Partial redundancy and functional specialization of E-class SEPALLATA genes in an early-diverging eudicot.

Plant MADS-box genes have duplicated extensively, allegedly contributing to the immense diversity of floral form in angiosperms. In Arabidopsis thaliana (a core eudicot model plant), four SEPALLATA (SEP) genes comprise the E-class from the extended ABCE model of flower development. They are redundantly involved in the development of the four types of floral organs (sepals, petals, stamens and carpels) and in floral meristem determinacy. E-class genes have been examined in other core eudicots and monocots, but have been less investigated in non-core eudicots. Our goal was to functionally characterize the E-class genes in the early-diverging eudicot Thalictrum thalictroides (Ranunculaceae), whose flowers are apetalous. We identified four SEP orthologs, which when placed in a phylogenetic context, resulted from a major gene duplication event before the origin of angiosperms and a subsequent duplication at the origin of the Ranunculales. We used Virus-Induced Gene Silencing (VIGS) to down-regulate the three expressed paralogs individually and in combination to investigate their function and to determine the degree of conservation versus divergence of this important plant transcription factor. All loci were partially redundant in sepal and stamen identity and in promoting petaloidy of sepals, yet the SEP3 ortholog had a more pronounced role in carpel identity and development. The two other paralogs appear to have subfunctionalized in their cadastral roles to keep the boundaries between either sepal and stamen zones or stamen and carpel zones. Double knockdowns had enhanced phenotypes and the triple knockdown had an even more severe phenotype that included partial to complete homeotic conversion of stamens and carpels to sepaloid organs and green sepals, highlighting a role of E-class genes in petaloidy of sepals in this species. While no floral meristem determinacy defects were observed, this could be due to residual amounts of gene expression in the VIGS experiments being sufficient to perform this function or to the masking role of a redundant gene.

Sub-functionalization to ovule development following duplication of a floral organ identity gene.

Gene duplications result in paralogs that may be maintained due to the gain of novel functions (neo-functionalization) or the partitioning of ancestral function (sub-functionalization). Plant genomes are especially prone to duplication; paralogs are particularly widespread in the floral MADS box transcription factors that control organ identity through the ABC model of flower development. C class genes establish stamen and carpel identity and control floral meristem determinacy, and are largely conserved across the angiosperm phylogeny. Originally, an additional D class had been identified as controlling ovule identity; yet subsequent studies indicated that both C and D lineage genes more commonly control ovule development redundantly. The ranunculid Thalictrum thalictroides has two orthologs of the Arabidopsis thaliana C class gene AGAMOUS (AG), ThtAG1 and ThtAG2 (Thalictrum thalictroides AGAMOUS1/2). We previously showed that ThtAG1 exhibits typical C class function; here we examine the role of its paralog, ThtAG2. Our phylogenetic analysis shows that ThtAG2 falls within the C lineage, together with ThtAG1, and is consistent with previous findings of a Ranunculales-specific duplication in this clade. However, ThtAG2 is not expressed in stamens, but rather solely in carpels and ovules. This female-specific expression pattern is consistent with D lineage genes, and with other C lineage genes known to be involved in ovule identity. Given the divergent expression of ThtAG2, we tested the hypothesis that it has acquired ovule identity function. Molecular evolution analyses showed evidence of positive selection on ThtAG2-a pattern that supports divergence of function by sub-functionalization. Down-regulation of ThtAG2 by virus-induced gene silencing resulted in homeotic conversions of ovules into carpel-like structures. Taken together, our results suggest that, although ThtAG2 falls within the C lineage, it has diverged to acquire "D function" as an ovule identity gene, and does not appear to require a direct interaction with the ThtAG1 protein. We therefore present a functional example of ovule identity being specified by either a single gene or a gene pair within the C lineage, with no D lineage contribution. In conclusion, following a Ranunculales-wide duplication in the AG lineage, functional divergence has led to the evolution of ovule identity-specificity in a T. thalictroides C lineage gene.

Complete plastome sequence of Thalictrum coreanum (Ranunculaceae) and transfer of the rpl32 gene to the nucleus in the ancestor of the subfamily Thalictroideae.

