Identification, characteristics and phylogenetic analysis of the PP2C gene family in Apocynum.
In this study, we used BLASTp to identify 68 members of the AvPP2C gene family in Apocynum venetum genome and 68 members of the AhPP2C gene family in the Apocynum hendersonii genome, respectively, using the sequences of 80 AtPP2C proteins from Arabidopsis thaliana as a reference. We used TBtools to map the positional distribution of the PP2C genes on chromosomes of Apocynum venetum and Apocynum hendersonii respectively, which were named AvPP2C1-AvPP2C68 and AhPP2C1-AhPP2C68 according to their chromosomal positions(Fig. 1). Except for AhPP2C68 which was not assembled, the remaining 68 AvPP2C genes and 67 AhPP2C genes were located on 11 chromosomes, respectively. There were fewer genes on chromosomes 4, 8 and 10.
We analyzed the physicochemical properties such as molecular weight (MW), theoretical isoelectric point (pI), and subcellular localization of the 68 AvPP2C proteins and 68 AhPP2C proteins (Table S1). The 68 AvPP2C genes encoded proteins were located at lengths ranging from 278-1086, theoretical isoelectric points ranging from 4.38-9.78, and molecular weights ranging from 30369.58-123024.02 Da. The 68 AhPP2C genes encoded proteins with lengths ranging from 138-1092, theoretical isoelectric points ranging from 4.62-9.8, and molecular weights of 15540.2-123582.74 Da. The results of subcellular localization show that PP2C genes have the ability to appear in various areas of the cell, including the nucleus, chloroplast, cell wall, cell membrane, cytoplasm, mitochondria, and peroxisome. Some specific PP2C genes, such as AvPP2C9, 10, 16, 19, 20, 21, 29, 31, 35, 38, 47, 53, 62 and AhPP2C9, 10, 21, 29, 31, 35, 37, 38, 39, 48, 53, 54, 58 are predicted to be present in multiple cellular structures.
To investigate the phylogenetic relationship between Apocynum venetum, Apocynum hendersonii and Arabidopsis thaliana PP2C genes, a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on 80 AtPP2C genes of Arabidopsis thaliana, 68 AvPP2C genes of Apocynum venetum and 68 AhPP2C genes of Apocynum hendersonii (Fig. 2). Based on phylogenetic analysis, the PP2C genes in Apocynum were categorized into 13 subgroups (A-L). These subgroups contained 9, 4, 4, 8, 7, 6, 4, 6, 3, 2, 2, 7, 2 AvPP2C genes and 9, 4, 4, 9, 7, 6, 3, 6, 3, 2, 2, 7, 2 AhPP2C genes, with similar groupings to those found in Arabidopsis. Notably, PP2C proteins from both Apocynum and Arabidopsis were present in each subfamily, suggesting a common ancestor. Interestingly, AvPP2C3, AvPP2C7, AvPP2C31, AvPP2C61, AhPP2C3, AhPP2C7, AhPP2C31, and AhPP2C60 of Apocynum did not cluster with any other group. The evolutionary tree also showed that genes in Apocynum venetum and Apocynum hendersonii were tightly clustered, indicating high homology and similar gene functions.
Gene structures and conserved motifs analyses of the AvPP2C genes and the AhPP2C genes
Based on their phylogenetic relationships, the structures and conserved domains of the AvPP2C and AhPP2C genes were studied. The gene structures include positional information such as coding sequence (CDS), untranslated regions (UTR), introns and PP2C structural domains(Fig. 3A and C). Genes within the same subgroup share similar gene structures, but there are variations in the length and arrangement of their CDSs and UTRs. For instance, all PP2C genes in group C contained UTR and CDS regions, but their lengths varied. AvPP2C42 had the lengthiest UTR region, while 16 genes in Apocynum venetum had no UTR regions and 14 genes in Apocynum hendersonii had no UTR regions. AvPP2C39 and AhPP2C40 had the highest number of CDS regions, totaling 16. After examining the number of introns, it was found that three AvPP2C genes and four AhPP2C genes in Apocynum had no introns, while the remaining 129 PP2C genes contained introns.
