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PubMed1997

Characterization of a Light-Controlled Anion Channel in the Plasma Membrane of Mesophyll Cells of Pea.

In leaf mesophyll cells of pea (Pisum sativum) light induces a transient depolarization that is at least partly due to an increased plasma membrane conductance for anions. Several channel types were identified in the plasma membrane of protoplasts from mesophyll cells using the patch-clamp technique. One of these was an anion channel with a single-channel conductance of 32 picasiemens in symmetrical 100/100 KCl solutions. In asymmetrical solutions the reversal potential indicates a high selectivity for Cl- over K+ at high cytoplasmic Cl-. At negative membrane voltages the channel openings were interrupted by very short closures. In the open channel conductance several substrates were identified. At a cytoplasmic negative logarithm of Ca concentration higher than 6.3, no channel openings were observed. When the protoplast was illuminated in the cell-attached configuration, at least one channel type had a higher opening probability. This channel can tentatively be identified as the above-described anion channel based on conductance and the characteristic short closures at negative membrane potentials. This light activation of the 32-picasiemen anion channel is a strong indication that this channel conducts the light-induced depolarizing current. Because channel activity is strongly Ca2+-dependent, a role of cytoplasmic Ca2+ concentration changes in the light activation of the conductance is discussed.

Journal Article↗
PubMed1975

C(4) Photosynthesis: Light-dependent CO(2) Fixation by Mesophyll Cells, Protoplasts, and Protoplast Extracts of Digitaria sanguinalis.

Mesophyll cells, protoplasts, and protoplast extracts of Digitaria sanguinalis were used for comparative studies of light-dependent CO(2) fixation. CO(2) fixation was low without the addition of organic substrates. Pyruvate, oxaloacetate, and 3-phosphoglycerate induced relatively low rates (10 to 90 mumoles/mg chlorophyll.hr) of CO(2) fixation when added separately. However, a highly synergistic relationship was found between pyruvate + oxaloacetate and pyruvate + 3-phosphoglycerate for inducing light-dependent CO(2) fixation in the mesophyll preparations. Highest rates of CO(2) fixation were obtained with protoplast extracts. Pyruvate, in combination with oxaloacetate or 3-phosphoglycerate induced light-dependent rates from 150 to 380 mumoles of CO(2) fixed/mg chlorophyll.hr which are equivalent to or exceed reported rates of whole leaf photosynthesis in C(4) species. Concentrations of various substrates required to give half-maximum velocities of CO(2) fixation were determined, with the protoplast extracts generally saturating at the lowest substrate concentrations. Chloroplasts separated from protoplast extracts showed little capacity for CO(2) fixation. The results suggest that CO(2) fixation in C(4) mesophyll cells is dependent on chloroplasts and extrachloroplastic phosphoenolpyruvate carboxylase.The stimulation of pyruvate-induced CO(2) fixation by oxaloacetate and 3-phosphoglycerate is thought to be due to induction of noncyclic electron transport which generates ATP for the conversion of pyruvate to phosphoenolpyruvate by pyruvate Pi dikinase. The primary products of the substrate-induced CO(2) fixation were oxaloacetate and malate, which provides further evidence for carbon fixation through the beta-carboxylation pathway. High rates of light-dependent CO(2) fixation with a significant percentage of (14)C fixed into malate suggest an efficient operation of both photosystems I and II.The substrate inductions are discussed with respect to the proposed role of the mesophyll cell in C(4) photosynthesis, and schemes suggesting the stoichiometry of energy requirements for photosynthetic carbon metabolism in C(4) mesophyll cells are presented.

Journal Article↗
PubMed2005

Death of mitochondria during programmed cell death of leaf mesophyll cells.

The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.

Apoptosis↗
PubMed1989

Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves.

Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO(2) fixation. Maximal CO(2) fixation rate was obtained when the osmotic potential of both cell isolation and CO(2) fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO(2) fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl(-) and Na(+) accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels.

Journal Article↗
PubMed1987

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells.

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO(4). Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H(+)/10(6) cells.minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-(14)C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K(+) efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.

Journal Article↗
PubMed1984

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.).

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.

Journal Article↗
PubMed1989

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.

