Search

The recruitment of a bacterial consortium by the host is a strategy not limited to animals but is also used in plants. A maize aerial root mucilage has been found that harbors nitrogen fixing bacteria that are attracted to the carbohydrate rich environment. This synbiotic relationship is facilitated by a polysaccharide, whose complicated structure has been previously unknown. In this report, we present the characterization of the maize polysaccharide by employing new analytical strategies combining chemical depolymerization, oligosaccharide sequencing, and monosaccharide and glycosidic linkage quantitation. The mucilage contains a single heterogeneous polysaccharide composed of a highly fucosylated and xylosylated galactose backbone with arabinan and mannoglucuronan branches. This unique polysaccharide structure may select for the diazotrophic community by containing monosaccharides and linkages that correspond to the glycosyl hydrolases associated with the microbial community. The elucidation of this complicated structure illustrates the power of the analytical methods, which may serve as a general platform for polysaccharide analysis in the future.

This study was performed to clarify the strategies of Penicillium digitatum during pathogenesis on citrus, assessing, on albedo plugs, the effects of treatment with sodium bicarbonate (NaHCO3 ), at two different pH values (5 and 8.3), on cell-wall-degrading enzyme activity over a period of 72 h.; Results: Treatment with NaHCO3 , under alkaline pH, delayed the polygalacturonase activity for 72 h, or 48 h in the case of the pectin lyase, compared with the control or the same treatment at pH 5. In contrast, pectin methyl esterase activity rapidly increased after 24 h, in plugs dipped in the same solution. In this case, the activity remained higher than untreated or pH 5-treated plugs up to 72 h.; Conclusion: The rapid increase in pectin methyl esterase activity under alkaline conditions is presumably the strategy of the pathogen to lower the pH, soon after the initiation of infection, in order to restore an optimal environment for the subsequent polygalacturonase and pectin lyase action. In fact, at the same time, a low pH delayed the enzymatic activity of polygalacturonase and pectin lyase, the two enzymes that actually cleave the α-1,4-linkages between the galacturonic acid residues. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

Fruit cracking is an important problem in horticultural crop production. Polygalacturonase (SlPG) and expansin (SlEXP1) proteins cooperatively disassemble the polysaccharide network of tomato fruit cell walls during ripening and thereby, enable softening. A Golden 2-like (GLK2) transcription factor, SlGLK2 regulates unripe fruit chloroplast development and results in elevated soluble solids and carotenoids in ripe fruit. To determine whether SlPG, SlEXP1, or SlGLK2 influence the rate of tomato fruit cracking, the incidence of fruit epidermal cracking was compared between wild-type, Ailsa Craig (WT) and fruit with suppressed SlPG and SlEXP1 expression (pg/exp) or expressing a truncated nonfunctional Slglk2 (glk2). Treating plants with exogenous ABA increases xylemic flow into fruit. Our results showed that ABA treatment of tomato plants greatly increased cracking of fruit from WT and glk2 mutant, but not from pg/exp genotypes. The pg/exp fruit were firmer, had higher total soluble solids, denser cell walls and thicker cuticles than fruit of the other genotypes. Fruit from the ABA treated pg/exp fruit had cell walls with less water-soluble and more ionically and covalently-bound pectins than fruit from the other lines, demonstrating that ripening-related disassembly of the fruit cell wall, but not elimination of SlGLK2, influences cracking. Cracking incidence was significantly correlated with cell wall and wax thickness, and the content of cell wall protopectin and cellulose, but not with Ca2+ content., Crop genetics: Keeping tomato skins together Researchers in the USA reveal a link between cell wall composition and splitting skin in ripe tomatoes, known as cracking. To unravel the mechanism behind cracking, a team led by Elizabeth Mitcham of the University of California, Davis, compared wild-type tomato plants, mutant plants with increased soluble solids, and mutant plants with altered cell wall structure. They treated the three lines with the hormone ABA to induce cracking and measured several traits in the fruit. ABA treatment did not affect cracking incidence in the cell wall mutant but increased its frequency in the other two lines. Cell wall composition and thickness were the traits with the strongest correlation with cracking rate. Altogether, these findings show that cell wall structure is a major determinant of cracking and point the way towards reducing skin splitting in tomatoes.

Lignocellulosic food processing residues are suitable feedstocks for bioenergy generation, however, seasonal production of these materials requires storage, and their cell wall pectin and hemicellulose must be disrupted to improve enzymatic hydrolysis of cellulose. The objectives of this study were to examine ensilage as a method to facilitate preservation and pretreatment and to determine if these processes are influenced by the secretion of carbohydrate-active enzymes. Production of xylanase (Xase), endo-β-1,4-glucanase (EGase), and polygalacturonase (PGase) was monitored during ensilage of tomato pomace (TP) and sugar beet pulp (SBP). During two months of ensilage of TP, secreted Xase and EGase activities increased gradually with peak activities occurring at day 30. For SBP, Xase and EGase activities were detected only at day 14. Higher sugar yields were observed for ensiled feedstocks upon post-storage enzymatic saccharification, indicating ensilage had a pretreatment effect on these feedstocks. Lactic acid bacteria (LAB) were the dominant organisms in the ensilage community. , , and were detected at relatively high levels in SBP; while was the prominent LAB species in TP. Ensilage had a positive effect on the post-storage enzymatic treatment of SBP and TP. This may have been due to bacterial production of carbohydrate-active enzymes during ensilage. Incorporating ensilage as part of a bioconversion process will likely reduce necessary pretreatment severity and inputs for enzymatic hydrolysis.

