The role of the cell wall in the mechanism of lead and zinc tolerance in Anthoxanthum odoratum L

The respiration rate and viability of cultured cells and protoplasts isolated from two clones of Anthoxanthum odoratum tolerant to both zinc and lead were unaffected by the presence of zinc. Although intact cells were largely unaffected by the presence of lead, protoplasts isolated from cultured cells were susceptible, showing a reduced respiration rate and a high mortality. In contrast cultured cells and protoplasts of non-tolerant clones of A. odoratum were susceptible to both zinc and lead. The results provide direct evidence that in A. odoratum the cell wall is part of the mechanism of tolerance to lead, but not to zinc.     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

Light-enhanced dark respiration in mesophyll protoplasts from leaves of pea

The respiratory oxygen uptake by mesophyll protoplasts of pea (Pisum sativum cv Arkel) was stimulated up to threefold after 15 minutes of illumination at an intensity of 1250 microeinsteins per square meter per second in the presence of 5 millimolar bicarbonate at 30°C. The extent of light-enhanced dark respiration (LEDR) increased progressively with duration of preillumination. The LEDR exhibited two phases. The initial high rate of respiration decreased in about 10 minutes to a lower steady value similar to that before illumination. The promotion of LEDR by the presence of bicarbonate and inhibition by glyceraldehyde or 3-(3,4-dichlorophenyl)-1,1-dimethylurea suggested that LEDR was dependent on products of photosynthetic carbon assimilation/electron transport. Thus, the photosynthetic products exert a markedly quick influence on dark respiration in mesophyll protoplasts.     

Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors

The microviscosity of the plasmalemma of protoplasts isolated from rose (Rosa hyb. cv. Golden Wave) petals was measured by fluorescence depolarization. The plasmalemma's microviscosity was found to increase in petals which were allowed to age on cut flowers or after isolation as well as in isolated protoplasts aged in an aqueous medium. Increasing the temperature of the cut flowers or the isolated protoplasts enhanced the increase of the microviscosity of the protoplast plasmalemma. The mole ratio of free sterol to phospholipid was greater in protoplasts isolated from old flowers or in protoplasts aged after isolation than in protoplasts isolated from younger flowers. Microviscosity was greatest when protoplasts were aged at pH 4.4 and in the presence of Ca²⁺. Artificial alterations of the sterol to phospholipid ratio in the protoplasts, induced by treatment with liposomes, caused similar changes in their measured microviscosity.

These findings strongly suggest that the increase in the petal plasmalemma microviscosity with age is associated with an increase in the sterol to phospholipid ratio which results, at least partially, from the activity of endogenous phospholipases.

     

Preparation of Metabolically Active Protoplasts and Particle Preparations from the Blue-Green Alga, Phormidium luridum

A method is described for the preparation of metabolically active protoplasts from the blue-green alga, Phormidium luridum. The isolated protoplasts are stable and are capable of endogenous respiration and photoassimilation of carbon dioxide at rates not appreciably different from the untreated filaments. The protoplasts can be ruptured by dilution of the stabilizing osmoticum and, therefore, they provide a convenient starting point for the production of cell-free preparations within a biochemical reaction mixture of interest.     

甘蔗幼叶原生质体分离过程中呼吸的变化及其影响因素

甘蔗幼叶呼吸途径以ＥＭＰ－ＴＣＡＣ为主,ＰＰＰ活力不强;游离原生质体ＥＭＰ活力与幼叶组织相近,但ＴＣＡＣ活力略有下降,ＰＰＰ活力明显提高,约为幼叶组织的两倍。甘蔗幼叶原生质体在用酶法分离过程中,其组织呼吸速率开始时呈现短暂跃升后即迅速下降。导致呼吸下降的主因是细胞壁降解产物中的某些物质,它们通过钝化呼吸途径中某些关键酶的活性而降低了呼吸活力。     

Analysis of respiration during germination and enlargement of spores of Bacillus megaterium and of the fungus Myrothecium verrucaria

