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Genome-wide investigation and expression profiling of polyphenol oxidase (PPO) family genes uncover likely functions in organ development and stress responses in Populus trichocarpa

Abstract

Background

Trees such as Populus are planted extensively for reforestation and afforestation. However, their successful establishment greatly depends upon ambient environmental conditions and their relative resistance to abiotic and biotic stresses. Polyphenol oxidase (PPO) is a ubiquitous metalloproteinase in plants, which plays crucial roles in mediating plant resistance against biotic and abiotic stresses. Although the whole genome sequence of Populus trichocarpa has long been published, little is known about the PPO genes in Populus, especially those related to drought stress, mechanical damage, and insect feeding. Additionally, there is a paucity of information regarding hormonal responses at the whole genome level.

Results

A genome-wide analysis of the poplar PPO family was performed in the present study, and 18 PtrPPO genes were identified. Bioinformatics and qRT-PCR were then used to analyze the gene structure, phylogeny, chromosomal localization, gene replication, cis-elements, and expression patterns of PtrPPOs. Sequence analysis revealed that two-thirds of the PtrPPO genes lacked intronic sequences. Phylogenetic analysis showed that all PPO genes were categorized into 11 groups, and woody plants harbored many PPO genes. Eighteen PtrPPO genes were disproportionally localized on 19 chromosomes, and 3 pairs of segmented replication genes and 4 tandem repeat genomes were detected in poplars. Cis-acting element analysis identified numerous growth and developmental elements, secondary metabolism processes, and stress-related elements in the promoters of different PPO members. Furthermore, PtrPPO genes were expressed preferentially in the tissues and fruits of young plants. In addition, the expression of some PtrPPOs could be significantly induced by polyethylene glycol, abscisic acid, and methyl jasmonate, thereby revealing their potential role in regulating the stress response. Currently, we identified potential upstream TFs of PtrPPOs using bioinformatics.

Conclusions

Comprehensive analysis is helpful for selecting candidate PPO genes for follow-up studies on biological function, and progress in understanding the molecular genetic basis of stress resistance in forest trees might lead to the development of genetic resources.

Peer Review reports

Background

Aggravated global warming induces more frequent and severe the local stress in whole ecosystems [1, 2]. Plants have evolved complex mechanisms to defend themselves against various biotic and abiotic stresses [3, 4]. For example, plants frequently encounter insect pests and have evolved an efficient immune response that mainly depends on producing specific metabolites and defense proteins [5, 6]. At the same time, plants competently transform their morphological structure, synthesize favorable metabolites, and evolve complex molecular mechanisms to deal with abiotic stresses [7,8,9], such as drought, cold, heat, and salt stress.

Polyphenol oxidase (PPO) is a widely distributed metalloproteinase that primarily exists in plants, fungi, and insects [10]. It can not only catalyze the oxidation of catechol to quinones but also acts on monophenol monooxygenase substrates. In a broad sense, PPO can be classified into three categories, i.e., tyrosinase (EC.1.14.18.1), catechol oxidase (EC.1.10.3.2), and laccase (EC.1.10.3.1) [11]. Of these three types of PPO, catecholase is mainly distributed in plants, while laccase and tyrosinase are distributed in microorganisms [12]. The function of PPO in plants is largely determined by three conserved domains i.e., KFDV, tyrosinase, and DWL [13].

PPO proteins predominantly exist in terrestrial plants, such as apple, litchi, spinach, potato, legume, tea, mulberry leaf, tobacco, and grapevine, as well as in fungi and some bacteria [11]. Under normal circumstances, PPO is inactive and tightly binds to the inner capsule membrane; activated PPO plays an important role after tissue damage. In addition, several PPO genes have been identified in Musa acuminata, Malus domestica, Oryza sativa, Sorghum bicolor, Solanum tuberosum, Ananas comosus, and Cucumis sativus [11, 14,15,16,17,18,19]. However, there are considerable differences in the distribution and function of PPO proteins in different plants. The location of PPO varies with plant species and maturity level; nevertheless, in most plant leaves, PPO is primarily distributed in chloroplasts [13], while almost all sub-cells of potato tubers contain PPO [12]. Polyphenol oxidase in tea exists in dissociative and combinative states; the former is chiefly found in the cytoplasm, while the latter is mainly found in chloroplasts, mitochondria, and other organelles [11, 20]. Majority of studies have revealed that PPO activity and polyphenol content in tea shoots significantly affect the quality of red tea [21].

Extensive literature mining has revealed that the expression of PPO genes in plants is closely related to stress and response to insect and mechanical damage, diseases, and microorganism invasion [11, 22]. Additionally, the PPO gene family exhibits a heightened response to methyl jasmonate (MeJA) in Salvia miltiorrhiza and Nicotiana tabacum L [23, 24]. Moreover, overexpression of the PPO gene in tomato can enhance plant resistance to insect pests, including Spodoptera litura, Helicoverpa armigera, and Spodoptera exigua [25, 26]. Similarly, the overexpression of PPO genes in poplar trees can result in inhibition of the growth of Malacosoma disstria [27]. Furthermore, upregulation of PPO expression in Lycopersicon esculentum, Juglans regia, Taraxacum officinale, and Fragaria ananassa enhances plant resistance to fungi [11, 25]. Some key enzymes in the phenolic metabolic system play an essential role in mediating the resistance of plants to pathogenic microorganisms. For example, the pathogen-related protein PPO can catalyze the formation of lignin and quinones, thus promoting the formation of defensive barriers and protecting cells from invasion by strengthening the cell structure [28].