Quality and compositional changes were determined in noni fruit harvested at five ripening stages, from dark-green to thaslucent-grayish. Fruit ripening was accompanied by acidity and soluble solids accumulation but pH diminution, whereas the softening profile presented three differential steps named early (no significant softening), intermediate (significant softening), and final (dramatic softening). At early step the extensive depolymerization of hydrosoluble pectins and the significantly increment of pectinase activities did not correlate with the slight reduction in firmness. The intermediate step showed an increment of pectinases and hemicellulases activities. The final step was accompanied by the most significant reduction in the yield of alcohol-insoluble solids as well as in the composition of uronic acids and neutral sugars; pectinases increased their activity and depolymerization of hemicellulosic fractions occurred. Noni ripening is a process conducted by the coordinated action of pectinases and hemicellulases that promote the differential dissasembly of cell wall polymers.

Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co-culturing the green alga Chlorella minutissima with Escherichia coli under high-substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co-cultures produced under autotrophic and mixotrophic conditions. We found that co-culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9- to 3.1-fold and fatty acid content 1.5- to 2.6-fold compared to equivalent axenic C. minutissima cultures. These same co-cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen-limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes.

The carbohydrate composition of the algal cell wall was investigated for its role in cell flocculation. Cultures of Chlorella variabilis NC64A, which were found to have different levels of neutral sugar, uronic acid and amino sugar in the cell wall when cultured in different nitrogen sources and concentrations, were subjected to flocculation with chitosan at dosages of 0–69.6 mg/l and pH values of 5.5, 7 and 8.5. In addition, flocculations of another three strains of Chlorella , which have different levels of cell wall components, were tested. Flocculation improved for all strains at pH 8.5 suggesting that inter molecular forces such as hydrogen bonding might be more important than charge neutralization in the flocculation of Chlorella . Total carbohydrate content in the cell wall was the most significant factor positively affecting the flocculation efficiency of C. variabilis NC64A cells with different cell wall compositions and the other Chlorella strains. The results presented in this study suggest that chitosan flocculation can be improved by optimizing the cell culture conditions to achieve higher cell wall polysaccharide content or selecting an algal strain with higher cell wall polysaccharide content.

Bitter pit (BP), a Ca2+ deficiency disorder of apple fruit (Malus domestica), is a complex process that involves not only the total input of Ca2+ into the fruit, but also a proper Ca2+ homeostasis at the cellular level. The objective of this study was to test the hypothesis that Ca2+ accumulation into storage organelles and binding to the cell wall is associated with BP development in apple fruit. The experiment was carried out on ‘Granny Smith’ apples stored at 0 °C for 60 d. After storage, fruit were segregated into two lots for analysis, apples with the water-soaked initial visual symptoms of BP and those not showing this symptom. Cytochemical and ultrastructural observations showed an accumulation of Ca2+ in the vacuole of individual outer cortical cells of pitted fruit. We also observed an increase in the expression of genes encoding four pectin methylesterases, a greater degree of pectin deesterification and therefore more Ca2+ binding sites in the cell wall, and a higher fraction of the total cortical tissue Ca2+ content that was bound to the cell wall in pitted fruit compared with non-pitted fruit. Cells of the outer cortical tissue of pitted fruit consistently had higher membrane permeability than outer cortical cells of non-pitted fruit. The results provide evidence that Ca2+ accumulation into storage organelles and Ca2+ binding to the cell wall represent important contributors to BP development in apple fruit.

Cell wall disassembly in ripening climacteric fruit is a highly complex process where ethylene plays a crucial role. Ethylene inhibitors can be used to explore the changes in the cell wall matrix and cross-linked polysaccharides in ethylene-regulated processes. The results of applying the ethylene receptor blocking inhibitor 1-methylcyclopropene (1-MCP) and the ethylene-releasing compound ethephon (2-chloroethylphosphonic acid) indicate that softening of ‘Maradol’ papaya fruit is dependent on ethylene. When fruit were induced to ripen extensively by exposure to a high dose of ethephon, 1-MCP inhibited the subsequent softening dramatically, but when inhibition of the ethylene response was caused by application of 1-MCP, subsequent fruit treatment with ethephon promoted extensive loss of galactose from the water-soluble polysaccharides, but this was not accompanied by fruit softening. The cell wall changes accompanying normal fruit softening were pectin solubilization and polyuronide depolymerization and these processes occurred simultaneously. Polygalacturonase likely is responsible for the ripening-associated changes in ‘Maradol’ papaya fruit texture and pectin polymer integrity. An increase in extractable fruit polygalacturonase follows the increased presence of pectin-derived oligosaccharides.

Polygalacturonase (PG) activity found in the salivary gland apparatus of the western tarnished plant bug (WTPB, Lygus hesperus Knight) has been thought to be the main chemical cause of the damage inflicted by this mirid when feeding on its plant hosts. Early viscosity and thermal stability studies of the PG activity in L. hesperus protein extracts were difficult to interpret. Thus, it has been suggested that one or more PG protein(s) with different hydrolytic modes of action are produced by this mirid. In order to understand the quantitative complexity of the WTPB salivary PG activity, PG purification from a protein extract from salivary glands excised from L. hesperus insects was performed using affinity and ion exchange chromatography. To elucidate the qualitative complexity of the purified PGs, the digestion products generated by the PGs were separated using high performance anion exchange chromatography with pulsed amperometric detection. At least five PG proteins were detected; these differing in terms of their glycosylation, mass-to-charge ratios, and/or molecular mass. The characterization of the products generated by these PGs showed that endo- and exo-acting PGs are produced by WTPB. Although none of the PGs was purified to homogeneity, the present work provides biochemical evidence of a multiplicity of PGs that degrade the pectin component of the plant tissue in different fashions. The implications of these findings affect the understanding of WTPB feeding damage and, potentially, help identify ways to control this important crop pest. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

Two decades of research have provided deep insights into the regulation of cell wall disassembly in ripening fruit. These insights have been important in understanding ethylene-regulated processes of fruit softening in climacteric fruit but have also provided insights into non-climacteric fruit softening as well as cell wall disassembly that accompanies many plant developmental processes such as cell growth and abscission. Major results indicate that no single gene or enzyme can account for the major events that underlie fruit softening and that many of the potentially responsible genes and their corresponding cell wall modifying proteins are members of large gene families that exhibit overlapping patterns of expression and possibly redundant biochemical action. The cooperative action of cell wall modifying proteins and enzymes has been demonstrated to participate in fruit softening as well as to the ripening-regulated increase in necrotrophic pathogen susceptibility. A general model of cooperative cell wall disassembly is now firmly established and future research focusing on complex interactions between cell wall modifying proteins and enzymes and between the major cell wall matrices should be productive in elucidating a general model of ripening-regulated cell wall disassembly and fruit softening.

