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Expression variation of Viola APETALA3 orthologous genes is correlated with chasmogamous and cleistogamous flower development

BMC Plant Biology volume 25, Article number: 319 (2025) Cite this article

Abstract

Background

Viola philippica and V. prionantha develop chasmogamous (CH) flowers under ≤ 12-h daylight conditions and cleistogamous (CL) flowers under long daylight (> 12-h daylight) conditions (LD), whereas V. cornuta develops CH flowers regardless of the daylight conditions. APETALA3 (AP3) is a major floral B-function gene that regulates the organ identity and development of stamens and petals. Evolutionary changes in AP3 orthologous genes might involve in the dimorphic flower formation. In the present study, we compared AP3 orthologous genes among three Viola species.

Results

The AP3 sequences were highly conserved, and obligate AP3-PISTILLATA heterodimers were universally formed. However, the floral expression of VphAP3 in V. philippica and VprAP3 in V. prionantha changed in response to the photoperiod. Their expression was significantly higher under 12-h daylight conditions than under 16-h daylight conditions. In contrast, VcoAP3 expression in the floral buds of V. cornuta was comparable among photoperiods. In accordance with these variations in expression, correlated sequence divergences were observed in the putative regulatory regions of Viola AP3 orthologous genes.

Conclusions

Developmental inhibition of petals and stamens may result from AP3 downregulation by LD, which thereby induces CL flowers. Our study provides insight into the molecular basis underlying the developmental evolution of environmentally dependent mating systems in dimorphic CL plants.

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Introduction

Dimorphic cleistogamy is a specialized form of a mixed mating system in which a single plant produces potentially outcrossed chasmogamous (CH) and obligately self-pollinated cleistogamous (CL) flowers. CH flowers have bright, colorful petals and nectaries and remain open for cross-pollination, while CL flowers remain closed for self-fertilization [1]. Nectar and odor are absent in CL flowers, petals are rudimentary or completely missing, and stamens are often reduced in both number and size [2, 3]. The mixed mating system has important significance in the adaptive evolution of plants. CH flowers are advantageous in that they promote outcrossing and gene flow within and among populations and produce genetically diverse progenies, thus maintaining genetic diversity and adapting to the changing environment. CL flowers may be energetically less costly to produce, resulting in more available resources for seed production and extending the reproductive window of CH/CL species in adverse or extreme environmental conditions [4, 5]. A CL flower is a structurally modified form of a CH flower for autogamy and is an ecological innovation characteristic of plant adaptation to the environment [6, 7].

The floral ABC model explains how three major function class genes (A, B, and C class) determine the identity of the four floral organ types. B-class MADS-box genes in Arabidopsis include APETALA3 (AP3) and PISTILLATA (PI) [8, 9]. B-class MADS-box genes are involved not only in the specification of the organ identity of petals and stamens but also in the control of organ maturation [2, 10,11,12]. Mutations in either the AP3 or PI genes result in similar phenotypic variations, wherein petals are transformed into sepals and stamens into carpels [10, 13, 14]. In Arabidopsis flowers, AP3/PI knockdown at intermediate stages (stages 8–10) induces petal-to-sepal transformations that gradually occur in consecutive buds, but stamens in these flowers retain their identity, become increasingly underdeveloped, and do not dehisce pollen [15]. Changes in the expression level or expression pattern of B-class MADS-box genes can create new traits and defects in floral organs [16,17,18,19]. The differential expression of the AP3 gene is regulated by some upstream genes or environmental factors, such as the photoperiod [2, 3]. The upstream genes include GIBBERELLIN INSENSITIVE [20], LEAFY, UNUSUAL FLORAL ORGANS [21], AINTEGUMENTA, and AINTEGUMENTALIKE6 [22]. Moreover, AP3 regulates other genes that regulate petal and stamen development [15, 23,24,25]. AP3 and PI positively regulate SPOROCYTELESS/NOZZLE expression, which is required during the late stages of stamen development in Arabidopsis for microsporogenesis and consequent pollen formation [15, 26]. FaesAP3_1 regulates the FaesELF3 gene, which determines the filament length of long-homostyle Fagopyrum esculentum [25]. In addition, B-class genes AP3 and PI and C-class gene AGAMOUS (AG) act redundantly with each other and in combination with SEPALLATA genes to activate CRABS CLAW in nectaries and carpels [23]. Transcription factor complex CmAP3-CmPI-CmUIF1 modulates carotenoid metabolism by directly regulating the carotenoid cleavage dioxygenase gene CmCCD4a-2 in Chrysanthemum flowers [24]. The organ alterations of these molecular interactions resemble the floral organ variations in sizes and numbers of petals and stamens during the CH–CL transition.

