Comprehensive characterization of potato TBL genes reveals candidates for salt and drought stress tolerance
Chenqi Wang, Xiaofeng Zhou, Bo Wang, Jianying Qiao, Zhiyong Xiong, Lei Wu
TL;DR
This study identifies potato genes that may help improve its tolerance to salt and drought stress.
Contribution
The first genome-wide characterization of the TBL gene family in potato, identifying candidates for stress tolerance.
Findings
72 StTBL genes were identified in the potato genome, distributed across 12 chromosomes.
10 genes were identified as potential salt-responsive candidates and 11 as potential drought-responsive candidates.
Phylogenetic analysis grouped the genes into three subfamilies with conserved domains.
Abstract
The Trichome Birefringence-Like (TBL) gene family encodes polysaccharide acetyltransferases that modify polysaccharide properties, playing key roles in trichome development, cell wall acetylation, and responses to biotic and abiotic stresses. Potato, a globally important crop, frequently faces salinity and drought stress. However, the role of the potato TBL gene family in stress resistance remains unexplored. Using bioinformatics, we identified 72 StTBL genes in the potato DM1-3–516 R44 genome, unevenly distributed across 12 chromosomes. Phylogenetic analysis grouped them into three subfamilies with conserved domains including PC-Esterase, PMR5N, and DUF4283. We further examined gene structure, promoter cis-elements, predicted miRNA targets, GO annotations, and tissue-specific expression. Under both salt and drought stress, we identified several responsive candidate genes from the 72…
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Taxonomy
TopicsPlant Molecular Biology Research · Plant-Microbe Interactions and Immunity · Plant Surface Properties and Treatments
Introduction
1
Potato (Solanum tuberosum L.), a member of the Solanaceae family originating from the Andes of Peru and Bolivia (Spooner et al., 2005; Tang et al., 2022), is the world’s third largest food crop and a key contributor to global food security and agricultural economies. However, its production is severely constrained by soil salinization and recurrent drought. Climate change is making the weather more extreme, with problems of high temperatures, droughts, and high soil salinity becoming more frequent. Globally, salinity affects about 950 million hectares (about 7% of arable land) (Munns and Tester, 2008; Yang and Guo, 2018).
Traditional agronomic measures, such as applying organic fertilizer, water-saving irrigation, soil amendments, and grafting salt-tolerant rootstock, can partially mitigate these stresses but are costly, resource-intensive, and environmentally unsustainable. Therefore, the use of bioinformatics and molecular biology techniques to screen stress-resistant genes to provide theoretical targets for genetic improvement is an important channel for cultivating a new generation of super crops to cope with climate change.
The plant cell wall, composed mainly of cellulose, hemicellulose, and lignin, is essential for cell morphology, defense, signal transduction, and development (Keegstra, 2010). Xylan, interconnected with cellulose and lignin, forms a complex network that strengthens the cell wall (Wen et al., 2024). Its acetylation is vital for proper wall assembly and mechanical stability (Yuan et al., 2016a). The Trichome Birefringence-Like (TBL) family encodes polysaccharide O-acetyltransferases involved in the acetylation of xylan (Lunin et al., 2020; Urbanowicz et al., 2014; Zhong et al., 2017), xyloglucan (Zhong et al., 2018a, Zhong et al., 2020), mannan (Zhong et al., 2018b), and pectin (Stranne et al., 2018). The TBL gene family is mainly involved in the modification of cell wall polysaccharides via acetyltransferase activity. TBL3 (Bischoff et al., 2010b; Yuan et al., 2016c), TBL22/AXY4L (Gille et al., 2011), TBL27/AXY4 (Gille et al., 2011), and TBL28-35 (Yuan et al., 2016b) in Arabidopsis belong to structural proteins. These proteins have acetyltransferase activity and dynamically regulate cell wall properties through chemical modification. In A. thaliana, xylan O-acetyltransferase 1 (XOAT1) specifically acetylates the xylan backbone (Lunin et al., 2020), while DUF231 proteins (e.g., TBL3, TBL28, TBL29/ESK1, TBL30–35) utilize acetyl-CoA to acetylate xylooligomer (Grantham et al., 2017). Loss of TBL3 decreases crystalline secondary cell wall cellulose in trichomes and stems (Bischoff et al., 2010a), underscoring the family’s pivotal role in cell wall biosynthesis.
TBL genes also influence stress responses (Bischoff et al., 2010a; Xin et al., 2007) and disease resistance (Bi et al., 2024; Chen et al., 2020; Gao et al., 2017; Wen et al., 2025; Zhang et al., 2025a). In A. thaliana, the acetylation of xylan mediated by TBL29 (ESK1) is a necessary step to maintain the structural integrity and mechanical support function of the catheter. The esk1 mutant enhances frost tolerance without acclimation (Xin et al., 2007), acting as a negative regulator, while tbl44 shows resistance to powdery mildew (Chen et al., 2020), and tbl27 is hypersensitive to aluminum stress (Gao et al., 2017). In Nicotiana tabacum, NtTBL31 contributes to drought tolerance (Wang et al., 2025). In rice, mutations in OsTBL1 and OsTBL2 reduce wall acetylation and increase susceptibility to leaf blight (Zhang et al., 2025a). In roses, RcTBL16 mediates interactions with Botrytis cinerea (Kumar et al., 2016). The tbl10 mutant in A. thaliana showed reduced RG-I acetylation and enhanced drought tolerance (Stranne et al., 2018). Collectively, these findings suggest that TBL genes play diverse roles in stress adaptation and pathogen defense. The above AtTBL29, AtTBL44, OsTBL1, OsTBL2 and RcTBL16 are regulatory TBL proteins.
