Mangifera indica (mango) ICE1s confer flowering and tolerance to multiple stresses in Arabidopsis thaliana

Mangifera indica (mango) ICE1s confer flowering and tolerance to multiple stresses in Arabidopsis thaliana | 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 Mangifera indica (mango) ICE1s confer flowering and tolerance to multiple stresses in Arabidopsis thaliana Tianli Guo, Yili Zhang, Yanzhu Liu, Zhiqi Lai, Tingting Liang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7980258/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract The Inducer of CBF Expression 1 (ICE1) family is known to regulate plant responses to low-temperature stress, but its roles in flowering and other abiotic stresses remain unclear. In this study, the tissue-specific expression patterns and functional roles of Mangifera indica (mango) ICE1s ( MiICE1 s) were characterized in Arabidopsis thaliana ( Arabidopsis ). MiICE1a/aL showed high expression across multiple tissues, while MiICE1b/bL were up-regulated in flowering branch stems. MiICE1c and MiICE1d exhibited peak expression in flowers and in flowering branch stems/flowers, respectively, whereas MiICE1e/eL were most abundant in non-flowering branch leaves. Overexpression of most MiICE1 s (except MiICE1b ) in Arabidopsis accelerated flowering compared with the wild type, suggesting their involvement in flowering regulation. MiICE1 s also responded to NaCl, PEG, and methyl jasmonate treatments, with transgenic lines displaying superior root elongation and fresh weight under stress, indicating enhanced abiotic stress tolerance. Protein interaction analyses further revealed that MiICE1s interacted with flowering- and stress-related regulators, potentially coordinating these processes. Overall, this study highlights the dual role of MiICE1 s in promoting flowering and enhancing stress adaptation, providing insights for engineering stress-resilient crops with optimized flowering traits. ICE1 flowering time stress mango MiFLC MiFTs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction During growth and development, plants frequently encounter various environmental stresses, including salt, drought, and biotic stresses caused by pests and diseases (Jung et al. 2014; Khatamidoost et al. 2015 ; Kazemi-Shahandashti et al. 2018; Gong et al. 2020 ; Hajabdollahi et al. 2021 ; Hossain et al. 2023 ; Long et al. 2024 ). These stresses can severely impair plant growth, development, and yield (Gong et al. 2020 ; Saberi Riseh et al. 2024). Therefore, improving plant tolerance to environmental stress has become a central focus in plant research. One effective strategy is genetic engineering, which improves stress tolerance by identifying and manipulating resistance-related genes. Mango ( Mangifera indica L.) is an important tropical fruit crop with a wide cultivation range and important economic value. It ranks as the fifth most important fruit crop worldwide (Sandip et al. 2015 ). Despite the rapid development of the mango industry in China, both cultivation area and production have steadily increased (Chen et al. 2013; Kong et al. 2024 ), yet mango production continues to face numerous challenges. In particular, abiotic stresses such as high salt, low temperature, and drought, as well as biotic stresses from pathogens, remain major constraints on mango cultivation (Lao et al. 2010 ; Wei et al. 2017 ; Abdel-Aziz et al. 2023 ). Therefore, identifying stress-resistance genes and investigating their functional roles provides a theoretical foundation for enhancing mango stress tolerance and holds significant implications for the development of the mango industry in China. As a member of the basic Helix-Loop-Helix (bHLH) family, the Inducer of CBF Expression 1 (ICE1) contains the conserved bHLH domain characteristic of the MYC transcription factor family. ICE1 is known to be evolutionarily conserved and plays a key role in regulating plant tolerance to low-temperature stress (Tang et al. 2020 ; Wang et al. 2022 ). In addition to its role in cold stress resistance, ICE1 is also involved in flowering and responses to other abiotic stresses. Previous studies have shown that overexpression of ICE1 and its homologs confers tolerance to multiple stresses in Arabidopsis thaliana ( Arabidopsis ) (Xu et al. 2014 ; Li et al. 2014 ; Ding et al. 2015 ; Wei et al. 2018 ), chrysanthemum (Chen et al. 2012 ), banana (Li et al. 2022 ), and tobacco (Feng et al. 2013 ; Nagaveni et al. 2016 ; Zhang et al. 2018 ). Moreover, the ICE-CBF pathway has been implicated in the regulation of flowering in Arabidopsis (Lee et al. 2015 ). Unlike their roles in other plants, the functions of ICE1s in mango remain poorly understood. In this study, we investigated the effects of MiICE1 s on flowering and stress tolerance. By generating transgenic Arabidopsis plants and conducting flowering induction and stress treatment experiments, we elucidated the functional mechanisms of MiICE1 s in regulating flowering and mediating stress responses. Our results indicate that MiICE1 s are functional genes with potential applications in mango, which provides a new strategy for improving crop traits through genetic engineering. Materials and methods Plant materials and treatments The plant materials used in this study included mango 'Guiqimang', cultivated in the specimen garden of the College of Agriculture, Guangxi University, and Columbia wild-type (WT) Arabidopsis . For gene expression pattern analysis, two types of samples were collected: (1) Different tissue samples: The leaves and stems were collected from the three-year-old 'Guiqimang' trees and the non-flowering and flowering branches were obtained from the six-year-old trees; (2) Stress treatment samples: The grafted seedlings of 'Guiqimang' were subjected to the following treatments: 300 mmol L − 1 NaCl irrigation, 30% PEG 6000 irrigation to simulate drought stress, and foliar spray with 200 µmol L − 1 methyl jasmonate (MeJA). Samples were collected at 0, 6, 12, 24, 48, and 72 h after treatment. All samples were immediately frozen in liquid nitrogen and stored at -80°C. Subcellular localization analysis The full-length cDNA sequences of MiICE1 s were cloned into the pBI221-EGFP vector under the control of the Cauliflower mosaic virus 35S (CAMV35S) promoter. Agrobacterium tumefaciens (GV3101) carrying the fusion constructs was then introduced into onion ( Allium cepa ) inner epidermal cells, and fluorescence was observed using a laser confocal microscope (TCS-SP8MP, Leica). The empty vector served as a control, and nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Transcriptional activation analysis Transcriptional activation assays were performed to identify the presence of activation domains in MiICE1 proteins. The coding sequences of MiICE1 s were fused to the Gal4 DNA-binding domain in the pGBKT7 vector to generate pGBKT7-MiICE1s constructs. AH109 yeast cells transformed with these recombinant vectors were evaluated for transcriptional activation activity, following previously described methods (Liu et al. 2024 ; Xue et al. 2025 ). Production of transgenic Arabidopsis plants The pBI121-MiICE1s fusion expression plasmids were introduced into Agrobacterium rhizogenes EHA105. The resulting Agrobacterium cells were used to transform Arabidopsis via the floral-dip method (Clough et al. 1998; Wang et al. 2017 ). Positive seedlings were selected on 1/2 MS medium containing 50 mg L − 1 kanamycin, confirmed by PCR, and advanced to the T3 generation for subsequent experiments. Flowering phenotype analyses WT and empty vector-transformed Arabidopsis plants were used as controls. The time from sowing to the opening of the first flower was recorded, and flowering phenotypes were documented. Stress treatments To evaluate the responses of WT and homozygous T3 transgenic Arabidopsis to environmental stress, seeds were surface-sterilized and germinated on standard MS medium. Stress treatments were performed following a modified protocol from Du et al. ( 2024 ). After 3 days, seedlings were transferred to either normal MS medium or MS medium supplemented with NaCl (100 or 150 mM), mannitol (300 or 400 mM), or MeJA (5 or 10 µM). Mannitol was used to simulate drought stress (Zhu et al. 2023 ). After 5 days of treatment, root length and fresh weight were measured for WT and transgenic plants under each condition. Quantitative real-time PCR (qRT-PCR) analysis qRT-PCR was performed using a LightCycler® 96 real-time PCR detection system (Roche Ltd. Basel, Switzerland) with the ChamQ SYBR qPCR Master Mixture (Vazyme Biotech Co. Ltd. Nanjing, China). MiActin1 and AtActin were used as internal reference genes to normalize cDNA concentration and PCR efficiency across samples (Luo et al. 2013 ). Relative expression levels were calculated using the 2 −∆∆Ct method (Livak et al. 2001). The results are presented in arbitrary units, with the expression ratio of the target gene to the reference gene in the control group set to 1. All experiments were conducted with three biological replicates. The primers used for qRT-PCR are listed in Appendix A. . Protein interaction analysis Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted following the methods described by Liu et al. ( 2024 ) and Xue et al. ( 2025 ). Statistical analysis All experiments included biological replicates. Statistical analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA), and graphs were generated with SigmaPlot 12.0 (Systat Software, Inc. San Jose, CA, USA). Data were subjected to one-way analysis of variance (ANOVA), and differences among means were determined using Tukey’s test at a significance