Basal Angiosperm Species Research Articles

Gene identification and tissue expression analysis inform the floral organization and color in the basal angiosperm Magnolia polytepala (Magnoliaceae).

In Magnolia polytepala, the formation of floral organization and color was attributed to tissue-dependent differential expression levels of MADS-box genes and anthocyanin biosynthetic genes. In angiosperms, the diversity of floral morphology and organization suggests its value in exploring plant evolution. Magnolia polytepala, an endemic basal angiosperm species in China, possesses three green sepal-like tepals in the outermost whorl and pink petal-like tepals in the inner three whorls, forming unique floral morphology and organization. However, we know little about its underlying molecular regulatory mechanism. Here, we first reported the full-length transcriptome of M. polytepala using PacBio sequencing. A total of 16 MADS-box transcripts were obtained from the transcriptome data, including floral homeotic genes (e.g., MpAPETALA3) and other non-floral homeotic genes (MpAGL6, etc.). Phylogenetic analysis and spatial expression pattern reflected their putative biological function as their homologues in Arabidopsis. In addition, nine structural genes involved in anthocyanin biosynthesis pathway had been screened out, and tepal color difference was significantly associated with their tissue-dependent differential expression levels. This study provides a relatively comprehensive investigation of the MADS-box family and anthocyanin biosynthetic genes in M. polytepala, and will facilitate our understanding of the regulatory mechanism underlying floral organization and color in basal angiosperms.

The complete chloroplast genome sequence of Knema conferta (Myristicaceae)

Knema conferta is a member of Myristicaceae. The K. conferta chloroplast genome is found to be 155,744 bp in length and has a base composition of A (30.02%), G (19.30%), C (19.90%), and T (30.78%). The genome contained two short inverted repeat (IRa and IRb) regions (48,052 bp) which were separated by a large single copy (LSC) region (86,926 bp) and a small single copy (SSC) region (20,770 bp). The genome encoded a total of 128 unigenes, including 89 protein-coding genes, 31 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of K. conferta was aligned together with 2 species of Knema and 5 basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of K. conferta.

The complete chloroplast genome sequence of Knema linifolia (Myristicaceae)

Knema linifolia is a member of Myristicaceae. The K. linifolia chloroplast genome is found to be 155,754 bp in length and has a base composition of A (30.02%), G (19.30%), C (19.89%), and T (30.79%). The genome contained two short inverted repeat (IRa and IRb) regions (48,080 bp) which were separated by a large single copy (LSC) region (86,991 bp) and a small single copy (SSC) region (20,683 bp). The chloroplast genome has 89 protein-coding genes, 31 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of K. linifolia was aligned together with 2 species of Knema and 5 basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of K. linifolia.

Large-scale survey reveals pervasiveness and potential function of endogenous geminiviral sequences in plants.

The family Geminiviridae contains viruses with single-stranded DNA genomes that have been found infecting a wide variety of angiosperm species. The discovery within the last 25 years of endogenous geminivirus-like (EGV) elements within the nuclear genomes of several angiosperms has raised questions relating to the pervasiveness of EGVs and their impacts on host biology. Only a few EGVs have currently been characterized and it remains unclear whether any of these have influenced, or are currently influencing, the evolutionary fitness of their hosts. We therefore undertook a large-scale search for evidence of EGVs within 134 genome and 797 transcriptome sequences of green plant species. We detected homologues of geminivirus replication-associated protein (Rep) genes in forty-two angiosperm species, including two monocots, thirty-nine dicots, and one ANITA-grade basal angiosperm species (Amborella trichopoda). While EGVs were present in the members of many different plant orders, they were particularly common within the large and diverse order, Ericales, with the highest copy numbers of EGVs being found in two varieties of tea plant (Camellia sinensis). Phylogenetic and clustering analyses revealed multiple highly divergent previously unknown geminivirus Rep lineages, two of which occur in C.sinensis alone. We find that some of the Camellia EGVs are likely transcriptionally active, sometimes co-transcribed with the same host genes across several Camellia species. Overall, our analyses expand the known breadths of both geminivirus diversity and geminivirus host ranges, and strengthens support for the hypothesis that EGVs impact the biology of their hosts.

