Microtubules in statocytes from roots of cress (Lepidium sativum L.)

Summary Statocytes in root caps ofLepidium sativum L. were examined by means of ultrathin serial sections to evaluate the amount and distribution of cortical microtubules. The microtubules encircle the cell, oriented normal to the root length axis. In the distal cell edges, microtubules form a network, separating the distal complex of endoplasmic reticulum from the plasmalemma. Preprophase bands in meristem cells are observable rarely, structures which can be regarded as nucleating sites for microtubules are lacking. During ageing of the root cap cells, the number of microtubules increases in combination with a decrease of microtubule length. Development of the roots on a horizontal clinostat preserves a younger developmental stage of the microtubule system regarding amount and length of the individual microtubules. Evidence for an involvement of microtubules in graviperception is low, whereas their role in orienting cellulose microfibrils cannot be ruled out. Compression of the distal network of microtubules after centrifugation of the roots indicates that microtubules in statocytes ofLepidium sativum L. roots might function in stabilizing the distal complex of endoplasmic reticulum.     

A role of microtubules in the polarity of statocytes from roots of Lepidium sativum L.

When roots of Lepidium sativum L. are immersed in a colchicine solution (10^-4 mol l^-1), the cortical microtubules of statocytes are affected such that the dense network ofmicrotubules at the distal cell edges, between the endoplasmic reticulum and the plasma membrane, disappears almost completely, whereas the microtubules, lining the anticlinal cell walls are reduced only to a limited extent. Upon inversion of colchicine-pretreated roots, the distal complex of endoplasmic reticulum sinks into the interior of the statocyte. Germination of seeds in the cold (3–4°C) leads to a retardation of statocyte development; the elaborated system of endoplasmic reticulum is lacking, and only a few microtubules are observable, lining the plasma membrane along the anticlinal cell walls. During an additional 4 h at 24°C, groups of microtubules develop near the plasma membrane in the distal one-third of the statocytes, coaligning with newly synthesized cisternae of the endoplasmic reticulum. It is proposed that, particularly at the distal statocyte pole, microtubules in coordination with cross-bridging structures, act in stabilizing the polar arrangement of the distal endoplasmic reticulum and, in turn, facilitate an integrated function of amyloplasts, endoplasmic reticulum and plasma membrane in graviperception.Abbreviations ER endoplasmic reticulum - MT microtubule     

Cytodifferentiation of polar plant cells Use of anti-microtubular agents during the differentiation of statocytes from cress roots (Lepidium sativum L.)

The development of the structural polarity of statocytes from cress roots (Lepidium sativum L.) was studied in a time- and stage-dependent manner. Outgrowing radicles had statocytes with abundant lipid droplets, sparsely developed endoplasmic reticulum (ER) and nuclei located at the proximal cell poles. During differentiation, coincidentally the lipid droplets disappeared, while rough ER increased in length. The ER was translocated into the distal cell pole to establish a complex of stacked ER. Microtubules occurred first at the distal cell edges. As a second step, ER was produced in the vicinity of the nucleus and was also translocated distally. By application of the antimicrotubular agents heavy water (90%), colchicine (10^-4 mol·l^-1) and triethyl lead chloride (20 mol·l^-1), the involvement of microtubules in these events was studied. Triethyl lead chloride led to a complete cessation of differentiation; root-cap cells remained at a stage without polar arrangement of the ER. Colchicine affected the development of structural polarity slightly, as shown by a higher density of cortical ER cisternae. Heavy water inhibited the translocation of ER almost completely and yielded ER located also in the cell center. All anti-microtubular agents inhibited cell division and the differentiation of the distal cell layer of the dermatocalyptrogen into statocytes. It is hypothesized that microtubules serve as anchoring sites for microfilaments, which actually mediate the translocation of the ER. Hence, an intact system of microtubules and microfilaments is necessary for the expression of structural polarity.Abbreviations DC dermatocalyptrogen - ER endoplasmic reticulum - M meristem cell layer - MT microtubule - pI prospective story I - TrEl triethyl lead chloride     

Fractionation and characterization of cellular membranes from root tips of garden cress (Lepidium sativum L.)