Because cell walls are the first barrier against environmental stress (Bi et al., 2024; Zhang et al., 2025b), their composition and modification are central to plant resilience. Despite this significance, the TBL gene family has not been systematically chara-cterized in potatoes. To address this gap, we performed a genome-wide identification and analysis of stTBLs, including gene structure, phylogeny, cis-regulatory elements, GO annotation, and stress-responsive expression in the Atlantic tetraploid variety under salt (200 mM NaCl) and drought (200 mM mannitol) stress for 0–96 h. Several candidate genes associated with salt and drought tolerance were identified. This study revealed the role of TBL-mediated o-acetylation in potato stress response, screened potential salt- responsive and drought-responsive candidates, and provided targets and directions for molecular breeding.
Materials and methods
2
Identification of StTBL genes, physicochemical properties, chromosome localization, and phylogenetic analyses
2.1
The complete genome and protein sequences of the potato DM variety were obtained from the SpudDB database (https://spuddb.uga.edu/dm_v6_1_download.shtml, accessed 4 January 2025). Candidate StTBL genes were identified using the HMM profile for the PC-Esterase (PF 13839), with protein domain downloaded from the Pfam database (https://pfam.xfam.org/, accessed 30 October 2024). Physicoche-mical properties of the encoded membrane proteins were analyzed with TBtools v2.210 (Chen et al., 2020), and subcellular localization was predicted using Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed 24 April 2024). Genes’ positions on chromosomes were visualized with TBtools, and members of the StTBL family were renamed according to their chromosomal order.
Protein sequences of Arabidopsis thaliana were retrieved from TAIR (https://www.arabidopsis.org/, accessed 2 June 2024) and extracted using the Fasta Extract Filter (Quick) function in Tbtools v2.210 (Bischoff et al., 2010a; Chen et al., 2020; Gao et al., 2017). Multiple sequence alignments of potato and Arabidopsis TBL proteins were performed with ClustalW using default parameters. Phylogenetic analysis of the StTBL gene family was conducted in MEGA 7.0 software (Kumar et al., 2016) using the maximum likelihood (ML) method with 1,000 bootstrap replicates and default settings. The resulting phylogenetic tree was visualized and refined with Evolview (https://www.evolgenius.info/evolview/#/treeview, accessed 6 June 2025).
Analysis of conserved motifs, domains, and gene structure of StTBL genes
2.2
Exon-intron structures of StTBL genes were analyzed by aligning cDNA sequences with their corresponding genomic DNA sequences. Conserved motifs were identified using MEME (Multiple Em for Motif Elicitation; https://meme-suite.org/meme/tools/meme, accessed 28 April 2025) (Bailey et al., 2006), with the maximum number of motifs set to 10 and other parameters left at default. Conserved domains were annotated using the NCBI CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed 28 April 2025). Gene structures were analyzed and visualized with TBtools v2.210 (Chen et al., 2020) using GTF/GFF3 files. Finally, the phylogenetic tree, conserved motifs, and gene structures were integrated using the Gene Structure View (Advanced) function in TBtools v2.210 (Chen et al., 2020).
Analysis of gene duplication, collinearity, and the ratio of nonsynonymous (Ka) to synonymous (Ks) nucleotide substitutions
2.3
Genome, genome annotation, and CDS sequence files of Solanum lycopersicum, A. thaliana, O. sativa, Triticum aestivum, and Zea mays were downloaded from Ensemblplants (https://plants.ensembl.org/index.html, accessed 11 May 2025). The ge-nome and annotation data of Nicotiana benthamiana were obtained from the Sol Genomics Network (https://solgenomics.net/, accessed 11 May 2025). CDS sequences of N. benthamiana were extracted from the genome and annotation files using Linux commands.
Gene duplication and collinearity analyses of the TBL gene family were performed using the Advanced Cricos plug-in in TBtools v2.210 (Chen et al., 2020). Interspecies collinearity of TBL genes of S. tuberosum with S. lycopersicum, N. benthamiana, A. thaliana, O. sativa, T. aestivum, and Z. mays was analyzed using TBtools v2.210 (Chen et al., 2020) and MCScanX (http://chibba.pgml.uga.edu/mcscan2/, accessed 11 May 2025) (Wang et al., 2012). The Ka and Ks substitution rates of duplicated gene pairs were calculated using TBtools v2.210 (E-value cut-off < 1 × 10^-10^ and num of BlastHits with 5) (Chen et al., 2020; Yuan et al., 2024), and the Ka/Ks ratio was used to infer the evolutionary patterns of StTBL genes. Ka/Ks values within and between potato species were plotted using OriginPro 2024 (OriginLab Corporation, Northampton, MA, USA).
