A cornucopia of diversity-Ranunculales as a model lineage.

The Ranunculales are a hyperdiverse lineage in many aspects of their phenotype, including growth habit, floral and leaf morphology, reproductive mode, and specialized metabolism. Many Ranunculales species, such as opium poppy and goldenseal, have a high medicinal value. In addition, the order includes a large number of commercially important ornamental plants, such as columbines and larkspurs. The phylogenetic position of the order with respect to monocots and core eudicots and the diversity within this lineage make the Ranunculales an excellent group for studying evolutionary processes by comparative studies. Lately, the phylogeny of Ranunculales was revised, and genetic and genomic resources were developed for many species, allowing comparative analyses at the molecular scale. Here, we review the literature on the resources for genetic manipulation and genome sequencing, the recent phylogeny reconstruction of this order, and its fossil record. Further, we explain their habitat range and delve into the diversity in their floral morphology, focusing on perianth organ identity, floral symmetry, occurrences of spurs and nectaries, sexual and pollination systems, and fruit and dehiscence types. The Ranunculales order offers a wealth of opportunities for scientific exploration across various disciplines and scales, to gain novel insights into plant biology for researchers and plant enthusiasts alike.

Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

BACKGROUND AND AIMS: Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. METHODS: In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). KEY RESULTS: We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. CONCLUSIONS: Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

Evolution of major flowering pathway integrators in Orchidaceae.

The Orchidaceae is a mega-diverse plant family with ca. 29,000 species with a large variety of life forms that can colonize transitory habitats. Despite this diversity, little is known about their flowering integrators in response to specific environmental factors. During the reproductive transition in flowering plants a vegetative apical meristem (SAM) transforms into an inflorescence meristem (IM) that forms bracts and flowers. In model grasses, like rice, a flowering genetic regulatory network (FGRN) controlling reproductive transitions has been identified, but little is known in the Orchidaceae. In order to analyze the players of the FRGN in orchids, we performed comprehensive phylogenetic analyses of CONSTANS-like/CONSTANS-like 4 (COL/COL4), FLOWERING LOCUS D (FD), FLOWERING LOCUS C/FRUITFULL (FLC/FUL) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) gene lineages. In addition to PEBP and AGL24/SVP genes previously analyzed, here we identify an increase of orchid homologs belonging to COL4, and FUL gene lineages in comparison with other monocots, including grasses, due to orchid-specific gene lineage duplications. Contrariwise, local duplications in Orchidaceae are less frequent in the COL, FD and SOC1 gene lineages, which points to a retention of key functions under strong purifying selection in essential signaling factors. We also identified changes in the protein sequences after such duplications, variation in the evolutionary rates of resulting paralogous clades and targeted expression of isolated homologs in different orchids. Interestingly, vernalization-response genes like VERNALIZATION1 (VRN1) and FLOWERING LOCUS C (FLC) are completely lacking in orchids, or alternatively are reduced in number, as is the case of VERNALIZATION2/GHD7 (VRN2). Our findings point to non-canonical factors sensing temperature changes in orchids during reproductive transition. Expression data of key factors gathered from Elleanthus auratiacus, a terrestrial orchid in high Andean mountains allow us to characterize which copies are actually active during flowering. Altogether, our data lays down a comprehensive framework to assess gene function of a restricted number of homologs identified more likely playing key roles during the flowering transition, and the changes of the FGRN in neotropical orchids in comparison with temperate grasses.

Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

Expression and Functional Studies of Leaf, Floral, and Fruit Developmental Genes in Non-model Species.

Researchers working on evolutionary developmental plant biology are inclined to choose non-model taxa to address how specific features have been acquired during ontogeny and fixed during phylogeny. In this chapter we describe methods to extract RNA, to assemble de-novo transcriptomes, to isolate orthologous genes within gene families, and to evaluate expression and function of target genes. We have successfully optimized these protocols for non-model plant species including ferns, gymnosperms, and a large assortment of angiosperms. In the latter, we have ranged a large number of families including Aristolochiaceae, Apodanthaceae, Chloranthaceae, Orchidaceae, Papaveraceae, Rubiaceae, Solanaceae, and Tropaeolaceae.

THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog.

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog ß-ethynylserine (ßES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding ßES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.

Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Role of Oxidative Stress and Lipid Peroxidation in the Pathophysiology of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is characterised by an excess of hepatic fat that can progress to steatohepatitis, fibrosis, cirrhosis and hepatocarcinoma. The imbalance between lipid uptake/lipogenesis and lipid oxidation/secretion in the liver is a major feature of NAFLD. Given the lack of a non-invasive and reliable methods for the diagnosis of non-alcoholic steatohepatitis (NASH), it is important to find serum markers that are capable of discriminating or defining patients with this stage of NASH. Blood samples were obtained from 152 Caucasian subjects with biopsy-proven NAFLD due to persistently elevated liver enzyme levels. Metabolites representative of oxidative stress were assessed. The findings derived from this work revealed that NAFLD patients with a NASH score of ≥ 4 showed significantly higher levels of lipid peroxidation (LPO). Indeed, LPO levels above the optimal operating point (OOP) of 315.39 µM are an independent risk factor for presenting a NASH score of ≥ 4 (OR: 4.71; 95% CI: 1.68−13.19; p = 0.003). The area under the curve (AUC = 0.81, 95% CI = 0.73−0.89, p < 0.001) shows a good discrimination ability of the model. Therefore, understanding the molecular mechanisms underlying the basal inflammation present in these patients is postulated as a possible source of biomarkers and therapeutic targets in NASH.

Evolution and expression of LEAFY genes in ferns and lycophytes.

BACKGROUND: The LEAFY (LFY) transcription factors are present in algae and across land plants. The available expression and functional data of these genes in embryophytes suggest that LFY genes control a plethora of processes including the first zygotic cell division in bryophytes, shoot cell divisions of the gametophyte and sporophyte in ferns, cone differentiation in gymnosperms and floral meristem identity in flowering plants. However, their putative plesiomorphic role in plant reproductive transition in vascular plants remains untested. RESULTS: We perform Maximum Likelihood (ML) phylogenetic analyses for the LFY gene lineage in embryophytes with expanded sampling in lycophytes and ferns. We recover the previously identified seed plant duplication that results in LEAFY and NEEDLY paralogs. In addition, we recover multiple species-specific duplications in ferns and lycophytes and large-scale duplications possibly correlated with the occurrence of whole genome duplication (WGD) events in Equisetales and Salviniales. To test putative roles in diverse ferns and lycophytes we perform LFY expression analyses in Adiantum raddianum, Equisetum giganteum and Selaginella moellendorffii. Our results show that LFY genes are active in vegetative and reproductive tissues, with higher expression in early fertile developmental stages and during sporangia differentiation. CONCLUSIONS: Our data point to previously unrecognized roles of LFY genes in sporangia differentiation in lycophytes and ferns and suggests that functions linked to reproductive structure development are not exclusive to seed plant LFY homologs.

Complete Mitogenomes of Two Aragoa Species and Phylogeny of Plantagineae (Plantaginaceae, Lamiales) Using Mitochondrial Genes and the Nuclear Ribosomal RNA Repeat.

Aragoa, comprising 19 high-altitude North Andean species, is one of three genera in the Plantagineae (Plantaginaceae, Lamiales), along with Littorella and Plantago. Based primarily on plastid data and nuclear ITS, Aragoa is sister to a clade of Littorella + Plantago, but Plantagineae relationships have yet to be assessed using multigene datasets from the nuclear and mitochondrial genomes. Here, complete mitogenomes were assembled for two species of Aragoa (A. abietina and A. cleefii). The mitogenomes of both species have a typical suite of genes for 34 proteins, 17 tRNAs, and three rRNAs. The A. abietina mitogenome assembled into a simple circular map, with no large repeats capable of producing alternative isoforms. The A. cleefii mitogenomic map was more complex, involving two circular maps bridged by a substoichiometric linear fragment. Phylogenetics of three mitochondrial genes or the nuclear rRNA repeat placed Aragoa as sister to Littorella + Plantago, consistent with previous studies. However, P. nubicola, the sole representative of subg. Bougueria, was nested within subg. Psyllium based on the mitochondrial and nuclear data, conflicting with plastid-based analyses. Phylogenetics of the nuclear rRNA repeat provided better resolution overall, whereas relationships from mitochondrial data were hindered by extensive substitution rate variation among lineages.

Evolution and expression of the MADS-box flowering transition genes AGAMOUS-like 24/SHORT VEGETATIVE PHASE with emphasis in selected Neotropical orchids.

In angiosperms the reproductive transition results in the transformation of a vegetative apical meristem (SAM) into an inflorescence meristem (IM), capable of forming floral meristems (FM). Two key players in the flowering transition are AGAMOUS-like 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP). They are eudicot MADS-box paralogs performing opposite roles, as AGL24 positively regulates flowering while SVP represses the reproductive transition in Arabidopsis. We confirm that the Arabidopsis functional reference cannot be readily extrapolated to all eudicots as there are additional duplications of AGL24 in early divergent eudicots and core eudicots with significant sequence variation. In addition, we found that in monocots, two additional independent duplication events have resulted in at least three clades of AGL24/SVP homologs, some only found in Orchidaceae. Protein sequence analyses and comparative evolutionary rates point to higher rates of relaxed negative selection in the Core Eudicot AGL24 B and the Orch SVP-like B clades, in eudicots and monocots respectively. On the other hand, expression data points to plesiomorphic pleiotropic roles of AGL24/SVP genes likely similar to SVP core eudicot genes, and the acquisition of new roles as flowering positive regulators in Core Eudicot AGL24 A genes. Our research presents evidence on the diversification and recruitment of AGL24/SVP homologs in flowering transition in orchids. Although, broad expression of most copies does not allow to determine if they act as flowering repressors or promoters, the restricted expression of some homologs in the SAM suggests putative roles in maintaining the vegetative phase. If so studying in detail the function of AGL24/SVP homologs in orchids is critical to identify putative flowering repressors in a lineage where other canonical repressors remain elusive.

