Evolution of Conifer Diterpene Synthases: Diterpene Resin Acid Biosynthesis in Lodgepole Pine and Jack Pine Involves Monofunctional and Bifunctional Diterpene Synthases

Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant’s stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K⁺ current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H⁺-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K⁺ current inactivation, and H⁺-ATPase phosphorylation were not sensitive to ABA.The phytohormone abscisic acid (ABA), which is synthesized in response to abiotic stresses, plays a key role in the drought hardiness of plants. Reducing transpirational water loss through stomatal pores is a major ABA response (Schroeder et al., 2001). ABA promotes the closure of open stomata and inhibits the opening of closed stomata. These effects are not simply the reverse of one another (Allen et al., 1999; Wang et al., 2001; Mishra et al., 2006).A class of receptors of ABA was identified (Ma et al., 2009; Park et al., 2009; Santiago et al., 2009; Nishimura et al., 2010). The sensitivity of stomata to ABA was strongly decreased in quadruple and sextuple mutants of the ABA receptor genes PYRABACTIN RESISTANCE/PYRABACTIN RESISTANCE-LIKE/REGULATORY COMPONENT OF ABSCISIC ACID RECEPTOR (PYR/PYL/RCAR; Nishimura et al., 2010; Gonzalez-Guzman et al., 2012). The PYR/PYL/RCAR receptors are involved in the early ABA signaling events, in which a sequence of interactions of the receptors with PROTEIN PHOSPHATASE 2Cs (PP2Cs) and subfamily 2 SNF1-RELATED PROTEIN KINASES (SnRK2s) leads to the activation of downstream ABA signaling targets in guard cells (Cutler et al., 2010; Kim et al., 2010; Weiner et al., 2010). Studies of Commelina communis and Vicia faba suggested that the ABA receptors involved in stomatal opening are not the same as the ABA receptors involved in stomatal closure (Allan et al., 1994; Anderson et al., 1994; Assmann, 1994; Schwartz et al., 1994). The roles of PYR/PYL/RCAR in either stomatal opening or closure remained to be elucidated.Blue light induces stomatal opening through the activation of plasma membrane H⁺-ATPase in guard cells that generates an inside-negative electrochemical gradient across the plasma membrane and drives K⁺ uptake through voltage-dependent inward-rectifying K⁺ channels (Assmann et al., 1985; Shimazaki et al., 1986; Blatt, 1987; Schroeder et al., 1987; Thiel et al., 1992). Phosphorylation of the penultimate Thr of the plasma membrane H⁺-ATPase is a prerequisite for blue light-induced activation of the H⁺-ATPase (Kinoshita and Shimazaki, 1999, 2002). ABA inhibits H⁺-ATPase activity through dephosphorylation of the penultimate Thr in the C terminus of the H⁺-ATPase in guard cells, resulting in prevention of the opening (Goh et al., 1996; Zhang et al., 2004; Hayashi et al., 2011). Inward-rectifying K⁺ currents (I_Kin) of guard cells are negatively regulated by ABA in addition to through the decline of the H⁺ pump-driven membrane potential difference (Schroeder and Hagiwara, 1989; Blatt, 1990; McAinsh et al., 1990; Schwartz et al., 1994; Grabov and Blatt, 1999; Saito et al., 2008). This down-regulation of ion transporters by ABA is essential for the inhibition of stomatal opening.A series of second messengers has been shown to mediate ABA-induced stomatal closure. Reactive oxygen species (ROS) produced by NADPH oxidases play a crucial role in ABA signaling in guard cells (Pei et al., 2000; Zhang et al., 2001; Kwak et al., 2003; Sirichandra et al., 2009; Jannat et al., 2011). Nitric oxide (NO) is an essential signaling component in ABA-induced stomatal closure (Desikan et al., 2002; Guo et al., 2003; Garcia-Mata and Lamattina, 2007; Neill et al., 2008). Alkalization of cytosolic pH in guard cells is postulated to mediate ABA-induced stomatal closure in Arabidopsis (Arabidopsis thaliana) and Pisum sativum and Paphiopedilum species (Irving et al., 1992; Gehring et al., 1997; Grabov and Blatt, 1997; Suhita et al., 2004; Gonugunta et al., 2008). These second messengers transduce environmental signals to ion channels and ion transporters that create the driving force for stomatal movements (Ward et al., 1995; MacRobbie, 1998; Garcia-Mata et al., 2003).In this study, we examined the mobilization of second messengers, the inactivation of I_Kin, and the suppression of H⁺-ATPase phosphorylation evoked by ABA in Arabidopsis mutants to clarify the downstream signaling events of ABA signaling in guard cells. The mutants included a quadruple mutant of PYR/PYL/RCARs, pyr1/pyl1/pyl2/pyl4, and a mutant of a SnRK2 kinase, srk2e.     

The Putative Factors Involved in the Induction of Embryogenesis in Angiosperms

We quantified various endogenous cytokinins during wheat (Triticum aestivumL.) and dandelion (Taraxacum officinaleWeb.) ovary development. Wheat ovaries were studied at the following developmental stages: the mature embryo sac with eight nuclei (stage 1), the interphasic zygote 12 h and 24 h after fertilization (stage 2), and the onset of zygote division (stage 3). The dandelion ovaries were studied at the stage of the mature embryo sac (stage 1), in the interphase of the parthenogeneticaly developing ovule (stage 2), and during its first division (stage 3). The material was analyzed by the method of competitive solid-phase immunoenzyme assay (ELISA) using peroxidase-labeled anti-rabbit antibodies. The onset of embryogenesis in wheat and dandelion ovules was accompanied by the substantial rearrangement of their hormonal complexes, which preceded the morphogenetic processes leading to seed formation. This implies that the hormonal system of the whole maternal plant is involved in the induction of embryogenesis. The final stages of embryogenesis depend on the hormonal systems in the flower, ovary, and ovule.     