Analysis of cis-acting elements in the promoter region of StTBL genes
2.4
The 2000 bp upstream promoter sequences of potato StTBL coding regions were extracted using TBtools v2.210 (Chen et al., 2020). Cis-regulatory elements related to plant growth and development, hormone response, and stress response were predicted using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed 15 April 2025). Elements lacking clear annotation or biological relevance were excluded. The distribution of cis-acting elements was visualized as heatmaps using TBtools v2.210 (Chen et al., 2020).
Gene ontology annotation and miRNA target prediction of StTBL genes
2.5
The Gene Ontology (GO) enrichment file (DM_1-3_516_R44_potato.v6.1. working_models.go.txt) was downloaded from SpudDB (https://spuddb.uga.edu/dm_v6_1_download.shtml, accessed 21 May 2025). GO enrichment analysis was performed using the clusterProfiler package in R, and results were ranked by enrichment factor (Yu et al., 2012).
CDS of 72 StTBLs were extracted from the SpudDB file (DM_1-3_516_R44_ potato.v6.1.working_models.cds.fa, https://spuddb.uga.edu/dm_v6_1_download.shtml, accessed 4 January 2025) using the seqkit command in Linux. Potential miRNAs targeting StTBL genes were predicted with psRNATarget (https://www.zhaolab.org/ps-RNATarget/, accessed 18 May 2025) using default parameters (The expectation is set to 5). Interaction networks between miRNAs and StTBL genes were visualized with Cytoscape 3.9.1.
Expression analysis of StTBL genes in different tissues
2.6
The expression patterns of StTBL genes in various tissues of DM potato were comprehensively analyzed using RNA-seq data retrieved from the PGSC database (https://spuddb.uga.edu/dm_v6_1_download.shtml, accessed on 4 January 2025). Ba-sed on the publicly available transcriptome data of potato tissues, the expression status of potato TBL family genes in different tissues and organs was studied by FPKM (Fragments Per Kilobase of transcript per Million mapped reads) value analysis method. The log2 (FPKM + 1) formula is used for calculation and the heat map is drawn. Raw sequences are available in the National Center for Biotechnology Information Sequence Read Archive under BioProject PRJNA753086 (Brose et al., 2025).
Expression of StTBL genes under drought and salt stress
2.7
Stem segments of potato (Solanum tuberosum L. cv. Atlantic) were cultured on MS solid medium (TQ-AL-PL361, Techisun, Shenzhen, China) for 35 days under controlled conditions (22 ± 2°C, light intensity 25.0-37.5 μmol m^-2^ s^-1^, 16 h light/8 h dark). MS solid medium containing 200 mM NaCl (4.41 g MS solid medium and 11.688 g NaCl (HDM-7647-14-5A5, TIANJINKEMAO, Tianjin, China) were added to 1L of distilled water), MS solid medium containing 200 mM mannitol (4.41 g MS basic medium and 36.4 g mannitol (CM7091-500g, Coolaber, Beijing, China) were added to 1L of distilled water) and ordinary MS solid medium (4.41 g solid medium was added to 1L of distilled water) were prepared (Garner and Blake, 1989; Zhu et al., 2020). The pH was adjusted to 5.8 and sub-packed in a test tube with a bottom diameter of 2.4 cm and a height of 18 cm. The height of the solid medium was 5 cm. High pressure sterilization 121°C 15min. The potato tissue culture seedlings with consistent growth were selected, and the roots were rinsed with sterile water, dried with filter paper, and transferred to MS test tube medium containing 200 mM mannitol or 200 mM NaCl. The potato tissue culture seedlings in the control group were transferred to ordinary MS solid medium. Drought stress was simulated using 200 mM mannitol. Nine replicate samples were set for each of the six groups (0, 12, 24, 36, 72 and 96 h). Samples were collected at 0,12,24,36,72 and 96 h after the start of stress. Three potato tissue culture seedlings with consistent growth were selected, and their leaf tissues were taken, frozen in liquid nitrogen and stored at − 80°C for further analysis. This experiment was repeated three times. Atlantic was gifted by the Inner Mongolia Potato Virus-free Seed Potato Breeding Center, China.
RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
2.8
Total RNA was extracted from potato leaves using standard protocols. First-strand cDNA was synthesized with the All-in-One First-Strand Synthesis MasterMix (with dsDNase) for qPCR (Cat#EG15133S, BestEnzymes, Nanjing, Jiangsu, China). Primers were designed using SnapGene software (https://www.snapgene.com). qRT-PCR was performed on a QuantStudio^®^ 3 Real-Time PCR Instrument (96-well 0.2 ml Block; Cat#A28567, Thermo Fisher Scientific, USA) using F488 SYBR qPCR Mix (Universal) (Cat#EG23111L, BestEnzymes, Nanjing, Jiangsu, China). The housekeeping gene StTubulin (PGSC0003DMC400020469) served as the internal control. Amplification conditions were 95°C for 30 s followed by 40 cycles of 95°C for 10 s, and 60°C for 30 s. Relative expression levels of target genes were calculated using the 2^-ΔΔCt^ method. Ct values were obtained from four biological replicates, with two technical replicates. The average relative expression of each group of genes was used to draw the heat map of cluster analysis using the HeatMap plug-in of TBtools v2.210 (Chen et al., 2020). The standard curves of cDNA (1, 2, 4, 10,10^2^,10^3^,10^4^,10^5^ × dilutions) diluted in a series of control groups were constructed, and the amplification efficiency of each pair of primers was calculated. The formula was E (%) = [10 ^(− 1/slope)^ − 1] × 100. The average cycle threshold (Ct) of each gene was obtained by four biological repeats, and each biological repeat was composed of two technical repeats (Fan et al., 2022; Livak and Schmittgen, 2001; Radonic et al., 2004). The primer sequences and amplification efficiency of 72 StTBL genes and reference genes were shown in Supplementary Table S8. The result of qRT-PCR melting curve analysis showed that there was a single smooth curve with a TM of the target gene around 80°C (Supplementary Figure S1).
