Identification and Evolution Analysis of PAL and CHS Genes in 18 Plant Species
Genome-wide identification of PAL and CHS genes in 18 plant species, which not only included major classes like monocots and dicots, but also represent diverse groups such as cereals (Sorghum bicolor, Oryza sativa, Setaria italic and Zea mays), fruits (Vitis vinifera), trees (Populus trichocarpa, Amygdalus persica, Citrus sinensis and Malus pumila), vegetables (Brassica oleracea, Brassica rapa, Cucumis sativus and Lycopersicon esculentum), legumes (Glycine max), Gossypium, Solanum tuberosum and model dicot and monocot species (Arabidopsis thaliana and Brachypodium distachyon). Shown in Table 1, the 13 dicots used in this study have an average genome size of 618.86 Mb compared with that of 780.54 Mb average genome size of the 5 monocots. In addition, higher number of chromosomes were observed in 13 dicots (14.2 in average) compared with that in 5 monocots (9.2 in average). In this study, 135 PAL and 479 CHS genes were identified in the 18 plant species (Table 1 and Table S2). For PAL genes, the maximum number of PAL genes was found in Zea mays (12) and Vitis vinifera (11), respectively. More number of PAL genes were found in monocots (9.4 in average) than that in dicots (6.8 in average). Similar results were also obtained in CHS genes, namely more gene number in monocots (38.2 in average) than that in dicots (22.2 in average). The maximum number of CHS genes was detected in Glycine max (43) and Sorghum bicolor (55) in dicots and monocots, respectively. Notably, 12 CHS and 8 PAL genes were identified in Solanum tuberosum.
Table 1
Genome-wide identification of PAL and CHS genes in 18 plant species.
Species | Chromosomes | Genome size (MB) | Gene nmber | PAL | CHS |
Arabidopsis thaliana | 5 | 119.167 | 39,551 | 4 | 4 |
Brassica oleracea | 9 | 514.431 | 56,687 | 6 | 24 |
Brassica rapa | 10 | 314.865 | 47,942 | 7 | 29 |
Cucumis sativus | 7 | 323.986 | 25,668 | 9 | 10 |
Glycine max | 20 | 984.88 | 71,219 | 7 | 43 |
Gossypium | 26 | 2189.14 | 90,927 | 8 | 22 |
Vitis vinifera | 19 | 427.191 | 76,662 | 11 | 22 |
Malus pumila | 17 | 702.961 | 51,695 | 8 | 29 |
Citrus sinensis | 9 | 323.528 | 45,387 | 4 | 25 |
Solanum tuberosum | 24 | 705.934 | 37,966 | 8 | 12 |
Populus trichocarpa | 19 | 434.29 | 51,717 | 5 | 37 |
Amygdalus persica | 8 | 212.767 | 32,595 | 2 | 14 |
Solanum lycopersicum | 12 | 792.038 | 37,660 | 9 | 18 |
Brachypodium distachyum | 5 | 218.345 | 37,892 | 8 | 21 |
Zea mays | 10 | 2182.61 | 52,470 | 12 | 33 |
Setaria italica | 9 | 441.705 | 35,844 | 10 | 39 |
Sorghum bicolor | 10 | 687.75 | 39,248 | 8 | 54 |
Oryza sativa | 12 | 372.31 | 67,393 | 9 | 43 |
Based on protein sequence similarities, the evolutionary trees of PAL and CHS gene family were constructed using MEGA 7.0 software (Fig. 1). For the PAL family, 135 protein sequences were divided into 8 subgroups (A-H). The members of subgroups A and B were monocots specific, while the others belonged to dicots (Fig. 1A). For the CHS family, 479 CHS protein sequences were divided into six subgroups, of which B and D branches belonged only to monocotyledons, while the other subgroups were common in both monocotyledons and dicotyledon (Fig. 1B).
