Delphinieae flowers originated from the rewiring of interactions between duplicated and diversified floral organ identity and symmetry genes.

Species of the tribe Delphinieae (Ranunculaceae) have long been the focus of morphological, ecological, and evolutionary studies due to their highly specialized, nearly zygomorphic (bilaterally symmetrical) spiral flowers with nested petal and sepal spurs and reduced petals. The mechanisms underlying the development and evolution of Delphinieae flowers, however, remain unclear. Here, by conducting extensive phylogenetic, comparative transcriptomic, expression, and functional studies, we clarified the evolutionary histories, expression patterns, and functions of floral organ identity and symmetry genes in Delphinieae. We found that duplication and/or diversification of APETALA3-3 (AP3-3), AGAMOUS-LIKE6 (AGL6), CYCLOIDEA (CYC), and DIVARICATA (DIV) lineage genes was tightly associated with the origination of Delphinieae flowers. Specifically, an AGL6-lineage member (such as the Delphinium ajacis AGL6-1a) represses sepal spur formation and petal development in the lateral and ventral parts of the flower while determining petal identity redundantly with AGL6-1b. By contrast, two CYC2-like genes, CYC2b and CYC2a, define the dorsal and lateral-ventral identities of the flower, respectively, and form complex regulatory links with AP3-3, AGL6-1a, and DIV1. Therefore, duplication and diversification of floral symmetry genes, as well as co-option of the duplicated copies into the preexisting floral regulatory network, have been key for the origin of Delphinieae flowers.

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

Water lilies belong to the angiosperm order Nymphaeales. Amborellales, Nymphaeales and Austrobaileyales together form the so-called ANA-grade of angiosperms, which are extant representatives of lineages that diverged the earliest from the lineage leading to the extant mesangiosperms1-3. Here we report the 409-megabase genome sequence of the blue-petal water lily (Nymphaea colorata). Our phylogenomic analyses support Amborellales and Nymphaeales as successive sister lineages to all other extant angiosperms. The N. colorata genome and 19 other water lily transcriptomes reveal a Nymphaealean whole-genome duplication event, which is shared by Nymphaeaceae and possibly Cabombaceae. Among the genes retained from this whole-genome duplication are homologues of genes that regulate flowering transition and flower development. The broad expression of homologues of floral ABCE genes in N. colorata might support a similarly broadly active ancestral ABCE model of floral organ determination in early angiosperms. Water lilies have evolved attractive floral scents and colours, which are features shared with mesangiosperms, and we identified their putative biosynthetic genes in N. colorata. The chemical compounds and biosynthetic genes behind floral scents suggest that they have evolved in parallel to those in mesangiosperms. Because of its unique phylogenetic position, the N. colorata genome sheds light on the early evolution of angiosperms.

Mechanisms underlying the formation of complex color patterns on Nigella orientalis (Ranunculaceae) petals.

Complex color patterns on petals are widespread in flowering plants, yet the mechanisms underlying their formation remain largely unclear. Here, by conducting detailed morphological, anatomical, biochemical, optical, transcriptomic, and functional studies, we investigated the cellular bases, chromogenic substances, reflectance spectra, developmental processes, and underlying mechanisms of complex color pattern formation on Nigella orientalis petals. We found that the complexity of the N. orientalis petals in color pattern is reflected at multiple levels, with the amount and arrangement of different pigmented cells being the key. We also found that biosynthesis of the chromogenic substances of different colors is sequential, so that one color/pattern is superimposed on another. Expression and functional studies further revealed that a pair of R2R3-MYB genes function cooperatively to specify the formation of the eyebrow-like horizontal stripe and the Mohawk haircut-like splatters. Specifically, while NiorMYB113-1 functions to draw a large splatter region, NiorMYB113-2 functions to suppress the production of anthocyanins from the region where a gap will form, thereby forming the highly specialized pattern. Our results provide a detailed portrait for the spatiotemporal dynamics of the coloration of N. orientalis petals and help better understand the mechanisms underlying complex color pattern formation in plants.

Phylogenomic and population genomic analyses reveal the spatial-temporal dynamics of diversification of the Nigella arvensis complex (Ranunculaceae) in the Aegean archipelago.