Homology analysis of StTBL and AtTBL proteins
2.9
Through the BLAST plug-in of TBtools v2.210 (Chen et al., 2020), the sequences of 72 StTBL proteins and 46 AtTBL proteins were compared. Plotting using DNAMAN (version 8.0, Lynnon Biosoft, Quebec, Canada).
Results
3
Identification, physicochemical properties, phylogenetic analysis, and chromosomal distribution of StTBL proteins
3.1
Based on the potato DM genome, 102 candidate StTBL proteins were initially identified using the Hidden Markov Model (HMM) profile of the PC-Esterase domain (PF13839). After domain validation with SMART and Pfam, redundant sequences (When the amino acid sequence similarity of the protein encoded by the gene is > 90% and the coverage length is > 90%, it is judged to be a redundant gene.) were removed, yielding 72 StTBL proteins (Figure 1, Supplementary Table S1). Phylogenetic analysis was conducted using 72 StTBL and 46 AtTBL protein sequences Supplementary Table S2. The neighbor-joining tree divided the proteins into three groups: Group I (31 potato, 22 Arabidopsis), Group II (29 potato, 15 Arabidopsis), and Group III (12 potato, 9 Arabidopsis). Detailed protein sequence data are provided in Supplementary Table S2.
Phylogenetic analysis of potato and Arabidopsis TBL proteins resolved 72 StTBLs and 46 AtTBLs into three groups (I-III), with distinct color-coding demarcating cladistic affiliations. The maximum likelihood phylogeny was constructed using MEGA 7, implementing 1000 bootstrap replicates based on TBL amino acid sequence alignments.
Analysis of physicochemical properties of StTBL proteins revealed that StTBL proteins ranged from 98 (StTBL39) to 846 (StTBL30) amino acid residues, with molecular weights from 10727.21 Da (StTBL39) to 98974.08 Da (StTBL30). Theore-tical isoelectric points (pI) ranged from 5.43 (StTBL7) to 10.04 (StTBL43), with 67% of proteins being basic (pI > 7.0). Instability index varied from 20.15 (StTBL45) to 59.8 (StTBL25), with 41 proteins classified as stable (index < 40). The aliphatic index ranged from 62.14 (StTBL65) to 98.56 (StTBL7). All proteins were hydrophilic, with GRAVY values ranging from –0.742 (StTBL21) to –0.107 (StTBL67). Subcellular localization predicted 24 proteins in the plasma membrane, 8 in the cell wall, 33 in the chloroplast, 1 in the cytoplasm, 1 in the mitochondria, and 5 in the nucleus (Supplementary Table S1). Chromosomal mapping showed that the 72 StTBL genes were unevenly distributed across 12 chromosomes (Figure 2). Chr01, Chr02, and Chr07 each contained 11 genes, while Chr04 and Chr08 had only one gene each. Genes were named StTBL1–StTBL72 according to their chromosomal order (Supplementary Table S1).
Chromosome distribution of StTBL gene family members. The colored rectangular bars represent the chromosomes of DM potatoes, marking the number and length of chromosomes (Mb).
Conserved motifs, domains, and gene structures of StTBL genes
3.2
Ten conserved motifs (motifs 1 − 10) were identified using the MEME program (Supplementary Figure 2). The predominant motif arrangement was motif 8 → motif 9 → motif 1 (GCD) → motif 6 → motif 4 → motif 7 → motif 5 → motif 10 → motif 3 → motif 2 (Asp-X-X-histidine, DXXH). Notably, Group III contained both the GCD and motif 4 (Figures 3A, B). The amino acid sequences of GCD, motif 4, and DXXH were DYLKWRWQPNDCELPRFBAKQFLEKQFLEKLRGKRJMFVGDSLNRNQ WZSLVCLL, WKGADVLIFNTGHWWW, and QDCSHWCLPGVPDTWNELLYAL L, respectively (Supplementary Figure 2). Conserved domain analysis showed that 57 genes carried a PC-esterase domain, and 43 of these also contained a PMR5N domain. Group I included 26 PC-esterase and 20 PMR5N domain genes; Group II comprised 26 PC-esterase and 16 PMR5N domain genes; and Group III contained 5 PC-esterase and 6 PMR5N domain genes. StTBL30 had the highest exon count, 15, whereas StTBL39 contained only one exon. Most StTBL genes carried 3 − 6 exons (Figure 3D).
