The Progesterone 5ß-Reductase/Iridoid Synthase Family: A Catalytic Reservoir for Specialized Metabolism across Land Plants.

Iridoids are plant-derived terpenoids with a rich array of bioactivities. The key step in iridoid skeleton formation is the reduction of 8-oxogeranial by certain members of the progesterone 5ß-reductase/iridoid synthase (PRISE) family of short-chain alcohol dehydrogenases. Other members of the PRISE family have previously been implicated in the biosynthesis of the triterpenoid class of cardenolides, which requires the reduction of progesterone. Here, we explore the occurrence and activity of PRISE across major lineages of plants. We observed trace activities toward either 8-oxogeranial or progesterone in all PRISEs, including those from nonseed plants and green algae. Phylogenetic analysis, coupled with enzymatic assays, show that these activities appear to have become specialized in specific angiosperm lineages. This broad analysis of the PRISE family provides insight into how these enzymes evolved in plants and also suggests that iridoid synthase activity is an ancestral trait in all land plants, which might have contributed to the rise of iridoid metabolites.

Oxygen response of leaf CO2 compensation points used to determine Rubisco specificity factors of gymnosperm species.

Rubisco specificity factor (Sc/o), a measure of the relative capacities of an enzyme to catalyze carboxylation and oxygenation of ribulose-1,5-bisphosphate, determines the extent of photosynthetic CO2 assimilation and photorespiratory CO2 release. The current model of C3 photosynthesis, the Farquhar-von Caemmerer-Berry (FvCB) model, requires a species-specific Sc/o. However, Sc/o values have never been reported in conifers, likely because in vitro kinetic analysis of conifer Rubisco presents difficulties. To estimate the Sc/o of conifers and compare it with angiosperm Sc/o, we measured changes in leaf CO2 compensation points (Γ) in response to O2 partial pressure for a variety of leaves, with different rates of day respiration (Rday) and maximum Rubisco carboxylation (Vcmax) in gymnosperms (Ginkgo biloba), conifers (Metasequoia glyptostroboides and Cryptomeria japonica), and angiosperms (Nicotiana tabacum and Phaseolus vulgaris). As predicted by the FvCB model, the slope of a linear function of Γ vs O2 partial pressure, d, increased alongside increasing Rday/Vcmax. The Sc/o was obtainable from this relationship between d and Rday/Vcmax, because the d values at Rday/Vcmax = 0 corresponded to α/Sc/o, where α was the photorespiratory CO2 release rate per Rubisco oxygenation rate (generally assumed to be 0.5). The calculated Sc/o values of N. tabacum and P. vulgaris exhibited good agreement with those reported by in vitro studies. The Sc/o values of both conifers were similar to those of the two angiosperm species. In contrast, the Sc/o value of G. biloba was significantly lower than those of the other four studied species. These results suggest that our new method for Sc/o estimation is applicable to C3 plants, including those for which in vitro kinetic analysis is difficult. Furthermore, results also suggest that conifer Sc/o does not differ significantly from that of C3 angiosperms, assuming α remains unchanged.

Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms.

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

Alternative Oxidase Capacity of Mitochondria in Microsporophylls May Function in Cycad Thermogenesis.

Cone thermogenesis is a widespread phenomenon in cycads and may function to promote volatile emissions that affect pollinator behavior. Given their large population size and intense and durable heat-producing effects, cycads are important organisms for comprehensive studies of plant thermogenesis. However, knowledge of mitochondrial morphology and function in cone thermogenesis is limited. Therefore, we investigated these mitochondrial properties in the thermogenic cycad species Cycas revoluta Male cones generated heat even in cool weather conditions. Female cones produced heat, but to a lesser extent than male cones. Ultrastructural analyses of the two major tissues of male cones, microsporophylls and microsporangia, revealed the existence of a population of mitochondria with a distinct morphology in the microsporophylls. In these cells, we observed large mitochondria (cross-sectional area of 2 µm2 or more) with a uniform matrix density that occupied >10% of the total mitochondrial volume. Despite the size difference, many nonlarge mitochondria (cross-sectional area <2 µm2) also exhibited a shape and a matrix density similar to those of large mitochondria. Alternative oxidase (AOX) capacity and expression levels in microsporophylls were much higher than those in microsporangia. The AOX genes expressed in male cones revealed two different AOX complementary DNA sequences: CrAOX1 and CrAOX2 The expression level of CrAOX1 mRNA in the microsporophylls was 100 times greater than that of CrAOX2 mRNA. Collectively, these results suggest that distinctive mitochondrial morphology and CrAOX1-mediated respiration in microsporophylls might play a role in cycad cone thermogenesis.

