Cell Wall Metabolism in Ripening Fruit (VI. Effect of the Antisense Polygalacturonase Gene on Cell Wall Changes Accompanying Ripening in Transgenic Tomatoes)

Cell walls of tomato (Lycopersicon esculentum Mill.) fruit, prepared so as to minimize residual hydrolytic activity and autolysis, exhibit increasing solubilization of pectins as ripening proceeds, and this process is not evident in fruit from transgenic plants with the antisense gene for polygalacturonase (PG). A comparison of activities of a number of possible cell wall hydrolases indicated that antisense fruit differ from control fruit specifically in their low PG activity. The composition of cell wall fractions of mature green fruit from transgenic and control (wild-type) plants were indistinguishable except for trans-1,2-diaminocyclohexane-N,N,N[prime],N[prime]-tetraacetic acid (CDTA)-soluble pectins of transgenic fruit, which had elevated levels of arabinose and galactose. Neutral polysaccharides and polyuronides increased in the water-soluble fraction of wild-type fruit during ripening, and this was matched by a decline in Na2CO3-soluble pectins, equal in magnitude and timing. This, together with compositional analysis showing increasing galactose, arabinose, and rhamnose in the water-soluble fraction, mirrored by a decline of these same residues in the Na2CO3-soluble pectins, suggests that the polyuronides and neutral polysaccharides solubilized by PG come from the Na2CO3-soluble fraction of the tomato cell wall. In addition to the loss of galactose from the cell wall as a result of PG activity, both antisense and control fruit exhibit an independent decline in galactose in both the CDTA-soluble and Na2CO3-soluble fractions, which may play a role in fruit softening.     

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.     

Use of FT-IR Spectra and PCA to the Bulk Characterization of Cell Wall Residues of Fruits and Vegetables Along a Fraction Process

This study focuses on the analysis of polysaccharide residues from the cell walls of fruits and vegetables: tomato, potato, pumpkin, carrot and celery root. An alcohol-insoluble residue was prepared from plant material by extraction using the hot ethyl alcohol method and then cell wall fractions soluble in trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetate, sodium carbonate and alkaline solution were sequentially extracted. Infrared spectroscopy combined with Fourier transform (FT-IR) was used to evaluate differences among cell wall residues and among species after each step of sequential extraction of pectins and hemicelluloses. Additionally, pectic substances were identified using an Automated Wet Chemistry Analyser. Principal component analysis (PCA) was applied to FT-IR spectra in two regions: 1,800–1,200 cm^?1 and 1,200–800 cm^?1 in order to distinguish different components of cell wall polysaccharides. This method also allowed us the possibility of highlighting the most important wavenumbers for each type of polysaccharide: 1,740, 1,610 and 1,240 cm^?1 denoting pectins or 1,370 and 1,317 cm^?1 denoting hemicelluloses and cellulose, respectively.     

Cell wall metabolism in ripening fruit. VIII. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe cv. Jackpot tomatoes

Pericarp discs were excised from mature green and red ripe tomato (Lycopersicon esculentum Mill. cv. Jackpot) fruit and kept in sterile tissue culture plates for 4 days, including 2 days of incubation with D-[U-¹³C]-glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA). Na₂CO₃, 4 M KOH and 8 M KOH. Cell wall noncellulosic neutral sugar (NS) composition and cell wall synthetic capacity (i.e. incorporation of density label into cell wall sugars) were determined by using a gas chromatograph coupled to a flame ionization detector and a mass spectrometer, respectively. In the crude cell wall, there was significantly less galactose (Gal) and glucose (Glc) in the “outer”2-mm pericarp region, including the cuticle, compared to the “inner”2-mm region immediately below it (closer to the locules). In the CDTA-soluble pectin, rhamnose (Rha), arabinose (Ara) and Gal accounted for approximately 90% of the total NS. The ratios of these sugars were very similar in the total (¹²C plus ¹³C) sugars, and also in the newly synthesized ([¹³C]-labeled) sugars, suggesting that newly synthesized NS associated with the chelator-extractable pectic fraction has a composition very similar to that of preexisting NS. In the 4 M KOH-soluble material, xylose (Xyl) and Glc accounted for approximately 70% of the total NS. The ratio of these sugars was very similar in the total sugars, but much lower in the newly synthesized portion. This suggests that the hemicellulosic polymers synthesized during the ripening process are different in type and/or proportion from those present in the developing fruit. Because the outer pericarp of tomatoes contains at least two distinct tissue types and these have a distinct cell wall composition, analysis of tomato cell wall polysaccharide composition by homogenization of the entire outer pericarp will obscure subtle changes associated with ripening/softening within specific tissue types.     

