Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress

Abstract

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

1. Introduction

In eukaryotes, the ubiquitin/26S proteasome system (UPS) is responsible for the selective degradation of most intracellular proteins [1]. There are three major enzymes involved in ubiquitin degradation, including the ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2), and ubiquitin-ligase (E3). E3 are divided into the following four categories based on their mechanism of action and subunit positions: the HETC structural domain type, RING-finger structural domain type, U-box structural domain type, and the SCF (Skp1-Cullin -F-box) domain type [2]. F-box proteins play a key role in recruiting substrates to UPS [3,4]. F-box proteins carry at least one 40–50 residue F-box/F-box-like domain at its N-terminus, which is responsible for binding to Skp1/Skp1-like proteins. At the same time, one or more conserved domains can also be found at the C-terminus, which are associated with substrate-specific recognition, including F-box-associated (FBA), kelch repeat and leucine-rich repeat (LRR) domains, etc. [5]. To date, F-box proteins have been discovered in almost all plants, and these F-box members form a larger protein family, of which the FBA subfamily is one of the largest subgroups [6]. According to previous studies, researchers found 94 F-box proteins containing FBA domains in Triticum aestivum [7], 46 in Gossypium hirsutum [8], 25 in Cicer arietinum [9], 14 in Dioscorea esculenta [10], 34 in Juglans regia [11], 278 in Brassica rapa [12], 17 in Zea mays [5], 196 in Arabidopsis thaliana [6], and 133 in Malus domestica [13].

A large number of F-box genes in plants are involved in the regulation of many biological processes, including hormone responses, lateral root formation, meristem formation, photomorphogenesis, senescence and stress response (abiotic and biotic processes) [9,14]. For example, the F-box protein TIR1 is an auxin receptor in Arabidopsis thaliana. Auxin-induced interaction between the Aux/IAA transcriptional repressor proteins and the ubiquitin–ligase complex SCF^TIR1 mediates Aux/IAA degradation and auxin-regulated transcription [15]. The Arabidopsis F-box protein UFO is more likely to be a co-regulator that functions together with LFY in controlling organ-identity genes [16]. EID1 possibly operates by targeting activated components of the phyA signaling pathway to ubiquitin-dependent proteolysis [17]. TaFBA1-OE plants improve drought tolerance by increasing antioxidant competence and decreasing ROS accumulation [18,19]. The information on plant F-box genes was mainly obtained from the research on Arabidopsis, rice and wheat, while the research on poplar F-box genes (PtrFBXs) is quite limited, and the research on F-box/FBA proteins is even more sparse. Hua et al. conducted a phylogenetic comparison of F-box proteins in 18 plants including poplar in 2011, and the results revealed the expansion, evolutionary selection, and functional relatedness of the F-box gene family. The results also indicate that the diversification of F-box genes may be achieved through genome drift [4]. In 2008, Yang et al. performed a comparative genomics analysis of the woody perennial plant poplar with the herbaceous annual plants Arabidopsis thaliana and rice, and elucidated the functional significance of this gene family [6]. Nonetheless, genome-wide analysis of PtrFBXs in poplar needs to be further studied.

Drought severely restricts plant growth and crop yields, and poplars are ideal materials for studying the response of trees to drought stress [20]. Some studies have been carried out on stress resistance genes in poplars. Previous studies have shown that plants have some intrinsic mechanisms to alleviate drought stress, including closing stomata, reducing transpiration, producing abscisic acid (ABA) and increasing surface wax. For example, PeCHYR1 enhanced drought tolerance by promoting H₂O₂-mediated stomatal closure in poplar [21]. The overexpression of PePYL4 led to higher water-use efficiency (WUE) and drought tolerance in poplar [22]. PeABF3 improves drought tolerance by promoting ABA-induced stomatal closure by regulating the expression of PeADF5 under drought conditions [23]. PeSHN1 regulates both WUE and drought tolerance in poplar by modulating wax biosynthesis [24]. In recent years, whole genome sequencing of P. trichocarpa has been carried out for the fourth generation of resequencing, providing a valuable reference resource for the analysis of whole gene families, such as the PPO polyphenol oxidase gene family and the gibberellin-stimulated Arabidopsis (GASA) protein under abiotic stress [25,26]. However, the F-box family and its subfamily features have not been reported in poplar.