The softening that accompanies ripening of commercially important fruits exacerbates damage incurred during shipping and handling and increases pathogen susceptibility. Thus, postharvest biologists have studied fruit softening to identify ways to manage ripening and optimise fruit quality. Studies, generally based on the premise that cell wall polysaccharide breakdown causes ripening-associated softening, have not provided the insights needed to genetically engineer, or selectively breed for, fruits whose softening can be adequately controlled. Herein it is argued that a more holistic view of fruit softening is required. Polysaccharide metabolism is undoubtedly important, but understanding this requires a full appreciation of wall structure and how wall components interact to provide strength. Consideration must be given to wall assembly as well as to wall disassembly. Furthermore, the apoplast must be considered as a developmentally and biochemically distinct, dynamic 'compartment', not just the location of the cell wall structural matrix. New analytical approaches for enhancing the ability to understand wall structure and metabolism are discussed. Fruit cells regulate their turgor pressure as well as cell wall integrity as they ripen, and it is proposed that future studies of fruit softening should include attempts to understand the bases of cell- and tissue-level turgor regulation if the goal of optimising softening control is to be reached. Finally, recent studies show that cell wall breakdown provides sugar substrates that fuel other important cellular pathways and processes. These connections must be explored so that optimisation of softening does not lead to decreases in other aspects of fruit quality. ! 2007 Society of Chemical Industry

Uronic acid oxidase activity was found in an extract from harvested peaches that was incubated with citrus pectin at pH 8.5. The product of this reaction was identified by GC–MS analysis to be galactaric acid. The reaction was linear at 37 °C for up to 20 h, and the pH optimum was 8.5. The activity found in firm peaches one day after harvest did not change as the peaches softened over 5 days to eating softness. The incubation conditions were those suitable for monitoring the activity of pectate lyase, but instead of finding an increase in galacturonosyl residue reducing groups due to generation of pectin-derived oligosaccharides, uronic acid oxidase catalyzed the oxidation of the aldehyde reducing functions to carboxyl groups.

An analytical approach using matrix-assisted laser desorption/ionization mass spectrometry for the structural characterization and assessment of the degree of polymerization of cell wall pectin-derived oligosaccharides (PDOs) in three regions of Botrytis cinerea-infected tomato fruit tissue is described. The PDOs were isolated from lesion centers (extensively macerated tissue), the area just beyond visible lesion margins, and healthy and intact tissue of an inoculated fruit, sampled at a distance from developing lesions. PDO mixtures were directly analyzed by mass spectrometry without chromatographic separation, after minimum cleanup by membrane drop dialysis. The structures identified implied the action of three different pathogen pectin-modifying enzymes. Modifications such as methyl esterification were identified by determination of exact PDO molecular masses and tandem mass spectrometry via collision-induced dissociation. We have identified four PDO series that were generated through the breakdown of homogalacturonan pectins. The decayed and lesion edge areas had fewer and less diverse PDOs than healthy tissues, possibly due to metabolic by-products of the pathogen. This analytical technique provides a simple and rapid method to characterize the pectin-derived oligosaccharides produced by in vivo digestion during pathogen infection.

Cell wall changes were examined in fruit of a melting flesh peach (Prunus persica L.) allowed to ripen on the tree. Three phases to softening were noted, the first of which began prior to the completion of flesh colour change and an increase in ethylene evolution. Softening in young mature fruit, prior to ripening, was associated with a depolymerization of matrix glycans both loosely and tightly attached to cellulose and a loss of Gal from all cell wall fractions. After the initiation of ripening, but before the melting stage, softening was associated with continuing, progressive depolymerization of matrix glycans. A massive loss of Ara from the loosely bound matrix glycan fraction was observed, probably from side chains of glucuronoarabinoxylan, pectin, or possibly arabinogalactan protein firmly bound into the wall and solubilized in this extract. An increase in the solubilization of polyuronides also occurred during this period, when softening was already well advanced. The extensive softening of the melting period was marked by substantial depolymerization of both loosely and tightly bound matrix glycans, including a loss of Ara from the latter, an increase in matrix glycan extractability, and a dramatic depolymerization of chelator-soluble polyuronides which continued during senescence. Depolymerization of chelator-soluble polyuronides thus occurred substantially after the increase in their solubilization. Ripening-related increases were observed in the activities of exo- and endo-polygalacturonase (EC 3.2.1.67; EC 3.2.1.15), pectin methylesterase (EC 3.1.1.11), endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), alpha-arabinosidase (EC 3.2.1.55), and beta-galactosidase (EC 3.2.1.23), but the timing and extent of the increases differed between enzymes and was not necessarily related to ethylene evolution. Fruit softening in peach is a continuous process and correlated closely with the depolymerization of matrix glycans, which proceeded throughout development. However, numerous other cell wall changes also took place, such as the deglycosylation of particular polymers and the solubilization and depolymerization of chelator-soluble polyuronides, but these were transient and occurred only at specific phases of the softening process. Fruit softening and other textural changes in peach appear to have a number of stages, each involving a different set of cell wall modifications.

