Anthocyanins are a class of water-soluble flavonoid pigments that play crucial roles in plant physiology and human health. Their biosynthesis is regulated by the MYB-bHLH-WD40 transcriptional complex, in which MYB transcription factors serve as the key determinants. However, the function of repressor-type MYBs in tuber crops such as potato remains poorly understood. Here we show that the R2R3-MYB transcription factor StMYB3 acts as a repressor of anthocyanin biosynthesis in potato. Transient-expression assays revealed that co-expression of StMYB3 with StAN2 (an R2R3 MYB activator) markedly reduced anthocyanin accumulation in leaves, and tuber skins of StMYB3-over-expressing lines exhibited a significant decrease in anthocyanin content. Mechanistic analyses demonstrate that both StMYB3 and StAN2 interact with StAN1 (an anthocyanin related bHLH). StMYB3 not only suppresses the StAN2-mediated transcriptional activation of anthocyanin biosynthesis genes (StCHS, StF3H, StF3'5'H, and StGST) but also directly represses the promoter activity of bHLH transcription factor StAN1. Moreover, StMYB3 and StAN2 form a mutually reinforcing positive-feedback loop at the transcriptional level. These results uncovers a StMYB3-StAN1-StAN2 regulatory module whose dynamics, driven by competition and bidirectional feedback, precisely control anthocyanin accumulation. This finding provides a reference for breeding potatoes with high anthocyanin content.

Potato, the fourth largest food crop in the world, stores nutrients in underground tubers. However, light exposure induces tuber accumulation of chlorophyll and toxic steroidal glycoalkaloids (SGAs); this unwanted trait called tuber greening causes a decline in potato quality and renders parts of tuber inedible. Despite progress in enzymatic cascades governing SGAs biosynthesis, the regulatory scheme of SGAs and chlorophyll metabolism in light-exposed tubers persists as a critical knowledge gap. Here, we identify that the blue light receptor StCRY1 plays a predominant role in light-induced tuber greening and SGA elevation, functioning as a light-controlled transcriptional switch for genes involved in SGAs and chlorophyll biosynthesis. We show that the transcription factor StHY5 acts downstream of StCRY1 to coregulate both chlorophyll and SGA metabolism. However, StMYB4, a transcription factor regulated by StHY5, promotes SGA synthesis by directly binding to SGA biosynthetic genes without affecting chlorophyll homeostasis. Furthermore, StCRY1 employs a dual strategy by modulating StMYB4 expression and physically interacting with StMYB4 to regulate its transcriptional activity. Collectively, these findings uncover a modularly coordinated control of SGA accumulation and chlorophyll biosynthesis by bifurcation of the StCRY1-StHY5 axis, providing a promising strategy to concurrently curb light-induced tuber greening and glycoalkaloids accumulation.

The plant hormone abscisic acid (ABA) is historically recognized as a tuberization stimulator, with exogenous application significantly enhancing potato tuber formation. However, the physiological significance of endogenous ABA signaling in tuberization and its underlying molecular mechanisms remain poorly understood. Here, by using ABA-insensitive hypermorphic StHAB1G276D-overexpression mutant and StHAB1 knockdown mutant, we demonstrate that ABA signaling is essential for normal tuber formation in potato. Blocking of ABA signaling by StHAB1G276D-overexpression reduces underground stolon sensitivity to leaf-derived tuber-inducing signals. Notably, StHAB1 directly interacts with the tuber-forming signal StSP6A and reduces its phosphorylation level. Transcriptomic and quantitative phosphoproteomic analyses proved that StHAB1G276D modulates gene expression and phosphorylation of key players in the tuberization pathway and gibberellic acid signaling. Collectively, our findings uncover a critical role of endogenous ABA signaling in potato tuber formation and identify a mechanism linking the conserved FLOWERING LOCUS T and ABA pathways, offering molecular insights for accelerating potato tuberization by modulating hormone signaling.

