BoMYC2 and BoAREB1 interact with BoMYB2 to respond to ABA signaling and drought stress in cabbage

The MYB family has been extensively studied in model organisms, but research on them in vegetable crops such as cabbage ( Brassica oleracea L. var. capitata L. ) still needs to be supplemented and improved. We cloned BoMYB2 and further determined its role in the response of cabbage to drought stress. We found that the overexpression of BoMYB2, which is induced by ABA, can significantly enhance the drought stress tolerance of cabbage, and the opposite phenotype was observed in BoMYB2 - silenced lines. Two proteins, BoMYC2 and BoAREB1, that interact with BoMYB2 were verified through yeast two - hybrid, luciferase complementation assay, pull - down assay, etc. Overexpressing them in Arabidopsis thaliana significantly improved the drought stress tolerance of A. thaliana . Transient silencing of them in cabbage seedlings reduced the plant’s drought tolerance. Both of them are also induced by ABA, and they cooperate with BoMYB2 to promote the antioxidant capacity of plants under drought stress, affect the expression of downstream genes, and increase the survival rate of plants in drought environments. In summary, our results indicate that BoMYB2 - BoMYC2 and BoMYB2 - BoAREB1 play important roles in the ABA - signal - mediated drought regulation process of cabbage.

Cabbage ( Brassica oleracea L. var. capitata L. ) is a common vegetable crop rich in soluble sugars and vitamins, which is widely cultivated around the world. The quality of cabbage is closely related to environmental factors. Abiotic stresses such as salinity, drought, and high temperature can significantly affect its quality and even lead to plant death (Y. Zhang et al., 2024). Among them, drought stress is an abiotic stress caused by water deficiency. When plants lack water, they cannot maintain the physical and chemical balance in their bodies (G. Xie, Xu, Chong, & Zhu, 2024), resulting in the accumulation of a large amount of reactive oxygen species (ROS) (Tenorio Berrio, Nelissen, Inze, & Dubois, 2022; P. Wang et al., 2024). Excessive ROS affects the protein activity of plants, hinders their development, causes oxidative damage, and ultimately leads to plant death (Fang et al., 2025). During the process of plants resisting drought stress, they also respond to and resist oxidative stress (Ren et al., 2021; X. Y. Zhao, Wang, Shi, Zhang, & Zhao, 2025). During the process of plant adaptation to variable environments, many complex mechanisms have evolved to respond to and resist various abiotic stresses (Xiong & Zhu, 2003), including the classic plant hormone signal transduction (Luhua et al., 2025; Sato, Mizoi, Shinozaki, & Yamaguchi-Shinozaki, 2024; H. Zhang, Zhu, Gong, & Zhu, 2022). Among them, abscisic acid (ABA) is one of the most important responsive hormones during drought stress (Kim, Kidokoro, Yamaguchi-Shinozaki, & Shinozaki, 2024). ABA is a plant hormone that inhibits growth and is widely distributed in higher plants, playing an important role in seed germination and growth and development. In a drought environment (Waadt et al., 2022), ABA transmits drought signals to ABA receptor proteins in plant cells (Yoshida et al., 2010). After the interaction between the receptor proteins and protein phosphatase type 2C (PP2C), the activity of PP2C is inhibited, releasing SnRK2s (G. J. Li, Chen, Sun, & Zhao, 2024). The kinase phosphorylates and activates downstream genes to further transmit signals (X. Chen et al., 2021), forming the classic PYR/PYL - PP2Cs - SnRK2s signaling pathway (R. Zhang et al., 2025; H. Zhao et al., 2020). During this process, many transcription factors are also involved in signal transduction to regulate plant drought tolerance (Singh & Laxmi, 2015). Transcription factor proteins are a class of key proteins for plants to improve drought tolerance (J. Chen et al., 2017; Yu et al., 2024). They mainly affect plant drought tolerance by antagonistic or synergistic effects to weaken or amplify drought signals and regulate the expression of downstream stress-related genes (Kim et al., 2024; J. Xie et al., 2025). For example, the basic helix-loop-helix (bHLH) protein MYC2, a negative regulator of the classic jasmonic acid (JA) signaling pathway (Mu et al., 2025; X. Zhao, He, Liu, Wang, & Zhao, 2024), regulates the expression of EPF2/EPFL4/EPFL9 in poplar to control the change of stomatal density, thereby controlling the water use efficiency of poplar to adapt to drought stress (Xia, Jiang, Wu, Du, & Kang, 2024). Studies have shown that MYC2 also responds to ABA signals (Abe et al., 2003). When maize suffers from drought stress and the ABA content increases, the JA content also increases simultaneously, promoting