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

α-l-Arabinofuranosidases (α-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different α-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. α-Af I and II are active throughout fruit ontogeny. α-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. α-Af II activity accounts for over 80% of the total α-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, α-Af III is ethylene dependent and specifically active during ripening. α-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas α-Af II and III acted on Na2CO3-soluble pectins. Different α-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. α-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only α-Af III activity. Results suggest that tomato α-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.

A 96-well plate method was developed for analysis of total hexosamine content in biological samples. Four hexosamine monomer derivatives-glucosamine hydrochloride, glucosamine sulfate, galactosamine hydrochloride, and mannosamine hydrochloride-were examined for the linearity of their spectra in the concentration range specified in the assay. The hexosamine concentration analysis range was linear from 0.1 to 1 mM. The quantification of hexosamines from chitin and chitosan upon acid hydrolysis was also tested. Accurate quantification of glucosamine content in chitin and chitosan with different molecular sizes and degrees of acetylation was demonstrated using the new method.

© 2014 Elsevier Inc. All rights reserved. Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

Microalgae have been proposed as a potential feedstock for biofuel production; however, cell disruption is usually required for collection and utilization of cytoplasmic polysaccharides and lipids. Virus infection might be one approach to disrupt the cell wall. The concentration of yeast extract and presence of KNO3 in algae cultivation media were investigated to observe their effects on Chlorella variabilis NC64A physiology and composition and the subsequent effect on production of Chlorella virus and disruption of infected cells. Cytoplasmic starch accumulation increased from 5% to approximately 35% of the total dry weight when yeast extract decreased from 1 to 0.25 g L(-1). When cells were cultured with the lowest nitrogen levels, the total polysaccharide accounted for more than 50% of the cell wall, which was 1.7 times higher than the content in cells cultured with the highest nitrogen levels. The C/N ratio of the algal biomass decreased by a factor of approximately 2 when yeast extract increased from 0.25 to 1 g L(-1). After virus infection, cells with a low C/N ratio produced a 7.6 times higher burst size than cells with a high C/N ratio, suggesting that the nitrogen content in C. variabilis has a large influence on viral production and cell lysis. The results have implications on management of nitrogen for both the synthesis of products from algae and product recovery via viral lysis.

Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth), secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes) identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue. © 2014 Blanco-Ulate, Morales-Cruz, Amrine, Labavitch, Powell and Cantu.

Plant polygalacturonase inhibitor proteins (PGIPs) bind fungal polygalacturonases (PGs), but inhibition specificities and kinetics vary within and among species. Purified bean PGIP inhibited all fungal PGs we tested, including Fusarium moniliforme PG. Pear PGIP, however, was only effective against Botrytis cinerea PG. Moreover, tomato PGIP inhibited B. cinerea PG more than Aspergillus niger PG. Models of codon evolution for 22 dicot PGIPs and 19 fungal PGs indicated that advantageous substitutions dominate the molecular evolution of these genes and identified 9 amino acid residues, each, that are likely to evolve adaptively in response to natural selection. Many of these residues are within the β-strand/β-turn region of the PGIP LRR, including two sites known to alter inhibition specificities of bean PGIPs, but others lie outside this region. Our results complement existing molecular and biochemical studies of resistance specificity, and suggest new target amino acids for manipulating PG-inhibition.

The Charentais variety of melon (Cucumis melo cv Reticulatus F1 Alpha) was observed to undergo very rapid ripening, with the transition from the preripe to overripe stage occurring within 24 to 48 h. During this time, the flesh first softened and then exhibited substantial disintegration, suggesting that Charentais may represent a useful model system to examine the temporal sequence of changes in cell wall composition that typically take place in softening fruit. The total amount of pectin in the cell wall showed little reduction during ripening but its solubility changed substantially. Initial changes in pectin solubility coincided with a loss of galactose from tightly bound pectins, but preceded the expression of polygalacturonase (PG) mRNAs, suggesting early, PG-independent modification of pectin structure. Depolymerization of polyuronides occurred predominantly in the later ripening stages, and after the appearance of PG mRNAs, suggesting the existence of PG-dependent pectin degradation in later stages. Depolymerization of hemicelluloses was observed throughout ripening, and degradation of a tightly bound xyloglucan fraction was detected at the early onset of softening. Thus, metabolism of xyloglucan that may be closely associated with cellulose microfibrils may contribute to the initial stages of fruit softening. A model is presented of the temporal sequence of cell wall changes during cell wall disassembly in ripening Charentais melon.

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- 13 C]-glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), Na 2 CO 3 , 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 ( 12 C plus 13 C) sugars, and also in the newly synthesized ([ 13 C]-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.

