2.1. Data retrieval of DCL, AGO, and RDR genes

To identify DCL, AGO, and RDR genes in Solanum tuberosum L., we retrieved protein sequences from the Phytozome database (v13; https://phytozome.jgi.doe.gov/) using the reference genome of S. tuberosum (Assembly: PGSC DM v4.03) [33]. For comparative analysis, reference sequences of AtDCLs, AtAGOs, and AtRDRs from Arabidopsis thaliana (L.) were obtained from The Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org) [34].

A Hidden Markov Model (HMM)-based Basic Local Alignment Search Tool (BLASTP; https://blast.ncbi.nlm.nih.gov/) search was conducted against the S. tuberosum proteome using the customized parameters (E-value ≤1 × 10^-15, identity ≥ 35%, BLOSUM62 matrix) (Fig 1) [35]. Only primary transcripts were retained to avoid redundancy. Genomic coordinates, transcript lengths, and protein sequences were extracted from Phytozome. Gene nomenclature followed phylogenetic clustering with A. thaliana orthologs.

2.2. Physicochemical characterization

Protein properties including amino acid (AA) length, molecular weight (MW; kDa) and isoelectric point (pI) were predicted using ProtParam (https://web.expasy.org/protparam/) [36]. These parameters provided fundamental insights into protein stability and functional characteristics.

2.3. Phylogenetic and sequence alignment analysis

Multiple sequence alignments of S. tuberosum and A. thaliana proteins were generated using ClustalW (https://www.clustal.org/clustal2/) implemented in Molecular Evolutionary Genetics Analysis (MEGA) version 11.0 [37]. Phylogenetic reconstruction was performed using Neighbor-Joining methods with 1,000 bootstrap replicates [38].

2.4. Conserved domain and motif identification

Protein domains were annotated using the Protein family (Pfam; https://pfam.xfam.org) database [39]. Conserved motifs were predicted using Multiple Expectation Maximization for Motif Elicitation (MEME; v5.5.2; http://meme-suite.org/) with parameters set to 6 ≤ width residues ≤ 50 and maximum 20 motifs per protein [40].

2.5. Gene structure and chromosomal mapping

Exon-intron structures were analyzed with Gene Structure Display Server (GSDS; v2.0; https://gsds.cbi.pku.edu.cn) [41]. Chromosomal locations were mapped using MapGene2Chromosome (MG2C; v2; http://mg2c.iask.in/) [42], revealing genomic distribution patterns of RNAi pathway genes.

2.6. Evolutionary rate (Ka/Ks) analysis

The evolutionary dynamics of duplicated gene pairs, including both segmental and tandem duplications, were analyzed through Ka/Ks (non-synonymous/synonymous substitution rate) analysis. The Ka and Ks values were calculated using MEGA version 11.0 [37]. The Ka/Ks ratio was employed to assess the type of selection pressure acting on each duplicated gene pair [43]. Divergence time for each duplicated gene pair was estimated using the formula T = Ks/(2 × 6.56 × 10⁻⁹) [44], with the resulting values expressed in million years ago (MYA). This analysis enabled the identification of evolutionary pressures and divergence patterns among segmental and tandem duplicated genes in the genome.

2.7. Synteny and collinearity analysis

Syntenic blocks between S. tuberosum and A. thaliana were identified using TBtools (v2.010) [45]. This analysis revealed conserved gene arrangements across evolutionarily diverse species.

2.8. PPI network analysis

PPI networks were inferred using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; v12.0; https://string-db.org) [46] and visualized in Cytoscape (v3.10.0; https://cytoscape.org/) [47]. The networks highlighted potential functional associations among RNAi pathway components.

2.9. GO and subcellular localization

GO terms were annotated using Plant Transcription Factor Database (PlantTFDB; http://planttfdb.cbi.pku.edu.cn) [48], providing functional and compartmentalization insights. Subcellular localization of the encoded proteins was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/), a protein localization predictor [49], enabling the determination of their probable cellular compartments and contributing to the understanding of their functional context.

2.10. CREs analysis

Promoter regions (2.0 kb upstream of transcription start sites) were scanned for CREs using Plant Cis-acting Regulatory Element (PlantCARE; https://bioinformatics.psb.ugent.be/webtools/plantcare/) database [50]. Identified elements were categorized based on their regulatory functions in plant stress responses and development.

2.11. TFs prediction

TFs were predicted with Plant Regulatory Map (PlantRegMap; https://plantregmap.gao-lab.org) using a stringent significance threshold (p-value (1 × 10⁻⁴)) [51]. The analysis identified potential transcriptional regulators of RNAi pathway genes.

2.12. miRNA target prediction

Putative miRNA interactions were identified using plant sRNA target analysis server (psRNATarget; https://www.zhaolab.org/psRNATarget/) [52] with miRNA sequences from miRNA Base (miRBase; v22; https://www.mirbase.org/) [53]. Interaction networks were visualized in Cytoscape (v3.10.0; https://cytoscape.org/) [47].

2.13. Comprehensive gene expression profiling across tissues, developmental stages, and stress conditions

RNA-seq data from 29 tissues of potato plants (NCBI BioProject: PRJNA753086, PRJNA489943, PRJNA588378, PRJNA1093478, PRJNA1093480, PRJNA957457, PRJEB52340, PRJNA882516, PRJNA879907 and PRJNA851775) were analyzed using SpudDB Potato Genomics Resource (https://solanaceae.plantbiology.msu.edu/) [54]. Genes with Transcripts Per Million (TPM) > 0.2 were considered expressed, revealing organ-specific expression patterns of RNAi components. The expression patterns were visualized through heatmap generation in TBtools (v2.010) [45], revealing differential expression profiles under Tissues, Developmental Stages, and Stress conditions.

3.1. In Silico identification of RNAi-related genes in potato genome

Using protein sequences of A. thaliana (AtDCL, AtAGO, and AtRDR) as query sequences, a HMM was constructed to identify RNA silencing genes in the potato genome. Through this analysis, we identified 6 DCL (StDCLs), 14 AGO (StAGOs), and 9 RDR (StRDRs) genes in the potato genome. The basic information of these genes, including chromosomal location, structural features (ORF length, gene length, and intron number), and protein profiles (MW, protein length, and isoelectric point (pI)), is summarized in Table 1.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Basic information of Solanum tuberosum DCL, AGO and RDR gene families.

https://doi.org/10.1371/journal.pone.0339021.t001

6 StDCL genes were identified in the potato genome, with ORF lengths ranging from 3093 bp (StDCL3, Soltu.DM.08G015780.1) to 7443 bp (StDCL2c, Soltu.DM.11G004160.1). The encoded protein lengths vary from 1031 aa (StDCL3) to 2481 aa (StDCL2c), with MWs ranging from 116.87 kD (StDCL3) to 281.40 kD (StDCL2c). The genomic lengths of StDCL genes range from 31,672 bp (StDCL3) to 42,980 bp (StDCL4, Soltu.DM.07G000050.1). All StDCL proteins exhibit acidic characteristics, with pI values ranging from 6.03 to 6.57. The acidic isoelectric values (pI) of RNAi pathway proteins (e.g., AGO, DCL, RDR) are critical for their subcellular localization and functional stability, particularly in acidic compartments like the lysosome, where low pI adaptations enhance PPIs and siRNA binding efficiency [55]. Moreover, these pI values guide optimized protein purification strategies, such as ion-exchange chromatography, during in vitro characterization of potato RNAi machinery [56], underscoring their relevance in both functional genomics and biotechnological applications. The presence of conserved domains such as DEAD, Helicase_C, Dicer_dimer, PAZ, RNase III, and DSRM confirms the functional role of StDCLs in the RNAi pathway. The acidic pI values of StDCL proteins suggest their potential involvement in interactions with other molecules, such as RNA or proteins, in the RNA silencing machinery [57].

A total of 14 StAGO genes were identified, with ORF lengths ranging from 2430 bp (StAGO9, Soltu.DM.01G035930.1) to 3384 bp (StAGO1b, Soltu.DM.03G019130.1). The encoded protein lengths vary from 810 aa (StAGO9) to 1128 aa (StAGO1b), with MWs ranging from 90.51 kD (StAGO9) to 124.70 kD (StAGO1b). The genomic lengths of StAGO genes range from 4,084 bp (StAGO9) to 13,800 bp (StAGO10a, Soltu.DM.09G025190.1). All StAGO proteins exhibit basic characteristics, with pI values ranging from 7.98 to 9.49. The basic pI values of StAGO proteins are consistent with their role in binding sRNAs, such as siRNAs and miRNAs, which are critical for RNA silencing [58]. Interestingly, the variation in ORF lengths, genomic spans, and protein sizes among StAGOs implies structural diversity, which may underlie functional divergence. Such diversity has also been reported in other species (e.g., Arabidopsis thaliana and Oryza sativa), where specific AGO clades specialize in distinct RNAi pathways, including miRNA-mediated regulation, tasiRNA biogenesis, and antiviral defense [59]. The larger genomic length of StAGO10a, compared with the compact structure of StAGO9, may reflect differences in intron content or regulatory elements, potentially influencing transcriptional regulation.

Moreover, the wide range in MWs and AA lengths suggests that certain StAGOs, such as StAGO1b, could act as central regulators with broader substrate recognition, while smaller AGOs like StAGO9 may have more specialized functions. This interpretation is supported by studies in Arabidopsis, where AGO1 is indispensable for miRNA function and acts broadly across developmental and regulatory processes, whereas AGO9 is more restricted [60].

