Biomedical subjects

Stephen C Fry

Publications and source records attributed to Stephen C Fry.

At least 19 recordsLinked to original sources

PubMed2006

O-oligosaccharidyl-1-amino-1-deoxyalditols as intermediates for fluorescent labelling of oligosaccharides.

Reducing monosaccharides were efficiently converted to stable 1-amino-1-deoxyalditols (=glycamines; distinguished from glycosylamines by mass-spectrometry) during incubation at 20 degrees C in saturated aqueous NH(4)HCO(3) containing NaCNBH(3). Potentially useful by-products included a novel, fully-reduced dimer (the corresponding secondary glycamine) and several relatively long-lived, unreduced products. With increasing incubation time, monomers exceeded dimers. Reducing disaccharides and oligosaccharides underwent similar reactions at their reducing termini; the yield of dimers decreased with increasing oligosaccharide M(r). The O-oligosaccharidyl-1-amino-1-deoxyalditols (OADs) obtained by reductive amination of oligosaccharides reacted readily with lissamine rhodamine sulfonyl chloride to yield OAD-sulforhodamine conjugates linked by a stable sulfonamide bond. Conditions for this reaction were optimised (borate buffer, pH9.0-9.5). The highly fluorescent OAD-sulforhodamine products were purified on a C(18) cartridge. They were electrophoretically immobile at pH2.0 and 6.5, and migrated towards the anode in borate buffer, pH9.4. The OAD-sulforhodamines were amenable to TLC and were excellent substrates for enzymic transglycosylation and for glycosylhydrolase action.

Carbohydrate Conformation↗

PubMed2006

Effect of ascorbate and its oxidation products on H2O2 production in cell-suspension cultures of Picea abies and in the absence of cells.

Dehydroascorbate and traces of ascorbate were present apoplastically in living spruce (Picea abies) twigs. Since the proposed apoplastic ascorbate degradation pathway contains several steps that possibly generate H(2)O(2), the effects of ascorbate and some of its degradation products were tested on apoplastic H(2)O(2) concentrations in a cell culture of P. abies as a model and on non-enzymic H(2)O(2) production in vitro. Ascorbate scavenged H(2)O(2) in the culture medium of lignin-producing Picea cells and in spent and boiled spent medium; in the presence of Cu(2+) or fresh medium, ascorbate led to the non-enzymic generation of H(2)O(2). Preparations of dehydroascorbate (the initial oxidation-product of ascorbate), and diketogulonate (the hydrolysis-product of dehydroascorbate) induced H(2)O(2) accumulation both non-enzymically and enzymically in Picea cell-suspensions. Paper electrophoresis showed that the dehydroascorbate and diketogulonate preparations contained several degradation products; some of these probably contributed to H(2)O(2) production and/or scavenging in these experiments, and would also do so in vivo. These results indicate a complex ability of apoplastic ascorbate, dehydroascorbate, diketogulonate, and further products to modulate H(2)O(2) concentrations, with potential consequences for the control of growth, development and lignification.

2,3-Diketogulonic Acid↗

PubMed2006

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis.

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K (m) (for UDP-glucose) 0.5-1.0 mM; there was also a minor activity with an unusually high K (m) of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K (m) values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E(L), E(M) and E(H) respectively). E(M) was the single major contributor to extractable UDPGDH activity when assayed at 0.6-9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E(L)-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E(H) activity is not due to ADH. At 30 microM UDP-glucose, 20-150 microM UDP-xylose inhibited UDPGDH activity, whereas 5-15 microM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.

Alcohol Dehydrogenase↗

PubMed2006

Why are Chloris gayana leaves shorter in salt-affected plants? Analyses in the elongation zone.

