Geranyllinalool Synthases in Solanaceae and Other Angiosperms Constitute an Ancient Branch of Diterpene Synthases Involved in the Synthesis of Defensive Compounds

Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the terpene synthase (TPS) family and two Fabaceae GLSs that belong to the TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (Solanum lycopersicum) TPS-e/f gene, TPS46, encoding GLS (SlGLS) and its homolog (NaGLS) from Nicotiana attenuata. The K_m value of SlGLS for geranylgeranyl diphosphate was 18.7 µm, with a turnover rate value of 6.85 s^–1. In leaves and flowers of N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides, NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato, SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the TPS-e/f clade that diverged from kaurene synthases, also in the TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the GLS activity of genes in this branch in diverse eudicot families suggest that GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a TPS sequence belonging to this GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants.Geranyllinalool is an acyclic diterpene alcohol with a wide distribution in the plant kingdom; it has been identified as component of essential oils of distantly related plant species such as Jasmin grandiflorum, Michelia champaca, and Homamelis virginiana (Sandeep, 2009). Geranyllinalool is the precursor of 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), a volatile C₁₆-homoterpene emitted from the foliage of many angiosperm species including Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), maize (Zea mays), fava bean (Vicia faba), lima bean (Phaseolus lunatus), alfalfa (Medicago sativa), and Eucalyptus spp. (Van Poecke et al., 2001; Ament et al., 2004; Williams et al., 2005; Hopke et al., 1994; Leitner et al., 2010; Webster et al., 2010). In addition, various hydroxygeranyllinalool glycosides have been isolated from many Solanaceous species such as Capsicum annuum, Lycium chinense, and at least 26 Nicotiana species (Yahara et al., 1993; Iorizzi et al., 2001; Snook et al., 1997).The biosynthetic pathway leading to geranyllinalool, as for all other terpenoids, begins with the condensation of isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate. Sequential condensation of one isopentenyl diphosphate molecule with three dimethylallyl diphosphate molecules produces geranylgeranyl diphosphate (GGPP), the C-20 intermediate of the diterpenoid pathway. Next, a terpene synthase (TPS) catalyzes a two-step reaction in which carbocation formation of the C20 precursor is followed by an allylic rearrangement that results in the production of the tertiary alcohol geranyllinalool (Herde et al., 2008).Although geranyllinalool and its derivatives, TMTT and geranyllinalool glycosides, have been reported in a wide variety of plant species, a geranyllinalool synthase (GLS) involved in TMTT biosynthesis was only recently identified in Arabidopsis (Herde et al., 2008). AtTPS04 belongs to the TPS-e/f subfamily along with the previously identified Clarkia spp. linalool synthases (Chen et al., 2011). More recently, two TPSs from Vitis vinifera and one from the daisy Grindelia hirsutula, also members of the TPS-e/f subfamily, were found to exhibit GLS activity in vitro (Martin et al., 2010; Zerbe et al., 2013). However, no in planta information has been presented for these, nor any evidence showing their involvement in TMTT biosynthesis.The common characteristic of the TPS-e/f GLSs so far identified is that they lack a predicted plastidial transit peptide, and direct evidence for nonplastidial localization was obtained in Arabidopsis by observing the AtTPS04-GUS fusion protein in the cytosol and endoplasmic reticulum (Herde et al., 2008). On the other hand, two TPS-g subfamily proteins from the closely related Fabaceae species Medicago truncatula and Phaseolus lunata (MtTPS03 and PlTPS2, respectively) were shown to be plastidic and to catalyze the formation of geranyllinalool in vitro when GGPP was provided as a substrate and also when expressed in a heterologous plant species (Arimura et al., 2008; Brillada et al., 2013). However, the same enzymes also produced linalool and nerolidol when supplied with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively (Arimura et al., 2008; Brillada et al., 2013). Given the present paucity of in vivo and in vitro studies of geranyllinalool biosynthesis in plants, it is not clear whether geranyllinalool in plants is typically produced via TPS-g or TPS-e/f type TPSs, or both.The role of geranyllinalool itself in plant tissues is not well established. Often geranyllinalool coexists in floral or vegetative tissues with its homoterpene derivative TMTT. The contribution of TMTT to the floral scent of insect-pollinated species suggests a putative role in attraction of pollinators (Tholl et al., 2011). On the other hand, in many angiosperm species, including tomato, TMTT is a component of volatile blends released from vegetative tissues upon herbivore attack, sometimes in parallel with its constitutive emission from floral tissues (Hopke et al., 1994; Ament et al., 2004; de Boer et al., 2004; Kant et al., 2004; Williams et al., 2005, Herde et al., 2008). The latter case suggests that TMTT might play a defensive role in both vegetative and floral tissues. TMTT production from insect-infested plants is considered as an indirect defense mechanism because TMTT attracts insect predators of the insect herbivores (Brillada et al., 2013). Interestingly, production of TMTT, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene, from herbivore-attacked lima bean plants has been found to correlate with enhanced expression of defense genes in neighboring nonaffected control plants (Arimura et al., 2000). In these cases, homoterpenes are believed to act as stress-responsive signals that enable intraspecies plant-to-plant communication.A plant defense role has also been suggested for 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) present in leaves and flowers of Nicotiana species, with higher concentrations measured in buds (Heiling et al., 2010; Jassbi et al., 2010). Several studies have found negative correlation between total leaf HGL-DTG content and the mass of the larvae that feed on them (Jassbi et al., 2008; Dinh et al., 2013). Eleven HGL-DTGs that differ in sugar moieties and number of malonylesters have been isolated from Nicotiana attenuata. The sugar groups of these compounds are Glc and rhamnose and are conjugated to the hydroxygeranyllinalool skeleton via bonds at C3 and C17 hydroxylated carbons. Additional sugars may be added to these sugars on their hydroxyl groups at C2, C4, and C6, and manolyl esters are typically formed at the C6 hydroxyl group of the glucoses. The concentrations of these HGL-DTGs are higher in young and reproductive tissues. While their total levels appear to be constant, the concentration of individual compounds change upon herbivore attack, with a proportionally greater increase in malonylated compounds. Unlike many other defense-related specialized metabolites, the N. attenuata HGL-DTGs are not found on the leaf surface or the trichomes, but, instead, they accumulate inside the leaves (Heiling et al., 2010).Here, we show that in the Solanaceae species cultivated tomato and N. attenuata, geranyllinalool is synthesized by TPSs that belong to the TPS-e/f subfamily and that the corresponding genes are related to Arabidopsis TPS04. The tomato and N. attenuata enzymes were biochemically characterized, and the kinetic parameters were determined. We also describe a detailed quantitative expression of these genes in different parts of the plant. In addition, we establish that the expression of the geranyllinalool synthase genes correlates well with the induced emission of TMTT in tomato leaves after alamethicin and methyl jasmonate (MeJA) treatments and with the total concentrations of HGL-DTGs in N. attenuata leaves and floral organs.     

