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Article

Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpa bungei

by
Erqin Fan
1,2,
Caixia Liu
1,3,
Zhi Wang
2,
Shanshan Wang
1,
Wenjun Ma
2,
Nan Lu
2,
Yuhang Liu
1,
Pengyue Fu
1,2,
Rui Wang
1,2,
Siyu Lv
1,2,
Guanzheng Qu
1 and
Junhui Wang
2,*
1
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
2
State Key Laboratory of Tree Genetics and Breeding, Key Laboratory of Tree Breeding and Cultivation of National Forestry and Grassland Administration, National Innovation Alliance of Catalpa bungei, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
3
College of Life Science, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(1), 97; https://doi.org/10.3390/ijms25010097
Submission received: 31 October 2023 / Revised: 27 November 2023 / Accepted: 28 November 2023 / Published: 20 December 2023
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Review Reports Versions Notes

Abstract

:
As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. Although the SPL gene family has been identified in diverse plant species, there have been no genome-wide identification or systematic study reports on the SPL gene family in Catalpa bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome. According to the phylogenetic relationship, they can be divided into eight groups, and the genes in the same group have a similar gene structure and conserved motifs. Synteny analysis showed that fragment duplication played an important role in the expansion of the CbuSPL gene family. At the same time, CbuSPL genes have cis-acting elements and functions related to light response, hormone response, growth and development, and stress response. Tissue-specific expression and developmental period-specific expression analysis showed that CbuSPL may be involved in flowering initiation and development, flowering transition, and leaf development. In addition, the ectopic expression of CbuSPL4 in Arabidopsis confirmed that it can promote early flowering and induce the expression of related flowering genes. These systematic research results will lay a foundation for further study on the functional analysis of SPL genes in C. bungei.

1. Introduction

Gene expression plays a crucial role in plant growth and development, and transcription factors (TFs), a class of proteins that can bind to specific nucleotide sequences upstream of genes, can greatly affect gene expression. This gene family is a collection of multiple genes with similar structures and functions, and they play a specific role in organisms. At present, many plant-specific transcription factor families have been identified in plants, such as NAC (NAM, ATAF, and CUC) [1], AP2/ERF [2], and SPL (SQUAMOSA promoter-binding protein-like) [3].
The SQUAMOSA promoter-binding protein-like (SPL) genes form a major family of plant-specific transcription factors, mainly related to flower development. SPL genes were first identified to regulate the expression of MADS-box genes during early flower development in Antirrhinum majus [4]. The SPL gene family encodes a highly conserved SBP domain containing approximately 76 amino acid residues, including 2 tandem zinc fingers (Cys-Cys-His-Cys and Cys-Cys-Cys-His) and a nuclear localization signal (NLS) at the C-terminus [3,5,6].
Research shows that SPL genes are widely involved in plant growth and development processes, including the initiation of flowering [7,8], phase transition from juvenile to adult, vegetative growth to reproductive growth [9,10,11], floral organ development [12], fruit development [13], phytohormone signal transduction [14], and response to abiotic stress [15,16,17]. In Arabidopsis, AthSPL3, SPL4, and SPL5 are closely related members, and the overexpression of all three genes promotes vegetative phase changes and flowering [10,18]. It was found that the Arabidopsis SPL protein can specifically bind to the conserved sequence motif in the promoter region of the A. thaliana floral meristem identity gene AP1 [5,18]. Although SPL has been shown to be involved in a variety of biological processes, the functional study of SPL in Catalpa bungei is still very limited.
Catalpa bungei is a perennial woody plant belonging to the genus Catalpa (Bignoniaceae) [19,20]. It is an ancient tree species with ornamental, economic, and medicinal values unique to China [21,22,23]. It takes 5–7 years to bloom, which seriously limits the work of hybrid breeding and genetic improvement [20,24]. Catalpa ‘Bairihua’ is an excellent hybrid with multi-season flowering obtained through the hybrid breeding of Catalpa bungei ‘Luo Qiu 4′ and C. fargesii Bur. f. duclouxii (http://rif.caf.ac.cn/News.aspx?ItemID=6009, accessed on 20 June 2022) [25,26]. Moreover, it can bloom in the same year through asexual reproduction methods such as grafting, which is very rare in woody plants [25]. The acquisition of Catalpa ‘Bairihua’ provides excellent materials for the study of flowering development and hybrid breeding of C. bungei and other woody plants. Therefore, it is crucial to explore the molecular mechanisms of flowering time regulation and reproductive transition of Catalpa ‘Bairihua’ for its genetic improvement and utilization.
As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. The SPL gene family has been identified in diverse plant species, such as A. thaliana [9,27], rice (Oryza sativa) [6], soybean (Glycine max) [28], maize (Zea mays) [29], tomato (Solanum lycopersicum L.) [30], grape (Vitis vinifera) [31], strawberry (Fragaria vesca) [32], poplar (Populus trichocarpa) [33], tea plant (Camellia sinensis) [34], apple (Malus domestica Borkh.) [35], Ziziphus jujuba [36], Fraxinus mandshurica [37], sweet cherry (Prunus avium L.) [13], and orchids [38]. However, there have been no genome-wide identification or systematic study reports on the SPL gene family in C. bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome. Subsequently, chromosome localization, phylogenetic analysis, gene structure analysis, conserved motif analysis, cis-acting elements analysis, and synteny analysis were performed. In addition, the expression patterns of 19 SPL genes in different tissues of C. bungei and flower buds of C. bungei and Catalpa ‘Bairihua’ at different developmental stages were analyzed. Finally, the CbuSPL4 gene was ectopically expressed in Arabidopsis to explore its gene function. Exploring the regulatory function of SPL gene family in the process of flowering and development of C. bungei is of great significance for the breeding and genetic improvement of C. bungei. The systematic research results will lay a foundation for further study on the functional analysis of SPL genes in C. bungei, and also provide new insights for further study on the biological function of SPL genes in the process of flowering transition and flower development of Catalpa ‘Bairihua’.

