Showing papers by "Institut national de la recherche agronomique published in 1998"

ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control

Abstract: To cope with environmental fluctuations and to prevent invasion by pathogens, plant metabolism must be flexible and dynamic. Active oxygen species, whose formation is accelerated under stress conditions, must be rapidly processed if oxidative damage is to be averted. The lifetime of active oxygen species within the cellular environment is determined by the antioxidative system, which provides crucial protection against oxidative damage. The antioxidative system comprises numerous enzymes and compounds of low molecular weight. While research into the former has benefited greatly from advances in molecular technology, the pathways by which the latter are synthesized have received comparatively little attention. The present review emphasizes the roles of ascorbate and glutathione in plant metabolism and stress tolerance. We provide a detailed account of current knowledge of the biosynthesis, compartmentation, and transport of these two important antioxidants, with emphasis on the unique insights and advances gained by molecular exploration.

A microsatellite map of wheat.

Abstract: Hexaploid bread wheat (Triticum aestivum L. em. Thell) is one of the world's most important crop plants and displays a very low level of intraspecific polymorphism. We report the development of highly polymorphic microsatellite markers using procedures optimized for the large wheat genome. The isolation of microsatellite-containing clones from hypomethylated regions of the wheat genome increased the proportion of useful markers almost twofold. The majority (80%) of primer sets developed are genome-specific and detect only a single locus in one of the three genomes of bread wheat (A, B, or D). Only 20% of the markers detect more than one locus. A total of 279 loci amplified by 230 primer sets were placed onto a genetic framework map composed of RFLPs previously mapped in the reference population of the International Triticeae Mapping Initiative (ITMI) Opata 85 x W7984. Sixty-five microsatellites were mapped at a LOD >2.5, and 214 microsatellites were assigned to the most likely intervals. Ninety-three loci were mapped to the A genome, 115 to the B genome, and 71 to the D genome. The markers are randomly distributed along the linkage map, with clustering in several centromeric regions.

Identifying Populations for Conservation on the Basis of Genetic Markers

Abstract: To select candidate populations of wild species to be given priority for conservation, genetic criteria gained from the study of molecular markers may be useful. Traditionally, diversity measures such as ex- pected heterozygosity or percentage of polymorphic loci have been considered. For conservation we propose instead that priority should be given to measures of allelic richness. To standardize the results of allelic rich- ness across populations, we used the technique of rarefaction. This technique allows evaluation of the ex- pected number of different alleles among equal-sized samples drawn from several different populations. We also show how the contribution of each population to total diversity can be partitioned into two components. The first is related to the level of diversity of the population and the second to its divergence from the other populations. For conservation purposes the uniqueness of a population-in terms of its allelic composition- may be at least as important as its diversity level. These new descriptors are illustrated by means of isozyme and chloroplast DNA data obtainedfor an endangered tree species, the argan tree of Morocco (Argania spinosa (L.) Skeels). With these analyses the conservation value of the argan tree populations, especially those of two isolates present in the north of the country, can be better appreciated. The methods proposed to identify prior- ity areas for conservation of the genetic resources of the argan tree are compared to those sometimes advo- cated in the case of reserve design, where one of the goals is to maximize species richness.

Functional food science and gastrointestinal physiology and function

Abstract: The gut is an obvious target for the development of functional foods, acting as it does as the interface between diet and the metabolic events which sustain life. The key processes in digestive physiology which can be regulated by modifying diet are satiety, the rate and extent of macronutrient breakdown and absorption from the small bowel, sterol metabolism, the colonic microflora, fermentation, mucosal function and bowel habit, and the gut immune system. The intestinal microflora is the main focus of many current functional foods. Probiotics are foods which contain live bacteria which are beneficial to health whilst prebiotics, such as certain non-digestible oligosaccharides which selectively stimulate the growth of bifidobacteria in the colon, are already on the market. Their claimed benefits are to alleviate lactose maldigestion, increase resistance to invasion by pathogenic species of bacteria in the gut, stimulate the immune system and possibly protect against cancer. There are very few reports of well-designed human intervention studies with prebiotics as yet. Certain probiotic species have been shown to shorten the duration of rotavirus diarrhoea in children but much more work is needed on the mechanism of immunomodulation and of competitive exclusion and microflora modification. The development of functional foods for the gut is in its infancy and will be successful only if more fundamental research is done on digestive physiology, the gut microflora, immune system and mucosal function.