Functional Genomics Reveals That a Compact Terpene Synthase Gene Family Can Account for Terpene Volatile Production in Apple

Terpenes are specialized plant metabolites that act as attractants to pollinators and as defensive compounds against pathogens and herbivores, but they also play an important role in determining the quality of horticultural food products. We show that the genome of cultivated apple (Malus domestica) contains 55 putative terpene synthase (TPS) genes, of which only 10 are predicted to be functional. This low number of predicted functional TPS genes compared with other plant species was supported by the identification of only eight potentially functional TPS enzymes in apple ‘Royal Gala’ expressed sequence tag databases, including the previously characterized apple (E,E)-α-farnesene synthase. In planta functional characterization of these TPS enzymes showed that they could account for the majority of terpene volatiles produced in cv Royal Gala, including the sesquiterpenes germacrene-D and (E)-β-caryophyllene, the monoterpenes linalool and α-pinene, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene. Relative expression analysis of the TPS genes indicated that floral and vegetative tissues were the primary sites of terpene production in cv Royal Gala. However, production of cv Royal Gala floral-specific terpenes and TPS genes was observed in the fruit of some heritage apple cultivars. Our results suggest that the apple TPS gene family has been shaped by a combination of ancestral and more recent genome-wide duplication events. The relatively small number of functional enzymes suggests that the remaining terpenes produced in floral and vegetative and fruit tissues are maintained under a positive selective pressure, while the small number of terpenes found in the fruit of modern cultivars may be related to commercial breeding strategies.Terpenes (also referred to as terpenoids or isoprenoids) constitute a large class of plant natural products with highly diversified functionality. Terpenes serve as precursors for the biosynthesis of essential plant metabolites (Croteau et al., 2000), including those involved in growth regulation (GAs, abscisic acid, and strigolactones), membrane stabilization (sterols), photosynthesis (carotenoids and the phytol side chain of chlorophyll), and electron transport coenzymes (ubiquinone and plastoquinone). Primarily, however, terpenes function as specialized plant metabolites that operate as attractants to pollinators or as defensive compounds against pathogens and herbivores (Kessler and Baldwin, 2001; Pichersky and Gershenzon, 2002). Terpenes are also important in determining the quality of horticultural food products, including the taste and aroma of wine (Styger et al., 2011) and fruit crops such as Citrus spp. (Vora et al., 1983; Maccarone et al., 1998; Aharoni et al., 2004) and strawberry (Fragaria spp.; Aharoni et al., 2004).Terpenes are derived from linear assemblages of prenyldiphosphates, including the C10 monoterpene precursor geranyl diphosphate (GDP), the C15 sesquiterpene precursor farnesyl diphosphate (FDP), and the C20 diterpene precursor geranylgeranyl diphosphate (GGDP). The catalytic conversion of these relatively simple precursors to the diverse array of terpenes seen in nature is carried out by terpene synthase (TPS) enzymes, which have the capacity to direct myriad precursor-binding conformations through subtle variations in their conserved catalytic fold (Yoshikuni et al., 2006; O’Maille et al., 2008; Miller and Allemann, 2012).TPS gene families have been explored in Arabidopsis (Arabidopsis thaliana; Aubourg et al., 2002; Lange and Ghassemian, 2003), grape (Vitis vinifera; Martin et al., 2010), poplar (Populus trichocarpa; Tuskan et al., 2006), rice (Oryza sativa; Goff et al., 2002), and sorghum (Sorghum bicolor; Paterson et al., 2009) as well as most recently in cultivated tomato (Solanum lycopersicum; Bleeker et al., 2011; Falara et al., 2011). The identification of 44 tomato TPS genes concurs with the comparative genome analysis work of Chen et al. (2011), indicating that the plant TPS gene family is midsized, with gene numbers ranging from approximately 20 to 150. The only exception so far appears to be the moss Physcomitrella patens, which has a