SPIKE1 Activates the GTPase ROP6 to Guide the Polarized Growth of Infection Threads in Lotus japonicus

In legumes, rhizobia attach to root hair tips and secrete nodulation factor to activate rhizobial infection and nodule organogenesis. Endosymbiotic rhizobia enter nodule primordia via a specialized transcellular compartment known as the infection thread (IT). The IT elongates by polar tip growth, following the path of the migrating nucleus along and within the root hair cell. Rho-family ROP GTPases are known to regulate the polarized growth of cells, but their role in regulating polarized IT growth is poorly understood. Here, we show that LjSPK1, a DOCK family guanine nucleotide exchange factor (GEF), interacts with three type I ROP GTPases. Genetic analyses showed that these three ROP GTPases are involved in root hair development, but only LjROP6 is required for IT formation after rhizobia inoculation. Misdirected ITs formed in the root hairs of Ljspk1 and Ljrop6 mutants. We show that LjSPK1 functions as a GEF that activates LjROP6. LjROP6 enhanced the plasma membrane localization LjSPK1 in Nicotiana benthamiana leaf cells and Lotus japonicus root hairs, and LjSPK1 and LjROP6 interact at the plasma membrane. Taken together, these results shed light on how the LjROP6-LjSPK1 module mediates the polarized growth of ITs in L. japonicus.     

Vesicular trafficking, cytoskeleton and signalling in root hairs and pollen tubes

Root hairs and pollen tubes show strictly polar cell expansion called tip growth. Recent studies of tip growth in root hairs and pollen tubes have revealed that small GTPases of the Rab, Arf and Rho/Rac families, along with their regulatory proteins, are essential for spatio-temporal regulation of vesicular trafficking, cytoskeleton organization and signalling. ROP/RAC GTPases are involved in a multiplicity of functions including the regulation of cytoskeleton organization, calcium signalling and endocytosis in pollen tubes and root hairs. One of the most exciting recent discoveries is the preferential localization of vesicles of the trans-Golgi network (TGN), defined by specific RAB GTPases, in the apical "clear zone" and the definition of TGN as a bona fide organelle involved in both polarized secretion and endocytosis. The TGN is thought to serve the function of an early endosome in plants because it is involved in early endocytosis and rapid vesicular recycling of the plasma membrane in root epidermal cells.     

Root hair sweet growth

Root hairs are single cells specialized in the absorption of water and nutrients from the soil. Growing root hairs require intensive cell-wall changes to accommodate cell expansion at the apical end by a process known as tip or polarized growth. We have recently shown that cell wall glycoproteins such as extensins (EXTs) are essential components of the cell wall during polarized growth. Proline hydroxylation, an early posttranslational modification of cell wall EXTs that is catalyzed by prolyl 4-hydroxylases (P4Hs), defines the subsequent O-glycosylation sites in EXTs. Biochemical inhibition or genetic disruption of specific P4Hs resulted in the blockage of polarized growth in root hairs. Our results demonstrate that correct hydroxylation and also further O-glycosylation on EXTs are essential for cell-wall self-assembly and, hence, root hair elongation. The changes that O-glycosylated cell-wall proteins like EXTs undergo during cell growth represent a starting point to unravel the entire biochemical pathway involved in plant development.Key words: cell wall, O-glycoproteins, extensins, proline hydroxylation, polarized growth, root hairs, P4H     

Nitrogen source interacts with ROP signalling in root hair tip‐growth

Root hairs elongate in a highly polarized manner known as tip growth. Overexpression of constitutively active Rho of Plant (ROP)/RAC GTPases mutants induces swelling of root hairs. Here, we demonstrate that Atrop11^CA‐induced swelling of root hairs depends on the composition of the growth medium. Depletion of ammonium allowed normal root hair elongation in Atrop11^CA plants, induced the development of longer root hairs in wild‐type plants and suppressed the effect of Atrop11^CA expression on actin organization and reactive oxygen species distribution, whereas membrane localization of the protein was not affected. Ammonium at concentrations higher than 1 mM and the presence of nitrate were required for induction of swelling. Oscillations in wall and cytoplasmic pH are known to accompany tip growth in root hairs, and buffering of the growth medium decreased Atrop11^CA‐induced swelling. Fluorescence ratio imaging experiments revealed that in wild‐type root hairs, the addition of NH₄NO₃ to the growth medium induced an increase in the amplitude of extracellular and intracellular pH oscillations and an overall decrease in cytoplasmic pH at the cell apex. Based on these results, we suggest a model in which ROP GTPases and nitrogen‐dependent pH oscillations function in parallel pathways, creating a positive feedback loop during root hair growth.     

