Systematic Analysis of Stay-Green Genes in Six Ipomoea Species Reveals the Evolutionary Dynamics, Carotenoid and Anthocyanin Accumulation, and Stress Responses of Sweet Potato

Abstract

Background/Objectives: Stay-green proteins (SGRs) play a vital role in regulating plant chlorophyll degradation and senescence. However, this gene family has not been explored in Ipomoea species and sweet potato. Methods: A total of 19 SGR family genes (SGRs) were identified using Basic Local Alignment Search Tool (BLAST) methods. The proteins’ physiological properties, evolutionary and phylogenetic relationships, conserved domain and motifs, gene structures, collinearity, and promoter cis-elements were systematically analyzed. Moreover, expression patterns and protein interaction network analyses were performed for sweet potato. Results: In this study, we identified 19 SGRs in six Ipomoea species. These SGRs were divided into four subgroups according to their phylogenetic relationships. Domian analysis revealed that SGRs had the conserved “stay-green” domain. Gene structure analysis showed that SGRs had similar structures. The collinearity analysis revealed that the SGRs originated from two genes, with one gene undergoing duplication during evolution history; moreover, the SGRs experienced rearrangement throughout the evolutionary process in the Ipomoea species. Cis-elements related to pigment biosynthesis and hormone and stress responses were found. In addition, expression pattern analysis showed that IbSGRs, especially IbSGR1, IbSGR2, and IbSGR3, might play an important role in pigment accumulation. The SGRs could also respond to stress responses (i.e., cold, drought, and salt) and take part in hormone crosstalk (i.e., abscisic acid (ABA), methyl jasmonate (MeJA), salicylic acid (SA)). Conclusions: Taken together, the findings of this study provide new insights for further understanding the functions of SGRs and candidate genes for pigment accumulation and stress tolerance in sweet potatoes.

1. Introduction

Chlorophyll is one of the most vital pigments on earth and a key component of photosynthesis [1]. Chlorophyll metabolism is a complex and coordinated process completed by a series of proteins that directly affect the life activities of plants, such as leaf senescence and fruit ripening [2,3]. However, excess chlorophyll generates reactive oxygen species (ROS) that are harmful to plants [3]. Thus, chlorophyll metabolic processes must be finely controlled. Stay-green proteins (SGRs) play a central role in the regulation of plant chlorophyll degradation and senescence, which are relatively conserved in higher plants [4,5]. The SGR superfamily is divided into two subfamilies, including the SGR subfamily and the SGR-like subfamily (SGRL) [6]. So far, there has been genome-wide identification of some of the SGR family genes (SGRs) in different plant species. For example, two, three, and three SGRs were identified in rice [7], Arabidopsis [8], and tomato [9], respectively. CsSGR1, CsSGR2, and CsSGRL were identified in tea [10]. Four candidate SGRs were identified in melon (Cucumis melo L.) [11]. Five SGRs were found in each of the genomes of the kiwifruit species [Actinidia chinensis (Ac) and Actinidia eriantha (Ae)] [6]. However, SGRs have not been identified in sweet potato or other Ipomoea species. The overexpression of the SGR gene causes reductions in chlorophyll contents and cell death in the model species Arabidopsis [12] and rice [4,13,14]. In rice, the overexpression of OsSGRL reduces the levels of chlorophyll and accelerates its degradation in dark-induced senescence leaves [7]. In fall fescue, the SGR family gene FaNYE1 might play a significant regulatory role in chlorophyll degradation during senescence [15]. MsSGR-RNAi transgenic plants enhanced the forage quality of Alfalfa [16]. In addition to the leaves, chlorophyll also acts in other organs. In Arabidopsis, SGR-mediated chlorophyll degradation is critical for detoxification during seed maturation [17]. Additionally, SGRs were observed to enhance chlorophyll breakdown in the ripening processes of both tomato and pepper [18,19].

According to previous reports, SGRs usually act as regulators in carotenoid accumulation. For example, SlSGR1 directly interacts with a key carotenoid biosynthesis enzyme protein SlPSY1 to regulate lycopene and β-carotene accumulation in tomato [20]. In addition, SGRs also play pivotal roles in stress response. The natural mutation of OsSGR enhanced resistance to rice sheath blight [21]. The loss of the expression of CsSGR (Cucumis sativus) improved disease resistance [22]. The overexpression of the CfSGR1 and CfSGR2 genes enhanced the stress tolerance of transgenic Arabidopsis [23]. The plant hormone abscisic acid (ABA) plays a critical role in leaf senescence and chlorophyll degradation [24]. In Arabidopsis, ABA-responsive-element (ABRE) binding transcription factors (TFs), ABF2, ABF3, ABF4, and ABSCISIC ACID INSENSITIVE 3 (ABI3) could directly bind the promoter of SGR1 and activate its expression [24,25]. In tomatoes, SlABI5 and SlABI5-LIKE could bind to the SlSGRL promoter and activate its expression in ABA-induced chlorophyll degradation [26].

