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Despite intensive prophylactic and pre-emptive measures, infections remain an important cause of morbidity and mortality in pediatric recipients of allogeneic hematopoietic cell transplantation (allo-HCT). Disruption of the gut microbiota has been linked to clinical outcomes after adult allo-HCT. The objective was to evaluate whether these or differing microbiota disruptions or signatures were associated with risk of infection in pediatric allo-HCT.In a prospective observational study, fecal samples from 74 children were collected prior to conditioning and at the time of neutrophil recovery and profiled by means of 16S ribosomal rRNA sequencing. The associations between microbiome signatures and infections or acute graft-versus-host disease (aGVHD) were examined using Cox proportional-hazards analysis.Previously associated indices of microbiome disruption in adults, including diversity and butyrate producer frequency, did not predict infection risk in pediatric allo-HCT. Unique microbiota signatures were associated with different infections or aGVHD. A ratio of strict and facultative anaerobes (e.g.Lachnoclostridium, Parabacteroides, Clostridium spp.) prior to conditioning predicted likelihood of bacteremia (cox hazards ratio 3.89) in first year post HCT. A distinct ratio of oral (e.g.Rothia, Veillonellaspp.) to colonic anaerobes (e.g.Anaerobutyricum, Dorea, Romboutsiaspp.) at neutrophil recovery predicted likelihood of bacterial infections (cox hazards ratio 1.81) and viral enterocolitis (cox hazards ratio 1.96) through first year post transplant.Interactions between medical interventions, pediatric hosts and microbial communities may be responsible for these consistent microbiota signatures that predict infections. A future multi-center investigation will be needed to demonstrate whether these ratios can be generalized to other pediatric cohorts.Key Points(1 or 2

Streptococcus pneumoniae is a major human pathogen of global health concern, causing a range of mild to severe infections, including acute otitis media, pneumonia, sepsis, and meningitis. The rapid emergence of antibiotic resistance among S. pneumoniae isolates poses a serious public health problem worldwide. Resistant pneumococcal strains have rendered the mainstay treatment with beta-lactams, fluoroquinolones, and macrolides, ineffective. Antibiotic resistance in S. pneumoniae has spread globally via the emergence of de novo mutations and horizontal transfer of resistance. Fluoroquinolone resistance in S. pneumoniae is an intriguing case because the prevalence of fluoroquinolone resistance does not correlate with increasing usage, as is often the case with other classes of antibiotics. In this study, we demonstrated that deleterious fitness costs constrain the emergence of individual fluoroquinolone resistance mutations in either topoisomerase IV or gyrase A in S. pneumoniae. Generation of double point mutations in the target enzymes in topoisomerase IV and gyrase A conferred high-level fluoroquinolone resistance while restoring fitness comparable to the sensitive wild-type. During an in vivo model of antibiotic resistance evolution, S. pneumoniae was able to circumvent deleterious fitness costs imposed by resistance determinants through development of antibiotic tolerance through metabolic adaptation that reduced the production of reactive oxygen species, an effect that could be recapitulated pharmacologically. The metabolic mutants conferring tolerance resulted in a fitness benefit during infection following antibiotic treatment with fluroquinolones. These data suggest that emergence of fluoroquinolone resistance is tightly constrained in S. pneumoniae by host fitness tradeoffs and that mutational pathways involving metabolic networks to enable tolerance phenotypes may be an important contributor to the evasion of antibiotic mediated killing.

Water lilies belong to the angiosperm order Nymphaeales. Amborellales, Nymphaeales and Austrobaileyales together form the so-called ANA-grade of angiosperms, which are extant representatives of lineages that diverged the earliest from the lineage leading to the extant mesangiosperms1–3. Here we report the 409-megabase genome sequence of the blue-petal water lily (Nymphaea colorata). Our phylogenomic analyses support Amborellales and Nymphaeales as successive sister lineages to all other extant angiosperms. The N. colorata genome and 19 other water lily transcriptomes reveal a Nymphaealean whole-genome duplication event, which is shared by Nymphaeaceae and possibly Cabombaceae. Among the genes retained from this whole-genome duplication are homologues of genes that regulate flowering transition and flower development. The broad expression of homologues of floral ABCE genes in N. colorata might support a similarly broadly active ancestral ABCE model of floral organ determination in early angiosperms. Water lilies have evolved attractive floral scents and colours, which are features shared with mesangiosperms, and we identified their putative biosynthetic genes in N. colorata. The chemical compounds and biosynthetic genes behind floral scents suggest that they have evolved in parallel to those in mesangiosperms. Because of its unique phylogenetic position, the N. colorata genome sheds light on the early evolution of angiosperms., The genome of the tropical blue-petal water lily Nymphaea colorata and the transcriptomes from 19 other Nymphaeales species provide insights into the early evolution of angiosperms.

