Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae

Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS’s profiled, 12 TPS’s were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.     

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.     

Cell-free Synthesis and Functional Characterization of Sphingolipid Synthases from Parasitic Trypanosomatid Protozoa

The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1–4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser²⁵²; TbSLS3, Phe²⁵²) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.     

Focus on Metabolism: Something Old,Something New: Conserved Enzymes and the Evolution of Novelty in Plant Specialized Metabolism

Plants produce hundreds of thousands of small molecules known as specialized metabolites, many of which are of economic and ecological importance. This remarkable variety is a consequence of the diversity and rapid evolution of specialized metabolic pathways. These novel biosynthetic pathways originate via gene duplication or by functional divergence of existing genes, and they subsequently evolve through selection and/or drift. Studies over the past two decades revealed that diverse specialized metabolic pathways have resulted from the incorporation of primary metabolic enzymes. We discuss examples of enzyme recruitment from primary metabolism and the variety of paths taken by duplicated primary metabolic enzymes toward integration into specialized metabolism. These examples provide insight into processes by which plant specialized metabolic pathways evolve and suggest approaches to discover enzymes of previously uncharacterized metabolic networks.The plant kingdom collectively produces hundreds of thousands of low molecular weight organic molecules traditionally known as secondary metabolites, some of which have been shown to play roles in abiotic and biotic stress responses (e.g. herbivory defense), beneficial insect interactions (e.g. pollinator attraction), and communication with other plant and nonplant species (e.g. allelopathy and legume-rhizobia interactions; Saito and Matsuda, 2010; Pichersky and Lewinsohn, 2011; Wink, 2011). These metabolites have been widely used throughout the course of human history as medicines, spices, perfumes, cosmetics, and pest-control agents as well as in religious and cultural rituals. For the past 150 years, there has been a strong focus on documenting the chemical diversity of secondary metabolites in the plant kingdom, leading to the discovery of diverse classes of compounds such as terpenes, flavonoids, alkaloids, phenylpropanoids, glucosinolates, and polyketides. These secondary compounds were historically differentiated from products of primary metabolism, such as sugars, amino acids, nucleic acids, and fatty acids, as being nonessential for plant survival (Sachs, 1874; Kossel, 1891; Hartmann, 2008). However, by the 1980s, important functional roles began to be elucidated for metabolites previously classified as secondary, such as the phenolics (e.g. plant-microbe interactions and UV-B light protection; Bolton et al., 1986; Peters et al., 1986; Li et al., 1993; Landry et al., 1995), alkaloids (defense against herbivory; Steppuhn et al., 2004), and terpenes (defense against herbivory, antimicrobial activities, and volatile pollinator attractants; Papadopoulou et al., 1999; Schiestl and Ayasse, 2001; Erbilgin et al., 2006; Nieuwenhuizen et al., 2009). This change in our understanding of their roles has led to the coining of a new term, specialized metabolites, for these compounds, both to acknowledge their importance and to reflect the fact that many of them are phylogenetically restricted (Pichersky et al., 2006; Pichersky and Lewinsohn, 2011).Although the structural diversity of specialized metabolites far exceeds that of primary metabolites, all specialized metabolite classes are ultimately derived from primary metabolic precursors (Wink, 2011). For example, phenylpropanoids are derived from the amino acid Phe (Vogt, 2010), while the biosynthetic blocks of terpenes, isopentenyl diphosphate and dimethylallyl diphosphate, originate from mevalonate, a sterol precursor, and alternatively from methylerythritol phosphate, which is derived from glycolytic pathway precursors (Kirby and Keasling, 2009). Nitrogen-containing alkaloids are derived from a variety of primary metabolites, including amino acids and purine nucleosides (Facchini, 2001). Over the past 20 years, an increasing number of specialized metabolic enzymes have also been found to have their origins in primary metabolic pathways (Weng, 2014). Such shifts in enzyme function are made possible primarily by the process of gene duplication, which is very common in plants.     

Molecular Cloning,Functional Characterization,and Evolutionary Analysis of Vitamin D Receptors Isolated from Basal Vertebrates

The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication (3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC₅₀ values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D₃ acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D₃ throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.     

Monoterpene Alcohol Metabolism: Identification, Purification, and Characterization of Two Geraniol Dehydrogenase Isoenzymes from Polygonum minus Leaves

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).     

