Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides

Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na₂CO₃ at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ~4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO₃-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na₂CO₃ at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.     

Host-Pathogen Interactions : XXII. A Galacturonic Acid Oligosaccharide from Plant Cell Walls Elicits Phytoalexins

Elicitors of phytoalexin accumulation in soybean (Glycine max L. Merr., cv Wayne) cotyledons were released from soybean cell walls and from citrus pectin by partial acid hydrolysis. These two hydrolysates yielded nearly identical distributions of elicitor activity when fractionated on anion-exchange columns. Chromatography of the pectin elicitor on gel filtration and high-pressure anion-exchange columns did not further purify the elicitor. Elicitor activity of the preparation was lost by treatment with either endo-α-1,4-polygalacturonase or pectate lyase. Glycosyl residue compositions of the purified elicitors from cell walls and pectin were both found to be approximately 98% galacturonosyl residues. Linkage analysis of the pectin elicitor showed that most, if not all, of the galacturonosyl residues were α-1,4-linked. The high-mass molecular ions detected by fast atom bombardment-mass spectrometry of the most active elicitor fractions from cell walls and pectin both corresponded precisely to a molecule composed of 12 galacturonosyl residues. These results suggest that dodeca-α-1,4-d-galacturonide is the active elicitor, but the possibility remains that the active component could be a slightly modified oligogalacturonide present, but not detected, in the purified fractions.     

Host-Pathogen Interactions: VII. Plant Pathogens Secrete Proteins which Inhibit Enzymes of the Host Capable of Attacking the Pathogen

The results presented demonstrate that microbial pathogens of plants have the ability to secrete proteins which effectively inhibit an enzyme synthesized by the host; an enzyme whose substrate is a constituent of the cell wall of the pathogen. The system in which this was discovered is the anthracnose-causing fungal pathogen (Colletotrichum lindemuthianum) and its host, the French bean (Phaseolus vulgaris). An endo-β-1, 3-glucanase present in the bean leaves is specifically inhibited by a protein secreted by C. lindemuthianum. The cell walls of C. lindemuthianum are shown to be composed largely of a 1, 3-glucan.     

Host-Pathogen Interactions: X. Fractionation and Biological Activity of an Elicitor Isolated from the Mycelial Walls of Phytophthora megasperma var. sojae

An elicitor of phytoalexin production in soybean (Glycine max L.) tissues was isolated from purified Phytophthora megasperma var. sojae mycelial walls by a heat treatment similar to that used to solubilize the surface antigens from the cell walls of Saccharomyces cerevisiae. The wall-released elicitor is a discrete, minor portion of the P. megasperma var. sojae mycelial walls. The elicitor released from the mycelial walls was divided by diethylaminoethylcellulose and concanavalin A-Sepharose chromatography into four fractions, each having different chemical characteristics. The four fractions were obtained from each of the three races of P. megasperma var. sojae. The corresponding fractions from each of the three races are very similar in composition and elicitor activity. The results suggest that the elicitor activity of each fraction resides in the glucan component of the fraction. Evidence is presented to demonstrate that the elicitors are not race-specific and that the accumulation of glyceollin is not sufficient to account for race-specific resistance.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: I. Polysaccharides Associated with Cellulose

Cell walls of suspension-cultured cells of Rosa glauca were fractionated by two different extraction procedures. The first involved a stepwise fractionation scheme based on alkaline extraction. The second took advantage of the powerful cellulose solvent system N-methylmorpholine N-oxide/dimethyl sulfoxide which is capable of solubilizing whole cell walls. From the analytical composition of each solubilized fraction and of the corresponding residues, the fate of each type of cell wall polysaccharide constituent was followed at each step of the extraction scheme and the mode of action of the extractant was interpreted. Although the two fractionation procedures were very different, they yielded very similar cellulosic complex residues and extracts, thus delimiting two blocks of polysaccharides in the cell wall. The cellulose residues still comprised uronic acid-containing polysaccharides and hemicelluloses in association with cellulose. Graded acid hydrolysis provided evidence for the central role of a homogalacturonan core interconnecting xyloglucans and arabinogalactans. A tentative model showing the possible interaction existing between the constituent polysaccharides still associated to cellulose after alkaline extraction is presented. Hydrogen bonding between xyloglucan and cellulose is confirmed, and glycosidic linkages between xyloglucans and pectic polymers are suggested.     