level of P < 0.05. Results Subcellular localization and transcriptional activity of MiICE1s Nucleic acid and amino acid sequence comparisons showed high similarity between MiICE1a and MiICE1aL , MiICE1b and MiICE1bL , and MiICE1e and MiICE1eL (Appendix B-G). Therefore, MiICE1a , MiICE1b , MiICE1c , MiICE1d , and MiICE1e were selected for further analysis. To investigate the subcellular localization of these MiICE1s , Green Fluorescent Protein (GFP) fusion proteins were expressed and observed under confocal microscopy. The free GFP protein was distributed throughout the cell, whereas the MiICE1s-GFP fusion proteins were exclusively localized to the nucleus (Fig. 1 A). Transcriptional activity was assessed by transforming Y2HGold yeast cells with pGBKT7-MiICE1a, pGBKT7-MiICE1b, pGBKT7-MiICE1c, pGBKT7-MiICE1d, and pGBKT7-MiICE1e constructs. On synthetic dropout medium lacking tryptophan (SD/-Trp), colonies containing pGBKT7-MiICE1a, pGBKT7-MiICE1b, and pGBKT7-MiICE1c appeared pink, and they also produced blue colonies on SD/–Trp/X-α-gal medium, with or without AbA. In contrast, pGBKT7-MiICE1d and pGBKT7-MiICE1e grew pink colonies on SD/-Trp and SD-Trp/X-α-gal medium, but only a small number of colonies survived with AbA. No growth was observed for the diluted cultures (Fig. 1 B). These results suggest that MiICE1a, MiICE1b, and MiICE1c exhibit transcriptional activity, while MiICE1d and MiICE1e do not. Tissue-specific expression patterns of MiICE1 s and their effects on flowering in Arabidopsis Tissue-specific expression analysis revealed that MiICE1 s were expressed in all examined tissues, although their expression levels varied considerably. MiICE1a/aL exhibited the highest expression in the leaves and stems of non-flowering branches of adult trees, moderate expression in the leaves and stems of flowering branches and in juvenile leaves, and the lowest expression in flowers and juvenile stems. In contrast, MiICE1b/bL was most highly expressed in flowering stems, with intermediate levels in leaves and the lowest levels in non-flowering leaves. Notably, MiICE1c displayed peak expression in flowers, with expression approximately 100-fold higher than in the stems of non-flowering branches. Similarly, MiICE1d was predominantly expressed in the stems and flowers of flowering branches, with expression in these tissues 124-fold higher than in the leaves of non-flowering branches. MiICE1e/eL were predominantly expressed in the leaves of non-flowering branches, followed by moderate expression in juvenile stems, with significantly lower expression in adult stems (Fig. 2 A). These results indicate that MiICE1 s display tissue-specific regulatory patterns, highlighting their potential roles in distinct physiological processes during mango development and stress responses. Given the high expression of MiICE1s in floral organs, we hypothesized that these genes contribute to flowering regulation. To test this, the flowering times of WT and T3 transgenic Arabidopsis plants were compared under different temperature conditions (8°C, 16°C, and 23°C). For each line, the duration from sowing to the opening of the first flower was recorded. Under the low-temperature condition of 8℃, compared with the wild-type (WT) and pBI121 control plants (about 75 days), the flowering of the MiICE1a and MiICE1c transgenic plants was significantly earlier, approximately 6–8 days and 6–11 days earlier, respectively. MiICE1d and MiICE1e also exhibited an early flowering phenotype, occurring 4 to 14 days and 3 to 8 days earlier respectively, while MiICE1b did not cause significant changes. At 16℃, some strains of MiICE1d and MiICE1e still promoted flowering (up to 4–5 days earlier), while the effects on MiICE1a , MiICE1b and MiICE1c were not significant. At 23℃, there was no significant difference in the flowering time among all genotypes. In summary, MiICE1a , MiICE1c , MiICE1d , and MiICE1e transgenic Arabidopsis plants exhibited early flowering phenotypes under low-temperature conditions (8°C), with MiICE1d and MiICE1e also promoting earlier flowering at 16°C. These findings suggest that MiICE1 s play a role in regulating flowering time in response to temperature, particularly under suboptimal growing conditions. This temperature-dependent regulation of flowering may provide a mechanism for plants to adapt to environmental stresses by modulating their developmental timing. MiICE1s specifically interact with flowering proteins Previous studies have demonstrated that MiICE1s play a significant role in modulating flowering time. To further elucidate the molecular mechanisms underlying this process, Y2H assays were performed to examine potential protein-protein interactions between MiICE1s and key flowering-related regulators. The screening revealed the following specific interactions: MiICE1a interacted with MiFT1b and MiFT4. MiICE1b interacted with MiFLC, MiFT1a, MiFT1b, and MiFT4; MiICE1c interacted with MiFLC, MiFT1a, MiFT1b, MiFT2, and MiFT4; MiICE1d interacted with MiFLC, MiFT1a, MiFT1b, and MiFT4; and MiICE1e interacted with MiFLC, MiFT1a, and MiFT1b (Fig. 3 A). The BiFC assay further confirmed these interactions, showing that the proteins were co-localized in the nucleus of onion epidermal cells (Fig. 3 B). Expression pattern of MiICE1 s under salt stress and enhanced salt tolerance in Arabidopsis expressing MiICE1 s To investigate the transcriptional regulation of MiICE1 family members under salt stress, their expression profiles were examined in mango leaves treated with 300 mmol L − 1 NaCl. Time-course analysis revealed distinct response patterns among different MiICE1 s. MiICE1a/aL exhibited transient down-regulation, reaching the lowest level at 6–12 h post-treatment (hpt), followed by gradual recovery and a 1.2-fold increase over the initial level (0 hpt) at 72 hpt. MiICE1b/bL were significantly suppressed at 6 hpt, but recovered to near-baseline levels at 24 hpt. In contrast, MiICE1c remained relative stable for the first 48 hpt, then showed a marked induction, peaking at 72 hpt with a 2.5-fold increase compared with 0 hpt. MiICE1d displayed a delayed but dramatic response, with expression rising 23.6-fold at 24 hpt before declining, yet remaining above baseline. MiICE1e/eL exhibited a complex expression pattern, with an initial decline at 6 hpt, a sharp peak at 12 hpt, a subsequent decrease, and gradual recovery after 24 hpt. These differential expression patterns suggest that MiICE1 family members contribute distinctively to the salt-stress response in mango, likely through diverse regulatory mechanisms and signaling pathways (Fig. 4 A). Given that MiICE1 s respond to salt stress, they are presumed to play a crucial role in enhancing plant salt tolerance. To test this, transgenic Arabidopsis lines expressing MiICE1a , MiICE1b , MiICE1c , MiICE1d , and MiICE1e were subjected to salt stress treatment. Under normal growth conditions for 5 days, no significant differences in root length or fresh weight were observed between WT and MiICE1 s transgenic Arabidopsis lines. However, when grown on 1/2 MS medium supplemented with NaCl, plant growth was significantly inhibited. After 5 days on 1/2 MS medium containing 100 mmol L − 1 NaCl, WT seedlings showed severe growth retardation, while the transgenic MiICE1s lines maintained significantly greater root length and fresh weight than WT. Moreover, with increasing NaCl concentration, MiICE1a , MiICE1b , MiICE1d , and MiICE1e transgenic lines exhibited enhanced salt tolerance compared with WT (Fig. 4 B). Expression pattern of MiICE1 s under PEG 6000 treatment and drought stress tolerance in transgenic Arabidopsis To investigate the response of MiICE1 family members to drought stress, polyethylene glycol (PEG)-induced osmotic stress was applied to simulate drought conditions, and the expression patterns of MiICE1 s were analyzed in mango leaves. The time-course analysis revealed distinct expression profiles among the MiICE1 s under PEG treatment. Specifically, MiICE1a/aL and MiICE1b/bL exhibited a transient down-regulation, reaching their lowest levels at 12 hpt, followed by gradual recovery. A similar pattern was observed for MiICE1c , which also reached its minimum expression at 12 hpt. In contrast, MiICE1d displayed a progressive increase in expression, peaking at 48 hpt with a 2.3-fold elevation compared with the control (0 hpt), after which the expression level stabilized. MiICE1e/eL exhibited maximum induction at 12 hpt, reaching 1.9-fold above baseline, followed by a gradual decline. These differential expression patterns suggest distinct regulatory mechanisms and potential functional diversification among MiICE1 family members in response to drought stress in mango (Fig. 5 A). Based on the observed responses of MiICE1 s to PEG-induced osmotic stress, we hypothesized that these genes contribute to drought stress tolerance. To test this, WT and transgenic Arabidopsis lines expressing MiICE1a , MiICE1b , MiICE1c , MiICE1d , and MiICE1e were subjected to D-mannitol treatment, and root growth and fresh weight were compared under normal and drought-simulated conditions. Under normal growth conditions, no significant differences in root length and fresh weight were observed between WT and transgenic lines. However, under D-mannitol-induced osmotic stress, transgenic lines exhibited significantly enhanced root growth and greater fresh weight compared with WT plants, suggesting that MiICE1s contribute to drought stress adaptation (Fig. 5 B). Expression pattern of MiICE1 s under MeJA treatment and MeJA tolerance in transgenic Arabidopsis To examine the response of MiICE1 family