Significance Chimeric terpene synthases, termed PTTSs, are a unique family of enzymes occurring only in fungi. Characterizing PTTSs is challenging due to the complex reactions they catalyze and the structural complexity of their products. Here, by devising an efficient precursor-providing yeast chassis and incorporating a high-throughput automated platform, we identified 34 active PTTSs, which was considerably more than the number of known functional PTTSs. This effective and rapid pipeline can be employed for the characterization of other PTTSs or related terpenoid biosynthetic enzymes. By systematically analyzing the presence/absence of PTTS genes together with phylogenetic analysis, the ancestral PTTS gene was inferred to have undergone duplication and functional divergence, which led to the development of two distinct cyclization mechanisms., Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of PTTS genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that PTTS genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of PTTS genes. One was that PTTS genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 PTTS genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 PTTS genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.

Background Human astroviruses (HAstV) are a leading cause of acute gastroenteritis in children, particularly those under the age of 2 or with immunosuppressive conditions. Indeed, our studies suggest that children with hematological malignancies are at high risk of infection. However, it has become increasingly clear that HAstV infections can also be associated with respiratory and even central nervous system (CNS) diseases. In the last decade, there have been at least 12 cases of astrovirus-induced CNS disease resulting in encephalitis and meningitis, including 2 St Jude patients. The CNS-associated infections are overwhelmingly fatal and are primarily caused by a novel astrovirus genotype, VA1, that is genetically more similar to animal astroviruses that also induce CNS-associated infections. The recent ability to propagate the VA1 strain in cell lines affords us the opportunity to better understand VA1 infection and how it compares to the better-studied HAstV1 strain. Methods Viral kinetics were determined by infection of human intestinal adenocarcinoma Caco-2 cells with either HAstV1 or VA1 and monitored for 24 hours post-infection (hpi). Infected cells were identified via immunofluorescent microscopy using antibodies against double-stranded RNA and viral capsid. Epithelial permeability of astrovirus-infected Caco-2 cells was measured using transepithelial electrical resistance (TER) and monitored for 24hpi. Nitazoxanide efficacy was identified by immunofluorescent analysis as described previously. Results Our findings demonstrate that HAstV1 and VA1 replicate in intestinal epithelial cells without inducing cell death or a pro-inflammatory cytokine response. However, these two strains have distinct replication kinetics. VA1 appears to have an ~8 hour lag in the expression of dsRNA and capsid protein compared to HAstV1 and does not require exogenous proteases to process the viral outer coat (capsid) protein, Additionally, we demonstrated that the increase in epithelial barrier permeability associated with HAstV1-infection is not found in VA1-infected cells, which is intriguing and may explain why HAstV1 is more likely to cause diarrhea. Of clinical importance, we have revealed a similar susceptibility of VA1 to the antiviral drug, nitazoxanide, which we have recently demonstrated its effectiveness inhibiting classical HAstV strains. Conclusion Overall, our studies highlight that VA1 pathogenesis is distinct from HAstV1, which could explain the differences in vivo. Future studies will be necessary to investigate viral replication and pathogenesis in distinct neuronal cells and in vivo.

Key message Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Abstract Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting fulllength genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert l-tyrosine, l-tryptophan, and l-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that l-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from l-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species.

