Thigmomorphogenesis: XXIII. Promotion of foliar senescence by mechanical perturbation of Avena sativa and four other species

Mechanical perturbation (MP, gentle tubbing) promoted the senescence of detached oat ( Avena sativa L. cv. Victory) leaf segments in the dark. The promotion of senescence increased with increase in the number of rubbings and could be seen after 24 h of dark incubation; the maximum effect was reached on day 3. The effect (% of control) of MP on the loss of protein was greater than the effect on chlorophyll (Chl) loss on day 1. However, on day 3 the effect of MP on the loss of Chl became greater than the effect on the loss of protein. Ethephon and 1-aminocyclopropane-1-carboxylic acid (ACC) marginally promoted the loss of Chl by both control and rubbed oat leaf segments, and the effect was additive with MP. Chloramphenicol (CAP), spermine, aminoethoxyvinylglycine (AVG) and Ca²⁺ marginally delayed the loss of Chl and protein in both control and rubbed segments. Kinetin greatly retarded the senescence of all segments. Even in the presence of these substances, the amounts of Chl and protein in the rubbed segments were always less than in their respective controls, thus retaining the effect of the MP. However, abscisic acid (ABA) and cycloheximide (CHI) caused the rubbed oat leaf segments to retain more Chl and protein than their respective control segments. The effect of CHI was actually enhanced by MP. Rubbing promoted the senescence of attached leaves of oats ( Avena sativa L. cv. Victory), maize ( Zea mays L. cv. Early Belle) and pumpkin ( Cucurbita pepo L. cv. Jack-o-lantern) cotyledons in the dark. Rubbing promoted the senescence of oat leaf segments even in light, although to a lesser extent compared to the effect in the dark. The senescence of leaves of pumpkin and cocklebur ( Xanthium strumarium Wallr. var. Pennsylvanicum ) in situ was also enhanced by MP.     

Thigmomorphogenesis: Evidence for a translocatable thigmomorphogenetic factor induced by mechanical perturbation of beans (Phaseolus vulgaris)

Mechanical perturbation of bean (Phaseolus vulgaris L.) internodes results in reduced elongation and increased diameter of the internodes (thigmomorphogenesis). Perturbation of a single lower internode results in thigmorphogenesis in that internode and all of those internodes above it, the degree of which depends on the age (size) of the internodes and the frequency of perturbation. Application of ethephon to the internodes mimics mechanical perturbation. Early removal of the shoot tip or the cotyledons does not effect thigmomorphogenesis, indicating that those organs do not exert control over the response. Mechanical perturbation of one plant of a pair grafted together at the first internodes results in thigmomorphogenesis in both plants. This indicates the transport of some factor from the mechanically perturbed donor to the non-treated receiver. Evidence is presented to support the contention that ethylene is not this transportable factor.     

Polyribosomes in protoplasts isolated from tobacco leaves

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.Abbreviations EGTA ethylene glycol bis (2-aminoethyl ether)-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - mRNA messenger ribonucleic acid - RNase ribonuclease - Tris tris(hydroxymethyl)aminomethane - TCA trichloroacetic acid     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa)

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 M indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl₂) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA-or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.     

Grain yield of common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) varieties is markedly increased by rhizobial inoculation and phosphorus application in Ethiopia

A field experiment was conducted to assess plant growth, symbiotic performance and grain yield of common bean in response to rhizobial incoculation and phosphorus application at Galalicha in Southern Ethiopia during the 2012 and 2013 cropping seasons under rain-fed conditions. The treatments consisted of 2 released common bean varieties (Hawassa Dume and Ibbado), 3 levels of Rhizobium inoculation (uninoculated, inoculated with strain HB-429 or GT-9) and 4 levels of phosphorus application (0, 10, 20 and 30 kg P ha^?1) using a split-split plot design with four replications. Here, phosphorus levels, Rhizobium inoculation and common bean varieties were assigned as main, sub- and sub-sub treatments, respectively. The results revealed marked varietal differences in plant growth, grain yield and symbiotic performance. Of the two common bean varieties studied, Hawassa Dume generally showed superior performance in most measured parameters in 2013. Rhizobium inoculation significantly (p?≤?0.05) increased plant growth, symbiotic performance and grain yield. Applying Rhizobium strain HB-429 to bean crop respectively increased plant growth, %Ndfa, amount of N-fixed and grain yield by 19, 17, 54 and 48% over uninoculated control. Similarly, the application of 20 kg P ha^?1 to bean plants respectively resulted in 36, 20, 96 and 143% increase in plant growth, %Ndfa, N-fixed and grain yield when compared to the control. These results clearly indicate that plant growth, symbiotic performance and grain yield of common bean can be significantly increased by Rhizobium inoculation and phosphorus fertilization in Ethiopia. Rhizobium inoculants are a cheaper source of nitrogen than chemical fertilizers and when combined with moderate phosphorus application can markedly increase grain yield for resource-poor farmers.     

