Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L

A mass spectrometric method combining ¹⁶O/¹⁸O and ¹²C/¹³C isotopes was used to quantify the unidirectional fluxes of O₂ and CO₂ during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O₂ uptake and CO₂ evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O₂ (61 micromoles of O₂ per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O₂ per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO₂ in darkness at a rate of 27 micromoles of CO₂ per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O₂ evolution and CO₂ fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO₂ evolution by guard cell protoplasts was sharply decreased (37%), while O₂ uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO₂ assimilation and may be dissipated for other purposes such as ion uptake.     

Transport of anions in isolated barley vacuoles : I. Permeability to anions and evidence for a cl-uptake system

The anion contents of young barley leaves and of mesophyll protoplasts from the leaves was compared. Anion loss from the protoplasts during isolation was small. Although only about 60% of the leaf cells were mesophyll cells, phosphate and sulfate contents of the mesophyll cells accounted for almost 90% of the leaf contents. Chloride accumulated in the leaf epidermis. The rapid isolation of vacuoles from mesophyll protoplasts permitted the determination of vacuolar ion concentrations. Sodium and nitrate levels were very low in the cytoplasm, and much higher in the vacuole. When barley plants were grown in the presence of low NaCl levels, chloride concentrations were comparable in cytoplasm and vacuole, and similar observations were made with sulfate. Cytoplasmic phosphate concentrations were close to 30 millimolar and potassium concentrations 100 millimolar. During a 30 minute incubation period at room temperature, anion contents of isolated vacuoles decreased considerably. Efflux of NO₃⁻ was faster than that of Cl⁻. Phosphate and sulfate crossed the tonoplast only slowly. 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid partially inhibited the efflux of nitrate and, to a lesser extent, that of chloride. Decreased efflux was also observed in the presence of MgATP. In remarkable contrast, p-chloromercuribenzene sulfonate and HgCl₂ stimulated the efflux of nitrate and chloride, but not of phosphate. Labeled chloride was taken up by isolated vacuoles. The apparent K_m for chloride uptake at low chloride concentrations was 2.3 millimolar. At elevated chloride concentrations, chloride did not display saturation characteristics but, rather, characteristics of a diffusional process. Uptake was stimulated by ATP.     

Photosynthetic and Carbohydrate Metabolism in Isolated Leaf Cells of Digitaria pentzii

Mesophyll cells and bundle sheath strands were isolated rapidly from leaves of the C₄ species Digitaria pentzii Stent. (slenderstem digitgrass) by a chopping and differential filtration technique. Rates of CO₂ fixation in the light by mesophyll and bundle sheath cells without added exogenous substrates were 6.3 and 54.2 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of pyruvate or phosphoenolpyruvate to the mesophyll cells increased the rates to 15.2 and 824.6 micromoles of CO₂ per milligram of chlorophyll per hour, respectively. The addition of ribose 5-phosphate increased the rate for bundle sheath cells to 106.8 micromoles of CO₂ per milligram of chlorophyll per hour. These rates are comparable to those reported for cells isolated by other methods. The K_m(HCO₃⁻) for mesophyll cells was 0.9 mm; for bundle sheath cells it was 1.3 mm at low, and 40 mm at higher HCO₃⁻ concentrations. After 2 hours of photosynthesis by mesophyll cells in ¹⁴CO₂ and phosphoenolpyruvate, 88% of the incorporated ¹⁴C was found in organic acids and 0.8% in carbohydrates; for bundle sheath cells incubated in ribose 5-phosphate and ATP, more than 58% of incorporated ¹⁴C was found in carbohydrates, mainly starch, and 32% in organic acids. These findings, together with the stimulation of CO₂ fixation by phosphoenolpyruvate for mesophyll cells and by ribose 5-phosphate plus ATP for bundle sheath cells, and the location of phosphoenolpyruvate and ribulose bisphosphate carboxylases in mesophyll and bundle sheath cells, respectively, are in accord with the scheme of C₄ photosynthesis which places the Calvin cycle in the bundle sheath and C₄ acid formation in mesophyll cells.     