Ipomoea is one of the largest genera in the Convolvulaceae family. Among them, sweet potato (Ipomoea batatas (L.) Lam. [2n = B₁B₁B₂B₂B₂B₂ = 6x = 90]) is an important high-yield, nutritious, hexaploidy storage root crop worldwide [27,28]. It can provide carbohydrates, proteins, dietary fiber, vitamins, and other bioactive compounds, including carotenoids and anthocyanins, and has therefore been considered a vital crop for food and nutrition security [29,30,31,32]. The color of sweet potato flesh ranges from white to cream, yellow, orange, pink, and even purple [33]. Orange-fleshed cultivars, in particular, contain high levels of β-carotene, which could be used to combat vitamin A deficiency, and purple-fleshed cultivars are rich in anthocyanin, which has some beneficial properties such as anti-oxidant, anti-inflammatory, anti-tumor, and anti-mutagenic effects for preventing diabetes, cardiovascular disease, colon cancer, and other diseases [34,35,36,37]. Chlorophyll and carotenoid are two main photosynthetic pigments in plants that play various roles in plant growth and development [38]. Carotenoid accumulation is usually accompanied by the process of chlorophyll degradation [39]. Previous studies have shown that IbSGR1 might participate in carotenoid accumulation in sweet potatoes. The IbNAC29-IbMYB1R1-IbAITR5 module down-regulates the expression of IbSGR1 to positively regulate carotenoid accumulation [40]. However, the functions and molecular mechanism of SGRs are still unclear in sweet potato and other Ipomoea species.

In this study, we identified three, three, three, three, three, and four SGR genes (SGRs) in the six Ipomoea species (i.e., Ipomoea aquatic, I. aquatic; Ipomoea cairica, I. cairica; Ipomoea nil, I. nil; Ipomoea triloba, I. triloba; Ipomoea trifida, I. trifida and Ipomoea batatas, I. batatas), respectively. We identified and systematically analyzed the protein’s physiological properties, evolutionary and phylogenetic relationships, conserved domains and motifs, gene structure, collinearity, and promoter cis-elements in six Ipomoea species. Their expression patterns associated with carotenoid and anthocyanin contents and stress responses were analyzed using RNA-seq data in sweet potato. This study interpreted the evolutionary relationships of SGRs and potential functions of the six Ipomoea species, which was helpful to provide valuable insights for further understanding the functions of SGRs and candidate genes for enhancing quality traits and improving stress tolerance in sweet potato.

2. Materials and Methods

2.1. Identification of SGRs

The genome sequences of I. aquatic, I. cairica, and I. nil were obtained from the National Genomics Data Center (NGDC) (https://ngdc.cncb.ac.cn/gwh/Assembly/986/show, accessed on 1 August 2024), the Zenodo repository (https://zenodo.org/records/6792002#.Y90Mb3ZBy4Q, accessed on 1 August 2024), and the Shigen database (http://viewer.shigen.info/asagao/index.php, accessed on 1 August 2024), respectively. In addition, all genome sequences of I. triloba, I. trifida, and I. batatas were downloaded from the Ipomoea Genome Hub (www.sweetpotao.com, accessed on 1 August 2024). Basic Local Alignment Search Tool (BLAST) methods were used to identify the SGRs. The amino acid sequences of SGRs were identified by a previous study of Arabidopsis thaliana (A. thaliana) as queries (BLASTP, E value ≤ 1 × 10⁻⁵). Furthermore, all putative SGRs were checked using the CD-Search in National Center for Biotechnology Information database.

2.2. Property Prediction of SGRs

The protein characteristics (i.e., Number of Amino Acids, Molecular Weight, Theoretical pI, Instability Index, Aliphatic Index, and Grand Average of Hydropathicity) were calculated busing the ExPASy tool (https://web.expasy.org/protparam/, accessed on 28 August 2024). The subcellular localization was conducted using the DeepLoc 2.1 server (https://services.healthtech.dtu.dk/services/DeepLoc-2.1/, accessed on 28 August 2024). The secondary of the SGRs were predicted using NetSurfP-3.0 [41].

