Identification and classification of bHLH genes in the Rosaceae
To define the bHLH gene families in the Rosaceae, we used the HMMER3 software profile and BLASTP program to identify the members of bHLH genes in Pear and the three other Rosaceae species. SMART analysis was used to further discriminate and confirm the candidate bHLH proteins. Finally, a total of 198 bHLH genes were identified in Pear (Table S1), 129 in peach, 112 in strawberry and 122 in Chinese plum (Table S2), while 188 MdbHLHs of apple had been identified in a previous report [23]. The bHLH genes were named according to the method proposed for Arabidopsis [24]. Among these 198 candidate PbbHLH genes, 169 were mapped onto all but chromosomes 9 and 11 of the pear chromosomes, while 29 PbbHLH genes were located on the scaffold (Table S1 and Fig. S1). Six PbbHLHs were located on chromosomes 1, 4 and 12, twelve on chromosomes 10 and 17, 18; fourteen located in chromosomes 6 and 14; five on chromosomes 7 and 16,13 on chromosome 2, ten on chromosome 3, 21on chromosome 5, nine on chromosome 13, 25 on chromosome 15, but only three genes on chromosome 8.
Multiple sequence alignments, conserved residues in the bHLH domains and predicted DNA-binding ability
To examine sequence features of these Rosaceae bHLH domains, we performed multiple sequence alignments of the Rosaceae bHLH amino acid sequences and checked each alignment by hand. In the alignment, we identified 11 and 12 residues with at least 50% conservation in pear and apple bHLH sequences, respectively, and 13, 11 and 12 in peach, strawberry and Chinese plum, respectively. Among these conserved residues, six residues (Glu-13, Arg-16, Leu-27, Lys-36, Tyr-49 and Leu-53) (Fig. 1 and Table S3) were present in more than 75% of sequences, suggesting that these residues are extremely important for the function of bHLH proteins.
It is generally acknowledged that bHLH domains with at least five basic amino acids in the basic region are considered to bind DNA. A total of 167 DNA-binding proteins and 31 DNA non-binding proteins were identified in Pear, with 164, 109, 98 and 104 DNA-binding proteins identified in apple, peach, strawberry and Chinese plum, respectively, and 24, 20, 14 and 18 DNA non-binding proteins identified in apple, peach, strawberry and Chinese plum, respectively (Table 1). According to the presence or absence of residues Glu-13 and Arg-16 in the basic region, the DNA-binding bHLH proteins were further divided into two groups: E-box-binding proteins or non-E-box-binding proteins. Twenty-nine, 22, 12, 15 and 16 non-E-box-binding proteins were found in Pear, apple, peach, strawberry and Chinese plum, respectively. According to the presence or absence of the His-9 residue, the E-box-binding proteins can be further divided into two subgroups, G-box-binding proteins and non-G-box-binding proteins. A total of 113, 116, 73, 61 and 68 G-box-binding proteins were found in Pear, apple, peach, strawberry and Chinese plum, respectively (Table 1).
Intron/exon structure in the Rosaceae bHLH domains
To better analyze the intron distribution within the bHLH domain of all of the Rosaceae bHLH genes, we performed a multiple alignment analysis between all the Rosaceae bHLH coding sequences and genome sequences, and eleven different distribution patterns (designated I to XI), with intron numbers ranging from 0 to 3, were found within the bHLH domain. Our results showed that approximately 80% of Rosaceae bHLH genes (159 PbbHLHs (from Pear), 157 MdbHLHs (from apple), 101 PpbHLHs (from peach), 90 FvbHLHs (from wild strawberry) and 96 PmbHLHs (from Chinese plum)) have introns in their bHLH domains (Fig. 2). The most common pattern differed among the five species. The most common pattern in pear and peach was pattern VI (including one intron), while pattern I (including three introns) was the most common pattern in apple, strawberry and Chinese plum. The most common intron pattern in pear and peach (pattern VI) was the same as in Arabidopsis. Patterns I and VI were found to be the most common ones, present in most bHLH genes.
