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Various plants possess non-photosynthetic, hydrophilic chlorophyll (Chl) proteins called water-soluble Chl-binding proteins (WSCPs). WSCPs are categorized into two classes; Class I (photoconvertible type) and Class II (non-photoconvertible type). Among Class II WSCPs, only Lepidium virginicum WSCP (LvWSCP) exhibits a low Chl a/ b ratio compared with that found in the leaf. Although the physicochemical properties of LvWSCP have been characterized, its molecular properties have not yet been documented. Here, we report the characteristics of the LvWSCP gene, the biochemical properties of a recombinant LvWSCP, and the intracellular localization of LvWSCP. The cloned LvWSCP gene possesses a 669-bp open reading frame. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis revealed that the precursor of LvWSCP contains both N- and C-terminal extension peptides. RT-PCR analysis revealed that LvWSCP was transcribed in various tissues, with the levels being higher in developing tissues. A recombinant LvWSCP and hexa-histidine fusion protein (LvWSCP-His) could remove Chls from the thylakoid in aqueous solution and showed an absorption spectrum identical to that of native LvWSCP. Although LvWSCP-His could bind both Chl a and Chl b, it bound almost exclusively to Chl b when reconstituted in 40 % methanol. To clarify the intracellular targeting functions of the N- and C-terminal extension peptides, we constructed transgenic Arabidopsisthaliana lines expressing the Venus protein fused with the LvWSCP N- and/or C-terminal peptides, as well as Venus fused at the C-terminus of LvWSCP. The results showed that the N-terminal peptide functioned in ER body targeting, while the C-terminal sequence did not act as a trailer peptide. [ABSTRACT FROM AUTHOR]

In roots, the 'hidden half' of all land plants, gravity is an important signal that determines the direction of growth in the soil. Hence, positive gravitropism has been studied in detail. However, since the 19th century, the response of roots toward unilateral light has also been analyzed. Based on studies on white mustard ( Sinapis alba) seedlings, botanists have concluded that all roots are negatively phototropic. This ' Sinapis-dogma' was refuted in a seminal study on root phototropism published a century ago, where it was shown that less then half of the 166 plant species investigated behave like S. alba, whereas 53% displayed no phototropic response at all. Here we summarize the history of research on root phototropism, discuss this phenomenon with reference to unpublished data on garden cress ( Lepidium sativum) seedlings, and describe the effects of blue light on the negative bending response in Thale cress ( Arabidopsis thaliana). The ecological significance of root phototropism is discussed and the relationships between gravi- and phototropism are outlined, with respect to the starch-statolith-theory of gravity perception. Finally, we present an integrative model of gravi- and blue light perception in the root tip of Arabidopsis seedlings. This hypothesis is based on our current view of the starch-statolith-concept and light sensing via the cytoplasmic red/blue light photoreceptor phytochrome A and the plasma membrane-associated blue light receptor phototropin-1. Open questions and possible research agendas for the future are summarized. [ABSTRACT FROM AUTHOR]

While there is ample evidence for a role of auxin in root gravitropism, the seeming rapidity of gravi-induced changes in electrical parameters has so far been an argument against auxin being a primary signal in gravitropic signal transmission. To address this problem, we re-investigated the effect of gravistimulation on membrane voltages of Lepidium sativum L. and Vigna mungo L. root cells. In our hands, gravistimulation did not induce changes in membrane voltage in cells of the root cap statenchyma, root meristem or apical elongation zone that can be correlated with the orientation of the cells relative to the gravity vector. While these results challenge a model of rapid electrically based signal transmission, there is evidence for a slower signal propagation along gravistimulated L. sativum roots. Using multiple proton-selective microelectrodes to simultaneously measure surface pH on opposite root flanks at different distances from the root tip, we observed gravi-induced asymmetric pH changes at the surface of all investigated root zones. Upon gravistimulation, the surface pH decreased on the physically upper root flank and increased on the lower flank. The pH asymmetry appeared first [2.1±0.4 min (mean ± SD) after tilting] at the root cap and then – with incrementing lag times – at the meristem (after 2.5±0.3 min at 300 µm from root tip; after 3.7±0.4 min at 700 µm) and apical elongation zone (4.8±0.5 min at 1,000 µm), suggesting a basipetal progression of differential surface acidification at a rate of 250–350 µm min–1, consistent with reported auxin transport rates. [ABSTRACT FROM AUTHOR]