Fungi are successful eukaryotes of wide distribution. They are known as rich producers of secondary metabolites, especially terpenoids, which are important for fungi-environment interactions. Horizontal gene transfer (HGT) is an important mechanism contributing to genetic innovation of fungi. However, it remains unclear whether HGT has played a role in creating the enormous chemical diversity of fungal terpenoids. Here we report that fungi have acquired terpene synthase genes (TPSs), which encode pivotal enzymes for terpenoid biosynthesis, from bacteria through HGT. Phylogenetic analysis placed the majority of fungal and bacterial TPS genes from diverse taxa into two clades, indicating ancient divergence. Nested in the bacterial TPS clade is a number of fungal TPS genes that are inferred as the outcome of HGT. These include a monophyletic clade of nine fungal TPS genes, designated as BTPSL for bacterial TPS-like genes, from eight species of related entomopathogenic fungi, including seven TPSs from six species in the genus Metarhizium. In vitro enzyme assays demonstrate that all seven BTPSL genes from the genus Metarhizium encode active enzymes with sesquiterpene synthase activities of two general product profiles. By analyzing the catalytic activity of two resurrected ancestral BTPSLs and one closely related bacterial TPS, the trajectory of functional evolution of BTPSLs after HGT from bacteria to fungi and functional divergence within Metarhizium could be traced. Using M. brunneum as a model species, both BTPSLs and typical fungal TPSs were demonstrated to be involved in the in vivo production of terpenoids, illustrating the general importance of HGT of TPS genes from bacteria as a mechanism contributing to terpenoid diversity in fungi.

Dictyostelids, or social amoebae, have a unique life style in forming multicellular fruiting bodies from unicellular amoeboids upon starvation. Recently, dictyostelids were found to contain terpene synthase (TPS) genes, a gene type of secondary metabolism previously known to occur only in plants, fungi and bacteria. Here we report an evolutionary functional study of dictyostelid TPS genes. The number of TPS genes in six species of dictyostelids examined ranges from 1 to 19; and the model species Dictyostelium purpureum contains 12 genes. Using in vitro enzyme assays, the 12 TPS genes from D. purpureum were shown to encode functional enzymes with distinct product profiles. The expression of the 12 TPS genes in D. purpureum is developmentally regulated. During multicellular development, D. purpureum releases a mixture of volatile terpenes dominated by sesquiterpenes that are the in vitro products of a subset of the 12 TPS genes. The quality and quantity of the terpenes released from D. purpureum, however, bear little resemblance to those of D. discoideum, a closely related dictyostelid. Despite these variations, the conserved clade of dictyostelid TPSs, which have an evolutionary distance of more than 600 million years, has the same biochemical function, catalyzing the formation of a sesquiterpene protoillud-7-ene. Taken together, our results indicate that the dynamic evolution of dictyostelid TPS genes includes both purifying selection of an orthologous group and species-specific expansion with functional divergence. Consequently, the terpenes produced by these TPSs most likely have conserved as well as species-adaptive biological functions as chemical languages in dictyostelids.

The triterpene squalene is a key metabolic intermediate for sterols, hopanoids and various other triterpenoids. The biosynthesis of squalene is catalyzed by squalene synthase (SQS), which converts two molecules of farnesyl diphosphate to squalene. In this study, a lycophyte Selaginella moellendorffii was found to emit squalene as a volatile compound under a number of conditions that mimic biotic stresses. Searching the genome sequence of S. moellendorffii led to the identification of a putative squalene synthase gene. It was designated as SmSQS. SmSQS is homologous to known squalene synthases from other plants and animals at both the amino acid level and structural level. Recombinant SmSQS expressed in Escherichia coli catalyzed the formation of squalene using farnesyl diphosphate as substrate. The expression of SmSQS was significantly induced by the same set of stress factors that induced the emission of volatile squalene from S. moellendorffii plants. Taken together, these results support that SmSQS is responsible for the biosynthesis of volatile squalene and volatile squalene may have a role in the defense of S. moellendorffii plants against biotic stresses.

The discovery of artemisinin as an antimalarial agent by Professor Youyou Tu of China was awarded the Noble prize in Physiology and Medicine in 2015. Notably, artemisinin belongs to the terpenoids; more than 55 000 of them have been isolated from nature. Important for both cellular and ecological functions (Gershenzon and Dudareva, 2007), terpenoids are particularly rich in plants. It has been a long-standing question to chemists and biologists alike: how are enormously diverse terpenoids made by plants? One current understanding is that terpene synthases, which convert isoprenyl diphosphates to terpene skeletons, play a central role in generating structural diversity of terpenoids (Chen et al., 2011).

