Pesticides and heavy metals in Danish streambed sediment

The role of streambed sediment as a sink for pesticides and heavy metals was investigated in 30 Danish lowland streams. The investigated streams drain catchments varying in hydrology, topography, soil type and land use. The <250 m newly accumulated fraction of the uppermost 1–2 cm layer of streambed sediment was analysed for 19 old and modern pesticides and 9 heavy metals. DDE was present in the sediment of all the streams. Of the herbicides, fungicides and insecticides currently in use, the most frequently detected was diuron (50.0%), fenpropimorph (66.7%) and lambda-cyhalothrin (6.7%), respectively. The pesticides detected in the highest concentration were fenpropimorph (1700 ng g^–1), propiconazole (130 ng g^–1) and isoproturon (110 ng g^–1). The heavy metals are listed in order of increasing median concentration: Cd (0.80 g g^–1), Co (9.1 g g^–1), As (12.0 g g^–1), Ni (19.0 g g^–1), Cr (19.2 g g^–1), Pb (19.7 g g^–1), Cu (20.1 g g^–1), V (28.5 g g^–1), Zn (103 g g^–1). The average number of pesticides detected in the 27 streams draining predominantly agricultural catchments was (3.7±2.0) being higher (p=0.077) than in the three streams draining non-agricultural catchments (1.7±0.6). Pesticides were significantly related to catchment size, soil type and hydrological regime. Several heavy metals (Cr, Cu, Pb, V and Zn) were related to urban activity and soil type.     

Nonidentity of the cDNA sequence of human breast cancer cell malic enzyme to that from the normal human cell

A cDNA coding for human breast cancer cell cytosolic NADP⁺-dependent malic enzyme was obtained. This cDNA is composed of a length of 2084 base pairs, with 1698 base pairs coding for 565 amino acid residues and a length of 386 base pairs representing a 3-noncoding region. Comparing this nucleotide sequence with that from the normal human tissue [Loeber, G., Dworkin, M. B., Infante, A., and Ahorn, H. (1994),FEBS Lett. 344, 181–186] reveals that three nucleotides in the open reading frame and the length of 3-noncoding region of the cDNA are different. One of the changes results in a substitution of serine at position 438 for proline, which, however, may not cause significant changes in the predicted secondary structure. A partial cDNA lacking the first 84 nucleotides in the open reading frame was successfully cloned and expressed functionally inEscherichia coli cells. ItsK _m value forl-malate (1.21±0.11 mM) is four times higher than that for the natural human breast cancer cell malic enzyme (0.29±0.04 mM) but similar to that for the full-length recombinant enzyme (1.06±0.07 mM). TheK _m values for Mn²⁺ and NADP⁺ (0.26±0.03 and 0.97±0.4M, respectively) are similar to those for the natural enzyme (0.12±0.02 and 1.9±0.3M, respectively) or the recombinant wild-type enzyme (0.56±0.04 and 0.44±0.02M, respectively). A recombinant pigeon liver malic enzyme without the first 13 amino acid residues was used for comparison. TheK _m values forl-malate and Mn²⁺ of the truncated enzyme (11.2±0.9 mM and 61.2±4.6M, respectively) are over 40 times larger than those for the natural pigeon liver malic enzyme (0.21±0.02 mM and 1.06±0.08M, respectively) or the recombinant wild-type enzyme (0.25±0.01 mM and 1.48±0.05M, respectively). We suggest that the N-terminus of malic enzyme may be required for the substrate binding during the catalytic cycle.     

Conversion of phenylpyruvate to l-phenylalanine by immobilized Clostridium butyricum-alanine dehydrogenase-Micrococcus luteus under hydrogen high pressure

Summary Alanine was the best amino donor among various amino acids and NH₄Cl for the phenylalanine production of Micrococus luteus. l-Alanine was regenerated at the rate of 9.2 moles/min/g dry cells from NH₄Cl and pyruvate by immobilized Clostridium butyricum-alanine dehydrogenase. l-Phenylalanine was continuously produced from hydrogen, NH₄Cl and phenylpyruvate by coupling immobilized C. butyricum, alanine dehydrogenase and M. luteus. The rate of phenylalanine production was 1.74 moles/min/g dry cells.     

