Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls

Background and AimsThe programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.MethodsWe developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action.Key ResultsIn model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–GalA’), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol^–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.ConclusionsThe results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.     

Changes in the antioxidative systems in mitochondria during ripening of pepper fruits

The presence of enzymes of the ascorbate–glutathione cycle was studied in mitochondria purified from green and red pepper (Capsicum annuum L.) fruits. All four enzymes, ascorbate peroxidase (APX; EC 1.11.1.11), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were present in the isolated mitochondria of both fruit ripening stages. The activity of the reductive ascorbate–glutathione cycle enzymes (MDHAR, GR and DHAR) was higher in mitochondria isolated from green than from red fruits, while APX and the antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) were higher in the red fruits. The levels of ascorbate and L-galactono-γ-lactone dehydrogenase (GLDH; EC 1.3.2.3) activity were found to be similar in the mitochondria of both fruits. The higher APX and Mn-SOD specific activities in mitochondria from red fruits might play a role in avoiding the accumulation of any activated oxygen species generated in these mitochondria, and suggests an active role for these enzymes during ripening.     

Occurrence of cellobiose residues directly linked to galacturonic acid in pectic polysaccharides

The study carried out in this work concerns the structural characterization of pectic polysaccharides from plum (Prunus domestica L.) and pear (Pyrus communis L.) cell walls and commercial pectic polysaccharides, obtained from Citrus. The α-(1 → 4)-d-galacturonic acid backbone was submitted to a selective hydrolysis with endo-polygalacturonase (EPG) and the fractions with low molecular weight (<1 kDa) obtained by size-exclusion chromatography were analysed by mass spectrometry using electrospray ionisation (ESI-MS). The ESI-MS spectra obtained revealed the presence of several [M+Na]⁺ ions of pectic oligosaccharides identified as belonging to different series, including oligosaccharides constituted only by galacturonic acid residues (GalA_n, n = 1-5) and galacturonic acid residues substituted by pentose residues (GalA₃Pent_n, n = 1-2). Surprisingly, it was also observed the occurrence of galacturonic acid residues substituted by hexose residues (GalA_nHex_m, n = 2-4, m = 1-2). The fragmentation of the observed [M+Na]⁺ ions, obtained under ESI-MS/MS and MSⁿ allowed to confirm the proposed structures constituent of these pectic oligosaccharides. Furthermore, the ESI-MSⁿ spectra of the ions that could be identified as GalA_nHex_m (n = 2-4, m = 1-2) confirmed the presence of Hex or Hex₂ residues linked to a GalA residue. Methylation analysis showed the presence, in all EPG treated samples, of terminally linked arabinose, terminally and 4-linked xylose, and terminally and 4-linked glucose. The occurrence of GalA substituted by Glc, and Glc-β-(1 → 4)-Glc are structural features that, as far as we know, have never been reported to occur in pectic polysaccharides.     

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the β-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased β-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 β-galactosidases. Similar functions for other β-galactosidases in both GHFs 2 and 42 are suggested by genomic data.     

Dimeric calcium complexes of arabinan-rich pectic polysaccharides from Olea europaea L. cell walls

The study carried out in this work concerns the pectic polysaccharides of olive cell walls as present in olive pulp and that remained entrapped in the cellulosic residue after sequential extraction of the cell wall material (CWM) with imidazole, carbonate and KOH aqueous solutions. These polymers, obtained after neutralisation and dialysis of an aqueous suspension of the residue (sn-CR fraction), extracted with 4 M KOH, were arabinan-rich pectic polysaccharides. They accounted for 11–19% of the total pectic polysaccharides found in the olive pulp cell walls of fruits collected in two years and in three stages of ripening (green, cherry and black). The analysis by powder X-ray diffraction highlighted the existence, in all sn-CR fractions, of crystalline phases related with the presence of calcium-pectic polysaccharide complexes (CPPC) occurring in an amorphous carbohydrate network. The relative crystallinity of the CPPC varied linearly with the Ca²⁺/GalA molar ratio until a maximum of 0.57. Size-exclusion chromatography showed that sn-CR fractions possessed a bimodal molecular weight distribution. The lower molecular weight fraction of sn-CR (M_w = 70–135 kDa) was independent on the ripening stage of olive fruit, whereas the higher molecular weight fraction showed values of 1.1, 0.6–0.9 and 0.5–0.7 MDa, respectively, for green, cherry and black olives. Treatment of the sn-CR pectic polysaccharides with a 2 M imidazole solution disrupted the CPPC crystalline network showing the loss of low molecular weight galacturonan-rich material during dialysis (12–14 kDa cut off) and the decrease of molecular weight of the polymers to roughly half. These results allowed to infer the presence of oligogalacturonides held within cell walls by calcium ions and that the pectic polysaccharides of sn-CR fraction occurred in olive pulp cell walls as calcium bridged macrodimers.     

