Genome-wide identification of SCL gene family in potato (Solanum tuberosum L.) and expression analyses under phytohormones, drought and salt stress

Abstract

Background

Abiotic stresses, such as drought and salinity, severely limit the yield and quality of potato. As a pivotal subfamily of GRAS transcription factors, SCARECROW-like (SCL) proteins play essential roles in phytohormone signaling and stress adaptation; however, their systematic characterization and functional mechanisms in potato remain largely unexplored.

Results

In this study, we conducted a genome-wide identification of the SCL gene family and identified 71 StSCL genes in potato. Phylogenetic analysis revealed that potato StSCL proteins share the closest evolutionary relationship with their counterparts in tomato. Structural and promoter analyses showed that StSCL members contain conserved GRAS and DELLA domains and harbor abundant cis-acting elements responsive to ABA, SA, JA, GA, and diverse abiotic stresses. Protein-protein interaction (PPI) network analysis predicted StSCL13, StSCL24, and StSCL66 as potential hub genes. Expression profiling revealed obvious tissue-specific expression patterns and widespread responses to phytohormone and stress treatments. StSCL3 was selected for further functional characterization due to its intact and conserved GRAS domain, as well as the proven hormonal and pathogen-responsive functions of its homolog in our previous study. Subcellular localization assay indicated that StSCL3 is a nuclear-localized protein. Its expression was significantly induced by ABA, GA, SA, JA, and drought stress but repressed by salt stress. Such divergent expression responses indicate that StSCL3 may act as a critical signal integrator that coordinates multiple phytohormone pathways to regulate plant growth and stress adaptation.

Conclusions

Collectively, this study elucidates the evolutionary characteristics and regulatory mechanisms of the potato SCL family, highlighting the pivotal role of StSCL3 in hormone signaling and abiotic stress responses, and providing a theoretical foundation for improving stress resistance in potato.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12870-026-09490-x.

Background

As a globally important staple and vegetable crop, potato (Solanum tuberosum) frequently suffers from diverse abiotic stresses, which severely restrict yield stability and commodity quality [1]. Drought and high salinity are predominant adverse factors that largely limit high-efficiency potato production [2]. Accordingly, exploring stress-responsive molecular regulatory mechanisms and screening core functional genes are essential for breeding stress-tolerant potato cultivars and safeguarding food supply.

Transcription factors serve as core modulators that govern plant stress response networks. SCARECROW-like (SCL) proteins belong to the GRAS transcription factor family, named after three representative members: GAI, RGA and SCR [3]. The founding family gene SCR was first cloned from Arabidopsis thaliana root developmental mutants, and it participates in transcriptional regulation and determines root radial tissue patterning [4]. Typical SCL proteins possess a highly conserved C-terminal GRAS domain and a variable N-terminal region carrying nuclear localization signals [5]. The flexible N-terminal intrinsically disordered region confers functional versatility, making SCLs representative intrinsically disordered proteins in plants [6]. Found in over 60 plant species, GRAS/SCL families exhibit striking differences in gene number between species, with the count ranging from 33 in Arabidopsis to 188 in Triticum aestivum [7]. Evolutionary evidence indicates that GRAS genes originated from bacteria and facilitated the formation of regulatory networks during early terrestrial plant adaptation [8].

SCL transcription factors widely participate in plant growth, development and stress adaptation [9]. In developmental processes, they regulate meristem activity, root morphogenesis, somatic embryogenesis, leaf growth and secondary metabolism [10]. For example, AtSCL23 modulates shoot apical meristem development dependent on auxin signaling, and tomato SlSCL3 controls volatile terpene synthesis [11]. In stress adaptation, SCL members confer tolerance to abiotic adversity and pathogen infection. ThSCL32 improves salt resistance by activating downstream stress genes, while cotton GhSCL13-2 A enhances Verticillium wilt resistance via JA and SA signal crosstalk [12]. Gene expression of SCLs is mainly modulated by miRNA modules and protein complexes, and SCL proteins can also maintain ROS homeostasis by interacting with redox-related proteins [13].

Although SCL gene family have been characterized in numerous plant species, systematic research in potato remains insufficient. Genome-wide identification, evolutionary classification and structural features of the potato SCL gene family have not been comprehensively clarified [14, 15]. Meanwhile, expression responses of StSCL genes to phytohormone stimuli and major abiotic stresses lack systematic exploration, which restricts the understanding of their biological functions in potato. Genome-wide family analysis combined with expression profiling has been proven as a reliable strategy to excavate gene functional divergence in crops [16, 17].

Here, we systematically identified the StSCL gene family based on the annotated potato genome. This study focuses on genome-wide identification, bioinformatics functional prediction and expression profiling analysis of potato StSCL gene family. We comprehensively analyzed gene structure, phylogenetic evolution, chromosomal localization and conserved domains. qRT-PCR was further applied to detect expression variations of StSCL genes under GA, auxin, drought and salt treatments. This work aims to reveal the evolutionary characteristics and stress response patterns of potato SCL gene family, and lays a theoretical basis for dissecting their regulatory roles in plant growth and abiotic tolerance, as well as providing candidate genes for potato genetic resistance improvement.

Results and analysis

Identification and physicochemical properties of StSCLs

Initial candidate SCL genes in potato were validated using the BLASTp tool from the NCBI database (https://www.ncbi.nlm.nih.gov/) via sequence alignment to ensure their classification as SCL family members. In total, 71 StSCL genes were identified and designated StSCL01 to StSCL71 based on the increasing order of their gene IDs.

Physicochemical property analysis of the encoded proteins (Table S1) showed that the protein encoded by StSCL67 was the shortest (61 aa), while that encoded by StSCL36 was the longest (826 aa). The molecular weights of these StSCL proteins ranged from 15.23 kDa (encoded by StSCL28) to 90.15 kDa (encoded by StSCL36), and their isoelectric points (pI) varied between 4.49 (encoded by StSCL38) and 8.42 (encoded by StSCL51). With the exception of proteins encoded by StSCL1, StSCL21, StSCL28, StSCL29, StSCL39, StSCL48, StSCL57, and StSCL67, the instability indices of most other StSCL proteins exceeded 40, indicating relatively low structural stability. Subcellular localization prediction revealed that: StSCL17, StSCL18, StSCL57, and StSCL64 were localized in the chloroplast; StSCL20, StSCL29, StSCL34, StSCL39, StSCL43, StSCL66, and StSCL67 to the cytoplasm; StSCL28 to the mitochondrion; StSCL54 to the peroxisome; StSCL69 to the endoplasmic reticulum; and all other StSCL proteins to the nucleus. The prediction results of the 3D structures of the StSCL gene family (Fig. S1) demonstrated high structural homology among multiple family members, specifically including StSCL2 and StSCL13, StSCL17 and StSCL18, StSCL12 and StSCL3, StSCL32 and StSCL33, StSCL26 and StSCL27, StSCL49 and StSCL50, StSCL55 and StSCL56, StSCL61 and StSCL62, StSCL23 and StSCL51, StSCL41 and StSCL42, and StSCL45 and StSCL46.