Populus has many advantages in basic research as a typical model woody plant with relatively upper ecological, economic, and cultural significance [29,30,31]. Stress severely restricts poplar cultivation in plantations. There are few functional studies on the role of the PPO protein family in biotic and abiotic stress in poplar trees. Here, we identified 18 putative PPO proteins in the Populus trichocarpa genome. Comparative genomics, transcriptomics, and RT-qPCR were used to comprehensively analyze the PPO protein family in poplar to provide a theoretical basis for studies on the characteristics and functions of PPOs in poplar development and stress response.

Results

Identification and analysis of poplar PPOs

Eighteen putative PPO genes were identified from the published P. trichocarpa reference genome sequence and were successively designated PtrPPO1 to PtrPPO18 according to their location on the genome (Figure S1). In order to clearly understand the characteristics of the PPO family in poplar, we analyzed the gene length, transcriptional sequence length, CDS (coding sequence) length, the position of the conserved domain, amino acid (AA) length, protein molecular weight (MW), grand average of hydropathicity (GRAVY), and isoelectric point (PI) of the proteins encoded by these genes (Table 1).

Table 1 Summary of Populus PPO genes

Among the 18 PtrPPO proteins, PtrPPO2 was the shortest with 192 amino acids, whereas PtrPPO9 was the longest (606 AA). The MW of PtrPPO proteins was 22.200 to 68.678 kDa, the GRAVY of the proteins was − 0.821 (PtrPPO2) to − 0.337 (PtrPPO11), and PI was 5.21 (PtrPPO4) to 7.53 (PtrPPO14). Generally, identifying the molecular characteristics of PPOs will be helpful in studying their specific biological functions.

Evolutionary and phylogenetic analysis of the PPO family

To better understand the evolution and differentiation of PPO family proteins among species, 138 PPO amino acid sequences from 25 species were used to construct an unrooted tree (Fig. 1a). These sequences were then divided into 11 groups (I–XI), among which groups VIII and IX contained the most members, including 51 members accounting for 36.96% of the deduced PPO protein. PtrPPO protein was found in groups V, VIII, IX, and XI. Simultaneously, 14 PPO proteins in poplar were concentrated in group IX and clustered in the same branch as 7 PPO proteins in Salix purpurea.

Fig. 1
figure1

Evolutionary and phylogenetic analysis of the PPO family in diverse plant species. a The phylogenetic tree of PPO proteins from Aquilegia coerulea (AqcPPO), Ananas comosus (AcPPO), Amaranthus hypochondriacus (AhPPO), Carica papaya (CpPPO), Cucumis sativus (CsPPO), Eucalyptus grandis (EgPPO), Gossypium raimondii (GrPPO), Kalanchoe fedtschenkoi (KfPPO), Kalanchoe laxiflora (KlPPO), Linum usitatissimum (LuPPO), Musa acuminata (MaPPO), Malus domestica (MdPPO), Mimulus guttatus (MgPPO), Marchantia polymorpha (MpPPO), Oryza sativa (OsPPO), Oropetium thomaeum (OtPPO), Populus trichocarpa (PtrPPO), Panicum virgatum (PvPPO), Ricinus communis (RcPPO), Sorghum bicolor (SbPPO), Sphagnum fallax (SfPPO), Solanum lycopersicum (SlPPO), Spirodela polyrhiza (SpiPPO), Salix purpurea (SpPPO), and Solanum tuberosum (StPPO). The shape and color in front of the node represent the identified PPOs, in which the black circle represents MdPPOs, the black rectangle represents CsPPOs, the black pentagram represents AcPPOs, the black triangle represents MaPPOs, the red circle represents OsPPOs, the red rectangle represents SbPPOs, and the red pentagram represents StPPOs. b Comparisons of PPO protein numbers across 25 plant species. The accession numbers and gene names are all shown in Table S2

To determine the origin and evolution of PPO genes, we searched for PPO genes in 25 species of lower aquatic plants and higher terrestrial plants (Fig. 1b). Interestingly, this gene was absent in Arabidopsis, but four PPO genes were found in monocotyledonous O. sativa. It is worth noting that PPO genes are widely distributed in woody plants, especially in P. trichocarpa (18), M. domestica (15), and S. purpurea (10). However, Marchantia polymorpha (common liverwort, an herbaceous plant) contains 17 PPO genes. In conclusion, the distribution of PPO genes in woody plants was much greater than that in herbaceous plants, indicating that considerable differentiation and doubling of PPO genes might have occurred during the evolution of perennial woody plants.

Determination of gene structure, conserved domains, and motifs

To explore the evolutionary relationship between different members of the PPO gene family more clearly, a phylogenetic tree was constructed using 18 PPO proteins from Populus (Fig. 2a), and their gene structure, conserved protein motifs, and conserved structure were assessed. Most PtrPPO genes (approximately two-thirds of the genes) had no introns, while the other six genes (PtrPPO2/3/5/11/14/17) had at most three introns. Surprisingly, none of the genes with two and three introns had a UTR (Fig. 2b). For example, PtrPPO7/8/1 genes lacked an intron, but they all had UTRs. However, PtrPPO2/5/14 contained at least two introns but no UTR.

