Nuclear dynamics during the simultaneous and sustained tip growth of multiple root hairs arising from a single root epidermal cell

Nuclear dynamics in root hairs, which depends upon the actin cytoskeleton, appears to be an important factor in root-hair tip growth. Previous evidence suggests that there is an absolute requirement for the nucleus to be a fixed distance from the growing root-hair tip for tip growth to proceed. To test this hypothesis, nuclear dynamics were examined in root-hair cells bearing multiple root hairs. The majority of root-hair cells of transgenic plants overexpressing the ROP2 GTPase (ROP2 OX) bear multiple root hairs. Simultaneous and sustained fast tip growth occurred in multiple root hairs of ROP2 OX, with the continual presence of tip-localized cytoplasm in these growing hairs. Nuclear dynamics were imaged in ROP2 OX by co-expressing a transgene encoding a nuclear localization signal (NLS)-green fluorescent protein (GFP) fusion protein. The nucleus was in continual proximity to one of the growing root-hair tips, whilst the other tip elongated at a similar rate but in the absence of the nucleus from the shank of that root hair. To test whether this phenomenon was an artefact of ROP2 overexpression, nuclear dynamics were examined in wild-type and NLS-GFP transgenic plants. Multiple root hairs on the same cell underwent simultaneous and sustained fast tip growth, with the nucleus lying deep within the shank of only one of these hairs. The nucleus was also moved into the root-hair tip during the severe root-hair tip branching which is characteristic of ROP2 OX transgenic plants. These results suggest that fast tip growth can proceed in some multiple root hairs at extreme distances from the nucleus.     

Endoplasmic microtubules configure the subapical cytoplasm and are required for fast growth of Medicago truncatula root hairs

To investigate the configuration and function of microtubules (MTs) in tip-growing Medicago truncatula root hairs, we used immunocytochemistry or in vivo decoration by a GFP linked to a MT-binding domain. The two approaches gave similar results and allowed the study of MTs during hair development. Cortical MTs (CMTs) are present in all developmental stages. During the transition from bulge to a tip-growing root hair, endoplasmic MTs (EMTs) appear at the tip of the young hair and remain there until growth arrest. EMTs are a specific feature of tip-growing hairs, forming a three-dimensional array throughout the subapical cytoplasmic dense region. During growth arrest, EMTs, together with the subapical cytoplasmic dense region, progressively disappear, whereas CMTs extend further toward the tip. In full-grown root hairs, CMTs, the only remaining population of MTs, converge at the tip and their density decreases over time. Upon treatment of growing hairs with 1 microM oryzalin, EMTs disappear, but CMTs remain present. The subapical cytoplasmic dense region becomes very short, the distance nucleus tip increases, growth slows down, and the nucleus still follows the advancing tip, though at a much larger distance. Taxol has no effect on the cytoarchitecture of growing hairs; the subapical cytoplasmic dense region remains intact, the nucleus keeps its distance from the tip, but growth rate drops to the same extent as in hairs treated with 1 microM oryzalin. The role of EMTs in growing root hairs is discussed.     

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.     

Involvement of the mitogen-activated protein kinase SIMK in regulation of root hair tip growth

Mitogen-activated protein kinases (MAPKs) are involved in stress signaling to the actin cytoskeleton in yeast and animals. We have analyzed the function of the stress-activated alfalfa MAP kinase SIMK in root hairs. In epidermal cells, SIMK is predominantly nuclear. During root hair formation, SIMK was activated and redistributed from the nucleus into growing tips of root hairs possessing dense F-actin meshworks. Actin depolymerization by latrunculin B resulted in SIMK relocation to the nucleus. Conversely, upon actin stabilization with jasplakinolide, SIMK co-localized with thick actin cables in the cytoplasm. Importantly, latrunculin B and jasplakinolide were both found to activate SIMK in a root-derived cell culture. Loss of tip-focused SIMK and actin was induced by the MAPK kinase inhibitor UO 126 and resulted in aberrant root hairs. UO 126 inhibited targeted vesicle trafficking and polarized growth of root hairs. In contrast, overexpression of gain-of-function SIMK induced rapid tip growth of root hairs and could bypass growth inhibition by UO 126. These data indicate that SIMK plays a crucial role in root hair tip growth.     

