Plants were grown under natural condition in Jinzhong (36°85’ N, 111°77’ E), Shanxi province, China. Yugu1 (Setaria italica cv. Yugu1), a sequenced wild-type foxtail millet variety with a complete reference genome (Bennetzen et al., 2012), was employed in this study.

The pYLCRISPR was used to construct CRISPR vector targeting OsCST1 (rice homolog of SiCST1), which were subsequently employed for genetic transformation of rice variety Zhonghua11 (Oryza sativa L. ssp. japonica Zhonghua11). CRISPR-P 2.0 was utilized to design the base-pairing sequence of the sgRNA (5′-GCATCAGCAGCAGACGCCAC-3′) targeting the single exon of OsCST1.

To complement oscst1 mutant, genomic fragments of SiCST1 (3609 bp) were cloned into pCAMBIA1301 vector. The resulting construct was transformed into Agrobacterium tumefaciens EHA105 and subsequently transformed into rice callus induced from mature seeds of oscst1 mutant.

Cold stress treatment were implemented as previously described (Ma et al., 2009). The rice seedlings were cultivated in Kimura B nutrient solution under controlled condition (day/night temperature: 30°C/25°C and light/dark photoperiod: 10 h/14 h) until they reached the three-leaf stage. Subsequently, the seedlings were subjected to cold stress (4°C) for 96 h. After a recovery period of 7 days at 30°C/25°C (day/night), seedling survival rate were assessed. Three biological replicates were established, each comprising 32 rice seedlings.

For RNA-Seq analysis under cold stress, three-leaf-stage foxtail millet seedlings grown at 28°C/24°C (day/night) were transferred to 4°C. After 24 h of treatment, both cold-treated and untreated seedlings were harvested, immediately frozen in liquid nitrogen, and stored at -80°C for subsequent RNA isolation. Total RNA was extracted from three biological replicates (each comprising five pooled seedlings). High-throughput sequencing was performed on HiSeq 2500 (Novogene, Beijing). Clean reads were aligned to Setaria italica reference genome (v2.2, Phytozome).

For SiCST1 expression analysis during cold stress treatment, foxtail millet seedlings cultivated to the three-leaf stage at 28°C/24°C (day/night) were exposed to 4°C. The seedlings were collected at 0, 1, 3, 6, 12, and 24 h post-treatment, immediately frozen in liquid nitrogen, and stored at -80°C for RNA extraction. Total RNA was extracted from three biological replicates, with each consisting of a pooled sample of five seedlings.

Total RNA was extracted from tissues using the RNeasy plant mini kit (QIAGEN). The isolated RNA was reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). Quantitative RT-PCR (RT-qPCR) was performed utilizing SYBR Green Real-Time PCR Master Mixes (Invitrogen). Gene expression level was normalized with foxtail millet actin gene (Seita.7G294000). Primers used for RT-qPCR are listed in Supplementary Table S1 .

The full-length cDNA of SiCST1 was cloned into pBI221-GFP vector to create fusion protein with GFP at the C-terminus of SiCST1. SiCST1-GFP and the nucleus marker H2B-mCherry were co-transformed into foxtail millet protoplasts via the polyethylene glycol-mediated transformation method, as previously described (Chen et al., 2006). After culturing at 25°C for 16 h in darkness, the transformed protoplasts were observed using a confocal scanning microscope.

For bait construction, the full-length SiCST1 cDNA was cloned into pGBKT7 (Clontech) and transformed into yeast strain Y2HGold. For prey cDNA library construction, total RNA was isolated from 2-week-old foxtail millet seedlings (Yugu1) using the RNeasy Plant Kit (QIAGEN). Synthesized cDNA was co-transformed with linearized pGADT7-Rec vector (Clontech) into Saccharomyces cerevisiae strain Y187 via the Make Your Own Mate & Plate Library System (Clontech).

For Y2H screening, bait strain Y2HGold (harboring pGBKT7-SiCST1) and prey strain Y187 (containing the cDNA library) were mated in YPDA liquid medium at 30°C for 24 h with 40 rpm shaking. Mating diploids were then plated onto SD/-Leu/-Trp/-His/-Ade + X-α-Gal plates. Following 7-day incubation at 30°C, colonies exhibiting blue coloration were restreaked for confirmation. Validated positive clones underwent PCR amplification of insert fragments, followed by DNA sequencing. Sequence alignment against Setaria italica genome using BLASTn identified SiOFP1 (LOC101755245) as a high-confidence interactor detected in 12 out of 20 sequenced clones.

The full-length cDNA of SiCST1 was cloned into pUC-SPYNE173 vector and the full-length cDNA of SiOFP1 was cloned into pUC-SPYCE (M) vector. To evaluate potential false positives, the pUC-SPYNE173-SiCST1 construct was co-transformed with the empty pUC-SPYCE (M) vector as a negative control.