single functional TPS gene (Chen et al., 2011). This analysis has also extended the phylogenetics used to define the initial TPS subfamilies (TPS-a to TPS-g; Bohlmann et al., 1998; Dudareva et al., 2003; Martin et al., 2004), culminating in the merging of the TPS-e and TPS-f subgroups (comprising gymnosperm and angiosperm ent-kaurene genes associated with primary metabolism) and the TPS-h subgroup so far specific to the lycopod Selaginalla moellendorffii.The domestic apple (Malus domestica), which has long been recognized by consumers for its flavor, health, and nutritional properties (Harker et al., 2003), is one of the most widely cultivated fruit species in the world’s temperate regions. Apple belongs to the Rosaceae family, and while most Rosaceae have a haploid chromosome number of seven, eight, or nine, Malus spp., as the result of a relatively recent genome-wide duplication (GWD) event in the Pyreae, have transitioned from nine ancestral chromosomes to 17 chromosomes (Velasco et al., 2010).Apple fruit produce more than 300 volatile organic compounds (VOCs), including alcohols, aldehyde esters, and ketones (Dimick and Hoskin, 1983; Paillard, 1990; Dixon and Hewett, 2000). Various terpenes have also been identified, although they only contribute a relatively minor component of total VOCs produced (Rapparini et al., 2001; Fuhrmann and Grosch, 2002; Hern and Dorn, 2003; Rowan et al., 2009a). The specific VOC composition in apple depends on several factors, including cultivar, climacteric ethylene production levels, maturity, and environmental conditions (Loughrin et al., 1990; Dixon and Hewett, 2000; Rapparini et al., 2001; Vallat et al., 2005). While the acyclic branched sesquiterpene (E,E)-α-farnesene appears to be the predominant terpene volatile associated with ripe fruit (Sutherland et al., 1977; Bengtsson et al., 2001; Hern and Dorn, 2003; Ferreira et al., 2009; Rowan et al., 2009b), various monoterpenes, cyclic sesquiterpenes, and terpene derivatives have also been identified, particularly in floral and vegetative tissues (Takabayashi et al., 1991; Bengtsson et al., 2001; Rapparini et al., 2001; Vallat and Dorn, 2005). Although many of these compounds are constitutively produced in relatively low amounts, a subset of common apple terpenes either are induced in response to insect infestation or have been observed to affect apple pest behavior directly. For example, (E)-β-ocimene is induced in immature apple fruit following infestation by codling moth (Cydia pomonella) larvae (Hern and Dorn, 2002), while (E,E)-α-farnesene exerts a concentration-dependent sexually dimorphic response in adult codling moth, attracting mated females at low dosages and repelling them at high dosages, while only attracting mated males at high dosages (Hern and Dorn, 1999). The relative abundance and diversity of terpenes in floral and vegetative tissues compared with ripe fruit suggests that these compounds are more likely to participate in defense and pollinator attraction roles rather than to attract seed dispersers or contribute to fruit flavor and aroma.Despite numerous studies on apple terpene volatile production, to our knowledge the only reported apple TPS enzyme to be functionally characterized is that of the α-farnesene synthase (AFS1/MdAFS1; Rupasinghe et al., 2000; Pechous and Whitaker, 2004; Green et al., 2007). The recent availability of the draft sequence assembly of the apple genome (Velasco et al., 2010), combined with a large number of accessible apple ESTs, provided an opportunity to extend the current knowledge on the organization and functional annotation of the apple TPS gene family. In this study, we initially survey the volatile terpenes produced in cv Royal Gala and then use available apple genomic and EST information to identify candidate TPS genes for functional characterization and quantitative expression analysis. We show that only a small number of the TPS genes in the apple genome encode functional TPS enzymes, but these enzymes can account for the diversity of terpenes present in apple. Our results also provide insight into how this small but evolutionarily conserved family was shaped by GWD events and how the range of terpenes has been influenced by selective pressures and commercial breeding strategies.     

