Molecular evolution and expression divergence of the Populus polygalacturonase supergene family shed light on the evolution of increasingly complex organs in plants

Authors

  • Zhi-Ling Yang,

    1. State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China
    2. University of Chinese Academy of Sciences, Beijing, China
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  • Hai-Jing Liu,

    1. State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China
    2. University of Chinese Academy of Sciences, Beijing, China
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  • Xiao-Ru Wang,

    1. Department of Ecology and Environmental Science, UPSC, Umeå University, Umeå, Sweden
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  • Qing-Yin Zeng

    Corresponding author
    1. State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, China
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Summary

  • Plant polygalacturonases (PGs) are involved in cell separation processes during many stages of plant development. Investigation into the diversification of this large gene family in land plants could shed light on the evolution of structural development.
  • We conducted whole-genome annotation, molecular evolution and gene expression analyses of PG genes in five species of land plant: Populus, Arabidopsis, rice, Selaginella and Physcomitrella.
  • We identified 75, 44, 16 and 11 PG genes from Populus, rice, Selaginella and Physcomitrella genomes, respectively, which were divided into three classes. We inferred rapid expansion of class I PG genes in Populus, Arabidopsis and rice, while copy numbers of classes II and III PG genes were relatively conserved in all five species. Populus, Arabidopsis and rice class I PG genes were under more relaxed selection constraints than class II PG genes, while this selective pressure divergence was not observed in Selaginella and Physcomitrella PG families. In addition, class I PG genes underwent marked expression divergence in Populus, rice and Selaginella.
  • Our results suggest that PG gene expansion occurred after the divergence of the lycophytes and euphyllophytes, and this expansion was likely paralleled by the evolution of increasingly complex organs in land plants.

Introduction

The plant cell wall is involved in many essential biological processes, including mechanical support, regulation of cell expansion, the control of tissue cohesion, ion exchange and protection against pathogens (Popper, 2008; Wei et al., 2009). Polysaccharides represent up to 95% of the plant cell wall mass, whereas cell wall proteins account only for 5–10% (Jamet et al., 2008). The polysaccharides of plant cell walls are divided into three principal types: pectin, cellulose and hemicellulose (Yin et al., 2010). Pectin is the most structurally complex family of polysaccharides, making up c. 35% of primary walls in dicots and nongraminaceous monocots and has functions in cell wall porosity, cell adhesion, plant defense and so on (Mohnen, 2008).

Plant cell walls are highly dynamic structures that undergo changes in composition and configuration in response to functional requirements (Prieto-Alcedo et al., 2011). The pectin network is systematically disassembled during many stages of plant development, such as organ abscission, fruit ripening, and pod and anther dehiscence (Hadfield & Bennett, 1998; Ogawa et al., 2009). The dismantling of the pectin network occurs through the action of pectin-degrading enzymes, including polygalacturonase (PG), pectate lyase and pectin methylesterase (Bosch & Hepler, 2005). Among these hydrolytic enzymes, the PGs belong to one of the largest hydrolase families (Kim et al., 2006). PGs are important for pectin disassembly (Hadfield & Bennett, 1998; Munoz et al., 1998). A recent study showed that the knockout of three Arabidopsis PGs led to the failure of pollen grain separation and silique and anther dehiscence (Ogawa et al., 2009). Overexpression of a PG gene in transgenic apple (Malus domestica Borkh. cv Royal Gala) trees led to premature leaf shedding as a result of reduced cell adhesion in leaf abscission zones (Atkinson et al., 2002). The functions of PGs are not restricted to plant developmental processes, but also include wound responses and host–parasite interactions (Orozco-Cardenas & Ryan, 2003). Thus, plant PG genes have extensive functional divergence.

Plant PGs are multifunctional proteins encoded by a large gene family. The Arabidopsis and rice (Oryza sativa) genomes contain 66 and 42 members, respectively, which are divided into three distinct groups (Kim et al., 2006). To date, genome-wide analyses of the PG gene family have focused mainly on herbaceous annual plants, such as Arabidopsis and rice. The expansion and functional divergence of this large gene family in other land plants, however, have not been investigated. Thus, there is a lack of general understanding of the tempo and mode of PG gene family evolution in plants. Now with the completion of lycophyte Selaginella moellendorffii (Banks et al., 2011), bryophyte Physcomitrella patens (Rensing et al., 2008), and Populus trichocarpa (Tuskan et al., 2006) genome projects, a joint phylogenetic analysis of PG genes from bryophyte, lycophyte and angiosperm monocot (rice) and eudicot (Populus, Arabidopsis) will help us to understand the expansion and diversification of this large gene family in land plants. Bryophytes are the closest extant relatives of early land plants, which began to diverge c. 450 million yr ago (Rensing et al., 2008). Lycophytes are early vascular plants with a dominant sporophyte generation (Banks et al., 2011). Compared with lycophytes and bryophytes, the angiosperm body possesses more complex organ systems and structures, such as flowers and fruit. One of the major questions in plant evolution concerns the evolution of the body developmental program, which was modified through time so that sporophytes became larger and acquired the ability to branch, develop conducting tissues, and produce roots, leaves, seeds, and flowers (Bowman et al., 2007). Populus represents a woody perennial plant, and is the most important tree model system of plant genomics currently available. The relatively close phylogenetic relationship between Populus and Arabidopsis in the eurosid clade of eudicotyledonous plants facilitates studies of gene family evolution in eudicots (Jansson & Douglas, 2007). In this study, we conducted genome-wide annotation of PG genes in the Populus genome. Through a comprehensive analysis of gene sequences, gene structures, molecular evolution, gene expression patterns under normal growth conditions and in response to stress treatments, and subcellular localization of PG proteins, we provided detailed characterization of the composition, expansion and expression divergence of the PG gene family in Populus. By further comparative analyses of this gene family in different land plant species, our study sheds light on the evolution of increasingly complex organs in plants.

