A comparative full-length transcriptomic resource provides insight into the perennial monocarpic mass flowering.

Perennial monocarpic mass flowering represents as a key developmental innovation in flowering time diversity in several biological and economical essential families, such as the woody bamboos and the shrubby Strobilanthes. However, molecular and genetic mechanisms underlying this important biodiversity remain poorly investigated. Here, we generated a full-length transcriptome resource incorporated into the BlueOmics database (http://blueomics.iflora.cn) for two Strobilanthes species, which feature contrasting flowering time behaviors. Using about 112 and 104 Gb Iso-seq reads together with ~185 and ~75 Gb strand-specific RNA seq data, we annotated 80 971 and 79 985 non-redundant full-length transcripts for the perennial polycarpic Strobilanthes tetrasperma and the perennial monocarpic Strobilanthes biocullata, respectively. In S. tetrasperma, we identified 8794 transcripts showing spatiotemporal expression in nine tissues. In leaves and shoot apical meristems at two developmental stages, 977 and 1121 transcripts were differentially accumulated in S. tetrasperma and S. biocullata, respectively. Interestingly, among the 33 transcription factors showing differential expression in S. tetrasperma but without differential expression in S. biocullata, three were involved potentially in the photoperiod and circadian-clock pathway of flowering time regulation (FAR1 RELATED SEQUENCE 12, FRS12; NUCLEAR FACTOR Y A1, NFYA1; PSEUDO-RESPONSE REGULATOR 5, PRR5), hence provides an important clue in deciphering the flowering diversity mechanisms. Our data serve as a key resource for further dissection of molecular and genetic mechanisms underpinning key biological innovations, here, the perennial monocarpic mass flowering.

Arabidopsis AGAMOUS-LIKE16 and SUPPRESSOR OF CONSTANS1 regulate the genome-wide expression and flowering time.

Flowering transition is tightly coordinated by complex gene regulatory networks, in which AGAMOUS-LIKE 16 (AGL16) plays important roles. Here, we identified the molecular function and binding properties of AGL16 and demonstrated its partial dependency on the SUPPRESSOR OF CONSTANS 1 (SOC1) function in regulating flowering. AGL16 bound to promoters of more than 2,000 genes via CArG-box motifs with high similarity to that of SOC1 in Arabidopsis (Arabidopsis thaliana). Approximately 70 flowering genes involved in multiple pathways were potential targets of AGL16. AGL16 formed a protein complex with SOC1 and shared a common set of targets. Intriguingly, only a limited number of genes were differentially expressed in the agl16-1 loss-of-function mutant. However, in the soc1-2 knockout background, AGL16 repressed and activated the expression of 375 and 182 genes, respectively, with more than a quarter bound by AGL16. Corroborating these findings, AGL16 repressed the flowering time more strongly in soc1-2 than in the Col-0 background. These data identify a partial inter-dependency between AGL16 and SOC1 in regulating genome-wide gene expression and flowering time, while AGL16 provides a feedback regulation on SOC1 expression. Our study sheds light on the complex background dependency of AGL16 in flowering regulation, thus providing additional insights into the molecular coordination of development and environmental adaptation.

Molecular and genetic regulation of petal number variation.

Floral forms with an increased number of petals, also known as double-flower phenotypes, have been selected and conserved in many domesticated plants, particularly in ornamentals, because of their great economic value. The molecular and genetic mechanisms that control this trait are therefore of great interest, not only for scientists, but also for breeders. In this review, we summarize current knowledge of the gene regulatory networks of flower initiation and development and known mutations that lead to variation of petal number in many species. In addition to the well-accepted miR172/AP2-like module, for which many questions remain unanswered, we also discuss other pathways in which mutations also lead to the formation of extra petals, such as those involved in meristem maintenance, hormone signalling, epigenetic regulation, and responses to environmental signals. We discuss how the concept of 'natural mutants' and recent advances in genomics and genome editing make it possible to explore the molecular mechanisms underlying double-flower formation, and how such knowledge could contribute to the future breeding and selection of this trait in more crops.