Distribution of G-actin is related to root hair growth of wheat

BACKGROUND AND AIMS: Actin distribution in root hair tips is a controversial topic. Although the relationship between Ca2+ gradient and actin dynamics in plant tip-growth has been a focus of study, there is still little direct evidence on the exact relationship in root hair tip-growth. METHODS: G-actin was labelled by fluorescein isothiocyanate-DNase I. F-actin was labelled by tetramethylrhodamine isothiocyanate-phalloidin. Actin in root hairs of Triticum aestivum (wheat) was investigated using confocal laser-scanning microscopy. KEY RESULTS: Thick F-actin bundles did not extend into a region of approx. 5-10 microm from the tip of the growing root hairs, although they gave off branches of fine actin filaments in the hair tips. A tip-focused G-actin gradient was shown at the extreme apex of growing root hairs. In full-grown wheat root hairs, the tip-focused G-actin gradient disappeared while the thick F-actin bundles extended into the tips. BAPTA-AM, a Ca2+ disruption agent, also caused the tip-focused G-actin gradient to disappear and the diffuse F-actin bundles to appear in the tips of wheat root hairs. CONCLUSIONS: These results suggest that the tip-focused gradient of intracellular G-actin concentration at the extreme apex may be essential for root hair growth, and that preserving the tip-focused gradient needs a high Ca2+ concentration in the root hair tips.     

ACTIN2 is essential for bulge site selection and tip growth during root hair development of Arabidopsis

Root hairs develop as long extensions from root epidermal cells. After the formation of an initial bulge at the distal end of the epidermal cell, the root hair structure elongates by tip growth. Because root hairs are not surrounded by other cells, root hair formation provides an excellent system for studying the highly complex process of plant cell growth. Pharmacological experiments with actin filament-interfering drugs have provided evidence that the actin cytoskeleton is an important factor in the establishment of cell polarity and in the maintenance of the tip growth machinery at the apex of the growing root hair. However, there has been no genetic evidence to directly support this assumption. We have isolated an Arabidopsis mutant, deformed root hairs 1 (der1), that is impaired in root hair development. The DER1 locus was cloned by map-based cloning and encodes ACTIN2 (ACT2), a major actin of the vegetative tissue. The three der1 alleles develop the mutant phenotype to different degrees and are all missense mutations, thus providing the means to study the effect of partially functional ACT2. The detailed characterization of the der1 phenotypes revealed that ACT2 is not only involved in root hair tip growth, but is also required for correct selection of the bulge site on the epidermal cell. Thus, the der1 mutants are useful tools to better understand the function of the actin cytoskeleton in the process of root hair formation.     

Unstable F-actin specifies the area and microtubule direction of cell expansion in Arabidopsis root hairs

Plant cells expand by exocytosis of wall material contained in Golgi-derived vesicles. We examined the role of local instability of the actin cytoskeleton in specifying the exocytosis site in Arabidopsis root hairs. During root hair growth, a specific actin cytoskeleton configuration is present in the cell's subapex, which consists of fine bundles of actin filaments that become more and more fine toward the apex, where they may be absent. Pulse application of low concentrations of the actin-depolymerizing drugs cytochalasin D and latrunculin A broadened growing root hair tips (i.e., they increased the area of cell expansion). Interestingly, recovery from cytochalasin D led to new growth in the original growth direction, whereas in the presence of oryzalin, a microtubule-depolymerizing drug, this direction was altered. Oryzalin alone, at the same concentration, had no influence on root hair elongation. These results represent an important step toward understanding the spatial and directional regulation of root hair growth.     

A class I ADP-ribosylation factor GTPase-activating protein is critical for maintaining directional root hair growth in Arabidopsis

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs.     

The role of actin in root hair morphogenesis: studies with lipochito-oligosaccharide as a growth stimulator and cytochalasin as an actin perturbing drug