Effect of path or sink anoxia on sugar translocation in roots of maize seedlings

The subcellular and developmental distribution of β-cyanoalanine synthase (EC 4.4.1.9), which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, were investigated in barley (Hordeum vulgare) leaves. Total leaf activity was 1.1 micromoles per minute per gram fresh weight. Sucrose density gradients of lysed mesophyll protoplasts of barley revealed the exclusive or predominant localization of β-cyanoalanine synthase in the mitochondria. The enzyme was absent from both vacuole and chloroplast fractions.

β-Cyanoalanine synthase activity was distributed over the entire length of the barley leaf. Activity was dependent on the developmental stage, with a 3.5-fold higher activity in the oldest (apical) compared to the youngest (basal) parts of the leaf. The corresponding difference in activity for mesophyll protoplasts isolated from these parts was 7.5-fold. In younger leaf seagments, the nonchlorophyllous tissues accounted for up to 70% of the total β-cyanoalanine synthase activity. These results are discussed with reference to the formation of HCN as a substrate in barley leaves.

     

Cryopreservation of rye protoplasts by vitrification

A procedure has been developed for the vitrification of mesophyll protoplasts isolated from leaves of nonacclimated (NA) and cold-acclimated (ACC) winter rye seedlings (Secale cereale L. cv Puma). The procedure involves (a) equilibration (loading) of the protoplasts with an intermediate concentration (1.5, 1.75, or 2.0 molar) of ethylene glycol (EG) at 20°C; (b) dehydration of the protoplasts in a concentrated vitrification solution made of 7 molar EG + 0.88 molar sorbitol + 6% (w/v) bovine serum albumin (BSA) at 0°C; (c) placing the protoplasts into polypropylene straws and quenching in liquid nitrogen (LN₂); and (d) recovery of the protoplasts from LN₂ and removal (unloading) of the vitrification solution. For NA protoplasts, 47 + 1% survival was obtained following recovery from LN₂ if the protoplasts were first loaded with 1.75 molar EG prior to the dehydration step. However, to achieve this level of survival, NA protoplasts had to be unloaded in a hypertonic (2.0 osmolal [osm]) sorbitol solution. If they were unloaded in an isotonic solution (0.53 osm), survival was 3±2%. In contrast, survival of ACC protoplasts following recovery from LN₂ was 34 ± 10% when the protoplasts were loaded in a 2.0 molar EG solution and unloaded in an isotonic sorbitol solution (1.03 osm). If ACC protoplasts were unloaded in an hypertonic sorbitol solution (1.5 osm), survival was 51 ± 9%. These results indicate that the osmotic excursions incurred during the procedure are a major factor affecting survival.     

Tissue distribution of acetyl-coenzyme a carboxylase in leaves

Acetyl-CoA carboxylase [acetyl-CoA—carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C₃- and C₄-plants. The mesophyll tissues of the C₃-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using ¹²⁵I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.

In the C₄-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.

The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

     

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2)

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO₂ compensation points at different O₂ levels, which is typical of C₄ plants, yet it does show about 4% inhibition of net photosynthesis by 21% O₂ at 30°C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C₄ pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C₃ cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial ¹⁴C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO₂ is fixed into C₄ acids (malate and aspartate), whereas about 20% of the CO₂ directly enters the C₃ cycle. This is consistent with the high activity of enzymes for CO₂ fixation by the C₄ pathway and the substantial activity of enzymes of the C₃ cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C₃ photosynthesis in the mesophyll cells and should be considered a C₄-like species.     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Phospholipid synthesis and exchange in castor bean endosperm homogenates

Leaves on a bush of Hyptis emoryi Torr. varied in length from less than 1 cm when development occurred in full sunlight (e.g. 40 Mjoules m⁻²) to over 7 cm when the total daily solar irradiance was less than 3 Mjoules m⁻². The 1-cm sun leaves were 3-fold higher than the 7-cm shade leaves in chlorophyll per unit area, mesophyll thickness, and the internal to external leaf area ratio (A^mes/A). The higher A^mes/A caused a 1.2-cm leaf to have a 3-fold lower CO₂ liquid phase resistance than did a 7.1-cm leaf. Large thin shade leaves captured photosynthetically active radiation effectively (less than 7% passed through), but were not adapted to full sunlight. Specifically, when a 6.9-cm leaf was placed at 910 w m⁻² for 30 min, its temperature exceeded that of the air by nearly 8 C. For the common daytime air temperatures above 30 C for this desert shrub, large shade leaves would have temperatures far in excess of that optimum for photosynthesis for H. emoryi, 29 to 32 C.     

