Dehydrodimers of Caffeic Acid in the Cell Walls of Suspension-Cultured Mentha

Dehydrodicaffeic acid derivatives were found in the cell walls of suspension-cultured cells of Mentha. Using gas chromatography/mass spectrometry (GC-MS) in a single ion chromatography at m/z 790 and m/z 718, eleven peaks of trimethylsilylated dehydrodimers of caffeic acid were detected in the extracts from the cell walls of suspension-cultured cells of Mentha using sodium hydroxide. The result suggests that dehydrodicaffeates are formed in the cell walls from two molecules of caffeate, probably formed through C-C, and C-O-C coupling processes.     

Hydroxycinnamic acids and their dimers involved in the cessation of cell elongation in mentha suspension culture

The contents of wall-bound hydroxycinnamic acids and their dimers were compared between elongated and non-elongated cells of suspension-cultured Mentha. Wall-bound peroxidase activity was also investigated. The main hydroxycinnamic acids esterified to these two kinds of cell walls were ferulic and caffeic acids. Eleven dehydrodicaffeic acid isomers and six dehydrodiferulic acid isomers formed through C-C and C-O-C coupling processes, were detected by GC-MS from the extracts released from the walls of non-elongated cells. On the other hand, only four dehydrodicaffeic acid isomers and three dehydrodiferulic acid isomers were found in the walls of elongated cells. Amounts of monomers of ferulic and caffeic acids and their 5,5'-dehydrodimers in non-elongated cell walls were about ten and twenty times higher, respectively, than those in the elongated cell walls. There was a close correlation between the amount of 5,5'-dehydrodimers and activity of wall-bound peroxidase in non-elongated and elongated cells. The level of 5,5'-dehydrodimers accumulated at a higher rate than monomers in non-elongated cell walls. These results suggest that the dimerization of ester-linked ferulic and caffeic acids by peroxidase and the increase in amounts of their 5,5'-dehydrodimers are important factors in the cessation of cell elongation in Mentha suspension culture.     

Responses of Mentha suspension-cultured cells to 2,4-dichlorophenoxyacetic acid and accumulation of esterified phenolic acids in their cell walls.

Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 micrograms l-1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34-40 microns) was observed after division in the medium containing 1-200 micrograms l-1 2,4-D; and significant cell elongation (average cell length: 95-130 microns) was observed after cell division in the medium containing 500-2000 micrograms l-1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.     

The Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension-cultured Monocots

The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed β-1,3; β-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included.     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

The effect of pH on the transformation of syringic and vanillic acids by the laccases of Rhizoctonia praticola and Trametes versicolor

Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) from Rhizoctonia praticola and Trametes versicolor formed different products from syringic and vanillic acids at different pH values, but both enzymes generated the same chemicals at a particular pH. The products were separated by thin-layer and high-performance liquid chromatography. Four compounds were determined from syringic acid (m/z 168, 334, 350 and 486) at pH 6.9, but only two of these (m/z 168 and 334) were found at pH 3.5. In the case of vanillic acid two isomeric dimers (m/z 304) and 2-methoxy-1,4-benzoquinone (m/z 138) were identified. The dimers were formed at both pH values (pH 3.5 and 6.9), but 2-methoxy-1,4-benzoquinone appeared only at pH 3.5.     

Determination of 11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostan oic acid and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoi c acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

11 alpha-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid (PGE-M) and 9 alpha,11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m/z 349 and m/z 356 (2H7 standard) (daughter ions of m/z 637 and m/z 644 (2H7 standard] were used. The results of the PGE-M assay were compared with those of an assay using the [2H3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m/z 484 and m/z 491 (2H7 standard) with the parent ions m/z 682 and m/z 689 (2H7 standard) were chosen.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells

 Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. `Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on `Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [³H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeat-units of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins. Received: 5 November 1999 / Accepted: 18 January 2000     

Widespread occurrence of a covalent linkage between xyloglucan and acidic polysaccharides in suspension-cultured angiosperm cells

