Identification and classification of SnRK2 genes in Rosaceae
To investigate the SnRK2 gene family in Rosaceae, the protein sequences of SnRK2s from Arabidopsis and rice were used as queries. In addition, a Hidden Markov Model (HMM) search using the SnRK2 gene domain HMM profile (PF00069.1) was used to screen the Rosaceae genome. A total of 318 candidate SnRK2 genes were screened using these two strategies. Finally, the online program SMART (http://smart.embl-heidelberg.de/) was used to assess the Ser/Thr protein kinase catalytic domains, followed by the elimination of redundant sequences, incomplete gene sequences, and transcripts of the same gene. Subsequently, 71 nonredundant SnRK2 genes were identified in the Rosaceae genome. Among them, 10 SnRK2 proteins were identified in Chinese white pear (PbrSnRK2s), 14 in apple (MdSnRK2s), 8 in strawberry (FvSnRK2s), 7 in peach (PpeSnRK2s), 7 in Chinese plum (PmSnRK2s), 5 in black raspberry (RocSnRK2s), 7 in cherry (PavSnRK2s), and 13 in European pear (PcSnRK2s) (Fig. 1).
To classify and investigate the evolutionary relationships among SnRK2 genes, a phylogenetic tree was constructed using multiple sequence alignment of SnRK2 protein sequences from the eight Rosaceae species, Arabidopsis, and rice. The results of this analysis showed that the SnRK2 gene family was clustered into three well-supported clades (Groups I, II, and III; Fig. 1), which is consistent with the findings of a previous study performed in Arabidopsis and rice [15]. Among them, 22 members belonged to Group I, 25 to Group II, and 24 to Group III (Fig. 1).
Multiple sequence alignment of SnRK2 proteins
To gain insights into the structural features of PbrSnRK2 proteins, the amino acid sequences in all members and groups were aligned. Result of the amino acid sequence alignment showed that all members of SnRK2s had 54.87% sequence identity, of which the maximum and minimum percentage of amino acid sequence conserved was 79.78% and 29.33%, respectively (Table S1). Furthermore, the ratio of sequence conservation for subgroup I, II and III was 78.92%, 64.5% and 65.17% respectively (Table S1). Previous studies in Arabidopsis had identified two conserved kinase regions and two functional subdomains within SnRK2, i.e. ATP-binding site, Ser/Thr kinase activity domain, Domain I and Domain II. To characterize the biological functions of PbrSnRK2 genes and their conserved domains in pear, multiple sequence alignment of PbrSnRK2 and AtSnRK2 proteins was performed. According to the result, the amino acid sites were very similar in these conserved regions of pear and Arabidopsis SnRK2 proteins. Specifically, members of PbrSnRK2 proteins have two conserved kinase domains in the N-terminal regions: an ATP-binding signature containing a lysine residue as an ATP-binding site, expected for the PbrSnRK2.5/2.6/2/9/2/10; and a Ser/Thr protein kinase active site signature, expected for the PbrSnRK2.8 (Fig. 2). In addition, the C-terminal of PbrSnRK2 contains two distinct domains (Domain I and Domain II), which is identical with the SnRK2 members identified in Arabidopsis lineages that showed divergent C-terminal domains. Together, the results manifest that SnRK2 gene family originated before the divergence of Rosaceae and Cruciferae. Additionally, the kinase domain regions are highly conserved during long-term evolution in SnRK2 proteins, which may have gone through purification choices. Furthermore, the conserved regions of several PbrSnRK2 proteins were lost, indicating that pseudogenezation or subfunctionalization might have been occurred during long-term evolution in pear.