members to MeJA signaling, their expression patterns were analyzed in mango leaves following treatment with 200 µmol L − 1 MeJA. Time-course analysis revealed distinct transcriptional responses among different MiICE1 s. MiICE1a/aL exhibited a gradual increase under MeJA treatment, reaching maximum induction (1.9-fold relative to 0 hpt) at 48 hpt, followed by a slight decline at 72 hpt. MiICE1b/bL displayed an initial suppression within 12 hpt, followed by recovery. MiICE1c was significantly down-regulated, reaching its lowest expression at 12 hpt, then gradually recovered to baseline levels by 48 hpt. In contrast, MiICE1d demonstrated a biphasic response, with an initial induction within 6 hpt, a decrease to the lowest level at 12 hpt, a rapid increase to peak expression (1.8-fold of the untreated control) at 24 hpt, and a subsequent decline to 12 hpt levels by 72 hpt. MiICE1e/eL exhibited continuous up-regulation during the first 24 hpt, reaching 2.8-fold induction compared with the untreated control. These differential expression patterns suggest that MiICE1 family members may participate in distinct aspects of jasmonate signaling pathways, potentially contributing to different physiological responses in mango (Fig. 6 A). To further investigate the functional roles of MiICE1 s in jasmonate-mediated growth regulation, WT and transgenic Arabidopsis lines expressing MiICE1a , MiICE1b , MiICE1c , MiICE1d , and MiICE1e were compared for root growth and fresh plant weight under normal and MeJA treatment conditions. Under normal growth conditions, no significant differences in root length and fresh weight were observed between WT and transgenic lines. However, after treatment with 10 µmol L-1 MeJA, transgenic lines exhibited significantly enhanced root growth and greater fresh weight compared with WT plants, suggesting that MiICE1s may contribute to jasmonate-mediated stress responses (Fig. 6 B). Discussion A large number of studies have found that ICE1 improves plant tolerance to low temperature stress (Chen et al. 2012 ; Feng et al. 2013 ; Li et al. 2014 ; Ding et al. 2015 ; Zhang et al. 2018 ). ICE1 interacts with C-repeat Binding Factors ( CBFs ) to activate their expression, and the induced CBF s further activate downstream cold-regulated ( COR ) genes, forming the ICE1-CBF-COR signaling pathway that enhances plant cold tolerance (Chinnusamy et al. 2003 ; Ding et al. 2015 ; Kim et al. 2015 ; Li et al. 2017 ; Wang et al. 2022 ). ICE1 not only directly regulates CBF expression but also modulates other low-temperature-responsive genes. Overexpression of ICE1 greatly increases the expression of CBF1 , CBF2 , CBF3 , and COR genes, thereby enhancing plant cold tolerance (Tang et al. 2020 ; Zhou et al. 2020 ). Beyond cold tolerance, ICE1 contributes to resistance against other abiotic stresses. Arabidopsis overexpressing ICE1 shows enhanced drought tolerance compared with non-transgenic plants (Xu et al. 2014 ). Ectopic expression of CsICE1 , VvICE1a , and VvICE1b enhances tolerance to drought and salinity (Li et al. 2014 ; Ding et al. 2015 ). Similarly, the tomato MYC-type ICE1-like transcription factor SlICE1a confers osmotic and salt tolerance in transgenic tobacco (Feng et al. 2013 ). AtICE1 transgenic plants exhibit enhanced tolerance to NaCl and methyl viologen (MV)-induced oxidative stress. Transgenic tobacco expressing AtICE1 , BcICE1 , and OsICE1 exhibits improved resistance to NaCl- and PEG-induced stress (Nagaveni et al. 2016 ; Zhang et al. 2018 ). In banana, overexpression of the ICE1 -encoding transcription factor MpbHLH enhances resistance to Fusarium wilt (Li et al. 2022 ). In contrast to extensive studies on ICE1 in stress resistance, its role in flowering has been less explored. Loss of ICE1 function in Arabidopsis results in dehydration and decreased pollen viability and germination rate (Wei et al. 2018 ). The ICE-CBF pathway is also implicated in flowering regulation. Under short-term low-temperature conditions, ICE1 binds to the promoter of FLOWERING LOCUS C ( FLC ), inducing its expression. FLC is a key floral repressor in Arabidopsis , while FLOWERING LOCUS T (FT) acts as a flowering inducer (Michaels et al. 2001; Sheldon et al. 2000 ; Corbesier et al. 2007 ; Jaeger et al. 2007; Mathieu et al. 2007 ). FLC delays flowering by inhibiting FT activity (Helliwell et al. 2006 ). Thus, ICE1 can induce FLC expression, repress FT and SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 ( SOC1 ), eventually delaying flowering in Arabidopsis (Lee et al. 2015 ). In this study, we investigated the functions and mechanisms of mango ICE1s in flowering regulation and stress resistance. MiICE1 s were expressed at all developmental stages of mango. The expression of MiICE1a/aL showed little variation among different tissues at the same stage, but its level was highest in non-flowering branches of adult trees. MiICE1b/bL exhibited the highest expression in stems of flowering branches and the lowest in leaves of non-flowering branches. MiICE1c was most strongly expressed in the flowers of flowering branches, whereas MiICE1d showed the highest expression in flowering stems and flowers of adult trees. MiICE1e/eL was most abundantly expressed in the leaves of non-flowering branches. The relatively high expression of MiICE1 s in flowering branches suggests their involvement in the regulation of flowering in mango. Overexpression of MiICE1 s in Arabidopsis did not alter flowering time under optimal growth temperatures. However, at low temperatures, some transgenic lines overexpressing MiICE1 s flowered earlier than WT, indicating that MiICE1 s may participate in the signaling pathways regulating flowering. As ambient temperature affects flowering in Arabidopsis , with lower temperatures progressively delaying flowering, ICE1 appears to regulate flowering time by modulating the expression of flowering-related genes in response to temperature cues. Previous studies reported that the expression of FLC decreased in the ice1 mutant of Arabidopsis under long-day conditions, while the up-regulation of FT and SOC1 promoted early flowering. This suggests that ICE1 , FT , SOC1 , and FLC jointly participate in the regulation of flowering (Lee et al. 2015 ). Ambient temperature also regulates FT expression and protein transport, prevents premature flowering at low temperatures, and ensures reproductive success under favorable environmental conditions. Appropriate temperatures promote FT expression, which regulates flowering responses to both photoperiod and ambient temperature (Liu et al. 2020 ). Conversely, low temperatures may prolong flowering time by elevating FLC transcription levels (Susila et al. 2018 ). In order to further investigate the underlying mechanism, we screened the interacting proteins of MiICE1s and found that they interacted with MiFLC and MiFTs , but not with MiFT3 . In addition, MiICE1a did not interact with MiFLC , MiFT1a , and MiFT2 ; MiICE1e did not interact with MiFT2 and MiFT4 ; and MiFT2 interacted exclusively with MiICE1c . As MiFTs are known to promote early flowering in plants (Li et al. 2024 ), these interactions suggest that MiICE1s may regulate flowering time by directly or indirectly modulating the activity of central flowering-related proteins, such as MiFLC and MiFTs . The differential interaction patterns observed among MiICE1s highlight the complexity of the molecular mechanisms controlling flowering time, particularly in response to environmental and developmental signals. The expression of MiICE1 s enhanced the resistance of Arabidopsis to salt and drought stresses, as well as to MeJA treatment. This finding suggests that MiICE1 s may strengthen the plant's ability to adapt to adverse environments by regulating stress response mechanisms. Our previous research showed that MiFT1a and MiFT1b transgenic lines did not respond to abiotic stress, while MiFT2 , MiFT3 , and MiFT4 enhanced resistance to salt or drought stress in Arabidopsis (Li et al. 2024 ). The interactions between MiICE1s and MiFTs may therefore contribute to the enhanced stress tolerance conferred by MiICE1s . In conclusion, MiICE1s were found to be localized in the nucleus. Their expression in Arabidopsis promoted flowering and improved resistance to stress. These results indicate that MiICE1 s are functional genes with potential application value in mango, which provides new ideas and methods for improving crop varieties by genetic engineering. Declarations Competing interests The authors declare that they have no conflict of interest. Funding This work was supported by Guangxi Natural Science Foundation (2023GXNSFAA026268), Guangxi Key Technologies R&D Program (Guikenong AB241484007), National Natural Science Foundation of China (31860541), the CARSGIT-Guangxi Mango Industry Project (nycytxgxcxtd-2021-06-02), start-up funding for introduced talents in Guangxi University (ZX01080033124004, EE101762) and Guangxi Higher Education Institutions Young and Middle-aged Teachers' Research Fundamental Capability Enhancement Project (2025KY0026). Author Contributions Xinhua He and Cong Luo: experimental design and implementation, composition and review of manuscript, financial support for experiments and laboratory apparatus; Fangfang Xie, Canbin Chen, Xukang Huang and Xijin Zhou: composition and review of manuscript; Yanzhu Liu, Zhiqi Lai and Tingting Liang: experimental implementation; Tianli Guo and Yili Zhang: experimental design and implementation, manuscript composition. 