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Article

The FataAP2-FataAP1-FataTOE Regulatory Cascade Regulates Floral Organ Development in Fagopyrum tataricum

by
Shengchun Li
1,2,
Yuhan Yan
3,
Haonan Liu
3,
Anhu Wang
1,2,* and
Zhixiong Liu
3,*
1
Panxi Crop Improvement Key Laboratory of Sichuan Province, Xichang University, Xichang 615000, China
2
College of Agricultural Sciences, Xichang University, Xichang 615000, China
3
College of Horticulture and Gardening, Yangtze University, Jingzhou 434025, China
*
Authors to whom correspondence should be addressed.
Agronomy 2026, 16(17), 1635; https://doi.org/10.3390/agronomy16171635
Submission received: 8 July 2026 / Revised: 16 August 2026 / Accepted: 21 August 2026 / Published: 27 August 2026
(This article belongs to the Special Issue Reproductive Biology for Improving Crop Yield and Breeding Efficiency)
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Abstract

Arabidopsis class A genes APETALA 1 (AP1) and AP2 determine sepal and petal identity; AP2 also acts as a repressor that directly negatively regulates AP1 expression to control flowering time, while TOE1 represses flowering by inhibiting CONSTANS (CO) activity. Petunia AP2-type ROB is required for perianth and pistil development, as well as the repression of B-function and TOE-type BEN. However, how AP1 and AP2 orthologs from Fagopyrum tataricum work together to regulateflowering and floral organ development remains unclear. F. tataricum is an edible and medical crop rich in bioactive phytochemicals that have health benefits. This plant has only one perianth whorl, making it an excellent model for exploring the development and evolution of apetaly. In this study, we found that AP1-like FataAP1, AP2-type FataAP2, and TOE-type FataTOE were expressed in the roots, stems, leaves, flowers, and fruits of tartary buckwheat and in all stages detected during floral bud differentiation and development. The Y1H assay and DLR suggested that FataAP2 directly activates FataAP1 transcription and that FataAP1 directly activates the transcription of FataTOE. The VIGS silencing of FataAP2, FataAP1, and FataTOE resulted in flowers with increased tepal numbers and partly abnormal outer-whorl stamens with reduced filament lengths and shriveled anthers without pollen grains. A hierarchical regulatory cascade, FataAP2-FataAP1-FataTOE, was uncovered, which modulated tepal numbers and outer-whorl stamen development during tartary buckwheat flower development. Our current research provides new insights into the molecular mechanism of floral organ development in tartary buckwheat and may provide new evidence for exploring the floral phenotypic differences between tartary buckwheat and common buckwheat.

1. Introduction

The formulation of the ABC model of flower development explains how ABC genes specify distinct organ identities and how perfect flowers are formed [1]. According to this model, class A genes APETALA 1 (AP1) and AP2 determine sepal and petal identity [1]. In Arabidopsis, AP2 maintains shoot apical meristem (SAM) activity, in part through maintaining active WUSCHEL (WUS) expression and controlling meristem activity via abscisic acid (ABA) and cytokinin (CK) responses [2]. Arabidopsis AP2 belongs to the euAP2 lineage, a subgroup of the APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factor (TF) family, and is characterized by two AP2 domains and a microRNA172 (miR172) target site [3,4]. The euAP2 lineage consists of AP2- and TOE-type classes [4]. There are two sister clades within seed plants: euAP2 and AINTEGUMENTA (ANT). euAP2 genes originally contribute to spore and sporangium development and are subsequently recruited for ovule, fruit, and floral organ development (sepal and petal identity) and the control of flowering time [5]. In Arabidopsis, AP2 is involved in floral development and flower organ identity, while TOE1/3 (TARGET OF EAT1/3) is involved in regulating flowering [6,7]. AP2 acts as a flowering repressor and is suppressed by MADS-box TF FRUITFULL (FUL) and miR172 in the SAM and plays a key role in regulating the end of flowering via the FUL–AP2 pathway [8]. Moreover, AP2 suppresses flowering and regulates flower development by directly repressing the expression of AP1, AGAMOUS (AG), and the flowering integrator gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) [9,10], while TOE1 interacts with CONSTANS (CO) and inhibits CO activity to prevent premature flowering, and acts as a flowering-time repressor by suppressing CO and FT expression [6,11,12]. euAP2 genes are likely functionally divergent outside Brassicaceae [13]. Two alleles of TOE-type RcAP2L are present in the double-flower rose, and one allele harbours a transposable element inserted into intron 8, leading to the formation of a miR172-resistant RcAP2L variant correlating with double-flower formation [14]. Moreover, miR172 target site deletion in a TOE-type gene results in a strong dominant double-flower trait in Rosaceae [15]. Mutations overlying the miR172 target site of the TOE-type gene pmTOE are also linked to flower doubleness in Prunus mume (Rosaceae) [16]. In addition, recent studies suggested that an R2R3-MYB transcription factor, RhMYB17, was upregulated at low temperatures and directly binds to rose AP2 (RhAP2) and RhAP2L promoters, thereby activating both genes’ expression and specifying stamen–petal homeotic transformation at low temperatures [17]. In Robinia pseudoacacia (Fabaceae), RpTOE1 binds directly to the FT-like (FLOWERING LOCUS T-like) RpFT promoter and negatively regulates RpFT during vegetative propagation [18]. However, in the asterid species Petunia hybrida (Solanaceae), the AP2-type REPRESSOR OF B-FUNCTION (ROB) genes are involved in sepal, petal, and pistil development, while the TOE-type gene BLIND ENHANCER (BEN) is redundant in proper petal development and represses the B-function in the sepal, together with BEN [4]. Moreover, functional losses of ROB genes mainly result in decreased style length in petunia, while stamen development was not affected [4]. In Camellia japonica (Theaceae), miR172-mediated repression of AP2-like genes also leads to the formation of double flowers (with an increased petal number) [19]. In Hylocereus polyrhizus (Cactaceae), gibberellin-3 (GA3) treatment inhibited AP1 and AP2 genes and suppressed flower bud formation [20]. In Long-homostyle (LH) common buckwheat (Fagopyrum esculentum), FaesAP2 (AP2-type) plays a key role in determining filament and style lengths, while FaesTOE (TOE-type) plays an important role in determining filament length and regulating pollen grain development [21]. In addition, FaesTOE directly upregulates FaesAP1_2, and the downregulation of FaesAP1_2 expression resulted in a decrease in tepal numbers and filament length in LH common buckwheat [21]. All these studies suggested that euAP2 genes involved in regulating perianth number show certain conservation among some rosid, asterid, and eudicot species, and their regulatory module involved in floral development and flowering time control is likely divergent.
Tartary buckwheat (F. tataricum) (Caryophyllales: Polygonaceae) and common buckwheat produce flowers with single-whorl sepaloid tepals (generally assumed to be sepals), showing obvious differences from most core eudicot flowers with perianth differentiated into typical green sepals (which make up the calyx) and showy petals (which make up the corolla whorl) [22,23]. However, F. esculentum is a self-incompatible crop resulting from its heteromorphic flowers (pin and thrum), whereas tartary buckwheat is a self-pollinated crop and produces homostylous flowers [24,25]. Hence, this flower phenotypic difference may result from the molecular regulatory pathway changes between tartary buckwheat and common buckwheat. In this study, we found that AP2-type TF FataAP2 directly upregulates AP1 orthologous FataAP1 expression, and FataAP1 further directly upregulates TOE-type FataTOE expression during flower development in tartary buckwheat. The three transcription factors (TFs) formed a FataAP2-FataAP1-FataTOE regulatory cascade involved in floral organ development in F. tataricum. The downregulation of FataAP2, FataAP1, or FataTOE expression via virus-induced gene silencing (VIGS) redundantly results in similar flower phenotypic changes in tartary buckwheat, leading to flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains. Our current research provides new insights into the molecular mechanism of floral organ development in tartary buckwheat and may provide new evidence for exploring the floral phenotypic differences between tartary and common buckwheat.