Fig. 2
figure2

Phylogenetic, gene structure, conserved protein structure, and motif analyses of PtrPPOs. a Phylogenetic tree constructed using full-length amino acid sequences from 18 PtrPPOs. b Exon-intron structure analysis of PtrPPO genes. Blue boxes represent UTRs; yellow boxes indicate exons; black lines represent introns. c Motif distribution of PtrPPO members. d conserved protein domain analysis of PtrPPOs. The length of each pattern is displayed proportionally

Generally, genes with high similarity in amino acid sequences in the same family may have similar biological functions. We identified three conserved domains (PPO1-KFDV, tyrosinase, and PPO1_DWL) in the PPO protein sequence using CDD and SMART software (Fig. 2d and Table S3). All PPO proteins, except for PtrPPO10/12, had these three structures, which may endow them with similar biological functions. Meanwhile, 10 conserved motifs were identified in the PtrPPOs protein sequence using MEME (Fig. 2c). Amino acid sequence encode, and SeqLogo of the Motif are displayed in Table S4. Among them, two motifs (2 and 9) were related to the C-terminal PPO-KFDV domain, motif 4 was related to the intermediate PPO_DWL domain, and five motifs (1, 6, 8, 3, and 5) were related to the N-terminal tyrosinase domain. Moreover, we found two novel motifs (motifs 7 and 10) at the N-terminal of most PtrPPO protein sequences.

Analysis of chromosomal location and gene duplication

To further understand the evolution and differentiation of PPO family genes, we analyzed the chromosomal distribution, synteny, and evolution of the 18 PPO genes in Populus. The PPO genes were primarily distributed on chromosomes 1, 4, 11, and scaffold_64 (Fig. 3 and Figure S1). Moreover, half of the PPO genes in poplar were distributed on chromosome 1, while there were, at least, two PPO genes on the other chromosomes. It has been reported that the chromosome region within 200 kb containing two or more genes is defined as a tandem replication event [32]. The 18 genes (PtrPPO1/2/3/4/5/6/7/8/9, PtrPPO12/13, PtrPPO14/15, PtrPPO16/17/18) formed four tandem repeat regions distributed on chromosomes 1, 11, and scaffold_64 (Fig. 3). The high sequence similarity between repetitive gene pairs indicates that they are likely involved in regulating similar biological processes. Furthermore, nine PPO genes formed a complex tandem repeat on chromosome 1, indicating a hot spot for PPO gene distribution. We also found that six genes (PtrPPO1/14, PtrPPO8/15, PtrPPO10/12) formed three segmental duplication events using the MCScanX method. These results suggest that PtrPPOs might be produced by gene replication, while tandem duplication and repetitive fragment events collectively catalyze the evolution of PPO genes in poplar.

Fig. 3
figure3

Schematic diagram depicting PPO gene distribution and Inter-chromosomal relationships in Populus chromosomes. The gray lines represent all synteny blocks in the chromosome of Populus trichocarpa. Red, blue, and green lines indicate duplicated PPO gene pairs in Populus trichocarpa. Chromosome number is shown in the middle of the arc square. The length of each arc corresponds to the length of the chromosome (Mb)

We constructed comparative syntenic maps between poplar and four other species to analyze PPO gene evolution in woody plants further. Syntenic maps revealed that nine pairs of homologous genes were found between P. trichocarpa and S. purpurea, and three pairs of homologous genes were found between P. trichocarpa and M. domestica (Fig. 4 and Table S5). Some PtrPPO genes (PtrPPO10 and PtrPPO12) were discovered to be connected with at least two synonym gene pairs (especially between poplar and willow, and between poplar and apple PPO genes), suggesting that these genes may play essential roles in the evolutionary process of the PPO gene family. However, the findings of previous studies and those of the present study revealed that there was no synteny in Populus vs. O. sativa and Populus vs. A. thaliana [11], suggesting that multiple PPO genes may be formed during the differentiation of woody plants.

Fig. 4
figure4

Collinearity analysis of PPO genes in poplar and four typical plant species. The gray lines in the background represent collinear blocks between Populus and another species’ genomes, while the red lines highlight the syntenic PPO gene pairs. P.trichocarpa, Populus trichocarpa; S.purpurea, Salix purpurea; M.domestica, Malus domestica; O. sativa, Oryza sativa

PtrPPO cis-element analysis

To determine the expression pattern of PtrPPOs, the TSS promoter-upstream region (~ 2000 bp) sequences were extracted from the genomic DNA sequence of P. trichocarpa. The cis-elements of the PtrPPOs promoter were analyzed using the PlantCARE database (Fig. 5). The specific functions of these motifs (cis-elements) are annotated in Table S6. A series of cis-acting elements involved in all aspects of poplar development, including palisade mesophyll cell differentiation, seed-specific regulation, cell cycle regulation, endosperm expression, meristem expression, and gibberellin-responsive and auxin-responsive elements, were identified, indicating the crucial role of PtrPPOs in poplar development. For example, the promoter regions of many PPO genes were identified to contain gibberellin-responsive elements, and the PtrPPO5 promoter contained three gibberellin-responsive elements, indicating that this gene is likely involved in gibberellin-signal transduction (Table S7). Moreover, the promoter region of PtrPPO10 contains elements of cell cycle regulation, meristem expression, and endosperm expression, indicating that this gene may be involved in the regulation of poplar growth and development.

