The seeds for pepper cultivar AA3 and Nicotiana benthamiana(N. benthamiana)were obtained from the School of Horticulture Landscape Architecture at Henan Institute of Science and Technology, Xinxiang 453003, China. The pepper plants were grown in a controlled environment with a photoperiod of 16 hours of light and 8 hours of darkness, at temperatures of 22°C during dayand 18°C at night, and maintained at an optimal humidity level of 80%. Similarly, N. benthamiana was maintained at 25°C during the day and 18°C at night, with a preferred relative humidity of 60%.

In compliance with the tobacco rattle virus-induced gene silence (VIGS) technology elucidated by Wang in 2013, the CaSBP11 gene of pepper was silenced (Wang, 2013). The development of a virus-induced gene silencing test vector was based on the methodology by Zhang et al. (2020b). Specifically, a 224bp-specific fragment from the CaSBP11 CDS region was amplified by specific primers ( Supplementary Table 1 ), followed by cloning into TRV2 vector via a double enzyme digestion technique. The recombinant vector was sequenced by Shanghai Sangon Biotechnology Co., Ltd. and transferred to Agrobacterium strain GV3101 for storage.

During the formation of two true leaves in pepper seedlings, the silencing of the CaSBP11 gene was performed using Zhang’s methodology with a prepared bacterial suspension containing the CaSBP11 gene, and cultivated at 28°C until 0D600 = 0.8 (Zhang et al., 2013). It was mixed with an equal amount of TRV1 and inserted into the pepper cotyledon using a 1ml syringe without needles. After injection, the plants were maintained at 28°Cin darkness for 2 days.They were then cultured with a photoperiod of 16/8 hours, at temperatures of 22°C during the day and 18°C at night. Upon observing photo-bleaching in positive plants (TRV2:CaPDS), five random leaves from CaSBP11-silenced plants and control plants (TRV2:00) were chosen to assess the efficiency of silencing.

The vector used for CaSBP11 N. benthamiana overexpression was constructed based on the protocol outlined by Zhang et al. (2020b). Then, the re-engineered vector (pVBG2307:CaSBP11:GFP) was then employed for CaSBP11 overexpression in N. benthamiana. CaSBP11 overexpressing transgenic N. benthamiana were created through Agrobacterium tumefaciens-assisted leaf disc transformation (Oh et al., 2005). RNA analysis confirmed two kanamycin-resistant CaSBP11 transformants. The T1 progeny originated from T0 plant regeneration, with T2 progeny deriving from T1 plants. T3 progeny were chosen for subsequent research.

To evaluate the expression of the CaSBP11 gene in peppers under drought stress, 6–8 true leaf seedlings were obtained from substrate (constructed with a 3:1:1 blend of matrix, perlite, and vermiculite).The seedlings were then cultivated in 1/2 Hoagland’s solution, with 20% Polyethylene glycol (PEG6000) applied for three days. Control cultures remained solely in 1/2 Hoagland’s solution. Leaves were collected at 0 h, 3 h, 6 h, 12 h, and 24 h, and stored at −80°C.

For drought treatments of CaSBP11-silenced plants and CaSBP11 overexpression plants, procedures outlined by Zhang et al. (2024) are applied. CaSBP11-silenced and control plants, as well as CaSBP11 overexpression and wild-type plants, were cultivated under identical controlled conditions prior to and during drought treatment. The conditions for CaSBP11-silenced and control plants were 16 hours of light, 8 hours of darkness, 22°C during the day, and 18°C at night, with a humidity of 80%. For CaSBP11 overexpression and wild-type plants, the conditions were 16 hours of light, 8 hours of darkness, 25°C during the day, and 18°C at night, with a humidity of 60%. Prior to drought treatment of CaSBP11-silenced and control plants, sufficient irrigation is implemented one month ahead with watering repeated every third day until the final watering, marking the onset of drought stress (Day 0) three days past the treatment commencement. Sampling is performed on days 0, 1, 2, 3, and 4, with samples are stored at −80°C for future use. For drought treatment in CaSBP11 overexpressed and wild-type plants, the identical procedures involve pre-treating with sufficient irrigation, followed by watering every four days until the final watering, marking the onset of drought stress (day 0). Sampling is conducted on days 0, 2, and 4, with samples being stored at −80°C for future use.

