Function of leaf hamamelitol as a compatible solute during water stress treatment of Hedera helix L.

Hamamelitol is an unusual branched-chain sugar alcohol previously suggested to function as a leaf compatible solute. In this study, we have examined the leaf metabolism and intracelluiar compartmentalization of hamamelitol and other soluble sugars during long-term water stress treatment of Hedera helix (English ivy). Total leaf hamamelitol content was relatively low in greenhouse control plants, but increased 2-fold during water stress treatment to levels approaching those observed in field-grown plants (6–7 μmol g^?1 fresh weight). Using density gradient fractionation with non-aqueous solvents, we showed that hamamelitol occurs primarily in the cytoplasm and vacuoles of leaf mesophyll cells. During water stress treatment most of the increase in leaf hamamelitol occurred in the mesophyll cytoplasm, compensating osmotically for a decrease in cytoplasmic sucrose concentration. The maximum concentration of cytoplasmic hamamelitol was 155 mol m^?3 and occurred in field-grown plants. Labelling experiments showed that hamamelitol is slowly synthesized from ¹⁴CO₂ in leaves of H. helix, but is very long-lived (estimated t_1/2 of 4 years). Together, these data indicate that hamamelitol probably functions during long-term stress conditions as an osmotically active, compatible solute in plant leaves. We suggest that the signal for enhanced accumulation of hamamelitol during the water stress treatment was initiated by decreased plant growth and increased leaf sucrose hydrolysis.     

Role of K+ in leaf growth: K+ uptake is required for light-stimulated H+ efflux but not solute accumulation

The stimulation of dicotyledonous leaf growth by light depends on increased H⁺ efflux, to acidify and loosen the cell walls, and is enhanced by K⁺ uptake. The role of K⁺ is generally considered to be osmotic for turgor maintenance. In coleoptiles, auxin‐induced cell elongation and wall acidification depend on K⁺ uptake through tetraethylammonium (TEA)‐sensitive channels (Claussen et al., Planta 201, 227–234, 1997), and auxin stimulates the expression of inward‐rectifying K⁺ channels ( Philippar et al. 1999) . The role of K⁺ in growing, leaf mesophyll cells has been investigated in the present study by measuring the consequences of blocking K⁺ uptake on several growth‐related processes, including solute accumulation, apoplast acidification, and membrane polarization. The results show that light‐stimulated growth and wall acidification of young tobacco leaves is dependent on K⁺ uptake. Light‐stimulated growth is enhanced three‐fold over dark levels with increasing external K⁺, and this effect is blocked by the K⁺ channel blockers, TEA, Ba⁺⁺ and Cs⁺. Incubation in 10 mm TEA reduced light‐stimulated growth and K⁺ uptake by 85%, and completely inhibited light‐stimulated wall acidification and membrane polarization. Although K⁺ uptake is significantly reduced in the presence of TEA, solute accumulation is increased. We suggest that the primary role of K⁺ in light‐stimulated leaf growth is to provide electrical counterbalance to H⁺ efflux, rather than to contribute to solute accumulation and turgor maintenance.     

Efflux of sucrose from minor veins of tobacco leaves

Mature leaves import limited amounts of nutrient when darkened for prolonged periods. We tested the hypothesis that import is restricted by the apoplast-phloem loading mechanism, ie., as sucrose exits the phloem of minor veins it is retrieved by the same tissue, thus depriving the mesophyll of nutrient. When single, attached, mature leaves of tobacco (Nicotiana tabacum L.) plants were darkened, starch disappeared from the mesophyll cells, indicating that the supply of solute to the mesophyll was limited. Starch was synthesized in mesophyll cells of darkened tissue when sucrose was applied to the apoplast at 0.1–0.3 mM concentration. Efflux from minor veins was studied by incubating leaf discs on [¹⁴C]sucrose to load the minor veins and then measuring subsequent ¹⁴C release. Efflux was rapid for the first hour and continued at a gradually decreasing rate for over 13 h. Net efflux increased when loading was inhibited by p-chloromercuribenzene-sulfonic acid, anoxia, isotope-trapping, or reduction of the pH gradient. Neither light nor potassium had a significant effect on the rate of labeled sucrose release. The site of labeled sucrose release was investigated by measuring efflux from discs in which sucrose had previously been loaded preferentially by either the minor veins or mesophyll cells. Efflux occurred primarily from minor veins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - Mops 3(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SE-CC sieve element-companion cell complex     