The continental-shelf islands of the Aegean Sea provide an ideal geographical setting for evolutionary-biogeographical studies but disentangling the relationships between palaeogeographical history and the times, orders of modes of taxon divergence is not straightforward. Here, we used phylogenomic and population genomic approaches, based on orthologous gene sequences and transcriptome-derived SNP data, to reconstruct the spatial-temporal evolution of the Aegean Nigella arvensis complex (Ranunculaceae; 11 out of 12 taxa). The group's early diversification in the Early/Mid-Pliocene (c. 3.77 Mya) resulted in three main lineages (Greek mainland vs. central Aegean + Turkish mainland/eastern Aegean islands), while all extant taxa are of Late Plio-/Early Pleistocene origin (c. 3.30-1.59 Mya). Demographic modelling of the outcrossing taxa uncovered disparate modes of (sub)speciation, including divergence with gene flow on the Greek mainland, para- or peripatric diversification across eastern Aegean islands, and a 'mixing-isolation-mixing (MIM)' mode of subspeciation in the Cyclades. The two selfing species (N. stricta, N. doerfleri) evolved independently from the outcrossers. Present-day island configurations are clearly insufficient to explain the spatial-temporal history of lineage diversification and modes of (sub)speciation in Aegean Nigella. Moreover, our identification of positively selected genes in almost all taxa calls into question that this plant group represents a case of 'non-adaptive' radiation. Our study revealed an episodic diversification history of the N. arvensis complex, giving new insight into the modes and drivers of island speciation and adaption across multiple spatiotemporal scales.

Identification of the Key Regulatory Genes Involved in Elaborate Petal Development and Specialized Character Formation in Nigella damascena (Ranunculaceae).

Petals can be simple or elaborate, depending on whether they have lobes, teeth, fringes, or appendages along their margins, or possess spurs, scales, or other types of modifications on their adaxial/abaxial side, or both. Elaborate petals have been recorded in 23 orders of angiosperms and are generally believed to have played key roles in the adaptive evolution of corresponding lineages. The mechanisms underlying the formation of elaborate petals, however, are largely unclear. Here, by performing extensive transcriptomic and functional studies on Nigella damascena (Ranunculaceae), we explore the mechanisms underlying elaborate petal development and specialized character formation. In addition to the identification of genes and programs that are specifically/preferentially expressed in petals, we found genes and programs that are required for elaborate rather than simple petal development. By correlating the changes in gene expression with those in petal development, we identified 30 genes that are responsible for the marginal/ventral elaboration of petals and the initiation of several highly specialized morphological characters (e.g., pseudonectaries, long hairs, and short trichomes). Expression and functional analyses further confirmed that a class I homeodomain-leucine zipper family transcription factor gene, Nigella damascena LATE MERISTEM IDENTITY1 (NidaLMI1), plays important roles in the development of short trichomes and bifurcation of the lower lip. Our results not only provide the first portrait of elaborate petal development but also pave the way to understanding the mechanisms underlying lateral organ diversification in plants.

Petal development and elaboration.

Petals can be simple or elaborate, depending on whether they have complex basic structures and/or highly specialized epidermal modifications. It has been proposed that the independent origin and diversification of elaborate petals have promoted plant-animal interactions and, therefore, the evolutionary radiation of corresponding plant groups. Recent advances in floral development and evolution have greatly improved our understanding of the processes, patterns, and mechanisms underlying petal elaboration. In this review, we compare the developmental processes of simple and elaborate petals, concluding that elaborate petals can be achieved through four main paths of modifications (i.e. marginal elaboration, ventral elaboration, dorsal elaboration, and surface elaboration). Although different types of elaborate petals were formed through different types of modifications, they are all results of changes in the expression patterns of genes involved in organ polarity establishment and/or the proliferation, expansion, and differentiation of cells. The deployment of existing genetic materials to perform a new function was also shown to be a key to making elaborate petals during evolution.

Parallel evolution of apetalous lineages within the buttercup family (Ranunculaceae): outward expansion of AGAMOUS1, rather than disruption of APETALA3-3.

Complete loss of petals, or becoming apetalous, has occurred independently in many flowering plant lineages. However, the mechanisms underlying the parallel evolution of naturally occurring apetalous lineages remain largely unclear. Here, by sampling representatives of all nine apetalous genera/tribes of the family Ranunculaceae and conducting detailed morphological, expression, molecular evolutionary and functional studies, we investigate the mechanisms underlying parallel petal losses. We found that while non-expression/downregulation of the petal identity gene APETALA3-3 (AP3-3) is tightly associated with complete petal losses, disruptions of the AP3-3 orthologs were unlikely to be the real causes for the parallel evolution of apetalous lineages. We also found that, compared with their close petalous relatives, naturally occurring apetalous taxa usually bear slightly larger numbers of stamens, whereas the number of sepals remains largely unchanged, suggestive of petal-to-stamen rather than petal-to-sepal transformations. In addition, in the recently originated apetalous genus Enemion, the petal-to-stamen transformations have likely been caused by the mutations that led to the elevation and outward expansion of the expression of the C-function gene, AGAMOUS1 (AG1). Our results not only provide a general picture of parallel petal losses within the Ranunculaceae but also help understand the mechanisms underlying the independent originations of other apetalous lineages.

