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Article

Identification and Functional Analysis of the Phosphatidylethanolamine-Binding Protein (PEBP) Gene Family in Liriodendron Hybrids

by
Miao Hu
1,2,
Lipan Liu
1,
Ping Hu
1,
Xiaoling Yu
1,
Hua Zhou
1,
Shujuan Liu
1,
Tengyun Liu
1,
Faxin Yu
1 and
Aihong Yang
1,*
1
Key Laboratory of Horticultural Plant Genetic and Improvement of Jiangxi Province, Institute of Biological Resources, Jiangxi Academy of Sciences, No. 7777, Changdong Road, Gaoxin District, Nanchang 330096, China
2
College of Forestry, Jiangxi Agricultural University, Nanchang 330045, China
*
Author to whom correspondence should be addressed.
Forests 2023, 14(10), 2103; https://doi.org/10.3390/f14102103
Submission received: 8 September 2023 / Revised: 13 October 2023 / Accepted: 18 October 2023 / Published: 20 October 2023
(This article belongs to the Section Genetics and Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
The plant phosphatidylethanolamine-binding protein (PEBP) gene family plays important roles in regulating flowering time and vegetative growth. Compared with its parents, Liriodendron hybrids (Liriodendron chinense (Hemsl.) Sarg. × L. tulipifera L.), have obvious heterosis in terms of higher seed germination, fast growth, bright flower colors, and long growth seasons. However, the genome-wide identification and functional analysis of PEBP genes that contribute to the heterosis of Liriodendron hybrids have not been studied. In this study, we characterized four members of expressed LhPEBP genes in Liriodendron hybrids and divided them into three subfamilies based on their phylogenetic relationships: FT-like (LhFT), TFL1-like (LhTFL1), and MFT-like (LhMFT1 and LhMFT2). A functional analysis of Arabidopsis showed that the overexpression of LhFT significantly promoted flowering, and the LhTFL1 gene induced a wide dispersion of the flowering timing. LhMFTs function differently, with LhMFT2 suppressing flowering, while LhMFT1 accelerates it and had a stronger promoting effect on the early stage of seed germination. Additionally, the seed germination of the LhMFT lines was relatively less influenced by ABA, while the transgenic LhFT and LhTFL1 lines were sensitive to both ABA and GA3. These results provide valuable insights into the functions of LhPEBP genes in flowering and seed germination.

1. Introduction

The phosphatidylethanolamine-binding protein (PEBP) gene family is widely distributed among plants, animals, and microorganisms [1], and functions as a critical regulator of diverse signal transduction pathways, governing growth and differentiation [2,3]. The plant PEBP gene family is closely associated with flowering and differentiation, serving as a key modulator of the transition from vegetative to reproductive growth and contributing to the determination of plant architecture [4,5]. The family is classified into three principal branches: FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER1 (TFL1)-like, and MOTHER OF FT AND TFL1 (MFT)-like genes [6].
FT-like and TFL1-like genes are well studied and are mainly involved in plant flowering and phenological regulation [5,7,8]. FT and TFL1 share sequence similarity, and the related proteins encoded by FT and TFL1 have opposite effects on flowering [9]. The FT gene is a crucial regulator of flowering in plants, playing a pivotal role in integrating external and internal signals to modulate this developmental process [10,11]. Growing evidence has revealed that the FT homologous genes HvFT1 in barley [12], LsFT in lettuce [13], and RFT1 in rice [14] play crucial roles in plant flowering [15]. Notably, FT-like homologous genes also perform diverse functions in different species with respect to growth, development [16,17], seed germination, and response to temperature changes associated with seasonal characteristics [18,19]. TFL1 antagonizes FT [20]. The TFL1 homologous gene family plays a pivotal role in regulating the transition, controlling the switch from vegetative to reproductive growth, and regulating flowering time and inflorescence architecture [4,21]. TFL1 could inhibit the formation of flower primordia, thus delaying flowering [22]. Loss of TFL1 accelerates flowering in apples [23] and causes continuous flowering in roses [24]. Similarly, the overexpression of mango MiTFL1 genes in Arabidopsis delays flowering [25].
The MFT subfamily is the ancestor of the FT and TFL1 subfamilies [26]. In contrast with FT-like and TFL1-like genes, the function of MFT-like genes is less characterized. It is generally believed that MFT homologs participate in the regulation of plant flowering and seed germination. MFT has partial FT-like activity [27,28], but it mainly plays a critical role in regulating seed germination via the abscisic acid (ABA) and gibberellic acid (GA) signaling pathways [29]. For example, soybean GmMFT [30], cotton GhMFTs [31], and rice OsMFT2 [32] all inhibit seed germination by participating in GA and ABA signaling. In addition to its important roles in flowering and seed development, MFT is also involved in stress response and other functions. For example, OsMFT1 promotes drought tolerance in rice [33], MiMFT enhances stress response [28], and GmMFT positively regulates seed content and seed weight [34].
Liriodendron L. (Magnoliaceae) is a genus of perennial temperate deciduous trees. Species in this genus are tall trees with straight trunks and beautiful flowers and leaves. It is a Tertiary relic genus and now comprises only two species, Liriodendron chinense and L. tulipifera, showing a typical discontinuous distribution pattern in East Asia and North America [35]. L. chinense is an endangered species in China with a low germination rate and poor natural regeneration, while the Liriodendron hybrid (L. chinense × L. tulipifera), an interspecific hybrid offspring of L. tulipifera and L. chinense, has obvious heterosis in many aspects. Compared with its parents, it has higher seed germination, fast growth, stress resistance, and a longer growing season [36]. To date, no identification or functional analysis of PEBP genes that are partially related to the heterosis of Liriodendron hybrids has been explored. Therefore, in the present study, we characterized the expressed PEBP family genes in Liriodendron hybrids and performed a functional analysis by overexpressing the PEBP genes in Arabidopsis thaliana. The results of this study will provide valuable information for elucidating the characteristics and functions of LhPEBP in Liriodendron hybrids.

