The sweetpotato cold-tolerant variety Liaohanshu21 (LHS21) was employed to clone the IbXTH16 gene. The sweetpotato variety LHS21 and cold-susceptible variety Sushu28 (SS28) were employed to analyze the expression level of IbXTH16. The sweetpotato variety Lizixiang was used to identify the function of IbXTH16.

Total RNA for cDNA generation (TRIzol reagent, CWBIO, Beijing, China) and genomic DNA (Easy Pure Plant Genomic DNA Kit, Trans Gen, Beijing, China) were isolated from the leaves of LHS21 according to Fan et al. (2024). The coding sequence (CDS), genome sequence, and promoter region of IbXTH16 were obtained based on a homologous cloning approach. Phylogenetic analysis was performed with MEGA 11.0 software. The genomic structure of IbXTH16 was analyzed by GSDS 2.0 (http://gsds.gao-lab.org/). The cis-acting regulatory elements of the IbXTH16 promoter region were analyzed by PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). All primers in this study were showed in Supplementary Table S1.

The CDS of IbXTH16 (without the stop codon) was integrated into the pCAMBIA1300-GFP vector according to Zhang et al. (2020). The pCAMBIA1300-IbXTH16-GFP was introduced into Agrobacterium tumefaciens strain GV3101 and transiently inoculated into Nicotiana benthamiana leaf hypodermal cells. After 48 h of infection, the GFP signals were observed using a confocal fluorescence microscope (LSM880, Zeiss, Jena, Germany).

The four-week-old in vitro-grown LHS21 or SS28 plants was treated with cold (4°C) or 100 mM BR for 0, 1, 3, 6, 12, 24 h and the expression of IbXTH16 was quantified with Real-time quantitative polymerase chain reaction (RT-qPCR) (SYBR Green Master Mix, YEASEN, Shanghai, China). Expression of IbXTH16 in leaf, root, and stem tissues four-week-old in vitro-grown LHS21 was quantified with RT-qPCR. The IbACTIN was used as the internal control.

The CDS of IbXTH16 (without the stop codon) was integrated into the pCAMBIA1300 vector according to Fan et al. (2024). The pCAMBIA1300-IbXTH16 was introduced into A. tumefaciens strain EHA105, and then infected Lizixiang embryogenic suspension cultures as described by Yu et al. (2007). The transgenic plants were identified with PCR (LA Taq, TaKaRa, Tokyo, Japan) and RT-qPCR.

4-week-old IbXTH16-overexpressing sweetpotato plants and wide type (WT) with the same status were subjected to cold treatment after a week of acclimatization. The cold treated leaves of the IbXTH16-overexpressing sweetpotato plants and WT were used to determine the superoxide dismutase (SOD) (SOD-1-W, Cominbio, Suzhou, China) and peroxidase (POD) activities (POD-1-Y, Cominbio), proline (PRO-1-Y, Cominbio) and malondialdehyde (MDA) contents (MDA-1-Y, Cominbio) according to manufacturer’s instructions. The BR content and relative electrical conductivity were determined by Norminkoda Biotechnology Co., Ltd (Wuhan, China). The expression of IbDWF4, IbDET2, IbBRI1, IbBES1, IbBEE3, IbBIN2, IbP5CR, IbP5CS, IbP5CDH, and IbPDH were quantified with RT-qPCR.

Data are analyzed using one-way ANOVA followed by post-hoc Tukey’s test or Student’s t-test at P < 0.05 or P < 0.01.

To identify potential regulators of cold resistance in sweetpotato, we cloned IbXTH16 gene from cold-tolerant variety LHS21. Its 879 bp CDS encoded a 292 aa protein with a molecular weight of 32.983 kDa and a pI of 8.47 (Supplementary Table S2). Phylogenetic analysis showed that IbXTH16 shared the closest relationship with AtXTH16 among Arabidopsis homologs (Figure 1A). The 2039 bp genomic sequence of IbXTH16 contained two exons and one intron, which was different from the three exons and two introns of AtXTH16 (Figure 1B). The 800 bp IbXTH16 promoter region contained a low-temperature response element LTR and an ABA-response element ABRE (Figure 1C). To examine the subcellular location of IbXTH16, the IbXTH16-GFP fusion protein was conducted by transiently expressing in N. benthamiana leaf epidermal cells. The results showed that IbXTH16-GFP was localized in the cell membrane (Figure 1D).

To study the potential role of IbXTH16 in cold resistance of sweetpotato, the expression level of IbXTH16 was analyzed. RT-qPCR assay showed that the expression level of IbXTH16 in LHS21 was much higher than that in SS28 (Figure 2A). Tissue-specific expression assay revealed that the expression level of IbXTH16 was relatively high in the roots of in vitro-grown LHS21 plants (Figure 2B). The expression of IbXTH16 was significantly induced by 4°C and 100 mM BR (Figures 2C,D).

To investigate whether IbXTH16 contributes to cold tolerance in sweetpotato, this gene was transferred into sweetpotato variety Lizixiang via A. tumefaciens-mediated method, and 12 IbXTH16-overexpressing lines (OX-1 to OX-12) were generated (Figures 3A–F). There was no significant difference in the phenotype of sweetpotato storage roots between overexpression lines and WT (Figure 3G). IbXTH16 exhibited increased expression level in the overexpression lines compared with the WT (Figure 3H). Three overexpression lines (OX-2, OX-7, and OX-12) with higher expression levels of IbXTH16 were selected for further study. Furthermore, the overexpression and WT plants were treatment at 4°C and restored at 25°C. The degree of wilting in the overexpression lines at 24 h and 48 h under cold stress was lower compared to that of WT (Figures 4A–C). Additionally, the overexpression lines recovered more rapidly than WT when returned to 25°C (Figure 4D). These results indicated that IbXTH16 functions as a positive regulator of cold tolerance in sweetpotato.