The Arabidopsis Rop2 GTPase is a positive regulator of both root hair initiation and tip growth

Root hairs provide a model system for the study of cell polarity. We examined the possibility that one or more members of the distinct plant subfamily of RHO monomeric GTPases, termed Rop, may function as molecular switches regulating root hair growth. Specific Rops are known to control polar growth in pollen tubes. Overexpressing Rop2 (Rop2 OX) resulted in a strong root hair phenotype, whereas overexpressing Rop7 appeared to inhibit root hair tip growth. Overexpressing Rops from other phylogenetic subgroups of Rop did not give a root hair phenotype. We confirmed that Rop2 was expressed throughout hair development. Rop2 OX and constitutively active GTP-bound rop2 (CA-rop2) led to additional and misplaced hairs on the cell surface as well as longer hairs. Furthermore, CA-rop2 depolarized root hair tip growth, whereas Rop2 OX resulted in hairs with multiple tips. Dominant negative GDP-bound Rop2 reduced the number of hair-forming sites and led to shorter and wavy hairs. Green fluorescent protein-Rop2 localized to the future site of hair formation well before swelling formation and to the tip throughout hair development. We conclude that the Arabidopsis Rop2 GTPase acts as a positive regulatory switch in the earliest visible stage in hair development, swelling formation, and in tip growth.     

Cell polarity and PIN protein positioning in Arabidopsis require STEROL METHYLTRANSFERASE1 function

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1(orc) mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1(orc) root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5-beta-glucuronidase were aberrant in smt1(orc). Patterning defects in smt1(orc) resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1(orc), whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.     

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.     

Auxin and cell polarity: the emergence of AXR4

Polar localization of the Arabidopsis auxin influx facilitator protein AUX1 in certain cell types is essential for normal root gravitropism and lateral root formation. Sunethra Dharmasiri and colleagues recently reported that this polar localization requires the activity of the AXR4 gene. The sequence of the AXR4 gene suggests that novel mechanisms could be important for targeting certain proteins to specific cellular locations.     

Root hair defective4 encodes a phosphatidylinositol-4-phosphate phosphatase required for proper root hair development in Arabidopsis thaliana

Polarized expansion of root hair cells in Arabidopsis thaliana is improperly controlled in root hair-defective rhd4-1 mutant plants, resulting in root hairs that are shorter and randomly form bulges along their length. Using time-lapse fluorescence microscopy in rhd4-1 root hairs, we analyzed membrane dynamics after labeling with RabA4b, a marker for polarized membrane trafficking in root hairs. This revealed stochastic loss and recovery of the RabA4b compartment in the tips of growing root hairs, consistent with a role for the RHD4 protein in regulation of polarized membrane trafficking in these cells. The wild-type RHD4 gene was identified by map-based cloning and was found to encode a Sac1p-like phosphoinositide phosphatase. RHD4 displayed a preference for phosphatidylinositol-4-phosphate [PI(4)P] in vitro, and rhd4-1 roots accumulated higher levels of PI(4)P in vivo. In wild-type root hairs, PI(4)P accumulated primarily in a tip-localized plasma membrane domain, but in rhd4-1 mutants, significant levels of PI(4)P were detected associated with internal membranes. A fluorescent RHD4 fusion protein localized to membranes at the tips of growing root hairs. We propose that RHD4 is selectively recruited to RabA4b-labeled membranes that are involved in polarized expansion of root hair cells and that, in conjunction with the phosphoinositide kinase PI-4Kbeta1, RHD4 regulates the accumulation of PI(4)P on membrane compartments at the tips of growing root hairs.     

A generalized method for transfecting root epidermis uncovers endosomal dynamics in Arabidopsis root hairs

