interactions between IAA and these three alkaloids appear competitive and specific since these molecules interact with IAA but with neither 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D), two synthetic auxins. The data reported further support the hypothesis that hypaphorine brucine and yohimbine compete with IAA on some auxin-binding proteins likely to be auxin receptors and that 2,4-D and NAA are not always perceived by the same receptor as IAA or the same component of that receptor. At certain steps of plant development and in certain cells, endogenous indole alkaloids could be involved in IAA activity regulation together with other well-described mechanisms such as conjugation or degradation. Hypaphorine with other active indole alkaloids remaining to be identified, might be regarded as a new class of IAA antagonists.