Root hairs develop from bulges on root epidermal cells and elongate by tip growth, in which Golgi vesicles are targeted, released and inserted into the plasma membrane on one side of the cell. We studied the role of actin in vesicle delivery and retention by comparing the actin filament configuration during bulge formation, root hair initiation, sustained tip growth, growth termination, and in full-grown hairs. Lipochito-oligosaccharides (LCOs) were used to interfere with growth ( De Ruijter et al . 1998 , Plant J. 13, 341–350), and cytochalasin D (CD) was used to interfere with actin function. Actin filament bundles lie net-axially in cytoplasmic strands in the root hair tube. In the subapex of growing hairs, these bundles flare out into fine bundles. The apex is devoid of actin filament bundles. This subapical actin filament configuration is not present in full-grown hairs; instead, actin filament bundles loop through the tip. After LCO application, the tips of hairs that are terminating growth swell, and a new outgrowth appears from a site in the swelling. At the start of this outgrowth, net-axial fine bundles of actin filaments reappear, and the tip region of the outgrowth is devoid of actin filament bundles. CD at 1.0 μ m , which does not affect cytoplasmic streaming, does not inhibit bulge formation and LCO-induced swelling, but inhibits initiation of polar growth from bulges, elongation of root hairs and LCO-induced outgrowth from swellings. We conclude that elongating net-axial fine bundles of actin filaments, which we call FB-actin, function in polar growth by targeting and releasing Golgi vesicles to the vesicle-rich region, while actin filament bundles looping through the tip impede vesicle retention.     

Synchronously developing collet hairs in Arabidopsis thaliana provide an easily accessible system for studying nuclear movement and endoreduplication

Early Arabidopsis thaliana seedling growth includes the highly synchronous development of hairs from every epidermal cell of the collet (the root-hypocotyl transition zone). The dynamics of collet hair growth, and accompanying nuclear movement and endoreduplication, were followed using a combination of different fluorescent probes for time-lapse imaging and flow cytometry. Using laser-scanning confocal microscopy on the double-transgenic Arabidopsis hybrid line NLS-GFP-GUS × YPM, there appeared to be a correlation between nuclear position and the cell tip during growth of the collet hair cells, as occurs in asynchronously developing root hairs. However, disruption of nuclear movement in the growing collet hairs using low concentrations of cytoskeletal inhibitors demonstrated that nuclear positioning close to the tip of the cell is not essential for tip-directed growth of the hair. Nuclear DNA content increases from 4C to 16C during development of the collet hairs. Following cessation of growth, nuclei moved to the base of the hairs and then their movement became asynchronous and limited. Co-visualization of RFP-highlighted prevacuolar vesicles and GFP-labelled nuclei showed that, whereas small vesicles allowed unimpeded nuclear movement within the hair, transient stops and directional reversals coincided with the presence of larger vesicles in close proximity to the nucleus. Arabidopsis collet hairs provide a robust, easily accessible, naturally synchronized population of single tip-growing cells that can be used as a model cell type for studying nuclear movement and endoreduplication.     

Fungal hypaphorine reduces growth and induces cytosolic calcium increase in root hairs of Eucalyptus globulus

Summary. Root hairs are tubular cells resulting from a tip-localized growth in which calcium ions play a key role. Hypaphorine, an indole alkaloid secreted by the fungus Pisolithus microcarpus during the formation of ectomycorrhizae with the host plant Eucalyptus globulus, inhibits root hair tip growth. Hypaphorine-induced inhibition is linked to a transient depolarization of the plasma membrane and a reorganization of the actin and microtubule cytoskeletons. Here we investigated the activity of hypaphorine on calcium distribution in E. globulus root hairs with the ratiometric fluorochrome calcium indicator Indo-1. In 85% of actively growing root hairs, a significant but modest calcium gradient between the apex and the base was observed due to an elevated cytoplasmic calcium concentration at the apical tip. Following exposure to 1 mM hypaphorine, the apical and basal cytoplasmic Ca²⁺ concentration increased in 70 and 77% of the hairs, respectively, 10 min after treatment. This led to a reduced calcium gradient in 81% of the cells. The hypothetical links between calcium concentration elevation, regulation of actin cytoskeleton dynamics, and root hair growth inhibition in response to hypaphorine treatment are discussed. Correspondence and reprints: UMR 1136 Interactions Arbres–Microorganismes, Faculté des Sciences, Université Nancy I, BP 239, 54506 Vandoeuvre Cedex, France.     