Chromosomal distribution of StSCL genes

To clarify the distribution pattern of potato SCL genes on chromosomes, we generated a physical localization map of StSCLs (Fig. 1). The 71 StSCLs were unevenly distributed across the 12 chromosomes of potato: Chromosomes 06 and 07 contained the largest number of StSCLs (9 each), while chromosome 3 had the fewest (only 3). The remaining chromosomes (ch01, ch02, ch04, ch05, ch08, ch09, ch10, ch11, ch12) harbored 4 to 8 StSCLs each.

Fig. 1.

Distribution of 71 StSCLs on 12 chromosomes. The gene IDs on the right side of each chromosome corresponds to the approximate location of each StSCL gene, and the chromosome number is shown at the top of each chromosome. The scale on the left is megabits (Mb)

Phylogenetic tree of StSCLs, AtSCLs, CaSCLs, SlSCLs, and NtSCLs

To elucidate the evolutionary relationships of SCL family members among different plant species, a phylogenetic tree was constructed using 71 StSCLs, 34 AtSCLs, 52 SlSCLs, 65 CaSCLs and 56 NtSCLs full-length protein sequences (Fig. 2). All 278 identified SCL proteins were classified into four distinct clades, designated Group I, II, III and IV. Group IV contained the most family members, occupying 33.2% of total proteins, followed by Group II (31.65%), Group III (17.98%) and Group I (17.26%). Evolutionary comparison demonstrated that potato StSCL proteins shared the closest phylogenetic affinity with tomato SlSCL homologs. Most StSCL members were dispersedly distributed across different branches. By contrast, relatively far genetic distances were observed between potato SCL proteins and those from Arabidopsis thaliana, Capsicum annuum and Nicotiana tabacum.

Fig. 2.

Phylogenetic tree of SCL proteins from five plant species. Species abbreviations correspond to full Latin names: Solanum tuberosum (potato, StSCL), Arabidopsis thaliana (AtSCL), Solanum lycopersicum (tomato, SlSCL), Capsicum annuum (pepper, CaSCL), Nicotiana tabacum (tobacco, NtSCL). The neighbor-joining phylogenetic tree was built via MEGA 7.0.14 software based on ClustalW-aligned full amino acid sequences, with 1000 bootstrap replications. A total of 278 SCL proteins were divided into four subgroups. Group I, II, III and IV are separately marked with orange, blue, green and purple branch lines for visual differentiation

Synteny relationships of StSCLs and AtSCLs, CaSCLs, SlSCLs, as well as NtSCLs

To further explore the potential evolutionary mechanisms of the StSCLs, we evaluated the synteny relationships of SCL genes among the five species based on sequence homology and positional information (Table S2, Fig. 3). The results showed that there were 37, 22, 14, and 5 SCL members participating in the potato-pepper, potato-Arabidopsis, potato-tomato, and potato-tobacco synteny relations, respectively. Among the above collinear gene pairs, StSCL70 with CaSCL, CaSCL, SlSCL, and SlSCL, respectively. StSCL4, StSCL33, StSCL57, each corresponded to two 2 AtSCL members (AtSCL31/AtSCL33, AtSCL04/AtSCL07, AtSCL16/AtSCL32, AtSCL31/AtSCL33), respectively. In addition, StSCL17 and StSCL58 each corresponded to 3 AtSCL members (AtSCL09/AtSCL14/AtSCL33, AtSCL06/AtSCL22/AtSCL27), respectively, indicating simultaneous collinear relations.

Fig. 3.

Synteny of SCL family in potato, Arabidopsis, tomato, tobacco, and pepper. The pink line indicates the collinearity of SCL gene pairs between potato and Arabidopsis; the blue line represents the collinearity of SCL gene pairs between potato and pepper; the green line shows the collinearity of SCL gene pairs between potato and tomato; and the golden yellow line shows the collinearity of SCL gene pairs between potato and tobacco

Intraspecific synteny analysis revealed a large number of highly homologous syntenic gene pairs within the StSCL gene family, and these gene pairs exhibited significant regional clustered distributions on all 12 chromosomes. For instance, both StSCL47 and StSCL48 are located on chromosome 1, StSCL32 and StSCL33 on chromosome 2, StSCL55 and StSCL56 on chromosome 4, StSCL62 and StSCL63 on chromosome 5, StSCL66 and StSCL67 on chromosome 6, StSCL41 and StSCL42 on chromosome 7, StSCL05 and StSCL06 on chromosome 8, StSCL17 and StSCL18 on chromosome 9, StSCL36 and StSCL37 on chromosome 10, StSCL26 and StSCL27 on chromosome 11, and StSCL08 and StSCL09 on chromosome 12 (Figs. 1 and 3).

Phylogeny of StSCLs and gene structure analysis

The phylogenetic tree of the StSCL gene family reveals that the 71 StSCL genes can be categorized into three distinct groups (Fig. 4A). Analysis of the conserved motifs within the StSCL proteins shows diverse motif compositions among family members. In addition, StSCL28 comprises motifs 3 and 4; StSCL2, StSCL5, StSCL39, and StSCL71 each possess five motifs; StSCL60 contains six motifs; StSCL7, StSCL23, StSCL25, StSCL38, StSCL58, and StSCL69 each have seven motifs; StSCL11, StSCL21, StSCL30, and StSCL36 contain eight motifs; 33 StSCLs contain nine motifs; and the remaining 21 StSCLs harbor the complete set of motifs (Fig. 4B).

Fig. 4.

Phylogenetic relationships of StSCL proteins and structures of StSCL genes. A Phylogenetic tree of 71 StSCL proteins. Different subgroups are represented by different background colors. B Distribution of conserved motifs in StSCL proteins. The length of the black line represents the size of the protein, which can be estimated by the scale at the bottom. Motifs 1 ~ 10 are distinguished by different colored rectangular boxes. C Domain analysis of StSCL proteins. Different colors represent different domains. D Intron-exon structure of the StSCL genes. Coding sequences (CDS) and introns are represented by yellow rectangular boxes and black lines, respectively

Analysis of the conserved domains in the StSCL family (Fig. 4C) indicates that 48 members contain the conserved GRAS domain, while 23 members harbor the GRAS superfamily domain, supporting the classification of SCLs within the GRAS transcription factor family. Notably, StSCL35 additionally contains a DELLA domain, StSCL58 carries an Amelogenin superfamily domain, and StSCL29 possesses a calmodulin-binding superfamily domain.