Effects of light, magnesium and sucrose on leaf anatomy, photosynthesis, starch and total sugar accumulation, in kiwifruit culturedin vitro

Shootlets of kiwifruit plants (Actinidia deliciosa) were culturedin vitro. Combinations of light intensity, Mg and sucrose in the cultures showed that an increase of light intensity resulted in a corresponding increase of the relative size of the leaf mesophyll cells and in a decrease of the numbers of chloroplasts and contained starch grains. The addition of sucrose to the substrate media negatively affected the size of the mesophyll cells under normal Mg concentration (35 mg l⁻¹), and positively under high Mg concentration (105 mg l⁻¹ ). Sucrose further resulted in an increase in the numbers of chloroplasts and contained starch grains. The photosynthetic capacity of leaves greatly increased when Mg concentration was enhanced and sucrose was excluded from the nutrient substrate. Total sugar accumulation in all treatments was favoured by normal light intensity and addition of sucrose.     

Solute accumulation and electrical membrane potential in Agrobacterium tumefaciens-induced crown galls in Kalanchoë daigremontiana leaves

Leaves of Kalanchoë daigremontiana were infected with Agrobacterium tumefaciens strain C58 in order to determine the relative contributions of energy-dependent ion uptake to the observed higher solute concentrations in crown-gall tissue compared with unaffected tissue. The tumor tissue exhibited the following characteristics with respect to unaffected tissue: the content of most solutes was much higher: Na⁺, K⁺, Cl^–, soluble P_i, total N, protein, soluble amino acids, and soluble sugars. Yet NH⁻₄ and starch content was less. Malate did not fluctuate in a typical CAM rhythm and was lower. The respiration rate on a cytoplasmic volume basis was similar. Photosynthetic rates were much lower. The cytoplasmic ATP concentration was even less, while that of NAD(P)H was higher. Electrical membrane potential was lower (− 184 mV tumor vs. − 223 mV control) and was composed of a higher energy-independent component (− 125 vs. − 98 mV) and a smaller energy-dependent component (− 59 vs. − 125 mV). The response of the membrane potential upon addition of neutral, acidic and basic amino acids including the opines octopine and nopaline was similar in both tissue types. It is suggested that the stronger accumulation of solutes in tumor vs. mesophyll cells cannot be due to thermodynamically more favourable conditions at the plasmalemma, but more probably to a hormone-regulated solute transport across the tonoplast.     

Preferential Accumulation by Mesophyll Cells at Low and by Veins at High Exogenous Amino Acid and Sugar Concentrations in Commelina benghalensis L. Leaves

The contribution to solute uptake by mesophyll cells and veinsin leaf discs, was assessed through a study of uptake in relationto concentration for ¹⁴C-labelled substrates (sucrose, glucose,arginine, proline, valine and -aminoisobutyric acid) using isolatedmesophyll cells and stripped leaf discs of Commelina benghalensisL. Uptake per unit fresh weight was higher in mesophyll cellsthan in discs at low substrate concentrations (lower than about0·5 mol m^–3). At higher concentrations, uptakeby discs exceeded that by mesophyll cells except for glucoseuptake which was higher in mesophyll cells over the whole concentrationrange. The profiles of uptake versus concentration displayedbiphasic kinetics in mesophyll cells and discs. Comparison ofthe uptake characteristics obtained by iterative fitting confirmedthat the high-affinity systems of uptake prevail in the mesophyllcells, whereas the low-affinity systems are dominant in theveins. The results provide good evidence that, supplementaryto direct vein loading, a pathway via the mesophyll contributesstrongly to the photosynthate loading by veins in stripped discs. Key words: Commelina benghalensis L., amino acid uptake, mesophyll, minor veins, phloem loading, sugar uptake     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

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.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Effect of phaseolotoxin on the synthesis of arginine and protein