2. Results

2.1. Identification of CbuSPL Genes in C. bungei

To identify SPL genes in C. bungei, BLASTP analysis and HMM were performed against the whole genome sequence. A total of 19 putative SPL family genes identified in C. bungei were named from CbuSPL1 to CbuSPL19 according to their position from top to bottom on each corresponding chromosome and different chromosomes from chromosome 1 to chromosome 20 (Figure 1 and Supplementary Table S1). Among them, the six CbuSPL genes with the largest number were located on chromosome 18, followed by three genes on chromosomes 1 and 2 and two genes on chromosomes 13 and 15, respectively. There was only one gene on chromosomes 7, 10, and 17, respectively. The characteristics of these genes were analyzed and summarized in detail, including their gene length, CDS length, amino acid length, number of exons, Mw, pI, and subcellular localization prediction information. Their gene length ranged from 1180 to 6654 bp; the largest protein was encoded by CbuSPL17 with 1088 amino acids and a CDS length of 3267 bp, whereas the smallest protein was encoded by CbuSPL6 with 170 amino acids and a CDS length of 513 bp. The Mw of these proteins ranged from 18,894.06 (CbuSPL6) to 119,687.73 kDa (CbuSPL17) and pI varied from 6.12 (CbuSPL14) to 9.59 (CbuSPL16) (Table 1). In other analyses, we found significant differences in the number of exons ranging from 2 to 11 and the number of introns ranging from 1 to 10 for all CbuSPL genes; the mean number of exons was 4.47 (Table 1). Subcellular localization results showed that all CbuSPL proteins were localized in the nucleus (Table 1).

2.2. Sequence Alignments of CbuSPL Genes in C. bungei

The differences between the 19 CbuSPL proteins were analyzed through multiple-sequence alignment, and the results showed that all members of CbuSPL proteins contained a typical highly conserved SBP domain, which contained 74 amino acid residues, including two zinc finger motifs (Zn-1, Zn-2) and a nuclear localization signal (NLS) (Figure 2).

2.3. Phylogenetic Analysis of SPL Genes

To understand the phylogenetic relationships of the SPL family, we constructed a phylogenetic tree using all SPL full-length protein sequences from C. bungei (19 genes), Arabidopsis thaliana (17 genes), Populus trichocarpa (28 genes), Malus domestica (34 genes), Sesamum indicum (19 genes), Solanum lycopersicum (15 genes), and Oryza sativa (19 genes). A total of 151 SPL proteins from these seven species were classified into eight groups (I, II, III, VI, V, VI, VII, and VIII), each of which contained one or more CbuSPL proteins. The three largest groups have 40 (Group VIII), 28 (Group VII), and 18 (Group V) members, respectively (Figure 3).

2.4. Conserved Motifs and Gene Structure Analysis of CbuSPL Genes

In order to understand the differences in the gene structure of CbuSPL genes in C. bungei, we used TBtools v1.127 to visualize the gene structure of the CbuSPL genes, including the untranslated region (UTR), exons, and introns. A phylogenetic tree of 19 CbuSPLs was constructed to further analyze their evolutionary relationships (Figure 4a). To analyze the diversity and similarity of CbuSPL gene structures, 10 motifs were identified on the MEME website. The results show that all CbuSPL members contain more than three conserved motifs, including Motif 1, Motif 2, and Motif 3. Motif 8 only appears in CbuSPL16 and CbuSPL17 (in the same evolutionary branch). At the same time, CbuSPL genes with a close evolutionary relationship contain roughly the same conserved motifs, indicating that they may have similar functions (Figure 4b). All 19 CbuSPL proteins have a complete SBP conserved domain (Figure 4c). In the analysis of gene structure, the number of exons and introns contained in CbuSPL is very different. CbuSPL17 has the largest number of exons and introns, with 11 and 10, respectively, while CbuSPL4, CbuSPL5, and CbuSPL6 contain only 2 exons and 1 intron. In addition, a total of 12 CbuSPL genes have 5′-UTR and 3′-UTR and the remaining 7 members lack 5′-UTR and 3′-UTR. It can be found that CbuSPL genes in the same branch have similar gene structures (Table 1 and Figure 4d).