Evolution of the FLOWERING LOCUS T-like genes in angiosperms: a core-Lamiales-specific diversification.

Plant life-history is determined by two transitions, the germination and the flowering times, in which the phosphatidylethanolamine-binding proteins (PEBP) FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1) play key regulatory roles. Compared to the highly conserved TFL1-likes, FT-like genes vary in copy numbers significantly in gymnosperms and monocots of the angiosperms, while sporadic duplications can be observed in eudicots. Here, via a systematic analysis of the PEBPs in angiosperms with a special focus on twelve representative species featuring high-quality genomes in the Lamiales order, we identified a successive lineage-specific but systematic expansion of FT-like genes in the families of core Lamiales. The first expansion event generated FT1-likes mainly via a core-Lamiales-specific whole-genome-duplication (cL-WGD), while on the other hand, a likely random duplication produced the FT2-likes in the lineages containing Scrophulariaceae and rest of the core Lamiales. Both FT1- and FT2-like genes were further amplified tandemly in some families. These expanded FT-likes featured highly diverged expression patterns and structural variation, indicating functional diversification. Intriguingly, some core Lamiales contained the relict MOTHER OF FT AND TFL1 like 2 (MFT2) that likely expanded in the common ancestor of angiosperms. Our data showcase the highly dynamic lineage-specific expansion of the FT-like genes, thus provide important and fresh evolutionary insights into the gene-regulatory-network underpinning flowering time diversity in Lamiales, and more generally, in angiosperms.

Remarkable mitochondrial genome heterogeneity in Meniocus linifolius (Brassicaceae).

KEY MESSAGE: Detailed analyses of 16 genomes identified a remarkable acceleration of mutation rate, hence mitochondrial sequence and structural heterogeneity, in Meniocus linifolius (Brassicaceae). The powerhouse, mitochondria, in plants feature high levels of structural variation, while the encoded genes are normally conserved. However, the substitution rates and spectra of mitochondria DNA within the Brassicaceae, a family with substantial scientific and economic importance, have not been adequately deciphered. Here, by analyzing three newly assembled and 13 known mitochondrial genomes (mitogenomes), we report the highly variable genome structure and mutation rates in Brassicaceae. The genome sizes and GC contents are 196,604 bp and 46.83%, 288,122 bp and 44.79%, and 287,054 bp and 44.93%, for Meniocus linifolius (Mli), Crucihimalaya lasiocarpa (Cla), and Lepidium sativum (Lsa), respectively. In total, 29, 33, and 34 protein-coding genes (PCGs) and 14, 18, and 18 tRNAs are annotated for Mli, Cla, and Lsa, respectively, while all mitogenomes contain one complete circular molecule with three rRNAs and abundant RNA editing sites. The Mli mitogenome features four conformations likely mediated by the two pairs of long repeats, while at the same time seems to have an unusual evolutionary history due to higher GC content, loss of more genes and sequences, but having more repeats and plastid DNA insertions. Corroborating with these, an ambiguous phylogenetic position with long branch length and elevated synonymous substitution rate in nearly all PCGs are observed for Mli. Taken together, our results reveal a high level of mitogenome heterogeneity at the family level and provide valuable resources for further understanding the evolutionary pattern of organelle genomes in Brassicaceae.

Differential expressions of anthocyanin synthesis genes underlie flower color divergence in a sympatric Rhododendron sanguineum complex.