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant., 陸上植物の祖先の特徴をもつ苔類ゼニゴケの全ゲノム構造を解明. 京都大学プレスリリース. 2017-10-06.

Abscisic acid (ABA) is a phytohormone that has been extensively characterized in higher plants for its roles in seed and bud dormancy, leaf abscission, and stress responses. Genomic studies have identified orthologs for ABA-related genes throughout the Viridiplantae, including in unicellular algae; however, the role of ABA in algal physiology has not been characterized, and the existence of such a role has been a matter of dispute. In this study, we demonstrate that ABA is involved in regulating algal stress responses. Chlorella sorokiniana strain UTEX 1230 contains genes orthologous to those of higher plants which are essential for ABA biosynthesis, sensing, and degradation. RNAseq-based transcriptomic studies reveal that treatment with ABA induces dramatic changes in gene expression profiles, including the induction of a subset of genes involved in DNA replication and repair, a phenomenon which has been demonstrated in higher plants. Pretreatment of C. sorokiniana cultures with ABA exerts a protective effect on cell viability in response to ultraviolet radiation. Additionally, C. sorokiniana produces and secretes biologically relevant amounts of both ABA and the oxylipin 12-oxo-phytodienoic acid (OPDA) into the growth medium in response to abiotic stressors. Taken together, these phenomena suggest that ABA signaling evolved as an intercellular stress response signaling molecule in eukaryotic microalgae prior to the evolution of multicellularity and colonization of land.

Terpenes constitute a large class of plant secondary metabolites. It was once presumed that these compounds are biosynthesized by typical plant terpene synthases in all land plants. This view has changed with the identification of a new group of terpene synthase genes called MTPSLs for microbial terpene synthase-like genes. MTPSLs are structurally and phylogenetically more related to bacterial and fungal terpene synthases than to typical plant terpene synthases. They are widely distributed in nonseed plants but absent in seed plants and green algae. Much of the terpene diversity in nonseed plants is presumed to be determined by MTPSLs. Phylogenetic analysis suggests that ancestral MTPSL genes were acquired by early land plants from bacteria and fungi through horizontal gene transfer.

Microbial terpene synthase-like (MTPSL) genes are a type of terpene synthase genes only recently identified in plants. In contrast to typical plant terpene synthase genes, which are ubiquitous in land plants, MTPSL genes appear to occur only in nonseed plants. Our knowledge of catalytic functions of MTPSLs is very limited. Here we report biochemical characterization of the enzymes encoded by MTPSL genes from two closely related species of hornworts, Anthoceros punctatus and Anthoceros agrestis. Seven full-length MTPSL genes were identified in A. punctatus (ApMTPSL1-7) based on the analysis of its genome sequence. Using homology-based cloning, the apparent orthologs for six of the ApMTPSL genes, except ApMTPSL2, were cloned from A. agrestis. They were designated AaMTPSL1, 3–7. The coding sequences for each of the 13 Anthoceros MTPSL genes were cloned into a protein expression vector. Escherichia coli-expressed recombinant MTPSLs from hornworts were assayed for terpene synthase activities. Six ApMTPSLs and five AaMTPSLs, except for ApMTPSL5 and AaMTPSL5, showed catalytic activities with one or more isoprenyl diphosphate substrates. All functional MTPSLs exhibited sesquiterpene synthase activities. In contrast, only ApMTPSL7 and AaMTPSL7 showed monoterpene synthase activity and only ApMTPSL2, ApMTPSL6 and AaMTPSL6 showed diterpene synthase activity. Most MTPSLs from Anthoceros contain uncanonical aspartate-rich motif in the form of either ‘DDxxxD’ or ‘DDxxx’. Homology-based structural modeling analysis of ApMTPSL1 and ApMTPSL7, which contain ‘DDxxxD’ and ‘DDxxx’ motif, respectively, showed that ‘DDxxxD’ and ‘DDxxx’ motifs are localized in the similar positions as the canonical ‘DDxxD’ motif in known terpene synthases. To further understand the role of individual aspartate residues in the motifs, ApMTPSL1 and ApMTPSL7 were selected as two representatives for site-directed mutagenesis studies. No activities were detected when any of the conserved aspartic acid was mutated into alanine. This study provides new information about the catalytic functions of MTPSLs and the functionality of their uncanonical aspartate-rich motifs, and builds a knowledge base for studying the biological importance of MTPSL genes and their terpene products in nonseed plants.