2.5. Chromosome Distribution and Synteny Analysis of the CbuSPL Genes in C. bungei

Gene duplication events are common and widely occur in plant gene family formation, which is important for understanding the adaptive evolution of species. To understand the duplication events of all CbuSPL genes in C. bungei, we performed synteny analysis using MCscanX and visualized with Advanced Circos in TBtools v1.127 [39,40,41]. We analyzed tandem duplication events between CbuSPL genes and found that there was only one gene pair tandem duplication event on chromosome 15 (Supplementary Table S2). Furthermore, we identified a total of six gene pairs with segmental duplication events, which occurred on 7 of the 20 chromosomes (Figure 5 and Supplementary Table S2), suggesting that segmental duplication plays an important role in the expansion of the SPL gene family in C. bungei. We also performed gene selection pressure analysis, using TBtools v1.127 to calculate the non-synonymous substitution (Ka) and synonymous substitution (Ks) values of the SPL gene family segmental duplication and tandem duplication gene pairs in C. bungei. It was found that the Ka/Ks ratio of each gene pair was less than 1, indicating that the CbuSPL gene family may have experienced strong purification selection pressure during evolution (Supplementary Table S2).

2.6. Syntenic Relationships of SPL Genes between C. bungei and Other Species

To further explore the synteny relationships between CbuSPL genes and related genes from other six representative species, including five eudicots (Arabidopsis thaliana, Populus trichocarpa, Malus domestica, Sesamum indicum, and Solanum lycopersicum) and one monocot (Oryza sativa), we performed comparative synteny analysis. The numbers of orthologous gene pairs were 15 between C. bungei and Arabidopsis, 38 between C. bungei and poplar, 44 between C. bungei and apple, 29 between C. bungei and sesame, 19 between C. bungei and tomato, and 5 between C. bungei and rice (Figure 6). It can be seen that there are few gene pairs between C. bungei and rice, which may be due to the closer phylogenetic relationship between dicots than that between monocots.

2.7. Analysis of Cis-Acting Elements in CbuSPL Promoters

The promoter sequence located upstream of the gene coding sequence is distributed with many cis-acting elements for regulating gene-specific expression. In order to better understand the potential regulatory mechanisms of CbuSPL genes in growth and development, plant hormone response, and stress response in Catalpa bungei, we further analyzed the 2000 bp promoter sequences upstream of CbuSPL genes and found a total of 47 types of cis-acting elements, including 22 light-responsive, 11 phytohormone-responsive, 8 plant growth-related, and 6 stress-responsive elements, respectively (Figure 7 and Supplementary Table S3). Among these cis-acting elements, light-responsive regulatory elements accounted for the largest proportion, including G-Box and Box 4, and others were distributed in most CbuSPL promoter regions. In addition, phytohormone regulatory elements were identified in most of the CbuSPL promoters, among which TGA-element, ABRE, P-box, and TCA-element were involved in the auxin response, abscisic acid response, gibberellin response, and salicylic acid response, respectively, while CGTCA-motif and TGACG-motif were involved in the methyl jasmonate (MeJA) response. Among plant-growth-related elements, a large number of CAT-box elements related to meristem expression were detected, and circadian elements were also found on the promoter regions of CbuSPL5, CbuSPL7, and CbuSPL9. Regarding stress response, ARE elements related to anaerobic induction, TC-rich repeats elements related to defense and stress responses, and LTR elements related to low-temperature response were more prevalent. The above results indicate that CbuSPL genes have potential roles in photosynthesis, abscisic acid, MeJA, circadian rhythm, meristem expression, and stress response.

2.8. Tissue-Specific Expression Analysis of CbuSPL Genes in C. bungei

In order to explore the expression pattern of CbuSPL genes in different tissues of C. bungei, we analyzed the expression of CbuSPL genes in different tissues from flower buds, leaves, petioles, and stems of C. bungei based on the previous RNA-seq data in the laboratory (Supplementary Table S4) and used TBtools v1.127 to draw a cartoon heat map. The results showed that the expression levels of CbuSPL3, CbuSPL4, CbuSPL5, CbuSPL6, CbuSPL8, CbuSPL9, CbuSPL11, CbuSPL17, and CbuSPL18 genes were significantly higher in flower buds, while the expression levels of CbuSPL2, CbuSPL12, and CbuSPL19 genes were higher in leaves. The expression levels of all 19 CbuSPL genes in petioles were lower. In addition, except for CbuSPL2, CbuSPL4, CbuSPL5, CbuSPL8, CbuSPL9, CbuSPL11, CbuSPL12, and CbuSPL19, the expression levels of the other genes in stems were relatively high (Figure 8). The above results indicate that the function of CbuSPL genes has differentiated and may be involved in flowering development, flowering transition, leaf development, stem development, and secondary growth in C. bungei.

