Micromanipulation of statoliths in gravity-sensing Chara rhizoids by optical tweezers

Infrared laser traps (optical tweezers) were used to micromanipulate statoliths in gravity-sensing rhizoids of the green alga Chara vulgaris Vail. We were able to hold and move statoliths with high accuracy and to observe directly the effects of statolith position on cell growth in horizontally positioned rhizoids. The first step in gravitropism, namely the physical action of gravity on statoliths, can be simulated by optical tweezers. The direct laser microirradiation of the rhizoid apex did not cause any visible damage to the cells. Through lateral positioning of statoliths a differential growth of the opposite flank of the cell wall could be induced, corresponding to bending growth in gravitropism. The acropetal displacement of the statolith complex into the extreme apex of the rhizoid caused a temporary decrease in cell growth rate. The rhizoids regained normal growth after remigration of the statoliths to their initial position 10–30 m basal to the rhizoid apex. During basipetal displacement of statoliths, cell growth continued and the statoliths remigrated towards the rhizoid tip after release from the optical trap. The resistance to statolith displacement increased towards the nucleus. The basipetal displacement of the whole complex of statoliths for a long distance (>100 m) caused an increase in cell diameter and a subsequent regaining of normal growth after the statoliths reappeared in the rhizoid apex. We conclude that the statolith displacement interferes with the mechanism of tip growth, i.e. with the transport of Golgi vesicles, either directly by mechanically blocking their flow and/or, indirectly, by disturbing the actomyosin system. In the presence of the actin inhibitor cytochalasin B the optical forces required for acropetal and basipetal displacement of statoliths were significantly reduced to a similar level. The lateral displacement of statoliths was not changed by cytochalasin B. The results indicate: (i) the viscous resistance to optical displacement of statoliths depends mainly on actin, (ii) the lateral displacement of statoliths is not impeded by actin filaments, (iii) the axially directed actin-mediated forces against optical displacement of statoliths (for a distance of 10 m) are stronger in the basipetal than in the acropetal direction, (iv) the forces acting on single statoliths by axially oriented actin filaments are estimated to be in the range of 11–110 pN for acropetal and of 18–180 pN for basipetal statolith displacements.Abbreviation CB cytochalasin B This work was supported by the Bundesminister für Forschung und Technologie, and by Fonds der Chemischen Industrie. We thank Professor Dr. A. Sievers (Botanisches Institut, Universität Bonn, Germany) for helpful discussions.     

轮藻假根中的平衡石在回转器水平回转时的运动

利用回转器重现了在ＴＥＸＵＳ火箭抛物线飞行的微重力实验中轮藻假根内平衡石和假根基部方向的运动。在快速回转器上回转１５ｍｉｎ时,假根中的平衡石复合体中心离假根顶端的距离比在原来沿重力方向生长的假根中的距离增加了６０％。细胞松弛素Ｄ的实验证实平衡石的这种运动是和肌动蛋白丝相关,而且在重力场中作用于平衡石的向基力也是肌动蛋白丝产生的。因此回转器和细胞松弛素Ｄ的实验证实了在地球上,平衡石的位置取决于作用方     

The response to gravity is correlated with the number of statoliths in Chara rhizoids.

In contrast to higher plants, Chara rhizoids have single membrane-bound compartments that appear to function as statoliths. Rhizoids were generated by germinating zygotes of Chara in either soil water (SW) medium or artificial pond water (APW) medium. Differential-interference-contrast microscopy demonstrated that rhizoids form SW-grown plants typically contain 50 to 60 statoliths per cell, whereas rhizoids from APW-grown plants contain 5 to 10 statoliths per cell. Rhizoids from SW are more responsive to gravity than rhizoids from APW because (a) SW rhizoids were oriented to gravity during vertical growth, whereas APW rhizoids were relatively disoriented, and (b) curvature of SW rhizoids was 3 to 4 times greater throughout the time course of curvature. The growth rate of APW rhizoids was significantly greater than that of SW-grown rhizoids. This latter result suggests that APW rhizoids are not limited in their ability for gravitropic curvature by growth and that these rhizoids are impaired in the early stages of gravitropism (i.e. gravity perception). Plants grown in APW appeared to be healthy because of their growth rate and the vigorous cytoplasmic streaming observed in the rhizoids. This study is comparable to earlier studies of gravitropism in starch-deficient mutants of higher plants and provides support for the role of statoliths in gravity perception.     

