The water lily genome and the early evolution of flowering plants.

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.

Origin and early evolution of the plant terpene synthase family.

As a midsized gene family conserved more by lineage than function, the typical plant terpene synthases (TPSs) could be a valuable tool to examine plant evolution. TPSs are pivotal in biosynthesis of gibberellins and related phytohormones as well as in formation of the extensive arsenal of specialized plant metabolites mediating ecological interactions whose production is often lineage specific. Yet the origin and early evolution of the TPS family is not well understood. Systematic analysis of an array of transcriptomes and sequenced genomes indicated that the TPS family originated after the divergence of land plants from charophytic algae. Phylogenetic and biochemical analyses support the hypothesis that the ancestral TPS gene encoded a bifunctional class I and II diterpene synthase producing the ent-kaurene required for phytohormone production in all extant lineages of land plants. Moreover, the ancestral TPS gene likely underwent duplication at least twice early in land plant evolution. Together these two gave rise to three TPS lineages leading to the extant TPS-c, TPS-e/f, and the remaining TPS (h/d/a/b/g) subfamilies, with the latter dedicated to secondary rather than primary metabolism while the former two contain those genes involved in ent-kaurene production. Nevertheless, parallel evolution from the ent-kaurene­producing class I and class II diterpene synthases has led to roles for TPS-e/f and -c subfamily members in secondary metabolism as well. These results clarify TPS evolutionary history and provide context for the role of these genes in producing the vast diversity of terpenoid natural products observed today in various land plant lineages.

GmSABP2-1 encodes methyl salicylate esterase and functions in soybean defense against soybean cyst nematode.

KEY MESSAGE: The soybean gene GmSABP2-1 encodes methyl salicylate esterase and its overexpression led to significant reduction in development of pathogenic soybean cyst nematode. Soybean cyst nematode (SCN, Heterodera glycines) is one of the most devastating pests of soybean (Glycine max L. Merr.). In searching for SCN-defense genes, a soybean gene of the methylesterase (MES) family was found to be upregulated in an SCN-resistant soybean line and downregulated in an SCN-susceptible line upon SCN infection. This gene was designated as GmSABP2-1. Here, we report on biochemical and overexpression studies of GmSABP2-1 to examine its possible function in SCN resistance. The protein encoded by GmSABP2-1 is closely related to known methyl salicylate esterases. To determine the biochemical function of GmSABP2-1, a full-length cDNA of GmSABP2-1 was cloned into a protein expression vector and expressed in Escherichia coli. The resulting recombinant GmSABP2-1 was demonstrated to catalyze the demethylation of methyl salicylate. The biochemical properties of GmSABP2-1 were determined. Its apparent Km value was 46.2 ± 2.2 µM for methyl salicylate, comparable to those of the known methyl salicylate esterases. To explore the biological significance of GmSABP2-1 in soybean defense against SCN, we first overexpressed GmSABP2-1 in transgenic hairy roots of an SCN-susceptible soybean line. When infected with SCN, GmSABP2-1-overexpressing hairy roots showed 84.5% reduction in the development of SCN beyond J2 stage. To provide further genetic evidence for the role of GmSABP2-1 in SCN resistance, stable transgenic soybean plants overexpressing GmSABP2-1 were produced. Analysis of the GmSABP2-1-overexpressing lines showed a significant reduction in SCN development compared to non-transgenic plants. In conclusion, we demonstrated that GmSABP2-1 encodes methyl salicylate esterase and functions as a resistance-related gene against SCN.

Biomimetic liposomal nanozymes improve breast cancer chemotherapy with enhanced penetration and alleviated hypoxia.