2.3. Phylogenetic Analysis of SGRs

The phylogenetic analysis of SGRs was performed using MAFFT with the default parameters in the six Ipomoea species [42]. Subsequently, the software TrimAl (version 1.5.0) was used to align the trimming of the sequences [43]. Then, the phylogenetic tree was created using the IQ-TREE 2 with the bootstrap (1000 replicates) and best model being performed automatically [44]. The visualization of the phylogenetic trees was finished using the Interactive Tree of Life (ITOL) platform (https://itol.embl.de/index.shtml, accessed on 15 September 2024).

2.4. Domain Identification, Conserved Motif Analysis, Gene Structure Analysis of SGRs

All SGR proteins’ conserved domains were identified using the CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 15 August 2024). The conserved motif of SGR proteins was predicted using Multiple Em for Motif Elicitation (MEME, https://meme-suite.org/meme/tools/meme, accessed on 30 September 2024). The gene structures were visualized by the GSDC (https://gsds.gao-lab.org/, accessed on 30 September 2024).

2.5. Collinearity Analysis and Classification of Gene Duplication

The BLASTP results were used for the analysis by MCScanX, which conducted the collinearity blocks across the entire genome [45]. The collinearity pairs were extracted and the collinearity map was generated using the CIRCOS software (version 0.69-9) [46]. The duplicate_gene_classifier script of MCScanX was used to analyze the classification of gene duplication.

2.6. Cis-Acting Element Analysis of the Promoter of SGRs

The sequence 2000 bp upstream of the promoter regions of SGRs was extracted using the TBtools software (version 2.154) [47]. PlantCARE tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 15 October 2024) was employed to predict the cis-elements of the promoter of SGRs. The visualization of the cis-element was conducted using the Python package Seaborn (version 0.13.1).

2.7. Transcriptome Analysis and Construction of Protein Interaction Network

We downloaded the publicly available RNA sequencing (RNA-seq)-based expression data under the BioProject accession numbers PRJNA881010, PRJNA881014, PRJNA881013, PRJNA881012, PRJNA642259, and PRJNA511028 from the National Genomics Data [48,49,50]. PRJNA881010, PRJNA881014, PRJNA881013, and PRJNA881012 are transcriptome datasets for four sweet potato varieties with different flesh colors: 1143-1 (white flesh), HS (orange flesh), DZ88 (purple flesh), and DZ54 (purple flesh). PRJNA642259 is the transcriptome dataset for the I. batatas varieties XS-18 (white flesh) and XZS-3 (purple flesh). PRJNA511028 is the transcriptome dataset for the sweet potato variety Xu18’s tissues leaves, stems, and fibrous roots, treated under hormone treatments (ABA, MeJA, SA) and abiotic stress treatments (cold, drought, and salt). Firstly, the adapt and low-quality reads were removed by Fastp [51]. Secondly, the obtained reads were mapped to the sweet potato reference (downloaded from the Ipomoea Genome Hub genome) using STAR software (version 2.7.10b) [52]. Finally, the Featurecount was used to obtain the abundance matrix of the exons [53]. We used an in-house Python script to calculate the Transcripts Per Kilobase of the exon model per Million mapped reads (TPM) values for the SGRs. The multiple tissue expression patterns of sweet potato were normalized using log₂ (TPM+1). Furthermore, a heatmap was generated using the Seaborn package in Python.

Based on the IbSGR protein sequences, the interaction networks of the IbSGRs were constructed using the STRING database (https://string-db.org/, accessed on 20 November 2024) and the AlphaFold 3 database (https://alphafoldserver.com/, accessed on 10 December 2024). The interaction networks were visualized using PyMOL software (version 3.1.3).