Fourier-Transform Raman and Fourier-Transform Infrared Spectroscopy (An Investigation of Five Higher Plant Cell Walls and Their Components)

Infrared and Raman spectra of sequentially extracted primary cell walls and their pectic polymers were obtained from five angiosperm plants. Fourier-transform Raman spectrometry was shown to be a powerful tool for the investigation of primary cell-wall architecture at a molecular level, providing complementary information to that obtained by Fourier-transform infrared microspectroscopy. The use of an extraction procedure using imidazole instead of cyclohexane trans-1,2-N,N,N[prime],N[prime]-diaminotetraacetate allows the extension of the infrared spectral window for data interpretation from 1300 to 800 cm-1, to 2000 to 800 cm-1, and allows us to obtain Raman spectra from extracted cell-wall material. Wall constituents such as pectins, proteins, aromatic phenolics, cellulose, and hemicellulose have characteristic spectral features that can be used to identify and/or fingerprint these polymers without, in most cases, the need for any physical separation. The Gramineae (rice [Oryza sativa], polypogon [Polypogon fugax steud], and sweet corn [Zea mays]) are spectroscopically very different from the nongraminaceous monocotyledon (onion [Allium cepa]) and the dicotyledon (carrot [Daucus carota]); this reflects differences in chemical composition and cross-linking of the walls. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed.     

The polysaccharide structure of potato cell walls: Chemical fractionation

Cell walls of potato tubers were fractionated by successive extraction with various reagents. A slightly degraded pectic fraction with 77% galacturonic acid was extracted in hot, oxalate-citrate buffer at pH 4. A further, major pectic fraction with 38% galacturonic acid was extracted in cold 0.1 M Na₂CO₃ with little apparent degradation. These two pectic fractions together made up 52% of the cell wall. Most of the oxalate-citrate fraction could alternatively be extracted with cold acetate-N,N,N-tetracetic acid (CDTA) buffer, a non-degradative extractant which nevertheless removed essentially all the calcium ions. This fraction was therefore probably held only by calcium binding, and the remainder of the pectins by covalent bonds. Electrophoresis showed that both pectic fractions contained a range of molecular types differing in composition, with a high arabinose: galactose ratio as well as much galacturonic acid in the most extractable fractions. From methylation data, the main side-chains were 1,4-linked galactans and 1,5-linked arabinans, with smaller quantities of covalently attached xyloglucan. Extraction with NaOH-borate removed a small hemicellulose fraction and some cellulose. The main hemicelluloses were apparently a galactoxyloglucan, a mannan or glucomannan and an arabinogalactan.Abbreviations GLC gas-liquid chromatography - MS mass spectrometry - V₀ void volume - MW weight-average molecular weight - DMSO dimethylsulphoxide - EDTA ethylenediamine tetraacetic acid - TFA trifluoroacetic acid - CDTA N,N,N-tetraacetic acid     

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.     