Examination of the intron-exon structures in the StSCL gene family (Fig. 4D) shows that StSCL3, StSCL4, StSCL20, StSCL34, and StSCL70 contain two exons, while the remaining 66 StSCLs possess a single exon. Notably, StSCL16 was predicted to have a two-exon structure with an extremely long intron (~ 12.7 kb) in the early PGSC v4.03 genome annotation. This anomaly was caused by the inherent limitations of early ab initio gene prediction algorithms: a fortuitous GT-AG splice signal pair between two adjacent independent genes was misidentified as an intron boundary, and the lack of sufficient transcriptome data at that time prevented correction of this error. The latest high-quality SolTub_3.0 annotation, based on long-read sequencing and comprehensive RNA-seq datasets, has corrected this mistake and reannotated the region as two separate independent genes. Therefore, StSCL16 was excluded from all subsequent functional analyses to ensure the accuracy of our results. In terms of intron numbers, 10 StSCLs contain three introns, 38 StSCLs contain two introns, 6 StSCLs contain one intron, and 18 StSCLs lack introns entirely.

Analysis of cis-acting elements in the StSCL promoters

cis-acting elements in promoter regions serve as core molecular switches for transcriptional regulation. Analysis of the promoters revealed that the 2000 bp upstream sequences of StSCL genes contain CAAT-box, TATA-box, stress-response elements, hormone-response elements, light-response elements, and development-related elements (Fig. S2). Most StSCL genes harbor CAAT-box and TATA-box, and the number of TATA-boxes generally exceeds that of CAAT-boxes (Fig. 5A), reflecting a high density and regulatory activity of transcription start sites in the promoter regions.

Fig. 5.

Distribution of cis-acting elements of StSCLs.A Distribution of core promoter elements of StSCLs. The core promoter elements were divided into two groups: CAAT-box and TATA-box, indicated by blue and orange colors, respectively. Bcis-acting elements of StSCLs in response to stress, plant hormones, light, and plant growth and development

Stress-response elements encompass 15 types, including ARE, TC-rich repeats, DRE1, MYB, DRE, SARE, MBSI, as-1, WRE3, STRE, WUN-motif, LTR, MBS, GC-motif, and H-box. Among these, ARE (anaerobic response element) and STRE (stress response element) are present at relatively high frequencies (Fig. 5B).

Hormone-response elements comprise five categories: abscisic acid-response elements (ABRE), methyl jasmonate-response elements (CGTCA-motif), salicylic acid-response elements (TCA-element), gibberellin-response elements (TATC-box, GARE-motif, P-box), and auxin-response elements (TGA-element, TGACG-motif, ERE) (Fig. 5B). Notably, 54% of StSCL genes contain ABRE, and 51% possess auxin-response elements, which aligns with the finding that 48 StSCL members harbor the conserved GRAS domain and 23 members contain the GRAS superfamily domain (Fig. 4C).

Light-response elements include nine types: ACE, AE-box, box S, chs.CMA1a, I-box, MRE, G-box, CAT-box, and CCGTCC-box. Among these, 59% of StSCL genes contain the G-box.

Development-related elements comprise only four types: A-box, O2-site, circadian, and GCN4_motif, and each StSCL gene contains relatively few elements of this category (Fig. 5B). Specifically, 30% of StSCL genes contain the O2-site, 15% harbor the A-box, 14% possess GCN4_motif, and 8.5% include the circadian element.

GO enrichment analysis of StSCLs

Gene Ontology (GO) enrichment analysis revealed that StSCLs are primarily enriched in 28 biological processes (BPs) (Fig. 6). For example, 53 StSCLs are involved in DNA-templated transcription regulation, regulation of RNA biosynthetic processes, andnucleobase-containing compound metabolic processes, with extremely high -log₁₀ (p-value) scores, indicating their central role in regulating gene expression and various biomolecular metabolic cascades. In addition, several genes are enriched in plant developmental processes such as vascular sheath cell fate determination, radial pattern formation, and branch morphogenesis. Although each of these GO terms contains only two StSCLs, the results suggest that, beyond their core regulatory and metabolic functions, StSCLs also participate in cell fate specification and morphogenesis. Moreover, StSCLs are enriched in cellular components (CCs), including the nucleus (7 StSCLs), intracellular part (9 StSCLs), and intracellular region (9 StSCLs). In terms of molecular functions (MFs), only two StSCLs are enriched in sequence-specific DNA binding.

Fig. 6.

GO analysis of StSCLs. Gene Ontology (GO) terms of molecular function (MF), biological process (BP), and cellular component (CC) categories for StSCLs were enriched using the g: Profiler Analyzer tool. The number on the right shows the gene counts associated with the GO terms

Protein-protein interaction analysis of StSCLs

Based on the STRING database, the protein-protein interaction (PPI) network among members of the potato StSCL family was further analyzed (Fig. 7 and Table S3). The results revealed that 51 StSCL members formed a complex interaction network through 610 interaction edges. Among them, StSCL13, StSCL24, and StSCL66 had the highest number of interacting partners (35 each), followed by StSCL01 and StSCL54 (each interacting with 34 proteins), suggesting that these five genes may serve as potential central nodes within this predicted network. In contrast, StSCL52 exhibited the fewest interactions (only 8 partners: StSCL21, StSCL23, StSCL29, StSCL40, StSCL48, StSCL59, StSCL61, and StSCL68). Although StSCL52 lacks a high-homology 3D structure (Fig. S1) and contains relatively few types and numbers of response elements, its core interacting partners (such as StSCL13, StSCL24, and StSCL66) are enriched with elements like ABRE and STRE, which are associated with hormone response and stress adaptation. It should be noted that all predicted interactions are based on orthology relationships in the STRING database and require experimental validation (e.g., Y2H or Co-IP assays) in future studies.

Fig. 7.

Protein-protein interaction (PPI) network of the StSCLs. The nodes in the network represent StSCL family members, and the grey lines indicate the protein-protein interaction relationships between different StSCLs. The orange nodes highlight the core hub members in the PPI network (including StSCL01, StSCL06, StSCL13, StSCL24, StSCL27, StSCL28, StSCL30, StSCL46, StSCL54, StSCL65 and StSCL66), which exhibit more extensive interaction connections (more grey lines) compared to other members, suggesting their key regulatory roles in the StSCL family interaction network

Strong interaction relationships (| r | > 0.95) further indicated that StSCL13 strongly interacts with StSCL01, StSCL06, StSCL07, StSCL17, StSCL24, StSCL27, StSCL28, StSCL30, StSCL32, StSCL43, StSCL46, StSCL54, and StSCL55; StSCL24 strongly interacts with StSCL01, StSCL06, StSCL07, StSCL13,StSCL17, StSCL27, StSCL28, StSCL30, StSCL32, StSCL43, StSCL46, StSCL54, StSCL55, StSCL58, StSCL65, StSCL66, and StSCL70; and StSCL66 strongly interacts with StSCL01, StSCL06, StSCL13, StSCL15, StSCL24, StSCL27, StSCL28, StSCL30, StSCL46, StSCL50, StSCL54, StSCL65, and StSCL67 (Table S3). These predicted strong interaction clusters may participate in regulatory processes associated with the biological functions of the StSCL family. Additionally, 20 StSCL members (StSCL02, StSCL03, StSCL04, StSCL05, StSCL08, StSCL12, StSCL18, StSCL26, StSCL31, StSCL33, StSCL34, StSCL39, StSCL41, StSCL45, StSCL49, StSCL51, StSCL56, StSCL62, StSCL64, and StSCL69) showed no detectable interactions in the prediction, possibly due to sequence divergence or structural differences that preclude effective protein binding.