Journal Article↗
PubMed1999

The endogenous sulfated pentapeptide phytosulfokine-alpha stimulates tracheary element differentiation of isolated mesophyll cells of zinnia

Dispersed zinnia (Zinnia elegans) mesophyll cells cannot differentiate into tracheary elements (TEs) at low cell density conditions even if auxin and cytokinin are present in the medium, indicating the involvement of intercellular interactions during the initiation and/or subsequent progresses in TE differentiation. When zinnia cells were incubated at a low density (2.5 x 10(4) cells mL(-1)) in TE-inductive medium in the presence of various concentrations of phytosulfokine (PSK)-alpha, which was originally identified as an intercellular signal peptide involved in cell proliferation, TE differentiation was strongly stimulated in a dose-dependent fashion; more than 35% of the living cells differentiated into TEs by 5 d of culture in the presence of 10 nM PSK-alpha. Enzyme-linked immunosorbent assay and mass spectroscopy confirmed that cultured zinnia cells produce nanomolar levels of PSKs under inductive conditions. These results suggest that PSK-alpha is a factor responsible for TE differentiation of zinnia mesophyll cells.

Journal Article↗
PubMed2003

Ca2+-dependent cessation of cytoplasmic streaming induced by hypertonic treatment in Vallisneria mesophyll cells: possible role of cell wall-plasma membrane adhesion.

In mesophyll cells of the aquatic angiosperm Vallisneria gigantea Graebner, a rapid and transient inhibition of cytoplasmic streaming was induced by hypertonic treatment with sorbitol. Higher concentrations of sorbitol induced the response more rapidly and in more cells. The response to hypertonic treatment was strictly dependent on the presence of extracellular Ca2+ and was sensitive to Ca2+-channel blockers, including the stretch-activated Ca2+-channel blocker Gd3+. Deplasmolyzed cells never responded to a second hypertonic treatment administered immediately after plasmolysis and subsequent deplasmolysis. Responsiveness was gradually recovered during 24 h of incubation; however, cycloheximide, cordycepin, and trypsin completely suppressed the recovery. Although an Arg-Gly-Asp (RGD) hexapeptide markedly disturbed the pattern of cytoplasmic streaming, it exhibited no specific effects on the response to hypertonic treatment or on the recovery of responsiveness. Taken together, our results demonstrate that leaf mesophyll cells in a multicellular plant can respond to mechanical stimuli and that a Ca2+ influx through stretch-activated Ca2+ channels plays an indispensable role in the response. Furthermore, the possible involvement of RGD-insensitive but trypsin-sensitive protein factor(s), whose function is impaired by detachment of the plasma membrane from the cell wall, is suggested.

Calcium↗
PubMed1998

Cyst(e)ine is the transport metabolite of assimilated sulfur from bundle-sheath to mesophyll cells in maize leaves

The intercellular distribution of the enzymes and metabolites of assimilatory sulfate reduction and glutathione synthesis was analyzed in maize (Zea mays L. cv LG 9) leaves. Mesophyll cells and strands of bundle-sheath cells from second leaves of 11-d-old maize seedlings were obtained by two different mechanical-isolation methods. Cross-contamination of cell preparations was determined using ribulose bisphosphate carboxylase (EC 4.1.1.39) and nitrate reductase (EC 1.6.6.1) as marker enzymes for bundle-sheath and mesophyll cells, respectively. ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase activities were detected almost exclusively in the bundle-sheath cells, whereas GSH synthetase (EC 6.3.2.3) and cyst(e)ine, gamma-glutamylcysteine, and glutathione were located predominantly in the mesophyll cells. Feeding experiments using [35S]sulfate with intact leaves indicated that cyst(e)ine was the transport metabolite of reduced sulfur from bundle-sheath to mesophyll cells. This result was corroborated by tracer experiments, which showed that isolated bundle-sheath strands fed with [35S]sulfate secreted radioactive cyst(e)ine as the sole thiol into the resuspending medium. The results presented in this paper show that assimilatory sulfate reduction is restricted to the bundle-sheath cells, whereas the formation of glutathione takes place predominantly in the mesophyll cells, with cyst(e)ine functioning as a transport metabolite between the two cell types.

Journal Article↗
PubMed1982

Two-Dimensional Electrophoretic Mapping of Proteins of Bundle Sheath and Mesophyll Cells of the C(4) Grass Digitaria sanguinalis (L.) Scop. (Crabgrass).

