Whole-genome analysis of CGS, SAHH, SAMS gene families in five Rosaceae species and their expression analysis in Pyrus bretschneideri

Plant materials

The plant material collected from Pyrus bretschneideri Rehd. ‘Dangshan Su’, which is grown on horticulture orchards (Dangshan County, Anhui Province). The 40-year-old Dangshan crisp pear tree has been artificially pollinated. Uniform sized fruits were collected at eight-different development stages: 15 DAF (days after flowering), 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF, and 145 DAF. In addition, flowers, inflorescence stems, leaves, and buds samples were also collected in the same year. All of the samples were stored at −80 °C for further use.

Identification and database search of CGS, SAHH, SAMS family members

The Pyrus bretschneideri and plum (Prunus mume) genomes were downloaded from the Genome Database For Rosaceae (https://www.rosaceae.org/), and the sequence information of strawberry (Fragaria vesca), peach (Prunus persica), and apple (Malus domestica) was downloaded from the Phytozome database (phytozome.jgi.doe.gov/pz/portal.html). The already known Arabidopsis thaliana CGS, SAHH and SAMS gene sequences were used as query (Chiba et al., 2003; Du et al., 2014; Shen, Li & Tarczynski, 2002). Secondly, the HMM (hidden Markov model) was searched through Pfam (http://pfam.xfam.org/) website to obtain the specific characteristic domain of CGS, SAHH, and SAMS families. Then the sequence was compared with BLASTP (E = <1.0E−10) in pear, strawberry, peach, apple, and plum genome database by using BioEdit software, and three gene family members were screened out among five Rosaceae species. Subsequently, SMART (http://smart.embl-heidelberg.de/) and the Pfam database were used to analyze the conserved domains of these genes, and the sequences without conserved domains and redundant sequences were removed (Cheng et al., 2018). Finally, the family members of the five Rosaceae species were identified.

Comparative phylogeny analysis of CGS, SAHH, SAMS gene family

The amino acid and cds sequences of CGS, SAHH and SAMS genes were aligned by using clustalw tool in MEGA5.1 software, and the phylogenetic tree was constructed by Neighbor-Joining (N-J) method (bootstrap = 1,000; substitution model = p-distance) and Maximum Likelihood (ML) method (bootstrap = 1,000; substitution model = WAG). The evolutionary tree of these gene families was constructed between pear, strawberry, peach, apple, and plum.

Analysis of genetic parameters in CGS, SAHH, SAMS gene family

The EXPASY (https://web.expasy.org/protparam/) online tool was used to predict isoelectric point (pI) and molecular weight (Mw). WoLF PSORT (https://www.genscript.com/wolf-psort.html) was used to predict subcellular localization.

Conserved motifs and gene structure analysis of CGS, SAHH, SAMS

The online tool MEME (http://meme-suite.org/tools/meme), set the number of motifs to 20, the Minimum width, and Maximum width to 6 and 200. Finally use the TBTools (Chen et al., 2020) software to draw a conservative motif diagram. The gene structure was analyzed by GSDS (http://gsds.gao-lab.org/).

Chromosomal locations and mode of duplication events

The genomic annotation data of five species were collected for chromosomal locations between three gene families. Finally, chromosomal locations were mapped with MapInspect software. Furthermore, the duplicate_gene_classifier tool of MCScanX was used to identify the duplication events in each species (Wang et al., 2012). In addition, the DnaSP 5.0 software was used to analyze the non-synonymous substitution rate (Ka), synonymous substitution rate (Ks) of duplicated genes.

Promoter cis-acting elements and intraspecific microcollinearity analysis

The 2,000 bp promoter sequences upstream of the three gene family members were obtained from the pear genome database, and PlantCARE online webtool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to analyze the cis-acting elements on the promoter region. Then, TBtools (Chen et al., 2020) software was used to draw the intraspecies microcollinearity map.

