A novel wheat S1-bZIP gene, TabZIP11 confers stress resistance in Arabidopsis

A novel wheat S1-bZIP gene, TabZIP11 confers stress resistance in Arabidopsis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A novel wheat S1-bZIP gene, TabZIP11 confers stress resistance in Arabidopsis Li na Zhang, Zhen Yu, Xingyan Liu, Yaoyao Wang, Jing Luo, Yinghong Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4483341/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The majority of basic leucine zipper (bZIP) transcription factor (TF) subgroup S1 play significant regulatory role in response to abiotic stress. However, their functions and underlying molecular mechanisms in abiotic stress responses are less known in wheat (Triticumaestivum L.). In this study, we isolated a TabZIP11 TF, which is from S1 subgroup of wheat bZIP transcription factor. TabZIP11 encodes a nuclear protein without transcriptional activation activity. Transcript of TabZIP11 gene was induced by abscisic acid (ABA), NaCl, and cold stress treatments. Whereas compared with NaCl treatment, TabZIP11 showed a lower expression level under NaCl+LaCl3 condition. We found that calcium-dependent protein kinase1 (TaCDPK1), TaCDPK5, TaCDPK9-1, TaCDPK30 and calcineurin B-like protein (CBL)-CBL-interacting protein kinase31 (TaCIPK31) cooperated with TabZIP11. The overexpression of TabZIP11 ectopically improved salt and freezing tolerances in Arabidopsis. TabZIP11 contributed to salt and freezing tolerance by modulating soluble sugar, proline, hydrogen peroxide (H2O2), and malondialdehyde (MDA) productions and abiotic stress responsive gene expression levels. TabZIP11 can form both homodimers and heterodimers with itself and group C TabZIP members. The modified yeast one-hybrid analysis confirmed that TabZIP36 significantly enhanced the binding ability of TabZIP11 to the promotor of TaCBF1 gene. Thus, these results suggest that TabZIP11 interacts with TabZIP36 to modulate cold signaling by facilitating the transcriptional activity of c-repeat binding factor (TaCBF1) gene. TabZIP11 functions as a positive regulator of salt stress responses through interacting with TaCDPK1/5/9-1/30 and TaCIPK31. TabZIP11 transcription factor TaCDPK1/5/9 − 1/30 and TaCIPK31 Homodimers and heterodimers Salt and freezing tolerances TaCBF1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Key message TabZIP11 gene enhances freezing and salt tolerance in transgenic plants through TaCBF1 and is directly regulated by TaCDPK1/5/9-1/30 and TaCIPK31. Introduction Transcription factors (TFs) play vital regulatory roles in various plant biological processes (Pruneda-Paz et al. 2014). Basic leucine zipper (bZIP) is a member of transcription factor family in plants. The bZIP domain of bZIP TFs contain a highly conserved DNA-binding basic region and a leucine zipper region (Kouzarides and Ziff. 1989). And the members of bZIP family are clustered into 13 groups (group A-K, M, S) and have diverse roles, especially in plant stress-responsive and hormone signal transduction (Dröge-Laser et al. 2018). Group S is composed of three subgroups: S1, S2, and S3 (Ehlert et al. 2006). Among them, only a few group S bZIP proteins have been characterized to regulate abiotic stress response in plants. For example, the transcriptional level of AtbZIP1 was significantly induced by salt, osmotic, and cold stresses in Arabidopsis . Overexpression and knockout mutants of AtbZIP1 in transgenic plants revealed that AtbZIP1 is capable of enhancing or reducing tolerance to salt and drought stresses (Sun et al. 2012). AtbZIP53 was shown to participate in hypo-osmolarity stress. The Arabidopsis thaliana proline dehydrogenase (ProDH) was found to be directly targeted by AtbZIP53 (Weltmeier et al. 2006). Rice OsbZIP71 was involved in ABA-mediated drought and salt tolerance (Liu et al. 2014). Previous research has also showed that overexpression of OsbZIP16 contributed to drought resistance in rice (Chen et al. 2012). In tomato, SlbZIP1 raised salt and drought stress tolerance by enhancing expression of multiple ABA biosynthesis and signal transduction-related genes (Zhu et al. 2018). Likewise, sweetpotato IbbZIP1 gene conferred salt and drought tolerances in transgenic Arabidopsis (Kang et al. 2019). In apple, group S MdbZIP80 TF could heterodimerize with group C bZIP members and restrain the expression of MdIPT5b , which resulted in negatively modulating drought and salt tolerance (Feng et al. 2021). GsbZIP67 play a role in improving the bicarbonate alkaline tolerance (Wu et al. 2018). Under abiotic conditions, the group S bZIP TFs can also interact with and be phosphorylated by calcium-dependent protein kinases CDPKs or SNF1‐related protein kinases SnRK to mediate their transcriptional activation activities. For instance, the salt-induced AtbZIP1 transcription was found to be strongly up-regulated by SnRK1 signaling. However, AtbZIP53 expression partially hinged on the SnRK2 kinase signaling pathway (Hartmann et al. 2015). And Ca 2+ signaling also participated in inducing AtbZIP1 transcription through Ca 2+ blocker LaCl 3 (Hartmann et al. 2015). However, the interaction between wheat TaCDPK or TaCIPK and group S bZIP TF has not been revealed until now. And the exact function and regulation mechanism of group S bZIP TF are poorly known in wheat. In this study, we identified a novel subgroup S1 TabZIP11, which was strongly induced by ABA, salt, and cold stresses. We further investigated and found that TabZIP11 localized in nucleus. But it did not exhibit transcriptional activation activity in yeast. The expression level of salt-induced TabZIP11 was strongly decreased by Ca 2+ blocker LaCl 3 . A yeast two-hybrid assay and BiFC showed that TaCDPK5, TaCDPK9-1, TaCDPK30 were able to interplay with TabZIP11, respectively. Overexpression of TabZIP11 in Arabidopsis enhanced salt and freezing tolerance. And TabZIP11 was capable of homodimerizing by itself and heterodimerizing with the group C TabZIP proteins. TabZIP36 raised the binding ability of TabZIP11 to the promoter of TaCBF1 . These results indicate that TabZIP11 mediates positively the salt and cold stresses response. Materials and methods Plant materials The wheat cv. Longchun 27 was used to clone the full-length coding region of TabZIP11 and analyse the expression level of TabZIP11 gene. Arabidopsis thaliana (Col-0) were used for genetic transformation to generate transgenic overex-pressing TabZIP11 through Agrobacterium infection. Identification and analysis of gene sequences The coding region of TabZIP11 and the group C members ( TabZIP9 / 14 / 32 / 36 ) of TabZIP proteins were amplified by PCR from the wheat cv. Longchun 27. In my previous study, we cloned the full length sequences of TaCDPK5 , TaCDPK9-1 and TaCDPK30 (Zhang et al. 2022). Multiple sequence alignment of TabZIP11 and other downloaded bZIP proteins were achieved using ClustalW. The neighbor-joining phylogenetic tree was conducted using MEGA 5.2 software with 1000 bootstrap replications. RNA isolation and quantitative real-time PCR (RT-qPCR) analysis To explore the expression pattern of TabZIP11 in common wheat, the roots treated with PEG (16%), NaCl (100 mM) and ABA (100 µM) solutions, and the leaves treated with cold stress (4℃) were from ten-day-old wheat seedling. These samples were gained at 0, 1, 3, 6, 12, 24 and 48 h, respectively. To investigate the effect of calcium channel blocker LaCl 3 on the expression of TabZIP11 gene, wheat cv. Longchun 27 were treated with 100 mM NaCl and NaCl + 0.8% LaCl 3 for 1h, 3h, 6h, 12h, 24h and 48h. To investigate the expression levels of stress related genes in transgenic Arabidopsis , the two-week-old Arabidopsis plants were treated with 150 mM NaCl for 12h and cold stress for 3h. Total RNA was segregated and cDNA was generated as described previously (Zhang et al. 2020). The relative expression of TabZIP11 and Arabidopsis stress responsive genes were determined with reference to the expression of wheat TaGAPDH (AF251217.1) or Arabidopsis AtActin (AT3G18780) with the 2 −ΔΔCT method. The gene specific primers used in RT-qPCR analysis were listed in additional Table S1 . Analysis subcellular localization of TabZIP11 The TabZIP11 CDS sequence lacking a termination codon was cloned into 35S-GFP vector, producing TabZIP11:GFP fusion plasmid, which was extracted with TIANprep Mini Plasmid Kit (TIANGEN, China) according to the instructions. TabZIP11:GFP fusion plasmid and empty 35S-GFP vector were expressed transiently in the leaves of tobacco through Agrobacterium tumefaciens strain GV3101 mediated transformation, respectively. After 16 h of incubation in the dark at 25°C, green fluorescent protein (GFP) fluorescence was captured with a confocal laser-scanning microscope (Leica Sp8, Germany). The gene specific primers used in this study were listed in additional Table S1 . The transactivation assay of TabZIP11 and yeast two-hybrid assay In order to define the capacity of TabZIP11 in regulating transcription, full-length coding sequences of TabZIP11 or TabZIP60 was fused to the GAL4 DNA-binding domain (BD) in vector pGBKT7 to create TabZIP11-BD and TabZIP60-BD, respectively. All these constructs and the empty vector pGBKT7 were individually transformed into the AH109 yeast competent cells using yeast Frozen-EZ transformation kit (Zymo, USA). TabZIP60-BD and empty vector pGBKT7 were used as positive and negative controls (Zhang et al. 2015). These transformants were cultivated on SD/ Trp − plates at 30℃ for 2 days. The positive clones were coated onto fresh SD/His − Ade − Trp − plates and grown at 30℃ to detect the transactivation activity of TabZIP11. For yeast two-hybrid assay, the coding region of TaCDPK1 , TaCDPK5 , TaCDPK9-1 , TaCDPK30 , and TaCIPK31 were cloned into the vector pGADT7 containing GAL4 DNA-binding domain (AD) to generate TaCDPK1 -AD, TaCDPK5 -AD, TaCDPK9-1 -AD, TaCDPK30 -AD, and TaCIPK31 -AD fusion plasmids. The recombinant plasmid TabZIP11 -BD comes from the fusion of TabZIP11 and pGBKT7 (BD) vector. Co-transformed TaCDPK1 -AD and TabZIP11 -BD, TaCDPK5 -AD and TabZIP11 -BD, TaCDPK9-1 -AD and TabZIP11 -BD, TaCDPK30 -AD and TabZIP11 -BD, TaCIPK31 -AD and TabZIP11 -BD were coexpressed in the yeast AH109, then cultured on SD/Leu − Trp − plates at 30°C for 2–3 days. Positive clones were streaked onto fresh SD/His − Ade − Leu − Trp − plates at 30°C about 2–4 days. The gene specific primers of constructing recombinant plasmids in this study were listed in additional Table S1 . To investigate