Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Filtration stress-induced variations of peroxidase activity in cell suspension cultures of sycamore (Acer pseudoplatanus) cells

Filtration stress, consisting in the rapid filtration of Acer pseudoplatanus L. cell suspension cultures, resulted in significant differences between the peroxidases (EC 1.11.1.7) released during cell growth and those released after filtered cells were resuspended in fresh medium (recovery medium). These differences concerned mainly modifications of (i) the pH optimum of peroxidase activity (guaiacol as electron donor), (ii) the number and the pI values of the peroxidase isoenzymes as shown by isoelectric focusing, and (iii) the molecular weights of the different peroxidase fractions determined by gel filtration chromatography. The presence of 1 m M Li⁺ in the recovery medium inhibited the release of peroxidase and this effect was partially reversed by K⁺. The release of peroxidase by stressed cells was also strongly inhibited by Na₂CO₃ in the recovery medium. The results presented are consistent with the proposal that the characteristic isoperoxidase patterns induced by filtration stress might be used as a model to study the response of plant cells to stress.     

Purification and characterization of an anionic peroxidase from sycamore maple (Acer pseudoplatanus) cell suspension cultures

A 42 kDa anionic peroxidase (EC 1.11.1.7) having a pl of 3.6 was purified from suspension cultures of cells of sycamore maple ( Acer pseudoplatanus L.) grown in the dark by a combination of lectin-affinity, anion-exchange and gel permeation chromatography. The enzyme had an amino acid composition similar to that found for other anionic plant peroxidases, but the protein was blocked to amino-terminal protein sequencing. Commercially available antibodies against horseradish peroxidase were shown to cross-react with the sycamore maple enzyme on immunoblots. The purified peroxidase displayed differences in its affinity for each of the three monolignols, and these differences were compared to those found for a commercial preparation of horseradish peroxidase, as well as a laccase ( p -diphenol:O₂ oxidoreductase: EC 1.10.3.1) purified from sycamore maple cell suspension cultures. These results are discussed with respect to the role played by peroxidases in lignin deposition and host-pathogen response.     

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.     

Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

Interacting effects of warming and drought on regeneration and early growth of Acer pseudoplatanus and A. platanoides

Climate change is acting on several aspects of plant life cycles, including the sexual reproductive stage, which is considered amongst the most sensitive life‐cycle phases. In temperate forests, it is expected that climate change will lead to a compositional change in community structure due to changes in the dominance of currently more abundant forest tree species. Increasing our understanding of the effects of climate change on currently secondary tree species recruitment is therefore important to better understand and forecast population and community dynamics in forests. Here, we analyse the interactive effects of rising temperatures and soil moisture reduction on germination, seedling survival and early growth of two important secondary European tree species, Acer pseudoplatanus and A. platanoides. Additionally, we analyse the effect of the temperature experienced by the mother tree during seed production by collecting seeds of both species along a 2200‐km long latitudinal gradient. For most of the responses, A. platanoides showed higher sensitivity to the treatments applied, and especially to its joint manipulation, which for some variables resulted in additive effects while for others only partial compensation. In both species, germination and survival decreased with rising temperatures and/or soil moisture reduction while early growth decreased with declining soil moisture content. We conclude that although A. platanoides germination and survival were more affected after the applied treatments, its initial higher germination and larger seedlings might allow this species to be relatively more successful than A. pseudoplatanus in the face of climate change.     

The effect of samara wing presence on predation of Acer pseudoplatanus (Sapindaceae) seeds on the ground

The key selective pressure shaping the morphology of samaras is seen as enhancing primary wind-borne dispersal from the parent plant to the ground. However, the consequences of the samara wing of primarily wind-dispersed tree species for post-dispersal processes has not been well studied. We explored whether the presence of this wing in Acer pseudoplatanus either deters or promotes predation after dispersal, either by increasing the time and energy required to predate the seed or by increasing the seed's visibility to predators. We found that wing-removed fruits were preferred, suggesting that the presence of samaras makes seed handling more expensive for granivores. Further, we found that fewer seeds were consumed from treatments that contained the most winged seeds, thus there was no evidence of the samaras making seed finding easier for granivores. We conclude that the presence of the wing may offer an anti-predatory benefit as well as aiding primary dispersal.     

