Absence of chromosome axis protein recruitment prevents meiotic recombination chromosome-wide in the budding yeast Lachancea kluyveri.

Meiotic recombination shows broad variations across species and along chromosomes and is often suppressed at and around genomic regions determining sexual compatibility such as mating type loci in fungi. Here, we show that the absence of Spo11-DSBs and meiotic recombination on Lakl0C-left, the chromosome arm containing the sex locus of the Lachancea kluyveri budding yeast, results from the absence of recruitment of the two chromosome axis proteins Red1 and Hop1, essential for proper Spo11-DSBs formation. Furthermore, cytological observation of spread pachytene meiotic chromosomes reveals that Lakl0C-left does not undergo synapsis. However, we show that the behavior of Lakl0C-left is independent of its particularly early replication timing and is not accompanied by any peculiar chromosome structure as detectable by Hi-C in this yet poorly studied yeast. Finally, we observed an accumulation of heterozygous mutations on Lakl0C-left and a sexual dimorphism of the haploid meiotic offspring, supporting a direct effect of this absence of meiotic recombination on L. kluyveri genome evolution and fitness. Because suppression of meiotic recombination on sex chromosomes is widely observed across eukaryotes, the mechanism for recombination suppression described here may apply to other species, with the potential to impact sex chromosome evolution.

Variation of the meiotic recombination landscape and properties over a broad evolutionary distance in yeasts.

Meiotic recombination is a major factor of genome evolution, deeply characterized in only a few model species, notably the yeast Saccharomyces cerevisiae. Consequently, little is known about variations of its properties across species. In this respect, we explored the recombination landscape of Lachancea kluyveri, a protoploid yeast species that diverged from the Saccharomyces genus more than 100 million years ago and we found striking differences with S. cerevisiae. These variations include a lower recombination rate, a higher frequency of chromosomes segregating without any crossover and the absence of recombination on the chromosome arm containing the sex locus. In addition, although well conserved within the Saccharomyces clade, the S. cerevisiae recombination hotspots are not conserved over a broader evolutionary distance. Finally and strikingly, we found evidence of frequent reversal of commitment to meiosis, resulting in return to mitotic growth after allele shuffling. Identification of this major but underestimated evolutionary phenomenon illustrates the relevance of exploring non-model species.

miRkwood: a tool for the reliable identification of microRNAs in plant genomes.

BACKGROUND: MicroRNAs (miRNAs) play crucial roles in post-transcriptional regulation of eukaryotic gene expression and are involved in many aspects of plant development. Although several prediction tools are available for metazoan genomes, the number of tools dedicated to plants is relatively limited. RESULTS: Here, we present miRkwood, a user-friendly tool for the identification of miRNAs in plant genomes using small RNA sequencing data. Deep-sequencing data of Argonaute associated small RNAs showed that miRkwood is able to identify a large diversity of plant miRNAs and limits false positive predictions. Moreover, it outperforms current tools such as ShortStack and contrary to ShortStack, miRkwood provides a quality score allowing users to rank miRNA predictions. CONCLUSION: miRkwood is a very efficient tool for the annotation of miRNAs in plant genomes. It is available as a web server, as a standalone version, as a docker image and as a Galaxy tool: http://bioinfo.cristal.univ-lille.fr/mirkwood.

Isolation and functional characterization of a τ-cadinol synthase, a new sesquiterpene synthase from Lavandula angustifolia.

In this paper we characterize three sTPSs: a germacrene D (LaGERDS), a (E)-ß-caryophyllene (LaCARS) and a τ-cadinol synthase (LaCADS). τ-cadinol synthase is reported here for the first time and its activity was studied in several biological models including transiently or stably transformed tobacco species. Three dimensional structure models of LaCADS and Ocimum basilicum γ-cadinene synthase were built by homology modeling using the template structure of Gossypium arboreum δ-cadinene synthase. The depiction of their active site organization provides evidence of the global influence of the enzymes on the formation of τ-cadinol: instead of a unique amino-acid, the electrostatic properties and solvent accessibility of the whole active site in LaCADS may explain the stabilization of the cadinyl cation intermediate. Quantitative PCR performed from leaves and inflorescences showed two patterns of expression. LaGERDS and LaCARS were mainly expressed during early stages of flower development and, at these stages, transcript levels paralleled the accumulation of the corresponding terpene products (germacrene D and (E)-ß-caryophyllene). By contrast, the expression level of LaCADS was constant in leaves and flowers. Phylogenetic analysis provided informative results on potential duplication process leading to sTPS diversification in lavender.

The homomorphic self-incompatibility system in Oleaceae is controlled by a hemizygous genomic region expressing a gibberellin pathway gene.