Identification of the target genes of AqAPETALA3-3 (AqAP3-3) in Aquilegia coerulea (Ranunculaceae) helps understand the molecular bases of the conserved and nonconserved features of petals.

Identification and comparison of the conserved and variable downstream genes of floral organ identity regulators are critical to understanding the mechanisms underlying the commonalities and peculiarities of floral organs. Yet, because of the lack of studies in nonmodel species, a general picture of the regulatory evolution between floral organ identity genes and their targets is still lacking. Here, by conducting extensive chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), electrophoretic mobility shift assay and bioinformatic analyses, we identify and predict the target genes of a petal identity gene, AqAPETALA3-3 (AqAP3-3), in Aquilegia coerulea (Ranunculaceae) and compare them with those of its counterpart in Arabidopsis thaliana, AP3. In total, 7049 direct target genes are identified for AqAP3-3, of which 2394 are highly confident and 1085 are shared with AP3. Gene Ontology enrichment analyses further indicate that conserved targets are largely involved in the formation of identity-related features, whereas nonconserved targets are mostly required for the formation of species-specific features. These results not only help understand the molecular bases of the conserved and nonconserved features of petals, but also pave the way to studying the regulatory evolution between floral organ identity genes and their targets.

A role for the Auxin Response Factors ARF6 and ARF8 homologs in petal spur elongation and nectary maturation in Aquilegia.

The petal spur of the basal eudicot Aquilegia is a key innovation associated with the adaptive radiation of the genus. Previous studies have shown that diversification of Aquilegia spur length can be predominantly attributed to variation in cell elongation. However, the genetic pathways that control the development of petal spurs are still being investigated. Here, we focus on a pair of closely related homologs of the AUXIN RESPONSE FACTOR family, AqARF6 and AqARF8, to explore their roles in Aquileiga coerulea petal spur development. Expression analyses of the two genes show that they are broadly expressed in vegetative and floral organs, but have relatively higher expression in petal spurs, particularly at later stages. Knockdown of the two AqARF6 and AqARF8 transcripts using virus-induced gene silencing resulted in largely petal-specific defects, including a significant reduction in spur length due to a decrease in cell elongation. These spurs also exhibited an absence of nectar production, which was correlated with downregulation of STYLISH homologs that have previously been shown to control nectary development. This study provides the first evidence of ARF6/8 homolog-mediated petal development outside the core eudicots. The genes appear to be specifically required for cell elongation and nectary maturation in the Aquilegia petal spur.

Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-Like Kinase EMS1.

Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana Here, we report the identification of ß-carbonic anhydrases (ßCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with ßCA proteins. Loss of function of ßCA genes caused defective tapetal cell differentiation, while overexpression of ßCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates ßCA1 at four sites, resulting in increased ßCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of ßCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. ßCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of ßCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.

The making of elaborate petals in Nigella through developmental repatterning.

Elaborate petals are present in many flowering plants lineages and have greatly promoted the success and evolutionary radiation of these groups. How elaborate petals are made, however, remains largely unclear. Petals of Nigella (Ranunculaceae) have long been recognized as elaborate and can thus be an excellent model for the study of petal elaboration. Here, by conducting detailed morphological, micromorphological, anatomical, developmental and evolutionary studies on the petals of Nigella species, we explored the processes, general patterns and underlying mechanisms of petal elaboration. We found that petals of Nigella are highly complex, and the complexity can be reflected at various levels. We also found that evolutionary elaboration of the Nigella petals is a gradual process, involving not only modifications of pre-existing structures but also de novo origination of new characters. Further investigations indicated that the elaboration and diversification of Nigella petals were accomplished by modifying the ancestral trajectory of petal development, a process known as developmental repatterning. Our results not only provide new insights into the development and evolution of elaborate petals, but also highlight the necessity of conducting multiple-level investigations for understanding the processes, patterns and underlying mechanisms of plant evolution.

Chloroplast genomic data provide new and robust insights into the phylogeny and evolution of the Ranunculaceae.

