Isolation and Characterization of APETALA3 Orthologs and Promoters from the Distylous Fagopyrum esculentum

Common buckwheat (Fagopyrum esculentum) produces distylous flowers with undifferentiated petaloid tepals, which makes it obviously different from flowers of model species. In model species Arabidopsis, APETALA3 (AP3) is expressed in petal and stamen and specifies petal and stamen identities during flower development. Combining with our previous studies, we found that small-scale gene duplication (GD) event and alternative splicing (AS) of common buckwheat AP3 orthologs resulted in FaesAP3_1, FaesAP3_2 and FaesAP3_2a. FaesAP3_2 and FaesAP3_2a were mainly expressed in the stamen of thrum and pin flower. Promoters functional analysis suggested that intense GUS staining was observed in the whole stamen in pFaesAP3_2::GUS transgenic Arabidopsis, while intense GUS staining was observed only in the filament of stamen in pFaesAP3_1::GUS transgenic Arabidopsis. These suggested that FaesAP3_1 and FaesAP3_2 had overlapping functions in specifying stamen filament identity and work together to determine normal stamen development. Additionally, FaesAP3_2 and FaesAP3_2a owned the similar ability to rescue stamen development of Arabidopsis ap3-3 mutant, although AS resulted in a frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif of FaesAP3_2a. These suggested that the MIK region of AP3-like proteins was crucial for determining stamen identity, while the function of AP3-like proteins in specifying petal identity was gradually obtained after AP3 Orthologs acquiring a novel C-terminal euAP3 motif during the evolution of core eudicots. Our results also provide a clue to understanding the early evolution of the functional specificity of euAP3-type proteins involving in floral organ development in core eudicots, and also suggested that FaesAP3_2 holds the potential application for biotechnical engineering to develop a sterile male line of F. esculentum.


Introduction
Common buckwheat (Fagopyrum esculentum) is a gluten-free pseudo-cereal crop mainly cultivated in Europe, North America and Asian for human consumption and forage [1,2]. Buckwheat grains are rich in bioactive compounds, such as rutin, quercetin, polysaccharides and dietary fiber, etc., but with low-calories. Hence, common buckwheat is increasingly demanded and recognized for its highly effective as a functional food with health benefits and illness prevention throughout recent years [1]. However, common buckwheat is an obligate outcrossing crop with heteromorphic self-incompatibility due to its distylous flowers, with population being equally composed of plants with thrum flowers (short pistil combined with long stamen and large pollen grains) and plants with pin flowers (long pistil combined with short stamen and small pollen grains) (Figure 1), and legitimate cross-pollinations occur strictly between anthers and stigmas of equivalent height in different floral morphs, which make it hard for hybrid breeding and result in low yield [3,4]. Improving the yield stability and efficiency of breeding programs requires a better understanding the developmental genetics of distylous flowers in common buckwheat.
Exploring the underlying molecular mechanism of heteromorphic self-incompatibility of common buckwheat has continued for a quarter of a century and has made considerable progress in understanding of the molecular basis of heteromorphic SI in recent years [4][5][6]. However, the molecular mechanisms responsible for the form and development of the dimorph flower remain to be elucidated in common buckwheat. Furthermore, F. esculentum belongs to family Polygonaceae in the order Caryophyllales, an early diverging core eudicots clade, and produces distylous flowers with undifferentiated petaloid tepals, representing a considerable difference with most core eudicots and major crops flowers, which make it an excellent model for exploring floral organ specification and evolution [7][8][9].
In Arabidopsis thaliana, the B-class MADS-box genes APETALA3 (AP3) and PISTILLATA (PI) work together to control the formation of petals and stamens during Arabidopsis flower development [10]. The AP3-like and PI-like genes originated from a duplication event of ancient class B genes that preceded the origin of extant angiosperms [11]. In addition, another major gene duplication event in the AP3/DEFICIENS (DEF) lineage occurred close to, or at the base of, the core eudicot origination, and resulted in the euAP3-like and TM6-like genes [12]. Moreover, most euAP3-like genes express in petals and stamens and specify petal and stamen development, while most TM6-like genes show a broader expression zone in flowers but mainly regulate stamen development [13,14]. Moreover, more and more studies suggested that small-scale gene duplication could also lead to subor neo-functionalization of AP3-like genes in different taxa [14][15][16].
Here, we found that a small-scale gene duplication (GD) event of buckwheat AP3 ortholog resulted in FaesAP3_1 and FaesAP3_2. Furthermore, alternative splicing (AS) generates two transcript isoforms from the FaesAP3_2 after the gene duplication. Both AS isoforms, FaesAP3_2 and FaesAP3_2a, differ by 32 nucleotides in length and result from alternative 5 splice-site selection. Our previous study suggested that FaesAP3_1 is involved only in stamen development in F. esculentum [9]. In the present study, the functional diversification of buckwheat AP3-like gene was analyzed by characterizing their promoters. In addition, the function of FaesAP3_2 and FaesAP3_2a specifying floral organ identity were analyzed by assessing their ability to rescue the phenotype of Arabidopsis ap3-3 mutant. The possible impacts of GD and AS on the function of three buckwheat AP3-like genes were investigated, and the functional diversification among them was proposed. Our findings also provide clues for tracing the structure and functional evolution of euAP3-type genes in the early diverging core eudicots.

