Genome-wide identification and chromosomes location of 14-3-3 genes family in the apple genome
To identify 14-3-3 family members in apple, previously published 15 Arabidopsis 14-3-3 protein sequences were served as queries against the Apple Genome Database using BLASTp program (E-value <1e-5). After manually removing sequences containing the incomplete 14-3-3 domain, 18 putative Md14-3-3 genes were identified, named MdGF14a-MdGF14r based on their chromosomal positions (Table 1; Additional file 1: Figure S1). The 18 Md14-3-3 genes identified were located on 9 of the 17 chromosomes of apple, and 2 genes (MdGF14a and MdGF14b) were still mapped on unanchored scaffolds. The basic information of these Md14-3-3 genes is provided in Table 1. The putative Md14-3-3 proteins contained 252 (MdGF14f and MdGF14h) to 302 (MdGF14q) amino acid residues.
Gene structure and multiple sequence alignment of 14-3-3 genes
To determine the gene structures of Md14-3-3 family members, we investigated the divergence of Md14-3-3s exon-intron structures (Fig. 1), revealing the evolutionary relationships. The full-length amino acid sequences of Md14-3-3s was used to construct the phylogenetic tree by using maximum likelihood method in MEGA. As shown in Fig. 1, the family members of Md14-3-3s grouped into two major evolutionary branches, the ɛ group and the non-ɛ group. The ɛ group is itself split into the isoforms MdGF14k, MdGF14o, MdGF14d, MdGF14j, MdGF14b, MdGF14r, MdGF14f and MdGF14m. The non-ɛ group is made up of the isoforms MdGF14a, MdGF14i, MdGF14g, MdGF14n, MdGF14e, MdGF14p, MdGF14q, MdGF14h, MdGF14c and MdGF14l (Fig. 1). Moreover, the ɛ group breaks into four subbranches. The non-ɛ group breaks down into three very distinct subbranches. The ɛ and non-ɛ groups are well supported by intron-exon structure. The ɛ members have six exons and six introns (including an additional C-terminal intron). Different from the ɛ group, most non-ɛ members contain four exons and three introns, except for MdGF14c with three exons and MdGF14e, MdGF14p and MdGF14p containing an extra in the 5’ leader (Fig. 1). Besides, to detect the sequence conservation of 14-3-3 family members, we performed multiple sequence alignment of the 18 full-length Md14-3-3 protein sequences (Additional file 2: Figure S2). It was worth noting that the amino acid sequences of the N-terminal and C-terminal regions are significantly different (little amino acid conservation), while other central regions composed of nine antiparallel α-helices (α1-α9) are relatively conserved (Additional file 2: Figure S2), especially α1, α3, α5, α7, and α9 domains which possibly play a very conservative function during the evolution.
Phylogenetic conduction and synteny analysis
To gain further insights into the evolutionary relationships of 14-3-3 proteins in different species, we constructed a phylogenetic tree by maximum likelihood method using the 14-3-3 protein sequence alignments of six plant species: Arabidopsis thaliana, Malus domestica, Oryza sativa, Medicago trucatula, Glycine max and Populus trichocarpa (Fig. 2). The detailed information of all 14-3-3 genes identified in this study was provided in Additional file 3: Table S1. As shown in the phylogenetic analysis (Fig. 2), the 14-3-3 family members from the six plant species were divided into two major classes (ɛ class and non-ɛ class), the same as described previously [3]. Most 14-3-3 members from apple and Populus clustered together (Fig. 2), exhibiting a closer phylogenetic relationship.
The evolution and expansion of gene families are closely related with the occurrence of tandem duplication and segmental duplication events. Tandem duplication usually was characterized as multiple family members forming gene clusters. Segmental duplication, which occurs most frequently in plants, might cause scattered family members on different chromosomes [46]. To understand the expansion patterns of the Md14-3-3 genes in apple genome, we performed the tandem and segmental duplicated analysis. As shown in Fig. 3A, four Md14-3-3 genes (MdGF14m/MdGF14n and MdGF14g/MdGF14f) were clustered into two tandem duplication regions on apple 08 and 15 linkage groups. Besides, the MdGF14l/MdGF14c, MdGF14k/MdGF14o and MdGF14j/MdGF14d gene pairs may be generated by segmental duplications because they are located on different and non-homologous chromosomes (Fig. 3A). Additionally, a syntenic map of 14-3-3s in apple and Arabidopsis were also created. A total of four pairs of orthologous genes (MdGF14o-AtGRF10, MdGF14f/MdGF14g-AtGRF12/AtGRF13, MdGF14c-AtGRF6, MdGF14c-AtGRF8) were found (Fig. 3B). These results indicated that some Md14-3-3 genes were possibly generated by gene duplication which plays a major driving force for Md14-3-3 evolution. In a word, synteny analysis and phylogenetic comparison of Md14-3-3 genes provided deep insight into the evolutionary characteristics of apple 14-3-3 genes.