In this study, we aimed to identify and characterize the functions of PtrFBAs from the P. trichocarpa genome. We searched the P. trichocarpa genome using the HMMER website and identified 74 PtrFBA candidate genes. We predicted and analyzed the chromosomal localization, gene amplification, gene structure and upstream promoter cis-acting elements of the candidate PtrFBA genes. The chemical properties, subcellular localization, motifs and phylogenetic relationships of the proteins they encode were also predicted and analyzed. Focusing on identifying PtrFBAs associated with drought stress, the subcellular localization of the representative gene PtrFBA60 and its germination rate and physiological phenotype under drought stress were analyzed. In this study, the PtrFBA gene was comprehensively analyzed by genomics, transcriptomics, and qRT-PCR for the first time, and the representative gene PtrFBA60 was screened. We performed subcellular localization of PtrFBA60 in tobacco and drought stress experiments in Arabidopsis thaliana, and analyzed the seed germination rate and physiological phenotype under drought stress to further study PtrFBA’s family properties and its possible role in drought tolerance in poplar.

2. Results

2.1. Genome-Wide Identification of PtrFBXs and PtrFBAs Families in P. trichocarpa

We searched the genome-wide protein database for the conserved F-box (PF00646) and FBA1(F-box associated 1) (PF07734) domains. The motif maps (Supplementary Figure S1) and hmm model files of the two domains were downloaded; 459 sequences of the whole genome of P. trichocarpa were screened. Redundant sequences without F-box structural domains were then manually removed, resulting in a total of 337 F-box genes. These genes were named according to the order of corresponding chromosomal positions identified in the NCBI database. Different alternative splice forms from the same locus were assigned under the same gene names, such as PtrFBA16 and PtrFBA16.1 (Supplementary Table S1). The domain analysis showed that 101 out of 337 F-box genes contained only F-box domains (accounting for about 30%), 74 genes contained FBA domains (accounting for about 21%) and the other genes showed FBD, PP2, WD40, Kelch-type, LRR, FIST, NBD, DUF, AMN, TUB, RBD, PLN, PP1, or other domains (Figure 1). In different plants, the types and numbers of C-terminus domains are different, which indicates that the evolutionary history of different domains at the C-terminus may be more complex. Previous studies have found that the number of clans in the F-box family and the FBA subfamily is different in different plants. We performed an evolutionary developmental analysis of the FBA subgroup in P. trichocarpa and divided it into four subgroups (Figure 2a). We next compared the number of F-box family and FBA subfamily genes in ten species; the results showed that the gene number of FBA subfamilies and F-box family varied between species. The highest number of FBA genes was found in Brassica rapa with 278 genes, and the number of F-box genes in wheat reached 1013 (Figure 2b). F-box motifs and FBA domains usually contain about 50 amino acids, and several residues are conserved. We screened PtrFBA60 as a representative gene from poplar’s FBA family. Malus domestica FBA protein MdFBX3, Antirrhineum hispanicum FBA protein AhSLF4, Zea mays FBA protein FBX230.1, Arabidopsis thaliana FBA protein AtFOA2 and Triticum aestivum FBA protein TaFBA1 for sequence alignment by using CLUSTALX (Supplementary Figure S2). The results showed that they were conserved in F-box and FBA motifs.

We next analyzed the physicochemical properties of the 74 PtrFBAs genes in the en-tire PtrFBAs gene family. Except for PtrFBA34, which encodes about 191 amino acids, the PtrFBAs genes in P. trichocarpa generally encode 277–466 amino acids, with an average of 396 amino acids. The molecular weights of PtrFBAs proteins were both larger than 30 KDa, except for PtrFBA34. The theoretical pI of PtrFBAs was 4.74–9.51; 31 genes belonged to acidic proteins and 43 belonged to basic proteins. In addition, 30% of these FBA proteins were stable with the instability index <40. The overall hydrophilicity of the proteins is relatively strong (Supplementary Table S2). According to the prediction of the Plant-mPLoc database (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 15 July 2021), most of the PtrFBAs proteins are located in the nucleus, and a few in the membrane system.