Variability among species of stomatal control under fluctuating soil water status and evaporative demand: modelling isohydric and anisohydric behaviours

Abstract: Stomatal control of species with contrasting stomatal behaviours have been investigated under natural fluctuations of evaporative demand and soil water status. Sunflower and barley (anisohydric behaviour) have a daytime leaf water potential (ψ l ) which markedly decreases with evaporative demand during the day and is lower in droughted than in watered plants. In contrast, maize and poplar (isohydric behaviour) maintain a nearly constant ψ l during the day at a value which does not depend on soil water status until plants are close to death. Plants were also subjected to a range of soil water potentials under contrasting air vapour pressure deficits (VPD, from 0.5 to 3 kPa) in the field, in the greenhouse or in a growth chamber. Finally, plants or detached leaves were fed with varying concentrations of artificial ABA. Stomatal conductance of well-watered plants had no response to VPD when plants were grown in natural soils, suggesting that the opposite result observed in many laboratory experiments might be linked to the low unsaturated hydraulic conductivity of usual potting substrates. The response of stomatal conductance of all studied species to the concentration of ABA in pressurized xylem sap ([ABA] xyl ) was the same whether ABA had an endogenous origin (droughted plants) or was artificially fed. However stomatal response of maize and poplar to [ABA] xyl markedly changed with varying evaporative demand or ψ l , whereas this was not the case in sunflower or barley. This suggests that isohydric behaviour is linked to an interaction between hydraulic and chemical information, while anisohydric behaviour is linked to an absence of interaction. In all cases, [ABA] xyl was related to soil water status with common relationships for different experimental conditions, but with markedly different responses among species. Diurnal variations of [ABA] xyl with evaporative demand were small in all studied species. Results are synthesized in a model which accounts for observed behaviours of g s , ψ l and [ABA] xyl in fluctuating conditions and for several species. The validity of this model, in particular the physiological meaning of [ABA] xyl , is discussed.

In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration.

Abstract: In this chapter, we describe a new protocol for obtaining transgenic Arabidopsis thaliana plants by a so-called in planta transformation method. Such methods, with no in vitro culture or regeneration step, have already been described. Briefly, germinating seeds (Feldmann and Marks 1987) or wounded plants (Chang et al. 1994) are inoculated with an appropriate Agrobacterium strain; plants are then grown to maturity and their seeds collected. Transformants are selected at low frequency among the progeny of inoculated plants. The main advantages of these methods are the simplicity of the procedure and the low frequency of somaclonal variants in the transgenic lines, which is particularly useful for T-DNA mutagenesis strategies (Feldmann 1991).

STICS : a generic model for the simulation of crops and their water and nitrogen balances. I. Theory, and parameterization applied to wheat and corn

Abstract: STICS (Simulateur mulTJdiscplinaire pour les Cultures Standard) is a crop model constructed as a simula- tion tool capable of working under agricultural conditions. Outputs comprise the production (amount and quality) and the environment. Inputs take into account the climate, the soi1 and the cropping system. STICS is presented as a model exhibiting the following qualities: robustness, an easy access to inputs and an uncomplicated f~~ture evolution thanks to a modular (easy adaptation to various types of plant) nature and generic. However, STICS is not an entirely new model since most parts use classic formalisms or stem from existing models. The main simulated processes are the growth, the development of the crop and the water and nitrogenous balance of the soil-crop system. The seven modules of STICS - development, shoot growth, yield components, root growth, water balance, thermal environment and nitrogen balance - are presented in tum with a discussion about the theoretical choices in comparison to other models. These choices should render the model capable of exhibiting the announced qualities in classic environmental contexts. However, because some processes (e.g. ammoniac volatilization, clrought resistance, etc.) are not taken into account, the use of STICS is presently limited to several cropping systems. (O InraIElsevier, Paris.) crop modelling / wheat / corn / water balance / nitrogen balance

