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.     

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.     

Lipochito‐oligosaccharides re‐initiate root hair tip growth in Vicia sativa with high calcium and spectrin‐like antigen at the tip

Lipochito-oligosaccharides, Nod factors secreted by Rhizobium bacteria, are signal molecules that induce deformation of root hairs of their host plant. A bioassay was used for deformation, and the cytological changes induced by specific lipochito-oligosaccharides in root hairs of Vicia sativa L. (vetch), grown between glass slides, were examined. In the assay, root hairs of a particular developmental stage, those that were terminating growth, were susceptible to deformation. These hairs obtained characteristics of tip-growing cells again: (i) a polar cytoplasmic organization and reverse fountain streaming, (ii) an accumulation of a spectrin-like antigen at the tip, and (iii) a tip-focused calcium gradient. Calcium gradients were visualized in Indo-1 loaded root hairs with UV confocal microscopy and ratio-imaging. The results show that hairs respond to the bacterial signal by recovering cytoplasmic polarity and exocytosis.     

Nod factors modulate the concentration of cytosolic free calcium differently in growing and non-growing root hairs of Medicago sativa L.

Using Ca²⁺-selective microelectrodes, the concentration of free calcium ([Ca²⁺]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca²⁺] gradient, i.e. 604–967 nM in the tip compared with 95–235 nM in the basal region. When tested within the first 5 to 10 μm of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca²⁺], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca²⁺] gradient. The Ca²⁺ response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S), but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs without a tip-to-base cytosolic [Ca²⁺] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca²⁺] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development of the root hairs, and that changes in cytosolic [Ca²⁺] may play different roles in Nod-factor signaling: changes of cytosolic [Ca²⁺] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca²⁺] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger downstream events. Received: 5 January 1999 / Accepted: 14 April 1999     

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D₂O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.Abbreviations DMSO dimethylsulfoxide - CB cytochalasin B Dedicated to Prof. Dr. A. Pirson on the occasion of his 70. birthday     

Immunolocalization of myosin in rhizoids ofChara globularis Thuill

Summary Myosin-related proteins have been localized immunocytochemically in gravity-sensing rhizoids of the green algaChara globularis using a monoclonal antibody against the heavy chain of myosin from mouse 3T3 cells and a polyclonal antibody to bovine skeletal and smooth muscle myosin. In the basal zone of the rhizoids which contain a large vacuole, streaming endoplasm and stationary cortical cytoplasm, the monoclonal antibody stained myosin-related proteins as diffusely fluorescing endoplasmic strands. This pattern is similar to the arrangement of subcortical actin filament bundles. In the apical zone which contains an aggregation of ER membranes and secretory vesicles for tip growth, diffuse immunofluorescence was detected; the intensity of the signal increasing towards the apical cell wall. The most prominent myosin-staining was associated with the surface of statoliths in the apical zone. The polyclonal antibody produced a punctate staining pattern in the basal zone, caused by myosin-related proteins associated with the surface of drganelles in the streaming endoplasm and the periphery of the nucleus. In the apical zone, this antibody revealed myosin-immunofluorescence on the surface of statoliths in methacrylate-embedded rhizoids. Neither antibody revealed myosin-immunofluorescence on the surface of organelles and vesicles in the relatively stationary cytoplasm of the subapical zone. These results indicate (i) that different classes of myosin are involved in the various transport processes inChara rhizoids; (ii) that cytoplasmic streaming in rhizoids is driven by actomyosin, corresponding to the findings onChara internodal cells; (iii) that actindependent control of statolith position and active movement is mediated by myosin-related proteins associated with the statolith surfaces; and (iv) that myosin-related proteins are involved in the process of tip growth.     

