Spinning of a gigantic bundle of hollow fibrils by a spirally moving higher plant protoplast

A unique fiber spinning was found in protoplasts from white birch (Betula platyphylla) leaves under an acidic medium containing high concentration of Ca²⁺. After expanding from 10 to 100 μm in diameter under the culture condition, the protoplast started secreting a gigantic fiber while moving in a spiral way. Real time video analyses elucidated that the orientation, rate and pattern of the motion were directed due to the inverse force of the fiber spinning. Moreover, observation using several microscopic methods accompanied with histochemical staining and nuclear magnetic resonance (NMR) analysis indicated that the fiber was composed of 400–500 nm wide (1→3)-β-glucan hollow sub-fibrils. This entire phenomenon may be a response against the stress imposed. The observation presented provides an understanding of the unique relationship between fiber spinning and the bottom-up fiber fabrication from nano to micro scales.     

Intergeneric gene transfer mediated by plant protoplast fusion

Summary In attempts at somatic transfer of plant genomes of reduced size, X-irradiated leaf protoplasts of parsley (Petroselinum hortense, 2n=22) were fused with cell culture protoplasts of a nuclear albino mutant of carrot (Daucus carota, 2n=18). Introduction of genes from the irradiated parsley nuclei into the carrot genome was shown by the correction of the albino defect and by the appearance of parsley isoenzymes in selected green tissues and plants. The cytological studies provided information on significant deviation from the amphidiploid chromosome number. The high frequency of cells with 2n=19, 2n=38 and regeneration of plants with 2n=19 chromosomes can indicate that the elimination of parsley chromosomes is incomplete. A correlation was found between the lethality of selected tissues and differentiated or undifferentiated stages of the cells.     

Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

植物原生质体培养研究进展

自从Ｃｏｃｋｉｎｇ(196 0 )用酶法首次分离出有活性的原生质体 ,1971年Ｔａｋｅｂｅ等首次从烟草叶片分离原生质体 ,经培养获得再生植株 ,原生质体的研究和应用进入了一个新阶段。1　原生质体培养影响因子的研究1 1　培养基种类及成分不同植物原生质体培养的基本培养基不尽相同。培养基种类影响到原生质体的分裂频率、植板率以及小愈伤组织的出现等[1,2 ] 。潘增光[3 ] 等比较了 4种培养基 (ＭＳ、ＭＴ、改良ＭＴ、ＢＨ3)对苹果叶肉原生质体培养的影响 ,结果表明 ,只有在改良ＭＴ培养基中能观察到多细胞团形成 ,而在ＭＳ、ＭＴ、ＢＨ3作为…     

Selection and enrichment of plant protoplast heterokaryons of Brassicaceae by flow sorting

The experimental conditions for an efficient and reproducible enrichment of fusion products by flow cytometry, using protoplasts of different Brassica species as hybridization material, have been investigated. The heterokaryons were identified by the endogenous chlorophyll autofluorescencence of mesophyll protoplasts of one parent and the fluorescense of exogenously supplied carboxyfluorescin to the hypocotyl protoplasts of the other parent. By using a low head drive frequency (11 kHz), a large nozzle (110 μm) and a low nozzle pressure (30–35 kPa) good survival of the protoplasts was obtained after sorting. Heterokaryons were sorted using these parameters and on average 80% of the protoplasts were fusion products as judged by microscopy. They were cultured in small volumes, 150 μl, and started to divide after 3–5 days and regenerated calli easily. Isozyme analysis of the calli confirmed that 81% had the pattern typical for a hybrrid. Differentiation into shoots have been obtained from some of the hybrid calli; these shoots were also confirmed to be true hybrids.     

Filter paper as a support and carrier for plant protoplast cultures

Summary Filter paper laid upon an agar medium provided a convenient and advantageous physical substrate for the culture of plant protoplasts. The technique simplified the changing of media and, inPteridium aquilinum, enhanced the regenerative response of the protoplasts, This work was carried out primarily in the Department of Botany, University of Nottingham, England, while the author was on sabbatical leave, 1978–1979.     

Calculation of protoplast freezing strain in multicellular plant tissues

A method for calculating the contraction strain (or the converse stress) to protoplasts of frozen multicellular plant tissues is described. The method requires (i) a nuclear magnetic resonance (NMR) measure of the quantity of bound water per gram dry weight (K), (ii) a gravimetric measure of grams of H₂O per gram dry weight (L₀), (iii) a measure of solute concentration in non-frozen cells (C₀), (iv) an estimate of the specific volume of tissue dry matter (0.625 ml/g), (v) an NMR measure of the fraction of tissue water that is intracellular osmotic water (P_a), and (vi) a measure of the fraction of the dry weight that is cell wall (f_cw). This method is a refinement of previous methods that calculate cell contraction strain from four (L₀, K, C₀, and 0.625) of the above six measurements. Comparison of the calculated protoplast strain to the calculated cell strain indicates that the two measures are quite similar, however, the measure of protoplast strain is, in theory, a more appropriate measure of the freezing strain. It is also demonstrated that derivation of a measure of strain from the parameters controlling it is useful, because it allows one to evaluate the relative contribution of each parameter in preventing the development of strain.     

Genetic changes induced in higher plant cells by a laser microbeam

Introducing foreign genes into higher plants has proved to be complicated, with the exception of transformation of protoplasts of some plants (Negrutiu et al. 1987). In particular, culture of protoplasts and regeneration to plants are difficult in many monocotyledonous crops. Therefore, it would be desirable to avoid extensive tissue culture by introducing cloned genes directly into cells. A laser microbeam can perforate plant cell walls, thus facilitating uptake of genes into cells.     

