Roles of the APETALA3-3 ortholog in the petal identity specification and morphological differentiation in Delphinium anthriscifolium flowers.

The genus Delphinium (Ranunculaceae) with its unique and highly complex floral structure is an ideal system to address some key questions in terms of morphological and evolutionary studies in flowers. In D. anthriscifolium, for example, the original eight petal primordia differentiate into three types at maturity (i.e., two dorsal spurred, two lateral flat, and four ventral reduced petals). The mechanisms underlying their identity determination and morphological differentiation remain unclear. Here, through a comprehensive approach combining digital gene expression (DGE) profiles, in situ hybridization, and virus-induced gene silencing (VIGS), we explore the role of the APETALLATA3-3 (AP3-3) ortholog in D. anthriscifolium. Our findings reveal that the DeanAP3-3 not only functions as a traditionally known petal identity gene but also plays a critical role in petal morphological differentiation. The DeanAP3-3 gene is expressed in all the petal primordia before their morphological differentiation at earlier stages, but shows a gradient expression level difference along the dorsventral floral axis, with higher expression level in the dorsal spurred petals, intermediate level in the lateral flat petals and lower level in the ventral reduced petals. VIGS experiments revealed that flowers with strong phenotypic changes showed a complete transformation of all the three types of petals into non-spurred sepals. However, in the flowers with moderate phenotypic changes, the transformation of spurred petals into flat petals is associated with moderate silencing of the DeanAP3-3 gene, suggesting a significant impact of expression level on petal morphological differentiation. This research also shed some insights into the role of changes in gene expression levels on morphological differentiation in plants.

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.

Transcription Factors ZjMYB39 and ZjMYB4 Regulate Farnesyl Diphosphate Synthase- and Squalene Synthase-Mediated Triterpenoid Biosynthesis in Jujube.

Jujube (Ziziphus jujuba Mill.) is rich in valuable bioactive triterpenoids. However, the regulatory mechanism underlying triterpenoid biosynthesis in jujube remains poorly studied. Here, we characterized the triterpenoid content in wild jujube and cultivated jujube. The triterpenoid content was higher in wild jujube than in cultivated jujube, triterpenoids were most abundant in young leaves, buds, and later stages of development. The transcriptome analysis and correlation analysis showed that differentially expressed genes (DEGs) were enriched in the terpenoid synthesis pathways, and triterpenoids content was strongly correlated with farnesyl diphosphate synthase (ZjFPS), squalene synthase (ZjSQS), and transcription factors ZjMYB39 and ZjMYB4 expression. Gene overexpression and silencing analysis indicated that ZjFPS and ZjSQS were key genes in triterpenoid biosynthesis and transcription factors ZjMYB39 and ZjMYB4 regulated triterpenoid biosynthesis. Subcellular localization experiments showed that ZjFPS and ZjSQS were localized to the nucleus and endoplasmic reticulum and ZjMYB39 and ZjMYB4 were localized to the nucleus. Yeast one-hybrid, glucuronidase activity, and dual-luciferase activity assays suggested that ZjMYB39 and ZjMYB4 regulate triterpenoid biosynthesis by directly binding and activating the promoters of ZjFPS and ZjSQS. These findings provide insights into the underlying regulatory network of triterpenoids metabolism in jujube and lay theoretical and practical foundation for molecular breeding.

Methyl Jasmonate- and Salicylic Acid-Induced Transcription Factor ZjWRKY18 Regulates Triterpenoid Accumulation and Salt Stress Tolerance in Jujube.