In flowering plants, outcrossing is commonly ensured by self-incompatibility (SI) systems. These can be homomorphic (typically with many different allelic specificities) or can accompany flower heteromorphism (mostly with just two specificities and corresponding floral types). The SI system of the Oleaceae family is unusual, with the long-term maintenance of only two specificities but often without flower morphology differences. To elucidate the genomic architecture and molecular basis of this SI system, we obtained chromosome-scale genome assemblies of Phillyrea angustifolia individuals and related them to a genetic map. The S-locus region proved to have a segregating 543-kb indel unique to one specificity, suggesting a hemizygous region, as observed in all distylous systems so far studied at the genomic level. Only one of the predicted genes in this indel region is found in the olive tree, Olea europaea, genome, also within a segregating indel. We describe complete association between the presence/absence of this gene and the SI types determined for individuals of seven distantly related Oleaceae species. This gene is predicted to be involved in catabolism of the gibberellic acid (GA) hormone, and experimental manipulation of GA levels in developing buds modified the male and female SI responses of the two specificities in different ways. Our results provide a unique example of a homomorphic SI system, where a single conserved gibberellin-related gene in a hemizygous indel underlies the long-term maintenance of two groups of reproductive compatibility.

Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture.

BACKGROUND: Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. RESULTS: Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. CONCLUSIONS: SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.

Transposable elements maintain genome-wide heterozygosity in inbred populations.

Elevated levels of inbreeding increase the risk of inbreeding depression and extinction, yet many inbred species are widespread, suggesting that inbreeding has little impact on evolutionary potential. Here, we explore the potential for transposable elements (TEs) to maintain genetic variation in functional genomic regions under extreme inbreeding. Capitalizing on the mixed mating system of Arabidopsis lyrata, we assess genome-wide heterozygosity and signatures of selection at single nucleotide polymorphisms near transposable elements across an inbreeding gradient. Under intense inbreeding, we find systematically elevated heterozygosity downstream of several TE superfamilies, associated with signatures of balancing selection. In addition, we demonstrate increased heterozygosity in stress-responsive genes that consistently occur downstream of TEs. We finally reveal that TE superfamilies are associated with specific signatures of selection that are reproducible across independent evolutionary lineages of A. lyrata. Together, our study provides an important hypothesis for the success of self-fertilizing species.

Detecting MicroRNAs in Plant Genomes with miRkwood.

We present miRkwood, a comprehensive software tool developed to identify microRNAs and their precursor in plant genomes, with or without small-RNA-seq sequencing data. We describe how to install the software, how to set up and run it, and how to explore and analyse the results: genomic annotations, secondary structure of the precursor, alignments, reads distribution.

Integrated sRNA-seq and RNA-seq Analyses Reveal a microRNA Regulation Network Involved in Cold Response in Pisum sativum L.

(1) Background: Cold stress affects growth and development in plants and is a major environmental factor that decreases productivity. Over the past two decades, the advent of next generation sequencing (NGS) technologies has opened new opportunities to understand the molecular bases of stress resistance by enabling the detection of weakly expressed transcripts and the identification of regulatory RNAs of gene expression, including microRNAs (miRNAs). (2) Methods: In this study, we performed time series sRNA and mRNA sequencing experiments on two pea (Pisum sativum L., Ps) lines, Champagne frost-tolerant and Térèse frost-sensitive, during a low temperature treatment versus a control condition. (3) Results: An integrative analysis led to the identification of 136 miRNAs and a regulation network composed of 39 miRNA/mRNA target pairs with discordant expression patterns. (4) Conclusions: Our findings indicate that the cold response in pea involves 11 miRNA families as well as their target genes related to antioxidative and multi-stress defense mechanisms and cell wall biosynthesis.

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.).

BACKGROUND: In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies 1 showed that the use of the beta-D-glucosyl Yariv reagent (beta-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that beta-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation 2. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used beta-GlcY to block SE in order to identify genes potentially involved in this process. RESULTS: Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. beta-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by beta-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by beta-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. CONCLUSION: The use of two different chicory genotypes differing in their responsiveness to SE induction, together with beta-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory.

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development.

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub-groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.

Identification of Genes Differentially Expressed in Response to Cold in Pisum sativum Using RNA Sequencing Analyses.

