Changes in Cell Wall Composition and Water-soluble Polysaccharides During Kiwifruit Development

Changes in both cell wall and water-soluble polysaccharide compositionduring the growth of kiwifruits [Actinidia deliciosa (A. chev.) C. F. Liang and A. R. Ferguson var. deliciosa ‘Hayward’]were investigated. Cellulose was the major wall polysaccharide,with galactose and uronics the main non-cellulosic sugars. Muchsolubilization of cell wall pectic polysaccharides was detected.While wall-galactose solubilization started 3 months after anthesis,polyuronide degradation did not start until the fifth month,1 month prior to the harvest date. Parallel to these processes,a linear increase in water-soluble polysaccharides was detected.These mainly comprised galactose-rich polymers in the first3 months and little-branched polyuronides after the fifth month.Two different mechanisms for galactose and uronic acid solubilizationfrom kiwifruit cell walls during fruit development are proposed. Actinidia deliciosa ; cell wall; fruit growth; kiwifruit; water-soluble polysaccharides     

Inhibition of the Breakdown of Xyloglucans in Azuki Bean Epicotyls by Concanavalin A

Indole-3-acetic acid at 10 µM caused a 30% decrease inthe weight-average molecular mass of xyloglucans extracted with24% KOH from the cell walls of epicotyl segments of azuki bean(Vigna angularis Ohwi et Ohashi cv. Takara). Concanavalin A(Con A) at 2 g liter^–1 completely inhibited the IAA-inducedchange in the molecular mass of the xyloglucans. Con A alsosuppressed the autolysis of pectin-depleted cell walls, as wellas the breakdown of xyloglucans by a protein fraction that hadbeen extracted with 1 M NaCl from the cell walls of azuki beanepicotyls. These results indicate that Con A is a potent inhibitorof the breakdown of xyloglucans both in vivo and in vitro. Mostof the activity responsible for the decrease in staining byiodine and the increase in reducing power of solution of xyloglucansin the protein fraction from cell walls bound to a column ofCon A-Sepharose and was eluted by the specific hapten, methyl     

Wall loss and origin of soluble carbohydrates during mating of Chlamydomonas reinhardi

In the mating reaction between gametes of the green alga Chlamydomonas reinhardi, a lytic factor which solubilizes the cell wall is released. It has been shown that carbohydrates accumulate in the supernatant of mating gametes. We present here data which support the notion that the released carbohydrates arise from solubilized gametic cell walls. The evidence is based, in part, on the comparison of the carbohydrates and amino acids in the acid hydrolysates of cell-free supernatants to the reported composition of isolated cell walls. In both cases the three predominant sugars are mannose, arabinose and galactose, and also, in both cases, large amounts of the amino acid hydroxyproline are present. In addition, it is shown that if gametic cell walls are removed prior to mating interactions by treatment with a preparation of lytic factor, much less carbohydrate is subsequently released into the supematant, when such ‘nake’ gametes are mated.     

The interaction in planta of polygalacturonases from Botrytis cinerea with a cell wall-bound polygalacturonase-inhibiting protein (PGIP) in raspberry fruits

The activities of four fungal polygalacturonases (endo-PGI,endo-PGII, exo-PGI, exo-PGII), detected when Botrytis cinereawas grown on immature fruits of red raspberry (Rubus idaeus),were fractionated into soluble and wall-bound fractions. Westernblots and plate-trapped antigen ELISA showed that endo-PGI andendo-PGII were most abundant in the cell wall-bound fractionsof the host. Immuno-inhibition studies using a polyclonal antiserumagainst polygalacturonase-inhibiting protein (PGIP), purifiedfrom immature raspberry fruits, showed that the low level offungal PG activity detected in fractions containing endo-PGIwas due to the presence of PGIP. When a purified preparationof endo-PGI and endo-PGII from B. cinerea was allowed to reactin vitro with either a crude host cell wall preparation, orone which had previously been treated to remove cell wall-boundproteins, both endo-PG isozymes had a greater binding capacitytowards the former wall preparation. Endo-PGI and endo-PGIIalso had an affinity for fungal cell walls. Exo-PGI and exo-PGIIbound to both fungal and host cell walls. Greater quantitiesof fungal endo-PGs were detected by ELISA in fruits previouslyfrozen and thawed (‘freeze-thawed’) and inoculated,than in fresh inoculated fruit. This result paralleled the extentof fungal growth in these tissues as assessed by chitin assayand suggests that the resistance shown by raspberries is dependenton continual replacement of inhibitory substances or inducedresistance mechanisms. Key words: Polygalacturonase-inhibiting protein, Rubus idaeus, red raspberry, Botrytis cinerea, pectinases     