2. Materials and Methods

2.1. Identification, Characteristics and Phylogenetic Analysis of LhPEBPs

The annotated genome data of L. chinense were obtained from Hardwood Genomics (https://treegenesdb.org/ (accessed on 3 June 2021)). Arabidopsis PEBP gene data were obtained from TAIR (http://www.arabidopsis.org/ (accessed on 10 June 2021)). Then, the HMM profile of the PEBP gene (PF01161) was downloaded from the Pfam website (http://pfam.xfam.org/family/PF01161 (accessed on 10 June 2021)) and used as a query to identify all PEBP-containing domains in L. chinense by retrieving against the genome (E-value ≤ 1E-5) using HMMER v3.3.2. Finally, all candidate PEBP genes were verified by Pfam (http://pfam.xfam.org/search (accessed on 15 June 2021)), CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 15 June 2021)), and SMART (http://smart.emblheidelberg.de/ (accessed on 15 June 2021)) for further verification.
The protein sequences of FT, TFL1, and MFT from A. thaliana, Populus alba, and Liriodendron hybrids were initially aligned using muscle methods in Clustal W v. 2.1 [37]. The sequence differences and conserved motif analysis were performed in Jalview software v. 2.11.1.0 [38].
The 2000 bp upstream promoter sequences of PEBP genes were extracted from the L. chinense genomic database, which were used to search for putative cis-elements using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 3 July 2021)). The physical distribution of cis-acting elements was visualized using TBtools v. 1.089 [39].
PEBP family proteins covering herbaceous angiosperms, tree species of gymnosperms and angiosperms, were downloaded from the NCBI database. The protein sequences were first aligned and then used to construct the phylogenetic tree by maximum-likelihood (ML) methods in MEGA 6 v. 7.0.26 [40]. The bootstrap replication value was set to 1000, and the substitution model was the JTT+G substitution model.

2.2. Plant Materials and RNA Extraction

The plant materials used in the experiment were young buds with mixed flowers and young leaves of Liriodendron hybrids from the campus of Jiangxi Academy of Sciences (28.698° N, 115.992° E) in May 2020. Samples were first frozen in liquid nitrogen and stored at −80 °C.
Total RNA was extracted using a TRIzol kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions, dissolved in sterile ultrapure water, digested with gDNase, assessed by 1% agarose gel electrophoresis, and finally stored at −80 °C.

2.3. LhPEBP Gene Cloning and Vector Construction

The expressed LhPEBP genes were first amplified by reverse transcription polymerase chain reaction (RT‒PCR) methods as follows: the total RNA of the Liriodendron hybrid was used as a template and the first strand of cDNA was synthesized using the PrimeScriptTM II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The reaction system and conditions were set according to the kit instructions. The cDNA after the reverse transcription was used as a template for CDS amplification of LhPEBPs. The primer sequences are listed in the supplementary material (Table S1). The PCR products were purified by agarose gel electrophoresis, cloned, and inserted into the pMD 18-T vector (Takara) and confirmed by sequencing. Finally, the full-length coding sequences of LhPEBP genes were cloned and inserted into pBWA(V)BS expression vectors with the cauliflower mosaic virus 35S promoter.
Arabidopsis [Columbia-0 (Col-0)] was used in this study. The seeds were sown in plastic pots and grown in a phytotron under a long-day photoperiod (16 h of light/8 h of dark) at 24 °C. The constructed vectors were transformed into Col-0 by the Agrobacterium-mediated flower dip method. Transgenic Arabidopsis plants were screened by both spraying 15 mg/L ammonium-glufosinate on transgenic seedlings and PCR amplification verification using the corresponding primer sequences in Table S1. Seeds of transgenic lines were harvested and then sown in plastic pots under the above conditions and screened by ammonium-glufosinate and PCR amplification. The positive seeds were harvested and sown until T3 seeds were obtained.