BACKGROUND: Doxorubicin (Dox) has been recommended in clinical guidelines for the standard-of-care treatment of breast cancer. However, Dox therapy faces challenges such as hypoxia, acidosis, H2O2-rich conditions and condensed extracellular matrix in TME as well as low targeted ability. METHODS: We developed a nanosystem H-MnO2-Dox-Col NPs based on mesoporous manganese dioxide (H-MnO2) in which Dox was loaded in the core and collagenase (Col) was wrapped in the surface. Further the H-MnO2-Dox-Col NPs were covered by a fusion membrane (MP) of inflammation-targeted RAW264.7 cell membrane and pH-sensitive liposomes to form biomimetic MP@H-MnO2-Dox-Col for in vitro and in vivo study. RESULTS: Our results shows that MP@H-MnO2-Dox-Col can increase the Dox effect with low cardiotoxicity based on multi-functions of effective penetration in tumor tissue, alleviating hypoxia in TME, pH sensitive drug release as well as targeted delivery of Dox. CONCLUSIONS: This multifunctional biomimetic nanodelivery system exhibited antitumor efficacy in vivo and in vitro, thus having potential for the treatment of breast cancer.

Sexually dimorphic age-related molecular differences in the entorhinal cortex of cognitively intact elderly: Relation to early Alzheimer's changes.

INTRODUCTION: Women are more vulnerable to Alzheimer's disease (AD) than men. The entorhinal cortex (EC) is one of the earliest structures affected in AD. We identified in cognitively intact elderly different molecular changes in the EC in relation to age. METHODS: Changes in 12 characteristic molecules in relation to age were determined by quantitative immunohistochemistry or in situ hybridization in the EC. They were arbitrarily grouped into sex steroid-related molecules, markers of neuronal activity, neurotransmitter-related molecules, and cholinergic activity-related molecules. RESULTS: The changes in molecules indicated increasing local estrogenic and neuronal activity accompanied by a higher and faster hyperphosphorylated tau accumulation in women's EC in relation to age, versus a mainly stable local estrogenic/androgenic and neuronal activity in men's EC. DISCUSSION: EC employs a different neurobiological strategy in women and men to maintain cognitive function, which seems to be accompanied by an earlier start of AD in women. HIGHLIGHTS: Local estrogen system is activated with age only in women's entorhinal cortex (EC). EC neuronal activity increased with age only in elderly women with intact cognition. Men and women have different molecular strategies to retain cognition with aging. P-tau accumulation in the EC was higher and faster in cognitively intact elderly women.

Genome-wide identification of AP2/EREBP in Fragaria vesca and expression pattern analysis of the FvDREB subfamily under drought stress.

BACKGROUND: Drought is a common phenomenon worldwide. It is also one of the main abiotic factors that affect the growth and quality of strawberry. The dehydration-responsive element binding protein (DREB) members that belong to the APETALA2/ethylene-responsive element binding protein (AP2/EREBP) superfamily are unique transcription factors in plants that play important roles in the abiotic stress response. RESULTS: Here, a total of 119 AP2/EREBP genes were identified in Fragaria vesca, and the AP2/EREBP superfamily was divided into AP2, RAV, ERF, DREB, and soloist subfamilies, containing 18, 7, 61, 32, and one member(s), respectively. The DREB subfamily was further divided into six subgroups (A-1 to A-6) based on phylogenetic analysis. Gene structure, conserved motifs, chromosomal location, and synteny analysis were conducted to comprehensively investigate the characteristics of FvDREBs. Furthermore, transcriptome analysis revealed distinctive expression patterns among the FvDREB genes in strawberry plants exposed to drought stress. The expression of FvDREB6 of the A-2 subgroup was down-regulated in old leaves and up-regulated in young leaves in response to drought. Furthermore, qRT-PCR analysis found that FvDREB8 from the A-2 subgroup had the highest expression level under drought stress. Together, analyses with the expression pattern, phylogenetic relationship, motif, and promoter suggest that FvDREB18 may play a critical role in the regulation of FvDREB1 and FvDREB2 expression. CONCLUSIONS: Our findings provide new insights into the characteristics and potential functions of FvDREBs. These FvDREB genes should be further studied as they appear to be excellent candidates for drought tolerance improvement of strawberry.

