Subcellular Localization of 2-(beta-d-Glucosyloxy)-Cinnamic Acids and the Related beta-glucosidase in Leaves of Melilotus alba Desr

The distribution of the glucosides of trans- and cis-2-hydroxy cinnamic acid and of the β-glucosidase which hydrolyzes the latter glucoside was examined in preparations of epidermal and mesophyll tissue obtained from leaves of sweet clover (Melilotus alba Desr.). The concentrations of glucosides in the two tissues were about equal when compared on the basis of fresh or dry weight. Inasmuch as the epidermal layers account for no more than 10% of the leaf volume, the mesophyll tissue contains 90% or more of the glucosides. Vacuoles isolated from mesophyll protoplasts contained all of the glucosides present initially in the protoplasts.     

Subcellular Localization of a UDP-Glucose:Aldehyde Cyanohydrin beta-Glucosyl Transferase in Epidermal Plastids of Sorghum Leaf Blades

Epidermal and mesophyll protoplasts, prepared from leaf blades of 6-day-old light-grown Sorghum bicolor seedlings were separated by differential sedimentation and assayed for a number of enzymes. The epidermal protoplasts contained higher levels of NADPH-cytochrome c reductase (EC 1.6.2.4), triose phosphate isomerase (EC 5.3.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.31), and a UDP-glucose:cyanohydrin β-glucosyl transferase (EC 2.4.1.85), but lower levels of NADP⁺ triosephosphate dehydrogenase (EC 1.2.1.13) than did mesophyll protoplasts. When protoplast preparations were lysed and applied to linear sucrose density gradients, triosephosphate isomerase was found to be present in epidermal plastids. A significant fraction (41%) of the glucosyl transferase activity was also associated with the epidermal plastids.     

Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L

The blue emission anisotropy, r, of two lipophilic probes, diphenylhexatriene (DPH) and its trimethyl-ammonium derivative (TMA-DPH), has been measured in foliar Lupinus albus L. protoplasts for the first time by flow cytometry. Distinctive values have been obtained for protoplasts of epidermal and mesophyll origin, identified by their intensities of chlorophyll fluorescence. Fluorescence microscopy confirmed that TMA-DPH remained in the plasma membrane while DPH penetrated into intracellular lipid domains. Typical emission anisotropy values at 22°C for mesophyll and epidermal protoplasts, respectively, were 0.225 and 0.312 with TMA-DPH, and 0.083 and 0.104 with DPH. This indicates that epidermal cells—and notably their plasma membranes (TMA-DPH)—have higher lipid microviscosity and/or more ordered lipid structure. Two lupin genotypes characterized as resistant or susceptible to drought were analyzed with or without 9 days of water stress shown to increase ion leakage from foliar discs. Water stress greatly increased the apparent fluidity, and more so in the susceptible genotype; the effect was more pronounced in the chlorophyll-containing mesophyll cells than in the epidermal cells.     

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.

     

Tissue Distributions of Chlorogenic Acid and of Enzymes Involved in Its Metabolism in Leaves of Sorghum bicolor

The tissue distributions of cholorgenic acid, chlorogenic acid oxidase, and three other enzymes involved in the metabolism of this secondary (natural) product have been investigated in leaf-blades of light-grown seedlings of Sorghum bicolor. Cholorogenic acid was found only in epidermal and mesophyll protoplasts isolated from the leaf; 60% of the chlorogenic was contained in the epidermal fraction. Nearly all (90%) of the chlorogenic acid oxidase was found in the mesophyll protoplasts. The bundle-sheath strands, on the other hand, contained no chlorogenic acid and essentially none of the oxidase. Three other enzymes required for the synthesis of chlorogenic acid, but also for other plant products, were found in all three tissue fractions.     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

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.

     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco

It is known that protoplasts derived from either leaves or suspension cultures of a citrus genotype vary greatly in their regeneration capacities; however, the underlying physiological mechanisms are not well known. In this study, oxidative stress and antioxidant systems during in vitro culture of callus-derived protoplasts and leaf mesophyll-derived protoplasts of Ponkan (Citrus reticulata Blanco) were analyzed to gain insights into observed physiological differences. Morphological observations using light microscopy and scanning microscopy have shown that new cell wall materials appeared within 2–3 days, and the integrate cell walls were regenerated approximately after 6 days of culture of the callus protoplasts, whereas no cell wall formation was observed in the mesophyll protoplasts after culture. During the culture, higher levels of H₂O₂ and malondialdehyde were detected in the mesophyll protoplasts as compared with the callus ones. On the contrary, the callus protoplasts possessed higher activities of antioxidant enzymes (SOD, POD and CAT) and larger amount of glutathione and ascorbic acid (at one time point) than the mesophyll protoplasts during the culture process. The current data indicate that the mesophyll and callus protoplasts displayed remarkable difference in the degree of oxidative stress and the antioxidant systems, suggesting that high levels of antioxidant activities might play an important role in the regeneration of protoplasts.     

Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis

In all organisms synthesising phenylalanine and/or tyrosine via arogenate, a prephenate aminotransferase is required for the transamination of prephenate into arogenate. The identity of the gene encoding this enzyme in the organisms where this activity occurs is still unknown. Glutamate/aspartate-prephenate aminotransferase (PAT) is thus the last homeless enzyme in the aromatic amino acids pathway. We report on the purification, mass spectrometry identification and biochemical characterization of Arabidopsis thaliana prephenate aminotransferase. Our data revealed that this activity is housed by the prokaryotic-type plastidic aspartate aminotransferase (At2g22250). This represents the first identification of a gene encoding PAT.     

Pressure Effects on Membrane Potentials of Mesophyll Cell Protoplasts and Epidermal Cell Protoplasts of Commelina communis L.

Pantoja, O. and Willmer, C. M. 1986. Pressure effects on membranepotentials of mesophyll cell protoplasts and epidermal cellprotoplasts of Commelina communis L.—J. exp. Bot. 37:315–320. Membrane potentials of epidermal cell protoplasts and mesophyllcell protopiasts of Comnelina communis were measured when theprotoplasts were immobilized in a suction micropipette. Whenzero suction was employed, membrane potentials of both protoplasttypes were near to zero. As suction pressure was increased,membrane potentials became increasingly more negative with gradientsof 14·3 mV/kPa and 10·5 mV/kPa for mesophyll cellprotoplasts and epidermal cell protoplasts, respectively. Theplasma membrane is stretched when suction pressure is appliedto protoplasts and it is considered that this simulates cellturgor pressure which is associated with negative membrane potentialsof intact cells. The results help to explain why some investigatorsobtain positive membrane potentials for protoplasts while othersobtain negative values. The results also indicate that considerablecaution is needed in the interpretation of ion flux data whenprotoplasts are used. Key words: Commelina communis, membrane potentials, pressure, protoplasts     

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.     

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Multiple forms of phosphoenolpyruvate carboxylase in mesophyll, epidermal and guard cells of Vicia faba

The distribution of phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in different leaf‐cell‐types and tissues of Vicia faba L. cv. 3‐fach Weiße was studied. The highest specific PEPCase activity was found in guard cell protoplasts (16.3 µmol mg⁻¹ protein h⁻¹) whereas for epidermal and mesophyll protoplasts remarkably lower specific activities were found (1.6 and 1.0 µmol mg⁻¹ protein h⁻¹, respectively). On chlorophyll and protoplast basis, a similar distribution of enzyme activity was observed. Compared with epidermal extracts, the specific PEPCase activity of mesophyll tissue was 17‐fold lower. Immunological studies with polyclonal antibodies to PEPCase indicated 3 immunoreactive proteins in epidermal tissue and guard cell protoplasts with molecular masses of 107 000, 110 000, and 112 000. Only the M_r 107 000 protein was found in extracts of mesophyll and epidermis protoplasts. Western immunoblots after native electrophoresis of epidermal and mesophyll proteins showed a significant difference in PEPCase mobility. It is assumed, that the immunostained proteins of M_r 110 000 and 112 000 represent isoforms or subunits of the PEPCase and that they are involved in stomatal movements.     

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.     

Localization of partially methylated flavonol glucosides in Chrysosplenium americanum. II. Immunofluorescence

Chrysosplenium americanum Schwein. ex Hooker (Saxifragaceae) accumulates a variety of partially methylated flavonol glucosides. Specific antibodies to tri- and tetramethylated flavonol-2′-O-glucosides, located using fluorescein isothiocyanate (FITC) goat antirabbit antibody, were used for the localization of the flavonol glucosides in leaf epidermis, cross sections and protoplasts. The results indicated that flavonoid accumulation occurred mainly in the walls of epidermal cells and, to a much lesser extent, in mesophyll cell walls. The weak fluorescence observed in the vacuoles of protoplasts suggested a minor role of this compartment in the accumulation process. The significance of flavonoid deposition within epidermal cell walls is discussed in relation to the lipophilic nature of these compounds and their possible role in the physiology of the plant.     

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.     

Secondary phenolic products in isolated guard cell,epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination

Summary Guard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.Abbreviations uE adaxial (upper) epidermis - IE abaxial (lower) epidermis - GCP guard cell protoplasts - ECP epidermal cell protoplasts - MCP mesophyll cell protoplasts - PP protoplasts - HPLC high performance liquid chromatography - TLC thin layer chromatography - CC column chromatography - HOAc acetic acid