The Arabidopsis Phosphatidylinositol Phosphate 5-Kinase PIP5K3 is a key regulator of root hair tip growth

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] functions as a site-specific signal on membranes to promote cytoskeletal reorganization and membrane trafficking. Localization of PtdIns(4,5)P2 to apices of growing root hairs and pollen tubes suggests that it plays an important role in tip growth. However, its regulation and mode of action remain unclear. We found that Arabidopsis thaliana PIP5K3 (for Phosphatidylinositol Phosphate 5-Kinase 3) encodes a phosphatidylinositol 4-phosphate 5-kinase, a key enzyme producing PtdIns(4,5)P2, that is preferentially expressed in growing root hairs. T-DNA insertion mutations that substantially reduced the expression of PIP5K3 caused significantly shorter root hairs than in the wild type. By contrast, overexpression caused longer root hairs and multiple protruding sites on a single trichoblast. A yellow fluorescent protein (YFP) fusion of PIP5K3, driven by the PIP5K3 promoter, complemented the short-root-hair phenotype. PIP5K3-YFP localized to the plasma membrane and cytoplasmic space of elongating root hair apices, to growing root hair bulges, and, notably, to sites about to form root hair bulges. The signal was greatest in rapidly growing root hairs and quickly disappeared when elongation ceased. These results provide evidence that PIP5K3 is involved in localizing PtdIns(4,5)P2 to the elongating root hair apex and is a key regulator of the machinery that initiates and promotes root hair tip growth.     

Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.     

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.     

Actin-based motility of endosomes is linked to the polar tip growth of root hairs

Plant tip growth has been recognized as an actin-based cellular process requiring targeted exocytosis and compensatory endocytosis to occur at the growth cone. However, the identity of subcellular compartments involved in polarized membrane trafficking pathways remains enigmatic in plants. Here we characterize endosomal compartments in tip-growing root hair cells. We demonstrate their presence at the growing tip and differential distribution upon cessation of tip growth. We also show that both the presence of endosomes as well as their rapid movements within the tip region depends on an intact actin cytoskeleton and involves actin polymerization. In conclusion, actin-propelled endosomal motility is tightly linked to the polar tip growth of root hairs.     

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.     

Actin organization during eucalyptus root hair development and its response to fungal hypaphorine

The fungus Pisolithus microcarpus establishes an ectomycorrhiza with Eucalyptus globulus. This symbiosis involves a fungal synthesis and secretion of hypaphorine, an indolic compound. Previous studies have shown that hypaphorine induces an alteration in the actin cytoskeleton of elongating root hairs and inhibits hair elongation. Using an alternative approved method, we analyzed the effects of hypaphorine on the E. globulus root hair cyto-architecture and actin configuration in more detail and provide new results. One mM hypaphorine stops root hair elongation within 20 min, and changes the hair cyto-architecture. Semi-quantitative analysis of the actin cytoskeleton before and after treatment with hypaphorine shows that hypaphorine induces a shift from fine F-actin to F-actin bundles in the sub-apex of the hair, which occurs first in the mid-plane of the cell. This creates a sub-apical cell centre free of filamentous actin, an actin configuration that differs from that during developmental growth arrest. The mechanism of action of hypaphorine is discussed.     

The maize actin-depolymerizing factor, ZmADF3, redistributes to the growing tip of elongating root hairs and can be induced to translocate into the nucleus with actin

The maize actin depolymerizing factor, ZmADF3, binds G-and F-actin, and increases in vitro actin dynamics. Polyclonal antibodies have been raised against ZmADF3 and these detect a single band of approximately 17 kDa in all maize tissues examined, with the exception of pollen. In the development of root hairs, the distribution of ZmADF3 is related to actin reorganization. In the early stages of hair development, ZmADF3 is distributed throughout the cytoplasm. As the hair emerges and the microfilament bundles redirect to the outgrowth there is a simultaneous redistribution of ZmADF3 which now concentrates at the tip of the emerging hair and remains in this position as elongation proceeds. These observations show that ZmADF3 localizes to a region where actin is being remodelled during tip growth. After cytochalasin D treatment which disrupts actin filaments, short rods of ZmADF3 and actin appear in the nucleus suggesting that ZmADF3 may function by guiding actin to sites of actin polymerization.     

Production of branched root hairs under progressive drought stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, data on the production of branched root hairs in the seedlings of A. thaliana under progressive water deficit were presented. The overall production of branched hairs was quite high under stress conditions and amounted to 8.27%. On the contrary, this form of root hairs was almost absent in the control group (0.27%). The highest number of branched hairs was produced at the beginning of the stress action. Branched root hairs are quite uniform structures in the sense of their morphology. To solve the question of how the branched hairs grow, the structure of actin cytoskeleton was explored. This structure was different in the root hair and in its branch, which is an indication that the hair stops its growth at the moment when the branching starts. We have also characterized the production of branched root hairs in hormonal mutants of Arabidopsis and found the involvement of auxin in this process.