Reduced hydraulic conductance calculated from growth data was suggested to be the main reason for reduced leaf expansion in salt-stressed Chloris gayana (Rhodes grass). In this work, xylem vessel cross-sections and wall enzyme activities were analysed to re-examine the effects of salinity on leaf growth in this species. Maximal segmental growth rates were 20% lower and the growth zone was 23% shorter in leaves from salinized plants than in controls; however, growth rates between 0 mm and 15 mm from the ligule were similar in both types of leaves. Xylem cross-sectional areas in this region were about 65% smaller in leaves of salinized plants, suggesting that hydraulic restrictions in the leaves of salinized plants were much higher than overall growth reductions. Extractable xyloglucan endotransglucosylase activity in this zone was twice as high in leaves of salinized plants as in leaves of controls. Nevertheless, the activity of the extracted enzyme was not affected by up to 1 M NaCl added to the reaction medium. Therefore, increased xyloglucan endotransglucosylase activity under salinity may be due to a promotion of transcription of XTH (xyloglucan endotransglucosylase/hydrolases) genes and/or translation of preformed transcripts. These results suggest that, as in drought stress, increased activity of cell wall enzymes associated with wall loosening may contribute to the maintenance of growth under saline conditions despite hydraulic restrictions.

Cell Wall↗

PubMed2005

Do polyamines contribute to plant cell wall assembly by forming amide bonds with pectins?

Two new reducing glycoconjugates [N-D-galacturonoyl-putrescinamide (GalA-Put) and N,N'-di-D-galacturonoyl-putrescinamide (GalA-Put-GalA)] and homogalacturonan-putrescine (GalAn-Put) conjugates were synthesised as model compounds representing possible amide (isopeptide) linkage points between a polyamine and either one or two pectic galacturonate residues. The amide bond(s) were stable to cold acid and alkali (2M TFA and 0.1M NaOH at 25 degrees C) but rapidly hydrolysed by these agents at 100 degrees C. The amide bond(s) were resistant to Driselase and to all proteinases tested, although Driselase digested GalAn-Put, releasing fragments such as GalA3-Put-GalA3. To trace the possible formation of GalA-polyamine amide bonds in vivo, we fed Arabidopsis and rose cell-cultures and chickpea internodes with [14C]Put. About 20% of the 14C taken up was released as 14CO2, indicating some catabolism. An additional approximately 73% of the 14C taken up (in Arabidopsis), or approximately 21% (in rose), became ethanol-insoluble, superficially suggestive of polysaccharide-Put covalent bonding. However, much of the ethanol-inextractable 14C was subsequently extractable by acidified phenol or by cold 1M TFA. The small proportion of radioactive material that stayed insoluble in both phenol and TFA was hydrolysable by Driselase or hot 6M HCl, yielding 14C-oligopeptides and/or amino acids (including Asp, Glu, Gly, Ala and Val); no free 14C-polyamines were released by hot HCl. We conclude that if pectin-polyamine amide bonds are present, they are a very minor component of the cell walls of cultured rose and Arabidopsis cells and chickpea internodes.

Arabidopsis↗

PubMed2005

UDP-glucose dehydrogenases of maize: a role in cell wall pentose biosynthesis.

UDPGDH (UDP-D-glucose dehydrogenase) oxidizes UDP-Glc (UDP-D-glucose) to UDP-GlcA (UDP-D-glucuronate), the precursor of UDP-D-xylose and UDP-L-arabinose, major cell wall polysaccharide precursors. Maize (Zea mays L.) has at least two putative UDPGDH genes (A and B), according to sequence similarity to a soya bean UDPGDH gene. The predicted maize amino acid sequences have 95% similarity to that of soya bean. Maize mutants with a Mu-element insertion in UDPGDH-A or UDPGDH-B were isolated (udpgdh-A1 and udpgdh-B1 respectively) and studied for changes in wall polysaccharide biosynthesis. The udpgdh-A1 and udpgdh-B1 homozygotes showed no visible phenotype but exhibited 90 and 60-70% less UDPGDH activity respectively than wild-types in a radiochemical assay with 30 microM UDP-glucose. Ethanol dehydrogenase (ADH) activity varied independently of UDPGDH activity, supporting the hypothesis that ADH and UDPGDH activities are due to different enzymes in maize. When extracts from wild-types and udpgdh-A1 homozygotes were assayed with increasing concentrations of UDP-Glc, at least two isoforms of UDPGDH were detected, having K(m) values of approx. 380 and 950 microM for UDP-Glc. Leaf and stem non-cellulosic polysaccharides had lower Ara/Gal and Xyl/Gal ratios in udpgdh-A1 homozygotes than in wild-types, whereas udpgdh-B1 homozygotes exhibited more variability among individual plants, suggesting that UDPGDH-A activity has a more important role than UDPGDH-B in UDP-GlcA synthesis. The fact that mutation of a UDPGDH gene interferes with polysaccharide synthesis suggests a greater importance for the sugar nucleotide oxidation pathway than for the myo-inositol pathway in UDP-GlcA biosynthesis during post-germinative growth of maize.