2.2. Genomic Localization and Collinearity Analysis of PtrFBA Genes

The 74 PtrFBA genes were unevenly distributed on poplar chromosomes 1–19, with the largest distribution on chromosome 1 and none on chromosome 9 (Figure 3). PtrFBA repeat gene clusters were found on chromosomes 1, 3, 5, 6, 8, 10, 11, 12, 13, 15, 16, and 17 of P. trichocarpa. The largest repeat gene cluster had five genes, and is located on chromosome 6. To illustrate the expansion pattern of PtrFBA genes, we analyzed duplication events in the P. trichocarpa genome. PtrFBAs have undergone multiple repetitive events during evolution. PtrFBA2 and PtrFBA15, PtrFBA7 and PtrFBA68, PtrFBA12 and PtrFBA24, PtrFBA12 and PtrFBA21, PtrFBA11 and PtrFBA60, PtrFBA20 and PtrFBA29, PtrFBA26 and PtrFBA41, PtrFBA27 and PtrFBA42, PtrFBA25 and PtrFBA65, PtrFBX118 and PtrFBA71 all form an entire genome duplication (WGD) pair, indicating that they all share a common ancestor with each other (Supplementary Table S3). The Ka/Ks values of PtrFBA7, PtrFBA68, PtrFBA65 and PtrFBA25 were all greater than 1, indicating that these genes had positive selection effects (Supplementary Table S4). The Ka/Ks values of the remaining three groups of genes are all less than 1, indicating that these genes have the effect of purification selection. In poplar, we found 19 genes that formed 10 segmental repeat events, which were distributed on 11 chromosomes, with two additional tandem repeat regions on chromosomes 6 and 10 (Figure 4). Next, we constructed comparative syntenic maps of the F-box gene family of P. trichocarpa and four typical plants (Arabidopsis, wheat, maize and apple) (Supplementary Figure S3). The syntenic maps showed that there were 129 pairs of FBX homologous genes between P. trichocarpa and Arabidopsis thaliana, 48 pairs of FBX homologous genes with maize, 124 pairs of FBX homologous genes with wheat, and 172 pairs of FBX homologous genes with apple. We then constructed comparative syntenic maps of the FBA gene families of P. trichocarpa and four species (Figure 5); it was found that P. trichocarpa has 8 pairs of FBA homologous genes with Arabidopsis, 1 pair of FBA homologous genes with maize, no FBA homologous genes with wheat, but 26 pairs of FBA homologous genes with apple. In addition, it was found that some PtrFBA genes are related to at least two synonymous gene pairs, especially FBA genes between poplar and apple, indicating that these genes may play an important role in the evolution of plants. The results of this study show that PtrFBAs have fewer homologs in wheat, Arabidopsis and maize and more homologs in the woody plant apple. This suggests that multiple FBA homologs may have been formed in woody plants during the differentiation process.

2.3. Gene Structure and Motif Identification of PtrFBA Gene Family

To more clearly explore the evolutionary relationship between different members of the PtrFBA gene family, their gene structures, conserved protein motifs, and conserved structures were evaluated (Figure 6b). Most PtrFBA genes (about three fifths of the genes) have no introns, six genes have two introns (PtrFBA19/24/26/27/37/53) and twenty-one genes have one intron. Moreover, 15 genes did not contain UTR, and 8 genes contained neither introns nor UTRs (PtrFBA5/16.1/23/43/46/47/56/73). At the same time, 10 conserved motifs were found in the PtrFBA protein sequence using MEME (Figure 6a), of which 2 motifs (1 and 8) were associated with the N-terminal F-box domain and 6 motifs (2, 5, 6, 9, 4, and 10) were associated with the C-terminal FBA domain (Supplementary Table S5).