Protoplasts were isolated from 10-day-old etiolated foxtail millet leaves (Yugu1) by enzymatic digestion. Leaves were sliced into 0.5-mm strips and incubated in digestion solution [1.5% (w/v) Cellulase R10, 0.4% (w/v) Macerozyme R10, 0.5 M D-mannitol, 20 mM KCl, 20 mM MES (pH 5.7), 10 mM CaCl₂, 0.1% BSA] at 28°C for 4 h with gentle shaking (40 rpm). Protoplasts were filtered through 75-μm nylon mesh, washed twice with W5 solution [154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM MES (pH 5.7)], and resuspended in MMg solution [0.5 M mannitol, 15 mM MgCl₂, 4 mM MES (pH 5.7)] at a density of 1 - 5 × 10⁶ cells/mL.

For transformation, 10 μg each of pUC-SPYNE173-SiCST1 (N-terminal YFP fragment) and pUC-SPYCE(M)-SiOFP1 (C-terminal YFP fragment) plasmids were added to 100 μL protoplast suspension, followed by 110 μL PEG solution (40% PEG4000, 0.4 M mannitol, 0.1 M CaCl₂). After 15-min incubation at 25°C, reactions were quenched with 1 mL W5 solution. Transfected protoplasts were cultured in WI solution [0.5 M D-mannitol, 4 mM MES (pH 5.7), 20 mM KCl] at 25°C for 16 h in darkness (Walter et al., 2004). Following incubation, YFP fluorescence was observed using a confocal scanning microscope.

The pA7-SiOFP1-GFP and FLAG-SiCST1 plasmids were co-transformed into foxtail millet protoplasts with FLAG-SiCST1 and the empty pA7-GFP vector serving as control to assess false positives. Transfected protoplasts were incubated at 25°C in darkness for 14–16 h. Upon microscopic confirmation of GFP fluorescence expression, the protoplasts were lysed with 400 μL IP buffer (10 mM HEPES, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, 1×protease inhibitor cocktail, pH 7.5). The lysate was centrifuged at 5,000 g for 5 min at 4°C. The resultant supernatant was incubated overnight at 4°C with 20 μL of anti-FLAG agarose beads (MBL). The beads were collected and washed five times with IP buffer, followed by boiling in SDS buffer. The samples were examined by western blotting using anti-GFP or anti-FLAG antibody.

Lamina joint bending assays were performed on the second fully expanded leaves of two-week-old WT and oscst1 rice seedlings as previously described (Feng et al., 2016). Excised leaf segments were placed in media supplemented with 0, 0.01, 0.1, or 1 μM 24-epibrassinolide, with three biological replicates per concentration and five seedlings per replicate. After 48 h incubation in darkness at 28°C, the lamina joint angle was quantified using ImageJ software.

Significant differences between control and treatment were analyzed using Student’s t-test within SPSS version 25 software (IBM SPSS Statistics). Differences were considered significant at P < 0.05 and highly significant at P < 0.01.

To identify genes associated with cold stress tolerance in foxtail millet at the seedling stage, we conducted transcriptome deep sequencing (RNA-Seq) analysis on three-leaf-stage seedlings of Yugu1, which were exposed to 4°C cold stress for 24 h. We thus identified SiCST1 (LOC101781117) was up-regulated 4-fold by cold stress treatment. To explore the possible involvement of SiCST1 in cold stress tolerance, we developed CRISPR/Cas9 knockout mutant line, oscst1, targeting the homolog of SiCST1 in rice (OsCST1, LOC4329262). OsCST1 was identified by conducting a blastp search of SiCST1 protein sequence against rice protein database (Oryza sativa ssp. japonica). Genomic analysis confirmed that OsCST1 is a single-copy gene in rice. Knockout of OsCST1 yielded oscst1 mutant. Sequencing of PCR products from oscst1 T1 generation plants confirmed a 1-bp insertion in the single exon of OsCST1, causing a frameshift and premature termination ( Supplementary Figures S1A–C ). The truncated protein consisting of 405 amino acids lacks the C-terminal domain ( Supplementary Figure S1D ).

We then proceeded to evaluate the cold stress tolerance of T1 generation plants of oscst1 by exposing seedlings to 4°C cold stress followed by recovery at 30°C. We defined plants possessing cold stress tolerance as those that displayed continued leaf growth or newly differentiated leaves upon being returned to normal condition following cold stress treatment. A significant difference in survival rate (percentage of seedlings that survived the treatment) and seedling height was observed between wild-type (WT) and oscst1 mutant ( Figures 1A–C ; Supplementary Figure S2 ). Notably, oscst1 mutant seedlings demonstrated cold sensitivity compared to WT. Furthermore, we genetically transformed oscst1 with FLAG-tagged genomic fragments of SiCST1. The resultant complementary line (Cp) rescued the impaired cold stress tolerance observed in oscst1 ( Figures 1D–F ; Supplementary Figure S2 ). Taken together, these findings imply a pivotal regulatory role for SiCST1 in modulating cold stress tolerance.