Fig. 5
figure5

Cis-element analysis of the PtrPPOs. The relative locations of stress and growth-related cis-elements in the PtrPPO promoter region. Different colors represent different cis-acting elements, and their position corresponds to the corresponding position of the promoter

Furthermore, PtrPPO promoter regions were associated with secondary metabolism processes, including zein metabolism regulation and flavonoid biosynthetic gene regulation. PtrPPO8 contained elements related to the aforementioned processes, suggesting that this gene might be involved in secondary metabolism in Populus. Besides, some stress-related elements were present in the promoter region, including MeJA responsiveness, salicylic acid responsiveness, abscisic acid responsiveness, drought inducibility, defense and stress responsiveness, anoxic specific inducibility elements, and low-temperature responsiveness, which implied that PtrPPOs might also respond to stress. Except for PtrPPO4/6/9/12/18, all other PPO genes contained MeJA-responsive elements (Table S7), indicating that most PPO genes were involved in MeJA signal transduction. Moreover, PtrPPO13 harbored multiple elements associated with stress, including two MeJA-responsive elements, one salicylic-acid-responsive element, four abscisic-acid-responsive elements, and two defense and stress responsive elements (Table S7), indicating that PtrPPO13 may respond to both biological and abiotic stresses.

RNA-seq analysis of Populus PPO genes

To explore the role of PPO genes in poplar growth and development, we analyzed the tissue expression patterns of 18 PtrPPOs in the transcriptomic dataset. First, we found that PtrPPO2 was not detected in all tissues and that it might be a pseudogene or might have a specific spatiotemporal expression pattern (not included in the library). Next, we constructed a dual clustering heat map (sample and gene) to investigate the expression profile of these genes in 15 Populus tissues (Fig. 6a and Table S8). Interestingly, some genes were preferentially expressed in specific plant tissues. Eight genes were highly expressed in young expanding leaves (PtrPPO14/15/18/3/7/16/4/5), two genes in whole suckers and freshly expanding leaves (PtrPPO14/15), five genes in dormant and prechilled buds (PtrPPO18/3/7/4/5), and one gene in mature seeds (PtrPPO9). It was further revealed that most of these genes were found in young plant tissues or seeds. For example, PtrPPO18 was expressed at high levels in young tissues but at low levels in mature tissues (such as mature leaves and petioles).

Fig. 6
figure6

Expression profile of PPO genes under developmental and stress conditions. a Heat maps showing the expression levels of 18 PtrPPO genes in different tissues at different developmental stages based on transcriptome data. b Heat maps depicting the expression levels of 18 PtrPPO genes following drought-, beetle-, and mechanical damage. The color bar represents the range of maximum and minimum values of relative expression in the heat map

To further understand the role of PPO genes in the response of poplar plants to stress, we mapped the expression configurations of the 18 poplar PPO genes following exposure to drought and beetle- and mechanical-damage using the uploaded transcriptome dataset (Fig. 6b and Table S9). The heat map revealed that the expression of PtrPPO9 was significantly induced in leaves in response to drought stress, and all three treatments significantly induced the expression of PtrPPO13. However, the expression of PtrPPO11/14/15 was significantly inhibited under conditions of beetle- and mechanical-damage-induced stress. These results will be helpful for future research on gene function.

Expression profile of Populus PPO genes in different plant tissues and treatments

The level of PPO gene expression in plants is closely related to stress and primarily reflects disease, insect and mechanical damage, and microorganism invasion [11, 22]. Results showed that the expression of five genes, i.e., PtrPPO9/11/13/14/15, was significantly induced or inhibited under conditions of stress. To verify the reliability of PPO gene expression in the transcriptome, the expression of 5 PPO (PtrPPO9/11/13/14/15) genes was investigated using RT-qPCR (Fig. 7a). Consistent with the previous results, most genes were expressed in young tissues. For example, except for PtrPPO13, which was preferentially expressed in mature leaves, the other four genes (PtrPPO9/11/14/15) were expressed primarily in the young leaves. Notably, PtrPPO9/11/13/14/15 were expressed at high levels in leaves (compare to other tissues, such as xylem, phloem, and root).

Fig. 7
figure7

RT-qPCR analysis of the expression profile of PPO genes. a Transcript-level analysis of five PtrPPOs in various tissues. YL, young leaf; AL, adult leaf; OL, old leaf; P, phloem; X, xylem; R, root. Transcript levels of the five PtrPPOs in response to PEG (b), ABA (c), and MeJA (d) were determined using RT-qPCR. Values are means + SE (n = 20). Asterisks denote significant differences: *P ≤ 0.05; **P ≤ 0.01

To further validate whether PPO abundance was affected by abiotic stress and hormone treatment, the expression of 5 PPO members—from among the 18 PtrPPOs genes—was carefully investigated in response to PEG, ABA, and MeJA treatment using qRT-PCR (Fig. 7b–d). When poplar plants were treated with PEG, the expression of the other four genes was significantly induced except PtrPPO11 (Fig. 7b). PEG treatment significantly upregulated the expression of PtrPPO9/13 that peaked 6 h after treatment. PtrPPO14 was significantly upregulated after 3 h of treatment, and its expression level remained at approximately 2-fold. Furthermore, PtrPPO15 was upregulated following PEG treatment, with a maximum expression of approximately 8-fold (compared to pre-treatment levels) after 9 h.