Expression patterns and transcriptomic analysis of CGS, SAHH, SAMS gene family

Download the transcriptome data of eight different developmental stages (15 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 79 DAF, 102 DAF, and 145 DAF) of four pear varieties: Yali (P. bretschneideri), Starkrimson (P. communis), Nanguoli (P. ussuriensis) and Kuerlexiangli (P. sinkiangensis) fragrant pear from the NCBI database (Accession no. SRP070620). The expression profiles of CGS, SAHH and SAMS in the development of pear fruit were analyzed.

According to the RNA extraction kit of Tiangen Biotechnology Effective Company, the total RNA of Dangshansu pear fruit is extracted, and then cDNA is synthesized by the PrimeScript^RT reagent Kit with gDNA Eraser (Perfect Real Time) kit produced by the company. The Primer Premier 5.0 software was used to design specific primers. Detailed information about the primers is shown in Table S1. The relative expression levels of CGS, SAHH and SAMS genes in pear fruits were analyzed by qRT-PCR. The experiment uses the CFX96 fluorescent quantitative PCR instrument (Bio-Rad, Hercules, CA, USA), and the reaction system is 10 μL of SYBR® Premix Ex TaqTM II (2×) (TaKaRa, Kusatsu, Shiga, Japan), 2 μL of cDNA, 6.4 μL of water, and 0.8 μL of the upstream and downstream primers. This experiment was conducted with three biological and technical replication for each sample, and the relative expression of genes was calculated by 2^−ΔΔCT method (Livak & Schmittgen, 2001).

Identification and amino acid characteristics of CGS, SAHH, SAMS family members

The members of CGS (6), SAHH (6), and SAMS (28) were identified from five Rosaceae species, which renamed in pear: PbCGS1, PbSAHH1-PbSAHH2, PbSAMS1-PbSAMS6; peach: PpCGS1, PpSAHH, PpSAMS1-PpSAMS4; plum: PmCGS1, PmSAHH, PmSAMS1-PmSAMS4; strawberry: FvCGS1, FvSAHH, FvSAMS1-FvSAMS6; and apple: MdCGS1-MdCGS2, MdSAHH, MdSAMS1-MdSAMS8 (Table 1). The amino acid length of CGS members is between 412–618 aa, most of SAHH is 485 aa; only PbSAHH2 is 502 aa; and the length of SAMS is between 253–448 aa, which has the biggest difference in length among apples. The molecular weights of CGS and SAHH are relatively high, mainly between 53.3–66.4 kDa, PmCGS1 is relatively low, 45.9 kDa; SAMS is mainly in the range of 41.1–44.6 kDa, only FvSAMS3, MdSAMS4, and MdSAMS6 individual members are different. Except for the isoelectric points of PbCGS1, FvSAMS2, and MdCGS2 greater than 7, the other members are all less than 7, indicating that the amino acids are generally acidic. Prediction of subcellular location shows that four members of CGS gene family are found in the chloroplast, PbCGS1 is in the chloroplast or endoplasmic reticulum, and PmCGS1 may be in various positions of cells; 31 members of SAHH and SAMS gene family are in cytoplasmic, MdSAMS4 is shown in the chloroplast or extracellular, while FvSAMS2 and MdSAMS6 are displayed in the chloroplast.

Phylogeny, classification, conservative motifs, and gene structure analysis of CGS, SAHH, SAMS genes