2.4. Flowering and Germination Analysis in Arabidopsis

For flowering observation, T3 seeds of transgene lines overexpressing LhPEBP genes were sown in plastic pots and grown in a phytotron under a long day (LD) photoperiod pattern (16 h of light/8 h of dark) at 24 °C. The positive plants were screened by ammonium-glufosinate as described above.
For the germination analysis, T3 transgenic Arabidopsis seeds were first sterilized with 75% (v/v) alcohol for 8 min and washed three times with sterile water. The seeds were sown onto 1/2 MS. After three days of darkness at 4 °C, seeds were then transferred to a 16-h light/8-h dark photoperiod at 24 °C to examine seed germination. To explore the response to hormones, abscisic acid (ABA) and gibberellic acid (GA) were added to 1/2 MS at the following concentrations: GA3 1 μM and ABA 10 μM. Each type of transgenic line was represented by at least 100 seeds, and three independent experiments were conducted. Mutants with a loss-of-function in the MFT gene in Arabidopsis (mft mutants) and Col-0 were also sown as controls for flowering and germination analysis.

2.5. Data Collection and Statistical Analysis

The number of days from sowing to flowering for transgenic lines overexpressing LhPEBP genes, Col-0, and mft mutants of Arabidopsis was recorded. Additionally, the number of rosette leaves was recorded to compare flowering phenologies. Seed germination was also calculated under three different conditions: 1/2 MS, 1 μM GA3, and 10 μM ABA. To further clarify the germination progress, seed germination from day 1 to day 8 was recorded daily. Germination was defined as the number of germinated seeds divided by the total number of seeds sown.
All data were analyzed using SPSS v21.0. Significant differences were determined by ANOVA tests with a significance value of 0.05. All error bars in the figures represent the standard deviation (SD) values.

3. Results

3.1. Identification of PEBP Family Members in Liriodendron

Five PEBP genes were identified from the genome of L. chinense. Detailed information is shown in Table S1. The BLAST verification showed that the five PEBP genes belong to four types, including two MFTs, one FT, one TFL1, and one BFT. However, except for LcMFT1 (Lchi05661), the other four PEBP family genes of L. chinense were incomplete. The published CDS of FT (Lchi27713) was only 267 bp, and the CDS of TFL1 (Lchi10509), MFT2 (Lchi25487), and BFT (Lchi11903) was only 327 bp, 321 bp, and 258 bp, respectively (Table S1). Therefore, we further located those genes in the genome data of L. chinense and then extracted the 2000 bp sequences upstream and downstream of the reported gene location. Finally, we found the CDS through FGENESH (http://www.softberry.com/berry.phtml?topic=fgenesh&group=programs&subgroup=gfind (accessed on 20 June 2021)) and checked the domain by the CD-Search confirmation method. The full-length CDSs of the five PEBP gene family members of L. chinense were then obtained. We used primer3 software [41] (http://frodo.wi.mit.edu/primer3/ (accessed on 3 July 2021)) to design primers that covered the gene promoter and the terminator. Finally, four PEBP gene family genes of Liriodendron hybrids were successfully cloned from the mRNA, including one FT, one TFL1, and two MFT genes (Table 1), while the mRNA of the BFT gene failed to be amplified in either L. chinense or Liriodendron hybrids. The cDNA sequences of the four LhPEBPs were submitted to the NCBI database under the accession numbers OR347693-OR347696.
Nevertheless, we successfully amplified the gDNA sequences of BFT in all Liriodendron species, including one Liriodendron hybrid, one L. tulipifera, and two L. chinense individuals from the southern (JP) and northern (BZ) regions. We found that the gDNA sequences of BFT were 1114 bp for Liriodendron hybrids and 1115 bp for the two sister species (Table S2). The gDNA sequences of BFT were submitted to NCBI under the accession numbers OR347689-OR347692. The BFT gDNA sequences of Liriodendron hybrids showed a 98.30%, 99.55%, and 98.57% identity to L. tulipifera and the BZ and JP populations of L. chinense, respectively. The predicted amino acid sequence of Liriodendron hybrids was the same as that of L. chinense from BZ, while there were four amino acid differences between Liriodendron hybrids and L. chinense from JP and two amino acid differences between Liriodendron hybrids and L. tulipifera. A phylogenetic analysis of DNA sequences and amino acids showed that Liriodendron hybrids have a closer relationship with L. chinense from the BZ population in the northern region (Figure S1).

3.2. Amino Acid Alignment and Conserved Domain Analysis

The LhFT cDNA encoded a 174-amino acid protein and shared a 78.74% and 87.93% amino acid sequence identity with the FT genes of A. thaliana and Populus alba, respectively (Table 1). LhTFL1 has 173 amino acid residues and shares a 75.72% similarity to A. thaliana and a 79.19% similarity to Populus PaTFL1. The cDNAs of LhMFT1 and LhMFT2 encoded 173 and 174 amino acid proteins, respectively. LhMFT1 had a 70.52% and 69.94% amino acid sequence identity to A. thaliana and P. alba, respectively, while LhMFT2 shared a 63.79% and 64.94% amino acid sequence identity with A. thaliana and P. alba, respectively (Table 1).
The multiple amino acid sequence alignment showed that PEBP family proteins of Liriodendron hybrids have conserved DPDxP and GxHR motifs, which are critical for anion binding (Figure 1). Motif GxHR is localized in segment A and has a preference for the Ile residue in all three species. LhFT had the key amino acid residue Tyr84 (Y), but it was replaced by His85 (H) in LhTFL1 and Trp (W) in LhMFTs, which are likely the most critical residues for distinguishing FT and TFL1 activity. The conserved motif L/IYN in segment C was LYN for LhFT. The preferred Pro (P) residues in most MFT genes were found in both LhMFTs in segment D, and the remaining amino acid sequences in LhMFT1 were more similar to those in PaMFT than to those in LhMFT2 (Figure 1).