The complete chloroplast genome sequence of Knema elegans (Myristicaceae)

Knema elegans is a member of Myristicaceae. The K. elegans chloroplast genome is found to be 155,691 bp in length and has a base composition of A (30.02%), G (19.31%), C (19.89%), and T (30.78%). The genome contained two short inverted repeat (IRa and IRb) regions (48,122 bp) which were separated by a large single-copy (LSC) region (86,883 bp) and a small single-copy (SSC) region (20,686 bp). The chloroplast genome has 85 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of K. elegans was aligned together with two species of Myristicaceae and five basal angiosperms species for which the complete chloroplast sequence have been reported. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of K. elegans.

The complete chloroplast genome sequence of Knema furfuracea (Myristicaceae)

Knema furfuracea is a member of Myristicaceae. The K. furfuracea chloroplast genome is found to be 154,527 bp in length and has a base composition of A (29.99%), G (19.31%), C (19.92%), and T (30.78%). The genome contained two short inverted repeat (IRa and IRb) regions (48,110 bp) which were separated by a large single copy (LSC) region (86,188 bp) and a small single copy (SSC) region (20,229 bp). The chloroplast genome has 87 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of K. furfuracea was aligned together with two species of Myristicaceae and five basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of K. furfuracea.

The complete chloroplast genome sequence of Horsfieldia amygdalina (Myristicaceae)

Horsfieldia amygdalina is a member of Myristicaceae. The H. amygdalina chloroplast genome is found to be 155,683 bp in length and has a base composition of A (29.99%), G (19.32%), C (19.92%), and T (30.77%). The genome contained two short inverted repeat (IRa and IRb) regions (37,754 bp) which were separated by a large single copy (LSC) region (86,931 bp) and a small single copy (SSC) region (30,998 bp). The genome encodes 121 unique genes, including 86 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of H. amygdalina was aligned together with Horsfieldia pandurifolia, Myristica yunnanensis and other Magnoliales and basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of H. amygdalina.

The complete chloroplast genome sequence of Horsfieldia kingii (Myristicaceae)

Horsfieldia kingii is a member of Myristicaceae. The H. kingii chloroplast genome is found to be 155,655 bp in length and has a base composition of A (30.03%), G (19.52%), C (19.72%), and T (30.73%). The genome contained two short inverted repeat (IRa and IRb) regions (48,052 bp) which were separated by a large single copy (LSC) region (86,912 bp) and a small single copy (SSC) region (20,691 bp). The genome encodes 123 unique genes, including 85 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of H. kingii was aligned together with other 2 species of Myristicaceae and other 5 basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of H. kingii.

The complete chloroplast genome sequence of Myristica yunnanensis (Myristicaceae)

Myristica yunnanensis is a member of Myristicaceae. The M. yunnanensis chloroplast genome is found to be 155,923 bp in length and has a base composition of A (30.04%), G (19.28%), C (19.91%), and T (30.77%). The genome contained two short inverted repeat (IRa and IRb) regions (48,004 bp) which were separated by a large single-copy (LSC) region (87,088 bp) and a small single-copy (SSC) region (20,731 bp). The genome encodes 120 unique genes, including 85 protein-coding genes, 27 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Further, complete chloroplast sequence of M. yunnanensis was aligned together with Horsfieldia pandurifolia and other Magnoliales and basal angiosperms species which have reported the complete chloroplast sequence. This complete chloroplast genome will provide valuable information for the development of DNA markers for future species resource development and phylogenetic analysis of M. yunnanensis.