1. There are certainly two, and probably three, stages in the development of B. megaterium from the spore to inception of cell division. The rapid increase in rate of respiration during the initial 10 minutes on glucose-peptone-yeast extract medium coincides with decrease in optical density and with increase in stainability. From about 10 to 100 minutes, the rate increases linearly, coinciding with swelling of the spores and ending at approximately the time of rupture of the spore case. From about 100 to 180 minutes, there is a second and steeper linear increase in respiration rate coinciding with cell elongation. These physiological and morphological phenomena are discussed as criteria for germination. 2. The rate of respiration of M. verrucaria spores also increases linearly up to about 300 minutes in sucrose-yeast extract medium. No breaks in the curves are observed during formation of the germ tubes. 3. Oxygen uptake follows the parabolic curve See PDF for Equation within the limits of experimental error for both types of spores. 4. It is postulated that metabolism during these stages of linear increase may be regulated by processes occurring at cellular or intracellular surfaces or by synthesis of a limiting enzyme at constant rate.     

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum)

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.     

Stimulation of respiration by Pb2+ in detached leaves and mitochondria of C3 and C4 plants

The exposure of detached leaves of C₃ plants (pea, barley) and C₄ plant (maize) to 5 m M Pb (NO₃)₂ for 24 h caused a reduction of their photosynthetic activity by 40–60%, whereas the respiratory rate was stimulated by 20–50%. Mitochondria isolated from Pb²⁺-treated pea leaves oxidized substrates (glycine, succinate, malate) at higher rates than mitochondria from control leaves. The respiratory control (RCR) and the ADP/O ratio were not affected. Pb²⁺ caused an increase in ATP content and the ATP/ADP ratio in pea and maize leaves. Rapid fractionation of barley protoplasts incubated at low and high CO₂ conditions, indicated that the increased ATP/ADP ratio in Pb²⁺-treated leaves resulted mainly from the production of mitochondrial ATP. The measurements of membrane potential of mitochondria with a TPP⁺-sensitive electrode further showed that mitochondria isolated from Pb²⁺-treated leaves had at least as high membrane potential as mitochondria from control leaves. The activity of NAD-malate dehydrogenase in the protoplasts from barley leaves treated with Pb²⁺ was 3-fold higher than in protoplasts from control leaves. The activities of photorespiratory enzymes NADH-hydroxypyruvate reductase and glycolate oxidase as well as of NAD-malic enzyme were not affected. The presented data indicate that stimulation of respiration in leaves treated by lead is in a close relationship with activation of malate dehydrogenase and stimulation of the mitochondrial ATP production. Thus, respiration might fulfil a protective role during heavy metal exposure.     

Ploidy Effects in Isogenic Populations of Alfalfa : II. Photosynthesis, Chloroplast Number, Ribulose-1,5-Bisphosphate Carboxylase, Chlorophyll, and DNA in Protoplasts

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraploid-octoploid alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chl), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number of chloroplasts and amount of Chl per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploid population. The rate of CO₂-dependent O₂ evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraploid to octoploid level. Whereas leaves and protoplasts had similar increases in RuBPCase, DNA, and Chl with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.     

Effect of cold acclimation on intracellular ice formation in isolated protoplasts

When cooled at rapid rates to temperatures between −10 and −30°C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation—cooling to −15 or −20°C at rates of 10 or 16°C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at −5°C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of −15°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of −42°C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.     

Light and Dark Controls of Nitrate Reduction in Wheat (Triticum aestivum L.) Protoplasts

Protoplasts were isolated from the leaves of nitrate-cultured wheat (Triticum aestivum L. var. Frederick) seedlings. When incubated in the dark, protoplasts accumulated nitrite under anaerobic, but not under aerobic, conditions. The assimilation of [¹⁵N]nitrite by protoplasts was strictly light-dependent, and no loss of nitrite from the assay medium was observed under dark aerobic conditions. Therefore, the absence of nitrite accumulation under dark aerobic conditions was the result of an O₂ inhibition of nitrate reduction and not a stimulation of nitrite reduction. In the presence of antimycin A, protoplasts accumulated nitrite under dark aerobic conditions. The oxygen inhibition of nitrate reduction was apparently due to a competition between nitrate reduction and dark respiration for cytoplasmic-reducing equivalents.     