水稻和其他禾本科植物基因组多倍体起源的证据

基因加倍(Gene duplication)被认为是进化的加速器。古老的基因组加倍事件已经在多个物种中被确定,包括酵母、脊椎动物以及拟南芥等。本研究发现水稻基因组同样存在全基因组加倍事件,大概发生在禾谷类作物分化之前,距今约7000万年。在水稻基因组中,共找到117个加倍区段(Duplicated block),分布在水稻的全部12条染色体,覆盖约60％的水稻基因组。在加倍区段,大约有20％的基因保留了加倍后的姊妹基因对(Duplicated pairs)。与此形成鲜明对照的是加倍区段的转录因子保留了60％的姊妹基因。禾本科植物全基因组加倍事件的确定对研究禾本科植物基因组的进化具有重要影响,暗示了多倍体化及随后的基因丢失、染色体重排等在禾谷类物种分化中扮演了重要角色。     

Ribonuclease and Other Factors Involved in the Respiratory Senescence of Maize Scutellum

The scutella from seedlings of Zea mays L. germinated at 28–30°C increase in respiration rate to the third day, followed bya decline which is quite noticeable by the fifth day. A searchhas been made for factors responsible for the respiratory decline.The electronmicroscope shows the five-day mitochondria to benormal in appearance. Very active preparations are obtainedby isolating the mitochondria at pH 7.6 with inclusions of bovineserum albumin and ethylenediamine-tetraacetic acid. A solubleribonuclease (RNase A) which increases rapidly with age impairsboth oxidation and phosphorylation. The largely particle-boundribonuclease (RNase B) is not inhibitory. Plant ribonucleaseis resistant to the proteolysis occurring during senescence.It is suggested that the soluble ribonuclease contributes tothe respiratory decline, but that other factors may also beinvolved.     

Hybridism and the rate of evolution in Angiosperms

Ohne Zusammenfassung

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Hybridism and the rate of evolution in Angiosperms

     

Prebiotic Synthesis of Methionine and Other Sulfur-Containing Organic Compounds on the Primitive Earth: A Contemporary Reassessment Based on an Unpublished 1958 Stanley Miller Experiment

Original extracts from an unpublished 1958 experiment conducted by the late Stanley L. Miller were recently found and analyzed using modern state-of-the-art analytical methods. The extracts were produced by the action of an electric discharge on a mixture of methane (CH₄), hydrogen sulfide (H₂S), ammonia (NH₃), and carbon dioxide (CO₂). Racemic methionine was formed in significant yields, together with other sulfur-bearing organic compounds. The formation of methionine and other compounds from a model prebiotic atmosphere that contained H₂S suggests that this type of synthesis is robust under reducing conditions, which may have existed either in the global primitive atmosphere or in localized volcanic environments on the early Earth. The presence of a wide array of sulfur-containing organic compounds produced by the decomposition of methionine and cysteine indicates that in addition to abiotic synthetic processes, degradation of organic compounds on the primordial Earth could have been important in diversifying the inventory of molecules of biochemical significance not readily formed from other abiotic reactions, or derived from extraterrestrial delivery.     

Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.     

Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.Lignin is a major structural and protective component of plant cell walls. Lignin exists as a polymer of mainly three hydroxycinnamyl alcohols and related compounds, referred to as monolignols. The most common monolignols are coniferyl, sinapyl, and p-coumaryl alcohol (Ralph et al., 2004; Vanholme et al., 2010). After polymerization, structures derived from those compounds are referred to as guaiacyl, syringyl, and p-hydroxyphenyl subunits, respectively. The specific composition of lignin subunits varies among species, tissues, and developmental stages. Gymnosperm trees produce lignin that is primarily made of guaiacyl subunits, angiosperm trees contain guaiacyl and syringyl subunits, whereas grasses contain guaiacyl and syringyl subunits with small amounts (approximately 5%) of p-hydroxyphenyl residues. This observed variation in subunit composition across species may reflect the heterogeneity in substrate specificity and kinetic parameters among various monolignol biosynthetic enzymes (Weng et al., 2008).Biosynthesis of the monolignols occurs via the phenylpropanoid pathway using Phe precursors (Vanholme et al., 2010). Phe ammonia lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from Phe (Vanholme et al., 2010). Grasses can bypass cinnamate-4-hydroxylase by using Tyr as a substrate for Phe ammonia lyase (Neish, 1961; Rösler et al., 1997). The hydroxycinnamoyltransferase (HCT) enzymes exchange the CoA functionality esterified to p-coumaric acid with shikimic or quinic acid to allow for the subsequent conversion of the p-coumaroyl moiety to a caffeoyl moiety by p-coumarate-3′-hydroxylase (C3′H). The hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferases (HSTs) exhibit preference for shikimate, whereas the hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferases prefer quinate as a substrate (Sander and Petersen, 2011). Subsequent reactions ultimately lead to coniferyl and sinapyl alcohol via reduction of the γ-carbon on the propane side chain and substitution of the C3 and C5 positions of the phenol ring (Boerjan et al., 2003).Sorghum (Sorghum bicolor) is an attractive bioenergy crop with typical dry biomass yields between 20 and 25 Mg ha⁻¹ and yields as high as 40 Mg ha⁻¹ possible under optimal conditions (Venuto and Kindiger, 2008). Moreover, sorghum utilizes nitrogen-based fertilizer more efficiently than maize (Zea mays) and sugarcane (Saccharum officinarum), leading to less groundwater contamination and lower CO₂ emission (Propheter and Staggenborg, 2010; Wortmann and Regassa, 2011). Overall, sorghum has a higher sugar yield potential per land area and requires less water for growth than maize, allowing it to grow in a more diverse range of environments (Saballos, 2008). The sorghum genome sequence has been released (Paterson et al., 2009), and Targeting Induced Local Lesions in Genomes populations exist (Xin et al., 2008) in which various cell wall mutants have been identified (Sattler et al., 2012; Vermerris and Saballos, 2012).A detailed understanding of the catalytic mechanism of phenylpropanoid-related enzymes will enable the targeted modification of lignin subunit composition. The presence of lignin poses a major obstacle to the production of biofuels and chemicals from lignocellulosic biomass, because of its ability to hinder the activity of enzymes required to degrade cellulose to sugars that can be fermented for ethanol production (Yang and Wyman, 2004; Berlin et al., 2006). Genetic modification of plant cell wall composition, especially lignin content and subunit composition, has been shown to improve biomass conversion to fermentable sugars (Chen and Dixon, 2007; Vermerris et al., 2007; Jung et al., 2012). In particular, HCT silencing in Arabidopsis (Arabidopsis thaliana) causes an accumulation of p-hydroxyphenyl residues in the lignin and decreased content of guaiacyl and syringyl residues, leading to a dwarf phenotype (Li et al., 2010). Down-regulation of HCT has also been shown to result in decreased plant growth in alfalfa (Medicago sativa; Shadle et al., 2007). Concomitantly, ruminant digestibility and the yield of fermentable sugars following enzymatic saccharification increased (Chen and Dixon, 2007; Shadle et al., 2007). Reduced HCT activity may alter cell wall polymer interactions and allow better access of cellulolytic enzymes to the cellulose. Therefore, it has the potential to reduce the energy and processing costs associated with the conversion of biomass to fuels and chemicals. However fine-tuning will be necessary to limit the negative impacts on plant growth, which will require a detailed understanding of the catalytic mechanism of HCT.Given the difference in lignin subunit composition among different species and the prominence of grasses among dedicated bioenergy crops, we have focused on elucidating the crystal structure and activity of monolignol-related enzymes of sorghum, starting with the HST-like HCT. HCT belongs to the BAHD superfamily of plant-specific acyl-CoA-dependent acyltransferases (Ma et al., 2005; D’Auria, 2006). However, the BAHD superfamily has functionally and structurally diverse members that frequently possess little (as low as 10%) sequence identity among them (St-Pierre and Luca, 2000). Recent studies led to the crystal structure of the HST-like HCT from robusta coffee (Coffea canephora), an angiosperm dicot with a binding pocket elucidated by molecular docking and mutagenesis (Lallemand et al., 2012). In this report, we present the three-dimensional structures of HCT in its apo-form and ternary complex, supplemented by mutagenic studies to elucidate its reaction mechanism and structural relationship to other members in this growing functional class.     

Identification and Functional Characterization of Monofunctional ent-Copalyl Diphosphate and ent-Kaurene Synthases in White Spruce Reveal Different Patterns for Diterpene Synthase Evolution for Primary and Secondary Metabolism in Gymnosperms