Competition for nitrogen sources between European beech (Fagus sylvatica) and sycamore maple (Acer pseudoplatanus) seedlings

To investigate the short‐term consequences of direct competition between beech and sycamore maple on root N uptake and N composition, mycorrhizal seedlings of both tree species were incubated for 4 days (i.e. beech only, sycamore maple only or both together) in an artificial nutrient solution with low N availability. On the fourth day, N uptake experiments were conducted to study the effects of competition on inorganic and organic N uptake. For this purpose, multiple N sources were applied with a single label. Furthermore, fine roots were sampled and analysed for total amino acids, soluble protein, total nitrogen, nitrate and ammonium content. Our results clearly show that both tree species were able to use inorganic and organic N sources. Uptake of inorganic and organic N by beech roots was negatively affected in the presence of the competing tree species. In contrast, the presence of beech stimulated inorganic N uptake by sycamore maple roots. Both the negative effect of sycamore maple on N uptake of beech and the positive effect of beech on N uptake of sycamore maple led to an increase in root soluble protein in beech, despite an overall decrease in total N concentration. Thus, beech compensated for the negative effects of the tree competitor on N uptake by incorporating less N into structural N components, but otherwise exhibited the same strategy as the competitor, namely, enhancing soluble protein levels in roots when grown under competition. It is speculated that enhanced enzyme activities of so far unknown nature are required in beech as a defence response to inter‐specific competition.     

Separation of membranes from semiprotoplasts of suspension-cultured sycamore maple (Acer pseudoplatanus) cells

Semiprotoplasts were produced from suspension-cultured Acer pseudoplatanus (sycamore maple) cells prior to cell disruption by passing them through a 60 μm nylon screen. Cell membranes from homogenates were separated by ultracentrifugation on linear sucrose density gradients. Samples were collected by gradient fractionation and subcellular fractions were assayed for membrane markers and glycosyl transferase activities. Results of standard marker assays (cytochrome c reductase for endoplas-mic reticulum. uridine and inosine diphosphatases for Golgi. and eosin-5'-maleimide binding for plasma membrane) showed partial separation of these three membrane types. Golgi and plasma membrane markers overlapped in most gradients. Incorporation of ¹⁴C-labeled sugars from UDP-glucose and UDP-xylose into ethanol precipitated polysaccharides was used to detect glucan synthases I & II (glucosyl transferases) and xylosyl transferase activities in Golgi membrane fractions. All three glycosyl transferases overlapped in fractions corresponding to both Golgi and plasma membrane markers, although peak activities for all three occurred in different fractions. More than one peak of glucan synthase I activity was found. Glucan synthase II, associated with ß-l.3 glucan (cullose) synthesis in plasma membranes, was also detected and exhibited a 10-fold stimulation in the presence of Ca²⁺.     

Enhanced K+-stimulated pyrophosphatase activity in NaCl-adapted cells of Acer pseudoplatanus

Cell suspension cultures of Acer pseudoplatanus L. (Bligny cell line) adapted to growth in the presence of NaCl, are a useful tool for investigating mechanisms for cellular salt tolerance. We compared the activities of vanadate-sensitive (plasma membrane) and nitrate-sensitive (tonoplast) ATPases, and tonoplast K⁺-stimulated PPase in microsomal fractions (8000–108 000 g) from unadapted and NaCl-adapted (80 m M ) cells of A. pseudoplatanus . Since NaCl reduces the growth rate of the culture, the two cell lines were harvested and compared at both the same cellular density and at the same growth phase (middle exponential phase or beginning of the stationary phase). The ATPase activity of the plasma membrane (expressed both on the basis of protoplast number and in relation to protein content) was not affected by the adaptation to salinity. The two enzyme activities of the tonoplast (mainly as expressed on a protein basis) were higher in adapted than in unadapted cells. However, a preferential increase in PPase activity took place, although the pH dependence, ionic requirements, and apparent K_m of the PPase activity were the same in the two cell lines. The three enzyme activities showed different sensitivities to detergents such as Triton X-100, Brij 58 and lysophosphatidylcholine (LPC). The stimulation of K⁺-stimulated PPase activity by detergents was higher in adapted than in unadapted cells. This suggests that the salt-induced enhancement of the PPase activity might partially depend on a modification of the lipid component of the tonoplast.     

The fusicoccin-stimulated phosphorylation of a 33 KDa polypeptide in cells of Acer pseudoplatanus as influenced by extracellular and intracellular pH

Abstract In a previous study, it was shown that the fungal toxin fusicoccin (FC) is able to stimulate the in vivo phosphorylation of a 33 KDalton polypeptide (33 KP) independently of protein synthesis. Here we show that the stimulation by FC of both proton efflux and 33 KP phosphorylation are strongly enhanced when the external medium contains K⁺ or Na⁺, suggesting that the two phenomena are related. The stimulatory effect of FC is higher in unbuffered than in buffered media; moreover, in the absence of FC, 33 KP is more phosphorylated at an acidic than at a basic pH of the medium, suggesting that the effect of FC may depend, to a certain extent, on the acidification of the free space caused by FC-promoted proton efflux. Treatments reported to alter the intracellular pH influence 33 KP phosphorylation even more strongly than the external pH does. The acidifying agents isobutyric acid and trimethylacetic acid decrease 33 KP phosphorylation, while the alkalinizing agents, ammonia and procaine, increase it. FC partially counteracts the inhibition by the weak acids, whereas the stimulatory effect of FC is not additive with that of the weak bases. The results indicate that 33 KP phosphorylation senses both the external and internal pH. The stimulatory effect of cytoplasm-alkalinizing treatments, which mimics that of FC, agrees with the reported capacity of FC to cause cytoplasmic alkalinization, following activation of the plasmalemma proton pump.     