Large-Scale Phylogenetic Classification of Fungal Chitin Synthases and Identification of a Putative Cell-Wall Metabolism Gene Cluster in Aspergillus Genomes

The cell wall is a protective and versatile structure distributed in all fungi. The component responsible for its rigidity is chitin, a product of chitin synthase (Chsp) enzymes. There are seven classes of chitin synthase genes (CHS) and the amount and type encoded in fungal genomes varies considerably from one species to another. Previous Chsp sequence analyses focused on their study as individual units, regardless of genomic context. The identification of blocks of conserved genes between genomes can provide important clues about the interactions and localization of chitin synthases. On the present study, we carried out an in silico search of all putative Chsp encoded in 54 full fungal genomes, encompassing 21 orders from five phyla. Phylogenetic studies of these Chsp were able to confidently classify 347 out of the 369 Chsp identified (94%). Patterns in the distribution of Chsp related to taxonomy were identified, the most prominent being related to the type of fungal growth. More importantly, a synteny analysis for genomic blocks centered on class IV Chsp (the most abundant and widely distributed Chsp class) identified a putative cell wall metabolism gene cluster in members of the genus Aspergillus, the first such association reported for any fungal genome.     

Focus on Metabolism: Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such as vasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase (CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, which has led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerial tissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 and S. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormone biosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome of the CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues, may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functional diterpenoids in plants.Salvia miltiorrhiza, a Lamiaceae species known as red sage or tanshen, is a traditional Chinese medicinal herb that is described in Shen Nong Ben Cao Jing, the oldest classical Chinese herbal book, which dates from between 25 and 220 C.E. The lipophilic pigments from the reddish root and rhizome consist of abietane quinone diterpenoids (Nakao and Fukushima, 1934), largely tanshinone IIA, cryptotanshinone, and tanshinone I (Zhong et al., 2009). These are highly bioactive. For example, tanshinone IIA exerts vasorelaxative activity, has antiarrhythmic effects, provides protection against ischemia reperfusion injury (Zhou et al., 2005; Gao et al., 2008; Sun et al., 2008), and exhibits anticancer activities (Efferth et al., 2008; Lee et al., 2008; Wang et al., 2008; Gong et al., 2011). In addition, tanshinones have been reported to have a broad spectrum of antimicrobial activities against various plant pathogens, including rice (Oryza sativa) blast fungus Magnaporthe oryzae (Zhao et al., 2011). Although tanshinones are mainly accumulated in the roots, trace amounts of tanshinones have been detected in aerial organs as well (Hang et al., 2008).Diterpenoid biosynthesis is initiated by diterpene synthases (diTPSs), which catalyze cyclization and/or rearrangement of the general acyclic precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to form various hydrocarbon backbone structures that are precursors to more specific families of diterpenoids (Zi et al., 2014). Previous work has indicated that tanshinone biosynthesis is initiated by cyclization of GGPP to copalyl diphosphate (CPP) by a CPP synthase (SmCPS1) and subsequent further cyclization to the abietane miltiradiene by a kaurene synthase-like cyclase (SmKSL1), so named for its homology to the ent-kaurene synthases (KSs) required for GA plant hormone biosynthesis (Gao et al., 2009). Miltiradiene is a precursor to at least cryptotanshinone (Guo et al., 2013), and RNA interference (RNAi) knockdown of SmCPS1 expression reduces tanshinone production, at least in hairy root cultures (Cheng et al., 2014). The identification of SmCPS1 and SmKSL1 has been followed by that of many related diTPSs from other Lamiaceae plant species (Caniard et al., 2012; Sallaud et al., 2012; Schalk et al., 2012; Brückner et al., 2014; Pateraki et al., 2014). These largely exhibit analogous activity, particularly the CPSs, which produce CPP or the stereochemically related 8α-hydroxy-labd-13E-en-15-yl diphosphate (LDPP) rather than the enantiomeric (ent) CPP relevant to GA biosynthesis.To further investigate diterpenoid biosynthesis in S. miltiorrhiza, we report here a more thorough characterization of its diTPS family. A previously reported whole-genome shotgun sequencing survey (Ma et al., 2012) has indicated that there are at least five CPSs, although only two KSL genes in S. miltiorrhiza (Supplemental Table S1). Intriguingly, based on a combination of biochemical and genetic (RNAi gene silencing) evidence, we find that these diTPSs nevertheless account for at least four different diterpenoid biosynthetic pathways, each dependent on a unique CPS, with the KS presumably involved in GA biosynthesis seeming to be responsible for alternative diterpenoid metabolism as well. In addition, our studies clarify the evolutionary basis for the observed functional diversity, with investigation of gene structure, positive selection, molecular docking, and mutational analysis used to explore the driving force for the functional divergence of these diTPSs. Moreover, we report metabolomic analysis, also carried out with SmCPS1 RNAi lines, which enables prediction of the downstream steps in tanshinone biosynthesis.     