The Viola genus (true violets) is well known for its large number of species displaying a CH/CL mixed breeding system [2, 3, 27, 28]. Viola philippica and V. prionantha possess significant and unique efficacy in clinical antiviral therapy. Viola philippica and V. prionantha extract from whole plants possess a wide range of pharmacological and biological activities, including antiviral, antifungal, anticoagulant, and anticancer functions [29,30,31]. Photoperiods play an important role in the development of CH and CL flowers in Viola, such as Viola philippica, V. prionantha, and V. odorata [2, 3, 27]. In Viola, CH flowers are fully developed and typically feature large, showy petals, while CL flowers have reduced stamens and undeveloped petals [2, 3, 27, 28]. CH and CL flowers are developed on the same individual plants at the different times of the season in Viola, and intermediate cleistogamous (inCL) flowers are occasionally developed under 12-h daylight conditions, which display variable characteristics, with poorly or less developed petals and stamens [2, 3]. In V. philippica and V. prionantha, CH flowers are induced under short-day or intermediate daylight conditions, and photoperiod extension is intended to induce CL flower formation [2, 3]. But, the V. cornuta CH flower type is not changed under varying photoperiod conditions (> 10 h of daylight), nonetheless, it hardly blooms under short (≤ 10-h) daylight conditions. The variation in the expression of the B-class MADS-box genes TOMATO MADS BOX GENE 6 (TM6) and PI during CH–CL development has been observed in V. philippica [2]. AP3 is the paralogous gene of TM6 [32]. These homologous genes are partially redundant and play a role in the diversification of floral morphology during evolution [33]. However, whether photoperiod regulates the expression of AP3 in Viola, and its implication in the CH–CL transition remain unknown. The aims of this study were to isolate the open reading frame (ORF), gDNA, and promoter of AP3 orthologous genes in three Viola species to investigate their sequence conservation and divergence, protein–protein interactions (PPIs), and mRNA expression levels and to show their potential roles in the CH–CL transition. This work provides additional evidence for the involvement of floral B-class genes in the developmental evolution of dimorphic flower in Viola.

Materials and methods

Plant materials and growth conditions

Seeds of V. philippica, V. prionantha, and V. cornuta were collected on the Northwest Normal University (Lanzhou, Gansu, China) campus. The plants were grown at 22–26 °C under 12 or 16 h of daylight in growth chambers. The procedures and conditions for plant cultivation followed the previous description by Li et al. [2].

Flower morphology observation

The morphology of V. philippica, V. prionantha, and V. cornuta flower buds was observed under a stereomicroscope (Olympus SZ61, Tokyo, Japan). Mature flowers of each type (CH and CL) were analyzed. Images were captured using a camera (Olympus, DP20-DRV, Tokyo, Japan) linked to a stereomicroscope (Olympus SZ61, Tokyo, Japan).

Sequence isolation

The cDNA fragments of the targeted genes were isolated using specific primers (Table S1) designed based on the full-length transcriptome data from V. prionantha [3]. The full-length cDNA was assembled according to the previous description by Li et al. [2].

Phylogenetic analysis

The amino acid sequences of these genes were aligned using CLUSTALW2.0 [34] with default settings and manual adjustments. Gaps were introduced for proper alignment. A maximum likelihood phylogeny tree based on amino acid sequences was constructed [35]. Bootstrap values were based on 1000 replicates.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

According to naked-eye observations of the floral buds, CH and CL flower buds from stages 1, 2, 3, 4, and 5 (also defined as F1, F2, F3, F4, and F5) [3] and floral organs from stage 5 (F5) were collected under 12- and 16-h light periods. Total RNA was extracted using TRIzol reagent (TIANGEN, Beijing, China). qRT-PCR was performed using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China), SYBR Premix EX Taq II (TaKaRa, Dalian, China), and gene-specific primers (Table S1). An 18S ribosomal RNA gene was used as an internal reference (AB354544.1). The amplification conditions were same as the previous study [2]. Expression levels were calculated according to a previous description [36].

RNA in situ hybridization

Floral buds at different developmental stages were fixed in 4% (wt/vol = 4 g/100 mL) paraformaldehyde and embedded in Paraplast (Sigma P3683, St. Louis, MO, USA). When ATG was set as 1, a 300-bp fragment of VphAP3 (positions 397–696), VprAP3 (positions 397–696), and VcoAP3 (positions 394–693) were used as templates for both sense and antisense probe synthesis using the DIG RNA labeling kit (Roche, Mannheim, Germany) and T7 RNA polymerase (Roche, Mannheim, Germany). Hybridization was performed as previously described by Javelle et al. [37]. Sections of floral tissues of CH and CL flowers from the three Viola species were incubated in the same hybridization solution for each probe. Images were captured with a microscope (Nexcope, Ningbo, Zhejiang, China).