The first step in the gravitropic reaction chain, i.e. perception, is known to occur in the statenchyma of the root cap. Because of the importance of the root tip in graviperception, a procedure has been developed to isolate root tips from garden cress (Lepidium sativum L.). The root tip fraction contains the tissues of the root cap plus the lower half of the meristem zone, but is clearly separated from the tissues of the elongation zone, the zone of gravitropic response. Membranes from the root tip and root base fractions have been centrifuged on sucrose density gradients and the marker enzyme profiles analyzed. These results show that the marker enzyme profiles for vacuoles, dictyosomes, mitochondria, and plasma membranes are similar in the root tip or root base fractions. The endoplasmic reticulum (ER) has a shoulder of cytochrome c reductase activity at a density of 1.16 g cm^-3 which is distinct from the other enzyme activities and is only observed in root tip preparations. The specific enzyme activity for ER, cytochrome c reductase, was enriched in root tip membranes 1.7 fold. This latter increase is interpreted as at least in part an increased ER content in the root tip.Abbreviations ASG 6-acyl-steryl glucoside - ER endoplasmic reticulum - IDP inosine-5-diphosphate - INT 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride - PM plasma membrane - SG steryl glucoside     

Demonstration by heavy-meromyosin of actin microfilaments in extracted cress (Lepidium sativum L.) root statocytes

When statocytes of cress root-caps were incubated in an extraction medium containing phalloidin, bundles of microfilaments (6.6±2.4 nm in diameter) were found in the cortical cytoplasm. The inclusion of heavy-meromyosin in this medium demonstrated an arrowhead pattern on the filaments indicative of the presence of actin.     

In cress roots (Lepidium sativum L.), abscisic acid prevents the development of the central cap cells into statocytes

Summary If seeds of Lepidium sativum are soaked in a 10^-4M solution of abscisic acid (ABA) for 30 min, the structural development of the central cap cells (C.C.) deviates strongly from the norm. ABA inhibits the elongation of the C.C., the sedimentation of the amyloplasts which may be caused by a higher viscosity of the cytoplasm, the formation of the stacked ER-cisternae at the distal cell pole, and the polar arrangement of the cell organelles along the gravity vector. The statistical distribution of the organelles and the arrangement of lipid droplets near the plasmalemma indicate that ABA prevents the normal development of the C.C. into statocytes.Abbreviations ABA abscisic acid - C.C. central cap cells     

Phototropism and geotropism in hypocotyls of cress (Lepidium sativum L.)

Abstract. The phototropic and the geotropic sensitivities of cress hypocotyls differed in etiolated and in green seedlings. In etiolated seedlings, phototropism was poorly developed and geotropism seemed to be the dominant orientation mechanism. In green seedlings, although geotropic sensitivity was slightly greater, phototropic responses were markedly enhanced, such that photo-signals could over-ride, or even reverse, geocurvature. The 'de-etiolation' light pretreatments required to bring about these changes in the photo-and geo-systems were different. The slight enhancement of geocurvature could be achieved by short-term exposure to red light and was reversed by far-red irradiation. The marked stimulation of photocurvature required extensive irradiation pretreatments.     

Hormone treatment of roots causes not only a reversible loss of starch but also of structural polarity in statocytes

Treatment of cress (Lepidium sativum L.) roots with phytohormones (4.3 x 10(-5) M gibberellic acid plus 4.3 x 10(-5) M kinetin, 30 h; T.H. Iversen, 1969, Physiol. Plant. 22, 1251-1262) caused not only complete destarching of amyloplasts but also destruction of the polar arrangement of cell organelles in statocytes. The nucleus was not positioned exclusively near the proximal cell pole as in the controls but was also found near the distal cell pole. The endoplasmic reticulum (ER) was no longer organized in parallel sheets at the distal cell pole but instead the ER-cisternae were randomly distributed. Additionally, the statocytes from hormone-treated roots contained a large central vacuole instead of numerous small ones as in the controls. The starch-free plastids had a reduced volume and an amoeboid shape. They did not sediment but were randomly distributed in the statocytes. The loss of structural polarity was accompanied by loss of graviresponsiveness although root growth still occurred. Twenty-two hours after removal of the hormones, structural polarity was restored and starch was resynthesized. The newly formed starch grains were smaller and more numerous per amyloplast compared to the controls. It is concluded that loss of gravisensitivity of roots after hormone treatment cannot be solely attributed to the loss of amyloplastic starch because there is a concomitant loss in the polar organisation of the statocyte.     