Expression profile analysis of StSCL genes

The SCL gene family plays a central role in maintaining meristems, regulating root development, orchestrating secondary metabolism, and mediating diverse stress responses. However, functional information on StSCLs remains limited. To investigate their roles, we analyzed transcriptome data of the StSCL gene family across different tissues and under various treatments, visualizing the expression patterns of 55 differentially expressed StSCLs (Fig. 8A, Table S4).

Fig. 8.

Relative expression profiles of StSCLs. A Expression profiles of StSCLs in different tissues or developmental stages; B Expression profiles of StSCLs during tuber development; C Expression profiles of StSCLs under abiotic stress and plant hormone treatments; D Expression profiles of StSCLs under biotic stress. The color scale below the heat map represents the expression levels: red indicates high transcript abundance, and blue indicates low transcript abundance

Tissue development heatmap analysis (Fig. 8A) showed an overall marked transcriptional downregulation of StSCLs. Specifically, 22 StSCLs were downregulated across all six tissues examined, with StSCL14, StSCL17, StSCL32, and StSCL35 showing pronounced downregulation, especially StSCL32 and StSCL35. In leaves, whole mature flowers, mature whole fruit, tubers, roots, and stolons, only 2, 5, 5, 9, 10, and 16 StSCLs were upregulated, respectively. These findings align with the observation that the StSCLs contains only four types of development-related cis-acting elements (A-box, O₂-site, circadian, and GCN4_motif), each present in low copy numbers per gene (Fig. 5B).

The tuber development heatmap (Fig. 8B) revealed a relatively balanced up- and downregulation of StSCLs across seven tuber developmental stages. Fourteen StSCLs (StSCL6, StSCL7, StSCL15, StSCL19, StSCL21, StSCL34, StSCL47, StSCL48, StSCL51, StSCL59, StSCL61, StSCL68, StSCL69, and StSCL70) were upregulated at all stages, with StSCL6 and StSCL59 showing the most pronounced upregulation. Thirteen StSCLs (StSCL14, StSCL16, StSCL22, StSCL25, StSCL29, StSCL32, StSCL42, StSCL50, StSCL54, StSCL55, and StSCL57) were downregulated across all stages, with StSCL54 and StSCL55 displaying the strongest downregulation. StSCL35 was notably upregulated in young tubers but significantly downregulated in the other six stages, which correlates with its DELLA domain, a region often involved in stage-specific regulation of plant growth and development (Fig. 4C). This pattern suggests that StSCL35 may function during early tuber differentiation but becomes inactive during tuber maturation.

Heatmap analysis under abiotic stresses and hormone treatments (Fig. 8C) indicated that, except under BAP (6-Benzylaminopurine, a synthetic plant growth regulator) treatment, StSCLs were overall markedly upregulated under the other seven treatments, particularly under mannitol (27 StSCLs) and salt stress (25 StSCLs). Eleven StSCLs were commonly upregulated under both water stress and mannitol-induced drought, suggesting that these genes represent core family members in potato drought response. In contrast, the responsive genes under heat and NaCl stress were relatively distinct, reflecting stress-specific regulation within the StSCL gene family. Under BAP treatment, only StSCL46 was upregulated, while the remaining StSCLs were downregulated or unchanged, consistent with the tissue development heatmap results (Fig. 8A). The StSCLs exhibited clear functional divergence in response to three plant hormones: 12 genes (StSCL1, StSCL4, StSCL22, StSCL25, StSCL27, StSCL40, StSCL42, StSCL46, StSCL54, StSCL60, StSCL65, and StSCL66) were upregulated, whereas seven (StSCL6, StSCL9, StSCL11, StSCL14, StSCL17, StSCL36, and StSCL58) were downregulated, likely to maintain hormonal homeostasis and avoid physiological damage from overresponse. StSCL46 showed strong upregulation, indicating that it may be recognized and bound by transcription factors from all three hormone pathways to activate transcription and mediate cross-talk among them, consistent with the enrichment of corresponding hormone-response cis-elements in its promoter region (Fig. 5B) and the predominance of stress-responsive genes among its strong interaction partners (Fig. 7). StSCL6 was downregulated under all eight treatments, which correlates with the limited number of stress-response elements in its promoter (Fig. 5), suggesting that it primarily functions in tuber development (Fig. 8B) and shows low sensitivity to stress and exogenous hormones.

Heatmap analysis under biotic stresses (Fig. 8D) revealed that after inoculation with Phytophthora infestans, StSCLs showed differential up-/down-regulation, indicating potential involvement in basal or induced immune pathways against pathogens. Notably, StSCL23 was upregulated, while StSCL54 was downregulated. Given that both can interact via StSCL6 or StSCL20 (Fig. 7), we speculate that StSCL23 acts as a positive regulator of late blight resistance by activating defense responses, whereas StSCL54 serves as a negative regulator that relieves suppression of resistance pathways. They may form protein complexes with StSCL23 or StSCL54 to finely tune the immune response to P. infestans. Under treatment with BABA and BTH (both disease-signal inducers), 29 StSCLs were upregulated under both conditions, suggesting that they may represent conserved core responders in disease resistance regulation. Moreover, 21 StSCLs were synchronously upregulated under all three biotic stresses, aligning with the enrichment of TC-rich repeats (a defense-related element) in their promoter regions (Fig. 5).

Expression analysis of StSCL3 under hormone, drought and salt stress

Among the 16 StSCL members that showed no significant transcriptional differences in the previous heatmap analysis (Fig. 8), StSCL3 contains a complete conserved GRAS domain (Fig. 4). Since GRAS family proteins have been widely confirmed to participate in phytohormone signaling and stress response regulation, the structural integrity of StSCL3 supports the execution of its disease-related functions. Furthermore, cis-element analysis revealed that the promoter region of StSCL3 is enriched with multiple hormone-responsive elements, such as abscisic acid (ABRE), salicylic acid (TCA-element), and methyl jasmonate (CGTCA-motif) (Fig. 5). The reason for selecting StSCL3 as the representative of this gene family is as follow: Our earlier work [18] demonstrated that its homolog Stsl-1 was upregulated upon Ralstonia solanacearum challenge and treatments with abscisic acid, salicylic acid and methyl jasmonate. Since the original diploid potato genotype DM13 carrying sl-1 cannot be used owing to patent constraints, we selected the homologous gene StSCL3 from our present material, the diploid line Solanum phureja DM1-3 516 R44. Consequently, StSCL3 was selected for RT-qPCR analysis under four phytohormone treatments (ABA, GA, SA, MeJA) as well as salt and drought stress (Fig. 9, Table S5). The results showed that StSCL3 responded most rapidly to GA treatment, peaking on day 1 after treatment (Fig. 9B). Under ABA and SA treatments, the expression peaks of StSCL3 appeared on day 3 (Fig. 9A, C). For SA, the delay may be attributed to processes such as cytoplasmic-nuclear translocation of the NPR1 protein and assembly of transcriptional complexes [19]. Both pathways involve additional molecular steps, leading to longer response times. In contrast, the response to MeJA was slowest, peaking on day 4 (Fig. 9D). Among the treatments, ABA induced the most pronounced upregulation of StSCL3, followed by MeJA, GA, and SA. These findings suggest that StSCL3 is involved in multiple plant hormone related signaling pathways.

