Genome-Wide Identiﬁcation and Expression Analysis of the SHI -Related Sequence Family in Cassava

: The SHORT INTERNODES (SHI) -related sequences ( SRS ) are plant-speciﬁc transcription factors that have been quantitatively characterized during plant growth, regeneration, and stress responses. However, the genome-wide discovery of SRS family genes and their involvement in abiotic stress-related activities in cassava have not been documented. A genome-wide search strategy was used to identify eight family members of the SRS gene family in cassava ( Manihot esculenta Crantz). Based on their evolutionary linkages, all MeSRS genes featured homologous RING-like zinc ﬁnger and IXGH domains. Genetic architecture and conserved motif analysis validated the categorization of MeSRS genes into four groups. Eight pairs of segmental duplications were detected, resulting in an increase in the number of MeSRS genes. Orthologous studies of SRS genes among cassava and three different plant species ( Arabidopsis thaliana , Oryza sativa , and Populus trichocarpa ) provided important insights into the probable history of the MeSRS gene family. The functionality of MeSRS genes was elucidated through the prediction of protein–protein interaction networks and cis -acting domains. RNA-seq data demonstrated tissue/organ expression selectivity and preference of the MeSRS genes. Furthermore, qRT-PCR investigation of MeSRS gene expression after exposure to salicylic acid (SA) and methyl jasmonate (MeJA) hormone treatments, as well as salt (NaCl) and osmotic (polyethylene glycol, PEG) stresses, showed their stress-responsive patterns. This genome-wide characterization and identiﬁcation of the evolutionary relationships and expression proﬁles of the cassava MeSRS family genes will be helpful for further research into this gene family and its function in stress response. It may also assist future agricultural efforts to increase the stress tolerance of cassava.


Introduction
Cassava (Manihot esculenta Crantz) is a vital source of food and income for over a hundred million people in tropical countries [1]. Due to its high starch content, it is also considered a promising biofuel crop for ethanol production [2]. According to the The Food and Agriculture Organization (FAO), global cassava production was expected to reach 306 million tons on approximately 34.23 million hectares by 2021 [3]. As cassava is the world's sixth-largest staple crop [4,5], maintaining high yields is crucial for ensuring food security worldwide. However, biotic challenges such as insect, bacterial, fungal, and viral hazards, as well as abiotic stresses such as water deficit, high salinity, and cold temperatures, can significantly affect plant growth and productivity, including that of cassava. To cope with these adverse conditions, plants have developed a various molecular and physiological defense mechanisms by activating a range of stress-responsive genes. Increasingly, research is focusing on enhancing cassava yields by discovering and charactering key functional genes associated with yield development and environmental adaptation [6]. Although the availability of whole-genome cassava sequences has facilitated the identification of

Characterization of Cassava SRS Genes and Proteins
The protein length, MW, pI, and GRAVY of each identified MeSRS protein were calculated using the ProtParam program listed on the Expasy website (https://expasy.org, access date: 12 November 2022) [40]. The subcellular localizations of MeSRS proteins were predicted using the WoLF PSORT web server (https://wolfpsort.hgc.jp/, access date: 12 November 2022) [41]. The MeSRS protein sequences were aligned using DNAMAN ver. 7. The exon-intron structure of the MeSRS genes was visualized on the Gene Structure Display Server (GSDS) website (http://gsds.cbi.pku.edu.cn/, access date: 1 December 2022) [42], and the data were obtained from the cassava GFF annotation files. The conserved motifs of MeSRS proteins were predicted using the multiple expectation maximization for motif elicitation (MEME) ver. 5.4.1 online tools (http://meme-suite.org/index.html, access date: 10 December 2022) as follows: the maximum motif number was six, and the others set as default [43]. The secondary structure of the deduced polypeptide was predicted by SOPMA (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, access date: 15 December 2022).

Chromosome Location, Gene Duplication, and Synteny Analysis
Information about the chromosome location of MeSRS genes was obtained from the cassava GFF annotation files, and the physical locations of MeSRS genes on chromosomes were visualized using TBtools [32]. Gene duplication pairs were identified from the plant genome duplication database (http://chibba.agtec.uga.edu/duplication/index/locket, access date: 15 February 2023). Synteny analysis and chromosomal location diagrams were generated using the Circos program in the TBtools software [32]. The Ka and Ks substitution rates were estimated using the TBtools software [32].

Protein Interaction Network Diagram and Cis-Regulatory Element Analysis
The functional relationship of the MeSRS proteins was predicted using the STRING protein interaction database (http://string-db.org/, access date: 20 November 2022) [44,45]. To analyze the regulatory elements in the MeSRS genes, the TBtools software was used to  [32]. Possible cis-acting elements were identified using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/index.html, access date: 29 November 2022) [46].