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of expressed sequence tag, full-length cDNA, genomic DNA, and targeted bacterial artificial chromosome sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologs in Sitka spruce (Picea sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.Conifers (Coniferophyta) are well known for producing an abundant and diverse assortment of oleoresin diterpenoids, predominantly in the form of diterpene resin acids from specialized (or secondary) metabolism, that play roles in conifer defense (Trapp and Croteau, 2001a; Keeling and Bohlmann, 2006a; Bohlmann, 2008) and are an important source of biomaterials (Bohlmann and Keeling, 2008). Several conifer diterpene synthases (diTPSs) that biosynthesize these compounds have been functionally characterized (Stofer Vogel et al., 1996; Peters et al., 2000; Martin et al., 2004; Keeling and Bohlmann, 2006b; Ro and Bohlmann, 2006). The formation of diterpene resin acids of conifer specialized metabolism parallels the formation of ent-kaurenoic acid in the biosynthesis of the gibberellin diterpenoid phytohormones (Fig. 1; Keeling and Bohlmann, 2006a; Yamaguchi, 2008). In gibberellin biosynthesis, geranylgeranyl diphosphate (GGPP) is cyclized by diTPS activity to ent-copalyl diphosphate (ent-CPP), and the ent-CPP is further cyclized by diTPS activity to ent-kaurene. A cytochrome P450 (P450)-dependent monooxygenase (CYP701) oxidizes ent-kaurene to ent-kaurenoic acid (Davidson et al., 2006), paralleling the activity of a P450 (CYP720B1) that oxidizes abietadiene to abietic acid in conifer diterpene resin acid biosynthesis (Ro et al., 2005). Other P450s further functionalize ent-kaurenoic acid to form the biologically active gibberellins. Surprisingly, no conifer diTPS involved in the general (or primary) metabolism of gibberellins has been reported to date, while metabolite profiles of gibberellins have been well characterized in conifers for their role in flowering (Moritz et al., 1990).Open in a separate windowFigure 1.Comparison of the biosynthesis of gibberellins, as it is known in angiosperm and lower plants, with the biosynthesis of diterpene resin acids in conifers, a large group of gymnosperm trees. In conifers, the formation of diterpene resin acids involves bifunctional diTPS (e.g. abietadiene synthase) for the stepwise cyclization of GGPP into diterpenes such as abietadiene via a copalyl diphosphate intermediate that moves between the two active sites of the bifunctional diTPS (Peters et al., 2001). The products of the diTPS are subsequently oxidized by P450 to the resin acids. In contrast, gibberellin biosynthesis in angiosperms requires two monofunctional diTPSs to convert GGPP into ent-kaurene, which is subsequently modified by P450s. The two monofunctional diTPSs in angiosperm gibberellin biosynthesis are CPS and KS. In the lower plant P. patens, the CPS and KS activities are combined in a bifunctional diTPS similar to the bifunctional diTPS in conifer diterpene resin acid biosynthesis. Prior to this work, to our knowledge, it was not known if the formation of gibberellins in a gymnosperm involves two monofunctional diTPSs, as in angiosperms, or a bifunctional diTPS, as in gymnosperm diterpene resin acid biosynthesis and in P. patens gibberellin biosynthesis. (Figure adapted from Keeling and Bohlmann [2006a].)In the fungi Gibberella fujikuroi (Toyomasu et al., 2000) and Phaeosphaeria species L487 (Kawaide et al., 1997) and in the primitive land plant Physcomitrella patens (Bryophyta; Hayashi et al., 2006; Anterola and Shanle, 2008), the formation of ent-kaurene from GGPP is catalyzed by bifunctional diTPS enzymes. These enzymes contain two active sites. The N-terminal active site domain harbors a conserved DXDD motif and catalyzes the protonation-initiated cyclization of GGPP to ent-CPP (Prisic et al., 2007). In the C-terminal active site domain, a conserved DDXXD motif is essential for the diphosphate ionization-initiated cyclization of ent-CPP to ent-kaurene (Christianson, 2006). The presence of two active sites with their characteristic DXDD and DDXXD motifs resembles the structure of conifer bifunctional diTPSs in specialized metabolism of diterpene resin acid biosynthesis (Fig. 1), such as the grand fir (Abies grandis) abietadiene synthase (AgAS) and Norway spruce (Picea abies) levopimaradiene/abietadiene synthases (PaLAS; Peters et al., 2001; Martin et al., 2004; Keeling and Bohlmann, 2006a). In contrast, the formation of ent-kaurene from GGPP in angiosperms is catalyzed by two separate monofunctional enzymes, one with only the DXDD motif and having ent-copalyl diphosphate synthase (ent-CPS) activity and the other with only the DDXXD motif and having ent-kaurene synthase (ent-KS) activity (Yamaguchi, 2008).A previously published model for the evolution of plant diTPS (Trapp and Croteau, 2001b) suggests that genes encoding the monofunctional CPS and KS enzymes known in angiosperms originated by gene duplication and subfunctionalization (Lynch and Force, 2000) of an ancestral bifunctional CPS/KS gene that may have been similar to the gene for the CPS/KS enzyme of the moss P. patens. The same model also suggests that genes for diTPSs of gymnosperm specialized diterpene resin acid metabolism arose from duplication and subsequent neofunctionalization of an ancestral bifunctional diTPS of the gibberellin pathway (Trapp and Croteau, 2001b). The pathways to specialized oleoresin diterpenes existed in ancient plants prior to the differentiation of gymnosperms and angiosperms (Bray and Anderson, 2009). Vascular plants split from nonvascular plants approximately 500 million years ago, and angiosperms split from gymnosperms approximately 300 million years ago (Palmer et al., 2004). As there has been no report to date of genes involved in gibberellin biosynthesis in gymnosperms, it remains unresolved and cannot be predicted whether conifers have a bifunctional CPS/KS for the formation of ent-kaurene similar to the primitive land plant P. patens and paralleling the diTPSs for conifer specialized diterpene resin acid biosynthesis or whether they have separate monofunctional CPS and KS enzymes, as is the case in angiosperms.In this study, we made use of the extensive EST resources for spruce species (Pavy et al., 2005; Ralph et al., 2008), combined with isolation and sequencing of full-length cDNAs, genomic (g)DNA, and targeted bacterial artificial chromosome (BAC) clones, as well as enzyme assays with recombinant proteins to search for, and functionally characterize, possible monofunctional or bifunctional diTPS for ent-kaurene biosynthesis in a gymnosperm. In summary, we successfully isolated and characterized monofunctional ent-CPS (PgCPS) and ent-KS (PgKS) from white spruce (Picea glauca) and isolated orthologous cDNAs from Sitka spruce (Picea sitchensis). Comparison of enzyme functions and gene structures support common ancestry but different routes of evolution of monofunctional and bifunctional diTPS in conifer general and specialized metabolism, respectively.     

Duplicate Genes and the Root of Angiosperms, with an Example Using Phytochrome Sequences

The root of the angiosperm tree has not yet been established. Major morphological and molecular differences between angiosperms and other seed plants have introduced ambiguities and possibly spurious results. Because it is unlikely that extant species more closely related to angiosperms will be discovered, and because relevant fossils will almost certainly not yield molecular data, the use of duplicate genes for rooting purposes may provide the best hope of a solution. Simultaneous analysis of the genes resulting from a gene duplication event along the branch subtending angiosperms would yield an unrooted network, wherein two congruent gene trees should be connected by a single branch. In these circumstances the best rooted species tree is the one that corresponds to the two gene trees when the network is rooted along the connecting branch. In general, this approach can be viewed as choosing among rooted species trees by minimizing hypothesized events such as gene duplication, gene loss, lineage sorting, and lateral transfer. Of those gene families that are potentially relevant to the angiosperm problem, phytochrome genes warrant special attention. Phylogenetic analysis of a sample of complete phytochrome (PHY) sequences implies that an initial duplication event preceded (or occurred early within) the radiation of seed plants and that each of the two resulting copies duplicated again. In one of these cases, leading to thePHYAandPHYClineages, duplication appears to have occurred before the diversification of angiosperms. Duplicate gene trees are congruent in these broad analyses, but the sample of sequences is too limited to provide much insight into the rooting question. Preliminary analyses of partialPHYAandPHYCsequences from several presumably basal angiosperm lineages are promising, but more data are needed to critically evaluate the power of these genes to resolve the angiosperm radiation.     