Combinatorial Evolution of a Terpene Synthase Gene Cluster Explains Terpene Variations in Oryza.

Terpenes are specialized metabolites ubiquitously produced by plants via the action of terpene synthases (TPSs). There are enormous variations in the types and amounts of terpenes produced by individual species. To understand the mechanisms responsible for such vast diversity, here we investigated the origin and evolution of a cluster of tandemly arrayed TPS genes in Oryza In the Oryza species analyzed, TPS genes occur as a three-TPS cluster, a two-TPS cluster, and a single TPS gene in five, one, and one species, respectively. Phylogenetic analysis revealed the origins of the two-TPS and three-TPS clusters and the role of species-specific losses of TPS genes. Within the three-TPS clusters, one orthologous group exhibited conserved catalytic activities. The other two groups, both of which contained pseudogenes and/or nonfunctional genes, exhibited distinct profiles of terpene products. Sequence and structural analyses combined with functional validation identified several amino acids in the active site that are critical for catalytic activity divergence of the three orthologous groups. In the five Oryza species containing the three-TPS cluster, their functional TPS genes showed both conserved and species-specific expression patterns in insect-damaged and untreated plants. Emission patterns of volatile terpenes from each species were largely consistent with the expression of their respective TPS genes and the catalytic activities of the encoded enzymes. This study indicates the importance of combinatorial evolution of TPS genes in determining terpene variations among individual species, which includes gene duplication, retention/loss/degradation of duplicated genes, varying selection pressure, retention/divergence in catalytic activities, and divergence in expression regulation.

Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthases from Dictyostelium purpureum and Streptomyces coelicolor.

The main product of DpTPS9 from the social amoeba Dictyostelium purpureum was identified as (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol that is also known as an intermediate of bacterial geosmin synthase, but the experimentally verified cyclisation mechanisms differ. Together with the low sequence identity this points to convergent evolution. The functionality of selected residues in DpTPS9 was investigated via site-directed mutagenesis experiments.

The reconstruction and biochemical characterization of ancestral genes furnish insights into the evolution of terpene synthase function in the Poaceae.

KEY MESSAGE: Distinct catalytic features of the Poaceae TPS-a subfamily arose early in grass evolution and the reactions catalyzed have become more complex with time. The structural diversity of terpenes found in nature is mainly determined by terpene synthases (TPS). TPS enzymes accept ubiquitous prenyl diphosphates as substrates and convert them into the various terpene skeletons by catalyzing a carbocation-driven reaction. Based on their sequence similarity, terpene synthases from land plants can be divided into different subfamilies, TPS-a to TPS-h. In this study, we aimed to understand the evolution and functional diversification of the TPS-a subfamily in the Poaceae (the grass family), a plant family that contains important crops such as maize, wheat, rice, and sorghum. Sequence comparisons showed that aside from one clade shared with other monocot plants, the Poaceae TPS-a subfamily consists of five well-defined clades I-V, the common ancestor of which probably originated very early in the evolution of the grasses. A survey of the TPS literature and the characterization of representative TPS enzymes from clades I-III revealed clade-specific substrate and product specificities. The enzymes in both clade I and II function as sesquiterpene synthases with clade I enzymes catalyzing initial C10-C1 or C11-C1 ring closures and clade II enzymes catalyzing C6-C1 closures. The enzymes of clade III mainly act as monoterpene synthases, forming cyclic and acyclic monoterpenes. The reconstruction and characterization of clade ancestors demonstrated that the differences among clades I-III were already present in their ancestors. However, the ancestors generally catalyzed simpler reactions with less double-bond isomerization and fewer cyclization steps. Overall, our data indicate an early origin of key enzymatic features of TPS-a enzymes in the Poaceae, and the development of more complex reactions over the course of evolution.