Yeast two-hybrid (Y2H) analysis

The ORFs of the involved Viola B-class MADS-box genes were cloned into the pGADT7 or PGBKT7 vector (Clontech, Mountain View, CA, USA). The co-transformed yeast cells were selected by growth on synthetic drop-out medium (SD) plates lacking leucine (Leu) and tryptophan (Trp). Interactions were analyzed on SD plates lacking Leu, Trp, adenine (Ade), and histidine (His) and further confirmed by the non-lethal ß-galactosidase activity assay in yeast strain AH109. Before checking the PPIs, the toxicity and self-activation capability of these Viola B-class MADS-domain proteins were checked. Briefly, these experiments followed the previous description by Gong and He [38].

Bimolecular fluorescence complementation (BiFC) assay

A BiFC assay was conducted using Nicotiana benthamiana leaf cells. The ORFs of the involved Viola B-class MADS-box genes lacking the stop codon were cloned into the pSPYNE-35S and pSPYCE-35S vector pair using the Xba I/Xho I and Xba I/Kpn I (TaKaRa, Dalian, China) restriction sites. These paired vectors were designed to express either the N- or C-terminal halves of the yellow fluorescence protein (YFP). The construct combination of two proteins fused with the N- or C-terminal halves of YFP was agroinfiltrated into N. benthamiana leaf epidermal cells. After 48 h, the YFP fluorescence signals were observed using a laser confocal microscope (Leica, Wetzlar, Germany).

Promoter isolation and prediction

The VphAP3 and VcoAP3 genomic sequences were isolated via PCR using the KOD-Plus enzyme (Takara, Dalian, China) and gene-specific primers (Table S1). The putative promoter sequences of each gene were acquired using the Genome Walking Kit (Takara, Dalian, China) based on the obtained genomic sequences. Cis-element prediction was performed using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Sequencing and statistical analyses

Construct sequencing and primer synthesis were performed by Qingke (Xian, China). The mean ± standard deviation was calculated from at least three independent replicates. Significance differences were analyzed with Duncan's test using SPASS27.0 (SPSS Inc., Chicago, IL, USA).

Results

Floral morphology of V. philippica, V. prionantha, and V. cornuta

Viola philippica and V. prionantha developed both CH and CL flowers under different photoperiods. CH flowers were induced under short (10 h) and intermediate daylight (12 h) conditions, whereas CL flowers were triggered under long daylight (16 h) conditions (Fig. 1A–J) [2, 3]. The inCL flowers were occasionally formed in V. philippica and V. prionantha under 12-h daylight conditions and they had 1–3 poorly developed petals and 2–5 developed stamens (Fig. 1E and J). However, Viola cornuta hardly bloomed under ≤ 10 h of daylight and only developed CH flowers under ≥ 10 h (12 and 16 h) of daylight, and no CL and inCL flowers were developed under any photoperiod conditions (Fig. 1K–L). The CH flowers of the three Viola species had five large and showy petals, with the lowest protruding at the base into a spur, and five stamens. Each stamen had four pollen sacs, and the lowest two stamens had noticeable nectar glands attached to them (Fig. 1A, B, E, F, G, K, and L). The CL flowers of V. philippica and V. prionantha had two stamens without nectar glands, each stamen had two pollen sacs, and the five petals were all undeveloped (Fig. 1C, D, H, and I). The filament of CL stamens was distinct compared with that of CH flowers, which were undetectable (Fig. 1B, D, G, and I). Thus, V. cornuta is a suitable control for revealing the genetic variations and mechanisms of dimorphic flower formation.