Mechanisms of potassium absorption by higher plant roots

Potassium, as a plant macronutrient, is accumulated in plant cells from relatively dilute soil solutions and is indispensable for many vital processes. Studies characterising potassium uptake by roots stretch back over many decades. However, it is only with the introduction of modern electrophysiological and molecular techniques that investigations have been possible at a molecular level. Such approaches have confirmed the existence of discrete high and low affinity uptake systems at the root plasma membrane and have greatly enhanced our understanding of the underlying molecular nature of these uptake systems.
High affinity K⁺ uptake from micromolar external K⁺ levels is coupled to H⁺ transport as demonstrated independently by patch clamping of single root protoplasts and by studying the transport system after expression in Xenopus oocytes . The measured coupling ratio between the two ions is 1:1 and is sufficient to account for an accumulation ratio in excess of 10⁶, a value which encompasses experimental observations on K⁺ accumulation.
Low affinity K⁺ uptake activates at relatively high external K⁺ levels in the millimolar range and is 'passive' i.e. down the electrochemical gradient for potassium. In two higher plant species single cell inward potassium currents have been identified which are associated with low affinity potassium uptake. Furthermore, specific ion channels which underlie these potassium influxes and form a major constituent of the low affinity potassium uptake pathway have been identified and characterised.     

Molecular characterization of glyoxalase-I from a higher plant; upregulation by stress

A cDNA, GLX1, encoding glyoxalase-I was isolated by differential screening of salt-induced genes in tomato. Glyoxalases-I and-II are ubiquitous enzymes whose functions are not clearly understood. They may serve to detoxify methylglyoxal produced from triosephosphates in all cells. The protein encoded by GLX1 shared 49.4% and 58.5% identity with glyoxalase-I isolated from bacteria and human, respectively. Furthermore, yeast cells expressing GLX1 showed a glyoxalase-I specific activity 20-fold higher than non-transformed cells. Both GLX1 mRNA and glyoxalase-I polypeptide levels increased 2- to 3-fold in roots, stems and leaves of plants treated with either NaCl, mannitol, or abscisic acid. Immunohistochemical localization indicated that glyoxalase-I was expressed in all cell types, with preferential accumulation in phloem sieve elements. This expression pattern was not appreciably altered by salt-stress. We suggest that the increased expression of glyoxalase-I may be linked to a higher demand for ATP generation and to enhanced glycolysis in salt-stressed plants.     

l-Canavanine: a higher plant insecticidal allelochemical

Summary. l-Canavanine, l-2-amino-4-(guanidinooxy)butyric acid, is a potentially toxic nonprotein amino acid of certain leguminous plants. Many species are prolific canavanine producers; they divert enormous nitrogen resource to the storage of this single natural product. Canavanine, a highly effective protective allelochemical, provides a formidable chemical barrier to predation and disease. The accumulated experimental evidence leaves little doubt that the key element in the ability of canavanine to function as an effective protective allelochemical is its subtle structural mimicry of arginine which makes it an effective substrate for amino acid activation and aminoacylation, and its marked diminution in basicity relative to arginine which mediates the production of structural aberrant, dysfunctional canavanyl proteins. The biological burdens of canavanyl protein formation by canavanine-treated Manduca sexta larvae were carried throughout their remaining life cycle. Protein-based sequestration of canavanine prevented turnover and clearance of the free amino acid, and undoubtedly contributed significantly to the antimetabolic character of this protective allelochemical. Received February 1, 2000 Accepted June 4, 2000     

Manganese superoxide dismutase from a higher plant

A manganese-containing superoxide dismutase (EC 1.15.1.1) was purified to homogeneity from a higher plant for the first time. The enzyme was isolated fromPisum sativum leaf extracts by thermal fractionation, ammonium sulfate salting out, ion-exchange and gel-filtration column chromatography, and preparative polyacrylamide gel electrophoresis. Pure manganese superoxide dismutase had a specific activity of about 3,000 U mg^-1 and was purified 215-fold, with a yield of 1.2 mg enzyme per kg whole leaf. The manganese superoxide dismutase had a molecular weight of 94,000 and contained one g-atom of Mn per mol of enzyme. No iron and copper were detected. Activity reconstitution experiments with the pure enzyme ruled out the possibility of a manganese loss during the purification procedure. The stability of manganese superoxide dismutase at-20°C, 4°C, 25°C, 50°C, and 60°C was studied, and the enzyme was found more labile at high temperatures than bacterial manganese superoxide dismutases and iron superoxide dismutases from an algal and bacterial origin.Abbreviations NBT nitro blue tetrazolium - SOD superoxide dismutase (EC 1.15.1.1)     

Enrichment for heterokaryocytes by the use of iso-osmotic density gradients after plant protoplast fusion

Summary Buoyant density differences between different types of protoplasts were used in an iso-osmotic density gradient system to enrich protoplast fusion mixtures for heterokaryocytes. Protoplasts of maize stem and wheat mesophyll, as well as epidermis, stem parenchyma and mesophyll protoplasts of two amphihaploid, light sensitive tobacco mutants were fused with polyethylene glycol using conventional methods and a new rolling tube technique. The protoplast combinations used for fusion involved protoplast types with considerably different buoyant densities. Enriched fractions of maize-wheat heterokaryocytes of intermediate density were recovered which contained up to 31% mostly binucleate heterokaryocytes (a 2–7 fold relative enrichment). Tobacco heterokaryocytes recovered analogously from enriched intermediate fractions readily divided and gave rise to an increased number of light resistant calluses when compared with cultures from non-fractionated fusion mixtures. Maize-wheat fusion products, however, failed to divide.