Root hairs: Specialized tubular cells extending root surfaces

Root hairs are tubular extensions of epidermal cells that have their origin either in any protoderm cell or in specialized protoderm cells called trichoblasts. These latter cells are the result of an asymmetric cytokinesis determined by the positioning of a pre-prophase band of microtubules. The smaller sibling cell is the trichoblast and specializes physiologically and structurally prior to root hair outgrowth. Several genes are involved in the initiation and outgrowth of root hairs. Elongation of root hairs is by tip growth, and, correlated with this, cytoplasmic organelles and cytoskeletal elements show a polarized distribution; the apical dome consists of numerous vesicles, many associated with cell wall synthesis. The relationship between cellulose microfibril deposition and the pattern of cortical microtubules has received considerable attention, as has the role of the cytoskeleton and calcium in controlling cytoplasmic streaming. Root hairs extend the absorbing surface of the root and therefore have been studied in terms both of physiological characteristics of the plasma membrane and uptake of water and of various ions in the soil solution. Many plant species develop soil sheaths (rhizosheaths) which protect the root surface from desiccation and harbour various microorganisms; root hairs are intimately involved in these sheaths. Various growth regulators have been studied in terms of their effect on the structure and function of root hairs. Root hairs play a significant role in the interaction between plants and nitrogen-fixing microorganisms (e.g.,Rhizobium, Frankia) and symbiotic mycorrhizal fungi.     

Growth and ultrastructure ofArabidopsis root hairs: therhd3 mutation alters vacuole enlargement and tip growth

The root hairs of plants are tubular projections of root epidermal cells and are suitable for investigating the control of cellular morphogenesis. In wild-typeArabidopsis thaliana (L.) Heynh, growing root hairs were found to exhibit cellular expansion limited to the apical end of the cell, a polarized distribution of organelles in the cytoplasm, and vesicles of several types located near the growing tip. Therhd3 mutant produces short and wavy root hairs with an average volume less than one-third of the wild-type hairs, indicating abnormal cell expansion. The mutant hairs display a striking reduction in vacuole size and a corresponding increase in the relative proportion of cytoplasm throughout hair development. Bead-labeling experiments and ultrastructural analyses indicate that the wavy-hair phenotype of the mutant is caused by asymmetric tip growth, possibly due to abnormally distributed vesicles in cortical areas flanking the hair tips. It is suggested that a major effect of therhd3 mutation is to inhibit vacuole enlargement which normally accompanies root hair cell expansion.     

Endocytosis and vesicle trafficking during tip growth of root hairs

Summary. The directional elongation of root hairs, “tip growth”, depends on the coordinated and highly regulated trafficking of vesicles which fill the tip cytoplasm and are active in secretion of cell wall material. So far, little is known about the dynamics of endocytosis in living root hairs. We analyzed the motile behaviour of vesicles in the apical region of living root hairs of Arabidopsis thaliana and of Triticum aestivum by live cell microscopy. For direct observation of endocytosis and of the fate of endocytic vesicles, we used the fluorescent endocytosis marker dyes FM 1-43 and FM 4-64. Rapid endocytosis was detected mainly in the tip, where it caused a bright fluorescence of the apical cytoplasm. The internalized membranes proceeded through highly dynamic putative early endosomes in the clear zone to larger endosomal compartments in the subapical region that are excluded from the clear zone. The internalized cargo ended up in the dynamic vacuole by fusion of large endosomal compartments with the tonoplast. Before export to these lytic compartments, putative early endosomes remained in the apical zone, where they most probably recycled to the plasma membrane and back into the cytoplasm for more than 30 min. Endoplasmic reticulum was not involved in trafficking pathways of endosomes. Actin cytoskeleton was needed for the endocytosis itself, as well as for further membrane trafficking. The actin-depolymerizing drug latrunculin B modified the dynamic properties of vesicles and endosomes; they became immobilized and aggregated in the tip. Treatment with brefeldin A inhibited membrane trafficking and caused the disappearance of FM-containing vesicles and putative early endosomes from the clear zone; labelled structures accumulated in motile brefeldin A-induced compartments. These large endocytic compartments redispersed upon removal of the drug. Our results hence prove that endocytosis occurs in growing root hairs. We show the localization of endocytosis in the tip and indicate specific endomembrane compartments and their recycling. Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dubravska cesta 14, 845 23 Bratislava, Slovak Republic.     