Materials and Methods

Identification of PG genes from the Populus genome

To identify Populus PG gene family members, the Populus genome database version 2.2 (http://www.phytozome.net/) was searched using 66 Arabidopsis PG protein sequences in the TBLASTN program with default algorithm parameters. Analysis of the collected Populus PG candidates indicated that some sequences were partially misannotated during the automated genome annotation process. Thus, manual reannotation was performed to rectify incorrect start codon predictions, splicing errors, missed or extra exons. Previous studies showed that all PG proteins contained glycosyl hydrolase family 28 (GH28) domains (Kim et al., 2006). In this study, all Populus PG candidates collected were primarily analyzed using the protein families database (Pfam) to confirm the presence of GH28 domains in their protein structures. To verify the intron/exon structures of Populus PGs, genes were amplified from Populus cDNA, cloned into pEASY-T3 vector (TransGen, Beijing, China) and sequenced in both directions. In this study, the cDNAs of 12 Populus PGs were not detected by PCR. The gene structures of these 12 Populus PGs were assumed to be identical to that of their closest relative on the phylogenetic tree. This approach was adapted from other studies (Meyers et al., 2003).

Phylogenetic and molecular evolution analyses

Full-length PG protein sequences were aligned with the MUSCLE software (http://www.drive5.com/muscle/) and manually adjusted using BioEdit (Hall, 1999). Phylogenetic relationships were reconstructed using a maximum-likelihood (ML) procedure in PHYML software (Guindon & Gascuel, 2003) with the Jones, Taylor, and Thornton (JTT) amino acid substitution model. One-thousand bootstrap replicates were performed in each analysis.

The synonymous substitution (Ks) rates among all pairwise comparisons within Populus PG genes were calculated with the YN00 program in the PAML package (Yang, 2007). To evaluate variation in selective pressures between the Populus classes I and II PG genes, the branch models of CODEML in PAML were used to estimate ω (= dN/dS) under two assumptions: a one-ratio model that assumes the same ω ratio for two classes; and a two-ratio model in which the two classes were assigned to different ω ratios. To verify which of the models best fits the data, likelihood ratio tests (LRTs) were performed by comparing twice the difference in log-likelihood values between pairs of the models using a χ2 distribution (Yang & Nielsen, 2000).

Expression of PG genes under different treatments

To investigate the expression patterns of the Populus PG genes under normal and abiotic stress conditions, seedlings of Populus were cultivated in potting soil at 25°C under 14 : 10 h light : dark coniditions in a growth chamber for 2 months before treatment. For salt and ABA treatments, seedlings were drenched and sprayed once a day using 150 mM NaCl solution for 1 wk and 100 μM ABA solution for 12 h, respectively. Drought stress was induced by withholding water for 2 wk. Cold stress was induced by growth at 4°C for 12 h. Each treatment consisted of three replicates. After treatment, total RNAs were isolated from roots, shoots, leaves, buds, phloem and leaf abscission zones (the narrow portion from the petiole to stem). In addition, total RNAs were isolated from the flower of c. 15-yr-old Populus trees. To investigate the expression patterns of the P. patens PG gene family, Physcomitrella plants were cultured in BCDATG solidified medium at 25°C under an 18 : 6 h light : dark cycle for c. 6 months. Total RNAs were isolated from rhizoids, stems and leaves, respectively. To investigate the expression patterns of Selaginella PG gene family, Selaginella plants were cultivated in potting soil for 2 yr. Total RNAs were isolated from root, stem and leaf tissues. Total RNAs isolated from Populus, Physcomitrella and Selaginella were treated with RNase-free DNase I (Promega, Madison, WI, USA) and reverse-transcribed into cDNA using an RNA PCR Kit (AMV) version 3.0 (TaKaRa, Dalian, China). Based on the multiple sequence alignment of PG gene sequences, specific PCR primers were designed (Supporting Information, Table S1). PCR conditions were optimized to consist of an initial denaturation of 3 min at 94°C, followed by 35 cycles of 30 s at 94°C, 40 s at 65°C and 1 min at 72°C with a final extension of 3 min at 72°C. In all PCR analyses, the Actin gene was used as an internal control. PCR products from each sample were analyzed using a 1% agarose gel and were validated by DNA sequencing. Independent biological triplicates were used in all gene expression analyses.

To evaluate the significance of differences in the gene expression patterns of class I and II PGs, we conducted a multiple response permutation procedure (MRPP) test. MRPP is a nonparametric test and is flexible over unequal sample sizes and violation of normality assumptions. In the MRPP test, positive and negative detection of gene expression in the corresponding tissue were defined as 1 and 0, respectively. We used Euclidean distance to calculate dissimilarity and ran 10 000 permutations to calculate the P value.

Subcellular localization of Populus PGs

We selected nine Populus PG proteins and investigated their subcellular localizations by generating C-terminal green fluorescent protein (GFP) fusions. The nine full-length PG genes were subcloned into the modified pCAMBIA1302 transgenic vector (Fig. S1). The primers used to construct the PG subcellular localization vectors are listed in Table S2. Colonies containing the appropriate insert were identified by sequencing. The modified pCAMBIA1302 vectors containing PG sequences were transformed into Agrobacterium tumefaciens LBA4404. Cultures were infiltrated into epidermal cells of tobacco (Nicotiana benthamiana) leaves as described by Sparkes et al. (2006). Transformed tissues were harvested 3–5 d later and immediately observed under a confocal laser microscope (Olympus FV1000MPE, Tokyo, Japan). GFP fusion fluorescence was excited with a 488 nm laser. Localization in the epidermal cell wall was confirmed by plasmolysis. Plasmolysis was induced by infiltrating 0.3 g ml−1 sucrose into the leaves of tobacco using a needleless syringe.