Restitution of polarity in statocytes from centrifuged roots*

Abstract The structural polarity of statocytes from cress roots is changed by centrifugation. Upon low- dose centrifugation (3000 g min), the extent of stratification depends on statocyte position, i.e., central statocytes are affected more than lateral ones. Upon higher doses of centrifugation (60,000 and 360,000 g min), a uniform density gradient is established in all statocytes. If, after centrifugation, the roots are exposed to gravity again, the endoplasmic reticulum (ER) cisternae are relocated parallel to the periclinal cell walls within a few minutes; this relocation is independent of the direction of gravity in relation to the root axis, and independent of the previously applied centrifugation dose. This supports the notion that polarity is determined genetically. Cytochalasin B treatment, before and during centrifugation, totally inhibits the relocation of ER. After removing the drug by rinsing the roots, the statocytes restore cell polarity and relocate ER. These results indicate that relocation of ER cisternae may be mediated by microfilaments. When centrifuged roots are exposed to 1 g in the horizontal position, the latent period of gravitropism increases by 8–10 min relative to controls, regardless of the previously applied centrifugation doses. The kinetics of curvature are virtually identical. Since the increase in the latent period coincides with the time needed for most statocytes to restore the distal cell pole, it is evident that perception of gravity is correlated to the integrity of the distal cell pole.     

Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Identification of tonoplast and plasma membrane in membrane fractions from garden cress (Lepidium sativum L.) with and without filipin treatment

Membranes from roots of Lepidium sativum L. were investigated in situ and after fractionation by applying morphological and biochemical methods. After freeze-fracture combined with filipin labelling the tonoplast and the plasma membrane could be easily characterized by the frequency of intramembranous particles and the arrangement of filipin-induced lesions. On tonoplast vesicles, the filipin-induced lesions were arranged in clusters of different size whereas they were evenly distributed on plasma membrane vesicles. Enrichment of tonoplast and plasma membrane in different fractions was documented by filipin labelling, phosphotungstic acid staining and by the profiles of marker enzyme activities and ATP-dependent H⁺-transport. Additionally, the presence of rightside-out and inside-out vesicles of both tonoplast and plasma membrane could be demonstrated. It was found that filipin labelling used in combination with freeze-fracturing is suitable for quantitative determinations of the percentages of tonoplast and plasma membrane in membrane fractions, which have been found to be more than 40% for the tonoplast and about 40% for plasma membrane in the respective enriched fractions.Abbreviations EF extraplasmatic fracture face - FIL filipin induced lesion - IMP intramembranous particle - PF plasmatic fracture face - PTA phosphotungstic acid-chromic acid stain - UDPG uridine 5-diphosphate glucose A preliminary report was presented at the joint Annual Meeting of the Belgian and German Societies for Cell Biology, Bonn, March 1985Dedicated to Professor Augustin Betz on the occasion of his 66th birthday     

Construction and characterization of a de novo draft genome of garden cress (Lepidium sativum L.)

Functional & Integrative Genomics - Garden cress (Lepidium sativum L.) is a Brassicaceae crop recognized as a healthy vegetable and a medicinal plant. Lepidium is one of the largest genera in...     

Membrane-potential responses following gravistimulation in roots of Lepidium sativum L.