9 StRDR genes were identified, with ORF lengths ranging from 636 bp (StRDR6c, Soltu.DM.05G004010.1) to 3516 bp (StRDR6b, Soltu.DM.04G009340.1). The encoded protein lengths vary from 212 aa (StRDR6c) to 1172 aa (StRDR6b), with MWs ranging from 24.68 kD (StRDR6c) to 133.34 kD (StRDR6b). The genomic lengths of StRDR genes range from 1,162 bp (StRDR6c) to 24,941 bp (StRDR3, Soltu.DM.06G009870.1). The pI values of StRDR proteins range from 6.49 to 9.53, with most proteins exhibiting basic characteristics. The conserved RdRP domain in StRDR genes and their basic pI values not only underscore their critical role in amplifying RNAi signals and RNA binding [61,62] but also highlight their evolutionary specialization in potato, where genome-wide diversification of AGO, DCL, and RDR families may fine-tune antiviral defense and gene regulation. However, the potential for off-target silencing due to uncontrolled RdRP activity [63] necessitates further exploration of regulatory mechanisms governing these gene families in Solanum tuberosum to optimize RNAi efficiency without compromising cellular homeostasis. The pI values of the identified proteins in potato are consistent with those observed in other plant species, ranging widely from 1.99 to 13.96 [57]. These variations in pI values, from acidic to basic, are critical for post-translational modifications and biochemical interactions within the RNAi machinery [64].

3.2. Phylogenetic relationships of RNAi-related genes in Solanum tuberosum and Arabidopsis thaliana

By investigation of the phylogenetic relationship between Solanum tuberosum and Arabidopsis thaliana, we identified total of 29 RNAi-associated genes in potato where 6 are StDCLs, 14 are StAGOs and 9 are StRDRs (S1–S3 Files and Fig 2A–2C). The analysis revealed both conserved and divergent evolutionary patterns, offering insights into the functional roles of these proteins in potato.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. (A–C): Phylogenetic tree for (A) DCL proteins (B) AGO proteins and (C) RDR proteins from Solanum tuberosum and Arabidopsis.

In phylogenetic tree, different groups are represented by different colors; red circles are mentioned genes of Solanum tuberosum and green circles are mentioned genes of A. thaliana.

https://doi.org/10.1371/journal.pone.0339021.g002

The DCL phylogenetic tree identified three groups (Group I–III) with strong bootstrap support (Fig 2A). Group I included StDCL2a, StDCL2b, and StDCL2c, which clustered with AtDCL2, confirming their classification into the DCL2 subfamily. These proteins are likely involved in processing long dsRNAs into siRNAs, a critical step in RNA silencing [65]. Group II comprised StDCL4, which showed high sequence similarity to AtDCL4, suggesting its role in ta-siRNA biogenesis and antiviral defense [66,67]. Such conservation implies that DCL4 may function as the primary initiator of phased siRNA production, contributing to PTGS and developmental regulation under pathogen challenge. Group III contained StDCL1 and StDCL3, closely related to AtDCL1 and AtDCL3, respectively, implying their involvement in miRNA processing and stress responses [68]. The co-clustering of these isoforms supports the notion that DCL1-driven miRNA pathways and DCL3-mediated heterochromatic siRNA systems jointly regulate epigenetic homeostasis during stress adaptation.

The AGO phylogenetic tree divided the AGO genes into three groups (Group I–III) (Fig 2B). Group I included StAGO1a, StAGO1b, StAGO10a, StAGO10b, and StAGO5, which clustered with AtAGO1, AtAGO10, and AtAGO5, respectively. These proteins are likely involved in miRNA-mediated gene silencing, meristem development, and plant growth regulation [69,70]. Their strong evolutionary conservation indicates that these AGOs are core effectors in miRNA loading and target slicing, thereby maintaining developmental patterning and tissue differentiation. Group II comprised StAGO7 and StAGO3a–c, closely related to AtAGO7 and AtAGO3, suggesting roles in siRNA-mediated silencing and developmental transitions [71]. This implies that potato AGO7 homologs could regulate trans-acting siRNAs (ta-siRNA) that coordinate leaf polarity and morphogenesis, similar to mechanisms observed in Arabidopsis [72]. Group III included StAGO6a, StAGO6b, StAGO9, StAGO4a, and StAGO4b, clustering with AtAGO6, AtAGO9, and AtAGO4, respectively. These proteins are predicted to function in epigenetic silencing, reproductive development, and stress responses [73].

The RDR phylogenetic analysis identified three groups (Group I–III) (Fig 2C). Group I included StRDR1a, StRDR1b, and StRDR2, clustering with AtRDR1 and AtRDR2, respectively, suggesting roles in salicylic acid-induced RNA silencing and chromatin modification [74]. Group II comprised StRDR6a, StRDR6b, and StRDR6c, closely related to AtRDR6, indicating involvement in ta-siRNA biogenesis and antiviral defense [75]. Group III included StRDR5, StRDR3, and StRDR4, clustering with AtRDR5, AtRDR3, and AtRDR4, respectively, potentially functioning in stress-related RNA silencing [76]. These the presence of multiple stress-responsive RDR clades suggests an expanded defensive toolkit in potato, possibly linked to its adaptation to varied environmental stresses and pathogen pressures [72].

The phylogenetic analysis of DCL, AGO, and RDR proteins in Solanum tuberosum and Arabidopsis thaliana revealed both conserved and divergent evolutionary patterns. The functional predictions for potato proteins, based on their Arabidopsis counterparts, align with established roles in RNA silencing, development, and stress responses [65]. The absence of certain genes in the potato genome (StAGO2) highlights species-specific adaptations, potentially reflecting unique evolutionary pressures such as pathogen resistance or abiotic stress tolerance [77,78]. Experimental validation—such as gene expression profiling under stress conditions (e.g., viral infection or drought) and functional characterization via CRISPR/Cas9 knockout studies—will be essential to confirm these predictions and elucidate the precise roles of these proteins in potato biology [68,75].

3.3. Multiple sequence alignment of DCL, AGO, and RDR proteins in potato and Arabidopsis

We performed multiple sequence alignment of the predicted StDCL, StAGO, and StRDR protein sequences with their Arabidopsis orthologs (AtDCL, AtAGO, AtRDR) using Clustal-W in MEGA 11 shown at Fig 3A–3C).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Comparative Multiple Sequence Alignment in StDCL, StAGO, StRDR Gene families.

(A) Multiple sequence alignment of RNase III domains (RIBOc I and II) from S. tuberosum and Arabidopsis DCL proteins. Red downward arrows indicate conserved EDDE (glutamate-aspartate-glutamate-aspartate) positions critical for RNase III activity, (B) Alignment of PIWI domains from S. tuberosum and Arabidopsis AGO proteins showing conserved catalytic residues. Red arrows highlight the DDH triad (H798 position indicated) essential for slicer activity, (C) RdRP domain alignment of S. tuberosum and Arabidopsis RDR proteins. The red box encloses the conserved DxDGD catalytic motif required for RDR activity.

https://doi.org/10.1371/journal.pone.0339021.g003

Alignment of the RNase III domains revealed strict conservation of the EDDE catalytic motif (glutamate, aspartate, aspartate, glutamate) across all StDCLs proteins (StDCL1, StDCL2a, StDCL2b, StDCL2c, StDCL3, and StDCL4) and AtDCLs (Fig 3A). Minor variations in flanking regions did not affect the catalytic residues, indicating strong evolutionary pressure to maintain dsRNA-processing activity. This universal conservation highlights the fundamental role of the EDDE motif in initiating the gene silencing cascade by generating the primary sRNA effectors. Notably, StDCL3 exhibited full conservation of the EDDE motif, unlike AtDCL3 (Fig 3A). This high conservation suggests StDCLs retain canonical RNase III functionality, analogous to Arabidopsis DCLs in RNAi pathways.

Comparative analysis of the PIWI domains between Solanum tuberosum (StAGO) and Arabidopsis thaliana (AtAGO) revealed both conserved catalytic motifs and lineage-specific variations (Fig 3B, Table 2). The essential DDH/H catalytic triad, required for slicer activity, was fully conserved in StAGO1a, StAGO1b, StAGO5, StAGO7, StAGO10a, and StAGO10b - mirroring their functional counterparts AtAGO1, AtAGO5, AtAGO7 and AtAGO10 in Arabidopsis. The preservation of this triad is critical for the effector step of PTGS, enabling the cleavage of target mRNAs. However, several key divergences point to significant functional diversification within the potato AGO family.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Comparison of the AGO proteins in PIWI domains between Solanum tuberosum and A. thaliana.

https://doi.org/10.1371/journal.pone.0339021.t002

Clade-specific variations were particularly evident in certain subgroups. The StAGO3a and StAGO3b paralogs feature a DDD/H motif, matching the configuration found in AtAGO3, which is known to bind phased siRNAs without exhibiting slicing activity. StAGO4a and StAGO4b display a DDH/P substitution, differing slightly from Arabidopsis AtAGO4 (DDH/S), though both variants likely impair slicer activity while maintaining functionality in RNA-directed DNA methylation (RdDM) pathways. These specific substitutions effectively partition the AGO family into distinct functional classes, with some members specializing in binding and guiding rather than catalytic cleavage. A particularly striking divergence was observed in StAGO6b, which uniquely possesses a GQA/P motif – a configuration not seen in either AtAGO6 (DDH/P) or other StAGO proteins, suggesting potential neofunctionalization in this lineage.

