Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

NtMYB1 and NtNCED1/2 control abscisic acid biosynthesis and tepal senescence in Chinese narcissus (Narcissus tazetta).

Chinese narcissus (Narcissus tazetta var. chinensis cv. 'Jinzhanyintai') is one of the 10 most famous traditional flowers of China, having a beautiful and highly ornamental flower with a rich fragrance. However, the flower longevity affects its commercial appeal. While petal senescence in Narcissus is ethylene-independent and abscisic acid-dependent, the regulatory mechanism has yet to be determined. In this study, we identified a R2R3-MYB gene (NtMYB1) from Narcissus tazetta and generated oeNtMYB1 and Ntmyb1 RNA interference mutants in Narcissus as well as an oeNtMYB1 construct in Arabidopsis. Overexpressing NtMYB1 in Narcissus or Arabidopsis led to premature leaf yellowing, an elevated level of total carotenoid, a reduced level of chlorophyll b, and a decrease in photosystem II fluorescence (Fv/Fm). A dual-luciferase assay and chromatin immunoprecipitation-quantitative PCR revealed that NtMYB1 directly binds to the promoter of NtNCED1 or NtNCED2 and activates NtNCED1/2 gene expression both in vitro and in vivo. Moreover, overexpressing NtMYB1 accelerated abscisic acid biosynthesis, up-regulated the content of zeatin and abscisic acid, and down-regulated the level of ß-carotene and gibberellin A1, leading to petal senescence and leaf yellowing in Narcissus. This study revealed a regulatory process that is fundamentally different between non-photosynthetic organs and leaves.

Synonymous Codon Usage Analysis of Three Narcissus Potyviruses.

Narcissus degeneration virus (NDV), narcissus late season yellows virus (NLSYV) and narcissus yellow stripe virus (NYSV), which belong to the genus Potyvirus of the family Potyviridae, cause significant losses in the ornamental value and quality of narcissus. Several previous studies have explored the genetic diversity and evolution rate of narcissus viruses, but the analysis of the synonymous codons of the narcissus viruses is still unclear. Herein, the coat protein (CP) of three viruses is used to analyze the viruses' phylogeny and codon usage pattern. Phylogenetic analysis showed that NYSV, NDV and NLSYV isolates were divided into five, three and five clusters, respectively, and these clusters seemed to reflect the geographic distribution. The effective number of codon (ENC) values indicated a weak codon usage bias in the CP coding region of the three narcissus viruses. ENC-plot and neutrality analysis showed that the codon usage bias of the three narcissus viruses is all mainly influenced by natural selection compared with the mutation pressure. The three narcissus viruses shared the same best optimal codon (CCA) and the synonymous codon prefers to use codons ending with A/U, compared to C/G. Our study shows the codon analysis of different viruses on the same host for the first time, which indicates the importance of the evolutionary-based design to control these viruses.

Transcriptome-Based WGCNA Analysis Reveals Regulated Metabolite Fluxes between Floral Color and Scent in Narcissus tazetta Flower.

A link between the scent and color of Narcissus tazetta flowers can be anticipated due to their biochemical origin, as well as their similar biological role. Despite the obvious aesthetic and ecological significance of these colorful and fragrant components of the flowers and the molecular profiles of their pigments, fragrant formation has addressed in some cases. However, the regulatory mechanism of the correlation of fragrant components and color patterns is less clear. We simultaneously used one way to address how floral color and fragrant formation in different tissues are generated during the development of an individual plant by transcriptome-based weighted gene co-expression network analysis (WGCNA). A spatiotemporal pattern variation of flavonols/carotenoids/chlorophyll pigmentation and benzenoid/phenylpropanoid/ monoterpene fragrant components between the tepal and corona in the flower tissues of Narcissus tazetta, was exhibited. Several candidate transcription factors: MYB12, MYB1, AP2-ERF, bZIP, NAC, MYB, C2C2, C2H2 and GRAS are shown to be associated with metabolite flux, the phenylpropanoid pathway to the production of flavonols/anthocyanin, as well as related to one branch of the phenylpropanoid pathway to the benzenoid/phenylpropanoid component in the tepal and the metabolite flux between the monoterpene and carotenoids biosynthesis pathway in coronas. It indicates that potential competition exists between floral pigment and floral fragrance during Narcissus tazetta individual plant development and evolutionary development.

NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

BACKGROUND: Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. RESULTS: In this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6. CONCLUSIONS: Our results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

Relative expression of putative genes involved in galanthamine and other Amaryllidaceae alkaloids biosynthesis in Narcissus field and in vitro tissues.

The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20 mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4 mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310 µg Gal/g FW) and bulb (980-1150 µg Gal/g FW) than the culture derived samples; callus (1.0-7.0 µg Gal/g FW) and bulblets (12-215 µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpO4OMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.

NtMYB3, an R2R3-MYB from Narcissus, Regulates Flavonoid Biosynthesis.

R2R3-MYB transcription factors play important roles in the regulation of plant flavonoid metabolites. In the current study, NtMYB3, a novel R2R3-MYB transcriptional factor isolated from Chinese narcissus (Narcissus tazetta L. var. chinensis), was functionally characterized. Phylogenetic analysis indicated that NtMYB3 belongs to the AtMYB4-like clade, which includes repressor MYBs involved in the regulation of flavonoid biosynthesis. Transient assays showed that NtMYB3 significantly reduced red pigmentation induced by the potato anthocyanin activator StMYB-AN1 in agro-infiltrated leaves of tobacco. Over-expression of NtMYB3 decreased the red color of transgenic tobacco flowers, with qRT-PCR analysis showing that NtMYB3 repressed the expression levels of genes involved in anthocyanin and flavonol biosynthesis. However, the proanthocyanin content in flowers of transgenic tobacco increased as compared to wild type. NtMYB3 showed expression in all examined narcissus tissues; the expression level in basal plates of the bulb was highest. A 968 bp promoter fragment of narcissus FLS (NtFLS) was cloned, and transient expression and dual luciferase assays showed NtMYB3 repressed the promoter activity. These results reveal that NtMYB3 is involved in the regulation of flavonoid biosynthesis in narcissus by repressing the biosynthesis of flavonols, and this leads to proanthocyanin accumulation in the basal plate of narcissus.

Developmental Regulation of the Expression of Amaryllidaceae Alkaloid Biosynthetic Genes in Narcissus papyraceus.

Amaryllidaceae alkaloids (AAs) have multiple biological effects, which are of interest to the pharmaceutical industry. To unleash the potential of Amaryllidaceae plants as pharmaceutical crops and as sources of AAs, a thorough understanding of the AA biosynthetic pathway is needed. However, only few enzymes in the pathway are known. Here, we report the transcriptome of AA-producing paperwhites (Narcissus papyraceus Ker Gawl). We present a list of 21 genes putatively encoding enzymes involved in AA biosynthesis. Next, a cDNA library was created from 24 different samples of different parts at various developmental stages of N. papyraceus. The expression of AA biosynthetic genes was analyzed in each sample using RT-qPCR. In addition, the alkaloid content of each sample was analyzed by HPLC. Leaves and flowers were found to have the highest abundance of heterocyclic compounds, whereas the bulb, the lowest. Lycorine was also the predominant AA. The gene expression results were compared with the heterocyclic compound profiles for each sample. In some samples, a positive correlation was observed between the gene expression levels and the amount of compounds accumulated. However, due to a probable transport of enzymes and alkaloids in the plant, a negative correlation was also observed, particularly at stage 2.

Transcriptome Sequencing and Biochemical Analysis of Perianths and Coronas Reveal Flower Color Formation in Narcissus pseudonarcissus.

Narcissus pseudonarcissus is an important bulbous plant with white or yellow perianths and light yellow to orange-red coronas, but little is known regarding the biochemical and molecular basis related to flower color polymorphisms. To investigate the mechanism of color formation, RNA-Seq of flower of two widely cultured cultivars ('Slim Whitman' and 'Pinza') with different flower color was performed. A total of 84,463 unigenes were generated from the perianths and coronas. By parallel metabolomic and transcriptomic analyses, we provide an overview of carotenoid biosynthesis, degradation, and accumulation in N. pseudonarcissus. The results showed that the content of carotenoids in the corona was higher than that in the perianth in both cultivars. Accordingly, phytoene synthase (PSY) transcripts have a higher abundance in the coronas than that in perianths. While the expression levels of carotenoid biosynthetic genes, like GGPPS, PSY, and LCY-e, were not significantly different between two cultivars. In contrast, the carotenoid degradation gene NpCCD4 was highly expressed in white-perianth cultivars, but was hardly detected in yellow-perianth cultivars. Silencing of NpCCD4 resulted in a significant increase in carotenoid accumulation, especially in all-trans-ß-carotene. Therefore, we presume that NpCCD4 is a crucial factor that causes the low carotenoid content and color fading phenomenon of 'Slim Whitman' by mediating carotenoid turnover. Our findings provide mass RNA-seq data and new insights into carotenoid metabolism in N. pseudonarcissus.