Phylogenetic relationship, conserved motifs, and gene structure of StTBLs. (A) A phylogenetic tree was constructed based on the full-length sequences of 72 potato TBL family proteins. Color-coding: Group I (blue), Group II (pink), Group III (green). (B) The conserved motif of the StTBL protein. The gray horizontal line represents the amino acid length of the sequence, and the different colors on the sequence represent various motif types. (C) StTBLs protein domain. The colored part marks different conserved domains, while the gray area indicates an area without a specific domain. (D) The way the StTBL genes are put together, with the exon and intron parts. UTR, untranslated region. The gray horizontal line represents the intron region, the green corresponds to the UTR, and the yellow corresponds to the CDS region.
Duplication and collinearity analysis of StTBL genes between potato and six other plants
3.3
In potato, eight tandemly duplicated StTBL gene pairs and 23 segmental duplication events were detected across different chromosomes (Figure 4; Supplementary Table S3). These duplications likely contributed to the expansion of the StTBL family (Figure 4). We calculated Ka/Ks ratios of duplicate gene pairs, and all values were < 1 (Figure 5). Collinearity analysis was then conducted between potato and six representative species. We identified 91 orthologous pairs with S. lycopersicum, 126 with N. benthamiana, 62 with A. thaliana, 19 with O. sativa, 31 with T. aestivum, and 8 with Z. mays (Figure 6; Supplementary Table S4). Among these, 17 StTBL genes showed collinearity with 3–8 N. benthamiana genes. Similarly, 13 genes had collinearity with three S. lycopersicum genes, while 6 genes were collinear with 3–4 A. thaliana genes. Fewer relationships were observed with monocots: one potato gene was collinear with three O. sativa genes, and several had collinearity with T. aestivum. No potato gene exhibited collinearity with multiple Z. mays genes (Supplementary Table S4). Ka/Ks values for orthologous pairs between potato and A. thaliana, S. tuberosum, and S. lycopersicum were consistently < 1, with some cases showing Ka=0 or undefined Ka/Ks values, likely due to extreme evolutionary conservation. By contrast, comparisons with monocots (O. sativa, T. aestivum, and Z. mays) yielded undefined values, likely due to low sequence homology preventing reliable estimation of substitution rates (Supplementary Table S4).
The synthetic relationship of the StTBL gene in potatoes was shown. The gray circle inside represents the potato chromosome, and the position of the StTBL gene is displayed on the circle. The internal white band delineates the syntenic region blocks in the potato genome, whereas the red band denotes segmental duplication events.
Ka/Ks values of TBL genes within potato species (tandem repeats and segmental repeats) and between species (S. tuberosum and S. lycopersicum, S. tuberosum and N. benthamiana, S. lycopersicum and A. thaliana).
A comparison of TBL genes between S. tuberosum and six other plants was performed using identical linear analysis. The grey line represents the gene blocks orthogonal to other genomes in S. tuberosum. The red line depicts the same line TBL gene pair.
Analysis of cis-acting elements in the StTBL genes’ promoters
3.4
The 2000 bp upstream sequences of StTBL genes were analyzed for cis-acting elements. A total of 1,218 elements were identified, grouped into four major categories: defense and stress response (51, 4.1%), growth and development (111, 9.1%), hormone response (239, 19.6%), and light response (817, 67.0%). Light-responsive elements were the most abundant, accounting for two-thirds of all elements. Among the defense and stress response category, drought-inducible elements were the most frequent (39, 3.2%), with MBS occurring 28 times; MBS is known to regulate drought-induced gene expression (Tan et al., 2025). Hormone-responsive elements included auxin response (53, 4.4%), gibberellin response (53, 4.4%), and MeJA response (130, 10.7%), with MeJA elements being most abundant. In the growth and development category, elements associated with zein metabolism regulation (31, 3.5%), meristem expression (28, 2.3%), endosperm expression (17, 1.4%), and circadian rhythm control (16, 1.3%) were relatively enriched (Figure 7; Supplementary Table S5).
Cis-regulatory architecture of StTBL promoters.
Gene Ontology annotation and miRNA analysis of potato TBL genes
3.5
GO enrichment analysis revealed eight biological processes, two molecular fun-ctions, and two cellular components associated with StTBL genes (Figure 8; Supplementary Table S6). Enriched biological processes included xylan biosynthetic process (11), plant-type cell wall modification (6), plant-type secondary cell wall biogenesis (6), pectin biosynthetic process (5), cellulose biosynthetic process (5), circadian rhythm (6), response to freezing (3), and xyloglucan metabolic process (2). Molecular functions terms were O-acetyltransferase activity (72) and xylan O-acetyltransferase activity (14). Cellular components included the trans-Golgi network (6) and Golgi trans cisterna (3). Functionally, StTBL15/16/25/31/33/50/51/61/62/63/71 exhibited xylan O-acetyltransferase activity and were involved in xylan biosynthesis. Genes such as StTBL2/64/65 were linked to cell wall modification and secondary wall biogenesis, while StTBL2/50/62/64/65 participated in both pectin biosynthesis and cellulose biosynthesis. Cold stress-related processes included StTBL25, StTBL31, and StTBL33. Localization analysis showed that StTBL/38/40/64/65/70 were enriched in the trans-Golgi network, with StTBL38/40/70 also enriched in the Golgi trans cisterna (Supplementary Table S6).
Gene Ontology analysis of the StTBL genes. Bubble diameter scales with quantitative magnitude, while color saturation encodes adjusted p-value significance.