Results

Sequence and structural characteristics of Populus PG genes

Seventy-five full-length genes encoding putative PG proteins were identified in the Populus genome (Table S3). All collected Populus PG candidates were analyzed primarily using Pfam to confirm the presence of GH28 domains in the protein structure. All 75 genes contained GH28 domains, confirming that these genes belong to the PG family. In addition to full-length PG genes, 21 partial PG fragments were identified in the Populus genome (Table S4). The length of these PG fragments ranged from 40 to 385 amino acids. Pfam analysis showed that these fragments contained partial GH28 domains. In this study, these PG fragments were considered to be pseudogenes.

Phylogenetic relationships among the 75 Populus PGs were reconstructed using an ML procedure. The phylogenetic tree showed that the PGs formed three distinct clades (blue, yellow and brown clades) with 100% bootstrap support (Fig. 1a). In this study, the PG genes in the blue, yellow and brown clades are termed classes I, II and III PGs, respectively. Populus classes I, II and III PG genes contained 60, 14 and one member, respectively.

Figure 1.

Phylogenetic relationships among Populus polygalacturonases (PGs) (a), their expression patterns (b) and gene structures (c). Numbers on branches indicate the bootstrap percentage values calculated from 1000 replicates, and only values higher than 50% are shown. Classes I, II and III PGs are shaded blue, yellow and brown, respectively. In (b), the green box indicates positive detection of gene expression in the corresponding tissue under normal growth conditions (NC) and following drought (DR), abscisic acid (AB), cold (CO) and salt (ST) stress treatments. In (c), the GH28 domains are highlighted by blue boxes. Introns are shown as lines.

Pairwise comparisons of the 75 full-length PG protein sequences revealed some notable features. All class I PGs showed > 23% pairwise sequence identity and all class II PGs showed > 35% pairwise sequence identity. A box plot showed that the protein sequence identities of class II PGs were higher than those of class I PGs (independent-sample t-test, P < 0.0001) (Fig. 2), indicating that the degree of sequence divergence among class I PGs was higher than that among class II PGs. In a pairwise protein sequence comparison, low sequence identities between the PG classes were found: < 24% between class I and class II PGs, < 18% between class I and class III PGs, and < 20% between class II and class III PGs. An independent sample t-test showed that the protein sequence identities within each class were higher than that between classes (P < 0.0001).

Figure 2.

Pairwise sequence identity of full-length Populus polygalacturonase (PG) proteins. A and B represent pairwise sequence identities of classes I and II PG proteins, respectively. AB, AC and BC represent pairwise sequence identities between classes I and II PG proteins, classes I and III PG proteins, and classes II and III PG proteins, respectively. The box plot shows the median (black line), interquartile range (box), and maximum and minimum scores (whiskers) of each data set. Outliers are shown as circles outside of the whiskers.

Highly variable gene structures were observed in Populus class I PGs (Fig. 1c). Among 60 class I PGs, two had two introns, 25 had three introns, nine had four introns, six had five introns, four had six introns, four had seven introns and 10 had eight introns. Compared with class I PGs, conservative gene structures were found among class II PG genes. Among 14 class II PGs, five had a five-intron/six-exon structure and nine had a four-intron/five-exon structure.

Expansion of the PG family in Populus and Arabidopsis

To investigate the extent of lineage-specific expansion of the PG genes in Populus and Arabidopsis, we performed a joint phylogenetic analysis of all Populus and Arabidopsis PGs. The 75 Populus and 66 Arabidopsis PGs fell into three distinct classes (Fig. 3). We identified the nodes that led to Populus- and Arabidopsis-specific clades (circles in Fig. 3). These nodes indicated the divergence point between Populus and Arabidopsis, and thus represented the most recent common ancestral genes before the split. Some PG genes might have been present in the most recent common ancestor (MRCA) of Populus and Arabidopsis but were later lost in either Populus or Arabidopsis. We found that five clades contained only Populus PG genes (Fig. 3, black arrows) and 10 clades contained only Arabidopsis PG genes (Fig. 3, red arrows), indicating that gene loss might have occurred in these clades. The number of clades indicated that there were at least 39 ancestral PG genes before the PopulusArabidopsis split. In addition, four clades (Fig. 3; clades a14, a15, a16 and b6) had low bootstrap support (< 50%). If we assume these less well-supported nodes are correct, there were at least 43 ancestral PG genes before the PopulusArabidopsis split.

Figure 3.

Phylogenetic tree of Populus and Arabidopsis polygalacturonases (PGs). Numbers on branches indicate the bootstrap percentage values calculated from 1000 replicates, and only values higher than 50% are shown. Classes I, II and III PGs are shaded blue, yellow and brown, respectively. The nodes that represent the most recent common ancestral genes before the Populus and Arabidopsis split are indicated by red circles (bootstrap support > 50%) and white circles (bootstrap support < 50%). Clades that contain only Populus or Arabidopsis PGs are indicated by black or red arrows, respectively.

Populus and Arabidopsis had 60 and 53 class I PG genes, respectively. There were at least 31 ancestral class I PG genes in the MRCA of Populus and Arabidopsis (Fig. 4). After the split, Populus and Arabidopsis have gained 36 and 26 genes and lost seven and four genes, respectively, resulting in the rapid expansion of class I PG genes in these two plants. Clearly, the number of genes gained in the Populus lineage was greater than that in the Arabidopsis lineage.

