Comparative genomic analysis of the PAL genes in five Rosaceae species and functional identification of Chinese white pear

Plant materials and treatments

The buds, stems, leaves, flowers, roots and fruits were collected from 60 years old pear trees, which managed on a farm in Dangshan, Anhui, China. Uniformly sized fruits were collected at eight time points: 15 DAF (days after flowering), 39 DAF, 47 DAF, 55 DAF 63 DAF, 79 DAF, 102 DAF and 145 DAF. All of the fruit samples were stored at −78 °C for further use.

To investigate the effect of hormone treatments on the gene expression levels of genes related to lignin biosynthesis pathway in pear fruit, we seected pests-free of the pear trees of same age and plant height. The concentration of the hormone treatment (the 0.5 mmol/L abscisic acid (ABA), 0.5 mmol/L methyl jasmonate (MeJA), or 0.2 mmol/L salicylic acid (SA)) were sprayed onto the fruits 39 DAF (Cheng et al., 2019). All samples were treated for 3 h under the same conditions. The pear flesh was weighed about 100 g and frozen at −20 °C for 24 h. The lignin content was measured using the Klason method (Cai et al., 2010).

Acquisition and identification of PAL family members

In this study, we have identified the number of PAL members in five Rosaceae plants. Pear genome database was obtained from (http://gigadb.org/dataset/100083) (Wu et al., 2013). The sequence information of Prunus mummer (mei), Malus domestica (apple), Prunus persica (peach) and Fragaria vesca (strawberry) gene were obtained from the Phytozomes database (https://phytozome.jgi.doe gov/pz/portal.html) (Jung et al., 2014). Initially, we acquired the Hidden Markov Model (HMM) profile of PAL proteins from the Pfam database (http://pfam.sanger.ac.uk/). Subsequently, we utilized the HMM profile as a query to identify all PAL-containing sequences by searching against the three of Rosaceae species genomes (E-value = 0.001). Then, all candidate PALs are validated using Pfam (http://pfam.xfam.org/) (Punta, 2015) and SMART database (http://smart.embl-heidelberg.de/) (Letunic, Doerks & Bork, 2012) to confirm that they contain core domains. Finally, we removed all potentially redundant PAL sequences according to the results of the sequence alignments.

Conserved motif, cis-element and feature analyses of the PALs

The online analysis tool ExPASy (http://web.expasy.org/compute_pi/) is used to predict the isoelectric point (pI) and protein molecular weight of (kDa) of each PAL based on their amino acid sequences. Prediction of subcellular localization was performed using the online tool MBC (http://cello.life.nctu.edu.tw/). Phylogenetic trees were constructed by the N-J method (bootstrap = 1,000) in MEGA5.0 software (Tamura et al., 2011). Analysis of exons and introns were carried out using the gene structure display server (GSDS) program (Liu et al., 2016). Conserved protein motifs were confirmed by MEME (http://meme-suite.org/) (Bailey et al., 2015), which following parameters: the maximum number of motifs is 20, and the base length is between 6 and 200.

The 2,000 bp promoter sequence of the PbPAL family members were obtained from the genome database of ‘Dangshan Su’ and then the online software PLANT CARE database was employed to analyze the cis-acting elements in the promoter regions (Lescot et al., 2002).

Chromosomal locations and Ka (nonsynonymous)/Ks (synonymous) analysis

The chromosomal locations of the PALs in five Rosaceae plants were obtained from genome annotation documents. The data were then plotted using the Circos software (Krzywinski et al., 2009). The duplication events were categorized into segmental and tandem duplication (Cao et al., 2018). Ka and Ks were calculated by DnaSPv5.0 software (Wang et al., 2010). Sliding window analysis was also carried out using this software.

RNA extraction and qRT-PCR analysis for PbPALs

Extraction of total RNA from the fresh pear fruit was performed using a Plant RNA Isolation Kit (Tiangen, China) for qRT-PCR analysis, and the fresh pear fruit here is not from the material used for lignin determination. Next, DNA was reverse transcribed from 1 µg of RNA using the transcriptase M-MLV system (Tiangen, Beijing, China) according to the manufacturer’s instructions. Primers (Table S1) were designed for real-time quantitative PCR (qRT-PCR) using the Beacon Designer 7 software. Tubulin (GenBank accession no. AB239680.1) (Wu et al., 2013) was used internal reference. The transcript levels were measured using a CFX96 Touch™ Real-Time PCR Detection System (BIO-RAD). The total volume of the reaction mixture was 20 µL, consisting of 10 µL of SYBR Premix Ex Taq II (2x), 2 µL of template cDNA, 0.8 µL of the forward and reverse primers and ddH₂O up to 20 µL. The relative expression levels of the genes were calculated using the 2^−ΔΔCT method.