Membrane potentials were measured in lateral statocytes of vertically and nonvertically growing roots of Lepidium sativum L. using conventional glass-microelectrode techniques. Statocytes in vertically growing roots showed a stable resting potential of-118±5.9 mV without spontaneous fluctuations. Upon tilting the root 45° from the vertical, an electrical asymmetry was observed. Statocytes on the physically lower side of the root depolarized by approx. 25 mV. This depolarization occurred following a latent period of 8 s reaching a minimum (approx.-93 mV) after 170 s. This depolarization is the earliest event in graviperception ever recorded. After this depolarization, the cell repolarized within 60 s to a potential approx. 10 mV more positive than the original resting potential. Statocytes on the upper flank showed a slow hyperpolarization (t _1/2h=half time for hyperpolarization=168 s) reaching a final, stable potential at a level 10 mV more negative. These effects of gravistimulation were statenchyma-specific, since cells in the cortex and rhizodermis showed no similar effects. The gravi-electrical responses were observed in 25% of all roots tested. Roots which showed no gravi-electrical response had a reduced elongation growth, lacked gravity-induced bending and lacked the typical structural polarity in punctured statocytes. This observed transition from a symmetrical pattern of resting potential in the statenchyma to an asymmetrical pattern following gravistimulation supports the results observed with external current measurements (Behrens et al., Plant Physiol. 70, 1079–1083, 1982) and extends these results to the cellular level and to considerably improved temporal resolution. The asymmetry in the gravi-electrical response extends the graviperception model of Sievers and Volkmann (Planta 102, 160–172, 1972) which comprises an asymmetrical sedimentation of the amyloplasts on the distal endoplasmic reticulum of statocytes. This generates an intraorgan signal which then must be transmitted to the growth zone.Abbreviation ER endoplasmic reticulum Preliminary reports were presented at the 11^th International Conference on Plant Growth Substances, Aberystwyth, July 1982, and International Symposium, Membranes and Compartimentation in Regulation of Plant Functions, Toulouse, September 1983     

Polarity of statocytes in lentil seedling roots grown in space (Spacelab D1 Mission)

A morphometric analysis of root statocytes was performed on seedlings of lentil ( Lens culinaris L., cv. Verte du Puy) in order to determine the effects of microgravity on the polarity of these cells. Seedlings were grown: (1) on the ground, (2) in microgravity, (3) on a 1 g centrifuge in space, (4) first in microgravity and then placed on a 1 g centrifuge for 3 h. Dry seeds were hydrated in space (except for the ground control) for 25 h in darkness at 22°C in the Biorack facility developed by the European Space Agency. At the end of the experiment, the seedlings were photographed and fixed in glutaraldehyde in the Biorack glove box. The average shape of the statocytes and the location of endoplasmic reticulum, amyloplasts and nucleus in the cells were analysed in the four samples. By considering the cell shape, it appears that the morphology of the statocytes on the ground was different from that observed in the space samples. Cell polarity was similar in microgravity and in the centrifuged samples except for the distribution of the amyloplasts. These organelles were not distributed at random in near zero gravity, and they were more numerous in the proximal than in the distal half. Moreover, the statoliths were more voluminous in microgravity than in the centrifuged samples. The nucleus was closer to the cell center in the statocytes of roots grown in microgravity than in statocytes of roots grown in microgravity and then placed on the 1 g centrifuge for 3 h. It is hypothesized that the nucleus is attached to the cell periphery and that its location is dependent upon gravity.     

Lectin-gene expression in pea (Pisum sativum L.) roots

The expression of a lectin gene in pea (Pisum sativum L.) roots has been investigated using the copy DNA of a pea seed lectin as a probe. An mRNA which has the same size as the seed mRNA but which is about 4000 times less abundant has been detected in 21-d-old roots. The probe detected lectin expression as early as 4 d after sowing, with the highest level being reached at 10 d, i.e. just before nodulation. In later stages (16-d- and 21-d-old roots), expression was substantially decreased. The correlation between infection by Rhizobium leguminosarum and lectin expression in pea roots has been investigated by comparing root lectin mRNA levels in inoculated plants and in plants grown under conditions preventing nodulation. Neither growth in a nitrate concentration which inhibited nodulation nor growth in the absence of Rhizobium appreciably affected lectin expression in roots.Abbreviation cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Propagated fluctuations of the electric potential in the apoplasm of Lepidium sativum L. roots

