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Genome-wide identification and comparative evolution of 14–3-3 gene family members in five Brassicaceae species

BMC Genomics volume 26, Article number: 309 (2025) Cite this article

Abstract

Background

The 14–3-3 proteins are highly conserved regulatory eukaryotic proteins, which are crucial in growth, development, and stress responses. However, systematic characterization of the 14–3-3 gene family in Brassicaceae species and their evolutionary relationships have not been comprehensively reported.

Results

This study conducted genome-wide identification, structural characteristics, and comparative evolutionary analysis of 14–3-3 gene family members in Arabidopsis thaliana, A. lyrata, A. pumila, Camelina sativa, and Brassica oleracea using comparative genomics. Overall, a total of 108 14–3-3 genes, which were phylogenetically classified into ε and non-ε groups were identified in the five species, with the non-ε members exhibiting more similar exon–intron structures and conserved motif patterns. Collinearity analysis revealed that the Brassicaceae 14–3-3 gene family members underwent varying degrees of expansion following whole-genome duplication (WGD) events. Notably, the number of 14–3-3 gene family members between A. lyrata and A. thaliana remained similar despite the former having approximately 1.66-fold larger genome size. In contrast, the number of 14–3-3 gene family members in A. pumila and C. sativa increased in proportionately to their genome size, while gene members in the more distantly related species to A. thaliana, B. oleracea, showed irregular expansion patterns. Selection pressure analysis revealed that 14–3-3 homologs in all the five species underwent purifying selection, with the group ε members experiencing relatively weaker purifying selection. Cloning of ApGRF6-2 gene from A. pumila indicated that the ApGRF6-2 protein was localized in the cell membrane and cytoplasm, while ectopic overexpression of ApGRF6-2 in A. thaliana could promote early flowering by upregulating the expression of floral meristem identity genes.

Conclusion

This study provides a comprehensive and systematic identification of the 14–3-3 gene family members in five Brassicaceae species using updated genome sequences, and the results could form a basis for further validation of functional and molecular mechanisms of 14–3-3 genes in plant growth, development, abiotic stress responses, as well as flowering regulation.

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Introduction

The 14–3-3 protein family contains highly conserved acidic and water-soluble proteins that are ubiquitously distributed in eukaryotes. The family was initially identified in bovine brain tissue and functionally crucial regulators of cellular life activities and plant growth and development [1]. The plant 14–3-3 proteins are composed of two monomers that form homo- or heterodimers and are considered part of the G-box protein complex, thus, they are also referred to as the G-box Factor 14–3-3 homologs (GF14) or General Regulatory Factors (GRF) [2, 3].

The 14–3-3 proteins in plants function as adaptor/scaffold proteins, interacting with target proteins to modulate their activity, dephosphorylation, subcellular localization, and expression [4,5,6]. Based on their sequence homology and introns count, the 14–3-3 gene family can be divided into ε and non-ε subfamilies. The ε subfamily typically contains more introns, while the non-ε members exhibit stronger H+-ATPase binding and activation [7]. Structurally, the highly conserved 14–3-3 proteins are generally categorized into three regions, including the variable N-terminal, the conserved core region, and the multifunctional C-terminal, with the pSer or pThr that interacts with target proteins typically located within the polar groove of the conserved core region [8, 9]. Moreover, 14–3-3 proteins can form dimers at the N-terminal site, while the C-terminal interacts with target proteins [10]. The motifs within target proteins that interact with 14–3-3 proteins are mainly categorized into three distinct types: motif I (R/K)SX(S/T)PXP, motif II (R/R)XΦX(S/T)PXP (with Φ indicating an aromatic or aliphatic amino acid and X representing any amino acid) [11, 12], and the C-terminal motif III (S/T)P X 1–2-COOH [13, 14]. Motif I and II are the main modes that recognize 14–3-3 proteins, while motif III exhibits a relatively weaker binding affinity [15].

Recent years have witnessed increased identification of the 14–3-3 gene family members in several species, including rice (Oryza sativa) [16], grape vine (Vitis vinifera) [17], cotton (Gossypium hirsutum) [18], potato (Solanum tuberosum) [19], and tobacco (Nicotiana tabacum) [20]. Numerous studies have shown that 14–3-3 proteins can form complexes with their interacting proteins to regulate various aspects of plant growth and development. For example, 14–3-3 acts as a receptor for the florigen FLOWERING LOCUS T (FT) in the shoot apical meristem (SAM). The resulting complex is transported to the nucleus where it binds with the bZIP transcription factor FD to form the florigen activation complex (FAC) that regulates the transition to flowering [21, 22]. In potato, 14–3-3/74 protein interacts with SELF-PRUNING protein (a homolog of A. thaliana TERMINAL FLOWER1), and overexpression of the 14–3-3/74 gene can rescue for the loss of SELF-PRUNING function [23]. Moreover, complexes formed by 14–3-3 and its interacting proteins exhibit significant regulatory roles in plant responses to abiotic stresses, including drought, salt, cold, and heat. For example, transgenic Arabidopsis plants overexpressing At14-3-3λ exhibited reduced cold tolerance [24]. Moreover, proteomic studies on Brassica napus have shown that 14–3-3 proteins participate in the responses to salt and drought stresses [25].