In Silico mRNA Levels of MeSRS Genes in Different Tissues
The transcriptome data were retrieved from the NCBI database (accession number: GSE82279) submitted by Wilson et al. [5] and used to investigate the expression profiles of MeSRS genes in different tissues, including leaves, mid veins, lateral buds, somatic embryos (OES), friable embryogenic callus (FEC), fibrous roots (FR), storage root (SR), stems, petioles, root apical meristem (RAM), and shoot apical meristem (SAM). Fragments per kilobase of transcript per million reads mapped (FPKM) values were calculated to evaluate the gene expression. Log 2 -transformed heatmaps of all MeSRS genes were produced using TBtools software [47].

Plant Materials and Stress Treatments
South China 9 (SC9) cassava, a common edible cultivar in China that has good performance in terms of properties of starch and stress tolerance, was used in this research. The stems, approximately 15 cm in length with two to three buds, were subcultured for approximately 30 days on Murashige and Skoog (MS) medium at 25 • C under a 16 h light/8 h dark cycle at Hainan University (Haikou, Hainan, China). Uniform seedlings were chosen to study the transcriptional response of cassava MeSRS genes under abiotic stresses and phytohormone treatments. The seedlings were transferred to MS medium containing 20% PEG, 400 mM NaCl, 100 µM SA, and 100 µM MeJA, respectively, and four treatment time points (0, 4, 12, and 24 h) were selected. Leaf samples were collected with three replications per treatment at each time point. All samples were flash-frozen in liquid nitrogen and stored at −80 • C until use.

RNA Isolation and qRT-PCR
RNAprep Pure Plan Plus Kit (TIANGEN Biotech Co., Ltd., Beijing, China) was used to isolate RNA from the leaves according to the manufacturer's instructions. Total RNA (1 ug) was used for first-strand cDNA synthesis using a reverse transcriptase kit (M1631, Thermo Fisher Scientific, Waltham, MA, USA). The elongation factor 1 α (EF1α) gene was used as an endogenous control. Quantitative primers were designed using the website Primer3 (http://bioinfo.ut.ee/primer3-0.4.0/) based on the identified MeSRS gene sequences, and the primers used in this study are listed in Table S5. Real-time PCR was performed on a 7500 Real Time PCR System (Thermo Fisher Scientific) with a total reaction volume of 20 µL, containing 2 µL cDNA template, 1 µL forward primer, 1 µL reverse primer, 10 µL qPCR Master Mix, and 6 µL sterilized ddH 2 O. The real-time PCR amplification protocol was set at 95 • C for 30 s for 40 cycles, 95 • C for 5 s, 55 • C for 30 s, 72 • C for 30 s, and 72 • C for 10 min [48]. Relative fold expression changes of the eight MeSRS genes were normalized to the EF1α gene and calculated using the 2 −∆∆Ct method described by Schmittgen and Livak [49]. Each sample was analyzed in three independent biological replications. Differences between groups were analyzed using Duncan's multiple range tests with SPSS Statistics ver. 20.0 (IBM, Armonk, NY, USA), and a p ≤ 0.05 was considered statistically significant.

Identification and Phylogenetic Analysis of MeSRS Genes in Cassava
The cassava genome revealed eight non-redundant SRS family members (MeSRS1 to MeSRS8) ( Table 1). Figure 1 shows a comprehensive sequence alignment of the MeSRS proteins. All of the MeSRS proteins had the conserved RING-like zinc finger domain CX 2 CX 7 CX 4 CX 2 CX 7 C and the IXGH domain, which are basic features of the SRS gene family ( Figure 1). Additionally, the physicochemical characteristics of these MeSRSs were investigated. The CDS lengths for MeSRS genes ranged from 642 bp (MeSRS7) to 1080 bp (MeSRS2), while the sizes of the MeSRS proteins ranged from 213 a.a (MeSRS7) to 359 a.a (MeSRS2). The molecular weights (MWs) ranged from 23.05 kDa (MeSRS7) to 37.22 kDa (MeSRS2), and the projected isoelectric points (pIs) ranged from 6.13 (MeSRS6) to 8.98 (MeSRS7). All of the MeSRS proteins had a negative grand average of hydropathicity (GRAVY), suggesting that they were hydrophilic. The WoLF PSORT web service was used to identify MeSRSs' subcellular localizations. Except for MeSRS2, which was predicted to be localized in the endoplasmic reticulum, almost all MeSRSs were predicted to be localized in the nucleus ( Table 1). The SPOMA online program was employed to estimate the structural features of eight MeSRS proteins to better comprehend their molecular function (Table S1). According to our findings, the random coil is the primary component of the secondary structure of MeSRS proteins, accounting for more than 60% of all MeSRS proteins. The secondary structure α helix of MeSRS1 and MeSRS3 proteins, on the other hand, accounted for a substantial amount (more than 17%), whereas the portion of other proteins was minimal, with the lowest at 8.36% for MeSRS2. In the MeSRS protein structure, the fraction of extended chains was less than 18%.