3. Results

3.1. Identification and Characterization of SGRs in the Six Ipomoea Species

In this study, a total of 19 SGR members were extensively identified using Basic Local Alignment Search Tool (BLAST) searches in I. aquatica (IaqSGRs), I. cairica (IcaSGRs), I. nil (InilSGRs), I. triloba (ItbSGRs), I. trifida (ItfSGRs) and I. batatas (IbSGRs) (Table S1). The protein physiological properties of SGRs were analyzed using the sequences in the six Ipomoea species. The CDS length of the SGRs varied from 663 bp (InilSGR3) to 1029 bp (IbSGR1) (Table S1). The length of putative proteins ranged from 220 aa (InilSGR3) to 342 aa (IbSGR1), with a molecular weight (MW) of 24.56 kDa (InilSGR3) to 38.32 kDa (IbSGR1) and a theoretical isoelectric point (pI) of 6.3 (InilSGR3) to 9.27 (IaqSGR2) (Figure 1A–C, Table S1). Except for InilSGR3, IbSGR4, ItfSGR2, and ItbSGR3, the instability index of other proteins was more than 40 (Figure 1D, Table S1). The aliphatic index of SGRs ranged from 76.7 (IbSGR1) to 90.41 (InilSGR3) (Figure 1E, Table S1). The grand average of hydropathicity (GRAVY) values of SGRs varied from −0.497 to −0.121, indicating that they are hydrophilic proteins (Figure 1F, Table S1). Subcellular localization prediction showed that most of the SGRs were located in plastids except for ItbSGR1 and InilSGR1 in the cytoplasm, IbSGR3 in the endoplasmic reticulum, and InilSGR3 in the nucleus (Table S1). A secondary structure analysis of SGRs showed that the coil structure was dominant in all SGRs and the ratio of the coil structure was between 47.73% (InilSGR3) and 64.69% (IbSGR2) (Figure 1G).

3.2. Evolutionary and Phylogenetic Relationship Analysis of SGRs in the Six Ipomoea Species

Constructing the species evolutionary tree and counting the number of SGR members in different species showed that the gene number of SGRs was increased during the evolutionary history (Figure 2A). In order to characterize the evolutionary relationships of SGRs in Arabidopsis thaliana (A. thaliana) and the six Ipomoea species, we constructed a phylogenetic tree for 23 SGRs of these seven species (i.e., four in A. thaliana, three in I. aquatica, three in I. cairica, three in I. nil, three in I. triloba, three in I. trifida, and four in I. batatas). These SGRs were divided into three subgroups together with their orthologous SGRs from Arabidopsis, including Group I (AtSGR1/2/3, InilSGR1, IaqSGR1, lcaSGR1, ItfSGR1, ItbSGR1, and IbSGR1), Group II (AtSGR4, lcaSGR3, IaqSGR3, InilSGR3, ItbSGR3, ItfSGR3, and IbSGR4) and Group III (lcaSGR2, IaqSGR2, InilSGR2, IbSGR3, ItfSGR2, ItbSGR2, and IbSGR2) (Figure 2B). In addition, we constructed a phylogenetic tree with evolutionary distances of the six Ipomoea species, and all SGRs were divided into four groups with uneven distribution in each group. Interestingly, both IbSGR2 and IbSGR3 were in Group B, revealing that gene expansion occurred within this species during the evolution process (Figure 2C).

3.3. Conserved Domain and Motif Analysis of SGRs in the Six Ipomoea Species

We analyzed the domain of SGR proteins, and the results showed that all SGRs contained the stay-green domain (Figure 3A). Moreover, we analyzed the sequence motifs of SGR proteins through the MEME website and identified the ten conserved motifs (Figure 3B). The majority of the SGRs contained most of the motifs except for motif 9 and motif 10, including IaqSGR1/2, IcaSGR1/2, ItbSGR2, ItfSGR2, and IbSGR1/2/3. In addition, IaqSGR3, IcaSGR3, ItbSGR3, ItfSGR3, and IbSGR4 contained seven motifs except for 2, 5, and 8 (Figure 3B). In addition, InilSGR1/2, ItbSGR1, and ItfSGR1 had seven motifs except for 2, 9, and 10. InilSGR3 contained the least number of motifs, only including motifs 3, 4, 6, 7, and 10 (Figure 3B).

3.4. Gene Structure Analysis of SGRs in the Six Ipomoea Species

To better understand the structural features of SGR genes, gene structures were analyzed based on their evolutionary relationships. Overall, the gene structure of SGRs showed small variations. The number of CDS and introns is 4, 5, or 6 and 3, 4, or 5, respectively (Figure 4). Here, 10 of 19 members of SGRs contained two UTRs, four CDS, and three introns in the six Ipomoea species, including all members of group A (IaqSGR1 and InilSGR1), most of the members of group B (except for IcaSGR2 and IbSGR3) and ItbSGR1, ItfSGR1, and IbSGR1 in group C (Figure 4). Most of the members of group D contained five CDSs except for IbSGR4, which had six CDSs (Figure 4). Interestingly, no UTR of the gene structure of SGRs was detected in I. cairica. In general, most of the SGRs in the same groups showed similar gene structures, which supports their close evolutionary relationship (Figure 4). IbSGR1/2, IbSGR3, and IbSGR4 contained four, five, and six CDSs, respectively. These results indicate that the SGR gene family might become more complex in the evolution process.