Fig. 1
figure 1

Floral variations under different photoperiods in three Viola species. A–E Viola philippica. A Chasmogamous (CH) flower under 12-h daylight conditions. B Stamen of a CH flower. C Cleistogamous (CL) flower under 16-h daylight conditions. D Stamens of a CL flower. E Intermediate CL (inCL) flower under 12-h daylight conditions. F–J Viola prionantha. F CH flower under 12-h daylight conditions. G Stamen of a CH flower. H CL flower under 12-h daylight conditions. I Stamens of a CL flower. J inCL flower under 12-h daylight conditions. K, L Viola cornuta. K Normal CH flower under 12- and 16-h daylight conditions. L Stamen of a CH flower. No CL and inCL flowers were developed in V. cornuta under 12-h daylight conditions. Bars = 500 µm in A–L. se, sepal; pe, petal; st, stamen; ca, carpel; sg, stigma; an, anther; sc, stamen cap; fi, filament; and ng, nectar gland

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According to naked-eye observations of the floral buds, roughly five floral developmental stages (F1–F5) after floral organogenesis were defined for CH and CL formation in V. prionantha [3], which seemed to be conserved in other Viola species. At F1, CH flowers had five obvious petals and stamens, and CL flowers had two stamens, with other stamens and all petals retained as rudimentary structures (Fig. S1A–E). At F2, the nectar glands at the base of the two stamens began to appear in CH flowers. In CL flowers, the five petals and the other three stamens remained in the organ rudimentary state, and the filaments of the stamen began to appear visibly (Fig. S1F–J). At F3, the nectar glands at the base of the two stamens were obvious in CH flowers, and the stigma was higher than in the stamen. In CL flowers, the filaments of the stamen became distinct at F3 (Fig. S1K–O). At F4, the petals outside the flower buds were visible and pigmented, and the stamen cap became white in CH flowers. In CL flowers, the stamen cap also became white, and the development of the three stamens and all petals was completely arrested as primordial structures (Fig. S1P–T). At F5, in CH flowers, the petals turned purple, and the stamen cap became yellow. In CL flowers, the stamen cap also became yellow, and the development of the three stamens and all petals was completely arrested as primordial structures (Fig. S1U–Y).

Sequencing analysis of AP3 orthologous genes in three Viola species

Using the full-length transcriptome data of V. prionantha and RACE, we acquired the cDNA of the AP3 lineages of the MADS-box genes from V. philippica, V. prionantha, and V. cornuta. The ORFs of VphAP3, VprAP3, and VcoAP3 were 696, 696, and 693 bp in length, putatively encoded polypeptides of 231, 231, and 230 amino acids, and had corresponding genomic regions of 1838, 1844, and 1552 bp in length, respectively. A maximum likelihood (ML) phylogenetic tree showed that the AP3 lineage proteins of the three Viola species clustered and that these AP3 lineage proteins were grouped with previously reported AP3-like proteins, such as closely homologous proteins from Arabidopsis (Fig. 2A). Multiple sequence alignment of these genes and representatives of previously functionally inferred AP3-like proteins showed that these hypothetical AP3 proteins had a euAP3 motif at the C-terminal end (Fig. 2B), clearly confirming that these isolated sequences were orthologous to AP3. The sequencing identity of Viola AP3 orthologous genes was 95.4–98.7% among VphAP3, VprAP3, and VcoAP3 (Fig. S2), indicating that they were highly conserved and implying the conserved biochemical functions of these Viola AP3 orthologs and hence a conserved developmental role.

Fig. 2
figure 2

Sequence characterization of AP3 in Viola. A Maximum Likelihood (ML) tree of AP3-like proteins. VphAP3, VprAP3, and VcoAP3 are in red. The accession numbers of other sequences used are VphAP3 (in this study), VprAP3 (in this study), VcoAP3 (in this study), VphTM6-1 (KU318322), VphTM6-2 (KU318323), VprTM6-1 (in this study), VcoTM6-1 (in this study), LeTM6 (X60759.1), LeAP3 (DQ674532.1), PhTM6 (AF230704.1), PhDEF (X69946.1), MtTM6 (JN412097.1), MtNMH7 (JN412096.1), AtAP3 (NM115294.5), AmDEF (AB516402.1), NtDEF (X96428.1), PfDEF (KC174703.1), and PfTM6 (KC174704.1). OsMADS16 (AF077760.1) was used as the outgroup. The species name is listed before the gene name. The numbers next to the nodes are bootstrap values. B Multiple sequence alignment. Gaps are introduced for proper alignment. The M, I, K, and C domains of each MADS-domain protein are indicated by the light blue box, the green box, the dark blue box, and the orange box, respectively. The pink and purple boxes at the end of C-domain respectively indicate the euAP3 and paleoAP3 motifs

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Heterodimerization of B-class MADS-box transcription factors

B-function MADS-box genes perform essential roles in flower development by forming obligate AP3-PI heterodimers [39, 40]. We then investigated protein–protein interactions (PPIs) among these Viola B-class MADS-domain proteins. In a yeast GAL4 two-hybrid system (Y2H), activating both His3 and LacZ reporters enabled cell growth of the transformed yeast and generated the blue coloration in the non-lethal ß-galactosidase assay, demonstrating PPIs. No toxicity or self-activation of these proteins was observed in yeast (Fig. S3) [2]. All B-class proteins from Viola formed AP3-PI, TM6-PI, and AP3-TM6 heterodimers, while no homodimers were formed in yeast (Fig. 3A and B; Fig. S4). In the BiFC assay, in vivo interactions occurred between the B-class MADS-domain proteins from the examined plant species (Fig. 3C–T; Fig. S5), thus confirming the observed PPIs. These results suggest the functional conversation of these Viola B-class MADS-domain proteins.