Identification and Functional Characterization of a Xenorhabdus nematophila Oligopeptide Permease

The bacterium Xenorhabdus nematophila is a mutualist of Steinernema carpocapsae nematodes and a pathogen of insects. Presently, it is not known what nutrients the bacterium uses to thrive in these host environments. In other symbiotic bacteria, oligopeptide permeases have been shown to be important in host interactions, and we therefore sought to determine if oligopeptide uptake is essential for growth or symbiotic functions of X. nematophila in laboratory or host environments. We identified an X. nematophila oligopeptide permease (opp) operon of two sequential oppA genes, predicted to encode oligopeptide-binding proteins, and putative permease-encoding genes oppB, oppC, oppD, and oppF. Peptide-feeding studies indicated that this opp operon encodes a functional oligopeptide permease. We constructed strains with mutations in oppA₁, oppA₂, or oppB and examined the ability of each mutant strain to grow in a peptide-rich laboratory medium and to interact with the two hosts. We found that the opp mutant strains had altered growth phenotypes in the laboratory medium and in hemolymph isolated from larval insects. However, the opp mutant strains were capable of initiating and maintaining both mutualistic and pathogenic host interactions. These data demonstrate that the opp genes allow X. nematophila to utilize peptides as a nutrient source but that this function is not essential for the existence of X. nematophila in either of its host niches. To our knowledge, this study represents the first experimental analysis of the role of oligopeptide transport in mediating a mutualistic invertebrate-bacterium interaction.     

The Influence of Leaf Development on the Expression of C4 Metabolism in Flaveria trinervia, a C4 Dicot

The initial products of ¹⁴CO₂ assimilation were determined understeady state illumination of leaves of Flaveria trinervia, aC₄ dicot of the NADP-mialic enzyme subgroup. Leaf age influencedthe partitioning of ¹⁴CO₂ between the C₄ cycle and the reductivepentose phosphate (RPP) pathway. An estimated 10 to 12%of theCO₂ entered the RPP pathway directly in leaves about 20% fullyexpanded, whereas CO₂ was apparently fixed entirely throughthe C₄ pathway in leaves 75% or more expanded. This partitioningpattern was attributed to the bundle sheath compartment in youngleaves having a relatively high conductance to CO₂ (i.e., beingsomewhat leaky). Of the initially labelled C₄ acids, the proportion that wasmalate, relative to aspartate, increased continuously duringleaf expansion (from 60 : 40 to 87 : 13 at full expansion).Concurrently, there was an increase in the whole leaf activityof NADP malate dehydrogenase and a decrease in the activitiesof aspartate and alanine aminotransferases. Low chlorophylla/b values were observed in young leaves, which may coincidewith an enhanced capacity for non-cyclic electron transportin the bundle sheath chloroplasts of such tissue. Both enhancedaspartate metabolism and direct fixation of CO₂ in the bundlesheath could provide a greater sink for utilization of photochemicallyderived NADPH in the bundle sheath of young leaves. Such metabolicchanges are discussed in relation to a possible decrease inCO₂ conductance of the bundle sheath during leaf development. (Received March 4, 1986; Accepted June 25, 1986)     

Aromatic Polyketide Synthases (Purification,Characterization, and Antibody Development to Benzalacetone Synthase from Raspberry Fruits)

p-Hydroxyphenylbutan-2-one, the characteristic aroma compound of raspberries (Rubus idaeus L.), is synthesized from p-coumaryl-coenzyme A and malonyl-coenzyme A in a two-step reaction sequence that is catalyzed by benzalacetone synthase and benzalacetone reductase (W. Borejsza-Wysocki and G. Hrazdina [1994] Phytochemistry 35: 623-628). Benzalacetone synthase condenses one malonate with p-coumarate to form the pathway intermediate p-hydroxyphenylbut-3-ene-2-one (p-hydroxybenzalacetone) in a reaction that is similar to those catalyzed by chalcone and stilbene synthases. We have obtained an enzyme preparation from ripe raspberries that was preferentially enriched in benzalacetone synthase (approximately 170-fold) over chalcone synthase (approximately 14-fold) activity. This preparation was used to characterize benzalacetone synthase and to develop polyclonal antibodies in rabbits. Benzalacetone synthase showed similarity in its molecular properties to chalcone synthase but differed distinctly in its substrate specificity, response to 2-mercaptoethanol and ethylene glycol, and induction in cell-suspension cultures. The product of the enzyme, p-hydroxybenzalacetone, inhibited mycelial growth of the raspberry pathogen Phytophthora fragariae var rubi at 250 [mu]M. We do not know whether the dual activity in the benzalacetone synthase preparation is the result of a bifunctional enzyme or is caused by contamination with chalcone synthase that was also present. The rapid induction of the enzyme in cell-suspension cultures upon addition of yeast extract and the toxicity of its product, p-hydroxybenzalacetone, to phytopathogenic fungi also suggest that the pathway may be part of a plant defense response.     

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.     

ATP Synthases With Novel Rotor Subunits: New Insights into Structure,Function and Evolution of ATPases

ATPases with unusual membrane-embedded rotor subunits were found in both F₁F₀ and A₁A₀ ATP synthases. The rotor subunit c of A₁A₀ ATPases is, in most cases, similar to subunit c from F₀. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with “normal” F₀ c subunits in the Na⁺ F₁F₀ ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.