Identification and Phylogenetic Analysis of MeSRS Genes in Cassava
The cassava genome revealed eight non-redundant SRS family members (MeSRS1 to MeSRS8) ( Table 1). Figure 1 shows a comprehensive sequence alignment of the MeSRS proteins. All of the MeSRS proteins had the conserved RING-like zinc finger domain CX2CX7CX4CX2CX7C and the IXGH domain, which are basic features of the SRS gene family ( Figure 1). Additionally, the physicochemical characteristics of these MeSRSs were investigated. The CDS lengths for MeSRS genes ranged from 642 bp (MeSRS7) to 1080 bp (MeSRS2), while the sizes of the MeSRS proteins ranged from 213 a.a (MeSRS7) to 359 a.a (MeSRS2). The molecular weights (MWs) ranged from 23.05 kDa (MeSRS7) to 37.22 kDa (MeSRS2), and the projected isoelectric points (pIs) ranged from 6.13 (MeSRS6) to 8.98 (MeSRS7). All of the MeSRS proteins had a negative grand average of hydropathicity (GRAVY), suggesting that they were hydrophilic. The WoLF PSORT web service was used to identify MeSRSs' subcellular localizations. Except for MeSRS2, which was predicted to be localized in the endoplasmic reticulum, almost all MeSRSs were predicted to be localized in the nucleus ( Table 1). The SPOMA online program was employed to estimate the structural features of eight MeSRS proteins to better comprehend their molecular function (Table S1). According to our findings, the random coil is the primary component of the secondary structure of MeSRS proteins, accounting for more than 60% of all MeSRS proteins. The secondary structure α helix of MeSRS1 and MeSRS3 proteins, on the other hand, accounted for a substantial amount (more than 17%), whereas the portion of other proteins was minimal, with the lowest at 8.36% for MeSRS2. In the MeSRS protein structure, the fraction of extended chains was less than 18%.  Multiple sequence alignments of MeSRS proteins. The RING-like region (CX 2 CX 7 CX 4 CX 2 CX 7 C) and the IXGH domain are marked by the red and blue boxes, respectively. An unrooted phylogenetic tree was generated using MAGA 7.1 software by utilizing multiple sequence alignment of SRS protein sequences from six distinct plant species, including eight in cassava, ten in A. thaliana, six in O. sativa, four in Spinacia oleracea (S. oleracea), ten in Populus trichocarpa (P. Trichocarpa), and five in Ricinus communis (R. Communis) (Table S2). Based on their evolutionary relationships, we grouped the 43 SRS proteins into six different groups (I-VI) ( Figure 2). Group VI had the most SRS proteins, with nine from all six species, whereas Group IV only had three from monocot plants. The eight MeSRS genes were divided into five groups: I, II, III, V, and VI, each with one, two, two, one and two members, respectively. MeSRSs are more strongly connected to SRSs from dicots, particularly R. communis and P. trichocarpa ( Figure 2).

MeSRS2
Manes.01G179500. An unrooted phylogenetic tree was generated using MAGA 7.1 software by utilizing multiple sequence alignment of SRS protein sequences from six distinct plant species, including eight in cassava, ten in A. thaliana, six in O. sativa, four in Spinacia oleracea (S. oleracea), ten in Populus trichocarpa (P. Trichocarpa), and five in Ricinus communis (R. Communis) (Table S2). Based on their evolutionary relationships, we grouped the 43 SRS proteins into six different groups (I-VI) ( Figure 2). Group VI had the most SRS proteins, with nine from all six species, whereas Group IV only had three from monocot plants. The eight MeSRS genes were divided into five groups: I, II, III, V, and VI, each with one, two, two, one and two members, respectively. MeSRSs are more strongly connected to SRSs from dicots, particularly R. communis and P. trichocarpa ( Figure 2).   Table S2. Figure 3A depicts a distinct cluster of cassava SRS proteins. The results showed that the tree's structure matched that of the phylogenetic tree constructed using SRS sequences from the six plant species displayed in Figure 2. A detailed comparison of exon-intron structure was performed to further understand the structural properties of MeSRS genes ( Figure 3B). Six MeSRS genes (MeSRS1, MeSRS2, MeSRS3, MeSRS4, MeSRS5, and MeSRS7) had two exons and one intron, but two MeSRS genes in group II (MeSRS6 and MeSRS8) had three exons and two introns. Exon-intron organization was generally comparable within the same group, supporting their tight evolutionary ties ( Figure 3B). The MEME online program discovered six motifs to further investigate structural diversity and anticipate the function of the MeSRS protein ( Figure 3C). The preserved motif lengths ranged from 18 (motif 3) to 50 (motif 2) a.a., while their logo symbol is illustrated in Figure 3D. A RING-like zinc finger motif CX 2 CX 7 CX 4 CX 2 CX 7 C appears in motif 1, while the IXGH conservation domain exists in motif 2. Motifs 1 and 2 were found in all MeSRS proteins, which is in line with the findings of multiple alignment of sequence analyses (Figure 1). It is worth mentioning that several conserved motifs were discovered in particular groups. For example, motif 3 was found only in groups VI and V, motifs 4 and 5 in groups III, IV, and VI, and motif 6 in two MeSRSs from group I. ( Figure 3C). This finding implies that the distinct roles of each subgroup may be linked to certain motifs.