2.4. PtrFBAs cis-Element Analysis

To determine the expression pattern of PtrFBAs, the sequence upstream of the PtrFBA family promoter (~2000 bp) was extracted from the P. trichocarpa genomic DNA sequence. The cis-elements of the PtrFBA promoter were analyzed using the PlantCARE database (Figure 7). We visualized the promoter elements in promoter positions and species (Supplementary Figure S4). The specific functions of these elements (cis-elements) are annotated (Supplementary Table S6). These elements are involved in abiotic and biotic stresses, phytohormonal responses, and plant growth and development. Elements of the PtrFBA promoter are mainly involved in abiotic and biotic stresses, as well as phytohormone response factors. Stress-related cis-elements (Myb, Myc, ARE and STRE) were extracted, suggesting that PtrFBAs may play a key role in coping with adverse conditions. In addition, some PtrFBA promoters are enriched in ABRE (involved in the ABA response), such as PtrFBA42/PtrFBA61/PtrFBA/65, and these genes may be responsive to ABA hormones. PtrFBA22/PtrFBA37 have more LTR elements (involved in low-temperature stress responses) in their promoters, suggesting that these genes may be responsive to low-temperature induction.

2.5. Transcriptome Analysis of PtrFBA Gene in P. trichocarpa

To further investigate the role of PtrFBA genes in growth and development, we examined the tissue expression profiles of 30 PtrFBA genes from transcriptome data (Supplemental Table S7). We created a heat map of the samples and genes clustered in both directions to explore the expression characteristics of PtrFBA genes in 15 poplar tissues (Figure 8a). Most of the genes were highly expressed in mature sites, while a few were highly expressed in young sites (e.g., PtrFBA1/4/6). Some genes were highly expressed in phloem (e.g., PtrFBA49/50/55/72), which may be related to the growth and development of poplar. We downloaded transcriptomic datasets to visualize the expression of 30 PtrFBAs genes after drought, beetle and mechanical damage (Supplemental Table S8) and performed transcriptomic data analysis and visualization to analyze the effects of PtrFBA genes on stress response in poplar (Figure 8b). Most of the genes in the PtrFBA gene family are sensitive to drought stress and their expression is up-regulated under drought stress. Some genes were up-regulated under leaves beetle damage stress (e.g., PtrFBA11/26/65), and some were up-regulated under leaves mechanical damage stress (e.g., PtrFBA26/40/42). PtrFBA26, PtrFBA42 and PtrFBA65 were up-regulated under both types of stress, indicating that these genes may respond to leaf injury stress.

2.6. Expression Patterns of Some PtrFBAs under Different Treatments

To further explore the function of PtrFBAs, we investigated the response of some PtrFBAs to drought stress by qRT-PCR in P. trichocarpa leaves (Figure 9). Three different gradients were set according to soil moisture content. PtrFBA15/45/60/72 were up-regulated under drought stress, while PtrFBA6/11/13/36/50 were down-regulated, and PtrFBA61 showed no significant change. We also found that PtrFBA60 was significantly sensitive to ABA (Figure S5a), and weakly sensitive to ethylene and NaCl (Figure S5b,c).

2.7. Drought Treatment of Transgenic Arabidopsis

We selected the PtrFBA60 gene for follow-up experiments. To determine the subcellular localization of PtrFBA60, the Super:: PtrFBA60-GFP (green fluorescent protein) fusion protein was transiently transfected into tobacco leaves. It was found to be likely located in the cytoplasm; however, it was not uniformly distributed in the cytosol, but rather formed punctate aggregates (Figure 10a). The PtrFBA60 aggregates were found to correspond to P-bodies by co-localization experiments between PtrFBA60-GFP and the P-body markers AtTZF1-mCherry and AtAGO-mCherry (Figure 10b). The results indicate that PtrFBA60 is localized in the P-bodies [27,28]. Since drought stress can induce the expression of PtrFBA60 (Figure 9), we hypothesized that PtrFBA60 plays a vital role in the drought response. The seed germination rate experiments showed that there was no significant difference in the germination rate of the OE-PtrFBA60, WT, and mutants fba60 on 1/2 MS medium, but the germination rate of OE-PtrFBA60 was significantly higher for the 200 mM mannitol cultured medium (Figure S6). The OE-PtrFBA60, mutant fba60, and WT Arabidopsis were cultured under the same conditions and then subjected to drought treatment. After 20 days, the leaves of mutant fba60 were severely wilted and the leaves of WT turned yellow and wilted, while those of OE-PtrFBA60 lines remained green and turgid. Furthermore, OE-PtrFBA60 trangenic plants recovered normally after being rewatered, while the mutant and WT Arabidopsis plants did not fully recover (Figure 11a). The survival rate of OE-PtrFBA60 was 85.8%, while the survival rate of WT and mutant fba60 was 51.6% and 36.6%, respectively (Figure 11b). The REC (relative electrical conductance) characterizes the degree of damage to plant cell membranes and the REC of OE-PtrFBA60, mutant fba60, and WT both increased after drought treatment, but the values for fba60 and WT were three times larger than those for OE-PtrFBA60 (Figure 11c). These results indicated that OE-PtrFBA60 transgenic Arabidopsis had better drought stress tolerance.