3.5. Collinearity Analysis of SGRs in the Genomes of the Six Ipomoea Species

To further investigate the evolution of SGR genes of the Ipomoea genus, we conducted interspecies and intraspecies collinearity analyses (Figure 5 and Figure S1). The results revealed that IaqSGR3, IcaSGR3, InilSGR3, ItbSGR3, ItfSGR3, and IbSGR4 exhibited collinearity, indicating that they originated from a common ancestral gene (Figure 5A). Additionally, IaqSGR1 and IcaSGR1, as well as IcaSGR2, exhibited collinearity with IaqSGR2 and IcaSGR1, suggesting a shared evolutionary origin. Furthermore, IcaSGR1, IcaSGR2, InilSGR1, InilSGR2, ItbSGR1, ItbSGR2, ItfSGR1, ItfSGR2, IbSGR1, IbSGR2, and IbSGR3 also exhibited collinearity. Moreover, the analysis of collinearity within I. aquatica showed that IaqSGR1 and IaqSGR2 exhibit synteny, suggesting that IaqSGR1, IaqSGR2, IcaSGR1, IcaSGR2, InilSGR1, InilSGR2, ItbSGR1, ItbSGR2, ItfSGR1, ItfSGR2, IbSGR1, IbSGR2, and IbSGR3 originated from a common ancestral gene (Figure 5A and Figure S1A). In addition, we performed synteny analysis within the I. batatas species and found that IbSGR1 and IbSGR3 exhibited collinearity, as did IbSGR3 and IbSGR2 (Figure 5B). However, no collinearity was observed between IbSGR1 and IbSGR2, indicating more complex chromosomal variations in the IbSGR1 and IbSGR2 regions within I. batatas (Figure 5B). Furthermore, we conducted a collinearity analysis between the SGR genes of I. batatas and the other five species, revealing that the SGR genes of I. batatas are highly conserved within the Ipomoea genus (Figure 5C). The Duplicate_gene_classifier tool in MCScanX was employed to ascertain the duplication modes of SGR genes in Ipomoea species. The primary duplication mode of SGR genes in Ipomoea species was identified as WGD or Segmental, and dispersed duplication (Figure 5D). To determine the within-species collinearity relationships of the other five Ipomoea species, we conducted a collinearity analysis. The results showed that within the SGR genes of these five species, only one pair of SGR genes exhibited collinearity in each species, while two pairs of genes in sweet potato displayed collinearity. This difference may be due to the duplication of SGR genes within sweet potato (Figure S1).

3.6. Cis-Element Analysis in the Promoters of SGRs in the Six Ipomoea Species

The cis-acting regulatory elements were analyzed using 2000 bp promoter sequences upstream of the coding region to explore the potential functions of SGRs in the six Ipomoea species. The results showed that all of the SGR promoters had a large number of core/binding elements, including CAAT-box and TATA-box, and the light-responsive elements existed in all of the SGR promoters (Figure 6A). Some growth, development, and pigment biosynthesis elements were detected in the promoters of SGRs. For example, the O2-site (zein metabolism regulatory element) was found in the promoters of IcaSGR3, IbSGR4, and ItfSGR1; the MBSI motif (MYB binding site involved in flavonoid biosynthetic gene regulation) was found in the promoters of ItfSGR2/3 and ItbSGR3 (Figure 6A). In addition, most of the SGRs contained abundant hormone-responsive elements such as abscisic acid-responsive elements ABRE, ABRE3a, and ABRE4; MeJA-responsive elements CGTCA-motif and TGACG-motif; gibberellin-responsive elements P-box and GARE-motif; auxin-responsive elements TGA-element and AuxRR-core; and the salicylic-acid-responsive element TCA-element. In addition, the biotic/abiotic elements including TC-rich repeats (defense- and stress-responsive elements), LTR (low-temperature responsive elements), ARE (aerobic induction-responsive elements), and MBS (MYB binding site involved in drought inducibility) were present in the promoter of SGRs. Furthermore, all promoters of IbSGRs contained ABA-responsive elements (Figure 6A). We detected the W-box in the promoter of IaqSGR1/3, InilSGR2/3, ItbSGR3, ItfSGR1/2/3, and IbSGR2/4, which might be bounded by WRKY TFs. Some of the promoters of SGRs (i.e., laqSGR1, lcaSGR1, ltbSGR3, ltfSGR3, and IbSGR3) had the MYB-binding site, which might be regulated by MYB TFs (Figure 6A). We then classified the functions of the elements and found that the SGR genes contain the highest number of light-responsive elements, followed by ABA-responsive elements. Some genes, such as IbSGR1, contain a relatively simple set of elements, indicating that this gene has simpler transcriptional regulation compared to other SGR genes (Figure 6B). These results suggested that SGRs might be involved in the regulation of plant growth and development and pigment biosynthesis, and may participate in phytohormone and abiotic/biotic stress adaption signaling pathways.