Glutathione homeostasis in plants: implications for environmental sensing and plant development

Abstract: Glutathione (GSH; gamma-glutamylcysteinyl glycine) is an abundant and ubiquitous thiol with proposed roles in the storage and transport of reduced sulphur, the synthesis of proteins and nucleic acids and as a modulator of enzyme activity. The level of glutathione has also been shown to correlate with the adaptation of plants to extremes of temperature, in the tolerance of plants to xenobiotics and to biotic and abiotic environmental stresses. In addition, the size of the reduced glutathione pool shows marked alterations in response to a number of environmental conditions. Taken together, these findings have prompted intense efforts to characterize in detail the mechanisms underlying glutathione homeostasis in plants and to elucidate the role of these responses in the strategies plants have evolved to adapt to environmental stresses,The aim of this review is to assess recent biochemical, molecular, genetic, and physiological advances which are increasing our understanding of the mechanisms by which plant glutathione homeostasis is controlled and the role of glutathione in the integration of cellular processes with plant growth and development under stress.

Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants

Abstract: Introduction Crucial roles in sulphur metabolism and plant defence Glutathione is found in the vast majority of prokaryotichave been described in recent years for the tripeptide and eukaryotic cells, where it often represents the majorthiol glutathione. In spite of this, the metabolism of pool of non-protein reduced sulphur (Kunert and Foyer,glutathione and its response to stress conditions 1993). The reduced form of glutathione (GSH) is aremained only partly understood. In many plants, one tripeptide thiol with the formula c-glu-cys-gly. The path-of the major difficulties in studying the control of gluta- way of GSH biosynthesis (Fig. 1) is well established: twothione synthesis is the low extractable activities of the sequential ATP-dependent reactions allow the synthesisenzymes involved. Consequently, several groups have of c-glutamylcysteine (c-EC) from l-glutamate and l-exploited transformation technology using genes for cysteine, followed by the formation of GSH by additionthe enzymes of glutathione synthesis or reduction. of glycine to the C-terminal end of c-EC (Meister, 1988).This approach has allowed the production of plants These reactions are catalysed by c-glutamylcysteinewith systematically enhanced levels of glutathione (up synthetase (c-ECS) and glutathione synthetase (GS).to 4-fold higher than untransformed controls) and has Several plant species produce analogous tripeptide deriv-permitted numerous insights into the control of atives of c-EC in addition to, or in place of, GSH (c-EC-glutathione synthesis or reduction state and its inter- ala or homoglutathione: Klapheck, 1988; c-EC-ser:action with other areas of primary or defensive Klapheck et al., 1992; c-EC-glu: Meuwly et al., 1993).metabolism. Since all functions thus far described for GSH in plantsare related to the cysteine moiety of the tripeptide, thesehomologues may exercise similar biochemical roles to c-Key words: Oxidative stress, photorespiration, glutathione, EC-gly.ascorbate, poplar. Numerous physiological functions have been attributed

A plasma membrane‐bound putative endo‐1,4‐β‐D‐glucanase is required for normal wall assembly and cell elongation in Arabidopsis

Abstract: Endo‐1,4‐β‐d‐glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non‐crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN ( KOR ) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane‐anchored member of the EGase family, which is highly conserved between mono‐ and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane–cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark‐grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR ‐mRNA levels. However, reduced KOR ‐mRNA levels were observed in det2 , a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose–hemicellulose network in the expanding cell wall.

Transgene-induced gene silencing in plants.