the accumulation of ZmMYC2. ZmMYC2 promotes the transcription and translation of ZmHsf28, which is phosphorylated by ZmSnRK2.2 released by ABA, promoting the transcription of downstream genes and ultimately improving the drought tolerance of plants (L. Liu et al., 2025). In addition, studies have shown that there is an interaction between MYC2 and ABI5, a key factor in the ABA signaling pathway (C. Du et al., 2025; Vittozzi, Kruger, Majee, Nee, & Wenkel, 2024). MYC2 and ABI5 play an important role in regulating the balance between JA and ABA hormones under plant stress conditions (Wan, Yao, Zhao, & Xu, 2025). Among the basic leucine zipper (bZIP) transcription factors, a class of ABA binding factors/ABA response element binding proteins (ABFs/AREBs) are activated under drought conditions and bind to the promoters of downstream genes to promote their expression in response to stress (Song et al., 2023). In the study of trifoliate orange, PtrABF4 and PtrABR1 (ABA repressor 1) synergistically promote the transcription of downstream PtrBAM3, promote starch decomposition and the accumulation of soluble sugars, and actively respond to drought stress (Y. Zhang et al., 2023). IbDof2.1-IbABF2 responds to and regulates ABA responses, promotes stomatal closure and proline biosynthesis in sweet potato, and activates the reactive oxygen species (ROS) scavenging system to improve the drought resistance of sweet potato (Y. Wang et al., 2025). As one of the largest transcription factor families in plants, MYB is widely involved in the regulation of hormones and abiotic stresses and also plays an important role in the ABA signal transduction process under drought stress (Cao et al., 2024; Su et al., 2025; D. Zhang et al., 2025). For example, in the study of the model plant Arabidopsis thaliana, MYB41-BRM regulates the stomatal movement of A. thaliana under the mediation of ABA to adapt to drought stress (L. Gao et al., 2024), and MYB96-HHP integrates ABA-dependent and non-ABA-dependent signals in A. thaliana to activate the CBF signaling pathway to adapt to various abiotic stresses (Lee & Seo, 2015). In recent years, the research on transcription factors and abiotic stresses in cruciferous vegetables has been deepening. However, there are still few reports on the drought stress response and ABA signaling pathway in cabbage. Exploring and verifying the ABA response mode of cabbage is helpful for the breeding of drought-tolerant varieties and for supplementing and improving the research on abiotic stresses in cruciferous vegetables. In this study, we found that the drought stress tolerance of cabbage seedlings overexpressing BoMYB2 was significantly improved, while the BoMYB2 - silenced lines of cabbage showed drought sensitivity. Additionally, through yeast two - hybrid screening, we identified two proteins in the ABA signaling pathway, BoMYC2 and BoAREB1, that interacted with BoMYB2, and the interaction was verified by luciferase complementation assay, BiFC assay, etc. After genetic transformation and gene silencing of BoMYC2 and BoAREB1, we found that the overexpression of both proteins promoted plant drought tolerance. They interacted with BoMYB2 respectively to transmit ABA signals, regulated the transcriptional activation of downstream genes, enhanced the reactive oxygen species scavenging ability of plants, and made plants more adaptable to oxidative stress and drought stress. Based on the BoMYC2 - BoMYB2 and BoMYB2 - BoAREB1 regulatory modules, our study proposed a possible ABA signal transduction and antioxidant regulation mechanism during drought stress in cabbage. Characterizations of BoMYB2 Previous research in our laboratory identified a MYB family gene associated with drought stress. The gene sequence showed high homology to AtMYB2, and thus it was named BoMYB2 (Figure 1D). Tissue expression analysis revealed that BoMYB2 was preferentially expressed in roots and seeds (Figure 1A). Results from detached leaf experiments indicated that the expression of BoMYB2 was induced by PEG and ABA (Figure 1B, C), suggesting that BoMYB2 may respond to ABA signals and participate in regulating drought stress in plants. Sequence alignment of BoMYB2 with those from A. thaliana, cabbage, and rapeseed showed that BoMYB2 had a conserved R2R3 domain, indicating that the biological functions of BoMYB2 may be similar to those in other species (Figure 1D). Overexpression and silencing of BoMYB2 affect plant drought resistance To investigate the biological function of BoMYB2, we overexpressed BoMYB2 in A. thaliana and cabbage. After identification by qRT- PCR, the expression level of the BoMYB2 in the overexpression lines was significantly higher than that in the control group (Figure 2B; Supporting