2. Materials and Methods

2.1. Plant Material

The tartary buckwheat cultivar ‘Chuanqiao 1’ was planted in 5-litre plastic pots on the campus of Yangtze University in Jingzhou, China, and it was grown under natural conditions in the spring of 2025 and 2026 and the autumn of 2025. The roots, stems, young leaves, flowers, and young fruits (achenes) of tartary buckwheat, as well as floral buds from different development stages, were separately sampled, frozen in liquid nitrogen, and stored at −80 °C according to the method described by Yang et al. [21]. The cytomorphological examination of floral buds and Nicotiana benthamiana was performed using the dual-luciferase reporter assay according to our previous work [21].

2.2. Isolation and Identification of AP2-Type Genes, FataAP1, and Their Promoters from Tartary Buckwheat

The total RNA of tartary buckwheat inflorescence and the first-strand cDNA were prepared according to our previous work [21]; then, the cDNA sequences of FataAP2, FataTOE, and FataAP1 were separately amplified with the gene-specific primer pairs FataAP1F/R, FataAP2F/R, and FataTOEF/R (Supplementary Table S1). The primer pairs were separately designed according to the ortholog sequences of F. esculentum FaesAP2(KM386628.1), FaesTOE(PP357007), and FaesAP1_2(PP357009). Phylogenetic trees were separately constructed with the software MEGA12.0.11 using the ML Maximum Likelihood (ML) method for selecting the Jones–Taylor–Thornton (JTT) model and 1000 bootstrap replications. All the AP2 homologous TFs and AP1/FUL-like proteins with complete sequences were selected for ML trees from NCBI GenBank (Supplementary Tables S2 and S3). In addition, the AGL6-like and SEP-like TFs were selected as outgroups because previous works, grouping them into the AP1/SEP/AGL6 superclade [26,27].
Tartary buckwheat genomic DNA was extracted from young leaves according to Yang et al. [21]. The FataAP1 promoter (pFataAP1) and FataTOE promoter (pFataTOE) were searched based on the FataAP1 and FataTOE sequences from F. tataricum genome sequence (accession number: GWHFQEG00000000.1) in the National Genome Science Data Center (https://ngdc.cncb.ac.cn/); then, the primer pairs pFataAP1F/R and pFataTOEF/R (Supplementary Table S1) were designed separately according to the sequences of FataAP1 and FataTOE promoter regions, respectively. The putative transcription start sites of FataAP1 and FataTOE were searched with the methods suggested by Solovyev et al. [28], and the cis-acting elements of the FataAP1 and FataTOE promoters were separately searched in the PLACE database [29].

