Flowering, the symbol of vegetative development to reproductive development is an important factor to ensure the success of plant reproduction (Khan et al., 2014). The transition from vegetative stage to reproductive stage is precisely regulated by external environmental signals and internal developmental states (Albani and Coupland, 2010; Wang et al., 2021). Appropriate flowering time is crucial for the crop yield and quality (Roux et al., 2006). Flowering studies are the most extensive in Arabidopsis, and mainly involved photoperiod, vernalization, gibberellin, autonomic, ambient temperature and age-related pathways (Albani and Coupland, 2010; He, 2012; Cheng et al., 2017). Flowering is a very complex physiological process, which is regulated and coordinated by multiple genes (Liljegren et al., 1999).

BBX transcription factors have a wide range of functions and play important roles in flower initiation, light morphogenesis, anthocyanin synthesis and abiotic stress tolerance (Almada et al., 2009; An et al., 2020; Li et al., 2021). For example, CmBBX24 regulates flowering time and tolerance to freezing and drought stress in chrysanthemum by regulating GA biosynthesis (Yang et al., 2014). AtBBX20 interacts with HY5 to activate the gene expression and promote light morphogenesis (Wei et al., 2016). MdBBX22 and MdBBX33 in apple are involved in MDHY5-mediated signal transduction and regulate anthocyanin accumulation (An et al., 2019; Plunkett et al., 2019). CONSTANS (CO) in Arabidopsis is the first BBX protein to be identified in plants. Flowering locus T (FT) genes receive signals from the photoperiodic regulatory center CO under long day conditions, which drives the transition from vegetative to reproductive growth in Arabidopsis (Putterill et al., 1995). However, CO could also inhibit FT-induced flowering by affecting TERMINAL FLOWER1 (TFL1) expression under short-day conditions (Luccioni et al., 2019). Interestingly, in rice, Hd1 is a CO homolog that promotes flowering under short-day conditions but inhibits flowering under long-day conditions (Yano et al., 2000; Kojima et al., 2002; Hayama et al., 2003). In flower formation pathway, BBX protein is strongly conserved among different plants. In addition to CO, other BBX proteins are also involved in the regulation of flower formation in plants. In Arabidopsis, both AtBBX6 and AtBBX24 are positive regulators of flower formation (Hassidim et al., 2009; Li et al., 2014), while AtBBX4, AtBBX7 and AtBBX32 delay flower formation (Cheng and Wang, 2005; Tripathi et al., 2017). OsCOL4, OsBBX14 and OsCOL9 in rice delayed the heading through repressing the Ehd1 pathway under SD and LD conditions (Lee et al., 2010; Bai et al., 2016; Liu et al., 2016). BvCOL1 in sugar beet also causes early flowering under LD conditions (Chia et al., 2008).

Pineapple is one of the world’s famous tropical fruits. Spraying ethephon to induce flower formation is currently the most widely used method in pineapple production (Min and Bartholomew, 1997). At present, there are some excellent pineapple varieties that cannot be widely promoted in the market due to the difficulty in regulating the maturation time (Turnbull et al., 1999). Therefore, studying the molecular mechanism of flower formation induced by ethylene in pineapple can provide theoretical support for perinatal regulation and new variety cultivation. In recent years, several genes regulating flowering such as AcERS and AcETR (Li et al., 2016), AcERF (Zhang et al., 2021a), AcTrihelix (Wang et al., 2022), AcBBX (Ouyang et al., 2022) have been isolated from pineapple, but however, the research on its functional mechanism is still not in-depth.

BBX genes are involved in the determination of flowering time through photoperiod, and there are few studies on flower formation in response to the hormone regulation (Putterill et al., 1995; Yano et al., 2000). Previous studies have found that AcBBX5 was found in the pineapple BBX family study to have an expression peak at 12 h and 7 w after ethylene induced flowering of pineapple. AcBBX5 may be involved in ethylene induced flower formation and flower morphogenesis in pineapple (Ouyang et al., 2022). Here, AcBBX5 was identified from the BBX family analysis of pineapple, and found it may regulate flower formation of pineapple. The sequence characteristics and expression characteristics of AcBBX5 were analyzed by bioinformatics, subcellular localization and transcriptional activation assay. AcBBX5 overexpression, expression characteristics and regulatory mechanism were further analyzed. All these data lay a foundation for the further study of floral formation regulation network in pineapple.