Redistribution of actin, profilin and phosphatidylinositol-4,5-bisphosphate in growing and maturing root hairs

The continuously changing polar cytoplasmic organization during initiation and tip growth of root hairs is reflected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein, profilin, which is known to be a widely expressed, potent regulator of actin dynamics, was specifically localized at the tip of root hairs and co-distributed with a diffusely fluorescing apical cap of actin, but not with subapical actin microfilament (MF) bundles. Profilin and actin caps were present exclusively in the bulge of outgrowing root hairs and at the apex of elongating root hairs; both disappeared when tip growth terminated, indicating a tip-growth mechanism that involves profilin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP₂), a ligand of profilin, was localized almost exclusively in the bulge and, subsequently, formed a weak tip-to-base gradient in the elongating root hairs. When tip growth was eliminated by the MF-disrupting inhibitor cytochalasin D, the apical profilin and the actin fluorescence were lost. Mastoparan, which is known to affect the PIP₂ cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct actin MFs replacing the diffuse apical actin cap and, concomittantly, tip growth stopped. This suggests that mastoparan interferes with the PIP₂-regulated profilin-actin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs. Received: 11 March 1999 / Accepted: 27 May 1999     

Differential cytoplasm-plasma membrane-cell wall adhesion patterns and their relationships to hyphal tip growth and organelle motility

Summary Plasmolysis of hyphae of the oomycetesSaprolegnia ferax andAchlya ambisexualis and the ascomyceteNeurospora crassa produced abundant cytoplasmic strands between the retracted cytoplasm and punctate adhesions of the plasma membrane to the cell wall. These strands formed throughout the length of mature hyphae and are the first demonstration of Hechtian strands in hyphae. In contrast to similar strands in various plant cells, the strands inSaprolegnia lacked endoplasmic reticulum but contained F-actin, suggesting similarity between their adhesion sites and focal contacts in animal cells. However, strand adhesion to the wall was insensitive to RGD-containing peptides, suggesting that the trans-membrane adhesion molecules differ from animal integrins. The pattern of plasma membrane-cell wall adhesion varied in different zones along hyphae, with broad, irregular connections in the extreme apex, uniform and continuous connection in a transition zone, and small, punctate adhesions in the mature subapical zone, suggesting differential functions in these different regions. The apical adhesions are important in tip growth, as diverse inhibitors induced concomitant changes in hyphal growth and the adhesions in the apical and transition zones. Plasmolysis also induced cytoplasmic migrations throughout hyphae. Such migrations were dominated by the central cytoplasm, and produced distorted organelles which spanned central and peripheral cytoplasm, thus supporting the idea that the adhesions in mature zones of hyphae anchor the peripheral cytoplasm and facilitate cytoplasmic and organelle migrations.Abbreviations OM organic medium - RP rhodamine phalloidin - DIC differential interference contrast - PIPES piperazine-N,N-bis-2-ethanosulphonic acid     

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 m from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.     

Myosin XIK of Arabidopsis thaliana Accumulates at the Root Hair Tip and Is Required for Fast Root Hair Growth

Myosin motor proteins are thought to carry out important functions in the establishment and maintenance of cell polarity by moving cellular components such as organelles, vesicles, or protein complexes along the actin cytoskeleton. In Arabidopsis thaliana, disruption of the myosin XIK gene leads to reduced elongation of the highly polar root hairs, suggesting that the encoded motor protein is involved in this cell growth. Detailed live-cell observations in this study revealed that xik root hairs elongated more slowly and stopped growth sooner than those in wild type. Overall cellular organization including the actin cytoskeleton appeared normal, but actin filament dynamics were reduced in the mutant. Accumulation of RabA4b-containing vesicles, on the other hand, was not significantly different from wild type. A functional YFP-XIK fusion protein that could complement the mutant phenotype accumulated at the tip of growing root hairs in an actin-dependent manner. The distribution of YFP-XIK at the tip, however, did not match that of the ER or several tip-enriched markers including CFP-RabA4b. We conclude that the myosin XIK is required for normal actin dynamics and plays a role in the subapical region of growing root hairs to facilitate optimal growth.     