3.3. Cis-Elements Analysis in Promoter of the PEBP Genes

We conducted an analysis of the 2000 bp promoter sequences upstream of each PEBP gene in L. chinense. This analysis revealed multiple cis-acting elements that can be classified into four groups: hormone responses, light responsiveness, plant development, and stress responses. Among these regulatory elements, hormone-responsive elements are the most abundant, primarily dominated by MeJA-responsive and abscisic acid-responsive elements (Figure 2). Additionally, we identified gibberellin-responsive elements, auxin-responsive elements, and salicylic acid-responsive elements. The presence of light-responsive elements was also notable. Furthermore, the plant development elements included cell cycle regulation, circadian control, meristem expression, and seed-specific regulation. The stress-responsive elements comprised elements associated with drought stress, low-temperature response, anaerobic induction, defense, and other stress responses. It is noteworthy that all the genes contained abscisic acid-responsive and light-responsive elements in their promoters. However, the promoters of two MFT genes, namely Lchi05661 and Lchi25487, possessed seed-specific regulation elements that were not found in the promoters of other L. chinense PEBP genes. Additionally, only the FT (Lchi27713) and MFT1 (Lchi05561) elements featured GA-responsive elements in their promoter regions.

3.4. Phylogenetic Analysis

To explore the phylogenetic relationships of the PEBP gene family in Liriodendron, we investigated the four characterized PEBP members from Liriodendron hybrids and 33 PEBP genes from A. thaliana, Populus tomentosa, Cinnamomum micranthum, etc. (Figure 3). The phylogenetic tree divided the 37 proteins into three subgroups: the FT-like subgroup, the TFL1-like subgroup, and the MFT-like subgroup. LhFT, LhTFL1, and LhMFTs belonged well to the eudicot group of the relative clades. LhFT had the closest relationship with CmFT genes from C. micranthum. LhTFL1 and LtTFL1 from L. tulipifera were clustered together and then clustered with ZmTFL1 of Zea mays, forming a subgroup of TFL1. MFT-like genes were divided into three subgroups. LhMFT1 and LhMFT2 were clustered into two different groups, with LhMFT2 clustering with CmMFT from C. micranthum, while LhMFT1 was clustered with most herbs and gymnosperms and seemed to have a more ancient origin than LhMFT2.

3.5. Flowering Analysis of LhPEBPs in Transgenic Arabidopsis Plants

To examine the function of LhPEBP genes, the coding sequences of four LhPEBPs were introduced into A. thaliana (Col-0). We successfully obtained several transgenic lines for the four LhPEBPs. To explore the function of the four LhPEBPs in flower regulation, we compared the flowering phenotype of LhPEBPs in overexpressing lines and other control lines. Through phenotypic observation, we found that the overexpression of LhPEBPs did not result in obvious floral morphology changes in the stem height, the number of lateral branches, the shape of leaves, and the feature of flowers and siliques, but resulted in different flowering times and rosette leaves in transgenic Arabidopsis under long-day conditions.
As shown in Figure 4, the LhFT lines flowered earlier than the wild type, approximately six days earlier (18.70 ± 2.21 DAS vs. 24.72 ± 3.66 DAS), which was significantly earlier than the other phenotypes. The function of promoting flowering was further verified by the result that the LhFT transgenic lines had the fewest rosette leaves. For the LhTFL1 lines, the flowering time was the most flexible (24.29 ± 3.95 DAS; Figure 4 and Table S3) but showed no significant differences from that of the wild type and other transgenic types. However, the average number of rosette leaves of the LhTFL1 transgenic lines was significantly greater than that of Col-0.
The mft mutants showed no significant differences in either bolting time or rosette leaves compared with the wild type in the present study, proving that the AtMFT gene in A. thaliana may have a weak function in flowering. Although weak, the function of the two LhMFTs in flowering showed two contrasting directions, with the LhMFT1 lines flowering slightly earlier than the wild type and the LhMFT2 plants flowering later than the wild type, but the flowering time of LhMFT2 was significantly later than that of LhMFT1. This was further confirmed by the rosette leaves (Figure 4). The number of rosette leaves of LhMFT2 was not only significantly greater than that of LhMFT1 but also greater than that of the Col-0 and mft mutants. On the other hand, the LhMFT1 lines have relatively fewer rosette leaves than the Col-0 and other transgenic lines except for LhFT (Figure 4).