Arabidopsis transformation

The full-length CDS of PbPAL1 (GenBank: MF346686) and PbPAL2 (GenBank: MF346687) were cloned from pear (Table S2). The correct pMD18-T-PbPAL plasmid and pCAMBIA1304 (GenBank: AF234300.1) vector plasmid were digested by restriction endonuclease Bgl II and Spe I (Takara, Japan) (Table S3), respectively. Subsequently, the recombinant eukaryotic expression plasmid pCAMBIA1304-PbCPAL was constructed and successfully obtained by ligation with T4 DNA ligase. Transformation of recombinant plasmid pCAMBIA1304-PbPAL into Agrobacterium tumefaciens EHA105. The A. tumefacien culture at 28 °C in medium with recombinant plasmid pCAMBIA1304-PbPAL. The bacteria were suspended in infection buffer (0.02% Silwet L-77, 1/2 MS, 5% sucrose). The OD₆₀₀ value of the infection solution was approximately 0.7–0.8, which is acceptable for subsequent infection.

The seeds of A. thaliana were sterilized (75% ethanol for 1 min, 10% sodium hypochlorite for 13 min). After 4 washes with sterile water, the seeds were evenly sown on MS solid medium plates containing hygromycin. After approximately 15 days, seedlings with 4 true leaves were transplanted into nutrient soil for further cultivation.

Selected pCAMBIA1304-PbPAL plants and wild type plants of some lotus leaves were grown for approximately 20 days. At the same time, leaf of DNA was extracted and tested by PCR with gfp specific primers (Table S4).

Determination of lignin content in A. thaliana

Dried mature stems were collected after removal of the leaves. The lignin levels of wild-type and overexpression plants were determined using the acetyl bromide method (Anderson et al., 2015). The lignin content was expressed as a percentage (calculated lignin content/calculated dry weight of test sample × 100%) (Tian et al., 2017).

Histochemical staining of A. thaliana inflorescence stems

Inflorescence stem segments of 60-day-old transgenic T₃ generation and wild type A. thaliana plants in the same position were collected (the sections were from the bottom approximately 4 cm of the inflorescence stems). The samples were fixed in 50% (v/v) ethanol, 100% (v/v) glacial acetic acid and 95% (v/v) methanol solution overnight and then embedded in paraffin for lateral and transverse sectioning at a thickness of 4 µm using a pathology slicer (RM2016). Plant tissue sections are placed in the dye solution (1% toluidine blue and Wiesner reagent) for about 2–5 min, washed with water, and the slices are placed in the oven and baked. Transparent sealing with neutral gum, and then directly observed with a microscope (Pradhan & Loque, 2014). Photographs were taken under a binocular microscope.

Collection and identification of PALs in five Rosaceae plants

Based on the HMM sequence on the Pfam website (http://pfamxfamorg/) and BLASTP strategies, PAL family members were identified from five Rosaceae species. The target sequence was compared with the DNAMAN software in the genome database, then remove repetitive redundant sequences. Finally, in our study, we identified 16 non-redundant and complete PALs in five Rosaceae species (Table 1). The correspondent proteins displayed that their lengths, molecular weights, isoelectric points (pI), were within the ranges of 414–753 amino acids, 44.42–87.75 kDa, 5.79–8.79, respectively (Table 1).

Conserved motifs and gene structure of PAL family members of five Rosaceae species

To investigate the evolutionary relationships of the PAL family of five Rosaceae species, we constructed a phylogenetic tree using the MEGA5.0 (Fig. 1A). Phylogenetic analysis of PbPALs revealed that the existence of highly differentiated PALs in P. bretschneideri and some other Rosaceae plants, which the 16 PALs were clustered into three major clades. Conservative gene structures may provide a record of key events in the evolution of genes. Furthermore, PALs structure analysis also supported clustering of occurrence groups. We found that in the same subfamily, the structure of PAL is usually very similar (Fig. 1B). But sometimes there are special phenomena, for example, in Cluster II members, the results shown that FvPAL2 structure is longer and contains more than one exon and intron, while FvPAL1 only contains three exons. Besides, the number, length and location of exons and introns are also different in PAL. In this study, we found that most members of PALs in five Rosaceae species contain two or three exons, which means that these genes are highly conserved during evolution.

To better understand the structural diversity of PALs, we captured twenty conversed motifs in PAL with the PAL protein sequences using MEME software (Fig. 1C). The conserved motif analysis of PALs proved the reliability of the phylogenetic relationship. Moreover, our results also suggested that most of PAL proteins have similar motifs in the same subfamily. Besides, the number of motifs involved in PAL protein sequence was quite uncertain. Coincidentally, motifs 1, 2, 3, 7 and 19 were found in all PAL protein sequences of five Rosaceae species. However, some of the motifs were found to be unique to a subfamily. For example, motif 20 only was found in Cluster I. PbPAL3 had fewer motifs, indicating the PAL domain may be incomplete.

