Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.     

Acclimation to Drought in Acer pseudoplatanus L. (Sycamore) Seedlings

A glasshouse experiment was conducted with well-watered andwater-stressed seedlings of sycamore (Acer pseudoplatanus L.)grown in soil columns. Water was withheld when the seedlingswere 82-d-old. Effects of soil drying on stomatal behaviour,water relations, xylem cavitation, and growth of leaves androots were evaluated. Stomatal conductance declined well before any observable changein bulk leaf water potentials, and was correlated with soilwater status. At seven weeks, osmotic potential had declinedby 0·51 MPa and 0·44 MPa at full and zero turgor,respectively. Drought significantly increased both bulk elasticmodulus and leaf dry weight to turgid weight ratio of water-stressedplants. Drought had no effect on relative water content at zeroturgor. Water cavitation in the xylem was detected as ultrasonic acousticemissions (AE). Water-stressed plants displayed significantlyhigher rates of AE than well-watered plants. Maximum rate ofAE coincided with the minimum level of stomatal conductanceand apparent rehydration of the leaves. Drought caused changes in the root distribution profile andit increased the root weight. The increase in root weight wasmainly due to a substantial shift in assimilates allocated infavour of roots with total biomass being unaffected. Leaf growthwas maintained for six weeks without any significant declinein expansion rate. However, the development of severe waterstress reduced both leaf production and expansion.     

Protein Phosphorylation in Amyloplasts Isolated from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Highly purified amyloplasts were isolated from cultured cells of sycamore (Acer pseudoplatanus L.). Incubation of amyloplasts with [γ-³²P]-ATP resulted in the labeling of more than ten polypeptides. Pulsechase experiments showed the reversibility of the process with some but not all of the polypeptides. The phosphorylation reaction of one polypeptide, M_r 100, was shown to be calcium dependent. Although exogenously added pig brain calmodulin had no effect, the calmodulin antagonist W-7 strongly inhibited phosphorylation of the 100 kilodaltons polypeptide. The presence of endogenous calmodulin, about 1 to 3 micrograms per milligram protein, in the amyloplast preparation was estimated by activation of phosphodiesterase in vitro.     

Effect of Iron Deficiency on the Respiration of Sycamore (Acer pseudoplatanus L.) Cells

The effects of iron deficiency on cell culture growth, cell respiration, mitochondrial oxidative properties, and the electron transport chain were studied with suspension-cultured sycamore (Acer pseudoplatanus L.) cells. Iron deprivation considerably decreased the initial growth rates and limited the maximum density of the cells. Under these conditions, the cells remained swollen throughout their growth. The absence of iron led to a steady decline in the uncoupled rate of O2 consumption. When the uncoupled rate of O2 uptake closely approximated the respiratory rate, the cells began to collapse. At this stage, the level of all the cytochromes and electron paramagnetic resonance-detectable Fe-S clusters of the mitochondrial inner membrane were dramatically decreased. Nevertheless, it appeared from substrate oxidation measurements that this overall depletion in iron-containing components solely disturbed the functioning of complex II, whereas neither complexes I, III, or IV, nor the machinery involved in ATP synthesis, was apparently impaired in iron-deficient mitochondria. However, our results suggest that the impairment of complex II resulted in a strong reduction of the overall capacity of the mitochondrial electron transport chain, which was responsible for determining the rate of endogenous respiration in sycamore cells. Finally, this situation led to a depletion of various energy metabolites that could contribute to the premature cell death.     

Isolation and Characterization of Golgi Membranes from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

A simple and rapid technique was developed for the isolationof the vesicular Golgi membranes from suspension-cultured cellsof sycamore (Acer pseudoplatanus L.). The procedure involvespreparation of protoplasts and differential centrifugation ofdisrupted protoplasts followed by the sucrose density gradientcentrifugation. Starting from broken protoplasts, sedimentableat two different centrifugal forces (10,000g and 100,000 g),two Golgi-enriched fractions of lower density, GF₁ and GF'₁,and higher density, GF₂ and GF'₂, were separated. Purity ofthe fraction was assessed by determining the marker enzyme activitiesas well as the electron microscopy of the specimens obtained. Inosine diphosphatase was enriched about 15- and 6-fold, respectively,in the GF₂ fraction from 10,000g and the GF'₂ one from 100,000gpellets, whereas the enrichment in GF₁ and GF'₁ was approximately6–7 fold. Galactosyl-transferase in GF₂ was enriched about25-fold. GF₁ and GF₂ account for 3–4% of the total proteinof 10,000g pellets, and GF'₁ and GF'₂ for about 6–7%of the total protein of 100,000g pellets. Electron microscopicobservations show that GF₂ and GF'₂ consisted principally ofvesicular Golgi membranes without an internal matrix althoughGF₁ and GF'₁ were contaminated with ER membranes and ribosomes. (Received March 11, 1985; Accepted June 17, 1985)     