3.6. Germination Analysis of LhPEBPs in Transgenic Arabidopsis Plants

In view of the likely function of PEBP genes in seed germination, we analyzed the seed germination process of LhPEBPs in overexpressing Arabidopsis lines. The average final seed germination ranged from 0.872 to 0.972 under 1/2 MS (Figure 5 and Table S4). All the LhPEBP transgenic lines increased the germination of seeds, although it was not significant in LhFT lines. When treated with abscisic acid (ABA) and gibberellic acid (GA), the transgenic LhPEBP lines largely had the same trend, with seed germination delayed one to two days under a 10 μM ABA treatment and largely promoted in the early germination stage under a 1 μM GA3 addition. Although not significant, the 1 μM GA3 treatment increased the seed germination except for the LhMFT2 transgenic lines. The 10 μM ABA treatment significantly decreased the final seed germination percentage of the LhFT and LhTFL1 lines and Col-0, while it had a relatively weak influence on the LhMFT lines and mft mutants (Figure 5).
The seed germination rate of the LhMFT1 lines and mft mutants was significantly higher than that of the other lines in the early stage of seed germination (Figure 5 and Table S4). When treated with 1 μM GA3, the seed germination rate of LhFT and LhTFL1 in the early stage increased significantly compared with that under 1/2 MS; in particular, the LhFT lines showed a sudden outbreak of seed germination on the second day under the hormone action of GA3. When treated with 10 μM ABA, in accordance with Col-0 lines, the seed germination of the LhFT and LhTFL1 was rare on the third day, and their germination was suppressed during the whole process, but the degree of decrease was reduced in the later germination stage for the LhMFTs transgenic seeds and mft mutants.

4. Discussion

4.1. The Characteristics and Evolution of PEBP Genes in Liriodendron Hybrids

Collectively, we identified five members of the LcPEBP genes from the reported genome [42]. Unlike the traditional identification of gene family members from the reported genome, we characterized four potentially functional LhPEBP members from the expressed genes in Liriodendron hybrids. As the PEBP genes were mainly formed by gene duplication from their ancient ancestor, and functions diverged from each other after duplication [26], the number of PEBP genes varies in different species. The number of LhPEBPs is less than that in Populus nigra [43], Perilla frutescens [44], sugarcane [45], and soybean [46], and it is largely the same as that in moso bamboo [47], Pyrus communis [48], and Picea abies [3].
Our phylogenetic analysis showed that the four PEBP genes fall into three typical clades: one each into the FT-like clade and TFL1-like clade, and two into the MFT-like clades (Figure 3). This result was consistent with the common ancestor duplication and diverged orders of PEBP genes [26,49]. LhFT and LhTFL share more sequence similarity with Populus tree species than with the herb Arabidopsis. Nevertheless, LhMFTs shared largely the same sequence similarity with the two species (Table 1). Furthermore, LhMFT1 showed more sequence consistency with Populus and Arabidopsis than LhMFT2. This was further proven by the ML tree of 37 PEBP proteins (Figure 3). LhMFT1 was located in the largest subclade of the MFT-like clades, including most herbs and gymnosperms, such as A. thaliana, Populus alba, and Picea abies. However, LhMFT2 was separated into a relatively small clade. Evolutionary analyses of the PEBP family have also provided us with valuable information to better understand functional diversification. From this perspective, the observed divergent functions of LhMFTs in flowering, seed germination, and hormone response (Figure 4 and Figure 5) may largely indicate a different adaptive evolution after ancient gene duplication. Such functional divergence of PEBP genes within the same clade was also found in other species. For example, in Populus tremula, FT2 paralogs control summer growth, and the FT1 paralog controls the release of winter dormancy in response to winter temperatures [17,50,51]; OsMFT2 delays germination [32], whereas OsMFT1 also maintains flowering regulation in a manner similar to FT-like or TFL1-like genes [33].
In the present study, although we successfully amplified the gDNA sequences of BFT in the genomes of all Liriodendron species, we failed to obtain the expressed mRNA of BFT genes in L. chinense or Liriodendron hybrids. The tissue of flowers and the mixture of young and old leaves were all screened in the present study. In addition, Sheng [52] also searched for the PEBP family gene in the transcriptome dataset of L. chinense, covering five developmental time points, including autumn bud, spring bud, and flower buds at three successive developmental phases, and no BFT transcripts were found. We argued that except for a special case not captured, for example, peaking in the early evening [53], this gene was more likely functionally redundant in Liriodendron. Loss of the BFT gene occurs in many species, such as grapevine [54], Picea abies [3], moso bamboo [47], and sugarcane [45]. In the process of evolution, gene functions are repeated and redundant, sometimes leading to the formation or selection of pseudogenes. For example, AcBFT2 from kiwifruit has the potential to reduce plant dormancy with no adverse effect on flowering [55]. Wang [26] reported that BFT, TFL1, and ATC were three daughter lineages created by two separate duplication events of the common TFL1 ancestor. From this aspect, BFT may function like TFL1; for example, BFT of Arabidopsis possesses TFL1-like activity and functions redundantly with TFL1 in inflorescence meristem development [53].