Phylogenetic analysis of the bHLH proteins
To examine the evolutionary relationships among Rosaceae bHLH proteins, and further predict the function of PbbHLHs, a neighbor-joining phylogenetic tree was conducted, using the alignment sequences of 749 Rosaceae bHLH proteins and 150 Arabidopsis bHLH proteins. The phylogenetic tree showed that these bHLH proteins were categorized into 34 groups (Fig. 3). The smallest group was group 19, having only six members, while the largest group was group 30, containing 65 members. Interestingly, the phylogenetic tree showed that group 20 contained 33 bHLH proteins which were from only Rosaceae species, indicating the Rosaceae-specificity of those bHLH proteins from group 20.
Evolutionary pattern analyses of the Rosaceae bHLH gene family
Different patterns of gene duplication events have drived the evolution of gene families, including singleton duplication (SD), WGD duplication, tandem duplication (TD), proximal duplication (PD) or dispersed duplication (DSD) [25]. To understand whether gene duplication events (and, if so, what type) occurred during the evolution of the Rosaceae bHLH gene families, we detected the origins of bHLH genes in the five Rosaceae genomes using the MCScanX package. As shown in Fig. 4 and table 2, 55.6% (110) of the bHLH genes in pear and 48% (108) in apple were duplicated from WGD or segmental events, compared to 41.9% (54) in peach, 62.5% (70) in strawberry and 51.6% (63) in Chinese plum being duplicated as a result of dispersed duplication events. This may be due to the recent lineage-specific WGD events (30–45 MYA) occurred in pear and apple pear and apple, whereas recent WGD events did not occur in peach, strawberry or Chinese plum. In addition, genome rearrangements, gene losses, and RNA- and DNA-based transposed gene duplications may contribute to the larger proportions of dispersed duplicates in peach, strawberry or Chinese plum. These results showed that WGD or segmental duplication and dispersed gene duplication played critical roles in the expansion of the bHLH gene family in the Rosaceae.
To investigate the potential evolutionary mechanisms of the bHLH gene family, we performed the paralogous relationships across the entire pear genome, using a local synteny-based method. We observed that most PbbHLH genes were distributed on 16 chromosomes with an uneven distribution, and 63 paired homologous relationships from segmental duplications appeared in pear (Fig. 5A and Table S4). Interestingly, Chromosomes 15 has the most duplicated genes (Fig. 4A), indicating that it plays important roles in duplication events of the PbbHLH gene family. Furthermore, take two homologous gene pairs duplicated in chromosomes 15, we performed an all-vs.-all local BLASTP based on a method similar to the one used for PGDD across the whole pear genome to identify synteny blocks. Highly Conserved synteny was observed in the regions, several of which contained over 100 syntenic gene pairs (Fig. 4B).
In addition, 158 paired orthologous relationships were found in pear and apple, 173 in pear and peach, 135 in pear and strawberry, and 130 in pear and Chinese plum (Fig S2.). The numbers of the orthologous relationships among pear, apple and peach were greater than those in pear, strawberry and Chinese plum, suggesting that bHLH genes in pear, apple and peach may have originated from a common ancestor.
Ka/Ks ratio and Ks value drive the selective pressure on the evolution of bHLH gene family
Ka/Ks ratio is used to explore the selective pressure on duplicated genes, the ratio being an indication of negative selection (where Ka/Ks<1) or positive selection (where Ka/Ks>1) [26,27]. Positive selection is associated with functional divergence. Our result showed that the Ka/Ks ratio of most bHLH gene pairs in Rosaceae was less than one (Fig 6A), suggesting these bHLH genes mainly experienced negative selection. However, several bHLH gene pairs exhibited Ka/Ks>1, including three gene pairs in pear, 12 gene pairs in apple, one gene pair in peach and four gene pairs in strawberry (Fig 6A), suggesting positive selection possibly playing an important role in the functional divergence of these genes.
