Cell Wall-associated Enzymes Research Articles

Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation.

Soft rot causes significant economic losses in the kiwifruit industry. This study isolated strain CTXW 7-6-2 from healthy kiwifruit tissue; this was a gram-positive bacterium that produced the red pigment pulcherrimin. The phylogenetic tree based on 16S ribosomal RNA, gyrA, rpoB, and purH gene sequences identified CTXW 7-6-2 as a strain of Bacillus subtilis. CTXW 7-6-2 inhibited hyphal growth of pathogenic fungi that cause kiwifruit soft rot, namely, Botryosphaeria dothidea, Phomopsis sp., and Alternaria alternata, by 81.76, 69.80, and 32.03%, respectively. CTXW 7-6-2 caused the hyphal surface to become swollen and deformed. Volatile compounds (VOC) produced by the strain inhibited the growth of A. alternata and Phomopsis sp. by 65.74 and 54.78%, respectively. Whole-genome sequencing revealed that CTXW 7-6-2 possessed a single circular chromosome of 4,221,676 bp that contained 4,428 protein-coding genes, with a guanine and cytosine (GC) content of 43.41%. Gene functions were annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, Carbohydrate-Active enZYmes, and Rapid Annotations using Subsystem Technology databases, revealing non-ribosomal pathways associated with antifungal mechanisms, biofilm formation, chemotactic motility, VOC 3-hydroxy-2-butanone, cell wall-associated enzymes, and synthesis of various secondary metabolites. antiSMASH analysis predicted that CTXW 7-6-2 can produce the active substances bacillaene, bacillibactin, subtilosin A, bacilysin, and luminmide and has four gene clusters of unknown function. Quantitative real-time PCR (qRT-PCR) analysis verified that yvmC and cypX, key genes involved in the production of pulcherrimin, were highly expressed in CTXW 7-6-2. This study elucidates the mechanism by which B. subtilis strain CTXW 7-6-2 inhibits pathogenic fungi that cause kiwifruit soft rot, suggesting the benefit of further studying its antifungal active substances.

Fruit From Two Kiwifruit Genotypes With Contrasting Softening Rates Show Differences in the Xyloglucan and Pectin Domains of the Cell Wall.

Fruit softening is controlled by hormonal and developmental cues, causing an upregulation of cell wall-associated enzymes that break down the complex sugar matrices in the cell wall. The regulation of this process is complex, with different genotypes demonstrating quite different softening patterns, even when they are closely related. Currently, little is known about the relationship between cell wall structure and the rate of fruit softening. To address this question, the softening of two Actinidia chinensis var. chinensis (kiwifruit) genotypes (a fast ‘AC-F’ and a slow ‘AC-S’ softening genotype) was examined using a range of compositional, biochemical, structural, and molecular techniques. Throughout softening, the cell wall structure of the two genotypes was fundamentally different at identical firmness stages. In the hemicellulose domain, xyloglucanase enzyme activity was higher in ‘AC-F’ at the firm unripe stage, a finding supported by differential expression of xyloglucan transglycosylase/hydrolase genes during softening. In the pectin domain, differences in pectin solubilization and location of methyl-esterified homogalacturonan in the cell wall between ‘AC-S’ and ‘AC-F’ were shown. Side chain analyses and molecular weight elution profiles of polyuronides and xyloglucans of cell wall extracts revealed fundamental differences between the genotypes, pointing towards a weakening of the structural integrity of cell walls in the fast softening ‘AC-F’ genotype even at the firm, unripe stage. As a consequence, the polysaccharides in the cell walls of ‘AC-F’ may be easier to access and hence more susceptible to enzymatic degradation than in ‘AC-S’, resulting in faster softening. Together these results suggest that the different rates of softening between ‘AC-F’ and ‘AC-S’ are not due to changes in enzyme activities alone, but that fundamental differences in the cell wall structure are likely to influence the rates of softening through differential modification and accessibility of specific cell wall polysaccharides during ripening.

Bacteria from the endosphere and rhizosphere of Quercus spp. use mainly cell wall-associated enzymes to decompose organic matter.