The analysis also revealed notable differences between paralogous genes. While StAGO6a maintains the canonical DDH/H motif, its counterpart StAGO6b (GQA/P) and StAGO9 (DDH/P) show substitutions that may redirect their functional roles toward transcriptional silencing or structural scaffolding functions. These motif variations, summarized in Table 2, clearly divide StAGO proteins into two functional subgroups: those retaining slicer activity (DDH/H) and those with impaired or altered catalytic potential (DDD/H, DDH/P, GQA/P).

The functional implications of these variations are significant. The DDH/P substitution in StAGO6b and StAGO9 parallels the configuration in Arabidopsis AtAGO6, which lacks endonucleolytic activity but participates in RdDM. Similarly, the DDD/H motif in StAGO3a/b resembles AtAGO3, known to bind siRNAs without cleavage activity. The diversification of catalytic motifs highlights an evolutionary strategy to expand the functional repertoire of the RNA-induced silencing complex (RISC) without gene family expansion. These observations suggest substantial functional specialization within the potato AGO family, with StAGO1a/b and StAGO10a/b likely maintaining their role in PTGS through slicing activity, while variants like StAGO4a/b and StAGO6b may have evolved specialized functions in transcriptional silencing (TGS) or non-catalytic RNA binding. The unique GQA/P motif in StAGO6b, absent in Arabidopsis, highlights a potential case of lineage-specific innovation in potato AGO evolution. Above results and discursive direction suggest functional specialization within the AGO family. While some members likely maintain slicing activity, others may have evolved alternative roles in RdDM [79]. This functional partitioning allows a single protein family to regulate gene expression at multiple levels, from transcript degradation to epigenetic modification. Future transient expression assays could help elucidate how these variations impact RNA silencing efficiency in planta.

Alignment of StRDR and AtRDR sequences confirmed strict conservation of the DxDGD motif in StRDR1a, StRDR1b, StRDR2, StRDR5, and StRDR6a, StRDR6b, StRDR6c, (Fig 3C), critical for RDR activity. This motif’s conservation is fundamental for amplifying the RNAi signal, as it enables the synthesis of double-stranded RNA from single-stranded templates, thereby propagating the silencing response. Truncations near the catalytic site in StRDR3 and StRDR6c (likely due to incomplete gene models) require cDNA sequencing for resolution. The widespread DxDGD conservation suggests StRDRs retain robust dsRNA synthesis roles, similar to AtRDRs, in siRNA amplification and antiviral defense.

The DDH/H triad, essential for in vitro endonuclease activity in AtAGOs [80–82], is disrupted in several StAGOs, potentially altering RNA silencing mechanisms. While StAGO1a and StAGO1b likely retain slicing activity, substitutions in StAGO4a and StAGO4b and StAGO6a may redirect their roles to transcriptional gene silencing (TGS). The strategic alteration of catalytic residues represents a mechanism for functional specialization within the gene silencing machinery, allowing different AGO paralogs to undertake distinct regulatory tasks.

3.4. Conserved domain analysis of RNAi-related genes in potato and Arabidopsis

The analysis of conserved domains in RNAi-related gene families (DCL, AGO, and RDR) in potato and Arabidopsis provides valuable insights into the evolutionary conservation and functional diversification of these proteins. We identified conserved domains in StDCL, StAGO, and StRDR gene families and compared them with their Arabidopsis counterparts shown at Fig 4.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. The conserved domains of the predicted DCL, AGO, and RDR proteins in Potato and Arabidopsis.

https://doi.org/10.1371/journal.pone.0339021.g004

The DCL proteins family in potato revealed significant conservation of functional domains compared to Arabidopsis. StDCL1, StDCL2a, StDCL2c, and StDCL4 contain all essential domains, including Res-III, Helicase-C, Dicer-dimer, PAZ, Ribonuclease-3, and DND1-DSRM, mirroring their Arabidopsis counterparts [83,84]. The presence of these core domains confirms their fundamental role in the initiation of the gene silencing pathway by processing double-stranded RNA into siRNAs. However, StDCL2b lacks the Ribonuclease-3 domain, which may impair its endonuclease activity. This absence could result from evolutionary divergence or adaptation to specific stressors in potato, as Ribonuclease-3 is critical for cleaving dsRNA during RNAi [68,85]. The loss of this catalytic domain suggests StDCL2b may have a specialized, non-canonical role or requires interaction with other proteins to function in silencing. Additionally, StDCL3 exhibits a rearranged domain order compared to AtDCL3, indicating potential functional novelty. This rearrangement might reflect species-specific adaptations, possibly related to potato’s unique developmental processes or environmental challenges [68,86]. Previous studies have highlighted the role of DCL proteins in plant defense against viral infections, and the conservation of these domains in potato underscores their importance in RNAi pathways [84,87]. However, the structural variations observed in StDCL2b and StDCL3 warrant further investigation to elucidate their functional implications through targeted mutagenesis and complementation assays in model systems [88].

The AGO family in potato displays a high degree of domain conservation with Arabidopsis, particularly in the PAZ and PIWI domains, which are essential for RNA binding and RNase activity [70,71]. All StAGO proteins retain these domains, emphasizing their conserved role in RNA silencing. These domains are indispensable for the effector phase of silencing, as the PAZ domain anchors the sRNA while the PIWI domain provides the ‘slicer’ activity for target cleavage. In contrast, StAGO1b lacks the Gly-rich Ago1 domain, suggesting functional divergence or specialization within the AGO family [77,89]. Furthermore, StAGO3b, StAGO4b, StAGO5, StAGO7, and StAGO10b contain the Argo-Mid domain, which is critical for sRNA binding. However, this domain is absent in several other StAGO proteins, indicating potential functional specialization [73,90]. The differential presence of the Argo-Mid domain likely influences the specificity and affinity for different classes of sRNAs, thereby directing distinct silencing outcomes. The diversity in domain composition among StAGO proteins may reflect their involvement in distinct biological processes, such as stress responses, developmental regulation, or pathogen defense [69,91]. For instance, the presence of the Argo-Mid domain in StAGO5 and StAGO7 suggests their role in gene regulation, while its absence in other StAGO variants may indicate redundancy or alternative regulatory mechanisms [92]. These findings highlight the functional complexity of the AGO family in potato and underscore the need for further research to characterize their roles in RNAi pathways through comprehensive PPI studies and tissue-specific expression analyses [82,93].

RDR proteins in potato exhibit a high degree of conservation in the RdRP domain, similar to their Arabidopsis counterparts [74,78]. All StRDR proteins contain this domain, which is essential for synthesizing dsRNAs from ssRNAs, a critical step in RNAi silencing. This domain is fundamental for amplifying the RNAi response by creating secondary double-stranded RNA substrates, which reinforces and sustains the gene silencing signal. However, shorter isoforms may represent truncated variants or products of alternative splicing [75,94]. These shorter isoforms could play regulatory roles or act as dominant-negative inhibitors of RNAi pathways [76,95]. Such truncated variants could modulate the efficiency of the silencing pathway by competing with full-length RDRs, thus providing a potential layer of regulation. The presence of multiple RDR isoforms in potato suggests functional diversification, possibly related to stress responses, viral defense, or developmental regulation [96,97]. Previous studies have shown that RDR proteins are essential for amplifying RNAi signals, and their conservation in potato underscores their importance in RNAi-mediated gene regulation [98,99]. However, the functional significance of the shorter isoforms requires further investigation to determine their roles in potato biology through detailed biochemical characterization and genetic complementation experiments [100,101].

3.5. Conserved motif analysis of RNAi-related genes in potato and Arabidopsis

We identified conserved motifs in the DCL, AGO, and RDR protein families of Solanum tuberosum (St) and compared them with their Arabidopsis thaliana (At) homologs which shown at Fig 5. The analysis revealed both conserved and divergent motifs, providing insights into the structural and functional similarities and differences between the two species.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. The conserved motifs of the predicted DCL, AGO and RDR protein families in Potato and Arabidopsis.

Each color represents different motifs in the predicted proteins domains.

https://doi.org/10.1371/journal.pone.0339021.g005

In the DCL family, we identified up to 20 conserved motifs in both S. tuberosum and A. thaliana proteins. StDCL1, StDCL2a, StDCL2b, and StDCL4 exhibited complete conservation of 18–20 motifs with their Arabidopsis counterparts, AtDCL1 and AtDCL4, indicating strong functional similarity [83,87]. For instance, StDCL1 and StDCL4 shared identical motif arrangements with AtDCL1 and AtDCL4, respectively, suggesting that these proteins likely perform similar roles in the RNAi pathway [84,85]. This high degree of conservation underscores their fundamental and non-redundant roles in initiating the gene silencing process by processing double-stranded RNA precursors into sRNAs. However, StDCL3 displayed only 10 motifs, significantly fewer than AtDCL3, which has 14 motifs. Notably, StDCL3 lacked motifs 19 (DEAD) and 20 (Dicer_dimer), which are present in AtDCL3 [86,102]. This absence may indicate structural or functional differences in the RNAi machinery of potato compared to Arabidopsis [68]. The high conservation of motifs in StDCL1, StDCL2, and StDCL4 suggests that these proteins are likely essential for RNAi pathways in potato, similar to their roles in Arabidopsis [65]. The DEAD and Dicer_dimer domains are critical for RNA processing and cleavage, and their absence in StDCL3 may imply a reduced or specialized role in potato [102]. The absence of these core motifs suggests a potential divergence in the molecular mechanism of StDCL3, which could influence the biogenesis or function of specific sRNA classes. This could result in altered RNAi efficiency or specificity, potentially affecting the processing of sRNAs [84]. Further experimental validation is needed to determine whether StDCL3 has a unique function in potato or if it represents a functional divergence from Arabidopsis DCL proteins.