Isolation and Characterization of FaesAP3_2 and FaesAP3_2a from F. esculentum
The 891 bp FaesAP3_2 cDNA contains a 732 bp ORF (Open Reading Frame, ORF) encoding 243 amino acids (aa) (Genbank accession number: MN016949.1), while the ORF of the FaesAP3_2a is only 615 bp and encodes 204 aa (Genbank accession number: MN016950.1). Moreover, genomic DNA sequence of FaesAP3_2 (Genbank accession number: MN016951.1) is 2214 bp consisting of seven exons and six introns. Sequences alignment revealed that FaesAP3_2 and FaesAP3_2a were derived from the consensus pre-mRNA, and both variants differed at the exon 6-intron 6 splice junction sites which resulted in a 32 bp nucleotides addition in the sixth exon of FaesAP3_2a from alternative 5 splice-site selection than the sixth exon of the FaesAP3_2 (Figure 2) [17,18]. Phylogenetic tree analysis grouped FaesAP3_2 into euAP3 lineage (Figure 3), and the protein sequence also showed 67.89% identity with the FaesAP3_1 (Genbank accession number: AFO83616.1), another common buckwheat AP3-like MADS-box transcription factor. The gene was designated as FaesAP3_2 (Fagopyrum esculentum APETALA3_2). Proteins alignment shows that FaesAP3_2 protein comprises a 27 amino acids (aa) seldom seen N-terminal extension region, a 57 aa highly conserved MADS-box domain (28-84), a 82 aa moderately conserved K domain  in the middle region and a 48 aa variable C-terminal region (196-243), but with two conserved motifs: PI-derived motif and an euAP3 motif ( Figure 3) [12,19]. Moreover, FaesAP3_2 includes three putative amphipathic α-helices designated as K1 (114-135), K2 (148-162) and K3 (170-205) subdomains containing conserved hydrophobic amino acids residues at the a and d positions in the (abcdefg)n heptad repeats [19]. The 32 bp nucleotides addition in the sixth exon of FaesAP3_2a results in frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif (Figure 4), two key conserved motifs located at the C-terminal region of the euAP3-like transcription factors that are involved in mediating protein-protein interactions [12].