Comparative anatomy and genetic bases of fruit development in selected Rubiaceae (Gentianales).

PREMISE: The Rubiaceae are ideal for studying the diversity of fruits that develop from flowers with inferior ovary. We aimed to identify morpho-anatomical changes during fruit development that distinguish those derived from the carpel versus the extra-carpellary tissues. In addition, we present the fruit genetic core regulatory network in selected Rubiaceae species and compare it in terms of copy number and expression patterns to model core eudicots in the Brassicaceae and the Solanaceae. METHODS: We used light microscopy to follow morphoanatomical changes in four selected species with different fruit types. We generated reference transcriptomes for seven selected Rubiaceae species and isolated homologs of major transcription factors involved in fruit development histogenesis, assessed their homology, identified conserved and new protein motifs, and evaluated their expression in three species with different fruit types. RESULTS: Our studies revealed ovary-derived pericarp tissues versus floral-cup-derived epicarp tissues. Gene evolution analyses of FRUITFULL, SHATTERPROOF, ALCATRAZ, INDEHISCENT and REPLUMLESS homologs suggest that the gene complement in Rubiaceae is simpler compared to that in Brassicaceae or Solanaceae. Expression patterns of targeted genes vary in response to the fruit type and the developmental stage evaluated. CONCLUSIONS: Morphologically similar fruits can have different anatomies as a result of convergent tissues developed from the epicarps covering the anatomical changes from the pericarps. Expression analyses suggest that the fruit patterning regulatory network established in model core eudicots cannot be extrapolated to asterids with inferior ovaries.

Molecular framework underlying floral bilateral symmetry and nectar spur development in Tropaeolum, an atypical member of the Brassicales.

PREMISE: Floral spurs are key innovations associated with elaborate pollination mechanisms that have evolved independently several times across angiosperms. Spur formation can shift the floral symmetry from radial to bilateral, as it is the case in Tropaeolum, the only member of the Brassicales with floral nectar spurs. The genetic mechanisms underlying both spur and bilateral symmetry in the family have not yet been investigated. METHODS: We studied flower development and morphoanatomy of Tropaeolum longifolium. We also generated a reference transcriptome and isolated all candidate genes involved in adaxial-abaxial differential growth during spur formation. Finally, we evaluated the evolution of the targeted genes across Brassicales and examined their expression in dissected floral parts. RESULTS: Five sepals initiate spirally, followed by five petals alternate to the sepals, five antesepalous stamens, three antepetalous stamens, and three carpels. Intercalary growth at the common base of sepals and petals forms a floral tube. The spur is an outgrowth from the adaxial region of the tube, lined up with the medial sepal. We identified Tropaeolum specific duplications in the TCP3/4L and STM gene lineages, which are critical for spur formation in other taxa. In addition, we found that TM6 (MADS-box), RL2 (RAD-like7), and KN2/6L2 and OSH6L (KNOX1 genes), have been lost in core Brassicales but retained in Tropaeolum. CONCLUSIONS: Three genes are pivotal during the extreme adaxial-abaxial asymmetry of the floral tube, namely, TlTCP4L2 restricted to the adaxial side where the spur is formed, and TlTCP12 and TlSTM1 to the abaxial side, lacking a spur.

Transcriptomic analyses of cacao flavonoids produced in photobioreactors.