4.2. The Function of LhFT/LhTFL1 Was Conserved, and LhMFTs Functioned Differently

We identified one FT and one TFL gene in Liriodendron hybrids. This enabled them to perform the basic functions of promoting and inhibiting flowering [5,9,56]. LhFT has a significant flowering promotion effect (Figure 4). Similar early-flowering phenotypes of transgenic Arabidopsis appeared upon the ectopic overexpression of FT genes from heterologous plants. For example, GbFT in Ginkgo biloba [57], PbFT in Pyrus betuleafolia [48], and PfFT1 in Perilla frutescens [44] all promote flowering in transgenic Arabidopsis. Furthermore, the function of FT splicing functioned contrarily, prolonging the blooming period [52] or negatively inducing flowering [58]. In Liriodendron hybrids, it showed a wide range of flowering timing in LhTFL1 transgenic lines, although the average time was similar with Col-0. The function of TFL was usually to delay the flowing time, such as in MiTFL1 genes in mangos [25] and ScFT1 genes in sugarcanes [59], but it was also found to function oppositely in alpine snow tussocks [60]. The machinery determining flower bud initiation in Arabidopsis may be disturbed by the activity of LhTFL1. On the other hand, the number of rosette leaves was increased in LhTFL1 overexpressing plants, partially showing the strengthening of its early vegetative growth, and indicating a certain regulation of the transition from vegetative growth to reproductive growth conducted by LhTFL1. In addition to the original flowering time control, FT/TFL1-like genes have been found to be involved in more diverse functions. For example, the balance between vegetative and reproductive growth is regulated in pears [48] and tomatoes [11]. A cis-element analysis of the promoter of the PEBP genes in L. chinese also showed circadian control elements, indicating the possibility of their function in the growth cycle. For example, the FT paralogs in Populus tremula regulate different aspects of the tree’s yearly growth cycle [17,51]. In Picea abies, PaFT4 correlates with both the photoperiod-controlled bud set and temperature-mediated bud burst [3]. In Arabidopsis, FT interacts with the transcription factor BRANCHED1 (BRC1) to regulate branching [61].
As the sister clade of the common ancestor of the FT and TFL1 subfamilies [26], MFT-like genes have maintained their potential to function like FT and TFL1. A few MFT-like genes regulate flowering in a manner similar to FT-like or TFL1-like genes [27,28]. Hence, previous studies have proven that MFTs from different species have diverse functions in flowering time. For example, in Arabidopsis, the overexpression of AtMFT caused slightly early flowering under long-day conditions [27]. In contrast, the overexpression of the orchid DnMFT [62] and rubber tree HbMFT1 also delayed floral initiation [63]. However, many studies also found that MFT had no effect on flowering; for example, GmMFT [30], PaMFT1, and PaMFT2 [3], GhMFT1, GhMFT2 [31], and MiMFT [28] did not affect the flowering time of transgenic plants. Consistent with the analysis results of ginkgophytes, cycadophytes, and pinophytes [49], we also identified two distinct clades of MFT proteins (LhMFT1/LhMFT2) in Liriodendron hybrids. Regarding their functions in flowering, the functions of LhMFTs seem to be contradictory. The overexpression of LhMFT1 caused slightly early flowering under long-day conditions, similar to the functions of MFT in Arabidopsis, which closely clustered together in the phylogenetic tree (Figure 3), while LhMFT2 delayed flowering, in accordance with the opposite examples, including rubber tree HbMFT1 [63], which is located in the same subclade of MFT-like genes in our constructed phylogenetic tree.
In addition to being involved in flowering regulation, the function of MFT-like genes in seed dormancy and germination was prominent. In the present study, we found seed specific cis-acting elements in the promoter of the two MFT genes in L. chinense (Figure 2). Furthermore, the function of MFTs in seed germination is different. AtMFT was reported to promote seed germination in Arabidopsis [29]. Yang also reported that the overexpression of PhFT5 (MFT-like clade) from moso bamboo promoted the seed germination rate in Arabidopsis [47]. However, more studies have proven that the overexpression of MFTs inhibits seed germination in transgenic Arabidopsis, such as cotton GhMFT1 and GhMFT2 [31], mango MiMFT [28], and wheat TaMFT [64]. In the present study, we found that LhMFTs promote seed germination, and the effect of LhMFT1 is higher than that of LhMFT2 (Table S4). The functional trend of LhMFT1 in determining the flowering time and seed germination in transgenic Arabidopsis was roughly the same. This consistent functional trend in flowering and seed germination was also found in other species, such as MFTs in cotton [31], mangos [28] and rubber trees [63].
Abscisic acid (ABA) and gibberellic acid (GA) are two antagonistic phytohormones that regulate plant growth and seed germination in response to biotic and abiotic environmental stresses [28,29,64,65]. We found that LhPEBPs were hormone-responsive genes. The seed germination of the LhMFT lines was relatively less influenced by ABA, and the transgenic LhFT and LhTFL1 lines were sensitive to both ABA and GA3 (Figure 5 and Table S4). The involvement of MFTs and seed germination in ABA signaling was actually controversial [29,32,66]. In Liriodendron hybrids, the application of exogenous ABA significantly inhibited the germination of PEBP transgenic lines, but the germination inhibition of overexpressed LhMFT lines was less than that for others (Figure 5). In mangos, the overexpression of MiMFT also increased the tolerance to ABA stresses [28]. The function of LhFT in seed germination was strongly promoted by GA3 in the early stage, and we also found GA responsive cis-acting elements in the promoter region of the FT and MFT1 gene in L. chinense, which may promote flowering and seed germination. We argue that the sensitivity of LhFT to GA and the relative insensitivity of LhMFTs to ABA may contribute to heterosis in Liriodendron hybrids. Nevertheless, further experiments are needed to elucidate their actual functions in Liriodendron hybrids.