For ABA treatment, seedlings were exposed to 20μM ABA in accordance with Yin et al. (2014). Controls consisted of a 0.5% tween and 0.1% ethanol solution. Leaves were harvested at 0h, 3h, 6h, 12h, 24h, and 48h, and stored at −80°C for future use. The statistical data for germination and root length of CaSBP11 overexpressed plants are performed according to the procedure delineated by Ma et al. (2011). Four ABA concentration gradients (0g/L ABA, 0.1g/L ABA, 0.5g/L ABA, and 1.0g/L ABA) were used for these experiments. The seeds of CaSBP11-overexpression plants were soaked in these ABA solutions for 24 hours, then placed on a culture dish with two layers of moistened filter paper under 25°C, 16 hours light, and 8 hours dark conditions. Ventilation was performed daily for 10 minutes, and water was added to maintain moisture. During the third, fifth, and tenth days, germination data were documented. For the root length statistics of CaSBP11-overexpression plants, the same method was employed, with the root length being counted on the tenth day.

Total RNA was extracted via the RNAprep Pure Micro Kit (Tiangen Beijing, China), in accordance with manufacturer’s guidelines. Reverse transcription was accomplished with the transcriptor First Strand cDNA Synthesis Kit (Tiangen Beijing, China). Diluted cDNA was used for quantitative real-time (qPCR) analysis at a concentration of 50 ng/L. Utilizing the iQ5.0 Bio-Rad iCycler thermocycler (Bio-Rad, Hercules, CA, USA), we implemented real-time Qpcr according to the methodology outlined by Zhang et al. (2020a). This involved a pre-denaturation phase at 95 °C for 1 min, followed by 40 cycles of denaturation (95 °C, 10 s), annealing (56 °C, 30 s), and extension (72°C, 30s). End-of-cycle fluorescence detection ensured PCR primer accuracy through post-PCR profile analysis ranging from 56 to 95 °C. The specificity of all primers was rigorously validated using NCBI Primer BLAST ( Supplementary Table 1 ). Gene expression was assessed and standardized against CaUBI3 (GenBank: AY486137.1), and Nbactin-97 (GenBank: XM_019369243.1) (Schmittgen and Livak, 2008; Du et al., 2015; Zhang et al., 2016).

Percentage drought index analysis employed Zhang’s methodology (Zhang et al., 2020b). Upon 5 days of drought stress, the drought phenotypes of CaSBP11-silenced and control plants were classified, and the percentage drought indices of both groups were calculated. The classification criteria were as follows: level 0, no symptoms; level 1, lower leaves wither; level 2, all leaves except the growing point wither; level 3, entire plant withers. For drought stress of 13 days, the drought phenotypes of CaSBP11 overexpression and wild-type plants were classified, and the percentage drought indices of both groups were calculated. The classification criteria were as follows: level 0, no symptoms; level 1, lower leaves wither or yellow; level 2, lower leaf death; level 3, entire plant death except the growing point.

Malondialdehyde (MDA), relative water content, and relative electrical conductivity evaluations followed Zhang et al. (2018) and Pan et al. (2012) protocols. Water loss rate was calculated based on Ma et al. (2021) method. Total chlorophyll content was determined via Arkus et al. (2005) methodology. Assays for peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) activities were conducted following Zhang et al. (2018); Stewart and Bewley (1980) methodology.

For hydrogen peroxide (H₂O₂) and oxygen (O₂ ^-) radical analysis, diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) stains were utilized as per Choi and Hwang (2012) and Kim et al. (2012) protocol, respectively. Quantification of these stained regions was executed based on Sekulska-nalewajko et al. (2016),while H₂O₂ content determination utilized Liu et al. (2010). Superoxide anion (O₂ ^-) detection is accomplished in accordance with the Solarbio Superoxide Anion kit guidelines (Solarbio, Beijing, China).

The analysis of ABA content utilizes the Plant Hormone Abscisic Acid (ABA) Enzyme Immuno Fluorometric Assay Kit (KeLu, Wuhan, China). The procedure is carried out as per the enclosed instructions.

The stomatal morphologies were observed at high magnification using a scanning electron microscope (FEI Quanta 200, USA). The stomatal density was accurately measured via the NIH (National Institutes of Health)-endorsed Image J software Stomatal apertures were determined via SEM, with the aperture dimensions encompassing both pore length (dumbbell-shaped aperturae) and width (maximum perpendicular to the dumbbell-shaped aperturae). Besides, SEM examination unveiled the dimensions of the stomatal apertures. The stomatal aperture encompasses both the pore length and width of the pore. The dumbbell-shaped aperturaeis identified as the latter, while its maximal perpendicular value represents the former.

