The study of many AP1/FUL-like genes from various species has demonstrated that AP1/FUL genes play key roles in flowering time, flower and fruit development. Like genes APETALLA3 (AP3, B class gene) and AGAMOUS (AG, C class gene), the AP1/FUL genes undergo several duplication events, resulting in the occurrence of euAP1 and euFUL clade in core eudicots [13, 14]. In this study, five AP1/FUL-like genes were obtained from marigold. Sequence alignment analysis indicated that all these 5 AP1/FUL-like proteins are typical MIKC proteins, and they contained conserved motif at their C terminal domain (Fig. 1). TeAP1-1 and TeAP1-2 were clustered into euAP1 clade proteins harboring an acidic domain and a farnesylation motif (Fig. 1), and the TeFUL1, TeFUL2 and TeFUL3 possessed a conserved FUL motif (Fig. 1) that was demonstrated to be all members of FUL clade proteins [13, 14]. Such changes in amino acid sequence have been explained by a frameshift mutation in an ancestral AP1/FUL-like gene [13, 43] and are responsible for gene-specific functions.
Our phylogenetic analysis indicated that TeAP1-1 and TeAP1-2 were members of AP1 clade, and seemed to be homologous to antirrhinum SQUA which was previously reported to be involved in regulating the floral meristem development and specifying the sepal and petal identity [44]. TeFUL1, TeFUL2 and TeFUL3 were clustered into the FUL clade, and TeFUL1 and TeFUL3 proteins were closer to the euFUL group. TeFUL2 belonged to the euFULII group (Fig. 2). The TeFUL2 was orthologous to the antirrhinum gene DEFH28 which was also clustered into euFULII group, and this gene participated in regulation of floral meristem development, fruit development, and flowering time [34]. Base on the expression pattern analysis, the TeFUL2 was mainly expressed at the early stage of inflorescence development (Fig. 3, S1), which was similar to the expression pattern of the early function genes represented by Arabidopsis FUL [21] and petunia PFG [36], implying a role of TeFUL2 in meristem identity. However, TeFUL1 and TeFUL3 were expressed in vegetative tissues, different stages of floral buds, and floral organs (Fig. 3, S1). Based on these findings, it could be speculated that TeFUL2 and TeFUL1(or TeFUL3) might arise due to gene duplication, and that this duplication event might cause the change in their expression patterns. Many previous studies reveal that functional divergence is caused by gene duplication which further drives evolution [10, 45]. Therefore, we speculated that the duplication events and the modification of transcript pattern of TeFUL genes might imply the diversification of their functions in marigold.
Conserved function of AP1/FUL genes in flowering performance
Function analysis of the AP/FUL-like genes in core eudicots and non-core eudicots revealed that AP1/FUL-like genes displayed conserved roles in regulating the flowering time. For example, overexpression of AP1 or FUL in Arabidopsis both leads to early flowering [25, 46]. Furthermore, the similar phenomena were also observed in the case of ectopic overexpression of AP1-like or FUL-like genes from Asteraceae species, such as C. morifolium (CDM111) [39], C. lavandulifolium (ClM8) [38] and G. hybrida (GSUQA2 ) [37]. In this study, heterologous expression of TeAP1-2 and TeFUL2 into Arabidopsis resulted in early flowering without affecting floral organ identity (Fig. 4d, f, h). Additionally, ectopic expression of TeAP1-2 also led to the curl of rosette leaf and cauline leaf (Fig. 4e, g), which was similar to the function of the AP1/FUL-like gene MBP20 [47]. The MADS-box transcription factors possess a DNA-binding domain to regulate their downstream gene expression [45]. Therefore, we speculated that the early flowering phenotypes observed in 35S:TeAP1-2 and 35S:TeFUL2 transgenic lines might be related to the change in endogenous gene expression level. In this study, AP1, FT, LFY, SOC1, SPE3, and TFL were significantly up-regulated in 10-day-old seedlings of transgenic lines containing 35S:TeAP1-2 and 35S:TeFUL fusion vectors (Fig. 5), suggesting TeAP1-2 and TeFUL2 might share overlapping regulation network of a series of downstream genes in Arabidopsis. In Arabidopsis, AP1 directly represses SVP, AGL24, and SOC1 to partially specify floral meristem identities [48]. However, in our study, no remarkable change in the expression level of AGL24 was observed in both 35S:TeAP1-2 and 35S:TeFUL2 transgenic lines (Fig. 5). Additionally, the expression level of the flowering repressor gene SVP was significantly activated in 35S:TeFUL2 transgenic lines, but not in 35S:AP1-2 transgenic lines (Fig. 5). In contrast to SVP, SPL9 was significantly upregulated in 35S:TeAP1-2 transgenic lines, but not in 35S:FUL2 transgenic lines (Fig. 5). These results revealed that TeAP1-2 and TeFUL2 had a divergent function in regulating downstream genes, which was further supported by their difference in protein interaction manners (Table 1, Supplementary Fig. S2b-e).