Mesophyll cells in discs cut from primary leaves of Phaseolus vulgaris L. were exposed to a concentration of phaseolotoxin that inhibited ornithine carbamoyltransferase (OCTase) measured in an extract of the tissue. This treatment also blocked incorporation of exogenous [¹⁴C] ornithine into protein-arginine of the mesophyll cells. By contrast more than 80% of the [¹⁴C]ornithine supplied to untreated tissue was incorporated into protein-arginine in 565 minutes. Protein synthesis in mesophyll cells was unaffected by phaseolotoxin because treated tissue continued to incorporate [¹⁴C]leucine into protein at the same rate as the untreated control. The phaseolotoxin-treated tissue should therefore remain metabolically competent and this prediction was reinforced by the finding that the rate of photosynthetic O₂ evolution per unit chlorophyll was similar for tissue from the phaseolotoxin-induced chlorosis and from green healthy tissue. Phaseolotoxin also blocked OCTase but not protein synthesis in exponentially growing cell suspension cultures. Phaseolotoxin rapidly inhibited growth of Escherichia coli and this effect was rapidly reversed by arginine. Thus, the toxic effects of phaseolotoxin may be attributed to the inhibition of OCTase which, in turn, blocks arginine synthesis. Protein accumulation is blocked as a consequence, but protein synthesis is unaffected. Chlorosis is due to reduced chlorophyll synthesis and this is presumably a consequence of the lower protein level in affected tissue.     

Effect of mannitol and glucose-induced osmotic stress on growth,water relations,and solute composition of cell suspension cultures of poplar (Populus deltoides var.Occidentalis) in relation to anthocyanin accumulation

Summary A cell suspension culture of poplar (Populus deltoides (Marsh.) Bartr. var.occidentalis Rydb.), accumulating the anthocyanin pigment, cyanidin 3-glucoside, in the lag phase of culture growth, was subjected to osmotic stress with glucose and mannitol. Osmotic stress treatments resulted in growth suppression and higher anthocyanin accumulation compared with unstressed cells. Both an increase in the proportion of pigmented cells and an increase in the concentration of anthocyanin in the pigmented cells were responsible for high anthocyanin content of cultured cells subjected to osmotic stress. The osmotic stress induced by glucose suppressed growth more than that by mannitol and produced higher anthocyanin levels. Only small amounts of [U-¹⁴C]mannitol were taken up and metabolized by the cells. Stressed cells accumulated sugars and free amino acids to a different extent resulting in altered cell sugar-to-amino acid ratios. The accumulation of osmotically active solutes and cell growth suppression may both be responsible for the accumulation of anthocyanin in stressed cells.     

Vacuoles of Mesophyll Cells as a Transient Reservoir for Assimilates

We studied the efflux of radioactive photosynthetic products from the central vacuole into the cytosol of protoplasts isolated from the mesophyll tissue of the sugar beet (Beta vulgaris L.) after their darkening and subsequent cessation of photosynthesis. Among the products accumulated in the vacuole were the ¹⁴C-labelled sugars malate and alanine, small amounts of citric, glutamic, and aspartic acids, and some other amino acids. During the initial 20–30 min of darkness, there was no substantial utilization of photoassimilates accumulated in the vacuole during the preceding light period. An efflux of assimilates occurred later, after 30–40 min of darkness. A decrease in the vacuolar ¹⁴C-sucrose occurred not only due to its exit into the cytosol but also because of its conversion into ¹⁴C-monosaccharides by the vacuolar invertase. In fact, this decrease in the sucrose content correlated well with the accumulation of monosaccharides. Immediately after photosynthesis ceased, the chloroplastic ¹⁴C-starch was utilized for the maintenance of cytoplasmic metabolism. After 30-min darkness, the content of starch in the chloroplasts decreased by several times. We believe that the vacuoles of sugar-beet mesophyll cells are transient reservoirs for assimilates and the products of their conversion (glucose and fructose), which can rapidly leave the vacuole to maintain homeostasis in the cytosol under varying environmental conditions.     