3. Discussion

As a component of the SCF E3 ligase complex, the F-box protein family plays an im-portant role in plant growth, development and stress resistance [29,30,31,32,33,34]. The FBA subfamily is a ubiquitous and abundant subfamily in the F-box family, consisting of an N-terminal F-box conserved domain and a C-terminal FBA conserved domain [18,19]. The FBA gene family is related to many plant physiological activities, such as plant growth and development, hormone signal transduction, abiotic and biotic stresses and plays a significant role [3,15,17,35,36,37]. In many plants (wheat, maize, Arabidopsis, etc.), the biological function and bioinformatic analysis of the FBA gene family has been widely reported, but this is not the case for the woody plant poplar [6]. In this study, 74 candidate FBA genes were identified in P. trichocarpa, and bioinformatics and expression patterns of these genes were analyzed. As is the case with maize, apple and Arabidopsis, the FBA genes in P. trichocarpa are unevenly distributed, and distributed on several chromosomes (Figure 3). These 74 proteins are all high-molecular-weight proteins, except PtrFBA34. The experimental results show that in agreement with other species, the predicted subcellular localization varies, but most are located in the nucleus. However, the subcellular localization of our target gene, PtrFBA60, showed that PtrFBA60 was localized in the P-body (Figure 10) [27,28]. Due to the gap between their occurrence positions and distances, the FBA gene families were discretely distributed in the phylogenetic tree, and were mainly divided into four clusters (Figure 2). The origin and evolution of the PtrFBAs genes were also further determined.

The increase in gene family members and the mechanism of genome evolution mainly depend on gene duplication events, including tandem duplication and segmental duplication [38]. In the present study, 74 PtrFBAs genes were unevenly distributed on 19 poplar chromosomes, and this phenomenon of uneven distribution of chromosomes suggested that this change occurred before species differentiation (Figure 3). A total of 10 pairs of whole genome duplicate genes and 12 tandem duplicate genomes were detected in poplar. This indicates that both tandem repeat sequences and whole genome repeats are involved in the evolution of poplar PtrFBAs genes (Figure 4). Previous gene family studies have suggested that tandemly repeated genes may have similar functions and expression patterns. Most PtrFBAs have no introns, while some have up to two introns (Figure 6). In order to quickly respond to stress, organisms need to stimulate the expression of genes, and gene structures with few or no introns contribute to the rapid expression of mRNA [39,40]. Genes such as PtrFBA3/4/7 lack introns but contain UTRs, so they can be transcribed faster to form mRNA.

A large number of genes for FBAs were present in each species (Figure 2b). Covariance analysis with four different species revealed that the F-box gene family and FBA gene family of poplar are more closely related and homologous to the woody plant apple than other species (Supplementary Figure S3, Figure 5) [11,13,31,41,42,43,44,45]. This suggests that considerable FBA divergence and doubling may have occurred during the evolution of perennial woody plants.

The expression patterns of FBA genes in various tissues have been confirmed in many species [10]. Due to differences in the number of FBA genes in different species, there is no uniform FBA gene expression profile in plants. According to the RNA-seq data of poplar, some genes were preferentially expressed in the phloem and shoots (Figure 8a), indicating that these genes are essential for plant growth [3]. Some genes are highly expressed throughout the growth and development process, indicating that the FBA gene family plays an important role in the entire plant growth and development process [14,18,32,33,35].