BACKGROUND: The Rhododendron sanguineum complex is endemic to alpine mountains of northwest Yunnan and southeast Tibet of China. Varieties in this complex exhibit distinct flower colors even at the bud stage. However, the underlying molecular regulations for the flower color variation have not been well characterized. Here, we investigated this via measuring flower reflectance profiles and comparative transcriptome analyses on three coexisting varieties of the R. sanguineum complex, with yellow flush pink, bright crimson, and deep blackish crimson flowers respectively. We compared the expression levels of differentially-expressed-genes (DEGs) of the anthocyanin / flavonoid biosynthesis pathway using RNA-seq and qRT-PCR data. We performed clustering analysis based on transcriptome-derived Single Nucleotide Polymorphisms (SNPs) data, and finally analyzed the promoter architecture of DEGs. RESULTS: Reflectance spectra of the three color morphs varied distinctively in the range between 400 and 700 nm, with distinct differences in saturation, brightness, hue, and saturation/hue ratio, an indirect measurement of anthocyanin content. We identified 15,164 orthogroups that were shared among the three varieties. The SNP clustering analysis indicated that the varieties were not monophyletic. A total of 40 paralogous genes encoding 12 enzymes contributed to the flower color polymorphism. These anthocyanin biosynthesis-related genes were associated with synthesis, modification and transportation properties (RsCHS, RsCHI, RsF3H, RsF3'H, RsFLS, RsANS, RsAT, RsOMT, RsGST), as well as genes involved in catabolism and degradation (RsBGLU, RsPER, RsCAD). Variations in sequence and cis-acting elements of these genes might correlate with the anthocyanin accumulation, thus may contribute to the divergence of flower color in the R. sanguineum complex. CONCLUSIONS: Our results suggested that the varieties are very closely related and flower color variations in the R. sanguineum complex correlate tightly with the differential expression levels of genes involved in the anabolic and catabolic synthesis network of anthocyanin. Our study provides a scenario involving intricate relationships between genetic mechanisms for floral coloration accompanied by gene flow among the varieties that may represent an early case of pollinator-mediated incipient sympatric speciation.

Genome-wide identification of WD40 genes reveals a functional diversification of COP1-like genes in Rosaceae.

KEY MESSAGE: Genome-wide identification of WD40-like genes reveals a duplication of COP1-like genes, one of the key players involved in regulation of flowering time and photomorphogenesis, with strong functional diversification in Rosaceae. WD40 proteins play crucial roles in a broad spectrum of developmental and physiological processes. Here, we conducted a systematic characterization of this family of genes in Rosa chinensis 'Old Blush' (OB), a founder genotype for modern rose domestication. We identified 187 rose WD40 genes and classified them into 5 clusters and 15 subfamilies with 11 of RcWD40s presumably generated via tandem duplication. We found RcWD40 genes were expressed differentially following stages of vegetative and reproductive development. We detected a duplication of CONSTITUTIVE PHOTOMORPHOGENIC1-like genes in rose (RcCOP1 and RcCOP1L) and other Rosaceae plants. Featuring a distinct expression pattern and a different profile of cis-regulatory-elements in the transcriptional regulatory regions, RcCOP1 seemed being evolutionarily conserved while RcCOP1L did not dimerize with RcHY5 and RcSPA4. Our data thus reveals a functional diversification of COP1-like genes in Rosacaeae plants, and provides a valuable resource to explore the potential function and evolution of WD40-like genes in Rosaceae plants.

Comparative transcriptomics identifies patterns of selection in roses.