Complete chloroplast genome sequence of an endangered tree species, Magnolia sieboldii (Magnoliaceae)

The characteristic of complete chloroplast (cp) genome of Magnolia sieboldii, an endangered species in China, was first sequenced in this study. It showed a typical quadripartite structure with a length of 160,177 bp. It composed of a large single copy (LSC) region of 88,238 bp, a small single copy (SSC) region of 18,763 bp, and a pair of inverted repeat (IR) regions of 26,588 bp. Further maximum parsimony phylogenetic analysis was performed based on 38 complete plastomes from 30 magnoliids and basal angiosperm species. The result supported a close relationship among M. grandiflora, M. officinals, M. sieboldii, and M. tripetala.

Oil biosynthesis in a basal angiosperm: transcriptome analysis of Persea Americana mesocarp.

BackgroundThe mechanism by which plants synthesize and store high amounts of triacylglycerols (TAG) in tissues other than seeds is not well understood. The comprehension of controls for carbon partitioning and oil accumulation in nonseed tissues is essential to generate oil-rich biomass in perennial bioenergy crops. Persea americana (avocado), a basal angiosperm with unique features that are ancestral to most flowering plants, stores ~ 70 % TAG per dry weight in its mesocarp, a nonseed tissue. Transcriptome analyses of select pathways, from generation of pyruvate and leading up to TAG accumulation, in mesocarp tissues of avocado was conducted and compared with that of oil-rich monocot (oil palm) and dicot (rapeseed and castor) tissues to identify tissue- and species-specific regulation and biosynthesis of TAG in plants.ResultsRNA-Seq analyses of select lipid metabolic pathways of avocado mesocarp revealed patterns similar to that of other oil-rich species. However, only some predominant orthologs of the fatty acid biosynthetic pathway genes in this basal angiosperm were similar to those of monocots and dicots. The accumulation of TAG, rich in oleic acid, was associated with higher transcript levels for a putative stearoyl-ACP desaturase and endoplasmic reticulum (ER)-associated acyl-CoA synthetases, during fruit development. Gene expression levels for enzymes involved in terminal steps to TAG biosynthesis in the ER further indicated that both acyl-CoA-dependent and -independent mechanisms might play a role in TAG assembly, depending on the developmental stage of the fruit. Furthermore, in addition to the expression of an ortholog of WRINKLED1 (WRI1), a regulator of fatty acid biosynthesis, high transcript levels for WRI2-like and WRI3-like suggest a role for additional transcription factors in nonseed oil accumulation. Plastid pyruvate necessary for fatty acid synthesis is likely driven by the upregulation of genes involved in glycolysis and transport of its intermediates. Together, a comparative transcriptome analyses for storage oil biosynthesis in diverse plants and tissues suggested that several distinct and conserved features in this basal angiosperm species might contribute towards its rich TAG content.ConclusionsOur work represents a comprehensive transcriptome resource for a basal angiosperm species and provides insight into their lipid metabolism in mesocarp tissues. Furthermore, comparison of the transcriptome of oil-rich mesocarp of avocado, with oil-rich seed and nonseed tissues of monocot and dicot species, revealed lipid gene orthologs that are highly conserved during evolution. The orthologs that are distinctively expressed in oil-rich mesocarp tissues of this basal angiosperm, such as WRI2, ER-associated acyl-CoA synthetases, and lipid-droplet associated proteins were also identified. This study provides a foundation for future investigations to increase oil-content and has implications for metabolic engineering to enhance storage oil content in nonseed tissues of diverse species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0586-2) contains supplementary material, which is available to authorized users.

Appearance and elaboration of the ethylene receptor family during land plant evolution.