the dimerization of between TabZIP11 and TabZIP9/11/14/32/36 (group C), the ORF of TabZIP9/11/14/32/36 and TabZIP11 were subcloned into AD or BD vectors, respectively. The yeast two-hybrid assay was used to compare the dimerization among the different combinations by prototrophic growth of the yeast strains on plates. BiFC assay in N. benthamiana leaves To carry out biomolecular fluorescent complementation (BiFC) assays, the CDS of TaCDPK5 , TaCDPK9-1 , TaCDPK30 without TAG were cloned into the BiFC-YN vector to construct TaCDPK5:NYFP, TaCDPK9-1:NYFP, and TaCDPK30:NYFP. And the TabZIP11 CDS was fused into the BiFC-YC vector to obtain TabZIP11:CYFP. These different recombination constructs and TabZIP11:CYFP were introduced into Agrobacterium tumefaciens strain GV3101 and co-transformed into in the leaves of tobacco. A confocal microscopy (Leica Sp8, Germany) was used to observe the fluorescent signal to determine the TaCDPK5/9 − 1/30-TabZIP11 interactions. The gene primers sequences used in this study were listed in additional Table S1 . Generation of transgenic arabidopsis plants To generate the overexpression transgenic lines, the open reading frame of TabZIP11 without a termination codon was inserted into the pDONR-Zeocin vector and then recombined into the plant overexpression vector 35S-GFP with a cauliflower mosaic virus (CaMV) 35S promoter through Gateway method. The identification of TabZIP11 overexpression lines was conducted by PCR amplification and RT-qPCR. Three overexpression lines L1, L2, and L3 were used to perform the phenotypic analysis. Phenotypic analysis of TabZIP11 transgenic plants under salt and freezing stresses For freezing stress treatment at the seedling stage, three-week-old homozygous transgenic lines and wild-type (WT) seedlings were treated at 4°C for 4–5 days, and then at -10°C for 7 hours after which plants were recovered at 22°C for 3 days. For salt stress, the seven-day-old WT and transgenic lines were planted 1/2 MS medium with 0 and 100mM NaCl for 4–5 days. The growth phenotype were photographed and the root length was measured. All experiments were replicated three times. Determination of H 2 O 2 accumulation, malondialdehyde (MDA) content, soluble sugar, and proline contents H 2 O 2 content was determined through the trichloroacetic acid and potassium iodide (KI) assays described previously (Velikova et al. 2000). The 0.5g prepared fresh Arabidopsis seedlings were mixed with 5 ml of 0.1% (w/v) trichloroacetic acid (TCA), then the mixture was ground and centrifuged at 12,000g for 20 min. Then, 0.7 ml potassium phosphate buffer (10 mM, pH 7.0) and 1.4 ml KI (1 M) were added to 0.7 ml of the supernatant and mixed evenly. The absorbance of samples was measured at 390 nm. The H 2 O 2 content was calculated according to a standard curve. The MDA, soluble sugar, and proline contents were measured according to previous method (Bates et al. 1973; Hodges et al. 1999; Cui and Wang. 2006). The modified yeast one-hybrid assay To further investigate how to regulate the transcript level of TaCBF1 gene by TabZIP11 and TabZIP36. The coding sequences of TabZIP36 and TabZIP11 were cloned into the pGADT7 or pB42AD vectors, respectively, generating TabZIP36 -AD and TabZIP11 - pB42AD. The promoter sequence of TaCBF1 gene (not shown) was inserted into the pLacZi vector, producing TaCBF1- promoter-pLacZi. The TabZIP36 -AD, TabZIP11 -pB42AD and TaCBF1- promoter-pLacZi were transfected into yeast EGY48. Three biological replicates were performed. Statistical analysis The statistical analysis was carried out with SPSS 26.0. The Origin 9.0. software was used to generate these figures. The significant differences are represented by different lowercase letters. Results Characterization of TabZIP11 gene The cDNA sequence of TabZIP11 was amplified from the wheat cv. Longchun 27 through RT-PCR. The cDNA sequence analysis revealed that the ORF is 435bp in length, encodes a protein, which is speculated to contain 144 amino acids with a molecular weight of 15.62 kDa and a theoretical pI of 10.56. The TabZIP11 protein harbours a typical bZIP domain (4-68Aa). The Arabidopsis bZIP TFs members are known to be divided into 13 groups, and S group includes S1, S2 and S3 subgroups. The neighbor-joining phylogenetic tree indicated that TabZIP11 was gathered into group S1 (Fig. 1 ). The expression profiles of TabZIP11 under different treatments In order to better investigate the expression pattern of TabZIP11 gene, we analyzed the expression level of the TabZIP11 gene in wheat root and leaf tissues under non-stressed condition through RT-qPCR. As shown in Fig. 2 a, the expression level of TabZIP11 gene presented no significant difference in untreated roots and leaves at different times. Nevertheless, after ABA and NaCl treatments, the transcription levels of TabZIP11 in the roots were rapidly and strongly induced. In the same way, the expression of TabZIP11 in the leaves was also significantly increased in response to cold stress. After PEG treatment, there was no significant change in the expression of TabZIP11 gene. Previous study has demonstrated that Ca 2+ signaling is required for AtbZIP1-S transcription in Arabidopsis (Hartmann et al. 2015). To investigate whether the TabZIP11 gene is also involved in the Ca 2+ signaling pathway, we examined the expression of TabZIP11 gene in the presence of the Ca 2+ blocker LaCl 3 under NaCl treatment. The transcriptional level of TabZIP11 was upregulated by NaCl stress alone. However, in the presence of LaCl 3 , TabZIP11 expression level was significantly reduced (Fig. 2 b). These data implied that Ca 2+ signaling is also necessary for salt-induced TabZIP11 transcription. TabZIP11 is a nuclear-localized TF without transactivation activity In order to investigate the subcellular localization of the TabZIP11 protein, a 35S: TabZIP11-GFP recombinant plasmid or a negative control ( 35S-GFP ) was transformed and transiently expressed in the leaves of tobacco. The signals of GFP alone were present in the plasma membrane and nucleus (Fig. 3 a). Whereas in 35S: TabZIP11-GFP transformed tobacco leaves cells, the GFP fluorescence was visualized in the nucleus, suggesting the TabZIP11 is a nuclear-localized protein. Because most transcription factors exhibit transcriptional activation activity. Consequently, we also determined whether TabZIP11 possess the potential transcriptional activity in yeast cells. The full-length coding sequence of TabZIP11 or TabZIP60 was cloned into the pGBKT7 (BD) vector, respectively. The pGBKT7 vector alone and BD-TabZIP60 were used as negative and positive controls. As shown in Fig. 3 b, only yeast cell expressing BD-TabZIP60 grew well on SD/Trp − His − Ade − selective media. Compared with the pGBKT7 vector alone, the BD-TabZIP11 also failed to exhibit any detectable yeast growth. This finding indicated that TabZIP11 did not show any transactivation property in yeast. TaCDPK1/5/9 − 1/30 and TaCIPK31 interact with TabZIP11 Our previous research has indicated that TaCDPK5, TaCDPK9-1 and TaCDPK30 were interacting proteins of group A TabZIP60 (Zhang et al. 2022, 2023). In this study, salt-induced TabZIP11 transcript level decreases through Ca 2+ blocker LaCl 3 . So we examined whether TabZIP11 interacts with TaCDPK1, TaCDPK5, TaCDPK9-1, TaCDPK30 and TaCIPK31 individually by yeast two-hybrid method. A significant interactions between TabZIP11 and the closely Ca 2+ related protein kinase members TaCDPK5/9 − 1/30/TaCIPK31 could be observed. A weaker interaction with TabZIP11 and TaCDPK1 could be examined. No significant interaction were determined in other yeasts cell expressing negative controls (Fig. 4 a). To further confirm the data obtained by yeast two-hybrid, biomolecular fluorescent complementation (BiFC) studies were executed by expressing TaCDPK5: NYFP, TaCDPK9-1: NYFP, TaCDPK30: NYFP and TabZIP11: CYFP in tobacco leaves cell. These results indicated that only samples expressing the combination TaCDPK5: NYFP and TabZIP11: CYFP, TaCDPK9-1: NYFP and TabZIP11: CYFP, or TaCDPK30: NYFP and TabZIP11: CYFP showed strong YFP complementation signals in the nucleus of tobacco leaves cells. No fluorescence in the negative controls were examined, which are in agreement with the results gained by yeast two-hybrid (Fig. 4 b). In conclusion, these results showed that TabZIP11 interacts with TaCDPK5, TaCDPK9-1 and TaCDPK30 in vivo, respectively. Overexpression of TabZIP11 enhances the freezing and salt tolerances of transgenic Arabidopsis seedlings To investigate the physiological function of TabZIP11 gene, we produced transgenic Arabidopsis plants overexpressing TabZIP11 (hereafter, TabZIP11 - OX ). The overexpression level of TabZIP11 in four independent TabZIP11 - OX lines was demonstrated by RT-qPCR method (Figure S1 ). We examined the phenotypes of TabZIP11 - OX plants in response to salt stress by transferring 7-day-old seedlings to the 1/2 MS medium with 100 mM NaCl. After 5 days of salt stress treatment, the TabZIP11 - OX plants showed much better growth than WT, exhibiting longer root length and more lateral roots. While a similar growth states in seedling phenotypes between TabZIP11 - OX and WT plants was observed under growth condition (Fig. 5 a, b). In accordance with the visible salt resistant phenotypes, the soluble sugar and proline contents remained high in TabZIP11 - OX (Fig. 5 c, d). But MDA and H 2 O 2 contents became low in TabZIP11 - OX during salt stress (Fig. 5 e, f). In addition, we find that TabZIP11 transcript level was strongly induced within 1–12 h of freezing treatment (Fig. 2 a). We therefore examined whether TabZIP11 regulate responses to freezing stress by comparing the phenotypes of 3-week-old TabZIP11 - OX and WT plants, which were placed in freezing stress (-10°C) for 7 hours and subsequently restored to appropriate temperature (22°C). It is worth noting that WT plants were more sensitive to freezing stress than the TabZIP11 - OX , exhibiting some leaves of WT seedlings died. Nevertheless, almost all transgenic lines were able to survive and their leaves retained green (Fig. 6 a). We also examined the levels of the soluble sugar and proline, which were significantly improved in TabZIP11 - OX lines compared to WT under freezing stress (Fig. 6 b, c), and yet MDA and H 2 O 2 turned lower than WT plants (Fig. 6 d, e). These results indicated that overexpression of TabZIP11 significantly improves tolerance to freezing stress. Overexpression of TabZIP11 results in increased