Figure 4.

The copy number changes of Populus and Arabidopsis polygalacturonase (PG) genes. Numbers in circles and rectangles represent the numbers of PG genes in extant and ancestral species, respectively. Numbers on branches with plus and minus symbols represent the numbers of gene gains and losses, respectively.

Based on phylogenetic analysis, there were at least 11 ancestral class II PG genes in the MRCA of Populus and Arabidopsis (Fig. 4). Since the split, Populus and Arabidopsis have gained six and two genes and lost three and one gene, respectively, resulting in the conserved copy numbers of class II PG genes in the two species. Each Populus and Arabidopsis genome had one class III PG gene and neither gain nor loss of genes was observed in either species.

Duplication mechanisms accounting for the Populus PG family expansion

We examined the distribution of the PG genes on the Populus chromosomes. Seventy-three PG genes were localized on 19 Populus chromosomes (Fig. 5), whereas the other two were on one as-yet-unattributed scaffold fragment (Table S3). The distribution of the PG genes on the chromosomes appears to be nonrandom. Three clusters (clusters I, II and III) with high densities of PG genes were observed on chromosomes 7, 17 and 19. Four pairs of PG genes (PtPG10/11, PtPG37/38, PtPG39/40, and PtPG44/45) were arranged in tandem repeats in four positions on chromosomes 2 and 10. Chromosomes 4, 11, 12, 13 and 15 each harbored only one PG gene.

Figure 5.

Genomic localization of Populus polygalacturonase (PG) genes. Schematic view of chromosome reorganization by the most recent whole-genome duplication in Populus (adapted from Tuskan et al., 2006, with permission from AAAS). Regions that are assumed to correspond to homologous genome blocks are shaded gray and connected by lines. Paralogous PG genes and clusters are indicated by dashed lines within the gray-shaded trapezoids.

Whole-genome analysis showed a recent whole-genome duplication event (salicoid duplication, 60–65 million yr ago) that affected c. 92% of the Populus genome (Tuskan et al., 2006). Paralogous segments created by this whole-genome duplication event were identified in previous analyses of the Populus genome (Tuskan et al., 2006). Fourteen duplicate pairs, including PtPG1/12, PtPG6/47, PtPG8/50, PtPG9/51, PtPG14/57, PtPG15/49, PtPG16/25, PtPG17/55, PtPG18/69, PtPG19/68, PtPG28/43, PtPG29/42, PtPG30/41 and PtPG32/36, are each located in a pair of paralogous blocks and can be considered to be direct results of the salicoid duplication event (Fig. 5). Similarly, PG gene clusters I and II correspond to paralogous blocks created by the salicoid duplication. The phylogenetic tree showed that the 15 PG genes in clusters I and II were grouped together (Fig. 1a), indicating that these genes descended from a single common ancestor.

Based on the phylogenetic tree and the positions of these 15 PG genes, we attempted to reconstruct the expansion history of the two clusters. The most parsimonious scenario for gene duplication, loss and rearrangement is presented in Fig. S2. It is likely that six ancestral genes created by three rounds of tandem duplications existed in the Populus genome before salicoid whole-genome duplication. After whole-genome duplication, one round of tandem duplication, loss and rearrangement took place in cluster I, while cluster II might have undergone complex tandem duplication, loss and rearrangement events.

Rapid expansion of class I PG genes in angiosperm plants

The newly available plant genomes (e.g. the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens) allow new insight into the evolution of the PG gene family in land plants. In this study, we identified 16 and 11 PG genes from the Selaginella and Physcomitrella genomes (Table S5), respectively. A previous study showed that rice and Arabidopsis possess 42 and 66 PG genes, respectively (Kim et al., 2006). In this study, based on the latest version of the rice genome sequence (version 7, released on 31 October 2011), we identified 44 PG genes in the rice genome. Using the ML method, we reconstructed the phylogenetic relationships among 212 PGs from Physcomitrella, Selaginella, rice, Arabidopsis and Populus. The phylogenetic tree showed that all the PGs were grouped into three distinct classes (classes I, II and III) with 100% bootstrap support (Fig. 6).

Figure 6.

Phylogenetic tree of the 212 polygalacturonases (PGs) from five land plant species. Classes I, II and III PGs are shaded blue, yellow and brown, respectively. The tree was constructed using the maximum-likelihood (ML) procedure. Numbers at the internal branches leading to the three PG classes indicate the bootstrap support from 1000 replicates.

The marked difference in PG gene family size among bryophyte, lycophyte and angiosperms (Fig. 7) suggests that PG gene expansion occurred after the divergence of the lycophytes and euphyllophytes. Owing to the lack of genome information for gymnosperms and monilophytes, the tempo of PG gene family evolution in land plants cannot yet be precisely defined. We found highly conserved copy numbers of the class III PGs in the each of the five species: each Physcomitrella and Selaginella genome had two class III PG genes, and each of rice, Arabidopsis and Populus had only a single class III PG gene. Similar to class III PGs, conserved copy numbers of the class II PGs in the five species were also observed: Physcomitrella and Selaginella had six and five copies, respectively, and rice, Arabidopsis and Populus had 11, 12 and 14 copies, respectively. However, pronounced copy number variations of class I PG genes were observed in these plant species: Physcomitrella and Selaginella contained three and nine genes, respectively, while rice, Arabidopsis and Populus had 32, 53 and 60 genes, respectively. Thus, the expansion of the PG gene family in angiosperm plants is mainly the result of the rapid expansion of class I PG genes.

Figure 7.

Comparison of the copy numbers of polygalacturonase (PG) genes in five land plant species. The blue, yellow and brown boxes represent classes I, II and III PGs, respectively.