Study of the methyl ester distribution in pectin with endo-polygalacturonase and high-performance size-exclusion chromatography

A method was developed that enables the study of the methyl ester distribution in the polymers of pectin on a molecular level. Endo-polygalacturonase was used to extensively degrade three 70% methyl esterified pectins. The molecular weight distribution of the non- and enzymatically degraded pectins was determined with high-performance size-exclusion chromatography. Next, the molecular weight distribution was converted into a degree of polymerization distribution of galacturonan fragments. Monte Carlo methods were employed for the reconstruction of the parental polymers from their enzymatic degradation products. The results for the random methyl esterified pectin revealed that the enzyme-degradable sites were indeed randomly distributed, which confirmed the correctness of the procedure developed. The two other pectins studied differed greatly in the amount of non-, low-, and high-esterified regions present in the reconstructed pectic molecules of a given molecular mass. That the approach developed is able to reveal such detailed information makes it unique. The information on the fragmental composition of pectic polymers obtained is an important addition to the study of the methyl ester distribution and the functional properties of pectin.     

Metabolism of cell wall polysaccharides from persimmon fruit. Pectin solubilization during fruit ripening occurs in apparent absence of polygalacturonase activity

Pectins from persimmon ( Diospyros kaki L.) fruit pericarp were sequentially extracted with 0. 05 M trans -1,2-diaminocyclohexane-N,N, N', N'-tetraacetic acid (CDTA), 0. 05 M Na₂CO₃ (1°C) and Na₂CO₃ (20°C) and the carbohydrate composition and metabolism during development determined. Young persimmon fruits contained a large proportion of pectins, 46% by dry weight, that decreased to 20% with ripening. This decrease occurred in the CDTA and Na₂CO₃ (1°C) fractions, mainly composed of uronic acids, and represents a net loss of uronic acids, arabinose and galactose. The amount of non-cellulosic neutral sugars was especially high in the Na₂CO₃ (20°C) fraction. The loss of pectins was also accompanied by a depolymerisation of the polysaccharides extracted in the three pectic fractions. However, none of these changes can be attributed to the action of polygalacturonase activity. Proteins were extracted from the pericarp tissue, but endopolygalacturonase (EC 3. 2. 1. 15) activity, determined as a decrease in viscosity of polygalacturonic acid, was not observed in the extract. Determination of exopolygalacturonase (EC 3. 2. 1. 67) activity by measuring the release of reducing groups from polygalacturonic acid was also negative. The results presented indicate that polygalacturonase is not responsible for the metabolism of pectins during persimmon fruit ripening.     

Molecular size and separability features of pea cell wall polysaccharides : implications for models of primary wall structure

Relative molecular size distributions of pectic and hemicellulosic polysaccharides of pea (Pisum sativum cv Alaska) third internode primary walls were determined by gel filtration chromatography. Pectic polyuronides have a peak molecular mass of about 1100 kilodaltons, relative to dextran standards. This peak may be partly an aggregate of smaller molecular units, because demonstrable aggregation occurred when samples were concentrated by evaporation. About 86% of the neutral sugars (mostly arabinose and galactose) in the pectin cofractionate with polyuronide in gel filtration chromatography and diethylaminoethyl-cellulose chromatography and appear to be attached covalently to polyuronide chains, probably as constituents of rhamnogalacturonans. However, at least 60% of the wall's arabinan/galactan is not linked covalently to the bulk of its rhamnogalacturonan, either glycosidically or by ester links, but occurs in the hemicellulose fraction, accompanied by negligible uronic acid, and has a peak molecular mass of about 1000 kilodaltons. Xyloglucan, the other principal hemicellulosic polymer, has a peak molecular mass of about 30 kilodaltons (with a secondary, usually minor, peak of approximately 300 kilodaltons) and is mostly not linked glycosidically either to pectic polyuronides or to arabinogalactan. The relatively narrow molecular mass distributions of these polymers suggest mechanisms of co- or postsynthetic control of hemicellulose chain length by the cell. Although the macromolecular features of the mentioned polymers individually agree generally with those shown in the widely disseminated sycamore cell primary wall model, the matrix polymers seem to be associated mostly noncovalently rather than in the covalently interlinked meshwork postulated by that model. Xyloglucan and arabinan/galactan may form tightly and more loosely bound layers, respectively, around the cellulose microfibrils, the outer layer interacting with pectic rhamnogalacturonans that occupy interstices between the hemicellulose-coated microfibrils.     