2.9. Expression Pattern Analysis of CbuSPL Genes in Flower Buds of C. bungei and Catalpa ‘Bairihua’ at Different Developmental Stages

In order to explore the function of CbuSPL genes in the flowering transition and flowering development of Catalpa ‘Bairihua’, we analyzed the expression pattern of CbuSPL genes in the flower buds of C. bungei (normal flowering) and Catalpa ‘Bairihua’ (early flowering) during vegetative period (Vp), transition period (Tp), and reproductive period (Rp). Using the previous RNA-seq data from the laboratory [42], the heat map analysis was carried out (Supplementary Table S5). The results showed that except for CbuSPL16, CbuSPL7, CbuSPL14, and CbuSPL17, the expression levels of all other genes in EF vegetative period were significantly higher than those in the same period of NF, indicating that the CbuSPL gene family plays an important role in the flowering initiation. The expression levels of these four genes during the EF transition period were higher than those at the same period as NF, suggesting that these four genes may play a role in the flowering transition process. In addition, CbuSPL2, CbuSPL18, CbuSPL9, CbuSPL5, CbuSPL12, CbuSPL6, CbuSPL10, CbuSPL4, and CbuSPL13 genes have higher expression levels in the EF reproductive period, indicating that these genes may play a specific function when the flower buds of Catalpa ‘Bairihua’ are in the reproductive growth stage (Figure 9).

2.10. Ectopic Expression of CbuSPL4 in Arabidopsis

To explore the function of CbuSPL4 in flower development, we transformed CbuSPL4 driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter into wild-type Arabidopsis (35S::CbuSPL4). It was found that the transgenic plants showed obvious morphological changes compared with the wild type, including flowering time, plant height, leaf number, and leaf size (Figure 10a,b). The ectopic expression of the CbuSPL4 gene in wild-type Arabidopsis advanced the phase transition, accelerated the transition from juvenile to the adult phase, and led to early flowering under LD conditions, indicating that CbuSPL4 is roughly equivalent to its homologous gene AthSPL3/4/5 in A. thaliana [43] (Supplementary Figure S1). We next examined the expression of flowering-related genes in the inflorescences of 35S::CbuSPL4 transgenic plants. As expected, the expression levels of AGL24, LFY, SOC1/AGL20, FT, FUL/AGL8, and AP1/AGL7 were significantly induced in transgenic plants compared with WT (Figure 10c).

3. Discussion

Catalpa bungei is an ancient tree species with ornamental, economic, and medicinal values unique to China [21,22,23]. It takes a long time to flower; generally, it takes 5–7 years for it to blossom, which seriously limits the work of hybrid breeding and genetic improvement [20,24]. Catalpa ‘Bairihua’ is an excellent hybrid with multi-season flowering obtained through the hybrid breeding of Catalpa bungei ‘Luo Qiu 4′ and C. fargesii Bur. f. duclouxii (http://rif.caf.ac.cn/News.aspx?ItemID=6009, accessed on 20 June 2022) [25,26]. Moreover, it can bloom in the same year through asexual reproduction such as grafting, which is very rare in woody plants [25]. Therefore, it is crucial to explore the molecular mechanisms of flowering time regulation and the reproductive transition of Catalpa ‘Bairihua’ for its genetic improvement and utilization. As a plant-specific transcription factor, the SPL gene family plays a critical role in plant growth and development. Although the SPL gene family has been identified in diverse plant species, there have been no genome-wide identification or systematic study reports on the SPL gene family in Catalpa bungei. In this study, we identified 19 putative SPL gene family members in the C. bungei genome, which is consistent with the number of SPL genes identified in sesame (Figure 3), which is similar to the homologous relationship of C. bungei. In addition, the amino acid number of these 19 CbuSPL proteins ranged from 170 to 1088, which was basically consistent with the number of Arabidopsis SPL proteins [27].
Through the tissue-specific expression analysis of CbuSPL genes, it was found that there were significant differences in the expression levels of different genes in flower buds, leaves, petioles, and stems (Figure 8), indicating that the function of the CbuSPL genes has undergone tissue differentiation, which may be involved in the flowering development, flowering transition, leaf development, stem development, and secondary growth of C. bungei. In addition, we explored the expression patterns of the CbuSPL genes in flower buds during the vegetative period (Vp), transition period (Tp), and reproductive period (Rp) of C. bungei (normal flowering) and Catalpa ‘Bairihua’ (early flowering). It was found that specific genes were highly expressed at different developmental stages (EF-Vp, EF-Tp, and EF-Rp) of Catalpa ‘Bairihua’. For example, CbuSPL16, CbuSPL7, CbuSPL14, and CbuSPL17 genes were significantly higher in the EF transition period (EF-Tp), which may be related to the flowering transition. However, the expression of CbuSPL genes fluctuated in the flower buds at different developmental stages (NF-Vp, NF-Tp, and NF-Rp) of C. bungei. That is to say, although C. bungei did not bloom in the same year, in fact, the expression of CbuSPL genes was different at different stages of vegetative growth, and the genes that may control flowering development and leaf development were differentially expressed (Figure 9). In summary, this is consistent with previous reports that the SPL gene is involved in flowering initiation [7,8], phase transition from juvenile to adult, vegetative growth to reproductive growth [9,10,11], floral organ development [12], and fruit development [13].
The CbuSPL4 gene was ectopically expressed in Arabidopsis to explore its function. The results showed that the transgenic plants had significant differences in flowering time, plant height, leaf number, and size compared with the wild type (Figure 10a,b). By analyzing the best homologous hits of CbuSPL genes in Arabidopsis thaliana, it was found that CbuSPL4 and AthSPL3/4/5 were classified into one cluster (Supplementary Figure S1). This study also confirmed that the function of CbuSPL4 is roughly equivalent to that of AthSPL3/4/5 and can promote flowering [43]. AGL24 encodes a MADS-box protein involved in flowering, regulates the expression of SOC1, and is also upregulated by SOC1. AGL20/SOC1 controls flowering and is required for CO to promote flowering. AGL20/SOC1 acts with AGL24 to promote flowering and inflorescence meristem identity. LFY encodes a transcriptional regulator that promotes flowering transition and is involved in floral meristem development. FT together with LFY promotes flowering. FUL/AGL8 overexpression flowered early under SD and LD conditions and was negatively regulated by APETALA1 [44,45]. AP1 specifies the identity of floral meristem and sepals. The overexpression of AP1 promotes flowering, and there is a transition from apical and lateral branches to flowers [46]. We examined the expression of AGL24, LFY, SOC1/AGL20, FT, FUL/AGL8, and AP1/AGL7 genes in 35S::CbuSPL4 transgenic plants and found that the expression levels of these positively regulated flowering genes were significantly induced. In the future, we will further verify its gene function in C. bungei and conduct research on the regulatory relationship between CbuSPL4 and these genes to improve its regulatory network.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Catalpa bungei is a perennial woody plant. Catalpa ‘Bairihua’ is an excellent hybrid with multi-season flowering obtained through the hybrid breeding of Catalpa bungei ‘Luo Qiu 4′ and C. fargesii Bur. f. duclouxii (http://rif.caf.ac.cn/News.aspx?ItemID=6009, accessed on 20 June 2022) [25]. Moreover, it can bloom in the same year through asexual reproduction such as grafting, which is very rare in woody plants. Catalpa ‘Bairihua’ plants under standard water management and pest control were grown via grafting in the Catalpa test forest base of Luoyang City, Henan Province, China. Flower bud samples at different developmental stages were collected from 24 January to 24 March 2021 for expression analysis and transcriptome sequencing. The Arabidopsis were grown in the greenhouse under 16 h/8 h, light/dark at 22 °C. All samples were collected in the morning in 5 mL cryovials and immediately stored in liquid nitrogen.