The vectors of pSPYCE-35S and pSPYNE-35S were used for these experiments. Besides, the target gene was ligated into the vector using the homologous recombination method, and the primers for vector construction are listed in Supplementary Table 1 . The constructed recombinant vectors were transferred into Agrobacterium (GV3101) for the BiFC and Co-IP experiments. For the BiFC experiment, the above transformed Agrobacterium bacterial solution was cultured overnight at 28°C and 200 rpm until the optical density (OD) reached 0.5. The culture was then centrifuged, and the supernatant was discarded. The pellet was re-suspended in an equal volume of suspension buffer. After resuspension, the bacterial solution was left to stand at room temperature for 3 hours. The bacterial suspension was then injected into Nicotiana Benthamiana leaves using a 1 ml syringe without a needle. After injection, the plants were maintained in darkness at 28°C for one day, followed by cultivation under a photoperiod of 16/8 hours, with temperatures of 22°C during the day and 18°C at night. Fluorescence was observed using a confocal laser scanning microscope (FV10-ASW, Olympus Corporation, Japan) three days after infection. The excitation wavelengths used were 515 nm for the yellow fluorescence field, 488 nm for the chloroplast auto fluorescence field, and 358 nm for the DAPI field (nuclear staining). The Co-IP experiments were conducted according to the method described by Song et al. (2023).

The SPSS 22.0 statistical software was utilized to evaluate the effects of different treatment alterations using a one-way ANOVA test. Post hoc Tukey analysis revealed potential significant differences at P ≤ 0.05 and P ≤ 0.01. Data is presented as mean ± standard deviation (SD). Experiments necessitate at least three biological replicas in rigorously designed protocols.

To investigate the role of CaSBP11 in resilience to drought and ABA stress its expression pattern in pepper was analyzed. As shown in Supplementary Figure 1 , CaSBP11 transcripts initially decreased at 3h post drought stress, subsequently significantly increasing at 12h. Besides, the expression of CaSBP11 was suppressed during ABA treatment ( Supplementary Figure 1 ). These findings indicating that CaSBP11 responds to both drought and ABA stress.

Virus-induced gene silencing was applied for elucidation of CaSBP11’s function in pepper’s drought stress response (Wang, 2013).The established positive control,TRV2:CaPDS, silenced the CaPDS gene, resulting in photo-bleached leaf symptoms, while TRV2:00 served as the negative control. Upon observing photo-bleaching in positive control plants, the silencing efficiency of TRV2:CaSBP11 was evaluated ( Supplementary Figure 2 ). The results in Supplementary Figure 2 show that CaSBP11-silenced (TRV2:CaSBP11) and control (TRV2:00) plants displayed no remarkable phenotypic variations under regular conditions. Additionally, the silencing efficiency of the CaSBP11 gene exceeds 84%. Therefore, both CaSBP11-silenced and control plants were used for subsequent analyses.

