Protoplasts from pectinolytic fungi: isolation, regeneration and pectinolytic enzyme production

S. Solís, M.E. FLORES AND C. HUITRON. 1996. Protoplast release in pectinolytic strain mutants of Aspergillus sp. CH-Y-1043 (A13) and Aspergillus flavipes ATCC-16795 (F7) is described. Optimum yield of protoplasts A13 was obtained in a lapse of 1 h when commercially lytic enzymes of Trichoderma harzanium (2 mg ml⁻¹) were added in 0.05 mol 1⁻¹ citrate-phosphate buffer pH 5.0 containing 0.7 mol 1⁻¹ KCl and 10 mg ml⁻¹ BSA. Best results in F7 were obtained when the protoplasting system of A13 was supplemented with 10 mg ml⁻¹ Aureobasidium sp. lytic enzymes. Isolated protoplasts in A13 and F7 were capable of a high regeneration frequency of 87% and 53% when 0.7 mol 1⁻¹ KCl and sorbitol were used as osmotic stabilizers. Endo-P, Exo-P and pectin lyase production were not modified during the process of regeneration.     

Phosphate accumulation in leaves of wheat seedlings measured in leaf cell protoplasts isolated after root or foliar treatment with orthophosphate

Leaf cell protoplasts were isolated from wheat seedlings ( Triticum aestivum L. cv. Urquie) after orthophosphate (Pi) treatment of the plant to determine the capacity for intracellular phosphate accumulation. Seedlings were treated with Pi concentrations near the phytotoxic level to maximize the Pi concentration in the leaf prior to protoplast isolation 1 day later. Both foliar and root treatment of seedlings with Pi increased the phosphate content of leaf protoplasts by approximately 20 μmol (mg chlorophyll)⁻¹ over Pi levels in untreated controls. Phosphate-loaded protoplasts from treated seedlings had similar photosynthetic rates and starch content but 50% more soluble reducing sugar than protoplasts from untreated seedlings. Protoplast dark respiration decreased after treatments which increased protoplast potassium content. The results suggest that similar amounts of Pi can be accumulated by leaf cells of wheat after foliar or root application of Pi to the seedling without hindering Pi-sensitive processes such as photosynthesis and starch synthesis.     

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Abstract An efficient protoplast transformation system and a procedure of plasmid transfer by means of protoplast fusion is described for Streptococcus lactis . Protoplasts of S. lactis IL1403 and S. lactis MG1363 were transformed by pGK12 [2.9 MDa erythromycin resistance (Em^r)] with an efficiency of 3 × 10⁵ transformants per μg plasmid DNA. This high efficiency was obtained by the inclusion in the transformation mixture of liposomes composed of cardiolipin and phosphatidyl choline in a molar ratio of 1 to 6 in the presence of 22.5% polyethylene glycol (PEG). This paper also reports an efficient plasmid transfer method between lactic and streptococci and Bacillus subtilis by means of protoplast fusion. When S. lactis and B. lactis protoplasts undergo fusion mediated by exposure to 37.5% polyethylene glycol, plasmid pGKV21 (3.2 MDa; Em^r) was transfered from one host to the other with a frequency of 10⁻³−10⁻⁵ transformants per regenerating recipient protoplast.     

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce ( Picea glauca ) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA-responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β -glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em-uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4-day-old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7-day growth cycle of suspension cultures. Racemic ABA [R.S-(±)-ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10⁻⁵ M , but maximum GUS activity was found at 1 × 10⁻³ M , ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S-(+)-ABA] was an effective inducer at a concentration as low as 1 × 10⁻⁷ M , but a concentration of greater than 1 × 10⁻⁴ M was required for induction by [R-(—)-ABA]. Moreover, analogs with the same configuration at C-l¹ as that of natural ABA were more effective for induction of expression from the Em-uidA . insert at 1 × 10⁻⁴ M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co-electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.     

Photosynthesis and photoassimilation of glucose during photomixotrophic growth of Marchantia polymorpha cells in suspension culture

Even in the presence of glucose the growth of Marchantia polymorpha L. (cell line HYH-2F) requires light, and growth is more sensitive to 10⁻⁶ M 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea than to 10⁻⁴ Antimycin A. The inability of the cells to grow in the dark is due to the low level of respiration. The respiration rate under light increased to four times the dark value. The values of the compensation ratio (the photosyntehtic rate/the respiration rate) for the oxygen exchange were below 1.0 daring the growth period, although oxygen evolution was found. At the early exponential phase, oxygen evolution was 0.373 μmol (mg cell dry weight)⁻¹ h⁻¹ [61.7 μmol (mg chlorophyll)⁻¹ h⁻¹]. M. polymorpha cells are unable to grow anaerobically in the light without a supply of carbon dioxide. When 1% carbon dioxide in nitrogen is supplied, photochemically produced oxygen and energy are sufficient for sustained growth although at significantly reduced yields in both cell dry weight and chlorophyll. Photosyntehtic CO₂ assimilation rate was 0.13 μmol (mg cell dry weight)⁻¹ h⁻¹[11.3 μmol (mg chlorophyll)⁻¹ h⁻¹]. At least one-third of the carbon atoms in cellular constituents seem to be derived from atmospheric carbon dioxide, which indicates that M. polymorpha cells grow photomixotrophicaily.     

