Identification and characterization of IbOPRs
Initially, the BLAST and HMMER search methods were used to identify putative OPR genes in sweetpotato, followed by further confirmation using the Pfam website. This approach resulted in the identification of nine IbOPR genes in the sweetpotato genome. The chromosomal location of these IbOPR genes was determined, showing that the nine identified IbOPR genes were unevenly distributed across LG3, 4, 7, 12, and 14 (Fig. 1). These nine genes were named IbOPR1-9 according to their position within the Linkage Groups (LGs).
Our analysis revealed that the length of IbOPR proteins varied slightly, with the shortest protein, IbOPR9, encoding 318 amino acids, and the longest protein, IbOPR4, encoding 442 amino acids. The molecular weights of the proteins ranged from 34.97 kDa to 44.15 kDa. The isoelectric point ranged from 5.43 (IbOPR9) to 8.78 (IbOPR2), with eight proteins being acidic and one protein being basic. The scores indicated that all IbOPR proteins were hydrophilic (GRAVY < 0) (Table S1). These findings provide a theoretical basis for further research into the purification, activity, and function of IbOPR proteins.
Furthermore, the predicted subcellular localization of the IbOPR proteins showed that the IbOPR2, IbOPR3, and IbOPR4 proteins were located in the peroxisome, while the IbOPR1, IbOPR5, IbOPR6, IbOPR7, IbOPR8, and IbOPR9 proteins were found in the cytoplasm (Table S1).
Phylogenetic analysis of OPR proteins
To elucidate the taxonomic and evolutionary connections within the IbOPR family, we utilized MEGA7.0 to construct an NJ phylogenetic tree. The tree consisted of 9 IbOPRs, 13 OsOPRs, 3 AtOPRs, 8 ZmOPRs, 4 SiOPRs, and 48 TaOPRs, as depicted in Fig. 2. The phylogenetic tree classified the 85 OPRs into five different groups. For reference, the names and protein sequences of OPRs from different species are provided in Table S2.
The results showed that the 85 OPR proteins were classified into five groups: Group I contained 16 OPR proteins, Group II contained 15, Group III contained 17, Group IV had 33, and Group V had 4. Group IV was the largest, and Group V was the smallest. Monocot species had OPR members in all five groups, while dicot species were only clustered in Groups I and II, consistent with a previous study [9]. Within the IbOPR proteins, a distinct grouping was observed. IbOPR1, IbOPR5, IbOPR6, IbOPR7, and IbOPR8 were classified under Group I and were found to cluster together with AtOPR1 and AtOPR2 in the same clade. On the other hand, IbOPR2, IbOPR3, IbOPR4, and IbOPR9 belonged to Group II and exhibited a close relationship with AtOPR3.
Gene structure and conserved motif analysis of IbOPR
Typically, genes clustered together in a subgroup have a similar structure. Our study found that the OPRs in Group I contained four introns, while Group II consisted of OPRs with three to five introns (Fig. 3a, and Fig. 3c). Different subgroups differed in the quantity and length of introns and exons. The OPR gene family in sweetpotato underwent loss and gain of introns and exons, particularly in Group II. Our results indicate that the exon-intron ratio of the IbOPR genes has mainly remained consistent throughout their evolutionary history.
To identify the conserved motifs of OPR proteins in sweetpotato, we used MEME online software combined with TBtools. Motifs 1-10 were identified as potential motifs (Fig. 3b, and Table S3). These motifs ranged in size from 21 to 50 amino acids, with 7 to 9 motifs found in IbOPR family members. The majority of the orthologous proteins in the same group shared comparable motif members. In Group I, for example, all IbOPRs possessed Motifs 1-9 except IbOPR8, which lacked Motif 8. Except for IbOPR9, which lacked Motif 9 and 10, all IbOPRs in Group II possessed Motifs 1-5 and 7-10. Notably, each group has its own specific motif, for example, Motif 6 in Group I and Motif 10 in Group II. Overall, our findings confirm the classification of IbOPR proteins and show that motifs in these proteins are conserved.
Collinear analysis, gene duplication, and Ka/Ks analysis
Four IbOPR genes (IbOPR5/IbOPR6, IbOPR6/IbOPR7, and IbOPR7/IbOPR8) were clustered into three tandem duplication events on LG12. Additionally, 2 WGD (whole genome duplication) or segmental duplication events involving 4 IbOPR genes (IbOPR3/IbOPR4 and IbOPR1/IbOPR5) were also identified with MCScanX methods (Fig. 4). These events suggest that tandem duplication, WGD or segmental duplication played an important role in the expansion of the IbOPR gene family. To understand the mechanism of gene divergence and evolutionary pressure, we also determined the Ka/Ks ratios (Table S4). A Ka/Ks ratio greater than 1 indicates positive selection, making the gene susceptible to nonsynonymous mutations. If the ratio equals 1, it means neutral selection, while a ratio less than 1 suggests purifying selection and preferential occurrence of synonymous mutations. All Ka/Ks values were less than 1, suggesting that the IbOPR genes underwent purifying solid selection during evolution.