BACKGROUND: Roses are important plants for human beings with pivotal economical and biological traits like continuous flowering, flower architecture, color and scent. Due to frequent hybridization and high genome heterozygosity, classification of roses and their relatives remains a big challenge. RESULTS: Here, to identify potential markers for phylogenetic reconstruction and to reveal the patterns of natural selection in roses, we generated sets of high quality and comprehensive reference transcriptomes for Rosa chinensis 'Old Blush' (OB) and R. wichuriana 'Basye's Thornless' (BT), two species exhibiting contrasted traits of high economical importance. The assembled reference transcriptomes showed transcripts N50 above 2000 bp. Two roses shared about 10,073 transcripts (N50 = 2282 bp), in which a set of 5959 transcripts was conserved within genera of Rosa. Further comparison with species in Rosaceae identified 4447 transcripts being common (Rosaceae-common) in Rosa, Malus, Prunus, Rubus, and Fragaria, while a pool of 164 transcripts being specific for roses (Rosa-specific). Among the Rosaceae-common transcripts, 409 transcripts showed a signature of positive selection and a clustered expression in different tissues. Interestingly, nine of these rapidly evolving genes were related to DNA damage repair and responses to environmental stimulus, a potential associated with genome confliction post hybridization. Coincident with this fast evolution pattern in rose genes, 24 F-box and four TMV resistant proteins were significantly enriched in the Rosa-specific genes. CONCLUSIONS: We expect that these Rosaceae-common and Rosa-specific transcripts should facilitate the phylogenetic analysis of Rosaceae plants as well as investigations of Rosa-specific biology. The data reported here could provide fundamental genomic tools and knowledge critical for understanding the biology and domestication of roses and for roses breeding.

miR824-Regulated AGAMOUS-LIKE16 Contributes to Flowering Time Repression in Arabidopsis.

The timing of flowering is pivotal for maximizing reproductive success under fluctuating environmental conditions. Flowering time is tightly controlled by complex genetic networks that integrate endogenous and exogenous cues, such as light, temperature, photoperiod, and hormones. Here, we show that AGAMOUS-LIKE16 (AGL16) and its negative regulator microRNA824 (miR824) control flowering time in Arabidopsis thaliana. Knockout of AGL16 effectively accelerates flowering in nonvernalized Col-FRI, in which the floral inhibitor FLOWERING LOCUS C (FLC) is strongly expressed, but shows no effect if plants are vernalized or grown in short days. Alteration of AGL16 expression levels by manipulating miR824 abundance influences the timing of flowering quantitatively, depending on the expression level and number of functional FLC alleles. The effect of AGL16 is fully dependent on the presence of FLOWERING LOCUS T (FT). Further experiments show that AGL16 can interact directly with SHORT VEGETATIVE PHASE and indirectly with FLC, two proteins that form a complex to repress expression of FT. Our data reveal that miR824 and AGL16 modulate the extent of flowering time repression in a long-day photoperiod.

Genome-wide analysis of gene expression reveals gene regulatory networks that regulate chasmogamous and cleistogamous flowering in Pseudostellaria heterophylla (Caryophyllaceae).

BACKGROUND: Pseudostellaria heterophylla produces both closed (cleistogamous, CL) and open (chasmogamous, CH) flowers on the same individual but in different seasons. The production of CH and CL flowers might be in response to environmental changes. To better understand the molecular mechanisms of CH and CL flowering, we compared the transcriptome of the two types of flowers to examine differential gene expression patterns, and to identify gene regulatory networks that control CH and CL flowering. RESULTS: Using RNA sequencing, we identified homologues of 428 Arabidopsis genes involved in regulating flowering processes and estimated the differential gene expression patterns between CH and CL flowers. Some of these genes involved in gene regulatory networks of flowering processes showed significantly differential expression patterns between CH and CL flowers. In addition, we identified another 396 differentially expressed transcripts between CH and CL flowers. Some are involved in environmental stress responses and flavonoid biosynthesis. CONCLUSIONS: We propose how the differential expression of key members of three gene regulatory modules may explain CH and CL flowering. Future research is needed to investigate how the environment impinges on these flowering pathways to regulate CH and CL flowering in P. heterophylla.

Genomes of Meniocus linifolius and Tetracme quadricornis reveal the ancestral karyotype and genomic features of core Brassicaceae.