Molecular Characterization, Chromosomal Location, Gene Duplication, Gene Structure and Motif Analysis of PAL and CHS Genes in Potato
Information of amino acid number, predicted molecular weight, theoretical isoelectric point (pI) and subcellular localization for StuPALs and StuCHSs were shown in Table 2. These StuPALs varied in length from 378 to 706 aa, with Mw ranging from 42.09 to 76.93 kDa, and pI from 5.50 to 6.28. And these genes encode proteins located mainly in cytoplasm (StuPAL4 and StuPAL8), endoplasmic reticulum membrane (StuPAL3, StuPAL5 and StuPAL7), peroxisome (StuPAL2) and plasma membrane (StuPAL1 and StuPAL6). For StuCHS proteins, they varied in length from 248 to 389 aa, with Mw ranging from 27.66 to 42.96 kDa, and pI from 5.38 to 9.57. Five of them were located in cytoplasm (StuCHS1, StuCHS3, StuCHS7, StuCHS11 and StuCHS12), four in endoplasmic reticulum membrane (StuCHS2, StuCHS4, StuCHS5 and StuCHS6), one in mitochondrial matrix space (StuCHS9), one in nucleus (StuCHS8) and one in plasma membrane (StuCHS10).
Table 2
Molecular characterization of StuPAL and StuCHS genes.
Gene name | Gene ID | Transcript | PR | MW | pI | Location | EN | SL |
StuPAL1 | PGSC0003DMG400005492 | PGSC0003DMT400014003 | 650 | 71.58 | 5.50 | chr05: 36342746-36347409 | 2 | plasma membrane |
StuPAL2 | PGSC0003DMG400019386 | PGSC0003DMT400049886 | 689 | 75.46 | 6.28 | chr10: 6301474-6305153 | 2 | microbody (peroxisome) |
StuPAL3 | PGSC0003DMG401021549 | PGSC0003DMT400055488 | 706 | 76.91 | 6.08 | chr09: 5510117-5512923 | 2 | endoplasmic reticulum |
StuPAL4 | PGSC0003DMG402021549 | PGSC0003DMT400055489 | 378 | 42.09 | 6.16 | chr09: 5502194-5505885 | 1 | cytoplasm |
StuPAL5 | PGSC0003DMG402021564 | PGSC0003DMT400055531 | 706 | 76.91 | 6.19 | chr09: 5530471-5533348 | 2 | endoplasmic reticulum |
StuPAL6 | PGSC0003DMG400023458 | PGSC0003DMT400060308 | 703 | 76.93 | 6.07 | chr05: 51694756-51698709 | 2 | plasma membrane |
StuPAL7 | PGSC0003DMG400031365 | PGSC0003DMT400080548 | 694 | 75.65 | 5.97 | chr10: 51926201-51930606 | 2 | endoplasmic reticulum |
StuPAL8 | PGSC0003DMG400031457 | PGSC0003DMT400080765 | 435 | 48.34 | 5.84 | chr03: 17248040-17249552 | 1 | cytoplasm |
StuCHS1 | PGSC0003DMG400001635 | PGSC0003DMT400004133 | 368 | 40.53 | 5.59 | chr01: 87143395-8714521 | 3 | cytoplasm |
StuCHS2 | PGSC0003DMG400008632 | PGSC0003DMT400022254 | 388 | 42.96 | 6.18 | chr12: 58758677-58761269 | 2 | endoplasmic reticulum |
StuCHS3 | PGSC0003DMG400008633 | PGSC0003DMT400022255 | 329 | 36.03 | 8.52 | chr12: 58741442-58742633 | 1 | cytoplasm |
StuCHS4 | PGSC0003DMG400008634 | PGSC0003DMT400022258 | 389 | 42.66 | 5.47 | chr12: 58746401-58747957 | 2 | endoplasmic reticulum |
StuCHS5 | PGSC0003DMG400012670 | PGSC0003DMT400032993 | 248 | 27.66 | 9.57 | chr02: 41086823-41089074 | 2 | endoplasmic reticulum |
StuCHS6 | PGSC0003DMG400016867 | PGSC0003DMT400043447 | 388 | 42.96 | 7.49 | chr12: 58934717-58937241 | 2 | endoplasmic reticulum |