Retention of Browning Compounds by Yeasts Involved in the Winemaking of Sherry Type Wines

Wine model solutions were used to study the ability of dehydrated yeasts to retain the brown products formed in the reaction between (+)-catechin and acetaldehyde. Saccharomyces cerevisiae races capensis and bayanus, two typical flor yeasts involved in the biological aging of sherry wines, had a higher capacity to retain coloured compounds than S. cerevisiae fermentative yeast. Of the flor yeasts, capensis exhibited a higher colour reduction capacity than bayanus. Such differences may account for the different rate at which browning compounds are removed at different times of year during the biological aging of wines.     

Oxidation of Free Methionine and Methionine Residues in Protein Involved in the Browning Reaction of Phenolic Compounds

The oxidation of methionine to its sulfoxide, as a possible cause of decrease in the biological value of red clover during drying with aeration, was examined using various model systems, in the presence or absence of polyphenol oxidase. The effects of catalase were also examined. Results indicated hydrogen peroxide as a possible intermediate that directly oxidizes methionine. The methionine oxidation can be one of the causes of the decrease in biological value of red clover during drying, beside the known damage of lysine in the same process.     

Lotus Cell Walls and the Genes Involved in its Synthesis and Modification

The lotus genome (Nelumbo nucifera (Gaertn.)) lacks the paleo-triplication found in other eudicots and has evolved remarkably slowly with fewer nucleotide mutations. It is thought to have greater retention of duplicated genes than other angiosperms. We evaluated the potential genes involved in cell wall synthesis and its modification, and ethylene synthesis and response. In many cell wall transferases and hydrolases families, lotus had fewer members in most families when compared to Arabidopsis. Lotus had similar or fewer members in each family as found in poplar, grape and papaya. The exceptions were in the sialyl and beta-glucuronsyl transferases where similar number were found as in the core eudicots. Lotus had similar numbers of polygalacturonase and pectin methyl esterases as found in Arabidopsis but fewer in all other hydrolases families. For starch degradation, lotus had only two alpha amylases predicted genes versus eight to ten in other eudicots, with similar numbers of beta amylase genes predicted. Lotus also had less than half the number of genes predicted for the enzymes involved in lignin and tannin synthesis compared to Arabidopsis. The stress plant growth regulator ethylene’s synthesis, reception and response predicted genes were fewer in lotus than other eudicots. Only two ethylene receptor genes were predicted in lotus with five reported for Arabidopsis and six for tomato. Our analysis does not supports the conclusion that this species has greater retention of duplicated genes though our data does support the conclusion that lotus split occurred at the base of the eudicots.     

Interactions Between Different Selenium Compounds and Essential Trace Elements Involved in the Antioxidant System of Laying Hens

Biological Trace Element Research - The purpose of this study was to investigate the interactions between different selenium (Se) compounds including sodium selenite (SS), selenium-enriched yeast...     

Antiglycation Effects of Carnosine and Other Compounds on the Long-Term Survival of Escherichia coli