4.2. Identification of SPL Gene Family in C. bungei

To identify the SPL gene family in the C. bungei genome, the amino acid sequences of 17 known SPL family genes [27] in Arabidopsis obtained from The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/, accessed on 6 July 2022) [47] were used as query sequences to perform a BLASTP search against the protein sequences of C. bungei data that annotated according to the C. bungei genome (the entire C. bungei genome was sequenced by our research group, and the related paper is in preparation) using a cutoff e-value of 1 × 10−5. After this initial screening, the HMM model file SBP domain (PF03110) was downloaded from the Pfam 35.0 database (http://pfam.xfam.org/, accessed on 6 July 2022) [48], and we searched the genome protein databases with an e-value cutoff of 1 × 10−5 using HMMER v3.3.2 software [49]. Subsequently, candidate SPL family genes and were then verified with the Batch CD-Search Tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 6 July 2022) [50], Pfam (http://pfam.xfam.org/, accessed on 6 June 2022) [48], and SMART (a Simple Modular Architecture Research Tool) (http://smart.embl.de/, accessed on 6 July 2022) [51] to ensure the completeness of the SBP domain. Redundant sequences or sequences with an incomplete SBP domain were excluded from the following analyses. Finally, each SPL gene was named according to the position on the chromosome from top to bottom. Molecular weight (Mw) and the theoretical isoelectric point (pI) of the SPL family genes in C. bungei were determined using the Compute pI/Mw tool (https://web.expasy.org/compute_pi/, accessed on 10 July 2022) of ExPASy (https://www.expasy.org/, accessed on 10 July 2022) [52,53]. Subcellular localization prediction was performed using DeepLoc-2.0 (https://services.healthtech.dtu.dk/services/DeepLoc-2.0/, accessed on 10 July 2022) [54].

4.3. Multiple-Sequence Alignment and Visualization of SBP Domain

Multiple-sequence alignment of SBP domains of CbuSPL proteins was performed using the Muscle program in MEGA11 v11.0.13 [55], and the results were visualized using Jalview v2.11.2.0 [56]. Sequence logos of SBP domains were drawn with TBtools v1.127 [40].

4.4. Phylogenetic Analysis

To infer the evolutionary history of CbuSPL genes, we selected six species, including five eudicots (Arabidopsis thaliana, Populus trichocarpa, Malus domestica, Sesamum indicum, and Solanum lycopersicum) and one monocot (Oryza sativa). Genome sequences and annotation files of Arabidopsis (Araport11) [57], Populus trichocarpa (v4.1) [58], apple (v1.1) [59], tomato (ITAG4.0) [60], and rice (v7.0) [61] were downloaded from Phytozome v13 (https://phytozome-next.jgi.doe.gov/, accessed on 20 October 2022) [62], while that of sesame (v1.0) [63] was downloaded from Ensembl Plants (http://plants.ensembl.org/index.html, accessed on 20 October 2022). Using the method mentioned above, the SPL gene family members from these species were identified for phylogenetic tree construction.
Multiple sequence alignments of SPL genes from C. bungei and 6 other species were performed using the Muscle program in MEGA11 [55], and then we used it to create maximum-likelihood phylogenetic trees with 5000 ultrafast bootstrap replicates using IQ-TREE v1.6.12 [64,65]. The tree was visualized using iTOL v 6.7.1 (https://itol.embl.de/, accessed on 10 March 2023) [66].