Divergence of selective pressure between the class I and class II PG genes

Compared with class II PGs, Class I PGs showed rapid expansion in Populus, Arabidopsis and rice. To infer the influence of selection on the expansion of class I PGs, we determined if there was a significant change in the selective pressure between the two classes using ML codon models in PAML software. Two assumptions were tested: a one-ratio model that assumes the same ω (= dN/dS) ratio for the two classes and a two-ratio model in which the two classes were assigned to a different ω ratio (Table 1). For Populus PG genes, the log-likelihood values under the one-ratio and two-ratio models were logeL = −36156.1315 and −36142.9111, respectively. The LRT showed that the two-ratio model rejected the null model (one-ratio model), indicating that the selective pressure differed significantly between the two classes (< 0.0001). Under the two-ratio model, the ω values for Populus classes I and II were 0.1879 and 0.1245, respectively, indicating that the Populus class I PG genes were under more relaxed selection constraints than the class II PG genes. Similar to Populus, class I PG genes in Arabidopsis and rice also had higher ω values than that of their class II PG genes. However, Selaginella class I PG genes showed similar selective pressure to class II PG genes, and Physcomitrella class II PG genes were under more relaxed selection constraints than class I PG genes.

Table 1. Summary statistics for detection of selection using branch-specific models of PAML
TreeModelEstimates of parametersLogeLl P
  1. All trees are shown in Fig. S5.

Tree 1 (Populus trichocarpa)One ratioω = 0.1727 for classes I and II PGs−36156.1315  
Two ratiosω1= 0.1879 for class I PGs−36142.911126.4408< 0.0001
ω0= 0.1245 for class II PGs   
Tree 2 (Arabidopsis thaliana)One ratioω = 0.1286 for classes I and II PGs−33806.1316  
Two ratiosω1= 0.1540 for class I PGs−33774.619763.0238< 0.0001
 ω0= 0.0619 for class II PGs   
Tree 3 (Oryza sativa)One ratioω = 0.2172 for classes I and II PGs−7381.8815  
Two ratiosω1= 0.2462 for class I PGs−7377.39468.9738< 0.003
 ω0= 0.1613 for class II PGs   
Tree 4 (Selaginella moellendorffii)One ratioω = 0.0065 for classes I and II PGs−15583.3174  
Two ratiosω1= 0.0065 for class I PGs−15583.22430.1862> 0.67
 ω0= 0.0120 for class II PGs   
Tree 5 (Physcomitrella patens)One ratioω = 0.1220 for classes I and II PGs−9626.8095  
Two ratiosω1= 0.0053 for class I PGs−9617.732418.1542< 0.0001
 ω0= 0.1260 for class II PGs   

Expression divergence of Populus PG genes under normal growth condition and abiotic stress

The expression patterns of Populus PG genes were investigated by PCR under normal growth conditions and in response to abiotic stress treatments (drought, ABA, cold and salt). We examined the expression of all 75 PG genes in seven tissues, including roots, shoots, leaves, buds, phloem, leaf abscission zones and flowers (Fig. 1b). Except for PtPG12, 14 and 57, which were selectively expressed either in a specific tissue and/or in response to a specific treatment, all class II PG genes were expressed in all tissues under all growth conditions. However, substantially higher variation in expression patterns was found among the class I members than among the class II members (Fig. 1b). Among the 60 class I PG genes, 11 (PtPG2, 30, 31, 34, 35, 39, 41, 47, 55, 62 and 64) were expressed in all tissues under all growth conditions, while 12 (PtPG3, 19, 21, 26, 33, 37, 40, 45, 53, 60, 66 and 68) were not expressed in any tissue or in response to any treatment applied in this study. The other 37 class I PG genes were selectively expressed either in a specific tissue and/or in response to a specific treatment. Among the selectively expressed class I PG genes, 11 (PtPG5, 10, 11, 38, 59, 70, 71, 72, 73, 74 and 75) were expressed only in flower tissue, suggesting specific roles for these genes in flower development, and two (PtPG58 and PtPG67) were specifically expressed in the leaf abscission zone under salt stress, suggesting a possible function in leaf abscission under this condition. An MRPP test showed a significant difference in gene expression patterns between Populus classes I and II PGs (MRPP test, < 0.0001).

With regard to the tandem-arrayed PG clusters I and II, marked expression divergence was observed among the members. Among the 10 PGs in cluster II, two genes (PtPG62 and 64) were expressed in all tissues, two (PtPG60 and 66) were not expressed in any tissue examined, while the other six (PtPG58, 59, 61, 63, 65 and 67) showed restricted tissue-specific expression patterns under normal and/or stress conditions (Fig. 1b). Among the five PG genes in cluster I, PtPG21 was not expressed in any tissue examined, while the other four genes (PtPG20, 22, 23 and 24) showed overlapping but distinct expression profiles. For example, PtPG20 was expressed in shoot tissue under drought stress, but PtPG22, 23 and 24 were not; PtPG22 was expressed in leaf tissue under ABA treatment, while the other three genes, PtPG20, 23 and 24, were not. For the tandem-arrayed PG cluster III, four genes in this cluster showed similar expression patterns, expressed only in flower tissue.