In the AGO family, we observed a maximum of 20 conserved motifs in both species. StAGO1a, StAGO1b, StAGO4a, StAGO4b, StAGO5, StAGO6a, StAGO9, and StAGO10a/b showed complete conservation of 18–20 motifs with their Arabidopsis homologs, indicating strong functional conservation [70,71]. For example, StAGO1a and StAGO1b shared identical motif arrangements with AtAGO1, suggesting that these proteins likely retain similar RNAi functions [89]. The conserved motifs in these AGO proteins are critical for forming the core of the RISC, which is directly responsible for the effector step of silencing through mRNA cleavage or translational repression. However, StAGO3a and StAGO3c exhibited fewer motifs (14 and 15, respectively) compared to AtAGO3 (18 motifs). Specifically, StAGO3a lacked motifs 13 (ArgoN) and 16 (ArgoN), which are critical for AGO protein function [73,90]. This may result in altered RNAi silencing efficiency or specificity in potato [77]. Additionally, StAGO6b and StAGO7 showed slight motif variations, with StAGO6b missing motif 3 (Piwi) and StAGO7 missing motif 12 (ArgoMid) [92]. The high conservation of motifs in StAGO1, StAGO4, StAGO5, and StAGO10 suggests that these proteins are likely essential for RNAi pathways in potato, similar to their roles in Arabidopsis [70]. The ArgoN domain is crucial for sRNA binding, and its absence in StAGO3a may impair the protein’s ability to bind target RNAs effectively [89]. Similarly, the missing Piwi and ArgoMid domains in StAGO6b and StAGO7 could affect their catalytic activity or interaction with other RNAi machinery components [73]. These specific motif losses could directly compromise the efficiency of RISC assembly and its gene silencing activity. These variations may indicate species-specific adaptations in the RNAi pathway, potentially influencing gene silencing efficiency or target specificity [90]. Experimental studies are needed to confirm whether these motif variations lead to functional differences in potato AGO proteins [77].

In the RDR family, we predicted 5–17 conserved motifs in S. tuberosum proteins. StRDR1a, StRDR1b, and StRDR2 showed complete conservation of 17 motifs with AtRDR1 and AtRDR2, indicating strong functional similarity [74,78]. Similarly, St This conservation highlights their essential role in amplifying the silencing signal by synthesizing double-stranded RNA, which serves as a substrate for DCL proteins to generate secondary sRNAs. RDR6a and StRDR6b exhibited high conservation with AtRDR6, suggesting similar roles in RNAi pathways [75]. However, StRDR3 and StRDR5 displayed significantly fewer motifs (5 and 6, respectively) compared to AtRDR3 (12 motifs). This suggests potential functional divergence or reduced activity in potato [96]. For instance, StRDR3 lacked motifs 11 (RdRP) and 14 (RdRP), which are present in AtRDR3 [76]. Additionally, StRDR4, while retaining 12 motifs, showed a unique arrangement, which may indicate functional specialization [95]. The high conservation of motifs in StRDR1, StRDR2, and StRDR6 suggests that these proteins are likely essential for RNAi pathways in potato, similar to their roles in Arabidopsis [74]. The RdRP domain is critical for RDR activity, and its absence in StRDR3 and StRDR5 may impair their ability to synthesize double-stranded RNA, a key step in the RNAi pathway [75]. This could result in reduced RNAi efficiency or altered target specificity in potato [96]. The unique motif arrangement in StRDR4 may indicate functional specialization, potentially enabling it to perform a unique role in potato RNAi pathways [100]. Further experimental studies are needed to explore the functional significance of these motif variations and their impact on RNAi mechanisms in potato [101].

3.6. Comparative analysis of gene structure in DCL, AGO, and RDR genes in potato and Arabidopsis

The gene structure analysis of StDCL, StAGO, and StRDR genes revealed a high degree of conservation with their orthologs in Arabidopsis thaliana (Fig 6). The exon-intron organization of these genes demonstrated structural similarities, suggesting functional conservation in the RNAi pathway.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Gene formation of the predicted DCL, AGO, and RDR proteins in S. tuberosum with Arabidopsis.

https://doi.org/10.1371/journal.pone.0339021.g006

The StDCL genes exhibited intron numbers comparable to their Arabidopsis counterparts. StDCL2a and StDCL2b contained 21 and 22 introns, respectively, closely aligning with AtDCL2 (21 introns). StDCL4 (22 introns) was structurally similar to AtDCL4 (24 introns), while StDCL1 and AtDCL1 both shared 19 introns. However, StDCL3 (17 introns) deviated from AtDCL3 (23 introns), indicating potential functional divergence. StDCL2c, with 41 introns, represented an outlier with significantly more introns than its Arabidopsis orthologs [68,102]. The presence of this unusually high intron number may contribute to alternative splicing events, potentially generating isoforms with distinct regulatory or stress-adaptive functions. Such intron expansion is often associated with increased transcriptomic plasticity in higher plants for adjusting to abiotic and biotic stresses [103]

Among StAGO genes, 12 out of 15 exhibited 19–22 introns, mirroring Arabidopsis homologs. For example, StAGO1a, StAGO1b, and AtAGO1 all contained 20 introns, while StAGO10b (21 introns) resembled AtAGO10 (18 introns). Exceptions included StAGO3a, StAGO3b, and StAGO7, which had 2, 2, and 3 introns, respectively, similar to AtAGO2 and AtAGO7. StAGO3c (3 introns) also deviated slightly from the typical intron range [104,105]. This pattern suggests that AGO genes with fewer introns, such as AGO2 and AGO7 orthologs, may have undergone intron loss to optimize rapid transcriptional responses during pathogen attack or developmental transitions. Reduced intron load is a known evolutionary strategy to enhance gene expression speed under stress [106,107].

StRDR genes showed conserved intron-exon structures, with most containing 2–10 introns. StRDR1a and StRDR1b (3 introns) matched AtRDR1 (2 introns) and AtRDR2 (3 introns). StRDR6a and StRDR6b (1 intron) mirrored AtRDR6, while StRDR6c (2 introns) showed minor variation. StRDR5 (6 introns) and StRDR3/StRDR4 (10 and 19 introns, respectively) aligned structurally with AtRDR5 (16 introns) and AtRDR3/AtRDR4 (17 and 16 introns) [78]. The conservation across RDR families underscores their stable evolutionary roles in RNA-dependent amplification of silencing signals. However, intron variation in StRDR3 and StRDR5 might indicate selective adaptation of RNAi components to potato-specific stress signaling pathways or virus-host interactions [108].

The structural conservation of StDCL, StAGO, and StRDR genes with their Arabidopsis counterparts underscores evolutionary conservation of RNAi pathways. The similarity in intron numbers between StDCL1, StDCL2 and AtDCL1, AtDCL2 supports their roles in miRNA and siRNA biogenesis, respectively [66,102]. The reduced intron count in StDCL3 and StAGO3c may reflect functional divergence, such as specialized stress-response roles [68,104]. Such divergence could provide evolutionary advantages by enabling faster transcription or alternative exon usage under abiotic and biotic stress.

The minimal introns in StAGO2a, StAGO2b and StAGO7 mirror AtAGO2, AtAGO7 respectively, which are critical for antiviral defense and developmental regulation [104]. Similarly, the conserved structure of StRDR1, StRDR6 with AtRDR1, AtRDR6 highlights their conserved roles in amplifying RNAi signals [78]. However, structural variations in StRDR3, StRDR5 suggest functional adaptations, potentially fine-tuning RNAi efficiency in potato-specific contexts [105].

These findings align with studies showing that intron-exon architecture influences RNAi machinery efficiency [65]. Overall, the observed structural dynamics reflect the evolutionary optimization of RNAi gene families, balancing stability with adaptability to ensure precise regulation of gene expression in diverse environmental and developmental conditions. Future work should explore how structural variations impact splicing efficiency, protein diversity, and stress adaptation in potato.

3.7. Genomic distribution of StDCL, StAGO, and StRDR genes

The chromosomal mapping of RNAi-related genes in Solanum tuberosum revealed non-random distribution patterns across 12 chromosomes (Fig 7).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. The genomic location of the predicted StDCL, StAGO,and StRDR genes.

The scale to indicate the chromosomal length is provided on the left.

https://doi.org/10.1371/journal.pone.0339021.g007

StDCL genes localized to chromosomes 6 (StDCL2a), 7 (StDCL4), 8 (StDCL3), 10 (StDCL1), and 11 (StDCL2b, StDCL2c), with notable clustering of StDCL2b and StDCL2c on chromosome 11 (positions 41.3 Mb and 42.1 Mb respectively). Duplication refers to a genetic mutation where a segment of DNA is replicated one or more times, leading to an increase in the amount of genetic material in the genome. Such duplications can lead to functional diversification or sub-functionalization of paralogs, as observed in other plant DCL families [109,110].

StAGO genes exhibited broader dispersion, occupying chromosomes 1 (StAGO4a, StAGO7, StAGO9), 2 (StAGO3a, StAGO3b, StAGO3c), 3 (StAGO1b, StAGO6b), 6 (StAGO1a, StAGO4b, StAGO5), 7 (StAGO6a), 9 (StAGO10a), and 12 (StAGO10b). Three gene clusters were identified: (i) a 1.8 Mb segment on chromosome 1 containing StAGO4a, StAGO7, and StAGO9; (ii) a 3.2 Mb region on chromosome 2 with three StAGO3 paralogs; and (iii) a 5.6 Mb segment on chromosome 6 harboring StAGO1a, StAGO4b, and StAGO5. Such clusters may facilitate coordinated regulation during stress responses [111].