Multiple Metal Resistance and Co-resistance in Acer pseudoplatanus L. (Sycamore) Callus Cultures

In vitro growth of Acer pseudoplatanus L. (sycamore) callustissue derived from shoot-tip explants and screened on metal-enrichedmedia was studied in an attempt to identify resistance traitswhich may explain the survival of trees at metal-contaminatedsites. Copper and Cd-resistance traits were identified in celllines originating from trees at a site with a relatively recenthistory of severe contamination by these metals, and Cd- andZn-resistance were identified in cell lines originating frommature trees at a mining spoil site with a much longer historyof exposure to elevated concentrations of these metals. In Zn-resistantcell lines, co-resistance to Ni was also found, even thoughthis metal was not elevated at the study site. This is the firstreport of multiple resistance and co-resistance to metals occurringat the cellular level in trees. The mechanisms of the measuredresistance traits remain unclear, although there was evidenceof reduced Cu and Ni uptake by resistant cell lines. It is concludedthat facultative adaptations allowing acclimation to metal stressmay be particularly significant for survival of mature trees;induction of metal resistance probably occurs in vivo in treesat metal-contaminated sites.Copyright 1995, 1999 Academic Press Acer pseudoplatanus, sycamore, callus, heavy metals, metal resistance, trees     

Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.)

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.     

The Involvement of Glycosidases in the Cell Wall Metabolism of Suspension-cultured Acer pseudoplatanus Cells

Several glycosidases have been isolated from suspensioncultured sycamore (Acer pseudoplatanus) cells. These include an α-galactosidase, an α-mannosidase, a β-N-acetyl-glucosaminidase, a β-glucosidase, and two β-galactosidases. The pH optimum of each of these enzymes was determined. The pH optima, together with inhibition studies, suggest that each observed glycosidase activity represents a separate enzyme. Three of these enzymes, β-glucosidase, α-galactosidase, and one of the β-galactosidases, have been shown to be associated with the cell surface. The enzyme activities associated with the cell surface were shown to possess the ability to degrade to a limited extent isolated sycamore cell walls. It was found that the activities of β-glucosidase and of one of the β-galactosidases increase as the cells go through a period of growth and decrease as cell growth ceases.     

Association of H-Translocating ATPase in the Golgi Membrane System from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

The Golgi complex and the disrupted vesicular membranes were prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) using protoplasts as the starting material and employing linear sucrose density gradient centrifugation followed by osmolysis (Ali et al. [1985] Plant Cell Physiol 26: 1119-1133). The isolated Golgi fraction was found to be enriched with marker enzyme activities and depleted of the activity of a typical mitochondrial marker enzyme, cytochrome c oxidase. Golgi complex, and vesicular membranes derived thereof were found to contain the specific ATPase (specific activity of about 0.5 to 0.7 micromoles per minute per milligram protein). Inhibitor studies suggested that the ATPase of Golgi was different from plasma membrane, tonoplast and mitochondrial ATPases as it was not inhibited by sodium vanadate, potassium nitrate, oligomycin and sodium azide. The sensitivity to N-ethylmaleimide further distinguished the Golgi ATPase from F₀ to F₁ ATPase of mitochondria. The internal acidification was measured by monitoring the difference in absorbance at 550 nanometers minus 600 nanometers using neutral red as a probe. The maximum rate detected with Golgi and disrupted membrane system was 0.49 and 0.61 optical density unit per minute per milligram protein, at pH 7.5, respectively, indicating that the proton pump activity was tightly associated with the Golgi membranes. In both cases, the acidification was inhibited 70 to 90% by various ionophores, indicating that the proton pump was electrogenic in nature. Both the Golgi ATPase activity and ATP-dependent acidification were profoundly inhibited by N,N′-dicyclohexylcarbodiimide, which also indicate that the two activities are catalyzed by the same enzyme.     

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [γ-³²P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg²⁺. ATP, and recovery of only [³²P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [γ-³²P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.     

Characterization of microsatellite markers in sycamore (Acer pseudoplatanus L.)

Sycamore (Acer pseudoplatanus L.) is a tetraploid European hardwood tree species. The reproduction system of the insect‐pollinated trees and patterns of genetic variation are largely unknown. We isolated and characterized eight polymorphic microsatellite markers for Acer pseudoplatanus L. The high degree of polymorphism observed at these markers makes them useful to observe genetic variation patterns at various spatial scales and to analyse gene flow and the mating system. Primers developed for the amplification of microsatellites in A. pseudoplatanus were tested for 21 different species of genus Acer. Amplification products of the expected size were obtained in most cases.     