High yield isolation of mesophyll protoplasts from wheat, barley and rye

Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO₂ fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO₂ fixation, which was in the order of 100 μmol CO₂ (mg chl)⁻¹h⁻¹.
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided.     

Thyrotrophin-Releasing Hormone Analogues Increase Dopamine Release from Slices of Rat Brain

Abstract: Rat brain slices were incubated with a high concentration of K⁺, thyrotrophin-releasing hormone (TRH), or one of two biologically stable TRH analogues (CG 3509 or RX 77368). Basal release of endogenous dopamine, measured by electrochemical detection, was increased by K⁺ (30 m M ) from slices of hypothalamus, septum, nucleus accumbens, and striatum. CG 3509 (10⁵–10⁻³ M ) increased the release of dopamine from slices of nucleus accumbens, septum, and hypothalamus in a dose-dependent fashion, whereas RX 77368 (10⁻⁴ M ) increased the release of dopamine from the septum only. Neither analogue increased the release of striatal dopamine. The results provide further evidence for specific regional interactions between TRH and dopamine in rat brain.     

Resistance to cadmium by pretreated rainbow trout alevins

A toxicity test with cadmium concentrations ranging from 0·1 to 100·0 mg Cd 1⁻¹ was used to assess the effect of cadmium pretreatment on rainbow trout ( Salmo gairdneri Richardson) alevins. The median period of survival for fish pretreated at 0·01 mg Cd 1⁻¹ was found to be increased at test concentrations up to 10mg Cd 1⁻¹ compared with alevins pretreated with dilution water. However, at concentrations above 10mg Cd 1⁻¹ pretreatment at 0·01 mg Cd 1⁻¹ reduced the median period of survival.     

Effect of auxins on spermidine uptake into carrot protoplasts

The effect of an auxin, indole-3-acetic-acid (IAA), on spermidine uptake into protoplasts of carrot ( Daucus carota L. cv. Ingrid) was studied. In the presence of 1 m M Ca²⁺, IAA (10⁻⁷ to 10⁻⁴ M ) enchances [¹⁴C]-spermidine uptake into carrot protoplasts, while no stimulation occurs in the absence of Ca²⁺. The time course of the uptake with and without IAA is very rapid and reaches saturation within 1 to 2 min. Preincubation of protoplasts with IAA inhibits the spermidine uptake. La³⁺, known not to penetrate the plasmalemma, exerts the same effect as Ca²⁺, but gives lower uptake values than Ca²⁺. The application of vanadate, an ATPase inhibitor, strongly inhibits IAA-stimulated spermidine uptake, suggesting that an energy-dependent mechanism may be involved in this transport. Neither spermidine nor Ca²⁺ alone stimulate IAA uptake. The synthetic auxin 2,4-dichlorophenoxyacetic acid, yields the same results as IAA with regard to time course of spermidine uptake with and without preincubation while, unlike IAA, no significant effect was observed on the Ca²⁺ -induced increase of spermidine uptake.     

The effect of dissolved oxygen and salinity on the toxicity of ammonia to smolts of salmon, Salmo salar L.

The survival of Atlantic salmon smolts on exposure to constant concentrations of ammonia has been measured under laboratory conditions. At concentrations of dissolved oxygen close to the air-saturation value, the 24-h LC50 of un-ionised ammonia is 0.15 mg NH₃1⁻¹ in fresh water (hardness 264 mg 1⁻¹ as CaCO₃) and 0.3 mg NH₃1⁻¹ in 30% sea water; at concentrations of dissolved oxygen of 3.5 mg 1⁻¹ in fresh water and 3.1 mg 1⁻¹ in 30% sea water, the 24-h LC50 is 0.09 mg NH₃ 1⁻¹ and 0.12 mg NH₃ 1⁻¹ respectively; for fish acclimated for 1 day to a concentration of ammonia close to the 24-h median for un-acclimated fish, the median is increased between 38 and 79%, depending on test conditions.     