Structural and conserved motifs analysis of SnRK2 genes in pear
Analysis of the arrangement of introns and exons can provide valuable information regarding evolutions and functions of gene families [25]. To better understand the structural diversity of PbrSnRK2 genes, an exon/intron analysis was performed by aligning gene sequences with their corresponding coding domains from SnRK2 in pear and Arabidopsis. The number of exons identified in the members of the SnRK2 gene family ranged from 4 to 10 in pear and Arabidopsis (Fig. 3). Most members of the individual groups exhibited different number of exons/introns and varying lengths of the coding sequence in pear, which also supported the phylogenetic classification of the SnRK2 gene family. For example, SnRK2 genes in subgroup I in pear contained 9 exons. Most members of subgroup II included 5–6 exons, except PbrSnRK2.4, which contained 9 exons. In addition, subgroup III contained a number of exons (i.e., 7–8), except PbrSnRK2.10, which contained 4 exons (Fig. 2). In addition, the MEME tool was used to further investigate the conserved motifs of SnRK2 proteins. A total of 12 conserved motifs were identified, denominating motif 1-12. The SnRK2 genes of each subgroup shared similar conserved motifs, nevertheless those in subgroup II were disorganized. Based on the result of motif analysis, motif 1/4/6/8/9/10 were identified to be the basic regions of the SnRK2 domain, as they were detected in each group of the gene family (Fig. S1). For example, it contains 11 of 12 conserved motifs in subgroup I, and only motif 12 is lacked (Fig. S1). Group III contains one specific motifs, i.e. it contains two motifs 12, expected for PbrSnRK2.9 (Fig. S1). In total, the conservation and specificity of the number of exons and motifs in each subgroup support the close evolutionary relationship of PbrSnRK2 genes. This may result from the replication events in evolution process of the gene family, indicating that these subgroups originated via different evolutionary paths.
Physicochemical features of the SnRK2 genes in Rosaceae
To further study the functions of the SnRK2 proteins, we performed systematic analysis of the physicochemical properties of the SnRK2 proteins in Rosaceae. We found that the SnRK2 protein sequences ranged from 198 to 891 amino acids, and that most of them contained 220 to 402 amino acids. The isoelectric point of 87.3% of the SnRK2 proteins was acidic, which indicates that SnRK2 proteins from Rosaceae are rich in acidic amino acids. Moreover, the molecular weights of these proteins ranged from 30.09 to 85.3 kDa (Table 1). The negative and positive GRAVY scores of proteins reflect their hydrophobicity and hydrophilicity, respectively [26]. The grand average of the hydropathy scores of all SnRK2 proteins was negative in Rosaceae, which indicates that these proteins are hydrophilic. In addition, we found that the aliphatic index ranged from 80.76 to 94.08 for the SnRK2 proteins from Rosaceae, which indicated that all of them are thermally stable (Table 1).
Evolutionary expansion and synteny analysis of SnRK2 genes in Rosaceae
Several gene duplication patterns drive the evolution of protein-coding gene families, which include whole-genome duplication (WGD) or segmental duplication, tandem and segmental duplications, and rearrangements at the gene and chromosomal levels [27]. The origins of duplicated genes were explored in the SnRK2 gene family in eight Rosaceae genomes using the MCScanX package. Each member of the SnRK2 gene family was allocated to one of five different categories: WGD or segmental, singleton, proximal, tandem, or dispersed. Five types of duplication events contributed to the expansion of the SnRK2 gene family in Rosaceae: 50% WGD, 19.7% dispersed, 15.2% transposed, 9% proximal, and 6% tandem (Fig. 4). Among them, WGD events occurred in each of the Rosaceae species; in particular, 60% of the SnRK2 genes in Chinese white pear, 86% in apple, 57.1% in peach, and 50% in strawberry were duplicated and retained from WGD events compared with only 40% in black raspberry, 28.6% in Chinese plum, 28.6% in cherry, and 7.7% in European pear (Table S2). Hence, WGD may have impacted the evolution of the SnRK2 gene family in Chinese white pear, apple, peach and strawberry. In addition, the proportions of dispersed SnRK2 gene duplication events in black raspberry (40%), European pear (30%), peach (28.6%), strawberry (28.6%), cherry (28.6%), and apple (7%) were assessed (Table S2). Transposed events were 10% in pear, 37.5% in strawberry, 28.6% in Chinese plum, 28.6% in cherry and 15.4% in European pear. These results indicate that transposed, WGD, and dispersed events impacted the evolution of the SnRK2 gene family in Chinese plum and cherry. In black raspberry, WGD and dispersed events were the main forces, while dispersed and transposed events played key roles in the evolution of European pear.