Ectopic Overexpression of a Novel R2R3-MYB, NtMYB2 from Chinese Narcissus Represses Anthocyanin Biosynthesis in Tobacco.

R2R3 MYB transcription factors play key functions in the regulation of secondary metabolites. In the present study, a R2R3 MYB transcriptional factor NtMYB2 was identified from Chinese narcissus (Narcissus tazetta L. var. Chinensis Roem) and functionally characterized. NtMYB2 belongs to subgroup 4 of the R2R3 MYB transcription factor family that are related to repressor MYBs involved in the regulation of anthocyanin and flavonoids. Transient expression confirmed that NtMYB2 strongly reduced the red pigmentation induced by MYB- anthocyanin activators in agro-infiltrated tobacco leaves. Ectopic expression of NtMYB2 in tobacco significantly reduced the pigmentation and altered the floral phenotypes in transgenic tobacco flowers. Gene expression analysis suggested that NtMYB2 repressed the transcript levels of structural genes involved in anthocyanin biosynthesis pathway, especially the UFGT gene. NtMYB2 gene is expressed in all examined narcissus tissues; the levels of transcription in petals and corona is higher than other tissues and the transcription level at the bud stage was highest. These results show that NtMYB2 is involved in the regulation of anthocyanin biosynthesis pathway and may act as a repressor by down regulating the transcripts of key enzyme genes in Chinese narcissus.

Transcriptome and metabolome profiling of Narcissus pseudonarcissus 'King Alfred' reveal components of Amaryllidaceae alkaloid metabolism.

Amaryllidaceae alkaloids (AAs) represent a diverse class of plant specialized metabolites and many display potent pharmacological activities. The AA metabolic pathway is poorly understood and resources are minimal. To enable AA pathway elucidation and novel biosynthetic enzymes discovery, we generated comprehensive metabolomic and corresponding transcriptomic datasets from different tissues of Narcissus pseudonarcissus 'King Alfred'. In this study, we performed untargeted UPLC-QTOF-MS metabolite analysis from different tissues, which generated exhaustive list of compounds, including several AAs, most predominant and diverse in bulbs. RNA sequencing of N. pseudonarcissus 'King Alfred' bulbs yielded 195,347 transcripts, after assembly. Top expressed genes belong to process like metabolism, survival, and defense including alkaloid biosynthetic genes. The transcriptome contained complete sequences for all proposed genes encoding AA-biosynthetic enzymes such as tyrosine decarboxylase (TYDC1 and TYDC2), phenylalanine ammonia-lyase (PAL1 and PAL2) and phenolic acids hydroxylases (C4H and C3H) to name a few. Furthermore, transcriptome data were validated using RT-qPCR analysis and expression study in different tissues of N. pseudonarcissus 'King Alfred' was performed. Here, we present the first comprehensive metabolome and transcriptome study from N. pseudonarcissus 'King Alfred' providing invaluable resources for metabolic engineering and biotechnological applications.

Secretory Expression and Characterization of Chinese Narcissus GNA-Like Lectin in Pichia pastoris.

Narcissus tazetta lectin (NTL) is a GNA-like lectin, which has a wide potential application in medicine, agriculture, and glycobiology. In the present paper, the codon-optimized ntl gene was transformed into the yeast Pichia pastoris; SDS-PAGE gel and western blotting analysis revealed that the recombinant lectin was expressed successfully in Pichia yeast. The similarity between the recombinant NTL and the native NTL was confirmed by circular dichroism (CD) and hemagglutination assay further. In the 5-L scale fermentator, the protein yield was as high as 1.2 g/L after fermentation for 96 h. In addition, the effect of metal ions (K+, Mg2+, Ca2+, and Cu2+), acid, and alkaline on hemagglutinating activity of NTL was tested, which provided biochemical characterizations of the mannose-binding lectin from Chinese Narcissus.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

Convergent recruitment of new pollinators is triggered by independent hybridization events in Narcissus.