Cell wall carbohydrates as signals in plants

Plant and fungal cells are surrounded by a cell wall rich in diverse polysaccharides and proteins. It has become apparent in recent years that the carbohydrates in the cell wall function not only to maintain cell shape and integrity, but also may serve as signals in plants. This review summarizes the evidence that biologically-active oligosaccharides (oligosaccharins) released from plant or microbial cell walls can serve as signals to regulate plant defense and plant growth and development. The oligosaccharins discussed include the fungal-derived hepta-β-glucoside and the plant cell wall-derived oligogalacturonides and xyloglucans. Possible mechanisms by which oligosaccharins may exert their effects on plant cells are discussed.     

Methods for obtaining neutral and acid oligosaccharides from flax pectins

Esterified acid soluble pectins from flax (Linun usitatissimum L.) were degraded either with HCl or pectin lyase. Centrifugation and 2-propanol precipitation led to the isolation of two low molecular weight polygalacturonates after acid hydrolysis of pectins. However, after pectin lyase digestion and purification by size-exclusion HPLC, ¹H NMR analyses indicated that acetylated hairy regions, large methylated and acetylated oligogalacturonides together with small unsubstituted oligogalacturonides were produced. Thus, in a few steps, a panel of substituted neutral and acidic oligosaccharides was produced from a raw plant material. Such oligosaccharides could be useful for further fractionations such as chemical saponification and enzymatic removal of neutral sugar chains from the hairy regions. The procedures used for pectin extraction, for degradation, and for the purification of fragments seem appropriate for large-scale production of biologically active oligosaccharides from flax.Revisions requested 24 September 2004; Revisions received 4 November 2004     

Cell Wall Metabolism in Ripening Fruit (VII. Biologically Active Pectin Oligomers in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits)

A water-soluble, ethanol-insoluble extract of autolytically inactive tomato (Lycopersicon esculentum Mill.) pericarp tissue contains a series of galacturonic acid-containing (pectic) oligosaccharides that will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs cut from mature green fruit. The concentration of these oligosaccharides in extracts (2.2 [mu]g/g fresh weight) is in excess of that required to promote ethylene synthesis. Oligomers in extracts of ripening fruits were partially purified by preparative high-performance liquid chromatography, and their compositions are described. Pectins were extracted from cell walls prepared from mature green fruit using chelator and Na2CO3 solutions. These pectins are not active in eliciting ethylene synthesis. However, treatment of the Na2CO3-soluble, but not the chelator-soluble, pectin with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit (according to high-performance liquid chromatography analysis) and are active as elicitors. The possibility that pectin-derived oligomers are endogenous regulators of ripening is discussed.     

Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia

Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes.     

Lipid-linked saccharides formed during pullulan biosynthesis in Aureobasidium pullulans

Radioactive glycolipids were extracted from cells of Aureobasidium pullulanspulsed with d-[¹⁴C]glucose. Labelled, alkali-stable lipids were resolved into one neutral and two acidic fractions. The neutral fraction was stable to mild hydrolysis with acid, whereas the acidic fractions could be hydrolysed, yielding d-glucose and a series of oligosaccharides having mobilities corresponding to those of isomaltose, panose, and isopanose. Amyloglucosidase (EC 3.2.1.3) catalysed the hydrolysis of 60% of the liberated radioactive oligosaccharides to d-glucose, indicating the presence of (1→4)-α- and (1 → 6)-α-d-glucosidic bonds. Since these lipid-linked saccharides are produced during pullulan biosynthesis in A. pullulans, it is proposed that they are intermediates in the biosynthetic pathway of that extracellular polysaccharide. A mechanism incorporating these glycolipids into a possible scheme of polysaccharide assembly is presented.     

Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening

A critical role in the initiation of ripening has been proposed for pectolytic enzymes which are known to be involved in fruit softening. The hypothesis that tomato (Lycopersicon esculentum Mill.) ripening is controlled by the initial synthesis of the cell-wall-degrading enzyme polygalacturonase (EC 3.2.1.15), which subsequently liberates cell-wall-bound enzymes responsible for the initiation of ethylene synthesis and other ripening events, has been examined. A study of kinetics of ethylene evolution and polygalacturonase synthesis by individual fruits in a ripening series, employing an immunological method and protein purification to identify and measure polygalacturonase synthesis, showed that ethylene evolution preceded polygalacturonase synthesis by 20h. Exogenous ethylene stimulated the synthesis of polygalacturonase and other ripening events, when applied to mature green fruit, whereas the maintenance of fruits in a low ethylene environment delayed ripening and polygalacturonase synthesis. It is concluded that enhanced natural ethylene synthesis occurs prior to polygalacturonase production and that ethylene is responsible for triggering polygalacturonase synthesis indirectly. Possible mechanisms for ethylene action are discussed.     

Solid-supported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Solid-phase biosynthetic reactions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF), was used to gain insight into the biosynthesis of pectin oligomers. Sepharose supports bearing long pectic oligogalacturonides (OGAs) anchored through a disulfide-containing cleavable linker, were prepared. The OGAs (degrees of polymerization of 13 and 14) were efficiently immobilized through the reducing end via formation of an oxime linkage. These OGA-derivatized matrices were subsequently employed in novel solid-phase enzymatic reactions, with the pectin biosynthetic enzyme, alpha-1,4-galacturonosyltransferase, GalAT (solubilized from Arabidopsis thaliana) and the glycosyl donor, uridine diphosphate-galacturonic acid (UDP-GalA). Solid-supported biosynthesis was followed by cleavage of the immobilized OGAs and direct analysis of the products released into the liquid phases by MALDI-TOF mass spectrometry. In time course studies conducted with an immobilized (alpha-D-GalA)14 and limiting amounts of the glycosyl donor, the predominant product was an OGA extended by one GalA residue at the non-reducing end (i.e., (GalA)15). When UDP-GalA was added in approximately excess compared to immobilized (GalA)13, OGAs up to the 16-mer were synthesized, confirming the non-processivity of the GalAT in vitro.     

Action of alpha-1,6-glucan glucohydrolase on oligosaccharides derived from dextran

The action of α-1,6-glucan glucohydrolase on α-(1→6)-D-glucosidic linkages in oligosaccharides that also contain an α-(1→2)-, α-(1→3)-, or α-(1→4)-D-glucosidic linkage has been investigated. The enzyme could hydrolyse α-(1→6)-D-glucosidic linkages from the non-reducing end, including those adjacent to an anomalous linkage. α-(1→6)-D-Glucosidic linkages at branch points were not hydrolysed, and the enzyme could neither hydrolyse nor by-pass the anomalous linkages. These properties of α-1,6-glucan glucohydrolase explain the limited hydrolysis of dextrans by the exo-enzyme. Hydrolysis of the main chain of α-(1→6)-D-glucans will always stop one D-glucose residue away from a branch point. The extent of hydrolysis by α-1,6-glucan glucohydrolase of some oligosaccharide products of the action on dextran of Penicillium funiculosum and P. lilacinum dextranase, respectively, has been compared. Differences in the specificity of the two endo-dextranases were revealed. The Penicillium enzymes may hydrolyse dextran B-512 to produce branched oligosaccharides that retain the same 1-unit and 2-unit side-chains that occur in dextran.     