Expression Analysis of FaesAP3_2 and FaesAP3_2a
FaesAP3_2 was mainly expressed in stamen of thrum and pin flower, but FaesAP3_2a transcript was detected only in stamen ( Figure 5A,B). Moreover, weak transcripts of FaesAP3_2 was also observed in tepal, gynoecia and 4-day-old fruit of pin and thrum flowers ( Figure 5A). FaesAP3_2 and FaesAP3_2a expressions became apparent after stamen primodium emerge in thrum and pin flower buds ( Figure 6A-C). In addition, FaesAP3_2 expression increased constantly and reached the peak until mononuclear microspore at Periphery formation and tepal enclosing in thrum flower buds (T4), but microspore mother cells meiosis and microspores tetrads formation occurred in the pin flower bud (P3), then FaesAP3_2 expression began to decline (T5, P4) ( Figure 6A,B). In addition, FaesAP3_2a expression was similar with the FaesAP3_2 during the thrum and pin floral bud development ( Figure 6A  The conserved PI-derived motif, euAP3 motif and paleoAP3 motif located at the various C-terminal regions are boxed. The dots represent identical amino acid residues with FaesAP3_2. Dashes introduced into the sequence to improve the alignment. Three red underlines represent k1, k2 and k3 subdomains with (abcdefg)n repeats and usually with hydrophobic amino acids at positions a and d [19].  buds; T1: stamen primodium emergence; T2: stamen filament elongation; T3: microspore mother cells meiosis; T4: mononuclear microspore at Periphery, tepal enclosing; T5: full maturity flower buds with mature pollen and embryo sac before anthesis; P1-P5: morphological differentiation and development of the pin flower buds; P1: stamen primodium appearance; P2: stamen filament rapid elongating; P3: microspore mother cells meiosis and formation of microspores tetrads; P4: microspore released from tetrads and tepal enclosing; P5: full maturity flower bud with mature pollen and embryo sac before anthesis; (B) FaesAP3_2 expression at sequential development stages of thrum and pin flower buds were detected by qRT-PCR, respectively; (C) FaesAP3_2a expression at sequential development stages of thrum and pin flower buds were detected by qRT-PCR, respectively; scale bar: 100 µm.  Figures S1-S2). Promoter pFaesAP3_1 contains a key CArG-box motif (−2160/−2151) for MADS-box transcription factor recognition and binding [20], which is also found in the promoter region of pFaesAP3_2 (−338/−329). In addition, each promoter contains two POLLEN2LELAT52 boxes and four GTGANTG10 boxes, which are essential for stamen-specific gene expression [21,22]. Furthermore, both promoters also contain binding sites (CCAATBOX1) for CONSTANS protein to regulate flowering [23]. Moreover, the MYCCONSENSUSAT box and ACGTATERD1 box are found in pFaesAP3_1 and pFaesAP3_2, which suggests that the corresponding gene expression could be induced by freezing/dehydration stress [24,25]. Some gibberellin-responsive elements, such as WRKY71OS-box, and PYRIMIDINEBOXOSRAMY1A/PYRIMIDINEBOXHVEPB box [26,27], are also found in pFaesAP3_1/2, which suggested that the both genes could be regulated by gibberellin-responsive gene. In addition, several mesophyll-specific elements CACTFTPPCA1 boxes are also lying at pFaesAP3_1 and pFaesAP3_2 region, which suggested that the corresponding gene expression may extend to leaves [28].

Isolation and Identification of FaesAP3_1 and FaesAP3_2 Promoters from F. esculentum
A GUS reporter gene driven by pFaesAP3_1 or pFaesAP3_2 was activated in the cauline leaf, inflorescence rachis and flower of transgenic Arabidopsis ( Figure 7B,C). Furthermore, GUS staining was observed in the flower where sepal, filament and stigma staining were intense, but was almost absent in petal, anther and stigmatic papillae of pFaesAP3_1::GUS transgenic Arabidopsis ( Figure 7E). However, GUS staining was observed in the flower where sepal, stamen (filament and anther), stigma and stigmatic papillae staining were intense, but was almost absent in the petal of pFaesAP3_2::GUS transgenic Arabidopsis ( Figure 7F).