expression of stress responsive genes To elucidate the transcriptional regulation of TabZIP11 gene, we examined the expression levels of abiotic stress-associated genes in WT and TabZIP11 - OX plants during NaCl and freezing stress treatments. After 12h hours of treatment in 150mM NaCl solution, several stress-related genes including AtRD29A , AtRD29B , AtSIZ1 , AtDREB2A , AtERD6 , and AtRAB18 were obviously enhanced in WT and TabZIP11 - OX plants but upregulated at higher levels in TabZIP11 - OX plants compared to WT (Fig. 7 a). We also found that during 4℃ stress, the transcript levels of AtCBF1 , AtCBF2 , AtCBF3 , AtMYB77 , AtRC12A , AtWRKY33 , AtCOR47 , AtCOR27 , and AtCOR15B were higher in TabZIP11 - OX lines compared to WT (Fig. 7 b). As shown in Fig. 7 a, b, the expression levels of these genes had no significant difference between the WT and TabZIP11 - OX under normal condition. TabZIP11 could form homodimer by itself and heterodimers with TabZIP9/ 14/32/36 The previous researches have uncovered that the group S1 bZIPs could form specific heterodimers or homodimers with group C bZIPs and itself to be involved in regulating gene expression (Weiste et al. 2014; Feng et al. 2021). This finding prompted us to determine the dimerization activity of TabZIP11 through a yeast two-hybrid method. As shown in Fig. 8 , all constructs that were transformed into the yeast AH109 grew well on SD/Leu − Trp − medium. But only the yeasts expressing BD-TabZIP11 and AD-TabZIP11, BD-TabZIP11 and AD-TabZIP9, BD-TabZIP11 and AD-TabZIP14, BD-TabZIP11 and AD-TabZIP32 or BD-TabZIP11 and AD-TabZIP36 were capable of growing on SD/Leu − Trp − Ade − His − medium. There were no yeast growth for other negative controls on the same medium. These results showed that TabZIP11 can form both homodimer as well as heterodimers with group C proteins in yeast. TabZIP36 enhances the binding of TabZIP11 to the promoter of TaCBF1 gene Our study demonstrated that there are the physical interactions between TabZIP36 and TabZIP11 (Fig. 8 ). AtCBF1 gene expression was significantly enhanced in TabZIP11 - OX lines under freezing stress compared to the WT plants (Fig. 7 b), which urged us to explore whether the dimer of TabZIP36 and TabZIP11 regulate the wheat homolog TaCBF1 transcript. As shown in Fig. 9 , we demonstrated that dimeric (TabZIP36: TabZIP11) proteins could strongly induced the transcription level of TaCBF1 gene, but lower activation level mediated by TabZIP11 alone on SD/Leu − Trp − Ura − with 20mM X-Gal and 200mM 3AT medium. However, in the presence of TabZIP36, the activation of TaCBF1 gene was significantly increased. Discussion Group S comprises the largest group of bZIP family in Arabidopsis . It can be separated into three subgroups, entitled S1, S2 and S3 (Dröge-Laser et al. 2018). The group S bZIPs play vital roles in regulating plant development, metabolic processes and stress response (Wang et al. 2022). However, some researches about group S1 bZIPs were performed in the model plants Arabidopsis and rice. No reports thus far have indicated a subgroup S1 bZIP protein playing a positive regulatory role in wheat abiotic stress. In this study, we isolated and identified a wheat TabZIP11 , which has high similarity with S1 subgroup of Arabidopsis bZIP TF. So the phylogenetic tree analysis showed that TabZIP11 belonged to S1 subgroup. TabZIP11 was found to be located in the nucleus and had no transactivation activity. In agreement with this result, several group S bZIP proteins have been shown to harbour nuclear localization (Satoh et al. 2004; Wu et al. 2018). And some other group S bZIPs also do not display transcriptional activation in yeast, such as OsbZIP71, LIP19, and CsbZIP44 (Shimizu et al. 2005; Liu et al. 2014; Sun et al. 2024). Furthermore, our studies also suggested that the transcription of TabZIP11 was dramatically reduced by NaCl stress together with Ca 2+ blocker LaCl 3 . This result revealed that TabZIP11 is involved in Ca 2+ signaling pathway, which is in line with the previous studies that Ca 2+ blocker LaCl 3 has been shown to decrease the salt-induced subgroup S1 AtbZIP1 transcription (Hartmann et al. 2015). These findings further suggest that TabZIP11 is a downstream substrate for CDPK or CIPK kinase and support the conception that the CDPK or CIPK alone might be insufficient for regulating the expression of TabZIP11 gene and the additional protein kinases are essential for its expression activity. Previous work identified that SnRK1 could act as a AtbZIP1-interacting kinase (Hartmann et al. 2015). But to date, few direct physical interactions between wheat CDPK or CIPK and group S1 bZIPs could be identified. We identified the TabZIP11 functions as a TaCIPK31/TaCDPK1/5/9 − 1/30-interacting protein in yeast. In accordance with the yeast-two hybrid experiment finding, TabZIP11 was showed to interplay with TaCDPK5/9 − 1/30 in planta through the BiFC assay. Several data also support the impact of suppressing CDPK kinase activity on downstream TabZIP gene expression (Zhang et al. 2020, 2022, 2023). In this study, it was then shown that TabZIP11 was strongly increased by cold and NaCl stresses, hinting that TabZIP11 may play a regulatory role in abiotic stress. Therefore, it may be interesting to study the potential function of TabZIP11 in response to different stresses, we overexpressed TabZIP11 in Arabidopsis and displayed that all TabZIP11 overexpression transgenic lines exhibited better ability to resist salt and freezing stresses than the WT plants, including higher soluble sugar and proline contents. Meanwhile, lower MDA and H 2 O 2 contents were found in TabZIP11 overexpression plants compared with WT under stress conditions. These results demonstrate that TabZIP11 contributes to freezing and salt tolerances in plant. To date, several bZIP-related overexpressed plants and a variety of abiotic-associated genes, which increase during stress treatment, have been identified and cloned. For example, AtDREB2A , AtSIZ1 , AtABF1 , AtABI1 , and AtPCS1 were significantly higher in GmbZIP152 - OX plants than those in WT plants under stress condition (Chai et al. 2022). Some stress-related genes, such as AtDREB2A, AtRD29A, AtERD10 and AtRD29B , indicated obviously higher expression levels in the AtPPRT3 - OX lines than in WT Arabidopsis (Liu et al. 2020). In line with these results, we found that the transcript level of abiotic-related genes ( AtRD29A , AtRD29B , AtSIZ1 , AtDREB2A , AtERD6 and AtRAB18 ) significantly upregulated in TabZIP11 -OX lines compared to WT plants after NaCl treatment. AtSIZ1, encoding a salt-induced C 2 H 2 type zinc finger protein, was involved in positively conferring salt tolerance of Arabidopsis through maintaining ionic and osmotic homeostasis (Han et al. 2019). AtRD29A , AtRD29B , and AtDREB2A participate in the ABA-mediated resistance pathway and are activated by several transcription factor (Hirayama et al. 2007). AtERD6 and AtRAB18 are the members of dehydrins (DHNs) protein family and accumulate by drought, salinity, and freezing stresses (Hanin et al. 2011). In addition, TabZIP11 also activated the expression of stress-related gene AtCOR47 , AtCOR27 , AtCOR15B , AtMYB77 , AtCBF1 , AtCBF2 , AtCBF3 , AtRCI2A , and AtWRKY33 under cold condition. C-repeat/DREB binding factors (CBFs), as a cold-induced transcription factor plays significant roles in cold stress by binding the promoter sequences of COR genes to activate their expression (Shi et al. 2018). AtRCI2A is involved in salt tolerance by reducing excessive accumulation of Na + (Medina et al. 2005). These results indicated that TabZIP11 is proposed to mediate transcriptional level of stress-related gene in response to salt and cold stresses. Transcriptional factors modulate the expressions of downstream target genes through binding to cis-element in promoter region. For group S bZIP proteins, the homodimerizations and heterodimerizations are regarded as the specific transcriptional regulation way of these bZIP TFs (Golldack et al. 2011). The heterodimerizations play an important function in the DNA binding affinity, transactivation activity, and cell physiology (Naar et al. 2001). For instance, strong heterodimerizations were achieved between members of the groups C (AtbZIP9,-10,-25,-63) and S1(AtbZIP1,-2,-11,-44,-53) bZIP proteins in Arabidopsis (Ehlert et al. 2006). OsbZIP71 homodimerized with itself as well as heterodimerized with group C proteins (OsbZIP15, OsbZIP20, OsbZIP33, and OsbZIP88) to enhance the transcript levels of downstream genes (Liu et al. 2014). TabZIP6 (group C) was able to shape heterodimers with Wlip19 or TaOBF1(group S) in yeast (Cai et al. 2018). TabZIP11 is also capable of forming a homodimer by itself and heterodimers with TabZIP9/14/32/36. But the modified yeast one-hybrid assay showed that TabZIP11 and only group C member TabZIP36 could help TabZIP11 bind the promoter of downstream TaCBF1 gene and upregulate its expression which might increase cold tolerance of TabZIP11 in vivo. We can deduce that TabZIP11 dimer might recruit other proteins necessary for transcriptional regulation to enhance binding capacity of the transcriptional complex. In a word, this study identified a novel subgroup S1 TabZIP11 , which was induced by ABA, NaCl, and cold stresses. TabZIP11 was involved in Ca 2+ signalling through interplaying TaCDPK/CIPKs kinases. The ectopic overexpression of TabZIP11 significantly improved plants tolerance to freezing and salt. Additionally, group C TabZIPs interact with TabZIP11 to form dimers, which are conducive to improving the expression of TaCBF1 gene. Abbreviations bZIP Basic leucine zipper TF Transcription factor ABA Abscisic acid CDPK Calcium-dependent protein kinase CIPK Calcineurin B-like protein (CBL)-CBL-interacting protein kinase CBF C-repeat binding factor H 2 O 2 Hydrogen peroxide MDA Malondialdehyde Declarations Author contribution statement LNZ, CX, LCZ, and XYK projected this paper. ZY, XYL, YYW, and JL implemented the experiments. YHW, NY, and YLY analyzed the data. LNZ and ZY wrote the paper. Conflict of interest The authors have no conflicts of interest to declare. Supporting information Table. Primers used in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China [32260471, 31660392], Academic Backbone Project from the Northwest Normal University [2019GG-3], Young Teachers Improving Program from the Northwest Normal University [NWNU-LKQN-14-11]. Data availability statement The published article and its supplementary data contain all data of this study. References Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant and Soil 39: 205-207. Cai W, Yang Y, Wang W, Guo G, Liu W, Bi C (2018) Overexpression of a wheat (Triticum aestivum L.) bZIP transcription factor gene, TabZIP6 , decreased the freezing tolerance of transgenic Arabidopsis seedlings by down-regulating the expression of CBFs. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4483341","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":310660177,"identity":"a73b2224-7752-444b-bc56-39e93a4fc764","order_by":0,"name":"Li na 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08:13:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4483341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4483341/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58574319,"identity":"7d72997a-cad5-43c1-ae7e-c44916152ac1","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":311033,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phylogenetic tree of TabZIP11 and Arabidopsis group C and S bZIP proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree was carried out using MEGA 5.2 software by the Neighbor-Joining method with 1000 bootstrap replicates.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/e9bdb2e243ae760f114f6147.jpeg"},{"id":58575067,"identity":"4f875ba0-8086-4414-afe5-be501e224ad5","added_by":"auto","created_at":"2024-06-18 11:56:56","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":255528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTabZIP11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e tested using RT-qPCR method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea The \u003cem\u003eTabZIP11\u003c/em\u003e transcript levels in wheat seedlings under different treatment, including NaCl, ABA, cold, and PEG compared to control. b Expression of \u003cem\u003eTabZIP11\u003c/em\u003e gene in response to NaCl and LaCl\u003csub\u003e3\u003c/sub\u003e treatment. The relative expression levels of \u003cem\u003eTabZIP11\u003c/em\u003e was normalized to control gene wheat \u003cem\u003eTaGAPDH \u003c/em\u003e(NCBI accession number: AF251217.1). Data are from three experiments. Different letters above the bars indicate significant differences between different treatment times with 0 h (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/ff38c0ee054ff4bc9c9501f6.jpeg"},{"id":58574324,"identity":"3f0c2c36-9d4a-4469-ade3-573cc82bce4f","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":614787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and transcriptional activity of TabZIP11\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea TabZIP11 localized in the nuclei of \u003cem\u003eN. benthamiana\u003c/em\u003eleaves. Scale bars=45 mm. b The empty pGBKT7 vector and TabZIP60 served as negative and positive controls.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/180bf265c2504431f616cb17.jpeg"},{"id":58574322,"identity":"3cadbdcf-0990-4eec-a1c9-10a2d17a1a10","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":535227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTaCDPK1/5/9-1/30/TaCIPK31 directly interact with TabZIP26 in vitro and in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea Yeast two-hybrid analysis showing interactions of TaCDPK1/5/9-1/30/TaCIPK31 and TabZIP26. TaCDPK1/5/9-1/30/TaCIPK31-AD and TabZIP26-BD were grown on the SD/Ade\u003csup\u003e-\u003c/sup\u003eTrp\u003csup\u003e-\u003c/sup\u003eLeu\u003csup\u003e-\u003c/sup\u003eHis\u003csup\u003e-\u003c/sup\u003e plate to examine their interactions. Two empty vectors pGADT7 and pGBKT7 served as negative controls. b BiFC assays carried out in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves proves the interactions of TaCDPK5/9-1/30 and TabZIP26 \u003cem\u003ein vivo\u003c/em\u003e. The YFP fluorescence signals were tested 48 hours after Agrobacterium infection. Two empty vectors BiFC-YN and BiFC-YC were used as controls. Scale bars: 45 mm.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/33cfe6231289b9cf1825126a.jpeg"},{"id":58575068,"identity":"fc1727fa-f165-4a78-8405-94accdba1291","added_by":"auto","created_at":"2024-06-18 11:56:56","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396222,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTabZIP11 is responsible for salt tolerance in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea The phenotypes of \u0026nbsp;\u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines and WT under different conditions, which were cultivated on MS medium containing 100 mM NaCl or nonsupplemented medium as control. b-f The root growth, soluble sugar, proline content, \u0026nbsp;MDA, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents of \u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines and WT under salt stress in (a). Different letters above the bars show significant differences compared with the wild type and transgenic lines (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/92f4bdc1d92a7ce65210679c.jpeg"},{"id":58574325,"identity":"bfddc031-513a-49df-b800-b4ca0dc6f665","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":599147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTabZIP11 is required for freezing tolerance in Arabidopsis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea Freezing treatment of \u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines and WT at -10°C for 7 hours and recovery at 23°C for 3 days. b-e Soluble sugar, proline content, MDA, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents of \u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines and WT under freezing stress. Different letters display significant differences between \u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines with WT plants under freezing stress conditions (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/84510c8b75149b3a4c662138.jpeg"},{"id":58575070,"identity":"cf4f7be9-8b24-40f6-8bf1-2fa06874019b","added_by":"auto","created_at":"2024-06-18 11:56:56","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":678870,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eTabZIP11\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-OE lines with higher expression levels of stress-responsive genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea The \u0026nbsp;transcript levels of \u003cem\u003eAtDREB2A\u003c/em\u003e, \u003cem\u003eAtERD6\u003c/em\u003e, \u003cem\u003eAtRAB18\u003c/em\u003e, \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e and \u003cem\u003eAtSIZ1\u003c/em\u003e genes of WT and \u003cem\u003eTabZIP11\u003c/em\u003e-OE plants before and after salt stress. b The \u0026nbsp;RT-qPCR analysis of \u003cem\u003eAtCBF1\u003c/em\u003e, \u003cem\u003eAtCBF2\u003c/em\u003e, \u003cem\u003eAtCBF3\u003c/em\u003e, \u003cem\u003eAtCOR15B\u003c/em\u003e, \u003cem\u003eAtCOR27\u003c/em\u003e, \u003cem\u003eAtCOR47\u003c/em\u003e, \u003cem\u003eAtMYB77\u003c/em\u003e,\u003cem\u003e AtWRKY33\u003c/em\u003e, \u003cem\u003eAtRCI2A \u003c/em\u003eThe relative expression levels of are normalized with the \u003cem\u003eAtActin\u003c/em\u003e. Different letters represent significant differences between \u003cem\u003eTabZIP11\u003c/em\u003e transgenic lines with WT plants under freezing and salt stress conditions (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/e7d6f6025462a0432a9966c4.jpeg"},{"id":58575069,"identity":"63ff897f-aa86-4876-b57f-940c3a12da31","added_by":"auto","created_at":"2024-06-18 11:56:56","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":730171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterodimerization and homodimerization analysis of TabZIP9/11/14/32/36 and TabZIP11 proteins by the yeast two-hybrid method.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe growth status of yeast AH109 on SD /His\u003csup\u003e-\u003c/sup\u003eAde\u003csup\u003e-\u003c/sup\u003eLeu\u003csup\u003e-\u003c/sup\u003eTrp\u003csup\u003e-\u003c/sup\u003e medium indicate dimerization of TabZIP9/11/14/32/36 and TabZIP11 proteins.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/ab1e0e0305e360debc9d970b.jpeg"},{"id":58574329,"identity":"3bab806d-57b8-4094-84bb-3cb2b2232945","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":639070,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTabZIP11and group C TabZIP36 together enhance TabZIP11 mediated transcript of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTaCBF1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TabZIP36 and TabZIP11 together promote TabZIP11 to bind the promoter sequence of \u003cem\u003eTaCBF1\u003c/em\u003e using modified yeast-one hybrid assays. Three empty vectors pGADT7, pB42AD, and pLacZi were selected for negative controls. Yeast cell were trained in liquid medium and diluted in a 10 × dilution series. The experiments were done three times with similar results.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/023f63b8f77bd9003f8415fd.jpeg"},{"id":60026930,"identity":"06f117be-81d9-4748-9791-f987b14154ad","added_by":"auto","created_at":"2024-07-10 17:42:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5762944,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/4d4be6ac-2245-409a-b735-d9b2e8acf4f9.pdf"},{"id":58574327,"identity":"105b9f39-94e6-4395-b353-f74a6cd0a72f","added_by":"auto","created_at":"2024-06-18 11:48:56","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":98068,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTAYMATERIALS.docx","url":"https://assets-eu.researchsquare.com/files/rs-4483341/v1/88f10721f8cd87cb6ae29879.docx"}],"financialInterests":"","formattedTitle":"A novel wheat S1-bZIP gene, TabZIP11 confers stress resistance in Arabidopsis","fulltext":[{"header":"Key message ","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTabZIP11\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene enhances freezing and salt tolerance in transgenic plants through TaCBF1 and is directly regulated by TaCDPK1/5/9-1/30 and TaCIPK31.