Effect of Phosphorus Deficiency on Phosphatase Activity of Cell Walls from Roots of Subterranean Clover

Clover (Trifolium subterraneum L. cv. Mt. Barker) was grownin solution culture with adequate (+P) or no phosphate (–P).Cell walls were extracted from roots in such a way that theywere uncontaminated by other cellular materials. Phosphataseactivity was assayed using p-nitro-phenylphosphate (NPP). Phosphatasebound to cell walls had a pH optimum between 5.0 and 6.0, irrespectiveof the P supply to the plants. Activity of phosphatase boundto cell walls increased with electrolyte concentration of theassay medium at pH 6.5 but not at pH 5.5. This increase in activitywas probably due to a higher degree of ionization of the cellwall at pH 6.5 than at pH 5.5, and to effects of high ionicstrength in decreasing the mutual repulsion of negatively chargedNPP from negative charges on the cell walls. Cell wall-boundphosphatase did not exhibit Michaelis-Menten kinetics: the concentrationof NPP at which activity was half the maximum rate (S_0.5) was0.7 mM for cell walls extracted from roots of both +P and –Pplants. Up to 30% of the phosphatase activity bound to cellwalls could be removed using buffer solutions of high pH andhigh ionic strength which contained Triton X100. Both soluble and cell wall-bound phosphatase(s) of roots increasedin activity with P deficiency. The phosphatase activity of cellwalls increased 1.5 fold as the P concentration in the rootsfell from 0.4–0.2% dry weight. Experiments with sterileroots of clover showed that increases in cell wall-bound phosphataseactivity associated with P deficiency were not due to microbialcontamination. It is argued that phosphatase(s) in cell wallsof roots could make a substantial contribution to the P nutritionof clover in soils deficient in inorganic phosphate by hydrolysingorganic phosphate compounds in the soil. Key words: Phosphatase, Clover, Roots, Phosphorus deficiency, Cell walls     

Glycanases Associated with Cell Walls of Cicer arietinum L: Arabinogalactan Degradation

The proteins extracted from Cicer arietinum cell walls showedendo-glycanase activity against the water-soluble hemicellulosicpolysaccharides extracted with 4% KOH. Such endo-glycanase activitywas able to shift the molecular mass distribution of the arabinogalactancausing a decrease in its average molecular mass, as well asto release small oligosaccharides constituted exclusively ofarabinose. Exo-glycanase activities such as glucosidase andgalactosidase were also present. Key words: Arabinogalactan, cell wall, Cicer arietinum, glycanase, hemicellulose     

Substrate Degradation Patterns of Polygalacturonic Acid Lyase from Erwinia carotovora and Bacillus polymyxa and Release of Phytoalexin-eliciting Oligosaccharides from Potato Cell Walls

The patterns of substrate degradation by purified pectate lyase(PGL) (E.C. 4.2.2.2 [EC] ) from Erwinia carotovora and Bacillus polymyxawere compared. Reaction products released by both enzymes frompotato cell walls, sodium polypectate and citrus pectin wereseparated by anion exchange chromatography using a TSK DEAE-5PWcolumn and measured quantitatively. The relative amounts ofoligomers released by both enzymes varied, especially the levelof unsaturated tetramers. Degradation patterns also varied accordingto the substrate used and results with citrus pectin suggestedthat methylation reduced the ability of E. carotovora PGL torelease wall fragments. Oligomers released from potato cell walls by E. carotovora PGLwere pooled separately and assayed for phytoalexin elicitoractivity using the soybean cotyledon bioassay. Fractions containingdeca- and undecagalacturonides had the highest elicitor activitywhen tested at 5.0µg of uronic acid per cotyledon. Key words: Pectic enzyme, elicitor, phytoalexin     

Injury-induced Cyanogenesis in Vegetative and Reproductive Parts of Two Grevillea Species and their F1 Hybrid