Structure of the Primary Cell Walls of Suspension-Cultured Rosa glauca Cells: II. Multiple Forms of Xyloglucans

Xyloglucans, characteristic hemicellulosic polysaccharides of plant primary walls, have been isolated from Rosa glauca suspension-cultured cells. The cell wall material was fractionated by two sequences of extraction based on solubilization of the hemicelluloses in alkaline and organic solvent systems, respectively. In both cases, only a part (about 50%) of the total xyloglucan could be extracted, the rest remaining tightly associated with cellulose and necessitating the use of acid to be solubilized. Purification of xyloglucans was effected by formation of a gel in appropriate mixtures of dimethyl sulfoxide and water. Further fractionation could be achieved on a cellulose column eluted with chaotropic solvents. This demonstrated the heterogeneity of xyloglucans in the primary cell walls. Analytical data show that all fractions are constituted with the same sugars: l-arabinose, l-fucose, d-galactose, d-xylose, and d-glucose, but their relative proportions differ, particularly the ratio of glucose to xylose which varies from 1.2 to 2 within the different xyloglucans. The structure of these hemicelluloses was established by methylation analysis and shown to consist of a (1 → 4)-linked glucan backbone which carries substituents on the O-6 of glucose. Here again, the multiple forms of xyloglucans was suggested by the various patterns of substitutions found on the different fractions. The configuration of the linkages were established by ¹³C nuclear magnetic resonance spectroscopy and shown to be β for the glucan backbone, α for the xylosyl and fucosyl substituents, and β for the galactosyl substituents. These configurations agree with the specific rotation of the xyloglucan.     

Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans

The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants.     

Effect of Jasmonic, Salicylic, and Abscisic Acids on [14C]Leucine Incorporation into Proteins of Pea Leaves

All investigated exogenous phytohormones (jasmonic, salicylic, and abscisic acids) induced the appearance of ¹⁴C-label in a polypeptide with molecular mass 29 kD that was not found in the control; these acids also increased [¹⁴C]leucine incorporation into a 25-kD polypeptide and decreased such incorporation into a 45-kD polypeptide. This can be considered as a nonspecific response of the plants to the action of these hormones. Salicylic and abscisic (but not jasmonic) acids induced the synthesis of a 19-kD polypeptide, and jasmonate induced the synthesis of a 96-kD polypeptide.     

The Incorporation of 14C into the Protein of Particles Isolated from Plant Cells

The incorporation of ¹⁴C from labelled fructose, succinate,urea, and proline, by particulate preparations from dormantand tissue-cultured carrot cells, is examined. It is shown that¹⁴C is incorporated readily from proline, and less readily fromfructose. No significant incorporation occurs from succinateor urea. No differences are noted between the two kinds of preparation.It is concluded that the incorporation of ¹⁴C does not dependon prior transfer of the label to carbon dioxide followed byfixation of carbon dioxide, since the particles do not incorporate¹⁴C from supplied carbon dioxide. Incorporation of ¹⁴C by various fractions of dormant carrottissue is examined, and it is established that the greatestincorporation per mg. nitrogen occurs in particles isolatedat 10,000 g. A total cell homogenate fails completely to incorporate¹⁴C from proline into protein, and this may be due to suppressionof the activity of the particles by a constituent of the supernatantliquid. The presence of coconut milk reduces the incorporationof ¹⁴C from proline by particles sedimented at 10,000 g, andaddition of a protein hydrolysate reduces it further. Hydroxy-prolinedoes not appear to compete with proline for incorporation, andin this respect the paniculate preparations contrast with wholecells. Particles from carrot tissue are shown to be more active inincorporating ¹⁴C from proline than are particles extractedby the same procedure from red beet roots, potato tubers, andskunk cabbage inflorescences. They are, however, considerablyless active than a mitochondrial preparation from rat liver. It is demonstrated by paper chromatography that the bulk ofthe ¹⁴C incorporated in the particles from carrot cells remainsin proline and there is little or no conversion of proline tohydroxyproline in the preparations. The nature of the particlesemployed in this investigation is discussed, and their metabolismconsidered, in relation to the structure and activity of wholecells.     