California rice straw is being evaluated as a feedstock for production of power and fuel. This paper examines the initial steps in the process: pretreatment of rice straw and enzymatic hydrolysis of the polysaccharides in the pretreated material to soluble sugars. Rice straw was subjected to three distinct pretreatment procedures: acid-catalyzed steam explosion (Swan Biomass Company), acid hydrolysis (U.S. DOE National Renewable Energy Laboratory), and ammonia fiber explosion or AFEX (Texas A & M University). Standard conditions for each pretreatment were used, but none was optimized for rice straw specifically. Six commercial cellulases, products of Genencor International (USA), Novo (Denmark), Iogen (Canada) and Fermtech (Russia) were used for hydrolysis. The Swan- and the acid-pretreatments effectively removed hemicellulose from rice straw, providing high yields of fermentable sugars. The AFEX-pretreatment was distinctly different from other pretreatments in that it did not significantly solubilize hemicellulose. All three pretreatment procedures substantially increased enzymatic digestibility of rice straw. Three commercial Trichoderma-reesei-derived enzyme preparations: Cellulase 100L (Iogen), Spezyme CP (Genencor), and A1 (Fermtech), were more active on pretreated rice straw compared than others tested. Conditions for hydrolysis of rice straw using Cellulase 100L were evaluated. The supplementation of this enzyme preparation with cellobiase (Novozyme 188) significantly improved the parameters of hydrolysis for the Swan- and the acid-pretreated materials, but did not affect the hydrolysis of the AFEX-pretreated rice straw. The three pretreatment techniques were compared on a basis of a total yield and distribution of fermentable carbohydrates released by enzymatic hydrolysis (the highest possible substrate concentrations were used, 150 g/l for the Swan- and the acid- and 100 g/l for the AFEX-pretreated straw; enzyme loading of 6.7 filter paper units (FPU) and 6.7 cellobiase units (CBU) per gram of dry straw was the same for all pretreated materials). A combined yield of monosaccharides produced by a pretreatment step and by enzymatic hydrolysis was found to be 46, 42 and 37 g/l for the Swan-, the acid- and the AFEX-pretreated rice straw, respectively.

Fourteen species of crop plants which differ in their reported tissue boron requirements were grown in B-replete or B-deflcient medium. Leaf samples were collected and analysed for B and cell wall components. There was a significant positive correlation among the species between B concentration in the leaf or the cell wall and uronic acid, rhamnose and galactose (indicative of pectin) in the cell wall. The concentration of cell wall pectin was also positively related with reported tissue-B requirements and observed sensitivity to B deficiency. Boron deficiency did not alter the amount of uronic acid present in cell walls, suggesting that there was no effect of B deficiency on pectin metabolism. Under B-deflcient conditions the amount of 'soluble' B (i.e. B not associated with the cell wall) declined dramatically while the proportion of cellular B that was 'insoluble' (i.e. B associated with the cell wall) increased. The positive relationship between pectin content, insoluble B and tissue-B requirement of diverse species suggests that the amount of cell wall pectin may be significant in determining the relative tissue-B requirements of the species. These results indicate that either (1) species with high cell wall pectin contents require greater amounts of B for the construction of the cell wall, or (2) pectin in cell walls forms an insoluble complex with B, thereby reducing its availability for other putative B-requiring metabolic functions. Thus, species with a high pectin content would have a higher tissue-B requirement.

The ripening of discs cut from the pericarp of green tomato (Lycopersicon esculentum Mill.) fruits is inhibited by treatments with GA3 and several divalent cations, including calcium. Normal ripening is marked by an increase in the solubility of wall pectins. Calcium and GA3 alter the pattern of pectin solubility changes. In part this may be because polygalacturonase synthesis and/or secretion to the apoplast is reduced. The impact of divalent cations on ripening-related tissue softening appears to have a nonmetabolic component. Ripening-inhibiting ions rapidly reduce tissue softening, pectin solubilization and the normal ripening-related decrease in cellular turgor.

We have used a tomato (Lycopersicon esculentum Mill.) pericarp disc system to study the inhibition of ripening caused by diazocyclopentadiene (DACP), gibberellic acid (GA3) and calcium (CaCl2), and to compare their modes of action. Treatment of discs with 1-aminocyclopropane carboxylic acid (ACC) accelerated their ripening while DACP treatment eliminated ripening in the absence or presence of a treatment with the ethylene precursor. Both GA3 and calcium retarded color development and tissue softening. Calcium delayed the onset of the climacteric rise in respiration and ethylene evolution, but GA3 had no consistent effect in this respect. The different modes of action of the two inhibitors were emphasized by the synergistic effect on inhibition of ripening obtained when both were simultaneously applied. Inhibition of color development and softening was also achieved by applying other divalent cations such as strontium and barium. However, no such effect was observed with manganese or with the monovalent cations potassium and sodium. We concluded that calcium inhibition of ripening is not specific and is not necessarily mediated by the calcium calmodulin complex.

The firmness of carrot root disks was measured after various periods of heating in boiling water. Tissues lost more than half of their initial firmness within a few minutes. A second, much slower, phase of firmness loss continued for 10-15 min. The early phase of firmness loss could be mimicked by plasmolyzing carrot tissues in mannitol solution, and plasmolyzed disks showed only the second, slower, phase of firmness loss during cooking. Measurements of cellular turgor (turgor pressure probe) and membrane integrity (vital dye) indicate that turgor and membrane function are quickly lost during heating. When disks were incubated in a range of mannitol solutions, tissue firmness and cell turgor were linearly related

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.

The influence of polygalacturonase inhibitor protein (PGIP) from mature green Bartlett pears on the action of a mixture of cell wall hydrolases from Botrytis cinerea on isolated pear cell walls, and on a soluble extract of these walls, was studied. The aim of this work was to test the hypothesis of Cervone et al. that PGIP may modify cell wall degradation to result in the production of an increased proportion of pectic oligomers in the particular size class most effective for elicitation of defence responses. Products of the partially inhibited and uninhibited enzyme system from B. cinerea culture supernatant were compared as elicitors of ethylene production by pear fruit cells in suspension culture (which would indicate the presence of oligogalacturonides in the size class known to elicit plant defence responses), and on the basis of direct analytical data relating to oligomer length. Pear PGIP caused partial inhibition of the crude mixture of Botrytis enzymes, and increased the ratio of dimeric to monomeric uronide products. However, no accumulation of larger oligomeric breakdown intermediates was detected, and no impact on ethylene elicitor activity of the digestion products was observed. Differential inhibition of the B. cinerea polygalacturonase isozymes by pear PGIP was demonstrated following their separation by isoelectric focusing. The fact that some isozymes remained relatively uninhibited may have accounted for the absence of any accumulation of oligomeric breakdown intermediates.