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J Fruit Sci 34:594–602 Xu FH, Liu ZX, Xie HY, Zhu J, Zhang J, Kraus J, Blaschnig T, Nehls R, Wang H (2014) Increased Drought Tolerance through the Suppression of ESKMO1 Gene and Overexpression of CBF-Related Genes in Arabidopsis. PLoS ONE 9:106509 Xue XS, Li L, Wang DH, Zhou W, Wang ZZ, Cao XY (2025) SmJAZ1/8 inhibits the stimulation of SmbHLH59, which limits the accumulation of salvianolic acids and tanshinones in Salvia miltiorrhiza. Int J Biol Macromol 285:138348 Zhang T, Mo J, Zhou K, Chang Y, Liu Z (2018) Overexpression of Brassica campestris BcICE1 gene increases abiotic stress tolerance in tobacco. Plant Physiol Biochem 132:515–523 Zhou L, He YJ, Li J, Li LZ, Liu Y, Chen HY (2020) An eggplant SmICEla gene encoding MYC-type ICEl-like transcription factor enhances freezing tolerance in transgenic Arabidopsis thaliana. Plant Biol 22:450–458 Zhu XL, Wang BQ, Liu WY, Wei XH, Wang X, Du XF, Liu HX (2023) Genome-wide analysis of AP2/ERF gene and functional analysis of CqERF24 gene in drought stress in quinoa. Int J Biol Macromol 253:127582 Supplementary Files AppendixFigure.docx AppendixTable.docx Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2026 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviewers agreed at journal 04 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 02 Nov, 2025 First submitted to journal 30 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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12:46:27","extension":"html","order_by":50,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":150143,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/16c0b2ffd4c7d06c7bec1335.html"},{"id":95832845,"identity":"97ced1ce-0c6d-4743-bb33-b8afd7485500","added_by":"auto","created_at":"2025-11-13 12:46:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":222233,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization and transcriptional activity of MiICE1s. \u003cstrong\u003e(A)\u003c/strong\u003e Subcelluar location of MiICE1 protein in \u003cem\u003eAllium cepa\u003c/em\u003e L.. The fluorescent green signal in the dark field or merged field shows the localization of GFP or MiICE1s-GFP fusion protein. Bars, 100 µm. \u003cstrong\u003e(B)\u003c/strong\u003e An auto-activation assay was conducted for the constructs pGBKT7-MiICE1s and pGADT7-MiICE1s. The results were observed using microscopy.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/8e405807c387a14ec5536806.jpg"},{"id":96239466,"identity":"4b6d1d37-f3db-4b98-8803-06db5baa919b","added_by":"auto","created_at":"2025-11-19 07:06:44","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":478857,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specific expression patterns of \u003cem\u003eMiICE1\u003c/em\u003es and their effects on flowering in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The tissue expression pattern analysis of \u003cem\u003eMiICE1\u003c/em\u003es genes. Different letters indicate significant differences of \u003cem\u003eMiICE1\u003c/em\u003es expression level according to one-way ANOVA Tukey's multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e The plants phenotypes of overexpressing \u003cem\u003eMiICE1\u003c/em\u003es under different temperatures. Representative phenotypes of first flower bud appearing in control and\u003cem\u003eMiICE1\u003c/em\u003es transgenic lines; Time from seed to first flower bud. Different letters indicate significant differences between WT and transgenic plants on the same day of different treatments, according to one-way ANOVA Tukey’s multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Data are means of three replicates with SD.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/e0b19de30503d0cb51100136.jpg"},{"id":95832831,"identity":"e801c5fb-8f66-43be-8f64-fc843bd252a6","added_by":"auto","created_at":"2025-11-13 12:46:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":382368,"visible":true,"origin":"","legend":"\u003cp\u003eMiICE1s specifically interact with flowering proteins. \u003cstrong\u003e(A)\u003c/strong\u003e Interaction analysis of MiICE1s protein with others protein. DDO: SDO/-Leu/-Trp medium. Q/X/A: SDO/-Ade/-His/-Leu/-Trp/X-a-gal/AbA media. OD600 = 0.2. \u003cstrong\u003e(B)\u003c/strong\u003e The interaction of MiICE1s by BiFC. Bars, 250 μm.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/0560c7e99866ab84c96ff5db.jpg"},{"id":96239715,"identity":"f792e812-be2f-43fc-9aee-a71d35017e1b","added_by":"auto","created_at":"2025-11-19 07:07:25","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":400800,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under salt stress and enhanced salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003eexpressing MiICE1s. \u003cstrong\u003e(A)\u003c/strong\u003e Expression analysis of \u003cem\u003eMiICE1\u003c/em\u003es gene in mango under salt treatment. Different letters indicate significant differences of \u003cem\u003eMiICE1\u003c/em\u003es expression level on the different day of treatments, according to one-way ANOVA Tukey's multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e Effects of salt treatment on root length and fresh weight of \u003cem\u003eMiICE1\u003c/em\u003es transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. Different letters indicate significant differences between WT and transgenic plants on the same day of different treatments, according to one-way ANOVA Tukey’s multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Data are means of three replicates with SD.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/fbdc01ce1254108e6f827a69.jpg"},{"id":95832829,"identity":"99e7c4de-3e03-4ff4-b69a-1f254316497c","added_by":"auto","created_at":"2025-11-13 12:46:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":396561,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under PEG 6000 treatment and drought stress tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Expression analysis of \u003cem\u003eMiICE1\u003c/em\u003es gene in mango under drought treatment. Different letters indicate significant differences of \u003cem\u003eMiICE1\u003c/em\u003es expression level on the different day of treatments, according to one-way ANOVA Tukey's multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e Effects of drought treatment on root length of \u003cem\u003eMiICE1\u003c/em\u003es transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. Different letters indicate significant differences between WT and transgenic plants on the same day of different treatments, according to one-way ANOVA Tukey’s multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Data are means of three replicates with SD.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/e6669a24d5b8fc82187fb051.jpg"},{"id":95832837,"identity":"2b7fea31-3a0d-4f9e-8eac-e0a237f5a12a","added_by":"auto","created_at":"2025-11-13 12:46:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":413544,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under MeJA treatment and MeJA tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Expression analysis of \u003cem\u003eMiICE1\u003c/em\u003es gene in mango under MeJA treatment. Different letters indicate significant differences of \u003cem\u003eMiICE1\u003c/em\u003es expression level on the different day of treatments, according to one-way ANOVA Tukey's multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e Effects of MeJA treatment on root length of \u003cem\u003eMiICE1\u003c/em\u003es transgenic \u003cem\u003eArabidopsis\u003c/em\u003e. Different letters indicate significant differences between WT and transgenic plants on the same day of different treatments, according to one-way ANOVA Tukey’s multiple range tests (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Data are means of three replicates with SD.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/c58720dc2dec8accda3e73ff.jpg"},{"id":104251521,"identity":"c73a4e48-8797-464f-9765-2e015724f4ba","added_by":"auto","created_at":"2026-03-09 16:13:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3272728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/eac8a001-cd44-48e7-8315-757f68e387bf.pdf"},{"id":96239567,"identity":"7dc57183-4ede-40ff-a36e-7d347d58efe1","added_by":"auto","created_at":"2025-11-19 07:06:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":214498,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/a8757f68a01fb4bfcf24a3c9.docx"},{"id":95832828,"identity":"54982a6c-7558-4444-b678-ba647ab8c655","added_by":"auto","created_at":"2025-11-13 12:46:26","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14309,"visible":true,"origin":"","legend":"","description":"","filename":"AppendixTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-7980258/v1/8bd5dced2ee454c029af2e84.docx"}],"financialInterests":"","formattedTitle":"Mangifera indica (mango) ICE1s confer flowering and tolerance to multiple stresses in Arabidopsis thaliana","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDuring growth and development, plants frequently encounter various environmental stresses, including salt, drought, and biotic stresses caused by pests and diseases (Jung et al. 2014; Khatamidoost et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kazemi-Shahandashti et al. 2018; Gong et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hajabdollahi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Hossain et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Long et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These stresses can severely impair plant growth, development, and yield (Gong et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saberi Riseh et al. 2024). Therefore, improving plant tolerance to environmental stress has become a central focus in plant research. One effective strategy is genetic engineering, which improves stress tolerance by identifying and manipulating resistance-related genes.