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including β-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.

The vast abundance of terpene natural products in nature is due to enzymes known as terpene synthases (TPSs) that convert acyclic prenyl diphosphate precursors into a multitude of cyclic and acyclic carbon skeletons. Yet the evolution of TPSs is not well understood at higher levels of classification. Microbial TPSs from bacteria and fungi are only distantly related to typical plant TPSs, whereas genes similar to microbial TPS genes have been recently identified in the lycophyte Selaginella moellendorffii. The goal of this study was to investigate the distribution, evolution, and biochemical functions of microbial terpene synthase-like (MTPSL) genes in other plants. By analyzing the transcriptomes of 1,103 plant species ranging from green algae to flowering plants, putative MTPSL genes were identified predominantly from nonseed plants, including liverworts, mosses, hornworts, lycophytes, and monilophytes. Directed searching for MTPSL genes in the sequenced genomes of a wide range of seed plants confirmed their general absence in this group. Among themselves, MTPSL proteins from nonseed plants form four major groups, with two of these more closely related to bacterial TPSs and the other two to fungal TPSs. Two of the four groups contain a canonical aspartate-rich “DDxxD” motif. The third group has a “DDxxxD” motif, and the fourth group has only the first two “DD” conserved in this motif. Upon heterologous expression, representative members from each of the four groups displayed diverse catalytic functions as monoterpene and sesquiterpene synthases, suggesting these are important for terpene formation in nonseed plants.

MicroRNAs (miRNA) participate in regulating diverse biological pathways by translational repression in animals. They have attracted increasing attention recently. However, little work has been done on the miRNA genes in agriculturally important pests. Because the transcripts of most miRNA genes are the products of type-II RNA polymerase, pri-miRNA has a poly(A) tail and appears in expressed sequence tags (EST). We developed a computational pipeline to identify miRNA genes from insect ESTs. First, 980,697 ESTs from 63 insects were collected and used to search the nr database. The ESTs which did not share significant similarities with any known protein-coding genes were treated as non-coding ESTs. Next, known mature miRNAs were used to align with non-coding ESTs. The ESTs which contain the sequence of mature miRNA were treated as candidate ESTs. Finally, putative precursors were extracted flanking the mature miRNA region in candidate ESTs and evaluated by the Triplet-SVM algorithm. As a result, 86 miRNAs from 30 insect species were found based on a strict criterion while 330 miRNAs from 51 species were found based on a loose criterion. Evolution analysis indicated that mir-467, mir-297 and mir-466 were the highest conserved miRNA families in insects. To confirm the reliability of putative insect miRNAs, the expression profile of nine predicted miRNAs in Locusta migratoria was investigated. Eight miRNAs were successfully detected by RT-PCR. Most miRNAs were expressed ubiquitously at all examined tissues and developmental stages whereas Lmi-mir-509 was specifically expressed in the thorax of the 2nd, 4th and 5th instars and adult locust. In all, our work reported an efficient computational strategy for predicting miRNA genes from insect ESTs and presented tens of miRNAs in diverse insect species which are expected to participate in many important physiological processes.

BACKGROUND: Previous studies have indicated that increased caboxylesterase (CarE) activity is a major mechanism of malathion resistance in field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), in China. The aim of the present study was to screen CarE-like genes from a large locust expressed sequence tag (EST) database and to assess their potential roles in malathion resistance. RESULTS: Twenty-five ESTs derived from different CarE-like genes in the locust EST database were identified, and 12 candidate genes with significantly increased expressions, ranging from 2.6- to 11.6-fold in a field-derived resistant (FR) colony of the locust, were found. These candidate genes were constitutively expressed in all nymph and adult stages, and most of them were predominantly expressed in the gastric caeca and the midgut. Among the 12 genes, two representative genes (LmCarE9 and LmCarE25) were chosen for RNAi followed by malathion bioassay. The nymph mortalities increased from 34.3 to 65.2 and 54.2% respectively after LmCarE9 and LmcarE25 were silenced. These results indicated significant roles of these CarE-like genes in conferring malathion resistance in the locust. CONCLUSION: Multiple CarE-like genes were involved in malathion resistance in the locust. As validated by RNAi followed by malathion bioassay, LmCarE9 and LmcarE25 played a significant role in conferring malathion resistance.

