The Regulation of Synthesis of Phenolics in Stationary Phase Cell Cultures of Acer pseudoplatanus L.

The addition of 2/ (w/v) sucrose to stationary phase suspensioncultures of sycamore (Acer pesudoplatanus) in medium depletedof nitrogen and phosphorus leads to the accumulation of highlevels of phenolics after a 5 d lag period. Urea and 2,4-D inhibitthis accumulation of phenolics. Urea results in a stimulationin cell protein content coincident with phenolics in hibition,and leads to a substantial diversion of the common precursorphenylalanine from phenolics synthesis into protein synthesis.2,4-Dichlorophenoxyacetic acid (2,4-D) does not lead to proteinaccumulation since the cultures are nitrogen-limited, but stimulatesan increase in protein turnover coincident with phenolics inhibition.The role of protein synthesis in the regulation of phenolicsmetabolism through competition for common precursors is discussed.     

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 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)     

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.     

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.     

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.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-H 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-(3)H 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.Apparently, processes involved in biosynthesis of primary cell wall continued to produce pectic substance during cell enlargement while processes leading to biosynthesis of typically secondary cell wall polysaccharide such as 4-0-methyl glucuronoxylan were not activated.     

The Effect of Growth Hormones on Cell Division and Expansion in Liquid Suspension Cultures of Acer pseudoplatanus

The effects on cell division and cell size of indole-3-aceticacid (IAA), gibberellic acid (GA), and kinetin were studiedin liquid suspension cultures of cambial cells derived fromAcer pseudoplatanus. It was shown that all three hormones promotecell division and that the effects of both GA and kinetin areadditive to those of IAA, but the effects of GA and kinetintogether are not additive. Treatment with IAA resulted in anincrease of mean cell size (indicating that cell expansion ispromoted), but after GA or kinetin treatment the mean cell sizewas smaller, indicating that little cell expansion had takenplace after each division. The results are discussed in relationto previous work on the effects of hormones in the intact cambiumand to current theories on the interactions of growth hormones.     

Seed dormancy in Acer: Endogenous germination inhibitors and dormancy in Acer pseudoplatanus L.

Summary Dormant seeds of Acer pseudoplatanus L. contain two zones of inhibition on paper chromatograms in 10:1:1 as detected by the lettuce and cress seed germination, and the wheat coleoptile bioassays. One zone at R_f 0.6–0.8 was partitioned into ethyl acetate at acid pH and was shown to contain ABA by its behaviour on GLC and isomerization under ultra-violet light. The other zone at R_f 0.9 was detected only in the germination bioassays and was partitioned into ethyl acetate over a range of pH indicating the presence of one or more neutral compounds.The inhibitors present in the embryo of dormant sycamore seeds inhibited the germination of non-dormant sycamore seeds at relatively low concentrations. A comparison with the effects of application of exogenous ABA indicated that endogenous ABA could not solely account for the inhibitory activity of seed extracts, which appeared to be due partly to the presence of ABA and partly to that of neutral compounds present in the embryo. Leaching treatments that removed dormancy led to a decrease in the level of inhibitors present mainly in the basic fraction. The exogenous application of kinetin to dormant sycamore seeds increased germination whereas gibberellic acid had no effect. Similar responses were obtained with lettuce seeds inhibited by the basic fraction of dormant sycamore seeds.It is suggested that an inhibitor-cytokinin interaction may be involved in the dormancy of sycamore seeds.     

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.     

Effect of Benzyladenine, 2,4-Dichlorophenoxyacetic Acid, and d-Glucose on myo-Inositol Metabolism in Acer pseudoplatanus L. Cells Grown in Suspension Culture

Suspension cultures of Acer pseudoplatanus L. cells grown for 15 days in medium (T. Murashige and F. Skoog. 1962. Physiol. Plant. 15: 473-497) contained 3% sucrose, 1 mg/l 6-benzylaminopurine (BA), and 0.1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), referred to here as normal media, removed newly added myo-inositol-2-(3)H up to 100 mg/l in 24 hours and utilized up to 20% of this cyclitol for pectin biosynthesis. When the BA content of the growth medium was raised 10-fold, uptake of myo-inositol was drastically reduced and very little was available for pectin biosynthesis. Neither cell growth as measured by packed cell volume or by dry weight, nor monomer composition of pectic polysaccharides was affected by the increased level of cytokinin. Increasing, 2,4-d 10-fold instead of BA had little or no effect on myo-inositol uptake, although it did reduce the amount of myo-inositol utilized for pectin biosynthesis. Cells grown 15 days in normal media failed to remove added myo-inositol if 3% d-glucose was included. The net result was similar to that found in cells grown in the high BA condition. If a trace amount of d-galactose-1-(14)C was supplied to cells after 15 days of growth in normal, high BA, or high 2,4-d media, there was no significant variation in uptake and utilization of label among the three growth conditions.     

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells

Acer pseudoplatanus cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of [³H]leucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the [³H]leucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20°C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.     

Rapid Degradation of Abnormal Proteins in Vacuoles from Acer pseudoplatanus L. Cells

In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([¹⁴C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([¹⁴C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole.     

Acer pseudoplatanus (Sapindaceae): Heterodichogamy and thrips pollination

One of the pathways to dioecy is via heterodichogamy, a system including protandrous (flowering male first) and protogynous (female first) plants. Using a research crane the reproductive ecology of the heterodichogamous Acer pseudoplatanus was studied in 74 mature trees over 2 years. The synchronized flowering phenology of the trees resulted in reciprocal pollination between the two morphs. Protandrous trees were more numerous (3:1), had more female flowers (2–3:1), had much less pollen on their stigmas (1:15) and had a much lower seed to fruit ratio (1:3–4). The pollinators were probably breeding thrips. The heterodichogamy of A. pseudoplatanus is confirmed and underlined as a functioning ecological system. Depending on the way pollination efficiency changes in time, either of the morphs can be interpreted as “more female” or “more male”. The evolution of heterodichogamy towards dioecy thus depends on more components of the reproductive ecology than have been assumed.     

The Occurrence of Peroxiredoxins and Changes in Redox State in Acer platanoides and Acer pseudoplatanus During Seed Development

Journal of Plant Growth Regulation - Norway maple (Acer platanoides L.) and sycamore (A. pseudoplatanus L.) are genetically closely related species that produce desiccation-tolerant (orthodox) and...     

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.     

Isolation and Partial Purification of Prophenoloxidase from Daucus carota L. Cell Cultures

The enzyme, phenoloxidase, was isolated and partially purified as an inactive enzyme, a proenzyme, from plant cell cultures of Daucus carota, Nicotiana tabacum, and Haplopappus gracilis. The prophenoloxidase was found to be specifically activated by Ca²⁺ or Mn²⁺ ions in concentrations above 1 millimolar. Calmodulin was not involved in this activation. Concentrations of Ca²⁺ or Mn²⁺ below 1 millimolar could not induce activation of the prophenoloxidase, but if trypsin was added simultaneously with Ca²⁺ or Mn²⁺ at a concentration of 1 millimolar or below, the proenzyme was converted to its active form. The inactive form of phenoloxidase was found to be a soluble enzyme, whereas after activation the enzyme aggregated, and a significant amount of the enzyme activity could become pelleted.