Fig. 9.

Expression of StSCL3 under hormone, salt, and drought stress. Expression analysis of StSCL3 under ABA (A), GA (B), SA (C), MeJA (D), drought (E), and salt (F) treatment by RT-qPCR. According to the One-way ANOVA analysis and multiple comparison tests, expression of StActin was used as a reference gene, and expression of StSCL3 at 0 time-point was set as 1. The x-axis represents the time points after treatment. Error bars represent the standard deviations from three biological replicates. Asterisks indicate that the expression levels of treatment groups show a significant difference in transcript abundance compared with the control. (ns: no significant difference, p < 0.05, p < 0.01)

Under drought stress, StSCL3 was significantly upregulated on day 2 (Fig. 9E). Combined with the presence of stress-response elements in the StSCL3 promoter (Fig. 5B, which are more inclined to respond to osmotic stress and hypoxia signals caused by drought) and the GO enrichment results (Fig. 6, indicating that the StSCL gene family is primarily involved in hormone-mediated stress responses), we speculate that drought stress activates StSCL3 expression via ABRE elements, enabling it to play an important role in potato drought adaptation.

In contrast, StSCL3 showed consistent downregulation under salt stress (Fig. 9F). This downregulation may involve two mechanisms: first, salt stress may suppress hormone pathways that normally upregulate StSCL3; second, the plant may downregulate genes unrelated to salt-tolerance mechanisms to better adapt to the saline environment.

The StSCL3 protein localizes in the nucleus

Based on bioinformatics online prediction, the StSCL3 protein is mainly localized in the nucleus. To further verify this prediction, the full-length coding sequence of the target gene StSCL3 (1608 bp, with 3 bp as the stop codon) was amplified from potato cDNA by PCR, and the primers are listed in Supplementary Table 6. The StSCL3 gene was digested with restriction enzymes Sal I and Kpn I (Fig. S3). Subsequently, this fragment was cloned into the pCAMBIAsuper1300 empty vector digested with the same two enzymes (Fig. 10). The recombinant plasmid was transformed into Escherichia coli competent cells TOP10. After sequence verification, the recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101. Agrobacterium-mediated infiltration was used for transient expression of the recombinant plasmid in tobacco epidermal cells to perform subcellular localization. The p1300-35 S-H2B-mCherry vector was used as a nuclear marker (red fluorescence) to detect the GFP fluorescence signal. Fluorescence signal of the 35 S:StSCL3-GFP fusion vector (Fig. S3) was detected in the nucleus, indicating that the StSCL3 protein is localized in the nucleus (Fig. 10). This result is consistent with previous bioinformatics predictions and functional analysis conclusions, confirming that StSCL3 can regulate the transcription of genes related to hormone signal transduction and stress response by binding to cis-acting elements in the promoter region of target genes in the nucleus.

Fig. 10.

Subcellular localization analysis of 35 S: StSCL3-GFP. The 35 S: StSCL3-GFP fusion protein were delivered into cells via A. tumefaciens and transiently expressed in tobacco leaves, then visualized after 18 h of transformation using confocal laser scanning microscopy. p1300-35 S-H2B-mCherry (nuclear marker) red fluorescence was used as control. Under the green fluorescence excitation channel, StSCL3-GFP clearly localized to the nucleus (green), which appears yellow in the merged image. Scale bars = 25 μm

Discussion

Soil salinization, drought stress, and pathogen infection represent major abiotic and biotic constraints limiting the growth, development, and yield formation of potato [20–22]. The SCL gene family, a pivotal subfamily of plant-specific GRAS transcription factors, is known to play critical and conserved roles in phytohormone signaling, stress responses, and developmental regulation, including root patterning and gibberellin signaling [23]. Although SCL genes have been extensively studied in species such as Arabidopsis [7], cucumber (Cucumis sativus) [24], pear (Pyrus bretschneideri) [25], pine (Pinus taeda) [26], and wheat (T. aestivum) [27], comprehensive genome-wide functional characterization of the StSCL gene family in potato remains limited. Our preliminary analysis previously showed that a member of the SCL family in potatoes (named Stsl-1) is elicted by the exogenous phytohormones such as MeJA, ABA, and SA. Building on this preliminary finding, the present study performed a systematic genome-wide analysis of the StSCL family, covering evolutionary conservation, gene structural features, cis-regulatory elements, protein interaction networks, and expression patterns under phytohormone, drought, and salt stress, aiming to clarify the potential functional diversity and stress response characteristics of potato SCL genes.

Characteristics and evolution of the StSCLs

In this study, a total of 71 StSCL genes were identified (Table S1). These genes were found to be unevenly distributed across the 12 chromosomes of potato (Fig. 1). This pattern, which is consistent with the non-uniform distribution of SlSCL genes [28], is common among plant multi-gene families and is often associated with genomic evolutionary events such as chromosomal segment duplications and tandem repeats [29]. Intraspecific synteny analysis revealed a large number of highly homologous syntenic gene pairs within the StSCL gene family, and these gene pairs exhibited significant regional clustered distribution characteristics on all 12 chromosomes (Figs. 1 and 3). These results indicated that tandem duplication is a major driving force for the expansion of the StSCL gene family. Meanwhile, the regional clustered distribution pattern on chromosomes also suggests that these homologous genes may originate from a common ancestral gene, which have been retained through gene duplication events during evolution. They are likely to maintain similar functions or participate in coordinated regulatory pathways, providing an evolutionary basis for the stability and diversity of the functions of the StSCL gene family.

To further explore the potential evolutionary mechanisms of the StSCLs, we evaluated the synteny relationships of SCL genes among the five species based on sequence homology and positional information (Table S2, Fig. 3). The results showed that there were 37, 22, 14, and 5 SCL members participating in the potato-pepper, potato-Arabidopsis, potato-tomato, and potato-tobacco synteny relations, respectively. Among these collinear pairs, StSCL70 has orthologs in pepper and tomato. StSCL4, StSCL33, StSCL57 and StSCL66 each match two Arabidopsis SCL genes, while StSCL17 and StSCL58 each correspond to three Arabidopsis SCL members. The difference in the number of such interspecific syntenic gene pairs indicates that SCLs are relatively conserved during plant evolution, and also represents one of the origin characteristics of homologous genes [23]. This is consistent with the phenomenon that multiple syntenic SCL gene pairs exist between the genomes of wheat and Arabidopsis, as well as Oryza sativa, further confirming that the conservation of SCL genes is cross-species universal [30]. Collectively, this suggests that these genes may play important roles during plant evolution and indicates that whole-genome or segmental duplications have been major drivers for the expansion of the StSCL gene family in solanaceous species [31].

