1. Introduction
The Bromeliaceae family, which consists of 3248 species in 58 genera [
1,
2], is one of the most morphologically diverse families with a pantropical distribution [
3]. Due to the astonishing flavour and fragrance of certain fruits, some species, such as pineapple (
Ananas comosus), were domesticated in northern South America possibly more than 6000 years ago [
4,
5]. Except for the edible pineapple, the vast majority of bromeliads cultivated worldwide are appreciated mainly for their ornamental value. Many bromeliads utilize crassulacean acid metabolism (CAM), a photosynthetic carbon fixation pathway that permits them to be highly productive in water-limited environments [
5,
6]. Thus, bromeliads, such as pineapple, are subtropical and tropical flowering plants with significant economical importance [
7].
The onset of flowering is critical during the transition from the vegetative to reproductive phase in the plant lifestyle [
8]. To date, five genetic pathways relevant to flowering have been identified in the model species
Arabidopsis thaliana, namely, the photoperiod, vernalization, gibberellic acid, autonomous and ageing pathways [
9]. During this complex developmental process, a subset of flowering-promoter and -suppressor genes participate in multiple pathways, such as
APETALA2 (
AP2) and five other
AP2-like transcription factors (TFs), including
TARGET OF EAT 1 (
TOE1),
TOE2,
TOE3,
SCHLAFMÜTZE (
SMZ), and
SCHNARCHZAPFEN (
SNZ) [
10,
11,
12,
13]. microRNA172 (miR172), a small, non-coding RNA that can complement a region of its target genes, can post-transcriptionally repress all members of the AP2 family [
14,
15,
16,
17].
toe1 mutants have previously been shown to flower significantly early, and this effect was enhanced in
toe1toe2 double-mutants [
11,
14]. Additionally, neither
smz nor
snz mutants display any notable early flowering phenotypes, whereas
toe1toe2smzsnz quadruple mutants were found to flower even earlier than
toe1toe2 double mutants but continued to flower significantly later than plants that constitutively express miR172 [
12,
14,
15]. The hextuple mutant lacking all six
AP2 family genes exhibits even an earlier flowering phenotype than
toe1toe2smzsnz quadruple mutants, but its phenotype is similar to that of miR172-overproducing plants [
18]. The overexpression of all of these genes, except
TOE3, results in delayed flowering [
11,
12,
14,
18]. As floral repressors, the expression of
TOE1,
TOE2,
SMZ and
SNZ transcripts displays circadian periodicity with a peak in the early morning under long day conditions, repressing the expression of
Flowering Locus T (
FT), which is considered to be the florigen, in the early morning and afternoon [
13]. Further investigations demonstrated that at least TOE1 and SMZ were able to bind directly to the promoter sites of
FT, and the repression of
FT and other flowering integrators is essential to prevent flowering [
12,
13]. These investigations indicated that all six AP2 family members act redundantly to repress flowering.
As a gaseous hormone, ethylene is essential for regulating variable plant developmental processes, including flowering [
19,
20,
21]. In
Arabidopsis, the activation of ethylene production can reduce bioactive gibberellin acid (GA) levels by inhibiting the protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) and increasing the levels of the downstream TF Ethylene Insensitive 3 (EIN3) [
20,
22]. Reduced bioactive GA levels can then increase the accumulation of DELLA proteins, a subfamily of GRAS TFs, and delay flowering by inhibiting the upregulation of the floral inducers
LEAFY (
LFY) and
SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (
SOC1) [
20]. Interestingly, exogenous ethylene can induce the flowering of Bromeliads, such as
A. comosus and
Aechmea fasciata. Aviglycine ((
S)-
trans-2-amino-4-(2 aminoethoxy)-3-butenoic acid hydrochloride, AVG), an inhibitor of ethylene biosynthesis, can delay the natural flowering of pineapple (
A. comosus var.
comosus) [
23,
24]. The silencing of one of 1-amino-cyclopropane-1-carboxylate synthase (ACC synthase) genes,
ACACS2, can also delay flowering in
A. comosus [
25]. These studies demonstrated that exogenous ethylene mediates the flowering of
A. comosus by stimulating the synthesis of endogenous ethylene. However, the precise mechanism by which ethylene induces flowering in bromeliads remains unknown, posing a large obstacle to shorten the long generation cycle and flowering time and culture new varieties using attractive genetic engineering tools [
6,
26].
In this study, we provide the first report of the isolation and functional characterization of AfAP2-1, an AP2-like gene in A. fasciata, a popular ornamental flowering bromeliad. Transactivation assays in yeast demonstrated that the activation domain is located in the C-terminal region. Furthermore, the expression of AfAP2-1 transcripts responded to plant age and ethylene treatment in A. fasciata and that the overexpression of AfAP2-1 in Columbia-0 (Col-0) background Arabidopsis (Wild Type, WT) significantly delayed flowering in both short-day (SD) and long-day (LD) conditions. These results all support the conclusion that AfAP2-1, a TF of the AP2 family, may play a pivotal role in regulating flowering time in Arabidopsis.
3. Discussion
The members of the APETALA2/Ethylene Responsive Element Binding Protein (AP2/EREBP) superfamily, which contain at least one AP2 domain, are responsive to multiple environmental stimuli and represent conservatively widespread TFs in the control of growth and developmental programs of the plants, protists, cyanobacteria and fungi [
27,
28]. The AP2/EREBP superfamily can be divided into the AP2 and EREBP families according to the number of AP2 domains. Specifically, members of this superfamily that are characterized by a tandem repeat of two AP2 domains are part of the AP2 family [
28]. The AP2 family is further divided into two groups, AP2 and ANT, based on differences in the amino acid residues of the double AP2 domains and the NLS [
28,
29].