The family Ranunculaceae, a member of early-diverging eudicots that is increasingly being used as a model for the study of plant developmental evolution, has been the focus of systematic studies for centuries. Recent studies showed that the family can be divided into 14 tribes, with Glaucideae, Hydrastideae, and Coptideae being the successive basal-most lineages. The relationships among the remaining 11 tribes, however, remain controversial, so that a clear picture of character evolution within the family is still lacking. In this study, by sequencing, assembling and analyzing the chloroplast (cp) genomes of 35 species representing 31 genera of the 14 tribes, we resolved the relationships among the tribes and genera of the Ranunculaceae and clarified several long-standing controversies. We found that many of the characters that were once widely used for taxonomic and systematic considerations were actually results of parallel, convergent or even reversal evolution, suggestive of unreliability. We also found that the family has likely experienced two waves of radiative evolution, through which most of the extant tribes and genera were generated. Notably, both waves of radiation were correlated with the increase in the temperature of the earth, suggesting that global warming may have been the driving force of the radiation events. Based on these observations, we hypothesize that global warming and the associated decrease in the type and number of animal pollinators may have been the main reason why taxa with highly elaborate petals as well as those without petal were generated during each of the two waves of radiation.

Gains and Losses of Cis-regulatory Elements Led to Divergence of the Arabidopsis APETALA1 and CAULIFLOWER Duplicate Genes in the Time, Space, and Level of Expression and Regulation of One Paralog by the Other.

How genes change their expression patterns over time is still poorly understood. Here, by conducting expression, functional, bioinformatic, and evolutionary analyses, we demonstrate that the differences between the Arabidopsis (Arabidopsis thaliana) APETALA1 (AP1) and CAULIFLOWER (CAL) duplicate genes in the time, space, and level of expression were determined by the presence or absence of functionally important transcription factor-binding sites (TFBSs) in regulatory regions. In particular, a CArG box, which is the autoregulatory site of AP1 that can also be bound by the CAL protein, is a key determinant of the expression differences. Because of the CArG box, AP1 is both autoregulated and cross-regulated (by AP1 and CAL, respectively), and its relatively high-level expression is maintained till to the late stages of sepal and petal development. The observation that the CArG box was gained recently further suggests that the autoregulation and cross-regulation of AP1, as well as its function in sepal and petal development, are derived features. By comparing the evolutionary histories of this and other TFBSs, we further indicate that the divergence of AP1 and CAL in regulatory regions has been markedly asymmetric and can be divided into several stages. Specifically, shortly after duplication, when AP1 happened to be the paralog that maintained the function of the ancestral gene, CAL experienced certain degrees of degenerate evolution, in which several functionally important TFBSs were lost. Later, when functional divergence allowed the survival of both paralogs, CAL remained largely unchanged in expression, whereas the functions of AP1 were gradually reinforced by gains of the CArG box and other TFBSs.

F-box proteins regulate ethylene signaling and more.

Ethylene regulates several aspects of plant development, such as fruit ripening. In this issue of Genes & Development, Qiao and colleagues (pp. 512-521) report that the stability of the ethylene signaling protein EIN2 is modulated by two F-box proteins ETP1/2, reminiscent of the finding that another regulator of ethylene response, EIN3, is also targeted by the F-box proteins EBF1/2. ETP1/2 and EBF1/2 show distinct phylogenetic patterns, suggesting that they have different evolutionary constraints.

Disruption of the petal identity gene APETALA3-3 is highly correlated with loss of petals within the buttercup family (Ranunculaceae).

Absence of petals, or being apetalous, is usually one of the most important features that characterizes a group of flowering plants at high taxonomic ranks (i.e., family and above). The apetalous condition, however, appears to be the result of parallel or convergent evolution with unknown genetic causes. Here we show that within the buttercup family (Ranunculaceae), apetalous genera in at least seven different lineages were all derived from petalous ancestors, indicative of parallel petal losses. We also show that independent petal losses within this family were strongly associated with decreased or eliminated expression of a single floral organ identity gene, APETALA3-3 (AP3-3), apparently owing to species-specific molecular lesions. In an apetalous mutant of Nigella, insertion of a transposable element into the second intron has led to silencing of the gene and transformation of petals into sepals. In several naturally occurring apetalous genera, such as Thalictrum, Beesia, and Enemion, the gene has either been lost altogether or disrupted by deletions in coding or regulatory regions. In Clematis, a large genus in which petalous species evolved secondarily from apetalous ones, the gene exhibits hallmarks of a pseudogene. These results suggest that, as a petal identity gene, AP3-3 has been silenced or down-regulated by different mechanisms in different evolutionary lineages. This also suggests that petal identity did not evolve many times independently across the Ranunculaceae but was lost in numerous instances. The genetic mechanisms underlying the independent petal losses, however, may be complex, with disruption of AP3-3 being either cause or effect.