Hybridization can generate new species if some degree of isolation prevents gene flow between the hybrids and their progenitors. The recruitment of novel pollinators by hybrids has been hypothesized to be one way in which such reproductive isolation can be achieved. We tested whether pollinators contributed to isolation between two natural Narcissus hybrids and their progenitors using pollination experiments, observations, plus morphological and floral-volatile measurements. These hybrids share the same maternal but different paternal progenitors. We found that only the hybrids were visited by and pollinated by ants. The two hybrids exceeded their progenitors in floral-tube aperture size and nectar production. The emission of floral volatiles by hybrid plants was not only equal to or higher than the progenitor species, but also contained some new compounds not produced by the progenitors. The recruitment of ants as novel pollinators in the hybrids involved the combination of increased nectar secretion and the production of novel floral scent compounds. A breakdown of chemical defence against ants may also be involved. This study provides support for the hypothesis that the recruitment of novel pollinators can contribute to reproductive isolation between hybrids and their progenitors.

Disassortative mating prevails in style-dimorphic Narcissus papyraceus despite low reciprocity and compatibility of morphs.

Evolution to reduce inbreeding can favor disassortative (intermorph) over assortative (intramorph) mating in hermaphroditic sexually polymorphic plant species. Heterostyly enhances disassortative pollination through reciprocal placement of stigmas and anthers of morphs and appropriate pollinators. Stylar dimorphism in which there is not reciprocal anther placement may compromise disassortative mating, particularly when there is not intramorph incompatibility. Variable rates of disassortative mating along with differential female fecundity or siring success among floral morphs could lead to variation in morph ratio. We investigated mating patterns, female fecundity, and siring success of style-length morphs in Narcissus papyraceus, a self-incompatible but morph-compatible species with dimorphic (long- and short-styled) and monomorphic (long-styled) populations in central and north regions of its range, respectively. We established experimental populations in both regions and exposed them to ambient pollinators. Using paternity analysis, we found similar siring success of morphs and high disassortative mating in most populations. Female fecundity of morphs was similar in all populations. Although these results could not completely explain the loss of dimorphism in the species' northern range, they provided evidence for the evolutionary stability of stylar dimorphism in N. papyraceus in at least some populations. Our findings support the hypothesis that prevailing intermorph mating is key for the maintenance of stylar dimorphism.

Range-wide population genetics and variation in morph ratio in style-dimorphic Narcissus papyraceus.

UNLABELLED: • PREMISE OF THE STUDY: Theoretical models state that natural selection and mating patterns account for floral morph ratio in style-polymorphic plants. However, the demographic history of populations can also influence variation in morph ratios. If so, we hypothesize an association between the morph ratios and the genetic structure across populations.• METHODS: We used nuclear microsatellites to assess genetic variation and structure in populations of Narcissus papyraceus, a style-dimorphic plant whose floral morph ratios (L-morph to S-morph) gradually vary throughout its distribution range in the southwestern Mediterranean Basin. We implemented analyses to relate the genetic features of populations with their morph ratios.• KEY RESULTS: We found greater frequencies of the S-morph in central populations and declining frequencies toward the periphery. This geographic pattern was not associated with the genetic structure of populations. Instead, we found two distinct genetic groups, mainly separated by the Strait of Gibraltar, with a mixture of morph ratios within each one. Overall, there was a weak genetic structure. Genetic diversity was greater in central and southern dimorphic populations than in northern L-monomorphic populations.• CONCLUSIONS: Altogether, our results do not support the hypothesis that the demographic history of populations can account for the observed geographical pattern of morph ratios in N. papyraceus. We suggest that adaptive processes shown in previous studies in the species are the main determinant of the existing variation in the morph composition of populations.

Narcissus tazetta SVP-like gene NSVP1 affects flower development in Arabidopsis.

SHORT VEGETATIVE PHASE (SVP) related genes have important functions in regulating floral transition and inflorescence structure in many plant species. Some SVP related genes have been shown associated with dormancy transition. Narcissus tazetta var. chinensis exhibits summer dormancy release and floral transition promoted by extended high temperature exposure. However, the molecular mechanism underlying such development remains unknown. In this study, we isolated and characterized one SVP-like gene, NSVP1 from N. tazetta var. chinensis. The results of RT-PCR and in situ hybridization assay showed that NSVP1 was expressed in both vegetative and floral tissues. The highest level of NSVP1 in the bulb apices was detected when the above-ground just senesced and its transcripts declined gradually during endo-dormany. The lowest level was found at the beginning of flower differentiation and the release of endo-dormancy. These data suggest that NSVP1 is differentially regulated coordinately with endo-dormancy induction and release. Ectopic expression of NSVP1 neither complemented the early flowering phenotype of svp mutant nor altered the rosette leaf number in Col background. However, NSVP1 in svp mutant and Ler plants increased the number of lateral inflorescence and caused abnormal floral morphologies. In addition, strong expression of NSVP1 in Ler background affected plastochron. These results suggest that NSVP1 might play a role in the regulation of flower development.