2.3. Yeast One-Hybrid Assay

A yeast one-hybrid (Y1H) assay was conducted using the Yeast One-Hybrid Media Kit (Coolaber, Beijing, China). The pFataAP1 region (−1729/−1281) harbouring TTTGTT motifs was cloned into the pAbAi plasmid to create pBait-AbAi vectors with the primers Y1HpFataAP1F and Y1HpFataAP1R for pFataAP1 (Supplementary Table S1) [30]. Tartary buckwheat FataAP2 cDNA with a full-length Open Reading Frame (ORF) and FataTOE cDNA were separately cloned into the pGADT7 plasmid to construct a prey vector with the primer pairs Y1HFataAP2F/R for FataAP2, but with the primer pairs Y1HFataTOEF/R for FataTOE (Supplementary Table S1). pAbAi-pFataAP1 was introduced into the Y1H Gold competent cells to create bait-reporter strains. The transformants were cultured on SD/-Ura medium to assess the minimal inhibitory concentration of Aureobasidin A (AbA). The prey plasmids pGADT7-FataAP2 and pGADT7-FataTOE were separately transformed into the bait yeast strains for the Y1H assay following the manufacturer’s protocol. Colonies were cultured on SD/-Leu/AbA medium with bait minimum AbA resistance for 3 d at 30 °C.
The pFataTOE region (−265/+250) harbouring the CArG-box motif was cloned into the pAbAi plasmid to create pBait-AbAi vectors with the primers Y1HpFataTOEF and Y1HpFataTOER (Supplementary Table S1) [31]. Tartary buckwheat A class MADS-box gene FataAP1 cDNAs containing full-length ORFs were cloned into the pGADT7 plasmid to construct a prey vector with the primer pairs Y1HFataAP1F and Y1HFataAP1R (Supplementary Table S1). The specific experimental steps for this process are the same as those mentioned above.

2.4. Dual-Luciferase Reporter Assay

The pFataAP1 region containing TTTGTT motifs was cloned into the pGreen0800-LUC vector to create the reporter plasmid pGreen0800-pFataAP1 with the primers Dual-pFataAP1F and Dual-pFataAP1R (Supplementary Table S1). The ORF of FataAP2 was cloned into the pGreenII 62-SK vector to create the effector vectors with primers Dual-FataAP2F and Dual-FataAP2R (Supplementary Table S1). The reporter vectors and effector were separately transformed into the Agrobacterium strain GV3101 (pSoup). The effector and the reporter Agrobacterium were prepared and infiltrated into N. benthamiana leaves according to the method described by Jiao et al. [32]. Empty pGreenII 62-SK was cotransformed with the pGreen0800-pFataAP1 reporter as a negative control. The treated tobacco plants were cultured, and firefly luciferase (LUC) and Renilla luciferase (REN) were assayed according to Jiao et al. [32]. In addition, the pFataTOE region (−265/+250) containing CArG-box motifs was cloned into the pGreen0800-LUC vector to create the reporter plasmid pGreen0800-pFataTOE with the primers Dual-pFataTOEF and Dual-pFataTOER (Supplementary Table S1). The ORF of FataAP1 was cloned into the pGreenII 62-SK vector to create the effector vectors with the primers Dual-FataAP1F and Dual-FataAP1R (Supplementary Table S1). The reporter vectors and effector were separately transformed into the Agrobacterium strain GV3101 (pSoup). The effector and the reporter Agrobacterium were infiltrated into N. benthamiana leaves. The treated tobacco plants were cultured, and firefly luciferase and Renilla luciferase were assayed, and a negative control was prepared according to the above work.

2.5. Expression Analysis of FataAP2, FataTOE, and FataAP1

The total RNA of each sample extracted, first-strand cDNA synthesized for qRT-PCR, and expression of FataAP2, FataTOE, and FataAP1 was separately detected in the roots, stems, young leaves, flowers, and young fruits of tartary buckwheat using qRT-PCR, according to Liu et al. [33], with the gene-specific primer pair qFataAP2F/R for FataAP2, qFataTOEF/R for FataTOE, and qFataAP1F/R for FataAP1, respectively (Supplementary Table S1). Moreover, FataAP2, FataTOE, and FataAP1 expression was evaluated in floral buds of tartary buckwheat at different development stages with qRT-PCR, according to Liu et al. [33]. Paraffin sections were utilized to explore the developmental stages of floral buds with the method described by Jiao et al. [32]. The amplicons of the F. tataricum actin gene were amplified as the internal control with the primers qFataactinF and qFataactinR.

2.6. VIGS Assay in Tartary Buckwheat

To assess the roles of FataAP2, FataAP1, and FataTOE in the floral development of F. tataricum, the virus-induced gene silencing (VIGS) technique was performed using the Tobacco Rattle Virus (TRV) system to downregulate their expression. A 583 bp FataAP2 cDNA, a 453 bp FataAP1 cDNA, and a 343 bp FataTOE cDNA were separately cloned into the TRV2 vectors with XbaI and SacI enzymes, with the primers TRV2-FataAP2F and TRV2-FataAP2R for FataAP2, TRV2-FataTOEF and TRV2-FataTOER for FataTOE, and TRV2-FataAP1F and TRV2-FataAP1R for FataAP1, respectively.
Then, the TRV2-FataAP2, TRV2-FataTOE, TRV2-FataAP1, and TRV1- and TRV2-empty vector plasmids were transformed into the Agrobacterium strain GV3101. The transformants were infiltrated into the two-true-leaf-stage leaves of tartary buckwheat seedlings, and the infected seedlings were cultivated with the method suggested by Liu et al. [34]. All the infected seedlings were cultivated in darkness for 24 h and then moved to a greenhouse with a temperature of 22 °C under short-day conditions. Infiltration treatment was repeated once a week until flowering to observe the phenotype. The expression levels of FataAP2, FataTOE, FataAP1, and their target genes were detected in the flowers of TRV2-treated plants with qRT-PCR.