Divergence of duplicate genes in exon-intron structure.

Gene duplication plays key roles in organismal evolution. Duplicate genes, if they survive, tend to diverge in regulatory and coding regions. Divergences in coding regions, especially those that can change the function of the gene, can be caused by amino acid-altering substitutions and/or alterations in exon-intron structure. Much has been learned about the mode, tempo, and consequences of nucleotide substitutions, yet relatively little is known about structural divergences. In this study, by analyzing 612 pairs of sibling paralogs from seven representative gene families and 300 pairs of one-to-one orthologs from different species, we investigated the occurrence and relative importance of structural divergences during the evolution of duplicate and nonduplicate genes. We found that structural divergences have been very prevalent in duplicate genes and, in many cases, have led to the generation of functionally distinct paralogs. Comparisons of the genomic sequences of these genes further indicated that the differences in exon-intron structure were actually accomplished by three main types of mechanisms (exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion), each of which contributed differently to structural divergence. Like nucleotide substitutions, insertion/deletion and exonization/pseudoexonization occurred more or less randomly, with the number of observable mutational events per gene pair being largely proportional to evolutionary time. Notably, however, compared with paralogs with similar evolutionary times, orthologs have accumulated significantly fewer structural changes, whereas the amounts of amino acid replacements accumulated did not show clear differences. This finding suggests that structural divergences have played a more important role during the evolution of duplicate than nonduplicate genes.

Spur development and evolution: An update.

Floral spurs, widely recognized as a classic example of key morphological and functional innovation and thought to have promoted the origin and adaptive evolution of many flowering plant lineages, have attracted the attention of researchers for centuries. Despite this, the mechanisms underlying the development and evolution of these structures remain poorly understood. Recent studies have discovered the phytohormones and transcription factor genes that play key roles in regulating patterns of cell division and cell expansion during spur morphogenesis. Spur morphogenesis was also found to be tightly linked with the programs specifying floral zygomorphy, floral organ identity determination, and nectary development. Independent origins and losses of spurs in different flowering plant lineages, therefore, may be attributed to changes in the spur program and/or its upstream ones.

The mechanism underlying asymmetric bending of lateral petals in Delphinium (Ranunculaceae).

During the process of flower opening, most petals move downward in the direction of the pedicel (i.e., epinastic movement). In most Delphinium flowers, however, their two lateral petals display a very peculiar movement, the mirrored helical rotation, which requires the twist of the petal stalk. However, in some lineages, their lateral petals also exhibit asymmetric bending that increases the degree of mirrored helical rotation, facilitating the formation of a 3D final shape. Notably, petal asymmetric bending is a novel trait that has not been noticed yet, so its morphological nature, developmental process, and molecular mechanisms remain largely unknown. Here, by using D. anthriscifolium as a model, we determined that petal asymmetric bending was caused by the localized expansion of cell width, accompanied by the specialized array of cell wall nano-structure, on the adaxial epidermis. Digital gene analyses, gene expression, and functional studies revealed that a class I homeodomain-leucine zipper family transcription factor gene, DeanLATE MERISTEM IDENTITY1 (DeanLMI1), contributes to petal asymmetric bending; knockdown of it led to the formation of explanate 2D petals. Specifically, DeanLMI1 promotes cell expansion in width and influences the arrangement of cell wall nano-structure on the localized adaxial epidermis. These results not only provide a comprehensive portrait of petal asymmetric bending for the first time but also shed some new insights into the mechanisms of flower opening and helical movement in plants.

Diversification of ranunculaceous petals in shape supports a generalized model for plant lateral organ morphogenesis and evolution.

Peltate organs, such as the prey-capturing traps of carnivorous plants and nectary-bearing petals of ranunculaceous species, are widespread in nature and have intrigued and perplexed scientists for centuries. Shifts in the expression domains of adaxial/abaxial genes have been shown to control leaf peltation in some carnivorous plants, yet the mechanisms underlying the generation of other peltate organs remain unclear. Here, we show that formation of various peltate ranunculaceous petals was also caused by shifts in the expression domains of adaxial/abaxial genes, followed by differentiated regional growth sculpting the margins and/or other parts of the organs. By inducing parameters to specify the time, position, and degree of the shifts and growth, we further propose a generalized modeling system, through which various unifacial, bifacial, and peltate organs can be simulated. These results demonstrate the existence of a hierarchical morphospace system and pave the way to understand the mechanisms underlying plant organ diversification.