Information S2: Figure S2). After drought treatment, the survival rate of plants overexpressing BoMYB2 was significantly higher than that of the wild type (Figure 2A, C; Figure S3, S4). After detecting the physiological indicators of the plant samples after drought treatment, it was found that the damage degree and water loss rate of the plants overexpressing BoMYB2 were significantly reduced (Figure 2D, E; Figure S5). The experimental results indicate that the overexpression of BoMYB2 significantly enhances the tolerance of cabbage to drought stress. By silencing BoMYB2 in cabbage through VIGS, the qRT- PCR results showed that the expression level of the BoMYB2 gene in PCVA: BoMYB2 was significantly lower than that in PCVA: 00 (Figure 2G). After drought treatment, the survival rate of PCVA: BoMYB2 was significantly lower than that of PCVA: 00 (Figure 2F, H). The detection results of physiological indicators indicated that the oxidative damage of PCVA: BoMYB2 was more severe (Figure 2I, J). Silencing BoMYB2 reduced the drought resistance of cabbage seedlings, which was opposite to the results of overexpressing BoMYB2. BoMYB2 interacts with BoMYC2 Previous reports have shown that MYB2 regulates the expression of downstream genes in response to ABA signals through interaction with other proteins. To understand the mechanism by which BoMYB2 regulates drought tolerance in response to ABA signals in cabbage, we identified BoMYC2, which interacts with BoMYB2, through yeast two- hybrid (Y2H) assay (Figure 3A, B). To further validate the protein - protein interaction, we conducted luciferase complementation (LCI) assay and bimolecular fluorescence complementation (BiFC) assay in Nicotiana benthamiana leaves to verify the interaction between BoMYB2 and BoMYC2 in vivo (Figure 3C, E). Additionally, we induced the prokaryotic expression of BoMYB2 and BoMYC2 proteins using IPTG and verified the direct interaction between BoMYC2 and BoMYB2 in vitro through Pull - down assay (Figure 3D). Overexpression and silencing of BoMYC2 affect plant drought resistance To investigate the biological function of BoMYC2 under drought stress, we obtained A. thaliana mation. After identification, the expression level of BoMYC2 in the overexpression lines was significantly higher than that in the wild- type (Figure 4B). The survival rate of the BoMYC2 overexpression lines after drought stress was significantly higher than that of the wild type (Figure 4A, C). By detecting the contents of malondialdehyde and proline in the control group and the experimental group, we found that the damage degree of the BoMYC2 overexpression lines was smaller, and the content of the osmotic regulatory substance proline was higher (Figure 4D, E). PCVA: BoMYC2 with significantly lower expression of the BoMYC2 than the control group was obtained through VIGS (Figure 4G). After drought stress, the survival rate of PCVA: BoMYC2 was significantly lower than that of PCVA: 00 (Figure 4F, H). Physiological experiment results showed that PCVA: BoMYC2 had a higher degree of oxidative damage (Figure 4I, J). The above experimental results indicate that BoMYC2 plays a positive regulatory role in the drought stress process of cabbage seedlings. BoMYB2 interacts with BoAREB1 In addition to BoMYC2, we also identified the ABA response element binding protein BoAREB1 that interacts with BoMYB2 through Y2H assay (Figure 5A, B). We confirmed the interaction between BoMYB2 and BoAREB1 in plants and found that they interact in the nucleus through LCI and BiFC assays (Figure 5C, E). Additionally, we verified that BoMYB2 and BoAREB1 can interact in vitro through Pull-down assay (Figure 5D). Overexpression and silencing of BoAREB1 affect plant drought tolerance BoAREB1 was overexpressed in A. thaliana . After identifying the transgenic lines, it was determined that the gene expression level of BoAREB1 was significantly higher than that of the wild type (Figure 6B). After subjecting three - week - old A. thaliana to drought stress for 14 days, the survival rate of the BoAREB1 overexpression lines was significantly higher than that of the wild type (Figure 6A, C). After measuring the malondialdehyde content and proline content of the plants, we found that the damage degree of the BoAREB1 overexpression lines was lower and their antioxidant capacity was stronger (Figure 6D, E). After subjecting PCVA: BoAREB1, with significantly lower BoAREB1 expression levels than PCVA: 00 (Figure 6G), and the control to drought stress, statistics showed that the survival rate of PCVA: BoAREB1 was significantly lower than that of PCVA: 00 (Figure 6F, H). Moreover, the detection of malondialdehyde content and reactive oxygen species staining results indicated that PCVA: BoAREB1 had a higher degree of oxidative damage (Figure 6I, J). The above experimental results suggest that BoAREB1 positively regulates the drought resistance of cabbage seedlings. BoMYB2 synergistically enhanced the drought tolerance of cabbage with BoMYC2 and BoAREB1 qRT-PCR was used to detect the responses of BoMYC2 and BoAREB1 to PEG and ABA treatments. It was found that the expression of BoAREB1 was induced by PEG and ABA (Figure 7D, E), and the expression of BoMYC2 was induced by ABA (Figure 7B) but inhibited by PEG (Figure 7A). When ABA treatment was performed 6 h in advance in the luciferase complementation assay, it was found that ABA promoted the interactions between BoMYB2 and BoMYC2 as well as BoAREB1 (Figure 7C, F). The expression levels of drought stress-related genes in PCVA: 00, PCVA: BoMYB2, PCVA: BoMYC2, and PCVA: BoAREB1 plants after drought stress were detected, and it was found that the expression levels in the silenced lines were significantly lower than those in the control group (Figure 7G, H). The experimental results indicate that ABA promotes the interactions between BoMYB2 and BoMYC2 as well as BoAREB1, affects the expression of downstream genes, and thereby regulates the drought tolerance of cabbage.

Drought stress is one of the major problems faced by agricultural production. It reduces the water content in plants, leading to an increase in reactive oxygen species, disruption of redox balance, damage to plant cells, and even plant death in severe cases (Jiang, van Zanten, & Sasidharan, 2025). Mining and identifying drought-resistant genes and understanding the mechanisms of plant response and improvement of drought resistance are of great significance for agricultural issues such as the breeding of drought-resistant varieties (Kim et al., 2024; Xiong & Zhu, 2003). Transcription factors play an important role in regulating the drought stress tolerance of plants (J. Chen et al., 2017; H. Zhang et al., 2022). For example, transcription factors such as MYB, MYC, and AREB have been verified to respond to and participate in the regulation of drought stress in various plants (S. Chen et al., 2025; Z. Liu et al., 2025), and the biological functions of many transcription factors in cruciferous crops have also been identified (W. Li et al., 2025; X. Liu et al., 2025). However, there are still few reports on their roles in cabbage. We identified a transcription factor related to drought stress in cabbage. Since it is highly homologous to AtMYB2 in A. thaliana, we named it BoMYB2 (Figure 1A). Previous reports in A. thaliana indicated that AtMYB2 responds to ABA signals and plays an important role in drought stress in A. thaliana (Abe et al., 2003), and preliminary verification has also been conducted in rapeseed (S. Gao et al., 2024). To verify the biological function of BoMYB2 during drought stress in cabbage, we overexpressed and transiently silenced BoMYB2 in cabbage and then subjected them to drought stress compared with the control group (Figure 2). The experimental results showed that BoMYB2 plays a positive regulatory role during drought stress in cabbage. Additionally, we heterologously expressed BoMYB2 in A. thaliana and verified the role of BoMYB2 during drought stress in A. thaliana (Figure S2-7). The results showed that BoMYB2 also plays a positive regulatory role during drought stress in A. thaliana . To gain an in-depth understanding of the mechanism by which BoMYB2 regulates drought stress in cabbage, we screened for proteins that interact with BoMYB2 during drought stress using Y2H screening. Subsequently, we verified these interactions in vivo and in vitro through LCI, BiFC, and Pull - down assays (Figure 3, 5). Ultimately, we confirmed that BoMYC2 and BoAREB1 interact with BoMYB2 respectively. We compared the protein sequences of BoMYC2 and BoAREB1 with those of A. thaliana and cruciferous crops (Figure S8, S9) and found that BoMYC2 and BoAREB1 are highly conserved. Previous studies have shown that MYC2 and AREB1 respond to ABA signals and participate in the regulation of drought resistance in many crops (L. Du et al., 2024; Hu et al., 2024; Ma et al., 2025). Moreover, in the LCI assay, we found that the addition of exogenous ABA significantly enhanced the interaction between BoMYC2 and BoMYB2, as well as between BoAREB1 and BoMYB2 (Figure 7C, F). This implies that BoMYC2 and BoAREB1 may cooperate with BoMYB2 to regulate the drought stress tolerance of cabbage. Research shows that MYC2 is the core transcription factor in the plant JA signaling pathway (Kim et al., 2024), and JAZ protein is a key suppressor of MYC2 . Under drought stress, JAZ protein is ubiquitinated and degraded, releasing MYC2 and activating the expression of a series of