Amino Acid Sequence↗

PubMed2005

Oxaziclomefone, a new herbicide, inhibits wall expansion in maize cell-cultures without affecting polysaccharide biosynthesis, xyloglucan transglycosylation, peroxidase action or apoplastic ascorbate oxidation.

BACKGROUND AND AIMS: Oxaziclomefone (OAC), a new herbicide, inhibits cell expansion, especially in roots and cell-cultures of gramineous monocots. OAC does not affect turgor in cultured maize cells, and must therefore inhibit wall-loosening or promote wall-tightening. METHODS: The effects of OAC in living cultured maize cells on various biochemical processes thought to influence wall extension were studied. KEY RESULTS: OAC did not affect 14C-incorporation from D-[U-14C]glucose into the major sugar residues of the cell wall (cellulosic glucose, non-cellulosic glucose, arabinose, xylose, galactose, mannose or uronic acids). OAC had no effect on 14C-incorporation from trans-[U-14C]cinnamate into wall-bound ferulate or its oxidative coupling-products. OAC did not influence the secretion or in-vivo action of peroxidase or xyloglucan endotransglucosylase activities-proposed wall-tightening and -loosening activities, respectively. The herbicide did not affect the consumption of extracellular L-ascorbate, an apoplastic solute proposed to act as an antioxidant and/or to generate wall-loosening hydroxyl radicals. CONCLUSIONS: OAC decreased wall extensibility without influencing the synthesis or post-synthetic modification of major architectural wall components, or the redox environment of the apoplast. The possible value of OAC as a probe to explore aspects of primary cell wall physiology is discussed.

Ascorbic Acid↗

PubMed2005

Oxidative coupling of a feruloyl-arabinoxylan trisaccharide (FAXX) in the walls of living maize cells requires endogenous hydrogen peroxide and is controlled by a low-Mr apoplastic inhibitor.

Feruloyl-polysaccharides can be oxidatively coupled in isolated cell walls by peroxidase plus exogenous H(2)O(2) in vitro, but the extent to which similar reactions may occur in the apoplast in vivo was unclear. Numerous cellular factors potentially control feruloyl coupling in vivo, and their net controlling influence is not readily studied in vitro. Therefore, we have monitored apoplastic feruloyl coupling in cultured maize cells in vivo using a radiolabelled model substrate, 5-O-feruloyl-alpha-L: -arabinofuranosyl-(1-->3)-beta-D: -xylopyranosyl-(1-->4)-D: -xylose (FAXX). FAXX was expected to permeate the wall and to undergo reactions analogous to those normally exhibited by apoplastic feruloyl-polysaccharides in vivo. Little difference was found between the fates of [feruloyl-(14)C]FAXX and [pentosyl-(3)H]FAXX, indicating negligible apoplastic hydrolase or transferase activities. Very little radioactivity entered the protoplasm. Maize cells that had recently been washed in fresh medium were able to bind most of the FAXX (90%) in their cell walls, regardless of the age of the culture. During wall-binding, the [(14)C]feruloyl groups were converted to [(14)C]dehydrodiferulates and larger coupling products, as revealed by TLC after alkaline hydrolysis. As expected for an oxidative reaction, wall-binding was delayed by added anti-oxidants (ascorbate, ferulate, sinapate, chlorogenate or rutin). It was also completely inhibited by iodide, an H(2)O(2)-scavenger, indicating a role for peroxidase rather than oxidase. The observations indicate that oxidative coupling of feruloyl groups occurred within the cell wall, dependent on endogenous apoplastic H(2)O(2) and wall-localised peroxidase, in vivo. Cells that had not recently been washed in fresh medium were much less able to bind FAXX, indicating the presence in the apoplast of an endogenous inhibitor of oxidative coupling. This inhibitor was of low M(r), was destroyed by heating, and remained in the aqueous phase (pH approximately 3.5) when shaken with ethyl acetate. Its effectiveness was not altered by ascorbate oxidase. It is thus a small, heat-labile, hydrophilic inhibitor (not ascorbate) which we suggest plays a natural role in the control of wall cross-linking, and thus potentially in the control of cell growth.