Two-dimensional electrophoresis was performed on proteins of bundle sheath and mesophyll cells isolated from the C(4) grass Digitaria sanguinalis (L.) Scop. Two-dimensional maps of these proteins were constructed and ribulose-1,5-biphosphate carboxylase and phosphoenolpyruvate carboxylase were identified. Of the total number of proteins found in both cell types, 36% were found only in bundle sheath cells, 17% only in mesophyll cells, and 47% in both cell types. By comparison, the distributions of 48 enzymes assayed in these cell types were 35%, 21%, and 44%, respectively.Protein patterns were also compared with C(4) plants exhibiting different decarboxylation pathways and, in both bundle sheath and mesophyll cells, proteins were found which were unique to each species. Bundle sheath proteins of one C(4) species were found to be more like bundle sheath proteins of another C(4) species than like mesophyll proteins of the same species.

Journal Article↗
PubMed2001

Involvement of local intercellular communication in the differentiation of zinnia mesophyll cells into tracheary elements.

The transdifferentiation of isolated mesophyll cells of zinnia (Zinnia elegans L.) into tracheary elements (TEs) has been well studied as a model of plant cell differentiation. In order to investigate intercellular communication in this phenomenon, two types of culture method were developed, in which mesophyll cells were embedded in a thin sheet of agarose gel and cultured on solid medium, or embedded in microbeads of agarose gel and cultured in liquid medium. A statistical analysis of the two-dimensional distribution of TEs in the thin-sheet cultures demonstrated their aggregation. In the microbead cultures, the frequency of TE differentiation was shown to depend on the local cell density (the cell density in each microbead): TE differentiation required local cell densities of more than 10(5) cells ml(-1). These results suggest that TE differentiation involves cell-cell communication mediated by a locally acting diffusible factor. This presumptive factor was characterized by applying a modified version of the sheet culture, which used two sheets of different cell densities, a low-density sheet and a high-density sheet. Differentiation of TEs in the former could be induced only by bringing it into contact with the latter. Insertion of a 25-kDa-cutoff membrane between the high-density and low-density sheets severely suppressed such induction of TEs in the low-density sheet while a 300-kDa-cutoff membrane suppressed induction only slightly. Insertion of agarose sheets containing immobilized pronase E or trypsin also interfered with the induction of TEs in the low-density sheets. Thus, a proteinaceous macromolecule of 25-300 kDa in molecular weight was assumed to mediate the local intercellular communication required for TE differentiation. This substance was designated "xylogen" with reference to its xylogenic activity. The time of requirement for xylogen during TE differentiation was assessed by experiments in which cells in the low-density sheet were separated from xylogen produced in the high-density sheet at various times by insertion of a 25-kDa-cutoff membrane between the two sheets, and was estimated to be from the 36th hour to the 60th hour of culture (12-36 h before visible thickening of secondary cell walls of TEs).

Asteraceae↗
PubMed1980

The effect of glyoxylate on photosynthesis and photorespiration by isolated soybean mesophyll cells.

Incubating isolated soybean leaf mesophyll cells with glyoxylate increased the rates of CO(2) fixation by as much as 150%. In order to cause this stimulation, the glyoxylate must be presented to the cells before the NaHCO(3). Significant stimulation was observed 15 seconds after beginning the glyoxylate treatment. The glyoxylate-dependent stimulation was increased by high O(2) concentrations and decreased by high CO(2) concentrations. Glyoxylate treatment resulted in a 71% inhibition in the rate of CO(2) incorporation into glycolate and glycine. Glyoxylate may be stimulating net photosynthesis solely by decreasing photorespiration or it may be increasing the amount of CO(2) fixed by both decreasing photorespiration and increasing gross photosynthesis. Ribulose bisphosphate carboxylase, when preactivated and assayed in situ, was unaffected by the glyoxylate treatment.

Journal Article↗
PubMed1973

Oxaloacetate as the hill oxidant in mesophyll cells of plants possessing the c(4)-dicarboxylic Acid cycle of leaf photosynthesis.

Isolated mesophyll cells from leaves of plants that use the C(4) dicarboxylic acid pathway of CO(2) fixation have been used to demonstrate that oxaloacetic acid reduction to malic acid is coupled to the photochemical evolution of oxygen through the presumed production of NADPH. The major acid-stable product of light-dependent CO(2) fixation is shown to be malic acid. In the presence of phosphoenolpyruvate and bicarbonate the stoichiometry of CO(2) fixation into acid-stable products to O(2) evolution is shown to be near 1.0. Thus oxaloacetic acid acts directly as the Hill oxidant in mesophyll cell chloroplasts. The experiments are taken as a firm demonstration that the C(4) dicarboxylic acid cycle of photosynthesis is the major pathway for the fixation of CO(2) in mesophyll cells of plants having this pathway.