Cloning and characterization of a norbelladine 4'-O-methyltransferase involved in the biosynthesis of the Alzheimer's drug galanthamine in Narcissus sp. aff. pseudonarcissus.

Galanthamine is an Amaryllidaceae alkaloid used to treat the symptoms of Alzheimer's disease. This compound is primarily isolated from daffodil (Narcissus spp.), snowdrop (Galanthus spp.), and summer snowflake (Leucojum aestivum). Despite its importance as a medicine, no genes involved in the biosynthetic pathway of galanthamine have been identified. This absence of genetic information on biosynthetic pathways is a limiting factor in the development of synthetic biology platforms for many important botanical medicines. The paucity of information is largely due to the limitations of traditional methods for finding biochemical pathway enzymes and genes in non-model organisms. A new bioinformatic approach using several recent technological improvements was applied to search for genes in the proposed galanthamine biosynthetic pathway, first targeting methyltransferases due to strong signature amino acid sequences in the proteins. Using Illumina sequencing, a de novo transcriptome assembly was constructed for daffodil. BLAST was used to identify sequences that contain signatures for plant O-methyltransferases in this transcriptome. The program HAYSTACK was then used to identify methyltransferases that fit a model for galanthamine biosynthesis in leaf, bulb and inflorescence tissues. One candidate gene for the methylation of norbelladine to 4'-O-methylnorbelladine in the proposed galanthamine biosynthetic pathway was identified. This methyltransferase cDNA was expressed in E. coli and the protein purified by affinity chromatography. The resulting protein was found to be a norbelladine 4'-O-methyltransferase (NpN4OMT) of the proposed galanthamine biosynthetic pathway.

Phenotypic integration in style dimorphic daffodils (Narcissus, Amaryllidaceae) with different pollinators.

Different pollinators can exert different selective pressures on floral traits, depending on how they fit with flowers, which should be reflected in the patterns of variation and covariation of traits. Surprisingly, empirical evidence in support of this view is scarce. Here, we have studied whether the variation observed in floral phenotypic integration and covariation of traits in Narcissus species is associated with different groups of pollinators. Phenotypic integration was studied in two style dimorphic species, both with dimorphic populations mostly visited by long-tongued pollinators (close fit with flowers), and monomorphic populations visited by short-tongued insects (loose fit). For N. papyraceus, the patterns of variation and correlation among traits involved in different functions (attraction and fit with pollinators, transfer of pollen) were compared within and between population types. The genetic diversity of populations was also studied to control for possible effects on phenotypic variation. In both species, populations with long-tongued pollinators displayed greater phenotypic integration than those with short-tongued pollinators. Also, the correlations among traits involved in the same function were stronger than across functions. Furthermore, traits involved in the transfer of pollen were consistently more correlated and less variable than traits involved in the attraction of insects, and these differences were larger in dimorphic than monomorphic populations. In addition, population genetic parameters did not correlate with phenotypic integration or variation. Altogether, our results support current views of the role of pollinators in the evolution of floral integration.

Comparative transcriptional analysis reveals differential gene expression between Sand Daffodil tissues.

Sand Daffodil (Pancratium maritimum) is a world-wide endangered Amayllidaceae species and represents an important anti-cancer medicinal resource due to alkaloids production. Despite its increasing pharmaceutical importance, there are not molecular resources that can be utilized toward improving genetic traits. In our research, the suppression subtractive hybridization (SSH) method conducted to generate large-scale expressed sequence tags (EST), was designed to identify gene candidates related to the morphological and physiological differences between the two tissues, leaves and bulbs, since lycorine, the main anti-cancer compound, is there synthesized. We focused on identification of transcripts in different tissues from Sand Daffodil using PCR-based suppression SSH to identify genes involved in global pathway control. Sequencing of 2,000 differentially screened clones from the SSH libraries resulted in 136 unigenes. Functional annotation and gene ontology analysis of up-regulated EST libraries showed several known biosynthetic genes and novel transcripts that may be involved in signaling, cellular transport, or metabolism. Real time RT-PCR analysis of a set of 8 candidate genes further confirmed the differential gene expression.