The electric potential on the surface of the Lepidium sativum L. root apex was recorded by means of six non-polarizable electrodes. Nonevoked fluctuations of the potential with amplitudes below 0.1 mV were observed. The fluctuations could be reversibly inhibited either by ether vapor or by anoxia caused by N₂. They did not occur in killed roots. Cross-correlation analysis of the fluctuations from six electrodes located one above another along the 3-mm apical region showed a pattern of time delay which indicates that the fluctuations may be the consequence of signals propagated in the root with a velocity of 3–9 mm · s^–1 in a basipetal direction from the root cap. We hypothesize that the fluctuations are due to signals of an unknown nature propagated along an intrasymplasmic continuous system, the symreticulum, composed of the cortical ER of individual cells and desmotubules passing through the plasmodesmata.Abbreviations AC alternating current - AP action potential - ACF autocorrelation function - CCF cross-correlation function - DC direct current - EEP extracellular electric potential This research was supported by Bundesminister für Forschung und Technologie, Bonn, and Ministerium für Wissenschaft und Forschung, Düsseldorf, (AGRAVIS). We are grateful to Mr. Dipl.-Ing. P. Blasczyk for constructing the amplifiers and for advice in instrumentation, and to Mr. H. Laubach for constructing the mechanical assembly.     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots

The presence of the glycolytic enzymes from hexokinase to pyruvate kinase in plastids of seedling pea (Pisum sativum L.) roots was investigated. The recoveries, latencies and specific activities of each enzyme in different fractions was compared with those of organelle marker enzymes. Tryptic-digestion experiments were performed on each enzyme to determine whether activities were bound within membranes. The results indicate that hexokinase (EC 2.7.1.2) and phosphoglyceromutase (EC 5.4.2.1) are absent from pea root plastids. The possible function of the remaining enzymes is considered.Abbreviations GADPH glyceraldehyde 3-phosphate dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate: fructose 6-phosphate 1-phosphotransferase Bronwen A. Trimming gratefully acknowledges the award of a studentship from the Science and Engineering Research Council     

哈尔滨医科大学附属第一医院神经外科

血脑屏障的是人体自然屏障之一。其主要作用是阻止有害物质通过颅内血管进入脑实质,并同时辅助排出脑内代谢物质等。对相当多的颅内恶性肿瘤术后患者,血脑屏障在一定程度上阻碍了化疗药物进入脑实质,从而影响化疗效果。因此近年来越来越多的学者将研究重点放在如何开放血脑屏障这个问题上。血脑屏障构成主要为毛细血管的内皮细胞、基膜周细胞和星状胶质细胞的足突,其中血管内皮细胞处于最重要的地位。原因归结于它自身的一个特殊结构--紧密连接。紧密连接是否完整,功能是否可以正常发挥关系到内皮细胞的完整性,因此对血脑屏障的开放有着举足轻重的作用。维持紧密连接结构中功能蛋白功能的能量物质为葡萄糖。脑血管中的葡萄糖进入脑实质需载体或通道,脑组织负责此过程的物质为葡萄糖转运蛋白1(GLUT1)。本文作者通过松胞菌素B抑制葡萄糖转运蛋白1,降低能量供应从而影响紧密连接功能,最终引起血脑屏障开放角度做一综述。     

Mitochondrial inner membrane particles seen in sections ofin situ large amplitude swollen mitochondria in rhizodermal cells of cress (Lepidium sativum L.)

Addition of 5–40 mM sodium acetate to root explants of cress (Lepidium sativum L.) growing in a nutrient medium causes large amplitude swelling of mitochondria in rhizodermal cells. On the average, 10 mM sodium acetate causes a fourfold increase in mitochondrial volume, with 40 mM sodium acetate producing an up to tenfold increase in mitochondrial volume. During swelling, however, the mitochondrial membranes remain predominantly intact, and only the outer membrane occasionally appears to be broken. Two types of swelling can be observed: an overall swelling of mitochondrial matrices and a less frequent local swelling which leads to clearly different matrical regions. These regions may sometimes be separated by a septum formed from the inner mitochondrial membrane. After large amplitude swelling in 10 or 40 mM sodium acetate, the visibility of lollipop-like particles on the matrix side of inner mitochondrial membranes is strongly enhanced. These particles are suggested to be identical with mitochondrial inner membrane particles as visualized by negative staining. The distribution of these particles is described. Possible mechanisms which may cause enhanced visibility are discussed.