Ethylene is perceived following binding to endoplasmic reticulum-localized receptors, which in Arabidopsis thaliana, include ETR1, ERS1, EIN4, ETR2, and ERS2. These receptors fall into two subfamilies based on conservation of features within their histidine kinase domain. Subfamily 1 contains ETR1 and ERS1 whereas subfamily 2 contains EIN4, ETR2, and ERS2. Because ethylene receptors are found only in plants, this raises questions of when each receptor evolved. Here it is shown that subfamily 1 receptors encoded by a multigene family are present in all charophytes examined, these being most homologous to ETR1 based on their evolutionary relationship as well as containing histidine kinase and receiver domains. In charophytes and Physcomitrella patens, one or more gene family members contain the intron characteristic of subfamily 2 genes, indicating the first step in subfamily 2 receptor evolution. ERS1 homologs appear in basal angiosperm species after Amborella trichopoda and, in some early and basal angiosperm species and monocots in general, it is the only subfamily 1 receptor present. Distinct EIN4 and ETR2 homologs appear only in core eudicots and ERS2 homologs appear only in the Brassicaceae, suggesting it is the most recent receptor to evolve. These findings show that a subfamily 1 receptor had evolved and a subfamily 2 receptor had begun to evolve in plants prior to the colonization of land and only these two existed up to the appearance of the first basal angiosperm. The appearance of ERS2 in the Brassicaceae suggests ongoing evolution of the ethylene receptor family.

Understanding the ecosystem implications of the angiosperm rise to dominance: leaf litter decomposability among magnoliids and other basal angiosperms

Summary Litter decomposition has been a key driver of carbon and nutrient cycling in the present and past. Based on extant species data, there is a great deal of variation in litter decomposability among major plant lineages, suggesting potential shifts in plant effects on carbon and nutrient cycling during the early evolutionary history of angiosperms. Existing data suggest that eudicot species produce faster decomposing litter compared to gymnosperms, ferns and mosses. One of the missing puzzle pieces in this transition is the basal angiosperms, the functional role of which in past carbon and nutrient cycling has seldom been investigated. We hypothesized that owing to constraints on leaf and plant design related to hydraulic capacity, basal angiosperm trees should generally have resource conservative leaves of low decomposability and that fast‐decomposing leaves may only be found in short‐statured taxa. We performed a litterbag experiment with simultaneous outdoor incubation of leaf litters in a common environment, including 86 basal angiosperm species (including the magnoliid lineage), 33 eudicots, five gymnosperms and four ferns. We fit a nonlinear model to the decomposition data, and each species’ decomposability was estimated using the proportional rate of mass loss through the experiment. The mass loss rates were 59.2% lower in basal angiosperms than in eudicot trees. There was one exceptional group within basal angiosperms: the Piperales had higher k values than other magnoliid lineages, but all of the free‐standing species were short. Eudicots had higher k values overall and covered a range of plant statures from small‐statured herbs to big woody trees. Synthesis. Understanding the ecosystem‐level effects of the angiosperm rise to dominance is a crucial goal. Our results indicated that, among generally slow‐decomposing magnoliid lineages, only the Piperales have fast decomposition rate associated with small plant statures. Thus it is unlikely that early magnoliid trees were both forest canopy dominants and produced resource acquisitive leaves turning into fast decomposable litter during the evolutionary history of angiosperms.

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.

Investigation of genome structure of a cinnamyl alcohol dehydrogenase locus in a basal angiosperm hardwood species, Liriodendron tulipifera L., reveals low synteny