Due to the ability of soil bacteria to solubilize minerals, fix N2 and mobilize nutrients entrapped in the organic matter, their role in nutrient turnover and plant fitness is of high relevance in forest ecosystems. Although several authors have already studied the organic matter decomposing enzymes produced by soil and plant root-interacting bacteria, most of the works did not account for the activity of cell wall-attached enzymes. Therefore, the enzyme deployment strategy of three bacterial collections (genera Luteibacter, Pseudomonas and Arthrobacter) associated with Quercus spp. roots was investigated by exploring both cell-bound and freely-released hydrolytic enzymes. We also studied the potential of these bacterial collections to produce enzymes involved in the transformation of plant and fungal biomass. Remarkably, the cell-associated enzymes accounted for the vast majority of the total activity detected among Luteibacter strains, suggesting that they could have developed a strategy to maintain the decomposition products in their vicinity, and therefore to reduce the diffusional losses of the products. The spectrum of the enzymes synthesized and the titres of activity were diverse among the three bacterial genera. While cellulolytic and hemicellulolytic enzymes were rather common among Luteibacter and Pseudomonas strains and less detected in Arthrobacter collection, the activity of lipase was widespread among all the tested strains. Our results indicate that a large fraction of the extracellular enzymatic activity is due to cell wall-attached enzymes for some bacteria, and that Quercus spp. root bacteria could contribute at different levels to carbon (C), phosphorus (P) and nitrogen (N) cycles.

XTHs from Fragaria vesca: genomic structure and transcriptomic analysis in ripening fruit and other tissues

BackgroundFragaria vesca or ‘woodland strawberry’ has emerged as an attractive model for the study of ripening of non-climacteric fruit. It has several advantages, such as its small genome and its diploidy. The recent availability of the complete sequence of its genome opens the possibility for further analysis and its use as a reference species. Fruit softening is a physiological event and involves many biochemical changes that take place at the final stages of fruit development; among them, the remodeling of cell walls by the action of a set of enzymes. Xyloglucan endotransglycosylase/hydrolase (XTH) is a cell wall-associated enzyme, which is encoded by a multigene family. Its action modifies the structure of xyloglucans, a diverse group of polysaccharides that crosslink with cellulose microfibrills, affecting therefore the functional structure of the cell wall. The aim of this work is to identify the XTH-encoding genes present in F. vesca and to determine its transcription level in ripening fruit.ResultsThe search resulted in identification of 26 XTH-encoding genes named as FvXTHs. Genetic structure and phylogenetic analyses were performed allowing the classification of FvXTH genes into three phylogenetic groups: 17 in group I/II, 2 in group IIIA and 4 in group IIIB. Two sequences were included into the ancestral group. Through a comparative analysis, characteristic structural protein domains were found in FvXTH protein sequences. In complement, expression analyses of FvXTHs by qPCR were performed in fruit at different developmental and ripening stages, as well as, in other tissues. The results showed a diverse expression pattern of FvXTHs in several tissues, although most of them are highly expressed in roots. Their expression patterns are not related to their respective phylogenetic groups. In addition, most FvXTHs are expressed in ripe fruit, and interestingly, some of them (FvXTH 18 and 20, belonging to phylogenic group I/II, and FvXTH 25 and 26 to group IIIB) display an increasing expression pattern as the fruit ripens.ConclusionA discrete group of FvXTHs (18, 20, 25 and 26) increases their expression during softening of F. vesca fruit, and could take part in cell wall remodeling required for softening in collaboration with other cell wall degrading enzymes.

Localization of functional β-xylosidases, encoded by the same single gene, xlsIV (xlnD), from Aspergillus niger E-1.

Cell wall-associated β-xylosidase was isolated from Aspergillus niger E-1 and identified as XlsIV, corresponding to the extracellular enzyme XlnD reported previously. xlsIV was transcribed only in the early cultivation period. Cell wall-associated enzyme activity gradually decreased, but extracellular activity increased as the strain grew. These results indicate that XlsIV (XlnD) was secreted into culture after localizing at cell wall.

Identification of appressorial and mycelial cell wall proteins and a survey of the membrane proteome of Phytophthora infestans