通气状况、某些呼吸中间产物和抑制剂对甘蔗幼叶原生质体酶解释离的影响

甘蔗幼叶原生质体酶解释离期间呼吸速率逐渐降低。通气良好者原生质体产量高,但各处理原生质体存活率差异不大。以无机盐为稳压剂者,苹果酸、α-酮戊二酸、丙酮酸钠、琥珀酸钠、柠檬酸钠等呼吸中间产物均能提高原生质体产量,尤以琥珀酸钠和丙酮酸钠效果最好。而以糖醇为稳压剂者,苹果酸、α-酮戊二酸、丙酮酸钠和琥珀酸钠等有促进作用,柠檬酸钠却有严重抑制效应,产物几全部为具壁细胞。 呼吸抑制剂如碘乙酸、氟化钠、丙二酸、叠氮化钠和2,4-二硝基酚等对原生质体产量和存活率均有抑制作用。随抑制剂浓度增加,呼吸受抑制越甚,原生质体产量和存活率的降低也越甚。琥珀酸能消除丙二酸对呼吸的抑制,并减轻丙二酸对原生质体释离的不利影响。 用酶法从具壁细胞中分离原生质体是既受细胞的代谢水平所调控,又受组成酶液的稳压剂的组分所影响。其原因需进一步研究。     

Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

The Mechanics of Injury to Isolated Protoplasts following Osmotic Contraction and Expansion

Micro-osmotic manipulation was used to determine the influence of osmotic contraction on the expansion potential of individual protoplasts isolated from rye (Secale cereale L. cv Puma) leaves. For protoplasts isolated from leaves of nonacclimated plants (NA protoplasts), osmotic contraction in sufficiently hypertonic solutions (>1.53 osmolal) predisposed the protoplasts to lysis during osmotic expansion when they were returned to isotonic conditions (0.53 osmolal). In contrast, for protoplasts isolated from leaves of cold acclimated plants (ACC protoplasts), osmotic contraction in either 2.6 or 4.0 osmolal solutions was readily reversible. Following osmotic contraction, the resting tension (γ_r) of NA protoplasts was similar to that determined for protoplasts in isotonic solutions (i.e. 110 ± 22 micronewtons per meter). In contrast, γ_r of ACC protoplasts decreased from 164 ± 27 micronewtons per meter in isotonic solutions to values close to zero in hypertonic solutions. Following expansion in hypotonic solutions, γ_r's of both NA and ACC protoplasts were similar for area expansions over the range of 1.3 to 1.6. Following osmotic contraction and reexpansion of NA protoplasts, hysteresis was observed in the relationship between γ_r and surface area—with higher values of γ_r at a given surface area. In contrast, no hysteresis was observed in this relationship for ACC protoplasts. Direct measurements of plasma membrane tension (γ) during osmotic expansion of NA protoplasts from hypertonic solutions (1.53 osmolal) revealed that γ increased rapidly after small increments in surface area, and lysis occurred over a range of 1.2 to 8 millinewtons per meter. During osmotic expansion of ACC protoplasts from hypertonic solutions (2.6 osmolal), there was little increase in γ until after the isotonic surface area was exceeded. These results are discussed in relation to the differences in the behavior of the plasma membrane of NA and ACC protoplasts during osmotic contraction (i.e. endocytotic vesiculation versus exocytotic extrusion) and provide a mechanistic interpretation to account for the differential sensitivity of NA and ACC protoplasts to osmotic expansion from hypertonic solutions.     