Evolutionary History of the Glycoside Hydrolase 3 (GH3) Family Based on the Sequenced Genomes of 48 Plants and Identification of Jasmonic Acid-Related GH3 Proteins in Solanum tuberosum.

Glycoside Hydrolase 3 (GH3) is a phytohormone-responsive family of proteins found in many plant species. These proteins contribute to the biological activity of indolacetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA). They also affect plant growth and developmental processes as well as some types of stress. In this study, GH3 genes were identified in 48 plant species, including algae, mosses, ferns, gymnosperms, and angiosperms. No GH3 representative protein was found in algae, but we identified 4 genes in mosses, 19 in ferns, 7 in gymnosperms, and several in angiosperms. The results showed that GH3 proteins are mainly present in seed plants. Phylogenetic analysis of all GH3 proteins showed three separate clades. Group I was related to JA adenylation, group II was related to IAA adenylation, and group III was separated from group II, but its function was not clear. The structure of the GH3 proteins indicated highly conserved sequences in the plant kingdom. The analysis of JA adenylation in relation to gene expression of GH3 in potato (Solanum tuberosum) showed that StGH3.12 greatly responded to methyl jasmonate (MeJA) treatment. The expression levels of StGH3.1, StGH3.11, and StGH3.12 were higher in the potato flowers, and StGH3.11 expression was also higher in the stolon. Our research revealed the evolution of the GH3 family, which is useful for studying the precise function of GH3 proteins related to JA adenylation in S. tuberosum when the plants are developing and under biotic stress.

Naturally fragmented but not genetically isolated populations of Podocarpus sellowii Klotzsch (Podocarpaceae) in southeast Brazil.

In southeastern Brazil, the majority of the riparian ecosystems are fragmented and degraded mainly due to human activities. The perennial gymnosperm Podocarpus sellowii Klotzch is a typical tree species from a gallery forest in the rupestrian area. Ten alloenzymatic loci were used to estimate the allelic frequency of 232 individuals distributed in eight subpopulations naturally divided by rock outcrops. The results indicated high genetic variability for the species in all subpopulations, with HO varying from 0.593 to 0.658, and HE, from 0.484 to 0.502. No endogamy was observed within (f = -0.292) and for the population set (f = -0.264). Genetic divergence of the species between subpopulations was 2.1%. Historic gene flow was low between subpopulations located in different water streams, corroborating the positive and significant correlation between genetic and geographical distance (rm = 0.496, P = 0.022). Co-ancestry revealed that only population A presents continuous distribution of the genotypes up to 94 m. Sp statistics did not indicate significant spatial genetic structure in the populations. In all the subpopulations, values of effective sizes were higher than the numbers of sampled individuals. The balance between mutation and drift was not verified, indicating the occurrence of a recent population bottleneck. These data can be used to determine the most effective strategies for the genetic conservation of this species.

Molecular evolution and sequence divergence of plant chalcone synthase and chalcone synthase-Like genes.

Plant chalcone synthase (CHS) and CHS-Like (CHSL) proteins are polyketide synthases. In this study, we evaluated the molecular evolution of this gene family using representative types of CHSL genes, including stilbene synthase (STS), 2-pyrone synthase (2-PS), bibenzyl synthase (BBS), acridone synthase (ACS), biphenyl synthase (BIS), benzalacetone synthase, coumaroyl triacetic acid synthase (CTAS), and benzophenone synthase (BPS), along with their CHS homologs from the same species of both angiosperms and gymnosperms. A cDNA-based phylogeny indicated that CHSLs had diverse evolutionary patterns. STS, ACS, and 2-PS clustered with CHSs from the same species (late diverged pattern), while CTAS, BBS, BPS, and BIS were distant from their CHS homologs (early diverged pattern). The amino-acid phylogeny suggested that CHS and CHSL proteins formed clades according to enzyme function. The CHSs and CHSLs from Polygonaceae and Arachis had unique evolutionary histories. Synonymous mutation rates were lower in late diverged CHSLs than in early diverged ones, indicating that gene duplications occurred more recently in late diverged CHSLs than in early diverged ones. Relative rate tests proved that late diverged CHSLs had unequal rates to CHSs from the same species when using fatty acid synthase, which evolved from the common ancestor with the CHS superfamily, as the outgroup, while the early diverged lineages had equal rates. This indicated that late diverged CHSLs experienced more frequent mutation than early diverged CHSLs after gene duplication, allowing obtaining new functions in relatively short period of time.