Progress in analysing the cellular functions of many structural proteins has accelerated through the use of confocal microscopy together with transient gene expression. Several methods for transient expression have been developed in the past few years, but their application has seen limited success beyond a few tractable species and tissues. We have developed a simple and efficient method to visualize fluorescent proteins in Arabidopsis root epidermis using co-cultivation of seedlings with Agrobacterium rhizogenes. The method is equally suitable for transient gene expression in other species, including Thellungiella, and can be combined with supporting molecular and biochemical analyses. The method promises significant advantages for study of membrane dynamics, cellular development and polar growth in root hairs without interference in the development of the plant. Since the method targets specifically the root epidermis, it also offers a powerful tool to approach issues of root-rhizosphere interactions, such as ion transport and nutrient acquisition. As a proof of principle, we carried out transfections with fluorescent markers for the plasma membrane (NpPMA2-GFP, Nicotiana plumbaginifolia L. Plasma Membrane H(+)-ATPase 2), the endoplasmic reticulum (YFP-HDEL), and the Golgi apparatus (sialyl transferase-GFP) to trace their distribution in growing Arabidopsis root hairs and epidermis. The results demonstrate that, in Arabidopsis root hairs, movement of the Golgi is faster than previously reported for tobacco leaf epidermal cells, consistent with the high secretory dynamics of the tip growing cell; they show a pattern to the endoplasmic reticulum within the cytoplasm that is more diffuse than found in tobacco leaf epidermis, and they confirm previous findings of a polarized distribution of the endoplasmic reticulum at the tip of growing root hairs.     

Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.     

The Arabidopsis Rab GTPase RabA4b localizes to the tips of growing root hair cells

Spatial and temporal control of cell wall deposition plays a unique and critical role during growth and development in plants. To characterize membrane trafficking pathways involved in these processes, we have examined the function of a plant Rab GTPase, RabA4b, during polarized expansion in developing root hair cells. Whereas a small fraction of RabA4b cofractionated with Golgi membrane marker proteins, the majority of this protein labeled a unique membrane compartment that did not cofractionate with the previously characterized trans-Golgi network syntaxin proteins SYP41 and SYP51. An enhanced yellow fluorescent protein (EYFP)-RabA4b fusion protein specifically localizes to the tips of growing root hair cells in Arabidopsis thaliana. Tip-localized EYFP-RabA4b disappears in mature root hair cells that have stopped expanding, and polar localization of the EYFP-RabA4b is disrupted by latrunculin B treatment. Loss of tip localization of EYFP-RabA4b was correlated with inhibition of expansion; upon washout of the inhibitor, root hair expansion recovered only after tip localization of the EYFP-RabA4b compartments was reestablished. Furthermore, in mutants with defective root hair morphology, EYFP-RabA4b was improperly localized or was absent from the tips of root hair cells. We propose that RabA4b regulates membrane trafficking through a compartment involved in the polarized secretion of cell wall components in plant cells.     

RPA, a class II ARFGAP protein, activates ARF1 and U5 and plays a role in root hair development in Arabidopsis

The polar growth of plant cells depends on the secretion of a large amount of membrane and cell wall materials at the growing tip to sustain rapid growth. Small GTP-binding proteins, such as Rho-related GTPases from plants and ADP-ribosylation factors (ARFs), have been shown to play important roles in polar growth via regulating intracellular membrane trafficking. To investigate the role of membrane trafficking in plant development, a Dissociation insertion line that disrupted a putative ARF GTPase-activating protein (ARFGAP) gene, AT2G35210, was identified in Arabidopsis (Arabidopsis thaliana). Phenotypic analysis showed that the mutant seedlings developed isotropically expanded, short, and branched root hairs. Pollen germination in vitro indicated that the pollen tube growth rate was slightly affected in the mutant. AT2G35210 is specifically expressed in roots, pollen grains, and pollen tubes; therefore, it is designated as ROOT AND POLLEN ARFGAP (RPA). RPA encodes a protein with an N-terminal ARFGAP domain. Subcellular localization experiments showed that RPA is localized at the Golgi complexes via its 79 C-terminal amino acids. We further showed that RPA possesses ARF GTPase-activating activity and specifically activates Arabidopsis ARF1 and ARF1-like protein U5 in vitro. Furthermore, RPA complemented Saccharomyces cerevisiae glo3Delta gcs1Delta double mutant, which suggested that RPA functions as an ARFGAP during vesicle transport between the Golgi and the endoplasmic reticulum. Together, we demonstrated that RPA plays a role in root hair and pollen tube growth, most likely through the regulation of Arabidopsis ARF1 and ARF1-like protein U5 activity.     

SNARE VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole and is essential for cell wall organization and root hair growth in arabidopsis

Background and Aims

Root hairs are responsible for water and nutrient uptake from the soil and their growth is responsive to biotic and abiotic changes in their environment. Root hair expansion is a polarized process requiring secretory and endosomal pathways that deliver and recycle plasma membrane and cell wall material to the growing root hair tip. In this paper, the role of VTI13 (AT3G29100), a member of the VTI vesicular soluble NSF attachment receptor (SNARE) gene family in Arabidopsis thaliana, in root hair growth is described.