StuCHS7 | PGSC0003DMG400016867 | PGSC0003DMT400043449 | 293 | 31.85 | 5.38 | chr12: 58934717-58937241 | 1 | cytoplasm |
StuCHS8 | PGSC0003DMG400016873 | PGSC0003DMT400043464 | 388 | 42.57 | 7.49 | chr12: 58954773-58956693 | 2 | nucleus |
StuCHS9 | PGSC0003DMG400019110 | PGSC0003DMT400049165 | 385 | 42.17 | 5.96 | chr05: 48886960-48888729 | 2 | mitochondrial matrix space |
StuCHS10 | PGSC0003DMG400027146 | PGSC0003DMT400069814 | 382 | 42.09 | 7.71 | chr05: 48437160-48440351 | 2 | plasma membrane |
StuCHS11 | PGSC0003DMG400029620 | PGSC0003DMT400076178 | 385 | 42.15 | 6.27 | chr09: 58356999-58359082 | 2 | cytoplasm |
StuCHS12 | PGSC0003DMG400029621 | PGSC0003DMT400076179 | 377 | 41.56 | 8.47 | chr09: 58361561-58364070 | 2 | cytoplasm |
| Note: PR: Peptide residues; MW: Molecular weight (kDa); EN: Exon number; SL: Subcellular localization. |
StuPALs and StuCHSs genes were unevenly located on chromosomes and they were distributed on four (Chr3. Chr5, Chr9 and Chr10) and five chromosomes (Chr1. Chr2, Chr5, Chr9 and Chr12), respectively (Fig. 2). StuPALs and StuCHSs genes were mainly distributed on chromosome Chr9 and Chr12, respectively (Fig. 2). Tandem and segmental duplication were the major duplication patterns, which led the expansion of gene families in the process of plant evolution [24, 25]. In this study, tandem duplicated genes of StuPALs (StuPAL3 and StuPAL4) and StuCHSs (StuCHS2, StuCHS3, StuCHS4, StuCHS6, StuCHS7 and StuCHS8) were indicated by yellow blocks in Fig. 2, which suggested that gene duplication played critical roles in the expansion of StuPALs and StuCHSs gene families.
To better explore the structural features of the StuPAL and StuCHS genes, the coding domain sequences and corresponding genomic sequences of the same StuPALs and StuCHSs gene were submitted to GSDS website together to display their exon/intron features. The results revealed the number of introns per gene varied from zero to a maximum of two (Fig. 3). All StuPAL and StuCHS genes had only one or zero intron except StuCHS1, which had two introns. Six StuPAL and nine StuCHS genes had one intron and two StuPAL (StuPAL4 and StuPAL8) and two StuCHS (StuCHS3 and StuCHS7) genes had no intron.
To further understand the potential functions of StuPAL and StuCHS genes, 10 motifs were screened within each protein sequence using the MEME website. The detailed information of predicted motifs was showed in Fig. 4 and Table 3. The length of predicted motifs was 21-50 aa and 8-50 aa for StuPAL and StuCHS proteins, respectively. Among them, motif 1, 2, 3, 4, 10 were detected in all eight StuPAL genes, StuPAL1 and StuPAL4 lacked motif 5 as well as StuPAL4 and StuPAL8 lacked motif 6, 7, 8, 9. Interestingly, the same motif appears more than once in the same StuPAL member, such as motif 1, 4 and 6. The same patterns were also detected in StuCHS proteins. To be noteworthy, only three motifs (motif 2, 4, 10) were screened in StuCHS5 (Fig. 4).