Kinematographische Studien zur Wasserpermeabilität von Tonoplasten

Summary With means of cinemicrographic methods it is tried to determine absolute values (2Kinwo) for the water permeability of tonoplasts of the inner epidermis cells ofAllium cepa bulbs as exactly as possible. Compared with uninjured protoplasts, tonoplasts show a water permeability which is mostly 20–30 times higher, largely independent on various kinds of pretreatment and/ or influences on the cell.Isoptin or EDTA for instance enhance the permeability for water as well of protoplasts as of tonoplasts. Slighter differences are found in cases of Alterungstonoplasten with controlexperiments. The water permeability of this kind of tonoplast is only 3 to 6 times higher as that of the corresponding protoplasts.The influence of various error sources on the value of the water permeability constant 2Kinwo is discussed guided by some select examples. To the facts influencing the value belong besides the position of the pair of points on the water permeability curve, especially also the selection of the exact value forl ₀. The influence of ion permeability on which also thel ₀-value is partly dependent is one fact to be considered.     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Rapid separation of the plastid,mitochondrial, and cytoplasmic fractions from intact leaf protoplasts of Avena

Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-m aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 l microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.Abbreviations BSA bovine serum albumin - Cyt c cytochrome c - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglyceric acid - PEP phosphoenol pyruvic acid - RuBP ribulose-1.5-bis-phosphate     

Scanning electron-microscopic study on the three-dimensional structure of motor endplates of the slow (tonic) muscle fibers in the frog,Rana n. nigromaculata

Summary The three-dimensional organization of the motor endplates of the slow fibers of the rectus abdominis muscle in the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) is visualized by use of a field-emission scanning electron microscope after removal of connective tissue components by HCl hydrolysis. Clusters of shallow oval depressions 1–3 m in diameter are seen in the postsynaptic membrane at intervals of about 150 m. On the surface of these depressions, a few low bulges of postsynaptic membrane are irregularly arranged. Terminal boutons, 1–3 m in diameter, occur along the length of nerve branches and terminals and fit into the shallow oval depressions of the postsynaptic membrane. The Schwann cells covering the terminal branches exhibit a simpler organization than those in twitch fibers.     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

Purification and characterization of phenylacetate-coenzyme A ligase from a denitrifying Pseudomonas sp., an enzyme involved in the anaerobic degradation of phenylacetate

The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP phenylacetyl-CoA+AMP+PP_i and requires Mg²⁺. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 mol min^-1 x mg^-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 mol min^-1 mg^-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn²⁺ can substitute for Mg²⁺. The apparent K _m values for phenylacetate, CoA, and ATP are 60, 150, and 290 M, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.     

Characterization of photosystem II in stroma thylakoid membranes

The functional state of the PS II population localized in the stroma exposed non-appressed thylakoid region was investigated by direct analysis of the PS II content of isolated stroma thylakoid vesicles. This PS II population, possessing an antenna size typical for PS II, was found to have a fully functional oxygen evolving capacity in the presence of an added quinone electron acceptor such as phenyl-p-benzoquinone. The sensitivity to DCMU for this PS II population was the same as for PS II in control thylakoids. However, under more physiological conditions, in the absence of an added quinone acceptor, no oxygen was evolved from stroma thylakoid vesicles and their PS II centers were found to be incapable to pass electrons to PS I and to yield NADPH. By comparison of the effect of a variety of added quinone acceptors with different midpoint potentials, it is concluded that the inability of PS II in the stroma thylakoid membranes to contribute to NADPH formation probably is due to that Q_A of this population is not able to reduce PQ, although it can reduce some artificial acceptors like phenyl-p-benzoquinone. These data give further support to the notion of a discrete PS II population in the non-appressed stroma thylakoid region, PS II, having a higher midpoint potential of Q_A than the PS II population in the appressed thylakoid region, PS II. The physiological significance of a PS II population that does not produce any NADPH is discussed.Abbreviations pBQ p-benzoquinone - Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCIP 2,6-dichloroindophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMBQ 2,5-dimethyl-p-benzoquinone - DQ duroquinone(tetramethyl-p-benzoquinone) - FeCN ferricyanide (potassium hexacyanoferrat) - MV methylviologen - NADPH,NADP⁺ reduced or oxidized form of nicotinamide adenine dinucleotide phosphate respectively - PpBQ phenyl-p-benzoquinone - PQ plastoquinone - PS II photosystem II - PS I photosystem I - Q_A primary quinone acceptor of PS II - Q_B secondary quinone acceptor of PS II - E microEinstein     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Effect of extracellular calcium on the contractility of warm-and cold-acclimated crucian carp heart