Cis-elements in the promoter of Md14-3-3 genes
To further explore the function and regulatory patterns of Md14-3-3 genes, the 2,000bp upstream intergenic regions from start codon of the 18 Md14-3-3 genes were scanned for putative cis-regulatory elements using the PlantCARE database. A series of cis-acting elements involved in hormonal responses, light and abiotic stress responses were found in the promoter region of these Md14-3-3s (Additional file 4: Table S2). Among the cis-acting regulatory elements involved in hormone responses, abscisic acid responsive element (ABRE) was present almost all members of Md14-3-3 family except MdGF14i. In addition, the numbers of hormone-related cis-regulatory elements showed great variance among different Md14-3-3 genes. For example, four of the gibberellin response elements (P-box) were present in MdGF14r promoter, but none in the promoters of MdGF14g, MdGF14h, MdGF14k and MdGF14n genes. MeJA-related element (CGTCA-motif and TGACG-motif), auxin-responsive element (AuxRR-core and TGA-element) and salicylic acid-related element (TCA-element) were also observed in the promoters of 14, 11 and 12 Md14-3-3 genes, respectively. Also, the numbers of light-responsive cis-elements were also found to be the most abundant among all 14-3-3 genes, including G-box, Box 4, AE-box, TCCC-motif, GATA-motif, I-box, TCT-motif and AT1-motif, which may reflect the response of the 14-3-3 involving light signals to regulate plant growth. Circadian-responsive element existed in the upstream flanking regions of MdGF14d, MdGF14m, MdGF14p and MdGF14q. Meanwhile, stress response (e.g., drought and low temperature) were also identifified in promoter sequences of a portion of the Md14-3-3 genes (Additional file 4: Table S2). The presence of abundant elements in the promoters suggested that 14-3-3s are involved in multiple biological processes.
Expression profiles of Md14-3-3 genes in RNA-seq datasets
Some reports claimed that 14-3-3 genes were involved in plant hormonal responses, such as cytokinins, GA, and ABA [16, 18, 21] as well as sugar metabolism [44, 47]. To further determine the potential role of Md14-3-3s genes in the context of apple flower induction, we performed a preliminary analysis of the transcript expressions of 18 Md14-3-3 genes in response to 6-benzylaminopurine (6-BA), glucose, and sucrose treatment based on the transcriptomic sequence databases. For 6-BA and glucose treatment, RNA-seq datasets were retrieved from NCBI Sequence Read Archive (SRA) datasets (SRR6510620 [48] and SRP226830, respectively). The fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM) values of Md14-3-3 genes were listed in Additional file 5: Table S3, and a heat map was generated to display the expression profiles of the Md14-3-3 genes (Fig. 4). In general, most genes with closer relationships of 14-3-3s exhibited similar expression patterns (Fig. 4). For example, the expression of MdGF14d was induced or inhibited by 6-BA and sugar at one or more time points, which is consistent with the expression pattern of MdGF14j. This indicated that they may have similar functions. MdGF14o showed no or very low expression levels (less than 1) during different flower bud developmental stages in all treatments, indicating that it did not function to a large extent in flower development. Besides, the expression levels of five genes (MdGF14c, MdGF14f, MdGF14m, MdGF14k and MdGF14q) were also significantly lower. On the contrary, the expression levels of genes, such as MdGF14a, MdGF14b, MdGF14d, MdGF14e, MdGF14g, MdGF14h, MdGF14i, MdGF14j, and MdGF14n, were significantly higher, indicating that they may play a major role in the flower induction phase. It is remarkable that MdGF14a, MdGF14i were down-regulated while MdGF14d, MdGF14j were up-regulated in response to 6-BA and glucose treatments at the early stage, which is a key stage for flower induction (Fig. 4). Overall, Md14-3-3s showed different and multiple expression patterns in transcriptome data, implying functional diversity.