Structural characterization, including 3D protein structure simulation (Fig. S1), physicochemical property prediction, conserved domain identification, and gene structure analysis (Fig. 4), revealed the structural basis for functional differentiation of StSCL members. All identified StSCL proteins harbor typical GRAS or GRAS superfamily domains, confirming their identity as canonical GRAS transcription factors. Consistent with the transcriptional regulatory function of GRAS family proteins, most StSCLs were predicted to localize in the nucleus [32]. High structural homology in conserved domains and 3D structures among StSCL members indicates potential functional redundancy and cooperative interaction, which is consistent with the conserved functional characteristics of SCL genes in other plant species [33]. For instance, tomato SlSCL3 shares high sequence and 3D structural homology with AtSCL3, both containing conserved GRAS domains (LRI, VHIID, LRII) that support conserved metabolic regulatory roles [34–35].

Phylogenetic analysis divided the StSCL family into four distinct subgroups (Fig. 2), and StSCL proteins showed the closest homology with tomato SCL members, which is consistent with the close genetic relationship between the two solanaceous species [36]. The subgroup classification is not random but corresponds to functional differentiation of the gene family. Group IV contains the largest number of StSCL members, implying that this clade retains core conserved functions in plant growth regulation and stress response during long-term evolution. In contrast, Groups I and III with fewer members may represent lineage-specific divergent genes that acquire specialized functions during potato genome evolution [37].

Gene structure and promoter analysis of the StSCLs

Consistent with their annotation as transcription regulators, most StSCL genes possess complete GRAS domains and are predicted to localize in the nucleus. Promoter cis-element analysis identified abundant phytohormone and stress-responsive elements, including ABRE, TCA-element, and CGTCA-motif, which are highly consistent with the known hormone-signaling regulatory functions of GRAS proteins [37]. The matching pattern between conserved structural domains and upstream cis-regulatory elements indicates that diverse hormone signals may act as upstream regulatory switches to control the transcriptional activity of StSCL genes.

Despite the conserved core GRAS domain, prominent structural divergence was observed among StSCL members, leading to functional specialization. Several genes carry unique additional domains: StSCL35 contains a DELLA domain associated with gibberellin signaling, StSCL58 harbors an Amelogenin superfamily domain, and StSCL29 possesses a calmodulin-binding superfamily domain (Fig. 4C) [38]. Such domain fusion events are typical evolutionary strategies for plant gene functional differentiation, similar to the pleiotropic function acquisition of rice Ghd7 [39].

Exon-intron structure analysis further revealed significant structural diversity in the StSCL family. Most StSCL genes are single-exon genes, while a small number contain 1–3 introns, consistent with the compact gene structure characteristics of the GRAS family in Arabidopsis [40]. Notably, StSCL67 only encodes an ultra-short protein of 61 amino acids and retains only motif 6 without complete GRAS conserved motifs. This structural defect indicates that is likely a truncated gene or a pseudogene generated during potato genome evolution, which may have lost canonical transcriptional regulatory functions or acquired new specialized regulatory roles [41]. Variable intron numbers among family members also suggest that intron gain and loss events occurred during StSCLs evolution, which may enrich transcript diversity through alternative splicing.

Promoter cis-element analysis demonstrated that StSCL promoters contain abundant light, hormone, and stress-responsive motifs. The widespread distribution of core transcription initiation elements (CAAT-box, TATA-box) confirms their transcriptional activity in vivo. G-box, the most abundant light-responsive element, endows StSCLs with potential light-signal transduction functions, consistent with the coordinated regulatory model of light and hormone signals in wheat TaSCL genes [42]. Combined with expression characteristics, the composition of cis-elements partially explains the tissue-specific and stress-induced expression patterns of StSCL genes, verifying the co-evolution relationship between gene structure, regulatory elements, and biological functions [43].

Expression patterns and functions of the StSCLs

The SCL subfamily of GRAS transcription factors is widely involved in plant growth, development, and environmental adaptation. Consistent with the reported functions of SCR, SHR, and SCL23 in root development, meristem regulation, and organ formation [44, 45], StSCL members also exhibit distinct tissue-specific expression patterns. For example, the specific high expression of StSCL46 in stolons implies its potential role in regulating stolon development. The tissue expression preference of StSCL members is generally consistent with their phylogenetic classification, indicating that evolutionary conservation largely determines functional differentiation.

Stress and hormone induction expression profiles further verify the regulatory potential of cis-elements. Most StSCL genes respond to at least one hormone or stress treatment, showing diversified expression trends. It is worth noting that all differential expression descriptions in this study only reflect expression trend changes, without statistical significance claims, due to the lack of differential expression threshold screening and statistical testing. The opposite response patterns of StSCL6 (universal downregulation) and StSCL46 (specific upregulation under single stress) reflect the functional division of labor within the StSCL family. Different members respond to distinct hormone and stress signals, enabling potato to adapt to complex variable environments through precise and layered regulatory networks [46].

Biotic stress expression analysis indicated that multiple StSCL genes are persistently downregulated under Phytophthora infestans infection, suggesting their potential roles in immune regulation. The diversified stress response modes of StSCL members lay a foundation for analyzing the molecular mechanism of potato disease resistance.

PPI network analysis predicted the interaction relationships among 51 StSCL proteins (Fig. 7). Notably, this PPI network is purely based on orthology prediction and lacks in vivo experimental verification in potato; all interaction results are preliminary predictions requiring further validation. The protein interaction capacity of StSCL members depends on the integrity of the GRAS domain. Genes with complete GRAS domains can form homodimers and heterodimers to stabilize transcription complexes, while truncated members with incomplete domains mainly function as monomers with weak interaction ability.

Genes including StSCL13, StSCL24, and StSCL66 showed relatively more predicted interaction partners in the network, which are speculated to be potential hub nodes in the StSCL regulatory network. However, their core functional status needs to be confirmed by subsequent yeast two-hybrid or Co-IP experiments. Consistent with transcriptional regulatory functions, most interactive StSCL proteins are localized in the nucleus. In addition, the expression co-regulation trend of partial StSCL genes is consistent with their predicted interaction relationships, supporting the correlation between co-expression and protein interaction [47, 48].

A total of 20 StSCL genes showed no predicted protein interaction relationships in this study, which can be attributed to multiple factors. Some genes have truncated sequences and incomplete functional domains, and some are classified as pseudogenes during iterative genome annotation, failing to generate functional proteins. Most of these genes exhibit extremely low expression levels across tissues and stress treatments, resulting in insufficient protein accumulation and losing the prerequisite for interaction. Certain genes lose core interaction domains during evolution and only bind DNA as monomers to perform regulatory functions. As potato is a non-model crop, homologous reference data in available databases is limited [43]. Besides, some proteins undergo transient specific interactions or function independently, whose connections cannot be detected by conventional bioinformatics approaches. Low-confidence genes were excluded in subsequent analyses to ensure credible research results.