StRDR genes primarily resided on chromosomes 3 (StRDR2), 4 (StRDR6b), 5 (StRDR1a, StRDR1b, StRDR6c), 6 (StRDR3, StRDR5), 8 (StRDR6a), and 12 (StRDR4). This chromosomal distribution pattern suggests both localized clustering and dispersed organization, reflecting the influence of tandem and segmental duplication events in the evolution of the RDR gene family. The close proximity of StRDR1a and StRDR1b on chromosome 5 implies recent duplication, a phenomenon frequently observed in RDRs of other Solanaceae species such as Solanum lycopersicum and Nicotiana benthamiana, where duplicated RDR1 and RDR6 members have undergone functional diversification to participate in antiviral defense and ta-siRNA biogenesis [14,112].

The scattered localization of StRDR2, StRDR3, and StRDR4 across separate chromosomes points toward sub-functionalization or condition-specific expression, consistent with findings from Arabidopsis thaliana and Oryza sativa, in which RDR paralogs display differential regulation under stress and developmental cues [113,114]. Such spatial organization may enhance the functional resilience of the potato RNA silencing machinery, ensuring redundancy and flexibility in response to diverse biotic and abiotic challenges. Collectively, the observed genomic pattern of StRDRs highlights the dynamic evolutionary trajectory of the potato RNAi system, maintaining a balance between conserved core function and adaptive specialization.

3.8. Sub-cellular localization of RNAi-related genes in potato

The subcellular localization of specific proteins is intricately connected to the biological functions of eukaryotic cells. The cellular positioning of these proteins offers valuable insights into their roles and activities within the cell, enabling a deeper understanding of the processes [115,116]. The detected StDCL, StAGO, and StRDR proteins were found in various subcellular compartments, including the nucleus, cytoplasm, chloroplast, mitochondria, cytoskeleton, plasma membrane, vacuole, peroxisome, endoplasmic reticulum, and Golgi apparatus which are shown at (Fig 8A and Fig 8B).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 8. Sub-cellular localization analysis for the (A) StDCL, StAGO, and StRDR proteins.

(B) The percentage of protein appeared in different cellular organelles.

https://doi.org/10.1371/journal.pone.0339021.g008

The StDCL proteins were distributed across multiple compartments, with all six StDCL proteins localized in the nucleus and cytoplasm, 66.67% in the chloroplast, 16.67% in the mitochondria, 16.67% in the cytoskeleton, 66.67% in the plasma membrane, 50% in the vacuole, and 50% in the endoplasmic reticulum. Notably, no StDCL proteins were found in peroxisomes, while 16.67% were localized in the Golgi apparatus. All StAGO proteins (14/14) were found in the nucleus and cytoplasm, with 71.43% localized in the chloroplast, 28.57% in the mitochondria, 64.29% in the cytoskeleton, 28.57% in the plasma membrane, 21.43% in the vacuole, and 14.29% in peroxisomes. No StAGO proteins were detected in the endoplasmic reticulum, while 7.14% were found in the Golgi apparatus. Similarly, all StRDR proteins (9/9) were localized in the nucleus, with 88.89% in the cytoplasm, 66.67% in the chloroplast, 33.33% in the mitochondria, 66.67% in the cytoskeleton, 33.33% in the plasma membrane, 11.11% in the vacuole, and 22.22% in peroxisomes. No StRDR proteins were found in the endoplasmic reticulum or Golgi apparatus.

Earlier research has indicated that RNAi proteins, such as AGO4 and DCL3 in Arabidopsis, are co-located in the nucleus and play a central role in the RNAi silencing process [117]. The multi-organellar localization of AGO, DCL, and RDR proteins, as evidenced by their presence in nuclei (gene silencing), cytoplasm (mRNA decay), and chloroplasts/mitochondria (organellar gene regulation), underscores their pleiotropic roles in potato RNAi pathways, mirroring findings in other systems where such proteins integrate oxidative phosphorylation, RNA processing, and stress responses [115,116]. However, the functional divergence of these proteins in potato particularly their potential roles in organelle-specific signaling or cytoskeletal RNA transport remains speculative without experimental validation of their spatiotemporal dynamics under biotic/abiotic stresses.

This widespread and overlapping distribution of StDCL, StAGO, and StRDR proteins across multiple subcellular compartments underscores their coordinated involvement in both transcriptional gene silencing (TGS) and PTGS pathways, wherein nuclear-localized components may participate in RdDM and chromatin remodeling, while cytoplasmic and organellar localizations facilitate siRNA biogenesis, mRNA degradation, and signal amplification, collectively reinforcing the spatial complexity of RNA silencing machinery in potato.

3.9. Evolutionary Ka/Ks ratio analysis of StDCL, StAGO, and StRDR in potato

The evolutionary dynamics of the StDCL, StAGO, and StRDR gene families were analyzed using the Ka/Ks ratio, which provides insights into the selective pressures acting on these genes (Fig 9). The Ka/Ks ratio measures the rate of non-synonymous (Ka) to synonymous (Ks) substitutions. We can identify purifying evolution, neutral evolution, and diversifying evolution from the ratio [118].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 9. Estimation of gene duplications, evoluation, duplication time and Ka/Ks analysis of StAGO, StRDR, and StDCL.

The ratio of Ka to Ks is represented by Ka/Ks, with divergence time (measured in MYA) also indicated. The color bar represents the data range. Visualization of Tandem duplications (dark blue) and segmental duplications (magenta) are mapped alongside purifying (orange) and diversifying (cyan) selection pressures.

https://doi.org/10.1371/journal.pone.0339021.g009

The Ka/Ks ratios for StDCL gene pairs ranged from 0.24 to 0.43. The lowest ratio was observed for StDCL2a-StDCL4 (0.24), while the highest ratios were for StDCL2a-StDCL3 and StDCL2b-StDCL3 (0.43). All StDCL gene pairs are under purifying evolution, consistent with functional constraints expected for RNAi pathway genes then the StAGO family showed Ka/Ks ratios from 0.06 to 1.42. Most pairs, such as StAGO1a-StAGO1b (0.06) and StAGO4a-StAGO4b (0.06), exhibited purifying evolution. However, StAGO3a-StAGO3c (1.13) and StAGO3b-StAGO4a (1.42) showed diversifying evolution, suggesting adaptive evolution in response to environmental or pathogenic challenges. This diversification reflects their roles in gene regulation and viral defense. Similar evolutionary patterns have been reported in Arabidopsis and rice, where AGO and DCL diversification is often linked to adaptation against lineage-specific viruses and stress-responsive pathways [119]. Such selective pressures indicate that certain AGO members may have undergone functional divergence to fine-tune sRNA-mediated silencing under biotic stress [120]. The StRDR family displayed Ka/Ks ratios from 0.22 to 2.04. Pairs like StRDR1a-StRDR2 (0.36) and StRDR5-StRDR6a (0.43) were under purifying evolution, while StRDR1a-StRDR6c (2.04) and StRDR2-StRDR6c (1.66) showed diversifying evolution. These adaptive changes may enhance RNA silencing efficiency against viral RNAs, whereas conserved pairs maintain core RNAi functions. Diversifying evolution was observed in StAGO3a-StAGO3c, StAGO3b-StAGO3c, StRDR1a-StRDR3, StRDR1a-StRDR6c, StRDR1b-StRDR3, StRDR1b-StRDR6c, StRDR2-StRDR3, StRDR2-StRDR5, and StRDR2-StRDR6c pairs, suggesting adaptive evolution in these gene pairs. In contrast, all remaining examined pairs exhibited purifying selection, indicating strong functional conservation.

We investigated most of the gene pairs are occurred segmental duplication. And the analysis revealed several gene pairs resulting from tandem duplication events, including StDCL2b-StDCL2c in the StDCL family, StAGO3a-StAGO3b, StAGO3a-StAGO3c, StAGO3b-StAGO4a, and StAGO4b-StAGO5 in the StAGO family, as well as StRDR1a-StRDR6c and StRDR1b-StRDR6c in the StRDR family. These duplication events represent important evolutionary mechanisms that have contributed to the expansion and functional diversification of these gene silencing gene families in plants, often followed by either sub-functionalization or neo-functionalization under selective constraints [121,122]. These duplication-driven expansions provide genetic plasticity that may allow differential gene regulation, adaptation to diverse environmental conditions, and reinforcement of silencing efficiency under stress.

3.10. Synteny relationship analysis of AGO, RDR, and DCL gene families in potato and Arabidopsis

To explore the evolutionary relationships and functional conservation of key gene families involved in RNAi pathways, synteny analysis was conducted between Solanum tuberosum and Arabidopsis thaliana. The study identified significant syntenic associations across the 12 chromosomes of S. tuberosum and the 5 chromosomes of A. thaliana, revealing conserved genomic regions and evolutionary patterns which are shown at Fig 10.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 10. Syntenic relationships between Solanum tuberosum and Arabidopsis thaliana for RNAi genes.

https://doi.org/10.1371/journal.pone.0339021.g010

The StAGO gene family demonstrated strong syntenic conservation, with multiple homologous regions identified between S. tuberosum chromosomes Chromosome 01, Chromosome 02, Chromosome 06, and Chromosome 09 and A. thaliana chromosomes including Chromosome 1, Chromosome 2 and Chromosome 5. These conserved syntenic blocks suggest a shared evolutionary origin and functional conservation of StAGO genes, which are known to play critical roles in RNAi and gene regulation pathways. The preservation of these syntenic relationships underscores the importance of AGO genes in maintaining RNAi machinery across plant species. Similar AGO syntenic conservation has been previously observed among diverse angiosperms, reflecting the evolutionary stability of the RISC components across monocots and dicots [119,123].

