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).     

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.     

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 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)     

The Tonoplast H+-ATPase of Acer pseudoplatanus Is a Vacuolar-Type ATPase That Operates with a Phosphoenzyme Intermediate

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+-ATPase was rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of radio-activity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.     

Polypeptide Compositions of Stroma, Internal Membranes, and Inner and Outer Envelope Membranes of Amyloplast from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Intact amyloplasts isolated from liquid-cultured white-wildcells of sycamore (Acer pseudoplatanus L.) were further subfractionatedinto internal membranes (d=1.05g/ml), envelope membranes (d=1.12g/ml)and stromal fraction, which contained each characteristic polypeptidecomposition as revealed by the Na-dodecyl sulfate polyacrylamidegel electrophoresis. Absorption spectra of internal and envelopemembranes were distinctly different. By the immunoblotting analysis,it was shown that the amyloplast envelope membranes contain31 kDa P_i-translocator, although it is not the predominant polypeptidecomponent in contrast to the case of chloroplast envelope. Onehundred kDa -l,4-glucan phosphorylase (plastid type) was detectedin the stromal fraction of amyloplasts using the specific antibodyraised against the major form of -l,4-glucan phosphorylase frompotato tuber. Amyloplast envelopes were further separated into inner and outermembrane fractions by the freezing-thawing method originallydeveloped for the separation of chloroplast envelope membranesby Cline and associates (1981) (Proc. Natl. Acad. Sci. USA 78:3595–3599). Nadodecylsulfate gel electrophoretic analysisrevealed that the inner and outer envelope membranes containthe distinctly different polypeptide compositions. ¹Supported by grants from the Ministry of Education, Scienceand Culture (Mombusho) of Japan. This is paper No. 77 in theSeries "Structure and Function of Chloroplast Proteins". ²Recipient of a predoctoral student fellowship from the Japanesegovernment (Mombusho). Permanent address: Department of Biochemistry,Faculty of Science, Kasetsart University, Bangkok 10900, Thailand ³Permanent address: Department of Biology, Southwest AgriculturalUniversity, BeiBei Chongqing, People's Republic of China. Holderof the Chinese Government Scholarship (1987) (Received May 27, 1988; Accepted August 30, 1988)     

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.     

Short-term Changes in Hexose Phosphates and ATP in Intact Cells of Acer pseudoplatanus L. Subjected to Anoxia

Endogenous concentrations of hexosemonophosphates and ATP decline sharply and rapidly in intact cells of Acer pseudoplatanus subjected to anoxia, whereas fructose-1,6-diphosphate and pyruvate accumulate markedly. In view of gas exchange data indicating an apparent acceleration of glycolysis under anoxic conditions, the observed changes in glycolytic metabolite concentrations indicate regulation of glycolysis by phosphofructokinase.     

Seed Dormancy in Acer pseudoplatanus L.: the Role of the Covering Structures

The present studies with Acer pseudoplatanus L. suggest thatthe covering structures play an important and multiple rolein the dormancy of the fruit. Whole fruits and seeds with thetesta intact required a period of chilling at 5 °C beforedormancy was broken whereas bare embryos germinated immediatelyat 20 °C without pretreatment. This suggested that dormancywas coat-imposed and that the testa was responsible for thiseffect. Germination of dormant seeds was inhibited by lightwhereas the non-dormant bare embryos showed little response.Studies on the manner in which the testa imposed dormancy onthe embryo indicated that restriction on oxygen uptake, wateruptake, mechanical restriction to embryo enlargement, and thepresence of germination inhibitors in the testa were not limitingfactors at this stage of dormancy. Results from leaching experimentssuggest that dormancy was the result of the restriction by thetesta of the outward diffusion of a germination inhibitor(s)present in the embryo. In seeds that had nearly completed theirstratification requirements, the covering structures seemedto act in a manner other than by preventing the leaching ofan inhibitor from the embryo. At this point the physical propertiesof the covering structures seem to determine any further delaysin germination by the mechanical restriction of embryo enlargementby the testa and by restriction of oxygen uptake by the pericarp.     

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.     

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.     

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.     

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.     

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.     

Immunochemical Analysis of Triton X-100-Insoluble Residues from Micrococcus lysodeikticus Membranes

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.     

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.