The synonymous (Ks) value is used to estimate the stage of evolution for the WGD or segmental duplication events. In our study, the paralogous gene pairs were used to calculate the Ks values for dating the evolutionary time of bHLH genes in Rosaceae. As shown in Fig. 6A, the Ks value of most bHLH duplicated genes in pear and apple ranged from 0.1-0.3, this consistent with a recent WGD duplication event in pear and apple. Interesting, the Ks values for duplicated gene pairs which the ratio of Ka/Ks was more than one almost ranged from 0.1 to 0.3 (Fig 6A), suggesting that the recent WGD event in pear and apple is an active stage of evolution for bHLH gene family. In peach, strawberry and Chinese plum, the Ks value of bHLH genes was singly ranged from 0.5-3, suggesting they might have duplicated from a more ancient duplication event. In addition, we characterized the mean Ks value of the PbbHLH gene pairs, showing that these PbbHLH gene pairs were distributed at the two Ks value peaks (Fig. 6B and Table S4), further suggesting that the recent WGD event and the ancient WGD event led to the expansion of PbbHLH gene family.
Expression profiles of PbbHLH genes in pear different tissues
To further understand the potential functions of the PbbHLH genes, we analyzed the expression profiles of PbbHLH genes in different tissues, including stem, leaf, bud, sepal, petal, ovary and fruit, based on the public RNA-seq data (Fig 7 and Table S5). The result of heatmap showed that the expression pattern of each PbbHLH gene varied greatly in the different tissues. However, we found that 93.9% of PbbHLHs were expressed in at least one tissue (stage) of pear. Among these PbbHLH genes, 48.5% of the genes (96 genes) were expressed in all tissues. Especially, PbbHLH195 was highly expressed in all tissues, suggesting its comprehensive functions in pear development. Furthermore, some PbbHLH genes are highly expressed in a specific tissue of pear, with low exoression in other tissues. For example, PbbHLH15, 18, 70, 84, 89, 93, 113 and 131 are accumulated mainly in leaf, suggesting these genes may play roles in leaf development or immunity. In addition, PbbHLH138, 167 and 176 are strongly expressed in fruit, with low expression in other tissues, suggesting the potential function in pear fruit development. On the other hand, several PbbHLH genes have high level of transcripts in reproductive organs. For example, PbbHLH13, 25 and 100 show mainly higher expression in ovary, suggesting these PbbHLH genes may be necessary for reproductive growth. These above results demonstrated that PbbHLH genes may have different functions in tissue-dependent manner.
Expression profiles of PbbHLHs under drought stress
Plants have formed a set of mechanisms to response for stresses during long-term evolution. Previous reports have shown that several bHLH genes play important roles in response to abiotic stress. However, information is limited on PbbHLH genes response to drought stress in pear. To investigate PbbHLHs response to drought stress in pear, the expression profiles of PbbHLH genes which under drought treatment for 0, 1h, 3h, 6h and re-watering 24h were obtained from the transcriptome data sets previously reported (Fig. 8 and Table S6). Most PbbHLH genes (143) showed differential gene expression. Among them, the expression of 13 PbbHLH genes were significantly up-regulated, while 23 PbbHLH genes were significantly down-regulated. Furthermore, we respectively selected 8 genes (PbbHLH26, 30, 77, 84, 113, 131, 152 and 166), 7 genes (PbbHLH1, 12, 15, 52, 58, 79, and 92) that were up-regulated and down-regulated by at least 10-fold after drought treatment for 6h and restore original expression after re-watering 24h, suggesting these genes may participate in drought stress response.
To verify the reliability of transcriptome data, quantitative real-time PCR (qRT-PCR) was further used to analyze the relative transcript abundance of six selected genes (PbbHLH22, 30, 39, 52, 58 and 131) (Fig 9). As shown in Fig. 7, the result of qRT-PCR is consistent with the transcriptome data. For example, the expression of PbbHLH30, 39 and 131 continuously rise under drought stress at certain time points and then down regulated 24 h of recovery. PbbHLH22, 52 and 58 displayed an obvious decrease in expression under drought stress for 1, 3 and 6h, and then up or down regulated 24 h of recovery. These results further suggested that these genes might play important roles in response to drought stress in pear.