77 putative miRNAs targeting 72 StTBLs genes were identified, while no miRNA targeted genes StTBL39/58/70 (Figure 9). Among them, 90 stu-miR395 family members targeted 9 StTBL genes, 20 stu-miR5303 family members targeted 10 StTBL genes, 19 stu-miR156 family members targeted 7 StTBL genes, 16 stu-miR172 family members targeted 9 StTBL genes, 17 stu-miR1886 family members targeted 10 StTBL genes, 16 stu-miR399 family members targeted StTBL25/32/41, 11 stu-miR482 family members targeted 10 StTBL genes (Supplementary Table S7).
The interaction network of miRNA-StTBL. StTBL gene paralogs are encoded by the leftward arrow, and the circular representing distinct miRNA families.
StTBL expression in potato tissues
3.6
To explore StTBLs gene expression across potato tissues, RNA-Seq transcriptome data were analyzed. The results showed that StTBL9/10/11/12/13/14/16/18/20/32/35/40/41/48/52/63/64/48/63/64/65/70 were expressed in all tissues, with StTBL14/20/40/48/63/64/65 showing consistently high expression. Specifically, StTBL14 was the most highly expressed in shoots, leaves, sepals, carpels, petals, and mature fruit (FPKM > 5); StTBL20 in tubers, stolons, petals, and mature fruit (FPKM > 5); and StTBL64 and StTBL66 in mature fruit (FPKM > 6). By contrast, StTBL3/6/8/43/53 were either undetected or expressed at very low levels (FPKM < 1) (Figure 10; Supplementary Table S8). The clustering results revealed multiple pairs of StTBL genes sharing identical expression patterns. These included: StTBL31 and StTBL41; StTBL35 and StTBL65; StTBL10 and StTBL48; StTBL19 and StTBL66; StTBL27 and StTBL64; StTBL11 and StTBL29; StTBL12 and StTBL32; StTBL7 and StTBL58; StTBL44 and StTBL45; StTBL18 and StTBL34; StTBL1 and StTBL28; StTBL30 and StTBL42; StTBL24 and StTBL57; StTBL4 and StTBL69; StTBL46 and StTBL62; StTBL36 and StTBL53; and StTBL51 and StTBL72. Additionally, StTBL33, StTBL25, and StTBL50 also exhibited the same expression pattern.
Differentially expressed StTBL genes over tissues in S. tuberosum.
Expression of StTBLs in potato under salt and drought treatment
3.7
To evaluate the role of StTBL gene in abiotic stress response, qRT-PCR was used to analyze the gene expression of StTBL gene in leaves under salt stress and drought stress. Under NaCl stress, 57 StTBL genes showed significant expression changes rela-tive to the control, while 15 genes remained unchanged. StTBL1/3/16/20/22/28/58/59/60/68 were consistently induced, and StTBL31/42/43/56 were induced during the early stage. StTBL15/22/28/41 reached peak expression at 36 h, whereas StTBL1/3/7/13/19/44/60/69 peaked at 72 h. StTBL68 was only induced at the late stage. These genes may function as positive regulators of salt stress responses. In contrast, StTBL10/11/25/33/46/50/51/62/66/70/72 was down-regulated (Figure 11; Supplementary Table 10). Overall, these findings indicate that many TBL genes participate in salt stress response, with individual members exhibiting distinct regulatory patterns.
The gene expression levels of 72 StTBL genes under salt stress and drought stress at 0–96 h, the horizontal axis represents the treatment conditions, and the vertical axis represents the gene.
Under drought stress, StTBL1/2/3/12/19/21/22/28/31/33/69 were significantly un-regulated, while StTBL4/13/14/50/70 showed no significant change. StTBL7/28/34 was induced early, and StTBL43/44 were induced late. StTBL21 exhibited the highest ex-pression at 36 h. In contrast, 43 genes (StTBL5/9/10/11/15/16/18/20/24/26/29/32/35/36/37/38/39/40/41/45/46/47/48/49/51/52/53/54/55/56/57/58/59/61/62/63/64/65/66/67/68/71/72) were down-regulated, showing a strong synergistic repression under drought stress (Figure 11). This suggests that many family members may contribute to stress sensitivity or to the function of metabolic or developmental pathways influenced by osmotic stress. Across both treatments, StTBL6/8/30 showed no detectable expression, probably due to extremely low transcript abundance in leaves (0, 0, and 0.14, respec-tively; Supplementary Table 8). Cluster analysis revealed several groups with similar expression dynamics, including StTBL1/3/59, StTBL16/20/58, StTBL19/22, StTBL28/33, StTBL60/21/31, and StTBL2/12/69. StTBL68 did not cluster with other stress-responsive genes.
Homology analysis of StTBL and AtTBL proteins
3.8
Protein sequence alignment revealed seven gene pairs with > 70% homology. Among them, AtTBL33–StTBL34 showed 78.261% identity (Supplementary Figure 3), followed by AtTBL29 and StTBL25 at 72.016% (Supplementary Figure 4), AtTBL33 and StTBL26 at 71.649% (Supplementary Figure 5), AtTBL28 and StTBL31 at 71.591% (Supplementary Figure 6), and AtTBL13 and StTBL11 at 71.023% (Supplementary Figure 7).