Gene Structure and Conserved Motifs of MeSRSs
had two exons and one intron, but two MeSRS genes in group II (MeSRS6 and MeSRS8) had three exons and two introns. Exon-intron organization was generally comparable within the same group, supporting their tight evolutionary ties ( Figure 3B). The MEME online program discovered six motifs to further investigate structural diversity and anticipate the function of the MeSRS protein ( Figure 3C). The preserved motif lengths ranged from 18 (motif 3) to 50 (motif 2) a.a., while their logo symbol is illustrated in Figure 3D. A RING-like zinc finger motif CX2CX7CX4CX2CX7C appears in motif 1, while the IXGH conservation domain exists in motif 2. Motifs 1 and 2 were found in all MeSRS proteins, which is in line with the findings of multiple alignment of sequence analyses (Figure 1). It is worth mentioning that several conserved motifs were discovered in particular groups. For example, motif 3 was found only in groups VI and V, motifs 4 and 5 in groups III, IV, and VI, and motif 6 in two MeSRSs from group I. ( Figure 3C). This finding implies that the distinct roles of each subgroup may be linked to certain motifs.

Chromosome Localization, Gene Duplication and Synteny Analysis
The eight MeSRS genes were spread across six cassava chromosomes. Two genes were found on chromosomes 1 and 2, and one gene on chromosomes 4, 5, 11, and 18. (Table 1; Figure 4A). We conducted a collinear analysis of MeSRS genes to better understand their evolutionary history. As segmental duplication genes, a total of eight homologous gene pairs including seven MeSRS genes were found. The duplicated pairings are MeSRS1: MeSRS8, MeSRS1: MeSRS3, MeSRS1: MeSRS6, MeSRS2: MeSRS4, MeSRS3: MeSRS6, MeSRS7: MeSRS8, MeSRS8: MeSRS3, and MeSRS8: MeSRS6 ( Figure 4B). These findings suggested that these genes were perhaps created by segmental duplication events, which operated as a primary driving factor in the development of the MeSRS family. Furthermore, the non-synonymous (Ka) and synonymous (Ks) substitution rates were calculated for each homologous gene pair using the TBtools program. All of the MeSRS orthologous gene pairs showed a Ka/Ks ratio < 1 ( Table 2). These findings point to the MeSRS genes' conserved evolution. We created three comparative syntenic maps of cassava compared to three representative species, including two dicot plants, A. thaliana and P. trichocarpa, and one monocot plant, O. sativa, to further infer the evolutionary pathways of the MeSRS family ( Figure 4C). Therefore, we identified pairwise homologues of the MeSRS genes in A. thaliana, P. trichocarpa, and O. sativa to detect 16, 27, and 2 pairs of homologous genes, respectively (Table S3) Figure 4B). These findings suggested that these genes were perhaps created by segmental duplication events, which operated as a primary driving factor in the development of the MeSRS family. Furthermore, the non-synonymous (Ka) and synonymous (Ks) substitution rates were calculated for each homologous gene pair using the TBtools program. All of the MeSRS orthologous gene pairs showed a Ka/Ks ratio < 1 ( Table 2). These findings point to the MeSRS genes' conserved evolution. We created three comparative syntenic maps of cassava compared to three representative species, including two dicot plants, A. thaliana and P. trichocarpa, and one monocot plant, O. sativa, to further infer the evolutionary pathways of the MeSRS family ( Figure 4C). Therefore, we identified pairwise homologues of the MeSRS genes in A. thaliana, P. trichocarpa, and O. sativa to detect 16, 27, and 2 pairs of homologous genes, respectively (Table S3).

Identification of Cis-Acting Elements in the Promoters of MeSRS Genes
PlantCARE was used to conduct a cis-elements study on the 2000 bp area upstream of the MeSRS genes ( Figure 5). Each component possesses its own unique sequence and serves a specific function. In this study, eighteen cis-acting elements were identified: eight stress-sensitive (MYB, MYC, TC-rich repeats, MBS, STRE, W box, WUN-motif, and LTR), ten hormone-responsive (ABRE, CGTCA motif, ERE, GARE-motif, P-box, TATC-box, TCAelement, TGACG-motif, AuxRR-CORE, and TGA-element) cis-acting elements, and nine growth and development related (ARE, CAT-box, as−1, O2-site, circadian, HD-Zip 1, MSAlike, OCT, and GC-motif) cis-acting elements. ERE and ARE were discovered in each MeSRS gene promoter. MeSRS3 is expected to have the largest amount of cis-elements ( Figure 5). Altogether, the promoter regions of each of these genes include a minimum of three types of stress-responsive and four types of hormone-responsive cis-acting elements, indicating that MeSRS genes may be engaged in abiotic and biotic stress responses.