3. Results

3.1. Isolation and Identification of FataAP2, FataTOE, FataAP1, and Their Promoters from Tartary Buckwheat

To explore AP1-like and euAP2 genes involved in the floral development of tartary buckwheat, three orthologous genes and both promoters of FataAP1 and FataTOE have been isolated from F. tataricum. The 1506 bp FataAP2 cDNA has a 1374 bp ORF encoding 457 amino acids (aa), while the 1086 bp FataTOE cDNA has a 1061 bp ORF encoding 353 aa and the 915 bp FataAP1 cDNA contains a 749 bp ORF encoding 249 aa. Phylogenetic tree and protein sequence alignment analyses suggested that FataAP2 and FataTOE are euAP2 lineage TFs (Figure 1A, Supplementary Figure S1A), and FataAP1 is an euAP1 lineage TF (Figure 1B, Supplementary Figure S1B). FataAP2 is an ortholog of the Arabidopsis AP2 TF and showed 93.58% identity with F. esculentum FaesAP2 (GenBank: AKI81900.1), and the FataAP2 gene was named FataAP2 (Fagopyrum tataricum AP2). FataTOE is an ortholog of Arabidopsis TOE1 TF and showed 90.36% identity with F. esculentum FaesTOE (GenBank: WWB03765.1), and the FataTOE gene was named FataTOE (Fagopyrum tataricum TOE). FataAP1 is an ortholog of Arabidopsis AP1 TF and showed 99.60% identity with F. esculentum FaesAP1_2 (GenBank: WWB03767.1), and the FataAP1 gene was designated as FataAP1 (Fagopyrum tataricum AP1).
A 2013 bp FataAP1 promoter (pFataAP1) fragment (−1944/+69) was isolated from tartary buckwheat, and the putative transcription start site and cis-acting regulatory elements of pFataAP1 are displayed in (Supplementary Figures S1 and S2). pFataAP1 has two CCAATBOX1s involved in flowering for CO binding [35] and five GTGANTG10-boxes and seven POLLEN1LELAT52-boxes associated with stamen and pollen development [36,37]. Moreover, pFataAP1 also contains two CArG-boxes for MADS-box TFs binding [38], and it harbours five AACAAA-/TTTGTT-motifs for floral homeotic AP2-like protein binding [30]. All these cis-acting elements suggest that FataAP1 may be involved in flowering and/or floral organ development in F. tataricum. In addition, there are two gibberellic acid (GA)-responsive elements (PYRIMIDINEBOXOSRAMY1A-boxes) [39] and seven abscisic acid (ABA)-responsive elements (six MYCCONSENSUSAT-boxes and a MYB1AT-box) [40] in the pFataAP1 region, which suggests that FataAP1 may regulate flowering and floral development via GA and ABA signal pathways.
A 1998 bp FataTOE promoter (pFataTOE) fragment (−1710/+288) was isolated from tartary buckwheat, and the putative transcription start site and cis-acting regulatory elements of the pFataTOE are displayed in (Supplementary Figures S2 and S3). Many floral development response cis-elements, such as two CCAATBOX1s [35], three GTGANTG10-boxes [36], two POLLEN1LELAT52-boxes [37], two CArG-boxes [38], five AACAAA-/TTTGTT-motifs [30], and two MYBCORE motifs for petal-epidermis-specific MYB protein binding [41], are also found in the pFataTOE region, which suggests that FataTOE may be involved in flowering and/or floral organ development in F. tataricum. In addition, there are four GA-responsive PYRIMIDINEBOXOSRAMY1A-boxes [39], an ABA-responsive MYB1AT element [40], and a low-temperature-responsive LTRE1HVBLT49-box element [42] in the pFataTOE region, which suggests that FataTOE may regulate flowering and floral development via GA, ABA, and low-temperature signal pathways.

3.2. The FataAP2-FataAP1-FataTOE Module Involved in F. tataricum Floral Development

Previous studies have shown that the floral homeotic TF AP2 can bind to the AACAAA-/TTTGTT-motifs and further regulate floral development [30]. To assess whether FataAP2 directly regulated FataAP1 and/or FataTOE or not, and whether FataTOE directly regulated FataAP1 or not, the pFataTOE promoter region (−265/+250) and the pFataAP1 fragment (−1729/−1281) containing AACAAA-/TTTGTT-motifs were screened. In addition, the pFataTOE promoter region (−265/+250) containing a CArG-box was assessed using the floral homeotic MADS-box TF FataAP1. The Y1H assay showed that FataAP2 could directly bind to the target fragment of the pFataAP1 (Figure 2A). In addition, DLR further suggested that FataAP2 upregulates FataAP1 expression in plants (Figure 2B). When the reporter construct carried the pFataAP1 region (−1729/−1281), the ratio of LUC/REN expression was significantly upregulated (p < 0.05) (Figure 2B,C). In addition, the Y1H assay and DLR further suggested that FataAP1 directly binds to the target fragment of pFataTOE (−265/+250) (Figure 3A) and activates FataTOE expression (Figure 3B,C).

3.3. Expression Analysis of FataAP2, FataTOE, and FataAP1 in Tartary Buckwheat

FataAP2, FataAP1, and FataTOE were expressed in the roots, stems, leaves, flowers, and fruits of tartary buckwheat (Figure 4A), and they were expressed in all stages examined during floral bud differentiation and development (Figure 4B). In addition, the expression level of FataAP2 in the roots, stems, leaves, flowers, and fruits showed no significant differences, and the FataAP1 expression level also showed no significant differences in the above organs. However, the highest expression level of FataTOE was observed in stems, followed by the expression level of FataTOE in leaves and flowers, which are significantly higher than the expression level of FataTOE in roots and fruits (p < 0.05, LSD). The expression of FataAP2, FataAP1, and FataTOE was separately detected during pistil and stamen emergence in tartary buckwheat floral buds (Figure 4B,C). In addition, the expression level of FataAP2 in the S1 (pistil and stamen emergence) to the S5 stage (floral bud with mature pollen and mature embryo sac before blossom) showed no significant differences, and the FataAP1 expression level also showed no significant differences in the above stages. However, FataTOE expression increased continuously during tepal elongation and stamen development in stages S1–S4 (mononuclear microspores at the periphery, rapid tepal elongation and closing), and reached its peak in anthers when mononuclear microspores were at the periphery and when rapid tepal elongation and closing occurred (Figure 4B,C(S4)). Moreover, the FataTOE expression increased sharply from S3 (microspore tetrad formation in the stamen) to S4 (p < 0.05, LSD). Then, the FataTOE expression began to drop significantly in floral buds with mature pollen and mature embryo sacs before blossom (p < 0.05, LSD) (Figure 4B,C(S4,S5)).