The expression of the FBA gene family in plants is closely related to stress and adversity, mainly in response to drought, insects and mechanical damage [5,18]. Transcriptome data showed that drought significantly induced some FBA genes in poplar (PtrFBA2/15/60) (Figure 8b), which was verified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (Figure 9). By analyzing the promoters of the FBA gene family, we found that there are many different elements in their promoters, especially ABRE and ARE, which are involved in biotic and abiotic stresses (Figure 7) [14,46,47,48]. Furthermore, ABRE elements play an important role in response to abiotic stress, which ensures that FBA genes can be rapidly induced under stress [49]. In order to verify whether the FBA gene can provide plants with the function of drought resistance, we overexpressed the PtrFBA60 gene in the model plant Arabidopsis thaliana and purchased Arabidopsis thaliana seeds with mutant fba60. It was found that overexpression of PtrFBA60 significantly improved the drought resistance of Arabidopsis (Figure 11), and the expression of PtrFBA60 also significantly increased under ABA treatment, which may improve plant drought resistance through the ABA pathway (Supplementary Figure S5) [19,46,50,51,52]. In summary, plants can adapt to various complex environments by regulating their expression level of FBA genes.

4. Materials and Methods

4.1. Identification and Evolutionary Analysis of the FBA Family of Populus Trichocarpa

The identification and analysis of the FBA gene family were based on the whole genome of P. trichocarpa. Relevant poplar genomic and protein data were downloaded from the Phytozome13.0 database (https://phytozome-next.jgi.doe.gov/, accessed on 12 July 2022). We downloaded the Hidden Markov model of the FBA1 structural domain (PF07734) and F-box structural domain (PF00646) from the Pfam database (http://pfam.xfam.org/, accessed on 30 January 2023), and searched the genome of P. trichocarpa using the Simple HMM Search program in TBtools software (TBtools_windows-x64_1_098685) and predicted the IDs of the PtrFBAs gene family. These sequences were further confirmed by the Protein BLAST function of the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 30 January 2023) [53,54]. The P. trichocarpa 4.1 genome, CDS, transcripts, polypeptides and 2000 bp upstream of the promoter region of the translation initiation site (ATG) were extracted from the Phytozome 13.0 database. The amino acid sequences of PtrFBX and PtrFBA target sequences were analyzed by ClustalX. In addition, using the neighbor-joining (NJ) method parameter on MEGA7.0 with pairwise deletion and 1000 bootstrap replicates, multiple alignments were applied to construct a phylogenetic tree among PtrFBAs [54,55]. PtrFBAs and PtrFBXs are named according to their positions on the scaffold and chromosomes, respectively.

4.2. Sequence Alignment, Physicochemical Properties, Subcellular Localization Prediction and Gene Positions

The amino acid sequences of each species were obtained from the NCBI database, the ClustalW (https://www.genome.jp/tools-bin/clustalw, accessed on 30 January 2023) website was used, the Clustal algorithm was used for sequence alignment, and the ENDscript/ESPript (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi, accessed on 30 January 2023) website was opened to map the sequence alignment results. The physicochemical properties of the PtrFBAs family were analyzed using the TBtools Protein Paramter Calc function. Subcellular localization was predicted by the site Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 30 January 2023) [56,57] and the predicted subcellular localization of the PtrFBAs family was obtained by entering their amino acid sequences into the website. Gene positions and chromosome sizes of PtrFBAs and PtrFBXs were obtained from the NCBI database and visualized by TBtools (South China Agricultural University, Guangzhou, China) [58].

4.3. Identification of Gene Duplication Events and Collinearity Relationship

The PtrFBX and PtrFBA duplication events in the poplar genome were evaluated using multiple collinear scanning toolkits (MCScanX) [59]. The poplar genome and gff3 files were applied to investigate the putative duplication events using TBtools with one-step MCScanX with default parameters. In addition, the ratio of non-synonymous (Ka) and synonymous (Ks) substitutions in PtrFBA gene pairs were measured to evaluate the selection pressure of the PtrFBAs evolutionary process. To further evaluate the collinearity relationship among poplar, Arabidopsis, apple, maize and wheat, the MCScanX with default parameters was also applied to identify the putative orthologs. In a similar manner to intra-poplar collinearity, the genome and gff3 files of poplar, Arabidopsis, apple, maize and wheat were aligned to achieve the three important files, including the control file (ctl), gff and collinearity formats. We manually removed unnamed chloroplasts and mitochondrial chromosomes from the ctl files and reordered them, and finally visualized them with TBtools.