Brassicaceae represents an important plant family from both a scientific and economic perspective. However, genomic features related to the early diversification of this family have not been fully characterized, especially upon the uplift of the Tibetan Plateau, which was followed by increasing aridity in the Asian interior, intensifying monsoons in Eastern Asia, and significantly fluctuating daily temperatures. Here, we reveal the genomic architecture that accompanied early Brassicaceae diversification by analyzing two high-quality chromosome-level genomes for Meniocus linifolius (Arabodae; clade D) and Tetracme quadricornis (Hesperodae; clade E), together with genomes representing all major Brassicaceae clades and the basal Aethionemeae. We reconstructed an ancestral core Brassicaceae karyotype (CBK) containing 9 pseudochromosomes with 65 conserved syntenic genomic blocks and identified 9702 conserved genes in Brassicaceae. We detected pervasive conflicting phylogenomic signals accompanied by widespread ancient hybridization events, which correlate well with the early divergence of core Brassicaceae. We identified a successive Brassicaceae-specific expansion of the class I TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) gene family, which encodes enzymes with essential regulatory roles in flowering time and embryo development. The TPS1s were mainly randomly amplified, followed by expression divergence. Our results provide fresh insights into historical genomic features coupled with Brassicaceae evolution and offer a potential model for broad-scale studies of adaptive radiation under an ever-changing environment.

Widespread interspecific divergence in cis-regulation of transposable elements in the Arabidopsis genus.

Transposable elements (TEs) are so abundant and variable that they count among the most important mutational sources in genomes. Nonetheless, little is known about the genetics of their variation in activity or silencing across closely related species. Here, we demonstrate that regulation of TE genes can differ dramatically between the two closely related Arabidopsis species A. thaliana and A. lyrata. In leaf and floral tissues of F1 interspecific hybrids, about 47% of TEs show allele-specific expression, with the A. lyrata copy being generally expressed at higher level. We confirm that TEs are generally expressed in A. lyrata but not in A. thaliana. Allele-specific differences in TE expression are associated with divergence in epigenetic modifications like DNA and histone methylation between species as well as with sequence divergence. Our data demonstrate that A. thaliana silences TEs much better than A. lyrata. For long terminal repeat retrotransposons, these differences are more pronounced for younger insertions. Interspecific differences in TE silencing may have a great impact on genome size changes.

Complex and reticulate origin of edible roses (Rosa, Rosaceae) in China.

While roses are today among the most popular ornamental plants, the petals and fruits of some cultivars have flavored foods for millennia. The genetic origins of these edible cultivars remain poorly investigated. We collected the major varieties of edible roses available in China, assembled their plastome sequences, and phased the haplotypes for internal transcribed spacers (ITS1/ITS2) of the 18S-5.8S-26S nuclear ribosomal cistron. Our phylogenetic reconstruction using 88 plastid genomes, of primarily maternal origin, uncovered well-supported genetic relationships within Rosa, including all sections and all subgenera. We phased the ITS sequences to identify potential donor species ancestral to the development of known edible cultivars. The tri-parental Middle-Eastern origin of R. × damascena, the species most widely used in perfume products and food additives, was confirmed as a descendent of past hybridizations among R. moschata, R. gallica, and R. majalis/R. fedtschenkoana/R. davurica. In contrast, R. chinensis, R. rugosa, and R. gallica, in association with six other wild species, were the main donors for fifteen varieties of edible roses. The domesticated R. rugosa 'Plena' was shown to be a hybrid between R. rugosa and R. davurica, sharing a common origin with R. 'Fenghua'. Only R. 'Jinbian' and R. 'Crimson Glory' featured continuous flowering. All remaining cultivars of edible roses bloomed only once a year. Our study provides important resources for clarifying the origin of edible roses and suggests a future for breeding new cultivars with unique traits, such as continuous flowering.

Structurally different alleles of the ath-MIR824 microRNA precursor are maintained at high frequency in Arabidopsis thaliana.