Discussion
Previous studies suggested that two paralogous lineages (euAP3 and TM6) of AP3-like genes in the core eudicots resulted from a duplication event of the ancestral paleoAP3 lineage within the basal eudicots [12]. Following the duplication, the euAP3 lineage acquired a novel C-terminal euAP3 motif instead of paleoAP3 motif and a new role in regulating petal development, while TM6 has preserved the C-terminal paleoAP3 motif [12,29]. In core eudicots, most euAP3-type genes, such as EjAP3 from Eriobotrya japonica [30], MtNMH7 from Medicago truncatula [31], PFDEF from Physalis floridana [32] and GDEF2 from Gerbera hybrid [33], were expressed only in petal and stamen, and were mainly involved in specifying petal and stamen identities during flower development. All these studies demonstrated that the functions of euAP3-type genes are highly correlated with their expression pattern in core eudicots. However, most TM6-like genes usually showed broader expression zones, but were involved only in stamen development in core eudicots. For examples, Medicago truncatula TM6-like gene MtTM6 was expressed predominantly in the outer cell layers of petal and stamen, but played a key role involving in stamen development [31]. Physalis floridana TM6-like gene PFTM6 was expressed in corolla, androecium and gynoecium, but was involved in pollen maturation [32]. Gerbera hybrid TM6-like GDEF1 was expressed in all four floral whorls of disk flower, but had a redundant role in determining stamen development [14]. Besides the broader expression patterns, small-scale gene duplication event of the paleoAP3-like genes were observed throughout basal eudicots with petaloid sepals and basal angiosperms with undifferentiated perianth (petaloid tepals). For examples, NdAP3-1, NdAP3-2 and NdAP3-3 were three paleoAP3type genes found in basal eudicots Nigella damascene (Ranunculaceae). NdAP3-3 was mainly expressed in petal and specified petal identity, while NdAP3-1 and NdAP3-2 have much broader expression domains (sepal, petal, stamen and carpel) and have overlapping functions in specifying stamen identity [33]. Three paleoAP3-type genes, AqAP3-1, AqAP3-2 and AqAP3-3, were also found in Aquilegia coerulea (Ranunculaceae). AqAP3-1 and AqAP3-2 were obviously expressed in sepals, petals, staminodia, stamens and carpels, and work together to specify stamen identity, while AqAP3-3 was mainly expressed in petals and was required only for petal identity [16]. Two paleoAP3-type genes, MAwuAP3_1 and MAwuAP3_2, were also found in basal angiosperms Magnolia wufengensis (magnoliids); both genes were mainly expressed in petaloid tepal and stamen, but were required only for stamen development [34]. All these data suggested that stamen-specific function of AP3-like genes antedate their petal-specific identity during angiosperm evolution.
Gene duplication events and AS are often associated with shifts in expression patterns and/or changes in coding sequence, giving rise to the diversification of gene function [12,16,35]. In F. esculentum, FaesAP3_1 was expressed only in stamen and exclusively required for stamen formation [9], while FaesAP3_2 expression extended to petaloid tepal and gynoecia although the expression was so slow. In addition, GUS staining was observed in the whole stamen of pFaesAP3_2::GUS transgenic Arabidopsis, while GUS staining was observed only in the filament of pFaesAP3_1::GUS transgenic Arabidopsis, but was absent in the anther. All these data may suggest that FaesAP3_1 and FaesAP3_2 had overlapping functions in specifying stamen filament identity, and FaesAP3_2 played a key role in regulating anther development. Both genes work together to control normal stamen development. Intense GUS staining was also observed in the stigma and stigmatic papillae of pFaesAP3_2::GUS transgenic Arabidopsis. Moreover, phenotype complementation analysis suggested that some 35S::FaesAP3_2 transgenic Arabidopsis ap3-3 mutants could produce flowers with a filament attached with stigmatic papillae in whorl 3. These data may suggest that FaesAP3_2 may be involved in style development. However, the AS isoform FaesAP3_2a was expressed only in the stamen of thrum and pin flowers. Phenotype complementation analysis suggested that FaesAP3_2a holds a similar ability with FaesAP3_2 to rescue stamen development of Arabidopsis ap3-3 mutant even without the C-terminal euAP3 motif, which suggested that both AS isoforms had overlapping functions in specifying stamen identity of common buckwheat though they showed different expression patterns. As most transcriptional factors are modular proteins with multiple functional modules, some truncated protein isoforms containing functional modules may still have function and act as dominant-negative regulators [36]. The AP3-like and PI-like transcriptional factors are closely related MADS domain proteins that are thought to act as obligate heterodimers, and their I and K domains were required for dimerization and protein stability [10,19]. Hence, truncated FaesAP3_2a isoform might still participate in dimerization, which can compete with FaesAP3_2. A future challenge remains in exploring how the both AS isoforms work together with other common buckwheat B genes to specify stamen identity. Moreover, previous studies also suggested that the petaloid tepal of common buckwheat were homologs to core eudicots sepal and showed a relative original trait of flowers [7,8]. All these data suggested that the MIK region of AP3-like proteins was crucial and essential for determining stamen identity, while the function of AP3-like proteins in specifying petal identity was gradually obtained accompanying sepal and petal differentiation after the AP3 orthologs acquired a novel C-terminal euAP3 motif during the evolution of core eudicots. Our data suggested early evolution of the functional specificity of euAP3-type proteins in floral organ development in core eudicots, and also provide an idea candidate gene for biotechnical engineering to develop a sterile male line of F. esculentum.