Abstract: The recent development of gene transfer methods for almost all eukaryotes has revealed that transgenes can undergo silencing after integration in the genome. Host genes can also be silenced as a consequence of the presence of a homologous transgene, thus limiting the potential application of genetic transformation. Despite this limitation, transgene-induced gene silencing events were considered originally as anecdotal phenomena. However, as more and more similarities were found between transgene-induced gene silencing and natural epigenetic phenomena, considerable interest has been devoted to this subject (for recent reviews see Depicker and Van Montagu, 1997; Stam et al., 1997b). Epigenetics is commonly defined as ‘the study of mitotically and/or meiotically heritable changes in the function of a gene that cannot be explained by changes in its DNA sequence’ (Russo et al., 1996). For a long time, DNA was considered as the only target for epigenetic modifications. Epigenetic changes corresponding to changes in chromatin structure and affecting transcription have been reported in almost all eukaryotes: yeast, fungi, Drosophila, plants and mammals (Dorer, 1997; Foss and Selker, 1991; Rossignol and Faugeron, 1994; Ye and Signer, 1996). However, recent studies have suggested that, besides DNA, other molecules can be modified in a manner that resembles epigenetic DNA changes. First, it was shown that proteins can be converted into molecules of aberrant conformation called prions in yeast and mammals (Lacroute, 1971; Prusiner, 1982). More recently, the involvement of RNA was hypothesized to explain post-transcriptional silencing in plants, fungi and nematodes (Cogoni et al., 1996; Fire et al., 1998; Napoli et al., 1990). This review will focus on transgene-induced silencing phenomena in plants. The number of copies of a transgene that integrate into the genome of a transformed plant and

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.

Abstract: . We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number an

Comparative analysis of microsatellite and allozyme markers: a case study investigating microgeographic differentiation in brown trout (Salmo trutta)

Abstract: A comparative study between microsatellite and allozyme markers was conducted on natural populations of resident brown trout (Salmo trutta) sampled over a reduced geographical scale and on hatchery strains. The higher level of polymorphism observed at microsatellite loci resulted in higher power of statistical tests for differentiation among population samples and for genotypic linkage disequilibrium. Genetic distances of Cavalli-Sforza and Edwards were on average two times larger for microsatellites than for allozymes but multilocus FST estimates computed over the entire set of populations were not significantly different for both categories of markers. Assignment tests of individual fish to the set of sampled populations demonstrated a much higher efficiency of microsatellites compared to allozymes. Pairwise multilocus FST estimates were significantly correlated to waterway distances and there was a significant tendency for the incorrectly classified individuals to be assigned to one of the nearest populations, indicating that isolation-by-distance acted significantly on brown trout populations. This increase of differentiation with distance was higher for allozymes than for microsatellites. Traditional measures of genetic differentiation (Cavalli-Sforza and Edwards' chord distance and FST) were compared for microsatellites to recently proposed statistics taking into account allele size differences (Goldstein's distance and PST). Using Goldstein's distance for neighbour-joining analysis did not improve the tree structure resolution. Multilocus estimates of PST and FST were not significantly different when computed over the entire set of populations but no significant correlation was detected between matrices of pairwise multilocus PST estimates and waterway distances.

Aligning male and female linkage maps of apple (Malus pumila Mill.) using multi-allelic markers

Abstract: Linkage maps for the apple cultivars ‘Prima’ and ‘Fiesta’ were constructed using RFLP, RAPD, isozyme, AFLP, SCAR and microsatellite markers in a ‘Prima’בFiesta’ progeny of 152 individuals. Seventeen linkage groups, putatively corresponding to the seventeen haploid apple chromosomes, were obtained for each parent. These maps were aligned using 67 multi-allelic markers that were heterozygous in both parents. A large number of duplicate RFLP loci was observed and, in several instances, linked RFLP markers in one linkage group showed corresponding linkage in another linkage group. Distorted segregation was observed mainly in two regions of the genome, especially in the male parent alleles. Map positions were provided for resistance genes to scab and rosy leaf curling aphid (Vf and Sd 1, respectively) for the fruit acidity gene Ma and for the self-incompatibility locus S. The high marker density and large number of mapped codominant RFLPs and some microsatellite markers make this map an ideal reference map for use in other progenies also and a valuable tool for the mapping of quantitative trait loci.