After enduring three drought days, CaSBP11-silenced plants demonstrated negligible phenotypic modifications while controls exhibited leaf wilting symptoms, including chlorosis in lower foliage ( Figure 1A ). Comparatively, there were no discernible differences in the phenotype of non-stressed CaSBP11-silenced and control plants ( Figure 1A ). Upon drought exposure, chlorophyll content decreased in both CaSBP11-silenced and control plants, yet the former retained significantly more ( Figure 1B ).Additionally, the rate of water loss in CaSBP11-silenced plants showed a decreasing trend after drought, notably outperforming controls at two and three days ( Figure 1C ). The electrical conductivity of CaSBP11-silenced plants varied consistently below the control plants, particularly on Days 1 and 3 ( Figure 1C ). Both plant types exhibited an overall increase in MDA content, with control plants showing higher values on Days 1 and 2 ( Figure 1C ). Besides, CaSBP11-silenced plant samples exhibited superabundant CAT and POD activities on Day 1, achieving statistical significance ( Figure 1C ). SOD activity exhibits a trend of initial increase and subsequent decline, while the peak time point in control plants precedes that of CaSBP11-silenced plants ( Figure 1C ). Upon drought stress for 5 days, we classified the drought phenotypes of CaSBP11-silenced and control plants to calculate their percent drought indices. The classifying criteria were: level 0, no symptoms; level 1, lower leaves of plant wilted; level 2, all plant leaves except the growing point wilted; level 3, entire plant wilted ( Supplementary Figure 3A ). Subsequently, we statistically analyzed the percent drought indices of CaSBP11-silenced and control plants. As demonstrated in Supplementary Figure 3B , at 5 days of drought stress, the drought index of CaSBP11-silenced plants was notably lower than that of control plants. These results indicated improved drought endurance in the CaSBP11-silenced plants. Subsequently, to assess reactive oxygen species (ROS) accumulation in drought-stressed plants with CaSBP11-silenced and control groups, H₂O₂ and O₂ ^- were detected via DAB and NBT staining ( Figures 2A, D ). After four days of drought exposure, significant DAB and NBT staining area was observed in control leaves compared to CaSBP11-silenced leaves ( Figures 2B, E ), with elevated H₂O₂ and O₂ ^-content ( Figures 2C, F ). This evidence suggests reduced ROS accumulation in CaSBP11-silenced foliage relative to control leaves. Furthermore, gene expression levels pertinent to ROS removal (CaAPX1, CaPOD, CaSOD, and CaCAT2) were evaluated ( Figure 2G ). On the fourth day of drought stress, except for elevated CaPOD expression, gene expression in CaSBP11-silenced and control plants decreased ( Figure 2F ). Nonetheless, these genes displayed significantly higher expression in CaSBP11-silenced plants compared to controls ( Figure 2G ).

The stomatal density of control plants was significantly higher than that of CaSBP11-silenced plants ( Figures 3A, B ). During four-day drought stress, ABA content increased in both CaSBP11-silenced and control plants; however, the increase was significantly greater in the former ( Figure 3C ). Additionally, post-drought stress, stomatal length and width decreased, but CaSBP11-silenced plants still exhibited noticeably larger dimensions compared to the controls ( Figures 3D, E ). Furthermore, key genes in the ABA signaling pathway (CaSNRK2.4, CaPYL9, CaAREB, and CaPP2C) were analyzed as shown in Figure 3F . These genes displayed elevated expression levels in CaSBP11-silenced plants under drought stress after four days ( Figure 3F ). Notably, even when untreated, their expression levels were lower in the CaSBP11-silenced plants than in the controls ( Figure 3F ).

To better understand CaSBP11’s role in plant drought resilience, we generated two transgenic lines (line 9 and line10) overexpressing this gene in N. benthamiana. The gene expression level in these lines is illustrated in Supplementary Figure 4 . In the absence of ABA, there was no discernible difference in germination rates between wild-type and CaSBP11 overexpression seeds ( Supplementary Table 2 ). However, under ABA treatment, the germination rates of CaSBP11 overexpression seeds exceeded those of wild-type seeds ( Supplementary Table 2 ). Specifically, at 0.1g/L ABA, CaSBP11 overexpression seeds demonstrated a distinctly increased germination rate at day 3 when compared to wild-types. Similarly, at 0.5g/L ABA, CaSBP11 overexpression seeds displayed a substantially superior germination rate when compared to wild-types by the third day. Lastly, under 1g/L ABA, CaSBP11 overexpression seeds demonstrated markedly enhanced germination rates compared to wild-types by day 5 ( Supplementary Table 2 ). Moreover, root length of CaSBP11 overexpression plants and wild-types diminished as the treatment intensity elevated across different concentration gradients. However, exposure to 0.1g/L ABA, 0.5g/L ABA, and 1g/L ABA significantly enhanced the root length of CaSBP11 overexpression plants compared to wild-types ( Supplementary Figures 5A, B ). These data show overexpression of CaSBP11 in N.benthamiana diminishing plant sensitivity to ABA.