In this study, NJ method was used to construct the phylogenetic tree (Figs. 2A–5A). The conserved motif analysis and gene structure analysis corresponds to the phylogenetic tree constructed by amino acid sequence and cds sequence. The analysis of the SAMS gene family phylogenetic tree showed that the SAMS genes of the five species were mainly divided into four subfamilies, namely Group I, II, III, and IV (Fig. 2A). In addition, FvSAMS2 and FvSAMS3 form independent branches. The SAMS family members formed 10 gene pairs, and only three of them had high bootstrap values (≥99). Twenty motifs of the SAMS gene family were identified by using the MEME tool (Fig. 2B). Except for the two independent branches of FvSAMS2 and FvSAMS3, the C-terminal conserved motifs of the four subfamilies include motif2, motif4, and motif6. Only PbSAMS6 does not include motif3; except for the conservative motifs of MdSAMS4, MdSAMS6, and MdSAMS8 at the N-terminal, most types and distributions are similar. The only PbSAMS6 exists in motif17, and the only FvSAMS2 exists in motif8 and motif20 indicating that they may have specific functions. The SAMS gene family is not all composed of CDS and UTR. Each subfamily and independent branch has UTR-free regions, and 16 of them have UTR regions (Fig. 3D). The C-terminal conserved motifs of the CGS gene family members are all consistent, including motif2, motif1, and motif3. Each member of the N-terminal contains a unique motif, which may contain some other functions; the UTR region and the non-UTR region each account for half (Fig. 4). Among the six members of the SAHH gene family, except for the PbSAHH2 conserved motif, which contains motif6, motif7, and motif8, the remaining conserved motifs are the same. These motifs may give the member unique functions. UTR region and the non-UTR region also account for half (Fig. 5). Taken together, the conserved motif composition and gene structure characteristics of each family are very similar, which supports their close evolutionary relationship and the reliability of constructing evolutionary trees.

Meanwhile, we also constructed the ML phylogenetic tree (Fig. 6), and it is clear from the comparison between the two trees that the gene pairs formed due to high homology are basically consistent, indicating that the construction of the phylogenetic trees are accurate. Only in the CGS gene family, the homology of PbCGS1, FvCGS1 and MdCGS2 were not quite consistent in the two phylogenetic trees. Since the NJ phylogenetic tree is a distance method, which indicates the degree of similarity between sequences. While the amino acid sequences of FvCGS1 and MdCGS2 are closer, so they form a gene pair. ML phylogenetic tree indicates a series of features that change over time, and from an evolutionary perspective, it is possible that PbCGS1 and MdCGS2 are more closely related. Thus leading to a less consistent situation of these three genes.

Characteristic domain analysis

Further analysis of the characteristic domains of five Rosaceae species from three families (Fig. S1) (Cheng et al., 2019). The result shows that the characteristic domain of CGS family members is Cys_Met_Meta_PP (PF01053). As can be seen from Fig. S1, according to Pfam’s annotation information (http://pfam.xfam.org/family/Cys_Met_Meta_PP), this domain uses pyridoxal phosphate (PLP) as a cofactor to participate in cysteine and methionine amino acid metabolism (Ferla & Patrick, 2014). PLP is the active form of vitamin B6. PLP is a versatile catalyst, as a coenzyme in many reactions, including decarboxylation, deamination, and transamination reactions (Hayashi, 1995; John, 1995; Kirsch & Eliot, 2004). The characteristic domain of SAHH family members is AdoHcyase_NAD (SM00997), according to Pfam’s annotation results (http://pfam.xfam.org/family/PF00670). S-adenosine-L-homocysteine acid hydrolase (AdoHcyase) is an activated methyl cycle enzyme responsible for the reversible hydration of S-adenosyl-L-homocysteine to adenosine and homocysteine. AdoHcyase is a ubiquitous enzyme that binds to and requires NAD⁺ as a cofactor (Sganga et al., 1992). The characteristic domains of SAMS family members mainly include S-AdoMet_synt_N (PF00438), S-AdoMet_synt_M (PF02772), and S-AdoMet_synt_C (PF02773). The three domains of S-adenosylmethionine synthetase have the same alpha + beta folding. S-adenosylmethionine synthase is an enzyme that catalyzes the formation of S-adenosylmethionine (AdoMet) from methionine and ATP (Horikawa et al., 1990). AdoMet is an important methyl donor for demethylation and a propylamine donor in polyamine biosynthesis. These characteristic domains indicate their important functions and further prove the accuracy of the identified members. However, the three members of FvSAMS2, FvSAMS3, and PbSAMS6 are missing one domain, which may change their functions.