Similarly, the StRDR gene family exhibited syntenic connections, primarily between S. tuberosum Chromosome 12 and A. thaliana Chromosome 2. This specific alignment highlights functional conservation of StRDR genes, which are essential for RDR activities. These enzymes are crucial for amplifying RNAi signals, contributing to antiviral defense and gene silencing mechanisms. The conserved positioning of RDR orthologs across species has been reported to correspond with stable transcriptional regulation and stress-responsive amplification of double-stranded RNA in higher plants [124,125]. The syntenic conservation of StRDR genes suggests that their roles in these pathways have been evolutionarily maintained.

The StDCL gene family did not show strong one-to-one syntenic alignment with A. thaliana chromosomes, though dispersed homologous regions were detected across several potato chromosomes, implying ancient segmental rearrangements and lineage-specific divergence [10,126].

The StDCL gene family not showed significant syntenic alignments, particularly between S. tuberosum Chromosome and A. thaliana but they are presented at various chromosome.

Overall, the synteny analysis reveals a high degree of evolutionary conservation among the RNAi gene families, emphasizing their critical roles in RNAi mechanisms like other studies [19]. The conservation of these RNA silencing components across taxonomic lineages reflects an evolutionary constraint imposed by their indispensable roles in post-transcriptional and transcriptional gene silencing. Such cross-species syntenic parallels strengthen the evidence that the molecular framework of RNAi is a deeply rooted regulatory system preserved throughout plant evolution [63,127]. These findings provide valuable insights into the evolutionary dynamics and functional conservation of RNAi-related genes in plants, highlighting the potential for cross-species functional studies to deepen our understanding of RNAi pathways.

3.11. PPI network analysis of RNAi-related genes in potato

The PPI analysis sought to explore the relationships of 29 RNAi-associated genes identified in potato. PPI network analysis visualized 20 genes (Fig 11) showcasing intricate reciprocal relations among RDR, AGO, and DCL gene families.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 11. PPI network of RNAi-related genes in potato, where red color, blue color and green color indicate StDCL, StRDR and StAGO respectively.

https://doi.org/10.1371/journal.pone.0339021.g011

In the PPI network, stronger interconnectivity was shown between StDCL1 and StDCL2a with several StRDR and StAGO genes, which may point toward their importance in silencing cascades [128]. StDCL1 specifically interacted with 15 genes, including StRDR6a, StRDR6b, StRDR1a, StRDR1b, as well as several members of the StAGO family (StAGO1a, StAGO1b, StAGO3a, StAGO3b, StAGO3c, StAGO4a, StAGO4b, StAGO10a, StAGO10b, and StAGO7).

StDCL2a was highly interconnected in a similar manner, interacting with 15 genes, including StRDR1a, StRDR1b, StRDR6a, StRDR6b, and various StAGO proteins (StAGO1a, StAGO1b, StAGO3a, StAGO3b, StAGO3c, StAGO4a, StAGO4b, StAGO10a, StAGO10b, and StAGO7).

Besides, StAGO7 was found to interact with StRDR1a, StRDR1b, and StRDR6b, furthering its role in bi-directionally bridging AGO and RDR proteins. Notably, StRDR1a and StRDR1b were found interacting together and with several other partners like StRDR6a, StRDR6b, StDCL1, StDCL2a, and StAGO7, implying their functional redundancies or possible bulk participation in the silencing pathway [129]. But beyond the core interactions, the PPI network shows StRDR4 to be a highly connected node, interacting with StRDR1a, StRDR1b, StRDR3, StRDR5, StRDR6a, and StRDR6b, which again suggests a regulatory or bridging role within the RDR family [130].

StRDR5 implicated in networks with StDCL2a, StRDR1a, StRDR1b, StRDR6a, and StRDR6b fosters further cross-talk within the RDR family and with DCL proteins. Unlike other AGOs, StAGO5 formed connections only with StDCL1 and StDCL2a, hinting at a specialization in linking AGO with DCL functions [131].

The extensive interactions among DCL, AGO, and RDR genes discovered are a reflection of the conserved RNAi mechanism where these proteins are involved in sRNA processing and gene silencing activities. The high connecting power of StDCL1, StDCL2a, StRDR1a, StRDR1b, and StAGO7 also signifies the key roles these components play in the network and perhaps as hubs for coordinating silencing efficiency [132,133].

These insights agree with the classical RNAi pathway models, where dsRNA is processed by DCL into siRNAs that are loaded into AGO proteins to target cleavage of complementary RNAs, while RDR provides enhancement for the silencing pathway by creating secondary siRNAs. The observed interactions point to a fine-regulated and cooperative Potato system, thereby adding to the evolutionarily conserved nature of these mechanisms [134,135].

3.12. GO analysis of StAGO, StDCL, and StRDR in Solanum tuberosum

GO analysis underscored the conserved roles of RNAi genes in antiviral defense and RNA silencing while highlighting their dynamic regulatory interplay in plant immunity, as evidenced by immune-related GO enrichment studies in other systems [136,137]. This reinforces their functional significance in Solanum tuberosum, where their characterization could unravel novel mechanisms of pathogen resistance and intracellular coordination.

The GO analysis identified 157 annotations across three categories: biological processes (BP), cellular components (CC), and molecular functions (MF) shown at Fig 12 and S4 File. Biological processes dominated with 126 terms (p-values: 5.4 × 10 ⁻ ²⁰ to 0.04789), while cellular components and molecular functions comprised 11 (p-value range: 0.00021–0.03323) and 20 terms (p-value range: 8.4 × 10 ⁻ ¹⁸–0.0344), respectively.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 12. StAGO, StDCL, and StRDR gene functions were analyzed by GO and visualized on the right of the circos plot, where gene counts per GO term, expected values, and rich factors are color-coded, and −log₁₀(p-value) is shown with three distinct colors.

https://doi.org/10.1371/journal.pone.0339021.g012

Here we also identified several key terms associated with gene silencing and RNAi, including posttranscriptional gene silencing by RNA (GO: 0035194), RNAi (GO: 0016246), and production of siRNA (GO: 0030422), highlighting the central role of RNAi in regulating gene expression. Additional terms such as virus-induced gene silencing (GO: 0009616) and gene silencing by miRNA (GO: 0035195) underscore the involvement of RNAi in antiviral defense and miRNA-mediated regulation. Molecular functions like RNA-directed RNA polymerase activity (GO: 0003968) and siRNA/miRNA binding (GO: 0035197, GO: 0035198) further emphasize the enzymatic and effector mechanisms of RNAi, involving proteins such as DCLs (StDCL1, StDCL2a), RDRs (StRDR6b, StRDR1b), and AGOs (StAGO1a, StAGO4a). These findings collectively illustrate the diverse roles of RNAi in gene regulation, stress response, and pathogen defense. Nucleic acid binding and protein binding (13 genes each) underscored critical interactions for RISC formation. StDCLs protein exhibited ribonuclease III activity, processing dsRNA into siRNAs, while StAGOs bound siRNAs/miRNAs for sequence-specific silencing. These conserved mechanisms underpin RNAi roles in antiviral defense, development, and stress responses [138,139].

The functional divergence of RNAi gene families reflects specialized roles in potato antiviral defense, as supported by studies integrating RNA silencing and CRISPR/Cas systems for engineered resistance [140,141]. Moreover, the enrichment of antiviral and silencing-related GO terms suggests that these gene families operate as central nodes in the RNA-based immune network, coordinating transcriptional and post-transcriptional repression during pathogen attack [142,143]. Such molecular crosstalk between sRNA pathways and stress-signaling cascades is increasingly recognized as a conserved defense paradigm across angiosperms, ensuring robust adaptation under viral and abiotic stress conditions [65,144]. These GO insights not only reinforce the evolutionary conservation of RNAi machinery but also highlight its potential exploitation for precision genome engineering and development of durable virus-resistant cultivars through combined RNAi–CRISPR strategies [145,146].

3.13. Collinearity relationship analysis of StAGO, StDCL, and StRDR in Solanum tuberosum

Collinearity analysis revealed 57 connections among StAGO, StDCL, and StRDR gene families in potato, elucidating their evolutionary and functional relationships which shown at Fig 13. These connections reflect duplication and divergence events shaping RNA silencing pathways [147,148].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 13. Collinearity relationships among StAGO, StRDR, and StDCL gene families.

https://doi.org/10.1371/journal.pone.0339021.g013

StDCL family exhibited 14 collinear connections, including StDCL1 (chromosome 10) linked to StDCL2a (chromosome 6), StDCL2b/StDCL2c (chromosome 11), and StDCL3 (chromosome 8). StDCL2a further interacted with StDCL3 and StDCL4 (chromosome 4), suggesting segmental duplications driving functional diversification [147]. StAGO family showed the highest collinearity (29 connections), indicating complex evolutionary dynamics. Key interactions included StAGO1a (chromosome 5) with StAGO1b (chromosome 5) and StAGO4a (chromosome 1), and StAGO4b (chromosome 6) with StAGO5 (chromosome 6) and StAGO10a (chromosome 9). These patterns suggest sub-functionalization of AGO members in sRNA-mediated regulation [148]. StRDR family displayed 14 connections, such as StRDR1a (chromosome 6) pairing with StRDR2 (chromosome 3) and StRDR6c (chromosome 5). StRDR2 linked to StRDR5 (chromosome 6) and StRDR6a (chromosome 8), reflecting conserved roles in dsRNA amplification and silencing enhancement [147]. The extensive collinearity among these RNAi gene families suggests that gene duplication and chromosomal rearrangements have contributed to the diversification of silencing pathways in potato. This genomic organization provides evolutionary flexibility, allowing functional specialization of AGO, DCL, and RDR proteins for distinct silencing processes such as antiviral RNAi, transposon suppression, and epigenetic regulation as like other Plants such as Rice, Maize [119,149]. Moreover, the co-localization of certain gene pairs across chromosomes may facilitate coordinated expression during pathogen attack or developmental transitions, strengthening the integrated silencing network in potato [150]. Similar collinearity-driven expansion of RNAi machinery has been reported in other plants such as Arabidopsis thaliana and Oryza sativa, emphasizing the evolutionary conservation of RNA silencing as a genomic defense system [151,152].