Notably, there is a numerical discrepancy between GO annotation (Figs. 6 and 53 StSCLs involved in transcriptional regulation) and GRAS domain statistics (Figs. 4 and 48 full-domain genes, 23 superfamily-domain genes). This inconsistency is caused by three key reasons: (1) different databases adopt distinct sequence alignment thresholds and domain boundary definition standards for GRAS family annotation [10]; (2) some StSCL members undergo domain truncation during evolution, retaining only superfamily conserved sequences but still maintaining basic nucleic acid binding and transcriptional regulation activity; (3) a few StSCLs participate in transcriptional regulation indirectly through interacting proteins and cis-regulatory pathways, independent of complete GRAS domains [11].

The roles of the StSCLs in hormone signal transduction and abiotic stress responses

The SCL family functions as a critical hub in the plant hormone-signaling network, coordinating growth, development, and stress responses [49]. Systematic promoter analysis reveals that upstream regulatory regions are enriched with cis-acting elements responsive to abscisic acid (ABRE), salicylic acid (TCA-element), methyl jasmonate (CGTCA-motif), and auxin (TGA-element) (Fig. 5). This co-occurrence suggests that StSCLs are cross-regulated by diverse hormonal signals, acting as downstream effectors that convert perceived signals into specific transcriptional programs [50, 51].

The candidate gene StSCL3 serves as a prime example of this integration through distinct temporal dynamics. StSCL3 responded most rapidly to GA treatment, peaking on day 1, likely via the nuclear degradation of DELLA repressor proteins [52]. Conversely, peaks under ABA and SA treatments appeared on day 3, reflecting the time required for signal transduction through the “PYR/PYL-SnRK2-ABF” cascade and NPR1 nuclear translocation, respectively [53, 54]. The JA treatment triggered the slowest response (day 4) (Fig. 9), indicating a late-response role [54]. This diversity, combined with the enrichment of multiple hormone-responsive elements in its promoter, suggests that StSCL3 may function as a signal integration node in ABA-GA crosstalk to balance stress adaptation and growth, although this hypothesis requires experimental validation [37]. Furthermore, members with special domains, such as the DELLA-containing StSCL35, define specialized hormonal functions, potentially fine-tuning GA signaling during tuber development [55]. These findings align with conserved GRAS functions in model plants.

Under abiotic stress, StSCL3 exhibits divergent regulation (Fig. 9). Under drought, endogenous ABA accumulation activates the classical signaling cascade: PYR/PYL receptors activate SnRK2 kinases, which phosphorylate ABF/bZIP transcription factors to bind ABRE elements in the StSCL3 promoter, thereby enhancing drought tolerance [56, 57]. In contrast, salt stress triggers significant downregulation of StSCL3. Although salt stress induces ABA, specific ionic signals triggered by Na⁺ toxicity (involving Ca²⁺, ROS, and the SOS pathway) may antagonize ABA signaling [58]. Moreover, the absence of salt-specific cis-elements (e.g., DRE) in the StSCL3 promoter renders it insensitive to osmotic components (Fig. 5). This downregulation likely represents an adaptive strategy reallocating limited resources toward urgent physiological processes like ion homeostasis rather than growth-related signals incompatible with the stress [59]. These findings highlight the capacity of the StSCL family to decode complex environmental crosstalk [60].

While this study performed a genome-wide identification and expression profiling of the SCL gene family in potato under phytohormone, drought and salt stresses, and further validated the function of StSCL3 through gene silencing, transient overexpression and stable overexpression experiments combined with physiological and biochemical assays, disease resistance-related gene expression analysis and phenotypic observation. In addition, the biological functions of StSCL65 and StSCL66 also require further investigation. We will systematically conduct similar functional validation experiments to characterize these candidate genes in our subsequent studies.

Conclusions

This study systematically identified 71 StSCLs in potato, confirming their evolutionary conservation with tomato and expansion via tandem duplication. Structural analysis revealed conserved GRAS domains alongside diverse cis-elements, implying broad regulatory potential. Network analysis identified key hub genes, while expression profiling highlighted tissue specificity and stress responsiveness. StSCL3 was localized to the nucleus. Based on its expression profiles, it is proposed to play a role in integrating GA, ABA, SA, and JA signals, although this needs further functional validation. Crucially, StSCL3 enhanced drought tolerance via ABA signaling but was suppressed under salt stress, reflecting distinct adaptive mechanisms for ionic versus osmotic stress. These findings elucidate the regulatory mechanisms of the StSCLs and provide a foundation for improving stress resistance in potato.

Materials and methods

Plant material and treatments

The potato plants used in this study were the diploid line Solanum phureja DM1-3 516 R44, which were provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (CAAS). These plants were grown in a growth chamber under the following conditions: 26 °C/18°C (day/night) temperature regime, 60–70% relative humidity, and a 16:8 h light: dark photoperiod. When seedlings reached the 7–8 leaf stage, the following treatments were applied separately: salt stress was induced by irrigating the roots with 200 mM NaCl, while drought stress was simulated by root irrigation with 300 mM mannitol [61]. For hormone treatments, leaves were sprayed with 1 mM salicylic acid (SA), 50 µM abscisic acid (ABA), 50 µM gibberellic acid (GA₃), or 25 µM jasmonate (JA) [62]. During the spraying process, both adaxial and abaxial surfaces of all leaves were uniformly moistened to form condensed water droplets without causing runoff. Immediately after spraying, the treated plants were covered with black plastic bags, and this treatment was performed only once. Subsequently, leaf samples were collected from the treated plants at 1, 2, 3, 4, and 5 days post-treatment (with day 0 set as the control group). Each set of samples was rapidly frozen in liquid nitrogen and then stored at -80 °C for subsequent analysis. Each treatment was performed in three biological replicates.

Genome-wide identification of the SCL transcription factor family in Solanum tuberosum, Capsicum annuum, Nicotiana tabacum, and S.lycopersicum

The potato genome sequence was obtained from the Potato Genome Sequencing Consortium (PGSC, http://potato.plantbiology.msu.edu/), while the genomes of pepper, tobacco, and tomato were retrieved from the Ensembl Plants database (https://plants.ensembl.org/). First, using AtSCL protein sequences (downloaded from The Arabidopsis Information Resource, TAIR) as queries, BLAST searches were performed against the genomes of potato, pepper, tobacco, and tomato with a cutoff E-value < 10^− 5 to identify potential SCL candidate genes. Subsequently, the hidden Markov model (HMM) profile of the GRAS domain (PF03514) was acquired from the Pfam database (http://pfam.sanger.ac.uk/). Further screening was conducted using BLASTP against SMART (http://smart.embl-heidelberg.de/) and HMMER 3.0 (https://www.ebi.ac.uk/Tools/Hmmer/) with default parameters to remove redundant sequences and eliminate candidates lacking the conserved PF03514 domain.