3.7. Expression Patterns of IbSGRs

3.7.1. Expression Patterns of IbSGRs in Different Flesh-Colored Sweet Potato Varieties

The RNA-seq data (PRJNA881010, PRJNA881014, PRJNA881013, and PRJNA881012) of sweet potato with different flesh colors, including the four sweet potato varieties 1143-1 (white flesh), HS (orange flesh), DZ88 (purple flesh), and DZ54 (purple flesh). The RNA-seq data (PRJNA642259) of XS-18 (white flesh) and XZS-3 (purple flesh) were analyzed to investigate whether IbSGRs had an effect on pigment biosynthesis. We discovered that the expression level of IbSGR1 and IbSGR2 in white-fleshed 1143-1 is lower than that of orange-fleshed HS, which might suggest that IbSGR1 and IbSGR2 play negative roles in carotenoid biosynthesis (Figure 7A). In addition, for the biosynthesis of anthocyanin, the expression level of IbSGR1, IbSGR2, and IbSGR3 was negatively correlated with anthocyanin accumulation (Figure 7A,B). The expression of IbSGR4 was relatively stable in sweet potato varieties with different-colored flesh (Figure 7A,B). Taken together, these results show that IbSGRs possess different expression patterns and play vital roles in the carotenoid and anthocyanin biosynthesis of sweet potato.

3.7.2. Expression Patterns of IbSGRs in Different Hormones and Stress Responses

We analyzed the previously published RNA-seq data of three tissues, including FR (fibrous root), leaf, and stem, for different hormones and stress responses (i.e., ABA, MeJA, SA, cold, drought, and salt) to discover the potential function of the IbSGRs. The IbSGRs were expressed in the FRs, leaves, and stems. The IbSGRs in different tissues were induced by various treatments to different degrees (Figure 7C). Above all, these results indicated that some IbSGRs are involved in the response to hormones and stresses in sweet potato.

3.8. Protein Interaction Network of IbSGRs in Sweet Potato

To investigate the potential regulatory network of IbSGRs, we constructed an IbSGR interaction network using the STRING database (Figure 8A). Consistent with the function of SGR proteins, IbSGRs usually interact with proteins in chloroplasts. For example, IbSGRs can interact with chlorophyll synthase (g18035 and g27011), chlorophyllase 2 (g13818), and protochlorophyllide reductase (g297 and g54429) (Figure 8). Interestingly, IbSGR4 can interact with IbSGR1, IbSGR2, and IbSGR3, which might mean that IbSGR4 synergistically works with other IbSGR proteins (Figure 8A). Then, we further investigated the interaction between IbSGR4 and the other three IbSGR proteins using AlphaFold 3, which indicated that IbSGR4 can interact with the other three IbSGR proteins (Figure 8B–D).

4. Discussion

4.1. Evolution of SGRs in the Six Ipomoea Species

So far, SGR genes have been identified in some plant species using comparative genome analysis methods [7,8,9,10,11,12]. Two SGRs were found in rice [7]. Arabidopsis, tomato, and tea all have three SGRs [8,9,10]. Melon has four SGRs, and kiwifruit has five SGRs [11,12]. In this study, three, three, three, three, three, and four SGRs were identified in I. aquatic, I. cairica, I. nil, I. triloba, I. trifida, and I. batatas, respectively. The results suggested that the number of SGRs was similar among different species, which indicated that SGR gene members are relatively conserved during the evolutionary process. In addition, the number of SGRs in sweet potato was one more than in other species. This indicated that the SGR gene family expanded during the evolutionary history of the six Ipomoea species. According to the phylogenetic tree analysis, the SGR gene family has undergone expansion during its evolutionary history and it occurs in group B (Figure 2). In order to further explore the origin and evolution of the SGR gene family, collinearity analysis was performed and revealed that the SGR gene family originated from two genes, with one gene undergoing duplication. WGD or Segmental duplication events play a dominant role in expanding the SGR gene family (Figure 5).