Expansive Evolution of the TREHALOSE-6-PHOSPHATE PHOSPHATASE Gene Family in Arabidopsis

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.The presence of trehalose in a wide variety of organisms and the existence of different biosynthesis pathways suggest a pivotal and ancient role for trehalose metabolism in nature. The most widely distributed metabolic pathway consists of two consecutive enzymatic reactions, with trehalose-6-phosphate (T6P) synthase (TPS) catalyzing the transfer of a glucosyl moiety from UDP-Glc to Glc-6-phosphate to produce T6P and UDP, and T6P phosphatase (TPP) catalyzing dephosphorylation of T6P to trehalose (Cabib and Leloir, 1958; Avonce et al., 2006). Apart from operating as a (reserve) carbon source and structural component in bacteria, fungi, and invertebrates, trehalose also functions as a major stress protectant of proteins and membranes during adverse conditions such as dehydration, high salinity, hypoxia, and nutrient starvation (Elbein et al., 2003). Trehalose accumulation is also observed in a few lower vascular resurrection plants (e.g. Selaginella lepidophylla). Until about a decade ago, higher vascular plants were believed to have lost the ability to produce trehalose, but with the emergence of more sensitive assays, genome sequencing, and the use of yeast (Saccharomyces cerevisiae) mutant complementation, minute amounts of trehalose and T6P, and functional plant enzyme orthologs were found (Goddijn et al., 1997; Vogel et al., 1998; Lunn et al., 2006). In addition, heterologous expression and disruption of trehalose metabolism in plants conferred pleiotropic effects, ranging from altered stress tolerance, leaf morphology, and developmental timing to embryo lethality (Holmström et al., 1996; Goddijn et al., 1997; Romero et al., 1997; Eastmond et al., 2002; Schluepmann et al., 2003; Avonce et al., 2004; Satoh-Nagasawa et al., 2006; Miranda et al., 2007; Chary et al., 2008), pointing to an important regulatory function. The intermediate T6P has been highlighted as a novel signal for carbohydrate status (for review, see Paul, 2008), positively correlating with Suc levels, redox-regulated ADP-Glc pyrophosphorylase activity, and starch biosynthesis (Lunn et al., 2006). Recently, it was reported that T6P inhibits the activity of the SnRK1 protein kinase to activate energy-consuming biosynthetic processes in growing tissue (Zhang et al., 2009) and that it is required for the onset of leaf senescence (Wingler et al., 2012).In most bacterial and eukaryotic species, the TPS and TPP activities are found on separate proteins. Recent phylogenetic and biochemical analyses showed that some archaea and bacteria, such as Cytophaga hutchinsonii, express proteins that have both active TPS and TPP domains resulting from gene fusion, suggesting that such prokaryotic bifunctional proteins are the evolutionary ancestors of the large eukaryotic trehalose biosynthesis enzymes in which one or both domains have subsequently lost their catalytic activity (Avonce et al., 2010). The yeast TPP enzyme Tps2, for example, harbors an inactive N-terminal TPS domain and an active C-terminal TPP domain. In contrast to the single TPS and TPP genes in most microorganisms, the genomes of higher plants encode a remarkably large family of putative trehalose biosynthesis enzyme homologs. These are commonly classified in three distinct subgroups, according to their similarity to the microbial TPS and TPP proteins and/or presence of specific motifs (e.g. conserved phosphatase boxes; Thaller et al., 1998; Leyman et al., 2001; Eastmond et al., 2003). Even primitive plants such as the alga Ostreococcus tauri and the moss Physcomitrella patens already contain members of each of these gene families, pointing to the early establishment and conservation of these proteins in plant evolution (Lunn, 2007; Avonce et al., 2010). In Arabidopsis (Arabidopsis thaliana), the class I TPS proteins (AtTPS1-4) show most similarity