Identification of Cis-Acting Elements in the Promoters of MeSRS Genes
PlantCARE was used to conduct a cis-elements study on the 2000 bp area upstream of the MeSRS genes ( Figure 5). Each component possesses its own unique sequence and serves a specific function. In this study, eighteen cis-acting elements were identified: eight stress-sensitive (MYB, MYC, TC-rich repeats, MBS, STRE, W box, WUN-motif, and LTR), ten hormone-responsive (ABRE, CGTCA motif, ERE, GARE-motif, P-box, TATC-box, TCA-element, TGACG-motif, AuxRR-CORE, and TGA-element) cis-acting elements, and nine growth and development related (ARE, CAT-box, as−1, O2-site, circadian, HD-Zip 1, MSA-like, OCT, and GC-motif) cis-acting elements. ERE and ARE were discovered in each MeSRS gene promoter. MeSRS3 is expected to have the largest amount of cis-elements ( Figure 5). Altogether, the promoter regions of each of these genes include a minimum of three types of stress-responsive and four types of hormone-responsive cis-acting elements, indicating that MeSRS genes may be engaged in abiotic and biotic stress responses.

Protein-Protein Interaction Network and GO Annotation Analysis
Utilizing protein-protein interaction network to connect unknown functional proteins can help us comprehend the many biological activities of proteins and the dynamic regulatory networks that exist among distinct biomolecules. In this study, the functional and physical interactions among potential MeSRS proteins were examined using the

Protein-Protein Interaction Network and GO Annotation Analysis
Utilizing protein-protein interaction network to connect unknown functional proteins can help us comprehend the many biological activities of proteins and the dynamic regulatory networks that exist among distinct biomolecules. In this study, the functional and physical interactions among potential MeSRS proteins were examined using the STRING program based on the A. thaliana association model ( Figure 6). Each node represents all proteins generated by a single protein-coding locus. The predicted number of boundaries and the average local clustering coefficient were 10 and 0.313, respectively, and the p-value for protein-protein interaction enrichment was 8.29e-11. We found nine SRS functional molecules (SHI, STY1, STY2, LRP1, SRS3, SRS4, SRS5, SRS6, and SRS7) and five putative interaction proteins (KIN13A, NGA3, YUC4, AT1G32730, and AT3G06840) directly connected to MeSRS proteins using statistical analysis of eight MeSRS proteins ( Figure 6). Among the seven discovered similar proteins in A. thaliana, SRS3 had the most interaction partners, including KIN13A, YUC4, AT1G32730, and AT3G06840.
The gene ontology (GO) functional analyses were carried out utilizing the PANNZER web server to identify the biological process, molecular function, and cellular component characteristics of the MeSRS proteins (Table S4). In the "biological process" category, auxin biosynthesis process (GO:0009851), auxin-activated signaling pathway (GO:0009734), regulation of DNA-templated transcription (GO:0006355), and positive regulation of nucleic acid-templated transcription (GO:1903508) characteristics of the MeSRS proteins were revealed. In the "molecular function" category, all proteins were classified as DNAbinding transcription factor activity (GO:0003700) and DNA binding (GO:0003677). Two GO terms related to cellular components, including nucleus (GO:0005634) and membrane (GO:0016020), were specifically enriched. These findings show that MeSRS proteins have a variety of roles in cell metabolism.
Genes 2023, 14, x FOR PEER REVIEW 10 of 18 STRING program based on the A. thaliana association model ( Figure 6). Each node represents all proteins generated by a single protein-coding locus. The predicted number of boundaries and the average local clustering coefficient were 10 and 0.313, respectively, and the p-value for protein-protein interaction enrichment was 8.29e-11. We found nine SRS functional molecules (SHI, STY1, STY2, LRP1, SRS3, SRS4, SRS5, SRS6, and SRS7) and five putative interaction proteins (KIN13A, NGA3, YUC4, AT1G32730, and AT3G06840) directly connected to MeSRS proteins using statistical analysis of eight MeSRS proteins ( Figure 6). Among the seven discovered similar proteins in A. thaliana, SRS3 had the most interaction partners, including KIN13A, YUC4, AT1G32730, and AT3G06840. The gene ontology (GO) functional analyses were carried out utilizing the PANNZER web server to identify the biological process, molecular function, and cellular component characteristics of the MeSRS proteins (Table S4). In the "biological process" category, auxin biosynthesis process (GO:0009851), auxin-activated signaling pathway (GO:0009734), regulation of DNA-templated transcription (GO:0006355), and positive regulation of nucleic acid-templated transcription (GO:1903508) characteristics of the MeSRS proteins were revealed. In the "molecular function" category, all proteins were classified as DNA-binding transcription factor activity (GO:0003700) and DNA binding (GO:0003677). Two GO terms related to cellular components, including nucleus (GO:0005634) and membrane (GO:0016020), were specifically enriched. These findings show that MeSRS proteins have a variety of roles in cell metabolism.