Stimulation of root development on buckwheat thin cell-layer explants by pectic fragments from pea stem cell walls

Buckwheat (Fagopyrum esculentum Moench.) thin cell-layers (TCLs) cultured individually in a liquid medium were used to test the root-inducing activity of pectic polysaccharides with a degree of polymerization (DP) of 20–25, isolated from pea (Pisum sativum L.) stem cell walls. These pectic fragments induced more rapid root formation on the explants in comparison with untreated controls. This pectic fragment treatment also promoted root growth as measured by both fresh and dry weights and about doubled the number of roots formed. This buckwheat TCL system is proposed as a new bioassay for oligosaccharins due to its sensitivity, reproducibility and ease of preparation.     

Partial Characterization of C(x) Cellulase and Cellobiase from Ripening Tomato Fruits

Cellulolytic enzymes were studied in extracts from the locular contents of ripening fruits of Lycopersicon esculentum var. KC-146. When acting on carboxymethyl cellulose, the enzyme preparations were capable of decreasing the viscosity of the reaction mixture and generating reducing groups, oligosaccharides, and glucose. Cellobiose cellotriose, cellotetrose, and cellopentose also served as substrates for glucose production.     

Microbial utilization and selectivity of pectin fractions with various structures

To evaluate the fermentation properties of oligosaccharides derived from pectins and their parent polysaccharides, a 5-ml-working-volume, pH- and temperature-controlled fermentor was tested. Six pectic oligosaccharides representing specific substructures found within pectins were prepared. These consisted of oligogalacturonides (average degrees of polymerization [DP] of 5 and 9), methylated oligogalacturonides (average DP of 5), oligorhamnogalacturonides (average DP of 10 as a disaccharide unit of galacturonic acid and rhamnose), oligogalactosides (average DP of 5), and oligoarabinosides (average DP of 6). The influence of these carbohydrates on the human fecal microbiota was evaluated. Use of neutral sugar fractions resulted in an increase in Bifidobacterium populations and gave higher organic acid yields. The Bacteroides-Prevotella group significantly increased on all oligosaccharides except oligogalacturonides with an average DP of 5. The most selective substrates for bifidobacteria were arabinan, galactan, oligoarabinosides, and oligogalactosides.     

A sensitive and rapid bioassay of homogalacturonan synthase using 2-aminobenzamide-labeled oligogalacturonides

Polygalacturonate 4-alpha-galacturonosyltransferase (GalA T) activity was detected in the microsomal fraction isolated from pumpkin (Cucurbia moschata Duchesne, cv. Tokyou-Kabocha) seedlings using UDP-GalA and 2-aminobenzamide (2AB)-labeled oligogalacturonides. A 2AB-labeled undecagalacturonide was elongated by the attachment of galacturonic acid (GalA) residues to give 2AB-labeled oligogalacturonides with a degree of polymerization (DP) between 12 and 17. Exogenous 2AB-labeled oligogalacturonide acceptors with a DP >3 are effective acceptor molecules for pumpkin GalA T.     

The Role of Polygalacturonase (PG) in the Ripening of Apple Fruits

Polygalacturonase (PG) activity and changes in respiratory intensity of apple fruits were investigated. The respiratory rate was decreased to a preclimacteric minimum from 30 Aug. to 20 Sept., Then increased to a climacteric peak (20–30 Sept.) and again drop down gradually with approaching the senescence stage. The PG activity was undetectable in a developing fruit until the onset climacteric phase. It rose rapidly after harvest, and reaching its highest level on 27 Oct. Just a month after the climacteric peak. The PG activity fell gradually. The amount of the fractions of pectic acid in fruits changed with the modifications of PG activity. With the ripening of fruits, the content of alcohol-soluble small molecules of pectic acids was increased from 12 to 13 5 mg/100 g of tissue, while the amount of alcohol- insoluble large molecules of pectic acids reduced from 530 to 280/100 g of tissue. PG activity would indicate the destruction of cell walls and the separation of cells. The onset of softening of fruits occurred 20 days after the rise of PG activity. It is supposed that the process of softening is directly controlled by PG activity.