Negative gravitropism in Chara protonemata: A model integrating the opposite gravitropic responses of protonemata and rhizoids

The unicellular protonema of Chara fragilis Desv. was investigated in order to establish a reaction chain for negative gravitropism in tip-growing cells. The time course of gravitropic bending after stimulation at angles of 45 degrees or 90 degrees showed three distinct phases of graviresponse. During the first hour after onset of stimulation a strong upward shift of the tip took place. This initial response was followed by an interval of almost straight growth. Complete reorientation was achieved in a third phase with very low bending rates. Gravitropic reorientation could be completely abolished by basipetal centrifugation of the cells, which lastingly removed conspicuous dark organelles from the protonema tip, thus identifying them as statoliths. Within minutes after onset of gravistimulation most or all statoliths were transported acropetally from their resting position 20-100 micrometers from the cell apex to the lower side of the apical dome. This transport is actin-dependent since it could be inhibited with cytochalasin B. Within minutes after arrival of the statoliths, the apical dome flattened on its lower side and bulged on the upper one. After this massive initial response the statoliths remained firmly sedimented, but the distance between this sedimented complex and the cell vertex increased from 7 micrometers to 22 micrometers during the first hour of stimulation and bending rates sharply declined. From this it is concluded that only statoliths inside the apical dome convey information about the spatial orientation of the cell in the gravitropic reaction chain. After inversion of the protonema the statoliths transiently arranged into a disk-shaped complex about 8 micrometers above the vertex. When this statolith complex tilted towards one side of the apical dome, growth was shifted in the opposite direction and bending started. It is argued that the statoliths intruding into the apical dome may displace a growth-organizing structure from its symmetrical position in the apex and may thus cause bending by bulging. In the positively gravitropic Chara rhizoids only a more stable anchorage of the growth-organizing structure is required. As a consequence, sedimented statoliths cannot dislocate this structure from the vertex. Instead they obstruct a symmetrical distribution of cell-wall-forming vesicles around the structure and thus cause bending by bowing.     

Relocalization of the calcium gradient and a dihydropyridine receptor is involved in upward bending by bulging of Chara protonemata, but not in downward bending by bowing of Chara rhizoids

The localization of cytoplasmic free calcium and a dihydropyridine (DHP) receptor, a putative calcium channel, was recorded during the opposite graviresponses of tip-growing Chara rhizoids and Chara protonemata by using the calcium indicator Calcium Crimson and a fluorescently labeled dihydropyridine (FL-DHP). In upward (negatively gravitropically) growing protonemata and downward (positively gravitropically) growing rhizoids, a steep Ca²⁺ gradient and DHP receptors were found to be symmetrically localized in the tip. However, the localization of the Ca²⁺ gradient and DHP receptors differed considerably during the gravitropic responses upon horizontal positioning of the two cell types. During the graviresponse of rhizoids, a continuous bowing downward by differential flank growth, the Ca²⁺ gradient and DHP receptors remained symmetrically localized in the tip at the centre of growth. However, after tilting protonemata into a horizontal position, there was a drastic displacement of the Ca²⁺ gradient and FL-DHP to the upper flank of the apical dome. This displacement occurred after the apical intrusion and sedimentation of the statoliths but clearly before the change in the growth direction became evident. In protonemata, the reorientation of the growth direction started with the appearence of a bulge on that site of the upper flank which was predicted by the asymmetrically displaced Ca²⁺ gradient. With the upward shift of the cell tip, which is suggested to result from a statolith-induced displacement of the growth centre, the Ca²⁺ gradient and DHP receptors became symmetrically relocalized in the apical dome. No major asymmetrical rearrangement was observed during the following phase of gravitropic curvature which is characterized by slower rates of bending. Labeling with FL-DHP was completely inhibited by a non-fluorescently labeled dihydropyridine. From these results it is suggested that FL-DHP labels calcium channels in rhizoids and protonemata. In rhizoids, positive gravitropic curvature is caused by differential growth limited to the opposite subapical flanks of the apical dome, a process which does not involve displacement of the growth centre, the calcium gradient or calcium channels. In protonemata, however, it is proposed that a statolith-induced asymmetrical relocalization of calcium channels and the Ca²⁺ gradient precedes, and might mediate, the rearrangement of the centre of growth, most likely by the displacement of the Spitzenk?rper, to the upper flank, which results in the negative gravitropic reorientation of the growth direction. Received: 13 February 1999 / Accepted: 25 June 1999     