In order to study the structure and function of PP2C proteins and their evolutionary relationships, the conserved domains of AvPP2Cs proteins and AhPP2Cs proteins were analyzed using the online MEME website. The analysis revealed 15 different conserved domains, which were given the names motif 1 to motif 15(Fig. 3A and B, Table S2). The results demonstrated that PP2C proteins in the same subgroups possess a similar distribution that may be linked to their functions. Motif 1 (except AvPP2C62, AhPP2C67, AhPP2C68), motif 2 (except AvPP2C31, AvPP2C44, AhPP2C45, AhPP2C31), and motif 3 (except AhPP2C68, AhPP2C19, AvPP2C19, AvPP2C48) were present in all subgroups. Motifs 6, 7, 11, 13 and 15 were absent in subgroups C and D, while motif 5 was only present in subgroups C and D, except AhPP2C68. Motif 9 and 10 were present only in subgroup D, and motif 11 was present only in subgroup E, but not in other groups.
Cis-element analysis of the PP2Cs promoter in Apocynum venetum and Apocynum hendersonii
The cis-acting elements were predicted in the promoter regions of Apocynum venetum and Apocynum hendersonii using analysis through the PlantCARE online website pairs and visualized using TBtools software, both of which retrieved a total of 68 cis-acting elements. Among them, there were 32 light-responsive elements, 13 phytohormone-responsive elements, 12 stress-responsive elements, and 11 elements related to plant growth and development (Fig. 4, Table S3). It was found that all the promoters of AvPP2Cs and AhPP2Cs contained light-responsive elements, phytohormone-responsive elements, and stress-responsive elements. However, elements associated with plant growth and development were missing in AvPP2C18, AvPP2C33, AvPP2C59, AvPP2C61, AvPP2C65, AhPP2C18, AhPP2C19, AhPP2C32, AhPP2C58, AhPP2C60, and AhPP2C66. Box 4 was identified as the most abundant light-responsive element, with 263 in Apocynum venetum and 290 in Apocynum hendersonii. Among the phytohormone response elements, a large number of abscisic acid response elements (ABRE) and AAGAA-motifs were found, with AvPP2Cs having 148 ABREs and 132 AAGAA-motifs, and AhPP2Cs having 141 ABREs and 129 AAGAA-motifs. The ethylene response element (ERE) was also found in large numbers, with AvPP2Cs having 147 and AhPP2Cs having 156. The stress-responsive elements MYB and MYC were found to be the most abundant with 247 and 235 in Apocynum venetum and 242 and 238 in Apocynum hendersonii, respectively. These elements are involved in drought and ABA-induced responses. Another stress-responsive element, ARE, was found to be essential for anaerobic induction, with 133 in Apocynum venetum and 130 in Apocynum hendersonii. The large number of these elements suggests that most PP2C genes may respond to various stresses and are closely related to plant stress resistance.
Collinear analysis and evolutionary analysis of AvPP2Cs proteins and AhPP2Cs proteins
The expansion of gene families in the genome is predominantly caused by tandem and segmental duplications. To gain insight into the PP2C gene family, 68 AvPP2C genes and 68 AhPP2C genes were analyzed for duplication (Fig. 5A and B, Table S4). It was discovered that in Apocynum venetum, one tandem duplication pair was located on chromosome LG06 (39/40), whereas 17 pairs of segmental duplication genes were unevenly distributed on chromosomes other than LG08 and LG10. In Apocynum hendersonii, there were 2 tandem replication gene pairs located on chromosome LG06 (38/39, 40/41), and 17 fragment replication gene pairs were unevenly distributed on 9 chromosomes except for LG08 and LG10. These findings suggest that the evolution of PP2Cs in Apocynum was primarily driven by segmental duplication events, as the number of segmental duplication gene pairs was significantly greater than that of tandem duplications.
Furthermore, the study investigated the homology between Apocynum venetum, Apocynum hendersonii, and Arabidopsis thaliana to understand the evolutionary relationship of PP2C genes(Fig. 5C, Table S5). The results indicated that there were 84 homologous gene pairs between Apocynum venetum and Arabidopsis thaliana, and 82 homologous gene pairs between Apocynum hendersonii and Arabidopsis thaliana. Among these pairs, Apocynum venetum and Apocynum hendersonii had the highest number of homologous gene pairs on chromosome LG06, with 13 pairs each. These findings suggest that Apocynum venetum and Apocynum hendersonii may have a high degree of homology with Arabidopsis.