When leaves of three Grevillea species were macerated, hydrogencyanide was only released from the soft, immature ones. Thebark but not wood of young stems was weakly cyanogenic. Thegreatest concentration of cyanoglucosides occurred in peduncles,flower buds, open flowers, immature seeds and germinants. Nocyanide was released from nectar, pedicels, dry fruit, elaiosomesand dry walls mature seeds. Addition of ß-glucosidase,and sometimes water only, increased cyanogenesis in young leaves,bark, some seed and fruit walls, and embryos of mature seeds.The endogenous levels of ß-glucosidase were sufficientto release all the cyanide present in at least 80% of cases. Young leaves and germinants of G. bipinnatifida (resprouter)released more cyanide than those of G. banksii, while flowerbuds, stigmatic region, styles, perianths and ovaries of G.banksii (non-sprouter) released far more cyanide than thoseof G. bipinnatifida. The interspecific and intraplant distributionof cyanoglucosides is consistent with the expected impact ofbeneficial and herbivorous animals on long term fitness of thesespecies. The F1 hybrid (G. 'Robyn Gordon') tended to have intermediatecyanoglucoside levels between the two putative parents.Copyright1993, 1999 Academic Press Grevillea bipinnatifida, G. banksii, G. 'Robyn Gordon', cyanide, herbivory, ß-glucosidase, cyanogenesis     

Effects of Ascorbate on the Oxidation of Derivatives of Hydroxycinnamic Acid and the Mechanism of Oxidation of Sinapic Acid by Cell Wall-Bound Peroxidases

A cell wall fraction isolated from epicotyls of Vigna angularis,which contained both ionically and covalently bound peroxidases,rapidly oxidized p-coumaric, caffeic and ferulic acids and slowlyoxidized sinapic acid. The oxidation of sinapic acid was greatlyenhanced in the presence of p-coumaric, caffeic or ferulic acid.Ascorbate (20 µM) inhibited the oxidation of ferulic acidby about 70% and completely inhibited the oxidation of p-coumaricand ferulic acids. The cell wall fraction was capable of bindingferulic and sinapic acids but not caffeic acid. p-Coumaric acidbound only slightly to cell walls. The oxidation of p-coumaricand ferulic acids by KCl-washed cell walls was inhibited byabout 60% and 10%, respectively, by 20 µM ascorbate, butthe oxidation of caffeic acid was completely inhibited by ascorbateat less than 20 µM. The oxidation of derivatives of hydroxycinnamicacid by peroxidases released from cell walls by washing with1 M KCl was completely inhibited by ascorbate. These resultssuggest that the inhibition by ascorbate depends on the substituentgroup of the phenyl ring of the derivatives of hydroxycinnamicacid when the oxidation reaction is catalyzed by cell wall-boundperoxidases and that the oxidation of sinapic acid is mediatedby phenoxyl radicals of derivatives of hydroxycinnamic acidother than sinapic acid. (Received December 2, 1993; Accepted March 3, 1994)     

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)¹ (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself², and is amenable to high-throughput analyses³. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes⁴. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase^{5, 11, 12, 13}, xylan using an endo-β-(1-4)-xylanase ^6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase ⁸. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined ⁹.     

Boron deficiency effects on peroxidase, hydroxyproline, and boron in cell walls and cytoplasm of Helianthus annuus L. hypocotyls

Boron-deficient sunflower hypocotyls have, on a fresh weightbasis, more cytoplasmic and cell wall peroxidase, more cytoplasmichydroxyproline but equal cell wall hydroxyproline, and slightlyless cell wall boron, than controls. Incubation of either Bdeficientor control cell wall suspensions with Sclerotium rolfsii culturenitrate released 80% of the peroxidase activity, but only 14to 30% of the hydroxyproline. This differential extraction suggeststhat the hydroxyproline-containing protein of cell walls isnot identical with peroxidase. Boron deficiency increased thesusceptibility of cell walls to degradation by fungal enzymes,as measured by release of peroxidase and hydroxyproline, butnot by reduction in dry weight. ¹Scientific article No. A1847, Contribution No. 4756 of theUniversity of Maryland Agricultural Experiment Station. Thisresearch represents part of a thesis for the M.S. degree, Universityof Maryland, by J.B.S., Jr. This paper is dedicated to ProfessorHugh G. Gauch on the occasion of his sixtieth birthday. (Received December 5, 1972; )     

Polygalacturonase isozyme 2 binding and catalysis in cell walls from tomato fruit: pH and β-subunit effects