Protein Changes in Response to Progressive Water Deficit in Maize: Quantitative Variation and Polypeptide Identification

Abstract: Three-week-old plants of two unrelated lines of maize (Zea mays L.) and their hybrid were submitted to progressive water stress for 10 d. Changes induced in leaf proteins were studied by two-dimensional electrophoresis and quantitatively analyzed using image analysis. Seventy-eight proteins out of a total of 413 showed a significant quantitative variation (increase or decrease), with 38 of them exhibiting a different expression in the two genotypes. Eleven proteins that increased by a factor of 1.3 to 5 in stressed plants and 8 proteins detected only in stressed plants were selected for internal amino acid microsequencing, and by similarity search 16 were found to be closely related to previously reported proteins. In addition to proteins already known to be involved in the response to water stress (e.g. RAB17 [Responsive to ABA]), several enzymes involved in basic metabolic cellular pathways such as glycolysis and the Krebs cycle (e.g. enolase and triose phosphate isomerase) were identified, as well as several others, including caffeate O-methyltransferase, the induction of which could be related to lignification.

Drought-Induced Effects on Nitrate Reductase Activity and mRNA and on the Coordination of Nitrogen and Carbon Metabolism in Maize Leaves

Abstract: Maize ( Zea mays L.) plants were grown to the nine-leaf stage. Despite a saturating N supply, the youngest mature leaves (seventh position on the stem) contained little NO 3 − reserve. Droughted plants (deprived of nutrient solution) showed changes in foliar enzyme activities, mRNA accumulation, photosynthesis, and carbohydrate and amino acid contents. Total leaf water potential and CO 2 assimilation rates, measured 3 h into the photoperiod, decreased 3 d after the onset of drought. Starch, glucose, fructose, and amino acids, but not sucrose (Suc), accumulated in the leaves of droughted plants. Maximal extractable phospho enol pyruvate carboxylase activities increased slightly during water deficit, whereas the sensitivity of this enzyme to the inhibitor malate decreased. Maximal extractable Suc phosphate synthase activities decreased as a result of water stress, and there was an increase in the sensitivity to the inhibitor orthophosphate. A correlation between maximal extractable foliar nitrate reductase (NR) activity and the rate of CO 2 assimilation was observed. The NR activation state and maximal extractable NR activity declined rapidly in response to drought. Photosynthesis and NR activity recovered rapidly when nutrient solution was restored at this point. The decrease in maximal extractable NR activity was accompanied by a decrease in NR transcripts, whereas Suc phosphate synthase and phospho enol pyruvate carboxylase mRNAs were much less affected. The coordination of N and C metabolism is retained during drought conditions via modulation of the activities of Suc phosphate synthase and NR commensurate with the prevailing rate of photosynthesis.

Melanocortin receptor 1 (MC1R) mutations and coat color in pigs.

Abstract: The melanocortin receptor 1 (MC1R) plays a central role in regulation of eumelanin (black/brown) and phaeomelanin (red/yellow) synthesis within the mammalian melanocyte and is encoded by the classical Extension (E) coat color locus. Sequence analysis of MC1R from seven porcine breeds revealed a total of four allelic variants corresponding to five different E alleles. The European wild boar possessed a unique MC1R allele that we believe is required for the expression of a wild-type coat color. Two different MC1R alleles were associated with the dominant black color in pigs. MC1R*2 was found in European Large Black and Chinese Meishan pigs and exhibited two missense mutations compared with the wild-type sequence. Comparative data strongly suggest that one of these, L99P, may form a constitutively active receptor. MC1R*3 was associated with the black color in the Hampshire breed and involved a single missense mutation D121N. This same MC1R variant was also associated with EP, which results in black spots on a white or red background. Two different missense mutations were identified in recessive red (e/e) animals. One of these, A240T, occurs at a highly conserved position, making it a strong candidate for disruption of receptor function.