The BBX gene family is widely involved in plant growth and development and response to the environment. AcBBX5 is a member of the pineapple AcBBX family (Ouyang et al., 2022). Here, conserved domain analysis confirmed that AcBBX5 and other orthologous proteins contain two conserved B-box domains at the N-terminus ( Figure 1A ). Motif analysis identified two conserved B-box domains containing a different conserved motif ( Figure 1B ). Based on the difference in amino acid sequence identity of the b-box motif and the specificity of zinc binding amino acid residues, it is divided into two types: B-box 1 and B-box 2 (Crocco and Botto, 2013). Plant B-box domains can form heterodimers within the BBX protein family or with other proteins, and play an important role in mediating protein interactions and regulating the gene expression (Gangappa et al., 2013). For example, PpBBX18 in pear forms a heterodimer with PpHY5 via two b-box domains, thereby inducing PpMYB10 transcription and regulating anthocyanin biosynthesis (Bai et al., 2019). Subcellular localization analysis showed that AcBBX5 protein was located in the nucleus of epidermal cells of Nicotiana benthamiana leaves ( Figure 2 ) and had transcriptional activation activity in yeast ( Figure 3 ), suggesting that AcBBX5 gene has general characteristics of transcription factor and may be involved in the transcription level of other genes. However, Figure S1 shows that the sequence length of AcBBX5 was significantly shorter than the others. In addition, except for AcBBX5, which has only 3 conserved motifs, other homologous proteins have at least 6 conserved motifs ( Figure 1A ). These imply that the function of AcBBX5 may be different from that of homologous genes in other species.

BBX genes are involved in the determination of flowering time (Putterill et al., 1995; Yano et al., 2000). AtBBX24, CmBBX8 and HvCO1 are the BBX genes that have been shown to promote flower formation. AtBBX24 overexpression not only reduces the expression level of FLC, but also activates FT and SOC1 expression, which leads to early flowering in Arabidopsis under long- and short-day conditions (Li et al., 2014). In summer chrysanthemum, overexpression of CmBBX8 accelerated flowering under long- and short-day conditions. CmFTL1 can act as floral inducer in long day conditions, and CmBBX8 promotes flowering through binding with CORE element (CCACA) of the CmFTL1 promoter (Wang et al., 2020). In barley, HvCO1 overexpression up-regulated HvFT1, which may promote flowering by activating HvFT1. But it has no promoting effect on Arabidopsis thaliana (Armstead et al., 2005). In this study, overexpression of AcBBX5 gene in Arabidopsis delayed flowering time ( Figure 4 ). BBX has also been reported to inhibit floral formation in other species. In rice, OsBBX14 delays heading date via different ways under long and short-day conditions. OsBBX14 delays heading date by promoting the expression of Hd1 under long day conditions. However, under short day conditions, it acts as a repressor of Ehd1 to delay heading date (Bai et al., 2016). CmBBX24 may inhibit flowering in Chrysanthemum Morifolium by negatively regulating the expression of GA biosynthetic genes (GA20ox and GA3ox) and photoperiodic flowering pathway genes (GI, PRR5, CO, FT and SOC1) (Yang et al., 2014). OsBBX14 and CmBBX24, which inhibit flower-forming genes, cluster on the same branch of the evolutionary tree with AcBBX5 ( Figure 1D ), suggesting that genes of the same classification or more closely related genes may have similar functions.

Molecular studies on flowering have focused on FT, FLC, CO and SOC1 genes. In addition to AtBBX24 (Li et al., 2014), which has been shown to regulate FLC, FT and SOC1, AtBBX7/AtCOL9 has been reported to inhibit CO and FT expression and delay floral transition in Arabidopsis (Cheng and Wang, 2005). The heterodimerization between BBX28 and CO affects the activation of FT transcription by CO, which negatively regulates Arabidopsis flower formation (Liu et al., 2020). In this study, the relative expression levels of positive regulators of flower formation (FT, SEP3, SOC1 and FUL) were inhibited in Arabidopsis transgenic lines, while negative regulators of flower formation (FLC and SVP) were higher than control lines ( Figure 5 ). This further confirmed that AcBBX5 overexpression inhibited flowering in Arabidopsis. However, the relative expression of CO in the Arabidopsis transgenic lines was almost no different from that in the control, indicating that AcBBX5 does not negatively regulate flower formation by inhibiting CO expression.