BACKGROUND: Plastids originated from cyanobacteria and the majority of the ancestral genes were lost or functionally transferred to the nucleus after endosymbiosis. Comparative genomic investigations have shown that gene transfer from plastids to the nucleus is an ongoing evolutionary process but molecular evidence for recent functional gene transfers among seed plants have only been documented for the four genes accD, infA, rpl22, and rpl32. RESULTS: The complete plastid genome of Thalictrum coreanum, the first from the subfamily Thalictroideae (Ranunculaceae), was sequenced and revealed the losses of two genes, infA and rpl32. The functional transfer of these two genes to the nucleus in Thalictrum was verified by examination of nuclear transcriptomes. A survey of the phylogenetic distribution of the rpl32 loss was performed using 17 species of Thalictrum and representatives of related genera in the subfamily Thalictroideae. The plastid-encoded rpl32 gene is likely nonfunctional in members of the subfamily Thalictroideae (Aquilegia, Enemion, Isopyrum, Leptopyrum, Paraquilegia, and Semiaquilegia) including 17 Thalictrum species due to the presence of indels that disrupt the reading frame. A nuclear-encoded rpl32 with high sequence identity was identified in both Thalictrum and Aquilegia. The phylogenetic distribution of this gene loss/transfer and the high level of sequence similarity in transit peptides suggest a single transfer of the plastid-encoded rpl32 to the nucleus in the ancestor of the subfamily Thalictroideae approximately 20-32 Mya. CONCLUSIONS: The genome sequence of Thalictrum coreanum provides valuable information for improving the understanding of the evolution of plastid genomes within Ranunculaceae and across angiosperms. Thalictrum is unusual among the three sequenced Ranunculaceae plastid genomes in the loss of two genes infA and rpl32, which have been functionally transferred to the nucleus. In the case of rpl32 this represents the third documented independent transfer from the plastid to the nucleus with the other two transfers occurring in the unrelated angiosperm families Rhizophoraceae and Salicaceae. Furthermore, the transfer of rpl32 provides additional molecular evidence for the monophyly of the subfamily Thalictroideae.

Timing and consequences of recurrent polyploidy in meadow-rues (thalictrum, ranunculaceae).

The discovery of ancient whole-genome duplications in eukaryotic lineages has renewed the interest in polyploidy and its effects on the diversification of organisms. Polyploidy has large-scale effects on both genotype and phenotype and has been linked to the evolution of genome size, dioecy, and changes in ecological interactions, such as pollinator visitation. Here, we take a molecular systematics approach to examine the evolution of polyploidy in the plant genus Thalictrum (Ranunculaceae) and test its correlation to changes in genome size, sexual system, and pollination mode. Thalictrum is an ideal study system due to its extensive ploidy range and floral diversity. Phylogenetic analyses were used for character reconstructions, correlation tests, and dating estimates. Our results suggest that polyploidization occurred frequently and recently in the evolution of Thalictrum, mostly within the last 10.6-5.8 My, coinciding with the diversification of particular clades. In spite of an overall trend of genomic downsizing accompanying polyploidy in angiosperms and proportional increases observed at finer scales, our genome size estimates for Thalictrum show no correlation with chromosome number. Instead, we observe genomic expansion in diploids and genomic contraction in polyploids with increased age. Additionally, polyploidy is not correlated with dioecy in Thalictrum; therefore, other factors must have influenced the evolution of separate sexes in this group. A novel finding from our study is the association of polyploidy with shifts to wind pollination, in particular, during a time period of global cooling and mountain uplift in the Americas.

Phylogenetic insights into the correlates of dioecy in meadow-rues (Thalictrum, Ranunculaceae).

Numerous studies have examined the evolution of sexual systems in angiosperms, but few explore the interaction between these and the evolution of pollination mode. Wind pollination is often associated with unisexual flowers, but which evolved first and played a causative role in the evolution of the other is unclear. Thalictrum, meadow-rues (Ranunculaceae), provides a unique opportunity to study the evolution of these traits because it contains insect and wind pollination and four sexual systems. We used a phylogenetic approach to reconstruct ancestral states for sexual system, pollination mode, and geographic distribution in Thalictrum, and tested for correlations to uncover the factors involved in the evolution of unisexuality and wind pollination. Our results show that dioecy, andro- and gynomonoecy evolved at least twice from hermaphroditism. Wind pollination, unisexual flowers, and New World distribution were all significantly correlated. Wind pollination may have evolved early in the genus, followed by multiple losses and gains, and likely preceded the origin of unisexual flowers in several cases; we found no evidence for unisexual flowers evolving prior to wind pollination. Given a broad scale study showing the evolution of dioecy before wind pollination, our results from a finer scale analysis highlight that different evolutionary pathways are likely to occur throughout angiosperms.