Fig. 3
figure 3

Protein–protein interactions (PPIs) of B-class MADS-box transcription factors among Viola philippica, V. prionantha,and V. cornuta. A, B Yeast-two hybrid assays showing PPIs of AP3 and other B-class MADS-box transcription factors in the indicated Viola species. C–T PPIs revealed by BiFC. C YCE-VphAP3:YNE-VphPI; D YCE-VphAP3:YNE-VprPI; E YCE-VphAP3:YNE-VcoPI; F YNE-VprAP3:YCE-VphPI; G YNE-VprAP3:YCE-VprPI; H YNE-VprAP3:YCE-VcoPI; I YNE-VcoAP3: YCE-VphPI; J YNE-VcoAP3:YCE-VprPI; K YNE-VcoAP3:YCE-VcoPI; L YCE-VphTM6-1:YNE- VprPI; M YCE-VphTM6-1:YNE-VcoPI; N YNE-VphTM6-1:YCE-VphPI; O YNE-VprTM6-1:YCE-VphPI; P YNE-VprTM6-1:YCE-VprPI; Q YNE-VprTM6-1:YCE-VcoPI; R YCE-VcoTM6-1:YNE-VphPI; S YCE-VcoTM6-1:YNE-VprPI; and T YNE-VcoTM6-1:YCE-VcoPI. Bars = 50 µm

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Floral expression levels of Viola AP3 orthologous genes in response to photoperiod

The potential role of AP3 in the formation of dimorphic flowers may be attributed to expression variation. CH and CL flower buds were respectively developed under 10 (12)-h and 16-h daylight conditions for V. philippica and V. prionantha, while V. cornuta exhibited minimal blooming under 10-h daylight conditions. To reveal the AP3 expression variation in the CH–CL transition of V. philippica, V. prionantha, and V. cornuta in response to the photoperiod, the CH and CL flower buds under 12-h and 16-h daylight conditions of V. philippica and V. prionantha and the CH flower buds of V. cornuta that corresponded to two photoperiods (12- and 16-h daylight conditions) were selected for the expression study. We determined that the expression levels of VphAP3 and VprAP3 were significantly higher in CH floral buds subjected to 12-h daylight than in CL floral buds subjected to 16-h daylight, especially at the F1–F3 stage (Fig. 4A and B), thereby implying that the expression level of VphAP3 and VprAP3 in V. philippica and V. prionantha decreased as the duration of daylight was extended. However, VcoAP3 expression in the CH floral buds of V. cornuta under 12-h daylight conditions was comparable to CH floral buds under 16-h daylight conditions (Fig. 4C). These results indicate that the expression of VphAP3 and VprAP3 may be regulated by photoperiod and correlate to CH and CL formation in these Viola species.

Fig. 4
figure 4

Floral expression of AP3 in response to photoperiod variation among Viola philippica, V. prionantha, and V. cornuta. A Expression of VphAP3 under 12- and 16-h daylight conditions. B VprAP3 expression under 12- and 16-h daylight conditions. C VcoAP3 expression under 12- and 16-h daylight conditions. Gene expression in flower buds around F1, F2, F3, F4, and F5 under 12- and 16-h daylight conditions. Gene expression in F1 under 12-h daylight conditions was set to 1. Three independent biological samples were used in the analyses. Means and standard deviation are presented. Different lowercase letters indicate a significant difference, and the same lowercase letters indicate no significant difference

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Temporal and spatial expression of Viola AP3 genes during flower development