downstream stress-responsive genes (X. Zhao et al., 2024). We detected the changes in the expression levels of some reported JAZ genes that interact with MYC2 in PCVA: 00 and PCVA: BoMYC2 after drought stress (Figure S1) and found that the expression of JAZ genes decreased to varying degrees. However, since BoMYC2 was silenced, cabbage showed drought sensitivity (Figure 4F). Additionally, interestingly, in the detached leaf induction experiment, we found that BoMYC2 was rapidly induced by ABA (Figure 7A), but the expression level of BoMYC2 showed a downward trend under short-term PEG induction (Figure 7B). We speculate that this is because the sequential order of different hormone responses or other inducing factors in cabbage affects the expression of BoMYC2 in the early stage of simulated drought. We will conduct more in-depth research and verification on this phenomenon in subsequent studies. Research indicates that AREB1 is activated during the ABA signal transduction process and functions as a transcription factor to regulate the expression of downstream genes and enhance plant drought resistance (S. Li et al., 2019; Soma et al., 2023). We transiently silenced BoAREB1 in cabbage seedlings through VIGS. After drought stress, the silenced lines showed drought sensitivity (Figure 6F-J), and the expression of stress-responsive genes was significantly lower than that in PCVA: 00 (Figure 7G, H). Silencing BoAREB1 significantly reduced the drought resistance of cabbage seedlings. Moreover, we verified in A. thaliana that BoMYC2 and BoAREB1 play positive regulatory roles in drought stress, affect the transcription of downstream genes (Figure 7G and H), and improve the antioxidant and drought resistance abilities of transgenic A. thaliana (Figure 4A-E, Figure 5A-E). In conclusion, we identified the positive regulatory role of BoMYB2 in the drought stress process of cabbage. BoMYB2 synergistically regulates the expression of downstream related genes with BoMYC2 and BoAREB1, respectively, thereby regulating the drought resistance of cabbage. Finally, we proposed a potential regulatory model for the response to drought and ABA signals in cabbage (Figure 8).

Plant materials and growth conditions The genetic background of cabbage used for overexpression in the experiment is SP362, and that used for gene silencing is SP323. The seeds of cabbage and N. benthamiana are stored in the Cruciferous Vegetable Breeding Laboratory of the College of Horticulture. The genetic background of A. thaliana used for genetic transformation is Col-0, and the seeds are stored in the Molecular Breeding Laboratory of Shaanxi Hybrid Rapeseed Research Center. The plants are grown in a greenhouse at 22 °C with a 16-hour light and 9-hour dark cycle. Gene acquisition and sequence alignment analysis All A. thaliana gene sequences were obtained from the TAIR database (https://www.arabidopsis.org), and the gene sequences of cruciferous crops were retrieved from the BRAD database (http://brassicadb.cn). The acquired genes were subjected to sequence alignment using DNAMAN software to analyze the conservation and correlation among the sequences. The primer sequences used in the processes of gene amplification and vector construction in this experiment can be found in Tab. S1. Genetic transformation of plants Wild-type A. thaliana plants that have grown for about 28 days are pre-treated by removing the siliques and open flowers one day in advance and watering them thoroughly. On the next day, the A. thaliana seedlings are infected using the Agrobacterium -mediated floral-dip method. After one-day dark treatment, the normal cultivation is resumed until the siliques mature and the seeds are harvested. Subsequently, A. thaliana is screened to homozygosity through methods such as antibiotic screening and quantitative detection for experiments. Select plump and crack-free SP362 cabbage seeds for disinfection, then sow them on MS medium. After the seedlings grow petiolate cotyledons, separate the cotyledons from the hypocotyls and conduct pre-culture. After pre-culture, perform Agrobacterium infection. After 2 days of dark culture, resume the culture for 7 days, and then transfer the explants to the selection medium containing antibiotics. When the positive seedlings have 4 true leaves, transfer them to the rooting medium. When the roots are well-developed, transfer the seedlings to soil for cultivation. After the seedlings grow 6 true leaves, conduct vernalization for 3 months, then perform self-pollination. Harvest the seeds after the plants are mature. Gene silencing in cabbage Select plump and crack-free SP323 cabbage seeds for disinfection, and evenly spread them on moistened filter paper. After the cotyledons emerge, soak