Table 3
Motif detail information of StuPAL and StuCHS genes predicted using MEME.
| Motif | Sequences | Width | Functional domains |
StuPALs | 1 | DYGFKGAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNS | 41 | L-Aspartase-like |
| 2 | KQDRYALRTSPQWLGPQIEVIRAATKMIEREINSVNDNPLIDVSRNKALH | 50 | L-Aspartase-like |
| 3 | ISARKTAEAVDILKLMSSTYLVALCQAIDLRHLEENLKNAVKNTVSQVAK | 50 | L-Aspartase-like |
| 4 | VRSPGEEIDKVFTAMCNGQIIDPLLECLK | 29 | - |
| 5 | GKPEFTDYLTHKLKHHPGQIEAAAIMEHILDGSSYVKAAQKLHEMDPLQK | 50 | L-Aspartase-like |
| 6 | GNGTETCHTLPHSATRAAMLVRINTLLQGYSGIRFEILEAI | 41 | Fumarase/histidase N-terminal, L-Aspartase-like |
| 7 | ASSDWVMDSMSKGTDSYGVTTGFGATSHRRTKNGGALQKELIRFLNAGVF | 50 | Fumarase/histidase N-terminal, L-Aspartase-like |
| 8 | GGFFELQPKEGLALVNGTAVGSGMASMVLFDSNILAVMSEVLSAIFAEVM | 50 | L-Aspartase-like |
| 9 | MAADSLRGSHLDEVKKMVDEFRKPIVKLGG | 30 | - |
| 10 | ANGELHPARFCEKELLRVVDR | 21 | Phenylalanine ammonia-lyase |
StuCHSs | 1 | ITHLVFCTTSGVDLPGADYQLTKLLGLEPSVKRFMMYQQGC | 41 | Chal_sti_synt_N |
| 2 | IGSDPIMNVEKPLFELVFATQTLLPDSEH | 29 | - |
| 3 | GLKVQIHKDTPMLISKNIERILVEAFQPLDISDWNSIFWVS | 41 | Chal_sti_synt_C |
| 4 | DQIELKLGLKPEKLKATRNVLSDYGNMASACVLFVLDEMRKTSIKAGLGT | 50 | Chal_sti_synt_C |
| 5 | PSNCVDQSTYPDYYFRITNSEHKTELKEKFKRMCDKSMIKKRYMHLTEEI | 50 | Chal_sti_synt_N |
| 6 | NPNICEYMAPSLDARQDIVVVEVPKLGKEAAQKAIKEWGQP | 41 | Chal_sti_synt_N |
| 7 | VCFRNPNETELEVLVAQALFSDGASAVI | 28 | Chal_sti_synt_N |
| 8 | TGEGLEWGVLFGFGP | 15 | - |
| 9 | EIRRTQRAMGPATVLAIGTAN | 21 | - |
| 10 | HPGGRAIL | 8 | - |
For StuPAL genes, seven motifs (1-3, 5-8) represented L-Aspartase-like (IPR008948) domain, and motif 10 represented Phenylalanine ammonia-lyase, shielding domain superfamily (IPR023144). While no known domain was detected in motif 4 and 9. Interestingly, Fumarase/histidase N-terminal, active site (IPR022313) domain was also detected in motif 6 and 7. The conserved motifs 1, 5, 6, and 7 represented the Chal-sti-synt-N domain, and motifs 3 and 4 possessed the Chal-sti-synt-C domain and no known domain was detected in the rest motifs among StuCHSs (Table 3).
MicroRNA Targeting Prediction of StuPAL and StuCHS Genes
To explore the potential roles of miRNAs involved in regulation of StuPALs and StuCHSs genes, the software psRNATarget Server was used to predict possible miRNAs based on the genomic sequences of all StuPALs and StuCHSs genes. In total, 68 and 61 putative miRNAs targeting all eight StuPALs and twelve StuCHSs genes, respectively, were detected and constructed the relationship network using Cytoscape software (Fig. 5). We further analysis the regulation network and found that StuPAL1, StuPAL6 and StuPAL7 were targeted by the top three miRNAs. The stu-miR8015-3p targeted StuPAL1, StuPAL2, StuPAL3 and StuPAL4 (Fig. 5A). For StuCHS genes, StuCHS10 was targeted by 23 miRNAs and stu-miR8040-3p targeted StuCHS8, StuCHS10 and StuCHS12. StuCHS6 and StuCHS7 are targeted by some common miRNAs. (Fig. 5B).