Ascorbic Acid↗

PubMed2005

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.

Cell Wall↗

PubMed2005

XTH acts at the microfibril-matrix interface during cell elongation.

Sulphorhodamine-labelled oligosaccharides of xyloglucan are incorporated into the cell wall of Arabidopsis and tobacco roots, and of cultured Nicotiana tabacum cells by the transglucosylase (XET) action of XTHs. In the cell wall of diffusely growing cells, the subcellular pattern of XET action revealed a 'fibrillar' pattern, different from the xyloglucan localization. The fibrillar fluorescence pattern had no net orientation in spherical cultured cells. It changed to transverse to the long axis when the cells started to elongate, a feature mirroring the rearrangements of cortical microtubules and the accompanying cellulose deposition. Interference with the polymerization of microtubules and with cellulose deposition inhibited this strong and 'fibrillar'-organized XET-action, whereas interference with actin-polymerization only decreased the intensity of enzyme action. Epidermal cells of a mutant with reduced cellulose synthesis also had low XET action. Root hairs (tip-growing cells) exhibited high XET-action over all their length, but lacked the specific parallel pattern. In both diffuse- and tip-growing cell types extraction of the incorporated fluorescent xyloglucans by a xyloglucan-specific endoglucanase reduced the fluorescence, but the 'fibrillar' appearance in diffuse growing cells was not eliminated. These results show that XTHs act on the xyloglucans attached to cellulose microfibrils. After incorporation of the fluorescent oligosaccharides, the xyloglucans decorate the cellulose microfibrils and become inaccessible to hydrolytic enzymes.

Arabidopsis↗

PubMed2005

The novel herbicide oxaziclomefone inhibits cell expansion in maize cell cultures without affecting turgor pressure or wall acidification.

Oxaziclomefone [OAC; IUPAC name 3-(1-(3,5-dichlorophenyl)-1-methylethyl)-3,4-dihydro-6-methyl-5-phenyl-2H-1,3-oxazin-4-one] is a new herbicide that inhibits cell expansion in grass roots. Its effects on cell cultures and mode of action were unknown. In principle, cell expansion could be inhibited by a decrease in either turgor pressure or wall extensibility. Cell expansion was estimated as settled cell volume; cell division was estimated by cell counting. Membrane permeability to water was measured by a novel method involving simultaneous assay of the efflux of (3)H(2)O and [(14)C]mannitol from a 'bed' of cultured cells. Osmotic potential was measured by depression of freezing point. OAC inhibited cell expansion in cultures of maize (Zea mays), spinach (Spinacia oleracea) and rose (Rosa sp.), with an ID(50) of 5, 30 and 250 nm, respectively. In maize cultures, OAC did not affect cell division for the first 40 h. It did not affect the osmotic potential of cell sap or culture medium, nor did it impede water transport across cell membranes. It did not affect cells' ability to acidify the apoplast (medium), which may be necessary for 'acid growth'. As OAC did not diminish turgor pressure, its ability to inhibit cell expansion must depend on changes in wall extensibility. It could be a valuable tool for studies on cell expansion.

Biological Transport, Active↗

PubMed2004

Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate.