In response to ABA treatment, PtrPPO9/13 showed similar expression patterns, with the expression of both genes significantly induced in response to ABA treatment, peaking after 3 h of treatment (approximately 20- and 15-fold pre-treatment levels, respectively) (Fig. 7c). However, the expression of PtrPPO11/14/15 was significantly inhibited by ABA. In response to MeJA treatment, the expression of PtrPPO13 was significantly induced, reaching a maximum value approximately 5-fold that of pre-treatment expression levels at 6 h of treatment, decreasing to approximately 3-fold at 9 h of treatment (Fig. 7d). In contrast, the expression of PtrPPO11/14/15 was significantly inhibited by MeJA, whereas that of PtrPPO9 was induced at 1 and 3 h but significantly inhibited at 9 and 12 h. Overall, each treatment may differentially induce and/or inhibit the expression of PPO genes in Populus.

Identification and expression pattern analysis of potential upstream transcription factors (TFs) of PtrPPOs

To further understand how PtrPPO9/11/13/14/15 function under stressful conditions, we identified potential upstream TFs of PtrPPOs using bioinformatics (Table S10). The regulatory network generated using Cytoscape (Fig. 8a) revealed that the expression of PtrPPO9/11/13/14/15 might be regulated by 10, 7, 19, 20, and 1 TFs, respectively. These TFs include Dof, ERF, BBR-BPC, MIKC_MADS, AP2, LBD, ARF, bHLH, B3, C2H2, GRAS, GATA, MYB, Nin-like, SBP, TALE, and WRKY family proteins. Moreover, Potri.002G026700, Potri.002G151700, Potri.010G181000, and Potri.014G074200 may simultaneously regulate the expression of PtrPPO9/13/14 genes. We also found that Potri.010G101400 may regulate the expression of three genes PtrPPO13/14/15.

Fig. 8
figure8

Bioinformatic analysis of potential upstream transcription factors (TFs) of PtrPPOs. a Molecular regulatory network analysis of TFs-PtrPPO9/11/13/14/15. The blue circles represent TFs upstream of PtrPPOs. The yellow square represents PtrPPO9/11/13/14/15. Cytoscape was used for visualization. Heat maps depicting the expression levels of potential upstream TFs of PtrPPO9 (b), PtrPPO11 (c), PtrPPO13 (d), PtrPPO14 (e) and PtrPPO15 (f) following drought-, beetle-, and mechanical damage. The color bar represents the range of maximum and minimum values of relative expressions in the heat map

We simultaneously evaluated the expression pattern of potential upstream TFs of PtrPPO9/11/13/14/15 under stress conditions (Fig. 8b–f and Table S11). The potential upstream MIKC_MADS TFs (Potri.002G151700) and bHLH TFs (Potri.003G207200) of PtrPPO9 were significantly upregulated in response to drought, beetle-, and mechanical-damage in poplar leaves (Fig. 8b). In addition, the expression of Potri.003G034200 (Dof family protein), Potri.009G064700 (bHLH family protein), and Potri.004G056900 (Dof family protein) was significantly induced in beetle-damaged leaves (Fig. 8b). When poplar trees were exposed to beetle- and mechanical-damage, the expression of potential upstream ARF TFs (Potri.005G236700 and Potri.012G106100) of PtrPPO11 was significantly induced, and that of the potential upstream Dof TFs (Potri.001G238400 and Potri.007G036400) of PtrPPO11 was significantly inhibited (Fig. 8c).

Exposure of poplar trees to beetle- and mechanical-damage-induced stress resulted in the significant downregulation of the potential upstream TALE (Potri.002G113300), bHLH (Potri.007G023600), Dof (Potri.007G036400), and B3 TFs (Potri.001G322700) of PtrPPO13 (Fig. 8d). Moreover, the expression of the potential upstream TFs of PtrPPO14 was significantly induced by stress, except for ERF TFs (Potri.018G028000) (Fig. 8e). The expression of potential upstream BBR-BPC TF (Potri.010G101400) of PtrPPO15 was significantly upregulated by drought stress in poplar leaves; however, it was significantly inhibited by beetle- and mechanical-damage induced stress (Fig. 8f).

Discussion

As widespread copper metalloenzymes in plants, PPOs play an important role in plant growth, development, and stress tolerance [10, 20, 24]. With the development of whole genome sequencing, an increasing number of PPO genes have been identified in different plants, including apple, litchi, opium, potato, legumes, tea, mulberry, and tobacco [11]. However, the PPO family in the woody poplar plants is yet to be fully understood.

In the present study, 18 PPO genes were identified in poplar plants, and the characteristics of all products after replication, transcription, and translation were analyzed; additionally, their systematic evolution and expression models were constructed. In line with previous gene family studies [32], PPO genes in poplars were designated PtrPPO1 to PtrPPO18 according to their chromosomal locations (Table 1 and Figure S1). Except for PtrPPO10/12, all PtrPPO proteins had three conserved domains (KFDV, tyrosinase, and DWL), which may endow them with similar biological functions. It has been reported that the loss of protein structure could lead to different gene functions [33]. As both PtrPPO10 and PtrPPO12 only had one KFDV domain (Fig. 2d), they were clustered in the same branch (Fig. 1a and 2a), and the length of gDNA and the number of motifs in the protein were significantly lower than those in other genes (Fig. 2c). Furthermore, PtrPPO10 and PtrPPO12 only contained the PPO1_KFDV domain (Fig. 2d), suggesting the two proteins may have similar functions. As shown above, PtrPPO10 and PtrPPO12 cluster together when tissue expression patterns are investigated (Fig. 6a). The two genes have similar tissue expression patterns, and both are expressed at high levels in flowers and seeds, indicating that they may play a role in the formation of poplar flowers and seeds.