Triterpenoids are important, pharmacologically active substances in jujube (Ziziphus jujuba Mill.), and play an important role in the plant's resistance to abiotic stress. However, regulation of their biosynthesis, and the underlying mechanism of their balance with stress resistance, remain poorly understood. In this study, we screened and functionally characterized the ZjWRKY18 transcription factor, which is associated with triterpenoid accumulation. The transcription factor is induced by methyl jasmonate and salicylic acid, and its activity was observed by gene overexpression and silencing experiments, combined with analyses of transcripts and metabolites. ZjWRKY18 gene silencing decreased the transcription of triterpenoid synthesis pathway genes and the corresponding triterpenoid content. Overexpression of the gene promoted the biosynthesis of jujube triterpenoids, as well as triterpenoids in tobacco and Arabidopsis thaliana. In addition, ZjWRKY18 binds to W-box sequences to activate promoters of 3-hydroxy-3-methyl glutaryl coenzyme A reductase and farnesyl pyrophosphate synthase, suggesting that ZjWRKY18 positively regulates the triterpenoid synthesis pathway. Overexpression of ZjWRKY18 also increased tolerance to salt stress in tobacco and Arabidopsis thaliana. These results highlight the potential use of ZjWRKY18 to improve triterpenoid biosynthesis and salt stress tolerance in plants, and provide a strong basis for metabolic engineering to improve the content of triterpenoids and breeding of jujube varieties that are resistant to stress.

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.

Identification of the key genes contributing to the LOX-HPL volatile aldehyde biosynthesis pathway in jujube fruit.

Jujube (Ziziphus jujuba Mill.) is a traditional popular fruit widely grown in China. The volatiles in jujube determine its unique flavor and the high fruit quality required by consumers. However, the biosynthesis of volatiles in jujube were remain unknown. By using gas chromatography-mass spectrometry, there were 46 volatile compounds were identified and determined from three representative jujube fruit types at six developmental stages, including the dry-used (Z. jujuba cv. 'Junzao'), the fresh-used (Z. jujuba cv. 'Dongzao'), and wild jujube (Z. jujuba var. spinosa Hu. cv. 'Qingjiansuanzao'). The aldehydes were identified as major volatile contributors to flavor, of which (E)-2-hexenal was the primary volatile in jujube fruit. Then LOX and HPL gene family were identified in jujube, which were involved in aldehyde biosynthesis through the lipoxygenase-hydroperoxide lyase (LOX-HPL) pathway. Gene expression analysis suggested that ZjLOX3, ZjLOX4, and ZjHPL1 were highly correlated with the accumulation of (E)-2-hexenal, and their proteins were localized to the nucleus and cytoplasm. Transient over-expression of ZjLOX3, ZjLOX4, and ZjHPL1 in jujube fruit significantly enhanced the accumulation of (E)-2-hexenal. Our study provides valuable information on the major volatiles and their biosynthesis in different types of jujube fruit. These results will help determine flavor improvements for future breeding.

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 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.

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.

Author Correction: The morphology, molecular development and ecological function of pseudonectaries on Nigella damascena (Ranunculaceae) petals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

The morphology, molecular development and ecological function of pseudonectaries on Nigella damascena (Ranunculaceae) petals.

Pseudonectaries, or false nectaries, the glistening structures that resemble nectaries or nectar droplets but do not secrete nectar, show considerable diversity and play important roles in plant-animal interactions. The morphological nature, optical features, molecular underpinnings and ecological functions of pseudonectaries, however, remain largely unclear. Here, we show that pseudonectaries of Nigella damascena (Ranunculaceae) are tiny, regional protrusions covered by tightly arranged, non-secretory polygonal epidermal cells with flat, smooth and reflective surface, and are clearly visible even under ultraviolet light and bee vision. We also show that genes associated with cell division, chloroplast development and wax formation are preferably expressed in pseudonectaries. Specifically, NidaYABBY5, an abaxial gene with ectopic expression in pseudonectaries, is indispensable for pseudonectary development: knockdown of it led to complete losses of pseudonectaries. Notably, when flowers without pseudonectaries were arrayed beside those with pseudonectaries, clear differences were observed in the visiting frequency, probing time and visiting behavior of pollinators (i.e., honey bees), suggesting that pseudonectaries serve as both visual attractants and nectar guides.

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.

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.

Plastomes resolve generic limits within tribe Clusieae (Clusiaceae) and reveal the new genus Arawakia.