In plants, the domain is evolutionarily conserved units of proteins, which are widely used to classify protein sequences and predict protein functions [54]. Furthermore, the domain structure of a protein is usually associated with its subcellular localization, interactions, and other functions of the protein [55,56]. Previous studies have suggested that single amino acid substitutions within this domain could obtain the mutant of SGRs [4,14,57]. In our study, all SGR proteins had the conserved stay-green domain and similar properties, which might mean that they have similar functions (Figure 1 and Figure 3). These results are consistent with previous reports, which found that the functions of SGR proteins are highly conserved in different plants [58]. Most of the SGRs are located in plastids, which is consistent with their work mainly in chloroplasts (Table S1). In addition, the different motif structure of IbSGR1/2/3 to that in IbSGR4 demonstrated the relative diversity of protein structures (Figure 3B).

Gene structure analysis is of great significance to the study of gene functions [59]. Based on the gene structure analysis, most of the SGRs had the same number of CDSs and introns. For example, all members of groups A, B, and C contained four CDSs and three introns, except IbSGR3 (Figure 4). These results showed that the gene structure pattern was relatively conservative in the six Ipomoea species. However, the three patterns in sweet potato include two UTRs–four CDSs–three introns, two UTRs–five CDSs–five introns, and two UTRs–six CDSs–five introns, which might result in their functional differences (Figure 4). These results indicate that IbSGRs went through structural diversification in the process of evolution. Taken together, the findings show that the SGR gene family members increased, the conserved domain structure was relatively stable, and the gene structure underwent some changes in the process of evolution.

4.2. IbSGRs Are Involved in Carotenoid and Anthocyanin Accumulation in Sweet Potato

SGRs are generally reported to participate in plant chlorophyll degradation and senescence, which are closely related to plant growth and development and stress responses [4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24]. The normal operation of chlorophyll metabolism is necessary for plants to maintain normal life activities [1,2,3]. SGR1 was demonstrated as the Mg-dechelatase responsible for catabolizing which was an important part of the chlorophyll breakdown [60]. The main function of SGRs is to regulate plant chlorophyll degradation and this has been confirmed in various species such as Arabidopsis [17], rice [13], tomato [26], and Medicago truncatula [16]. Chlorophylls and carotenoids are essential and beneficial substances for both plant and human health, and there is a certain connection between the synthesis mechanisms of these two pigments. Carotenoid accumulation is usually accompanied by the process of chlorophyll degradation [38,39]. In tomatoes and peppers, SGRs are involved in the regulation of color change during fruit ripening [18,19]. Interestingly, some studies have shown that SGRs also play a regulatory role in the biosynthesis of carotenoids. SlSGR1 directly interacts with SlPSY1 to play a pivotal regulatory role in color formation and fruit ripening regulation in tomato [20]. As reported by Biswal [61], the levels of both chlorophyll and carotenoid decline during leaf senescence. Hence, it was proposed that SGRs may act as a key factor in the regulation of carotenoid metabolism.

Sweet potato is an essential part of food and nutritional security [27,28]. The flesh of sweet potato comes in many colors and is rich in numerous pigments and nutrients [29,30,31,32,33,34,35,36]. Carotenoids and anthocyanins are important nutrients in sweet potato. Previous studies have shown that the downregulation of IbSGR1 expression increases carotenoid accumulation [40]. Previous research has also indicated that the expression of SGR genes is upregulated during leaf senescence and chlorophyll breakdown [4,14]. The analysis of gene expression levels is helpful for better functional prediction. In our study, according to the analysis of RNA-seq, IbSGR1 and IbSGR2 played negative roles in carotenoid biosynthesis, and IbSGR1, IbSGR2, and IbSGR3 acted as negative regulators in anthocyanin biosynthesis (Figure 7A). The results showed that SGRs might play an important role in carotenoid accumulation in sweet potato. The protein–protein interaction network is a key component in understanding protein functions and building protein regulatory networks [62]. The prediction of the protein interaction network showed that SGRs can interact with chlorophyll synthesis (Figure 8).