Maintenance of low cl concentrations in mesophyll cells of leaf blades of barley seedlings exposed to salt stress

The concentrations of vacuolar Na⁺ and Cl⁻ in the epidermal and mesophyll cells of the leaf blade and sheath of Hordeum vulgare seedlings (cv California Mariout and Clipper) were measured by means of quantitative electron probe x-ray microanalysis. A preferential accumulation of Cl⁻ in vacuoles of epidermal cells in both blade and sheath and a low level in mesophyll cells of the blade were evident in plants grown in full strength Johnson solution. The concentration of Cl⁻ in the mesophyll cells of the blade remained at a low level after exposure to 50 or 100 millimolar NaCl for 1 day or to 50 millimolar for 4 days, while at the same time the concentration of Cl⁻ in the epidermis and mesophyll of the sheath showed a dramatic increase. Clipper generally contained more Cl⁻ in the mesophyll cells of the blade than California Mariout. A greater accumulation of Na⁺ in the mesophyll of the sheath relative to that of the blade was only apparent after treatment with 100 millimolar NaCl for 1 day or 50 millimolar for 4 days. These results confirm the suggestion that sheath tissue is capable of accumulating excess Cl⁻ (and to a lesser extent Na⁺) and suggest that the site of regulation of Cl⁻ concentration in the barley leaf is located in the mesophyll cells of the blade.     

Accumulation of glycinebetaine and its synthesis from radioactive precursors under salt-stress in the cyanobacterium Aphanothece halophytica

Growth in salt-stressed (2.0 M NaCl) Aphanothece halophytica was initially delayed during the first two days of cultivation and eventually attained the same growth rate as the control (0.5 M NaCl) cells. Glycinebetaine accumulation increased slightly in control cells but a dramatic increase of glycinebetaine occurred in salt-stressed cells during a growth period of six days. There was no apparent increase in the synthesis of [¹⁴C] glycinebetaine in the control cells, in contrast to the marked increase in its synthesis in the salt-stressed cells. Increasing NaCl concentration in the growth medium induced both the accumulation and the synthesis of glycinebetaine. Time course experiments provided evidence that [¹⁴C] choline was first oxidized to [¹⁴C] betaine aldehyde which was further oxidized to [¹⁴C] glycinebetaine in A. halophytica. The supporting data for such a pathway were obtained from the presence of choline and betaine aldehyde dehydrogenase activities found in the membrane and cytoplasmic fractions, respectively. The activities of these two enzymes were also enhanced upon increasing NaCl concentration in the growth medium from 0.5 M to 2.0 M. Under this condition an increaseof approximately 1.5-fold was observed for choline dehydrogenase activity as compared to 2.5-fold for betaine aldehyde dehydrogenase activity, suggesting a preferable induction of the latter enzyme by salt stress. A. halophytica was able to utilize [¹⁴C] ethanolamine and [¹⁴C] glycine for the synthesis of [¹⁴C] glycinebetaine. This revised version was published online in August 2006 with corrections to the Cover Date.     

Light-induced membrane potential changes of epidermal and mesophyll cells in growing leaves of Pisum sativum

Light transiently depolarizes the membrane of growing leaf cells. The ionic basis for changes in cell membrane electrical potentials in response to light has been determined separately for growing epidermal and mesophyll cells of the argenteum mutant of pea (Pisum sativum L.). In mesophyll cells light induces a large, transient depolarization that depends on the external Cl^– concentration, is unaffected by changes in the external Ca²⁺ or K⁺ concentration, is stimulated by K⁺-channel blockers tetraethylammonium (TEA⁺) and Ba²⁺, and is inhibited by 3-(3-4-dichlorophenyl)-1,1-dimethylurea (DCMU). In isolated epidermal tissue, light induces a small, transient depolarization followed by a hyperpolarization of the membrane potential. The depolarization is enhanced by increasing the external Ca²⁺ concentration and by addition of Ba²⁺, and is not sensitive to DCMU. Epidermal cells in contact with mesophyll display a depolarization resembling the response of the underlying mesophyll cells. The light-induced depolarization in mesophyll cells seems to be mediated by an increased efflux of Cl^– while the membrane-potential changes in epidermal strips reflect changes in the fluxes of Ca²⁺ and in the activity of the proton-pumping ATPase.Abbreviations BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - CCCP carbonylcyanide m-chlorophenylhydrazone - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - LID _e light-induced depolarization in epidermal cells - LID _m light-induced depolarization in mesophyll cells - LIH light-induced hyperpolarization - TEA⁺ tetraethylammonium Ecotrans paper #43. This research was supported by National Science Foundation grants DCB-8903744 and MCB-9220110 to E.V.     