The Brassicaceae family, as one of the largest and most diverse group of flowering plants that harbors a critical position in plant systematics and evolutionary studies [26]. WGD events are the major driving force behind the evolution and diversification of Brassicaceae species. During gene evolution, redundant genes following WGD can evolve new functions through sub-functionalization and neofunctionalization to enhance plant adaptability to the environment. Similarly, WGD can also trigger reconstruction of gene expression regulatory networks, which can modify various aspects of plant growth, development, metabolic pathways, and stress responses [27, 28]. A. thaliana has undergone at least five WGD events [29], which provides a genetic basis for its adaptability and diversity, while A. lyrata has experienced a WGD event common to all Brassicaceae species (At-α), which has significantly modified its genome evolution and adaptability [30]. In contrast, Brassica plants experienced additional whole-genome triplication event (Br-β) approximately 17 million years ago, which through increased gene dosage, enhanced recombination, and polyploidy effects, contributed to a broader ecological niche distribution in these crops [31]. Moreover, an allopolyploid, C. sativa plant has also undergone an additional whole-genome triplication event [32], which not only shaped its genomic features, but also had potential significant implications for its physiology, ecology, and evolution.

Since the first release of A. thaliana genome sequence approximately 25 years ago [33], the genomes of nearly 1,500 plant species have subsequently been sequenced, with the genome sequences of species [34], such as A. lyrata [35], C. sativa [32], and B. oleracea [36] having also been completed. Comparative genomics studies indicate that the genome of A. lyrata (207 Mb) is approximately 1.66 times the size of that of A. thaliana (125 Mb) [35], while the genome size of the allopolyploid C. sativa is about three times the size of A. thaliana [32]. Interestingly, B. oleracea genome has been shown to have high degree of collinearity with the A. thaliana genome [36, 37]. A. pumila is a salt tolerant ephemeral plant in the Brassicaceae family with a relatively recent WGD that primarily occurs in the deserts of Xinjiang, China [38,39,40,41]. Although the 14–3-3 genes have been reported in numerous plant species, reaches on the identification, structural characterization, and comparative evolution of this gene family in Brassicaceae species using recently updated genome sequences remain limited.

Here, bioinformatic techniques were used to systematically identify the 14–3-3 gene family members, perform structural analysis of gene and protein domain features, as well as their evolutionary relationships from the complete genomes of five Brassicaceae species. In addition, the expression profiles of A. pumila 14–3-3 gene members in different tissues in response to salt stress as well as the role of ApGRF6-2 in the regulation of flowering transition were analyzed. Our results provide a foundation for further systematic analysis of the evolutionary history of the Brassicaceae 14–3-3 gene family, evaluation of their functional roles and molecular mechanisms in environmental adaptation, and prediction of species-specific functional genes.

Materials and methods

Plant materials

The seeds of wild-type A. thaliana (Col-0) and A. pumila were sterilized and cultured on 1/2 Murashige and Skoog (MS) medium for about 7 days at 23℃ in darkness [42]. Afterward, the seedlings were transferred to a plant growth room under long-day (LD) conditions (16 h light/8 h dark, 23 °C, 200 μmol m⁻2 s⁻1) for further growth. Samples were collected from various developmental stages of A. pumila: hypocotyls (8 d), cotyledons (8 d), roots (30 d), stems (30 d), rosette leaves (30 d), flowers (65 d), and pods (70 d). Four-week-old A. pumila plants were watered with 1/2 MS nutrient mixture supplemented with 250 mM NaCl as described previously [39]. Leaf tissues were collected at 7 different time points (0, 0.5, 3, 6, 12, 24, and 48 h) after stress treatment. Col-0 and transgenic A. thaliana SAM samples were collected at the stage of third true leaf expansion. All the collected materials were quickly frozen in liquid nitrogen and stored at −80 °C for subsequent gene expression analysis. Each treatment was conducted in three biological replicates.

Identification of 14–3-3 genes in Brassicaceae species

The genome data for A. lyrata [35], C. sativa [32], and B. oleracea [36] were downloaded from the Ensembl Plants genome database (https://plants.ensembl.org); while the A. thaliana genome data (TAIR10.1) was obtained from the A. thaliana Information Resource website (TAIR, https://www.arabidopsis.org). The A. pumila genome data were provided by our laboratory. The protein sequences of the A. thaliana 14–3-3 gene family were used as queries to perform BLASTP searches against the protein sequence databases of A. lyrata, A. pumila, C. sativa, and B. oleracea, with an E-value ≤ 1e−10. The retrieved protein sequences were then submitted to the PfamScan website (https://www.ebi.ac.uk/jdispatcher/pfa/pfamscan) for domain alignment. Genes corresponding to protein sequences containing the 14–3-3 protein domain (PF00244) were considered members of the 14–3-3 gene family in each species. The naming of genes was based on their similarity to A.thaliana 14–3-3 homologs and their gene structure features (Table S1). The molecular weight, isoelectric point, and grand average hydrophobicity (GRAVY) were estimated using the ExPASy (https://web.expasy.org/protparam/) protein analysis tool. Subcellular localization information was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).

Sequence alignment and phylogenetic analysis

The amino acid conservation of 14–3-3 protein sequences in five Brassicaceae species was analyzed with DNAMAN software. All acquired sequences were first aligned using ClustalX software [43] with the default parameters. An unrooted neighbor-joining (NJ) phylogenetic tree was constructed using MEGA11 [44] software with 1000 bootstrap replicates.

Gene structure and conserved motif analysis

Gene structure information of 14–3-3 genes in each species was extracted using the TBtools-II software [45] based on the genome gff files of the five species. The exon–intron distribution of the 14–3-3 genes was analyzed using the Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/). Conserved motifs in the 14–3-3 proteins from each species were identified using the MEME website (http://meme-suite.org/tools/meme), with motif lengths ranging from 10 to 60 amino acid residues and a maximum of 10 motifs. Other parameters were set to default values.