StuPAL s and StuCHSs Genes Cis-acting Element Analysis
Gene expression was mainly regulated at the transcriptional level and cis-acting elements regulated the precise initiation and transcriptional efficiency of gene transcription by binding to transcription factors. Cis-acting elements in StuPAL and StuCHS gene promoters were analyzed using the PlantCARE database. In our study, promoter sequences (1500 bp from the coding start) of StuPALs and StuCHSs genes were used to screen putative cis-acting elements, which involved in auxin (TGA-element and AuxRR-core), salicylic acid (TCA-element), abscisic acid responsiveness (ABRE), gibberellin (TATC-box, GARE-motif and P-box), MeJA (CGTCA-motif and TGACG-motif), defense and stress responsiveness (TC-rich repeats), drought-inducibility (MBS), low-temperature responsiveness (LTR), MYBHv1 binding site (CCAAT-box), endosperm expression (GCN4), cell cycle regulation (MSA-like) and the anaerobic induction (ARE). In total, 70 and 116 cis-acting elements were detected in StuPAL and StuCHS gene promoters (Fig. 6). Among them, 3 (StuPAL2)-12 (StuPAL5 and 7) and 4 (StuCHS8) -13 (StuCHS11) cis acting regulatory elements were varied in two gene families. In StuPAL gene promoters, 14 ABREs and 13 AREs counted the top two elements (Fig. 6A). And 21 ABA responsive and 31 MeJA responsive elements were detected in StuCHS promoters, indicating that StuCHS genes may be regulated by the two hormones (Fig. 6B).
Expression Analysis of StuPALs and StuCHSs Genes in Various Tissues of Potato
The expression patterns of genes are usually correlated with their functions. To analysis the expression patterns of StuCHSs and StuPALs genes in various tissues and organs, RNA-Seq data from PGSC were downloaded and analyzed. A heatmap was generated using the FPKM data of all StuCHS and StuPAL genes by the software Mev 11.0 (Fig. 7). Thirteen different tissues and organs were included in the analysis. All StuPALs genes showed high expression levels in all tissues except StuPAL1, which showed very low expression level in all tissues. Notably, StuPAL4 had the highest expression levels in all tissues except in stamens. StuPAL5 had relatively low expression levels in stamens (Fig. 7A). For StuCHS genes, their expression patterns were very diverse (Fig. 7B). StuCHS5, StuCHS9 and StuCHS11 (except in callus and stolons) showed constitutive expression in all detected tissues. StuCHS1 was only expressed in roots and tubers, while StuCHS6 and StuCHS7 were only highly expressed in shoots, sepals and petioles. StuCHS3, StuCHS4, StuCHS8 and StuCHS10 showed little expression in detecting tissues. Highly expressed in shoots, sepals, stolons, petioles, petals and fruits was detected in StuCHS2, while StuCHS12 showed high expression level in sepals, stamens, flowers, petals, carpels and fruit (Fig. 7B).