Human surfactant protein D: SP-D contains a C-type lectin carbohydrate recognition domain

Lung surfactant protein D (SP-D) shows calcium-dependent binding to specific saccharides, and is similar in domain structure to certain members of the calcium-dependent (C-type) lectin family. Using a degenerate oligomeric probe corresponding to a conserved peptide sequence derived from the amino-terminus of the putative carbohydrate binding domain of rat and bovine SP-D, we screened a human lung cDNA library and isolated a 1.4-kb cDNA for the human protein. The relationship of the cDNA to SP-D was established by several techniques including amino-terminal microsequencing of SP-D-derived peptides, and immunoprecipitation of translation products of transcribed mRNA with monospecific antibodies to SP-D. In addition, antibodies to a synthetic peptide derived from a predicted unique epitope within the carbohydrate recognition domain of SP-D specifically reacted with SP-D. DNA sequencing demonstrated a noncollagenous carboxy-terminal domain that is highly homologous with the carboxy-terminal globular domain of previously described C-type lectins. This domain contains all of the so-called "invariant residues," including four conserved cysteine residues, and shows high homology with the mannose-binding subfamily of C-type lectins. Sequencing also demonstrated an amino-terminal collagenous domain that contains an uninterrupted sequence of 59 Gly-X-Y triplets and that also contains the only identified consensus for asparagine-linked oligosaccharides. The studies demonstrate that SP-D is a member of the C-type lectin family, and confirm predicted structural similarities to conglutinin, SP-D, and the serum mannose binding proteins.     

In-vivo observations of a spherical aggregate of endoplasmic reticulum and of Golgi vesicles in the tip of fast-growing Chara rhizoids

In-vivo differential interference contrast microscopy was used to detect individual Golgi vesicles and a new structure in the tip of fast-growing rhizoids of Chara fragilis Desvaux. This structure is a spherical clear zone which is free of Golgi vesicles, has a diameter of 5 m and is positioned in the center of the apical Golgi-vesicle accumulation (Spitzenkörper). After glutaraldehyde fixation and osmium tetroxide-potassium ferricyanide staining of the rhizoid, followed by serial sectioning and three-dimensional reconstruction, the spherical zone shows a tight accumulation of anastomosing endoplasmic reticulum (ER) membranes. The ER membranes radiate from this aggregate towards the apical plasmalemma and to the membranes of the statolith compartments. Upon gravistimulation the ER aggregate changes its position according to the new growth direction, indicating its participation in growth determination. After treatment of the rhizoid with cytochalasin B or phalloidin the ER aggregate disappears and the statoliths sediment. It is concluded that the integrity of the ER aggregate is actin-dependent and that it is related to the polar organisation of the gravitropically growing cell tip.Abbreviations CB cytochalasin B - DIC differential interference contrast microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum     

Fixation procedure for transmission electron microscopy of Chara rhizoids under microgravity in a Spacelab (IML-2)

A special fixation device and fixation procedure have been developed to investigate for the first time the ultrastructure of gravity-sensing, unicellular Chara rhizoids grown for 30 h under microgravity (MG) conditions during the IML-2 mission. The fixation unit allowed culture, fixation and storage of Chara rhizoids in the same chamber without transferring the samples. The procedure was easy and safe to perform and required a minimum of crew time. Rhizoids fixated with glutaraldehyde in space and further processed for electron microscopy on ground showed that the fixation was of high quality and corresponded to the fixation quality of rhizoids in the ground controls. Thus, the equipment accomplished the manifold problems related to the physical effects of MG. The polarity of the rhizoids was maintained in MG. Well-preserved organelles and microtubules showed no obvious difference in ultrastructure or distribution after 30-h growth in MG compared to ground controls. The statoliths were more randomly distributed, however, only up to 50 microns basal to the tip. Thus, changing the gravity conditions does to disturb the cellular organisation of the rhizoids enabling the tip-growing cells to follow their genetic program in development and growth also under MG.     