the seedlings in the resuspension of PCVA series Agrobacterium and apply vacuum. After vacuum treatment, transfer the seedlings to soil. The gene silencing efficiency is determined by qRT-PCR according to the instructions of PerfectStart Universal Green qPCR SuperMix (TransGen Biotech, China). Analysis of expression patterns The roots, stems, and leaves were isolated from the four-leaf stage cabbage seedlings, and the flower buds, flowers, and seeds were isolated from the cabbage plants at the reproductive growth stage. The RNA of each component was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech, China). After reverse transcription to obtain cDNA, tissue expression analysis was performed by qRT-PCR. Leaves with similar growth states and in the same positions were selected from cabbage seedlings and soaked in water, 15% PEG6000, and 10 μM ABA respectively. Samples were collected at different time points. RNA was extracted from the samples at each time point, reverse-transcribed into cDNA, and then analyzed for exogenous responses by qRT-PCR. Drought stress experiment Three-week-old wild-type and overexpressed A. thaliana plants in good growth condition were selected, watered or subjected to drought stress for about 14 days. The survival rate of the plants was counted, and typical plants were selected for photography. Cabbage seedlings with about three true leaves in good growth condition were selected and subjected to drought treatment for about 20 days. The survival rate of the plants was counted, and typical plants were selected for photography before and after the treatment. Representative samples were selected to detect the contents of malondialdehyde and proline to reflect the degree of plant damage. Representative samples were also selected for chemical staining using 3,3’-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) chloride to reflect the degree of oxidative damage to the plants. Yeast two-hybrid (Y2H) assay Since BoMYB2 has self-activation activity, BoMYB2 sequences of different lengths were ligated into the pGBKT7 vector and co-transformed with the AD vector respectively to verify the self-activation domain and binding domain of BoMYB2 . The BoMYC2 and BoAREB1 sequences were ligated into the pGADT7 vector. According to different combinations, they were co-transformed into AH109 yeast competent cells. The transformed yeast strains were inoculated on different dropout media. -T/-L indicates that the medium lacks Trp and Leu, -T/-L/-H/-A indicates that the medium lacks Trp, Leu, His, and Ade, and X-α-gal indicates that the chromogenic substrate for yeast galactosidase (MEL1) is added. Firefly luciferase complementation imaging assay (LCI) assay The sequence of BoMYB2 was cloned into the pCAMBIA1300-cLUC vector, and the sequences of BoMYC2 and BoAREB1 were cloned into the pCAMBIA1300-nLUC vector respectively. Then, these vectors and the empty vector were transformed into Agrobacterium GV3101 respectively. Well-grown N. benthamiana seedlings were selected, and the Agrobacterium strains were co-injected into N. benthamiana leaves according to different combinations. Two days after cultivation, the leaves were picked, and the fluorescence was observed using an in-vivo imaging system (Princeton, America). Pull-down assay The sequence of BoMYB2 was cloned into the pMal-C2 vector with an MBP tag, and the sequences of BoMYC2 and BoAREB1 were cloned into the pGEX-4T vector with a GST tag. These constructs were then transformed into BL21 (DE3) E. coli competent cells respectively for prokaryotic expression of proteins fused with different tags. After inducing protein expression, the total protein was selected as the input. The target proteins were purified, co-incubated, and eluted. Subsequently, the eluate and input were detected using GST and MBP antibodies. Bimolecular Fluorescence Complementation (BiFC) assay The sequence of BoMYB2 was cloned into the pCAMBIA1300-cYFP vector, and the sequences of BoMYC2 and BoAREB1 were cloned into the pCAMBIA1300-nYFP vector respectively. Then, these vectors and the empty vector were transformed into Agrobacterium GV3101 respectively. Similar to the LCI experiment, appropriate N. benthamiana leaves were selected for co-injection according to different combinations. After two days of cultivation, the YFP fluorescence signals were observed using an upright fluorescence microscope (Olympus, Japan). Data analysis The bar graphs represent the mean values of three replicates. Student’s t-test was used to detect the significant differences between the data. A P-value less than 0.05 was considered to indicate a significant difference with statistical significance. The graphs were plotted using GraphPad Prism 10 (Beijing, China).