A full-length SiCST1 cDNA was synthesized using total RNA extracted from two-week-old seedlings of foxtail millet (Yugu1). Sequence analysis revealed that SiCST1 consists of a single exon and has an open reading frame (ORF) encoding 798 amino acid residues, with predicated molecular weight of 87032.1 and isoelectric point of 8.6. Domain analysis revealed that SiCST1 possesses a ribonuclease H‐like (RNase H‐like) domain, which was conserved in SiCST1 and its homologs ( Figure 2 ). Sequence homology searches indicated the presence of SiCST1 homologs in various spermatophytes, but notably absent in animals. Phylogenetic analysis distinctly categorized these sequences into separate clades corresponding to monocots and dicots ( Figure 3 ), suggesting that the emergence of these sequences may be associated with the differentiation of spermatophytes.

To identify the subcellular localization of SiCST1, we performed transient protoplast transformation of foxtail millet using constructs of either ubi::SiCST1-GFP (maize ubiquitin promoter-driven fusion) or H2B-mCherry (constituting a nucleus marker). We observed complete overlap between SiCST1-GFP and H2B-mCherry fluorescence signals ( Figure 4A ), indicating that SiCST1 is localized in nucleus. Constitutive expression of SiCST1 was observed in all examined tissues, with particularly high expression level detected in young tissues ( Figure 4B ). Additionally, exposure to cold stress (4°C) induced SiCST1 expression, with expression level more than three times higher after 24 h of treatment compared to the untreated control ( Figure 4C ). This discovery was consistent with the involvement of this gene in seedling cold stress tolerance.

To elucidate how SiCST1 regulates cold stress tolerance, potential SiCST1-interacting proteins were identified using prey cDNA library generated from foxtail millet seedling RNA and a bait expressing the full-length cDNA of SiCST1. The yeast two-hybrid system (Y2H) revealed a potential interaction between SiCST1 and SiOFP1 (LOC101755245) ( Figure 5A ). The OFPs are a group of OVATE family transcription factors characterized by a conserved OVATE domain. This interaction was further verified via bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays. In BiFC assay, SiCST1 and SiOFP1 were fused to the N- and C-termini of YFP, respectively, generating SiCST1-nYFP and SiOFP1-cYFP constructs. Co-expression of these constructs in foxtail millet protoplasts resulted in fluorescence signals, confirming the interaction between SiCST1 and SiOFP1 through YFP reconstitution. Conversely, protoplasts co-expressing SiCST1-nYFP and the control construct cYFP failed to generate any fluorescence signals ( Figure 5B ). In CoIP assay, co-expression of FLAG-SiCST1 and GFP-SiOFP1 in foxtail millet protoplasts revealed co-precipitation of FLAG-SiCST1 and GFP-SiOFP1, confirming that SiCST1 directly interacts with SiOFP1 in vivo ( Figure 5C ).

Several studies have reported that OFPs play regulatory roles in phytohormone signaling and biosynthesis pathways, particularly in BR signaling (Snouffer et al., 2020), leading us to formulate the speculation that SiCST1 might be implicated in BR signaling pathways. To rigorously test this hypothesis, a series of lamina joint bending assays were conducted on both oscst1 mutant and WT. The results revealed that in WT, the lamina joint angle gradually increased with rising concentrations of 24-epibrassinolide (epiBL). In contrast, oscst1 mutant demonstrated reduced sensitivity to exogenous BR treatment, even at high concentrations of epiBL ( Figures 6A, B ). These observations provide preliminary evidence supporting the involvement of SiCST1 in BR signaling pathway.

The exploration of cold stress tolerance in foxtail millet has become a central focus in agricultural research, driven by the urgent need to enhance crop resilience in the face of global climate change. To date, most research efforts concentrated on elucidating the physiological changes, transcriptomic response, and metabolic adaptations that occur in foxtail millet following cold stress exposure (Ananthi et al., 2023; Zhang et al., 2023; Zhao et al., 2023). These studies provided invaluable insights into the multifaceted nature of foxtail millet’s cold tolerance mechanisms. Physiological studies demonstrated that foxtail millet underwent a series of adaptive physiological changes in response to cold stress (Ananthi et al., 2023). Transcriptomic analysis further complemented these findings by revealing a complex network of genes that were either up- or down-regulated in response to cold stress, hinting at the intricate regulatory pathways involved in cold stress tolerance (Zhang et al., 2023). Additionally, metabolic profiling studies identified specific metabolites that accumulated or decreased under cold stress, offering clues into the metabolic reprogramming that occurred in foxtail millet to cope with cold stress (Zhao et al., 2023).