\u003c/p\u003e\u003cp\u003eMango (\u003cem\u003eMangifera indica\u003c/em\u003e L.) is an important tropical fruit crop with a wide cultivation range and important economic value. It ranks as the fifth most important fruit crop worldwide (Sandip et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite the rapid development of the mango industry in China, both cultivation area and production have steadily increased (Chen et al. 2013; Kong et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), yet mango production continues to face numerous challenges. In particular, abiotic stresses such as high salt, low temperature, and drought, as well as biotic stresses from pathogens, remain major constraints on mango cultivation (Lao et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Abdel-Aziz et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Therefore, identifying stress-resistance genes and investigating their functional roles provides a theoretical foundation for enhancing mango stress tolerance and holds significant implications for the development of the mango industry in China.\u003c/p\u003e\u003cp\u003eAs a member of the basic Helix-Loop-Helix (bHLH) family, the Inducer of CBF Expression 1 (ICE1) contains the conserved bHLH domain characteristic of the MYC transcription factor family. ICE1 is known to be evolutionarily conserved and plays a key role in regulating plant tolerance to low-temperature stress (Tang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition to its role in cold stress resistance, ICE1 is also involved in flowering and responses to other abiotic stresses. Previous studies have shown that overexpression of \u003cem\u003eICE1\u003c/em\u003e and its homologs confers tolerance to multiple stresses in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eArabidopsis\u003c/em\u003e) (Xu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), chrysanthemum (Chen et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), banana (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and tobacco (Feng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nagaveni et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, the ICE-CBF pathway has been implicated in the regulation of flowering in \u003cem\u003eArabidopsis\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnlike their roles in other plants, the functions of ICE1s in mango remain poorly understood. In this study, we investigated the effects of \u003cem\u003eMiICE1\u003c/em\u003es on flowering and stress tolerance. By generating transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants and conducting flowering induction and stress treatment experiments, we elucidated the functional mechanisms of \u003cem\u003eMiICE1\u003c/em\u003es in regulating flowering and mediating stress responses. Our results indicate that \u003cem\u003eMiICE1\u003c/em\u003es are functional genes with potential applications in mango, which provides a new strategy for improving crop traits through genetic engineering.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and treatments\u003c/h2\u003e\u003cp\u003eThe plant materials used in this study included mango 'Guiqimang', cultivated in the specimen garden of the College of Agriculture, Guangxi University, and Columbia wild-type (WT) \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eFor gene expression pattern analysis, two types of samples were collected: (1) Different tissue samples: The leaves and stems were collected from the three-year-old 'Guiqimang' trees and the non-flowering and flowering branches were obtained from the six-year-old trees; (2) Stress treatment samples: The grafted seedlings of 'Guiqimang' were subjected to the following treatments: 300 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl irrigation, 30% PEG 6000 irrigation to simulate drought stress, and foliar spray with 200 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e methyl jasmonate (MeJA). Samples were collected at 0, 6, 12, 24, 48, and 72 h after treatment. All samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSubcellular localization analysis\u003c/h3\u003e\n\u003cp\u003eThe full-length cDNA sequences of \u003cem\u003eMiICE1\u003c/em\u003es were cloned into the pBI221-EGFP vector under the control of the Cauliflower mosaic virus 35S (CAMV35S) promoter. \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101) carrying the fusion constructs was then introduced into onion (\u003cem\u003eAllium cepa\u003c/em\u003e) inner epidermal cells, and fluorescence was observed using a laser confocal microscope (TCS-SP8MP, Leica). The empty vector served as a control, and nuclei were stained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI).\u003c/p\u003e\n\u003ch3\u003eTranscriptional activation analysis\u003c/h3\u003e\n\u003cp\u003eTranscriptional activation assays were performed to identify the presence of activation domains in MiICE1 proteins. The coding sequences of \u003cem\u003eMiICE1\u003c/em\u003es were fused to the Gal4 DNA-binding domain in the pGBKT7 vector to generate pGBKT7-MiICE1s constructs. AH109 yeast cells transformed with these recombinant vectors were evaluated for transcriptional activation activity, following previously described methods (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Xue et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eProduction of transgenic Arabidopsis plants\u003c/h3\u003e\n\u003cp\u003eThe pBI121-MiICE1s fusion expression plasmids were introduced into \u003cem\u003eAgrobacterium rhizogenes\u003c/em\u003e EHA105. The resulting \u003cem\u003eAgrobacterium\u003c/em\u003e cells were used to transform \u003cem\u003eArabidopsis\u003c/em\u003e via the floral-dip method (Clough et al. 1998; Wang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Positive seedlings were selected on 1/2 MS medium containing 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e kanamycin, confirmed by PCR, and advanced to the T3 generation for subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eFlowering phenotype analyses\u003c/h3\u003e\n\u003cp\u003eWT and empty vector-transformed \u003cem\u003eArabidopsis\u003c/em\u003e plants were used as controls. The time from sowing to the opening of the first flower was recorded, and flowering phenotypes were documented.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStress treatments\u003c/h2\u003e\u003cp\u003eTo evaluate the responses of WT and homozygous T3 transgenic \u003cem\u003eArabidopsis\u003c/em\u003e to environmental stress, seeds were surface-sterilized and germinated on standard MS medium. Stress treatments were performed following a modified protocol from Du et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). After 3 days, seedlings were transferred to either normal MS medium or MS medium supplemented with NaCl (100 or 150 mM), mannitol (300 or 400 mM), or MeJA (5 or 10 \u0026micro;M). Mannitol was used to simulate drought stress (Zhu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). After 5 days of treatment, root length and fresh weight were measured for WT and transgenic plants under each condition.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative real-time PCR (qRT-PCR) analysis\u003c/h3\u003e\n\u003cp\u003eqRT-PCR was performed using a LightCycler\u0026reg; 96 real-time PCR detection system (Roche Ltd. Basel, Switzerland) with the ChamQ SYBR qPCR Master Mixture (Vazyme Biotech Co. Ltd. Nanjing, China). \u003cem\u003eMiActin1\u003c/em\u003e and \u003cem\u003eAtActin\u003c/em\u003e were used as internal reference genes to normalize cDNA concentration and PCR efficiency across samples (Luo et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method (Livak et al. 2001). The results are presented in arbitrary units, with the expression ratio of the target gene to the reference gene in the control group set to 1. All experiments were conducted with three biological replicates. The primers used for qRT-PCR are listed in Appendix A.\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\n\u003ch3\u003eProtein interaction analysis\u003c/h3\u003e\n\u003cp\u003eYeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays were conducted following the methods described by Liu et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and Xue et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments included biological replicates. Statistical analyses were performed using SPSS 21.0 (IBM, Armonk, NY, USA), and graphs were generated with SigmaPlot 12.0 (Systat Software, Inc. San Jose, CA, USA). Data were subjected to one-way analysis of variance (ANOVA), and differences among means were determined using Tukey\u0026rsquo;s test at a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSubcellular localization and transcriptional activity of MiICE1s\u003c/h2\u003e\u003cp\u003eNucleic acid and amino acid sequence comparisons showed high similarity between \u003cem\u003eMiICE1a\u003c/em\u003e and \u003cem\u003eMiICE1aL\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e and \u003cem\u003eMiICE1bL\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e and \u003cem\u003eMiICE1eL\u003c/em\u003e (Appendix B-G). Therefore, \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e, \u003cem\u003eMiICE1c\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e were selected for further analysis.