Abstract Basal angiosperms contain a wide diversity of floral and growth forms and gave rise to the largest recent angiosperm lineages. As none of the basal angiosperm genomes has been sequenced, examining large bacterial artificial chromosome (BAC) inserts remains the main approach to providing a first glimpse of the structure and organization of their genomes. In this study, we sequenced a 126.9‐kbp BAC contig harboring a cinnamyl alcohol dehydrogenase gene (LtuCAD1) in a basal angiosperm species, Liriodendron tulipifera L., an important timber tree species with significant ecological and economic values. A key enzyme in lignin biosynthesis, CAD catalyzes the final step in the synthesis of monolignols. We carried out phylogenetic analyses of seven full‐length CAD family genes (LtuCAD1–7) obtained from a comprehensive Liriodendron expressed sequence tag dataset. The phylogenetic tree suggests that LtuCAD1 is the primary CAD gene involved in lignifications as it is the only Liriodendron CAD grouped with the bona fide CADs class. As well as the LtuCAD1, the BAC contig contained fragmented sequences for one integrase, eight hypothetical proteins, two gag‐pol polyproteins, one RNase H family protein, and one chromatin binding protein. Comparative analysis with other angiosperm species suggests that the genomic segment in this BAC has undergone frequent arrangement. This study is our initial step in identifying and understanding lignin biosynthesis genes from basal angiosperm species. Such knowledge can help bridge the information gap between hardwood (angiosperm) and softwood (gymnosperm) species and benefit potential breeding and biotechnology application for enhanced production of biomass and digestibility in L. tulipifera.

Comparison of gene order ofGIGANTEAloci in yellow-poplar, monocots, and eudicots

GIGANTEA plays an important role in the control of circadian rhythms and photoperiodic flowering. The GIGANTEA gene has been studied in various species, but not in basal angiosperms. Moreover, to the best of our knowledge, no study of the genome organization of a basal angiosperm has yet been published. In this study, we sequenced a bacterial artificial chromosome (BAC) harboring GIGANTEA from yellow-poplar (Liriodendron tulipifera L.) and compared the genomic organization of this gene in yellow-poplar with that in other species from various angiosperm clades. This is the first report on the gene structure and organization of a large contig in any basal angiosperm species. The BAC clone, covering a region of approximately 122 kb from the yellow-poplar genome, was sequenced and assembled by coupling the 454 pyrosequencing technology with ABI capillary sequencing. In addition to GIGANTEA, the gene RPS18.A (encoding ribosomal protein S18.A) was found in this segment of the genome. We found that gene content and order in this region of the yellow-poplar genome were similar to those in the corresponding region in eudicots but not in Oryza sativa and Sorghum bicolor, implying that clustering of the GIGANTEA and RPS18.A genes is ancestral and separation of the genes occurred after the phylogenetic split of monocots from dicots. Phylogenetic analysis of GIGANTEA amino acid sequences placed yellow-poplar closer to eudicots than to monocots. In addition, evidence for transposition and large insertions and duplications was found, suggesting multiple and complex mechanisms of basal angiosperm genome evolution.

Comparative longevity and low-temperature storage of seeds of Hydatellaceae and temporary pool species of south-west Australia

The comparative longevity of seeds of species from the early-angiosperm group, Hydatellaceae, along with other temporary wetland aquatics from the South-west Australian Floristic Region were tested under standard experimental storage conditions. In contrast to recent hypotheses proposing that seeds from basal angiosperm species may be short-lived in storage, seeds of the Hydatellaceae species (Trithuria submersa Hook.f. and T. austinensis D.D.Sokoloff, Remizowa, T.Macfarlane and Rudall) were longer-lived than the other temporary wetland aquatic species tested. Seeds of Glossostigma drummondii Benth. (Scrophulariaceae), Myriophyllum petreaum Orchard and M. balladoniense Orchard (Haloragaceae), lost viability quickly and are thus predicted to be short-lived in seed bank storage. To assist seed bank conservation programs, the effect of seed moisture content on the viability of seeds stored for 1, 6 and 12 months at −18°C or in vapour phase cryopreservation (−150°C) was determined. Seeds of all species survived storage at both temperatures for up to 12 months, provided seed equilibrium relative humidity was below ~50%. Given the high conservation value of Hydatellaceae species and the potential short-lived nature of seeds of some of the species, we recommend that ex situ conservation programs for these aquatic species should consider cryopreservation as a means to maximise the longevity of their seeds.