The spatial and temporal expression patterns of AP3 were determined during flower development using in situ hybridization in three plant species. VphAP3, VprAP3, and VcoAP3 in V. philippica, V. prionantha, and V. cornuta, respectively, were continuously expressed during flower development. They were mainly expressed in petals, stamens, and carpels (Fig. 5). VphAP3 and VprAP3 were expressed in the developed petals and stamens of CH flowers under 12-h daylight conditions or CL flowers under 16-h daylight conditions (Fig. 5A–H). The AP3 expression in the undeveloped, rudimentary petals of CL flower buds was not demonstrated, although we assumed its expression in petal organs since the petal primordia is initiated [2]. In V. cornuta, the expression pattern of VcoAP3 was the same in normal CH flowers under 12- and 16-h daylight conditions, and this gene was mainly expressed in petals, stamens, and carpels (F ig. 5I–L). Furthermore, at the more mature development stage of floral buds in three species, the expression of three Viola AP3 orthologous genes in stamens was mainly observed in anthers and less in filaments and stamen caps (Fig. 5B, D, F, H, J, and L). However, the expression levels of VphAP3 and VprAP3 seemed to be higher in CH flower stamens than in CL flower stamens (Fig. 5N and O), and little difference was observed in VcoAP3 expression in the developed petals and stamens of flowers under both 12- and 16-h daylight conditions (Fig. 5P).

Fig. 5
figure 5

Expression pattern of AP3 orthologous genes in floral buds of Viola philippica, V. prionantha, and V. cornuta. A–D VphAP3 expression at the F1 and F2 stages of (A, B) CH and (C, D) CL flowers. E–H VprAP3 expression at the F1 and F2 stages of (E, F) CH and (G, H) CL flowers. I–L VcoAP3 expression at the F1 and F2 stages of normal CH flowers under (I, J) 12 and (K, L) 16-h daylight conditions. The gene expression levels were determined using in situ hybridization. The developmental stages are defined as those in Fig. S1. M Hybridization was performed using sense probes. VphAP3, VprAP3, and VcoAP3 are indicated by 1, 2, and 3, respectively. Bars = 100 μm. se, sepal; pe, petal; st, stamen; and ca, carpel. N–P Relative expression of (N) VphAP3, (O) VprAP3, and (P) VcoAP3 in the floral organs of mature flowers. No petal sample was harvested in CL floral buds since they were undeveloped and rudimentary in V. philippica and V. prionantha under 16-h daylight conditions in N and O. The level of gene expression in the petals of CH flowers subjected to 12-h daylight conditions was set to 1. Three independent biological samples were used in all analyses. Means and standard deviation are presented. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 in single factor ANOVA test

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Taken together, inhibition of petal and stamen development in the CL flowers of Viola might result from the downregulation of AP3 under long daylight conditions, suggesting that the expression variation in AP3 may play an essential role in the CH–CL transition of Viola species.

Promoter divergence of Viola AP3 may account for the differential expression

The gene structure was highly conserved (Fig. 6); therefore, the differential expression of AP3 in the three Viola species may be due to differences in the regulatory sequences. The upstream sequences of the ATG of VphAP3 and VcoAP3, as the putative promoter, were isolated using the genome walking method, and the length of the obtained sequences was 2822 and 2693 bp, respectively. In V. prionantha, the putative promoter of VprAP3 (2754 bp) was previously cloned [3]. Sequence comparison showed that the putative promoters could be roughly divided into three regions: the VphAP3-VprAP3-VcoAP3 conserved region (P1, − 1 to − 358 bp), the VphAP3-VprAP3 conserved region (P2, − 359 to − 1152 bp) relative to VcoAP3, and the variable region (P3, beyond − 1153 bp) among the three promoters (Fig. S6). In the P1 region, the three promoters shared more than 80% sequence identity. Beyond this three-gene conserved region, VphAP3 and VprAP3 further remained relatively conserved (80% sequence identity) in the P2 region, but they were different from that of VcoAP3, sharing approximately 52% sequence identity with VphAP3 and VprAP3. However, the further distal promoter sequences of VphAP3, VprAP3, and VcoAP3 in the P3 region were quite variable and different, and the sequence identity among them was less than 42% (Fig. S6). Therefore, the VcoAP3 promoter sequence deviated from the other two overall (VphAP3–VprAP3) (Fig. 6; Fig. S6). This might form a basis for the differential expression of these Viola AP3 orthologous genes in response to photoperiod variation.