Glycation, or nonenzymatic glycosylation, is a chemical reaction between reactive carbonyl-containing compounds and biomolecules containing free amino groups. Carbonyl-containing compounds include reducing sugars such as glucose or fructose, carbohydrate-derived compounds such as methylglyoxal and glyoxal, and nonsugars such as polyunsaturated fatty acids. The latter group includes molecules such as proteins, DNA, and amino lipids. Glycation-induced damage to these biomolecules has been shown to be a contributing factor in human disorders such as Alzheimer''s disease, atherosclerosis, and cataracts and in diabetic complications. Glycation also affects Escherichia coli under standard laboratory conditions, leading to a decline in bacterial population density and long-term survival. Here we have shown that as E. coli aged in batch culture, the amount of carboxymethyl lysine, an advanced glycation end product, accumulated over time and that this accumulation was affected by the addition of glucose to the culture medium. The addition of excess glucose or methylglyoxal to the culture medium resulted in a dose-dependent loss of cell viability. We have also demonstrated that glyoxylase enzyme GloA plays a role in cell survival during glycation stress. In addition, we have provided evidence that carnosine, folic acid, and aminoguanidine inhibit glycation in prokaryotes. These agents may also prove to be beneficial to eukaryotes since the chemical processes of glycation are similar in these two domains of life.One factor that may affect the long-term survival of bacterial cells in a population is the level of damage incurred by macromolecules via the nonenzymatic process of glycation, first described by Louis-Camille Maillard (16). The Maillard reaction is responsible for the formation of several compounds identified as advanced glycation end products (AGEs) (9). In vivo this reaction appears to play a role in the aging process, as it leads to slow degradation of molecules. The principal mechanisms of glycation-related damage involve cross-links between proteins and/or DNA, modifying or destroying their functional properties (2, 8, 38). Most studies of glycation have been performed with eukaryotes because of its relationship to aging and disorders such as Alzheimer''s disease and diabetes (6, 21, 30, 42). However, several studies (32, 33) have shown that glycation also takes place in Escherichia coli, affecting protein and DNA of this prokaryote.Many biochemical pathways produce reactive dicarbonyl intermediates, such as glyoxal and methylglyoxal (MG), which can further react with DNA, proteins, or other biomolecules to form AGEs (8, 36). Reaction of glucose with amino groups of proteins and subsequent formation of reactive dicarbonyls via a series of reactions involving Schiff base and Amadori product intermediates have been well documented (40). Methylglyoxal can be formed by spontaneous decomposition of glycolytic triose phosphates such as dihydroxyacetone phosphate (DHAP) (1) or can be produced enzymatically from DHAP by the E. coli enzyme methylglyoxal synthase (MgsA) (12). MG synthesis usually requires an environment low in phosphate and high in DHAP, a situation that occurs most frequently under high-glucose conditions (25, 26). If MG is not degraded, MG accumulation will lead to cell death (12). E. coli maintains pathways for the detoxification of methylglyoxal, including glyoxalase enzymes I and II (encoded by gloA and gloB, respectively), which convert MG to S-lactoyl glutathione and then to d-lactate (12). This system has been proposed to be the predominant MG detoxification system in E. coli (12, 29).Glyoxal is also a toxic dicarbonyl compound capable of damaging cells via AGE formation. One of the AGEs formed in the presence of glyoxal is carboxymethyl lysine (CML), which has been used extensively as a biomarker for aging (11, 20, 31, 39). CML can be formed by different pathways: glucose can be oxidized to glyoxal, which can react with protein to form CML (1, 17); glucose can also react with protein to form fructoselysine (an Amadori product), which can undergo oxidative cleavage to form CML (1). In this study, we investigated CML formation in E. coli growing under standard and glycation-prone laboratory conditions. Since AGE formation may negatively affect cell survival and reproduction during long-term batch culture (35), we hypothesized that CML would accumulate in these cultures as cells progress through stationary phase.One product that may interfere with AGE formation is carnosine (β-alanyl-l-histidine), a naturally occurring dipeptide in many organisms. Although its mechanism of action has not been fully determined, there is evidence that both the free amino group derived from the β-alanine and the imidazole ring of histidine compete with amino groups of proteins in the presence of reactive dicarbonyl compounds (7, 24). In this study we designed assays to determine the effect of carnosine (and other compounds) on survival of cultures of E. coli under a variety of experimental conditions. Additionally, since strains lacking glyoxalase enzymes I and II have a reduced ability to detoxify methylglyoxal, we hypothesized that gloA and/or gloB mutants would require larger amounts of carnosine than would wild-type strains to survive in the presence of this toxic electrophile.     

Identification of a Polyprenylphosphomannosyl Synthase Involved in the Synthesis of Mycobacterial Mannosides