3.4. Characterization of FataAP2, FataAP1, and FataTOE-Silenced Plants

To explore the functions of FataAP2, FataAP1, and FataTOE in floral development, the virus-induced gene silencing (VIGS) technique was performed using the Tobacco Rattle Virus (TRV) system to downregulate their expression in F. tataricum. Among 55 TRV2-FataAP2-treated tartary buckwheat plants, 34 (61.82%) TRV2-FataAP2-treated plants produced flowers with phenotypic changes (Figure 5A). A total of 19 (34.55%) FataAP2-silenced plants had flowers with partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type I), and 15 (27.27%) FataAP2-silenced plants had flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type II). In addition, similar flower phenotypic changes were also observed in the TRV2-FataAP1-treated and TRV2-FataTOE tartary buckwheat. Among 52 TRV2-FataAP1-treated tartary buckwheat plants, 31 (59.62%) TRV2-FataAP1-treated plants produced flowers with phenotypic changes (Figure 5A). A total of 15 (28.85%) FataAP1-silenced plants had flowers with partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type I), and 16 (30.77%) FataAP1-silenced plants had flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type II). Moreover, among 43 TRV2-FataTOE-treated tartary buckwheat plants, 35 (81.40%) TRV2-FataTOE-treated plants produced flowers with phenotypic changes (Figure 5A). A total of 23 (53.49%) FataTOE-silenced plants had flowers with partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type I), and 12 (27.91%) FataTOE-silenced plants had flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains (Figure 5B, Type II).
In addition, the expression levels of FataAP2, FataAP1, and FataTOE were separately detected using qRT-PCR in the gene-silenced plants. The results showed that FataAP2 expression level significantly decreased in the flowers of TRV2-FataAP2-silenced plants (Figure 5C). Correspondingly, the expression of the direct target FataAP1 and its downstream gene FataTOE significantly decreased in the TRV2-FataAP2-silenced plants (Figure 5C). In the flowers of TRV2-FataAP1-silenced plants, the expression level of FataAP1 and its direct target FataTOE significantly decreased (Figure 5C). Moreover, FataTOE expression was also significantly decreased in the flowers of TRV2-FataTOE-silenced plants (Figure 5D). These observations suggest that the downregulation of FataAP2 expression results in the decreased expression of the direct target FataAP1 and its downstream FataTOE (Figure 5D), while the downregulation of FataAP1 expression results in the decreased expression of its direct target FataTOE (Figure 5E).

4. Discussion

The expansion of the AP1-like and/or AP2-like genes leads to variations in petal (tepal) number in many species [43]. In the rosid species, miR172 target-deficient AP2-like genes, such as TOE-type RcAP2L from Rosa sp. (Rosaceae) [14], pmTOE from P. mume (Rosaceae) [16], and the euAP2 PETALOSA gene from P. persica (Rosaceae) [44], result in changes in expression zones in these genes and further lead to a double-flower phenotype (increased petal number and decreased stamen number). Moreover, RhMYB17 directly binds to RhAP2 and RhAP2L promoters, thereby activating their expression to regulate the petaloid stamen number and the normal stamen number in roses [17]. In the asterid species, miR172-mediated repression of AP2-like genes also leads to the formation of double flowers in Camellia japonica (Theaceae) [20]. Petunia (Solanaceae) AP2-type ROB genes are involved in sepal, petal, and pistil development, while the TOE-type gene BEN is redundant in proper petal development [4]. Solanum lycopersicum (Solanaceae) TOE1-like SLTOE1 knockout resulted in highly branched inflorescences and defective floral organs in tomato [45]. In Dianthus chinensis (Caryophyllaceae), another species from Caryophyllales (eudicots), an SNP mutation in the miR172 target site of the DcAP2L gene is associated with a double-flower phenotype in Carnation [46]. Even in the basal angiosperm species Nelumbo nucifera (Nymphaeaceae), AP2 orthologs (NnAP2a and NnAP2b) regulate tepal development and tepal number by working together with B-class MADS-box genes AP3 and PISTILLATA (PI) [47]. Ectopic expression of NnAP2a or NnAP2b in Arabidopsis led to increased petal and sepal numbers, and each of the two genes was able to remedy the defective sepal and petal phenotypes in the Arabidopsis ap2-6 mutant [47]. In LH F. esculentum, both FaesAP2 and FaesTOE are redundantly involved in style and/or filament length determination, and FaesTOE is also involved in anther development [21]. FaesAP2 and FaesTOE were further proved to control style and/or filament length by indirectly regulating other ABC-class MADS-box genes via the direct repression of FaesMYB15 expression in LH F. esculentum [33]. The AP1 orthologs show more conserved functions in regulating flowering and perianth development among angiosperms. In Castanea mollissima (Fagaceae), AP1-like gene CmAP1 promotes flowering and plays a key role in petal development [48]. Osmanthus fragrans (Oleaceae) AP1 ortholog also acts as a petal number modulator and plays important roles in flowering [49]. In F. esculentum, an AP1-like gene FaesAP1_1 activates flowering and regulates tepal (perianth) development [23], while another AP1-like gene FaesAP1_2 is mainly involved in filament length and tepal number determination [21]. FaesTOE (TOE-type) directly upregulates FaesAP1_2 and downregulates FaesAP1_2 expression, resulting in decreased tepal numbers and filament lengths [21]. Moreover, the VIGS silencing of FaesAP2 (AP2-type) or FaesTOE resulted in decreased style and stamen filament lengths, and the silencing of FaesTOE also led to empty anthers (male sterile anthers) attached to short filament tops [21].
Tartary buckwheat and common buckwheat are two major cultivar species from the genus Fagopyrum (Polygonaceae), but they produce flower phenotypes with obvious differences. Common buckwheat is a self-incompatible crop due to its heteromorphic flowers (pin and thrum), whereas tartary buckwheat is a self-pollinated crop producing homostylous flowers [21,22]. Hence, this flower phenotypic difference may result from the molecular regulatory pathway changes between tartary buckwheat and common buckwheat. In tartary buckwheat, we found that FataAP2 directly activates FataAP1 expression and FataAP1 further directly upregulates FataTOE expression during flower development. The three TFs formed a FataAP2-FataAP1-FataTOE regulatory cascade involved in floral organ development in F. tataricum, which shows a different regulatory cascade from LH common buckwheat (Figure 6). In LH common buckwheat, TOE-type FaesTOE directly activates FaesAP1_2 expression, and the two TFs form a FataTOE-FaesAP1_2 module to regulate stamen development and tepal number [15]. The downregulated expression of FataAP2, FataAP1, or FataTOE produces similar flower phenotypic changes in tartary buckwheat, which produces flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains. However, the downregulated expression of FataAP2, FataAP1, or FataTOE did not lead to early or delayed flowering. Our current research provides new insights into the molecular mechanism of floral organ development in tartary buckwheat and may provide new cues for exploring the floral phenotypic differences between tartary buckwheat and common buckwheat.