Low temperature stress affects growth and development in pea (Pisum sativum L.) and decreases yield. In this study, RNA sequencing time series analyses performed on lines, Champagne frost-tolerant and Térèse frost-sensitive, during a low temperature treatment versus a control condition, led us to identify 4981 differentially expressed genes. Thanks to our experimental design and statistical analyses, we were able to classify these genes into three sets. The first one was composed of 2487 genes that could be related to the constitutive differences between the two lines and were not regulated during cold treatment. The second gathered 1403 genes that could be related to the chilling response. The third set contained 1091 genes, including genes that could be related to freezing tolerance. The identification of differentially expressed genes related to cold, oxidative stress, and dehydration responses, including some transcription factors and kinases, confirmed the soundness of our analyses. In addition, we identified about one hundred genes, whose expression has not yet been linked to cold stress. Overall, our findings showed that both lines have different characteristics for their cold response (chilling response and/or freezing tolerance), as more than 90% of differentially expressed genes were specific to each of them.

Differential retention of transposable element-derived sequences in outcrossing Arabidopsis genomes.

BACKGROUND: Transposable elements (TEs) are genomic parasites with major impacts on host genome architecture and host adaptation. A proper evaluation of their evolutionary significance has been hampered by the paucity of short scale phylogenetic comparisons between closely related species. Here, we characterized the dynamics of TE accumulation at the micro-evolutionary scale by comparing two closely related plant species, Arabidopsis lyrata and A. halleri. RESULTS: Joint genome annotation in these two outcrossing species confirmed that both contain two distinct populations of TEs with either 'recent' or 'old' insertion histories. Identification of rare segregating insertions suggests that diverse TE families contribute to the ongoing dynamics of TE accumulation in the two species. Orthologous TE fragments (i.e. those that have been maintained in both species), tend to be located closer to genes than those that are retained in one species only. Compared to non-orthologous TE insertions, those that are orthologous tend to produce fewer short interfering RNAs, are less heavily methylated when found within or adjacent to genes and these tend to have lower expression levels. These findings suggest that long-term retention of TE insertions reflects their frequent acquisition of adaptive roles and/or the deleterious effects of removing nearly neutral TE insertions when they are close to genes. CONCLUSION: Our results indicate a rapid evolutionary dynamics of the TE landscape in these two outcrossing species, with an important input of a diverse set of new insertions with variable propensity to resist deletion.

Functional Analysis of Four Terpene Synthases in Rose-Scented Pelargonium Cultivars (Pelargonium × hybridum) and Evolution of Scent in the Pelargonium Genus.

Pelargonium genus contains about 280 species among which at least 30 species are odorant. Aromas produced by scented species are remarkably diverse such as rose, mint, lemon, nutmeg, ginger and many others scents. Amongst odorant species, rose-scented pelargoniums, also named pelargonium rosat, are the most famous hybrids for their production of essential oil (EO), widely used by perfume and cosmetic industries. Although EO composition has been extensively studied, the underlying biosynthetic pathways and their regulation, most notably of terpenes, are largely unknown. To gain a better understanding of the terpene metabolic pathways in pelargonium rosat, we generated a transcriptome dataset of pelargonium leaf and used a candidate gene approach to functionally characterise four terpene synthases (TPSs), including a geraniol synthase, a key enzyme responsible for the biosynthesis of the main rose-scented terpenes. We also report for the first time the characterisation of a novel sesquiterpene synthase catalysing the biosynthesis of 10-epi-γ-eudesmol. We found a strong correlation between expression of the four genes encoding the respective TPSs and accumulation of the corresponding products in several pelargonium cultivars and species. Finally, using publically available RNA-Seq data and de novo transcriptome assemblies, we inferred a maximum likelihood phylogeny from 270 pelargonium TPSs, including the four newly discovered enzymes, providing clues about TPS evolution in the Pelargonium genus. Notably, we show that, by contrast to other TPSs, geraniol synthases from the TPS-g subfamily conserved their molecular function throughout evolution.

Characterization of expressed sequence tags obtained by SSH during somatic embryogenesis in Cichorium intybus L.

BACKGROUND: Somatic embryogenesis (SE) is an asexual propagation pathway requiring a somatic-to-embryonic transition of differentiated somatic cells toward embryogenic cells capable of producing embryos in a process resembling zygotic embryogenesis. In chicory, genetic variability with respect to the formation of somatic embryos was detected between plants from a population of Cichorium intybus L. landrace Koospol. Though all plants from this population were self incompatible, we managed by repeated selfing to obtain a few seeds from one highly embryogenic (E) plant, K59. Among the plants grown from these seeds, one plant, C15, was found to be non-embryogenic (NE) under our SE-inducing conditions. Being closely related, we decided to exploit the difference in SE capacity between K59 and its descendant C15 to study gene expression during the early stages of SE in chicory. RESULTS: Cytological analysis indicated that in K59 leaf explants the first cell divisions leading to SE were observed at day 4 of culture. In contrast, in C15 explants no cell divisions were observed and SE development seemed arrested before cell reactivation. Using mRNAs isolated from leaf explants from both genotypes after 4 days of culture under SE-inducing conditions, an E and a NE cDNA-library were generated by SSH. A total of 3,348 ESTs from both libraries turned out to represent a maximum of 2,077 genes. In silico subtraction analysis sorted only 33 genes as differentially expressed in the E or NE genotype, indicating that SSH had resulted in an effective normalisation. Real-time RT-PCR was used to verify the expression levels of 48 genes represented by ESTs from either library. The results showed preferential expression of genes related to protein synthesis and cell division in the E genotype, and related to defence in the NE genotype. CONCLUSION: In accordance with the cytological observations, mRNA levels in explants from K59 and C15 collected at day 4 of SE culture reflected differential gene expression that presumably are related to processes accompanying early stages of direct SE. The E and NE library obtained thus represent important tools for subsequent detailed analysis of molecular mechanisms underlying this process in chicory, and its genetic control.