\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eTranscription factors (TFs) play vital regulatory roles in various plant biological processes (Pruneda-Paz et al. 2014). Basic leucine zipper (bZIP) is a member of transcription factor family in plants. The bZIP domain of bZIP TFs contain a highly conserved DNA-binding basic region and a leucine zipper region (Kouzarides and Ziff. 1989). And the members of bZIP family are clustered into 13 groups (group A-K, M, S) and have diverse roles, especially in plant stress-responsive and hormone signal transduction (Dr\u0026ouml;ge-Laser et al. 2018). Group S is composed of three subgroups: S1, S2, and S3 (Ehlert et al. 2006). Among them, only a few group S bZIP proteins have been characterized to regulate abiotic stress response in plants. For example, the transcriptional level of \u003cem\u003eAtbZIP1\u003c/em\u003e was significantly induced by salt, osmotic, and cold stresses in \u003cem\u003eArabidopsis\u003c/em\u003e. Overexpression and knockout mutants of \u003cem\u003eAtbZIP1\u003c/em\u003e in transgenic plants revealed that AtbZIP1 is capable of enhancing or reducing tolerance to salt and drought stresses (Sun et al. 2012). AtbZIP53 was shown to participate in hypo-osmolarity stress. The \u003cem\u003eArabidopsis thaliana\u003c/em\u003e proline dehydrogenase (ProDH) was found to be directly targeted by AtbZIP53 (Weltmeier et al. 2006). Rice OsbZIP71 was involved in ABA-mediated drought and salt tolerance (Liu et al. 2014). Previous research has also showed that overexpression of \u003cem\u003eOsbZIP16\u003c/em\u003e contributed to drought resistance in rice (Chen et al. 2012). In tomato, SlbZIP1 raised salt and drought stress tolerance by enhancing expression of multiple ABA biosynthesis and signal transduction-related genes (Zhu et al. 2018). Likewise, sweetpotato \u003cem\u003eIbbZIP1\u003c/em\u003e gene conferred salt and drought tolerances in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (Kang et al. 2019). In apple, group S MdbZIP80 TF could heterodimerize with group C bZIP members and restrain the expression of \u003cem\u003eMdIPT5b\u003c/em\u003e, which resulted in negatively modulating drought and salt tolerance (Feng et al. 2021). GsbZIP67 play a role in improving the bicarbonate alkaline tolerance (Wu et al. 2018). Under abiotic conditions, the group S bZIP TFs can also interact with and be phosphorylated by calcium-dependent protein kinases CDPKs or SNF1‐related protein kinases SnRK to mediate their transcriptional activation activities. For instance, the salt-induced \u003cem\u003eAtbZIP1\u003c/em\u003e transcription was found to be strongly up-regulated by SnRK1 signaling. However, \u003cem\u003eAtbZIP53\u003c/em\u003e expression partially hinged on the SnRK2 kinase signaling pathway (Hartmann et al. 2015). And Ca\u003csup\u003e2+\u003c/sup\u003e signaling also participated in inducing \u003cem\u003eAtbZIP1\u003c/em\u003e transcription through Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e (Hartmann et al. 2015). However, the interaction between wheat TaCDPK or TaCIPK and group S bZIP TF has not been revealed until now. And the exact function and regulation mechanism of group S bZIP TF are poorly known in wheat.\u003c/p\u003e \u003cp\u003eIn this study, we identified a novel subgroup S1 TabZIP11, which was strongly induced by ABA, salt, and cold stresses. We further investigated and found that TabZIP11 localized in nucleus. But it did not exhibit transcriptional activation activity in yeast. The expression level of salt-induced \u003cem\u003eTabZIP11\u003c/em\u003e was strongly decreased by Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e. A yeast two-hybrid assay and BiFC showed that TaCDPK5, TaCDPK9-1, TaCDPK30 were able to interplay with TabZIP11, respectively. Overexpression of \u003cem\u003eTabZIP11\u003c/em\u003e in Arabidopsis enhanced salt and freezing tolerance. And TabZIP11 was capable of homodimerizing by itself and heterodimerizing with the group C TabZIP proteins. TabZIP36 raised the binding ability of TabZIP11 to the promoter of \u003cem\u003eTaCBF1\u003c/em\u003e. These results indicate that TabZIP11 mediates positively the salt and cold stresses response.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eThe wheat cv. Longchun 27 was used to clone the full-length coding region of \u003cem\u003eTabZIP11\u003c/em\u003e and analyse the expression level of \u003cem\u003eTabZIP11\u003c/em\u003e gene. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Col-0) were used for genetic transformation to generate transgenic overex-pressing \u003cem\u003eTabZIP11\u003c/em\u003e through \u003cem\u003eAgrobacterium\u003c/em\u003e infection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and analysis of gene sequences\u003c/h2\u003e \u003cp\u003eThe coding region of \u003cem\u003eTabZIP11\u003c/em\u003e and the group C members (\u003cem\u003eTabZIP9\u003c/em\u003e/\u003cem\u003e14\u003c/em\u003e/\u003cem\u003e32\u003c/em\u003e/\u003cem\u003e36\u003c/em\u003e) of TabZIP proteins were amplified by PCR from the wheat cv. Longchun 27. In my previous study, we cloned the full length sequences of \u003cem\u003eTaCDPK5\u003c/em\u003e, \u003cem\u003eTaCDPK9-1\u003c/em\u003e and \u003cem\u003eTaCDPK30\u003c/em\u003e (Zhang et al. 2022). Multiple sequence alignment of TabZIP11 and other downloaded bZIP proteins were achieved using ClustalW. The neighbor-joining phylogenetic tree was conducted using MEGA 5.2 software with 1000 bootstrap replications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative real-time PCR (RT-qPCR) analysis\u003c/h2\u003e \u003cp\u003eTo explore the expression pattern of \u003cem\u003eTabZIP11\u003c/em\u003e in common wheat, the roots treated with PEG (16%), NaCl (100 mM) and ABA (100 \u0026micro;M) solutions, and the leaves treated with cold stress (4℃) were from ten-day-old wheat seedling. These samples were gained at 0, 1, 3, 6, 12, 24 and 48 h, respectively. To investigate the effect of calcium channel blocker LaCl\u003csub\u003e3\u003c/sub\u003e on the expression of \u003cem\u003eTabZIP11\u003c/em\u003e gene, wheat cv. Longchun 27 were treated with 100 mM NaCl and NaCl\u0026thinsp;+\u0026thinsp;0.8% LaCl \u003csub\u003e3\u003c/sub\u003e for 1h, 3h, 6h, 12h, 24h and 48h. To investigate the expression levels of stress related genes in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e, the two-week-old Arabidopsis plants were treated with 150 mM NaCl for 12h and cold stress for 3h. Total RNA was segregated and cDNA was generated as described previously (Zhang et al. 2020). The relative expression of \u003cem\u003eTabZIP11\u003c/em\u003e and Arabidopsis stress responsive genes were determined with reference to the expression of wheat \u003cem\u003eTaGAPDH\u003c/em\u003e (AF251217.1) or Arabidopsis \u003cem\u003eAtActin\u003c/em\u003e (AT3G18780) with the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The gene specific primers used in RT-qPCR analysis were listed in additional Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis subcellular localization of TabZIP11\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eTabZIP11\u003c/em\u003e CDS sequence lacking a termination codon was cloned into 35S-GFP vector, producing TabZIP11:GFP fusion plasmid, which was extracted with TIANprep Mini Plasmid Kit (TIANGEN, China) according to the instructions. TabZIP11:GFP fusion plasmid and empty 35S-GFP vector were expressed transiently in the leaves of tobacco through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 mediated transformation, respectively. After 16 h of incubation in the dark at 25\u0026deg;C, green fluorescent protein (GFP) fluorescence was captured with a confocal laser-scanning microscope (Leica Sp8, Germany). The gene specific primers used in this study were listed in additional Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eThe transactivation assay of TabZIP11 and yeast two-hybrid assay\u003c/h2\u003e \u003cp\u003eIn order to define the capacity of \u003cem\u003eTabZIP11\u003c/em\u003e in regulating transcription, full-length coding sequences of \u003cem\u003eTabZIP11\u003c/em\u003e or \u003cem\u003eTabZIP60\u003c/em\u003e was fused to the GAL4 DNA-binding domain (BD) in vector pGBKT7 to create TabZIP11-BD and TabZIP60-BD, respectively. All these constructs and the empty vector pGBKT7 were individually transformed into the AH109 yeast competent cells using yeast Frozen-EZ transformation kit (Zymo, USA). TabZIP60-BD and empty vector pGBKT7 were used as positive and negative controls (Zhang et al. 2015). These transformants were cultivated on SD/ Trp\u003csup\u003e\u0026minus;\u003c/sup\u003e plates at 30℃ for 2 days. The positive clones were coated onto fresh SD/His\u003csup\u003e\u0026minus;\u003c/sup\u003eAde\u003csup\u003e\u0026minus;\u003c/sup\u003eTrp\u003csup\u003e\u0026minus;\u003c/sup\u003e plates and grown at 30℃ to detect the transactivation activity of TabZIP11.\u003c/p\u003e \u003cp\u003eFor yeast two-hybrid assay, the coding region of \u003cem\u003eTaCDPK1\u003c/em\u003e, \u003cem\u003eTaCDPK5\u003c/em\u003e, \u003cem\u003eTaCDPK9-1\u003c/em\u003e, \u003cem\u003eTaCDPK30\u003c/em\u003e, and \u003cem\u003eTaCIPK31\u003c/em\u003e were cloned into the vector pGADT7 containing GAL4 DNA-binding domain (AD) to generate \u003cem\u003eTaCDPK1\u003c/em\u003e-AD, \u003cem\u003eTaCDPK5\u003c/em\u003e-AD, \u003cem\u003eTaCDPK9-1\u003c/em\u003e-AD, \u003cem\u003eTaCDPK30\u003c/em\u003e-AD, and \u003cem\u003eTaCIPK31\u003c/em\u003e-AD fusion plasmids. The recombinant plasmid \u003cem\u003eTabZIP11\u003c/em\u003e-BD comes from the fusion of \u003cem\u003eTabZIP11\u003c/em\u003e and pGBKT7 (BD) vector. Co-transformed \u003cem\u003eTaCDPK1\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e-BD, \u003cem\u003eTaCDPK5\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e-BD, \u003cem\u003eTaCDPK9-1\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e-BD, \u003cem\u003eTaCDPK30\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e-BD, \u003cem\u003eTaCIPK31\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e-BD were coexpressed in the yeast AH109, then cultured on SD/Leu\u003csup\u003e\u0026minus;\u003c/sup\u003eTrp\u003csup\u003e\u0026minus;\u003c/sup\u003e plates at 30\u0026deg;C for 2\u0026ndash;3 days. Positive clones were streaked onto fresh SD/His\u003csup\u003e\u0026minus;\u003c/sup\u003eAde\u003csup\u003e\u0026minus;\u003c/sup\u003eLeu\u003csup\u003e\u0026minus;\u003c/sup\u003eTrp\u003csup\u003e\u0026minus;\u003c/sup\u003e plates at 30\u0026deg;C about 2\u0026ndash;4 days. The gene specific primers of constructing recombinant plasmids in this study were listed in additional Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eTo investigate the dimerization of between TabZIP11 and TabZIP9/11/14/32/36 (group C), the ORF of \u003cem\u003eTabZIP9/11/14/32/36\u003c/em\u003e and \u003cem\u003eTabZIP11\u003c/em\u003e were subcloned into AD or BD vectors, respectively. The yeast two-hybrid assay was used to compare