Collinearity analysis, selection pressure, and duplication of the 14–3-3 gene family

The inter- and intra-species collinearity of 14–3-3 genes among A. thaliana and the other four Brassicaceae species was defined using the MCScanX method within TBtools-II software [45, 46]. The nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) were calculated using DnaSP 6.0 software [47]. Selective pressure on the 14–3-3 genes during evolution was assessed by analyzing the Ka/Ks ratios of collinear gene pairs of the 14–3-3 gene family in the five species of Brassicaceae. A Ka/Ks ratio > 1, < 1, or = 1 indicates positive, negative, or neutral selection, respectively [48]. Potential segmental duplication and tandem duplication events were investigated using TBtools-II software [45].

Analysis of cis-acting elements in the promoters of 14–3-3 genes

The 2000 bp DNA region upstream of the start codon (ATG) of the 14–3-3 genes was extracted from the genomes of the five species. The presumed promoter regions of each 14–3-3 gene were then submitted to Plant CARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for analysis of cis-acting elements in the promoters. Cis-acting elements were visualized in the promoter regions using TBtools-II software [45].

Expression profile analysis of A. pumila 14–3-3 genes using RNA‑seq data

RNA-seq data for seven tissues (cotyledons, hypocotyls, stems, rosette leaves, roots, flowers, and siliques) of A. pumila and leaf tissues under 250 mM NaCl stress at seven different time points (0, 0.5, 3, 6, 12, 24, and 48 h) were obtained from the SRA database (https://www.ncbi.nlm.nih.gov/sra) (Tables S2 and S3) [39]. The RNA-seq data were standardized using Cufflinks software [49]. The fragments per kilobase per million reads (FPKM) values were log-transformed using Log2 (FPKM + 1), and the results were visualized with heatmaps using TBtools-II software [45].

Total RNA extraction and expression analysis of Ap14-3–3 genes

Specific primers for the Ap14-3–3 gene were designed using Primer 3.0 (https://primer3.ut.ee/) (Table S4), and the ApGAPDH gene was used as the internal control for normalizing gene expression in real-time quantitative reverse transcription PCR (qRT-PCR) analysis [50]. Total RNA from each sample was extracted using the RNAprep Pure Plant Plus Kit (TianGen, China) according to the manufacturer's protocol, and cDNA was synthesized using the MonScriptTM RTIII AII-in-One Mix with dsDNase kit (Monad, Wuhan) as directed by the manufacturer. The qRT-PCR experiment was performed on an ABI ViiA7 Real-Time PCR System (Life Technologies, USA). The qRT-PCR system and methods used were as described by Jin et al. [50]. The relative expression levels of genes in different tissues were calculated using the 2 (-Delta C(T)) method, and the relative expression levels of genes under salt stress at different time points were calculated using the 2 (-Delta Delta C(T)) method [51]. For each sample, three biological repeats, with three technical replicates each, were performed to assess the reliability of the results. The results are presented as means ± standard deviation (SD). All statistical analyses were conducted using Duncan multiple-range test, and different letters were used to denote significant differences (P < 0.05).

Prediction of 3D structure and subcellular localization analysis

Based on the amino acid sequences of GRF6 homologous genes from five species of Brassicaceae, the initial models of the GRF6 proteins from these five species were predicted using AlphaFold 3 [52]. The 3D structures were then visualized using ChimeraX [53]. Based on the coding sequence (CDS) of the ApGRF6-2 gene, primers without the stop codon were designed (Table S4). The target fragment was amplified by PCR, and a 35S:ApGRF6-2-GFP green fluorescent protein (GFP) fusion overexpression vector was constructed following the method described by Guo et al. [54]. The ApGRF6-2 coding sequence, excluding the stop codon, was cloned and inserted into the pEASY-Blunt-Zero (TRAN, Beijing) vector and transformed into Escherichia coli DH5α. Using T4 ligase, the correctly sequenced gene fragment was ligated with the pCAMBIA2300-GFP vector [54] and transformed into E. coli DH5α. The correct plasmid, 35S:ApGRF6-2, was then transformed into Agrobacterium GV3101. The A. thaliana protoplast subcellular localization experiment was conducted as described by Liu et al. [55].

Genetic transformation of A. thaliana

The 35S:ApGRF6-2 construct was created by inserting the ApGRF6-2 CDS downstream of the Cauliflower mosaic virus (CaMV) 35S promoter into the binary vector pCAMBIA2300-OCS [54]. The recombinant plasmid 35S:ApGRF6-2 was introduced into Agrobacterium GV3101, and then transformed into wild-type A. thaliana (Col-0) using the floral dip method [56]. Transgenic A. thaliana positive plants were selected on 1/2 MS medium containing 50 mg/ml kanamycin, and homozygous 35S:ApGRF6-2 transgenic lines were obtained. Under LD conditions, the flowering time and the number of rosette leaves at flowering were observed and counted for Col-0 and two homozygous 35S:ApGRF6-2 transgenic lines. The expression levels of ApGRF6-2 and flowering-related genes in the SAM of Col-0 and the two homozygous 35S:ApGRF6-2 transgenic A. thaliana lines were measured by qRT-PCR, with AtActin2 (GenBank accession no. NM_180280) used as the internal reference gene (Table S4).

Results

Identification and comparative evolution of 14–3-3 gene family members in five Brassicaceae species

Analysis of the whole-genome sequences revealed a total of 13, 24, 39, and 19 14–3-3 gene members in A. lyrata, A. pumila, C. sativa, and B. oleracea, respectively (Table S1). Cumulatively, the five plant species contained 108 genes, with A. thaliana and A. lyrata having the fewest 14–3-3 genes (both with 13 genes), while C. sativa had most 14–3-3 genes, with 39 members. The proteins encoded by the 14–3-3 genes showed amino acid (aa) sequence length ranging from 166 (CsGRF3-5) to 413 (CsGRF12-2) aa, molecular weights ranging from 18.36 (CsGRF3-5) to 47.51 (CsGRF12-2) kDa, and isoelectric points (pI) ranging from 4.47 (AtGRF7) to 5.49 (AlGRF13). All proteins were found to be acidic, while the GRAVY for all 14–3-3 proteins was less than zero, indicating that all members were hydrophilic. Subcellular localization prediction showed that 14–3-3 proteins from the five species were primarily localized in the nucleus, with some members showing dual localization in both the nucleus and the cytoplasm (Table S1).