To further explore how IbXTH16 mediate the cold tolerance in sweetpotato, the contents of stress response-related components were measured. Under 4°C treatments for 24 h and 48 h, higher SOD and POD activities, higher proline and BR contents, and lower relative electrical conductivity and MDA content were found in the overexpression lines relative to WT (Figures 5A–F). In BR biosynthesis and signalling pathway, key enzyme genes IbDWF4 and IbDET2 and positive regulatory factors IbBRI1, IbBES1, and IbBEE3 in the transgenic plants were upregulated under 4°C treatment (Figures 6A–E), while negative regulatory factor IbBIN2 was downregulated (Figure 6F). In proline biosynthesis and signalling pathway, key enzyme genes IbP5CR and IbP5CS in the transgenic plants were upregulated under 4°C treatment (Figures 6G,H), while degradation pathway IbP5CDH and IbPDH were downregulated (Figures 6I,J). These results indicated that IbXTH16 positively regulates cold tolerance of sweetpotato by activating the BR and proline pathways.

Many crops are well-suited for growth in tropical or subtropical regions (Chinnusamy et al., 2007). However, the average minimum temperature of most land areas on Earth is <0°C (Rihan et al., 2017). Low temperatures adversely affect crop growth and development, limiting their geographical distribution (Pearce, 2001; Ding et al., 2019). Sweetpotato is an important crop for ensuring national food security, but it is vulnerable to yield reductions caused by low-temperature damage (Jin et al., 2017; Yu et al., 2022). Genetic engineering has emerged as an effective strategy for enhancing sweetpotato’s tolerance to cold stress (Jin et al., 2017; 2021; 2022; Yu et al., 2022). Nevertheless, the function of XTH in cold stress of sweetpotato remains to be further studied. In this study, a novel IbXTH16 gene was cloned from the cold-tolerant variety LHS21 (Figure 1). The plant cell membrane serves as a crucial barrier for maintaining stable cellular metabolism and also plays a key role in sensing low temperatures (Zhang et al., 2019). After plants are exposed to low temperatures, the permeability and fluidity of their cell membranes decline, activating cold-response genes (Muratan, 1997). The localization of IbXTH16 in the cell membrane and the induction of IbXTH16 by cold suggest that IbXTH16 might serve as a signalling molecule in cold tolerance of sweetpotato (Figures 1D, 2C). The expression of IbXTH16 was induced by the BR (Figure 2D), and its overexpression enhanced cold tolerance in sweetpotato (Figure 4). Therefore, IbXTH16 is believed to be involved in the cold tolerance of sweetpotato.

Under low-temperature stress, plants accumulate excessive reactive oxygen species (ROS), which can be detrimental to plant cells (Guo et al., 2022; Mittler et al., 2022). The ROS scavenging system can detoxify ROS by enhancing the activity of ROS-scavenging enzymes, such as SOD and POD, preventing oxidative damage to plant cells (Gill and Tuteja, 2010; Bose et al., 2014; Choudhury et al., 2017). In Zoysia japonica, overexpression of ZjICE1 conferred cold tolerance in transgenic plants by increasing SOD, POD, and proline contents, as well as decreasing MDA content (Zuo et al., 2019). In Betula platyphylla, overexpression of BpERF13 improved the cold tolerance of transgenic plants by binding to cis-elements of SOD and POD and increasing SOD and POD contents (Lv et al., 2020). In this study, the SOD and POD contents were significantly increased in the transgenic plants under 4°C (Figures 5A,E). It is suggested that overexpression of IbXTH16 enhances cold tolerance by activating the biosynthesis of SOD and POD in transgenic sweetpotato. Previous reports have indicated that the plant hormone ABA is also involved in the cold tolerance of plants (Huang et al., 2017;Ma et al., 2018; Li et al., 2021). The IbXTH16 promoter region contained an ABA-response element ABRE (Figure 1C). However, whether the XTH16 gene regulates cold tolerance in sweetpotato via the ABA pathway requires further investigation.

BR signalling not only participates in plant growth and development, but also has been reported in plant resistance to low temperature (Kinoshita et al., 2005; Vriet et al., 2012). In the initial phase when plants are exposed to cold stress, the activity of BIN2 kinase is suppressed, while OST1 kinase is activated, this synergistic regulation stabilizes ICE1, thereby enhancing the expression of CBF and improving the cold tolerance of plants (Barrero-Gil and Salinas, 2018; Ye et al., 2019). In Arabidopsis, compared with the WT, the cold resistance of overexpressing BRI1 plants was enhanced, while that of mutant BRI1 plants was decreased (Eremina et al., 2016). In this study, the transgenic plants showed a significant increase in BR content, which might be due to the overexpression of IbXTH16 increasing the BR biosynthesis of transgenic plants, thus conferring cold tolerance (Figure 5F). Interestingly, the BR content in the transgenic plants was significantly decreased after restoring the room temperature (Figure 5F). The BR biosynthetic pathway involves the participation of a series of genes (Zhao and Li, 2012). In this study, the expression levels of BR biosynthesis and signalling pathway-related positive regulatory factors were significantly upregulated, while negative regulatory factor was significantly downregulated (Figures 6A–F). More proline accumulation can protect plants from low-temperature stress and ROS damage (Ghosh et al., 2022; Kidokoro et al., 2022). In this study, the proline biosynthesis-related genes were significantly upregulated, while degradation pathway-related genes were significantly downregulated (Figures 6G–J). Collectively, these findings suggest that IbXTH16 positively regulates cold tolerance of sweetpotato by activiting BR signalling pathway and proline accumulation (Figure 7).