[14C]Leucine Incorporation into Brain Proteins in Gerbils After Transient Ischemia: Relationship to Selective Vulnerability of Hippocampus

Regional [14C]leucine incorporation into brain proteins was studied in gerbils after global ischemia for 5 min and recirculation times of 45 min to 7 days, using a combination of quantitative autoradiography and biochemical analysis. After recirculation for 45 min, incorporated radioactivity was reduced to approximately 20-40% of control values in all ischemic brain regions. Specific activity of the tracer, in contrast, was increased, a finding indicating that the reduced incorporation of radioactivity was not due to reduced tracer influx from plasma or a dilution of the tracer by increased proteolysis. After recirculation for 6 h, [14C]leucine incorporation returned to control levels in all regions except the CA1 sector of the hippocampus, where it amounted to less than 50%. After 1 day, protein synthesis in the CA1 sector returned to approximately 70% of control values, followed by a secondary decline to less than 50% after 3 days and returned to near control values after 7 days. Histological evaluations revealed selective neuronal death in the CA1 sector of the hippocampus after 3 days of recirculation. The complex time course of protein synthesis in the CA1 sector suggests a biphasic mode of injury, which may be related to similar changes of calcium homeostasis. The final return to near normal after CA1 neurons have disappeared is explained by astroglial proliferation and demonstrates that at this time protein synthesis is not a marker of neuronal viability.     

Studies on the Cell Wall of Chlorella IV. Incorporation of Glucose-Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

Uniformly ¹⁴C-labeIled glucose was fed to synchronously growingChlorella cells in the dark or in light. The rate of ¹⁴C-incorporationinto hemicellulose showed two maxima one in the growth phaseand one in the reproductive phase. Significant ¹⁴Cincorporationinto a "rigid wall" was found only in the reproductive phase. (Received April 14, 1983; Accepted June 15, 1983)     

Formation of Extracellular Enzyme Systems during the Growth of Geotrichum candidum3C on Cell Walls Isolated from Cereal Grain Coats

The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain coats, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, -L-arabinan, and 1,4--D-galactan), as well as -D-galactosidase and -L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl--D-glucurono--D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52–56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8–28 h of cultivation. Enzymes degrading -D-galacto--D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.     

Studies on the Cell Wall of Chlorella III. Incorporation of Photosynthetically Fixed Carbon into Cell Walls of Synchronously Growing Cells of Chlorella ellipsoidea

The carbon metabolism in cell walls of Chlorella ellipsoideawas studied by following ¹⁴C incorporation into cell wall constituentsin photosynthesizing, synchronously growing cells. The rateof incorporation was higher at an early growth phase of thecell cycle. The ¹⁴C was incorporated into both the major cellwall constituents, hemicellulose and ‘rigid wall’,and the radioactivity in the latter was distributed into itstwo components, glucosamine and amino acids. In pulse-chaseexperiments, the ¹⁴C fixed photosynthetically in the precedingcell cycle was rapidly transferred into the cell wall constituentsat the early growth phase of the ongoing cell cycle, and thereafterwas gradually released from the cell walls, although the totalamount of ¹⁴C in the cells remained constant. It was concludedthat the cell wall constituents are turned over during the growthphase of the algal cell cycle, and that the cell wall metabolismin the ongoing cell cycle is closely connected with the carbonmetabolism in the preceding cell cycle. (Received February 3, 1982; Accepted June 21, 1982)     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells: IV. Differential Subfractionation and Comparison of the Acidic Precursors

A series of ^l4C-arabinose-labelled cell wall precursors weredifferentially subfractionated to compare their properties andanalyze their sugar side chains. The ratio of radioactivity/proteinof the subfractions increased 100- to 5,000-fold from that ofthe starting homogenate. The less acidic fractions showed higherratio of radioactivity/protein. Molecular weights of these fractions'constituents were distributed mostly between 130,000 and 150,000and their dispersion in each fraction was small. The chemicalcomposition of the fractions, which was rather invariable, wasthat of the typical arabinogalactan protein, however, a lowcontent of protein and uronic acid was noticed in the less acidicfractions which showed the highest radioactivity/protein ratio. Gel chromatography of an alkaline hydrolysate of these subfractionsindicated that the precursors consisted of two kinds of molecules:1) a less acidic polysaccharide of high radioactivity/proteinratio, probably arabinogalactan, and 2) a more acidic arabinogalactanprotein of low specific radioactivity/protein ratio. The averagesize of the side chains of the arabinogalactan protein was about10,000 and linked to about 70% of the hydroxyproline residues.No small oligosaccharide side chain (DP<13) was found. Also,no precursor-product relationship was observed between thesesubfractions. (Received September 1, 1986; Accepted May 7, 1987)     