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively.

Extracellular localization of the diterpene sclareol in clary sage (Salvia sclarea L., Lamiaceae).

Sclareol is a high-value natural product obtained by solid/liquid extraction of clary sage (Salvia sclarea L.) inflorescences. Because processes of excretion and accumulation of this labdane diterpene are unknown, the aim of this work was to gain knowledge on its sites of accumulation in planta. Samples were collected in natura or during different steps of the industrial process of extraction (steam distillation and solid/liquid extraction). Samples were then analysed with a combination of complementary analytical techniques (gas chromatography coupled to a mass spectrometer, polarized light microscopy, environmental scanning electron microscopy, two-photon fluorescence microscopy, second harmonic generation microscopy). According to the literature, it is hypothesized that sclareol is localized in oil pockets of secretory trichomes. This study demonstrates that this is not the case and that sclareol accumulates in a crystalline epicuticular form, mostly on calyces.

The biosynthetic pathways and biological scopes of plant small RNAs.

RNA silencing plays a crucial role in coordinating the expression, stability, protection and inheritance of eukaryotic genomes. It comprises several mechanisms that invariably depend on core small RNA molecules (sRNAs) and that achieve dedicated sequence-specific functions. Biogenesis and function of sRNAs generally follow a framework that involves main protein families conserved among eukaryotes. The most recent studies have provided structural insights into the function of RNA silencing components, identified novel players of these pathways and highlighted possible emerging classes of sRNA regulators. In this review, we integrate these studies to provide updated views on the biosynthetic pathways and dedicated functions of the endogenous sRNA classes of plants, emphasizing their specialized scopes.

Lavender inflorescence: a model to study regulation of terpenes synthesis.

We analysed VOC composition of complete inflorescences and single flowers of lavender during the flowering period. Our analyses, focused on the 20 most abundant terpenes, showed that three groups of components could be separated according to their patterns of variation during inflorescence ontogeny. These three groups were associated with three developmental stages: flower in bud, flower in bloom and faded flower. The expression of two terpene synthases (TPS) was followed using qPCR during inflorescence ontogeny. A comparison of these chemical and molecular analyses suggested that VOC production in lavender spike is mainly regulated at the transcriptional level. These results highlighted that lavender could be a model plant for future investigations on terpene biosynthesis and regulation, and could be used to explore the functions of terpene metabolites.

One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L.

The outermost floral whorl, composed of sepals, is generally thought to function in the protection of reproductive tissues. In the plant family Lamiaceae, sepals are fused into a tube that is densely covered by hairs for mechanical defence and contains secondary metabolites for chemical defence against insects and abiotic stresses. Despite the importance of this tissue in plant fitness, virtually no study has addressed the basic aspects of sepal development and functioning. Because of its large size and its impressive metabolic activity (both in terms of quantity and diversity of secondary metabolites), we have used clary sage calyx as a model system to generate the first high throughput sequencing of the transcriptome of an angiosperm calyx. We applied massive parallel 454 pyrosequencing technology to a normalized cDNA extract and unveiled potential candidate genes for all steps of secondary metabolite pathways (phenylpropanoids and terpenoids). It also proved efficient in predicting the expression of large numbers of transcription factors and, with the use of bioinformatics tools, it predicted in the same sequencing run the presence of a novel class of gene transcription regulatory elements, miRNAs, without the need to generate a separate miRNA library. In our clary sage EST library, 18 conserved miRNAs were predicted. Among them, 15 were present in most studied plant species while the others were only shared with limited or discrete plant lineages. A separate data mining of the same clary sage EST library suggested the presence of 19 potential target genes to the 18 predicted conserved miRNAs. These coded for only 6 transcription factors or F-box proteins, 11 metabolism or abiotic stress response related proteins and 2 products with no known predicted function. All in all, this study provides novel genomic information on an angiosperm calyx and an experimental framework to predict in a single step metabolic pathway enzymes and regulator genes including miRNAs.