We report on the identification of a glycosyltransferase (GT) from Mycobacterium tuberculosis H37Rv, Rv3779, of the membranous GT-C superfamily responsible for the direct synthesis of polyprenyl-phospho-mannopyranose and thus indirectly for lipoarabinomannan, lipomannan, and the higher-order phosphatidyl-myo-inositol mannosides.The mycobacterial cell envelope consists of a multilayered structure of covalently linked peptidoglycan, arabinogalactan, and mycolic acids (the mAGP complex) and, among other important constituents, various noncovalently bound glycosylated lipids, notably the phosphatidyl-myo-inositol mannosides (PIMs) and their more glycosylated end products lipomannan (LM) and lipoarabinomannan (LAM) (6, 8). These glycolipids and lipoglycans exhibit a broad range of immunomodulatory activities implicated in the pathogenesis of tuberculosis and leprosy (for recent reviews, see references 5, 8, and 10).Many steps in the biosynthesis of these phosphoinositides have been defined (for recent reviews, see references 3 and 12). Mannosyltransferases (ManTs) responsible for the polymerization aspects of the synthesis of the higher-order extracytoplasmic PIMs (PIM₄ to PIM₆), LM and LAM, are of the glycosyltransferase C (GT-C) multi-transmembrane domain superfamily, whose members are dependent on polyprenyl-phospho-mannopyranose (polyprenyl-P-Manp), specifically C₃₅-P-Man or C₅₀-P-Man, as the Manp donor, which is in contrast with what occurs in the early steps of the synthesis of the simpler PIMs, which directly utilize GDP-Man (3, 4, 12). However, the biosynthetic origins of C₃₅/C₅₀-P-Man have not been fully explored. In this report, we identify Rv3779, an unassigned GT-C, as a ManT directly responsible for the origins of polyprenyl-P-Man and indirectly for the synthesis of the more polar PIMs, LM and LAM.A survey of the Mycobacterium tuberculosis H37Rv genome for genes with predicted (poly)saccharide-associated functions led to the identification of a cluster of 31 “cell wall biosynthetic genes” (2, 3), including not only arabinogalactan synthetic genes (embCA and embCB, glf, glfT1, and glfT2) but the genes for the putative ABC transporter proteins and mycolyl transferases and Rv3779, among five open reading frames, apparently encoding polyprenyl-P sugar-dependent GT-Cs (3). In particular, the integral membrane protein Rv3779 (666 amino acids) was identified as a putative GT-C, due to a conserved hallmark DXD motif and 12 to 14 predicted membrane-spanning domains (3). However, unlike with other GT-C enzymes, which typically carry the signature DXD motif on extracytoplasmic loops, the DLD motif of Rv3779 (at amino acid position 82) is predicted to map to the second loop and to be on the cytosolic side of the plasma membrane. This observation suggests that unlike other GT-C superfamily enzymes, Rv3779 utilizes a cytosolic sugar donor, presumably a nucleotide sugar. Rv3779 has orthologs in all slow-growing mycobacteria whose genome sequences are available but is not found in Mycobacterium smegmatis or in any fast-growing mycobacteria, with the notable exception of Mycobacterium abscessus. Orthologs of this gene are also not found in any other members of the suborder Corynebacterineae.Since Rv3779 is not naturally present in M. smegmatis, M. smegmatis strain mc²155 was transformed with a multicopy plasmid (pVV16-Rv3779), allowing the expression of a recombinant C-terminal His₆-tagged Rv3779 protein under the control of the phsp60 promoter (13). A cell-free ManT assay using membranes or whole lysate, similar to the one described by Korduláková et al. (13), was then conducted to determine if Rv3779 is involved in some aspect of PIM/LM/LAM biosynthesis. Thin-layer chromatography (TLC) autoradiography demonstrated an approximately threefold increase in a time-dependent manner in the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into C₃₅-P-Man and C₅₀-P-Man by mc²155/pVV16-Rv3779 extracts compared with that of the control (Fig. ​(Fig.1A).1A). The two accumulated Man-containing glycolipids were mildly alkali stable and mildly acid labile (data not shown), with TLC mobility properties confirming their identities as precursor glycolipids of the mycobacterial polyisoprenyl-P class (4). An important and substrate concentration-dependent increase in mannosyl transfer following Rv3779 overexpression was also observed when C₅₀-P, the only form of polyprenylphosphate apparently produced by M. tuberculosis (7), was added as an acceptor substrate to the reaction mixture (Fig. 1B and C). This increase in activity over the control was about 27-fold during the first 30 min of the reaction when 0.05 mM C₅₀-P was used in the assay (Fig. ​(Fig.1C).1C). The background polyprenyl-P-Manp syntase activity detected in the control strain most likely resulted from MSMEG_3859/MSMEG_3860 (Ppm1/Ppm2), an M. smegmatis ortholog of the Ppm1 synthase from M. tuberculosis (68% identity, as determined with a 781-amino-acid overlap) (1, 9, 11). Unfortunately but not unexpectedly in light of the reported difficulty of expressing polytopic membrane GTs in Escherichia coli (14), attempts to produce Rv3779 in an active form in E. coli using different expression systems, host strains, growth conditions, and induction protocols were unsuccessful.Open in a separate windowFIG. 1.Effect of Rv3779 overexpression on the incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into mannolipids by cell extracts from M. smegmatis. (A) TLC analysis of an in vitro cell-free assay using GDP-[¹⁴C]Man and crude cell lysates from mc²155/pVV16 and mc²155/pVV16-Rv3779. Lysates were incubated with GDP-[¹⁴C]Man at 37°C for the indicated periods of time. The synthesized mannolipids were extracted with CHCl₃-CH₃OH (2:1, vol/vol), and a 10% aliquot of each sample was analyzed by TLC followed by autoradiography. The TLC plate was developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). Lanes: C, mc²155/pVV16; O, mc²155/pVV16-Rv3779. (B and C) Incorporation of [¹⁴C]Man from GDP-[¹⁴C]Man into exogenous decaprenyl-phosphate (C₅₀)-P using membrane extracts from mc²155/pVV16 and mc²155/pVV16-Rv3779. (B) TLC analysis of the first 15 min of the in vitro cell-free assays using 0.5 mM of (C₅₀)-P. (C) Quantification of the Man incorporated by the control (open symbols) and overexpressor (filled symbols) into C₅₀-P-Man over time. The concentrations of (C₅₀)-P used in the assays were 0 (circles), 0.005 (rectangles), and 0.05 (diamonds) mM. The inset shows a closeup of results of the assays using 0 and 0.005 mM (C₅₀)-P.Incubation of whole-cell lysate with GDP-[¹⁴C]Man for more-extended periods (4 to 24 h) and extraction of products with more-polar solvents (e.g., hot phenol) provides a ready estimate of the degree of synthesis of at least the LM metabolic end product. A comparison of the products synthesized by mc²155/pVV16 and mc²155/pVV16-Rv3779 (Fig. ​(Fig.2)2) showed little synthesis of [¹⁴C]LM at 4 h but the production of some low-molecular-weight [¹⁴C]LM at 24 h by the control lysate. In contrast, there was a distinct production of [¹⁴C]LM populations by the lysate from mc²155/pVV16-Rv3779 at both times, indicative of a secondary stimulatory effect of Rv3779 overexpression on LM, and presumably LAM, synthesis.Open in a separate windowFIG. 2.In vitro LM biosynthesis in M. smegmatis overexpressing Rv3779. Crude cell extracts (4 mg protein) of mc²155/pVV16 and mc²155/pVV16-Rv3779 were incubated with 1.0 μCi of GDP-[¹⁴C]Man (specific activity, 305 mCi/mmol) for 4 and 24 h. The reaction was stopped by adding CHCl₃-CH₃OH (2:1, vol/vol), and the LM/LAM contained in the cell pellet was extracted with hot phenol. LM/LAM separated on 10 to 20% Tricine gel and subsequently blotted to a nitrocellulose membrane was revealed by autoradiography. The leftmost column shows molecular mass markers (in kilodaltons).To confirm these effects of Rv3779, an M. tuberculosis H37Rv Rv3779 knockout mutant (H37RvΔRv3779) was generated by homologous recombination using standard procedures (15) (see Fig. S1 in the supplemental material). H37RvΔRv3779 grew at a much lower rate than WT H37Rv (see Fig. S2 in the supplemental material). Moreover, when examined by scanning electron microscopy, the mutant cells were found to be significantly shorter than their wild-type (WT) parent (Fig. ​(Fig.3).3). Normal growth rate and cell length were restored in the mutant upon complementation with a WT copy of Rv3779 (Fig. ​(Fig.3;3; see Fig. S2 in the supplemental material). TLC profiles of lipid extracts and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses showed a profound decrease in the amounts of acyl-PIM₆ (AcPIM₆) and Ac₂PIM₆ in H37RvΔRv3779 compared to those of the WT strain (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). Importantly, the synthesis of the simpler PIMs, those arising directly from GDP-Man, was not altered in the mutant (Fig. ​(Fig.4A;4A; see Fig. S3 in the supplemental material). The examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with the monoclonal antibody CS-35 and concanavalin A of the phenol-extracted LM/LAM fraction from WT H37Rv and H37RvΔRv3779 also revealed considerably smaller amounts of both LM and LAM in the mutant (Fig. ​(Fig.4B).4B). Complementation of H37RvΔRv3779 with a WT copy of Rv3779 restored both a normal polar PIM and a normal LM/LAM profile to the mutant.Open in a separate windowFIG. 3.Scanning electron micrographs of M. tuberculosis H37Rv (WT), the mutant H37RvΔRv3779, and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 cultured in 7H9-oleic acid-albumin-dextrose-catalase-Tween 80 broth at 37°C. The length of the H37RvΔRv3779 mutant cells was on average 1.2 ± 0.1 μm, compared to 2.3 ± 0.1 μm for the WT strain and 2.1 ± 0.3 μm for the complemented mutant.Open in a separate windowFIG. 4.Analysis of polar PIMs LM and LAM from WT M. tuberculosis H₃₇Rv, the H37RvΔRv3779 mutant, and H37RvΔRv3779/pVV16-Rv3779. (A) Equal amounts of total cellular lipids from WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), the mutant carrying an empty plasmid, H37RvΔRv3779/pVV16 (lane 3), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 4) were analyzed by TLC developed in CHCl₃-CH₃OH-H₂O-NH₄OH (65:25:4:0.5). (B) LM and LAM extracted from equal-weight cells of WT H₃₇Rv (lane 1), the Rv3779 mutant H37RvΔRv3779 (lane 2), and the complemented mutant H37RvΔRv3779/pVV16-Rv3779 (lane 3) were separated on a 10 to 20% Tricine gel and revealed by periodic acid-Schiff staining. The Western blot analyses were performed on the same samples using concanavalin A (ConA) reacting with the t-Manp residues of LM/LAM, as well as the CS-35 monoclonal antibody known to react with the arabinan segment of LAM.From this evidence, we conclude that Rv3779 is involved in the synthesis of the polar PIMs LM and LAM through its primary role as a polyprenyl-P-Man synthase. The disruption of this gene, which results in important alterations in PIM, LM, and LAM profiles, also has profound effects on cell growth and shape. Rv3779 is the second polyprenyl-P-Man synthase involved in PIM/LM/LAM biosynthesis identified in M. tuberculosis (11).      