Thirty-one potential miRNAs that belong to 16 miRNA families were discovered from more than 324 000 EST sequences of apple (Malus domestica). In addition, precise sequences, especially terminal nucleotides of the 16 apple miRNAs (mdo-miRNAs) in 16 families were validated by miR-RACE, a newly developed method for the determination of the potential miRNAs predicted computationally. The expression of these 16 microRNAs could be detected in apple young leaf, old leaf, young stem, flower bud, flower and developing fruits by quantitative RT-PCR (qRT-PCR) and some of them showed tissue-specific expression. Fifty-six potential targets were identified for the 16 apple miRNAs, most of which were transcription factors that play important roles in apple development. Twelve target genes were experimentally verified by qRT-PCR, with some exhibiting different expression trends from their corresponding microRNAs, indicating the cleavage mode of miRNAs on their target genes.

Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

The known members of plant methyl esterase (MES) family catalyze the hydrolysis of a C-O ester linkage of methyl esters of several phytohormones including indole-3-acetic acid, salicylic acid and jasmonic acid. The genome of grapevine (Vitis vinifera) was found to contain 15 MES genes, designated VvMES1-15. In this report, VvMES5 was selected for molecular, biochemical and structural studies. VvMES5 is most similar to tomato methyl jasmonate esterase. E. coli-expressed recombinant VvMES5 displayed methyl jasmonate (MeJA) esterase activity, it was renamed VvMJE1. Under steady-state conditions, VvMJE1 exhibited an apparent Km value of 92.9 μM with MeJA. VvMJE1 was also shown to have lower activity with methyl salicylate (MeSA), another known substrate of the MES family, and only at high concentrations of the substrate. To understand the structural basis of VvMJE1 in discriminating MeJA and MeSA, a homolog model of VvMJE1 was made using the X-ray structure of tobacco SABP2, which encodes for methyl salicylate esterase, as a template. Interestingly, two bulky residues at the binding site and near the surface of tobacco SABP2 are replaced by relatively small residues in VvMJE1. Such a change enables the accommodation of a larger substrate MeJA in VvMJE1. The expression of VvMJE1 was compared in control grape plants and grape plants treated with one of the three stresses: heat, cold and UV-B. While the expression of VvMJE1 was not affected by heat treatment, its expression was significantly up-regulated by cold treatment and UV-B treatment. This result suggests that VvMJE1 has a role in response of grape plants to these two abiotic stresses.

Key message Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Abstract Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting full-length genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert l-tyrosine, l-tryptophan, and l-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that l-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from l-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species. Electronic supplementary material The online version of this article (doi:10.1007/s11103-017-0646-0) contains supplementary material, which is available to authorized users.

We review the evidence that upstream open reading frames (uORFs) function as RNA sequence elements for post-transcriptional control of gene expression, specifically translation. uORFs are highly abundant in the genomes of angiosperms. Their negative effect on translation is often attenuated by ribosomal translation reinitiation, a process whose molecular biochemistry is still being investigated. Certain uORFs render translation responsive to small molecules, thus offering a path for metabolic control of gene expression in evolution and synthetic biology. In some cases, uORFs form modular logic gates in signal transduction. uORFs thus provide eukaryotes with a functionality analogous to, or comparable to, riboswitches and attenuators in prokaryotes. uORFs exist in many genes regulating development and point toward translational control of development. While many uORFs appear to be poorly conserved, and the number of genes with conserved-peptide uORFs is modest, many mRNAs have a conserved pattern of uORFs. Evolutionarily, the gain and loss of uORFs may be a widespread mechanism that diversifies gene expression patterns. Last but not least, this review includes a dedicated uORF database for Arabidopsis.