Most PtrPPOs have no introns, whereas some have three introns at most (Fig. 2b). Research has shown that introns play essential roles in the regulation of the transcriptome [34]. In order to respond quickly to stress, organisms need to stimulate the expression of genes, and gene structures with a few or no introns contribute to the rapid expression of mRNA [35]. For example, PtrPPO7/8/1 genes lack introns, but contain UTRs; therefore, they can transcribe faster to form mRNA. In many plants, PPO genes respond quickly to both biological and abiotic stresses [11, 24].

We constructed an evolutionary tree from 25 model plants and divided it into 11 sections to further determine the origin and evolution of PPO genes. Consistent with the aforementioned results (Fig. 2a), PtrPPO protein was found in Groups V, VIII, IX, and XI (Fig. 1a). The phylogenetic tree analysis of family genes can clearly describe the evolutionary course of genes [36]. Because Populus (poplar) and Salix (willow) are both members of the family Salicaceae [37], most of their PPO genes are clustered in the same branch (IX). Notably, the PPO gene was not found in Arabidopsis, but four PPO genes were found in monocotyledonous O. sativa (Fig. 1b). It is worth noting that PPO genes are widely distributed in woody plants, especially in P. trichocarpa (18), M. domestica (15), and S. purpurea (10). No gene pairs have been found between Arabidopsis and poplar, whereas multiple gene pairs have been found between apple and poplar and between S. purpurea and poplar (Fig. 4), indicating that considerable differentiation and doubling of PPOs might have taken place during the evolution of perennial woody plants.

The increase in gene family members and the mechanism of genome evolution primarily depend on gene replication events, including tandem and segmented replications [38]. In the current study, 18 PtrPPO genes were unevenly distributed on 19 poplar chromosomes, and half of these PtrPPO genes were located on chromosome 1. This phenomenon of uneven distribution of chromosomes indicates that the change occurred before species differentiation. A total of three pairs of segmented replication genes and four tandem repeat genomes were detected in poplars, indicating that both tandem and segmental repeats contribute to the evolution of PtrPPO genes in Populus (Fig. 3). Previous gene family studies have shown that tandem repeat genes may have similar functions and expression patterns [34, 39]. For example, PtrPPO12/13 has a similar expression level in all tissues, while PtrPPO14/15 show a downward trend under adverse conditions (Fig. 6). Similar expression levels indicate similar functions and structures of tandem repeat genes.

The expression pattern of PPO genes in various tissues has been demonstrated in numerous species [22, 27, 28, 40]. Due to the difference in the number of PPOs in different species, there are no uniform gene expression profiles of PPO genes in plants. According to the RNA-seq data of poplar, some genes are preferentially expressed in young plant tissues and seeds (Fig. 6a), suggesting these tissues are more attractive to intruders. Consistent with the above results, fluorescence quantitative PCR results also showed that the PPO was expressed preferentially in young leaves than in other tissues (Fig. 7a). Research elsewhere has indicated that young leaves have higher PPO activity than old leaves [41].

The expression of PPO genes in plants is closely related to stress and mainly responds to insect and mechanical damage, diseases, and microorganism invasion [11, 22]. Transcriptome dataset analysis revealed that some PPO genes in poplar were significantly induced (PtrPPO 9/13) and inhibited (PtrPPO 11/14/15) by drought. A similar trend was observed using RT-qPCR. PEG and ABA treatments significantly upregulated PtrPPO 9/13 expression (Fig. 7b and c). By analyzing the promoters of PtrPPO 9/13, we found that there were multiple abscisic acid responsiveness elements in these two promoters (Fig. 5). PtrPPO9 was significantly upregulated under conditions of PEG and ABA treatment than under MeJA treatment, indicating that PtrPPO9 had a higher probability of being induced by abiotic stress. In addition, PPO genes in Nicotiana tabacum and Glycine max were significantly induced by ABA [24, 42]. Transcriptome analysis verified that PtrPPO13 was significantly upregulated, and PtrPPO11/14/15 was significantly inhibited in response to mechanical injury and damage caused by the poplar borer (beetle; Fig. 6b). MeJA is involved in signal transduction during insect attack and plant mechanical damage [23, 24]. MeJA responsiveness elements were identified on the PtrPPO13 promoter. Additionally, RT-qPCR revealed that PtrPPO13 was significantly induced by MeJA (Fig. 7d). PtrPPO13 was significantly induced by PEG, ABA, and MeJA treatments, reaching a peak at 3 or 6 h, suggesting that PtrPPO13 might be involved in biological and abiotic stress. Compared with the expression profiles revealed by RNA-seq data, the patterns of qRT-PCR expression were dissimilar. Various reasons might cause the observed differences in expressions.