Potato (Solanum tuberosum) breeding is severely hindered by its highly heterozygous autotetraploid genome, where complex allelic interactions impede precise trait selection. Reconstructing complete haplotype-resolved assemblies is crucial for genome-assisted breeding. However, current assembly methods for autopolyploids often generate fragmented sequences, haplotype-switch errors, and gaps in complex regions such as centromeres. To address these challenges, we develop PHap, a haplotype assembly pipeline tailored for autopolyploids, using only standard sequencing data, including long-reads and Hi-C. Applying PHap to the autotetraploid potato cultivar HuaShu4, we generate a haplotype-resolved, near telomere-to-telomere assembly of 3.12 Gb with an N50 of 32.7 Mb and 99.7

The pigmentation patterns of potato tubers are complex and diverse, often exhibiting significant tissue specificity. This study was conducted to elucidate the molecular mechanisms underlying the differential pigmentation in different parts of potato tubers using two cultivars, ‘Huashu 12’ and 15EM36-26, which exhibit opposite pigmentation patterns between the bud eyes and the tuber periderm. Metabolomic analysis revealed that cyanidin, pelargonidin, and malvidin are the key anthocyanin components responsible for the observed pigmentation differences. A total of 118 common differentially expressed genes in the differentially pigmented tissues of both cultivars were identified in transcriptomic analysis, including key structural genes of the anthocyanin biosynthesis pathway (such as StPAL, StCHS, and StDFR). Weighted gene co-expression network analysis was further employed to screen modules significantly correlated with pigmentation phenotypes, and 28 candidate genes associated with anthocyanin biosynthesis were identified. Expression validation demonstrated that the expression of StbHLH14 was significantly higher in non-pigmented tissues compared to pigmented tissues. Functional analysis revealed that StbHLH14 can inhibit the activation of structural gene promoters (such as StCHS and StDFR) via the MYB transcription factor StAN2, thereby negatively regulating anthocyanin biosynthesis. This study unveils the metabolic and transcriptional basis of tissue-specific pigmentation in potato tubers and clarifies the negative regulatory role of StbHLH14.

Potato is a globally important field crop used as in staple food, processing, starch extraction, and bio-based manufacturing. Developing cultivars with durable virus resistance is critical for maintaining yield stability and supporting commercial seed production. This study aimed to evaluate resistance to three major potato viruses, Potato virus Y (PVY), Potato virus A (PVA), and Potato virus X (PVX), in a diverse panel of potato genotypes and to understand the influence of virus resistance in potato yield along with identification of reliable molecular markers and underlying resistance gene sources to guide future breeding efforts. A total of 67 genetically diverse potato genotypes were assessed using an integrated approach combining phenotypic screening and genotyping. Resistance was evaluated under controlled conditions using both mechanical inoculation and grafting methods with the respective viruses. Genotyping was performed using conventional PCR and kompetitive allele-specific PCR (KASP) assays targeting known resistance genes. To determine the agronomic performance, 12 representative genotypes differing in virus resistance profiles were assessed for yield and stability across multiple environments. Phenotypic screening identified 28 genotypes with extreme resistance to PVY, 20 to PVA, and 17 to PVX. Four genotypes (HS2, HS4, HS15, and G22) exhibited broad-spectrum resistance to all three viruses. Genotyping revealed Rx2–2R2R (AUC = 0.900) for Rx2 and Rx1–5R (AUC = 0.869) for Rx1 as highly predictive markers for PVX resistance, with full concordance between KASP and PCR results. For PVY, the Ryadg-linked marker M6–994 showed the strongest association (AUC = 0.967), while the Ra-linked marker M10 was moderately predictive for PVA resistance (AUC = 0.730). Marker profiles indicated that 18 genotypes harbored Ryadg, 4 carried Rysto, 2 carried Rychc, 12 carried Rx1, 4 carried Rx2, and 19 carried Ra. Some resistant genotypes lacked known markers, suggesting novel resistance sources or allelic variation. Along with resistance evaluation, triple-resistant genotypes (HS2, HS4, HS15), carrying combinations of Ryadg or Rysto (PVY), Rx1 (PVX), and Ra (PVA), produced the highest yields with strong stability across environments (CV = 13.5–18.9 %) and significantly outperformed susceptible and single-resistance groups. In contrast, PVX-only genotypes (HS12, HS14; Rx1 carriers) produced moderate yields (30–36 t/ha) but showed high variability (CV ≈ 29–30 %), while susceptible lines recorded the lowest yields (23.3 t/ha; CV = 39.6 %). Finally, this study provides validated markers, identifies high-value resistance sources, and highlights the agronomic benefits of multi-virus resistance for breeding high-performing, industrial potato cultivars.