5. Conclusions

In summary, we isolated four members of the PEBP family from Liriodendron hybrids, including one LhFT, one LhTFL1, and two LhMFTs, and identified the functions of their products. Our results provide the first relatively comprehensive description of the PEBP family genes in Liriodendron species, and to our knowledge, this is the first report to demonstrate the function of PEBP genes in Liriodendron hybrids. This study provides insight into the characteristics and function of four PEBP family genes from Liriodendron hybrids in the regulation of flowering and seed germination in A. thaliana.
In accordance with the reported functions of most PEBP genes, LhFT promoted floral induction and exhibited a rapid response to GA3 in the seed germination of transgenic Arabidopsis. Conversely, the LhTFL1 gene induced a wide dispersion of the flowering timing. Both LhMFT genes promoted seed germination and they were less influenced by ABA. However, they functioned differently, with LhMFT2 inhibiting flowering and LhMFT1 slightly promoting it. This suggested that amino acid divergence following duplication drove the adaptive functional differentiation of PEBP family genes. However, further studies are needed to characterize the LhPEBP family genes by genetic approaches to understand their actual roles in flowering, germination, and other related functions related to heterosis in Liriodendron species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14102103/s1, Figure S1: Phylogenetic analysis of BFT genes in Liriodendron; Table S1: Primers and original accession information for LhPEBP genes in Liriodendron hybrids; Table S2: Information for BFT genes in Liriondendron. Table S3: Flowering traits of Col-0, LhPEBP transgenic lines and mft mutants. Table S4: Time course of the seed germination for Col-0, over expressed LhPEBPs transgenic lines and mft mutations in Arabidopsis under 1/2 MS, with or without 1 μM GA3 or 10 μM ABA treatment. Different letters in the right corner indicate significant differences of ANOVA tests under the Duncan method for all the gene lines with different treatment in the same day (p < 0.05)

Author Contributions

Conceptualization, A.Y.; Methodology, A.Y.; Validation, S.L.; Resources, X.Y. and T.L.; Data curation, M.H., L.L. and P.H.; Writing—original draft, M.H.; Writing—review & editing, A.Y.; Visualization, H.Z.; Project administration, F.Y. All authors have read and agreed to the published version of the manuscript.

Funding

The National Natural Science Foundation of China (No. 32060312); Training Program for Academic and Technical Leaders of Major Disciplines in Jiangxi Province (20212BCJ23032); Jiangxi Provincial Academy of Sciences Provincial-Level Comprehensive Responsibility Project (2021YSBG22019, 2023YSBG22002, 2021YSBG22016); National Government Guidance Fund for Regional Science and Technology Development (20192ZDD01004).

Data Availability Statement

Data is contained within the article or Supplementary Materials. All raw data have been submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/) and are accessible under the numbers OR347689-OR347696.

Conflicts of Interest

The authors declare no conflict of interest.