To explore the evolutionary process of SnRK2 genes, an intra-genomic synteny map was constructed for each analyzed Rosaceae specie in the study. The result showed that PbrSnRK2 genes were distributed on 4 out of the 17 pear chromosomes, with 4 SnRK2 genes anchored on chromosome 15, and 2 syntenic pairs were identified (Fig. 5). 13 SnRK2 genes were assigned to 7 of the 17 chromosomes in apple, with 5 genes anchored to chromosome 15, and 9 syntenic pairs were identified. Furthermore, 7 SnRK2 genes were distributed on 5 of the 8 chromosomes in peach, with 3 genes anchored to chromosome 1, and 1 syntenic pair was identified (Fig. 5). 8 SnRK2 genes were assigned to 4 of the 7 chromosomes in strawberry, with 4 genes anchored to chromosome 5, and 2 syntenic pairs were identified (Fig. 5). In addition, 7 SnRK2 genes were located on Chr1, Chr2, Chr4, Chr6 and Chr8 in Chinese plum, of these, 3 genes were co-located on Chr2, and 1 syntenic pair was identified (Fig. S2). 5 SnRK2 genes were located on Chr1, Chr2 and Chr5 in black raspberry, of these, 2 genes were co-located on Chr2 and Chr5, respectively, and 1 syntenic pairs was identified (Fig. S2). 7 SnRK2 genes were located on Chr1, Chr5, Chr6, Chr7 and Chr8 in cherry, of these, 3 genes were co-located on Chr1 and 1 syntenic pairs was identified (Fig. S2). 9 SnRK2 genes were located on Chr1, Chr2, Chr4, Chr8 and Chr15 in European pear, of these, 4 genes were co-located on Chr15 and 2 genes were co-located on Chr8, and 2 syntenic pairs were identified (Fig. S2).
Ks value and Ka/Ks ratio reveal dates and driving forces of evolution
Purifying selection (negative selection) is the process via which disadvantageous mutations are removed, whereas Darwinian selection (positive selection) accumulates new advantageous mutations and spreads them throughout the population [27]. To identify the selection process that drove the evolution of the SnRK2 gene family, the Ka value and Ka/Ks ratio of its paralogs were examined in the eight Rosaceae species based on coding sequences. We found that all values were <1 in the studied Rosaceae species (Fig. 6A), implying that this family underwent a purifying selection pressure during its evolution in Rosaceae and that its evolution was very conservative.
The Ks value is extensively used to evaluate the evolutionary dates of WGD or segmental duplication events. Previous studies have shown that the genome of pear and apple have undergone two genome-wide duplication events (ancient and recent). In Chinese white pear, the recent WGD event that is inferred to have occurred 30- 45 MYA (Ks ~0.15-0.3) and the ancient WGD event that is inferred to take place ~140 MYA (Ks ~1.5-1.8) [24]. Furthermore, the recent WGD event that is inferred to have occurred 30- 45 MYA (Ks ~0.2) and the ancient WGD event that is inferred to take place ~140 MYA (Ks ~1.6) in apple [28, 29]. To explore the evolutionary dates of the duplication events among the SnRK2 gene family members, Ks values were analyzed in the Rosaceae species. The results showed that the Ks values for the SnRK2 gene pairs ranged from 0.107 to 4.0487 in Rosaceae; moreover, the Ks values of WGD gene pairs PbrSnRK2.1–PbrSnRK2.2 (Ks, ~0.1028), PbrSnRK2.7–PbrSnRK2.8 (Ks, ~0.1363), PCP022180.1–PCP002062.1 (Ks, ~0.182), MD01G1035000–MD15G1321000 (Ks, ~0.1644), MD02G1166500–MD15G1279000 (Ks, ~0.1757), MD04G1054400–MD06G1046300 (Ks, ~0.1635), MD08G1187200–MD15G1373000 (Ks, ~0.1138), and MD08G1236500–MD15G1428500 (Ks, ~0.107) are close to the Ks peak corresponding to the recent WGD that was detected in pear genome, indicating that some SnRK2 genes were derived and retained from recent WGD events (30–45 MYA) [24]. Furthermore, other duplicated gene pairs (such as PbrSnRK2.5 and PbrSnRK2.6) possessed higher Ks values (2.26–4.0487), indicating that they might have stemmed from a more ancient duplication event (Fig. 6B).