Evolutionary analysis of the 14–3-3 gene family members in Brassicaceae species using multiple alignment of the amino acid sequences showed that the secondary structures of the 108 14–3-3 proteins contained 9 α-helices, with α3 and α4 as the longest helices (Fig. S1). Subsequent phylogenetic tree using the neighbor-joining method revealed that the 14–3-3 proteins could primarily cluster into ε and non-ε subgroups (Figs. 1 and 2A). In each of the five species, the number of non-ε group members was greater than the number of ε group members (Fig. S2). Notably, the homologous gene numbers in the other four species differed from those in A. thaliana, with GRF3 harboring highest number (6) of homologous genes. However, the homologous genes of GRF11 were absent in A. pumila, while those of GRF4, GRF5, and GRF13 were absent in B. oleracea.

Fig. 1
figure 1

Phylogenetic tree of the 14–3-3 gene family members from five Brassicaceae species (circle tree). The neighbor-joining tree was constructed using 108 aligned 14–3-3 amino acid sequences from A. thaliana (13), A. lyrata (13), A. pumila (24), C. sativa (39), and B. oleracea (19) in MEGA11 [44], with bootstrap values calculated from 1000 replicates and parameters set to default values

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Fig. 2
figure 2

Phylogenetic relationships and structural characterization of 14–3-3 genes and conserved motifs in five Brassicaceae species. A Phylogenetic tree (branching tree) of 108 14–3-3 genes from five Brassicaceae species; B Intron–exon structures of the14-3–3 genes in five Brassicaceae species; C and D Conserved motifs of 14–3-3 proteins in the five Brassicaceae species. The colors represent conserved motifs: 1–10

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Gene structure and protein conserved motifs of 14–3-3 gene family members

Comparative gene structure analysis revealed that the non-ε group members had conserved gene structures, consisting of 4 exons and 3 introns, whereas the ε group members had between 6 and 9 exons (Fig. 2B). Using the MEME (http://meme-suite.org/tools/meme) online tool, 10 conserved motifs were identified in the 108 14–3-3 proteins (Fig. 2C and D), with all members harboring motif3, 6, and 7, whereas motif8 and motif10 were only found in the non-ε and the ε subgroups, respectively. Additionally, AlGRF11, CsGRF11-2, and ApGRF13-2 lacked motif1, motif2, and motif4, respectively (Fig. 2C). The similarities and differences in gene structure and protein motif characteristics suggest that the 14–3-3 gene family has undergone both functional conservation and differentiation during its evolutionary history.

Inter-species collinearity of 14–3-3 gene members

To investigate the duplication and selective pressure of the 14–3-3 gene family members in Brassicaceae, MCScanX [46] was used to analyze the collinearity relationship between A. thaliana and the other four species. The results showed that collinear genes were only distributed on some chromosomes in each species (Fig. 3A). A total of 92 colinear gene pairs were found between A. thaliana and the other four Brassicaceae species (Table S5). Among them, each of the 13 A. thaliana 14–3-3 gene members shared colinear pairs with those of A. lyrata. An orthologous AtGRF11 gene was deleted in A. pumila, ApGRF1 had only one colinear gene pair with A. thaliana, while the remaining 11 A. thaliana 14–3-3 genes had two orthologous genes each in the A. pumila genome. For C. sativa, except for two homologs of AtGRF4 and AtGRF9, the other 11 14–3-3 homologous genes were three times their number in A. thaliana. B. oleracea had 4 GRF2, 3 GRF12, 2 GRF3, 2 GRF6, 2 GRF7, and 2 GRF8, and GRF1, GRF9, GRF10, and GRF11 colinear gene pairs, but no colinear gene pairs for GRF4, GRF5, and GRF13 (Fig. 3A). In summary, the 14–3-3 gene family members in the five studied Brassicaceae species have undergone both expansion and contraction during their evolutionary history, with expansion being more pronounced.

Fig. 3
figure 3

Inter-species and intra-species collinearity relationships and Ka/Ks values of 14–3-3 genes from five Brassicaceae species. A Analysis of inter-species collinearity relationships. Chromosomes are color-coded according to the corresponding species name. Gray lines represent all inter-species collinearity gene pairs, while colored lines represent collinear gene pairs between corresponding species and A. thaliana; B Ka/Ks values of ε group and non-ε group inter-species 14–3-3 collinearity gene pairs; C Intra-species collinearity of 14–3-3 genes. Colored blocks represent chromosomes of the five species. The outer circle represents gene density on chromosomes. Gray lines represent all intra-species collinearity gene pairs, while colored lines represent colinear gene pairs within the corresponding species; D Ka/Ks values of intra-species 14–3-3 collinearity gene pairs

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Analysis of the evolutionary selective pressure (Ka/Ks) on the orthologous 14–3-3 genes in the five Brassicaceae species revealed that 92 orthologous gene pairs had Ka/Ks values of < 1 (Table S5), indicating that the gene family has undergone strong purifying selection during evolution. Interestingly, average Ka/Ks value of the ε group colinear gene pairs was larger than that of the non-ε group colinear gene pairs, suggesting that the ε group genes have experienced relatively weaker purifying selection (Fig. 3B).