Terpene Biosynthesis in Red Algae Is Catalyzed by Microbial Type But Not Typical Plant Terpene Synthases.

Red algae (Rhodophyta) and land plants belong to the monophyletic clade Archaeplastida, and taxa of both groups are rich producers of terpene secondary metabolites. The terpene carbon skeletons of land plants are made by two types of terpene synthases: typical plant terpene synthases and microbial-type terpene synthases (MTPSLs); however, terpene biosynthesis in red algae is poorly understood. By systematic sequence analysis of seven genomes and 34 transcriptomes of red algae, MTPSL homologs were identified within one genome and two transcriptomes, whereas no homolog of typical plant terpene synthase genes was found. Phylogenetic analysis showed that red algae MTPSLs group with bacterial terpene synthases. Analysis of the genome assembly and characterization of neighboring genes demonstrated red algal MTPSLs to be bona fide red algal genes and not microbial contaminants. MTPSL genes from Porphyridium purpureum and Erythrolobus australicus were characterized via heterologous expression in Escherichia coli and demonstrated to have sesquiterpene synthase activities. We detected a number of volatile sesquiterpenes in the headspace of P. purpureum and E. australicus cultures, most identical to the in vitro products of the respective MTPSLs. Expression of the MTPSL gene in P. purpureum was found to be induced by methyl jasmonate, suggesting a role for this gene in host defense. In summary, this study indicates that the formation of terpene carbon skeletons in red algae is carried out by MTPSLs that are phylogenetically unrelated to typical plant terpene synthases and most likely originated in Rhodophyta via horizontal gene transfer from bacteria.

Molecular Diversity of Terpene Synthases in the Liverwort Marchantia polymorpha.

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.

Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae.

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 ß-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.

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not in seed plants.

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.

Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum.

Terpene synthases (TPSs) are pivotal enzymes for the production of diverse terpenes, including monoterpenes, sesquiterpenes, and diterpenes. In our recent studies, dictyostelid social amoebae, also known as cellular slime molds, were found to contain TPS genes for making volatile terpenes. For comparison, here we investigated Physarum polycephalum, a plasmodial slime mold also known as acellular amoeba. Plasmodia of P. polycephalum grown on agar plates were found to release a mixture of volatile terpenoids consisting of four major sesquiterpenes (α-muurolene, (E)-ß-caryophyllene, two unidentified sesquiterpenoids) and the monoterpene linalool. There were no qualitative differences in terpenoid composition at two stages of young plasmodia. To understand terpene biosynthesis, we analyzed the transcriptome and genome sequences of P. polycephalum and identified four TPS genes designated PpolyTPS1-PpolyTPS4. They share 28-73% of sequence identities. Full-length cDNAs for the four TPS genes were cloned and expressed in Escherichia coli to produce recombinant proteins, which were tested for sesquiterpene synthase and monoterpene synthase activities. While neither PpolyTPS2 nor PpolyTPS3 was active, PpolyTPS1 and PpolyTPS4 were able to produce sesquiterpenes and monoterpenes from the respective substrates farnesyl diphosphate and geranyl diphosphate. By comparing the volatile profile of P. polycephalum plasmodia and the in vitro products of PpolyTPS1 and PpolyTPS4, it was concluded that most sesquiterpenoids emitted from P. polycephalum were attributed to PpolyTPS4. Phylogenetic analysis placed the four PpolyTPSs genes into two groups: PpolyTPS1 and PpolyTPS4 being one group that was clustered with the TPSs from the dictyostelid social amoeba and PpolyTPS2 and PpolyTPS3 being the other group that showed closer relatedness to bacterial TPSs. The biological role of the volatile terpenoids produced by the plasmodia of P. polycephalum is discussed.

The kinome of pineapple: catalog and insights into functions in crassulacean acid metabolism plants.