5. Conclusions

Tartary buckwheat (Fagopyrum tataricum) and common buckwheat (F. esculentum) are two major cultivar species from the genus Fagopyrum (Polygonaceae); however, they produce flower phenotypes with obvious differences. Common buckwheat is a self-incompatible crop due to its heteromorphic flowers (pin and thrum), whereas tartary buckwheat is a self-pollinated crop with homostylous flowers. In Arabidopsis, APETALA 1 (AP1) and AP2 determine sepal and petal identity, and AP2 acts as a repressor by directly negatively regulating AP1 expression to control flowering timing, while TOE1 represses flowering by inhibiting CO activity. However, how the three homologs work together to regulate flowering and floral organ development remains unclear in F. tataricum. In this study, AP1-like FataAP1, AP2-type FataAP2, and TOE-type FataTOE were isolated from F. tataricum. Expression pattern analysis shows that FaesAP2, FataTOE, and FataAP1 were separately expressed in the roots, stems, leaves, flowers, and fruits of tartary buckwheat and that they were expressed in all stages examined during floral bud differentiation and development. The Y1H assay and DLR suggested that FataAP2 directly activates FataAP1 transcription, and FataAP1 further directly activates FataTOE transcription. FataAP2-silenced plants produced flowers with increased tepal numbers and partially abnormal outer-whorl stamens with reduced filament lengths and shrivelled anthers without pollen grains. Similar VIGS-silenced flower phenotypes were also observed in the flowers of the FataAP2 direct target, the FataAP1-silenced plants, and their downstream FataTOE-silenced plants. FataAP2, FataAP1, and FataTOE are redundantly involved in tepal and stamen development in tartary buckwheat and form a FataAP2-FataAP1-FataTOE regulatory cascade to regulate tepal numbers and outer-whorl stamen development. Our current research provides new insights into the genetic regulation of floral organ shape in tartary buckwheat and may provide new evidence for exploring the floral phenotypic differences between tartary # and common buckwheat.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy16171635/s1, Table S1: List of primer sequences used in this study; Table S2: AP1/SEP/AGL6-like proteins selected for phylogenetic analyses from NCBI GenBank; Table S3: AP2-like proteins selected for phylogenetic analyses from NCBI GenBank; Figure S1: Sequence alignments of euAP2 and AP1 orthologous proteins; Figure S2: FataAP1 promoter (pFataAP1) sequence; Figure S3: FataTOE promoter (pFataTOE) sequence.