Effects of the duplication mechanism on Populus PG gene expression

It was considered that more closely related genes would tend to have similar expression patterns. To test this, we investigated the expression patterns of recently duplicated gene pairs. Twenty-five recently duplicated gene pairs were identified at the terminal nodes of the phylogenetic tree shown in Fig. 1(a) (Table 2). Six categories of expression patterns were observed among these duplicate gene pairs. In the first category, both duplicates were expressed in all tissues under normal growth and stress treatments (AA model in Table 2), suggesting that the duplicate genes may have similar functions. Six duplicate gene pairs (PtPG8/50, 18/69, 32/36, 62/64, 16/25 and 30/41) showed this expression pattern. In the second category, one copy was selectively expressed either in a specific tissue and/or in response to a specific treatment, while the other was not detected in any tissue type under any growth condition (SN model in Table 2), suggesting that one duplicate gene may have become a pseudogene or evolved a new function not identified in this study. Three duplicate gene pairs (PtPG5/33, 44/45 and 66/67) belonged to this category. In the third category, one copy of each duplicate pair was expressed in all tissues under all growth conditions, while the other was expressed only following a specific treatment and/or in a specific tissue (AS model in Table 2). Four duplicate gene pairs (PtPG1/12, 6/47, 17/55 and 38/39) showed this expression pattern. In the fourth category, both duplicates were expressed only in flower tissue (EF model in Table 2). Three duplicate gene pairs (PtPG10/11, 71/75 and 72/73) showed this expression pattern. In the fifth category, both duplicates exhibited selective expression in the tissues examined, but the expression patterns of the two duplicates differed (SE model in Table 2). Seven duplicate gene pairs (PtPG9/51, 14/57, 15/49, 24/46, 28/43, 29/42 and 56/58) belonged to this category. In the last category, neither duplicate was expressed in any of the tested tissues (NN model in Table 2). Two duplicate gene pairs (PtPG19/68 and 37/40) showed this expression pattern. Among the 25 recently duplicated gene pairs, 14 (56%) duplicate gene pairs showed divergent expression patterns, indicating that the expression patterns of closely related genes have not always been similar.

Table 2. Divergence between paralogous polygalacturonase (PG) gene pairs in Populus
No.Gene1Gene2 K a K s Ka/KsGene expression
  1. The synonymous (Ks) and nonsynonymous (Ka) substitution rates between gene pairs were calculated by the K-Estimator program (Comeron, 1999). Gene pairs created by tandem duplication (T), whole-genome duplication (W) or other duplication (O) events are indicated in the first column of the table. Gene expression patterns were categorized into six classes (see text).

1(W) PtPG8 PtPG50 0.06110.17590.35AA
2(W) PtPG18 PtPG69 0.04420.24710.18AA
3(W) PtPG32 PtPG36 0.05310.20340.26AA
4(T) PtPG62 PtPG64 0.01010.02470.41AA
5(W) PtPG16 PtPG25 0.06020.19910.30AA
6(W) PtPG30 PtPG41 0.05350.26380.20AA
7(O) PtPG5 PtPG33 0.28850.82800.35SN
8(T) PtPG44 PtPG45 0.03190.12410.26SN
9(T) PtPG66 PtPG67 0.03070.06910.44SN
10(W) PtPG1 PtPG12 0.04740.20840.23AS
11(W) PtPG17 PtPG55 0.11370.24930.46AS
12(O) PtPG38 PtPG39 0.02180.09840.22AS
13(W) PtPG6 PtPG47 0.05430.25170.22AS
14(W) PtPG19 PtPG68 0.35590.97460.37NN
15(O) PtPG37 PtPG40 0.05110.13070.39NN
16(T) PtPG10 PtPG11 0.00470.00530.88EF
17(O) PtPG71 PtPG75 NA0.01090.00EF
18(T) PtPG72 PtPG73 0.00330.00740.45EF
19(W) PtPG9 PtPG51 0.05410.22820.24SE
20(W) PtPG14 PtPG57 0.16150.48310.33SE
21(W) PtPG15 PtPG49 0.06730.34320.20SE
22(O) PtPG24 PtPG46 0.09270.57800.16SE
23(W) PtPG28 PtPG43 0.05080.23840.21SE
24(W) PtPG29 PtPG42 0.04790.29600.16SE
25(T) PtPG56 PtPG58 0.00110.00350.32SE

To explore the relationship between the synonymous substitution rate (Ks) and the expression profile, Ks among all pairwise comparisons of the 75 Populus PG genes were calculated. Expression divergences between two duplicate genes were represented by the Hamming distance. In this study, the Hamming distance between duplicate genes was defined as the number of tissues in which one duplicate gene was expressed while the other was not. For example, if one duplicate gene was expressed in all 31 tissues examined in this study, while another duplicate gene was not expressed in these tissues, then the Hamming distance between the two duplicate genes was 31; if two duplicate genes were expressed in all tissues examined, or not expressed, the Hamming distance between the two duplicate genes was 0. Linear regression analysis was carried out between the Ks value and the Hamming distance. The dot plot showed there was not significant correlation between the Ks value and the gene expression divergence (R = 0.028, > 0.145) (Fig. S3).

Expression patterns of PG genes in Physcomitrella, Selaginella and rice

The expression patterns of Populus class I PG genes were distinct from those of class II PG genes. To investigate whether other land plants had similar gene expression patterns to Populus PGs, we examined the expression of PG genes in Physcomitrella, Selaginella and rice. The expression patterns of all 11 Physcomitrella and 16 Selaginella PG genes in different tissues were investigated using PCR (Fig. 8). The Physcomitrella genome contains three class I, six class II and two class III PGs. Except for one class I PG gene (PpPG9), which was not expressed in any of the tissues examined, the other 10 genes were expressed in all tissues examined. The expression patterns of classes I and II PG genes did not differ significantly (MRPP test, = 1).

Figure 8.

Expression patterns of polygalacturonase (PG) genes in Physcomitrella (a) and Selaginella (b). Numbers on branches indicate the bootstrap percentage values calculated from 1000 replicates, and only values higher than 50% are shown. Classes I, II and III PGs are shaded blue, yellow and brown, respectively. The blue box indicates positive detection of gene expression in the corresponding tissue under normal growth conditions.