Chromosomal localization, subcellular localization, and physicochemical property analysis of StSCLs

The physical positions of StSCL genes on each chromosome were retrieved from the potato genome database, and the chromosomal location map of these StSCL genes was plotted using TBtools software [63]. The subcellular localization of StSCL proteins was predicted via the Plants-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/). Basic physicochemical properties including sequence length, molecular weight, isoelectric point, and hydrophilicity were obtained using the ProtParam tool of ExPASy (http://web.expasy.org/Protparam/). Three-dimensional structural models of the StSCLs were predicted by UniProt (https://www.uniprot.org/).

Phylogenetic tree and collinearity analysis of StSCLs with those from four other species

First, the amino acid sequences of StSCL, AtSCL, CaSCL, SlSCL, and NtSCL were aligned using ClustalW. A phylogenetic tree was then constructed from the alignment file with MEGA7 software (http://www.megasoftware.net/) using the neighbor-joining method, and bootstrap analysis was performed with 1000 replicates. Finally, the tree was annotated and color-coded via the iTOL website (https://itol.embl.de). Additionally, homology analysis among potato, Arabidopsis, pepper, tobacco, and tomato was conducted using TBtools (v2.096), and synteny relationships among SCL family members across the five species were visualized with the Advanced Circos module (http://circos.ca/).

Analysis of phylogeny, conserved motifs, conserved domains, and gene structures of StSCLs

First, multiple sequence alignment of the StSCL gene family was performed using ClustalW. Based on the alignment results, a phylogenetic tree was constructed with the neighbor-joining (NJ) method in MEGA7 software (http://www.megasoftware.net/), and its topological reliability was assessed using 1,000 bootstrap replicates. Subsequently, conserved motifs of the StSCL protein family were predicted using the MEME online program (http://meme-suite.org/tools/meme). Sequence analysis was carried out with the EMBL-EBI Pfam 32.0 batch search tool (http://pfam.xfam.org/search#tabview=tab1). Combined with results retrieved from the NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), the conserved domains of the StSCL protein family were identified and validated. Furthermore, by aligning the DNA and cDNA sequences of StSCL genes, their intron-exon structures were predicted using the online Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn/). Finally, TBtools software was employed to integrate and visualize the results from phylogenetic tree construction, conserved motif prediction, conserved domain identification, and gene structure analysis.

Prediction of cis-acting elements, gene ontology (GO) enrichment analysis, and transcriptome-based expression profiling of StSCLs

First, upstream sequences (2000 bp from the start codon) of StSCL genes were extracted from the potato genome database. The cis-acting elements in the promoter regions of StSCLs were then predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Finally, the results were visualized via the OmicStudio platform (https://www.omicstudio.cn/home).

Gene Ontology (GO) enrichment analysis of StSCL gene sequences was performed using the online tool g: Profiler (https://biit.cs.ut.ee/gprofiler/gost) with a significance threshold of p < 0.001. The enriched terms were visualized using the Bioinformatics online platform (http://www.bioinformatics.com.cn/).

RNA-Seq data of the potato DM genotype were downloaded from the Spud DB database (http://spuddb.uga.edu/PotatoGenomicTools). The expression levels of StSCLs were analyzed across different tissues, tuber developmental stages, and under various biotic/abiotic stresses and hormone treatments. Finally, a gene expression heatmap was generated using TBtools software (version 2.0), with expression values represented as log10 (FPKM).

Protein-protein interaction (PPI) analysis of StSCLs

Using the online database STRING v12.0 (https://cn.string-db.org/), protein sequences interacting with 71 known sequences were retrieved, and a protein-protein interaction network was constructed with a confidence threshold of 0.150. The network was then visualized graphically using Cytoscape software (version 2.8.3).

RNA isolation and RT-qPCR analysis

Leaf samples were ground to a fine powder in liquid nitrogen, and total RNA was isolated using the TransZol Up Plus RNA kit (Trans, Beijing, China) following the manufacturer’s recommended protocol. Subsequently, the purified RNA was used as a template for first-strand cDNA synthesis with TransScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix for qPCR (Trans, Beijing, China), which concurrently eliminates genomic DNA contamination. All primer sequences employed in this study were designed via the NCBI Primer-BLAST tool 16 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and their detailed information is provided in Table S6. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Shanghai, China). The 20 µL reaction mixture consisted of 1 µL cDNA template, 0.4 µL of each specific primer (StSCL-F and StSCL-R), 10 µL SuperMix, 0.4 µL DyeII, and 7.8 µL nuclease-free water. The thermal cycling program was set as follows: initial denaturation at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s and annealing/extension at 60 °C for 30 s. Each sample was analyzed in triplicate, and the relative gene expression levels were calculated using the 2 − ΔΔCt method [64]. Three biological replicates were included for each sample. Following a previously described method, statistical analysis of the RT-qPCR data was performed using GraphPad Prism 10.0, where the Student’s t-test was adopted for evaluating the significance of observed differences at a threshold of p ≤ 0.05. One-way analysis of variance (ANOVA) was applied to evaluate differences between groups, with significance levels denoted as p < 0.05, p < 0.01, p < 0.001, p < 0.0001. Each experiment was repeated three times. The Ct values of StActin presented narrow distribution with a low coefficient of variation (CV = 0.99–2.3%), demonstrating that StActin was stably expressed and appropriate for use as the internal control in this study (Table S7).

Subcellular localization of StSCL3

To investigate the subcellular localization and expression pattern of StSCL3 in potato, the full-length coding sequence (CDS) of StSCL3 lacking the stop codon was cloned into the pCAMBIA1300 vector. Initially, the complete CDS of StSCL3 (1605 bp) was amplified from potato cDNA via PCR using a pair of primers with homologous arms. The amplified fragment was then inserted into the pCAMBIA1300 vector, which had been pre-linearized with the restriction enzymes Sal I and Kpn I. Subsequently, the resulting pSCL3-GFP fusion plasmid was transformed into E. coli DH5α competent cells. Positive clones were validated through bacterial liquid PCR and Sanger sequencing (Sangon Biotech, Shanghai, China). The confirmed recombinant plasmid was further introduced into A. tumefaciens strain GV3101, and single colonies were selected for PCR-based positive screening. Finally, the recombinant Agrobacterium harboring the pSCL3-GFP vector was infiltrated into tobacco leaves for transient expression assays, as described previously [65]. GFP fluorescence signals were observed and analyzed using a laser scanning confocal microscope (LSCM).

Authors' contributions

LL conceptualized and designed the study. SL interpreted and analyzed the data, and drafted the manuscript. XZ conducted the bioinformatics analysis and mapping, designed and executed the experimental work. YZ and LW assisted in enhancing the bioinformatics content and refining the visual representations. GG and WL provided project supervision and critically revised the manuscript. All authors contributed to the development of this article and approved the submitted version.

Data availability

The datasets supporting the conclusions of this article are included within the article and its supplementary files. All other relevant raw data supporting the results are available from the corresponding author. Further inquiries can be directed to the corresponding author. All related resources are publicly available.

Declarations

Ethics approval and consent to participate

All materials used in this study complies with both local and national regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.