Author Contributions

S.L. and Z.L. designed the study. S.L., Y.Y., and H.L. performed the experiments. S.L. and Z.L. wrote the manuscript. A.W. and Z.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province (grant number: SZKF202403); the Open Project Program of Panxi Crops Research and Utilization Key Laboratory of Sichuan Province (grant number: SZ24ZZ01); and the Sichuan Provincial Agricultural Department Innovative Research Team (grant number: SCCXTD-2024-11).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 16 01635 g001
Figure 1. The phylogenetic tree of AP1 and AP2 homologous TFs: (A) the ML tree of FataAP2, FataTOE, and other APETALA2-Like TFs; (B) the ML tree of FataAP1_2 with AP1/SEP/AGL6 TFs. Fagopyrum tataricum’s FataAP2, FataTOE, and FataAP1 TFs are marked in red.
Figure 1. The phylogenetic tree of AP1 and AP2 homologous TFs: (A) the ML tree of FataAP2, FataTOE, and other APETALA2-Like TFs; (B) the ML tree of FataAP1_2 with AP1/SEP/AGL6 TFs. Fagopyrum tataricum’s FataAP2, FataTOE, and FataAP1 TFs are marked in red.
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Figure 2. The positive regulation of FataAP1 by FataAP2 TF. (A) The Y1H assay suggests that FataAP2 TF directly binds to the FataAP1 promoter. (B) DLR shows that FataAP2 activates FataAP1 expression by directly binding to pFataAP1. The ratio of LUC/REN activity indicates the relative activity of the FataAP2 and FataAP1 promoters. (C) The DLR images of TF FataAP2 and pFataAP1 in tobacco leaves. Data are presented as means ± SD of three biological replicates. Asterisks (*) above the bar indicate statistical significance (p < 0.05).
Figure 2. The positive regulation of FataAP1 by FataAP2 TF. (A) The Y1H assay suggests that FataAP2 TF directly binds to the FataAP1 promoter. (B) DLR shows that FataAP2 activates FataAP1 expression by directly binding to pFataAP1. The ratio of LUC/REN activity indicates the relative activity of the FataAP2 and FataAP1 promoters. (C) The DLR images of TF FataAP2 and pFataAP1 in tobacco leaves. Data are presented as means ± SD of three biological replicates. Asterisks (*) above the bar indicate statistical significance (p < 0.05).
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Figure 3. The positive regulation of FataTOE by FataAP1 TF. (A) The Y1H assay suggests that FataAP1 TF directly binds to the FataTOE promoter. (B) DLR shows that FataAP1 activates FataTOE expression by directly binding to pFataTOE. The ratio of LUC/REN activity indicates the relative activity of the FataAP1 and FataTOE promoters. (C) The DLR images of TF FataAP1 and pFataTOE in tobacco leaves. Data are presented as means ± SD of three biological replicates (* p < 0.05).
Figure 3. The positive regulation of FataTOE by FataAP1 TF. (A) The Y1H assay suggests that FataAP1 TF directly binds to the FataTOE promoter. (B) DLR shows that FataAP1 activates FataTOE expression by directly binding to pFataTOE. The ratio of LUC/REN activity indicates the relative activity of the FataAP1 and FataTOE promoters. (C) The DLR images of TF FataAP1 and pFataTOE in tobacco leaves. Data are presented as means ± SD of three biological replicates (* p < 0.05).
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Figure 4. FataAP2, FataAP1, and FataTOE expression in different organs and different-development-stage floral buds of F. tataricum. (A) FataAP2, FataAP1, and FataTOE expression in the roots, stems, leaves, tepals, flowers, and fruits was detected using qRT-PCR. (B) FataAP2, FataAP1, and FataTOE expression in different-development-stage floral buds was separately detected using qRT-PCR. (C) The cytomorphological section of different-development-stage floral buds in F. tataricum; S1: pistil and stamen primordium emergence; S2: microspore mother cell formation and tepal and stamen filament elongation; S3: microspore tetrad formation; S4: mononuclear microspores at the periphery and rapid tepal elongation and closing; S5: floral buds with mature pollen and mature embryo sacs before blossom. Scale bar: 100 µm. Different lowercase letters indicate significant differences (p < 0.05, LSD).
Figure 4. FataAP2, FataAP1, and FataTOE expression in different organs and different-development-stage floral buds of F. tataricum. (A) FataAP2, FataAP1, and FataTOE expression in the roots, stems, leaves, tepals, flowers, and fruits was detected using qRT-PCR. (B) FataAP2, FataAP1, and FataTOE expression in different-development-stage floral buds was separately detected using qRT-PCR. (C) The cytomorphological section of different-development-stage floral buds in F. tataricum; S1: pistil and stamen primordium emergence; S2: microspore mother cell formation and tepal and stamen filament elongation; S3: microspore tetrad formation; S4: mononuclear microspores at the periphery and rapid tepal elongation and closing; S5: floral buds with mature pollen and mature embryo sacs before blossom. Scale bar: 100 µm. Different lowercase letters indicate significant differences (p < 0.05, LSD).
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Figure 5. The phenotypes and gene expression of FataAP2-, FataAP1-, and FataTOE-silenced F. tataricum. (A) The efficiency of gene silencing and phenotype statistics. (B) Flower phenotype comparison between TRV2-empty-treated tartary buckwheat and gene-silenced F. tataricum. (WT) Normal tartary buckwheat flower with five tepals, eight stamens (five outer-whorl stamens and three inner-whorl stamens), and three styles; (TRV2-FataAP2, Type I) TRV2-FataAP2-treated tartary buckwheat flower with normal five tepals and three styles, but with three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and one outer-whorl stamen with pollen sac abortion; (TRV2-FataAP1, Type I) TRV2-FataAP1-treated tartary buckwheat flower with normal five tepals and three styles, but with two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and one outer-whorl stamen with pollen sac abortion; (TRV2-FataTOE, Type I) TRV2-FataTOE-treated tartary buckwheat flower with normal five tepals and three styles, but with two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and two outer-whorl stamens with abnormal anthers; (TRV2-empty) TRV2-empty-treated tartary buckwheat flower with five tepals, eight stamens (five outer-whorl stamens and three inner-whorl stamens), and three styles; (TRV2-FataAP2, Type II) TRV2-FataAP2-treated tartary buckwheat flower with six tepals (increased tepal number), two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and three outer-whorl stamens with abnormal anthers and three normal styles; (TRV2-FataAP1, Type II) TRV2-FataAP1-treated tartary buckwheat flower with six tepals (increased tepal number), three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and two outer-whorl stamens with abnormal anthers and three normal styles; (TRV2-FataTOE, Type II) TRV2-FataTOE-treated tartary buckwheat flower with six tepals (increased tepal number), three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and two outer-whorl stamens with abnormal anthers and three normal styles. tep—tepal; ant—anther; fil—filament; sfi—short filament; san—shrivelled anther; spc—shrivelled pollen sac; sty—style. Scale bar = 1 mm. (C) Expression levels of FataAP2, FataTOE, and FataAP1 in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataAP2-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. (D) Expression levels of FataTOE and FataAP2 in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataAP2-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. (E) FataTOE expression level in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataTOE-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. The grey column shows gene expression in TRV2-empty-treated tartary buckwheat flowers; the blue column shows gene expression in FataAP2-silenced tartary buckwheat flowers; the green column shows gene expression in FataAP1-silenced tartary buckwheat flowers; and the pink column shows gene expression in FataTOE-silenced tartary buckwheat flowers (* p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 5. The phenotypes and gene expression of FataAP2-, FataAP1-, and FataTOE-silenced F. tataricum. (A) The efficiency of gene silencing and phenotype statistics. (B) Flower phenotype comparison between TRV2-empty-treated tartary buckwheat and gene-silenced F. tataricum. (WT) Normal tartary buckwheat flower with five tepals, eight stamens (five outer-whorl stamens and three inner-whorl stamens), and three styles; (TRV2-FataAP2, Type I) TRV2-FataAP2-treated tartary buckwheat flower with normal five tepals and three styles, but with three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and one outer-whorl stamen with pollen sac abortion; (TRV2-FataAP1, Type I) TRV2-FataAP1-treated tartary buckwheat flower with normal five tepals and three styles, but with two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and one outer-whorl stamen with pollen sac abortion; (TRV2-FataTOE, Type I) TRV2-FataTOE-treated tartary buckwheat flower with normal five tepals and three styles, but with two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains) and two outer-whorl stamens with abnormal anthers; (TRV2-empty) TRV2-empty-treated tartary buckwheat flower with five tepals, eight stamens (five outer-whorl stamens and three inner-whorl stamens), and three styles; (TRV2-FataAP2, Type II) TRV2-FataAP2-treated tartary buckwheat flower with six tepals (increased tepal number), two outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and three outer-whorl stamens with abnormal anthers and three normal styles; (TRV2-FataAP1, Type II) TRV2-FataAP1-treated tartary buckwheat flower with six tepals (increased tepal number), three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and two outer-whorl stamens with abnormal anthers and three normal styles; (TRV2-FataTOE, Type II) TRV2-FataTOE-treated tartary buckwheat flower with six tepals (increased tepal number), three outer-whorl stamens consisting of short filaments and abnormal anthers (shrivelled anthers without pollen grains), and two outer-whorl stamens with abnormal anthers and three normal styles. tep—tepal; ant—anther; fil—filament; sfi—short filament; san—shrivelled anther; spc—shrivelled pollen sac; sty—style. Scale bar = 1 mm. (C) Expression levels of FataAP2, FataTOE, and FataAP1 in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataAP2-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. (D) Expression levels of FataTOE and FataAP2 in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataAP2-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. (E) FataTOE expression level in the TRV2-empty-treated tartary buckwheat flowers and TRV2-FataTOE-treated tartary buckwheat flowers with obvious floral phenotypic changes, respectively. The grey column shows gene expression in TRV2-empty-treated tartary buckwheat flowers; the blue column shows gene expression in FataAP2-silenced tartary buckwheat flowers; the green column shows gene expression in FataAP1-silenced tartary buckwheat flowers; and the pink column shows gene expression in FataTOE-silenced tartary buckwheat flowers (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Figure 6. The regulatory cascade involved in tepal and stamen development in F. tataricum and LH F. esculentum. (A) The FataAP2-FataAP1-FataTOE regulatory cascade regulating tepal numbers and outer whorl stamen development in F. tataricum. (B) The FataTOE-FaesAP1_2 module regulating tepal numbers and stamen development in LH F. esculentum.
Figure 6. The regulatory cascade involved in tepal and stamen development in F. tataricum and LH F. esculentum. (A) The FataAP2-FataAP1-FataTOE regulatory cascade regulating tepal numbers and outer whorl stamen development in F. tataricum. (B) The FataTOE-FaesAP1_2 module regulating tepal numbers and stamen development in LH F. esculentum.
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MDPI and ACS Style