Homeostatic regulation of proteolytic activity is fundamental to plant cellular physiology, and dysregulated protease-like activities are frequently associated with cytotoxic or stress-induced cell death. Here, we identify RipBH, a previously uncharacterized type III-secreted protein from Ralstonia solanacearum, as an intracellular self-cleaving protease-like effector with the capacity to perturb host physiological balance. RipBH harbors a papain-like catalytic core and multiple ankyrin repeats; structural mutagenesis showed that conserved catalytic residues (C135, H244, D268, and N117) are indispensable for self-processing and cell death-inducing activity. RipBH undergoes auto-cleavage inside plant cells, producing smaller fragments that are detectable in both the cytoplasm and nucleus. Truncation of ankyrin repeats altered cleavage behavior and abolished cell-death induction, supporting the idea that ankyrin-mediated structural constraints function as a regulatory module required for activation. Importantly, RipBH-induced necrosis occurred largely independently of the tested canonical ETI-related signaling components, suggesting a physiology-centered disruption pathway rather than immune receptor-mediated recognition. We propose that RipBH operates as a pathogen-encoded proteolytic switch that destabilizes intracellular homeostasis, providing a potential mechanistic link between effector auto-processing and necrosis-like physiological collapse under biotic stress. Our findings contribute to the conceptual framework of proteolysis-associated plant cell dysfunction and highlight pathogen-driven interference with core physiological processes.

The shikimate pathway serves as a central metabolic route for the biosynthesis of aromatic amino acids and diverse secondary metabolites involved in plant growth and defense. While its contribution to structural and chemical immunity is well recognized, the roles of individual shikimate pathway enzymes in immune regulation remain poorly understood. Here, we characterize NbMEE32, a 3-dehydroquinate dehydratase/shikimate dehydrogenase (DHD/SDH), as a negative regulator of bacterial wilt resistance in Nicotiana benthamiana. Virus-induced gene silencing (VIGS) of NbMEE32 enhances resistance to Ralstonia solanacearum, accompanied by increased shikimate accumulation and lignin deposition in vascular tissues. Exogenous shikimate treatment partially recapitulates defense responses observed in NbMEE32-silenced plants. Transcriptome profiling indicates that NbMEE32 silencing is associated with extensive transcriptional reprogramming related to defense responses and shikimate-associated metabolism. Together, these findings reveal a previously unappreciated immune-regulatory role of shikimate pathway enzymes and suggest new strategies for engineering disease resistance through metabolic intervention.