Journal Article↗
PubMed1993

Characterization of a G-protein-regulated outward K+ current in mesophyll cells of vicia faba L.

Whole-cell voltage-dependent currents in isolated mesophyll protoplasts of Vicia faba were investigated by patch-clamp techniques. With 104 mM K+ in the cytosol and 13 mM K+ in the external solution, depolarization of the plasma membrane from -47 mV to potentials between -15 and +85 mV activated a voltage- and time-dependent outward current (Iout). The average magnitude of Iout at +85 mV was 28.5 +/- 3.3 pA.pF-1. No inward voltage-dependent current was observed upon hyperpolarization of the plasma membrane from -55 mV to potentials as negative as -175 mV. Time-activated outward current was blocked by Ba2+ (1 mM BaCl2) and was not observed when K+ was eliminated from the external and internal solutions, indicating that this outward current was carried primarily by K+ ions. The voltage dependency of outward K+ current revealed a possible mechanism for K+ efflux from mesophyll cells. A GDP analogue guanosine 5'-[beta-thio]diphosphate (500 microM) significantly enhanced outward K+ current. The outward K+ current was inhibited by the GTP analogue guanosine 5'-[gamma-thio]triphosphate (500 microM) and by an increase in cytoplasmic free Ca2+ concentrations. Cholera toxin, which ADP-ribosylates guanine nucleotide-binding regulatory proteins, also inhibited outward K+ current. These findings illustrate the presence in mesophyll cells of outward-rectifying K+ channels that are regulated by GTP-binding proteins and calcium.

Adenosine Diphosphate↗
PubMed2000

Metabolic relations of inositol 3,4,5,6-tetrakisphosphate revealed by cell permeabilization. Identification of inositol 3,4,5, 6-tetrakisphosphate 1-kinase and inositol 3,4,5,6-tetrakisphosphate phosphatase activities in mesophyll cells.

Using a permeabilization strategy to introduce Ins(3,4,5,6) P(4) into mesophyll protoplasts of Commelina communis, we have identified Ins(3,4,5,6) P(4) 1-kinase activity in mesophyll cells. Multiple InsP(3) isomers were identified in Spirodela polyrhiza and Arabidopsis. Only two of these, Ins(1,2,3) P(3) and Ins(3,4,6) P(3), have previously been identified in plants and only in monocots. The isomers detected in S. polyrhiza included D- and/or L-Ins(3,4,5) P(3), D- and/or L-Ins(3,5,6) P(3), and D- and/or L-Ins(2,4,5) P(3). Ins(1,4,5) P(3), if present, was only a tiny fraction of total InsP(3) species. We have also identified inositol polyphosphate phosphatase activities, Ins(3,4,5,6) P(4) 6-phosphatase and Ins(3,4, 5, 6) P(4) 4-phosphatase, whose action on endogenous inositol polyphosphates explains the presence of D- and/or L-Ins(3,4,5) P(3) and D- and/or L-Ins(3,5,6) P(3) in mesophyll cells. Inositol trisphosphates identified in Arabidopsis include Ins(1,2,3) P(3) and D- and/or L-Ins(3,4,6) P(3), suggesting that dicots may share pathways of InsP(6) biosynthesis and breakdown in common with monocots.

Cell Membrane Permeability↗
PubMed2004

Transition of G1 to early S phase may be required for zinnia mesophyll cells to trans-differentiate to tracheary elements.

We have used the Zinnia elegans mesophyll cell system, in which single isolated leaf mesophyll cells can be induced to trans-differentiate into tracheary elements in vitro, to study the relationship between the cell division cycle and cell differentiation. Almost all cells go through several rounds of division before characteristic features of tracheary element formation are observed. The addition of aphidicolin, a DNA synthesis inhibitor, blocks cell division but not cell differentiation in the zinnia system. Low concentrations of aphidicolin, which possibly delay cells in the early S phase, can significantly enhance levels of tracheary element formation. In contrast, roscovitine, an inhibitor of cyclin-dependent kinase activity, decelerates the cell division cycle and inhibits tracheary element formation with similar dose responses. Cells blocked in S phase and then transferred to roscovitine-containing medium can divide once, indicating that roscovitine may target the G1/S transition, but do not differentiate. Cells inhibited in G1/S in roscovitine-containing medium that are subsequently blocked in S phase by transfer to aphidicolin-containing medium, do not divide but do differentiate. Taken together, our results indicate that cells may be required to transit the G1/S checkpoint and enter early S phase to acquire competence to trans-differentiate to tracheary elements.

Aphidicolin↗