Antibodies to cell-surface antigens of plant protoplasts

Both mouse and rabbit polyclonal antibodies to plant cell-surface antigens were developed by immunization with cell membrane material from oat (Avena sativa L. cv. Garry) roots. We were able to quickly assess the activity of antisera by monitoring the degree of protoplast agglutination and by using an indirect immunofluorescence assay. Using polyclonal antibodies to cell-surface antigens, we have found that oat root protoplasts share common surface antigens with protoplasts from other plant tissues and species. From experiments with antisera treated with excess oat leaf or oat root protoplasts before our immunoassays, we have obtained evidence for the existence of organ-specific cell-surface antigens in higher plants.     

Intracellular protein phosphorylation in oat (Avena sativa L.) protoplasts by phytochrome action. 1. Measurement of action spectra for the protein phosphorylation

The effects of red light and wavelength dependency of the protein phosphorylation in oat protoplasts were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Red light (660 nm) irradiation of the protoplasts increased the phosphorylation of 15 different proteins, and the phosphorylation of 2 proteins (27 KDa, 32 KDa) out of 15 were observed to be dependent on the wavelength of the irradiating light. The phosphorylation densities of these two proteins increased up to two or three hundred percent during a three-minute period of irradiation. The phosphorylation of these two proteins revealed a red/far-red photoreversibility of phytochrome. When a calcium ion chelator (2 mM EGTA) was added into the cell suspension, the phosphorylations of all the proteins were reduced about 200%. These findings suggest that phytochrome action and Ca2+ influx are certainly involved in the in vivo phosphorylation of proteins in oat protoplasts.     

Specificity patterns indicate that auxin exporters and receptors are the same proteins

A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.     

Xylem development and hydraulic conductance in sun and shade shoots of grapevine (Vitis vinifera L.): evidence that low light uncouples water transport capacity from leaf area

Morphology, water relations, and xylem anatomy of high-light (sun)- and low-light (shade)-grown Vitis vinifera L. shoots were studied to determine the effects of shading on the hydraulic conductance of the pathway for water flow from the roots to the leaves. Shade shoots developed leaf area ratios (leaf area: plant dry weight) that were nearly threefold greater than sun shoots. Water-potential gradients (·m^–1) in the shoot xylem accounted for most of the ·m^–1 between soil and shoot apex at low and high transpiration rates in both sun and shade shoots, but the gradients were two- to fourfold greater in shade-grown plants. Low light reduced xylem conduit number in petioles, but had an additional slight effect on conduit diameter in internodes. The hydraulic conductance per unit length (Kh) and the specific hydraulic conductivity (k_s, i.e. Kh per xylem cross-sectional area) of internodes, leaf petioles, and leaf laminae at different developmental stages leaf plastochron index was calculated from measurements of water potential and water flow in intact plants, from flow through excised organs, and from vessel and tracheid lumen diameters according to Hagen-Poiseuille's equation. For all methods and conductance parameters, the propensity to transport water to sink leaves was severalfold greater in internodes than in petioles. The Kh and k_s increased logarithmically until growth ceased, independent of treatment and measurement method, and increased further in pressurized-flow experiments and Hagen-Poiseuille predictions. However, the increase was less in shade internodes than in sun internodes. Mature internodes of shade-grown plants had a two- to fourfold reduced Kh and significantly lower k_s than sun internodes. Except very early in development, leaf lamina conductance and k_s from shade-grown plants was also reduced. The strong reduction in Kh with only a slight reduction in leaf area (17% of sun shoots) in the shade shoots indicated a decoupling of water-transport capacity from the transpirational surface supplied by that capacity. This decoupling resulted in strongly reduced leaf specific conductivities and Huber values for both internodes and petioles, which may increase the likelihood of cavitation under conditions of high evaporative demand or soil drought.Abbreviations A_c total cross-sectional area (internodes, petioles, leaf laminae) - A_x xylem cross-sectional area - HV Huber value - Kh hydraulic conductance per unit length - k_s specific hydraulic conductivity - LPI leaf plastochron index - LSC leaf specific conductivity - water potential - water-potential gradient - q volume flow of water per unit time Hans R. Schultz was supported in part by the Deutsche Forschungsgemeinschaft (grant Ki-114/8-1). We wish to thank Dr. Thomas Geier, Institut für Biologie, Forschungsanstalt D-6222 Geisenheim, Germany for his advice on sample preparation and microscopy, and two anonomous reviewers for their helpful comments.     