Proteins embedded in the cell wall and plasma membrane of filamentous oomycetes and fungi provide a means by which these organisms can interact with their local environment. However, cell wall and membrane proteins have often proved difficult to isolate using conventional proteomic techniques. Here we have used liquid chromatography tandem mass spectrometry (LC-MS/MS) to facilitate rapid and sensitive quantification of the cell wall proteome. We report the use of LC-MS/MS to identify differentially regulated proteins from the cell walls of three different lifecycle stages of the oomycete plant pathogen Phytophthora infestans: non-sporulating vegetative mycelium, sporulating mycelium, and germinating cysts with appressoria. We have also used quantitative real-time RT-PCR to confirm that the transcripts corresponding to some of these proteins, namely those identified in cell walls of germinating cysts with appressoria, accumulate differentially throughout the lifecycle. These proteins may, therefore, be important for pre-infective development and early pathogenicity. Up to 31 covalently and non-covalently bound cell wall-associated proteins were identified. All of the proteins identified in germinating cysts with appressoria, and several of those from mycelial fractions, were classified as putative effector or pathogen-associated molecular pattern (PAMP) molecules, including members of the CBEL family, the elicitin family, the crinkler (CRN) family and two transglutaminases. Thus, the cell wall of P. infestans may represent an important reservoir for surface-presented, apoplastic effectors or defence activation molecules. Proteins predicted to be cell surface proteins included IPI-B like proteins, mucins, cell wall-associated enzymes and annexin family members. Additionally we identified up to 27 membrane-associated proteins from Triton X-114 phase partitioned mycelial membrane preparations, producing the first inventory of oomycete membrane-associated proteins. Four of these proteins are small Rab-type G-proteins and several are associated with secretion.

Characterization of PCEng2, a β-1,3-Endoglucanase Homolog in Pneumocystis carinii with Activity in Cell Wall Regulation

Pneumocystis jirovecii pneumonia is an opportunistic fungal infection that causes severe respiratory impairment in immunocompromised patients. The viability of Pneumocystis organisms is dependent on the cyst cell wall, a structural feature that is regulated by essential cell wall-associated enzymes. The formation of the glucan-rich cystic wall has been previously characterized, but glucan degradation in the organism-specifically, degradation during trophic excystment-is not yet fully understood. Most studies of basic Pneumocystis biology have been conducted in Pneumocystis carinii or Pneumocystis murina, the varieties of this genus that infect rats and mice, respectively. Furthermore, all known treatments for P. jirovecii were initially discovered through studies of P. carinii. Accordingly, in this study, we have identified a P. carinii beta-1,3-endoglucanase gene (PCEng2) that is demonstrated to play a significant role in cell wall regulation. The cDNA sequence contained a 2.2-kb open reading frame with conserved amino acid domains homologous to similar fungal glycosyl hydrolases (GH family 81). The gene transcript showed up-regulation in cystic isolates, and the expressed protein was detected within both cyst and trophic forms. Complementation assays in Eng2-deleted Saccharomyces cerevisiae strains showed restoration of the cell wall separation defect during proliferation, demonstrating the importance of PCEng2 protein. during fungal growth. These findings suggest that regulation of cyst cell wall beta-glucans is a fundamental process during completion of the Pneumocystis life cycle.

Analysis of xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and apple

Xyloglucan endotransglucosylase/hydrolase (XTH) enzymes are thought to play a key role in fruit ripening by loosening the cell wall in preparation for further modification by other cell wall-associated enzymes and through disassembly of xyloglucan. Twenty-five XTH genes were isolated and characterised from kiwifruit and apple databases containing >270,000 expressed sequence tags (ESTs). The XTH genes (14 from kiwifruit, 11 from apple) encoded putative proteins with similar molecular weights, but with isoelectric points ranging from acidic to basic. All 25 XTH amino acid sequences contained the two conserved glutamic acid residues at the active site of the XTH enzyme. Phylogenetic analysis produced trees with branches each containing kiwifruit, apple, tomato and Arabidopsis XTH sequences, suggesting the potential for functional orthology. EST expression profiling and quantitative PCR analysis were used to identify kiwifruit and apple XTH transcripts expressed in root tip, flower, young leaf and ripe fruit. In ripe apple fruit, the transcripts for two XTH genes ( Md-XTH2 and Md-XTH10) were the most abundant, whilst in ripe kiwifruit three transcripts predominated ( Ad-XTH4, 5 and 7). Ad-XTH7 was highly expressed in the outer pericarp of firm kiwifruit at commercial harvest and expression of this mRNA decreased during the rapid softening phase. Three kiwifruit XTH genes ( Ad-XTH5, Ad-XTH7 and Ac-XTH14) were expressed in Escherichia coli and the recombinant proteins were shown to have xyloglucan endotransglucosylase activity. This research lays the groundwork for understanding the role of XTHs during fruit ripening and storage in kiwifruit and apple.