Additionally, post-seven-day drought exposure, both CaSBP11 overexpression and wild-type plants displayed wilt ( Figure 4A ). However, the transgenic plants exhibited pervasive wilt and severe yellowing of lower leaves, while the wild-type ones only experienced wilt of lower leaves ( Figure 4A ). After 13 days of drought, when rewatering occurred, nearly all CaSBP11 overexpression plants, apart from the growing point, had died. In contrast wild-type plants displayed no growth anomalies except for leaf yellowing ( Figure 4A ). Besides, we categorized the drought phenotypes of CaSBP11 overexpression plants and wild-type plants after 13 days of drought stress. The classification criteria were as follows: level 0, no symptoms; level 1, wilting or yellowing of lower leaves; level 2, sublethal leaf death; level 3, plant death excluding the growing point ( Supplementary Figure 6A ). Subsequently, we calculated the percentage of drought index of CaSBP11 overexpression plants and wild-type plants. As shown in Supplementary Figure 6B , the percentage of drought index of CaSBP11 overexpression plants was notably elevated compared to wild-types. Additionally, at day 4 of drought, CaSBP11 overexpression plants displayed significantly reduced chlorophyll content compared to wild-types ( Figure 4B ). Similarly, their MDA content and relative electrical conductivity showed a significant increase ( Figure 4C ). Additionally, plants overexpressing CaSBP11 displayed a noticeable decrease in relative water content on day 9 of drought stress exposure, as depicted in Figure 4C . These data underscore improved drought stress sensitivity in CaSBP11 overexpressing plants.

In addition, on day 4 of drought stress, the CaSBP11 overexpression plants exhibited larger DAB and NBT staining regions than wild-types ( Figures 5A, B, D, E ). Their H₂O₂ and O₂ ^-content was appreciably higher than wild-type plants ( Figures 5C, F ). These data suggest a potential role for CaSBP11 in ROS signaling pathways during drought stress. Therefore, gene expressions associated with the ROS pathway (NbPOD, NbCAT, NbAPX, NbSOD) were examined. During drought, NbPOD expression was induced and notably elevated compared to wild-type ( Figure 5G ). Conversely, NbCAT expression in the CaSBP11 overexpression plants was notably reduced ( Figure 5G ). Moreover, during the fourth day of drought stress, NbAPX expression in CaSBP11 overexpressing plants displayed notably higher levels compared to wild-types ( Figure 5G ). However, NbSOD expression in these overexpressing plants is notably reduced ( Figure 5G )

Besides, the stomatal density of CaSBP11 overexpression plants significantly exceeded that of their wild-type counterparts ( Figures 6A, B ). Similarly, the ABA concentration in CaSBP11 overexpression plants significantly declined compared to wild-types ( Figure 6C ). Notably, drought stress resulted in diminished pore size in both CaSBP11 overexpression and wild-type plants after four days, with a remarkable disparity in pore length and width between these strains ( Figures 6D–F ). During this time, significant reductions in the transcript levels of key ABA signaling pathway genes like NbPYL9, NbAREB, NbPP2C, and NbSNRK2.4 were observed in the CaSBP11 overexpression plants compared to wild-types ( Figure 6G ). Notably, without treatment, the CaSBP11 overexpression plants exhibited higher expressions of NbPP2C, NbSNRK2.4, and NbSRK2E versus wild-types ( Figure 6G ). These results indicate that overexpression of CaSBP11 in N.benthamiana accentuates plant drought stress susceptibility, potentially linked to ROS and ABA signaling pathways.

In the previous study, it was found that under non-stress conditions, core ABA signaling cascade genes (CaPP2C, CaPYL9, CaSNRK2.4, CaAREB) exhibited lower expression levels in CaSBP11-silenced plants when compared to controls. Conversely, this trend was reversed for CaSBP11 overexpressed lines (NbPP2C, NbAREB, NbSNRK2.4, NbSRK2E). Therefore, we speculate that CaSBP11 may regulate the plant’s response to drought stress through interactions with CaPP2C, CaPYL9, CaSNRK2.4, and CaAREB. Further research was conducted on this hypothesis. The BiFC experiments demonstrated that CaSBP11 interacts with CaPP2C, CaPYL9, CaSNRK2.4, and CaAREB in the nucleus. This was evidenced by a significant YFP signal in the nucleus, while no YFP signal was observed in the control samples ( Figure 7 , Supplementary Figures 7 - 9 ). We also performed Co-IP assays by co-expressing CaPYL9-MYC and CaSBP11-CE, CaAREB-MYC and CaSBP11-CE, CaSnRK2.4-MYC and CaSBP11-CE, CaPP2C-MYC and CaSBP11-CE in N. benthamiana leaves respectively ( Figure 8 ). These results indicated that CaSBP11 interacts with CaPP2C, CaPYL9, CaSNRK2.4, and CaAREB. Additionally, CaSBP11 may participate in the response of pepper to drought stress by interacting with these genes.