\u003c/p\u003e\u003cp\u003eTo investigate the subcellular localization of these \u003cem\u003eMiICE1s\u003c/em\u003e, Green Fluorescent Protein (GFP) fusion proteins were expressed and observed under confocal microscopy. The free GFP protein was distributed throughout the cell, whereas the MiICE1s-GFP fusion proteins were exclusively localized to the nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTranscriptional activity was assessed by transforming Y2HGold yeast cells with pGBKT7-MiICE1a, pGBKT7-MiICE1b, pGBKT7-MiICE1c, pGBKT7-MiICE1d, and pGBKT7-MiICE1e constructs. On synthetic dropout medium lacking tryptophan (SD/-Trp), colonies containing pGBKT7-MiICE1a, pGBKT7-MiICE1b, and pGBKT7-MiICE1c appeared pink, and they also produced blue colonies on SD/\u0026ndash;Trp/X-α-gal medium, with or without AbA. In contrast, pGBKT7-MiICE1d and pGBKT7-MiICE1e grew pink colonies on SD/-Trp and SD-Trp/X-α-gal medium, but only a small number of colonies survived with AbA. No growth was observed for the diluted cultures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results suggest that MiICE1a, MiICE1b, and MiICE1c exhibit transcriptional activity, while MiICE1d and MiICE1e do not.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTissue-specific expression patterns of \u003cem\u003eMiICE1\u003c/em\u003es and their effects on flowering in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTissue-specific expression analysis revealed that \u003cem\u003eMiICE1\u003c/em\u003es were expressed in all examined tissues, although their expression levels varied considerably. \u003cem\u003eMiICE1a/aL\u003c/em\u003e exhibited the highest expression in the leaves and stems of non-flowering branches of adult trees, moderate expression in the leaves and stems of flowering branches and in juvenile leaves, and the lowest expression in flowers and juvenile stems. In contrast, \u003cem\u003eMiICE1b/bL\u003c/em\u003e was most highly expressed in flowering stems, with intermediate levels in leaves and the lowest levels in non-flowering leaves. Notably, \u003cem\u003eMiICE1c\u003c/em\u003e displayed peak expression in flowers, with expression approximately 100-fold higher than in the stems of non-flowering branches. Similarly, \u003cem\u003eMiICE1d\u003c/em\u003e was predominantly expressed in the stems and flowers of flowering branches, with expression in these tissues 124-fold higher than in the leaves of non-flowering branches. \u003cem\u003eMiICE1e/eL\u003c/em\u003e were predominantly expressed in the leaves of non-flowering branches, followed by moderate expression in juvenile stems, with significantly lower expression in adult stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results indicate that \u003cem\u003eMiICE1\u003c/em\u003es display tissue-specific regulatory patterns, highlighting their potential roles in distinct physiological processes during mango development and stress responses.\u003c/p\u003e\u003cp\u003eGiven the high expression of \u003cem\u003eMiICE1s\u003c/em\u003e in floral organs, we hypothesized that these genes contribute to flowering regulation. To test this, the flowering times of WT and T3 transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants were compared under different temperature conditions (8\u0026deg;C, 16\u0026deg;C, and 23\u0026deg;C). For each line, the duration from sowing to the opening of the first flower was recorded.\u003c/p\u003e\u003cp\u003eUnder the low-temperature condition of 8℃, compared with the wild-type (WT) and pBI121 control plants (about 75 days), the flowering of the \u003cem\u003eMiICE1a\u003c/em\u003e and \u003cem\u003eMiICE1c\u003c/em\u003e transgenic plants was significantly earlier, approximately 6\u0026ndash;8 days and 6\u0026ndash;11 days earlier, respectively. \u003cem\u003eMiICE1d\u003c/em\u003e and \u003cem\u003eMiICE1e\u003c/em\u003e also exhibited an early flowering phenotype, occurring 4 to 14 days and 3 to 8 days earlier respectively, while \u003cem\u003eMiICE1b\u003c/em\u003e did not cause significant changes. At 16℃, some strains of \u003cem\u003eMiICE1d\u003c/em\u003e and \u003cem\u003eMiICE1e\u003c/em\u003e still promoted flowering (up to 4\u0026ndash;5 days earlier), while the effects on \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e and \u003cem\u003eMiICE1c\u003c/em\u003e were not significant. At 23℃, there was no significant difference in the flowering time among all genotypes.\u003c/p\u003e\u003cp\u003eIn summary, \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1c\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants exhibited early flowering phenotypes under low-temperature conditions (8\u0026deg;C), with \u003cem\u003eMiICE1d\u003c/em\u003e and \u003cem\u003eMiICE1e\u003c/em\u003e also promoting earlier flowering at 16\u0026deg;C. These findings suggest that \u003cem\u003eMiICE1\u003c/em\u003es play a role in regulating flowering time in response to temperature, particularly under suboptimal growing conditions. This temperature-dependent regulation of flowering may provide a mechanism for plants to adapt to environmental stresses by modulating their developmental timing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMiICE1s specifically interact with flowering proteins\u003c/h2\u003e\u003cp\u003ePrevious studies have demonstrated that MiICE1s play a significant role in modulating flowering time. To further elucidate the molecular mechanisms underlying this process, Y2H assays were performed to examine potential protein-protein interactions between MiICE1s and key flowering-related regulators. The screening revealed the following specific interactions: MiICE1a interacted with MiFT1b and MiFT4. MiICE1b interacted with MiFLC, MiFT1a, MiFT1b, and MiFT4; MiICE1c interacted with MiFLC, MiFT1a, MiFT1b, MiFT2, and MiFT4; MiICE1d interacted with MiFLC, MiFT1a, MiFT1b, and MiFT4; and MiICE1e interacted with MiFLC, MiFT1a, and MiFT1b (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The BiFC assay further confirmed these interactions, showing that the proteins were co-localized in the nucleus of onion epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under salt stress and enhanced salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e expressing \u003cem\u003eMiICE1\u003c/em\u003es\u003c/h2\u003e\u003cp\u003eTo investigate the transcriptional regulation of \u003cem\u003eMiICE1\u003c/em\u003e family members under salt stress, their expression profiles were examined in mango leaves treated with 300 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl. Time-course analysis revealed distinct response patterns among different \u003cem\u003eMiICE1\u003c/em\u003es. \u003cem\u003eMiICE1a/aL\u003c/em\u003e exhibited transient down-regulation, reaching the lowest level at 6\u0026ndash;12 h post-treatment (hpt), followed by gradual recovery and a 1.2-fold increase over the initial level (0 hpt) at 72 hpt. \u003cem\u003eMiICE1b/bL\u003c/em\u003e were significantly suppressed at 6 hpt, but recovered to near-baseline levels at 24 hpt. In contrast, \u003cem\u003eMiICE1c\u003c/em\u003e remained relative stable for the first 48 hpt, then showed a marked induction, peaking at 72 hpt with a 2.5-fold increase compared with 0 hpt. \u003cem\u003eMiICE1d\u003c/em\u003e displayed a delayed but dramatic response, with expression rising 23.6-fold at 24 hpt before declining, yet remaining above baseline. \u003cem\u003eMiICE1e/eL\u003c/em\u003e exhibited a complex expression pattern, with an initial decline at 6 hpt, a sharp peak at 12 hpt, a subsequent decrease, and gradual recovery after 24 hpt. These differential expression patterns suggest that \u003cem\u003eMiICE1\u003c/em\u003e family members contribute distinctively to the salt-stress response in mango, likely through diverse regulatory mechanisms and signaling pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eGiven that \u003cem\u003eMiICE1\u003c/em\u003es respond to salt stress, they are presumed to play a crucial role in enhancing plant salt tolerance. To test this, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines expressing \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e, \u003cem\u003eMiICE1c\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e were subjected to salt stress treatment. Under normal growth conditions for 5 days, no significant differences in root length or fresh weight were observed between WT and \u003cem\u003eMiICE1\u003c/em\u003es transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines. However, when grown on 1/2 MS medium supplemented with NaCl, plant growth was significantly inhibited. After 5 days on 1/2 MS medium containing 100 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e NaCl, WT seedlings showed severe growth retardation, while the transgenic MiICE1s lines maintained significantly greater root length and fresh weight than WT. Moreover, with increasing NaCl concentration, \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e transgenic lines exhibited enhanced salt tolerance compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under PEG 6000 treatment and drought stress tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the response of \u003cem\u003eMiICE1\u003c/em\u003e family members to drought stress, polyethylene glycol (PEG)-induced osmotic stress was applied to simulate drought conditions, and the expression patterns of \u003cem\u003eMiICE1\u003c/em\u003es were analyzed in mango leaves. The time-course analysis revealed distinct expression profiles among the \u003cem\u003eMiICE1\u003c/em\u003es under PEG treatment. Specifically, \u003cem\u003eMiICE1a/aL\u003c/em\u003e and \u003cem\u003eMiICE1b/bL\u003c/em\u003e exhibited a transient down-regulation, reaching their lowest levels at 12 