* BACKGROUND AND AIMS: Covalent linkages between xyloglucan and rhamnogalacturonan-I (RG-I) have been reported in the primary cell walls of cultured Rosa cells and may contribute to wall architecture. This study investigated whether this chemical feature is general to angiosperms or whether Rosa is unusual. * METHODS: Xyloglucan was alkali-extracted from the walls of l-[1-3H]arabinose-fed suspension-cultured cells of Arabidopsis, sycamore, rose, tomato, spinach, maize and barley. The polysaccharide was precipitated with 50 % ethanol and subjected to anion-exchange chromatography in 8 m urea. Eluted fractions were Driselase-digested, yielding [3H]isoprimeverose (diagnostic of [3H]xyloglucan). The Arabidopsis cells were also fed [6-14C]glucuronic acid, and radiolabelled pectins were extracted with ammonium oxalate. * KEY RESULTS: [3H]Xyloglucan was detected in acidic (galacturonate-containing) as well as non-anionic polysaccharide fractions. The proportion of the [3H]isoprimeverose units that were in anionic fractions was: Arabidopsis, 45 %; sycamore, 60 %; rose, 44 %; tomato, 75 %; spinach, 70 %; maize, 50 %; barley, 70 %. In Arabidopsis cultures fed d-[6-(14)C]glucuronate, 20 % of the (galacturonate-14C)-labelled pectins were found to hydrogen-bond to cellulose, a characteristic normally restricted to hemicelluloses such as xyloglucan. * CONCLUSIONS: Alkali-stable, anionic complexes of xyloglucan (reported in the case of Rosa to be xyloglucan-RG-I covalent complexes) are widespread in the cell walls of angiosperms, including gramineous monocots.     

Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS

An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells.     

Interaction of Pseudomonas solanacearum with Suspension-Cultured Tobacco Cells and Tobacco Leaf Cell Walls In Vitro

Attachment of radiolabeled Pseudomonas solanacearum cells to suspension-cultured tobacco cells and tobacco leaf cell walls was measured in vitro by a filtration technique that allowed separation of attached and unattached bacteria. An avirulent strain (B1) attached more rapidly to suspension-cultured cells than did the virulent parent strain (K60), and B1 attachment was less sensitive to inhibition by high ionic strength than was K60. Attachment of B1 bacteria to suspension-cultured cells and to leaf cell walls was comparable (50 to 70%), but only a small proportion (10 to 20%) of K60 bacteria attached to leaf cell walls under optimal conditions. With high bacterial populations (10⁸ bacteria per ml), attachment of K60 to suspension-cultured cells was greatly reduced. Attachment of both strains was completely inhibited by pretreating bacterial cells with heat (41°C) or azide and was partially inhibited by EDTA and kanamycin. The mechanism of attachment is not known, but ionic forces may be involved.     

Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures

Maize(Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil(DCB), a cellulose biosynthesis inhibitor. Cellulose de ficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared(FTIR) spectroscopy.Cell wall compositional analysis indicated that the cellulosede ficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-de ficient cell walls showed a fivefold increase in Klason-type lignin.Thioacidolysis/GC-MS analysis of cellulose-de ficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to shortterm DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment.     

Cell wall proteins from sugar beet cells in suspension culture

Several proteins were extracted from the purified cell walls of suspension-cultured sugar beet cells with 0.5% EDTA (pH 6.8) after prior extraction of the walls with 0.5% deoxycholate and then with 2 molar NaCl. Two abundant proteins (P-I and P-II protein) were separately purified to homogeneity by procedures that included fractionation with ammonium sulfate, column chromatography on DEAE-cellulose and butyl Toyopearl, and preparative polyacrylamide electrophoresis. P-I exists as a dimer of identical subunits, and P-II is composed of four different subunits. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that quite different polypeptides are present in the culture medium and in the NaCl and EDTA extracts of the wall.     

Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells

The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified α-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.     

Influence of a specific xyloglucan-nonasaccharide derived from cell walls of suspension-cultured cells of Daucus carota L. on regenerating carrot protoplasts

A xyloglucan oligosaccharide was isolated from cell walls of Daucus carota L. suspension-cultured cells. From analytical data (gel-permeation chromatography, thin-layer chromatography, monosaccharide analysis, methylation analysis) it can be concluded that this oligosaccharide preparation consists mainly of a nonasaccharide known as XG9 (Glc₄Xyl₃GalFuc). This nonasaccharide showed excellent “anti-auxin” properties in the pea-stem bioassay, with 80% inhibition of 2,4-dichlorophenoxyacetic acid (2,4-D)-induced longitudinal growth of etiolated pea stem segments at concentrations of 1-0.1 nM. Applied in nanomolar concentrations to protoplasts regenerating in a medium containing 4.52 μM 2,4-D, the nonasaccharide influenced the viability of the protoplasts and the activities of glycan synthases in vitro. The effects were similar to those achieved by the omission of 2,4-D from the regeneration medium. The composition of the regenerated cell wall was not changed significantly by the use of 2,4-D-depleted medium or the addition of XG9 to 2,4-D-containing medium.     

Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation

The occurrence and function of the side chains occurring in the rhamnogalacturonan I domain of pectic poly- saccharides have been investigated during carrot cell development using monoclonal antibodies to defined epitopes of (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan. Immunolocalization studies of carrot root apices indicated that cell walls in the central region of the meristem contained higher levels of (1-->5)-alpha-arabinan than the cell walls of surrounding cells. In contrast (1-->4)-beta-galactan was absent from the cell walls of the central meristematic cells but appeared abundantly at a certain point during root cap cell differentiation and also appeared in cell walls of differentiating stele and cortical cells. This developmental pattern of epitope occurrence was also reflected in a suspension-cultured carrot cell line that can be induced to switch from proliferation to elongation by altered culture conditions. (1-->4)-beta-galactan occurred at a low level in cell walls of proliferating cells but accumulated rapidly in cell walls following induction, before any visible cell elongation, while (1-->5)-alpha-arabinan was present in cell walls of proliferating cells but was absent from cell walls of elongated cells. Immunochemical assays of the cultured cells confirmed the early appearance of (1-->4)-beta-galactan during the switch from cell proliferation to cell elongation. Anion-exchange chromatography confirmed that (1-->4)-beta-galactan was attached to acidic pectic domains and also indicated that it was separate from a distinct homogalacturonan-rich component. These results indicate that the neutral components of pectic polysaccharides may have important roles in plant cell development.     

Distribution of pectic polysaccharides throughout walls of suspension-cultured carrot cells

Summary A monoclonal antibody (2 F 4) recognizing a conformational epitope of polygalacturonic acid was used for immunogold localization of pectins in walls of suspension-cultured carrot (D. carota L.) cells at the electron microscopic level. In microcolonies of young or mature cells, polygalacturonic acid was essentially located on the middle lamella material expanded at three-way junctions between cells or lining intercellular spaces but was not found in primary walls. Middle lamellae far from junction zones and intercellular spaces were not recognized. Largely esterified pectic polymers, only detected by the 2 F 4 antibodies after on-grid de-esterification treatment by pectin methyl esterases, were present within all primary cell walls. Golgi bodies and associated vesicles were also labeled by the 2 F 4 antibodies only after de-esterification treatment, which indicates that pectic polymers are synthesized and secreted in a highly esterified form. A decrease of pectin esterification, which results probably from an in situ enzymatic de-esterification of the pectic polymers of the primary walls, was observed in senescent cells. These results are discussed in relation to biochemical analyses showing changes of the methyl ester content of pectins during the cell-wall growth.     

Metabolism of cell wall polysaccharides in cell suspension cultures of Populus alba in relation to cell growth

Changes in the composition of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Populus alba cells. Three growth phases, namely the cell division phase, cell elongation phase and stationary phase, were distinguished. The active deposition of polysaccharides in cell wall fractions (50 m M Na₂CO₃-, 1 M KOH-, 4 M KOH-soluble and 4 M KOH-insoluble) was observed during the elongation phase. A 50 m M Na₂CO₃-soluble pectic fraction mainly composed of 1,4-linked galactan and arabinan except acidic sugars. The 1,4-linked galactan decreased markedly during elongation. In 1 and 4 M KOH-soluble hemicellulosic fractions, non-cellulosic 1,4-glucan and xyloglucan were observed as major components, respectively. These polysaccharides also decreased during elongation. A large amount of polysaccharides was secreted into the medium as ECP. Neutral sugars were detected predominantly by sugar composition analysis. Acidic sugars, such as galacturonic acid, were less than 12% of total. In this study, active metabolism of pectic polysaccharides in addition to hemicellulosic polysaccharides, especially neutral side chains of pectin, during cell growth, was clarified.