Clusieae is an exclusively Neotropical tribe in the family Clusiaceae sensu stricto. Although tribes within Clusiaceae are morphologically and phylogenetically well-delimited, resolution among genera within these tribes remains elusive. The tribe Clusieae includes an estimated ∼500 species distributed among five genera: Chrysochlamys, Clusia, Dystovomita, Tovomita, and Tovomitopsis. In this study, we used nearly complete plastid genomes from 30 exemplar Clusieae species representing all genera recognized, plus two outgroups to infer the phylogeny of the tribe using Maximum Likelihood and Bayesian Inference. For comparison, we also inferred a phylogeny from the nuclear Internal Transcribed Spacer (ITS) region using the same methods. Our study corroborates earlier findings that Clusia is monophyletic while Tovomita is not. It also provides additional support to the hypothesis that Chrysochlamys and Tovomitopsis are not closely related despite gross morphological similarity. Tovomita is divided into three distantly related clades: (i) core Tovomita (including the type T. guianensis), (ii) T. croatii, and (iii) the T. weddelliana species complex. Members of the T. weddelliana complex are isolated from the core Tovomita, and placed in a well-supported clade that is sister to a clade composed of Chrysochlamys plus Clusia. Tovomita croatii is nested within Chrysochlamys. We propose taxonomic revisions to accommodate our phylogenetic findings, including the description of the new genus Arawakia, which includes the 18 species formerly recognized in the T. weddelliana species complex. Lectotypes are also designated for nine species (i.e., Arawakia angustata, A. lanceolata, A. lingulata, A. longicuneata, A. macrocarpa, A. oblanceolata, A. pithecobia, A. rhizophoroides, and A. weddelliana), and a taxonomic key for the identification of the six genera of Clusieae recognized is presented.

Prevalent Exon-Intron Structural Changes in the APETALA1/FRUITFULL, SEPALLATA, AGAMOUS-LIKE6, and FLOWERING LOCUS C MADS-Box Gene Subfamilies Provide New Insights into Their Evolution.

AP1/FUL, SEP, AGL6, and FLC subfamily genes play important roles in flower development. The phylogenetic relationships among them, however, have been controversial, which impedes our understanding of the origin and functional divergence of these genes. One possible reason for the controversy may be the problems caused by changes in the exon-intron structure of genes, which, according to recent studies, may generate non-homologous sites and hamper the homology-based sequence alignment. In this study, we first performed exon-by-exon alignments of these and three outgroup subfamilies (SOC1, AG, and STK). Phylogenetic trees reconstructed based on these matrices show improved resolution and better congruence with species phylogeny. In the context of these phylogenies, we traced evolutionary changes of exon-intron structures in each subfamily. We found that structural changes have occurred frequently following gene duplication and speciation events. Notably, exons 7 and 8 (if present) suffered more structural changes than others. With the knowledge of exon-intron structural changes, we generated more reasonable alignments containing all the focal subfamilies. The resulting trees showed that the SEP subfamily is sister to the monophyletic group formed by AP1/FUL and FLC subfamily genes and that the AGL6 subfamily forms a sister group to the three abovementioned subfamilies. Based on this topology, we inferred the evolutionary history of exon-intron structural changes among different subfamilies. Particularly, we found that the eighth exon originated before the divergence of AP1/FUL, FLC, SEP, and AGL6 subfamilies and degenerated in the ancestral FLC-like gene. These results provide new insights into the origin and evolution of the AP1/FUL, FLC, SEP, and AGL6 subfamilies.

Flexibility in the structure of spiral flowers and its underlying mechanisms.

Spiral flowers usually bear a variable number of organs, suggestive of the flexibility in structure. The mechanisms underlying the flexibility, however, remain unclear. Here we show that in Nigella damascena, a species with spiral flowers, different types of floral organs show different ranges of variation in number. We also show that the total number of organs per flower is largely dependent on the initial size of the floral meristem, whereas the respective numbers of different types of floral organs are determined by the functional domains of corresponding genetic programmes. By conducting extensive expression and functional studies, we further elucidate the genetic programmes that specify the identities of different types of floral organs. Notably, the AGL6-lineage member NdAGL6, rather than the AP1-lineage members NdFL1/2, is an A-function gene, whereas petaloidy of sepals is not controlled by AP3- or PI-lineage members. Moreover, owing to the formation of a regulatory network, some floral organ identity genes also regulate the boundaries between different types of floral organs. On the basis of these results, we propose that the floral organ identity determination programme is highly dynamic and shows considerable flexibility. Transitions from spiral to whorled flowers, therefore, may be explained by evolution of the mechanisms that reduce the flexibility.

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.