Yeast one-hybrid and dual luciferase assay results found that AcBBX5 is transcriptional activator ( Figure 3 ), but it can bind to the promoter of FT gene and inhibit its expression ( Figure 7 ). Similarly, previous studies on transcription factors in other species have also confirmed that transcription factors with transcriptional activation activity can also act as repressors under certain conditions. In grape hyacinth, MaBBX51 interfered with the binding of MaHY5 to the promoters of MaMybA and MaDFR, thereby inhibiting anthocyanin biosynthesis (Zhang et al., 2022). VvMYB30 in grapevine competes with activator VvMYB14 to bind to common binding sites in VvSTS15/21 promoter, controls stilbene biosynthesis in grapevine (Mu et al., 2022). In grape, VvMYB30 competed with the activator VvMYB14 to bind to characteristic binding sites in the VvSTS15/21 promoter to effect stilbene biosynthesis (Mu et al., 2022). In Arabidopsis, AtBBX24 inhibited anthocyanin accumulation by interfering with the binding of HY5 to the promoter of related genes in anthocyanin biosynthesis (Job et al., 2018). AtBBX19 negatively regulated flowering time by interfering with CO binding to FT through physical interaction with CO proteins (Wang et al., 2014). In view of this, BBX5 may act as a weak activator, which binds to the FT promoter of the target gene and occupies a limited binding site to block the binding of some strong activators, thus inhibiting the expression of FT gene. This speculation still needs further systematic experimental verification.

A trait is regulated by multiple genes, and a gene may be involved in the regulation of multiple traits. Here, AcBBX5 may not only regulate the flower formation of pineapple ( Figure 4 ), but also promote the growth and development of floral organs ( Figure 6 ). BBX gene is one of the many genes involved in flower organ growth and development. However, there are few studies on the BBX gene in flower organs. In rose, RhBBX28 is a key player in regulating petal senescence, and overexpression of RhBBX28 produces smaller flowers than WT (Zhang et al., 2021b). In the present study, AcBBX5 overexpression also showed differences in flower size in Arabidopsis. But in contrast to RhBBX28, overexpression of AcBBX5 produced larger flowers than the control ( Figure 6 ). Transcriptome and quantitative data showed that AcBBX5 was highly expressed in petals, suggesting that AcBBX5 may play a role in petal development. Further studies and sufficient experimental evidence are needed to prove the specific regulatory mechanism.

In this study, AcBBX5, a member of the BBX family, is identified as a negative regulator of floral formation in Arabidopsis and may be involved in floral organ development. Subcellular localization and transcriptional activation analysis showed that AcBBX5 was located in the nucleus and had transcriptional activation potential. The relative expressions of FT, SOC1, FUL and SEP3 were decreased, and FLC and SVP were increased in AcBBX5-overexpressing Arabidopsis. In addition, AcBBX5 was expressed in different floral organs of pineapple, but highly expressed in stamens, ovary and petals. Yeast one-hybrid and a dual luciferase assay results confirmed that AcBBX5 bound to AcFT promoter in pineapple and inhibited the expression of AcFT gene. These data will enrich the known regulatory network of BBX in different plants and provide information on the regulation of flowering and floral organ development in pineapple.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

YO and HZ wrote the manuscript and completed the experiments. XZ and HZ designed the experiment and contributed to data. YH took charge the experimental materials treatment and collection. YW investigated the data analyze of the study. YO and CW carried out RNA extraction and QRTPCR verification. XZ and ZL constructed the vectors and completed the transgenic experiments. HZ and XZ conceived the study. All authors contributed to the article and approved the submitted version.

The project was funded by the National Natural Science Fund of China (31872079 and 32160687), the National Key R&D Program of China (2019YFD1001105 and 2018YFD1000504), the Natural Science Foundation of Hainan Province (321RC467 and 322MS013), the major science and technology project of Hainan Province (ZDKJ2021014), and the Scientific Research Start-up Fund Project of Hainan University (KYQD-ZR-20090).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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