Microtubules and microfilaments coordinate to direct a fountain streaming pattern in elongating conifer pollen tube tips

This study investigates how microtubules and microfilaments control organelle motility within the tips of conifer pollen tubes. Organelles in the 30-m-long clear zone at the tip of Picea abies (L.) Karst. (Pinaceae) pollen tubes move in a fountain pattern. Within the center of the tube, organelles move into the tip along clearly defined paths, move randomly at the apex, and then move away from the tip beneath the plasma membrane. This pattern coincides with microtubule and microfilament organization and is the opposite of the reverse fountain seen in angiosperm pollen tubes. Application of latrunculin B, which disrupts microfilaments, completely stops growth and reduces organelle motility to Brownian motion. The clear zone at the tip remains intact but fills with thin tubules of endoplasmic reticulum. Applications of amiprophosmethyl, propyzamide or oryzalin, which all disrupt microtubules, stop growth, alter organelle motility within the tip, and alter the organization of actin microfilaments. Amiprophosmethyl inhibits organelle streaming and collapses the clear zone of vesicles at the extreme tip together with the disruption of microfilaments leading into the tip, leaving the plasma membrane intact. Propyzamide and oryzalin cause the accumulation of membrane tubules or vacuoles in the tip that reverse direction and stream in a reverse fountain. The microtubule disruption caused by propyzamide and oryzalin also reorganizes microfilaments from a fibrillar network into pronounced bundles in the tip cytoplasm. We conclude that microtubules control the positioning of organelles into and within the tip and influence the direction of streaming by mediating microfilament organization.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations APM Amiprophosmethyl - FITC Fluorescein isothiocyanate - LATB Latrunculin B     

The distributional changes and role of microtubules in Nod factor-challenged<Emphasis Type="Italic"> Medicago sativa</Emphasis> root hairs

The normal tip-growing pattern exhibited by root hairs of legumes is disrupted when the hair is exposed to Nod factors generated by compatible bacteria capable of inducing nodule formation. Since microtubules (MTs) play an important role in regulating directionality and stability of apical growth in root hairs [T.N. Bibikova et al. (1999) Plant J 17:657–665], we examined the possibility that Nod factors might affect the MT distribution patterns in root hairs of Medicago sativa L. We observed that Nod factor application caused rapid changes in the pattern of MTs starting as early as 3 min after perfusion. Within 3 to 10 min after Nod factor application, first endoplasmic and then cortical MTs depolymerised, initially at the proximal ends of cells. Twenty minutes after exposure to Nod factors, a transverse band of microtubules was seen behind the tip, while almost all other MTs had depolymerised. By 30 min, very few MTs remained in the root hair and yet by 1 h the MT cytoskeleton re-formed. When Nod factors were applied in the presence of 10 M oryzalin or 5 M taxol, the MTs appeared disintegrated while the morphological effects, such as bulging and branching, became enhanced. Compared to the treatments with oryzalin or taxol alone, the combinatory treatments exhibited higher growth rates. Since microtubule reorganization is one of the earliest measurable events following Nod factor application we conclude that microtubules have an important role in the early phases of the signalling cascade. Microtubule involvement could be direct or a consequence of Nod factor-induced changes in ion levels.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-003-1097-1Abbreviations BNM buffered nodulation medium - CLSM confocal laser scanning microscopy - MT microtubule     

Time Course of Cell Biological Events Evoked in Legume Root Hairs by Rhizobium Nod Factors: State of the Art

In many common legumes, when host-specific nodule bacteria meettheir legume root they attach to it and enter through root hairs.The bacteria can intrude these cells because they instigatein the hairs the formation of an inward growing tube, the infectionthread, which consists of wall material. Prior to infectionthread formation, the bacteria exploit the cell machinery forwall deposition by inducing the hairs to form a curl, in whichthe dividing bacteria become entrapped. In most species, Nodfactor alone (a lipochito-oligosaccharide excreted by bacteria)induces root hair deformation, though without curling, thusmost aspects of the initial effects of Nod factor can be elucidatedby studying root hair deformation. In this review we discussthe cellular events that host-specific Nod factors induce intheir host legume root hairs. The first event, detectable onlya few seconds after Nod factor application, is a Ca²⁺influxat the root hair tip, followed by a transient depolarizationof the plasma membrane potential, causing an increase in cytosolic[Ca²⁺] at the root hair tip. Also within minutes, Nod factorschange the cell organization by acting on the actin cytoskeleton,enhancing tip cell wall deposition so that root hairs becomelonger than normal for their species. Since the remodellingof the actin cytoskeleton precedes the second calcium event,Ca²⁺spiking, which is observed in the perinuclear area, we proposethat the initial cytoskeleton events taking place at the hairtip are related to Ca²⁺influx in the hair tip and that Ca²⁺spikingserves later events involving gene expression. Copyright 2001Annals of Botany Company Review, Nod factor, tip growth, root hair, Rhizobium, legume, cytoskeleton, calcium, symbiosis     

Microtubule dynamics in root hairs of Medicago truncatula

The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.     