In plants and animals, gene expression can be down-regulated at the posttranscriptional level by microRNAs (miRNAs), a class of small endogenous RNA. Comparative analysis of miRNA content across species indicates continuous birth and death of these loci in the course of evolution. However, little is known about the microevolutionary dynamics of these genetic elements, especially in plants. In this article we examine polymorphism at two miRNA-encoding loci in Arabidopsis thaliana, miR856 and miR824, which are not found in rice or poplar. We compare their diversity to other miRNA-encoding loci conserved across distant taxa. We find that levels of variation vary significantly across loci and that the two recently derived loci harbor patterns of diversity deviating from neutrality. miRNA miR856 shows a weak signature of a selective sweep whereas miR824 displays signs of balancing selection. A detailed examination of structural variation among alleles found at the miR824-encoding locus suggests nonrandom evolution of a thermoresistant substructure in the precursor. Expression analysis of pre-miR824 and its target, AGL16, indicates that these structural differences likely impact the processing of mature miR824. Our work highlights the relevance of RNA structure in precursor sequence evolution, suggesting that the evolutionary dynamics of miRNA-encoding loci is more complex than suggested by the constraints exerted on the interaction between mature miRNA fragments and their target exon.

Catalyst-free Synthesis of Aminomethylphenol Derivatives in Cyclopentyl Methyl Ether via Petasis Borono-Mannich Reaction.

OBJECTIVE: Aminomethylphenol molecules have wider applications in pharmaceuticals, agrochemicals, plant protection and promising functional materials. The development of an efficient and practical method to prepare this class of compound is highly desirable from both environmental and economical points of view. MATERIALS AND METHODS: In order to establish an effective synthetic method for preparing aminomethylphenol derivatives, the Petasis borono-Mannich reaction of salicylaldehyde, phenylboronic acid and 1,2,3,4- tetrahydroisoquinoline was selected as a model reaction. A variety of reaction conditions are investigated, including solvent and temperature. The generality and limitation of the established method were also evaluated. RESULTS AND DISCUSSION: It was found that model reaction can be carried out in cyclopentyl methyl ether at 80 oC under catalyst-free conditions. This protocol, with broad substrate applicability, the reaction of various arylboronic acid, secondary amine and salicylaldehyde proceeded smoothly under optimal reaction conditions to afford various aminomethylphenol derivatives in high yields. A practical, scalable, and high-yielding synthesis of aminomethylphenol derivatives was successfully accomplished. CONCLUSION: A catalyst-free practical method for the synthesis of minomethylphenol derivatives based on Petasis borono-Mannich (PBM) reaction of various arylboronic acid, secondary amine and salicylaldehyde in cyclopentyl methyl ether has been developed. The salient features of this protocol are avoidance of any additive/catalyst and toxic organic solvents, use of cyclopentyl methyl ether as the reaction medium, clean reaction profiles, easy operation, and high to excellent yield.

Accession-specific flowering time variation in response to nitrate fluctuation in Arabidopsis thalian a.

Flowering time, a key transition point from vegetative to reproductive growth, is regulated by an intrinsic complex of endogenous and exogenous signals including nutrient status. For hundreds of years, nitrogen has been well known to modulate flowering time, but the molecular genetic basis on how plants adapt to ever-changing nitrogen availability remains not fully explored. Here we explore how Arabidopsis natural variation in flowering time responds to nitrate fluctuation. Upon nitrate availability change, we detect accession- and photoperiod-specific flowering responses, which also feature a accession-specific dependency on growth traits. The flowering time variation correlates well with the expression of floral integrators, SOC1 and FT, in an accession-specific manner. We find that gene expression variation of key hub genes in the photoperiod-circadian-clock (GI), aging (SPLs) and autonomous (FLC) pathways associates with the expression change of these integrators, hence flowering time variation. Our results thus shed light on the molecular genetic mechanisms on regulation of accession- and photoperiod-specific flowering time variation in response to nitrate availability.

Expansion and expression diversity of FAR1/FRS-like genes provides insights into flowering time regulation in roses.