Increasing the L-ascorbate (vitamin C) content of crops could in principle involve promoting its biosynthesis or inhibiting its degradation. Recent progress has revealed biosynthetic pathways for ascorbate, but the degradative pathways remain unclear. The elucidation of such pathways could promote an understanding of the roles of ascorbate in plants, and especially of the intriguing positive correlation between growth rate and ascorbate oxidase (or its products). In some plants (Vitaceae), ascorbate is degraded via L-idonate to L-threarate (L-tartrate), with the latter arising from carbons 1-4 of ascorbate. In most plants, however (including Vitaceae), ascorbate degradation can occur via dehydroascorbate, yielding oxalate plus L-threonate, with the latter from carbons 3-6 of ascorbate. The metabolic steps between ascorbate and oxalate/L-threonate, and their subcellular location, were unknown. Here we show that this pathway operates extracellularly in cultured Rosa cells, proceeds via several novel intermediates including 4-O-oxalyl-L-threonate, and involves at least one new enzyme activity. The pathway can also operate non-enzymatically, potentially accounting for vitamin losses during cooking. Several steps in the pathway may generate peroxide; this may contribute to the role of ascorbate as a pro-oxidant that is potentially capable of loosening the plant cell wall and/or triggering an oxidative burst.

Ascorbic Acid↗

PubMed2004

N-[3H]Benzoylglycylglycylglycine as a probe for hydroxyl radicals.

N-[4-(3)H]Benzoylglycylglycylglycine ([(3)H]BzG(3)) was tested as a probe for detecting hydroxyl radicals (*OH). Aerated solutions of l-ascorbate generated *OH, which oxidized [(3)H]BzG(3), yielding hydrophilic (probably hydroxylated) derivatives plus tritiated water. The (3)H(2)O was separated from organic products and remaining [(3)H]BzG(3) on Dowex-1. (3)H(2)O production was much greater with *OH than with other reactive oxygen species (ROS) (e.g., H(2)O(2), superoxide). The slight (3)H(2)O production in the presence of H(2)O(2) or superoxide was blocked by *OH scavengers (e.g., glycerol, mannitol, butan-1-ol) that do not scavenge H(2)O(2) or superoxide. This indicates that (3)H(2)O production was caused by *OH and that other ROS only generated any (3)H(2)O by forming traces of *OH. Doses of *OH that caused detectable nonenzymic polysaccharide scission also caused (3)H(2)O production, indicating that [(3)H]BzG(3) is a sensitive *OH probe in studies of polymer scission. The ability of scavengers and chelators to protect against ascorbate-mediated polysaccharide scission paralleled their ability to inhibit concurrent (3)H(2)O production, indicating that both processes were due to *OH. Thus, [(3)H]BzG(3) is a simple, specific, sensitive, and robust probe for detecting *OH production in vitro. It may have applications for in vivo detection of extracellular *OH in arthritic joints and of apoplastic *OH in plant cell walls.

Ascorbic Acid↗

PubMed2004

Change in XET activities, cell wall extensibility and hypocotyl elongation of soybean seedlings at low water potential.

In dark-grown soybean (Glycine max [L.] Merr.) seedlings, exposing the roots to water-deficient vermiculite (psi(w)=-0.36 MPa) inhibited hypocotyl (stem) elongation. The inhibition was associated with decreased extensibility of the cell walls in the elongation zone. A detailed spatial analysis showed xyloglucan endotransglucosylase (XET; EC 2.4.1.207) activity on the basis of unit cell wall dry weight was decreased in the elongation region after seedlings were transplanted to low psi(w). The decrease in XET activity was at least partially due to an accumulation of cell wall mass. Since cell number was only slightly altered, wall mass had increased per cell and probably led to increased wall thickness and decreased cell wall extensibility. Alternatively, an increase in cell wall mass may represent a mechanism for regulating enzyme activity in cell walls, XET in this case, and therefore cell wall extensibility. Hypocotyl elongation was partially recovered after seedlings were grown in low-psi(w) vermiculate for about 80 h. The partial recovery of hypocotyl elongation was associated with a partial recovery of cell wall extensibility and an enhancement of XET activity in the hypocotyl elongation zone. Our results indicate XTH proteins may play an important role in regulating cell wall extensibility and thus cell elongation in soybean hypocotyls. Our results also showed an imperfect correlation of spatial elongation and XET activity along the hypocotyls. Other potential functions of XTH and their regulation in soybean hypocotyl growth are discussed.