4.4. Analysis of Conserved Motifs and Gene Structures

We uploaded gff3 annotation files that contained gene information to the TBtools (https://github.com/CJ-Chen/TBtools, accessed on 30 January 2023) software (TBtools_windows-x64_1_098685)to reveal gene conserved motifs and gene structures. In addition, the PtrFBA protein sequences were submitted to Motif’s Multiple Expectation Maximization (MEME) web version (https://meme-suite.org/meme/tools/meme, accessed on 30 January 2023) [60]. In addition, gff3 files and patterns stored in the Extensible Markup Language (XML) format were uploaded to the TBtools software for displaying conserved motifs and gene structures.

4.5. Analysis of Cis-Regulatory Elements

The cis-acting elements of promoters were predicted by PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 30 January 2023), and classified and analyzed according to the literature, and the results were visualized by EXCEL and TBtools.

4.6. Transcriptome Analysis and Visualization of the PtrFBA Family

To analyze the expression profile of the PtrFBA family, we searched and downloaded the transcriptome data of 30 PtrFBAs genes from the PopGenIE (https://plantgenie.org/, accessed on 30 January 2023) public database [61,62]. In this database, expression data were collected for three abiotic stresses and one biotic stress, as well as expression data for 15 different plant tissues. Heat maps of the relative expression quantification of PtrFBAs were created using TBtools, and Row Scale was chosen for homogenization, while all the other parameters were set as default parameters.

4.7. Plant Materials and Sample Collections

It was modified to: Annual poplar seedlings were planted in the nursery of Beijing Forestry University, with two poplar seedlings planted in each pot. P. trichocarpa seedlings were grown for about 3 to 4 months under the control of a greenhouse environment. Under different abiotic stresses, 5 potted seedlings with the same growth conditions were selected for each abiotic stress treatment. All collected samples were immediately frozen in liquid nitrogen and stored in a −80 °C freezer. After returning to the laboratory, RNA extraction (kit from Aidlab biotechnologies CO.LTD., Beijing, China) and reverse transcription (kit from TIANGEN BIOTECH CO.LTD.) were performed.

Arabidopsis ecotype Colombia (Col-0) was used in this study. The fba60 (SALK_019628.33.80.x) mutant was obtained from the Arabidopsis biological resource center ABRC (ABRC (osu.edu)). fba60 is a T-DNA insertional mutant. Plants were grown in soil or 1/2 MS medium and cultured in a constant temperature incubator (16 h light/8 h dark cycle, 22 °C).

4.8. Quantitative Real-Time (qRT-PCR) Analysis

qRT-PCR analysis-specific primers were designed and tested by NCBI Primer BLAST (https://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome, accessed on 30 January 2023). Real-time PCR was run with the CFX96 Touch™ instrument (Bio-Rad, Hercules, CA, USA) to detect the chemical SYBR Green. The reaction system established was as follows: 10 µL of 2× SuperReal Premix Plus (TIANGEN), 0.5 µL of each forward and reverse primer, 1 µL of diluted cDNA template (100 ng/µL), and finally, RNase-free ddH₂O was added until the total was 20 µL. The reaction procedures were as follows: 95 °C for 15 min, 45 cycles of 10 s at 95 °C and 30 s at 60 °C. We used two internal reference genes to conduct the experiment. The selection of internal reference genes and analysis of the experimental results were based on the research of Wang et al. [63,64]. SPSS (IBM, Armonk, NY, USA) was used for statistical analysis, and the LSD test was used to calculate the p value. All the primers used are listed in Supplemental Table S9.