Roles of Oxygen and the Intestinal Microflora in the Metabolism of Lignin-Derived Phenylpropanoids and Other Monoaromatic Compounds by Termites

Prompted by our limited understanding of the degradation of lignin and lignin-derived aromatic metabolites in termites, we studied the metabolism of monoaromatic model compounds by termites and their gut microflora. Feeding trials performed with [ring-U-(sup14)C]benzoic acid and [ring-U-(sup14)C]cinnamic acid revealed the general ability of termites of the major feeding guilds (wood and soil feeders and fungus cultivators) to mineralize the aromatic nucleus. Up to 70% of the radioactive label was released as (sup14)CO(inf2); the remainder was more or less equally distributed among termite bodies, gut contents, and feces. Gut homogenates of the wood-feeding termites Nasutitermes lujae (Wasmann) and Reticulitermes flavipes (Kollar) mineralized ring-labeled benzoic or cinnamic acid only if oxygen was present. In the absence of oxygen, benzoate was not attacked, and cinnamate was only reduced to phenylpropionate. Similar results were obtained with other, nonlabeled lignin-related phenylpropanoids (ferulic, 3,4-dihydroxycinnamic, and 4-hydroxycinnamic acids), whose ring moieties underwent degradation only if oxygen was present. Under anoxic conditions, the substrates were merely modified (by side chain reduction and demethylation), and this modification occurred at the same time as a net accumulation of phenylpropanoids formed endogenously in the gut homogenate, a phenomenon not observed under oxic conditions. Enumeration by the most-probable-number technique revealed that each N. lujae gut contained about 10(sup5) bacteria that were capable of completely mineralizing aromatic substrates in the presence of oxygen (about 10(sup8) bacteria per ml). In the absence of oxygen, small numbers of ring-modifying microorganisms were found (<50 bacteria per gut), but none of these microorganisms were capable of ring cleavage. Similar results were obtained with gut homogenates of R. flavipes, except that a larger number of anaerobic ring-modifying microorganisms was present (>5 x 10(sup3) bacteria per gut). Neither inclusion of potential cosubstrates (H(inf2), pyruvate, lactate) nor inclusion of hydrogenotrophic partner organisms resulted in anoxic ring cleavage in most-probable-number tubes prepared with gut homogenates of either termite. The oxygen dependence of aromatic ring cleavage by the termite gut microbiota is consistent with the presence, and uptake by microbes, of O(inf2) in the peripheral region of otherwise anoxic gut lumina (as reported in the accompanying paper [A. Brune, D. Emerson, and J. A. Breznak, Appl. Environ. Microbiol. 61:2681-2687, 1995]). Taken together, our results indicate that microbial degradation of plant aromatic compounds can occur in termite guts and may contribute to the carbon and energy requirement of the host.