Marchantia polymorpha is a basal terrestrial land plant, which like most liverworts accumulates structurally diverse terpenes believed to serve in deterring disease and herbivory. Previous studies have suggested that the mevalonate and methylerythritol phosphate pathways, present in evolutionarily diverged plants, are also operative in liverworts. However, the genes and enzymes responsible for the chemical diversity of terpenes have yet to be described. In this study, we resorted to a HMMER search tool to identify 17 putative terpene synthase genes from M. polymorpha transcriptomes. Functional characterization identified four diterpene synthase genes phylogenetically related to those found in diverged plants and nine rather unusual monoterpene and sesquiterpene synthase-like genes. The presence of separate monofunctional diterpene synthases for ent-copalyl diphosphate and ent-kaurene biosynthesis is similar to orthologs found in vascular plants, pushing the date of the underlying gene duplication and neofunctionalization of the ancestral diterpene synthase gene family to >400 million years ago. By contrast, the mono- and sesquiterpene synthases represent a distinct class of enzymes, not related to previously described plant terpene synthases and only distantly so to microbial-type terpene synthases. The absence of a Mg2+ binding, aspartate-rich, DDXXD motif places these enzymes in a noncanonical family of terpene synthases.

Previous studies have indicated that increased carboxylesterase (CarE) activity is a major mechanism of malathion resistance in field populations of the oriental migratory locust, Locusta migratoria manilensis (Meyen), in China. The aim of the present study was to screen CarE-like genes from a large locust expressed sequence tag (EST) database and to assess their potential roles in malathion resistance.Twenty-five ESTs derived from different CarE-like genes in the locust EST database were identified, and 12 candidate genes with significantly increased expressions, ranging from 2.6- to 11.6-fold in a field-derived resistant (FR) colony of the locust, were found. These candidate genes were constitutively expressed in all nymph and adult stages, and most of them were predominantly expressed in the gastric caeca and the midgut. Among the 12 genes, two representative genes (LmCarE9 and LmCarE25) were chosen for RNAi followed by malathion bioassay. The nymph mortalities increased from 34.3 to 65.2 and 54.2% respectively after LmCarE9 and LmcarE25 were silenced. These results indicated significant roles of these CarE-like genes in conferring malathion resistance in the locust.Multiple CarE-like genes were involved in malathion resistance in the locust. As validated by RNAi followed by malathion bioassay, LmCarE9 and LmcarE25 played a significant role in conferring malathion resistance.

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.

Background Terpenoids constitute the largest class of secondary metabolites made by plants and display vast chemical diversity among and within species. Terpene synthases (TPSs) are the pivotal enzymes for terpenoid biosynthesis that create the basic carbon skeletons of this class. Functional divergence of paralogous and orthologous TPS genes is a major mechanism for the diversification of terpenoid biosynthesis. However, little is known about the evolutionary forces that have shaped the evolution of plant TPS genes leading to terpenoid diversity. Results The orthologs of Oryza Terpene Synthase 1 (OryzaTPS1), a rice terpene synthase gene involved in indirect defense against insects in Oryza sativa, were cloned from six additional Oryza species. In vitro biochemical analysis showed that the enzymes encoded by these OryzaTPS1 genes functioned either as (E)-β-caryophyllene synthases (ECS), or (E)-β-caryophyllene & germacrene A synthases (EGS), or germacrene D & germacrene A synthases (DAS). Because the orthologs of OryzaTPS1 in maize and sorghum function as ECS, the ECS activity was inferred to be ancestral. Molecular evolutionary detected the signature of positive Darwinian selection in five codon substitutions in the evolution from ECS to DAS. Homology-based structure modeling and the biochemical analysis of laboratory-generated protein variants validated the contribution of the five positively selected sites to functional divergence of OryzaTPS1. The changes in the in vitro product spectra of OryzaTPS1 proteins also correlated closely to the changes in in vivo blends of volatile terpenes released from insect-damaged rice plants. Conclusions In this study, we found that positive Darwinian selection is a driving force for the functional divergence of OryzaTPS1. This finding suggests that the diverged sesquiterpene blend produced by the Oryza species containing DAS may be adaptive, likely in the attraction of the natural enemies of insect herbivores. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0239-x) contains supplementary material, which is available to authorized users.