The pattern of acropetal and basipetal cytoplasmic streaming velocities in Chara rhizoids and protonemata, and gravity effect on the pattern as measured by laser-Doppler-velocimetry

 The spatial pattern of acropetal and basipetal cytoplasmic streaming velocities has been studied by laser-Doppler-velocimetry (LDV) in the positively gravitropic (downward growing) rhizoids of Chara globularis Thuill. and for the first time in the negatively gravitropic (upward growing) protonemata. The LDV method proved to be precise and yielded reproducible results even when tiny differences in velocities were measured. In the apical parts of the streaming regions of both cell types, acropetal streaming was faster than basipetal streaming. Starting at the apical reversal point of streaming, the velocity increased basipetally with the distance from that point and became fairly constant close to the basal reversal point; subsequently, the velocity decreased slightly acropetally as the apical reversal point was again approached. There was no change in velocity at the basal reversal point. However, at the apical reversal point there was an abrupt decrease in velocity. The pattern of the ratio of acropetal to basipetal streaming velocity (VR) was a function of the relative distance of the site of measurement from the apical reversal point rather than a function of the absolute distance. Upon inversion of the rhizoids, the VR decreased on average by 3.8% (±0.4%), indicating that the effect of gravity on the streaming velocity was merely physical and without a physiological amplification. Rhizoids that had developed on the slowly rotating horizontal axis of a clinostat, and had never experienced a constant gravity vector, were similar to normally grown rhizoids with respect to VR pattern. In protonemata, the VR pattern was not significantly different from that in rhizoids although the direction of growth was inverse. In rhizoids, oryzalin caused the polar organization of the cell to disappear and nullified the differences in streaming velocities, and cytochalasin D decreased the velocity of basipetal streaming slightly more than that of acropetal streaming. Cyclopiazonic acid, known as an inhibitor of the Ca²⁺-ATPase of the endoplasmic reticulum, also reduced the streaming velocities in rhizoids, but had slightly more effect on the acropetal stream. It is possible that the endogenous difference in streaming velocities in both rhizoids and protonemata is caused by differences in the cytoskeletal organization of the opposing streams and/or loading of inhibitors (like Ca²⁺) from the apical/subapical zone into the basipetally streaming endoplasm. Received: 4 October 1999 / Accepted: 4 November 1999     

The evolution of root hairs and rhizoids

Background

Almost all land plants develop tip-growing filamentous cells at the interface between the plant and substrate (the soil). Root hairs form on the surface of roots of sporophytes (the multicellular diploid phase of the life cycle) in vascular plants. Rhizoids develop on the free-living gametophytes of vascular and non-vascular plants and on both gametophytes and sporophytes of the extinct rhyniophytes. Extant lycophytes (clubmosses and quillworts) and monilophytes (ferns and horsetails) develop both free-living gametophytes and free-living sporophytes. These gametophytes and sporophytes grow in close contact with the soil and develop rhizoids and root hairs, respectively.

Scope

Here we review the development and function of rhizoids and root hairs in extant groups of land plants. Root hairs are important for the uptake of nutrients with limited mobility in the soil such as phosphate. Rhizoids have a variety of functions including water transport and adhesion to surfaces in some mosses and liverworts.

Conclusions

A similar gene regulatory network controls the development of rhizoids in moss gametophytes and root hairs on the roots of vascular plant sporophytes. It is likely that this gene regulatory network first operated in the gametophyte of the earliest land plants. We propose that later it functioned in sporophytes as the diploid phase evolved a free-living habit and developed an interface with the soil. This transference of gene function from gametophyte to sporophyte could provide a mechanism that, at least in part, explains the increase in morphological diversity of sporophytes that occurred during the radiation of land plants in the Devonian Period.     