hpt, followed by gradual recovery. A similar pattern was observed for \u003cem\u003eMiICE1c\u003c/em\u003e, which also reached its minimum expression at 12 hpt. In contrast, \u003cem\u003eMiICE1d\u003c/em\u003e displayed a progressive increase in expression, peaking at 48 hpt with a 2.3-fold elevation compared with the control (0 hpt), after which the expression level stabilized. \u003cem\u003eMiICE1e/eL\u003c/em\u003e exhibited maximum induction at 12 hpt, reaching 1.9-fold above baseline, followed by a gradual decline. These differential expression patterns suggest distinct regulatory mechanisms and potential functional diversification among \u003cem\u003eMiICE1\u003c/em\u003e family members in response to drought stress in mango (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eBased on the observed responses of \u003cem\u003eMiICE1\u003c/em\u003es to PEG-induced osmotic stress, we hypothesized that these genes contribute to drought stress tolerance. To test this, WT and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines expressing \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e, \u003cem\u003eMiICE1c\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e were subjected to D-mannitol treatment, and root growth and fresh weight were compared under normal and drought-simulated conditions. Under normal growth conditions, no significant differences in root length and fresh weight were observed between WT and transgenic lines. However, under D-mannitol-induced osmotic stress, transgenic lines exhibited significantly enhanced root growth and greater fresh weight compared with WT plants, suggesting that \u003cem\u003eMiICE1s\u003c/em\u003e contribute to drought stress adaptation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eExpression pattern of \u003cem\u003eMiICE1\u003c/em\u003es under MeJA treatment and MeJA tolerance in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo examine the response of \u003cem\u003eMiICE1\u003c/em\u003e family members to MeJA signaling, their expression patterns were analyzed in mango leaves following treatment with 200 \u0026micro;mol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e MeJA. Time-course analysis revealed distinct transcriptional responses among different \u003cem\u003eMiICE1\u003c/em\u003es. \u003cem\u003eMiICE1a/aL\u003c/em\u003e exhibited a gradual increase under MeJA treatment, reaching maximum induction (1.9-fold relative to 0 hpt) at 48 hpt, followed by a slight decline at 72 hpt. \u003cem\u003eMiICE1b/bL\u003c/em\u003e displayed an initial suppression within 12 hpt, followed by recovery. \u003cem\u003eMiICE1c\u003c/em\u003e was significantly down-regulated, reaching its lowest expression at 12 hpt, then gradually recovered to baseline levels by 48 hpt. In contrast, \u003cem\u003eMiICE1d\u003c/em\u003e demonstrated a biphasic response, with an initial induction within 6 hpt, a decrease to the lowest level at 12 hpt, a rapid increase to peak expression (1.8-fold of the untreated control) at 24 hpt, and a subsequent decline to 12 hpt levels by 72 hpt. \u003cem\u003eMiICE1e/eL\u003c/em\u003e exhibited continuous up-regulation during the first 24 hpt, reaching 2.8-fold induction compared with the untreated control. These differential expression patterns suggest that \u003cem\u003eMiICE1\u003c/em\u003e family members may participate in distinct aspects of jasmonate signaling pathways, potentially contributing to different physiological responses in mango (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eTo further investigate the functional roles of \u003cem\u003eMiICE1\u003c/em\u003es in jasmonate-mediated growth regulation, WT and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines expressing \u003cem\u003eMiICE1a\u003c/em\u003e, \u003cem\u003eMiICE1b\u003c/em\u003e, \u003cem\u003eMiICE1c\u003c/em\u003e, \u003cem\u003eMiICE1d\u003c/em\u003e, and \u003cem\u003eMiICE1e\u003c/em\u003e were compared for root growth and fresh plant weight under normal and MeJA treatment conditions. Under normal growth conditions, no significant differences in root length and fresh weight were observed between WT and transgenic lines. However, after treatment with 10 \u0026micro;mol L-1 MeJA, transgenic lines exhibited significantly enhanced root growth and greater fresh weight compared with WT plants, suggesting that \u003cem\u003eMiICE1s\u003c/em\u003e may contribute to jasmonate-mediated stress responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA large number of studies have found that ICE1 improves plant tolerance to low temperature stress (Chen et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Feng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). ICE1 interacts with C-repeat Binding Factors (\u003cem\u003eCBFs\u003c/em\u003e) to activate their expression, and the induced \u003cem\u003eCBF\u003c/em\u003es further activate downstream cold-regulated (\u003cem\u003eCOR\u003c/em\u003e) genes, forming the ICE1-CBF-COR signaling pathway that enhances plant cold tolerance (Chinnusamy et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ICE1 not only directly regulates \u003cem\u003eCBF\u003c/em\u003e expression but also modulates other low-temperature-responsive genes. Overexpression of \u003cem\u003eICE1\u003c/em\u003e greatly increases the expression of \u003cem\u003eCBF1\u003c/em\u003e, \u003cem\u003eCBF2\u003c/em\u003e, \u003cem\u003eCBF3\u003c/em\u003e, and \u003cem\u003eCOR\u003c/em\u003e genes, thereby enhancing plant cold tolerance (Tang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Beyond cold tolerance, \u003cem\u003eICE1\u003c/em\u003e contributes to resistance against other abiotic stresses. \u003cem\u003eArabidopsis\u003c/em\u003e overexpressing \u003cem\u003eICE1\u003c/em\u003e shows enhanced drought tolerance compared with non-transgenic plants (Xu et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Ectopic expression of \u003cem\u003eCsICE1\u003c/em\u003e, \u003cem\u003eVvICE1a\u003c/em\u003e, and \u003cem\u003eVvICE1b\u003c/em\u003e enhances tolerance to drought and salinity (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Ding et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, the tomato MYC-type ICE1-like transcription factor \u003cem\u003eSlICE1a\u003c/em\u003e confers osmotic and salt tolerance in transgenic tobacco (Feng et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). \u003cem\u003eAtICE1\u003c/em\u003e transgenic plants exhibit enhanced tolerance to NaCl and methyl viologen (MV)-induced oxidative stress. Transgenic tobacco expressing \u003cem\u003eAtICE1\u003c/em\u003e, \u003cem\u003eBcICE1\u003c/em\u003e, and \u003cem\u003eOsICE1\u003c/em\u003e exhibits improved resistance to NaCl- and PEG-induced stress (Nagaveni et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In banana, overexpression of the \u003cem\u003eICE1\u003c/em\u003e-encoding transcription factor \u003cem\u003eMpbHLH\u003c/em\u003e enhances resistance to Fusarium wilt (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast to extensive studies on \u003cem\u003eICE1\u003c/em\u003e in stress resistance, its role in flowering has been less explored. Loss of \u003cem\u003eICE1\u003c/em\u003e function in \u003cem\u003eArabidopsis\u003c/em\u003e results in dehydration and decreased pollen viability and germination rate (Wei et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The ICE-CBF pathway is also implicated in flowering regulation. Under short-term low-temperature conditions, \u003cem\u003eICE1\u003c/em\u003e binds to the promoter of \u003cem\u003eFLOWERING LOCUS C\u003c/em\u003e (\u003cem\u003eFLC\u003c/em\u003e), inducing its expression. \u003cem\u003eFLC\u003c/em\u003e is a key floral repressor in \u003cem\u003eArabidopsis\u003c/em\u003e, while \u003cem\u003eFLOWERING LOCUS T (FT)\u003c/em\u003e acts as a flowering inducer (Michaels et al. 2001; Sheldon et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Corbesier et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jaeger et al. 2007; Mathieu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003eFLC\u003c/em\u003e delays flowering by inhibiting \u003cem\u003eFT\u003c/em\u003e activity (Helliwell et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Thus, ICE1 can induce \u003cem\u003eFLC\u003c/em\u003e expression, repress \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eSUPPRESSOR OF CONSTANS OVEREXPRESSION 1\u003c/em\u003e (\u003cem\u003eSOC1\u003c/em\u003e), eventually delaying flowering in \u003cem\u003eArabidopsis\u003c/em\u003e (Lee et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In this study, we investigated the functions and mechanisms of mango ICE1s in flowering regulation and stress resistance.