Nectarines (Prunus persica, cv Fiesta Red) were harvested and either ripened for five days at 20° or stored for eight weeks at 0° in air or in a controlled atmosphere (CA) of 10% CO2 and 15% O2 before ripening. Air-stored fruit developed the flesh disorder of mealiness, while CA-stored fruit did not. During storage, the degree of esterification of the cell wall decreased in air- but not CA-stored fruits. Air-stored fruits retained more pectin in the sodium carbonate fraction after ripening and the polymers in the trans-1,2-diaminocyclohexane-N,N,N′, N′-tetraacetic acid (CDTA) and water-soluble fractions were of smaller M, than unstored or CA-stored fruits. Air storage also led to loss of arabinose from the sodium carbonate and guanidine thiocyanate fractions, and a lower arabinose content was also found in the CDTA fraction when air-stored fruits were ripened. The size distribution of componentsof the pectic fractions from air-stored fruits differed from fruits after harvest or CA-storage. In comparison with air-stored fruit, the CA-stored fruit showed changes in cell wall components which were very similar to those in fruit ripened immediately after harvest.

We cloned and purified the major family 10 xylanase (Xyn10A) from Acidothermus cellulolyticus 11B. Xyn10A was active on oat spelt and birchwood xylans between 60°C and 100°C and between pH 4 and pH 8. The optimal activity was at 90°C and pH 6; specific activity and K m for oat spelt xylan were 350 μmol xylose produced min −1 mg of protein −1 and 0.53 mg ml −1 , respectively. Based on xylan cleavage patterns, Xyn10A is an endoxylanase, and its half-life at 90°C was approximately 1.5 h in the presence of xylan.

This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited., Industrial processes to produce ethanol from lignocellulosic materials are available, but improved efficiency is necessary to make them economically viable. One of the limitations for lignocellulosic conversion to ethanol is the inaccessibility of the cellulose and hemicelluloses within the tight cell wall matrix. Ferulates (FA) can cross-link different arabinoxylan molecules in the cell wall of grasses via diferulate and oligoferulate bridges. This complex cross-linking is thought to be a key factor in limiting the biodegradability of grass cell walls and, therefore, the reduction in FA is an attractive target to improve enzyme accessibility to cellulose and hemicelluloses. Unfortunately, our knowledge of the genes responsible for the incorporation of FA to the cell wall is limited. A bioinformatics prediction based on the gene similarities and higher transcript abundance in grasses relative to dicot species suggested that genes from the pfam family PF02458 may act as arabinoxylan feruloyl transferases. We show here that the FA content in the cell walls and the transcript levels of rice genes Os05g08640, Os06g39470, Os01g09010 and Os06g39390, are both higher in the stems than in the leaves. In addition, an RNA interference (RNAi) construct that simultaneously down-regulates transcript levels of these four genes is associated with a significant reduction in FA of the cell walls from the leaves of the transgenic plants relative to the control (19% reduction, P < 0. 0001). Therefore, our experimental results in rice support the bioinformatics prediction that members of family PF02458 are involved in the incorporation of FA into the cell wall in grasses. © 2009 The Author(s).

The effect of pectic oligomers and 1-aminocyclopropane carboxylic acid on ethylene biosynthesis and color change was studied in ripening tomato pericarp discs excised from mature-green tomato fruit (Lycopersicon esculentum Mill.). Pectic oligomers induced at least four distinct responses when added to pericarp discs: (a) a short-term, transient increase in ethylene biosynthesis; (b) a long-term, persistent increase in climacteric ethylene in discs excised from mature-green fruit; (c) an advance in ripening processes, as indicated by increased reddening of the disc surfaces; and (d) a darkening of the treated endocarp surface. Pectic oligomers appear to affect the ripening of exocarp and endocarp tissues by different mechanisms. In exocarp tissues, the acceleration of reddening by pectic oligomers might simply be a consequence of induced ethylene biosynthesis. In endocarp tissues, the acceleration of reddening appears to be a direct effect of oligomers on ripening processes. We suggest that the rate of ripening of endocarp tissues may be regulated, in part, by the release of pectic oligomers from the cell walls of adjacent exocarp tissues. Exocarp and endocarp tissues of pericarp discs appear to differ in their sensitivity to ethylene at each maturity stage, and to exhibit independent changes in sensitivity to ethylene as ripening progresses. The tissue-specific pattern of reddening in tomato pericarp may result from this differential sensitivity to endogenous ethylene concentrations.

Pectic oligomers induced a rapid, transient increase in ethylene biosynthesis when added to pear cells in suspension culture. The rate of ethylene biosynthesis increased within 30 to 40 minutes after oligomer addition, reached a maximum between 90 and 120 minutes after addition, and then decreased to basal rates of synthesis. Both the rapid increase and decrease in biosynthesis appear to be precisely regulated components of the ethylene response to oligomers. Induction of ethylene biosynthesis by pectic oligomers resulted in a reduced sensitivity of cells to further ethylene induction. This reduction in sensitivity occurred within 90 minutes after an oligomer treatment, slightly preceding the decline in ethylene synthesis. The degree of insensitivity induced was proportional to the concentration of oligomer in the first treatment. Induced insensitivity to elicitors appears to represent a novel mechanism which may limit continued ethylene biosynthesis after ethylene induction. Ethylene was produced by pear cells throughout the cell growth cycle, as cells increased in density over a 6 day period. Endogenous ethylene biosynthesis was at a maximum during the first 4 days of rapid cell growth, then declined to half the peak rate through day 10. Pectic oligomers could induce an increase in ethylene biosynthesis above this background rate only after day 5, as endogenous biosynthesis declined. Changes in sensitivity to added oligomer during the growth cycle may result from insensitivity to elicitors induced by growth processes.