Expression of MeSRS Genes in Different Tissues
Wilson et al. [5] made their cassava RNA-seq data publicly available, and their dataset was used to investigate the expression patterns of MeSRS genes across eleven tissues. All eight MeSRS genes were included in the hierarchical clustering analysis (Figure 7) and found to be expressed differently across different cassava tissues. MeSRS2 and MeSRS5 exhibited higher expression levels in almost all tissues than the other MeSRS genes, suggesting that they may play important roles in cassava development. In contrast, MeSRS7 expression was low in all tissues tested. Some MeSRS genes exhibited tissue-specific expression patterns. MeSRS1, MeSRS6, and MeSRS8 exhibited higher expression levels in friable embryogenic callus (FEC), organized embryogenic structures (OES), fibrous roots (FR), and lateral buds than in other tissues. MeSRS4 had the greatest expression in storage root (SR). The high levels of expression of these MeSRS genes in a specific tissue suggest that they may play crucial roles in tissue growth or functionality (Figure 7). gesting that they may play important roles in cassava development. In contrast, MeSRS7 expression was low in all tissues tested. Some MeSRS genes exhibited tissue-specific expression patterns. MeSRS1, MeSRS6, and MeSRS8 exhibited higher expression levels in friable embryogenic callus (FEC), organized embryogenic structures (OES), fibrous roots (FR), and lateral buds than in other tissues. MeSRS4 had the greatest expression in storage root (SR). The high levels of expression of these MeSRS genes in a specific tissue suggest that they may play crucial roles in tissue growth or functionality (Figure 7).

Response of MeSRS Genes to Hormone Treatments and Abiotic Stresses
To study the possible functions of the cassava MeSRS genes in response to various stimuli, qRT-PCR was used to examine the expression patterns of these genes in cassava seedlings treated with salicylic (SA), methyl jasmonate (MeJA), NaCl, and polyethylene glycol (PEG) (Figures 8 and 9). As a control, the expression level at 0 h was set as the baseline (CK). Except for MeSRS3, the seven MeSRS genes were strongly activated or repressed by SA treatment. MeSRS1 and MeSRS2 were suppressed after 12 h and 4 h, respectively. MeSRS4 and MeSRS6 demonstrated a similar dynamic, with upregulation beginning at 12 h. MeSRS5 and MeSRS7 were considerably upregulated at 12 h, but MeSRS8 showed no significant change from 0 to 12 h before being significantly upregulated at 24 h. MeSRS1 and MeSRS7 levels increased progressively under MeJA therapy and peaked at 24 h. MeSRS2 was significantly activated and sustained reasonably high levels of expression. MeSRS3 and MeSRS5 had a similar expression profile, with a strong increase after 12 h and a subsequent decrease after 24 h. MeSRS4 and MeSRS6 showed a similar

Response of MeSRS Genes to Hormone Treatments and Abiotic Stresses
To study the possible functions of the cassava MeSRS genes in response to various stimuli, qRT-PCR was used to examine the expression patterns of these genes in cassava seedlings treated with salicylic (SA), methyl jasmonate (MeJA), NaCl, and polyethylene glycol (PEG) (Figures 8 and 9). As a control, the expression level at 0 h was set as the baseline (CK). Except for MeSRS3, the seven MeSRS genes were strongly activated or repressed by SA treatment. MeSRS1 and MeSRS2 were suppressed after 12 h and 4 h, respectively. MeSRS4 and MeSRS6 demonstrated a similar dynamic, with upregulation beginning at 12 h. MeSRS5 and MeSRS7 were considerably upregulated at 12 h, but MeSRS8 showed no significant change from 0 to 12 h before being significantly upregulated at 24 h. MeSRS1 and MeSRS7 levels increased progressively under MeJA therapy and peaked at 24 h. MeSRS2 was significantly activated and sustained reasonably high levels of expression. MeSRS3 and MeSRS5 had a similar expression profile, with a strong increase after 12 h and a subsequent decrease after 24 h. MeSRS4 and MeSRS6 showed a similar expression profile, whereas MeSRS6 remained relatively low at 24 h. MeSRS8 exhibited almost no change during the first phase of MeJA treatment and began to be upregulated after 24 h (Figure 8).
Following NaCl treatment, the expression of MeSRS1, MeSRS2, MeSRS5, MeSRS7, and MeSRS8 increased considerably at 4 h or 12 h and then decreased after the peak time point. MeSRS3 was progressively suppressed, and its expression was downregulated at 24 h compared to 0 h. MeSRS4 levels were much lower after 12 h, then rose after 24 h and eventually peaked at 24 h. MeSRS6 was shown to be considerably up-regulated after 24 h (Figure 9). The expression of most MeSRS genes was considerably affected by PEG treatment, except for MeSRS7, whose expression remained relatively steady at all time periods. MeSRS3, MeSRS5, and MeSRS6 levels first increased but subsequently declined, peaking at 4 h or 12 h. MeSRS1, MeSRS4, and MeSRS8 all showed similar "down-up-down" tendencies from 0 h to 24 h. MeSRS3 was significantly upregulated at 24 h compared to 0 h, with a continuous rise in expression (Figure 9).  Following NaCl treatment, the expression of MeSRS1, MeSRS2, MeSRS5, MeSRS7, and MeSRS8 increased considerably at 4 h or 12 h and then decreased after the peak time point. MeSRS3 was progressively suppressed, and its expression was downregulated at 24 h compared to 0 h. MeSRS4 levels were much lower after 12 h, then rose after 24 h and eventually peaked at 24 h. MeSRS6 was shown to be considerably up-regulated after 24 h (Figure 9). The expression of most MeSRS genes was considerably affected by PEG treatment, except for MeSRS7, whose expression remained relatively steady at all time periods. MeSRS3, MeSRS5, and MeSRS6 levels first increased but subsequently declined, peaking at 4 h or 12 h. MeSRS1, MeSRS4, and MeSRS8 all showed similar "down-up-down" tendencies from 0 h to 24 h. MeSRS3 was significantly upregulated at 24 h compared to 0 h, with a continuous rise in expression (Figure 9).  Taken together, most MeSRS genes were significantly affected by a variety of treatments. It was hypothesized that different MeSRS genes had diverse roles and used different coping techniques in response to stress or phytohormone signals in cassava.