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Forests 14 02103 g001
Figure 1. Multiple sequence alignment of PEBP family proteins. Amino acid sequences of the PEBP proteins were isolated from Liriodendron hybrids (LhFT, OR347696.1; LhTFL1, OR347695.1; LhMFT1, OR347693.1; LhMFT2, OR347694.1), Arabidopsis thaliana (AtFT, AAF03936.1; AtMFT, OAP13671.1; AtTFL1, AAB41624.1), and Populus alba (PaFT, TKR74523.1; PaMFT, TKR98064.1; PaTFL1, TKR85832.1). Black boxes mark the conserved DPDxP and GxHR motifs. Underlines represent segments A, B, C, and D. The red arrow indicates the key amino acids distinguishing FT-like (Y), TFL1-like (H), and MFT-like (W) functions; the blue arrow indicates the preferred Pro (P) residue found in most MFT genes.
Figure 1. Multiple sequence alignment of PEBP family proteins. Amino acid sequences of the PEBP proteins were isolated from Liriodendron hybrids (LhFT, OR347696.1; LhTFL1, OR347695.1; LhMFT1, OR347693.1; LhMFT2, OR347694.1), Arabidopsis thaliana (AtFT, AAF03936.1; AtMFT, OAP13671.1; AtTFL1, AAB41624.1), and Populus alba (PaFT, TKR74523.1; PaMFT, TKR98064.1; PaTFL1, TKR85832.1). Black boxes mark the conserved DPDxP and GxHR motifs. Underlines represent segments A, B, C, and D. The red arrow indicates the key amino acids distinguishing FT-like (Y), TFL1-like (H), and MFT-like (W) functions; the blue arrow indicates the preferred Pro (P) residue found in most MFT genes.
Forests 14 02103 g001
Forests 14 02103 g002
Figure 2. Cis-acting elements within the 2000 bp upstream region of PEBP Genes in Liriodendron chinense. Within Figure 2, a spectrum of cis-acting elements is denoted by diverse, color-coded boxes. Notably, elements associated with light responsiveness, hormone responses, plant development, and stress responses are delineated by green, blue, brown, and red rectangles, respectively.
Figure 2. Cis-acting elements within the 2000 bp upstream region of PEBP Genes in Liriodendron chinense. Within Figure 2, a spectrum of cis-acting elements is denoted by diverse, color-coded boxes. Notably, elements associated with light responsiveness, hormone responses, plant development, and stress responses are delineated by green, blue, brown, and red rectangles, respectively.
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Figure 3. The phylogenetic tree of PEPB family proteins from Liriodendron hybrids and other plants. The tree was constructed using the maximum likelihood method in MEGA 6.0. The number at the nodes represents the bootstrap values based on 1000 replications (%). The FT-like, TFL1-like, and MFT-like subfamilies are marked by green, red, and blue, respectively. LhPEBPs are marked in bold font.
Figure 3. The phylogenetic tree of PEPB family proteins from Liriodendron hybrids and other plants. The tree was constructed using the maximum likelihood method in MEGA 6.0. The number at the nodes represents the bootstrap values based on 1000 replications (%). The FT-like, TFL1-like, and MFT-like subfamilies are marked by green, red, and blue, respectively. LhPEBPs are marked in bold font.
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Figure 4. Phenotype pattern of LhPEBP genes affecting flowering in Arabidopsis in a phytotron under LD conditions. (A) Flowering time of Col-0, four LhPEBP transgenic lines and the mft mutant. (B) Rosette leaf numbers of Col-0, four LhPEBP transgenic lines and the mft mutant.
Figure 4. Phenotype pattern of LhPEBP genes affecting flowering in Arabidopsis in a phytotron under LD conditions. (A) Flowering time of Col-0, four LhPEBP transgenic lines and the mft mutant. (B) Rosette leaf numbers of Col-0, four LhPEBP transgenic lines and the mft mutant.
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Figure 5. Time course of seed germination for Col-0, overexpressed LhPEBP transgenic lines, and mft mutants in Arabidopsis. Three situations, (A) under 1/2 MS, (B) 1 μM GA3, and (C) 10 μM ABA, are presented; a–c indicate the results based on Duncan’s test at a significance value of 0.05.
Figure 5. Time course of seed germination for Col-0, overexpressed LhPEBP transgenic lines, and mft mutants in Arabidopsis. Three situations, (A) under 1/2 MS, (B) 1 μM GA3, and (C) 10 μM ABA, are presented; a–c indicate the results based on Duncan’s test at a significance value of 0.05.
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Table 1. Characterization of PEBP genes and proteins from Liriodendron hybrid.
Table 1. Characterization of PEBP genes and proteins from Liriodendron hybrid.
Gene NameAccession No.CDS Length (bp)Putative Protein Length (aa)aa Identities to Arabidopsis thaliana (%)aa Identities to Populus alba (%)
LhFTOR34769652517478.7487.93
LhTFLOR34769552217375.7279.19
LhMFT1OR34769352217370.5269.94
LhMFT2OR34769452517463.7964.94
CDS, coding sequence; aa, amino acid.
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Hu, M.; Liu, L.; Hu, P.; Yu, X.; Zhou, H.; Liu, S.; Liu, T.; Yu, F.; Yang, A. Identification and Functional Analysis of the Phosphatidylethanolamine-Binding Protein (PEBP) Gene Family in Liriodendron Hybrids. Forests 2023, 14, 2103. https://doi.org/10.3390/f14102103

AMA Style

Hu M, Liu L, Hu P, Yu X, Zhou H, Liu S, Liu T, Yu F, Yang A. Identification and Functional Analysis of the Phosphatidylethanolamine-Binding Protein (PEBP) Gene Family in Liriodendron Hybrids. Forests. 2023; 14(10):2103. https://doi.org/10.3390/f14102103

Chicago/Turabian Style

Hu, Miao, Lipan Liu, Ping Hu, Xiaoling Yu, Hua Zhou, Shujuan Liu, Tengyun Liu, Faxin Yu, and Aihong Yang. 2023. "Identification and Functional Analysis of the Phosphatidylethanolamine-Binding Protein (PEBP) Gene Family in Liriodendron Hybrids" Forests 14, no. 10: 2103. https://doi.org/10.3390/f14102103

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