Chromosome location and gene duplication analysis of CGS, SAHH, SAMS family members

To understand the distribution of CGS, SAHH and SAMS families on chromosomes, the chromosome locations of five species were analyzed (Fig. 7). Except for PbSAHH1, all members of CGS, SAHH and SAMS gene family members were located on chromosomes. In Pyrus bretschneideri, eight members are distributed on chromosomes 4, 12, 13, 16, and 17. In Fragaria vesca, eight members were mainly distributed on chromosome 6, and the rest on chromosomes 1 and 4. The 11 members of Malus domestica were evenly distributed on chromosomes 4, 9, 12, 13, 16, and 17. In Prunus mume, six members are distributed on chromosomes 1, 2, 4, and 8. In Prunus persica, six members are distributed on chromosomes 1, 3, 6, and 7.

Subsequently, the evolutionary relationship analysis of these three gene families and the chromosome locations, it is found that five gene pairs have different modes of duplication events. They are all shown in Fig. 7 and each pair is connected together with a dotted line. The analysis showed that they have a high percentage of segmental duplication, indicating that the amplification of SAMS gene family through a segmental duplication event. According to Table 2 and Fig. 8, Ka is less than Ks and the peak value is not 1. Also we used functional motifs for analysis and comparison (Table S2), and found that the calculated Ka and Ks values of the two results are very close, and the results are consistent. It indicates that the gene has purification and selection effects, and the structure and function are not seriously affected.

Intraspecies microcollinearity

In order to further analyze the collinearity relationship of CGS, SAHH and SAMS gene families in pear, and we identified seven pairs of collinearity genes in a pear that have high homology, indicating that may be due to similar functions (Fig. 9). Respectively: Pbr008602.1/Pbr001686.1, Pbr018549.1/Pbr037756.1, Pbr026061.1/Pbr006002.1, Pbr026061.1/Pbr006707.1, Pbr018549.1/Pbr037756.1, Pbr037756.1/Pbr006707.1, Pbr006002.1/Pbr006707.1.

Identification of cis-acting elements analysis

To better understand the promoter characteristics of members of the CGS, SAHH and SAMS gene families in pear, we analyzed their cis-acting elements (Fig. 10). Among these nine genes, only PbSAMS3 contains MRE (light response element), indicating that only the expression of this member may be regulated by light. These members also contain elements related to defense and abiotic stress, such as TC-rich repeats (defense and stress response), MBS (drought induction), LTR (low-temperature response). Except for PbSAMS2 and PbSAMS6, all other members contained at least one element of MBS or LTR, indicating that PbCGS, PbSAHH, PbSAMS are closely related to pear drought and low-temperature stress. We also found many CGTCA-motif, TCA-element, ABRE elements and ERE elements related to MeJA, SA, ABA and ethylene hormone response. All of the nine members contained ABRE elements, a total of 32, which are the most abundant cis-acting elements, indicating that they have the most extensive response to abscisic acid. The cis-acting elements TCA-element and ERE related to SA and ethylene were identified in six different members. CGTCA-motif, a cis-acting element related to MeJA, was identified among the seven members, a total of 14, which is the second most abundant cis-acting element. In addition to these elements, we also found AC elements that bind to MYB, which are related to lignin synthesis (Cao et al., 2016). These results indicate that PbCGS, PbSAHH and PbSAMS not only play a role in the synthesis of lignin, but also participate in the light response process, abiotic stress, and hormone response.

Comparative phylogenetic analysis and function prediction

In this study, 10 species of CGS, SAHH, and six species of SAMS were constructed to form an interspecies complex phylogenetic tree (Fig. 11), and the members of the CGS, SAHH and SAMS families in pear were studied. We constructed 10 species of CGS, SAHH, and six species of SAMS to form an interspecific complex phylogenetic tree, and studied the members of the CGS, SAHH and SAMS families in pear. PbCGS1 and AtCGS are very close to the branch of the phylogenetic tree. In previous studies, it was found that overexpression of AtCGS significantly increased Met and SMM. CGS is the rate-limiting enzyme in this metabolic pathway (Gakière et al., 2002). PbCGS1 may also have a similar role.