In this study, we provided the first report of the isolation of an
AP2-like gene,
AfAP2-1, from
A. fasciata. A sequence analysis identified two conserved AP2 domains, a highly conserved putative NLS adjacent to AP2R1 and three other conserved motifs that have been identified in several AP2-like proteins (
Figure 1B). These conserved domains and motifs may be essential for the functional conservation and evolution of AfAP2-1 [
10,
29,
30,
31] (
Figure 1A,B).
3.1. Overexpression of AfAP2-1 in Arabidopsis Strongly Delayed Flowering under both LD and SD Conditions
Two copies of AP2 domains separated by a spacer region confer AP2 subfamily members with the ability to bind the promoter region of target genes [
32]. Our results indicated that AfAP2-1 plays a role as a transactivator and that the activation domain is located in the C terminus, which did not contain two AP2 domains (
Figure 4B). This result contradicted that obtained for AP2 isolated from
Brassica napus, whose transcriptional activity was localized to the N terminus [
33]. This difference indicates structural divergence during AP2 evolution.
TFs regulate the expression of numerous genes to mediate many patterning processes in the plant kingdom by binding to specific sites or motifs of DNA sequences [
34,
35]. The AP2/EREBP TF family, which contains nearly 7% of all TFs in
Arabidopsis, are involved in multiple signalling processes [
36]. Previous studies demonstrated that all six
AP2 family genes in
Arabidopsis act redundantly to repress flowering [
11,
12,
14,
18], and at least TOE1 and SMZ can repress floral induction by inhibiting the photoperiod pathway [
12,
13]. To investigate the role of
AfAP2-1 in floral induction, we overexpressed this gene in
Arabidopsis. Our results show that the heterogeneous overexpression of
AfAP2-1 in
Arabidopsis significantly delayed flowering time under both the LD and SD conditions (
Figure 6A,B). Furthermore,
AfAP2-1-OX transgenic lines contained significantly more rosette leaves than WT plants (
Figure 6C,D), indicating that
AfAP2-1 may play an important role in promoting the vegetative growth of
Arabidopsis. TOE1 can bind to an AT-rich element in the
FT promoter near the CONSTANS (CO)-binding site to convey a photoperiodic signal to regulate flowering time [
13]. Our study identified several AT-rich elements in the promoter of
AfFT2, a putative florigen in
A. fasciata, implying the putative direct binding site(s) of members of the AP2 subfamily, such as AfAP2-1 (data not shown). Furthermore, the relative expression of
AfAP2-1 was under the control of the circadian rhythm (
Figure 3C), similar to the control of
TOE1,
TOE2,
SMZ and
SNZ [
13]. This regulation implies that AfAP2-1 proteins may function not only in the morning and afternoon but also late at night (
Figure 3C). Further investigation should be focused on verifying the interactions between AfAP2-1 and
FT-like genes or other flowering integrators in
A. fasciata.
3.2. The Abundance of AfAP2-1 Transcripts Is Negatively Regulated by Exogenous Ethylene Treatment
Although ethylene is widely used to induce the flowering of bromeliads to both avoid the desynchrony of natural flowering and reduce time and manpower costs, this forced flowering also depends on the plant development age,
i.e., adult plants can successfully bolt, whereas juvenile plants cannot. To date, the precise mechanism by which ethylene induces flowering is unknown. Our previous study identified several members of the AP2/EREBP family that may be involved in both the ethylene-responsive and age-dependent flowering pathways [
37], indicating that
AP2-like genes may play a role in this complex process. Jung [
11] demonstrated that the transcript levels of
TOE1 and
TOE2 gradually decreased throughout plant growth.
CfTOE1 is also involved in age-dependent flowering in
Cardamine flexuosa [
38]. In our study, the expression of
AfAP2-1 also depended on the plant development age; it was relatively high in juvenile plants, decreased in adult plants prior to flower bud differentiation, and further decreased in 39-DAF adult plants (
Figure 3A). To date, the correlation between ethylene and AP2 subfamily members has not yet been investigated. Here, we showed that the transcripts levels of
AfAP2-1 in all detected organs dramatically decreased after treatment with 400 μL·L
−1 of ethrel for 1 h and were almost undetectable after 6 h, indicating the rapid response of
AfAP2-1 to exogenous ethylene treatment both in juvenile and in adult plants (
Figure 3D). Exogenous ethylene treatment after 6 h also strongly induced
AfFT2 expression in central leaves and stems (unpublished data). The response time delay of
AfFT2 compared with that of
AfAP2-1 implied that
AfAP2-1 may be upstream of
AfFT2. These results all indicate that a decrease in the expression of
AfAP2-1 below a threshold may induce bolting in adult bromeliads treated with exogenous ethylene, but not juveniles, or that some other regulators may be involved in this complex process. Further studies should focus on verifying the precise mechanism by which ethylene affects
AfAP2-1.
In conclusion, our results indicate that AfAP2-1 is an AP2 family gene expressed in A. fasciata. The overexpression of AfAP2-1 in Arabidopsis significantly delayed flowering, implying that the molecular mechanism by which AfAP2-1 affects flowering may be partially conserved between Arabidopsis and A. fasciata. Further analyses of the interactions between ethylene-responsive factors and AfAP2-1 and transgenic plants of A. fasciata will help us to better understand the function of AfAP2-1 in flowering and provide a theoretical basis for the genetic engineering of A. fasciata and possibly other species of bromeliads.