Highly purified tonoplast and plasma-membrane vesicles isolated from roots of Lepidium sativum L. (garden cress) were used as a starting material for generating a monoclonal antibody against plant tonoplast. Tonoplast vesicles were isolated by discontinuous-sucrose-gradient centrifugation followed by free-flow electrophoresis. The deglycosylated tonoplast fraction was used to generate monoclonal antibodies by immunization of Balb/c-mice and by fusion of their β-lymphocytes with the mouse cell line X 63 Ag 8.653. Using plasma membrane purified by two-phase partitioning and freeflow electrophoresis to define the negative signal in screening, and purified tonoplast to define the positive signal in screening, a monoclonal antibody (TOP 71) was obtained which recognized a tonoplast protein of 71 kDa by immunoblotting in cress-root membrane fractions. Two-dimensional gel electrophoresis, affinoblotting and binding to concanavalin A showed that the TOP 71-antigen was a glycosylated protein and had an isoelectric point (pI) of 4.5. The TOP 71-antigen was found in the different tissues of organs of several higher plants ( Glycine max L., Curcurbita pepo L., Zea mays L.) where it did not cross-react with the purified plasma-membrane fractions of these plants. Additionally, TOP 71 recognized its antigen in microsomal fractions of two lower plants ( Chara globularis Thuili., Matteucia struthiopteris Tod.). [ABSTRACT FROM AUTHOR]

Cress ( Lepidium sativum L.) roots were treated with 20 μM cyclopiazonic acid (CPA), an inhibitor of the Ca-transporting ATPases present in the sarcoplasmic/endoplasmic reticulum of animals and the endoplasmic reticulum of plants, in order to investigate its effect on the gravitropic response. Root growth was not significantly reduced by the applied dose of CPA, but the gravitropic response (curvature) was drastically inhibited. We hypothesize that the ER Ca-ATPase of statocytes is involved in transduction of the gravity stimulus and that CPA disturbs a cytosolic Ca signal necessary for graviperception. [ABSTRACT FROM AUTHOR]

Large-scale preparations of highly purified tonoplast and plasma-membrane vesicles were obtained from roots (garden cress, Lepidium sativum L.) and shoots (etiolated zucchini hypocotyl, Cucurbita pepo L.) of representative dicotyledonous seedlings. When tonoplast-enriched fractions of cress roots were prepared by centrifugation and then subjected to free-flow electrophoresis a highly purified tonoplast fraction was obtained. This fraction from cress roots was characterized by morphometry of filipin-treated freeze-fractured preparations and by enzymology to be about 90% homogeneous. Using latency of nitrate-inhibited ATPase and H-pumping as criteria we found that the majority of the tonoplast vesicles from both sources were oriented right(cytoplasmic)-side-out. Plasma-membrane vesicles were first purified by two-phase partitioning and then subjected to free-flow electrophoresis for further purification. From cress roots, the fraction of highest purity contained 89% plasma-membrane vesicles as judged by morphometry of filipin-treated, freeze-fractured preparations and by enzymology. From both sources, the major plasma-membrane subfraction in the upper phase after two-phase partitioning was shown to have the least electrophoretic mobility in free-flow electrophoresis and to be oriented right(extracytoplasmic)-side-out a slightly more mobile plasma-membrane subfraction was oriented inside-out and originated after freezing thawing from outside-out plasma-membrane vesicles. [ABSTRACT FROM AUTHOR]

During five rocket flights (TEXUS 18, 19, 21, 23 and 25), experiments were performed to investigate the behaviour of statoliths in rhizoids of the green alga Chara globularia Thuill. and in statocytes of cress ( Lepidium sativum L.) roots, when the gravitational field changed to approx. 10 · g (i.e. microgravity) during the parabolic flight (lasting for 301-390 s) of the rockets. The position of statoliths was only slightly influenced by the conditions during launch, e.g. vibration, acceleration and rotation of the rocket. Within approx. 6 min of microgravity conditions the shape of the statolith complex in the rhizoids changed from a transversely oriented lens into a longitudinally oriented spindle. The center of the statolith complex moved approx. 14 μm and 3.6 μm in rhizoids and root statocytes, respectively, in the opposite direction to the originally acting gravity vector. The kinetics of statolith displacement in rhizoids demonstrate that the velocity was nearly constant under microgravity whereas it decreased remarkably after inversion of rhizoids on Earth. It can be concluded that on Earth the position of statoliths in both rhizoids and root statocytes depends on the balance of two forces, i.e. the gravitational force and the counteracting force mediated by microfilaments. [ABSTRACT FROM AUTHOR]

Treatment of cress ( Lepidium sativum L.) roots with phytohormones (4.3-10 M gibberellic acid plus 4.3-10M 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. [ABSTRACT FROM AUTHOR]

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 μg\ml) or cytochalasin D (2.5 μg\ml) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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=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. [ABSTRACT FROM AUTHOR]