Extensor and flexor protoplasts from samanea pulvini : I. Isolation and initial characterization

Protoplasts were isolated from extensor and flexor regions of open pulvini of the nyctinastic tree Samanea saman. Both types of protoplasts undergo many changes during isolation. Extensor protoplasts are univacuolate in vivo, but some become multivacuolate. All flexor protoplasts are univacuolate. In an open pulvinus, extensor cells have a higher osmotic pressure than flexor cells. However, both types of protoplasts can be isolated with optimal yield using the same osmoticum (0.5 molar sorbitol) in the digestion medium. This suggests that some leakage of osmoticum occurs during harvest or digestion, especially from extensor tissue. Despite these changes, both types of protoplasts extrude protons in response to 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/10⁶ protoplasts/minute), demonstrating that the protoplasts are metabolically active and that proton transport mechanisms must be at least partially functional. The changes in vacuolar structure and osmotic pressure are what one might expect if the protoplasts, which are isolated from open pulvini, take on characteristics of cells in a closed pulvinus.     

The effect of heating on the respiration of MC7 and D23 tumour tissue

The sensitivity of the tissue respiration of two tumours and liver slices to heat has been studied. The tumour tissue is sensitive to in vitro preincubation at temperatures above 43°C, whereas liver slices were less temperature sensitive. The inclusion of 1 mM tetracaine during preincubation sensitizes the tumour tissue to heating. In vivo heating of the tumour tissue at 44°C for 1h was not inhibitory of respiration when subsequently measured at 37°C. Mitochondria isolated from the D23 hepatoma tissue showed coupled respiration, however mitochondria isolated from in vivo heated tumour did not show coupled respiration. It contrast to mitochondria isolated from unheated tissue, these mitochondria lacked cristae and contained electron-dense granules, indicators of damage. The lack of effect of an in vivo heat-dose, known to cause tumour regression, on respiration and the reports that ATP levels are unaffected by such heating, suggests that cell respiration is not a primary lesion in cellular heat injury. This implies that the observed impairment of mitochondrial function following in vivo heating is best explained if the heating sensitized the mitochondria to subsequent damage during isolation.     

The effects of oligomycin on content of adenylates in mesophyll protoplasts,chloroplasts and mitochondria from Pb<Superscript>2+</Superscript> treated pea and barley leaves

The effects of oligomycin on photosynthesis and respiration in relation to ATP production in chloroplasts and mitochondria were investigated in protoplasts isolated from the detached pea (Pisum sativum L cv. Iłowiecki.) and barley (Hordeum vulgare L. cv. Gunilla) leaves treated 5 mM Pb(NO₃)₂. The oligomycin (OM), an inhibitor of oxidative phosphorylation at 0.1 μM concentration caused the inhibition of photosynthesis rate in the protoplasts from both the control and the Pb-treated pea leaves. The respiration rate and ATP/ADP ratio in the protoplasts and the activity of ATPase in mitochondria, were also diminished in the control protoplasts. These effects were not observed in the protoplasts and mitochondria isolated from the Pb-treated leaves. Oligomycin, an inhibitor of photophosphorylation at 10 μM concentration decreased ATPase activity in chloroplasts from both the control and the Pb- treated leaves. Using the method of rapid fractionation of barley protoplasts it was shown that the ATP/ADP ratio in the mitochondria from Pb-treated leaves was largely suppressed (from 1.8 to 0.4) by OM under nonphotorespiratory conditions (high CO₂), whereas under photorespiratory conditions (low CO₂) this ratio was high (5.3) and under OM decreased less (to 3.1). Our results indicate that oligomycin, in organelle isolated from Pb-treated leaves, had no inhibitory effect on the mitochondrial ATPase, whereas it inhibited chloroplasts ATPase. We suggest that Pb ions affected the catalytic cycle and/or conformational changes of ATPase in pea chloroplasts differently than in mitochondria. The differences in Pb responses may reflect fine mechanisms for the regulation of ATP production in the plant cells under stress conditions.     

Cortical microtubules and protoplast fusion: Effect and fate of microtubular lattices

Cortical microtubular lattices of isolated plant protoplasts retain their pattern during PEG-induced protoplast fusion and integrate into each other as shown by indirect immunofluorescence for protoplasts of Nicotiana glutinosa fused with those of Daucus carota. In fused and unfused cultures, microtubules assume a parallel orientation and a pre-prophase band-like pattern develops. There is a negative correlation between the presence of a cortical microtubular net in protoplasts and the fusion rate.