Fig. 6
figure 6

Presence and distribution of cis-elements on the Viola AP3 orthologous genes. The putative promoter and gene structure of VphAP3, VprAP3, and VcoAP3 were compared. The gene structures of these AP3 genes were conserved and included seven exons (E1–E7) and six introns (I1–I6). The three regions (P1, P2, and P3) of their putative promoters were defined according to sequence identity analyses (Fig. S6). The cis-elements or motifs related to light and light response are highlighted using different colored symbols, as indicated. Detailed information on these cis-elements is presented in Tables S2 and S3. The sequence identities of different regions of each pair of genes are given

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To further endorse this presumption, we further predicted the cis-elements on these putative promoters. Many cis-elements, including CArG-box, ABRE, P-box, MBS, MYB, MYC, and CGTCA-motif, were distributed in these promoters across the three defined regions (P1, P2, and P3); however, some cis-elements were differently distributed in the putative promoters of VphAP3–VprAP3 or VcoAP3, especially cis-elements related to light or light response (Fig. 6; Table S2). The light-responsive element Sp1-motif was found in the promoter sequences of VphAP3 and VprAP3 but not in the VcoAP3 promoter, while a chs-CMA2a element, GA-motif, Gap-box, and LS7 element were present in the promoter sequences of VcoAP3 but not in the promoters of VphAP3 and VprAP3. Moreover, auxin responsiveness TGA elements were found in the promoter sequences of VphAP3 and VprAP3 but not in the VcoAP3 promoter. Furthermore, the circadian elements were revealed in the promoter sequences of VcoAP3 but not in the promoters of VphAP3 and VprAP3. In addition, sequence divergence in the introns was observed, which might be involved in the regulation of gene expression. The fourth intron of VcoAP3 was shorter than that of VphAP3 and VprAP3, which had different cis-elements (Fig. 6, Table S3). Overall, the presence and distribution of these cis-motifs in the promoters and introns of these AP3 orthologous genes might lead to the differential expression of these genes in response to the photoperiod in the three Viola species.

Discussion

In dimorphic cleistogamy plants, heterogeneity in the environment may lead to phenotype selection of floral buds, and the frequencies of CH and CL flowers depend on various environmental conditions [2, 3, 27, 41,42,43,44]. The photoperiod plays a key role in CH/CL flower development in Viola species. CH flowers are induced under short-daylight and intermediate daylight conditions, and long-daylight (LD) induces CL flower formation in V. philippica, V. prionantha, and Viola odorata [2, 3, 27]. However, V. cornuta rarely blooms under short-daylight conditions, and normal CH flowers are always induced under intermediate daylight and LD conditions. Therefore, V. cornuta was a control species for elucidating the molecular mechanisms of dimorphic flower development.

From an evolutionary developmental biology perspective, alterations in coding regions or expression variation of orthologous genes may account for developmental reprogramming, thus leading to morphological variation of homologous organs between species. The main differences between CH and CL flowers lie in petal and stamen growth and morphology, which may relate to the alteration of floral B-function MADS-box genes because these genes regulate stamen and petal identity and development [10,11,12, 15]. Moreover, the evolution of these floral MADS-box genes and their obligate heterodimers play an important role in petal formation and diversification in core eudicots [32, 40, 45,46,47,48]. Previous work revealed that other B-function homologous genes, VphPI and VphTM6, may function in dimorphic flower formation [2]. In this study, we compared the AP3 orthologous genes of these three Viola species and found that the sequences and obligate AP3-PI heterodimerization were highly conserved, indicating a high degree of conservation in biochemical and developmental roles for these Viola AP3 orthologous genes. However, their differential expression in response to the photoperiod may contribute to the CH–CL transition in Viola. The changes in the expression level and pattern of AP3-like genes lead to innovations in flower morphology [16,17,18,19, 49,50,51]. The long-lasting flowers of azalea cultivars exhibit small-sized corollas during long blooming because the transcript of the AP3 homolog was reduced [19]. Independent petal losses within the buttercup family (Ranunculaceae) are strongly associated with decreased or eliminated expression of a single floral organ identity gene, AP3-3 [16], while petal loss in B. frutescens is likely associated with the lack of AP3-3 expression and expanded AG expression [49]. In rice, mutants of the AP3-like gene SUPERWOMAN1 have normal stamens, but their lodicules are homeotically transformed to lodicule-glume mosaic organs, thereby engendering cleistogamy [52]. Moreover, some environmental factors, such as high temperature and drought, induce AP3 downregulation, thus leading to anther deformation and reduced male fertility [53, 54]. The differential floral expression of PI and TM6 in response to variations in the photoperiod is associated with CH–CL flower development in V. philippica [2]. In this study, the AP3 orthologous genes were mainly expressed in the petals, anthers of stamens, and carpels in three Viola species. The expression of AP3 orthologous genes in the carpels of three Viola species aligns with the observations made in basal angiosperms, such as Amborella, Nuphar, and Asarum [55, 56]. The AP3 and PI exhibited expression in the early stages of gynoecium development is likely conducive to carpel determinacy and development [57]. However, the main difference was that the expression levels of VphAP3 and VprAP3 in the petals and stamens of V. philippica and V. prionantha decreased as the daylight duration was extended. In contrast, the VcoAP3 expression in V. cornuta under 12-h daylight conditions did not change compared with that under 16-h daylight conditions. These results indicate that the significant decrease in the expression of AP3 orthologous genes was differentially induced by photoperiod extension, playing an essential role in CL and CH flower formation in Viola.