Enzymatic degradation of cell wall polysaccharides from soybean meal

Soybean meal, soybean water unextractable solids (WUS) and extracts thereof, which contain particular cell wall polysaccharides, were incubated with a number of cell wall degrading enzymes. The intact cell wall polysaccharides in the meal and WUS were hardly degradable, while the extracts from WUS were well degraded. The arabinogalactan side chains in the pectin-rich ChSS fraction (Chelating agent Soluble Solids) could to a large extent be removed from the pectins by the combined action of endo-galactanase, exo-galactanase, endo-arabinanase and arabinofuranosidase B. The remaining polymer was isolated and represented 30% of the polysaccharides in the ChSS fraction. Determination of the sugar composition showed these polymers to be very highly substituted pectic structures. It still contained 5 mol% of arabinose and 12 mol% of galactose, representing 7% and 12%, respectively, of the arabinose and galactose present in the ChSS fraction before degradation. Further, the presence of uronic acid (50 mol%) and of xylose (18 mol%) indicated the presence of a xylogalacturonan.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of carrot

Pectic and hemicellulosic polysaccharides were successively extracted from an alcohol-insoluble residue (AIR) from carrot root by the actions of Pronase, hot dilute acid, cold dilute alkali, and concentrated alkali in yields corresponding to 12.6, 13.5, 21.7, and 6.7% of AIR, respectively. The first two products were fractionated further by ion-exchange chromatography. Carrot pectins contained 61.3–66.0% of galacturonic acid and 16.0–19.9% of neutral sugars, mainly galactose, arabinose, and rhamnose. Except for the alkali-soluble pectins, the degrees of methylation were high (62.9–67.1) and there was a significant degree of acetylation (7.2–13.5). Pectin fractions were homogeneous in gel-filtration chromatography with viscosity-average molecular weights varying between 36,200 and 56,500. Methylation analysis indicated the presence of arabinogalactans in the pectins extracted during the proteolysis, and fairly long chains of (1→4)-linked galactan with a branched arabinan in the two other pectic fractions. The hemicellulose fraction was mainly composed of (1→4)-linked glucan, (1→4)-linked mannan, (1→4)-linked xylan, and small but significant amounts of pectic polysaccharides. The possible association of cell-wall polymers is discussed.     

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Summary Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca²⁺. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca²⁺ extract. Tracer experiments with³H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid - CWP cell-wall pellet - IAA indole-3-acetic acid - LSE low-salt extract - TCA trichloroacetic acid; Tris tris-(hydroxy-methyl)aminoethane     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

Comparison of the structural features of apple and citrus pectic substances

Pectic substances were extracted from Alcohol Insoluble Solids from lemon peel (albedo) and fractionated by ion exchange chromatography and gelfiltration. The pectin molecules contained rhamnose, arabinose, galactose, glucose and galacturonic acid residues; xylose residues were almost absent. Degradation with purified pectolytic enzymes and subsequent gelfiltration of the resulting pectin fragments showed that the neutral sugar side chains were present in ‘hairy regions’ (blocks of neutral sugar side chains). The distribution of the methoxyl groups was studied by HPLC analysis of enzyme-degraded pectins. Some influence of native pectinesterase on the distribution of the methoxyl groups was found. The results are compared with those of similarly extracted and purified apple pectic substances.     

Pectin Depolymerase Activities Associated with Cell Walls from Cicer arietinum L. Epicotyl

The hydrolytic activity of the proteins extracted with 3 M LiClfrom chick-pea (Cicer arietinum) cell walls to pec-tic fractionsextracted with 50 mM trans-l,2-diaminocy-clohexane-N,N,N,N,-tetraaceticacid (CDTA) and 50 mM sodium carbonate was studied. The pecticfractions contained acidic polysaccharides with high molecularmass (higher than 5 x 10³ kDa), mainly composed of uronic acids,galac-tose, arabinose and rhamnose. The extracted proteins depo-lymerizedthe pectic polysaccharides and also a commercial preparationof polygalacturonic acid from citrus, detected by a decreasein their viscosity and a shift of their molecular mass distribution.The extract was able to depolymerize a uronic acid-rich componentin all the cases, although in different extent. Also, with regardto the CDTA-soluble pectins, a degradation of polyuronide anda shift of the molecular mass distribution of arabinogalactanwas observed. (Received March 11, 1997; Accepted September 10, 1997)     