Escherichia coli contains a DNA replication compartment in the cell center

The active replication forks of E. coli B/r K cells growing with a doubling time of 210 min have been pulse-labeled with [(3)H] thymidine for 10 min. By electron-microscopic autoradiography the silver grains have been localized in the various length classes. From the known pattern of the DNA replication period in the cell cycle at slow growth and from the average position of grains per length class it was deduced that DNA replication starts in the cell center and that it remains there for a substantial part of the DNA replication period. This suggests the occurrence of a centrally located DNA replication compartment.     

Cholesteryl ester transfer protein is secreted by Hep G2 cells and contains asparagine-linked carbohydrate and sialic acid

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Cholesteryl ester transfer activity was found to accumulate in the medium of cultured Hep G2 cells. The transfer activity was removed by immunoprecipitation with specific antibodies to the plasma CETP. Sodium dodecyl sulfate gel electrophoresis of immunoprecipitates prepared from the medium of cells pulsed with [35S]methionine revealed a broad specific band of protein of Mr 72,000 to 76,000; by contrast, immunoprecipitates of cellular homogenates showed a sharp specific band of Mr 58,000. The Mr 72,000 to 76,000 band disappears, concomitant with the appearance of lower Mr products, upon neuraminidase or glycopeptidase F treatment of medium immunoprecipitates or of purified CETP. The results indicate that liver cells have the capacity to synthesize and secrete CETP. The CETP peptide acquires asparagine-linked carbohydrate and sialic acid during intracellular processing.     

The oligodendrocyte-myelin glycoprotein belongs to a distinct family of proteins and contains the HNK-1 carbohydrate

The complete primary structure of the human oligodendrocyte-myelin glycoprotein (OMgp), a glycophospholipid-linked membrane protein of oligodendrocytes and central nervous system myelin, has been determined. The deduced amino acid sequence predicts a polypeptide of 433 amino acids which includes a 17-amino acid leader sequence. OMgp consists of four domains: (a) a short cysteine-rich motif at the NH2 terminus; (b) a series of tandem leucine-rich repeats (LRs) present in several other proteins where they may play roles in adhesion; (c) a serine/threonine-rich region that contains probable attachment sites for O-linked carbohydrates; and (d) a hydrophobic COOH-terminal segment that is likely to be cleaved concomitant with the attachment of lipid during biosynthesis of OMgp. OMgp shares the first three of its four domains with the platelet glycoprotein Ib, which is responsible for the initial adhesion of platelets to the exposed subendothelium during hemostasis. Together with glycoprotein Ib and several other proteins, OMgp belongs to a family of proteins that contain both an NH2-terminal cysteine-rich motif and an adjacent series of LRs. In addition, we report that a subpopulation of OMgp molecules contains the HNK-1 carbohydrate, which has been shown to mediate interactions among cells in the central nervous system.     

Uptake and polar transport of indoleacetic acid in moss rhizoids

In the cucumber ( Cucumis sativus L. cv. Straight Eight) cotyledon expansion assay, cytokinin-stimulated ethylene production was separated from cytokinin-stimulated growth through the use of potassium and calcium salts. Low concentrations of KC1, which dramatically promoted growth induced by cytokinin, inhibited ethylene evolution, while CaCl₂ at a concentration that had no effect on growth, strongly promoted the cytokinin-induced ethylene evolution. In contrast to the growth response, stimulation of ethylene production was not directly related to the presence of potassium or calcium but to their relative concentrations. Concentrations of KCl and CaCl₂ which promoted ethylene evolution singly, strongly inhibited it when mixed together. Low rates of exogenous ethylene had no effect on the growth response. Both the growth and ethylene responses were found to be general cytokinin phenomena. Cotyledon respiration was promoted by KC1, CaCl₂ and cytokinin, but its stimulation was not correlated with either growth or ethylene production. In the presence of KClm cytokinin-induced respiration sharply lowered the content of certain sugars during the large growth response and followed KCl uptake. Analysis of KCl uptake showed that its growth promoting synergism with cytokinin was not due to osmotic effects.     

The cdc25 protein contains an intrinsic phosphatase activity.

Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.