Cell Wall↗

PubMed2004

Control of xyloglucan endotransglucosylase activity by salts and anionic polymers.

Crude extracts of cauliflower florets had high xyloglucan endotransglucosylase (XET) activity, but this was largely lost after partial purification and de-salting. Activity was restored (promoted up to 40-fold) by any of a wide variety of inorganic and organic salts. Optimum concentrations for Na+, K+ and NH4+ salts were typically approximately 300 mM. The chlorides of Ca2+, Mg2+, Al3+ and La3+ were optimally active at lower concentrations (e.g. 0.1 mM LaCl3), but became inhibitory at higher concentrations (e.g. 5 mM LaCl3). Some anionic polysaccharides at 0.04-0.2% w/v (e.g. gum arabic, pectin and hypochlorite-oxidised xyloglucan) promoted the XET activity of de-salted enzyme, especially if a sub-optimal concentration of NaCl was also present; others (e.g. homogalacturonan, 4- O-methyl-glucuronoxylan and alginate) were inhibitory. Similar ionic effects were noted on the XET activity of the Arabidopsis protein XTH24 (heterologously expressed by insect cells); in this case carboxymethylcellulose was also stimulatory. To look for endogenous modulators of XET activity, we prepared a cold-water extract of cauliflower florets; after boiling and centrifugation, the supernatant [boiled cauliflower preparation (BCP)] promoted the XET activity of de-salted cauliflower enzyme and of XTH24. About half the activator present in BCP was an ethanol-precipitable, anionic polymer of apparent Mr <5,000. After acid hydrolysis the polymer yielded much arabinose and galactose, and small amounts of galacturonic and glucuronic acids amino acids were also present. The polymer may thus contain arabinogalactan-proteins. We suggest that acidic polymers and/or other apoplastic ions are naturally occurring regulators of XET action in vivo, and may thus control cell wall assembly, loosening, and growth.

Anions↗

PubMed2004

Extracellular cross-linking of xylan and xyloglucan in maize cell-suspension cultures: the role of oxidative phenolic coupling.

Cell-suspension cultures of maize ( Zea mays L.) released soluble extracellular polysaccharides (SEPs) into their medium. Some or all of the SEPs had feruloyl ester groups. Pulse-labelling with [(3)H]arabinose was used to monitor changes in the SEPs' M(r) (estimated by gel-permeation chromatography) with time after synthesis. Newly released (3)H-SEPs were 1.3-1.6 MDa, but between 2 days and 3 days after radiolabelling (in one experiment) or between 5 days and 6 days (in another), the (3)H-SEPs abruptly increased to approximately 17 MDa, indicating extensive cross-linking. The cross-linking involved both [(3)H]xylan and [(3)H]xyloglucan components of the SEPs. The cross-links could be cleaved by alkali, returning the SEPs to their original M(r). In 0.1 M NaOH at 37 degrees C, 58% cleavage was effected within 24 h. The requirement for such prolonged alkali treatment indicates that ester-bonded (e.g. diferuloyl) groups were not solely responsible for the cross-linking. Bonds cleaved only by relatively severe alkali could include benzyl ether linkages formed between sugar residues and oxidised phenolics that had quinone methide structures. The ability of alkali to cleave the cross-links was independent of the age of the (3)H-SEP molecules. Cross-linking of (3)H-SEPs in vivo was delayed (up to approx. 7 days after radiolabelling) by exogenous sinapic acid, chlorogenic acid or rutin-agents predicted to compete with the oxidative coupling of feruloyl-polysaccharides. The cross-linking was promoted by exogenous ferulic acid or l-tyrosine, possibly because these compounds acted as precursors for polysaccharide feruloylation, thus providing additional partner substrates for the oxidative coupling of previously formed (3)H-SEPs. The ability of certain phenolics to prevent the cross-linking of (3)H-SEPs supports the idea that the cross-linking involved phenolic oxidation.

Cell Culture Techniques↗

PubMed2003

Solubilisation of tomato fruit pectins by ascorbate: a possible non-enzymic mechanism of fruit softening.