3.14. CREs analysis in the promoters of StDCL, StAGO, and StRDR

CREs refer to non-coding DNA sequences that include short motifs typically ranging from 5 to 20 base pairs. These motifs act as binding sites for TFs, allowing them to attach to specific target genes to initiate transcription and regulate gene expression [153,154]. The advancement of high-throughput genome sequencing technology has significantly increased the availability of sequencing data for commercially important crops every year [155]. Consequently, bioinformatics tools can be utilized to efficiently explore databases and identify functional regulatory regions within DNA sequences—most commonly in promoter and enhancer regions—linked to particular gene functions. The analysis of CREs in the promoters of StDCL, StAGO, and StRDR revealed 52 elements, with the most abundant category being Box 4, related to light responsiveness. CAREs were classified into four categories based on their functional regulation: light responsiveness, tissue-specific expression, phytohormone responsiveness, and stress responsiveness (Fig 14 and S5 File). The light response occurring in the leaf tissue of potato plants is significantly affected by photosynthesis, an essential physiological process [156].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 14. A heatmap represents the distribution of putative CAREs on the 2.0 kb promoter region of StDCLs, StAGOs and StRDRs.

The names of the StDCLs, StAGOs and StRDRs genes are shown on the right side of the heat map. Functions associated with CAREs of the corresponding genes, such as light responsiveness (green), phytohormone responsiveness (red), stress responsiveness (blue), and tissue-specific expression (magenta) are indicated at the bottom.

https://doi.org/10.1371/journal.pone.0339021.g014

The largest category of CAREs related to light responsiveness included 26 elements, such as 3-AF1 binding site, AAAC-motif, ACA-motif, ACE, AE-box, AT1-motif, ATC-motif, ATCT-motif, Box 4, Box II, Chs-CMA1a, Chs-CMA2a, GA-motif, GT1-motif, GATA-motif, G-Box, G-box, Gap-box1, I-box, LAMP-element, L-box, LS7, MRE, Sp1, TCCC-motif, and TCT-motif. The phytohormone responsiveness CAREs included 10 elements, such as ABRE, GC-motif, AuxRR-core, TGA-element, GARE-motif, P-box, CGTCA-motif, TGACG-motif, TCA-element, and RY-element. The stress responsiveness category included five elements, such as MBS, TC-rich repeats, DRE, LTR, and WUN-motif. The tissue specific expression category included 11 elements, such as ARE, AT-rich element, circadian, GCN4_motif, MBSI, AT-rich sequence, CAT-box, CCAAT-box, Box III, HD-Zip3, and O2-site. This comprehensive analysis highlights the diverse regulatory mechanisms governing the expression of StDCL, StAGO, and StRDR, emphasizing their roles in light signaling, tissue-specific functions, phytohormone responses, and stress adaptation.

In this analysis, the light-responsive elements Box 4 and G-box demonstrated the highest frequency of occurrence across multiple genes. Among tissue-specific elements, ABRE showed particularly widespread distribution, appearing in numerous gene promoters, while CGTCA-motif and TGACG-motif were also prominently represented. Within the phytohormone-responsive category, these same elements (CGTCA-motif and TGACG-motif) again displayed significant prevalence. Regarding stress-responsive elements, ARE and O2-site emerged as the most abundant, appearing frequently across various gene families in all analyzed promoter sequences.

The presence of these regulatory motifs in RNAi-associated gene promoters suggests an intricate transcriptional control network that links environmental cues to PTGS. CAREs such as ABRE and MBS are known to mediate abscisic acid and drought-responsive transcription, which can activate RDR and DCL expression during stress, enhancing sRNA biogenesis [157,158]. Similarly, light-responsive motifs (G-box, GT1-motif) may synchronize AGO and DCL gene activity with diurnal or developmental regulation, maintaining the stability of sRNA populations under varying light conditions [159]. The enrichment of stress- and hormone-responsive elements reflects the adaptive flexibility of RNAi genes in modulating defense signaling cascades under pathogen attack, consistent with the transcriptional upregulation of AGO and RDR members observed during viral infection and abiotic stress in other plants [65,160].

These findings collectively highlight that cis-element diversity contributes to the spatiotemporal expression of silencing genes in potato, integrating transcriptional and post-transcriptional layers of regulation to ensure robust RNA silencing and stress tolerance mechanisms.

3.15. Regulatory network between TFs and StDCL, StAGO, and StRDR in Solanum tuberosum

A comprehensive analysis of TFs regulating StAGOs, StDCLs, and StRDRs in Solanum tuberosum identified a total of 132 unique TFs, categorized into 25 distinct families (S6 File). Notably, TFs from major families such as ERF, Dof, MYB, bZIP, BRB-BPC, and MIKC-MADS play pivotal roles in regulating these gene groups which are shown at Fig 15.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 15. The regulatory network among the TFs and the predicted RNAi genes.

The StDCL, StAGO, and StRDR genes were represented by red, green, and pink node color, respectively, and the TFs were represented by yellow node color with different shape for different families of TFs.

https://doi.org/10.1371/journal.pone.0339021.g015

In the StDCL–StAGO–StRDR interaction set, five TFs establish connections across all three gene families. These include PGSC0003DMG401023951 (ERF family), PGSC0003DMG400019528 (Dof family), PGSC0003DMG400002507 (ERF family), PGSC0003DMG400000786 (Dof family), and PGSC0003DMG400037029 (Dof family). Additionally, PGSC0003DMG400018190 interacts within this set, though it belongs to a TF group outside the primary families considered.

Within the StDCL–StAGO interaction set, three TFs PGSC0003DMG400002350, PGSC0003DMG400009142, and PGSC0003DMG400030228 are identified as members of the ERF family, underscoring the prominent role of ERF TFs in regulating both DCL and AGO genes. Other TFs, such as PGSC0003DMG400015424 and PGSC0003DMG400004953, also show interactions but are classified into other families not central to this analysis.

In the StDCL–StRDR interaction set, PGSC0003DMG400009475 and PGSC0003DMG400013402, both from the ERF family, interact across these gene classes. Another TF, PGSC0003DMG400016148, is also involved but is associated with other less defined TF groups.

The StAGO–StRDR interaction set exhibits extensive regulatory connections involving multiple TFs from diverse families. These include PGSC0003DMG400004948 (MYB family), PGSC0003DMG400010075 and PGSC0003DMG400023426 (Dof family), PGSC0003DMG400002185 and PGSC0003DMG400014541 (ERF family), PGSC0003DMG402029444 (BRB-BPC family), PGSC0003DMG400000008 (MIKC-MADS family), PGSC0003DMG400004062 (Dof family), and PGSC0003DMG400007951 and PGSC0003DMG400026232 (ERF family). PGSC0003DMG400012660, originally classified in the bZIP family, also establishes connections within this group. Additional TFs like PGSC0003DMG400623717 and PGSC0003DMG400009914 are noted after sequence formatting corrections but require further validation.

Balloon plot analysis indicates that StAGO1A has the highest interaction frequency with the ERF family, with 20 interactions recorded, which shown at Fig 16. Strong interactions are also observed between StDCL1 and members of the ERF family, and between StAGO3c, StRDR6a, and StRDR5 with the ERF family, where StRDR6A exhibits a stronger interaction profile compared to StRDR5 and StRDR3C. StAGO3C additionally forms significant interactions with the C2H2 family, while StAGO4b, StAGO5, and StRDR1a are connected to the Dof family. StAGO5 further establishes links with the WRKY family. Moderate interaction levels (10–15 counts) are observed for TF families such as DOF, ERF, bHLH, and bZIP, with lower-level interactions occurring with MYB, NAC, and MIKC-MADS TFs across the DCL, AGO, and RDR gene members.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 16. Balloon plot represents the related number of TFs with the predicted StRNAi genes with StDCL, StAGO, and StRDR proteins on the left and TF names at the bottom.