Most recent studies have found that WRKY TFs play important roles in carotenoid and anthocyanin biosynthesis and usually act by regulating the expression of other genes. W-box acts as a binding site sequence for WRKY TFs [63]. In citrus, CrWRKY42 regulates chlorophyll degradation and carotenoid biosynthesis [39]. SlWRKY35 can directly activate the expression of the SlDXS1 gene to enhance carotenoid accumulation in tomato [64]. OfWRKY3 positively regulates the carotenoid cleavage dioxygenase gene OfCCD4 in Osmanthus fragrans [65]. The overexpression of MdWRKY75 enhances the accumulation of anthocyanins in apple (Malus domestica) [66]. In our study, the transcription factor (TF) binding sites were analyzed on the promoter of SGRs. IbSGR2 and IbSGR4 had the WRKY TFs binding site W-box in their promoter, which may mean that IbSGR2 and IbSGR4 might be bound and regulated by WRKY TFs to affect pigment accumulation (Figure 6). In addition, MYB TFs occupy a dominant position in the regulatory network of anthocyanin biosynthesis [67,68]. For example, in tomato, SlMYB75 promotes anthocyanin accumulation and enhances volatile aroma production [69]. MdMYB3 is involved in the regulation of anthocyanin biosynthesis and flower development in apple [70]. BoMYBL2b inhibits anthocyanin accumulation via directly repressing BoDFR1 gene transcription in kale [71]. PdMYB118 directly interacts with bHLH transcription factor PdTT8 to regulate wound-induced anthocyanin biosynthesis in poplar [72]. By analyzing the cis-acting elements in the promoters of SGRs, we found that IbSGR3 had an MYB-binding site, which may imply that MYB TFs regulate the expression of IbSGR3 to influence anthocyanin accumulation (Figure 6 and Figure 7). Taken together, these results indicate that IbSGRs may be involved in the response to carotenoid and anthocyanin accumulation in sweet potato.

4.3. IbSGRs Regulate Hormone and Stress Responses in Sweet Potato

SGRs are reported to regulate the response to ABA in plants. In Arabidopsis, ABF2, ABF3, ABF4, and ABI3 can all bind to the promoter of SGR1 to activate its expression [24,25]. SlSGRL can be activated by SlABI5 in tomato [26]. However, the regulation mechanism of SGRs in response to hormones is largely unexplored. In our research, we found that ABA-, MeJA-, gibberellin-, auxin-, and SA-responsive elements were present in the SGR promoters (Figure 6). The promoter of IbSGR1 and IbSGR2 had ABA-, MeJA- and auxin-responsive elements, the IbSGR3 promoter had ABA- and gibberellin-responsive elements, and IbSGR4 had gibberellin- and SA-responsive elements (Figure 6). The RNA-seq analysis results showed that IbSGRs were induced by ABA, MeJA, and SA (Figure 7). These results indicate that SGRs may participate in hormone crosstalk in sweet potato.

SGRs are the key regulator involved in keeping plants “stay-green”, which allows plants to keep their leaves on the active photosynthetic level under stress conditions [73]. OsSGR was a negative regulator in rice sheath blight [21]. CsSGR negatively regulates disease resistance in citrus [22]. In Arabidopsis, AtSGR1 and AtSGRL act synergistically for rapid chlorophyll degradation before senescence under salt stress [74]. However, there is no research reporting whether SGRs play a role in the stress responses of sweet potato and other Ipomoea species. In this research, drought inducibility-, low-temperature-, defense- and stress-, anaerobic induction-, wound-, and anoxic-specific-inducibility-responsive elements were present in the promoters of SGRs (Figure 6). Furthermore, the promoters of IbSGR2 had the defense- and stress- and the anaerobic induction responsiveness elements; the promoters of IbSGR3 contained low-temperature, anaerobic induction, and anaerobic induction responsiveness elements; and the promoters of IbSGR4 had wound responsiveness elements (Figure 6). Furthermore, IbSGRs were induced by cold, drought, and salt treatment based on the RNA-seq. Interestingly, the FRs, leaves, and stems of IbSGR3 were all downregulated under cold stress, which might mean that IbSGR3 is a key negative regulator factor at low temperatures (Figure 7C). Taken together, these results suggest that SGRs may regulate the response to biotic and abiotic stress.

5. Conclusions

In summary, our results demonstrated that a total of 19 SGR members were identified in I. aquatica, I. cairica, I. nil, I. triloba, I. trifida, and I. batatas. There is one more IbSGR member than in the other five species. The SGR proteins had the conserved stay-green domain and similar properties. The SGR genes of the six Ipomoea species originated from two distinct SGR ancestral genes. Most of the SGR genes had two exons and three introns. The promoters of the SGRs had the cis-elements of phytohormone and stress responsiveness. We found that some of the promoters of IbSGRs had pigment-associated TF (WRKY and MYB) binding factors. IbSGR2 and IbSGR4 had a WRKY TFs binding site and IbSGR3 had an MYB-binding site in their promoters. In addition, the expression of IbSGR1, IbSGR2, and IbSGR3 were related to the color of the sweet potato flesh. IbSGR3 might be a potential candidate gene to negatively regulate low-temperature responses according to our results. This work provides valuable insights into the evolution and functions of SGRs and also supplies valuable candidate genes for improving carotenoid and anthocyanin contents and stress tolerance in sweet potatoes.