Organelle Movement in the Germinated Plooen Grains and Pollen Tubes of Oenothera odorata

The movement of organelles in the germinated pollen of Oenothera odorata was studied in detail by video microscopy. The image of the organelle movement was processed by computergation. The pollen grain of Oenothera odorata is large and easy to germinate in vitro and is suitable for the study of organelle movement . The motion of organelles in the germinated pollen grains and pollen tubes is very vigorous. But the movement of organelles in the pollen tubes is more vigorous than that in the germinating pollen grains. Some of their motion is saltatory. A kind of fibrils was observed in the germinated pollen grains. They are supposed to be made up of actin filaments. Some of the fibrils have one end connected to the plasma membrane and others have both ends linked to the plasma membrane , forming a network. Organelles move along the fibrils continuosely and the speed changes constantly . The speed of movement of organelles is not related to their dimensions. Cytochalasin B can inhibit the movement of organelles. Our results suggest that the movement of organelles is independent of the cytoplasmic streaming in the germinated pollen grains and pollen tubes.     

Cytoplasmic streaming affects gravity-induced amyloplast sedimentation in maize coleoptiles

Living maize (Zea mays L.) coleoptile cells were observed using a horizontal microscope to determine the interaction between cytoplasmic streaming and gravity-induced amyloplast sedimentation. Sedimentation is heavily influenced by streaming which may (1) hasten or slow the velocity of amyloplast movement and (2) displace the plastid laterally or even upwards before or after sedimentation. Amyloplasts may move through transvacuolar strands or through the peripheral cytoplasm which may be divided into fine cytoplasmic strands of much smaller diameter than the plastids. The results indicate that streaming may contribute to the dynamics of graviperception by influencing amyloplast movement.     

Apical Structure of Actively Growing Fern Rhizoids Examined by DIC and Confocal Microscopy

The development and cytology of gametophyte primary rhizoidsof the fern Dryopteris affinis was examined using actively growingmaterial. During development an apical cytoplasmic ‘ accumulation’forms and is associated with active tip growth. This accumulationdeteriorates as terminal differentiation and cessation of growthapproaches. During early development the nucleus moves fromthe rhizoid cell base into the newly extending rhizoid. Later,during the active elongation phase, the nucleus takes up a relativelystable location approx. 100 µm behind the extending apex.Towards terminal differentiation the nucleus lags further behindthe tip. In actively growing rhizoids four distinct zones weredistinguished: a richly cytoplasmic ‘cap’; an apicalregion with tubular vacuolar intrusions; a region distinguishedby a peripheral sheath of cytoplasm and fine irregular cytoplasmicstrands connecting to the nucleus; and the main subapical vacuole.Confocal microscopy of gametophytes stained with fluorescentvital dyes, not previously used to examine fern rhizoid structure,confirmed that the tubular vacuolar system extends well intothe apical cytoplasm, and that the network of fine cytoplasmicstrands leads back from the apical cytoplasm to the nucleus.It also revealed that mitochondria are distributed throughoutthe rhizoid and are not excluded from the extreme apex. Membranestaining by FM 4-64 suggested a high density of membrane vesicleswithin the cytoplasm of the extreme apex. Uptake of this endocytosismarker into endomembranes also suggested rapid plasma membraneturnover in the rhizoid. This study highlights the similarityin the developmental stages and appearance of D. affinis rhizoidsto angiosperm root hairs and their much less distinct apicalzonation compared to pollen tubes. Copyright 2000 Annals ofBotany Company Rhizoid, root hair, confocal imaging, vital stains.