BACKGROUND: Theobroma cacao is a major source of flavonoids such as catechins and their monomers proanthocyanidins (PAs), widely studied for their potential benefits in cardiovascular diseases. Light has been shown to promote plant secondary metabolite production in vitro. In this study, cacao cells cultured in 7.5 L stirred tank photobioreactors (STPs) were exposed to a change of white to blue LED lights for 28 days (d). RESULTS: Transcriptomic analyses were performed in three time points comparing changing expression patterns, after cell exposure to white light (d0-VS-d14), after a shift from white to blue light (d14-VS-d15), and after an extended period of blue light for the following 15 days (d15-VS-d28). Under white light, there was enrichment in metabolic pathways associated with cell growth (carbon, glycolysis, and amino acid biosynthesis) accompanied by a significant increase in the PAs content. In the shift to blue light, further increase in PAs content was observed concomitantly with the significant expression of TWO-COMPONENT RESPONSE REGULATOR genes involved in the early stress responses via circadian clock and hormone pathways. Under blue light exposure, we observed a depletion of PAs content associated with ROS-mediated stress pathways. CONCLUSIONS: Light effects on large-scale cell cultures in photobioreactors are complex and pleiotropic; however, we have been able to identify key regulatory players upstream cacao flavonoid biosynthesis in STPs, including TWO-COMPONENT SYSTEM and ROS-signaling genes. The crosstalk between flavonoid biosynthesis and regulatory networks led to understand the dynamics of flavonoid production and degradation in response to light-driven ROS signals. This can be used to optimize the time, and the yield of in vitro targeted metabolites in large-scale culture systems.

Plastomes from tribe Plantagineae (Plantaginaceae) reveal infrageneric structural synapormorphies and localized hypermutation for Plantago and functional loss of ndh genes from Littorella.

Tribe Plantagineae (Plantaginaceae) comprises ~ 270 species in three currently recognized genera (Aragoa, Littorella, Plantago), of which Plantago is most speciose. Plantago plastomes exhibit several atypical features including large inversions, expansions of the inverted repeat, increased repetitiveness, intron losses, and gene-specific increases in substitution rate, but the prevalence of these plastid features among species and subgenera is unknown. To assess phylogenetic relationships and plastomic evolutionary dynamics among Plantagineae genera and Plantago subgenera, we generated 25 complete plastome sequences and compared them with existing plastome sequences from Plantaginaceae. Using whole plastome and partitioned alignments, our phylogenomic analyses provided strong support for relationships among major Plantagineae lineages. General plastid features-including size, GC content, intron content, and indels-provided additional support that reinforced major Plantagineae subdivisions. Plastomes from Plantago subgenera Plantago and Coronopus have synapomorphic expansions and inversions affecting the size and gene order of the inverted repeats, and particular genes near the inversion breakpoints exhibit accelerated nucleotide substitution rates, suggesting localized hypermutation associated with rearrangements. The Littorella plastome lacks functional copies of ndh genes, which may be related to an amphibious lifestyle and partial reliance on CAM photosynthesis.

Generation of excitatory and inhibitory neurons from common progenitors via Notch signaling in the cerebellum.

Brain neurons arise from relatively few progenitors generating an enormous diversity of neuronal types. Nonetheless, a cardinal feature of mammalian brain neurogenesis is thought to be that excitatory and inhibitory neurons derive from separate, spatially segregated progenitors. Whether bi-potential progenitors with an intrinsic capacity to generate both lineages exist and how such a fate decision may be regulated are unknown. Using cerebellar development as a model, we discover that individual progenitors can give rise to both inhibitory and excitatory lineages. Gradations of Notch activity determine the fates of the progenitors and their daughters. Daughters with the highest levels of Notch activity retain the progenitor fate, while intermediate levels of Notch activity generate inhibitory neurons, and daughters with very low levels of Notch signaling adopt the excitatory fate. Therefore, Notch-mediated binary cell fate choice is a mechanism for regulating the ratio of excitatory to inhibitory neurons from common progenitors.

Slit/Robo Signaling Regulates Multiple Stages of the Development of the Drosophila Motion Detection System.

Neurogenesis is achieved through a sequence of steps that include specification and differentiation of progenitors into mature neurons. Frequently, precursors migrate to distinct positions before terminal differentiation. The Slit-Robo pathway, formed by the secreted ligand Slit and its membrane bound receptor Robo, was first discovered as a regulator of axonal growth. However, today, it is accepted that this pathway can regulate different cellular processes even outside the nervous system. Since most of the studies performed in the nervous system have been focused on axonal and dendritic growth, it is less clear how versatile is this signaling pathway in the developing nervous system. Here we describe the participation of the Slit-Robo pathway in the development of motion sensitive neurons of the Drosophila visual system. We show that Slit and Robo receptors are expressed in different stages during the neurogenesis of motion sensitive neurons. Furthermore, we find that Slit and Robo regulate multiple aspects of their development including neuronal precursor migration, cell segregation between neural stem cells and daughter cells and formation of their connectivity pattern. Specifically, loss of function of slit or robo receptors in differentiated motion sensitive neurons impairs dendritic targeting, while knocking down robo receptors in migratory progenitors or neural stem cells leads to structural defects in the adult optic lobe neuropil, caused by migration and cell segregation defects during larval development. Thus, our work reveals the co-option of the Slit-Robo signaling pathway in distinct developmental stages of a neural lineage.