Identification of POD genes
To identify members of POD family in B. pendula, we used the 73 POD genes of Arabidopsis to obtain the best hits in the B. pendula genome by BLASTP. A total of 90 putative PODs were identified in the B. pendula genome. We further examined the conserve domains of proteins encoded by these genes using Pfam [27] and SMART [28] database. The results revealed that all the genes have classical POD domain structures, which demonstrates the reliability of the results. The B. pendula genome contains more PODs than Arabidopsis (73) [6], but fewer than Populus euphratica (93) [29], chinese pear (94) [26], and rice (138) [11]. We defined the BpPODs as BpPOD1 to BpPOD90. The isoelectric point (PI) varied from 4.28 to 9.6 with a mean of 7.25 and >7.0 of 52.2% POD proteins. Their detailed information, including chromosome location, gene name and molecular weight (MW) gene size of each BpPOD gene/protein, was listed in Table1.
Phylogenetic analyses of POD proteins in B. pendula
To investigate the evolutionary history and phylogenetic relationships among the members of POD family in B. pendula, a phylogenetic tree was constructed with the Neighbor-Joining method based on multiple sequence alignment of the 90 BpPODs, with 1000 bootstrap replicates (Figure 1). The BpPOD proteins were divided into twelve major subgroups with high bootstrap probabilities, designated group I to group XII. The POD genes of each subgroup is unevenly distributed, with the number of members varies from 4 to 15. Subgroup VIII contains the most members (15), subgroup X, XI, XII contains the least number of members, with only 4 members.
Analysis of conserved amino acid motifs
To understand the functional regions of BpPODs, conserved amino acid motifs analyses of BpPOD proteins were performed. A total of eight conserved amino acid motifs were identified in the BpPOD proteins (Figure 2). All BpPOD proteins contain at least one conserved amino acid motif. For example, BpPOD55 only contains motif 8, BpPOD83 contains motif 1 and 7, while BpPOD10 proteins contain all the eight conserved amino acid motifs.
Most of the closely related members have the same motif compositions, suggesting that there are functional similarities between POD proteins within the same subgroup [30]. We found that motifs 1, 2, 3, 4, 5 and 7 appeared in nearly all members of BpPOD proteins, these motifs might be important for the functions of BpPOD proteins.
Analysis of chromosomal location
To investigate the genome organization and distribution of BpPODs on different chromosomes of B. pendula, a chromosome map was constructed. The results show that the 90 BpPOD genes were distributed among 14 chromosomes, as shown in Figure 3, the physical locations of these BpPODs on chromosomes were scattered and uneven. Chromosome 1 and 8 contains the most BpPOD genes (14), followed by chromosome 13 (10). Eight BpPOD genes were simultaneously distributed on chromosomes 5 and 7, whereas chromosomes 14 had only one and chromosomes 11 does not include the POD genes. In addition, some chromosomes exhibit a relatively high density of BpPOD genes, such as the bottoms of chromosomes 13 and the top of chromosome 8.
Gene duplication, including segmental and tandem duplication, is considered to be one of the primary driving forces in the evolution of genomes [31, 32]. In this study, among the 90 BpPOD genes identified, a large number of BpPOD genes have the same duplicated regions (Figure 4). Generally, a gene cluster is the result of gene tandem duplication [33]. In this study, we found that some BpPOD genes were adjacent to each other (Figure 3). For instance, BpPOD17-20, BpPOD22-29 and BpPOD11-15 were located sequentially in tandem on chromosomes 5, 8, and 13, respectively, implying that these genes might arise from recent tandem duplication events [34]. The result indicated that tandem duplications play main contributors in the expansion of the BpPOD genes family. However, in previous studies, segmental duplication and tandem duplication were identified in maize POD family [30]. This indicates that there are significant differences in the POD genes expansion pattern in B. pendula and maize, which strongly implied that POD family members have different expansion patterns among different species. It may be the reason why the POD family members (90) in B. pendula were less than those in the maize (119) [26].
Tissue-specifc expression of BpPOD genes in B. pendula
To better understand the functions of POD genes in the growth and development of B. pendula, their expression profiles in different tissues (including root, xylem, young leaf and flower) were analyzed with publicly available transcriptome data. Of the 90 BpPOD genes, 69 genes were expressed in one or more birch tissues, while 21 BpPOD genes exhibited no expression in various individual tissues. The heat map (Figure 5) demonstrated that most BpPOD genes had tissue-specific or preferential expression patterns. BpPOD6, BpPOD21 and BpPOD37 were highly expressed in xylem, suggesting that they may play specific roles in xylem development. Several BpPOD genes were expressed in root during development, revealing the significant roles of these genes in root growth, such as BpPOD62, BpPOD63 and BpPOD65. BpPOD78 and BpPOD19 showed higher expression levels in young leaf and flower, respectively, implying their specific roles in leaf and flower development. The expression level of BpPOD6 was high in xylem and low in root, leaf and flower. In contrast, BpPOD67, BpPOD68, BpPOD80 and BpPOD81 had no expression in any of the investigated tissues, suggesting that these genes are not involved in the development of these tissues. BpPOD21, BpPOD59 and BpPOD62 were highly expressed in developing xylem, root, leaf and flower. In conclusion, the variations in the expression of BpPOD genes in different tissues revealed that POD genes may be involved in several processes during B. pendula growth and development.
Responses of BpPOD genes expression to cold treatment
Several roles have been attributed to plant peroxidases in response to biotic and abiotic stresses [35]. In recent years, the number of studies on POD genes response to abiotic stress have been reported [30]. For example, Arabidopsis overexpressing AtPOD3 showed an increase in dehydration and salt tolerance, whereas the antisense suppression of AtPOD3 exhibited dehydration and salt sensitive phenotypes [36]. The expression of POD genes is induced by various environmental stresses, such as metal, pathogens, humidity, temperature, anoxia and potassium deficiency [35], suggesting that POD genes are involved in plant defense. In this study, we examined the expression levels of the BpPOD genes in response to low temperature stress. As shown in Figure 6, the result indicated that the expression of most BpPOD genes was altered under cold treatment. After cold treatment, the expression levels of BpPOD4, BpPOD13, BpPOD15, BpPOD17 and BpPOD21 were significantly induced at a relatively early stage (0.5 h after treatment), this suggests that these genes play a more important role in B. pendula under cold treatment than other genes. The expression levels of BpPOD19, BpPOD21, BpPOD39 and BpPOD47 were increased after 1.5 h treatment of low temperature. BpPOD50 and BpPOD58 did not response to cold treatment at the beginning (0.5 h), and were slightly increased after 2 h exposure to low temperature. The low temperature responsive BpPODs may play important roles in birch under cold stress.