Chromosome location and gene duplication event analysis of PAL members family in five Rosaceae plants

To clarify the distribution of PAL family members on the chromosomes of five Rosaceae species. According to the genome information of each species, and we constructed a chromosomal location map (Fig. 2). The PALs are randomly distributed on 13 chromosomes. Two genes each are located on one chromosome in strawberry and plum blossom. Three genes each are located on one chromosome in P. brestschneideri. Two chromosomes containing three genes in P. persica. Four out of the 13 chromosomes harbored MdPALs, with 2 (chromosomes 1and 8) possessing one MdPAL and 2 (chromosomes 4 and 12) possessing two MdPALs.

Segmental or tandem duplication is the main way to increase the number of family members in plants. In order to further explore the driving forces of PAL evolution, we calculated the rate of nonsynonymous/synonymous substitution (Ka/Ks) among five gene paris. Five pairs of gene duplication events were found in sixteen PALs of five Rosaceae species (Fig. S1). Generally, Ka/Ks>1 indicates positive selection and accelerates evolution; Ka/Ks<1 indicates functional constraints of negative selection. Our results showed that all Ka/Ks pairs of PALs were less than 1 (Table 2), which illustrates that they have undergone strong evolutionary selection, and their functions have not been seriously differentiated. Except MdPAL3/MdPAL6 belonged to tandem duplication, the others were segmental duplication, which indicated that the expansion of PAL family of five Rosaceae species was mainly due to segmental duplication events.

Promoter analysis of PALs in pear

To further understand the regulation mechanism of PbPALs expression, we predicted possible cis-acting elements using PLANT CARE online software (Table S5 and Fig. 3). It was found that the promoter of PbPALs contained two types of stress response regulatory elements, such as MBS and LTR repetitive sequences, which responds to drought induction, and cold stress, respectively. Among which four kinds of hormone regulatory elements: ERE, ABRE, CGTAC-motif and TCA-element were associated with ethylene, ABA, MeJA and SA responses respectively. In addition, two members of the PbPALs family contain the MRE light-responsive element, which hinted that expression of PbPALs were closely related to light. Furthermore, we found that PbPAL1 and 3 gene contains at least one AC element, AC element can activate lignin monomer synthesis gene by binding with MYB transcription factor (Patzlaff, 2010). Therefore, we proposed that expression of PbPALs are closely related to lignin formation.

Phylogenetic analysis of PALs in pear and other plants

Previous research shown that NnPAL1 as an ancient member of the PAL family, and was found to be a polybasic origin in the evolution of PAL in angiosperms (Wu et al., 2014; Wu et al., 2017). To investigate the phylogenetic relationships of PbPALs with other plants and bacterias, which a neighbor-joining tree was created. The phylogenetic tree clustering results showed that PALs of fifteen species could be divided into three well-supported families (Fig. 4). Previous works have shown that the PAL family was divided into a subfamily of A. thaliana, which was consistent with our classification results (Barros et al., 2016). During the evolution of PAL, the recurrence of specific pedigrees occurred in A. thaliana, P. trichocarpa and Selaginella moellendorffii. This is supposed to be a universal phenomenon that promotes the diversity of polygenic families. In this study, the PbPALs were intimately related to dicotyledon plant PAL and belongs to the group. However, the three PbPALs were aggregated with each other and form a different subgroup. Interestingly, just as the results of PbPALs classification are resemble, most of plant PALs are clustered by species, and PALs are in one species are closer to each other than their homologues in another. Based on this evidence, PAL diversity occurs independently in each species. In addition, we found PtPAL2 and BoPAL2 are closer to tyrosine ammonia-mutase (TAM) and histidine ammonia-lyase (HAL). Based on peptide sequence similarity, we speculated that PtPAL2 and BoPAL2 may could encode putative ammonia-lyases.

Expression profiles of pear PALs at different tissues and developmental stages of fruits

The potential functions of gene families can be probed by means of gene expression analysis (Cao et al., 2016). To further describe the function of the pear PALs, and comparative gene expression analysis was carried out in different tissues or organs (leaves, stems, flowers, roots and buds) (Fig. 5). The results showed that transcript levels of PbPAL1 and 2 were higher in lignified tissues (roots and stems) than in less lignified tissues (leaves, buds and flowers) (Fig. 5A). Therefore, PbPAL1 and 2 are highly expressed in stems and roots, and we conjectured that they may be involved in lignin biosynthesis in pear. While expression level of PbPAL3 was relatively low in different tissues. These results suggest that different PbPALs may play key roles in the development of specific tissues.