Cell wall changes during the expansion and senescence of carnation (Dianthus caryophyllus) petals

Senescence of carnation (Dianthus caryophyllus L. ev. White Sim) petals coincided with a decrease on a per flower basis in the yield of cell wall and ethanol-insoluble solids. The decrease in cell wall yield per flower was due largely to a loss of neutral sugars, primarily galactose (45%) and arabinose (23%). On a per flower basis, water-and chelator-soluble pectins increased throughout development, comprising in senescent petals 18 and 58%, respectively, of total pectin. Alkali-soluble pectins ranged from 35 to 45% of the total pectin and decreased during senescence. Gel chromatography of chelator- and alkali-soluble pectins revealed no change in molecular size and polygalacturonase activity was not detected. Large-molecular-size hemicelluloses decreased during development, an observation reminiscent of the changes affecting hemicelluloses during the ripening of a number of fruit types. Compositional analysis of the large hemicellulosic polymers revealed a decrease in xylose and galactose content.     

Effect of GA3 on the Molecular Mass of Polyuronides in the Cell Walls of Alaska Pea Roots

The role of cell wall matrix polysaccharides in gibberellin-regulatedroot growth is unknown. We examined pectic polysaccharides frompea roots treated with or without gibberellin A₃ (GA₃) in thepresence of ancymidol, an inhibitor of gibberellin biosynthesis.Pectic polymers solubilized by CDTA (trans-l,2-cyclohexanediamine-N,N,N',N'-tetraaceticacid) at 23°C and subjected to gel permeation analysis exhibitedhigh polydispersity with a molecular mass in excess of 500 kDa.Subsequent extraction of cell walls with CDTA at 100°C solubilizedpolymers with an average mol mass of 10 to 40 kDa. Subjectingthe high molecular mass pectic polymers extracted at 23°Cto 70–100°C for 2h generated 10 to 40 kDa fragments,similar in size distribution to those solubilized directly fromcell walls by CDTA solutions at 100°C. Pectic polymers from(GA₃+Anc)-treated roots were of higher average mol mass thanthose from Anc-treated roots in both the elongation zone andin the basal maturation zone. Since (GA₃+Anc)-treated rootselongate more quickly than Anc-treated roots [Tanimoto (1994)Plant Cell Physiol. 35:1019], the slender, GA₃-treated rootsmay produce and deposit highly integrated pectins more rapidlythan the thicker, Anc-treated roots in the elongating or elongatedcell walls. ²Present address: Horticultural Sciences Department, POB 110690IFAS, University of Florida, Gainesville, FL 32611-0690 U.S.A.     

Degradation of different pectins by fungi: correlations and contrasts between the pectinolytic enzyme sets identified in genomes and the growth on pectins of different origin

ABSTRACT: BACKGROUND: Pectins are diverse and very complexe biomolecules and their structure depends on the plant species and tissue. It was previously shown that derivatives of pectic polymers and oligosaccharides from pectins have positive effects on human health. To obtain specific pectic oligosaccharides, highly defined enzymatic mixes are required. Filamentous fungi are specialized in plant cell wall degradation and some produce a broad range of pectinases. They may therefore shed light on the enzyme mixes needed for partial hydrolysis. RESULTS: The growth profiles of 12 fungi on four pectins and four structural elements of pectins show that the presence/absence of pectinolytic genes in the fungal genome clearly correlates with their ability to degrade pectins. However, this correlation is less clear when we zoom in to the pectic structural elements. CONCLUSIONS: This study highlights the complexity of the mechanisms involved in fungal degradation of complex carbon sources such as pectins. Mining genomes and comparative genomics are promising first steps towards the production of specific pectinolytic fractions.