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.     

Photosynthesis and photosynthate partitioning in n(2)-fixing soybeans

Leaf area, chlorophyll content, net CO₂ photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. A second set (Nod+/−) was inoculated and had N₂ fixation as its sole source of nitrogen. A third group (Nod⁻) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. The mean net micromole CO₂ uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/− test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/− utilized their chlorophyll more efficiently for photosynthetic CO₂ uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/− but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/− sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

Effects of Irradiance during Growth on Adaptive Photosynthetic Characteristics of Velvetleaf and Cotton

We grew velvetleaf (Abutilon theophrasti Medic.) and cotton (Gossypium hirsutum L. var. Stoneville 213) at three irradiances and determined the photosynthetic responses of single leaves to a range of six irradiances from 90 to 2000 μeinsteins m⁻²sec⁻¹. In air containing 21% O₂, velvetleaf and cotton grown at 750 μeinsteins m⁻²sec⁻¹ had maximum photosynthetic rates of 18.4 and 21.9 mg of CO₂ dm⁻²hr⁻¹, respectively. Maximum rates for leaves grown at 320 and 90 μeinsteins m⁻²sec⁻¹ were 15.3 and 10.3 mg of CO₂ dm⁻²hr⁻¹ in velvetleaf and 12 and 6.7 mg of CO₂ dm⁻²hr⁻¹ in cotton, respectively. In 1 O₂, maximum photosynthetic rates were 1.5 to 2.3 times the rates in air containing 21% O₂, and plants grown at medium and high irradiance did not differ in rate. In both species, stomatal conductance was not significantly affected by growth irradiance. The differences in maximum photosynthetic rates were associated with differences in mesophyll conductance. Mesophyll conductance increased with growth irradiance and correlated positively with mesophyll thickness or volume per unit leaf area, chlorophyll content per unit area, and photosynthetic unit density per unit area. Thus, quantitative changes in the photosynthetic apparatus help account for photosynthetic adaptation to irradiance in both species. Net assimilation rates calculated for whole plants by mathematical growth analysis were closely correlated with single-leaf photosynthetic rates.     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

Spinach Leaf Chloroplast CO(2) and NO(2) Photoassimilations Do Not Compete for Photogenerated Reductant: Manipulation of Reductant Levels by Quantum Flux Density Titrations

Potential competition between CO₂ and NO₂⁻ photoassimilation for photogenerated reductant (e.g. reduced ferredoxin and NADPH) was examined employing isolates of mesophyll cells and intact chloroplasts derived from mature `source' spinach leaves. Variations in the magnitude of incident light energy were used to manipulate the supply of reductant in situ within chloroplasts. Leaf cell and plastid isolates were fed with saturating CO₂ and/or NO₂⁻ to produce the highest demand for reductant by CO₂ and/or NO₂⁻ assimilatory processes (enzymes). Even in the presence of CO₂ fixation, NO₂⁻ reduction in intact leaf cell isolates as well as plastid isolates was maximal at light energies as low as 50 to 200 microeinsteins per second per square meter. Simultaneously, 500 to 800 microeinsteins per second per square meter were required to support maximal CO₂ assimilation. Regardless of the magnitude of the incident light energy, CO₂ assimilation did not repress NO₂⁻ reduction, nor were these two processes mutually repressed. These observations have been interpreted to mean that reduced ferredoxin levels in situ in the plastids of mature source leaf mesophyll cells were adequate to supply the concurrent maximal demands exerted by enzymes associated with CO₂ as well as with inorganic nitrogen photoassimilation.     