Intra-species collinearity of 14–3-3 gene members

Intra-species collinearity analysis of the 14–3-3 genes in A. thaliana, A. lyrata, A. pumila, C. sativa, and B. oleracea identified 127 colinear gene pairs (Fig. 3C and Table S6). Of these, the C. sativa genome had the largest number of colinear gene pairs, with 75 pairs; A. lyrata only had 3 pairs of paralogous homologs. A. thaliana, A. pumila, and B. oleracea had 6, 32, and 13 intra-species colinear gene pairs, respectively. Selective pressure analysis on the intra-species paralogous 14–3-3 gene pairs revealed that 122 pairs had Ka/Ks values of < 1, indicating that most genes experienced purifying selection during evolution (Fig. 3D and Table S6). Only ApGRF13-2/ApGRF13-1 colinear gene pair had Ka/Ks values slightly greater than 1, suggesting that it has almost not been subjected to natural selection pressure during evolution. Additionally, the Ka values for the ApGRF6-2/ApGRF6-1, CsGRF4-1/CsGRF4-2, CsGRF6-1/CsGRF6-2, and CsGRF9-1/CsGRF9-2 gene pairs were zero, indicating high sequence similarity and the absence of non-synonymous substitutions.

Duplication history of the 14–3-3 gene family members in Brassicaceae species

Analysis of gene duplication revealed that the 108 14–3-3 genes could fall into four categories, with the majority occurring as whole-genome/segmental duplicates (WGD/S), while the remaining few occurring as dispersed, proximally, and tandemly duplicated genes (Table S7). WGD/S duplicated genes in A. thaliana, A. lyrata, A. pumila, C. sativa, and B. oleracea occurred in the frequency of 7 (53.3%), 6 (46.2%), 24 (100%), 14 (73.7%), and 39 (100%), respectively. Dispersed duplicated genes in A. thaliana, A. lyrata, and B. oleracea occurred in the frequency of 4 (30.8%), 7 (53.8%), and 5 (26.3%), respectively (Table S7).

Cis-elements screening in the 14–3-3 gene promoters

Screening of the 2.0 kb upstream promoter sequence regions of 108 14–3-3 genes identified 53 cis-acting element categories, including those responsive to light, growth and development, hormones, and abiotic stress. Light-responsive elements, such as Box 4, G-box, GT1-motif, and TCT-motif were predominantly detected in most of the 14–3-3 genes (Table S8). Similarly, cis-elements associated with growth and development-related, such as GCN4-motif and O2-site as well as hormone-responsive elements, such as ABRE, TGA-element, CGTCA-motif, and TCA-element were predominantly detected in 14–3-3 genes. Other cis-elements involved in abiotic stress responses, such as GC-motif, TC-rich repeats, ARE, MBS, LTR, and WUN-motif were also detected. Among the identified cis-elements, light-responsive elements were the most abundant across the five Brassicaceae species, while growth and development-related elements were the least abundant (Fig. S3). Moreover, the enrichment patterns of cis-elements in the 14–3-3 gene promoters varied among different species (Fig. S4), which reflected the diverse functions of this gene family in Brassicaceae.

Tissue-specific expression patterns of 14–3-3 genes in A. pumila

To explore the potential roles of A. pumila 14–3-3 genes in plant growth and development, RNA-seq data from seven tissues, including cotyledons, hypocotyls, stems, rosette leaves, roots, flowers, and siliques were used to generate tissue expression heatmaps for 24 A. pumila 14–3-3 genes (Fig. 4A). The results showed that the expression patterns of these genes could be clustered into three main groups, designated C1, C2, and C3. Most members of the C1 group were highly expressed in the cotyledons and hypocotyls, but with lower expression in other tissues. However, one of the C1 members, ApGRF2-2, was specifically expressed in the hypocotyls and nearly undetectable in other tissues (Fig. 4A). Most of the C2 class genes showed high expression in siliques, with some also being highly expressed in flowers, while ApGRF7-1 was highly expressed in the stems (Fig. 4A). The C3 class members, including ApGRF1-1/2, ApGRF10-1/2, and ApGRF12-1/2, showed tissue-specific expression in stems, roots, and flowers, respectively, but with diminished expression in other tissues (Fig. 4A). Additionally, qRT-PCR analysis confirmed that ApGRF3-1/2 were predominantly expressed in siliques (Fig. 4B). Notably, ApGRF4-1/2 and ApGRF6-1/2 were highly expressed in cotyledons and hypocotyls, but with diminished profiles in other tissues, while ApGRF7-1/2 and ApGRF9-1/2 showed expression patterns consistent with the heatmap results. The tissue-specific expression of different Ap14-3–3 genes suggests that they may have functional specificity.

Fig. 4
figure 4

Tissue expression characteristics of Ap14-3–3 genes. A RNA-seq based expression heatmap of 24 Ap14-3–3 genes in cotyledons, hypocotyls, stems, rosette leaves, roots, flowers, and siliques; B qRT-PCR analysis of the gene names highlighted in red in (A). Different lowercase letters represent statistically significant differences (Duncan multiple-range test, P < 0.05)

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Expression patterns of Ap14-3–3 genes under 250 mM NaCl stress

The expression profiles of 24 Ap14-3–3 genes were analyzed in the transcriptome data of A. pumila under salt stress, and the profiles revealed three main patterns in response to salt stress, including C1, C2, and C3 (Fig. 5A). The C1 class showed a gradual increase in expression from 0 to 12 h under salt stress, followed by a gradual decrease from 12 to 48 h. In contrast, members of the C2 class were gradually downregulated after salt treatment. Most C3 class members showed gradual upregulated profiles after salt stress. However, ApGRF1-2 and ApGRF12-1 were significantly upregulated from 0 to 6 h before exhibiting gradual downregulation. ApGRF2-1 and ApGRF10-2 were upregulated from 0 to 24 h, then downregulated at 48 h (Fig. 5A). qRT-PCR results and RNA-seq data generally showed consistent expression profiles, however some genes exhibited inconsistent patterns (Fig. 5A and B). For instance, ApGRF3-2, ApGRF4-1, and ApGRF7-1 showed induced expression from 0 to 12 h, but with diminished profiles from 12 to 48 h under salt stress. The profiles of ApGRF4-2, ApGRF6-1/2, and ApGRF9-1/2 were gradually downregulated after salt treatment, while ApGRF3-1 and ApGRF7-2 showed gradual upregulation in response to salt stress (Fig. 5B). The differential expression patterns of Ap14-3–3 genes in response to prolonged salt stress suggested their crucial roles in salt stress response in A. pumila.