Expressions of PbrSnRK2 genes in different tissues
To understand the expression patterns and functional properties of SnRK2 genes in different tissues, we constructed a heat map at the transcriptional level using MeV to depict the overall expression patterns of SnRK2 genes in pear based on the transcriptome data from pear root, stem, leaf, fruit, petal, sepal, ovary, bud, pollen, pollen tube and stop-growth pollen tube. The transcriptome data was obtained from previous studies conducted by our group [30-32]. The results showed that SnRK2 genes were expressed in most organizations of pear, i.e., three genes (PbrSnRK2.2/2.5/2.7) were exhibited high expression in fruits, two (PbrSnRK2.3/2.8) in leaves, and one (PbrSnRK2.4) in roots (Fig. S3). Furthermore, PbrSnRK2.1 and PbrSnRK2.6 were highly expressed in petal, PbrSnRK2.9 was highly expressed in bud, and PbrSnRK2.10 was highly expressed in ovary.
In addition, those PbrSnRK2 genes were also examined by qRT-PCR using gene-specific primers and in diverse pear tissues including root, stem, leaf, flower, fruit, PG, PT and style (Table S3). The results showed that SnRK2 genes have diverse expression patterns in different pear tissues, although some genes have the same expression pattern between the two methods, and some genes have different expression characteristics in different tissues. For example, PbrSnRK2.3 and PbrSnRK2.8 were also highly expressed in leaf, and PbrSnRK2.6 also exhibited high expression in the flower, which is consistent with transcriptome data (Fig. 7 and Fig. S3). PbrSnRK2.2, PbrSnRK2.4 and PbrSnRK2.7 were highly expressed in flower based on the data of qRT-PCR, nevertheless transcriptome data showed that PbrSnRK2.2, PbrSnRK2.4 and PbrSnRK2.7 were highly expressed in fruit, root and root, respectively (Fig. 7 and Fig. S3). PbrSnRK2.5 and PbrSnRK2.9 were highly expressed in leaf by qRT-PCR; PbrSnRK2.1 was highly expressed in PG, and PbrSnRK2.10 was highly expressed in style by qRT-PCR (Fig. 7). These results indicated that SnRK2 genes may play important roles in these pear tissues and respond to abiotic in different organizations. In addition, these genes may have different expression characteristics in different developmental stages of plant tissues.
Expression profiles of PbrSnRK2 genes under ABA treatment
Many studies have shown that SnRK2s play key roles in response to multiple abiotic stresses such as salinity, dehydration, and hyperosmotic stress [14]. Moreover, the members of the SnRK2 gene family are involved in the regulation of phytohormone pathway responses, particularly ABA signal transduction [4]. To explore the dynamic transcriptional changes in PbrSnRK2 genes in response to ABA treatment, the expression levels of these genes in leaves of pear were evaluated by qRT-PCR under ABA (50 μM) treatment at four time points: 0, 3, 6, and 9 h. The results showed that in response to exogenous ABA application, the SnRK2 genes exhibited different expression patterns. For example, eight genes (PbrSnRK2.1/2.2/2.3/2.4/2.6/2.7/2.8/2.9) were activated by ABA, whereas the expressions of two genes (PbrSnRK2.5/10) remained unchanged at each time point (Fig. 8). Among them, the expression levels of PbrSnRK2.1, PbrSnRK2.3, and PbrSnRK2.4 were significantly upregulated at 3 h after ABA treatment, and the expression pattern of PbrSnRK2.1 shows a wavy trend with ABA treatment (Fig. 8), whereas PbrSnRK2.4’s expression pattern shows a parabolic trend under ABA treatment. PbrSnRK2.7 and PbrSnRK2.9 showed similar expression patterns. Specifically, they were both significantly upregulated at 6 h after ABA treatment, and reached their peak value at 9 h after ABA treatment (Fig. 8). The expression levels of PbrSnRK2.2/2.6 were significantly upregulated at 9 h after ABA treatment. However, PbrSnRK2.8 was downregulated over time after ABA treatment (Fig. 8).