Concerning the high conservation in the coding regions and obligate AP3-PI heterodimerization, the differential expression of AP3 orthologous genes in V. cornuta and V. philippica–V. prionantha in response to photoperiod variation may be essential for the CH–CL transition. The differential expression of the AP3 orthologous genes among Viola species may be influenced by the presence and distribution of cis-acting elements related to light in their regulatory regions. We found similar putative promoter sequences for AP3 orthologous genes in V. philippica and V. prionantha, but they were quite diverged from those in V. cornuta. Light can significantly regulate gene expression in the presence of light-responsive elements, such as Gap-boxes in the promoter [58, 59]. PHYTOCHROME A, 3AF1, and GAF1 factors can bind to the chs-CMA2a element, GA-motif, and Gap-box, respectively, and regulate target gene expression [58, 60,61,62]. In our study, differences in the presence and distribution of Sp1-motif, chs-CMA2a element, GA-motif, Gap-box, and LS7 element were observed between VcoAP3, VphAP3, and VprAP3, which might correlate to expression variations in these AP3 orthologous genes in response to photoperiod variation. Moreover, extensive crosstalk occurs between the light and auxin signaling pathways, and light may affect auxin-responsive gene expression [63, 64]. Auxin-responsive elements bound by auxin response transcription factors (ARFs) regulate the expression of target genes and promote petal expansion, stamen development, and petal spur elongation and nectary maturation [65,66,67,68]. The changes in auxin levels and differential expression of ARFs are associated with dimorphic flower development in Sinoswertia tetraptera [1]. Furthermore, the photoperiod regulates the circadian clock [69], and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) regulates a variety of genes by directly binding to their promoters [70,71,72]. An auxin-responsive element was only found in the putative promoters of VphAP3 and VprAP3, and a circadian element was only found in that of VcoAP3, which might also be related to the differential expression of these genes in response to photoperiod variation. In addition, introns may be involved in the regulation of gene expression [73,74,75]. We found that the intron regions, particularly the fourth intron of VcoAP3, differed from that of VphAP3 and VprAP3 in length and cis-element presence. Further functional investigation is required to identify the precise role of the observed sequence divergence of putative promoters and introns in the expression variations of these Viola AP3 orthologous genes in response to the photoperiod. Because AP3 conservatively formed the obligate heterodimers with other B-class MADS-domain proteins, such as PI and TM6, in the studied Viola species, we conclude that the differential expression of AP3 orthologous genes and the expression alteration of PI and TM6 homologous genes are altogether involved in regulating the CH–CL transition in Viola.

Data availability

All data is available in the manuscript or the supplementary materials. Sequences reported in this work have been deposited in the NCBI database, with accession numbers PQ147835–PQ147841.

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Acknowledgements

We sincerely thank the assistance of Hailong Pang (Life Science College, Northwest Normal University) in BiFC analysis. This work was supported by the grants (32160055, 32360059, and 31930007) from the National Natural Science Foundation of China.

Funding

This work was supported by the grants (32160055, 32360059, and 31930007) from the National Natural Science Foundation of China.

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  1. Life Science College, Northwest Normal University, Anning East Road 967, Anning, Lanzhou, Gansu, 730070, China

    Qiaoxia Li, Yuanyuan Zhu, Youlong Li, Chenlong Chen, Jigang Li & Kun Sun

  2. State Key Laboratory of Plant Diversity and Specialty Crops / State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing, 100093, China

    Chaoying He

  3. China National Botanical Garden, Beijing, 100093, China

    Chaoying He

  4. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China

    Chaoying He

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Contributions

QXL conceived and designed the work. QXL, JGL, KS, and CYH analyzed the data. QXL, YYZ, YLL, and CLC performed experiments. QXL and CYH wrote the manuscript. All authors have read and approved the manuscript.

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Li, Q., Zhu, Y., Li, Y. et al. Expression variation of Viola APETALA3 orthologous genes is correlated with chasmogamous and cleistogamous flower development. BMC Plant Biol 25, 319 (2025). https://doi.org/10.1186/s12870-025-06348-6

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  • DOI: https://doi.org/10.1186/s12870-025-06348-6

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BMC Plant Biology

ISSN: 1471-2229

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