The Selaginella genome contains nine class I, five class II and two class III PGs. All five class II PGs were expressed in all tissues examined, while substantially greater variation in expression pattern was found among the nine class I PGs. Among these, two (SmPG11 and 14) were expressed in all tissues examined, three (SmPG7, 8 and 15) were selectively expressed in specific tissues and four (SmPG5, 9, 10 and 16) were not expressed in any of the tissues examined. A MRPP test showed a significant difference in the gene expression pattern between Selaginella classes I and II PGs (MRPP test, < 0.02).

The expression patterns of rice PGs were investigated using RNA-seq data from the Michigan State University (MSU) Rice Genome Annotation (http://rice.plantbiology.msu.edu) databases (Fig. S4). With the exception of LOC_Os05 g50960, all rice class II PG genes were expressed in all tissues examined in this study. However, compared with class II PG genes, rice class I PGs showed extensive expression divergences. Among the 32 class I PGs, one (LOC_Os05 g14150) was specifically expressed in pistil tissue, two (LOC_Os03 g11760 and LOC_Os06 g40880) in inflorescence tissue, five (LOC_Os06 g35320, LOC_Os06 g35370, LOC_Os06 g35300, LOC_Os06 g40890 and LOC_Os01 g33300) in the anther, pistil and inflorescence, while five (LOC_Os06 g16810, LOC_Os07 g10680, LOC_Os07 g10740, LOC_Os06 g31270 and LOC_Os11 g14410) were not expressed in any of tissues examined. The expression patterns of rice classes I and II PG genes differed significantly (MRPP test, < 0.0001).

Subcellular localization of Populus PG proteins

Polygalacturonase proteins are involved in the degradation of pectin, which are considered to be localized in the plant cell walls. In this study, we first predicted the subcellular localization of Populus PG proteins using TargetP 1.1 Server (http://www.cbs.dtu.dk/services/TargetP). TargetP 1.1 was used to predict the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal presequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP) (Emanuelsson et al., 2007). Among the 75 Populus PGs, 66 proteins were predicted to contain SP, indicating these proteins might be localized to the cell wall, three (PtPG1, 12 and 54) to contain mTP, one (PtPG43) to contain cTP and the other five (PtPG16, 18, 21, 25 and 69) to contain none of the three signal peptides. To examine the accuracy of the prediction of subcellular locations of Populus PGs, we selected nine PG proteins and investigated their subcellular localization by generating C-terminal GFP fusions and visualization by confocal microscopy after transient expression of the fusions in N. benthamiana. Among the nine proteins from the three PG classes, five (PtPG22, 28, 34, 38 and 47) were predicted to contain SP, two (PtPG1 and 54) to contain mTP and the other two (PtPG18 and 69) were predicted not to contain any of the three signal peptides. Confocal microscopy analysis showed that the fluorescent signal for the nine fusion proteins was only detected at the peripheral regions of the N. benthamiana epidermal cells, suggesting a plasma membrane and/or cell wall localization. To distinguish the plasma membrane and/or cell wall localization, the epidermal cells were plasmolyzed by treatment with 0.3 g ml−1 sucrose. After plasmolysis, the fluorescent signal was retained in the cell wall, as illustrated by PtPG22, 18 and 54–GFP (Fig. 9). Thus, these nine PG–GFP fusion proteins were localized to cell wall.

Figure 9.

Subcellular localizations of Populus polygalacturonase (PG)–green fluorescent protein (GFP) fusion proteins. GFP signal (green) was detected using confocal laser-scanning microscopy. The plasma membrane separated from the cell wall after plasmolysis is indicated by red arrows. Bars, 10 μm.

Discussion

Polygalacturonases were first identified over 35 yr ago and have been suggested to play important roles in the disassembly of pectin that accompanies many stages of plant development, particularly in various cell separation processes (Hadfield & Bennett, 1998). PGs belong to a large gene family with > 44 members in the rice, Arabidopsis and Populus genomes. A previous study showed that Arabidopsis and rice PGs were divided into three groups (Kim et al., 2006). In this study, we found that the 212 PGs from the Physcomitrella, Selaginella, rice, Arabidopsis and Populus genomes fell into three distinct groups (Fig. 6). In addition, the protein sequence identities within each group of Populus PGs were higher than those between groups (Fig. 2). Thus, in this study, the PG genes in the three groups were classified as three classes (classes I, II and III).

Compared with classes II and III PGs, rapid expansion of class I PG genes were observed in angiosperms (Fig. 7). Why did this rapid expansion of class I PG genes occur in angiosperms? One possible explanation is functional requirement. Class I PGs play important roles in plant developmental processes, such as organ abscission, pod and anther dehiscence, pollen grain maturation and fruit softening (Atkinson et al., 2002; Fabi et al., 2009). Compared with bryophytes and lycophytes, angiosperms possess more complex organ systems and structures, such as flowers and fruit. New organ systems and structures might require more PGs to maintain their biological functions. The expansion of class I PGs might provide new raw materials for specific expression of PG genes in the abscission zone of these new organs or structures. If this assumption is correct, angiosperm class I PGs may be under either relaxed selection constraints or positive selection, which allows new duplicate genes to diverge into new specific expression patterns or functions. In fact, the Populus, Arabidopsis and rice class I PG genes are under more relaxed selection constraints than the class II genes. The class I PG genes in these three species showed extensive expression divergence. In particular, some class I PG genes in these three species were expressed in specific organs, such as flowers (Figs 1b, S4) (Kim et al., 2006). Thus, functional requirements could explain the rapid expansion of class I PG genes in angiosperms.