Palisade mesophyll cell expansion during leaf development in Zinnia elegans (Asteraceae)

Temporal and spatial patterns of palisade mesophyll cell expansion in Zinnia elegans were characterized as a basis for developing a suspension culture model for mesophyll cell expansion. Our objectives were to 1) identify the leaf regions from which cells in various stages of expansion could be selectively isolated for culture, and 2) develop a basis for comparison of rate and extent of mesophyll cell expansion in culture with that in the leaf. Palisade mesophyll cells were isolated from expanding leaves by gentle physical maceration without the use of enzymes. Isolated cells from leaves in different stages of expansion were then measured by computer image analysis. Analysis of size frequency distributions showed that unexpanded cells can be isolated from the entire blade of small leaves or the basal regions of partially expanded leaves. Fully expanded cells can be obtained from the apical and middle regions of partially expanded leaves. Within the leaf, Zinnia mesophyll cells expanded from about 400 μm² to about 2.300 μm² at an estimated rate of 160 μm² d^-1. The percent increase in cell length exceeded the percent increase in cell width. Expansion of mesophyll cells continued for 6–8 d after epidermal expansion ceased. This difference in the timing of cell expansion in epidermal and mesophyll cells indicates that different regulatory factors may be operating in these adjacent tissues and underscores the importance of investigating the regulation of mesophyll cell expansion at the cellular level.     

Effects of lithium and potassium on recovery of solute uptake capacity of Acer pseudoplatanus cells after gas-shock

At concentrations of 10-^?3M, Li⁺ inhibits the recovery of solute uptake capacity of Acer pseudoplatanus L. cell suspension cultures after gas-shock (i.e. after rapid exchange of the atmosphere in the culture flasks for ambient air). It also reduces solute uptake capacity of cells having already attained high rates of uptake during recovery from gas-shock. The effects of Li⁺ are much greater in cells which have been cultivated in 7 mM K⁺ solution than in cells cultivated with higher K⁺ levels (19 mM). Increasing K⁺ concentration during recovery reverses the effect of 10^–3M Li⁺ and, with sufficiently high concentrations of K⁺ (≥ 10-^?2M) during recovery, the solute uptake capacity of the fully recovered cells can even become greater than that of the control, at least for the low values of substrate concentration (here sulphate 10-^?5M). Since Li⁺ does not affect the time course of solute uptake measured over 15–20 min, it is thought that it interacts with the synthesis and turnover of the solute uptake machinery of the Acer pseudoplatanus cells. Thermodynamic analysis of the flux data also supports the hypothesis that Li⁺ inhibits the biosynthesis of specific sites of solute permeation, but it does not rule out the possibility that K⁺ interferes rather on the forces acting on the transport of the considered solutes than on the catalytic structures of permeation.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Rubisco activity in guard cells compared with the solute requirement for stomatal opening

We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [¹⁴C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to ¹⁴CO₂ for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.     

Isolation of mesophyll and secretory cell protoplasts of the halophyte Ceratostigma plumbaginoides (L.): a comparison of ATPase concentration and activity

Summary A method is described for isolating mesophyll protoplasts from leaves and secretory cell protoplasts from salt glands of the facultative halophyte, Ceratostigma plumbaginoides (L.). Rates of ATP hydrolysis in both cell types were determined, and levels in secretory cell protoplast preparations were fourfold higher than those in mesophyll protoplast preparations, based on total protein. The rate of ATP hydrolysis was sensitive to azide and vanadate, and stimulated by Triton-X-100. Additionally, immunoblot procedures using an antibody to the plasma membrane H⁺/ATPase was used to compare ATPase levels of the mesophyll and secretory cell protoplasts. Results indicate that secretory cells have a higher concentration of H⁺/ATPase than mesophyll cells, consistent with their putative function in salt glands.Abbreviations ATP adenosine triphosphate - BSA bovine serum albumin - DIDS diisothiocyano-2,2'-disulfonic acid stilbene - DNP dinitrophenol - DTT dithiothreitol - FITC fluorescein isothiocyanate - NAD⁺/NADH nicotinamide adenine dinucleotide - SDS sodium dodecylsulfate