ABSTRACT Apical dominance is a key determinant of plant architecture and yield formation in crops. Auxin and jasmonic acid are crucial endogenous regulators of this process, yet their functions in the above‐ground shoots and underground modified stems (tubers) of asexually propagated potato plants remain largely unclear. Here, we demonstrate that the jasmonate pathway component StJAZ1‐like promotes the release of apical dominance in potato. StJAZ1‐like overexpression enhanced axillary bud outgrowth, increased shoot branching and triggered multi‐bud sprouting from individual tuber eyes. Transcriptome profiling of dormant axillary buds revealed pronounced alterations in hormone‐related pathways, including marked upregulation of the auxin efflux carrier StPIN3 , accompanied by reduced indole‐3‐acetic acid (IAA) and abscisic acid (ABA) levels in axillary buds. Consistently, functional analyses demonstrated that StPIN3 positively regulates lateral branching and tuber sprouting, indicating that StPIN3 acts downstream of StJAZ1‐like. Mechanistically, StJAZ1‐like physically interacts with StPIF02, a bHLH transcription factor. StPIF02 directly binds the StPIN3 promoter and activates its transcription. Collectively, our results suggest a StJAZ1‐like–StPIF02–StPIN3 regulatory module that links jasmonate with auxin to modulate apical dominance in potato, providing candidate pathways for designing an ideal multi‐branched plant architecture in potato.

Potato spindle tuber viroid (PSTVd) poses a major threat to potato production due to its stable, non-coding RNA genome and high infectivity. In this study, we harnessed the CRISPR/Cas13a system to confer targeted resistance against PSTVd in potato. Specific CRISPR RNAs (crRNAs) were designed to target conserved left terminal and central regions of the PSTVd genome, and two CRISPR/Cas13a vectors, A and B, were constructed to target these distinct functional domains. Using the Desiree potato cultivar, seven stable transgenic lines were generated via Agrobacterium-mediated transformation. Phenotypic evaluation confirmed that transgene expression did not affect normal plant growth. Quantitative assessment of PSTVd accumulation after mechanical friction inoculation showed that all transgenic lines exhibited significantly reduced viroid levels relative to wild-type controls. This reduction was observed in leaves sampled from 10 to 40 days post-inoculation and in tubers analyzed 30 days after harvest. These findings demonstrate that CRISPR/Cas13a efficiently and specifically suppresses PSTVd accumulation, establishing a durable strategy for engineering viroid-resistant potato. This study provides a successful example for applying RNA-targeting CRISPR technologies to control viroid infections in potato and advance breeding for viroid resistance in potato.

Light is a key factor for inducing anthocyanin biosynthesis; however, its regulatory mode in potato anthocyanin biosynthesis remains unclear. Previous research identified a specific genotype that causes the tuber skin to gradually turn purple when exposed to light of different wavelengths. In the present study, we conducted metabolome and transcriptome analyses on tuber samples during anthocyanin accumulation. The metabolome data showed that the contents of naringenin chalcone, naringenin, dihydrokaempferol, and cyanidin gradually increased during anthocyanin accumulation. The transcriptome data showed that the expression levels of most structural genes increased gradually during anthocyanin accumulation, especially the StF3'H gene that promotes cyanidin formation. Moreover, the photo-responsive transcription factor StHY5 was specifically expressed at high levels before anthocyanin accumulation, occurring 2 h after light induction. Establishment of transgenic lines demonstrated that StHY5 overexpression could promote the accumulation of anthocyanin in potato tubers, along with a parallel increase in the transcription levels of StAN2, StMYBA1, StCHI, StF3H, StF3'H, and StDFR. Electrophoretic mobility shift and dual luciferase assays showed that StHY5 can enhance the promoter activity of the MYB transcription factors StAN2 and StMYBA1 as well as the structural genes StCHI and StF3H through binding to the G-box motif. StAN2 activated the expression of StF3'H (a newly identified purple gene locus in potato) and StDFR by binding to the MYB-binding site in the promoters, thereby promoting anthocyanin biosynthesis. This study provides a theoretical basis for revealing the molecular mechanism of light-regulated anthocyanin biosynthesis in potatoes.