Plant Material
Thrum and pin floral buds at sequential developmental stages were sampled from common buckwheat 'Beizaosheng' growing under natural conditions in Jingzhou, China, respectively. In addition, each sample was divided into two aliquots; one was immediately frozen in liquid nitrogen, and then stored at −80 • C until used; another was fixed in FAA (38% formaldehyde: acetic acid: 70% ethanol = 1:1:18, by volume). The root, stem, juvenile leaf, tepal, stamen, gynoecium and 4-day-old fruit of thrum and pin plants were dissected, respectively, immediately frozen in liquid nitrogen and then stored at −80 • C until used. The Arabidopsis ap3-3 mutant line (CS3086, Landsberg ecotype) seeds were obtained from the ABRC (Arabidopsis Biological Resource Center, ABRC) at Ohio State University, Columbus, OH, USA.

Isolation and Characterization of FaesAP3_2 and Its AS Isoform FaesAP3_2a from F. esculentum
Total RNA from common buckwheat floral buds and the first-strand cDNA of 3 RACE was prepared according to Fang et al. [9]. The 3 end and 5 partial cDNA sequences of two buckwheat FaesAP3_2 isoforms (FaesAP3_2 and FaesAP3_2a) were obtained by using the 3 -full RACE Core Set Ver. 2.0 kit (TaKaRa, Shiga, Japan) with gene-specific primer GSPAP3 based on the manufacturer's protocol (Supplementary Table S1). The gene-specific primer GSPAP3 was designed based on F01.PB7802 (putative AP3-like MADS-box transcription factor gene) of the BioProject ID PRJNA517031 deposited in the NCBI. Common buckwheat genomic DNA was extracted from leaves by using the CTAB Plant Genomic DNA Rapid Extraction Kit (Aidlab, Beijing, China) following the manufacturer's protocol. The full length of genomic DNA sequence of FaesAP3_2 was isolated from common buckwheat genomic DNA with the forward primer DFaesAP3_2F and the reverse primer DFaesAP3_2R. The PCR amplification of FaesAP3_2 genomic DNA was performed in a 25 µL reaction volume containing 0.5 µL Phanta Max Super Fidelity DNA Polymerase (Vazyme, Nanjing, China). PCR was performed with a 3 min 94 • C denaturation step, followed by 30 cycles of 30 s at 94 • C, 30 s annealing at 58 • C, a 90 s extension at 72 • C, with a final extension period of 10 min. Sequence alignments and phylogenetic analysis of FaesAP3_2 were referenced to the method described by Liu et al. [37]. Putative FaesAP3_2 and FaesAP3_2a protein sequences, as well as B-class MADS-box transcription factors from different species, were selected for Phylogenetic trees from NCBI Genbank (Supplementary Table S2).