Numerous studies have found that down-regulating PvSAHH1 will increase SAH levels and reduce the SAM/SAH ratio, thereby reducing the accumulation of lignin (Bai et al., 2018). PvSAHH1 is in the same sub-branch as PbSAHH1 and PbSAHH2, indicating that these two genes may have this function. However, PbSAHH1 and PbSAHH2 are not in the same gene pair, and their functions may be slightly different. Four members of the SAMS family have been identified in Arabidopsis thaliana. Among these four members, AtSAMS3 shows different functions from other members. The reduction of AtSAMS3 results in the decrease of lignin content in plants, indicating that AtSAMS3 plays a role in lignin biosynthesis (Shen, Li & Tarczynski, 2002). The SAMS members in pear are evenly distributed near AtSAMS3, such as PbSAMS1, PbSAMS2, PbSAMS5, etc., indicating that these members may participate in lignin biosynthesis.

Expression profile analysis of PbCGS, PbSAHH and PbSAMS genes

PbCGS1 expressed higher in the first three periods (15 DAF, 30 DAF, and 55 DAF) in Nanguoli, but lower in the other three pear. PbSAMS2 was also only expressed higher in the first three periods in Yali (Fig. 12). The expression levels of PbSAHH1 and PbSAHH2 were lower in the first three periods of Kuerlexiangli, but higher in the other three pear. The expression of PbSAMS4 was only low in the first three stages of Starkrimson. The expression levels of PbSAMS5 and PbSAMS6 in the corresponding period are not consistent with the changing trend of lignin synthesis and stone cell development, indicating that there is no connection with it. These identical genes have different expression in different varieties of pear fruit. By promoter analysis predicts that these members are closely related to drought stress. It may be different due to the differences between geographical, cultivation and environmental conditions, and it is inconsistent in pear fruit (Arzani et al., 2008). The expression levels of PbSAMS1 and PbSAMS3 in Yali, Starkrimson, Nanguoli and Kuerlexiangli were higher in the first three stages, but lower in the four stages after flowering. This is consistent with the trend of lignin synthesis and stone cell development (Cai et al., 2010; Cao et al., 2016; Cheng et al., 2016). These two members may be involved in lignin synthesis and stone cell development.

Analysis of PbCGS, PbSAHH and PbSAMS genes expression patterns in Dangshan Su Pear

In order to further understand the different functions of the three gene families in pear, we studied the expression patterns of their different tissues (Fig. 13). All members have shown higher expression levels in stems, and three members of PbSAMS1, PbSAMS2 and PbSAMS5 have the highest level in flowers. PbCGS1 is only highly expressed in stems. PbSAHH1, PbSAHH2, PbSAMS2, PbSAMS4 and PbSAMS5 are mainly expressed in flowers and stems. PbSAMS1 is mainly expressed in flowers, stems and leaves. PbSAMS3 has high expression levels in flowers, stems, leaves and buds. PbSAMS6 is abundantly expressed in flowers, stems and buds, but hardly expressed in leaves. The content of stone cells is a key factor affecting the quality of pear fruit. The change of lignin content is closely related to the changes in stone cell content. Therefore, we studied the expression patterns of these nine genes in Dangshan Su pear at eight different development stages by qRT-PCR (Fig. 13). The expression levels of PbCGS1 was the highest in 15 DAF, and the expression levels were low in the remaining seven periods, indicating that they may play an important role in this period. The expression level of PbSAMS5 was the highest in 15 DAF and 145 DAF, and the expression level was lower in the middle stages of development. PbSAHH2 and PbSAMS2 have high expression levels in the early and later stages of development, but moderate expression levels in the middle stages of development. The expression levels of PbSAMS3, PbSAMS4, and PbSAMS6 were only highest in the first and middle stages of development but were not high in the other stages. The expression of PbSAHH1 and PbSAMS1 is the highest at 55 DAF, which is consistent with the changing trend of lignin content in pear fruits (Cai et al., 2010; Jin et al., 2013), indicating that these two genes may be involved in the synthesis and regulation of lignin in pear fruits.