to the yeast TPS Tps1, but also have a C-terminal domain with limited similarity to TPPs. However, only one of these, AtTPS1, appears to have heterologous enzymatic TPS activity in yeast (Blázquez et al., 1998; Vandesteene et al., 2010). Strikingly, AtTPS1 is the only class I enzyme with an N-terminal extension that seems to operate as an autoinhibitory domain (Van Dijck et al., 2002). The class II TPS proteins (AtTPS5-11) are similar bipartite proteins with a TPS-like domain but a more conserved TPP domain. They appear to lack both heterologous TPS and TPP activity (Ramon et al., 2009). The high level of conservation of putative substrate-binding residues in class I and class II proteins, however, suggests that substrates might still bind (Avonce et al., 2006; Lunn, 2007; Ramon et al., 2009; Vandesteene et al., 2010). Together with the specific expression patterns of the class I genes (van Dijken et al., 2004; Geelen et al., 2007; Vandesteene et al., 2010) and the extensive expression regulation of all class II members by plant carbon status (Baena-González et al., 2007; Usadel et al., 2008; Ramon et al., 2009), this suggests tissue-specific regulatory functions for these proteins in metabolic regulation of plant growth and development. Finally, Arabidopsis also harbors a family of 10 smaller proteins (AtTPPA-J; 320–385 amino acids) with limited similarity to the class I and class II proteins (795–942 amino acids). Like class II proteins, they contain the phosphatase box consensus sequences, characteristic of the l-2-haloacid dehalogenase (HAD) super family of enzymes, which includes a wide range of phosphatases and hydrolases (Thaller et al., 1998). It has been suggested that the origin of these plant TPP genes is different from the origin of the class I and II genes (Avonce et al., 2010) and that plants recruited the TPP genes after their divergence from fungi, most probably from proteobacteria or actinobacteria. Consistently, homologous TPP proteins are present in proteobacteria such as Rhodopherax ferrireducens (Avonce et al., 2010). To date, only a few of these single-domain plant TPP proteins have been subject to biochemical characterization, e.g. TPPA and TPPB from Arabidopsis (Vogel et al., 1998), OsTPP1 and OsTPP2 from rice (Oryza sativa; Pramanik and Imai, 2005; Shima et al., 2007), and RAMOSA3 (RA3) from maize (Zea mays; Satoh-Nagasawa et al., 2006).The phenotypic alterations observed in plants fed with trehalose or genetically modified in trehalose biosynthesis, suggest a pivotal role for trehalose metabolism in integrating the metabolic status with growth and development. Disruption of the only known active TPS enzyme in Arabidopsis (AtTPS1) results in embryo lethality (Eastmond et al., 2002) and, when rescued to bridge embryogenesis, causes a strong disruption of vegetative and generative development and abscisic acid (ABA) hypersensitivity (van Dijken et al., 2004; Gómez et al., 2010). Overexpressing AtTPS1 on the other hand renders seedlings sugar and ABA insensitive (Avonce et al., 2004, 2005). These observations strongly link trehalose metabolism with ABA signaling. Interestingly, a mutation of a TPP gene in maize, RA3, results in a distinct phenotype, with incorrect axillary meristem identity and determinacy in both male and female inflorescences (Satoh-Nagasawa et al., 2006). Arabidopsis plants with overall increased T6P levels, such as OtsA (Escherichia coli TPS) overexpression plants, similarly show increased inflorescence branching (Schluepmann et al., 2003; van Dijken et al., 2004).To better understand why higher plants harbor such a large number of putative TPP proteins, we have made a comprehensive study of the 10 Arabidopsis TPP genes and gene products, combining phylogenetic approaches and yeast growth complementation assays, together with a detailed analysis of all 10 TPP gene expression profiles in Arabidopsis, and a more detailed single AtTPP mutant phenotypic analysis.     