Fig. 5
figure 5

Expression characteristics of Ap14-3–3 genes in response to sustained high salt stress. A RNA-seq based expression heatmap of 24 Ap14-3–3 genes at seven different time points under sustained 250 mM NaCl salt stress; B qRT-PCR results of genes marked in red from (A). Different lowercase letters represent statistically significant differences (Duncan multiple-range test, P < 0.05)

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Three-dimensional protein structure prediction of GRF6 protein and subcellular localization of ApGRF6-2 protein

The 3D structure predictions of nine proteins, including AtGRF6, AlGRF6, ApGRF6-1/2, BoGRF6-1/2, and CsGRF6-1/2/3 using AlphaFold 3 [52] indicated that all the proteins contained nine α-helical structures, with α3 and α4 as the longest helices (Fig. 6A). These findings suggested that the GRF6 protein structure is highly conserved across different species, indicating that their functions have also been conserved during evolution. Subcellular localization of ApGRF6-2 protein was analyzed by transforming 35S:ApGRF6-2-GFP vector in A. thaliana protoplasts followed by fluorescence observation, which revealed both cytoplasm and nucleus localization of ApGRF6-2 protein (Fig. 6B).

Fig. 6
figure 6

3D structure prediction analysis of GRF6 protein and subcellular localization of ApGRF6-2 protein. A 3D structure prediction of AtGRF6, AlGRF6, ApGRF6-1/2, CsGRF6-1/2/3, and BoGRF6-1/2 proteins, with α1–9 representing conserved motifs; B Subcellular localization of ApGRF6-2 protein in A. thaliana protoplasts

Full size image

Ectopic overexpression of ApGRF6-2 gene promotes flowering in A. thaliana

Seven independent 35S:ApGRF6-2 transgenic wild-type A. thaliana (Col-0) lines with similar phenotypes were generated in this study. Notably, transgenic lines flowered earlier than the wild-type Col-0 plants (Fig. 7A, B, and Table S9). Under LD conditions, the homozygous T3 generation 35S:ApGRF6-2 transgenic plants flowered 6–8 d earlier than the wild-type Col-0, and with significantly reduced of rosette leaves at flowering (Fig. 7C, D, and Table S9). However, compared with Col-0, the 35S:ApGRF6-2 transgenic plants did not show obvious differences in leaf size, plant height, and seed yield. qRT-PCR confirmed that the expression of the ApGRF6-2 gene in the transgenic A. thaliana plants was significantly higher than in the wild-type plants (Fig. 7E). Additionally, the endogenous flowering genes, FRUITFULL (AtFUL), APETALA1 (AtAP1), and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1) in the 35S:ApGRF6-2 A. thaliana plants showed varying upregulated degrees under LD conditions (Fig. 7F-H).

Fig. 7
figure 7

Phenotypic analysis of transgenic A. thaliana plants overexpressing ApGRF6-2. A and B Phenotypes of transgenic A. thaliana plants overexpressing 35S:ApGRF6-2 under LD conditions; C Comparative flowering-time phenotypes between the transgenic and wild-type A. thaliana plants; D Comparison of rosette leaf number at flowering between transgenic and wild-type A. thaliana plants; E Relative expression profiles of ApGRF6-2 in the transgenic A. thaliana plants; F–H Relative expression levels of endogenous flowering genes in the transgenic and wild-type A. thaliana plants. The #2 and #6 represent two independent 35S:ApGRF6-2 transgenic lines. Scale bar: 1 cm. qRT-PCR was used to detect the expression levels of ApGRF6-2, AtFUL, AtAP1, and AtSOC1 (E–H). Different lowercase letters indicate statistically significant differences (Duncan multiple-range test, P < 0.05)

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Discussion

The 14–3-3 proteins are highly conserved homodimer or heterodimer eukaryotic proteins that can interact simultaneously with two different target proteins to form complexes with vital roles in various processes, such as plant growth, development, and stress responses [14, 57]. Despite their wide identification in numerous species [58, 59], systematic characterization and comparative evolutionary roles of 14–3-3 gene family in the Brassicaceae flowering process remain limited.

This study identified a total of 13, 24, 39, and 19 14–3-3 gene members from the genomes of A. lyrata, A. pumila, C. sativa, and B. oleracea (Table S1). Although A. lyrata has undergone a complete WGD event [30], the number of 14–3-3 gene family members in its genome remained similar to those of A. thaliana, suggesting that this gene family remained relatively conserved during A. lyrata evolution [35]. However, the number of 14–3-3 gene family members increased varyingly in A. pumila, C. sativa, and B. oleracea, indicating that this gene family has undergone significant expansion during the evolutionary process of these species, which has likely contributed to their divergence and adaptation to changing environmental conditions [60]. However, further evaluating the underlying mechanisms of their functional divergence is still warranted.