Cold-induced sweetening (CIS) in potato tubers represents a significant challenge for the potato processing industry, characterized by the accumulation of reducing sugars during cold storage that leads to undesirable browning and potential acrylamide formation during frying. This comprehensive review integrates current understanding of CIS mechanisms, from pre-harvest factors through molecular regulation to breeding strategies. Recent multi-omics studies have revealed complex regulatory networks involving transcriptional, post-transcriptional, and epigenetic modifications. Key metabolic pathways include starch degradation, primarily regulated by β-amylase (BAM) and starch phosphorylase (PHO1), sucrose biosynthesis controlled by UDP-glucose pyrophosphorylase (UGPase) and sucrose phosphate synthase (SPS), and sucrose degradation mediated by vacuolar invertase (StVInv). The discovery of VInvIn2En, a cold-responsive enhancer in StVInv's second intron, has provided new insights into transcriptional regulation. Notably, the interaction between StVInv and its inhibitor (StInvInh2), modulated by the SnRK1 complex, emerges as a central regulatory mechanism. The role of tonoplast sugar transporters (TST), particularly StTST1, has been identified as crucial in sugar compartmentalization. Environmental factors, including ethylene and hypoxia, significantly influence CIS through distinct molecular mechanisms. Breeding efforts have leveraged wild Solanum species and molecular tools, including CRISPR/Cas9-mediated gene editing, to develop CIS-resistant varieties. Despite these advances, challenges remain in understanding plastidial transport mechanisms, regulatory networks, and translating molecular insights into practical breeding applications. Future directions emphasize the need for integrated approaches combining genetic improvement, optimized storage conditions, and enhanced understanding of regulatory mechanisms to develop sustainable solutions for the potato industry.

The Rx1 and Rx2 genes from wild potato species confer extreme resistance to potato virus X (PVX). Accurate genotyping is essential for their integration into breeding programs. We developed tetra-primer ARMS-PCR markers targeting SNPs (G2515C in Rx1 and C2445G in Rx2) in the LRR regions to distinguish resistance alleles. These markers effectively genotyped 50 tetraploid potato varieties, identifying 16 as Rx1 heterozygous, 5 as Rx2 heterozygous, and 29 as homozygous susceptible consistent with phenotypic data. The T-ARMS-PCR outperformed existing markers and enabled efficient duplex detection of both genes. Although no homozygous resistant varieties were found, artificial DNA mixtures validated marker reliability for homozygous and heterozygous resistance detection. To our knowledge, this is the first use of T-ARMS-PCR for Rx1/Rx2 genotyping, offering a high-throughput tool for marker-assisted selection in developing PVX-resistant cultivars.

Potato virus X (PVX) seriously threatens global potato cultivation, necessitating a deeper understanding of the genetic mechanisms responsible for resistance. The Rx1 and Rx2 genes play a pivotal role in breeding potatoes with broad-spectrum PVX resistance. However, current DNA markers for these genes present uncertainties, as they are found in both resistant and susceptible materials, complicating the identification of the responsible resistance gene. This study aimed to develop robust high-throughput molecular markers, specifically Kompetitive Allele-Specific PCR (KASP) markers, to reliably detect the Rx1 and Rx2 genes. Genetic polymorphism analysis of seven resistant and four susceptible potato genotypes revealed seven single nucleotide polymorphisms (SNPs) and one insertion-deletion (Indel), along with corresponding amino acid changes in the Rx1 and Rx2 protein sequences. Based on these variations, high-throughput molecular markers were developed. Notably, the primer pairs Rx1-1R-F + Rx1-5R-R and Rx2-1R2-F + Rx2-2R2-R showed high accuracy, detecting Rx1 and Rx2 genes without false positives or false negatives. Optimized multiplex PCR protocols also demonstrated strong potential in simultaneously detecting both genes. KASP markers targeting SNPs at positions 2 515 (G to C) in the Rx1 CDS and 2 445 (C to G) in the Rx2 CDS efficiently differentiated between heterozygous resistant and homozygous susceptible genotypes. Furthermore, these markers effectively identified Rx1 and Rx2 genotypes in 243 and 133 progenies across multiple crosses. Progenies that tested positive for Rx1 and Rx2 through PCR or KASP assays exhibited extreme resistance (ER) to PVX, while negative progeny was susceptible. These findings highlight the potential of the developed markers to accelerate the breeding cycle and improve the production of PVX-resistant potato varieties.