The rapid expansion of the PG gene family in angiosperm plants is largely the result of the expansion of class I PG genes. Among the 60 Populus class I PG genes, 20 (33%) were created by tandem duplication and eight (13%) by the salicoid whole-genome duplication event, indicating that tandem duplication was the major factor responsible for the rapid expansion of the class I PG genes in Populus. This is similar to other gene families, such as Populus glutathione S-transferase (GST) (Lan et al., 2009) and late embryogenesis abundant protein family (Lan et al., 2012). In addition, we found 21 PG fragments in the Populus genome (Table S4), indicating that a large number of PG duplicates were lost. Our previous study of the Populus GST supergene family also identified a large number of GST fragments in the Populus genome (Lan et al., 2009). Thus, our data suggest that gene loss might be a general evolutionary patterns of large gene family evolution (see also Zou et al., 2009). This gene loss was considered an important mechanism in the generation of genome diversity among eukaryotic species (Li et al., 2005aa).

Although a large number of PG genes were lost, many duplicates have been retained in the Populus genome, bringing us to a central issue in the evolution of duplicate genes: why have so many duplicate genes been retained? At present, the neofunctionalization or subfunctionalization models are frequently invoked to explain the retention of duplicate genes (Li et al., 2005ab). In the neofunctionalization model, one duplicate copy accumulates beneficial mutations and acquires a new function, whereas another duplicate copy retains the original function. In the subfunctionalization model, each duplicate copy partitions the ancestral gene function. Neofunctionalization or subfunctionalization of duplicate genes is associated with the processes of tissue expression divergence (Ganko et al., 2007). In addition, the expansions of large gene family are often associated with high levels of tissue-specific expression divergence (Lan et al., 2009; Huerta-Cepas et al., 2011). Among the 60 Populus class I PGs, 11 were specifically expressed in flowers. The flower-specific expressions of class I PG genes were also observed in rice and Arabidopsis (Fig. S4) (Kim et al., 2006). Compared with bryophytes and lycophytes, flowers are a key innovation in angiosperms. The class I PG genes expressed specifically in flowers might acquire new functions related to flower development. Thus, the evolutionary fates of these class I PG genes might be neofunctionalization and could contribute to the retention of these class I PG genes. Among the 25 recently duplicated gene pairs, the six duplicate gene pairs (PtPG9/51, 14/57, 15/49, 24/46, 28/43 and 29/42) have overlapping but distinct expression profiles, indicating typical subfunctionalization. In the tandem-arrayed PG clusters I and II, we found that some PG genes were expressed in all tissues, some had restricted tissue-specific expression patterns under normal and/or stress conditions, while others were not expressed in any tissue examined. This markedly divergent expression pattern indicates that multiple evolutionary fates occurred among the PG genes in clusters I and II, which may contribute to the retention of these duplicate genes.

It has also been reported that expression divergence between duplicate genes is significantly correlated with their Ks value: when the Ks is relatively small, the duplicated genes tend to have more similar expression patterns (Gu et al., 2002). However, no such correlation was identified in the Populus PG gene family (Fig. S3). The Arabidopsis PG gene family did not show this correlation either (Kim et al., 2006). The lack of this correlation might be attributable to the small number of genes examined, resulting in a lack of statistical power. However, this correlation was also not observed upon examination of the expression divergences of 503 duplicate gene pairs in Arabidopsis (Ganko et al., 2007). Thus, there is a need to further study the relationship between expression divergence and synonymous substitutions of duplicate genes.

The duplicate genes created by large segmental duplication tend to maintain similar expression patterns, while those of genes created by nonsegmental duplications rapidly diverge (Ganko et al., 2007). One possible mechanism of this pattern is that large-scale segmental duplication results in the duplication of multiple genes together with their promoter and/or enhancer elements (Casneuf et al., 2006), while nonsegmental duplications, such as tandem duplication, may disrupt the regulatory regions of target genes, resulting in large variations in their expression responses. In this study, 14 segmental duplicate gene pairs and six tandem duplicate gene pairs were identified in the Populus PG gene family. Among the 14 segmental duplicate gene pairs, eight (57%) showed expression divergences between two duplicate genes. Among the six tandem duplicate gene pairs, three (50%) shared similar expression patterns. Thus, this study did not observe the significant effects resulting from the duplication mechanism on gene expression divergence.

Casneuf et al. (2006) reported a strong bias in the divergence of gene expression towards gene function: genes that are involved in signal transduction, hydrolase activity and response to external stimuli appear to have diverged very quickly after duplication, while those involved in nucleic acid and protein metabolisms appear to have diverged slowly after duplication. Because class I PGs have typical hydrolase activity, according to the report of Casneuf et al. (2006), the expression of class I PG duplicate genes should diversify after gene duplications. Indeed, we found rapid divergence in the expression of class I PG genes in Populus and rice. However, among the 36 class II PG genes from Populus, rice, Selaginella and Physcomitrella, except for five PG genes, the other 31 PG genes were expressed in all the tissues examined (Figs 1b, 8, S4), indicating that the function of class II PGs might be conserved in land plants. The maintenance of similar expression patterns in class II PGs in land plants might be the result of functional requirements.

By exploring the currently available genome information in land plants, our comparative evolutionary analysis coupled with expression assays provided new insights into the evolution of the PG gene family in land plants. The expansion of PG genes seems to be correlated with the evolution of increasingly complex organs in plants. Further sampling of ferns and gymnosperms would help us to achieve a better understanding of whether the expansion of the PG genes predated the split of gymnosperms and angiosperms, and of the functional significance of PG gene family evolution in plants.

Acknowledgements

This study was supported by grants from the National Basic Research Program of China (2009CB119104) and Vetenskapsrådet, Sweden.

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