Phylogenetic analysis showed that the 108 14–3-3 gene family members from the five species could be categorized into ε and non-ε groups, with fewer genes in the former than in the latter group (Figs. 1 and 2A). This finding is consistent with previous observation made in rice [61], A. thaliana [62], banana [63], and cotton [18]. Gene structure analysis revealed that the non-ε group members generally harbored fewer exons and introns compared to the ε group members. Conserved motif analysis indicated that ε and non-ε groups exhibited distinct motif structures, which was consistent with those observed in other species, suggesting their functional diversity (Fig. 2B-D) [18, 62]. Previous studies found that AtGRF1-8, the non-ε group 14–3-3 gene in Arabidopsis, mainly functions to regulate organ size and participate in response to low temperature stress [24, 64]. The ε group members, AtGRF9-13, mainly respond to phosphorus stress [65]. In this study, we found that there were specific motifs between the members of the ε and non-ε groups (Fig. 2C), suggesting that these motifs were highly likely the key factors in the functional differentiation of the ε and non-ε genes. For example, motif8 is only present in non-ε group proteins, while motif10 is only present in ε group proteins (Fig. 2C). We speculated that motif8 and motif10 may affect the functions of non-ε group and ε group proteins by involving them in different signaling pathways or metabolic processes, ultimately leading to functional differences between the two groups.

Collinearity analysis which can predict genetic patterns and potential gene functions was used to identify 92 inter- and 127 intra-species collinear gene pairs in the five species studied. Moreover, few 14–3-3 genes in A. pumila, C. sativa, and B. oleracea formed more than one collinear gene pair, suggesting that these genes might harbor crucial evolutionary functions (Fig. 3A and C, Tables S5 and S6). Ka/Ks values of < 1 was observed for all 14–3-3 orthologous genes between the five species (Table S5), indicating that purifying selection has potentially played a dominant role in maintaining the evolutionary functions of these genes, suggesting their critical basic physiological functions in these plants. When facing with sudden environmental changes, the ε genes can quickly respond and adjust the physiological state of plants by changing their own structure and function [60], but changes in gene sequences may not necessarily lead to functional changes [66]. In this study, we found that compared to non-ε group genes, the purification selection of ε group genes was relatively weaker (Fig. 3C). Therefore, during the evolution of Brassicaceae plants in response to rapidly changing environmental factors, the ε group genes may have evolved more flexible regulatory mechanisms. Nearly all intra-species collinear gene pairs in the five species had Ka/Ks values of < 1, except for the ApGRF13-2/ApGRF13-1 collinear gene pair, which had a value slightly greater than 1 (Table S6). The result suggested that this collinear gene pair might be under adaptive evolutionary process, potentially to generate new phenotypes that could be beneficial for plant survival and reproduction. The cis-elements identified in the promoters of the 108 14–3-3 genes showed association with various responses (Fig. S3 and Table S8), indicating their functional diversity. This study found that light-responsive elements accounted for the highest proportion in the prediction of promoter cis-acting elements of 108 14–3-3 genes (Fig. S3), suggesting that 14–3-3 proteins may be involved in regulating the adaptive responses of plants to different light qualities and intensities, thereby affecting important physiological processes in plants such as photosynthesis, growth direction, and flowering time. Previous studies have found that the At14-3–3 proteins in Arabidopsis regulates root growth and chloroplast development as a component of the light-sensing system [67]. In the light signal transduction pathway, 14–3-3 proteins are highly likely to interact with photoreceptors or downstream signaling molecules, achieving fine-tuning of light response by regulating the expression of related genes [68].

Tissue expression analysis showed differential expression of Ap14-3–3 genes in all the tissues studied, suggesting tissue-specific expression patterns (Fig. 4A), which is consistent with the profiles of 14–3-3 genes of other plants. For example, the A. thaliana AtGRF1 gene showed expression in roots, flowers, and fruits, but not in leaves or cotyledons [2]. Similarly, the V. vinifera VviGRF12 gene only exhibited high expression profiles in certain floral organs [17]. The observed tissue-specific expression of Ap14-3–3 genes suggests that functional differentiation might have occurred during evolution. Previous functional studies of 14–3-3 genes under salt stress have revealed that the gene family can either enhance or negatively regulate plant salt tolerance. For instance, the rice OsGRF6 could enhance salt tolerance by reducing H2O2 accumulation and increasing reactive oxygen species (ROS) scavenging enzyme activity [69]. Conversely, Malus pumila MdGRF6-overexpressing plants exhibited enhanced salt stress sensitivity compared to wild-type plants [70]. In this study, the expression of genes, such as ApGRF3-1 and ApGRF7-2 were continuously upregulated after salt stress treatment, while others, such as ApGRF4-2 and ApGRF6-1 showed continuous downregulation profiles (Fig. 5A). These results suggest that Ap14-3–3 genes could both positively and/or negatively regulate salt stress response in A. pumila. Overall, results demonstrated that different members of the 14–3-3 gene family had distinct roles in plant salt stress responses, suggesting their functional diversity for improved plant adaptation to the environment.