This research was funded by the National Key Research and Development Program of China (2024YFD1200400) and the National Natural Science Foundation of China (32472056), and Key Research and Development Projects of Henan Province (241111112400), and the National Key Research and Development Program of China (2023YFD1200100). Author contributions ZS and SH designed the project and experiments; ZS, MG and QP performed most of the experiments and analyzed the data; YZ and ZX provided the research platform; YZ and ZX provided financial support; ZS and SH directed the manuscript. All authors read, reviewed, and approved the final manuscript. Data availability All data supporting the results of this study are included in the article and additional files. Conflicts of interest The authors declare no conflicts of interest. Supplementary data Supplementary data is available at online. Figure legeneds Figure 1. Characterization analysis of BoMYB2 . (A) Tissue expression analysis of BoMYB2 . (B) Analysis of the expression level changes of BoMYB2 in detached leaves after treatment with 15% PEG6000 for different durations. (C) Analysis of the expression level changes of BoMYB2 in detached leaves after treatment with 10 μM ABA for different durations. (D) Alignment of MYB2 protein sequences from different cruciferous plants. The R2 and R3 domains are marked with red lines. Student’s t-test was used to detect significant differences (*P<0.05, **P<0.01). Figure 2. Overexpression and silencing of BoMYB2 affect the drought tolerance of plants. (A) Phenotypes of cabbage seedlings of the control and BoMYB2 overexpression lines before and after drought stress treatment. (B) Identification of BoMYB2 overexpression plants by qRT-PCR. (C) Statistics of plant survival rate after drought stress. (D) Analysis of malondialdehyde content in the control and BoMYB2 overexpression lines after drought stress. (E) Analysis of DAB and NBT staining in the control and BoMYB2 overexpression lines after drought stress. (F) Phenotypes of cabbage seedlings of PCVA: 00 and PCVA: BoMYB2 before and after drought stress treatment. (G) Identification of the expression level of BoMYB2 in PCVA: 00 and PCVA: BoMYB2 plants by qRT-PCR. (H) Statistics of the survival rate of PCVA: 00 and PCVA: BoMYB2 plants after drought stress. (I) Analysis of malondialdehyde content in PCVA: 00 and PCVA: BoMYB2 after drought stress. (J) Analysis of DAB and NBT staining in PCVA: 00 and PCVA: BoMYB2 after drought stress. Student’s t-test was used to detect significant differences (*P<0.05, **P<0.01). Figure 3. Interaction between BoMYB2 and BoMYC2 proteins. (A) Schematic diagram of BoMYB2 segmentation. BoMYB2, 1 - 272 aa; BoMYB2-N, 1 - 120 aa; BoMYB2-C, 121 - 272 aa. (B) Analysis of the interaction between BoMYB2 and BoMYC2 proteins by Y2H assay. -T/-L, medium lacking Trp and Leu; -T/-L/-H/-A, medium lacking Trp, Leu, His and Ade. (C) Analysis of the interaction between BoMYB2 and BoMYC2 proteins by LCI assay. (D) Analysis of the interaction between BoMYB2 and BoMYC2 proteins by Pull-down assay. (E) Analysis of the interaction between BoMYB2 and BoMYC2 proteins by BiFC assay. Bar = 500 μm. Figure 4. Overexpression and silencing of BoMYC2 affect plant drought tolerance. (A) Phenotypes of control and BoMYC2 -overexpressing A. thaliana lines with or without drought stress. (B) Identification of BoMYC2 -overexpressing A. thaliana lines by qRT-PCR. (C) Statistics of plant survival rates with or without drought stress. (D) Analysis of malondialdehyde content in control and BoMYC2 -overexpressing A. thaliana lines with or without drought stress. (E) Analysis of proline content in control and BoMYC2 -overexpressing A. thaliana lines with or without drought stress. (F) Phenotypes of cabbage seedlings of PCVA: 00 and PCVA: BoMYC2 before and after drought stress. (G) Identification of BoMYC2 expression levels in PCVA: 00 and PCVA: BoMYC2 plants by qRT-PCR. (H) Statistics of survival rates of PCVA: 00 and PCVA: BoMYC2 plants after drought stress. (I) Analysis of malondialdehyde content in PCVA: 00 and PCVA: BoMYC2 after drought stress. (J) Analysis of DAB and NBT staining in PCVA: 00 and PCVA: BoMYC2 after drought stress. Student’s t-test was used to detect significant differences (*P < 0.05, **P < 0.01). Figure 5. Interaction between BoMYB2 and BoAREB1 proteins. (A) Schematic diagram of BoMYB2 segmentation. BoMYB2, 