Cis-element analysis in the promoter of IbOPR genes in sweetpotato
Cis-elements are important elements located upstream of the gene start codon that play a crucial role in gene function involved in plant development and stress response. We extracted 2000 bp upstream sequences of IbOPR genes in sweetpotato and performed cis-element analysis. The elements were grouped into five categories: core/binding, development, light, hormone, and abiotic/biotic factors (Fig. 5 and Table S5). All nine IbOPR genes had many core promoter elements and binding sites, such as the TATA box, CCAAT box, AT-TATA box, W box, and MYB recognition site. Light-responsive elements, such as Box4, GT1-motif, G-box, and TCT-motif, were found in most IbOPR genes. Moreover, some development-related elements were found in IbOPR genes. For example, MSA-like, which is related to cell cycle regulation, was found only in IbOPR3; the CAT-box, which is associated with meristem formation and cell division, was found in IbOPR1 and IbOPR7; the O2-site, which is associated with zein metabolism, was found in IbOPR6 and IbOPR9. In addition, hormone-responsive elements were abundant in IbOPR genes, including the ABA-responsive elements ABRE and AAGAA-motif; MeJA-responsive elements CGTCA-motif and TGACG-motif; SA-responsive element TCA-element; GA-responsive elements F-box, P-box, and TATC-box; ETH-responsive element ERE; and IAA-responsive elements AuxRE, AuxRR-core, and TGA-element. In addition, some abiotic-responsive elements, such as drought and salt-responsive elements, including MBS, Myb, MYC, anaerobic induction element ARE, and low temperaturee-responsive element LTR, were found in most IbOPR genes. Overall, these results indicate that IbOPR genes are involved in regulating plant growth, development, and stress adaptation in sweetpotato.
Cloning and sequence analysis of IbOPR
According to the reference CDS of the nine IbOPR genes (Table S6), using “Haida HD7791” and “Haida HD7798” cDNA as templates, IbOPR primers were used for PCR amplification (Table S7). The electrophoresis bands were consistent with the expected sizes of the target fragments (Fig. 6). IbOPR2, IbOPR3, IbOPR6, and IbOPR7 were eventually cloned. The coding region sequences of IbOPR2, IbOPR3, IbOPR6, and IbOPR7 from “Haida HD7791” and “Haida HD7798” were obtained by sequencing and were 1203, 1200, 1134, and 1137 bp, encoding 400, 399, 377, and 378 amino acids, respectively (Table S6). The CDS similarity between “Haida HD7791” and “Haida HD7798” for IbOPR2, IbOPR3, IbOPR6, and IbOPR7 were found to be 98.25%, 99.92%, 95.06%, and 98.50%, respectively (Fig. S1). The protein sequence similarity values for IbOPR2, IbOPR3, IbOPR6, and IbOPR7 between “Haida HD7791” and “Haida HD7798” were 96.75%, 99.75%, 92.06%, and 98.68%, respectively (Fig. 7).
Subcellular localization of the IbOPR2 and IbOPR3 proteins
Previous studies have shown that JA is synthesized in peroxisomes, and our subcellular localization prediction indicated that IbOPR2 and IbOPR3 were located in peroxisomes. To further verify the subcellular localization of IbOPR2 and IbOPR3, the enhanced green fluorescent protein eGFP was fused to the N-terminus of IbOPR2 and IbOPR3, respectively, and the subcellular localization of the fusion proteins was analyzed by confocal laser scanning microscopy after transient expression in tobacco protoplast cells. Characteristic cytoplasm and nucleus staining were observed when eGFP was expressed alone (Fig. 8a-d). Likewise, the eGFP-IbOPR3 fusion protein showed diffuse cytoplasm and nucleus staining (Fig. 8i-l), indicating that IbOPR3 is localized in the cytoplasm and nucleus instead of peroxisomes. However, cells expressing the eGFP-IbOPR2 fusion exhibited punctuate staining in the cytoplasm (Fig. 8e-h), indicating the association of IbOPR2 with organellar structures. The fluorescence of eGFP-IbOPR2 was found to co-localize with that of the red fluorescent protein mKATE fused to the peroxisomal targeting signal SKL (Ser-Lys-Leu), which is present in hydroxypyruvate reductase, an endogenous enzyme of peroxisomes[32, 33]. The co-localization of IbOPR2 with a bona fide peroxisomal targeting signal indicates that IbOPR2 is also localized in peroxisomes.
Subcellular localization of the IbOPR6 and IbOPR7 proteins
Our subcellular localization prediction found that IbOPR6 and IbOPR7 were located in the cytoplasm. To further validate the result, the green fluorescent protein GFP was fused to the C-terminus of IbOPR6 and IbOPR7, respectively, and the subcellular localization of the fusion proteins was analyzed by confocal laser scanning microscopy after transient expression in tobacco protoplast cells. When GFP was expressed alone, characteristic staining patterns were observed in the cytosol and nucleus (Fig. 9a-c). Interestingly, the IbOPR6-GFP and IbOPR7-GFP fusion proteins exhibited similar staining patterns, showing diffuse distribution in both the cytoplasm and nucleus (Fig. 9d-f and Fig. 9g-i). These results provide further evidence that IbOPR6 and IbOPR7 are indeed localized in the cytoplasm and nucleus.
Expression analysis of IbOPR genes in response to salt stress
To examine the biological function of four IbOPR genes in response to salt stress, we analyzed the expression levels of these genes in the salt-tolerant variety “Haida HD7791” and the salt-sensitive variety “Haida HD7798” under 200 mM NaCl treatment by qRT‒PCR (Fig. 10 and Table S8). IbOPR2, IbOPR3, and IbOPR7 were not significantly induced by salt stress in “Haida HD7798” but were significantly influenced by 2-, 2- and 2-fold in “Haida HD7791” at 6 h (p < 0.05), respectively. Additionally, the expression of these three genes was significantly higher in “Haida HD7791” than in “Haida HD7798” at 0 h and 6 h (p < 0.05). On the other hand, IbOPR6 was significantly induced by salt stress in “Haida HD7798” at 48 h but was significantly repressed in “Haida HD7791” at 12 h, 24 h, 48 h, and 72 h (p < 0.05).