Crucian carp (Carassius carassius L.) were acclimated for at least 4 weeks to 2°C or 22°C, and the consequences of thermal acclimation on force development, time-course of contraction and action potential duration of the ventricular myocardium were studied. In cold-acclimated fish contraction was activated at much lower external [Ca] than in warm-acclimated fish: [Ca] for half-maximal force was 0.9±0.15 and 3.1±0.92 mmol·l^-1 (P<0.05) for cold- and warm-acclimated fish, respectively. Durations of contraction and relaxation were significantly longer in fish acclimated to 2°C than in fish acclimated to 22°C, especially at [Ca] below 2 mmol·l^-1. In low-Ca solution ventricular action potential was prolonged both in cold- and warm-acclimated fish. In 0.5 mmol·l^-1 Ca action potential duration at zero voltage level was longer in cold- than warm-acclimated fish. Although lengthening of action potential was evident in both acclimation groups, a marked prolongation of contraction duration by low-Ca solutions occurred only in cold-acclimated fish. This suggests that a plateau component of contraction is present in cold-acclimated fish but less well developed in warm-acclimated fish hearts. Contractions were strongly inhibited by sarcolemmal Ca-channel blocker, cadmium (100 and 300 mol·l^-1), in both warm- and cold-acclimated crucian carp hearts. However, the sarcoplasmic reticulum Ca release channel blocker, ryanodine (10 mol·l^-1), had no effect on the force of contraction in either acclimation group. These results suggest that the contraction of crucian carp heart is controlled by sarcolemmal mechanisms without contribution by sarcoplasmic reticulum Ca release. Since the Ca sensitivity of myofilaments was not altered by thermal acclimation, the results indicate that thermal acclimation alters Ca activation of contraction of the crucian carp heart at the level of sarcolemma.Abbreviations AP action potential - EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N-tetra-acetic acid - F _max maximum force - F _max maximum rate of contraction - F _min maximum rate of relaxation - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - pCa log [Ca] - Pl action potential plateau - SL sarcolemma - SR sarcoplasmic reticulum - TPF time to peak force - T1/2R time to half relaxation from the peak force     

Competence for gene transfer by electroporation in a sub-population of protoplasts from uniform carrot cell suspension cultures

We have investigated the basis for increased transient reporter gene expression following electroporation of protoplasts from uniform carrot cell suspension cultures at increasing DNA concentrations. Use of a combination of histochemical and fluorometric GUS gene assays allowed differentiation between increases due to a higher proportion of expressing protoplasts and increases due to higher expression by each expressing protoplast. A plateau of 20–25% expressing protoplasts was reached by 50 g ml^–1 DNA but total expression continued to increase in direct proportion to applied DNA concentration up to at least 100 g ml^–1. This indicates the existence of a subpopulation of protoplasts competent for the uptake and expression of genes by electroporation.     

