TaCSP ‐ H1 Regulates Flowering Time by Directly Promoting the Expression of TaTOE1 in Wheat

Maturity timing is a critical agronomic trait influencing both adaptation and productivity of wheat. In general, early maturation can be an advantageous trait that helps wheat evade terminal abiotic stresses (e.g., dry-hot winds, terminal high temperature, drought) and biotic threats (e.g., pre-harvest sprouting, Fusarium head blight), while delayed maturation extends photosynthetic accumulation and grain-filling duration, thereby boosting its yield and quality (Xiao et al. 2022). Flowering time is the key determinant of wheat maturity timing, which is orchestrated by various signals, such as vernalization (Vrn genes), photoperiod (Ppd genes), and autonomous pathway (Eps genes) (Cao et al. 2021). Our previous study identified a type of genes encoding cold-shock proteins (CSPs) in wheat (TaCSP-Hs), which were acquired from bacteria through horizontal gene transfer (HGT), could enhance wheat's environmental adaptability by improving its photosynthetic efficiency and stress resilience (Wang et al. 2025). However, whether these unique genes are involved in other physiological processes of wheat remains unclear. To investigate the potential roles of horizontally acquired TaCSP-H1 in wheat, we created two tacsp-h1 knockout mutants using CRISPR-Cas9 technology in wheat cultivar Fielder plants and examined the developmental phenotypes of these mutants (Figure 1a). Interestingly, the two mutant lines flowered approximately 4 days earlier and matured about 5 days earlier than WT, respectively (Figure 1b–e), while other agronomic traits, such as plant heights and tiller numbers, were comparable with WT (Figure 1f,g). Meanwhile, the TaCSP-H1 gene is mainly expressed in root, leaf, stem and spike, particularly highly induced during floral transition, which is in line with its role in flowering regulation (Figure S1). To further confirm the function of TaCSP-H1 in wheat flowering control, we also constructed two TaCSP-H1 knockout mutants in wheat cultivar Aikang58 (AK58), which is an elite cultivar and widely planted in China (Figure S2a). The mutants displayed similar early-flowering phenotypes to those in cultivar Fielder background (Figure S2b–e). Taken together, these results suggest that TaCSP-H1 is a negative regulator in wheat flowering control. TaCSP-H1 is mainly localised in the nucleus (Figure S3) and functions as a co-factor during transcription (Wang et al. 2025). In order to further reveal the downstream targets and molecular network of TaCSP-H1 in the regulation of wheat flowering, we performed a transcriptome assay using WT and tacsp-h1 mutant lines (Table S1). Numerous differentially expressed genes (DEGs) were identified between WT and tacsp-h1 mutants during floral transition (Figure S4a and Table S2). Notably, most DEGs were enriched in ‘regulation of multicellular organismal development’, ‘secondary metabolic process’ or ‘regulation of flower development’ (Figure 1h and Table S3). The expressions of many flowering regulatory genes were affected after TaCSP-H1 mutation (Figure S4b), indicating that TaCSP-H1 is a key factor during wheat floral transition. The quantitative PCR validation showed that the expressions of some core regulators in wheat flowering control were significantly altered in tacsp-h1 mutants compared with WT, including VRNs, PPD1, TaSOC1 and TaFUL3, implying that TaCSP-H1 may be involved in the regulation of flowering through these genes in wheat (Figure S5). We next performed RNA immunoprecipitation and quantitative PCR (RIP-qPCR) to identify its direct targets. Among them, TARGET OF EAT1 (TaTOE1), a gene encoding the transcription factor of APETALA2/Ethylene Responsive Factor (AP2/ERF) subfamily (Aukerman and Sakai 2003), was significantly enriched (Figure 1i). The yeast one-hybrid (Y1H) assay indicated that TaCSP-H1 could bind to the promoter region of TaTOE1 (Figure 1j). We further performed the MST and FITC assays and confirmed that TaCSP-H1 could directly bind to the 5′-UTR of TaTOE1 and unfold the hairpin structure in vitro (Figure S6a–c). In addition, the dual-luciferase (LUC) reporter assays indicated that TaCSP-H1 could strongly activate the reporters by increasing the transcript level of the LUC gene (Figure S6d–g). Meanwhile, the transcript abundance of three TaTOE1 homoeologs in A, B, and D sub-genomes largely decreased in tacsp-h1 mutants (Figure S7). Collectively, these results suggest that TaCSP-H1 can regulate TaTOE1 expression through directly increasing its transcript level. Recent studies have reported that the TOE genes could delay flowering by repressing the transcription of FT in soybean (Li et al. 2023). As the expression level of VRN3, the ortholog of FT in Arabidopsis and soybean (Yan et al. 2006), was significantly higher in the tacsp-h1 mutants than WT (Figure S5c), we speculate that TaCSP-H1 regulates VRN3 and is involved in flowering control via the TaTOE1-mediated pathway. To test this hypothesis, we performed the dual-LUC transient assay by fusing the VRN3 promoter with the LUC reporter gene. When the reporter and effector were co-transformed into Nicotiana benthamiana leaves, the transcription of VRN3 was significantly repressed in the presence of TaTOE1 (Figure S8a,b). Additionally, the Y1H assay further confirmed that TaTOE1 could directly bind to the promoter region of VRN3 (Figure S8c). We next investigated the genetic relationship between TaCSP-H1 and TaTOE1 using virus-induced transient gene silencing (VIGS) in WT and tacsp-h1 mutants (Figure S9). The WT and tacsp-h1 mutants exhibited similar early-flowering phenotypes after TaTOE1 knockdown (Figure 1k,l), and the expression levels of VRN3 were also comparable (Figure 1m). Taken together, these findings indicate that TaCSP-H1 represses wheat floral transition by reducing the expression of VRN3 via TaTOE1. We next evaluated the grain agronomic traits of WT and tacsp-h1 mutants. Intriguingly, although the TaCSP-H1 mutation accelerated flowering and maturity time in wheat, which may reduce the accumulation of carbohydrates due to a shortened photosynthetic period, it had no marked impacts on the yield traits (Figure 1n and Figure S10a–c). Moreover, grain quality-related traits, such as protein and starch content, also remained largely unchanged in WT and tacsp-h1 mutants (Figure 1o and Figure S10d,e). It is generally believed that optimal wheat production requires early flowering and maturity to avoid harsh environmental conditions that threaten grain development or harvest, such as drought, heat, or to escape from pathogen attack. However, some flowering-promoting factors in wheat negatively impact grain yield, likely due to reduced photosynthetic accumulation and shortened grain-filling duration period, thereby constraining their utility in breeding applications. In the present study, we demonstrate that TaCSP-H1 is involved in floral transition by directly binding to and increasing the transcripts of TaTOE1, which is a core regulator of flowering in wheat. Besides, the TaCSP-H1 mutation is able to promote flowering and maturity with no significant penalty in grain yield and quality under normal conditions, while the TaCSP-H1 overexpression has no adverse effects on the main agronomic traits (Figure S11), showcasing its great potential value in modern wheat breeding. This study was supported by the Biological Breeding–National Science and Technology Major Project (2023ZD040710109) and the National Natural Science Foundation of China (32472112). The authors declare no conflicts of interest. Data supporting the findings of this study is available in the Supporting Information—S1 of this article. Appendix S1. Data S1. Figures S1–S11. Tables S1–S4. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.