1-Methylcyclopropene Application and Modified Atmosphere Packaging Affect Ethylene Biosynthesis, Fruit Softening, and Quality of ‘Tegan Blue’ Japanese Plum During Cold Storage

This research was carried out to extend the postharvest storage of japanese plum (Prunus salicina Lindl. cv. Tegan Blue), which has a short shelf life limiting its export potential. The effects of 1.0 μL·L−1 1-methylcyclopropene (1-MCP) and modified atmosphere packaging (MAP), alone or in combination, on quality of mature japanese plum fruit during storage (0 ± 1 °C and 90% ± 5% relative humidity) were investigated. The activities of enzymes of ethylene biosynthesis [1-aminocyclopropane-1-carboxylic acid synthase (ACS), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), and 1-aminocyclopropane-1-carboxylic acid (ACC) content] and those of cell wall-associated enzymes [exo-polygalacturonase (exo-PG), endo-polygalacturonase (endo-PG), pectin esterase (PE), and endo-1,4-β-D-glucanase (EGase)] were also measured. 1-MCP-treated fruit stored in normal atmosphere or in MAP had lower ACC content and inhibited ethylene production with reduced ACS and ACO activities compared with fruit stored in MAP and in normal atmosphere. Similarly, 1-MCP-treated fruit, stored either in normal atmosphere or in MAP, were firmer with reduced exo-PG, endo-PG, PE, and EGase activities compared with fruit stored in MAP and in normal atmosphere. During storage as well as during ripening, fruit stored in MAP exhibited a higher rate of respiration compared with other treatments. MAP exacerbated the effect of 1-MCP in reduction of ethylene production and fruit softening. 1-MCP application in combination with MAP after 5 and 7 weeks of storage delayed the fruit ripening by 10 and 8 days in contrast with control fruit, respectively. During storage, and as well as in ripe fruit, weight loss was reduced in fruit stored in MAP either with or without 1-MCP application. Control fruit and 1-MCP-treated fruit, stored in a normal atmosphere or in MAP, had the same values for the following parameters: chromaticity value L*, C*, and hue angle, titratable acidity, and concentrations of soluble solids, ascorbic acid, and total antioxidants. In conclusion, 1-MCP application in combination with MAP can be used effectively to reduce the ethylene biosynthesis and fruit softening during cold storage and to extend the storage life up to 7 weeks followed by 8 d of ripening without any adverse effects on the quality of ripe fruit.

Phytase activity inCryptococcus laurentiiABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.

GENE EXPRESSION AND ACTIVITIES OF CELL WALL-ASSOCIATED ENZYMES IN COLD-STORED TOMATO FRUIT

Tomato fruit (Solanum lycopersicum L.) can develop mealiness and enhanced softening when exposed to chilling temperatures during storage, but the involvement of cell wall-associated enzymes in chilling injury development is not well understood. To study this aspect of injury development, we have exposed breaker-stage `Trust' tomato fruit to a chilling temperature of 3 °C for 0, 7, 14, and 21 days followed by storage at 20 °C for 12 days. Ethylene production was not affected by storage except after 21 days where production was greater at 20 °C. Exposure of fruit to chilling temperatures delayed the ripening-related color change (chroma and hue) and initially increased compression values, but percent extractable juice was not affected consistently. Increased polygalacturonase (PG) activity during ripening was reduced by about 50% after 7 days at 3 °C, and further inhibited with increasing storage periods. In contrast, the activities of pectin methylesterase (PME) and α-galactosidase were not significantly affected by the cold treatments. β-Galactosidase activity was greater in all chilled fruit compared with fruit ripened at harvest, whereas endo-β-1,4-glucanase activity was lower after 21 days at 3 °C. In chilled fruits, transcript accumulations for PG, PME (PME1.9), and expansin (Expt.1) were lower during storage at 20 °C compared with those of nonchilled fruits. Transcript accumulation for β-galactosidase (TBG4) was affected only at 14 days of cold storage, when transcript accumulation decreased. Cold treatment increased transcript accumulation of endo-β-1,4-glucanase (Cel1) after 12 days at 20 °C and decreased transcript accumulation after 7 days and 21 days at 21 °C. Cell wall analyses to investigate relationships among enzyme activities and cell wall disassembly are ongoing.