In recent years, promoters, TFs, and microRNAs (miRNAs) have been found to regulate PPO response to stress in plants [11]. miR528, miR12112, and miR058 from Banana, Salvia miltiorrhiza, and Grapevine can silence PPO genes and participate in cold stress and MeJA response, respectively [23, 43,44,45]. Similarly, TFs play a crucial role in the transcriptional regulation of genes [46]. The potential upstream TFs of PtrPPOs were identified using bioinformatics (Fig. 8a). Compared with the previous results (Fig. 6b), some potential upstream TFs of PtrPPOs were also induced significantly in stress (Fig. 8a-f). Recently, it has been reported that MnMYB3R1 in Mulberry can combine with the MSA element on the MnPPO1 promoter and then regulate MnPPO1 to increase plant drought resistance [28]. In conclusion, PPOs play a critical role in the regulation of stress response.

The finding of the current study allows us to infer the functional roles of PPO genes in poplar plants. Comprehensive analysis is useful to select candidate PPO genes for further functional characterization, while genetic improvement of stress resistance in forest trees provides genetic resources.

Conclusions

To summarize, whole-genome analysis of the poplar PPO family was accomplished, and 18 PtrPPO genes were identified. Bioinformatics and qRT-PCR were then used to analyze the gene structure, phylogeny, chromosomal localization, gene replication, cis-elements, and expression patterns of PtrPPOs. Finally, we found that PPO genes were preferentially expressed in young plant tissues and fruits, and some genes could be significantly induced by PEG, ABA, and MeJA, indicating that PPO plays an integral part in the stress resistance of poplars. This research will help to explore the function of PPO genes in Populus.

Methods

Genome-wide identification and retrieval of PPO genes in P. trichocarpa

The extraction and identification of poplar family members were conducted according to a previously described method [47]. The genome data of A. thaliana (Athaliana_447_TAIR10.fa.gz), O. sativa (Osativa_323_v7.0.fa.gz), and P. trichocarpa (Ptrichocarpa_533_v4.0.fa.gz) were obtained from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) [48]. In addition, BLAST and HMMER were used to identify 18 PPO proteins with conserved structures in poplar plants. Redundant sequences were manually discarded. Furthermore, the conservative structure, molecular weights, isoelectric points, and hydrophilicity analysis of the identified PPOs were conducted using SMART on the ExPasy server (https://web.expasy.org/protparam/) [32].

Evolutionary relationships of PPO genes

A total of 138 PPO proteins from Aquilegia coerulea, Ananas comosus, Amaranthus hypochondriacus, Carica papaya, Cucumis sativus, Eucalyptus grandis, Gossypium raimondii, Kalanchoe fedtschenkoi, Kalanchoe laxiflora, Linum usitatissimum, Musa acuminata, Malus domestica, Mimulus guttatus, Marchantia polymorpha, Oryza sativa, Oropetium thomaeum, Populus trichocarpa, Panicum virgatum, Ricinus communis, Sorghum bicolor, Sphagnum fallax, Solanum lycopersicum, Spirodela polyrhiza, Salix purpurea, and Solanum tuberosum were obtained from the Phytozome database [49]. They all had PPO-KFDV conserved domains. The amino acids of all PPO target sequences were analyzed using ClustalX, and then the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method in MEGA7.0. The accession numbers and gene names are all shown in Table S2.

Analysis of gene and protein structures and motifs

The Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/) [50] was used to analyze the introns, exons, and UTRs of the 18 poplar PPO genes. The conserved motifs and domains of candidate PPO proteins were identified using MEME (https://meme-suite.org/meme/tools/meme) [51] and Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi?) [52], respectively. Finally, the conserved motifs and structures of all PPO proteins in poplar were drawn using TBtools (https://github.com/CJ-Chen/TBtools). Previously described specific parameter settings were used [53].

Chromosomal locations and gene duplication

The chromosomal positions of PtrPPOs were collected from the Phytozome database [49]. MCScanX was used to analyze PPO gene duplication events [54]. TBtools was used to display the locations and collinearity of PtrPPO genes.

Analysis of cis-regulatory elements

The promoter sequences (2000 bp upstream of the start codon) of PPOs were analyzed online using Plant-CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [55], and all cis-regulatory elements related to hormones and stress were identified.

Transcriptomic data sets to analyze the expression patterns of PPOs

To evaluate PtrPPOs gene expression profiles, the publicly available transcriptomic data were obtained from PopGenIE (https://popgenie.org/) [56]. In the current study, transcriptome data were collected from stressed plants (drought, mechanical damage, insect beetle damage) and 15 different plant tissues (non-girdled twigs, dormant flowers, expanded flowers, mature leaves, expanding flowers, whole-sucker suckers, mature petiole, prechilling buds, dormant buds, freshly expanded leaves, non-girdled leaves, girdled leaves, mature seeds, young expanding leaves, and dormant cambium phloem) during the growth and development of P. trichocarpa. The relative expression of PtrPPOs was displayed as a heat map generated using TBtools [53].

Plant materials and treatments

Populus (Populus trichocarpa) plant seeds were acquired from Sichuan Agricultural University trail Populus planting base [48]. One-year-old Populus trichocarpa seedlings were planted in a plastic plot (16.0 h light; 20–25 °C; 70% air humidity) in Wenjiang, Chengdu, China (30°70′ N, 103°85′ E, 537.11 m above sea level). The plants were watered with 1 L Hoagland nutrient solution every 2 weeks for 2 months before treatment [46]. Previously, PEG, ABA, and MeJA treatments have been used to investigate the gene responses to abiotic stresses in plants [48, 57, 58]. Sixty-day-old seedlings were treated with PEG, ABA, and MeJA. At least five biological replicates were used for each treatment. For PEG treatments, similarly grown P. trichocarpa seedlings (40–50-cm height, with 30–35 leaves) were treated with 15% PEG6000 for 0, 1, 3, 6, 9, and 12 h. For phytohormone analysis, similarly grown P. trichocarpa seedlings were treated with a solution containing 200 μM ABA (Sigma-Aldrich, Santa Clara, CA, USA) and MeJA (Sigma-Aldrich), for 0, 1, 3, 6, 9, and 12 h. The leaves were separated from the plant at the different processing time points, rapidly frozen in liquid nitrogen, and stored in an ultra-low temperature freezer (Thermo Fisher Scientific, Waltham, MA, USA).