Several Cell Responses to Insulin of Cultured Cells Derived from Micromeres, Isolated from Sea Urchin Embryos at the 16 Cell Stage

In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of ³²P incorporation into protein (protein phosphorylation) followed by activation of ³²P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds. In cells treated with horse serum treated cells, high rates of protein phosphorylation and RNA synthesis were maintained even after the initiation of cable growth and about 5 h later, spicule rods were produced. Insulin treatment did not induce spicule rod formation. In cells treated with horse serum, actinomycin D treatment started at the time of initiation of cable growth, cables were formed but formation of spicule rods was blocked. These results suggest that horse serum contains some other substance besides insulin-like ones, which induces expression of genes that are indispensable for spicule rod formation.     

Malate Uptake into Isolated Vacuoles from Catharanthus roseus Cells: Role of the Membrane Potential

The mechanisms involved in the transport of malate into isolated vacuoles of Catharanthus roseus (L.) cells were investigated with special reference to the effects of induced changes in membrane potential and surface charges of the tonoplast. For this purpose, thiocyanate (SCN^?), a highly permeant anion often used as a membrane potential probe, was extensively exploited. In the absence of Mg-ATP, the low accumulation ratio of ¹⁴C SCN^? could be related to the presence of negative charges at the outer surface of the tonoplast exerting a screening effect on the displacement of lipophilic anionic species. Nevertheless, malate was taken up continuously by vacuoles supporting the concept of a transport component which facilitates its transfer through the tonoplast. From experiments showing the pH dependence of malata uptake, it is suggested that the protonated form of the transporter is implicated in this process. Moreover, when the vacuoles are energized by Mg-ATP, the study of the equilibrium distribution of ¹⁴C SCN^? indicated an inside positive membrane potential difference. Advantage was taken of these results to modulate the membrane potential with high levels of thiocyanate. The data obtained demonstrate that malate uptake results from electrophoretic movement in response to the positive potential difference.     

Interactions of the hyperparasitoidsCatolaccus aeneoviridis [Hym.: Pteromalidae] andSpilochalcis side [Hym.: Chalcididae] with the microsporidansNosema heliothidis andN. Campoletidis

The interactions of the hyperparasitoidsCatolaccus aeneoviridis (Girault) andSpilochalcis side (Walker) with the microsporidansNosema heliothidis Lutz & Splendor andN. campoletidis Brooks & Cranford are described. Neither hyperparasitoid species was infected upon parasitization of pupae of the primary parasitoidCampoletis sonorensis infected withN. heliothidis. However, upon development inCampoletis pupae infected withN. campoletidis, adults ofC. aeneoviridis were infected systemically; trassovarian transmission of the microsporidan to F₃ individuals was also shown.S. side was also infected byN. campoletidis but in this hyperparasitoid, microsporidan development was arrested in the sporoblast stage. Mature spores were not observed in infectedS. side adults and the microsporidan was not transmitted transovarially. The interactions of the hyperparasitoids with the two microsporidans were not demonstrably detrimental as data on the developmental period and adult longevity of infected or exposed hyperparasitoids were similar to those of control individuals.

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Résumé Les auteurs décrivent les interactions entre deux hyperparasitoides,Catolaccus aeneoviridis etSpilochalcis side, et les microsporidies,Nosema heliothidis etN. campoletidis. Aucune des espèces hyperparasitoides ne fut infectée quand elles ont parasité des nymphes du parasitoide primaire,Campoletis sonorensis infecté parN. hehothidis. Cependant, les adultes deC. aeneoviridis furent infectés systématiquement lors qu'ils se développèrent dans des nymphes deCampoletis infectées parN. campoletidis; la transmission transovarienne de la microsporidie jusqu'aux individus de la F3 a été également démontrée.S. side a été aussi infecté parN. campoletidis mais chez cet hyperparasitoide le développement de la microsporidie a été arrêté au stade de sporoblaste. Les spores mures n'ont pas été observées dans les adultes deS. side et la microsporidie n'est pas transmise par la voie transovarienne. Les interactions entre les hyperparasitoides et les deux microsporidies n'ont pas d'effet nuisible démontrable puisque la durée de développement et la longévité des adultes chez les hyperparasitoides contaminés ou exposés sont comparables à celles observées chez les témoins.

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Paper No. 4123 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, North Carolina.