The Role and Mechanism of Diacylglycerol-Protein Kinase C1 Signaling in Melanogenesis by Cryptococcus neoformans

The fungus Cryptococcus neoformans is an opportunistic human pathogen that causes a life-threatening meningoencephalitis by expression of virulence factors such as melanin, a black pigment produced by the cell wall-associated enzyme laccase. In previous studies (Heung, L. J., Luberto, C., Plowden, A., Hannun, Y. A., and Del Poeta, M. (2004) J. Biol. Chem. 279, 21144-21153) we proposed that the sphingolipid enzyme inositol-phosphoryl ceramide synthase 1 (Ipc1) regulates melanin production through the generation of diacylglycerol (DAG), which was found to activate in vitro protein kinase C1 (Pkc1). Here, we investigated the molecular mechanisms by which DAG regulates Pkc1 in vivo and the effect of this regulation on laccase activity and melanin synthesis. To this end we deleted the putative DAG binding C1 domain of C. neoformans Pkc1 and found that the C1 deletion abolished the activation of Pkc1 by DAG. Deletion of the C1 domain repressed laccase activity and, consequently, melanin production. Finally, we show that these biological effects observed in the C1 deletion mutant are mediated by alteration of cell wall integrity and displacement of laccase from the cell wall. These studies define novel molecular mechanisms addressing Pkc1-laccase regulation by the sphingolipid pathway of C. neoformans, with important implications for understanding and targeting the Ipc1-Pkc1-laccase cascade as a regulator of virulence of this important human pathogen.

Activity of Cell Wall-associated Enzymes in Cold-stored Tomato Fruit

Tomato fruit (Solanum lycopersicon L.) can develop mealiness and enhanced softening when exposed to chilling temperatures during storage, but the involvement of cell wall-associated enzymes in chilling injury development is not well understood. To study this aspect of injury development, we have exposed breaker stage tomato cv. Trust fruit to a chilling temperature of 3 °C for 0, 7, 14, and 21 days followed by storage at 20 °C for 12 days. Ethylene production was not affected by storage except after 21 days, where production was greater at 20 °C. Exposure of fruit to chilling temperatures delayed the ripening-related color change (chroma and hue) and initially increased compression values, but percentage of extractable juice was not affected consistently. Increased polygalacturonase activity during ripening was reduced by about 50% after 7 days at 3 °C, and further inhibited with increasing storage periods. In contrast, the activities of pectin methylesterase and α-galactosidase were not significantly affected by the cold treatments. β-Galactosidase activity was greater in all chilled fruit compared with fruit ripened at harvest, whereas endo-β-1,4-glucanase activity was lower after 21 days at 3 °C. These results will be compared with equivalent changes in the activities of cell wall enzymes that are associated with wooliness development in chilling-injured peach fruit.

Localization of Glucose Oxidase with Immunocytochemistry in the Biocontrol Fungus Talaromyces flavus

ABSTRACT Production of the enzyme glucose oxidase has been shown to be involved in the biological control of Verticillium wilt by Talaromyces flavus. In this study, the location of glucose oxidase was determined in T. flavus by immunocytochemistry using glucose oxidase-specific polyclonal antibody. Immunostaining revealed that glucose oxidase was both intracellular and extracellular. Old, as well as young, hyphal cells contained glucose oxidase, but labeling of the cell wall-associated enzyme decreased as the cells aged. Exocytosis rather than cell lysis was the primary means of release of glucose oxidase from cells. Enzyme stability studies confirmed that the glucose oxidase of T. flavus is an extremely stable enzyme, retaining 13% of its original activity after 2 weeks at 25 degrees C and retaining activity for several days at temperatures up to 50 degrees C.

Isolation and characterization of a gene encoding endo-beta-1,4-glucanase from pepper (Capsicum annuum L.).

The endo-beta-1,4-glucanases, or cellulases, of higher plants are cell wall-associated enzymes believed to function in cell wall changes associated with the diverse processes of fruit ripening, organ abscission and cell elongation. We have isolated and characterized cDNA and genomic clones encoding a cellulase, PCEL1, which is abundant in ripening pepper fruit. Genomic analysis indicates that PCEL1 is encoded by a single gene, PCEL1, which belongs to a small, structurally divergent gene family. In ripening fruit, PCEL1 transcription is initiated at two distinct sites which yields overlapping mRNA species of 1.7 and 2.1 kb. High-level accumulation of both transcripts occurs in red fruit, while the 1.7 kb transcript is detected at a much lower level in stem and petiolar tissue. The increase in cellulase activity which is measured during fruit ripening is the product of PCEL1 expression and is tightly coupled to fruit reddening. High-level applications of ethylene serve to enhance the rate of ripening and the accumulation of PCEL1 mRNA. A direct role for ethylene in regulating PCEL1 expression is shown by the exclusive induction, in immature green fruit, of the 1.7 kb transcript in response to prolonged high-level exposure to ethylene--a pattern of expression not observed in fruit development on the vine.