4.5. Conserved Motifs and Gene Structure Analysis

The online tool MEME v 5.5.1 (https://meme-suite.org/meme/tools/meme, accessed on 20 March 2023) was used to identify the conserved motifs of CbuSPL genes; the number of motifs was set to 10 and other parameters were default [67]. Based on the C. bungei genome annotation files, the gene structure was analyzed using TBtools v1.127, and the phylogenetic tree, conserved motifs, conserved domains, and gene structure were integrated and visualized using TBtools v1.127 [40].

4.6. Syntenic Analysis between C. bungei and Other Species

Syntenic analysis between C. bungei and other 6 species was performed by MCScanX program of TBtools v1.127 [39,40]. Using genome sequences and annotation files (gff3 or gtf format) as input data, the syntenic blocks for each pair of species were identified, with default parameters.

4.7. Cis-Elements Analysis

The 2000 bp upstream sequence of CDS of CbuSPL genes in C. bungei genome was obtained using TBtools v1.127 [40]. Then, the cis-elements of CbuSPL promoters were screened using the PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 July 2023) [68].

4.8. Expression Pattern Analysis

In order to explore the expression pattern of CbuSPL genes, we used the previous RNA-seq data to analyze the expression of CbuSPL genes in different tissues including flower buds, leaves, petioles, and stems, and explored the expression patterns of CbuSPL genes in flower buds during the vegetative period (Vp), transition period (Tp), and reproductive period (Rp) of C. bungei (normal flowering) and Catalpa ‘Bairihua’ (early flowering). The original data are shown in Supplementary Tables S4 and S5.

4.9. Ectopic Expression of CbuSPL4 in Arabidopsis

The full-length coding sequences of CbuSPL4 were cloned into pENTR/D-TOPO vector (Invitrogen, Waltham, MA, USA) after sequencing confirmation and inserted into the pBI121 binary vector [69] under the control of the CaMV 35S promoter with the In-Fusion HD Cloning Kit (TaKaRa, Dalian, China). The recombinant construct was introduced into Agrobacterium tumefaciens strain GV3101 and then transformed into wild-type Arabidopsis by the floral dip method [70,71]. The transgenic plants were screened on Murashige and Skoog (MS) medium [72] with 50 mg·mL−1 kanamycin until homozygous lines were obtained. The primers used for vector construction are given in Supplementary Table S6.

4.10. Total RNA Extraction and RT-qPCR

The total RNA from transgenic and wild-type Arabidopsis was extracted using an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). RNA quantity and purity were assessed with a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Reverse-transcription quantitative PCR (RT-qPCR) was performed to detect the expression of flowering-related genes in transgenic and wild-type Arabidopsis. One microgram of total RNA from each sample was used for reverse transcription to generate cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s protocol. RT-qPCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa) on a 7500 Fast Real-Time PCR System machine (Applied Biosystems, Waltham, MA, USA). Three biological and three technical replicates were performed, and the relative expression of the target gene was calculated with the 2−ΔΔCT method [73]. β-TUBULIN-2 (AT5G62690.1) was used as an internal reference gene [7]. The primers used for RT-qPCR are listed in Supplementary Table S6.

4.11. Heatmap Analysis

The heatmap analysis of cis-elements, tissue-specific expression, and developmental stage-specific expression was carried out using TBtools v1.127 [40]. The entire raw data are provided in the Supplementary Materials.

4.12. Statistical Analysis

All statistical analysis and graphing were performed using Excel and GraphPad Prism v9.0.0 (121).

5. Conclusions

To the best of our knowledge, this is the first report on the genome-wide analysis of the SPL genes in Catalpa bungei. A total of 19 CbuSPLs were identified in this study, all of which have a complete SBP domain. According to the phylogenetic relationship, they can be divided into eight groups, and the genes in the same group have similar gene structure and conserved motifs. Synteny analysis showed that CbuSPL genes were unevenly distributed on chromosomes, and also suggested that fragment duplication played an important role in the expansion of the CbuSPL gene family. At the same time, CbuSPL genes have cis-acting elements and functions related to light response, hormone response, growth and development, and stress response. Tissue-specific expression and developmental period-specific expression analysis showed that CbuSPL may be involved in flowering initiation and development, flowering transition, leaf development, and other processes. In addition, the ectopic expression of CbuSPL4 in Arabidopsis confirmed that it can promote early flowering and induce the expression of related flowering genes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25010097/s1.