The aim of this work was to test the hypothesis that endogenous ascorbate, released into the apoplast by membrane permeabilisation early in fruit ripening, could promote the solubilisation and depolymerisation of polysaccharides, and thus contribute to fruit softening. In vitro, ascorbate (1 mM), especially in the presence of traces of either Cu2+ or H2O2, solubilised up to 40% of the total pectin from the alcohol-insoluble residue of mature-green tomato (Lycopersicon esculentum Mill.) fruit. Solubilisation was due to the action of ascorbate-generated hydroxyl radicals (*OH), which can cause non-enzymic scission of polysaccharides. The pectins solubilised by ascorbate in vitro were polydisperse (4-1,000 kDa), partially esterified and galactose-rich. Excised pieces of living tomato fruit released ascorbate into the medium (apoplast); the ability of different tissues to do this increased in the order pericarp < placenta < locule. In all three tissues, but especially in the locule, the ability to release ascorbate increased during ripening. The Cu content of each tissue also increased during ripening, whereas neither Fe nor Mn showed a similar trend. We suggest that progressively increasing levels of Cu and ascorbate in the fruit apoplast would lead to elevated *OH production there and thus to non-enzymic scission of pectins during ripening. Such scission could contribute to the natural softening of the fruit. De-esterified citrus pectin was more susceptible to ascorbate-induced scission in vitro than methylesterified pectin, suggesting a possible new significance for pectin methylesterase activity in fruit ripening. In conclusion, non-enzymic mechanisms of fruit softening should be considered alongside the probable roles of hydrolases, xyloglucan endotransglucosylases and expansins.

Ascorbic Acid↗

PubMed2003

Pre-formed xyloglucans and xylans increase in molecular weight in three distinct compartments of a maize cell-suspension culture.

Cultured cells of maize ( Zea mays L.) were pulse-labelled with l-[1-(3)H]arabinose (Ara) and then monitored for 7 days. The (3)H-hemicelluloses present in three compartments (protoplasm, cell wall and culture medium) were size-fractionated and the fractions assayed for [(3)H]xyloglucans and [(3)H]xylans. Protoplasmic [(3)H]xylans and [(3)H]xyloglucans initially (15 min after [(3)H]Ara-feeding) had weight-average relative molecular masses ( M(w)) approximately 0.5x10(6) and 0.3x10(6), respectively, both rising to 2x10(6) by 30 min. Thus, newly formed hemicellulose molecules were joined to other polymers, or to each other, presumably within Golgi vesicles. New (3)H-hemicelluloses very rapidly bound to the cell wall; however, after 1 day, some [(3)H]xyloglucan and [(3)H]xylan was sloughed from the wall into the medium. The wall-bound [(3)H]xyloglucans were present in the form of extremely large complexes, of M(w)>17x10(6), even as early as 15 min after [(3)H]Ara-feeding. This M(w) is >70-fold greater than that observed by similar methods in cultures of a dicotyledon ( Rosa sp.). Thus, during wall-binding, newly secreted xyloglucans greatly increased in size, possibly by transglucosylation. Some modest degradation (trimming) of wall-bound [(3)H]xyloglucan occurred later. The earliest wall-bound [(3)H]xylan had M(w) approximately 2x10(6), similar to the protoplasmic [(3)H]xylan; this increased to approximately 4x10(6) by 6 h. For the first 2 days after [(3)H]Ara-feeding, the soluble extracellular (3)H-hemicelluloses present in the culture medium had M(w) approximately 1x10(6)-2x10(6), comparable to the protoplasmic hemicelluloses. However, between 2 and 3 days after [(3)H]Ara-feeding, the M(w) of the soluble extracellular [(3)H]xylans increased abruptly to approximately 10x10(6); the soluble extracellular [(3)H]xyloglucans underwent a similar but more gradual increase in M(w). Maize (3)H-hemicelluloses thus underwent increases in M(w) in three episodes: (i) intra-protoplasmically, (ii) during wall-binding (especially xyloglucans), and (iii) after sloughing into the medium. Possible mechanisms and roles of these increases are discussed.

Arabinose↗