Delivery of O2 to bacteroids in soybean nodule cells: Consideration of gradients of concentration of free,dissolved O2 in and near symbiosomes and beneath intercellular spaces

Summary Based on a simulation model of the structure of and distribution of O₂ within infected cells of soybean nodules, gradients of concentration of dissolved O₂ ([O₂]) have been calculated within and between symbiosomes embedded in host cytoplasm, through which the flux of O₂ to the symbiosomes is facilitated by leghaemoglobin. As a consequence of facilitation, gradients of [O₂] in cytoplasm between symbiosomes are very small. Within symbiosomes, from which leghaemoglobin is considered to be absent, respiration by bacteroids generates steeper gradients of [O₂], thus restricting respiration and N₂ fixation. However, if bacteroid mass is considered to be randomly distributed within a symbiosome, about 80% of this mass lies within about 0.6 m of the surface (the peribacteroid membrane). Consequently, respiration within a symbiosome was calculated to be between 65% and 92% of that attained if bacteroids were directly in contact with the cytoplasm. For N₂ fixation, the corresponding values were 44% to 91%. In cytoplasm, near the surface of a symbiosome, there is a boundary layer in which equilibrium between O₂, leghaemoglobin and oxyleghaemoglobin is perturbed by O₂ consumption within. Calculations of the thickness of the boundary layers gave values of only 3.65 to 3.75×10^–9 m, thus they had little effect on calculated gradients of [O₂] in cytoplasm. In contrast, perturbations of the leghaemoglobin oxygenation equilibrium affected layers of cytoplasm beneath intercellular spaces to a depth of 0.15 to 0.3×10^–6 m in the physiological range of volume average [O₂]. This affected gradients of [O₂] in the cytoplasm near intercellular spaces. Revisions have been made to the model cell, incorporating these new calculations. Results suggest that infected nodule cells may be able to withstand 1–2 M O₂ in the outermost layers of cytoplasm without inhibition of N₂ fixation caused by excessive O₂ within the symbiosomes.Abbreviations Lb leghaemoglobin - LbO₂ oxyleghaemoglobin - [O₂] concentration of free, dissolved O₂ - Y fractional oxygenation of Lb - Y _av volume averagedY     

Molecular environment of the phencyclidine binding site in the nicotinic acetylcholine receptor membrane

Summary Phencyclidine is a highly specific noncompetitive inhibitor of the nicotinic acetylcholine receptor. In a novel approach to study this site, a spin-labeled analogue of phencyclindine. 4-phenyl-4-(1-piperidinyl)-2.2.6.6.-tetramethylpiperidinoxyl (PPT) was synthesized. The binding of PPT inhibits⁸⁶Rb flux (IC₅₀=6.6m), and [³H] phencyclidine binding to both resting and desensitized acetylcholine receptor (IC₅₀=17 m and 0.22 m, respectively). From an indirect Hill plot of the inhibition of [³H]phencyclidine binding by PPT. a Hill coefficient of approximately one was obtained in the presence of carbamylcholine and 0.8 in -bungarotoxin-treated preparations. Taken together, these results indicate that PPt mimics phencyclidine in its ability to bind to the noncompetitive inhibitor site and is functionally active in blocking ion flux across the acetylcholine receptor channel. Analysis of the electron spin resonance signal of the bound PPT suggests that the environment surrounding the probe within the ion channel is hydrophobic, with a hydrophobicity parameter of 1.09. A dielectric constant for the binding site was estimated to be in the range of 2–3 units.     

Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Summary Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4–5.8 with citrate buffer; concentration of poly-L-ornithine, 0.8–1.0 g/ml; concentration of TYMV, 0.2–1.0 g/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced 1 to 2x10⁶ TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.     

Xylogenesis and lignification in plant tissue cultures differently affected by 8-hydroxyquinoline sulphate

Summary An established callus tissue derived from mesophyll cells ofSedum telephium L. was used to assess the influence of 8-hydroxyquinoline sulphate (Quinosol) on the differentiation of tracheary cells.Electron microscopic study of cells from calli grown in the presence of Quinosol (20.55 and 41.10 M) showed no marked ultrastructural differences compared to controls at the same developmental stages. Growth of calli diminished with increasing concentrations of Quinosol, being drastically affected by the maximum dose used (82.20 M). On the other hand, xylogenesis, expressed as the number of tracheary elements to parenchyma cells, increased with increasing concentrations of the drug. However, lignin and phenolic content responded differently, increasing at a low Quinosol dose (20.55 M) but decreasing at higher concentrations of the drug (41.10 and 82.20 M).The impaired growth of the culture was due to decreased cell proliferation in spite of an increase in differentiation of xylem-like cells. However, the calli with numerous tracheary elements synthesized less lignin than calli with a lower percentage of such cells.     