Methods

Genetic analysis and complementation of the vti13 root hair phenotypes of Arabidopsis thaliana were first used to assess the role of VTI13 in root hair growth. Transgenic lines expressing a green fluorescent protein (GFP)–VTI13 construct were used to characterize the intracellular localization of VTI13 in root hairs using confocal microscopy and immunotransmission electron microscopy.

Key Results

VTI13 was characterized and genetic analysis used to show that its function is required for root hair growth. Expression of a GFP–VTI13 fusion in the vti13 mutant background was shown to complement the vti13 root hair phenotype. GFP–VTI13 localized to both the vacuole membrane and a mobile endosomal compartment. The function of VTI13 was also required for the localization of SYP41 to the trans-Golgi network. Immunohistochemical analysis indicated that cell wall organization is altered in vti13 root hairs and root epidermal cells.

Conclusions

These results show that VTI13 plays a unique role in endosomal trafficking pathways associated with the vacuole within root hairs and is essential for the maintenance of cell wall organization and root hair growth in arabidopsis.     

Building a hair: tip growth in Arabidopsis thaliana root hairs

The Arabidopsis thaliana root hair is used as a model for studying tip growth in plants. We review recent advances, made using physiological and genetic approaches, which give rise to different, yet compatible, current views of the establishment and maintenance of tip growth in epidermal cells. For example, an active calcium influx channel localized at the tip of Arabidopsis root hairs has been identified by patch-clamp measurements. Actin has been visualized in vivo in Arabidopsis root hairs by using a green-fluorescent-protein-talin reporter and shown to form a dense mesh in the apex of the growing tip. The kojak gene, which encodes a protein similar to the catalytic subunit of cellulose synthase, is needed in the first stages of hair growth. A role for LRX1, a leucine-rich repeat extensin, in determining the morphology of the cell wall of root hairs has been established using reverse genetics. The new information can be integrated into a general and more advanced view of how these specialized plant cells grow.     

AtCSLD3, a cellulose synthase-like gene important for root hair growth in arabidopsis

A member of the cellulose synthase-like (subfamily D) gene family of Arabidopsis, AtCSLD3, has been identified by T-DNA tagging. The analysis of the corresponding mutant, csld3-1, showed that the AtCSLD3 gene plays a role in root hair growth in plants. Root hairs grow in phases: First a bulge is formed and then the root hair elongates by polarized growth, the so-called "tip growth." In the mutant, root hairs were initiated at the correct position and grew into a bulge, but their elongation was severely reduced. The tips of the csld3-1 root hairs easily leaked cytoplasm, indicating that the tensile strength of the cell wall had changed at the site of the tip. Based on the mutant phenotype and the functional conservation between CSLD3 and the genuine cellulose synthase proteins, we hypothesized that the CSLD3 protein is essential for the synthesis of polymers for the fast-growing primary cell wall at the root hair tip. The distinct mutant phenotype and the ubiquitous expression pattern indicate that the CSLD3 gene product is only limiting at the zone of the root hair tip, suggesting particular physical properties of the cell wall at this specific site of the root hair cell.     

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.     

The rhd6 Mutation of Arabidopsis thaliana Alters Root-Hair Initiation through an Auxin- and Ethylene-Associated Process

Root-hair initiation in Arabidopsis thaliana provides a model for studying cell polarity and its role in plant morphogenesis. Root hairs normally emerge at the apical end of root epidermal cells, implying that these cells are polarized. We have identified a mutant, rhd6, that displays three defects: (a) a reduction in the number of root hairs, (b) an overall basal shift in the site of root-hair emergence, and (c) a relatively high frequency of epidermal cells with multiple root hairs. These defects implicate the RHD6 gene in root-hair initiation and indicate that RHD6 is normally associated with the establishment of, or response to, root epidermal cell polarity. Similar alterations in the site of root-hair emergence, although less extreme, were also discovered in roots of the auxin-, ethylene-, abscisic acid-resistant mutant axr2 and the ethylene-resistant mutant etr1. All three rhd6 mutant phenotypes were rescued when either auxin (indoleacetic acid) or an ethylene precursor (1-aminocyclopropane-1-carboxylic acid) was included in the growth medium. The rhd6 root phenotypes could be phenocopied by treating wild-type seedlings with an inhibitor of the ethylene pathway (aminoethoxyvinylglycine). These results indicate that RHD6 is normally involved in directing the selection or assembly of the root-hair initiation site through a process involving auxin and ethylene.