Plant viruses, constrained by their limited genomic coding capacity, rely significantly on host factors for successful infection. Disruption of these essential host factors can confer resistance to viruses, with such factors categorized as susceptibility genes or recessive resistance genes. Recent research has identified developmentally regulated plasma membrane polypeptide (DREPP) proteins as susceptibility factors integral to the cell-to-cell movement of potyviruses. In the present study, we demonstrated that the silencing of StPCaP1, a DREPP gene in potato, confers novel resistance to both Potato virus Y (PVY, Potyvirus) and Potato virus S (PVS, Carlavirus). Interaction and subcellular localization analyses revealed that the movement proteins (MPs) of PVY (P3NPIPO) and PVS (TGB1) interact with StPCaP1, recruiting it to plasmodesmata (PD). Furthermore, transcriptome analysis and experimental validation indicated that compared to wild-type (WT) controls, StPCaP1-silenced lines exhibit significantly increased glucose content and elevated expression levels of several UDP-glucosyltransferases (UGTs), which are potential components of the callose synthesis complex. These findings suggest that StPCaP1 participates in callose deposition, as evidenced by the increased callose deposition at PD and reduced PD permeability observed in StPCaP1-silenced lines. Additionally, we found that StPCaP1 expression in Nicotiana benthamiana led to reduced callose deposition at PD and promoted PVY-GFP cell-to-cell movement in NbPCaP1-silenced plants in a concentration-dependent manner, which suggests the changes in callose deposition at PD induced by StPCaP1 relates to viral cell-to-cell movement. This study provides a deeper understanding of DREPP-mediated viral movement and highlights potential targets for developing virus-resistant crops.

Sugar transporters play pivotal roles in plant growth, development, and stress responses. However, the function of sugar transporters in potato (Solanum tuberosum) is still obscure. In this study, the function of potato tonoplast sugar transporter 1 (StTST1) in subcellular sugar compartmentation and abiotic stress tolerance was characterized. Heterologous expression assays in Saccharomyces cerevisiae (strain W303) demonstrated that StTST1 mediates sucrose transporting into the vacuole. Moreover overexpression and RNA interference (RNAi) of StTST1 in potato altered leafy sugar content including sucrose, glucose, and fructose without affecting the activity of key metabolic enzymes. Intriguingly, RNAi-mediated suppression of StTST1 enhanced freezing tolerance and compromised drought tolerance. In contrast, overexpression of StTST1 enhanced drought tolerance but reduced freezing tolerance. Our results demonstrate that StTST1 dynamically regulates subcellular sugar partitioning and differentially modulates freezing and drought stress responses. These findings highlight the potential of targeted manipulation of sugar transporters to modulate crop resilience to multiple abiotic stresses.

Frost stress poses a serious threat to the potato industry. C-repeat binding factors (CBFs) are key transcription factors involved in plant cold responses and the adaptive evolution of land plants. However, their function and underlying mechanisms in potato remain poorly understood. This study analyzed homologous CBF2 genes from 46 potato genotypes and revealed significant structural variations, including a critical site (site A) that is closely associated with cold tolerance. There are at least 2 site A types, including the cold-tolerant Solanum commersonii type and the cold-sensitive Solanum tuberosum type. Overexpression of ScCBF2 significantly enhanced potato cold tolerance, whereas StCBF2 overexpression had a limited effect. We demonstrated that both ScCBF2 and StCBF2 improve cold resistance by regulating glutathione S-transferase tau (GSTU)- and ZAT10-mediated reactive oxygen species scavenging systems. Notably, ScCBF2 uniquely upregulated Galactinol synthase 3 (GolS3), promoting raffinose biosynthesis. Compared with StCBF2, ScCBF2 exhibited a stronger binding affinity to the GolS3 promoter, resulting in higher transcriptional activation. Overexpression of ScGolS3 increased leaf raffinose content and cold tolerance. Furthermore, we confirmed the critical role of site A in the ScCBF2-GolS3 regulatory pathway. In summary, this study highlights the functional divergence caused by structural variations in CBF2, with differential regulation of GolS3 contributing to cold tolerance. Our work provides insights into the molecular mechanisms underlying cold tolerance in potato and offers potential targets for improving frost resistance in this vital crop.