Expression patterns of Md14-3-3 genes in various tissues and their responses to GA3 treatment by qRT-PCR
To investigate the possible roles of the Md14-3-3 genes, tissue-specific (leaves, stems, leaf buds, flower buds, flowers and fruits) gene expressions were determined by qRT-PCR (Fig. 5, Additional file 6: Table S4). As shown in Fig. 5, some Md14-3-3 genes exhibited similar expression patterns in different tissues, while other Md14-3-3s showed tissue-specific transcript accumulation patterns, potentially suggesting the functional divergence of Md14-3-3 genes during apple growth and development. For example, four Md14-3-3 (MdGF14b, MdGF14h, MdGF14q and MdGF14r) genes were ubiquitously high expressed in nearly all tested tissues, whereas MdGF14p genes were almost undetectable in leaf buds. It is noteworthy that the transcription level of MdGF14o alone cannot be detected in any selected tissues by qRT-PCR, due to its very low abundance of MdGF14o. Similarly, in soybean, both SGF14q and SGF14r, the closest homolog of MdGF14o, were not detected in the EST database [43]. Some Md14-3-3 genes showed a very high level in specific tissues (Fig. 5). For example, MdGF14e, and MdGF14k exhibited strong preferential expression in flowers, signifying their putative role in the regulation of flower development. Besides, genes with closer relationships (MdGF14a and MdGF14i) showed similar expressions, and both were expressed at a higher level in the tested tissues (Fig. 5), demonstrating that they play similar roles in tissue development. Furthermore, two pair Md14-3-3s in the segmental duplications also showed similar expression patterns (Fig. 5). For example, MdGF14d and MdGF14j with similar gene structure were mainly expressed in stems and flowers. MdGF14c and MdGF14l showed relatively high expression level in the stem. However, some genes in tandem duplicated regions displayed different expression patterns (Fig. 5). MdGF14m were expressed to very higher level in stem compared to other tissues, while MdGF14f were highly expressed in flowers. MdGF14g and MdGF14n displayed higher expression level in flowers and fruits, respectively. These results indicated that some Md14-3-3 genes maybe play multiple important roles in apple growth and development.
Gibberellin promotes vegetative growth, but inhibit floral transition, resulting a significant reduction of fruit load the following year in apple [40]. To gain insight into the potential roles of the Md14-3-3s in response to GA signal, qRT-PCR was used to analyze the expression of the Md14-3-3 genes under GA3 treatment (Fig. 6). It is noteworthy that MdGF14o was not expressed at various stages of flower bud development in GA3 treatment. In the early stages of GA induction, the expressions of 11 Md14-3-3 genes, including MdGF14b, MdGF14c, MdGF14d, MdGF14e, MdGF14f, MdGF14g, MdGF14h, MdGF14j, MdGF14k, MdGF14m and MdGF14p were extremely reduced and remained at a lower level. Rather, signifificant up-regulation of MdGF14a and MdGF14i was observed at 30 DAFB (Fig. 6). The transcription level of MdGF14n did not differ significantly at first, but subsequently increased by 4-fold at the second sampling point after the GA3 treatment (Fig. 6). Interesting, MdGF14l, MdGF14q and MdGF14r showed similar expression patterns during flower induction, which were up- or down-regulated at different time points after treatment (Fig. 6), indicating that they might have similar roles in hormonal stress responses or apple development.
Md14-3-3s can interact with MdTFL1, and MdFT
To address how Md14-3-3s participate in floral transition, we focused on the floral pathway integrators, TFL1 and FT. In fact, we previously used full-length MdTFL1 protein as a bait protein to conduct yeast two-hybrid screening in apple flower bud cDNA library, and screened the MdGF14a and MdGF14j protein. In addition, MdGF14i and MdGF14d are closely related to MdGF14a and MdGF14j, respectively (Fig. 1), and they exhibited prominent transcriptional responses to sugars and hormones. Therefore, we chose these four genes for further analysis. Previous studies reported that 14-3-3 protein can interact with TFL1 and FT [11, 35, 36]. In apple, there are two MdTFL1 encoding genes, MdTFL1-1 and MdTFL1-2 [50]. An experiment further confifirmed both MdTFL1 (MdTFL1-1 and MdTFL1-2) proteins can interact with four 14-3-3 isoforms (MdGF14a, MdGF14d, MdGF14i and MdGF14j) using the yeast system (Fig. 7A). Moreover, the 14-3-3 isoforms preference of MdTFL1 can also be comparable with that of MdFT, the four 14-3-3 isoforms also interact with MdFT in yeast two-hybrid assays (Fig. 7A).
Also, we used a BiFC assay to determine the interactions between Md14-3-3 proteins and MdTFL1 or MdFT in vivo (Fig. 7, Additional file 7: Figure S3). Different fusion protein combinations containing the expression vectors for MdTFL1-1, MdTFL1-2 and MdFT proteins fused to the pSPYNE and the four Md14-3-3 proteins fused to the pSPYCE were transiently introduced into the Nicotiana benthamiana leaves, yellow fluorescent protein (YFP) fluorescence signals from MdTFL1-1-Md14-3-3s (Fig. 7B), MdTFL1-2-Md14-3-3s (Fig. 7C) and MdFT-Md14-3-3s interactions (Fig. 7D) were detected both in the cytoplasm and nuclear, but mainly in the cytoplasm. Thus, these results clearly showed that Md14-3-3s can interact with MdTFL1 and MdFT in yeast and in plant cells.
Subcellular localization of 14-3-3 proteins
To determine the subcellular localization of Md14-3-3 proteins, we made a fusion construct green fluorescent protein (GFP)-linked Md14-3-3s driven by the cauliflower mosaic virus (CaMV) 35S promoter and analyzed the intracellular localization of the four Md14-3-3s. These constructs were introduced into Nicotiana benthamiana leaves and fluorescent signals were observed in the cytoplasm and nucleus (Fig. 8), consistent with previous studies [11].