Electrical transmembrane potential differences and resistances in different tissues of intact root tips of Lepidium sativum L. were investigated in a humid atmosphere by conventional glass-microelectrode techniques with the reference electrode at the surface (apoplast) of the root. The resting potential (inside negative) in cells of the root cap rose from-80 mV in external cell layers (secretion cells) to approx.-140 mV in central cells (statocytes). Measurements of the electric input resistance within the apoplast of the root tip (calyptra, meristem and elongation zone) yielded a preference for longitudinal contact (resistance per length of tissue approx. 3.4 GOhm m) compared with transversal contact (approx. 14 GOhm m). Similarly, the symplastic coupling expressed as the characteristic length ( L) where a signal is reduced to 1/c compared with the origin yielded L=390 μm in the longitudinal ( y) direction and L=140 μm in the transversal ( x) direction. Cable analytical treatment of the symplastic input resistances (approx. 10 MOhm) resulted in low membrane resistances in the y-direction at the ends of cells compared with the membrane resistances in the x-direction (approx. 0.2 Ohm m) of the lateral membranes in the approximately cylindrical cells. This anisotropy is discussed in terms of model calculations. The resistivity of the symplast was calculated to be about 2.5 Ohm m. The input current-voltage relationship displayed a slight curvature with increasing slope for the more negative membrane potential typical of membranes with electrogenic pumps. Even after massive electrical stimulation in the range from-50 to-150mV carried out to trace current-voltage curves, electrical excitations (action potentials) were not detected in the cells investigated. [ABSTRACT FROM AUTHOR]

Growing root hairs of cress ( Lepidium sativum L.) were investigated using freeze-fracture and electron-microscopic techniques. Three zones of differentiation could be detected: the tip zone, the zone of vacuolation and the foot zone. Corresponding to these zones, the plasmatic fracture face of the plasma membrane showed areas of pronounced differentiation with respect to the distribution and frequency of intramembranous particles (IMPs). The tip zone was characterized by an irregular fracture plane caused by a large number of blisters which were more or less free of IMPs. These blisters coincided in size and shape with Golgi vesicles accumulated in the ground cytoplasm near the very tip. Outside these blisters, IMPs were randomly distributed. The surrounding cell wall was very thin and mainly composed of amorphous material. The plasma membrane of the vacuolation zone often revealed areas of hexagonally ordered particles (HOPS). Such patterns of particles were observed in chemically fixed and unfixed root hairs with a maximum surface density of 1200 HOPS per area. Mostly, however, 15-50 HOPS per area were found. The number of such areas increased with increasing distance from the tip up to five areas per μm. Additionally, imprints of large cellulose microfibrils could be detected in unfixed material; they were mainly parallel to the root-hair axis and sometimes ended in areas of HOPS. However, HOPS were observed only in approximately 60% of the root hairs. Otherwise, large areas free of IMPs were interspersed between areas of randomly distributed IMPs. The particle frequency was relatively low and varied greatly in the tip as well as in the vacuolation zone, that is, from 1200 to 2000 IMPs μm. Finally, the plasma membrane of the foot zone showed a very constant number of approx. 2000 IMPs μm. These particles were mainly distinct and randomly distributed. In this zone, HOPS were never observed in spite of the fact that the cell wall was composed of numerous parallel-running cellulose microfibrils. Since membrane material is mainly incorporated in the tip zone where IMPs are statistically distributed, the results indicate that the plasma membrane of the outgrowing part of the root-hair cells is characterized by a high lateral mobility of its components. Furthermore, they indicate that specifically arranged particles are involved in the synthesis of cellulose microfibrils. These areas of HOPS seem to be locally restricted and - or limited with respect to their lifetime. [ABSTRACT FROM AUTHOR]

When roots of Lepidium sativum L. are immersed in a colchicine solution (10 mol l), 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. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum membranes were isolated from roots of garden cress ( Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca transport (ruthenium red, LaCl and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanide m-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca transport function located on the endoplasmic reciculum of garden cress roots. [ABSTRACT FROM AUTHOR]

Photographic observations on germinating seedlings of Lepidium sativum L., Cucumis sativus L., and Helianthus annuus L. showed that the hypocotyl hook is not present in the seed but forms during the early stages of growth. Evidence that gravity plays a major role in inducing curvature of the hypocotyl, and in maintaining the hook once it has been formed, was obtained from clinostat experiments, from the use of morphactin to remove geotropic sensitivity and from inversion of seedlings to change the direction of the geostimulus. In L. sativum and H. annuus gravity perception seemed to be the only mechanism responsible for hook formation. In C. sativus hook formation was additionally aided by the mode of emergence of the cotyledons from the seed coat but gravity played an indirect role in regulating such emergence. Further evidence that hook formation is linked to a georesponse was derived from a comparison of hypocotyl development in wild-type Arabidopsis thaliana seedlings with that of an ageotropic mutant, hook formation being found to occur only in the wild type. Hook formation and maintenance is discussed in terms of contrasting geosensitivity between the apical and basal ends of the hypocotyl and it is suggested that light-induced hook opening is a reversal to a condition of uniformly negative georesponse throughout the hypocotyl. [ABSTRACT FROM AUTHOR]