The phenylalanine ammonia lyase (PAL) gene family shows a gymnosperm-specific lineage.

BACKGROUND: Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda. METHODS: We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages. RESULTS: In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda were derived from a combination of duplication events predating and postdating the divergence of angiosperms and gymnosperms. CONCLUSIONS: Gymnosperms have a more phylogenetically diverse set of PAL genes than angiosperms. This inference has contrasting implications for the evolution of PAL gene function in gymnosperms and angiosperms.

The endo-1,4-ß-glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms.

The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions. A membrane-bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken. Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1-1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure. Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants.

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds.

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

Analysis of the soluble cell wall proteome of gymnosperms.

We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

Structural motifs of syringyl peroxidases predate not only the gymnosperm-angiosperm divergence but also the radiation of tracheophytes.

* The most distinctive variation in the monomer composition of lignins in vascular land plants is that found between the two main groups of seed plants. Thus, while gymnosperm lignins are typically composed of guaiacyl (G) units, angiosperm lignins are largely composed of similar levels of G and syringyl (S) units. * However, and contrary to what might be expected, peroxidases isolated from basal (Cycadales and Ginkgoales) and differentially evolved (Coniferales and Gnetales) gymnosperms are also able to oxidize S moieties, and this ability is independent of the presence or absence of S-type units in their lignins. * The results obtained led us to look at the protein database to search for homologies between gymnosperm peroxidases and true eudicot S-peroxidases, such as the Zinnia elegans peroxidase. * The findings showed that certain structural motifs characteristic of eudicot S-peroxidases (certain amino acid sequences and beta-sheet secondary structures) predate the gymnosperm-angiosperm divergence and the radiation of tracheophytes, since they are found not only in peroxidases from basal gymnosperms, ferns and lycopods, but also in peroxidases from the moss Physcomitrella patens (Bryopsida) and the liverwort Marchantia polymorpha (Marchantiopsida), which, as typical of bryophytes, do not have xylem tissue nor lignins.

Molecular basis for the evolution of xylem lignification.

The lignification of xylem is an adaptive trait of great significance. Gymnosperms and angiosperms share an ancient, conserved set of enzymes that are regulated by a conserved transcription factor and that are responsible for the formation of guaiacyl lignin. Angiosperms have evolved at least two enzymes that catalyze the production of syringyl lignin. Association genetics is now being used to explore the adaptive significance of sequence variation in the genes that encode these monolignol biosynthetic enzymes.

Functional characterization of nine Norway Spruce TPS genes and evolution of gymnosperm terpene synthases of the TPS-d subfamily.

Constitutive and induced terpenoids are important defense compounds for many plants against potential herbivores and pathogens. In Norway spruce (Picea abies L. Karst), treatment with methyl jasmonate induces complex chemical and biochemical terpenoid defense responses associated with traumatic resin duct development in stems and volatile terpenoid emissions in needles. The cloning of (+)-3-carene synthase was the first step in characterizing this system at the molecular genetic level. Here we report the isolation and functional characterization of nine additional terpene synthase (TPS) cDNAs from Norway spruce. These cDNAs encode four monoterpene synthases, myrcene synthase, (-)-limonene synthase, (-)-alpha/beta-pinene synthase, and (-)-linalool synthase; three sesquiterpene synthases, longifolene synthase, E,E-alpha-farnesene synthase, and E-alpha-bisabolene synthase; and two diterpene synthases, isopimara-7,15-diene synthase and levopimaradiene/abietadiene synthase, each with a unique product profile. To our knowledge, genes encoding isopimara-7,15-diene synthase and longifolene synthase have not been previously described, and this linalool synthase is the first described from a gymnosperm. These functionally diverse TPS account for much of the structural diversity of constitutive and methyl jasmonate-induced terpenoids in foliage, xylem, bark, and volatile emissions from needles of Norway spruce. Phylogenetic analyses based on the inclusion of these TPS into the TPS-d subfamily revealed that functional specialization of conifer TPS occurred before speciation of Pinaceae. Furthermore, based on TPS enclaves created by distinct branching patterns, the TPS-d subfamily is divided into three groups according to sequence similarities and functional assessment. Similarities of TPS evolution in angiosperms and modeling of TPS protein structures are discussed.