the dimerization among the different combinations by prototrophic growth of the yeast strains on plates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiFC assay in\u003c/b\u003e \u003cb\u003eN. benthamiana\u003c/b\u003e \u003cb\u003eleaves\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo carry out biomolecular fluorescent complementation (BiFC) assays, the CDS of \u003cem\u003eTaCDPK5\u003c/em\u003e, \u003cem\u003eTaCDPK9-1\u003c/em\u003e, \u003cem\u003eTaCDPK30\u003c/em\u003e without TAG were cloned into the BiFC-YN vector to construct TaCDPK5:NYFP, TaCDPK9-1:NYFP, and TaCDPK30:NYFP. And the \u003cem\u003eTabZIP11\u003c/em\u003e CDS was fused into the BiFC-YC vector to obtain TabZIP11:CYFP. These different recombination constructs and TabZIP11:CYFP were introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 and co-transformed into in the leaves of tobacco. A confocal microscopy (Leica Sp8, Germany) was used to observe the fluorescent signal to determine the TaCDPK5/9\u0026thinsp;\u0026minus;\u0026thinsp;1/30-TabZIP11 interactions. The gene primers sequences used in this study were listed in additional Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of transgenic arabidopsis plants\u003c/h2\u003e \u003cp\u003eTo generate the overexpression transgenic lines, the open reading frame of \u003cem\u003eTabZIP11\u003c/em\u003e without a termination codon was inserted into the pDONR-Zeocin vector and then recombined into the plant overexpression vector 35S-GFP with a cauliflower mosaic virus (CaMV) 35S promoter through Gateway method. The identification of \u003cem\u003eTabZIP11\u003c/em\u003e overexpression lines was conducted by PCR amplification and RT-qPCR. Three overexpression lines L1, L2, and L3 were used to perform the phenotypic analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhenotypic analysis of\u003c/b\u003e \u003cb\u003eTabZIP11\u003c/b\u003e \u003cb\u003etransgenic plants under salt and freezing stresses\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor freezing stress treatment at the seedling stage, three-week-old homozygous transgenic lines and wild-type (WT) seedlings were treated at 4\u0026deg;C for 4\u0026ndash;5 days, and then at -10\u0026deg;C for 7 hours after which plants were recovered at 22\u0026deg;C for 3 days. For salt stress, the seven-day-old WT and transgenic lines were planted 1/2 MS medium with 0 and 100mM NaCl for 4\u0026ndash;5 days. The growth phenotype were photographed and the root length was measured. All experiments were replicated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation, malondialdehyde (MDA) content, soluble sugar, and proline contents\u003c/h2\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was determined through the trichloroacetic acid and potassium iodide (KI) assays described previously (Velikova et al. 2000). The 0.5g prepared fresh Arabidopsis seedlings were mixed with 5 ml of 0.1% (w/v) trichloroacetic acid (TCA), then the mixture was ground and centrifuged at 12,000g for 20 min. Then, 0.7 ml potassium phosphate buffer (10 mM, pH 7.0) and 1.4 ml KI (1 M) were added to 0.7 ml of the supernatant and mixed evenly. The absorbance of samples was measured at 390 nm. The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was calculated according to a standard curve. The MDA, soluble sugar, and proline contents were measured according to previous method (Bates et al. 1973; Hodges et al. 1999; Cui and Wang. 2006).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eThe modified yeast one-hybrid assay\u003c/h2\u003e \u003cp\u003eTo further investigate how to regulate the transcript level of \u003cem\u003eTaCBF1\u003c/em\u003e gene by TabZIP11 and TabZIP36. The coding sequences of \u003cem\u003eTabZIP36\u003c/em\u003e and \u003cem\u003eTabZIP11\u003c/em\u003e were cloned into the pGADT7 or pB42AD vectors, respectively, generating \u003cem\u003eTabZIP36\u003c/em\u003e-AD and \u003cem\u003eTabZIP11\u003c/em\u003e- pB42AD. The promoter sequence of \u003cem\u003eTaCBF1\u003c/em\u003e gene (not shown) was inserted into the pLacZi vector, producing \u003cem\u003eTaCBF1-\u003c/em\u003epromoter-pLacZi. The \u003cem\u003eTabZIP36\u003c/em\u003e-AD, \u003cem\u003eTabZIP11\u003c/em\u003e-pB42AD and \u003cem\u003eTaCBF1-\u003c/em\u003epromoter-pLacZi were transfected into yeast EGY48. Three biological replicates were performed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was carried out with SPSS 26.0. The Origin 9.0. software was used to generate these figures. The significant differences are represented by different lowercase letters.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eTabZIP11\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe cDNA sequence of \u003cem\u003eTabZIP11\u003c/em\u003e was amplified from the wheat cv. Longchun 27 through RT-PCR. The cDNA sequence analysis revealed that the ORF is 435bp in length, encodes a protein, which is speculated to contain 144 amino acids with a molecular weight of 15.62 kDa and a theoretical pI of 10.56. The TabZIP11 protein harbours a typical bZIP domain (4-68Aa). The \u003cem\u003eArabidopsis\u003c/em\u003e bZIP TFs members are known to be divided into 13 groups, and S group includes S1, S2 and S3 subgroups. The neighbor-joining phylogenetic tree indicated that TabZIP11 was gathered into group S1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression profiles of\u003c/b\u003e \u003cb\u003eTabZIP11\u003c/b\u003e \u003cb\u003eunder different treatments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to better investigate the expression pattern of \u003cem\u003eTabZIP11\u003c/em\u003e gene, we analyzed the expression level of the \u003cem\u003eTabZIP11\u003c/em\u003e gene in wheat root and leaf tissues under non-stressed condition through RT-qPCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, the expression level of \u003cem\u003eTabZIP11\u003c/em\u003e gene presented no significant difference in untreated roots and leaves at different times. Nevertheless, after ABA and NaCl treatments, the transcription levels of \u003cem\u003eTabZIP11\u003c/em\u003e in the roots were rapidly and strongly induced. In the same way, the expression of \u003cem\u003eTabZIP11\u003c/em\u003e in the leaves was also significantly increased in response to cold stress. After PEG treatment, there was no significant change in the expression of \u003cem\u003eTabZIP11\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003ePrevious study has demonstrated that Ca\u003csup\u003e2+\u003c/sup\u003e signaling is required for \u003cem\u003eAtbZIP1-S\u003c/em\u003e transcription in \u003cem\u003eArabidopsis\u003c/em\u003e (Hartmann et al. 2015). To investigate whether the \u003cem\u003eTabZIP11\u003c/em\u003e gene is also involved in the Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathway, we examined the expression of \u003cem\u003eTabZIP11\u003c/em\u003e gene in the presence of the Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e under NaCl treatment. The transcriptional level of \u003cem\u003eTabZIP11\u003c/em\u003e was upregulated by NaCl stress alone. However, in the presence of LaCl\u003csub\u003e3\u003c/sub\u003e, \u003cem\u003eTabZIP11\u003c/em\u003e expression level was significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These data implied that Ca\u003csup\u003e2+\u003c/sup\u003e signaling is also necessary for salt-induced \u003cem\u003eTabZIP11\u003c/em\u003e transcription.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTabZIP11 is a nuclear-localized TF without transactivation activity\u003c/h2\u003e \u003cp\u003eIn order to investigate the subcellular localization of the TabZIP11 protein, a \u003cem\u003e35S: TabZIP11-GFP\u003c/em\u003e recombinant plasmid or a negative control (\u003cem\u003e35S-GFP\u003c/em\u003e) was transformed and transiently expressed in the leaves of tobacco. The signals of GFP alone were present in the plasma membrane and nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Whereas in \u003cem\u003e35S: TabZIP11-GFP\u003c/em\u003e transformed tobacco leaves cells, the GFP fluorescence was visualized in the nucleus, suggesting the TabZIP11 is a nuclear-localized protein.\u003c/p\u003e \u003cp\u003eBecause most transcription factors exhibit transcriptional activation activity. Consequently, we also determined whether TabZIP11 possess the potential transcriptional activity in yeast cells. The full-length coding sequence of \u003cem\u003eTabZIP11\u003c/em\u003e or \u003cem\u003eTabZIP60\u003c/em\u003e was cloned into the pGBKT7 (BD) vector, respectively. The pGBKT7 vector alone and BD-TabZIP60 were used as negative and positive controls. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, only yeast cell expressing BD-TabZIP60 grew well on SD/Trp\u003csup\u003e\u0026minus;\u003c/sup\u003eHis\u003csup\u003e\u0026minus;\u003c/sup\u003eAde\u003csup\u003e\u0026minus;\u003c/sup\u003e selective media. Compared with the pGBKT7 vector alone, the BD-TabZIP11 also failed to exhibit any detectable yeast growth. This finding indicated that TabZIP11 did not show any transactivation property in yeast.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTaCDPK1/5/9\u0026thinsp;\u0026minus;\u0026thinsp;1/30 and TaCIPK31 interact with TabZIP11\u003c/h2\u003e \u003cp\u003eOur previous research has indicated that TaCDPK5, TaCDPK9-1 and TaCDPK30 were interacting proteins of group A TabZIP60 (Zhang et al. 2022, 2023). In this study, salt-induced \u003cem\u003eTabZIP11\u003c/em\u003e transcript level decreases through Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e. So we examined whether TabZIP11 interacts with TaCDPK1, TaCDPK5, TaCDPK9-1, TaCDPK30 and TaCIPK31 individually by yeast two-hybrid method. A significant interactions between TabZIP11 and the closely Ca\u003csup\u003e2+\u003c/sup\u003e related protein kinase members TaCDPK5/9\u0026thinsp;\u0026minus;\u0026thinsp;1/30/TaCIPK31 could be observed. A weaker interaction with TabZIP11 and TaCDPK1 could be examined. No significant interaction were determined in other yeasts cell expressing negative controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eTo further confirm the data obtained by yeast two-hybrid, biomolecular fluorescent complementation (BiFC) studies were executed by expressing TaCDPK5: NYFP, TaCDPK9-1: NYFP, TaCDPK30: NYFP and TabZIP11: CYFP in tobacco leaves cell. These results indicated that only samples expressing the combination TaCDPK5: NYFP and TabZIP11: CYFP, TaCDPK9-1: NYFP and TabZIP11: CYFP, or TaCDPK30: NYFP and TabZIP11: CYFP showed strong YFP complementation signals in the nucleus of tobacco leaves cells. No fluorescence in the negative controls were examined, which are in agreement with the results gained by yeast two-hybrid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In conclusion, these results showed that TabZIP11 interacts with TaCDPK5, TaCDPK9-1 and TaCDPK30 in vivo, respectively.