Physiology of organic nitrogen acquisition by ectomycorrhizal fungi and ectomycorrhizas

Abstract: Ectomycorrhizal fungi are symbiotically associated microorganisms which ecological importance has been repeatedly demonstrated. There has been a considerable amount of research aimed at assessing the ability of ectomycorrhizal fungi and ectomycorrhizas to utilize organic nitrogen sources. The fate of soil proteins, peptides and amino acids has been studied from a number of perspectives. Exocellular hydrolytic enzymes have been detected and characterized in a number of ectomycorrhizal and ericoid fungi. Studies on amino acid transport through the plasma membrane have demonstrated the ability of ectomycorrhizal fungi to take up the products of proteolytic activities. Investigations on intracellular metabolism of amino acids have allowed the identification of the metabolic pathways involved. Possible intracellular compartmentation of amino acids will be examined by immunocytochemistry. Further translocation of amino acids in symbiotic tissues has been established by experiments using isotopic tracers, although the exact nature of the nitrogenous compounds transferred at the symbiotic interface remained unclear. One of the main future challenges in the physiology of organic nitrogen acquisition is to determine the nature, the regulation and the location of N-compound transporters at the soil-fungus and fungus-plant interfaces. The molecular approach which is just emerging in this particular research area will greatly improve our knowledge. Future research should also address the extent of competition between different ectomycorrhizal species and between different microbial populations for organic nitrogen.

Rotavirus RNA‐binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F

Abstract: Most eukaryotic mRNAs contain a 5′cap structure and a 3′poly(A) sequence that synergistically increase the efficiency of translation. Rotavirus mRNAs are capped, but lack poly(A) sequences. During rotavirus infection, the viral protein NSP3A is bound to the viral mRNAs 3′ end. We looked for cellular proteins that could interact with NSP3A, using the two‐hybrid system in yeast. Screening a CV1 cell cDNA library allowed us to isolate a partial cDNA of the human eukaryotic initiation factor 4GI (eIF4GI). The interaction of NSP3A with eIF4GI was confirmed in rotavirus infected cells by co‐immunoprecipitation and in vitro with NSP3A produced in Escherichia coli . In addition, we show that the amount of poly(A) binding protein (PABP) present in eIF4F complexes decreases during rotavirus infection, even though eIF4A and eIF4E remain unaffected. PABP is removed from the eIF4F complex after incubation in vitro with the C‐terminal part of NSP3A, but not with its N‐terminal part produced in E.coli . These results show that a physical link between the 5′ and the 3′ ends of mRNA is necessary for the efficient translation of viral mRNAs and strongly support the closed loop model for the initiation of translation. These results also suggest that NSP3A, by taking the place of PABP on eIF4GI, is responsible for the shut‐off of cellular protein synthesis.

Within‐population genetic structure in Quercus robur L. and Quercus petraea (Matt.) Liebl. assessed with isozymes and microsatellites

Abstract: The spatial distribution of alleles is described in a forest stand of natural origin of 5 ha comprising 355 mature Quercus petraea and Q. robur trees. Each tree was genotyped for six microsatellite loci. Previous studies on the same population based on isozymes allowed a comparison of different markers for the detection of spatial genetic structure. Different statistics were used: differentiation measures at different spatial scales, and spatial autocorrelation analysis based on Moran’s index I. For microsatellites, differentiation and autocorrelation were calculated with unordered alleles (identity in state) and with alleles ordered according to their size. Results showed the same tendency of a significant, but low, spatial genetic structure for markers and different statistics. Some differences could, however, be detected. First, microsatellites interpreted as unordered alleles exhibit stronger spatial structure than isozymes or microsatellite interpreted as ordered alleles. Second, differentiation and autocorrelation values were higher in Q. petraea than in Q. robur. These differences were attributed to species differences in gene flow via pollen or seed.

Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase from Aspergillus oryzae

Abstract: Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.