Pollen allergens: development and function

Pollen allergens interact with the human immune system and the resulting IgE antibodies provide specific probes for their identification and characterisation. In one case, grass allergenic proteins are expressed late in pollen development coincident with the laying down of reserves. Sequence similarity of allergens has indicated possible functions for some allergens. The major birch pollen allergen shows sequence similarity with pathogenesis-related proteins, which form a secondary response in plant host-pathogen interactions and show anti-microbial activity. Some allergens of unknown function are cysteine-rich proteins, while some others have cysteine-rich regions; for example, the major allergen from rye-grass pollen, Lol p 1, has a cysteine-rich N-terminal region, while at the C-terminal region four tryptophan residues together with tyrosine and phenylalanine residues resemble those of cellulose- or sugar-binding domains of other proteins. Several pollen allergens show sequence similarity to cell wall-associated enzymes, while others show hydrolytic enzyme activity often associated with cell walls.

Activity of cell wall-associated enzymes in ripening olive fruit

Activity of cell wall-associated enzymes in ripening olive fruit

Soluble and Cell Wall-Associated β-Galactosidases from Cold-Grown Winter Rape ( Brassica napus L., var. oleifera L.) Leaves

Summary Soluble and cell wall associated (solubilized from cell wall preparations by 0.5 M NaCl) β-galactosidases were found in non-acclimated or cold-acclimated leaves of winter rape (Brassica napus L., var. L. [itoleifera} L.). Gel filtration of soluble proteins on a Sephadex G-200 column revealed one fraction with β-galactosidase activity with a molecular weight close to 65 kDa. The temperature optimum for soluble and cell wall-associated β-galactosidases was 50 °C and 55 °C, respectively. The cell wall associated form showed higher thermostability at 60 °C. Both fractions had a similar pH optimum of 4.0. The Km against p-nitrophenyl β-D-galactopyranoside was 0.25 mM and 50 mM and the Vmax values 2.80 and 1.42 μmol (mg protein)-1 min-1 for the cell wall-associated and soluble form, respectively. The 3-week growth of plants in the cold (2 °C) brought about a 22 % and 38 % decrease in the total and specific activities of the soluble form, respectively. The activity of the cell wall-associated form decreased by ca. 70 % during the treatment, independently of the calculation basis. The cold-induced decrease in the enzyme activities was associated with the increase of the Km value for the soluble form and with the decrease of Vmax values for both soluble and wall-associated enzymes. The transient frost (-5 °C for 18 h followed by 6 h at 2 °C), applied in darkness, markedly stimulated the activity of the cell wall-associated enzyme in the cold-acclimated leaves. It resulted in decreased Km and increased Vmax values for both soluble and cell wall-associated forms. The possible meaning of cold- and frost-induced changes in the β-galactosidase activities for cold acclimation of winter rape leaves is discussed.

Xyloglucan Endotransglycosylase Activity Increases during Kiwifruit (Actinidia deliciosa) Ripening (Implications for Fruit Softening).

The activity of xyloglucan endotransglycosylase (XET) was as-sayed in three tissue zones of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var deliciosa cv Hayward) at harvest and at several softening stages following a postharvest ethylene treatment. At harvest, extractable XET activity per unit fresh weight in the inner pericarp (IP) and core tissue was 4.5 and 42 times higher, respectively, than in the outer pericarp (OP). Within 24 h of ethylene treatment there was an increase in the activity and specific activity of XET in all tissues that continued throughout softening. Activity increased most in the OP, where it showed a 12-fold rise 6 d after ethylene treatment compared with 4.5- and 2.5-fold increases in the IP and core tissues, respectively. Visible swelling of the cell wall in each tissue was observed 24 h after the first detectable rise in XET activity and was most pronounced in the OP, which showed the greatest percentage increase in XET activity. Xyloglucan, galactoglucomannan, and cell wall materials isolated and purified from kiwifruit OP were tested as donor substrates for kiwifruit XET. The enzyme showed activity against xyloglucan but was inactive against galactoglucomannan. XET was active against cell wall materials from unripe and ripe fruit, with swollen walls from the latter being the better substrate. The results indicate that XET may have a key role early in fruit ripening, loosening the cell wall in preparation for further modification by other cell wall-associated enzymes.

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.