While previous studies have significantly expanded our understanding of foxtail millet cold stress tolerance, a critical gap persists in our knowledge of the underlying molecular mechanisms. This limitation hinders the development of targeted genetic engineering or breeding strategies to enhance cold tolerance. In this context, the identification of SiCST1 as a novel plant-specific gene with a predicted ribonuclease H-like domain in foxtail millet represents a significant step forward in our understanding of cold stress tolerance mechanisms. Our results demonstrated that SiCST1 played a crucial role in conferring cold stress tolerance, as evidenced by the significant decrease in survival rate observed in oscst1 knockout mutant following cold stress treatment compared to WT and restored cold stress tolerance in complemented plants. Furthermore, we propose a potential molecular mechanism of SiCST1, suggesting its involvement in regulating key molecular processes during cold stress.

To bridge this discovery toward crop improvement, we propose two translational strategies: genetic engineering and marker-assisted selection (MAS) breeding (Xiaodi et al., 2023; Zhang et al., 2024; Wang et al., 2025b). Specifically: (1) We will construct ubiquitin promoter-driven SiCST1 overexpression lines in rice and foxtail millet to enhance cold tolerance; (2) Through haplotype analysis of diverse germplasm, we will develop SiCST1-linked molecular markers for efficient identification of cold-tolerant varieties via MAS. Thus, SiCST1 will serve as both a biotechnological target and a molecular breeding anchor bridging mechanistic insights in cold stress tolerance with practical crop resilience enhancement.

SiCST1 encodes a plant-specific protein harboring a conserved RNase-H-like domain. Proteins within the RNase-H-like superfamily (RNHLS), categorized as exonucleases or endonucleases, function in critical nucleic acid metabolism processes including DNA replication or repair, homologous recombination, and RNA interference (Majorek et al., 2014). Intriguingly, recent studies identified Reduced height 8 (Rht8), a key “Green Revolution” gene in wheat, as encoding an RNHLS protein that modulated plant architecture through gibberellin biosynthesis regulation (Lingling et al., 2022) (Hongchun et al., 2022). Notably, orthologs of Rht8 in Arabidopsis and maize exhibited conserved roles in height determination (Lingling et al., 2022). In our study, the identification of SiOFP1 as an interactor of SiCST1 through Y2H screening provided critical insights into the molecular mechanism underlying SiCST1-mediated cold stress tolerance. Recent studies have implicated OFPs in various aspects of plant growth, development, as well as response to biotic and abiotic stresses (Wang et al., 2007, 2011; An et al., 2024). The OFPs were implicated in BR signaling pathways, which are known to play pivotal roles in plant growth, development, and stress response (Xiao et al., 2020; Cheng et al., 2023; Wang et al., 2025a; Zhou et al., 2025). Notably, several OFPs have been demonstrated to interact with components of the BR signaling pathways, such as DLT and BES1/BZR1 transcription factors (Yang et al., 2016, 2018; Xiao et al., 2020). These OFPs act as negative regulators of BR response by inhibiting these BR-signaling transcriptional complexes (Yang et al., 2016, 2018; Xiao et al., 2020). These studies suggest that OFPs may function as regulator of BR signaling, modulating the downstream response to BR. We propose that SiCST1 indirectly participates in BR signaling pathways by interacting with SiOFP1.

Accumulating evidence suggests that BR signaling pathways were involved in the regulation of cold stress tolerance in plants (He et al., 2024). BR treatment has been shown to enhance the expression of cold-responsive genes and improve plant cold stress tolerance (Kagale et al., 2007; Anwar et al., 2018; Xia et al., 2018). Conversely, mutants defective in BR signaling exhibit reduced cold stress tolerance (Planas-Riverola et al., 2019; Chaudhuri et al., 2022). These findings collectively indicate that BR signaling plays a positive role in conferring cold stress tolerance. In our study, we speculate that SiCST1 may regulate cold stress tolerance by interacting with SiOFP1 and modulating BR signaling. The model is as follows: In WT plants, CST1 interacts with OFP1, to release its inhibition of BR signaling transcription complex, thereby activating BR signaling pathways and conferring cold stress tolerance. On the other hand, in mutant, the mutated CST1 fails to interact with OFP1, allowing OFP1 to maintain its inhibition of BR signaling transcription complex, which renders the mutant plants sensitive to cold stress ( Figure 7 ). Future studies aimed at elucidating the precise molecular mechanisms underlying the interaction between SiCST1 and SiOFP1, as well as their roles in BR signaling and cold stress tolerance, will be crucial for advancing our understanding of this important regulatory pathway.