1-272 aa; BoMYB2-N, 1-120 aa; BoMYB2-C, 121-272 aa. (B) Analysis of the interaction between BoMYB2 and BoAREB1 proteins by Y2H assay. -T/-L, Medium lacking Trp and Leu; -T/-L/-H/-A, Medium lacking Trp, Leu, His, and Ade. (C) Analysis of the interaction between BoMYB2 and BoAREB1 proteins by LCI assay. (D) Analysis of the interaction between BoMYB2 and BoAREB1 proteins by Pull-down assay. (E) Analysis of the interaction between BoMYB2 and BoAREB1 proteins by BiFC assay. Bar = 500 μm. Figure 6. Overexpression and silencing of BoAREB1 affect plant drought tolerance. (A) Phenotypes of control and BoAREB1 -overexpressing A. thaliana lines with or without drought stress. (B) Identification of BoAREB1 -overexpressing A. thaliana lines by qRT-PCR. (C) Statistics of plant survival rates with or without drought stress. (D) Analysis of malondialdehyde content in control and BoAREB1 -overexpressing A. thaliana lines with or without drought stress. (E) Analysis of proline content in control and BoAREB1 -overexpressing A. thaliana lines with or without drought stress. (F) Phenotypes of cabbage seedlings of PCVA: 00 and PCVA: BoAREB1 before and after drought stress. (G) Identification of BoAREB1 expression levels in PCVA: 00 and PCVA: BoAREB1 plants by qRT-PCR. (H) Statistics of survival rates of PCVA: 00 and PCVA: BoAREB1 plants after drought stress. (I) Analysis of malondialdehyde content in PCVA: 00 and PCVA: BoAREB1 after drought stress. (J) Analysis of DAB and NBT staining in PCVA: 00 and PCVA: BoAREB1 after drought stress. Student’s t-test was used to detect significant differences (*P<0.05, **P<0.01). Figure 7. Analysis of the expression patterns of BoMYC2 and BoAREB1 and the changes in the expression levels of downstream genes in the silenced lines after drought stress. (A) Analysis of the changes in the expression level of BoMYC2 in detached leaves treated with 15% PEG6000 for different durations. (B) Analysis of the changes in the expression level of BoMYC2 in detached leaves treated with 10 μM ABA for different durations. (C) LCI assay to analyze the effect of exogenous ABA on the interaction between BoMYB2 and BoMYC2. (D) Analysis of the changes in the expression level of BoAREB1 in detached leaves treated with 15% PEG6000 for different durations. (E) Analysis of the changes in the expression level of BoAREB1 in detached leaves treated with 10 μM ABA for different durations. (F) LCI assay to analyze the effect of exogenous ABA on the interaction between BoMYB2 and BoAREB1. (G) qRT-PCR analysis of the changes in the expression level of BoRAB18 in PCVA: 00, PCVA: BoMYB2, PCVA: BoMYC2, and PCVA:BoAREB1 plants after drought stress. (H) qRT-PCR analysis of the changes in the expression level of BoRD29B in PCVA: 00, PCVA: BoMYB2, PCVA: BoMYC2, and PCVA: BoAREB1 plants after drought stress. Student’s t-test was used to detect significant differences (*P < 0.05, **P < 0.01). Figure 8. A model of BoMYB2 -mediated ABA signaling response in drought stress response of cabbage. Under drought stress, the endogenous ABA content in plants increases, inducing the accumulation of BoMYC2, BoMYB2, and BoAREB1 proteins, which affects the expression of downstream genes. BoMYC2 and BoAREB1 synergize with BoMYB2 respectively to enhance the tolerance of cabbage to drought stress. Figure S1. Identification of JAZs expression in control as well as BoMYC2 silenced plants after drought stress Figure S2. qRT-PCR identification of Arabidopsis overexpressing BoMYB2 Figure S3. Overexpression of BoMYB2 improved drought stress tolerance in Arabidopsis Figure S4. Overexpression of BoMYB2 improved Arabidopsis survival in drought stress Figure S5. Overexpression of BoMYB2 improved tolerance to osmotic stress in Arabidopsis Figure S6. Arabidopsis overexpressing BoMYB2 has longer roots under osmotic stress Figure S7. MYC2 multiple sequence comparison Figure S8. AREB1 multiple sequence comparison

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Authors Metrics & Citations Metrics Article Usage 226views 149downloads Citations Download citation Zhen Shen, Shuhua Huang, Minghui Guo, et al. BoMYC2 and BoAREB1 interact with BoMYB2 to respond to ABA signaling and drought stress in cabbage. Authorea. 05 November 2025. DOI: https://doi.org/10.22541/au.176233177.79742647/v1 DOI: https://doi.org/10.22541/au.176233177.79742647/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.