The catalytic activity of endopolygalacturonase (PG, EC 3.2.1.15) against pectic polymers in vitro is typically not expressed in vivo. In the present study, the binding and catalytic properties of PG isozyme 2 and the influence of the β-subunit protein were investigated in cell walls prepared from tomato fruit expressing an antisense gene to the β-subunit protein. Cell walls prepared from mature-green fruit were employed for binding and assay of PG2. Walls were provided with rate-limiting quantities of purified PG2 and incubated at 100 mM KCl, pH 4.5, or 25 mM KCl, pH 6.0. Cell walls of both β-subunit antisense and wild-type fruit retained comparable quantities of added PG2. The release of pectin from PG2-loaded walls was proportional to the quantity of added enzyme, consistent with a finite catalytic capacity of individual PG proteins. β-Subunit-antisense cell walls released 2- to 3-fold higher levels of pectin in response to PG2 than did wild-type walls. Cell walls incubated at pH 6.0 released lower quantities and showed less extensive depolymerization of pectins than did walls incubated at pH 4.5. Pectins recovered from ripe fruit were similar in size distribution to polymers released by PG2 at pH 6.0, indicating that pH can influence both quantitative and qualitative aspects of pectin metabolism and may be responsible for the restricted hydrolysis of pectins in vivo. Molecular mass differences were not evident in the polymers rendered freely soluble in response to PG2-mediated hydrolysis of β-subunit-antisense compared with wild-type cell walls. The solubilization of pectin from cell walls was not the sole indicator of the extent of PG-mediated cell wall hydrolysis. Hydrolytic modifications were also evident in a pectic fraction extracted from postcatalytic cell walls with 50 mM CDTA (trans-1,2-cyclohexanediamine-N,N,N′,N′-tetraacetic acid), and were more extensive for the β-subunit-antisense cell walls compared with the wild-type walls. Pectic polymers derived from ethanol insoluble-powders showed molecular mass downshifts during ripening but differences between the β-subunit-antisense and wild-type fruits were not observed.     

Carbohydrate solutilization of tomato locule tissue cell walls: Parallels with locule tissue liquefaction during ripening

Carbohydrate solubilization and glycosidase activities were investigated in tomato ( Lycopersicon esculentum Mill. cv. Solar Set) locule cell walls to identity processes involved in the liquefaction of this tissue. Cell walls were prepared from the locule tissue of fruit at the immature green, mature green, and breaker stages of development. Enzymically active walls incubated in dilute buffer released high molecular mass pectins, oligomeric carbohydrate, and the neutral sugars rhamnose, glucose, galactose, arabinose, xylose, and mannose. The release was sustained for at least 50 h at 34°C and was inhibited more than 50% by 1 m M Hg²⁺. Pectins released from the cell walls of locule tissue at progressive stages of liquefaction were similar in molecular mass and showed no evidence of downshifts on a Sepharose CL–2B–300 column during prolonged incubation. A cell-free protein extract prepared from the locule tissue of mature-green fruit promoted a net release of polymeric and monomeric carbohydrates from high-temperature inactivated cell walls. Polygalacturonase activity was not detected in locule protein although glycosidases including β-mannosidase (EC 3.2.1.25), α- and β-galactosidases (EC 3.2.1.22–23), β-arabinosidase (EC 3.2.1.56) and β-glucosidase (EC 3.2.1.21) were present. Pectinmethylesterase (EC 3.1.1.1 1) activity was detected at the immature-green stage but declined to negligible levels in mature-green and breaker locule tissue. Parallels between the in vitro solubilization of carbohydrate from locule tissue cell walls and the changes occurring during locule liquefaction are discussed.     

Resistance of wheat aleurone cell walls to acid and xylanase action

Isolated wheat (Triticum aestivum var. Potam) aleurone layers have a high capacity to acidify their environment, and secrete hydrolytic enzymes (endoxylanase, glucanase, α-amylase, proteases, etc.) under the control of GA₃. Acidic pH and xylanases are found to be essential for cell wall relaxation in growing tissues, but aleurone is a non-growing, non-dividing tissue. In this tissue, we studied the effect of these loosening factors on aleurone cell walls.Exposure to pH 3.0 caused the release of carbohydrates and calcium ions from the pericarp, and a small amount of carbohydrates, mainly polysaccharides, from aleurone layers from which pericarp tissue had been removed. 50 percnt; of the total sugars released into the incubation medium by these isolated aleurone tissue was arabinose, but no xylose, calcium ions, or phenolic compounds were found. Acid preincubation decreased by 30 percnt; the susceptibility of aleurone cell walls to degradation by exogenously-applied endoxylanase, and also modified the architecture of cell wall as observed by autofluorescence of phenolic groups. These findings suggest that acid treatment and endoxylanase action, rather than having a loosening effect on aleurone cell wall, can have an opposite effect, increasing the resistance of aleurone cell walls to loosening.     