Saline crops and autoclaved municipal organic solid wastes were evaluated for their potential to be used as feedstock for fermentable sugar production through dilute acid pretreatment and enzymatic hydrolysis. The saline crops included two woods, athel (Tamarix aphylla L) and eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose tall wheatgrass (Agropyron elongatum), and creeping wild rye (Leymus triticoides). Each of the biomass materials was first treated with dilute sulfuric acid under selected conditions (acid concentration =1.4% (w/w), temperature =165 degrees C, and time =8 min) and then treated with the enzymes (cellulases and beta-glucosidase). The chemical composition (cellulose, hemicellulose, and lignin contents) of each biomass material and the yield of total and different types of sugars after the acid and enzyme treatment were determined. The results showed that among the saline crops evaluated, the two grasses (creeping wild rye and Jose tall wheatgrass) had the highest glucose yield (87% of total cellulose hydrolyzed) and fastest reaction rate during the enzyme treatment. The autoclaved municipal organic solid wastes showed reasonable glucose yield (64%). Of the two wood species evaluated, Athel has higher glucose yield (60% conversion of cellulose) than eucalyptus (38% conversion of cellulose).

The aim of this study was to analyze the changes in cell wall pectins in normally ripening (juicy) and in chilling-injured plum fruit (Prunus salicina cv. Fortune) showing mealiness. Total cell wall neutral sugars and uronic acids, solubilization and depolymerization of pectins in water-, CDTA- and Na2CO3-soluble fractions of the cell wall (WSF, CSF and NSF, respectively), non-cellulosic neutral sugar compositions of these fractions, and the activities of the cell wall-degrading enzymes polygalacturonase (PG), pectin methylesterase (PME), 1,4-β- d -glucanase/glucosidase and β-galactosidase (β-gal) were determined. No differences in the total content of pectin and neutral sugars between normally ripening and chilling-injured fruit were detected. However, the mealy plums presented a higher level of tightly bound pectin (NSF) and a lower proportion of loosely bound pectin (WSF) than the juicy controls. Lower pectin depolymerization and reduced solubilization of neutral sugars in the WSF and CSF were also detected in the chilling-injured tissues, confirming an alteration in the normal ripening-associated pattern of polyuronide disassembly. While no differences were found in the activities of PG, PME and 1,4-β- d -glucanase/glucosidase between normally ripening and mealy fruit, the latter had reduced β-gal activity. This might have led to differential solubilization of polymers with galactan side chains, but further studies are required to determine if there is a causal relationship between these events. Overall, results indicated that the development of chilling injury symptoms in ‘Fortune’ plums is associated with abnormalities in cell wall metabolism, including a reduction in pectin solubilization and depolymerization and decreased ripening-associated modification of galactose-rich pectin polymers.

Boysenberry fruit was harvested at five developmental stages, from green to purple, and changes in pectin and hemicellulose solubilisation and depolymerisation, polymer neutral sugar contents, and the activities of cell wall degrading enzymes were analysed. The high xylose to glucose ratio in the 4% KOH-soluble hemicellulose fraction suggests that xylans are abundant in the boysenberry cell wall. Although the cell wall changes associated with fruit development do not proceed in discrete stages and the cell wall disassembly is a consequence of highly regulated changes occurring in a continuum, the results suggest that the temporal changes in cell wall degradation in boysenberry account for at least three stages: an early stage (green to 75% red colour), associated with metabolism of cellulose and cross-linking glycans; an intermediate period (75 to 100% red colour), characterised by substantial pectin solubilisation without depolymerisation in which α-arabinofuranosidase increases markedly and 50% of the wall arabinose is lost; and a final stage (100% red colour to purple), characterised mainly by a reduction of pectic galactose content and a dramatic increase in pectin depolymerisation associated with higher polygalacturonase, pectin methylesterase, acetyl esterase and β-galactosidase activities. From a biotechnological perspective enzymes involved in pectin matrix disassembly seem to be the better candidates to affect boysenberry fruit late-softening by genetic intervention. A model for cell wall disassembly in boysenberry fruit is proposed.

Alfalfa and cotton flowers were pierced with small glass capillaries of an overall size and shape similar to that of Lygus stylets, and injected with small quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes. Crude and partially purified protein solutions from Lygus heads and isolated salivary glands showed substantial polygalacturonase (PG) activity, as has been previously reported. Following injection with both crude and partially purified protein solutions, as well as with pure fungal and bacterial PGs, flowers of both alfalfa and cotton exhibited damage similar to that caused by Lygus feeding. Injection with the same volume of a buffer control as well as a buffer control containing BSA at a comparable protein concentration (approximately 6 µg/mL) showed no symptoms. These results are consistent with a previously suggested hypothesis that the extensive tissue damage caused by Lygus feeding is primarily due to the action of the PG enzyme on the host tissue, rather than to mechanical damage caused by the insect stylet. Substantial genotypic variation for a PG inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest that breeding and selection for increased native PGIP levels, or transformation with genes encoding PGIP from other plant species, may be of value in obtaining alfalfa and cotton varieties that are more resistant to Lygus feeding damage. Arch. Insect Biochem. Physiol. 58:69–83, 2005. © 2005 Wiley-Liss, Inc.