Discussion
4
The TBL gene family plays a central role in the O-acetylation of plant cell wall polysaccharides, a modification essential for proper cell wall formation, plant growth, and responses to biotic and abiotic stresses (Gao et al., 2017; Xin et al., 2007; Zhao et al., 2021). However, a genome-wide analysis of the StTBL gene family in potato has not previously been conducted. In this study, we systematically analyzed the StTBL gene family using bioinformatics approaches. Temporal expression profiling under drought and salt treatments (0–96 h) identified 10 salt-responsive candidates and 11 drought-responsive candidates.
TBL genes represent the major plant enzyme family responsible for polysaccharide O-acetylation (Anantharaman and Aravind, 2010; Lunin et al., 2020). Comparative genomic studies have revealed varying numbers of TBL genes across species: 50 in Rose (Rosa chinensis) (Tian et al., 2021), 65 in Pyrus bretschneider (Ban et al., 2025), 64 in Populus trichocarpa (Zhong et al., 2018c), 37 in Dendrobium officinale (Si et al., 2022), 46 in A. thaliana (Bischoff et al., 2010a), 66 in O. sativa (Gao et al., 2017), 69 in S. lycopersicum (Zhong et al., 2020), 130 in Nicotiana tabacum (Wang et al., 2025), 49 in Eucalyptus grandis (Tang et al., 2024), 131 in Gossypium hirsutum, 130 in Gossypium barbadense (Zhu et al., 2024), and 37 in Dendrobium officinale (Si et al., 2022). Among these, A. thaliana has been the most extensively studied: of its 46 TBL genes, AtTBL3/28/29/30, AtTBL31/32/33/34/35 are associated with xylan (Xiong et al., 2013; Yuan et al., 2016a, Yuan et al., 2016c, Yuan et al., 2016b; Zhong et al., 2017), AtTBL19/20/21/22/27 with xyloglucan (Zhong et al., 2020) (Gille et al., 2011; Zhong et al., 2020; Zhu et al., 2014), AtTBL23/24/25/26 with mannan (Zhong et al., 2018b), and AtTBL46/TBR with pectin (Bischoff et al., 2010a). Our study identified 72 TBL genes in potato, a substantially higher number than in A. thaliana. This expansion likely reflects evolutionary replication events, including possible whole-genome duplication.
The TBL genes of S. tuberosum and A. thaliana are only separated by 3–5 genes on average, highlights the evolutionary conservation of this family among dicotyle-donous plants. The StTBL gene in potato is orthologous to the A. thaliana Group II TBL28–35 acetylation-related subfamily. It may have similar molecular functions, such as participating in acetylation modification. Collinearity analysis revealed stronger synteny among dicotyledonous plants compared with monocots, reflecting closer phylogenetic relationships. Chromosomal mapping revealed an uneven distribution, suggesting region-specific amplification. Moreover, all duplicated gene pairs exhibited Ka/Ks < 1, indicating strong purifying selection and functional conservation of StTBL genes during evolution, consistent with the results in R. chinensis (Tian et al., 2021), N. tabacum (Wang et al., 2025), and G. hirsutum (Zhu et al., 2024).
Length variation in StTBL proteins reflects functional diversity within the family, with their isoelectric point ranges suggesting heterogeneity that could affect protein-protein interactions and subcellular localization. Motifs 1–10 are essential for core biochemical activity (likely acetylation) and are evolutionarily conserved. All three subgroups contain GCD (motif 1), DXXH (motif 2), and motif 4, as also observed in N. tabacum (Wang et al., 2025). Many StTBL genes possess both PC-esterase and PMR5N domains, similar to findings in R. chinensis (Tian et al., 2021). Notably, StTBL33/49/60 may have roles beyond cell wall modification; StTBL33, for instance, could be involved in regulating cell wall formation, cellulose, and xylan synthesis. Sixteen genes contain the conserved PLN02629 domain, reported also in G. hirsutum, N. tabacum, and P. bretschneideri, indicating its potential importance. Group I, with frequent PC-Esterase and PMR5N domains, represents a classical acetyltransferase branch. In Group II, PC-esterase is more conserved than PMR5N, pointing to functional divergence. Group III may originate from recent tandem duplications or retrotranspositions, with higher PMR5N content possibly indicating positive selection. This subgroup uniquely retains the GCD and DXXH motifs, associated with glycosylation and acid catalytic activity respectively, suggesting distinct substrate specificity (Wang et al., 2025). StTBL33/49/60 belong to the PMR5N superfamily, while 16 genes are part of the PLN02629 superfamily, underscoring TBL multi-functionality in cell wall metabolism—a pattern also noted in G. hirsutum, N. tabacum, and P. bretschneideri (Ban et al., 2025; Wang et al., 2025; Zhu et al., 2024). Gene structure analysis shows considerable exon number variation (1–15). Single-exon genes like StTBL39 may arise via retrotransposition, whereas multi-exon genes (e.g., StTBL30 with 15 exons) could gain novel functions through exon shuffling. Fewer-exon genes likely perform core functions, while genes with more exons may undergo alternative splicing to produce isoforms responsive to environmental or endogenous signals (Lin et al., 2024).