Developing biotechnology tools for ‘beautiful’ vavilovia (<Emphasis Type="Italic">Vavilovia formosa</Emphasis>), a legume crop wild relative with taxonomic and agronomic potential

Beautiful vavilovia, the closest species to the common now extinct ancestor of the whole tribe Fabeae holds significant taxonomical interest and also for breeding within this group of species, which includes the most cultivated leguminous pulses in the world. In spite of this, vavilovia has attracted very scarce research to date and is in danger of complete extinction. Thus, as a part of the research carried out by an informal international group of researchers from various countries, we report here various experiments for the development and exploitation of a range of biotechnology tools for vavilovia, ranging from standard in vitro propagation, to plant regeneration from explant-derived callus, and also from protoplasts. Plants were successfully recovered following propagation from nodes, and by regeneration through organogenesis from callus derived from internodes (which provided the best responses) and leaves. Also, protoplasts were isolated from leaves and stems from in vitro shoots and from callus derived from these two explants, with the latter undergoing sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic fidelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, flow cytometry also permitted us to provide the first record on the relative nuclear DNA content and genome size for Vavilovia formosa.     

Preparation of a homogeneous coated vesicle fraction from bean leaves

Summary Using a procedure previously developed for suspension-cultured carrot cells, we have been able to isolate two different coated vesicle-containing fractions from green bean leaves (Vicia faba). The two fractions differ in their isopycnic densities in D₂O-Ficoll as well as in their diameters. One of the fractions (the less dense of the two) is almost 100% pure as judged by negative staining. Because of this the polypeptide pattern obtained from SDS-PAGE is most clear and has enabled a clear recognition of clathrin light chains, in addition to the 190 kDa heavy chain coat component. Significantly the 100k Da and 50k Da polypeptides typical of brain coated vesicles are absent from bean leaf coated vesicles. Due to a) the high degree of vacuolation b) the presence of large amounts of ribulose bisphosphate carboxylase in the postmicrosomal supernatant, the yield of coated vesicles from bean leaves, as compared to nongreen plant cells, or to bovine brain tissue is extremely low (1 mg coated vesicles from 2.4 kg leaf tissue).Abbreviations D₂O deuterium oxide - EGTA ethylene glycol-bis ( amino ethyl ether) N,N,N,N tetraacetic acid - MES, 2 (N-morpholino)-ethanesulfonic acid - PMSF phenyl methylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS Tris-hydroxy methyl amino methane     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Actin dynamics mediates the changes of calcium level during the pulvinus movement of Mimosa pudica

The bending movement of the pulvinus of Mimosa pudica is caused by a rapid change in volume of the abaxial motor cells, in response to various environmental stimuli. We investigated the relationship between the actin cytoskeleton and changes in the level of calcium during rapid contractile movement of the motor cells that was induced by electrical stimulation. The bending of the pulvinus was retarded by treatments with actin-affecting reagents and calcium channel inhibitors. The actin filaments in the motor cells were fragmented in response to electrical stimulation. Further investigations were performed using protoplasts from the motor cells of M. pudica pulvini. Calcium-channel inhibitors and EGTA had an inhibitory effect on contractile movement of the protoplasts. The level of calcium increased and became concentrated in the tannin vacuole after electrical stimulation. Ruthenium Red inhibited the increase in the level of calcium in the tannin vacuole and the contractile movement of the protoplasts. However, treatment with latrunculin A abolished the inhibitory effect of Ruthenium Red. Phalloidin inhibited the contractile movement and the increase in the level of calcium in the protoplasts. Our study demonstrates that depolymerization of the actin cytoskeleton in pulvinus motor cells in response to electrical signals results in increased levels of calcium.Key words: actin, calcium, pulvinus movement, the tannin vacuole, Mimosa pudica     

Effect of Xyloglucan Nonasaccharide on Cell Elongation Induced by 2,4-Dichlorophenoxyacetic Acid and Indole-3-acetic Acid