Color intensity and size indicates number of TF presence for each proteins.

https://doi.org/10.1371/journal.pone.0339021.g016

A comprehensive analysis of TFs regulating StAGOs, StDCLs, and StRDRs in Solanum tuberosum identified the TFs bHLH, bZIP, C2H2, Dof, ERF, MYB, WRKY, MIKC_MADS, and BRB-BPC families may play significant role in regulating RNAi genes (Fig 17). Specifically, only seven TF families (ERF, bZIP, C2H2, WRKY, Dof, MYB and bHLH) are connected 67.42% out of total connections. Additionally we also investigated that the single PGSC0003DMG402029444 (BBR-BPC) and PGSC0003DMG400000008 (MIKC_MADS) are connected with more than five RNAi genes. The ERF family emerged as the most prominent, comprising 34 TFs, followed by the bZIP family with 12 TFs. The C2H2 and WRKY families each included 11 TFs, while the Dof and MYB families consisted of 8 and 7 TFs, respectively. The bHLH family contributed 6 TFs, and the MIKC_MIDS and BRB-BPC families were represented by a single TF each. This diverse array of TFs highlights the complexity of the regulatory network governing these genes in potato.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 17. A heatmap depicts TF families regulating potato RNAi-related genes, with StDCL, StAGO, and StRDR proteins on the left and TF names at the bottom.

Color intensity indicates TF presence for each protein.

https://doi.org/10.1371/journal.pone.0339021.g017

These findings align with previous studies highlighting the roles of ERF [161], Dof [162], MYB [163], and bZIP [164] TF families in plant development and stress responses. Specifically, the ERF family is known for its involvement in various stress responses in potatoes [161], while the bZIP family has been implicated in gene diversification and spatiotemporal gene expression in Solanum tuberosum [165]. The MYB family also plays multiple roles in development and stress responses in potatoes [163].

The prominent enrichment of ERF, Dof, and MYB TFs within RNAi-associated networks underscores a regulatory bridge between transcriptional control and post-transcriptional silencing. ERF and WRKY TFs, for instance, are known to modulate RNA silencing genes under viral or abiotic stress, coordinating defense-related expression of AGO and RDR members [166,167]. Likewise, Dof and bZIP TFs are implicated in hormonal signaling and transcriptional reprogramming during pathogen challenge, which can activate or repress RNAi gene modules [164]. These multilayered connections reflect an integrated feedback loop where TFs modulate sRNA machinery to fine-tune gene silencing, thereby contributing to adaptive stress responses and genome defense in potato [163,168]. Such regulatory coupling may represent an evolutionary optimization that ensures transcriptional flexibility and RNAi robustness under environmental pressures.

3.17. Prediction of potential miRNAs targeting StDCL, StAGO and StRDR genes in Solanum tuberosum

In this analysis, a total of 117 miRNA-target interactions were identified, affecting 26 genes from the StDCL, StAGO, and StRDR families. The investigation uncovered 68 unique miRNA sequences which are shown at Fig 18, with stu-miR827, stu-miR5303 and stu-miRN3270 being the most prevalent, each appearing five times. Among the targeted genes, StDCL1 (17), StDCL2a (5), StDCL2c (7), StAGO1a (6), StAGO1b (5), StRDR1a (2), StRDR1b (2), and StRDR2 (5) were the most frequently targeted.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 18. miRNAs interaction network of RNAi-related genes in potato, where purple color, red color, blue color and green color indicate miRNAs, StDCL, StRDR and StAGO respectively.

https://doi.org/10.1371/journal.pone.0339021.g018

The findings indicate that stu-miR827 targets several genes, including StAGO5, StRDR3, StAGO6a, StDCL2a, and StAGO10a. This miRNA is not only evolutionarily conserved but also associated with phosphate starvation responses in plants, consistent with prior studies linking miR827 to nutrient stress regulation and homeostasis [169]. Its multiple interactions with AGO and RDR genes imply a role in adjusting RNA silencing pathways during nutrient stress, which may help balance growth and adapt to stressful conditions [90].

In contrast, stu-miR5303 targets genes are StDCL2c, StDCL1, StDCL2b, StDCL2a, and StRDR2 and the miRNA has previously been identified in Various Plants miRNA family [170], suggesting a lineage-specific adaptation mechanism. It is thought to regulate isoform-specific DCL activity, thereby fine-tuning miRNA biogenesis and reinforcing the dynamic feedback between miRNA productions and silencing control [171]. Notably, the presence of StRDR2 indicates potential crosstalk between miRNA and siRNA pathways, highlighting the cooperative regulation within the RNAi machinery..

Lastly, stu-miRN3270 interacts with StAGO1b, StDCL4, StDCL2b, StRDR6b, and StRDR6a and its targeting of StRDR6—a key amplifier of secondary siRNA production—suggests a conserved mechanism of miRNA-mediated feedback in PTGS [172]. This interaction pattern implies that miRN3270 could influence systemic silencing and antiviral defense, consistent with previous evidence that RDR6-associated miRNAs enhance long-distance silencing and viral resistance [173].

Overall, these findings reveal the intricate regulatory interplay between miRNAs and RNA silencing components in potato, providing insights into how sRNAs orchestrate stress adaptation and antiviral defense in crop plants and the highly connected miRNA genes are summarized in Table 3.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Information of highly connected potential miRNAs targeting StDCL, StAGO, and StRDR genes in Solanum tuberosum.

https://doi.org/10.1371/journal.pone.0339021.t003

3.18. Comprehensive gene expression profiling across tissues, developmental stages, and stress conditions of RNAi-related genes

The tissue-specific expression analysis of RNAi-associated genes revealed variable expression patterns across potato tissues, developmental stages and stress conditions which shown at Fig 19. In root tissues, StAGO1b, StAGO3b, StAGO4a, and StDCL2b were highly expressed under both control and stress conditions, particularly under salt and drought stress. Expression of StAGO3b remained consistently high in all root samples, suggesting a prominent role in root development and stress response. These findings align with previous studies indicating that genes such as StRD22 and StERD7 are upregulated under drought conditions in potato roots, contributing to stress tolerance mechanisms [162,174]. Such expression dynamics further reinforce the central role of RNAi machinery in modulating abiotic stress responses through post-transcriptional regulation [65].

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 19. Comprehensive gene expression pattern of StDCL, StRDR and StAGO across tissues, developmental stages, and stress conditions are represented at the bottom of the heat map.

The respective StDCL, StRDR and StAGO gene names are shown on the right side of the heat map. The color gradient from white-green-red indicates the expression levels on the right side of the heat map.

https://doi.org/10.1371/journal.pone.0339021.g019

In stolon tissues, especially during the hooked and swollen stages, StAGO4a and StAGO3b showed strong expression. These genes were also highly expressed in the stolon tip, suggesting involvement in early tuber formation. In contrast, StDCL2b, StRDR1b, and StRDR6b displayed lower or no expression in these tissues. Similar tissue-specific expression patterns have been observed in the StSOS1 gene family, where certain members are predominantly expressed in stolon tissues, indicating their role in tuber development [175]. This parallel expression between silencing-related genes and developmental regulators highlights how sRNA pathways coordinate hormonal and developmental signaling during tuber initiation [176].

Leaf tissues exhibited relatively uniform expression profiles. StAGO1b, StAGO4a, and StAGO6 showed higher expression levels under heat stress (3 h and 6 h), while cold-stressed leaves displayed reduced expression for most RNAi genes. StAGO4a and StDCL3a were among the few genes with moderate expression under cold conditions. This pattern is consistent with the expression of stress-responsive genes such as StEREBP1, which is upregulated under various environmental stresses, including low temperature, enhancing tolerance in transgenic potato plants [177]. The modulation of AGO and DCL members under temperature extremes supports the hypothesis that RNA silencing components fine-tune stress gene expression through temperature-sensitive regulatory loops [178].

In petiole tissue, StAGO1b, StAGO4a, and StAGO6 were actively expressed, along with moderate expression of StDCL2b and StDCL3a. Expression patterns in stem and shoot apex were similar, with StAGO1b, StAGO4a, and StAGO6 showing consistently higher expression. These observations are in line with studies demonstrating that genes involved in hormone signaling pathways exhibit tissue-specific expression, contributing to plant development and stress responses [19]. The consistent presence of AGO1b and AGO4a across vegetative organs indicates their likely housekeeping function in maintaining endogenous sRNA balance for growth regulation [179].

In reproductive organs, including seeding, flower, and fruit tissues, several genes showed stage-specific expression. StAGO1b, StAGO4a, and StDCL3a exhibited strong expression in flower bud, closed flower, and mature flower tissues. In fruit tissues, StAGO1b and StAGO4a were highly expressed in mature fruit, while StAGO6 showed moderate expression in both young and mature stages. These findings suggest that RNAi-related genes may play roles in reproductive development, as supported by previous research on gene expression during microtuberization under various stress conditions [175]. Such temporal expression of silencing-related genes during reproductive organ formation suggests a conserved mechanism by which miRNA and siRNA pathways orchestrate developmental phase transitions [180].

In tuber-related tissues, expression levels varied with developmental stage. StAGO1b, StAGO4a, and StDCL2b showed increased expression during tuber development, particularly in later stages (Stage 3 and Stage 5). Expression was also high in sprouting tubers and in the tuber cortex. These genes also displayed similar expression patterns in multiple tuber-associated tissues, including peel, pith, and young tuber. The involvement of RNAi-related genes in tuber development and stress adaptation is further supported by studies highlighting the role of genes like StProDH1 in enhancing drought tolerance through gene silencing approaches [181]. Such temporal expression of silencing-related genes during reproductive organ formation suggests a conserved mechanism by which miRNA and siRNA pathways orchestrate developmental phase transitions [180].

Overall, StAGO1b, StAGO4a, and StAGO3b were among the most widely expressed genes across all tissues, indicating broad functional roles in development and stress adaptation. Collectively, these expression profiles underscore the integrative role of RNAi-related genes as transcriptional and post-transcriptional regulators linking developmental plasticity, hormonal control, and stress resilience in potato [65,176,178–180].