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)     

Distribution of Glycosidases and Acid Invertase Activities in Relation to Elongation Growth in Pearl Millet Internode

The mean cell length along a differentiating internode and alliedchanges in the activities of ß-glucosidase, - andß-galactosidase. -mannosidase and acid invertase,together with the contents of reducing and non-reducing sugars,were examined in pearl millet (Pennisetum americanum L. Leekecv. BJ-104). The specific activities of cytoplasmic -mannosidase,wall ß-glucosidase, and cytoplasmic and wall acidinvertase showed close relationships with the rate of cell elongation.The linear regressions of the rate of cell elongation, and thespecific activities of wall ß-glucosidase and cytoplasmicand wall invertase showed significant positive correlations(P<0·05), whereas cytoplasmic -mannosidase was negativelycorrelated (P<0·01). The results are discussed in the light of cell wall looseningand the provision of carbon substrates for cell elongation. Key words: Glycosidases, acid invertase, sugars, cell elongation, Pennisetum americanum L., Leeke     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Seasonal Concentrations of Non-structural Carbohydrates of Five Actinidia Species in Fruit, Leaf and Fine Root Tissue

To characterize seasonal patterns of carbohydrate concentrationsin Actinidia species from different natural habitats, leaf,fruit and fine root tissue samples from five species (A. arguta,A. deliciosa, A. chinensis, A. polygama and A. eriantha) werecollected over one season, and analysed for fructose, glucose,sucrose, myo -inositol and starch concentrations. Sucrose andstarch peaked in leaf tissue around flowering time. In fruit,hexose sugars and/or myo -inositol transiently increased earlyin development. Starch accumulated in fruit of all species,beginning sooner after anthesis in A. arguta and A. polygamathan in the other species. Sucrose accumulation coincided withonset of net starch degradation in A. arguta but was delayedin the other species. At final fruit sampling, concentrationsof glucose and fructose were greater than sucrose in all speciesexceptA. arguta . myo -Inositol concentrations constituted >10%of total sugars for most of the season in leaf and fruit tissuesof all species except A. polygama. Fine roots of A. arguta andA. polygama contained significantly more starch and sucrosefor most of the year than those of the other species. Observeddifferences in seasonal carbohydrate patterns may reflect differentnatural habitats, with A. arguta and A. polygama growing naturallyin colder climates than the other species. Transient accumulationof sugars in fruit during early stages of development has beenconsidered to act as primary osmoticum for cell expansion. However,the presence of only low sugar concentrations in A. erianthaquestions this hypothesis. Copyright 2000 Annals of Botany Company Actinidia arguta, Actinidia deliciosa, Actinidia chinensis, Actinidia eriantha,Actinidia polygama , kiwifruit, carbohydrates, fruit, leaves, fine roots, seasonal     

Selected cell wall proteins from Zea mays: Assessment of their role in wall hydrolysis

Coleoptile cell wall proteins from Zea mays L. hybrid B 37 × Mo 17 were extracted and fractionated. Three enzymes identified in that extract were examined to determine their role in cell wall hydrolysis with a goal of evaluating the extent to which they participated in autohydrolytic reactions. Two separate proteins were identified as endo- and exo-glucanases. Incubation of these enzymes with heat inactivated cell walls, liberates products derived from the constitutive (1→3), (1→4)-β- d -glucan. The release of sugars from walls resembles that of cell wall autolysis. A third cell wall protein degraded polysaccharides in a more general manner, releasing carbohydrates containing xylose, arabinose, galactose and glucose. Polyclonal antibodies raised against the exoglucanase protein suppressed autolytic reactions of isolated cell wall.     

Pectolytic Activity by the Haustorium of the Parasitic PlantOrobancheL. (Orobanchaceae) in Host Roots

The possible involvement of enzymes in the penetration of intrusivecells of the parasitic angiospermOrobancheinto host root tissueswas studied using cytochemical and immunocytochemical methods.Pectin methyl esterase (PME) was detected, with specific antibodies,in the cytoplasm and cell walls ofOrobancheintrusive cells andin adjacent host apoplast. Depletion and chemical changes ofpectins in host cell walls were shown by histochemical stainingwith PATAg, which detects carbohydrates that are sensitive toperiodic acid, especially pectins, and with the monoclonal antibodiesJIM 5 and JIM 7 that label pectins with low and high rates ofesterification, respectively. Galacturonic sequences with lowrates of esterification were more abundant in host cell wallsadjacent to the parasite, which is consistent with pectin de-methylationby PME release from the parasite. Pectins were absent in middlelamellae and in host cell walls neighbouring mature intrusivecells of the parasite, consistent with further degradation ofpectins by other enzymes. These results provide the first directevidence for the presence and activity of a pectolytic enzymein the infection zone of the haustorium of a parasitic angiosperminsitu.Copyright 1998 Annals of Botany Company Broomrape;Orobanche; parasitic weed; haustorium; pectin methyl esterase; pectin; cell wall.