Partially tree-ripened ripe fruit of peach (Prunus persica L.) were stored for 1-4 weeks at 5 degrees C and then ripened at 20 degrees C for 3 d to induce chilling injury. With increasing cold storage the incidence and severity of mealiness symptoms increased progressively, manifested as reduced quantities of free juice and internal flesh browning. Relative to juicy fruit, tissue of mealy fruit showed altered intercellular adhesion when examined by microscopy and, upon crushing, a higher proportion of cells remained intact and did not release cellular contents. Substantial alterations in the metabolism of cell wall polysaccharides were observed. Chelator-soluble polyuronides from mealy fruit were partially depolymerized during cold storage in a manner dissimilar to that in unripe or ripe juicy fruit, and were not depolymerized further during the ripening period. The solubility of these high molecular weight pectins remained low, and did not show the increase characteristic of juicy fruit. Furthermore, in mealy fruit the dramatic decline in the polymeric Ara content of base-soluble, matrix glycan-enriched fractions occurring during normal ripening was absent, indicating diminished disassembly of an arabinan-rich polysaccharide firmly attached to cellulose. A corresponding rise in the polymeric Ara content of the most soluble pectin fraction was also absent, as was a decline in the Gal content of this extract. The depolymerization of matrix glycans showed only minor differences between juicy and mealy fruit. After cold storage and ripening, the activities of endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), beta-galactosidase (EC 3.2.1.23), alpha-arabinosidase (EC 3.2.1.55), and particularly endo-polygalacturonase (EC 3.2.1.15) were lower in mealy fruit than in juicy fruit, whereas pectin methylesterase activity (EC 3.1.1.11) was lower in slightly mealy and higher in very mealy fruit. The data suggest that cold storage affects the activities of numerous cell wall-modifying enzymes, with important consequences for pectin metabolism. These changes alter the properties of the primary wall and middle lamella, resulting in tissue breakage along enlarged air spaces, rather than across cells, which reduces the amount and availability of free juice upon tissue fragmentation.

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to

Recent work has shown that pectic oligosaccharides extracted from tomatoes are capable of promoting ripening in mature green tomato fruit explants (11). A water soluble, ethano-insoluble extract of autolytically-inactivated tomato pericarp tissue will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs from mature green fruit. Treatment of Na2CO3− soluble but not chelator-soluble pectin from tomato with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit. The endogeneous concentration of these oligomers is low, but in excess of that necessary to promote ethylene biosynthesis in this system (29). A major difficulty in assigning a regulatory role to such pectic oligomers is that at the onset of ripening in tomato fruit little evidence of polygalacturonase activity exists. However our work demonstrating the presence of neutral sugar-rich acidic oligomers suggests that other enzymatic activities capable of degrading pectic substances exist in developing tomatoes. The recent report (22) of a rhamnogalacturonase-type activity in developing fruit supports this supposition. The possibility of a role for pectic oligomers in fruit ripening regulation will be discussed.

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.

A polygalacturonase inhibitor glycoprotein with an apparent molecular mass of 43 kD was purified from pear (Pyrus communis L. cv Bartlett) fruit. Chemical deglycosylation of this protein decreased the molecular mass to 34 kD. Gas chromatographic analysis suggests that N-linked glycosylation accounts for the majority of sugar moieties. Partial amino acid sequence analysis of the purified polygalacturonase inhibitor protein provided information used to amplify a corresponding cDNA by polymerase chain reactions. Multiple cloned products of these reactions were sequenced and the same open reading frame was identified in all of the products. It encodes a 36.5-kD polypeptide containing the amino acid sequences determined by protein sequencing and predicts a putative signal sequence of 24 amino acids and seven potential N-glycosylation sites. The expression of polygalacturonase inhibitor is regulated in a tissue-specific manner. Activity and mRNA level were much higher in fruit than in flowers or leaves.

Acid hydrolysis-generated pectic oligomers have been shown to affect ripening of tomato fruit by inducing both acceleration of reddening and increased ethylene biosynthesis (Campbell & Labavitch, 1991 Plant Physiol 97:706-713). In the present work, homogeneous size classes of these oligomers were demonstrated to have different impacts on ethylene production of tomato fruit pericarp discs. Endogenous oligomeric material of the same size classes was isolated from ripening tomato tissues and also tested for biological activity. They promoted some aspects of ripening as shown by increased ACC and ethylene production, which suggests that pectic oligomers are potential regulators of the ripening process in tomatoes. A metabolic origin for these oligomers is suggested by the fact that they are produced by in vitro polygalacturonase I treatment of polygalacturonic acid or tomato pectin.

Higher plant inhibitors of fungal polygalacturonases are potential contributors to plant defense. To test this hypothesis we have raised antibodies against the `Bartlett' pear fruit polygalacturonase inhibitor (PGIP) and cloned a pear fruit PGIP cDNA. The pear PGIP cDNA was isolated by polymerase chain reactions based on our amino acid and nucleotide sequence information. Sequence analysis predicts a gene product of 34.5 kD with an isoelectric point of 6.02 in agreement with our biochemical data. Seven potential glycosylation sites are consistent with the glycoprotein character of these PGIPs. Southern blot analysis suggests the presence of 1 or 2 genes in the pear genome. Northern blot analysis indicates the presence of a transcript of 1.5 kb. Western blot analysis shows cross-reactivity of the anti-pear PGIP antibody to various dicot species as well as corn.

Commercially grown Granny Smith apples were stored at 0°C in air or 1% O2, and 2 sets of samples were taken every 4 weeks over a 28 week period. One set was immediately analysed for weight loss, firmness, color, soluble solids, pH and titratable acidity. Alcohol-insoluble substances were analysed for starch, water-soluble uronides, water-insoluble uronides, cellulose and neutral sugars. The second set of samples was kept in air at 20°C for an additional week, during which respiration and ethylene production rates were measured, prior to the above analyses. Storage in 1% O2 led to the improved maintenance of firmness, reduced respiration and ethylene production rates in ambient air, and a reduced content of water-soluble uronides, suggesting a reduced degree of hydrolysis. The correlation between firmness and water-soluble uronide content was not very strong. The predominant neutral sugars present in the wall were arabinose and galactose, and activities of putative hydrolyses that may be involved in the metabolism of polymers containing these sugars will be discussed.