Genome-wide identification and chromosomes location of 14-3-3 genes family in the apple genome
To identify 14-3-3 genes in apple, Arabidopsis 14-3-3 protein sequences were served as queries against the Apple Genome Database using BLASTp. 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 2: 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 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 two 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, the non-ɛ members contain four exons and three introns, except for 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 3: 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 3: 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 using the 14-3-3 protein sequence alignments of five plant species: Arabidopsis thaliana, Malus domestica, Oryza sativa, Medicago trucatula, Glycine max and Populus trichocarpa (Fig. 2). The detailed information of all14-3-3 genes identified in this study was provided in Additional file 4: Table S2. Phylogenetic analyses of 14-3-3s provide a robust tree and the 14-3-3 family members from the five plant species were divided into two major classes (ɛ class and non-ɛ class), the same as described previously [3].
To understand the expansion patterns of the Md14-3-3 genes in apple genome, we performed the tandem duplicated analysis. As shown in Fig. 3A, four Md14-3-3 genes (MdGF14m/MdGF14n and MdGF14g/MdGF14f) were clustered into two tandem duplication event 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, a 2,000bp promoter region of the 18 Md14-3-3 genes was scanned for putative cis-regulatory elements using the PlantCARE database. Notably, various cis-acting elements involved in hormonal responses such as ABA, GA, MeJA, auxin and salicylic acid were found in the promoter region of those Md14-3-3s (Additional file 5: Figure S3). Also, the numbers of light-responsive cis-elements were also found to be the most abundant among all 14-3-3 genes (Additional file 5: Figure S3). This 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 identified in promoter sequences of a portion of the Md14-3-3 genes (Additional file 5: Figure S3). The presence of abundant elements in the promoters suggested that 14-3-3s are involved in multiple biological processes.
Expression pattern of Md14-3-3 genes in different tissues
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. 4). Noticeably, our results showed that the transcription level of MdGF14o alone cannot be detected in all selected tissues by qRT-PCR, due to its very low abundance of MdGF14o. Therefore, we conclude that despite the presence of 18 14-3-3 genes in apple genome, 17 isoforms are transcribed. Similar results were also found in other species [43, 45]. Moreover, MdGF14d, MdGF14e, MdGF14f, MdGF14g, MdGF14j and MdGF14k expression patterns were consistent and exhibited strong preferential expression in flowers, while MdGF14m and MdGF14p were expressed to higher level in stem compared to other tissues (Fig. 4). However, MdGF14m has no transcript in leaf buds and flower buds. Besides, genes with closer relationships (MdGF14a and MdGF14i, MdGF14h and MdGF14l) showed similar expressions in all tissues (Fig. 4), suggesting that they act largely redundantly in the control of apple growth and development.
Expression of Md14-3-3 genes after hormone and sugar treatments
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, 46]. Moreover, 14-3-3 protein family also plays an important role in the regulation of flowering time. These suggested that 14-3-3 protein may act as a crosstalk point in signal transduction networks to regulate floral transition. Our previous study showed that 6-BA treatment contributes to the increase the proportion of short branches and promotes floral transition [47]. Sugar as an energy substance involved in the flowering regulation [48]. Consequently, to further determine the potential role of Md14-3-3s genes in the context of apple flower induction, research on their expressions in response to sugars and hormones is very necessary. We first performed a preliminary analysis of Md14-3-3s expression after 6-BA [47], sucrose treatment [48] and glucose treatment (data not published) by analyzing transcriptome data (Fig. 5, Additional file 6: Table S3). In general, most orthologous genes of 14-3-3s exhibited similar expression patterns. MdGF14o showed no or very low expression levels (less than 1), indicating that it did not function to a large extent in flower development. Besides, the expression levels of 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. Noticeably, at the early stage, which is a key stage for flower induction, MdGF14a, MdGF14i were down-regulated while MdGF14d, MdGF14j were up-regulated in response to 6-BA and glucose treatments. Overall, Md14-3-3s showed different and multiple expression patterns in transcriptome data, implying functional diversity.
Furthermore, to gain insight into the response of Md14-3-3s to GA signal, we examined their expression patterns using qRT-PCR under 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, significant 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 flowering induction, which were up- or down-regulated at different time points after treatment (Fig. 6), indicating that they might have roles in hormonal stress responses or apple development.
Subcellular localization of 14-3-3 proteins
Based on gene expression data, we selected four candidate Md14-3-3 isoforms (MdGF14a, MdGF14d, MdGF14i and MdGF14j) for further analysis, which may be related to flower induction in apple. To gain insight into the molecular function 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. 7).
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. Previously, TFL1 and FT were reported to interact with 14-3-3 proteins [11, 35, 36]. In apple, there are two MdTFL1 encoding genes, MdTFL1-1 and MdTFL1-2 [49]. We performed yeast two-hybrid assays and confirmed that both MdTFL1 (MdTFL1-1 and MdTFL1-2) proteins can interact with four 14-3-3 isoforms (MdGF14a, MdGF14d, MdGF14i and MdGF14j) (Fig. 8A). 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 system (Fig. 8A).
Also, we used a BiFC assay to determine the interactions between Md14-3-3 proteins and MdTFL1 or MdFT in vivo (Fig. 8B, 8C, 8D). 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. 8B), MdTFL1-2-Md14-3-3s (Fig. 8C) and MdFT-Md14-3-3s interactions (Fig. 8D) 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.