Expression Analysis of StuPAL and StuCHS Genes in Potato under Environmental Stresses
To further insight into the roles of StuPAL and StuCHS genes in response to abiotic (salt, drought and heat), biotic (PIL, BTH and BABA) and hormone (ABA, BAP, GA3 and IAA) stresses, we analysis the fold changes of FPKM using the expression data obtained from the Spud DB database (Fig. 8). For abiotic stresses, seven of eight StuPAL genes (except StuPAL8) were down-regulated after salt and drought stresses and StuPAL6 and StuPAL7 were strongly up-regulated in response to heat stress (Fig. 8A). The expression patterns were varied in StuCHS genes in response to abiotic stresses (Fig. 8B). StuCHS3 and StuCHS12 were up-regulated (log2 fold change>1) in both salt and drought stresses, while only StuCHS8 was significantly down-regulated in salt stress. StuCHS9 StuCHS11 and StuCHS12 were strongly up-regulated in heat stress. The rest StuCHS gene members have little response (|log2 fold change|<1). For biotic stresses, all StuPAL genes were down-regulated in response to PIL (except StuPAL4 and StuPAL8) and BTH (except StuPAL7) stresses while StuPAL4 and StuPAL6 were significantly up-regulated in response to BABA stress (Fig. 8A). All StuCHS genes were down-regulated in response to BABA and PIL (except StuCHS1) stress, while five and four StuCHS genes were up- and down-regulated in response to BTH stress and the rest three StuCHS genes had no response to BTH stress (Fig. 8B). For exogenous hormone treatments, all StuPAL genes had no express in response to ABA, GA3 (except StuPAL1) and IAA (except StuPAL6 and StuPAL7), while all except StuPAL1 were down-regulated in response to BAP treatment (Fig. 8A). No StuCHS genes were induced by BAP and IAA, while StuCHS11 and StuCHS12 were strongly up-regulated after GA3 treatment. For ABA treatment, five StuCHS genes, namely StuCHS1, StuCHS3, StuCHS8, StuCHS11 and StuCHS12, were up-regulated while StuCHS2, StuCHS4 and StuCHS10 were down-regulated (Fig. 8B).
Anthocyanin content analysis in three colored potato varieties
Three colored varieties of potato were selected in this study. Among them, JYS are purple peel with purple pulp, while both peel and pulp of 1-11 is red and 16-A2 shows yellow both peel and pulp (Fig. 9A). The content of anthocyanins in the peel and pulp were significant difference among the three materials. The anthocyanin content was the highest in JYS peel while it was the lowest in 16-A2 pulp (P < 0.05) (Fig. 9B).
Expression Analysis of StuPAL and StuCHS Genes in Peel and Pulp of Colored Potato
To further analysis the functions of StuPAL and StuCHS genes in anthocyanin biosynthesis pathways, the expression levels of StuPAL and StuCHS genes in three colored potato varieties were detected using qRT-PCR (Fig. 10). For StuPAL genes, StuPAL1-StuPAL8 were mainly expressed in the peel of the three potato varieties, but almost not expressed in the pulp (Fig. 10A). Among them, StuPAL2, StuPAL3, StuPAL4 and StuPAL6 had the highest expression in JYS peel, followed by 1-11 peel, and the lowest expression in 16-A2 peel. The expression patterns of the rest genes are different. StuPAL1, StuPAL5, StuPAL7 and StuPAL8 had the lowest expression in 1-11 peel. Among them, StuPAL1 had the highest expression in JYS peel, followed by 16-A2 peel. The expression levels of StuPAL5 and StuPAL8 in JYS and 16-A2 peel were basically the same, while StuPAL7 had the highest expression in 16-A2 peel, followed by JYS peel. However, the expression of StuCHS genes in the peel and pulp of the three varieties changed greatly (Fig. 10B). StuCHS1, StuCHS4, and StuCHS6 had almost no changes in the three different colored potatoes peel. StuCHS3 was highly expressed in JYS peel, followed by 1-11 peel, and almost no expression in 16-A2 peel. StuCHS5 had the highest expression in JYS peel, and there was little difference between 1-11 and 16-A2 peel. StuCHS7 had the highest expression in 16-A2 peel and almost no expression between JYS and 1-11 peel. However, StuCHS9 and StuCHS11 had the highest expression in 1-11 peel, followed by JYS peel, and almost no expression in 16-A2 peel. Most of the StuCHS genes were expressed at low levels in the pulp, except StuCHS1 was clearly expressed in JYS pulp. StuCHS3, StuCHS9 and StuCHS11 were hardly expressed in the pulp among three potato varieties. The expression of StuCHS5 in JYS, 1-11 and 16-A2 pulp did not change much. StuCHS4, StuCHS6 and StuCHS7 was obviously expressed in JYS and 16-A2 pulp, but almost not expressed in 1-11 pulp. The expression of StuCHS2, StuCHS8, StuCHS10 and StuCHS12 were lower in the three varieties, and the data was not shown.