Extensive cis-Regulatory Variation Robust to Environmental Perturbation in Arabidopsis

cis- and trans-acting factors affect gene expression and responses to environmental conditions. However, for most plant systems, we lack a comprehensive map of these factors and their interaction with environmental variation. Here, we examined allele-specific expression (ASE) in an F1 hybrid to study how alleles from two Arabidopsis thaliana accessions affect gene expression. To investigate the effect of the environment, we used drought stress and developed a variance component model to estimate the combined genetic contributions of cis- and trans-regulatory polymorphisms, environmental factors, and their interactions. We quantified ASE for 11,003 genes, identifying 3318 genes with consistent ASE in control and stress conditions, demonstrating that cis-acting genetic effects are essentially robust to changes in the environment. Moreover, we found 1618 genes with genotype x environment (GxE) interactions, mostly cis x E interactions with magnitude changes in ASE. We found fewer trans x E interactions, but these effects were relatively less robust across conditions, showing more changes in the direction of the effect between environments; this confirms that trans-regulation plays an important role in the response to environmental conditions. Our data provide a detailed map of cis- and trans-regulation and GxE interactions in A. thaliana, laying the ground for mechanistic investigations and studies in other plants and environments.     

Evolutionary Patterns and Coevolutionary Consequences of MIRNA Genes and MicroRNA Targets Triggered by Multiple Mechanisms of Genomic Duplications in Soybean

The evolutionary dynamics of duplicated protein-encoding genes (PEGs) is well documented. However, the evolutionary patterns and consequences of duplicated MIRNAs and the potential influence on the evolution of their PEG targets are poorly understood. Here, we demonstrate the evolution of plant MIRNAs subsequent to a recent whole-genome duplication. Overall, the retention of MIRNA duplicates was correlated to the retention of adjacent PEG duplicates, and the retained MIRNA duplicates exhibited a higher level of interspecific preservation of orthologs than singletons, suggesting that the retention of MIRNA duplicates is related to their functional constraints and local genomic stability. Nevertheless, duplication status, rather than local genic collinearity, was the primary determinant of levels of nucleotide divergence of MIRNAs. In addition, the retention of duplicated MIRNAs appears to be associated with the retention of their corresponding duplicated PEG targets. Furthermore, we characterized the evolutionary novelty of a legume-specific microRNA (miRNA) family, which resulted from rounds of genomic duplication, and consequent dynamic evolution of its NB-LRR targets, an important gene family with primary roles in plant-pathogen interactions. Together, these observations depict evolutionary patterns and novelty of MIRNAs in the context of genomic duplication and evolutionary interplay between MIRNAs and their PEG targets mediated by miRNAs.     

Genome-Wide Mapping of Structural Variations Reveals a Copy Number Variant That Determines Reproductive Morphology in Cucumber

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.     

MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.     

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.     

In Planta Stage-Specific Fungal Gene Profiling Elucidates the Molecular Strategies of Fusarium graminearum Growing inside Wheat Coleoptiles

The ascomycete Fusarium graminearum is a destructive fungal pathogen of wheat (Triticum aestivum). To better understand how this pathogen proliferates within the host plant, we tracked pathogen growth inside wheat coleoptiles and then examined pathogen gene expression inside wheat coleoptiles at 16, 40, and 64 h after inoculation (HAI) using laser capture microdissection and microarray analysis. We identified 344 genes that were preferentially expressed during invasive growth in planta. Gene expression profiles for 134 putative plant cell wall–degrading enzyme genes suggest that there was limited cell wall degradation at 16 HAI and extensive degradation at 64 HAI. Expression profiles for genes encoding reactive oxygen species (ROS)–related enzymes suggest that F. graminearum primarily scavenges extracellular ROS before a later burst of extracellular ROS is produced by F. graminearum enzymes. Expression patterns of genes involved in primary metabolic pathways suggest that F. graminearum relies on the glyoxylate cycle at an early stage of plant infection. A secondary metabolite biosynthesis gene cluster was specifically induced at 64 HAI and was required for virulence. Our results indicate that F. graminearum initiates infection of coleoptiles using covert penetration strategies and switches to overt cellular destruction of tissues at an advanced stage of infection.     

Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5′ coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.