Author Contributions

E.F. and J.W. conceived the research and designed the experiments. E.F., C.L. and Z.W. analyzed the data. E.F., C.L., S.W., Y.L., P.F., R.W. and S.L. performed the experiments. E.F. and C.L. wrote the manuscript with input from all coauthors. W.M., N.L., G.Q. and J.W. revised the manuscript. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Program of China (2021YFD2200301).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Distribution of CbuSPL genes on the C. bungei chromosomes.
Figure 1. Distribution of CbuSPL genes on the C. bungei chromosomes.
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Figure 2. Multiple sequence alignment of SBP domains from CbuSPL proteins. (a) Multiple alignment of SBP domains; (b) sequence logo of the SBP domains.
Figure 2. Multiple sequence alignment of SBP domains from CbuSPL proteins. (a) Multiple alignment of SBP domains; (b) sequence logo of the SBP domains.
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Figure 3. Maximum-likelihood phylogenetic tree of SPL family proteins in C. bungei, Arabidopsis thaliana, Populus trichocarpa, Malus domestica, Sesamum indicum, Solanum lycopersicum, and Oryza sativa. Branches and labels of different colors represent different groups, and the numbers at nodes represent bootstrap values.
Figure 3. Maximum-likelihood phylogenetic tree of SPL family proteins in C. bungei, Arabidopsis thaliana, Populus trichocarpa, Malus domestica, Sesamum indicum, Solanum lycopersicum, and Oryza sativa. Branches and labels of different colors represent different groups, and the numbers at nodes represent bootstrap values.
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Figure 4. Phylogenetic relationship, conserved motif, and gene structure analysis of CbuSPL genes in C. bungei. (a) Phylogenetic tree of all CbuSPL proteins constructed using maximum-likelihood method; (b) motif distribution of CbuSPL proteins; Motifs 1–10 are shown as rectangular boxes of different colors; (c) conserved domain distribution of CbuSPL proteins; the purple box indicates the SBP domain in the corresponding amino acid sequence; (d) gene structures of CbuSPL genes arranged according to phylogenetic relationship; yellow boxes represent 5′ UTR and 3′ UTR, green boxes represent exons, and gray lines represent introns.
Figure 4. Phylogenetic relationship, conserved motif, and gene structure analysis of CbuSPL genes in C. bungei. (a) Phylogenetic tree of all CbuSPL proteins constructed using maximum-likelihood method; (b) motif distribution of CbuSPL proteins; Motifs 1–10 are shown as rectangular boxes of different colors; (c) conserved domain distribution of CbuSPL proteins; the purple box indicates the SBP domain in the corresponding amino acid sequence; (d) gene structures of CbuSPL genes arranged according to phylogenetic relationship; yellow boxes represent 5′ UTR and 3′ UTR, green boxes represent exons, and gray lines represent introns.
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Figure 5. Collinearity analysis of the SPL gene family in C. bungei. From the inside to the outside, the gradient color rectangle represents chromosomes 01–20, the red dot represents the gap distribution on the genome, the black line represents the GC ratio on the genome, and the heat map of the outermost circle represents the gene density. In the center, the gray lines indicate synteny blocks in the C. bungei genome, while the red lines between chromosomes delineate segmental duplication gene pairs.
Figure 5. Collinearity analysis of the SPL gene family in C. bungei. From the inside to the outside, the gradient color rectangle represents chromosomes 01–20, the red dot represents the gap distribution on the genome, the black line represents the GC ratio on the genome, and the heat map of the outermost circle represents the gene density. In the center, the gray lines indicate synteny blocks in the C. bungei genome, while the red lines between chromosomes delineate segmental duplication gene pairs.
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Figure 6. Synteny analysis of the CbuSPL genes between C. bungei and six other plant species. The gray lines indicate gene blocks in C. bungei that are orthologous to the other genomes. The red lines delineate the syntenic SPL gene pairs.
Figure 6. Synteny analysis of the CbuSPL genes between C. bungei and six other plant species. The gray lines indicate gene blocks in C. bungei that are orthologous to the other genomes. The red lines delineate the syntenic SPL gene pairs.
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Figure 7. Analysis of cis-acting elements in the promoter region of the CbuSPL genes. Based on functional annotation, CbuSPL gene cis-acting elements can be classified into four categories: light-responsive, phytohormone-responsive, plant-growth-related, and stress-responsive.
Figure 7. Analysis of cis-acting elements in the promoter region of the CbuSPL genes. Based on functional annotation, CbuSPL gene cis-acting elements can be classified into four categories: light-responsive, phytohormone-responsive, plant-growth-related, and stress-responsive.