Li, S.; Yan, Y.; Liu, H.; Wang, A.; Liu, Z. The FataAP2-FataAP1-FataTOE Regulatory Cascade Regulates Floral Organ Development in Fagopyrum tataricum. Agronomy 2026, 16, 1635. https://doi.org/10.3390/agronomy16171635

AMA Style

Li S, Yan Y, Liu H, Wang A, Liu Z. The FataAP2-FataAP1-FataTOE Regulatory Cascade Regulates Floral Organ Development in Fagopyrum tataricum. Agronomy. 2026; 16(17):1635. https://doi.org/10.3390/agronomy16171635

Chicago/Turabian Style

Li, Shengchun, Yuhan Yan, Haonan Liu, Anhu Wang, and Zhixiong Liu. 2026. "The FataAP2-FataAP1-FataTOE Regulatory Cascade Regulates Floral Organ Development in Fagopyrum tataricum" Agronomy 16, no. 17: 1635. https://doi.org/10.3390/agronomy16171635

APA Style

Li, S., Yan, Y., Liu, H., Wang, A., & Liu, Z. (2026). The FataAP2-FataAP1-FataTOE Regulatory Cascade Regulates Floral Organ Development in Fagopyrum tataricum. Agronomy, 16(17), 1635. https://doi.org/10.3390/agronomy16171635

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