BACKGROUND: Crassulacean acid metabolism (CAM) plants use water 20-80% more efficiently by shifting stomata opening and primary CO2 uptake and fixation to the nighttime. Protein kinases (PKs) play pivotal roles in this biological process. However, few PKs have been functionally analyzed precisely due to their abundance and potential functional redundancy (caused by numerous gene duplications). RESULTS: In this study, we systematically identified a total of 758 predicted PK genes in the genome of a CAM plant, pineapple (Ananas comosus). The pineapple kinome was classified into 20 groups and 116 families based on the kinase domain sequences. The RLK was the largest group, containing 480 members, and over half of them were predicted to locate at the plasma membrane. Both segmental and tandem duplications make important contributions to the expansion of pineapple kinome based on the synteny analysis. Ka/Ks ratios showed all of the duplication events were under purifying selection. The global expression analysis revealed that pineapple PKs exhibit different tissue-specific and diurnal expression patterns. Forty PK genes in a cluster performed higher expression levels in green leaf tip than in white leaf base, and fourteen of them had strong differential expression patterns between the photosynthetic green leaf tip and the non-photosynthetic white leaf base tissues. CONCLUSIONS: Our findings provide insights into the evolution and biological function of pineapple PKs and a foundation for further functional analysis of PKs in CAM plants. The gene duplication, expression, and coexpression analysis helped us to rapidly identify the key candidates in pineapple kinome, which may play roles in the carbon fixation process in pineapple and help engineering CAM pathway into C3 crops for improved drought tolerance.

The rice terpene synthase gene OsTPS19 functions as an (S)-limonene synthase in planta, and its overexpression leads to enhanced resistance to the blast fungus Magnaporthe oryzae.

Rice blast disease, caused by the fungus Magnaporthe oryzae, is the most devastating disease of rice. In our ongoing characterization of the defence mechanisms of rice plants against M. oryzae, a terpene synthase gene OsTPS19 was identified as a candidate defence gene. Here, we report the functional characterization of OsTPS19, which is up-regulated by M. oryzae infection. Overexpression of OsTPS19 in rice plants enhanced resistance against M. oryzae, while OsTPS19 RNAi lines were more susceptible to the pathogen. Metabolic analysis revealed that the production of a monoterpene (S)-limonene was increased and decreased in OsTPS19 overexpression and RNAi lines, respectively, suggesting that OsTPS19 functions as a limonene synthase in planta. This notion was further supported by in vitro enzyme assays with recombinant OsTPS19, in which OsTPS19 had both sesquiterpene activity and monoterpene synthase activity, with limonene as a major product. Furthermore, in a subcellular localization experiment, OsTPS19 was localized in plastids. OsTPS19 has a highly homologous paralog, OsTPS20, which likely resulted from a recent gene duplication event. We found that the variation in OsTPS19 and OsTPS20 enzyme activities was determined by a single amino acid in the active site cavity. The expression of OsTPS20 was not affected by M. oryzae infection. This indicates functional divergence of OsTPS19 and OsTPS20. Lastly, (S)-limonene inhibited the germination of M. oryzae spores in vitro. OsTPS19 was determined to function as an (S)-limonene synthase in rice and plays a role in defence against M. oryzae, at least partly, by inhibiting spore germination.

An (E,E)-α-farnesene synthase gene of soybean has a role in defence against nematodes and is involved in synthesizing insect-induced volatiles.