Xyloglucan nonasaccharide (XG9) is recognized as an inhibitorof 2,4-D-induced long-term growth of segments of pea stems.In the presence of 10^–5 M 2,4-D, inhibition by 10^–9M XG9 of elongation of third internode segments of pea seedlingswas detected within 2 h after the start of incubation, in someexperiments. Analysis by double-reciprocal (Lineweaver-Burk)plots of elongation in the presence of various concentrationsof 2,4-D, with or without XG9, gave parallel lines, indicatingthat XG9 inhibited 2,4-D-induced elongation in an uncompetitivemanner. XG9 did not influence the 2,4-D-induced cell wall loosening.Thus, XG9 does not fulfill the proposed definition of an "antiauxin". XG9 at 10^–11 to 10^–6 M did not influence IAA-inducedelongation of segments from pea third internodes, azuki beanepicotyls, cucumber hypocotyls, or oat coleoptiles. Inhibitionof IAA-induced elongation by XG9 was not observed even whenthe segments from pea or azuki bean were abraded. Furthermore,fucosyl-lactose at 10^–11 to 10^–4 M did not affectthe IAA-induced elongation of segments of pea internodes orof azuki bean epicotyls. XG9 may be incapable of inhibitingthe IAA-induced cell elongation (especially in oat) or, alternatively,the endogenous levels of XG9 may be so high that exogenouslyapplied XG9 has no inhibitory effect on IAA-induced elongation. (Received February 28, 1991; Accepted May 25, 1991)     

Evaluation of parameters affecting the yield, viability and cell division of Pinus pinaster protoplasts

Various factors affecting the yield and viability of Pinus pinaster Ait. cotyledon protoplasts and the mitotic activity of or regenerated cells are described. A study of the effect of sterilization procedures of the plant material showed that whereas the organs collected from disinfested seedlings allow for good yield and viability of isolated protoplasts, germination under non-sterile conditions favours a greater germinating capacity and stronger mitotic activity. Numerous clusters of from 10 to 15 cells were formed after 20 days of culture when a 5% aqueous solution of calcium hypochlorite was used as a sterilizing agent.
The effects of an additional purification of the enzymes showed that although yield and viability of the protoplasts are only slightly improved, the more highly purified enzymes on the other hand enhanced the mitotic activity markedly. Between the two total enzyme concentrations used (0.2 and 0.4%, and in which the relative ratio of each element was unchanged), only the lowest level supplied a debris-free protoplast suspension; mitotic activity occurred only in that case.
Comparison of the populations of cotyledon protoplasts collected from seedlings at two different growth stages (not fully-developed or fully-expanded cotyledons) did not reveal any appreciable difference in their size distribution. Neither was the extent of cellular viability affected by the degree of cell differentiation at the time of collecting. On the other hand, the yield of protoplasts and the mitotic activity of the regenerated cells were greater when partially-developed organs were used. Moreover a pretreatment of the elongating cotyledons with a mineral (half-strength MS macronutrients and full-strength micronutrients) and hormonal (15 μ M BAP, 0.5 μ M NAA) solution improved cell division frequency.     

Isolation Conditions for High Yields of Protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae)

In an investigation of the main factors determining protoplastyield in Laminaria saccharina and L. digitata, protoplasts wereisolated from epidermal, cortical and medullary cells of vegetativethallus by incubation with commercial cellulases, crude andpurified mannuronate lyases and purified guluronate lyases.Treatment of the tissue with the calcium chelator EGTA beforeenzymatic digestion greatly increased the protoplast yield.Preplasmolysis was also necessary to obtain large numbers ofhealthy protoplasts and this was most effective when carriedout during chelation with EGTA. Purification of the mannuronatelyases by ion exchange chromatography reduced the toxicity ofthe crude enzyme preparation. The activities of the wall degradingenzymes were differentially influenced by pH and the optimumfor alginate-lyase activity (8.0) was higher than that for cellulaseactivity (<6.0). Protoplast yield decreased linearly withincreasing pH in the enzyme medium over the range tested (6.0–8.0),and this suggests that cellulases are more critical to walldigestion than alginate-lyases. Ionic osmotica gave improvedyields compared with sugar alcohols or sugars. Increasing thecalcium concentration of the enzyme medium brought about anexponential decrease in protoplast yield and wall digestionwas almost completely inhibited at concentrations exceeding8.0 mol m^–3. However, low levels of calcium (<2.0 molm^–3) were beneficial to protoplast viability. Yields of10⁷ to 10⁸ protoplasts g^–1 fr. wt. were consistently obtainedand 20% to 30% of these regenerated new cell walls within 1–2d of culture. Key words: Laminaria protoplasts, cell wall, alginate-lyases     