Norcoclaurine synthase is a member of the pathogenesis-related 10/Bet v1 protein family.

Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate-dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type-specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde.

Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.

An ortholog of MIXTA-like2 controls epidermal cell shape in flowers of Thalictrum.

Here, we investigated the genetic underpinnings of pollination-related floral phenotypes in Thalictrum, a ranunculid with apetalous flowers. The variable presence of petaloid features in other floral organs correlates with distinct adaptations to insect vs. wind pollination. Conical cells are present in sepals or stamens of insect-pollinated species, and in stigmas. We characterized a Thalictrum ortholog of the Antirrhinum majus transcription factor MIXTA-like2, responsible for conical cells, from three species with distinct floral morphologies, representing two pollination syndromes. Genes were cloned by PCR and analysed phylogenetically. Expression analyses were conducted by quantitative PCR and in situ hybridization, followed by functional studies in transgenic tobacco. The cloned genes encode R2R3 MYB proteins closely related to Antirrhinum AmMYBML2 and Petunia hybrida PhMYB1. Spatial expression by in situ hybridization overlaps areas of conical cells. Overexpression in tobacco induces cell outgrowths in carpel epidermis and significantly increases the height of petal conical cells. We have described the first orthologs of AmMIXTA-like2 outside the core eudicots, likely ancestral to the MIXTA/MIXTA-like1 duplication. The conserved role in epidermal cell elongation results in conical cells, micromorphological markers for petaloidy. This adaptation to attract insect pollinators was apparently lost after the evolution of wind pollination in Thalictrum.

Targeted metabolite and transcript profiling for elucidating enzyme function: isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species.

An integrated approach using targeted metabolite profiles and modest EST libraries each containing approximately 3500 unigenes was developed in order to discover and functionally characterize novel genes involved in plant-specialized metabolism. EST databases have been established for benzylisoquinoline alkaloid-producing cell cultures of Eschscholzia californica, Papaver bracteatum and Thalictrum flavum, and are a rich repository of alkaloid biosynthetic genes. ESI-FTICR-MS and ESI-MS/MS analyses facilitated unambiguous identification and relative quantification of the alkaloids in each system. Manual integration of known and candidate biosynthetic genes in each EST library with benzylisoquinoline alkaloid biosynthetic networks assembled from empirical metabolite profiles allowed identification and functional characterization of four N-methyltransferases (NMTs). One cDNA from T. flavum encoded pavine N-methyltransferase (TfPavNMT), which showed a unique preference for (+/-)-pavine and represents the first isolated enzyme involved in the pavine alkaloid branch pathway. Correlation of the occurrence of specific alkaloids, the complement of ESTs encoding known benzylisoquinoline alkaloid biosynthetic genes and the differential substrate range of characterized NMTs demonstrated the feasibility of bilaterally predicting enzyme function and species-dependent specialized metabolite profiles.

Structural basis of enzymatic (S)-norcoclaurine biosynthesis.

The enzyme norcoclaurine synthase (NCS) catalyzes the stereospecific Pictet-Spengler cyclization between dopamine and 4-hydroxyphenylacetaldehyde, the key step in the benzylisoquinoline alkaloid biosynthetic pathway. The crystallographic structure of norcoclaurine synthase from Thalictrum flavum in its complex with dopamine substrate and the nonreactive substrate analogue 4-hydroxybenzaldehyde has been solved at 2.1A resolution. NCS shares no common features with the functionally correlated "Pictet-Spenglerases" that catalyze the first step of the indole alkaloids pathways and conforms to the overall fold of the Bet v1-like protein. The active site of NCS is located within a 20-A-long catalytic tunnel and is shaped by the side chains of a tyrosine, a lysine, an aspartic, and a glutamic acid. The geometry of the amino acid side chains with respect to the substrates reveals the structural determinants that govern the mechanism of the stereoselective Pictet-Spengler cyclization, thus establishing an excellent foundation for the understanding of the finer details of the catalytic process. Site-directed mutations of the relevant residues confirm the assignment based on crystallographic findings.

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum.

Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 A resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 86.31, c = 118.36 A. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 A resolution. The model is under construction.

Cell type-specific localization of transcripts encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis.

Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type-specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.