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Figure 8. Expression analysis of CbuSPL genes in flower buds, leaves, petioles, and stems of C. bungei. Expression values are based on RNA-seq, visualized using TBtools v1.127. Red represents high expression levels and blue represents low expression levels.
Figure 8. Expression analysis of CbuSPL genes in flower buds, leaves, petioles, and stems of C. bungei. Expression values are based on RNA-seq, visualized using TBtools v1.127. Red represents high expression levels and blue represents low expression levels.
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Figure 9. Expression pattern analysis of CbuSPL genes in flower buds of C. bungei and Catalpa ‘Bairihua’ at different developmental stages. NF: Normal flowering (C. bungei), EF: Early flowering (Catalpa ‘Bairihua’); VP: Vegetative period, TP: Transition period, RP: Reproductive period.
Figure 9. Expression pattern analysis of CbuSPL genes in flower buds of C. bungei and Catalpa ‘Bairihua’ at different developmental stages. NF: Normal flowering (C. bungei), EF: Early flowering (Catalpa ‘Bairihua’); VP: Vegetative period, TP: Transition period, RP: Reproductive period.
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Figure 10. Ectopic expression of CbuSPL4 in Arabidopsis. (a,b) Phenotypic characterization of 35S::CbuSPL4 transgenic plants. (c) Expression analysis of flowering-related genes in transgenic Arabidopsis and wild-type plants through real time RT-PCR. Error bars represent SE values of three biological replicates. Asterisks indicate significant differences between transgenics and wild-type plants, determined with Student’s t test (**, p < 0.01). Bars = 2 cm.
Figure 10. Ectopic expression of CbuSPL4 in Arabidopsis. (a,b) Phenotypic characterization of 35S::CbuSPL4 transgenic plants. (c) Expression analysis of flowering-related genes in transgenic Arabidopsis and wild-type plants through real time RT-PCR. Error bars represent SE values of three biological replicates. Asterisks indicate significant differences between transgenics and wild-type plants, determined with Student’s t test (**, p < 0.01). Bars = 2 cm.
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Table 1. Characteristics properties of CbuSPLs in C. bungei.
Table 1. Characteristics properties of CbuSPLs in C. bungei.
Gene NameGene IDChromosomal
Location		Number of ExonNumber of
Intron		Gene Length (bp)CDS Length (bp)Number of Amino Acid (aa)Molecular Weight (kDa)Theoretical pISubcellular Localization
CbuSPL1evm.TU.group0.1477Chr01:55742624-55745394432771112837541,716.90 8.65 Nucleus
CbuSPL2evm.TU.group0.1766Chr01:58009013-58013912764900138346050,055.82 8.68 Nucleus
CbuSPL3evm.TU.group0.1777Chr01:58096915-5810094354402956418720,922.46 9.26 Nucleus
CbuSPL4evm.TU.group1.669Chr02:12828539-1283016721162952817520,058.44 8.18 Nucleus
CbuSPL5evm.TU.group1.1223Chr02:18112076-1811378121170657018921,333.84 8.60 Nucleus
CbuSPL6evm.TU.group1.1470Chr02:19850125-1985224921212551317018,894.06 9.38 Nucleus
CbuSPL7evm.TU.group14.282Chr07:2454793-2458481873689154851557,276.22 8.16 Nucleus
CbuSPL8evm.TU.group17.394Chr10:7319282-7324180654899143147651,933.40 8.56 Nucleus
CbuSPL9evm.TU.group2.188Chr13:10783205-1078507343186986128631,903.87 8.86 Nucleus
CbuSPL10evm.TU.group2.1122Chr13:24327368-24330828323461108336038,249.37 9.40 Nucleus
CbuSPL11evm.TU.group4.922Chr15:21772905-2177545432255097532435,676.03 8.16 Nucleus
CbuSPL12evm.TU.group4.923Chr15:21785690-21788384322695114338041,706.53 8.74 Nucleus
CbuSPL13evm.TU.group6.274Chr17:9460552-9463317322766126642147,028.82 8.69 Nucleus
CbuSPL14evm.TU.group7.217Chr18:2162703-21685461095844236478788,053.29 6.12 Nucleus
CbuSPL15evm.TU.group7.2043Chr18:20920539-20922742322204104134638,610.53 6.80 Nucleus
CbuSPL16evm.TU.group7.2477Chr18:24516899-2451807832118081327030,105.91 9.59 Nucleus
CbuSPL17evm.TU.group7.2481Chr18:24528473-245351261110665432671088119,687.73 6.78 Nucleus
CbuSPL18evm.TU.group7.2509Chr18:24721085-2472288132179797532436,178.42 9.31 Nucleus
CbuSPL19evm.TU.group7.3182Chr18:30778588-30781167322580112237340,566.42 9.35 Nucleus
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Fan, E.; Liu, C.; Wang, Z.; Wang, S.; Ma, W.; Lu, N.; Liu, Y.; Fu, P.; Wang, R.; Lv, S.; et al. Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpa bungei. Int. J. Mol. Sci. 2024, 25, 97. https://doi.org/10.3390/ijms25010097

AMA Style

Fan E, Liu C, Wang Z, Wang S, Ma W, Lu N, Liu Y, Fu P, Wang R, Lv S, et al. Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpa bungei. International Journal of Molecular Sciences. 2024; 25(1):97. https://doi.org/10.3390/ijms25010097

Chicago/Turabian Style

Fan, Erqin, Caixia Liu, Zhi Wang, Shanshan Wang, Wenjun Ma, Nan Lu, Yuhang Liu, Pengyue Fu, Rui Wang, Siyu Lv, and et al. 2024. "Genome-Wide Identification and Expression Analysis of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Catalpa bungei" International Journal of Molecular Sciences 25, no. 1: 97. https://doi.org/10.3390/ijms25010097

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