Cytomorphological Examination and Expression Analysis of FaesAP3_2 and FaesAP3_2a
The thrum and pin floral buds of F. esculentum fixed in FAA above were dehydrated in a graded ethanol series, cleared in a xylene series, infiltrated with molten paraffin, embedded into paraffin block, serially sectioned and then sections were stained according to Liu et al. [37]. The sections were observed under a CAIKON RCK-40C microscope and subsequently taken photomicrographs.
Total RNA of each sample was prepared for quantitative real-time PCR (qRT-PCR) by using the EASYspin plant RNA Rapid Extraction Kit (Aidlab, Beijing, China) following the manufacturer's protocol. The first-strand cDNA was synthesized by using the HiScript ® II Q RT SuperMix for qPCR kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. FaesAP3_2 and FaesAP3_2a expressions were detected in root, stem, juvenile leaf, tepal, stamen, gynoecium and 4-day-old fruit of thrum and pin plants by using qRT-PCR according to Liu et al. [8], but with the gene-specific forward primer qFaesAP3_2F and the gene-specific reverse primer qFaesAP3_2R for FaesAP3_2, and the gene-specific forward primer qFaesAP3_2aF and the gene-specific reverse primer qFaesAP3_2aR for FaesAP3_2a, respectively. Moreover, the expressions of FaesAP3_2 and FaesAP3_2a were also detected in different development stage floral buds of thrum and pin buckwheat through qRT-PCR suggested above, respectively. For qRT-PCR analysis, the reaction was performed on the Line-Gene 9600 Plus Real-time PCR Detection System by using 2 × ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). Amplification fragment of F. esculentum actin gene (Genbank accession number: HQ398855.1) was used as the internal control with the forward primers qFaesactinF and the reverse qFaesactinR. qRT-PCR was carried out with three biological replicates, the PCR program was cycled and relative expression levels were calculated according to Liu et al. [8] but with 30 s annealing at 57 • C.

Isolation and Identification of FaesAP3_1 and FaesAP3_2 Promoter from F. esculentum
The FaesAP3_1 5 flanking region was isolated according to Liu et al. [8], but with the gene-specific primer D1pAP3_1SP1, D1pAP3_1SP2 and D1pAP3_1SP3 for the first walking sequencing, and with the gene-specific primer D2pAP3_1SP1, D2pAP3_1SP2 and D2pAP3_1SP3 for the second walking sequencing. In addition, The FaesAP3_2 5 flanking region was isolated according to the method suggested above but with the gene-specific primer FLpAP3_2SP1, FLpAP3_2SP2 and FLpAP3_2SP2 for the walking sequences. Moreover, the putative transcription start site of FaesAP3_1 was found based on the 5 RACE according to Fang et al. [9]. The putative transcription start site of FaesAP3_2 was found based on the 5 RACE using the 5 RACE System for Rapid Amplification of cDNA Ends (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol and the genespecific primer 5RAP3GSP1, 5RAP3GSP2 and 5RAP3GSP3. The cis-acting elements lying at the pFaesAP3_1 and pFaesAP3_2 regions were searched in the PLACE database, respectively [38]. The 1.5 kb 5 flanking region upstream of FaesAP3_2 translation start was cloned into pCAMBIA1300 vector with the forward primer TpFaesAP3_2F and the reverse primer TpFaesAP3_2R, and restriction enzymes Xba I and Sac I. pFaesAP3_2::GUS construct was transformed into A. thaliana Col-0 plants (ecotype Columbia) using the floral-dip method described by Clough and Bent [39]. Transgenic Arabidopsis seedlings were selected, cultivated and prepared for histochemical GUS staining according to Liu et al. [8].