Changes in Metabolite Levels during Growth of Acer pseudoplatanus (Sycamore) Cells in Batch Suspension Culture

Measurements were made of the levels of key glycolytic intermediates and co-factors during the growth of Acer pseudoplatanus L. cells in batch culture. Mass action ratios were calculated for the enzymes phosphoglucoseisomerase, phosphofructokinase and pyruvate kinase. The ratio for phosphofructokinase was greatly displaced from equilibrium. Major increases in the level of NADPH and ATP and energy charge were observed in the initial or lag period of culture. The data indicate that the energy generating system of the cells is most active in the lag phase.     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

The Role of Intervessel Pits and Vessel Plugs in Determining Flow Pathways Through and Between Vessels in Sycamore (Acer pseudoplatanus L.)

It has previously been suggested from physical evidence thatvessels in twigs of sycamore (Acer pseudoplatanus L.) are connectedby small pores in intervessel pit membranes. From results obtainedusing a combination of physical techniques and electron microscopyit is clear that this is an oversimplification: almost all intervesselpit membranes are absent in sycamore twig and stem wood, yetphysical tests still suggest that vessels are connected by poressmaller than pit apertures. Where these pores are is less certain.They may be in plugs observed to fill vessel lumens. They maypossibly be in a small number of intact intervessel pit membranesin the terminal elements of vessels, but such membranes couldnot be found under the microscope. In either case, the resultsof physical tests suggest that vessels connected by membranelessintervessel pits must effectively end together at a particularpoint in the tree. Key words: Flow, vessels, sycamore     

Aspects of Growth and Metabolism in a Suspension Culture of Acer pseudoplatanus (L. ) Grown on a Glycerol Carbon Source

A suspension culture of Acer pseudoplatanus cells was transferredfrom medium containing 2% (w/v) glucose to an identical onecontaining glycerol at 2% (w/v) as the sole carbon source. Thepatterns of cell number increase, dry weight increase, and changesin packed cell volume showed marked differences as a resultof this transfer. The glucose-grown cultures contained a small proportion of cellsof exceptionally large diameter, and transfer to the glycerolcarbon source appeared to bring about a considerable increasein their number. These larger cells, in both glucose- and glycerol-containingcultures, exhibited considerable differences in cell wall architecturewhen compared with their smaller counterparts. They appearedmore irregular, and had much looser cellulose microfibril arrangementin their outer layers     

Enzyme Sets of Glycolysis, Gluconeogenesis, and Oxidative Pentose Phosphate Pathway Are Not Complete in Nongreen Highly Purified Amyloplasts of Sycamore (Acer pseudoplatanus L.) Cell Suspension Cultures

Differential centrifugation and Percoll-gradient centrifugation of protoplast lysates of suspension-cultured cells of sycamore (Acer pseudoplatanus L.) yielded pure amyloplasts. Contamination of the final amyloplast preparation by foreign compartments was assessed by measuring marker enzyme activities. The activity of alkaline pyrophosphatase was taken as a 100% plastid marker; relative to this marker, mitochondria (cytochrome c oxidase) averaged 0.34%, microbodies (catalase) 0.61%, and cytosol (alcohol dehydrogenase) 0.09%. Enzymatic activities of the glycolytic, gluconeogenic, pentose phosphate and the starch degradation pathways were found to be present in these amyloplast extracts in appreciable amounts. But the pyrophosphate-dependent phosphofructokinase and phosphoglyceromutase were judged to be essentially absent from amyloplasts because the activities of these enzymes were not enriched above the level of contaminating enzymatic activities in the amyloplast fractions. Additionally, the in vitro activities of starch phosphorylase, ATP dependent phosphofructokinase, NAD dependent glyceraldehyde-3 phosphate dehydrogenase, and glucose-6 phosphate dehydrogenase did not seem to support carbon fluxes from starch to triose phosphates as calculated from the rate of starch disappearance during carbon starvation of the cells. These results provide additional, indirect evidence for the recently emerged view that, in addition to the well known phosphate-triosephosphate translocator, another hexose phosphate and possibly also an ATP/ADP translocating system play major roles in nongreen plastids.     

Anatomical acclimation of mature leaves to increased irradiance in sycamore maple (Acer pseudoplatanus L.)

Photosynthesis Research - Trees regenerating in the understory respond to increased availability of light caused by gap formation by undergoing a range of morphological and physiological...