Pollen development in Annona cherimola Mill. (Annonaceae). Implications for the evolution of aggregated pollen

BackgroundIn most flowering plants, pollen is dispersed as monads. However, aggregated pollen shedding in groups of four or more pollen grains has arisen independently several times during angiosperm evolution. The reasons behind this phenomenon are largely unknown. In this study, we followed pollen development in Annona cherimola, a basal angiosperm species that releases pollen in groups of four, to investigate how pollen ontogeny may explain the rise and establishment of this character. We followed pollen development using immunolocalization and cytochemical characterization of changes occurring from anther differentiation to pollen dehiscence.ResultsOur results show that, following tetrad formation, a delay in the dissolution of the pollen mother cell wall and tapetal chamber is a key event that holds the four microspores together in a confined tapetal chamber, allowing them to rotate and then bind through the aperture sites through small pectin bridges, followed by joint sporopollenin deposition.ConclusionPollen grouping could be the result of relatively minor ontogenetic changes beneficial for pollen transfer or/and protection from desiccation. Comparison of these events with those recorded in the recent pollen developmental mutants in Arabidopsis indicates that several failures during tetrad dissolution may convert to a common recurring phenotype that has evolved independently several times, whenever this grouping conferred advantages for pollen transfer.

An EST database for Liriodendron tulipifera L. floral buds: the first EST resource for functional and comparative genomics in Liriodendron

Liriodendron tulipifera L. was selected by the Floral Genome Project for identification of new genes related to floral diversity in basal angiosperms. A large, non-normalized cDNA library was constructed from premeiotic and meiotic floral buds and sequenced to generate a database of 9,531 high-quality expressed sequence tags. These sequences clustered into 6,520 unigenes, of which 5,251 were singletons, and 1,269 were in contigs. Homologs of genes regulating many aspects of flower development were identified, including those for organ identity and development, cell and tissue differentiation, and cell-cycle control. Almost 5% of the transcriptome consisted of homologs to known floral gene families. Homologs of most of the genes involved in cell-wall construction were also recovered. This provides a new opportunity for comparative studies in lignin biosynthesis, a trait of key importance in the evolution of land plants and in the utilization of fiber from economically important tree species, such as Liriodendron. Also of note is that 1,089 unigenes did not match any sequence in the public databases, including the complete genomes of Arabidopsis, rice, and Populus. Some of these novel genes might be unique in basal angiosperm species and, when better characterized, may be informative for understanding the origins of diverged gene families. Thus, the Liriodendron expressed sequence tag database and library will help bridge our understanding of the mechanisms of flower initiation and development that are shared among basal angiosperms, eudicots, and monocots, and provide new opportunities for comparative analysis of gene families across angiosperm species.

Conservation and divergence of candidate class B genes in Akebia trifoliata (Lardizabalaceae)

There is evidence that gene duplication and diversification within the MADS-box gene family had significant impact on floral architecture. In this study, we report the isolation of four class B homologous genes from Akebia trifoliata, termed AktAP3_1, AktAP3_2, AktAP3_3, and AktPI. Phylogenetic analysis indicates that the three AktAP3 paralogs were produced by two gene duplication events and AktAP3_2 and AktAP3_3 are recent paralogs, which are yielded by the duplication before the origin of the genus Akebia. In situ hybridization demonstrates that these genes are mainly expressed in the stamens and carpels of A. trifoliata, but in differential patterns, similar to those in other basal eudicot and basal angiosperm species. AktAP3_3 and AktPI are expressed in the developing petaloid perianth, suggesting that the petaloidy of the perianth is caused by the expression of class B genes. Reverse transcriptase polymerase chain reaction analyses indicate that these genes are expressed in both male and female flowers, but at different levels. We explore the interaction behavior of the class B proteins in the basal eudicots using yeast two-hybrid system for the first time. The AktAP3_1/2/3 proteins and the AktPI protein can form obligate heterodimers, but at different strength. From the mRNA expression and protein interaction patterns of the duplicated copies of the AktAP3 genes, we conclude that subfunctionalization very likely contributes to the maintenance of multiple AP3-like gene copies in A. trifoliata.