Plant terpene synthase genes (TPSs) have roles in diverse biological processes. Here, we report the functional characterization of one member of the soybean TPS gene family, which was designated GmAFS. Recombinant GmAFS produced in Escherichia coli catalysed the formation of a sesquiterpene (E,E)-α-farnesene. GmAFS is closely related to (E,E)-α-farnesene synthase gene from apple, both phylogenetically and structurally. GmAFS was further investigated for its biological role in defence against nematodes and insects. Soybean cyst nematode (SCN) is the most important pathogen of soybean. The expression of GmAFS in a SCN-resistant soybean was significantly induced by SCN infection compared with the control, whereas its expression in a SCN-susceptible soybean was not changed by SCN infection. Transgenic hairy roots overexpressing GmAFS under the control of the CaMV 35S promoter were generated in an SCN-susceptible soybean line. The transgenic lines showed significantly higher resistance to SCN, which indicates that GmAFS contributes to the resistance of soybean to SCN. In soybean leaves, the expression of GmAFS was found to be induced by Tetranychus urticae (two-spotted spider mites). Exogenous application of methyl jasmonate to soybean plants also induced the expression of GmAFS in leaves. Using headspace collection combined with gas chromatography-mass spectrometry analysis, soybean plants that were infested with T. urticae were shown to emit a mixture of volatiles with (E,E)-α-farnesene as one of the most abundant constituents. In summary, this study showed that GmAFS has defence roles in both below-ground and above-ground organs of soybean against nematodes and insects, respectively.

Mechanisms of the Diterpene Cyclases ß-Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus.

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

Novel constructs for efficient cloning of sRNA-encoding DNA and uniform silencing of plant genes employing artificial trans-acting small interfering RNA.

KEY MESSAGE: TAS atasiRNA-producing region swapping used one-step, high efficiency, and high fidelity directional TC-cloning. Uniform silencing was achieved without lethality using miRNA trigger- TAS overexpression fusion cassettes to generate 21-nt atasiRNA. Plant transgenic technologies are very important for basic plant research and biotechnology. Artificial trans-acting small interfering RNA (atasiRNA) represents an attractive platform with certain advantages over other silencing approaches, such as hairpin RNA, artificial microRNA (amiRNA), and virus-induced gene silencing (VIGS). In this study, we developed two types of constructs for atasiRNA-mediated gene silencing in plants. To functionally validate our constructs, we chose TAS1a as a test model. Type 1 constructs had miR173-precursor sequence fused with TAS1a locus driven by single promoter-terminator cassette, which simplified the expression cassette and resulted in uniform gene silencing. Type 2 constructs contained two separate cassettes for miR173 and TAS1a co-expression. The constructs in each type were further improved by deploying the XcmI-based TC-cloning system for highly efficient directional cloning of short DNA fragments encoding atasiRNAs into TAS1a locus. The effectiveness of the constructs was demonstrated by cloning an atasiRNA DNA into the TC site of engineered TAS1a and silencing of CHLORINA 42 (CH42) gene in Arabidopsis. Our results show that the directional TC-cloning of the atasiRNA DNA into the engineered TAS1a is highly efficient and the miR173-TAS1a fusion system provides an attractive alternative to achieve moderate but more uniform gene silencing without lethality, as compared to conventional two separate cassettes for miR173 and TAS locus co-expression system. The design principles described here should be applicable to other TAS loci such as TAS1b, TAS1c, TAS2, or TAS3, and cloning of amiRNA into amiRNA stem-loop.

Bioinformatic analysis of microbial type terpene synthase genes in plants.

Plants are prolific producers of terpenoids. Terpenoid biosynthesis is initiated by terpene synthases (TPS). In plants, two types of terpenes synthase genes are recognized: typical plant TPS genes and microbial-terpene synthase like-genes (MTPSL). While TPS genes are ubiquitous in land plants, MTPSL genes appear to be restricted to non-seed land plants. Evolutionarily, TPS genes are specific to land plants, whereas MTPSL genes have related counterparts in other organisms, especially fungi and bacteria. The presence of microbial type TPS in plants, fungi and bacteria, with the latter two often being associated with plants, poses a challenge in accurately identifying bona fide MTPSL genes in plants. In this chapter, we present bioinformatic procedures designed to identify MTPSL genes in sequenced plant genomes and/or transcriptomes. Additionally, we outline validation methods for confirming the identified microbial-type TPS genes as genuine plant genes. The method described in this chapter can also be adopted to analyze microbial type TPS in organisms other than plants.