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eTabZIP11\u003c/b\u003e \u003cb\u003eenhances the freezing and salt tolerances of transgenic Arabidopsis seedlings\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the physiological function of \u003cem\u003eTabZIP11\u003c/em\u003e gene, we produced transgenic Arabidopsis plants overexpressing \u003cem\u003eTabZIP11\u003c/em\u003e (hereafter, \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e). The overexpression level of \u003cem\u003eTabZIP11\u003c/em\u003e in four independent \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e lines was demonstrated by RT-qPCR method (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). We examined the phenotypes of \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants in response to salt stress by transferring 7-day-old seedlings to the 1/2 MS medium with 100 mM NaCl. After 5 days of salt stress treatment, the \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants showed much better growth than WT, exhibiting longer root length and more lateral roots. While a similar growth states in seedling phenotypes between \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e and WT plants was observed under growth condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b). In accordance with the visible salt resistant phenotypes, the soluble sugar and proline contents remained high in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). But MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents became low in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e during salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eIn addition, we find that \u003cem\u003eTabZIP11\u003c/em\u003e transcript level was strongly induced within 1\u0026ndash;12 h of freezing treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). We therefore examined whether \u003cem\u003eTabZIP11\u003c/em\u003e regulate responses to freezing stress by comparing the phenotypes of 3-week-old \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e and WT plants, which were placed in freezing stress (-10\u0026deg;C) for 7 hours and subsequently restored to appropriate temperature (22\u0026deg;C). It is worth noting that WT plants were more sensitive to freezing stress than the \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e, exhibiting some leaves of WT seedlings died. Nevertheless, almost all transgenic lines were able to survive and their leaves retained green (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). We also examined the levels of the soluble sugar and proline, which were significantly improved in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e lines compared to WT under freezing stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, c), and yet MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e turned lower than WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). These results indicated that overexpression of \u003cem\u003eTabZIP11\u003c/em\u003e significantly improves tolerance to freezing stress.\u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003eTabZIP11\u003c/b\u003e \u003cb\u003eresults in increased expression of stress responsive genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo elucidate the transcriptional regulation of \u003cem\u003eTabZIP11\u003c/em\u003e gene, we examined the expression levels of abiotic stress-associated genes in WT and \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants during NaCl and freezing stress treatments. After 12h hours of treatment in 150mM NaCl solution, several stress-related genes including \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, \u003cem\u003eAtSIZ1\u003c/em\u003e, \u003cem\u003eAtDREB2A\u003c/em\u003e, \u003cem\u003eAtERD6\u003c/em\u003e, and \u003cem\u003eAtRAB18\u003c/em\u003e were obviously enhanced in WT and \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants but upregulated at higher levels in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). We also found that during 4℃ stress, the transcript levels of \u003cem\u003eAtCBF1\u003c/em\u003e, \u003cem\u003eAtCBF2\u003c/em\u003e, \u003cem\u003eAtCBF3\u003c/em\u003e, \u003cem\u003eAtMYB77\u003c/em\u003e, \u003cem\u003eAtRC12A\u003c/em\u003e, \u003cem\u003eAtWRKY33\u003c/em\u003e, \u003cem\u003eAtCOR47\u003c/em\u003e, \u003cem\u003eAtCOR27\u003c/em\u003e, and \u003cem\u003eAtCOR15B\u003c/em\u003e were higher in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e lines compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, the expression levels of these genes had no significant difference between the WT and \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e under normal condition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTabZIP11 could form homodimer by itself and heterodimers with TabZIP9/ 14/32/36\u003c/h2\u003e \u003cp\u003eThe previous researches have uncovered that the group S1 bZIPs could form specific heterodimers or homodimers with group C bZIPs and itself to be involved in regulating gene expression (Weiste et al. 2014; Feng et al. 2021). This finding prompted us to determine the dimerization activity of TabZIP11 through a yeast two-hybrid method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e, all constructs that were transformed into the yeast AH109 grew well on SD/Leu\u003csup\u003e\u0026minus;\u003c/sup\u003e Trp\u003csup\u003e\u0026minus;\u003c/sup\u003e medium. But only the yeasts expressing BD-TabZIP11 and AD-TabZIP11, BD-TabZIP11 and AD-TabZIP9, BD-TabZIP11 and AD-TabZIP14, BD-TabZIP11 and AD-TabZIP32 or BD-TabZIP11 and AD-TabZIP36 were capable of growing on SD/Leu\u003csup\u003e\u0026minus;\u003c/sup\u003eTrp\u003csup\u003e\u0026minus;\u003c/sup\u003eAde\u003csup\u003e\u0026minus;\u003c/sup\u003eHis\u003csup\u003e\u0026minus;\u003c/sup\u003e medium. There were no yeast growth for other negative controls on the same medium. These results showed that TabZIP11 can form both homodimer as well as heterodimers with group C proteins in yeast.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTabZIP36 enhances the binding of TabZIP11 to the promoter of\u003c/b\u003e \u003cb\u003eTaCBF1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOur study demonstrated that there are the physical interactions between TabZIP36 and TabZIP11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e). \u003cem\u003eAtCBF1\u003c/em\u003e gene expression was significantly enhanced in \u003cem\u003eTabZIP11\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e lines under freezing stress compared to the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eb), which urged us to explore whether the dimer of TabZIP36 and TabZIP11 regulate the wheat homolog \u003cem\u003eTaCBF1\u003c/em\u003e transcript. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e, we demonstrated that dimeric (TabZIP36: TabZIP11) proteins could strongly induced the transcription level of \u003cem\u003eTaCBF1\u003c/em\u003e gene, but lower activation level mediated by TabZIP11 alone on SD/Leu\u003csup\u003e\u0026minus;\u003c/sup\u003eTrp\u003csup\u003e\u0026minus;\u003c/sup\u003eUra\u003csup\u003e\u0026minus;\u003c/sup\u003e with 20mM X-Gal and 200mM 3AT medium. However, in the presence of TabZIP36, the activation of \u003cem\u003eTaCBF1\u003c/em\u003e gene was significantly increased.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGroup S comprises the largest group of bZIP family in \u003cem\u003eArabidopsis\u003c/em\u003e. It can be separated into three subgroups, entitled S1, S2 and S3 (Dr\u0026ouml;ge-Laser et al. 2018). The group S bZIPs play vital roles in regulating plant development, metabolic processes and stress response (Wang et al. 2022). However, some researches about group S1 bZIPs were performed in the model plants Arabidopsis and rice. No reports thus far have indicated a subgroup S1 bZIP protein playing a positive regulatory role in wheat abiotic stress. In this study, we isolated and identified a wheat \u003cem\u003eTabZIP11\u003c/em\u003e, which has high similarity with S1 subgroup of Arabidopsis bZIP TF. So the phylogenetic tree analysis showed that \u003cem\u003eTabZIP11\u003c/em\u003e belonged to S1 subgroup.\u003c/p\u003e \u003cp\u003eTabZIP11 was found to be located in the nucleus and had no transactivation activity. In agreement with this result, several group S bZIP proteins have been shown to harbour nuclear localization (Satoh et al. 2004; Wu et al. 2018). And some other group S bZIPs also do not display transcriptional activation in yeast, such as OsbZIP71, LIP19, and CsbZIP44 (Shimizu et al. 2005; Liu et al. 2014; Sun et al. 2024). Furthermore, our studies also suggested that the transcription of \u003cem\u003eTabZIP11\u003c/em\u003e was dramatically reduced by NaCl stress together with Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e. This result revealed that TabZIP11 is involved in Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathway, which is in line with the previous studies that Ca\u003csup\u003e2+\u003c/sup\u003e blocker LaCl\u003csub\u003e3\u003c/sub\u003e has been shown to decrease the salt-induced subgroup S1 \u003cem\u003eAtbZIP1\u003c/em\u003e transcription (Hartmann et al. 2015). These findings further suggest that TabZIP11 is a downstream substrate for CDPK or CIPK kinase and support the conception that the CDPK or CIPK alone might be insufficient for regulating the expression of \u003cem\u003eTabZIP11\u003c/em\u003e gene and the additional protein kinases are essential for its expression activity. Previous work identified that SnRK1 could act as a AtbZIP1-interacting kinase (Hartmann et al. 2015). But to date, few direct physical interactions between wheat CDPK or CIPK and group S1 bZIPs could be identified. We identified the TabZIP11 functions as a TaCIPK31/TaCDPK1/5/9\u0026thinsp;\u0026minus;\u0026thinsp;1/30-interacting protein in yeast. In accordance with the yeast-two hybrid experiment finding, TabZIP11 was showed to interplay with TaCDPK5/9\u0026thinsp;\u0026minus;\u0026thinsp;1/30 in planta through the BiFC assay. Several data also support the impact of suppressing CDPK kinase activity on downstream \u003cem\u003eTabZIP\u003c/em\u003e gene expression (Zhang et al. 2020, 2022, 2023).