Discussion
SRS is a plant TF family that has an important regulatory function in plant development and growth. In A. thaliana, SRS family genes have been well demonstrated to play vital roles in regulating photomorphogenesis [21,22], floral organ development [14,18], lateral root formation [17,21], carpel development [15], and phytohormone response [10,16]. Recent studies have further confirmed the involvement of SRS genes in abiotic stresses and phytohormone response [17,27,28,50], and their potential utilization value for genetic improvement of crop resistance. Although studies have been performed on SRS genes in several plant species [17,[27][28][29][30][31], no systematic research on the SRS gene family in cassava has been reported. It is necessary to investigate the possible role of SRS in cassava. In this study, eight SRS genes were discovered in the cassava genome and named MeSRS1 through MeSRS8 based on their chromosomal placement (Table 1; Figure 4). A higher number of SRS genes were found in the cassava genome than in O. sativa (n = 6) [29], but fewer than in A. thaliana (n = 10) [18,51], P. trichocarpa (n = 10) [52], Z. mays (n = Taken together, most MeSRS genes were significantly affected by a variety of treatments. It was hypothesized that different MeSRS genes had diverse roles and used different coping techniques in response to stress or phytohormone signals in cassava.

Discussion
SRS is a plant TF family that has an important regulatory function in plant development and growth. In A. thaliana, SRS family genes have been well demonstrated to play vital roles in regulating photomorphogenesis [21,22], floral organ development [14,18], lateral root formation [17,21], carpel development [15], and phytohormone response [10,16]. Recent studies have further confirmed the involvement of SRS genes in abiotic stresses and phytohormone response [17,27,28,50], and their potential utilization value for genetic improvement of crop resistance. Although studies have been performed on SRS genes in several plant species [17,[27][28][29][30][31], no systematic research on the SRS gene family in cassava has been reported. It is necessary to investigate the possible role of SRS in cassava. In this study, eight SRS genes were discovered in the cassava genome and named MeSRS1 through MeSRS8 based on their chromosomal placement (Table 1; Figure 4). A higher number of SRS genes were found in the cassava genome than in O. sativa (n = 6) [29], but fewer than in A. thaliana (n = 10) [18,51], P. trichocarpa (n = 10) [52], Z. mays (n = 11) [30], and G. max (n = 21) [17]. The number of SRS family genes between species may be related to gene duplication events and genome size [53].
The phylogenetic analysis of 43 SRS proteins from A. thaliana, O. sativa, S. oleracea, P. trichocarpa, and R. communis allowed us to analyze the evolutionary history and estimate the recurring occurrences during the growth of the SRS family. SRS proteins from six species were classified into six classes, with group VI having the most members (10) and being present in all six investigated species. Group IV was a monocot-specific group, whereas groups I, II, III and V had dicot members (Figure 2), which was consistent with He et al. [30]. It has been demonstrated that the four SRS protein subfamilies evolved through duplication of a common ancestor prior to the divergence of the monocot and eudicot lineages. The results of phylogenetic trees suggest that the grouping link is more likely when the functions are comparable [54]. Eight MeSRS genes, for example, are derived from groups I, II, III, V and VI, which have closer phylogenetic relationships and genetic structures with A. thaliana (Figures 2 and 3A). This implies that MeSRS members could be operationally redundant and regulate auxin homeostasis in a dose-dependent manner during plant development [18]. SRS genes in plants had highly conserved exon/intron architectures. Exons in MeSRS genes in cassava vary from 2 to 3 ( Figure 3B), which is comparable to O. sativa (2) [29], Z. mays (2-3) [30], G. max (2)(3)(4) [17], and M. sativa (2)(3)(4) [27]. We found six conserved motifs that helped us understand structural similarities among MeSRS proteins ( Figure 3C; Figure 3D). Motif 1 was linked to RING-like region domains, whereas motif 2 was linked to IXGH domains. Other motifs were either increased or removed and shifted, which might be connected to the distinct genetic variety created by natural selection in the face of varied adversities in cassava ( Figure 3D). This also suggested that the MeSRS proteins might have different functions.