Subcellular compartmentation of fructose 2,6-bisphosphate in oat mesophyll cells

Evacuolated mesophyll protoplasts from oat (Avena sativa L.) were fractionated by a membrane-filtration technique. This method of rapid quenching of metabolic reactions permitted estimation of the in-vivo pools of fructose 2,6-bisphosphate (Fru2,6bisP) in the cytosol, chloroplasts and mitochondria. Vacuolar Fru2,6bisP was calculated as the difference between control protoplasts and evacuolated ones. The results indicate that Fru2,6bisP is exclusively cytosol-located in oat mesophyll protoplasts. Assuming a cytosolic volume of about 2 pl per evacuolated protoplast, the cytosolic concentration there was 11 M if protoplasts were in darkness. Illumination of either control or evacuolated protoplasts resulted in a significant decrease in the Fru2,6bisP content within 5 min.Abbreviations EPs evacuolated protoplasts - Fru2,6bisP fructose 2,6-bisphosphate - PFP fructose 6-phosphate kinase (pyrophosphate-dependent), EC 2.7.1.90 - PEPCase phosphoenolpyruvate carboxylase, EC 4.1.1.31     

Effects of acid stress on polyamine levels,ion efflux,protective enzymes and macromolecular synthesis in cereal leaves

Wheat or oat leaf segments were floated on acid solutions and the changes in several parameters measured. The putrescine content of wheat leaves was 12.8-fold greater at pH 3.5 than at pH 6 after 72 h. The increase in putrescine was accompanied by an increase in the amount of K⁺ efflux from the leaf tissue to the external solution. The activities of superoxide dismutase and catalase of wheat leaves at pH 3.5 decreased to 51% and 40%, respectively, of their levels at pH 6 by 24 h. [³H]Uridine and [³H]leucine incorporation into macromolecules in oat leaves (with the lower epidermis removed) decreased to 58% and 28% of the control in response to acid stress in 8 h, at the same time as a 5-fold increase in putrescine. When DL--difluoro-methyarginine was added to the pH 3.5 buffered solution, the effect of acid was slightly less, with the incorporation into macromolecules being 64% and 35% of the pH 6 control. The results indicated that the putrescine accumulation under acid stress was concomitant with ion efflux, and a decrease in both macromolecular synthesis and the activities of superoxide dismutase and catalase in cereal leaves.Abbreviations Put putrescine - Spd spermidine - Spm spermine - SOD superoxide dismutase - CAT catalase - ADC arginine decarboxylase - DFMA DL--difluoromethylarginine This paper is dedicated to Dr. A.W. Galston in honor for his great achievements and in recognition of his continuing contributions to the study of plant science.     

Seasonal and physiological aspects of the isolation of tobacco protoplasts

The viability of tobacco ( N. tabacum – L. cv. Xanthi-nc) protoplasts isolated during the winter months is affected by membrane calcium, total calcium, plant age and supplementary lighting. Feeding calcium nitrate and calcium chloride helps to increase protoplast stability. Feeding is important because it increases membrane calcium, but it also has other beneficial effects which may be connected with increased nitrate and chloride (anion) uptake and cation/anion interaction. Whatever optimal feeding regime is chosen, it must be used in conjunction with the correct harvesting practice, which depends on whether or not supplementary light is given. Plants grown without supplementary light are best harvested between 8–9 weeks. If supplementary light is given, older plants (with and without supplementary calcium feeding) of approx. ten weeks are better for protoplast isolation. Plants grown under supplementary light have low leaf shape index (LSI) values (i.e. wider leaves) compared to plants in low light.
Spring and summer calcium feeding treatments had no significant effect on increasing total or membrane calcium. Likewise, there was no significant correlation between total/membrane calcium and percentage protoplast viability from days 0–5; and protoplasts isolated from plants grown in shade and fed with calcium salts, were no more stable than protoplasts from control plants. Irrespective of the feed treatments given, spring and summer plants older than about fifty days and grown under shaded and normal light conditions, produced poor protoplasts despite the fact that there was more calcium present in the membranes compared to protoplasts of younger plants. This decrease in percentage protoplast viability appears to be associated with changes in leaf shape. As plants age from 49–63 days, the leaf shape index (LSI) values increase (i.e. leaves are becoming narrower) and % protoplast viability decreases.