Ectopic Expression Analysis of FaesAP3_2 and FaesAP3_2a in Arabidopsis ap3-3 Mutant
Full-length FaesAP3_2 and FaesAP3_2a cDNAs in the sense orientation were separately cloned into pBI121 vector with Xba I and Sac I restriction enzymes, and the forward primer TFaesAP3_2/2aF and the reverse primer TFaesAP3_2/2aR under control of the CaMV35S promoter using the ClonExpress ® Ultra One Step Cloning Kit (Vazyme, Nanjing, China) according to the manufacturer's protocol. The 35S::FaesAP3_2 and 35S::FaesAP3_2a constructs were transformed into heterozygote Ap3/ap3-3 Arabidopsis using the floral-dip method described by Clough and Bent, respectively [39]. Transgenic Arabidopsis seeds were selected, and seedlings were cultivated according to Fang et al. [9]. Homozygous ap3-3 transgenic Arabidopsis lines were identified by dCAPS genotyping following the method suggested by Lamb and Irish [29]. The phenotypes of transgenic Arabidopsis were analyzed after flowering. In addition, the complementation degrees of independent transgenic lines of 35S::FaesAP3_2 and 35S::FaesAP3_2a homozygous ap3-3 Arabidopsis were categorized as 'no complementation', 'weak complementation', 'medium complementation' and 'strong complementation', respectively. Moreover, independent transgenic lines of each complementation degree were confirmed by qRT-PCR with the primers qTFaesAP3_2F and qTFaesAP3_2R for FaesAP3_2, and with the primers qFaesAP3_2a and qFaesAP3_2aR suggested above for FaesAP3_2a, respectively. Amplification fragment of A. thaliana ubiqui-tin 5 (Genbank accession number: NM_116090.3) with the primers qUBQ5F and qUBQ5R as the internal control.

Statistical Treatment
All experiments were carried out with three biological replicates, and data were expressed as mean ± SE (standard errors). Statistical significance was determined by oneway ANOVA followed by LSD, and statistical significance was declared at p-value ≤ 0.01 or 0.05 level, respectively.

Conclusions
Combined with our previous studies, we found that small-scale gene duplication (GD) event and alternative splicing (AS) of buckwheat AP3 orthologs resulted in FaesAP3_1, Fae-sAP3_2 and FaesAP3_2a. FaesAP3_2 and FaesAP3_2a were mainly expressed in the stamen of thrum and pin flowers. Promoters functional analysis suggested that intense GUS staining was observed in the whole stamen of pFaesAP3_2::GUS transgenic Arabidopsis, while intense GUS staining was observed only in the filament of pFaesAP3_1::GUS transgenic Arabidopsis. These suggested that FaesAP3_1 and FaesAP3_2 had overlapping functions in specifying stamen filament identity and FaesAP3_2 played a key role in regulating anther development. Both genes work together to determine normal stamen development. Additionally, FaesAP3_2 and FaesAP3_2a owned the similar ability to rescue stamen development in Arabidopsis ap3-3 mutant, although AS resulted in frameshift mutation and consequent omission of the complete PI-derived motif and euAP3 motif of FaesAP3_2a. In addition, previous studies also suggested that the petaloid tepal of common buckwheat were homologs to core eudicots sepal. All these suggested that the MIK region of AP3-like proteins was crucial for determining stamen identity, while the function of AP3-like proteins in specifying petal identity was gradually obtained after the AP3 orthologs acquired a novel C-terminal euAP3 motif during the evolution of core eudicots. Our results also provide a clue to understanding the early evolution of the functional specificity of euAP3-type proteins in floral organ development in core eudicots, and also suggest that FaesAP3_2 holds the potential application for biotechnical engineering to develop a sterile male line of F. esculentum.
Author Contributions: L.Z., writing-original draft preparation; L.Z., J.Z. and X.W., methodology; Z.L., writing-review and edition, supervision. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All data generated or analyzed during this study are included in this published article. Further inquiries can be addressed to the corresponding author.