Acute toxicity of copper, zinc and manganese in single and mixed salt solutions to juvenile longfin dace, Agosia chrysogaster

The acute toxicity of copper, zinc and manganese and copper-zinc and copper-manganese mixtures were determined for juvenile longfin dace, Agosia chrysogaster in hard water bioassays (mean=218 mg 1⁻¹ CaCO₃). Copper-zinc was the most lethal toxicant (96-h L.c.₅₀= 0.21 mg 1⁻¹ copper and 0.28 mg 1⁻¹ zinc) and exhibited a more than additive toxicity which was in contrast to the additive toxicity of copper-manganese mixtures (96-h L.c.₅₀= 0.45 mg 1⁻¹ copper and 64.0 mg 1⁻¹ manganese). The toxicity of copper (96-h L.c.₅₀= 0.86 mg 1⁻¹) and zinc (96-h L.c.₅₀= 0.79 mg 1⁻¹) to the fish was similar but both were considerably more lethal than manganese (96-h L.c.₅₀= 130 mg 1⁻¹).     

Simultaneous phototrophic and chemotrophic growth in the purple sulfur bacterium Thiocapsa roseopersicina M1

Abstract The anoxygenic phototrophic purple sulfur bacterium Thiocapsa roseopersicina was grown in illuminated continuous cultures with thiosulfate as growth limiting substrate. Aeration resulted in completely colorless cells growing chemotrophically, whereafter the conditions were changed to a 23 h oxic/1 h anoxic regime. After 11 volume changes at a dilution rate of 0.031 h⁻¹ (35% of μ_max) a time dependent equilibrium was established. During the 23 h oxic periods bacteriochlorophyll a synthesis (BChl a ) was not observed, whereas during the 1 h anoxic periods synthesis was maximal (i.e. 1.1 μg (mg protein)⁻¹ h⁻¹). As a result the BChl a concentration gradually increased from zero to an average value over 24 h of 1.9 μg (mg protein)⁻¹. Concomitantly, the protein concentration increased from 13.9 mg 1⁻¹ during continuous oxic conditions to 28.8 mg 1⁻¹. For comparison, the protein concentration during fully phototrophic growth at an identical thiosulfate concentration in the inflowing medium was 53.7 mg 1⁻¹. The specific respiration rate was 8 μmol O₂ (mg protein)⁻¹ h⁻¹ during full chemotrophic growth and gradually decreased to 3.5 μmol O₂ (mg protein)⁻¹ h⁻¹ after 11 volume changes at the regime employed. These data show that T. rosepersicina is able to simultaneously utilize light and aerobic respiration of thiosulfate as sources of energy. The ecological relevance of the data is discussed.     

Efficacy of certain disinfectants against infectious pancreatic necrosis virus

The virucidal properties of iodophor, chlorine (sodium hypochlorite), formalin, thimerosal (organic mercurial compound), malachite green, and acriflavine were tested on infectious pancreatic necrosis virus (IPNV). Iodine and chlorine showed good activity, but efficacy depended on the concentration of virus, the presence of organic matter (calf serum), and water _pH. Water hardness (0-300 mg 1⁻¹ as CaCO₃) did not affect virucidal activity. In a 5 min exposure, 4 mg 1⁻¹ available iodine inactivated 10^3.9 TCID₅₀ m1⁻¹ IPNV but 16 mg 1⁻¹ iodine were needed for inactivation of 10^6.3TCID₅₀m1⁻¹. The addition of 0-5% calf serum significantly reduced the iodine concentration and the virucidal activity. In comparison, 4 mg 1⁻¹ chlorine were needed to inactivate 10⁴⁶ TCID₅₀ m1⁻¹ IPNV in 5 min. However, the addition of 0-07 % serum greatly reduced the chlorine concentration and extended the virucidal contact time to 30 min or more. IPNV at 10^6.3 TCID₆₀ m1⁻¹ was not inactivated by exposures for 60 min to 0-2% formalin, 10 min to 0-2% thimerosal, 60 min to 5 mg 1⁻¹ malachite green, or 20 min to 500 mg 1⁻¹ acriflavine. However, acriflavine at 0-5 mg 1⁻¹ in cell culture media prevented the development of cytopathology caused by IPNV and may be useful in the treatment of the disease.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Oxygen uptake in developing eggs and larvae of the cod, Gadus morhua L.