The major driving mechanisms for gene family expansion in diverse plant species are gene duplication events, including segmental, tandem, and whole-genome duplications. Genetic mapping of MeSRS genes found that eight MeSRS genes were distributed across six chromosomes, with the number of genes ranging from one to two on each chromosome ( Figure 4A). Analysis of gene duplication events identified eight pairs of duplicated genes with strong nucleotide sequence similarity. Furthermore, both Ka/Ks ratios were lower than one, suggesting that all duplicated MeSRS gene family members were under purifying selection pressure after gene duplications ( Figure 4B; Table 2). In M. albus SRS genes, eight pairs of segmental duplication genes were identified, with gene duplication playing a key role in gene family expansion [28]. Two segmental duplications of OsSRS genes were observed in O. sativa [29]. Additionally, synteny analysis showed at least 16 orthologous gene pairs between cassava and the other two dicot species, P. trichocarpa and A. thaliana, but only two orthologous gene pairs between cassava and the monocot species O. sativa, suggesting that these gene pairs emerged after the divergence of dicot and monocot plants ( Figure 4C). The phylogenetic tree also supports this hypothesis, demonstrating that cassava was more closely linked to P. trichocarpa than to A. thaliana and O. sativa (Figure 2).
A cis-acting regulatory element is a non-coding DNA segment found in the promoter region of genes. Drought sensitive (MBS, MYB), low temperature responsive (LTR), and abscisic acid (ABA) responsive (ABRE) cis-regulatory elements were found in various MeSRS promoters ( Figure 5), showing that these genes might be activated by ABA and other abiotic stimuli [55,56]. Similarly, MYC responds to salt, drought, and ABA stresses by forming complexes with MYC-binding proteins [55,57,58]. In addition, three MeSRS genes had a W-box element ( Figure 5), which is the DNA binding site of the WRKY protein produced by salicylic acid and has a role in disease defense [59][60][61]. Phytohormones are also required for plant response to abiotic stresses [62]. In this work, we determined that the MeSRS gene contains a variety of hormone response elements, including ethylene (ERE), gibberellin (TATC-box), MeJA (CGTCA/TGACG-motif), and IAA (TGA-element) ( Figure 5). The cis-acting elements analysis suggests that the function of the MeSRS genes may play a role in abiotic and biotic stress responses, which is consistent with the findings of SRS gene family research in G. max [17].
Phytohormones and environmental stresses have a significant impact on crop development and growth. Previous research found that TFs play a role in many plants' abiotic and biotic stress responses [63][64][65][66]. Plants contain putative stress or phytohormonerelated SRS genes [28,30]. A recent study also discovered that GmSRS18 is present in the cell nucleus of G. max and can improve the susceptibility of transgenic A. thaliana to drought and salt stresses [17]. In this investigation, qRT-PCR was utilized to confirm the expression level of MeSRS genes under different conditions of phytohormone induction (SA and MeJA), osmotic pressure (PEG), and salt stress (NaCl) (Figures 8 and 9). MeSRS genes responded differently to various hormone treatments. For example, MeSRS1 and MeSRS2 may be inhibited by SA but stimulated by MeJA (Figure 8), implying that SA and MeJA trigger antagonistic pathways. Almost all MeSRS genes were upregulated following PEG and NaCl treatments (Figure 9), suggesting that they play a role in the regulation of diverse stress-related responses. Interestingly, the changes in gene expression and time across various treatments are not simple linear connections, indicating that the regulation of distinct MeSRS family members is a complex process, as corroborated by other studies [28,47]. Although our findings suggest that the MeSRS genes respond to phytohormone stimulation and abiotic stress, much more research is required to fully understand this response mechanism.
In all, we identified eight non-redundant MeSRS genes in the cassava genome. A comprehensive genome-wide investigation of the MeSRS gene family was carried out, including phylogenetic analysis and gene architecture, chromosomal position, evolutionary traits, and a functional network. Following that, the cis-elements in the promoter region were studied, as well as the expression patterns in different tissues and in response to diverse stimuli, using transcriptome or qRT-PCR analysis. Our findings will be valuable for future functional characterization of MeSRS genes in cassava, and for improving the adaptive ability of plants exposed to environmental challenges.