GO analysis revealed that all 72 genes participate in cell wall xylan O-acetylation, with the initial acetylation step likely occurring in the Golgi apparatus (Manabe et al., 2011). Specifically, StTBL2/38/40/64/65/70 may function in the initiation of xylan backbone acetylation, while StTBL25, StTBL31, and StTBL33 may be involved in cold stress responses. In addition, cis-acting elements in StTBL promoters also may play a pivotal role in regulating gene expression related to growth, hormonal signaling, and stress responses. StTBL genes are involved in abiotic stress responses (such as drought and salt) and hormone signaling pathways.
These StTBL-targeting miRNAs may integrate multiple signals related to potato growth, development, and stress responses. Specifically, developmental cues—such as stu-miR156/172-regulated morphogenesis, tuberization, and flowering time—and nutrition/stress signals—including miR395/miR399-mediated sulfur/phosphorus meta-bolism and salt adaptation—could converge on the regulation of specific StTBL genes (He et al., 2024; Luo et al., 2024; Pegler et al., 2020; Yang et al., 2022). As TBL proteins directly participate in modifications like cell wall acetylation, changes in their expression may finely tune the wall’s mechanical and chemical properties, positioning them as key hubs linking internal signaling with external morphogenesis or biotic/abiotic stress resistance. For example, during pathogen infection, stu-miR482-mediated regulation of NBS-LRR immune genes may occur alongside its targeting of certain StTBL genes (Luo et al., 2023), potentially enabling a dual defense strategy that combines classical immunity with rapid adjustment of cell wall barriers.
StTBL9/10/11/12/13/14/16/18/20/32/35/40/41/48/52/63/64/48/63/64/65/70 likely participate in fundamental physiological processes and function. Among these, StTBL14/20/40/48/63/64/65 may play a role in fundamental physiological processes. StTBL14 may play an important regulatory role in both vegetative growth and reproductive development, while StTBL20 may contribute to storage organ formation and reproductive development.
StTBL1 and StTBL3 may have a synergistic effect in response to salt and drought stress. StTBL1, StTBL3 and StTBL59, StTBL16, StTBL20 and StTBL5 may synergistic involvement in salt tolerance. Several gene pairs or groups (such as StTBL19/22, StTBL21/31, StTBL28/33, and StTBL69/2/12) indicate that they may function together in coordinated pathways to respond to drought. It is worth noting that both StTBL2 and StTBL12 contain MBS elements, which further provides a basis for their potential drought tolerance. Notably, the GO annotation for StTBL2 indicates a possible role in cell wall formation, supporting its involvement in stress-associated structural regulation. In addition, StTBL3 and StTBL69 also contained MBS elements. These results further speculated that StTBL3 responded to salt and drought stress and the potential drought tolerance of StTBL69. In addition, the tissue expression patterns were similar between StTBL1 and StTBL28; StTBL3 and StTBL31; and StTBL2 and StTBL21. This suggests that the genes within each pair may function together in the same biological pathway or as part of a complex.
StTBL31, like AtTBL28, may be a key xylan o-acetyltransferase involved in plant cell wall biosynthesis. StTBL31 showed a sustained increase in expression under drought stress. Together with its high sequence homology and collinearity with AtTBL28, as well as GO annotations indicating involvement in xylan synthesis and cold-stress responses, these findings suggest that StTBL31 participates in long-term protective responses rather than transient stress reactions. StTBL31 may function as a conserved xylan acetyltransferase induced by osmotic stress, contributing to drought and environmental adaptation by dynamically modifying cell wall xylans. This makes StTBL31 a strong candidate gene for molecular breeding of drought-resistant potato, with potential roles extending beyond cell wall synthesis to include integrating stress signaling and cell wall integrity pathways. No collinearity between the potato TBL gene and AtTBL33, although StTBL34 and StTBL26 proteins share high homology with AtTBL33. This suggests conserved functional evolution, while gene duplication and functional diversification in potato may enhance regulatory complexity despite loss of positional correspondence.
Collectively, this study shows that StTBL genes play central roles in cell wall modification, chloroplast function, and stress responses. These findings provide valuable candidate genes for improving potato stress resistance.
Conclusions
5
This study comprehensively characterized 72 TBL genes in potato, delineating their conserved PC-esterase domain. Phylogenetic analysis divided these genes into three groups based on distinct structures and motif compositions, while chromosomal mapping revealed an uneven distribution of TBL genes across the 12 potato chrom-osomes. Tandem and fragment duplication events were identified as key evolutionary forces driving the StTBL family expansion. Collinearity analysis with six representative species further provided insights into evolutionary conservation, offering a basis for future comparative functional studies. Expression profiling revealed tissue-specific patterns and dynamic responses of 72 genes under drought and salt stress. 10 genes (StTBL1/3/16/20/22/28/58/59/60/68) were identified as potential salt-responsive cand-idates, while 11 genes (StTBL1/2/3/12/19/21/22/28/31/33/69) were potential drought-responsive candidates. Notably, StTBL1/3/22/28 responded to both salt and drought stress. In addition, StTBL1/2/16/22/31/33 are likely involved in cell wall formation or modification. Collectively, these findings identify key StTBL genes as promising targets for further functional validation and the development of stress-resilient potato cultivars.
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