\u003c/p\u003e \u003cp\u003eIn this study, it was then shown that \u003cem\u003eTabZIP11\u003c/em\u003e was strongly increased by cold and NaCl stresses, hinting that TabZIP11 may play a regulatory role in abiotic stress. Therefore, it may be interesting to study the potential function of \u003cem\u003eTabZIP11\u003c/em\u003e in response to different stresses, we overexpressed \u003cem\u003eTabZIP11\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e and displayed that all \u003cem\u003eTabZIP11\u003c/em\u003e overexpression transgenic lines exhibited better ability to resist salt and freezing stresses than the WT plants, including higher soluble sugar and proline contents. Meanwhile, lower MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents were found in \u003cem\u003eTabZIP11\u003c/em\u003e overexpression plants compared with WT under stress conditions. These results demonstrate that TabZIP11 contributes to freezing and salt tolerances in plant.\u003c/p\u003e \u003cp\u003eTo date, several bZIP-related overexpressed plants and a variety of abiotic-associated genes, which increase during stress treatment, have been identified and cloned. For example, \u003cem\u003eAtDREB2A\u003c/em\u003e, \u003cem\u003eAtSIZ1\u003c/em\u003e, \u003cem\u003eAtABF1\u003c/em\u003e, \u003cem\u003eAtABI1\u003c/em\u003e, and \u003cem\u003eAtPCS1\u003c/em\u003e were significantly higher in \u003cem\u003eGmbZIP152\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e plants than those in WT plants under stress condition (Chai et al. 2022). Some stress-related genes, such as \u003cem\u003eAtDREB2A, AtRD29A, AtERD10\u003c/em\u003e and \u003cem\u003eAtRD29B\u003c/em\u003e, indicated obviously higher expression levels in the \u003cem\u003eAtPPRT3\u003c/em\u003e-\u003cem\u003eOX\u003c/em\u003e lines than in WT Arabidopsis (Liu et al. 2020). In line with these results, we found that the transcript level of abiotic-related genes (\u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, \u003cem\u003eAtSIZ1\u003c/em\u003e, \u003cem\u003eAtDREB2A\u003c/em\u003e, \u003cem\u003eAtERD6\u003c/em\u003e and \u003cem\u003eAtRAB18\u003c/em\u003e) significantly upregulated in \u003cem\u003eTabZIP11\u003c/em\u003e-OX lines compared to WT plants after NaCl treatment. AtSIZ1, encoding a salt-induced C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e type zinc finger protein, was involved in positively conferring salt tolerance of Arabidopsis through maintaining ionic and osmotic homeostasis (Han et al. 2019). \u003cem\u003eAtRD29A\u003c/em\u003e, \u003cem\u003eAtRD29B\u003c/em\u003e, and \u003cem\u003eAtDREB2A\u003c/em\u003e participate in the ABA-mediated resistance pathway and are activated by several transcription factor (Hirayama et al. 2007). \u003cem\u003eAtERD6\u003c/em\u003e and \u003cem\u003eAtRAB18\u003c/em\u003e are the members of dehydrins (DHNs) protein family and accumulate by drought, salinity, and freezing stresses (Hanin et al. 2011). In addition, TabZIP11 also activated the expression of stress-related gene \u003cem\u003eAtCOR47\u003c/em\u003e, \u003cem\u003eAtCOR27\u003c/em\u003e, \u003cem\u003eAtCOR15B\u003c/em\u003e, \u003cem\u003eAtMYB77\u003c/em\u003e, \u003cem\u003eAtCBF1\u003c/em\u003e, \u003cem\u003eAtCBF2\u003c/em\u003e, \u003cem\u003eAtCBF3\u003c/em\u003e, \u003cem\u003eAtRCI2A\u003c/em\u003e, and \u003cem\u003eAtWRKY33\u003c/em\u003e under cold condition. C-repeat/DREB binding factors (CBFs), as a cold-induced transcription factor plays significant roles in cold stress by binding the promoter sequences of COR genes to activate their expression (Shi et al. 2018). \u003cem\u003eAtRCI2A\u003c/em\u003e is involved in salt tolerance by reducing excessive accumulation of Na\u003csup\u003e+\u003c/sup\u003e (Medina et al. 2005). These results indicated that TabZIP11 is proposed to mediate transcriptional level of stress-related gene in response to salt and cold stresses.\u003c/p\u003e \u003cp\u003eTranscriptional factors modulate the expressions of downstream target genes through binding to cis-element in promoter region. For group S bZIP proteins, the homodimerizations and heterodimerizations are regarded as the specific transcriptional regulation way of these bZIP TFs (Golldack et al. 2011). The heterodimerizations play an important function in the DNA binding affinity, transactivation activity, and cell physiology (Naar et al. 2001). For instance, strong heterodimerizations were achieved between members of the groups C (AtbZIP9,-10,-25,-63) and S1(AtbZIP1,-2,-11,-44,-53) bZIP proteins in Arabidopsis (Ehlert et al. 2006). OsbZIP71 homodimerized with itself as well as heterodimerized with group C proteins (OsbZIP15, OsbZIP20, OsbZIP33, and OsbZIP88) to enhance the transcript levels of downstream genes (Liu et al. 2014). TabZIP6 (group C) was able to shape heterodimers with Wlip19 or TaOBF1(group S) in yeast (Cai et al. 2018). TabZIP11 is also capable of forming a homodimer by itself and heterodimers with TabZIP9/14/32/36. But the modified yeast one-hybrid assay showed that TabZIP11 and only group C member TabZIP36 could help TabZIP11 bind the promoter of downstream \u003cem\u003eTaCBF1\u003c/em\u003e gene and upregulate its expression which might increase cold tolerance of \u003cem\u003eTabZIP11\u003c/em\u003e in vivo. We can deduce that TabZIP11 dimer might recruit other proteins necessary for transcriptional regulation to enhance binding capacity of the transcriptional complex.\u003c/p\u003e \u003cp\u003eIn a word, this study identified a novel subgroup S1 \u003cem\u003eTabZIP11\u003c/em\u003e, which was induced by ABA, NaCl, and cold stresses. TabZIP11 was involved in Ca\u003csup\u003e2+\u003c/sup\u003e signalling through interplaying TaCDPK/CIPKs kinases. The ectopic overexpression of \u003cem\u003eTabZIP11\u003c/em\u003e significantly improved plants tolerance to freezing and salt. Additionally, group C TabZIPs interact with TabZIP11 to form dimers, which are conducive to improving the expression of \u003cem\u003eTaCBF1\u003c/em\u003e gene.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003ebZIP \u0026nbsp; \u0026nbsp; \u0026nbsp; Basic leucine zipper\u003c/p\u003e\n\u003cp\u003eTF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transcription factor\u003c/p\u003e\n\u003cp\u003eABA \u0026nbsp; \u0026nbsp; \u0026nbsp; Abscisic acid\u003c/p\u003e\n\u003cp\u003eCDPK \u0026nbsp; \u0026nbsp;Calcium-dependent protein kinase\u003c/p\u003e\n\u003cp\u003eCIPK \u0026nbsp; \u0026nbsp; Calcineurin B-like protein (CBL)-CBL-interacting protein kinase\u003c/p\u003e\n\u003cp\u003eCBF \u0026nbsp; \u0026nbsp; \u0026nbsp;C-repeat binding factor\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Hydrogen peroxide\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp;Malondialdehyde\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLNZ, CX, LCZ, and XYK projected this paper. ZY, XYL, YYW, and JL implemented the experiments. YHW, NY, and YLY analyzed the data. LNZ and ZY wrote the paper.\u003c/p\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eSupporting information\u003c/h2\u003e \u003cp\u003eTable. Primers used in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Natural Science Foundation of China [32260471, 31660392], Academic Backbone Project from the Northwest Normal University [2019GG-3], Young Teachers Improving Program from the Northwest Normal University [NWNU-LKQN-14-11].\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e \u003cp\u003eThe published article and its supplementary data contain all data of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. 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BMC Plant Biol 18(1):83.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"TabZIP11 transcription factor, TaCDPK1/5/9 − 1/30 and TaCIPK31, Homodimers and heterodimers, Salt and freezing tolerances, TaCBF1","lastPublishedDoi":"10.21203/rs.3.rs-4483341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4483341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The majority of basic leucine zipper (bZIP) transcription factor (TF) subgroup S1 play significant regulatory role in response to abiotic stress. However, their functions and underlying molecular mechanisms in abiotic stress responses are less known in wheat (Triticumaestivum L.). In this study, we isolated a TabZIP11 TF, which is from S1 subgroup of wheat bZIP transcription factor. TabZIP11 encodes a nuclear protein without transcriptional activation activity. Transcript of TabZIP11 gene was induced by abscisic acid (ABA), NaCl, and cold stress treatments. Whereas compared with NaCl treatment, TabZIP11 showed a lower expression level under NaCl+LaCl3 condition. We found that calcium-dependent protein kinase1 (TaCDPK1), TaCDPK5, TaCDPK9-1, TaCDPK30 and calcineurin B-like protein (CBL)-CBL-interacting protein kinase31 (TaCIPK31) cooperated with TabZIP11. The overexpression of TabZIP11 ectopically improved salt and freezing tolerances in Arabidopsis. TabZIP11 contributed to salt and freezing tolerance by modulating soluble sugar, proline, hydrogen peroxide (H2O2), and malondialdehyde (MDA) productions and abiotic stress responsive gene expression levels. TabZIP11 can form both homodimers and heterodimers with itself and group C TabZIP members. The modified yeast one-hybrid analysis confirmed that TabZIP36 significantly enhanced the binding ability of TabZIP11 to the promotor of TaCBF1 gene. Thus, these results suggest that TabZIP11 interacts with TabZIP36 to modulate cold signaling by facilitating the transcriptional activity of c-repeat binding factor (TaCBF1) gene. TabZIP11 functions as a positive regulator of salt stress responses through interacting with TaCDPK1/5/9-1/30 and TaCIPK31.","manuscriptTitle":"A novel wheat S1-bZIP gene, TabZIP11 confers stress resistance in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-18 11:48:51","doi":"10.21203/rs.3.rs-4483341/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa474284-d877-41b7-adfb-eb7c07eb3b9a","owner":[],"postedDate":"June 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-10T17:34:45+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-18 11:48:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4483341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4483341","identity":"rs-4483341","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}