The FAC is composed of the florigen FT, 14–3-3, and FD proteins, and is responsible for promoting flowering by activating downstream target genes [21, 71, 72]. The 3D structure of GRF6 homologous proteins from five Brassicaceae species showed a high degree of similarity, while subcellular localization confirmed that ApGRF6-2 protein is both localized in the cytoplasm and the nucleus (Fig. 6A and B), which was consistent with the subcellular localization of the cotton GhFT1 protein [54]. Moreover, 35S:ApGRF6-2 transgenic plants in this study had enhanced expression levels of the endogenous flowering genes AtFUL, AtAP1, and AtSOC1, resulting in reduced number of rosette leaves and activated early flowering (Fig. 7A-H and Table S9). AtGRF6 was relatively highly expressed in SAM and flower buds of Arabidopsis, but its expression level is very low in mature flowers. AtGRF6 may be involved in the process of flowering regulation [73]. These data suggest that 14–3-3, as an important component of the FAC in Brassicaceae, could regulate flowering time in plants. However, the molecular mechanism by which ApGRF6-2 regulates flowering in A. pumila requires further investigation. Moreover, we should explore the specific components of the A. pumila FAC and the molecular mechanisms underlying its adaptation to rapid flowering in desert environments. In addition, future studies will focus on the following directions: i) molecular mechanism of ApGRF6-2 in regulating flowering through the FAC, combined with gene editing techniques to verify the functional differences of the ε/non-ε subfamilies and the roles of motif8/10; ii) evolutionary driving forces and tissue-specific expression networks of 14–3-3 genes in Brassicaceae; iii) potential application of the 14–3-3 genes in Brassicaceae plants for crop stress resistance and flowering time regulation, as well as its interaction with other environmental factors.

Conclusion

In summary, this study identified 13, 24, 39, and 19 14–3-3 genes from the whole genomes of A. lyrata, A. pumila, C. sativa, and B. oleracea, respectively. The number of 14–3-3 genes were not directly proportional to the sizes of the plant genomes. Overall, the 108 14–3-3 proteins from the five species could be classified into ε and non-ε group, based on phylogenetic analysis. Structural features, inter-species and intra-species collinearity, gene duplication types, and cis-regulatory elements both revealed the conservation and diversity of 14–3-3 gene functions in Brassicaceae species. The Ap14-3–3 genes exhibited tissue-specific expression and showed functional differentiation by positively and/or negatively regulating salt stress responses. Overexpression of ApGRF6-2 could promote flowering by enhancing the expression of floral meristem identity genes. This study provides important insights into the functional mechanisms of the 14–3-3 gene family in growth, development, stress adaptation, and flowering regulation of Brassicaceae species. The results could lay crucial foundation for further exploration of the molecular mechanisms underlying rapid flowering transformation of the desert A. pumila plant.

Data availability

The genome data for A. lyrata, C. sativa, and B. oleracea were downloaded from the Ensembl Plants genome database (https://plants.ensembl.org), while the A. thaliana genome data (TAIR10.1) was obtained from the Arabidopsis Information Resource website (TAIR, https://www.arabidopsis.org). Accession numbers for the RNA-seq data of leaves exposed to salt stress at 0.5, 3, 6, 12, 24 and 48 h are SRR6320595, SRR6320596, SRR6320597, SRR6320598, SRR6320599, SRR6320600 and SRR6320601 under BioProject PRJNA417986 at the SRA database (https://www.ncbi.nlm.nih.gov/sra). Accession numbers for the RNA-seq data of cotyledons, hypocotyls, stems, rosette leaves, roots, flowers, and siliques are SRR14470721, SRR14470718, SRR14470723, SRR14470714, SRR14470717, SRR14470719 and SRR14470724 under BioProject PRJNA728218 at the SRA database (https://www.ncbi.nlm.nih.gov/sra).

Abbreviations

A. thaliana :

Arabidopsis thaliana

A. lyrata :

Arabidopsis lyrata

A. pumila :

Arabidopsis pumila

C. sativa :

Camelina sativa

B. oleracea :

Brassica oleracea

MW:

Molecular weight

pI:

Isoelectric points

At14-3–3 :

14-3–3 Genes of Arabidopsis thaliana

Al14-3–3 :

14-3–3 Genes of Arabidopsis lyrate

Ap14-3–3 :

14-3–3 Genes of Arabidopsis pumila

Cs14-3–3 :

14-3–3 Genes of Camelina sativa

Bo14-3–3 :

14-3–3 Genes of Brassica oleracea

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Funding

This work was supported by the National Natural Science Foundation of China (32270385), the Scientific and Technological Innovation Team of Anhui Science and Technology University (2023KJCXTD001), the Excellent Scientific Research and Innovation Team of the Education Department of Anhui Province (2022AH010087), the Talent Introduction Start-up Fund Project of Anhui Science and Technology University (NXYJ202001), the Key Discipline Construction Fund for Crop Science of Anhui Science and Technology University (XK-XJGF001).

National Natural Science Foundation of China,32270385,Scientific and Technological Innovation Team of Anhui Science and Technology University,2023KJCXTD001,Excellent Scientific Research and Innovation Team of the Education Department of Anhui Province,2022AH010087,Talent Introduction Start-up Fund Project of Anhui Science and Technology University,NXYJ202001,Key Discipline Construction Fund for Crop Science of Anhui Science and Technology University,XK-XJGF001

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  1. Jingya Zhao and Shengqin Liu contributed equally to this work.

Authors and Affiliations

  1. College of Life Sciences, Shihezi University, Shihezi, 832003, China

    Jingya Zhao, Shengqin Liu & Hui Ren

  2. Center for Crop Biotechnology, College of Agriculture, Anhui Science and Technology University, Chuzhou, 233100, China

    Jingya Zhao, Shengqin Liu, Hui Ren, Owusu Edwin Afriyie, Mengzhu Zhang, Dachao Xu & Xianzhong Huang

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  1. Jingya Zhao
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Contributions

XH conceived and designed the experiments. JZ and SL performed most of the experiments. HR, E O A, and MZ analyzed the data. JZ and DX draft the manuscript. XH supervised and complemented the writing. All authors have reviewed the manuscript.

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Correspondence to Xianzhong Huang.

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Zhao, J., Liu, S., Ren, H. et al. Genome-wide identification and comparative evolution of 14–3-3 gene family members in five Brassicaceae species. BMC Genomics 26, 309 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11513-0

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Keywords

BMC Genomics

ISSN: 1471-2164

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