4.9. Subcellular Localization of PtrFBA60 Proteins

To determine the subcellular localization of PtrFBA60 in plant cells, the full-length CDS of PtrFBA60 was cloned into the pSuper-1300 vector, generating a PtrFBA60-GFP fusion protein, and then introduced into A. tumefaciens GV3101. A plasmid that contained GFP alone served as a control. Similarly, the full-length CDS of the P-body markers gene AtAGO and AtTZF1 was cloned into the pSuper-1300 vector to generate AtAGO-mCherry and AtTZF1-mCherry fusion protein, and then introduced into A. tumefaciens GV3101. Transient expression assay in tobacco leaf epidermal cells was performed as previously described [65]. The GFP or mCherry signal was detected by laser confocal microscopy SP8 after two days of infestation (Leica Microsystems TCS SP8, Wetzlar, Germany).

4.10. Cultivation of Arabidopsis, Construction of Vector and Transformation of Arabidopsis and Identification of Transgenic Arabidopsis

The PtrFBA60 gene was cloned using P.trichocarpa as a template, and PtrFBA60 was inserted into the pBI121-GUS vector with XbaI and XmaI restriction sites. After fusion with the GUS in the pBI121 vector, the OE-PtrFBA60 vector was obtained and transformed into GV3101 Agrobacterium. The inflorescence method was used to transform Arabidopsis thaliana. We chose the activated monoclonal positive Agrobacterium containing the target gene and placed it into 10 mL fresh YEB medium (containing 100 mg/L Kan, 50 mg/L Rif), shaken overnight in a constant temperature incubator at 28 °C and 200 rpm cultivated for 12 h. The overnight cultured bacterial solution was added to 100 mL of YEB culture solution (containing 100 mg/L Kan, 50 mg/L Rif), shaken again to achieve the OD600 = 0.8 and centrifuged at 5000× g rpm for 10 min. The supernatant was discarded and we used the resuspension solution (containing 2.18 g 1/2 MS, 0.3–0.4% foaming agent, 50 g sucrose; 0.5 g MES per liter), resuspending the agrobacterium until the OD600 was 0.8. Arabidopsis thaliana inflorescence was invaded in the infestation solution for 2–3 min.After completion, the solution was incubated in the dark for 24 h. The second infection experiment was performed after 3–4 days. For the histochemical GUS analysis, the prepared material was immersed in GUS staining solution and incubated overnight at 37 °C.

4.11. Determination of Physiological Indicators Such as Drought Treatment and Germination Rate of Arabidopsis Thaliana

Wild-type and transgenic Arabidopsis seeds were sown simultaneously on 1/2 MS solid medium at a mannitol concentration of 0 or 250 mmol/L. Fifty-six seeds of each line were sown equidistantly on each medium, and three technical replicates were made. After 2 days of dark culture, the cells were placed in a constant temperature incubator at 22 °C for 16 h in the light, 8 h in the dark, and the germination rate was recorded every 12 h. Wild-type and transgenic Arabidopsis seeds were also sown simultaneously in soil and grown in a climate-controlled chamber with a light intensity of 180 μ mol/m²/s, a light duration of 14 h/dark duration of 10 h, and a temperature of 25 °C. Arabidopsis were subjected to natural drought treatment for 20 days after one month of growth. Wild-type and mutant Arabidopsis leaves were photographed after water loss and yellowing, and finally treated with rehydration for three days and counted for viability and REC.

4.12. Statistical Analyses

Statistical analyses were conducted using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and SPSS (version 25, IBM Corporation, Armonk, NY, USA). One-way ANOVA with the LSD multiple comparisons test (* p < 0.05, ** p < 0.01, *** p < 0.001.) was performed for the gene relative expression. Before applying the ANOVA test, the data were tested for normality and homogeneity of variance. Student‘s t test (* p < 0.05; ** p < 0.01) was performed for the survival rates and REC.

5. Conclusions

We believe that both tandem and WGD repeats were involved in the evolution of PtrFBAs genes in poplar, and that perennial woody plants may have undergone considerable FBA gene differentiation and doubling during evolution. Moreover, PtrFBA genes play a significant role in the process of plant growth and development, and also play a regulatory role in adversity stress. Among them, PtrFBA60 plays a positive regulatory role in plant drought resistance, which may allow plants to acquire drought resistance through the ABA pathway. Future experiments will aim to investigate the nature of these results from a genetic, biochemical and physiological perspective.