The Ra extreme resistance against potato virus A was mapped to the upper of chromosome 4 in tetraploid potato. Potato virus A (PVA) is one of the major viruses affecting potato worldwide and can cause serious disease symptoms and yield losses. Previously, we determined that potato cultivar Barbara harbors Rysto (genotype: Ryryryry) and Ra (genotype: Rararara) that each independently confer extreme resistance to PVA. In this study, employing a combination of next-generation sequencing and bulked-segregant analysis, we further located this novel Ra on chromosome 4 using a tetraploid BC1 potato population derived from a Ry-free progeny (Rararararyryryry) of Barbara (RarararaRyryryry) × F58050 (rararararyryryry). Using 29 insertion–deletion (InDel) markers spanning chromosome 4, Ra was delimited by the InDel markers M8-83 and M10-8 within a genetic interval of 1.46 cM, corresponding to a 1.86-Mb genomic region in the potato DM reference genome. The InDel marker M10-8, which is closely linked with the resistance against PVA in the Ry-free segregating populations, was then used to screen 43 selected Rysto-free tetraploid potato breeding clones. The phenotype to PVA was significantly correlated with the present/absent of the marker, albeit with a 9.3

The integration of molecular markers in the realm of potato genetics has opened new avenues for accelerating genotype analysis and developing improved varieties. Many markers linked to important features have been discovered so far and are consistently distributed across 12 chromosomes (× = 12) of potato. Notably, the genes allied to disease resistance stand out as significant and prevalent. Molecular markers associated with these genes have revolutionized selection processes, making them faster and more effective. Besides, advanced technologies such as kompetitive allele-specific PCR, high-resolution melting assay, SNP-array, genotyping by sequencing, and genome-wide association study, are emphasizing the use of those molecular markers with greater accuracy to detect R genes aligning with the phenotypes. This review discusses advances in potato breeding for resistance against common stresses, focusing on progress made through molecular marker-assisted selection.

Potato (Solanum tuberosum L.) is cultivated worldwide for its underground tubers, which provide an important part of human nutrition and serve as a model system for belowground storage organ formation. Similar to flowering, stolon-expressed FLOWERING LOCUS T-like (FT-like) protein SELF-PRUNING 6A (StSP6A) plays an instrumental role in tuberization by binding to the bZIP transcription factors StABI5-like 1 (StABL1) and StFD-like 1 (StFDL1), causing transcriptional reprogramming at the stolon subapical apices. However, the molecular mechanism regulating the widely conserved FT-bZIP interactions remains largely unexplored. Here, we identified a TCP transcription factor StAST1 (StABL1 and StSP6A-associated TCP protein 1) binding to both StSP6A and StABL1. StAST1 is specifically expressed in the vascular tissue of leaves and developing stolons. Silencing of StAST1 leads to accelerated tuberization and a shortened life cycle. Molecular dissection reveals that the interaction of StAST1 with StSP6A and StABL1 attenuates the formation of the alternative tuberigen activation complex (aTAC). We also observed StAST1 directly activates the expression of potato GA 20-oxidase gene (StGA20ox1) to regulate GA responses. These results demonstrate StAST1 functions as a tuberization repressor by regulating plant hormone levels; our findings also suggest a mechanism by which the widely conserved FT-FD genetic module is fine-tuned.