Tissue-specific expression of AvPP2C genes and AhPP2C genes in different tissues
To gain a better understanding of the function of PP2C genes in the growth and development of Apocynum venetum and Apocynum hendersonii, we analyzed the expression patterns of PP2C genes using RNA-Seq data of roots, stems, and leaves of both species(Fig. 6). Most of the PP2C genes were expressed in all tissues, except for four genes (AvPP2C19, AvPP2C38, AhPP2C39, and AhPP2C68), which were barely expressed. In Apocynum venetum, AvPP2C47 was highly expressed in roots, stems, and leaves. AvPP2C9, AvPP2C24, AvPP2C49, AvPP2C60, and AvPP2C68 were moderately expressed in roots; AvPP2C13 and AvPP2C60 were highly expressed in stems; AvPP2C13 was highly expressed in leaves, and AvPP2C7 and AvPP2C60 were moderately expressed in leaves. In Apocynum hendersonii, AhPP2C29 and AhPP2C67 were highly expressed in roots, while AhPP2C24, AhPP2C13, AhPP2C49, AhPP2C48, and AhPP2C59 were moderately expressed in roots. AhPP2C9, AhPP2C13, AhPP2C59, and AhPP2C48 were highly expressed in stems, and AhPP2C13, AhPP2C48, and AhPP2C59 were highly expressed in leaves.
Relative expression changes of AvPP2C genes and AhPP2C genes under NaCl treatment
In order to study the expression of AvPP2C genes and AhPP2C genes under salt stress conditions and to identify important PP2C genes related to salt tolerance. Based on phylogenetic analysis and homology of known PP2C genes in Arabidopsis thaliana, five genes in subgroup D and two genes in subgroup G were selected in Apocynum hendersonii and their expression patterns in different tissues of Apocynum venetum under salt stress were investigated by qRT-PCR(Fig. 7).
The results showed that the AvPP2C1 gene was down-regulated in roots after NaCl stress, with the lowest expression at 50 mM concentration. It was up-regulated in leaves and phloem, with the highest expression at 200 mM and 100 mM concentrations, respectively, reaching 2.14-fold and 1.40-fold of CK. AhPP2C1 gene was up-regulated in leaves and increased with increasing NaCl concentration; it showed down-regulation in bast and decreased with increasing NaCl concentration. AvPP2C2 gene showed up-regulation under NaCl stress, in which the expression of the gene in leaves varied the most with NaCl concentration, which was 10.44 times that of CK. The expression trend of AvPP2C2 in root, xylem and phloem was the same, and all of them reached the maximum expression at 100 mM concentration. While the AhPP2C2 gene was down-regulated in roots and up-regulated in leaves after NaCl stress, there was little change in the xylem and phloem, but the expression reached the maximum at 50 mM concentration in all. AvPP2C6 gene did not change significantly in roots, but showed up-regulation in leaves and xylem phloem, and it is noteworthy that its expression in leaves increased with the increase of NaCl concentration and reached the maximum at 200 mM, which was 13.55 times that of CK. And AhPP2C6 was different from AvPP2C6 in that it showed down-regulation in roots. The expression of AvPP2C14 was different in roots, leaves, xylem and phloem, with the highest in leaves and the lowest in roots; after NaCl stress, the AvPP2C14 gene showed a tendency of increasing and then de-creasing with the increase of NaCl concentration, and its expression reached the maximum in all tissues at the concentration of 50 mM. While the expression of AhPP2C14 did not change significantly under NaCl treatment, it only showed up-regulation in leaves, and the expression of the gene was the highest at 50 mM concentration, and its expression gradually declined with the increase of concentration. AvPP2C24 gene had the highest expression in leaves and showed up-regulation in all tissues after NaCl stress. AhPP2C24 gene was the same as AvPP2C24 gene in that the expression of AhPP2C24 gene was up-regulated in all tissues after NaCl stress, except in phloem at 50 mM concentration. The difference was that the expression of AhPP2C24 gene changed more significantly in leaves after NaCl treatment. Both AvPP2C27 and AhPP2C27 genes showed up-regulation after NaCl treatment, but the expression of AvPP2C27 gene in roots and leaves increased with increasing NaCl concentration, while the expression of AhPP2C27 gene in leaves reached a maximum at 50 mM concentration and then decreased with increasing concentration. The expression of AvPP2C41 and AhPP2C42 genes in roots increased with increasing NaCl concentration, AvPP2C41 gene showed down-regulation in leaves at 100 mM and up-regulation at 50 mM and 200 mM concentrations. While AhPP2C42 gene showed up-regulation after NaCl stress, the expression was maximum in leaves at 50 mM concentration, which was 8.20 times of CK, and then decreased with the increase of NaCl concentration.