The content of stone cell is an important factor affecting the quality of pear fruit. As one of the main components of stone cell wall, lignin synthesis directly affects the formation of stone cells rich in pear fruits (Cai et al., 2010; Jin et al., 2013b). Moreover, the change of lignin content is also closely related to the change of stone cell content. Afterward, the expression profiles of these PbPALs at different the stages of fruit development were also surveyed by using qRT-PCR (Fig. 5B). Previous studies showed that the content of stone cell and lignin in pear fruit first increase and then decrease during fruit development, reaching the peak at 55 DAF (Cai et al., 2010). It is noteworthy that the expression levels of PbPAL1 and 2 were proporational to the content of stone cell and lignin in pear fruits, indicating that these genes might be related to lignin aggregation and stone cell formation in pear fruits. This study implying that PbPAL1 and 2 are closely related to lignin synthesis and stone cell development. While PbPAL3 was highly expressed at the 79, 102 and 145 DAF, indicating that this gene might play important roles in the mature stage of pear fruit development.

Differential expression PbPALs under hormonal treatments

Previous studies have shown that the expression of PALs are subjected to abiotic stress (Scott et al., 2004). However, information on PALs involvement in pear hormone response is limited. Previous studies have found that spraying exogenous hormones on the pear fruits can regulate stone cell development and lignin synthesis in pear fruits to a certain extent (Yang et al., 2015). We through the analysis of cis-acting elements in promoters of PbPAL family members, and found that most of the promoters of PbPALs contain a variety of biological or abiotic stress-related elements (Table S5). Consequently, we hope to further study whether the hormones involved in these stress responses (SA, MeJA and ABA) could alter the expression of these genes (Fig. 6). After ABA treatments, the expression of PbPAL1 was obviously induced, while the expression of PbPAL2 was reversed, and the expression level was significantly inhibited. Interestingly, the expression of PbPAL3 was induced at 1 and 3 h of treatment, but inhibited at 2 h, which the lowest expression level was found in 2 h of treatments (Fig. 6A).

In the MeJA-treated pear fruit, PbPAL2 and PbPAL3 showed the same trend, and were inhibited in 1 h and 3 h of treatments. After 2 h of treatments, they were significantly induced and the expression level reached peak. However, the expression level of PbPAL1 showed an obvious opposite trend. The expression of PbPAL1 was induced at 1 and 3 h of treatments, and the expression level reached peak at 3 h after treatments. After 2 h of treatments, the expression level was significantly inhibited (Fig. 6B).

The response patterns of PbPALs to SA can be divided into two categories, including inhibiting gene expression and inducing gene expression. SA inhibited the expression of PbPAL1 and PbPAL2, which was the lowest at 1 h. The other PbPAL3 was induced by SA and peaked at 1 h with the prolongation of treatments time and the induction degree decreased (Fig. 6C).

Determination of lignin content in transgenic A. thaliana of PbPALs

To further determine the role of PbPAL genes in lignin synthesis, we obtained transgenic A. thaliana plants overexpressing these genes. Firstly, we constructed an eukaryotic expression vector (Fig. 7A). The DNA of the transgenic strain was amplified by GFP specific primers on pcambiA1304 vector (Fig. 7B). The successful cloning of the target fragment of approximately 700 bp indicated that the foreign gene has been successfully integrated into the A. thaliana genome (Fig. 7C). Subsequently, we successfully obtained three T₃ generation transgenic lines of PbPAL1 and PbPAL2. We determined the lignin level in A. thaliana inflorescence stems and leaves by acetyl bromide method (Fig. 8). The results showed that that the lignin content in inflorescence stems of transgenic plants of PbPAL1 (12.42%) and PbPAL2 (12.17%) was significantly higher than that of the wild-type plants (10.47%) (Fig. 8A). In addition, we determined that the lignin content in the leaves of transgenic PbPAL1 (7.15%) and PbPAL2 (7.01%) plants was also higher than that in wild-type A. thaliana (6.18%) (Fig. 8B). Our works demonstrated that both PbPAL1 and 2 genes were involved in plant lignin synthesis.

Lignin staining analysis

We next wanted to directly observe the distribution of lignin in inflorescence stems of transgenic A. thaliana plants. Thus, cross-sections of inflorescence stems of wild-type and transgenic plants were stained with phloroglucinol to identify possible changes in the content and/or distribution of lignified tissues. The Wiesner (phloroglucinol-HCl) staining results showed that the strongest staining of the xylem and intervascular fibers were observed in the stems of PbPAL1 and PbPAL2 transgenic A. thaliana than in wild-type plants (Fig. 9I). Furthermore, toluidine blue staining was used to examine the cell wall of a cross-sectional area of the A. thaliana pedicel (Fig. 9II). The cell wall thickness of PbPAL1 and PbPAL2 transgenic plants increased significantly. These two dyeing results showed that PbPAL1 and PbPAL2 can increase lignin synthesis. This is consistent with many previous studies, which PAL is related to the degree of lignification of plants (Chong et al., 2018).