Potential redundant function of TeAP1-1 and TeAP1-2 as class A genes
In Arabidopsis, AP1 is an early-acting gene and functions as an class A gene to specify sepal and petal identity [20, 49]. AP1 is expressed in floral meristems and developing sepal and petal primordia [20, 21, 24, 50]. However, in other core eudicots, the AP1-like genes can be also expressed in bracts and reproductive organs [27, 39, 51]. Similarly, TeAP1-1 and TeAP1-2 were both highly expressed in sepals of two-type florets and petals of disk florets as well as in bracts, receptables, and ovules (Fig. 3, S1). In Arabidopsis, AP1 only interacted with SEP3 to form heterodimer. Furthermore, in Asteraceae species, the AP1-like proteins C. morifolium CDM111 [39, 40], G. hybrida GSQUA1, and GSQUA3 [37] also had a limited protein interaction manner. In other words, they only formed heterodimers with SEP3 proteins. In this study, TeAP1-1 and TeAP1-2 shared a similar protein interaction pattern to form heterodimers with TeSEP3-2 and TeSEP3-3 (Table 1, Supplementary Fig. S2b, e), suggesting that euAP1-like proteins shared a conserved protein interaction manner. Taken together, TeAP1-1 and TeAP1-2 may play a redundant role as class A genes.
Divergent functions among TeFULs genes
The functions of FUL-like genes in the transition from vegetative meristems to reproductive meristems and in fruit development were well-known in many core eudicots and non-core eudicots. In model plant Arabidopsis, FUL regulates the cell differentiation during fruit development [31, 33, 52] and participates in specifying floral meristem identity with AP1 and CAL [21]. In basal eudicots, the Aquilegia coerulea FUL-like genes regulate leaf morphogenesis and inflorescence development [8]. Additionally, in monocots, the Oryza sativa homologues genes OsMADS14 and OsMADS15 are involved in specifying the meristem identity, palea and lodicule identity [7]. In contrast to the AP1-like genes, the FUL-like genes are widely expressed in vegetative and productive tissues [6, 31, 46].
In our study, TeFUL1 and TeFUL3 were expressed in stems and leaves as well as in productive tissues (Fig. 3, S1), which was consistent with the typical FUL-like expression pattern [6, 31, 46], implying that TeFUL1 and TeFUL3 might play a role as FUL genes. Furthermore, ectopic expression of TeFUL1 or TeFUL3 into Arabidopsis led to no visible phenotype changes. In Arabidopsis, FUL functions redundantly with CAL and AP1 to specify the floral meristem identity, and single ful mutation has no ability to affect floral organ identity [21]. In general, we speculated that TeFUL1 and TeFUL3 might function redundantly in regulating the floral meristem identity, or that TeFUL1 and TeFUL3 need to work together with AP1-like genes to regulate the floral meristem development. However, the striking difference in protein interaction manner was observed between TeFUL1 and TeFUL3 (Table 1, Supplementary Fig. S2b-e). TeFUL1 only interacted with TeAGL6, while TeFUL3 interacted with TeSEP1, TeSEP3-1, TeSEP3-2, TeSEP3-3, and TeSEP4 to form heterodimers (Table 1, Fig. S2b-e). Different protein interaction patterns might be related to their different conserved regions at C domains (Fig. 1). The above results suggested that TeFUL1 and TeFUL3 might be partially functionally redundant, but they might have their own specific functions in regulating floral organ identity.
In contrast to TeFUL1 and TeFUL3, TeFUL2 was highly expressed in floral buds and vegetative tissues, and weakly expressed or unexpressed in floral organs and ovules (Fig. 3, S1). Additionally, TeFUL2 could form homodimer, and heterodimers with TeAGL6 and TeSEP3-2 (Table 1, Supplementary Fig. S2b, e). Ectopic expression of TeFUL2 into Arabidopsis also led to early flowering with less number rosette leaves (Fig. 4h), which was consistent with phenotype of the overexpressed euFULII (DEFH28) clade genes from core eudicots and non-core eudicots [28, 37]. The above results suggested that FUL1 and FUL3 might lose some functions, but these functions might have been retained in FUL2. Overexpression of Antirrhinum DEFH28 (euFULII clade genes) into Arabidopsis resulted in early flowering, two to four carpel formation, and failure to silique dehiscence [28]. However, ectopic expression of TeFUL2 into Arabidopsis did not affect floral organ identity and silique dehiscence (Fig. 4), which was in line with the study results of Gerbera GSQUA2. In general, TeFUL2 might retain a conserved role in regulating the meristem transition rather than fruit ripping.