Unfertilised cod eggs showed a mean oxygen uptake rate at 5°C of 0.089 μl O₂, dry wt.⁻¹ h⁻¹; this gradually rose to 0.768 μl O₂ mg dry wt.⁻¹ h⁻¹ in eggs about to hatch. From hatching to complete yolk absorption larvae respired at 1.6 μl O₂, mg dry wt.⁻¹ h⁻¹. During starvation following yolk absorption, uptake fell significantly to 1.1 μl O₂, mg dry ⁻¹ h⁻¹. Much of this decrease in oxygen consumption was shown to be caused by reduction in activity. Loss of weight during the embryo and larval phases could not easily be reconciled with total oxygen consumption; it is suggested that cod embryos and larvae may not rely solely upon endogenous energy reserves during development.     

The interactions of water hardness and pH with the acute toxicity of zinc to the brown trout, Salmo trutta L.

Exposure of brown trout, Salmo trutta , to zinc under continuous flow conditions over 96 h showed that both water hardness and pH exert major influences on the toxicity of the metal. 96-h LC50 values for total zinc ranged from <0.14mg 1⁻¹ in alkaline soft water (pH 8; lOmg 1⁻¹ as CaCO₃) to 3.20 mg 1⁻¹ in acidic hard water (pH 5; 204 mg 1⁻¹ as CaCO₃). A variable reduction in zinc toxicity in hard water compared with soft water over the pH range 4–9 was attributed to high external calcium. Zinc toxicity was positively correlated with decreasing acidity over the pH range 5–7, the metal being most toxic at pH 8–9 where metal complexes predominate. Below pH 5 metal toxicity also increased, irrespective of hardness. Water hardness and pH interacted with zinc toxicity in a complex manner, apparently dependent on physical and chemical transformations of the metal, and as changes in uptake. detoxification and excretion by the fish.     

Cadmium toxicity to the freshwater amphipod Gammarus pulex (L.) during the moult cycle

SUMMARY. 1. The toxicity of cadmium to mature Gammarus pulex at different stages in the moult cycle is described.
2. Immediate post-moult animals are significantly more sensitive than intermoult specimens at cadmium concentrations between 1.0 and 0.1 mg 1⁻¹ but not at 0.03 or 0.01 mg Cd 1⁻¹.
3. At a calcium concentration of 40mgl⁻¹, post-moult animals undergo recalcification within 7 days and thereafter there is little variation in their response to cadmium.
4. External calcium concentrations of 40 and 115mg 1⁻¹ do not affect cadmium toxicity but at 180 mg Ca1⁻¹ the sensitivity of immediate post-moult specimens is significantly reduced.
5. The results are discussed with regard to the protection of G. pulex by present water quality standards.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Distribution of fingerling brook trout, Salvelinus fontinalis (Mitchill), in dissolved oxygen concentration gradients

A self-recording linear gradient tank and procedures are described in which individual brook trout fingerlings unstressed by recent transfer, unaccustomed surroundings or the presence of an observer could move freely in 16 oxygen concentration gradients within the limits of 1 and 8.9 mg O₂1⁻¹. They avoided oxygen concentrations below 4 mg 1⁻¹ most of the time and preferred 5 mg 1⁻¹ or higher more than half the time, which supports the field-derived belief that fish avoid oxygen concentrations below 5 mg l⁻¹ in the natural environment if they can.     

Characteristics of ATPase from sugarcane protoplast and vacuole membranes

Characteristics of membrane-associated ATPase from commercial Hawaiian varieties of sugarcane ( Saccharum spp. hybrids) were investigated in preparations from sugarcane cell suspension culture and from stalk tissues of the intact plant. In order to examine comparable preparations, protoplasts and vacuoles, in turn, were obtained from both sources. ATPase from preparations of crude protoplast membranes and tonoplast had a pH optimum of 6 to 6.5. The relative effectiveness of divalent cations in stimulating ATPase was Mg²⁺ > Mn²⁺≥ Co²⁺ > Ca²⁺≥ Zn²⁺. Enzyme activity was not stimulated by K⁺, nor by other monovalent cations. Protoplasts and vacuoles from both sources showed significant acid phosphatase activity. Acid phosphatase activity was inhibited by molybdate, but ATPase activity was unaffected. Membrane preparations from protoplasts contained inorganic pyrophosphatase, but enzyme activity was low or not present in tonoplast preparations. Cell suspension and stalk tissue preparations hydrolyzed a large number of nucleoside di- and triphosphates. The hydrolysis is most likely due to a series of enzymes rather than a single enzyme. ATPase from protoplast and tonoplast preparations was inhibited 30–50% by diethylstilbestrol and sodium ortho-vanadate and was unaffected by ionophores. This study illustrates the complexity of phosphohydrolase activities in membrane preparations from sugarcane. The study, however, also illustrates substantial similarity in the behavior of these enzymes, whether they are derived from the plant itself or from cell cultures originating from comparable tissues of the plant.