Roses are important horticultural plants with enormous diversity in flowers and flowering behavior. However, molecular regulation of flowering time variation in roses remains poorly characterized. Here, we report an expansion of the FAR1/FRS-like genes that correlates well with the switch to prostrate-to-erect growth of shoots upon flowering in Rosa wichuraiana 'Basye's Thornless' (BT). With the availability of the high-quality chromosome-level genome assembly for BT that we developed recently, we identified 91 RwFAR1/FRS-like genes, a significant expansion in contrast to 52 in Rosa chinensis 'Old Blush' (OB), a founder genotype in modern rose domestication. Rose FAR1/FRS-like proteins feature distinct variation in protein domain structures. The dispersed expansion of RwFAR1/FRS-like genes occurred specifically in clade I and II and is significantly associated with transposon insertion in BT. Most of the RwFAR1/FRS-like genes showed relatively higher expression level than their corresponding orthologs in OB. FAR1/FRS-like genes regulate light-signaling processes, shade avoidance, and flowering time in Arabidopsis thaliana. Therefore, the expansion and duplication of RwFAR1/FRS-like genes, followed by diversification in gene expression, might offer a novel leverage point for further understanding the molecular regulation of the variation in shoot-growth behavior and flowering time in roses.

Rose without prickle: genomic insights linked to moisture adaptation.

Prickles act against herbivores, pathogens or mechanical injury, while also preventing water loss. However, whether prickles have new function and the molecular genetics of prickle patterning remain poorly explored. Here, we generated a high-quality reference genome assembly for 'Basye's Thornless' (BT), a prickle-free cultivar of Rosa wichuraiana, to identify genetic elements related to stem prickle development. The BT genome harbors a high level of sequence diversity in itself and with cultivar 'Old Blush' (R. chinensis), a founder genotype in rose domestication. Inheritance of stem prickle density was determined and two QTL were identified. Differentially expressed genes in QTL were involved in water-related functions, suggesting that prickle density may hitchhike with adaptations to moist environments. While the prickle-related gene-regulatory-network (GRN) was highly conserved, the expression variation of key candidate genes was associated with prickle density. Our study provides fundamental resources and insights for genome evolution in the Rosaceae. Ongoing efforts on identification of the molecular bases for key rose traits may lead to improvements for horticultural markets.

MPF2-like-a MADS-box genes control the inflated Calyx syndrome in Withania (Solanaceae): roles of Darwinian selection.

The Chinese lantern, which is the inflated calyx syndrome (ICS) of Physalis, is formed by MPF2 in the presence of the plant hormones, cytokinin and gibberellin. MPF2 knockdown mutants of Physalis have small leaves, no ICS, and are male sterile, thus, revealing three MPF2-related functions. Of the close relatives of Physalis, Tubocapsicum has only a rudimentary calyx, whereas others, like the Withania species, have ICS. From all Withania samples tested, two classes of MPF2-like orthologs, MPF2-like-A and MPF2-like-B, were isolated, whereas only the latter class was obtained from tetraploid Tubocapsicum. Though distinct differences can be observed between MPF2-like-A and MPF2-like-B proteins, that is MPF2-like-A proteins have an aberrant structure in that they have a three amino acid deletion in their C-domain and an eight amino acid extension at the C-terminal end, MPF2-like-A genes are phylogenetically closer to the Physalis MPF2-like genes. Unlike MPF2-like-B, the overexpression of MPF2-like-A in Arabidopsis revealed extra large sepals thus suggesting that MPF2-like-A genes are very likely responsible for the ICS formation in Withania. This correlated with the expression pattern of MPF2-like-A in vegetative and flower tissues, whereas MPF2-like-B is expressed only in vegetative tissues of Withania. In Tubocapsicum, however, MPF2-like-B RNA is detectable in all tissues tested. Finally, positive Darwinian selection was observed in the branch leading to Physalis MPF2-like and Withania MPF2-like-A proteins, followed by purifying selection once the trait had evolved. By contrast, purifying selection was detected for all other MPF2-like proteins tested. The contribution of the MPF2-like gene duplication to subfunctionalization is discussed.