Poplar tissues and organs were sampled in accordance with a previously described method [48]. Plant tissues and organs were sampled under stress-free conditions; each plant tissue and organ had at least five biological replicates. We simultaneously collected different P. trichocarpa organs and tissues, including young, mature, and old leaves, xylem, phloem, and roots, and immediately immersed them in liquid nitrogen.

RNA extraction and quantitative real-time (qRT-PCR) analysis

Approximately 1 g of tissue was isolated for total RNA extraction. Total RNA was prepared using the plant total RNA extraction kit (Aidlab, Beijing, China) according to the manufacturer’s protocol, and 2 μg of total RNA was reverse transcribed into cDNA using the Tiangen Fast Quant RT Kit (Tiangen Biotech Co. Ltd., Beijing, China). qRT-PCR was conducted as per a previously described protocol [59]. Based on the target gene fragment, Primer Premier 6.0 was used to design primers online; all primers used are listed in Table S1. At least 20 replicates (5 biological replicates × 4 technical replicates) were used per experiment.

Bioinformatic analysis of potential TFs upstream of PtrPPOs

The plant regulatory network database PlantRegMap (http://plantregmap.gao-lab.org/) [60] was used to identify potential upstream transcriptional factors of PtrPPO9/11/13/14/15. The molecular network regulation map of target genes and upstream transcription factors was drawn using Cytoscape [61]. Transcriptome data for upstream transcription factors under stress conditions (drought and mechanical and insect damage) were obtained from PopGenIE (https://popgenie.org/) [56] and the heat maps were drawn using TBtools.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in this article and additional files. The nucleotide and protein sequences of PPO-related genes in Aquilegia coerulea, Ananas comosus, Amaranthus hypochondriacus, Carica papaya, Cucumis sativus, Eucalyptus grandis, Gossypium raimondii, Kalanchoe fedtschenkoi, Kalanchoe laxiflora, Linum usitatissimum, Musa acuminata, Malus domestica, Mimulus guttatus, Marchantia polymorpha, Oryza sativa, Oropetium thomaeum, Populus trichocarpa, Panicum virgatum, Ricinus communis, Sorghum bicolor, Sphagnum fallax, Solanum lycopersicum, Spirodela polyrhiza, Salix purpurea, and Solanum tuberosum are available in the Phytozome v12.1 database (JGI, https://phytozome.jgi.doe.gov/pz/portal.html).

Statistical analyses

Microsoft Excel 2020 (Microsoft Corporation, Redmond, WA, USA) and SPSS v.17.0 (SPSS Inc., Chicago, IL, USA) were used to analyze the experimental data. Both one-way analysis of variance was used to determine the significance of the differences among treatments. Student’s t-test was used to calculate P-values (*P < 0.05; **P < 0.01). The data were normalized, and all samples were normally distributed in terms of homogeneity of variance.

Availability of data and materials

All data supporting the conclusions of this article are provided within the article and its additional files. The genomics sequences data of A. thaliana, O. sativa, and P. trichocarpa are available in the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html). The public RNA-seq data are available on PopGenIE (https://popgenie.org/gene?id=Potri.001G388900).

Abbreviations

CDS:

Coding sequence

AA:

Amino acid

MW:

Molecular mass (kDa)

GRAVY:

Grand average of hydropathicity

PI:

Theoretical isoelectric point

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Acknowledgments

Not applicable.

Funding

This work was supported by the National Natural Science Foundation of China (31870645 to XW; 32101481 to FH), and supported by Sichuan Science and Technology Program (2021YFYZ0032 to XW). Furthermore, we appreciate the China Postdoctoral Foundation (2021 M692331 to FH) for supporting this research.

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FH, YS, QZ, LC, HY, FZ, KZ, XC, JM, JH, and XW conceived and performed the original research project. FH, YS, and QZ performed the experiments. FH, YS, and XW designed the experiments and analyzed the data. FH refined the project and wrote the manuscript with contributions from all authors. FZ and XW supervised the experiments and revised the writing. XW obtained the funding for the research project. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Fang He or Xue-Qin Wan.

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All experiments conducted in this research were in accordance with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The use of P. trichocarpa has been licensed.

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All the authors of this manuscript give their consent to publish the findings in BMC Genomics.

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The authors declare that there are no conflicts of interest.

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He, F., Shi, YJ., Zhao, Q. et al. Genome-wide investigation and expression profiling of polyphenol oxidase (PPO) family genes uncover likely functions in organ development and stress responses in Populus trichocarpa. BMC Genomics 22, 731 (2021). https://doi.org/10.1186/s12864-021-08028-9

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Keywords

  • Genome-wide
  • Populus
  • PPO gene family
  • Expression pattern
  • Stress