Effects of Polyploidy on Photosynthetic Rates, Photosynthetic Enzymes, Contents of DNA, Chlorophyll, and Sizes and Numbers of Photosynthetic Cells in the C(4) Dicot Atriplex confertifolia

Photosynthetic rates, chlorophyll content, and activities of several photosynthetic enzymes were determined per cell, per unit DNA, and per unit leaf area in five ploidal levels of the C₄ dicot Atriplex confertifolia. Volumes of bundle sheath and mesophyll protoplasts were measured in enzymatic digestions of leaf tissue. Photosynthetic rates per cell, contents of DNA per cell, and activities of the bundle sheath enzymes ribulose 1,5-bisphosphate carboxylase (RuBPC) and NAD-malic enzyme per cell were correlated with ploidal level at 99% or 95% confidence levels, and the results suggested a near proportional relationship between gene dosage and gene products. There was also a high correlation between volume of mesophyll and bundle sheath cells and the ploidal level. Contents of DNA per cell, activity of RuBPC per cell, and volumes of cells were correlated with photosynthetic rate per cell at the 95% confidence level. The mesophyll cells did not respond to changes in ploidy like the bundle sheath cells. In the mesophyll cells the chlorophyll content per cell was constant at different ploidal levels, there was less increase in cell volume than in bundle sheath cells with an increase in ploidy, and there was not a significant correlation (at 95% level) of phosphoenolpyruvate carboxylase activity or content and pyruvate,Pi dikinase activity with increase in ploidy. The number of photosynthetic cells per unit leaf area progressively decreased with increasing ploidy from diploid to hexaploid, but thereafter remained constant in octaploid and decaploid plants. Numbers of cells per leaf area were not correlated with cell volumes. The mean photosynthetic rates per unit leaf area were lowest in the diploid, similar in 4×, 6×, and 8×, and highest in the decaploid. The photosynthetic rate per leaf area was highly correlated with the DNA content per leaf area.     

Ribulosebisphosphate carboxylase activity and photosynthetic o(2) evolution rate in vicia guard-cell protoplasts

Activities of ribulose-1,5-bisphosphate carboxylase and rates of photosynthetic O₂ evolution were measured in guard-cell and mesophyll protoplasts from Vicia faba. The ribulose-1,5-bisphosphate carboxylase activity of guard-cell protoplasts was 30% of that of mesophyll protoplasts; however, the O₂ evolution rate was 3 times higher in guard-cell protoplasts than in mesophyll protoplasts on a chlorophyll basis. When the dark-adapted, guard-cell protoplasts were illuminated by red light, O₂ was evolved with an induction period, which became shorter when the protoplasts were reilluminated. High activity of irreversible NADP-glyceraldehyde-3-phosphate dehyrogenase was found in guard-cell protoplasts. Several lines of evidence revealed that there was virtually no contamination by mesophyll cells in guard-cell preparations. These results indicate that guard cells fix CO₂ photosynthetically and imply that the cells utilize a considerable proportion of reducing equivalents from water for reactions other than CO₂ fixation.     

Isolation and transport properties of protoplasts from cortical cells of corn roots

A procedure was developed for the enzymic isolation of large quantities of protoplasts from the cortex of Zea mays L. WF9 × MO 17 roots. Cortex was separated from the primary root, sectioned, and the cell walls digested for 3.5 hours in 2% (w/v) Cellulysin, 0.1% Pectolyase Y-23, 1 millimolar CaCl₂, 0.05% bovine serum albumin, 0.5 millimolar dithiothreitol in 0.6 molar mannitol (pH 5.6). Cortical cell protoplasts were collected by centrifugation and purified by flotation in a Ficoll step gradient. The yield of protoplasts was approximately 650 × 10³/gram fresh tissue. To obtain maximum yield it was essential to include an effective pectinase (Pectolyase Y-23) and protectants (bovine serum albumin and dithiothreitol) in the digestion medium.

Cortical cell protoplasts exhibited energy-dependent uptake of K⁺ (⁸⁶Rb), H₂³²PO₄⁻, and ³⁶Cl⁻ as well as net H⁺ extrusion. Ion fluxes were sustained for at least 3 hours. Influx of K⁺ was highest between pH 7.5 and 8.0, whereas the influx of H₂PO₄⁻ was greatest between pH 4.0 and 5.0. K⁺ and H₂PO₄⁻ influx and net H⁺ efflux were inhibited by respiratory poisons such as cyanide (0.1 millimolar) and oligomycin (5 micrograms per milliliter), and by inhibitors of plasma membrane ATPase such as diethylstilbestrol (50 micromolar). Calculated flux for Cl⁻ was low, but not greatly different from that observed for other plant cells. K⁺ flux was somewhat high, probably because the K⁺ concentration in the cortical cells was below steady-state. The results indicate that isolated cortical cell protoplasts retain transport properties which are similar to those of root tissue.