The effects of open-air fumigation with sulphur dioxide on the decomposition of sycamore (Acer pseudoplatanus L.) leaf litters from polluted and unpolluted woodlands

Sycamore (Acer pseudoplatanus L.) leaf litters from 15 woodlands exposed to a broad range of ambient sulphur dioxide (SO₂) concentrations were fumigated with environmentally realistic concentrations (ll-20nmol mol^?1) of SO₂, for 166 d in an open-air fumigation experiment. Fumigation of the sycamore litters significantly increased sulphate-S and proton leaching, and decreased calcium, magnesium and potassium concentrations in leachates and leaf tissues. Leaf litters from relatively unpolluted woodlands showed a tendency to lose higher amounts of sulphate-S, calcium, magnesium and nitrate-N in leachates than litters from polluted sites when exposed to elevated levels of SO₂ in treatment plots. Fumigation inhibited the decomposition rates (CO₂ evolution) of the leaf litters. Marked changes in the composition of the saprotrophic fungal communities in SO₂-fumigated leaf litters were also recorded, but fungal communities and responses to SO₂, were similar between woodlands. There was no evidence from our data to suggest that resistance to SO₂, was developed in decomposer mycofloras in woodlands more frequently polluted by the gas.     

Comparison between the effects of fusicoccin, Tunicamycin, and Brefeldin A on programmed cell death of cultured sycamore (Acer pseudoplatanus L.) cells

Summary. Programmed cell death occurs in plants during several developmental processes and during the expression of resistance to pathogen attack (i.e., the hypersensitive response). An unsolved question of plant programmed cell death is whether a unique signaling pathway or different, possibly convergent pathways exist. This problem was addressed in cultured sycamore (Acer pseudoplatanus L.) cells by comparing the effects of fusicoccin, Tunicamycin and Brefeldin A, inducers of programmed cell death with well-defined molecular and cellular targets, on some of the parameters involved in the regulation of this process. In addition to cell death, the inducers are able to stimulate the production of H₂O₂, the leakage of cytochrome c from mitochondria, the accumulation of cytosolic 14-3-3 proteins, and changes at the endoplasmic reticulum level, such as accumulation of the molecular chaperone binding protein and modifications in the organelle architecture. Interestingly, no additive effect on any of these parameters is observed when fusicoccin is administered in combination with Tunicamycin or Brefeldin A. Thus, these inducers seem to utilize the same or largely coincident pathways to induce programmed cell death and involvement of the endoplasmic reticulum, in addition to that of mitochondria, appears to be a common step.Correspondence and reprints: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.     

Effects of carbanilates on the growth and development of cell suspension cultures of Acer pseudoplatanus

The effects of three isopropyl carbanilate derivatives (propham, chlorpropham, 3,4-diCl isopropyl carbanilate) were studied on Acer pseudoplatanus L. cell suspension cultures (Lamport's strain). At 0.1 m M , three types of effects were observed: the 3,4-diCl derivative rapidly killed the cells; chlorpropham inhibited mitosis, but the cells remained alive without any significant increase in fresh or dry matter; and propham strongly inhibited mitosis, but not cell growth.
Ultrastructural analysis indicated that no observable changes in mitochondrial or plastid density occurred during the cytoplasmic expansion in the presence of propham. Size increase was also accompanied by vacuolar expansion and morphological changes of the nuclei. In Acer cell cultures, isopropyl carbanilates clearly show selective inhibition of mitosis only when the phenyl ring is not substituted by chlorine.     

长效碳酸氢铵缓效机理与环境效应研究

依据长效碳铵的光谱特征、理化特性及其对影响氨挥发的主要因子的分析 ,建立了土壤中氨挥发的数学模式 ,提出了长效碳铵中DCD与从碳铵中离解出来的游离态氨形成分子间氢键的新观点 ,这种弱相互作用力降低了土壤中氨的表观浓度 ,使土壤 pH在氨挥发期间降低了 0 .2～ 0 .4,延缓了NO-3 N形成时间 ,缓解了N损失 .长效碳铵能够调节N素供应的形态、时间与数量 ,减少施用化肥后引起的地面水和地下水硝酸盐的污染 .