The structural polarity of statocytes of Lepidium sativum L. is converted to a physical stratification by a root-tip-directed centrifugal acceleration. Sedimentation of amyloplasts and nucleus to the centrifugal (distal) cell pole and the lateral displacement of the distal endoplasmic reticulum (ER) complex occur after centrifugation for 20 min at an acceleration of 50 g. With higher doses (20 min, 100-2,000 g), smaller organelles become increasingly displaced. From the centrifugal to the centripetal cell pole, the following stratification is observed: 1) amyloplasts with mitochondria; 2) nucleus with mitochondria and a few dictyosomes, as well as laterally located ER; 3) dictyosomes with a few mitochondria; 4) vacuoles; and 5) lipid droplets. Within the first 7.5 min, after the roots have been returned to 1 g, the original arrangement of the amyloplasts sedimented on the underlying ER complex is reestablished in 66% of the statocytes. When roots previously centrifuged in an apical direction are exposed in a horizontal position to 1 g, the latent period of the graviresponse is increased by 7.5 min relative to the non-centrifuged controls. The kinetics of the response are identical to the controls. Roots centrifuged first in an apical direction and then for 2 h in a lateral direction (1,000 g) have statocytes with a physical stratification perpendicular to the root axis. A gravitropic curvature does not take place during the lateral centrifugation. These results support the hypothesis that the distal ER complex is necessary and sufficient for graviperception. [ABSTRACT FROM AUTHOR]

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 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. [ABSTRACT FROM AUTHOR]

Statocytes from roots of Lepidium sativum L., which developed after a 2-min soaking on a horizontal clinostat (2 rotations per min) for 44 h, exhibit the same polarity as in vertically grown roots, as indicated by a complex of endoplasmic reticulum (ER) cisternae at the distal cell pole. Amyloplasts are distributed randomly. The kinetics of graviresponse (=curvature) of such roots are identical to those of normally grown roots. Ten-minute exposure of the root, after 24 h development on the clinostat with gravity acting towards the root's basis (inversion), induces no changes in statocyte ultrastructure. However, corresponding exposure in normal orientation leads to subsequent disintegration of the distal ER complex, loss of amyloplast starch, confluence of lipid droplets, and an increase of the lytic compartment. These ultrastructural events thus appear to be induced by a physical contact - however short - between amyloplasts and the distal ER complex. [ABSTRACT FROM AUTHOR]

Using Concanavalin A (Con A) labeled with fluorescein isothiocyanate, we studied the intracellular localization of receptor molecules in the calyptra of 24-h dark-grown cress roots. Fixation in glutaraldehyde gave positive binding of the distal complex of the endoplasmic reticulum and the nucelus in the statocytes. In contrast, fixation in formaldehyde did not preserve the membrane-associated receptors, but revealed Con A affinity of the starch grain surface within the amyloplasts. Treatment of glutaraldehydefixed sections with non-ionic detergents led to partial solubilization of membrane components: the starch grain surface turned positive, though the positive binding of Con A to the endoplasmic reticulum and the nucleus remained unaffected. We therefore conclude that the Con A receptor in the membrane is a glycoprotein tightly inserted in other components of the compartment. [ABSTRACT FROM AUTHOR]

The amount of pectin held in cell walls by ionic bonds only was determined by extraction with cyclohexanediamine tetraacetic acid (CDTA) at room temperature, to remove calcium ions without degrading the galacturonan chains. Enzymic degradation was avoided by extracting the cell walls with phenol-acetic acid-water during preparation. From cell walls of celery petioles, cress hypocotyls and tomato and cucumber pericarp CDTA extracted 64-100 mg g pectin, leaving 80-167 mg g uronic acid in the residue. An additional extraction at high ionic strength was used to make the galacturonan chains more flexible and thus detach any pectins held by steric interactions, but the amount released in this way was small. Most of the residual uronic acid polymers could be extracted by cold alkali and remained soluble on neutralisation, showing that it was not water-insolubility that prevented their extraction with CDTA. Covalent bonding was thought more likely. [ABSTRACT FROM AUTHOR]