Measurement of guard-cell respiration rates using Cartesian-diver technique

Dark respiration rates of guard-cell protoplasts of Commelina communis L. were measured over a temperature range (15–30° C) using a Cartesian-diver microrespirometry technique. Measurements were made using a few microliters of suspension medium containing between 400 and 3 700 protoplasts. Respiration rates were approximately linear for at least 1 h at all temperatures. Respiration rates increased rapidly between 20 and 25° C to relatively high levels (6.11·10^-6 mol O₂ h^-1 protoplast^-1=1259 mol O₂ mg^-1 chlorophyll h^-1=22.97 mol O₂ mg^-1 protein h^-1) with no further increases above this temperature. Respiration rates were much lower in protoplasts 15–16 h old than in freshly prepared ones indicating considerable deterioration of their viability over this time period.     

Gamete Structure and Fertilization in the Barents Sea Sponge Leucosolenia complicata

The ultrastructure of male and female gametes of asconoid sponge Leucosolenia complicata(Calcispongiae, Calcaronea), a hermaphrodite species that reproduces in autumn, is described. The mature sponge's oocytes were up to 70 m in diameter, had no coatings, and contained a nucleus about 31 m in diameter with large nucleoli (up to 6.6 m). There were vacuoles with fibrillar contents typical of calcareous sponges in ooplasm. During vitellogenesis, a cluster of a great number of nurse cells developed above each oocyte from transformed choanocytes. Mature spermia of L. complicatalooked like orbicular cells about 2.5 m in diameter, with no acrosome or tail. The spermium nucleus (diameter about 2.2 m) was formed by incompletely condensed chromatin and was surrounded with a thin layer of cytoplasm of nonuniform thickness. In the thick layer of cytoplasm beyond the ribosomes, there were two or three mitochondria, dictyosomes, and electron-dense protein bodies lying freely under the nucleus. Fertilization occurred with the aid of a carrier cell. During spawning (mass release of spermia), any nurse cell complex can seize a spermium and transform into a carrier cell in situ. The transformation of a seized spermium into a spermiocyst was connected with the rapid isolation of the spermium nucleus from the protein body. Fertilization began with the penetration of the protein body into the oocyte cytoplasm. Only after this did the spermium's nucleus penetrate into the oocyte.     

Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts

A functional assay for gibberellin (GA) receptors is described based on the induction of -amylase gene expression in isolated aleurone protoplasts of Avena fatua L. by GA₄ immobilised to Sepharose beads. A 17-thiol derivative of GA₄, shown to be biologically active with aleurone protoplasts, has been coupled to epoxy-activated Sepharose 6B. This GA₄-17-Sepharose induces high levels of -amylase when incubated with isolated aleurone protoplasts, while cells of the intact aleurone layer do not respond appreciably to the immobilised GA₄. In order to eliminate the possibility that GA₄ may be released from the Sepharose when incubated with protoplasts, aleurone layers and isolated aleurone protoplasts have been co-incubated, and their responses to GA₄, GA₄-17-Sepharose and control Sepharose estimated by determining the relative amounts of -amylase mRNA induced in each tissue. Evidence from these experiments is consistent with the view that GA₄17-Sepharose induces -amylase gene expression in aleurone protoplasts by interacting with the protoplast surface. This indicates that GA receptors may be located at, or near, the external face of the aleurone plasma membrane.Abbreviation GA(n) gibberellin A(n) We thank Professor Jake MacMillan and Drs. Peter M. Chandler (CSIRO, Division of Plant Industry, Canberra, Australia), Peter Hedden and Johnathan Weir (Unilever, Port Sunlight, UK) for helpful discussions and suggestions. Computer graphics were performed by the University of Bristol Molecular Recognition Centre.