Expression of Chia4-Pa chitinase genes during somatic and zygotic embryo development in Norway spruce (Picea abies): similarities and differences between gymnosperm and angiosperm class IV chitinases.

The developmental pathway of somatic embryogenesis in Norway spruce involves proliferation of proembryogenic masses (PEMs), PEM-to-somatic embryo transition and further development of the somatic embryos. It has previously been shown that extracellular signal molecules, including arabinogalactan proteins, lipo-chitooligosaccharides and chitinases, regulate somatic embryogenesis. The Chia4-Pa1 gene from Norway spruce is described here. The Chia4-Pa1 encodes a typical basic class IV chitinase, although the intron-exon organization of this gymnosperm chitinase is different from that in angiosperm class IV chitinases. The Chia4-Pa1 belongs to a small gene family with highly similar members, and the expression pattern of Chia4-Pa1 cannot be distinguished from that of other Chia4-Pa members. Upon withdrawal of plant growth regulators, i.e. during a treatment that stimulates PEM-to-somatic embryo transition and massive programmed cell death, a significant increase in transcription and translation of Chia4-Pa genes takes place. The expression pattern analysis revealed that Chia4-Pa genes are expressed in a subpopulation of proliferating cells and at the base of the somatic embryo. Furthermore, in seeds, Chia4-Pa genes are expressed in the megagametophyte in the single cell-layered zone surrounding the corrosion cavity. Taken together these results suggest that the Chia4-Pa expressing cells have a megagametophyte signalling function and that CHIA4-Pa stimulates programmed cell death and promotes PEM-to-somatic embryo transition.

Xyloglucan endotransglucosylase action is high in the root elongation zone and in the trichoblasts of all vascular plants from Selaginella to Zea mays.

The endotransglucosylase action of the enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was localized in the roots of diverse vascular plants: club-mosses (lycopodiophytes), ferns, gymnosperms, monocots, and dicots. High action was always found in the epidermis cell wall of the elongation zone and in trichoblasts in the differentiation zone. Clearly XTH and its action in root development evolved before the evolutionary divergence of ferns and seed plants and also of the lycopodiophytes and euphyllophytes.

Endogenous protein phosphorylation and casein kinase activity during seed development in Araucaria angustifolia.

Protein kinases and phosphatases are responsible for several cellular events mediated by protein phosphorylation and dephosphorylation. Among these events are cell growth and differentiation and cellular metabolism. Casein kinase I (CKI) and casein kinase II (CKII) are involved in the phosphorylation of several substrates. Endogenous protein phosphorylation and casein kinase activity were investigated in the megagametophyte of the native Brazilian conifer Araucaria angustifolia, during seed development. It was observed that a number of different polypeptides are phosphorylated in vitro in the three megagametophyte stages of development tested (from globular, cotyledonary and mature embryos, respectively) and the phosphate was incorporated mainly in serine residues. The use of okadaic acid and vanadate in the phosphorylation reactions increased phosphate incorporation in several polypeptides suggesting the presence of serine/threonine as well as tyrosine phosphatases in the megagametophyte. Also, the results obtained in experiments with CKII inhibitor, GTP as phosphate donor, RNA hybridizations, and in-gel kinase assays indicate the presence of CKII in the A. angustifolia megagametophyte.

A desaturase-like protein from white spruce is a Delta(9) desaturase.

Gymnospermae seed lipids are characterized by a high degree of desaturation, most having a Delta(9) double bond. By degenerate polymerase chain reaction (PCR) we have isolated a white spruce (Picea glauca) cDNA clone that encodes an amino acid sequence sharing a high degree of homology with other putative plant acyl-coenzyme A (CoA) Des9 desaturases. Both in vivo and in vitro expression studies in a Delta(9) desaturase-deficient yeast strain demonstrated the desaturation functionality of the white spruce clone, and gas chromatography-mass spectrometry (GC-MS) analyses confirmed the regioselectivity of the encoded enzyme. This is the first report of the functional characterization of a plant membrane-bound acyl-CoA-like protein Delta(9) desaturase by heterologous expression in yeast.

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei.

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.