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMiICE1\u003c/em\u003es were expressed at all developmental stages of mango. The expression of \u003cem\u003eMiICE1a/aL\u003c/em\u003e showed little variation among different tissues at the same stage, but its level was highest in non-flowering branches of adult trees. \u003cem\u003eMiICE1b/bL\u003c/em\u003e exhibited the highest expression in stems of flowering branches and the lowest in leaves of non-flowering branches. \u003cem\u003eMiICE1c\u003c/em\u003e was most strongly expressed in the flowers of flowering branches, whereas \u003cem\u003eMiICE1d\u003c/em\u003e showed the highest expression in flowering stems and flowers of adult trees. \u003cem\u003eMiICE1e/eL\u003c/em\u003e was most abundantly expressed in the leaves of non-flowering branches. The relatively high expression of \u003cem\u003eMiICE1\u003c/em\u003es in flowering branches suggests their involvement in the regulation of flowering in mango. Overexpression of \u003cem\u003eMiICE1\u003c/em\u003es in \u003cem\u003eArabidopsis\u003c/em\u003e did not alter flowering time under optimal growth temperatures. However, at low temperatures, some transgenic lines overexpressing \u003cem\u003eMiICE1\u003c/em\u003es flowered earlier than WT, indicating that \u003cem\u003eMiICE1\u003c/em\u003es may participate in the signaling pathways regulating flowering. As ambient temperature affects flowering in \u003cem\u003eArabidopsis\u003c/em\u003e, with lower temperatures progressively delaying flowering, ICE1 appears to regulate flowering time by modulating the expression of flowering-related genes in response to temperature cues. Previous studies reported that the expression of \u003cem\u003eFLC\u003c/em\u003e decreased in the \u003cem\u003eice1\u003c/em\u003e mutant of \u003cem\u003eArabidopsis\u003c/em\u003e under long-day conditions, while the up-regulation of \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eSOC1\u003c/em\u003e promoted early flowering. This suggests that \u003cem\u003eICE1\u003c/em\u003e, \u003cem\u003eFT\u003c/em\u003e, \u003cem\u003eSOC1\u003c/em\u003e, and \u003cem\u003eFLC\u003c/em\u003e jointly participate in the regulation of flowering (Lee et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Ambient temperature also regulates \u003cem\u003eFT\u003c/em\u003e expression and protein transport, prevents premature flowering at low temperatures, and ensures reproductive success under favorable environmental conditions. Appropriate temperatures promote \u003cem\u003eFT\u003c/em\u003e expression, which regulates flowering responses to both photoperiod and ambient temperature (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Conversely, low temperatures may prolong flowering time by elevating \u003cem\u003eFLC\u003c/em\u003e transcription levels (Susila et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In order to further investigate the underlying mechanism, we screened the interacting proteins of \u003cem\u003eMiICE1s\u003c/em\u003e and found that they interacted with \u003cem\u003eMiFLC\u003c/em\u003e and \u003cem\u003eMiFTs\u003c/em\u003e, but not with \u003cem\u003eMiFT3\u003c/em\u003e. In addition, \u003cem\u003eMiICE1a\u003c/em\u003e did not interact with \u003cem\u003eMiFLC\u003c/em\u003e, \u003cem\u003eMiFT1a\u003c/em\u003e, and \u003cem\u003eMiFT2\u003c/em\u003e; \u003cem\u003eMiICE1e\u003c/em\u003e did not interact with \u003cem\u003eMiFT2\u003c/em\u003e and \u003cem\u003eMiFT4\u003c/em\u003e; and \u003cem\u003eMiFT2\u003c/em\u003e interacted exclusively with \u003cem\u003eMiICE1c\u003c/em\u003e. As \u003cem\u003eMiFTs\u003c/em\u003e are known to promote early flowering in plants (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), these interactions suggest that \u003cem\u003eMiICE1s\u003c/em\u003e may regulate flowering time by directly or indirectly modulating the activity of central flowering-related proteins, such as \u003cem\u003eMiFLC\u003c/em\u003e and \u003cem\u003eMiFTs\u003c/em\u003e. The differential interaction patterns observed among \u003cem\u003eMiICE1s\u003c/em\u003e highlight the complexity of the molecular mechanisms controlling flowering time, particularly in response to environmental and developmental signals.\u003c/p\u003e\u003cp\u003eThe expression of \u003cem\u003eMiICE1\u003c/em\u003es enhanced the resistance of \u003cem\u003eArabidopsis\u003c/em\u003e to salt and drought stresses, as well as to MeJA treatment. This finding suggests that \u003cem\u003eMiICE1\u003c/em\u003es may strengthen the plant's ability to adapt to adverse environments by regulating stress response mechanisms. Our previous research showed that \u003cem\u003eMiFT1a\u003c/em\u003e and \u003cem\u003eMiFT1b\u003c/em\u003e transgenic lines did not respond to abiotic stress, while \u003cem\u003eMiFT2\u003c/em\u003e, \u003cem\u003eMiFT3\u003c/em\u003e, and \u003cem\u003eMiFT4\u003c/em\u003e enhanced resistance to salt or drought stress in \u003cem\u003eArabidopsis\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The interactions between \u003cem\u003eMiICE1s\u003c/em\u003e and \u003cem\u003eMiFTs\u003c/em\u003e may therefore contribute to the enhanced stress tolerance conferred by \u003cem\u003eMiICE1s\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eIn conclusion, \u003cem\u003eMiICE1s\u003c/em\u003e were found to be localized in the nucleus. Their expression in \u003cem\u003eArabidopsis\u003c/em\u003e promoted flowering and improved resistance to stress. These results indicate that \u003cem\u003eMiICE1\u003c/em\u003es are functional genes with potential application value in mango, which provides new ideas and methods for improving crop varieties by genetic engineering.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003e\u0026nbsp;\u003c/h2\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by Guangxi Natural Science Foundation (2023GXNSFAA026268), Guangxi Key Technologies R\u0026amp;D Program (Guikenong AB241484007), National Natural Science Foundation of China (31860541), the CARSGIT-Guangxi Mango Industry Project (nycytxgxcxtd-2021-06-02), start-up funding for introduced talents in Guangxi University (ZX01080033124004, EE101762) and Guangxi Higher Education Institutions Young and Middle-aged Teachers' Research Fundamental Capability Enhancement Project (2025KY0026).\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\n\u003cp\u003eXinhua He and Cong Luo: experimental design and implementation, composition and review of manuscript, financial support for experiments and laboratory apparatus; Fangfang Xie, Canbin Chen, Xukang Huang and Xijin Zhou: composition and review of manuscript; Yanzhu Liu, Zhiqi Lai and Tingting Liang: experimental implementation; Tianli Guo and Yili Zhang: experimental design and implementation, manuscript composition.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll Data will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdel-Aziz HF, Hamdy AE, Sharaf A, Abd El-wahed AE, Elnaggar IA, Seleiman MF, Omar M, Al-Saif AM, Shahid MA, Sharaf M (2023) Effects of fogging system and nitric oxide on growth and yield of \u0026lsquo;naomi\u0026rsquo; mango trees exposed to frost stress. 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Int J Biol Macromol 253:127582\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ICE1, flowering time, stress, mango, MiFLC, MiFTs","lastPublishedDoi":"10.21203/rs.3.rs-7980258/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7980258/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Inducer of CBF Expression 1 (ICE1) family is known to regulate plant responses to low-temperature stress, but its roles in flowering and other abiotic stresses remain unclear. In this study, the tissue-specific expression patterns and functional roles of \u003cem\u003eMangifera indica\u003c/em\u003e (mango) \u003cem\u003eICE1s\u003c/em\u003e (\u003cem\u003eMiICE1\u003c/em\u003es) were characterized in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eArabidopsis\u003c/em\u003e). \u003cem\u003eMiICE1a/aL\u003c/em\u003e showed high expression across multiple tissues, while \u003cem\u003eMiICE1b/bL\u003c/em\u003e were up-regulated in flowering branch stems. \u003cem\u003eMiICE1c\u003c/em\u003e and \u003cem\u003eMiICE1d\u003c/em\u003e exhibited peak expression in flowers and in flowering branch stems/flowers, respectively, whereas \u003cem\u003eMiICE1e/eL\u003c/em\u003e were most abundant in non-flowering branch leaves. Overexpression of most \u003cem\u003eMiICE1\u003c/em\u003es (except \u003cem\u003eMiICE1b\u003c/em\u003e) in \u003cem\u003eArabidopsis\u003c/em\u003e accelerated flowering compared with the wild type, suggesting their involvement in flowering regulation. \u003cem\u003eMiICE1\u003c/em\u003es also responded to NaCl, PEG, and methyl jasmonate treatments, with transgenic lines displaying superior root elongation and fresh weight under stress, indicating enhanced abiotic stress tolerance. Protein interaction analyses further revealed that MiICE1s interacted with flowering- and stress-related regulators, potentially coordinating these processes. Overall, this study highlights the dual role of \u003cem\u003eMiICE1\u003c/em\u003es in promoting flowering and enhancing stress adaptation, providing insights for engineering stress-resilient crops with optimized flowering traits.\u003c/p\u003e","manuscriptTitle":"Mangifera indica (mango) ICE1s confer flowering and tolerance to multiple stresses in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-13 12:46:21","doi":"10.21203/rs.3.rs-7980258/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-04T06:50:47+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T17:59:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T04:15:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2025-10-31T02:17:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ef733af-0fe1-4069-bb6e-c66026b8e800","owner":[],"postedDate":"November 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:09:38+00:00","versionOfRecord":{"articleIdentity":"rs-7980258","link":"https://doi.org/10.1007/s11240-026-03375-5","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2026-03-05 15:58:55","publishedOnDateReadable":"March 5th, 2026"},"versionCreatedAt":"2025-11-13 12:46:21","video":"","vorDoi":"10.1007/s11240-026-03375-5","vorDoiUrl":"https://doi.org/10.1007/s11240-026-03375-5","workflowStages":[]},"version":"v1","identity":"rs-7980258","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7980258","identity":"rs-7980258","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}