To experimentally analyze the subcellular localization, the full length of CaSLP was inserted into the green fluorescent protein (GFP) reporter gene, and the fusion construct 35S:CaSLP:GFP and 35S::GFP were transiently expressed in tobacco leaves. The nuclei were stained with DAPI. N. benthamiana leaves were collected efficiently. Fluorescent signaling of the 35S:CaSLP:GFP fusion protein was was distributed throughout the cell nucleus and cell membrane of tobacco epidermal cells, showing that CaSLP was localized in the cell nucleus and cell membrane (Fig. 1A). The qRT-PCR was performed to characterize the expression pattern of CaSLP. After moisture stress, the expression of CaSLP was upregulated within 6 h (Fig. 1B). At 3 h after exogenous SA treatment, the expression of CaSLP progressively peaked (Fig. 1C). These results indicated that CaSLP is primarily induced by drought and SA.

CaSLP upregulation in response to drought revealed that it plays a function in drought stress response. VIGS studies were carried out to investigate this possibility.. As indicated in supplemental Figure S1, TRV2-treated plants: CaPDS solution revealed photo-bleaching in the leaves, confirming that the treatment was successful. Then, the silencing efficiency was measured through qRT-PCR, which was almost 85%. (Figure S1). No obvious morphological differences were found under normal growth conditions. Under drought stress, the CaSLP-silenced pepper plants showed a drought-resistant phenotype compared with control pepper plants (Fig. 2A). The results of the measurement of H₂O₂ and O₂^.− contents, the ROS indicators showed that the H₂O₂ and O₂^.−contents in the CaSLP-silenced pepper plants were significantly higher than those in the control pepper plants after drought treatment (Fig. 2C, F). The DAB and NBT staining of CaSLP-silenced pepper exhibited stains much darker and deeper than in the control pepper plants when plants were exposed to drought stress (Fig. 2B, E). When the plants were exposed to different mannitol concentrations, the CaSLP-silenced pepper showed more severe leaf wilting than did the control under each mannitol concentration (Fig. 2D). CaSLP-silenced pepper plants had lower chlorophyll content than the control (Fig. 2G). In addition, the stomatal aperture and water loss rate were measured. No apparent differences were found between the CaSLP-silenced and control cells under normal conditions. However, when subjected to drought, the VIGS plants showed slightly higher stomatal aperture, stomatal density and water loss rates than the control plants (Fig. 2H-K). All of these data demonstrated that silencing of CaSLP reduces drought tolerance.

To explore the function of CaSLP in response to drought stress tolerance, transient overexpression of CaSLP (CaSLP-To) in pepper was performed. The CaSLP-To and Mock plants were exposed to drought for 48 h. CaSLP-To expression was assessed. It was clearly taller than Mock pepper plants (Fig. 3A). The CaSLP-To plants maintained good growth and leaf turgor. Without stressful conditions, no clear differences were found in plant morphology between CaSLP-To and Mock pepper plants. However, drought stress caused severe leaf wilting and a significant increase in ROS levels. The H₂O₂ and O₂^.− contents of CaSLP-To plants were significantly lower than that of Mock pepper plants (Fig. 3B, E, F). Next, we measured the stomatal aperture. The stomatal aperture of CaSLP-To and Mock plants did not differ (Fig. 3C). Upon the drought exposure, the stomatal aperture of CaSLP-To plants showed a lower stomatal aperture and stomatal density compared with the control Mock pepper plants (Fig. 3G, H). Water loss rate was also assessed and observed that CaSLP-To plants lose less water than Mock pepper plants (Fig. 3D). We also detected the transcript levels of the stomatal development-related genes SDD1, YODA, and FAMA in CaSLP-To and the control plants. The transcript abundance of SDD1, YODA, and FAMA in CaSLP-To plants were higher than control plants (Fig. 3I-K). CaSLP transient expression increased drought stress tolerance. These findings showed that CaSLP transient expression improved drought stress tolerance.

To further determine the function of CaSLP in drought tolerance, Agrobacterium-mediated transformation was performed to conduct 35 s:CaSLP:GFP transgenic overexpression (OE) lines in Arabidopsis. A obvious upregulated in CaSLP expression level was confirmed in the OE lines than WT plants. CaSLP transgenic lines #1, #2, and #3 were chosen to study the roles of CaSLP in drought tolerance. Under well-watered conditions, no apparent difference was found between the CaSLP transgenic and the wild type (WT). After drought stress, the WT showed more severe leaf wilting than the CaSLP transgenic lines (Fig. 4A, B). Relative electrolyte leakage (REL), malondialdehyde (MDA), and chlorophyll contents supported better growth performance of CaSLP transgenic lines following drought exposure. The CaSLP transgenic lines demonstrated lower REL and MDA contents, and greater chlorophyll content than WT (Fig. 4C-E). These findings showed that CaSLP overexpression in Arabidopsis significantly increased drought tolerance.

Drought has become a dangerous threat to decrease crop yields. Exogenous spraying SA regulator has been effectively used to reduce drought tolerance in field crops. We anticipate CaSLP-mediated drought tolerance via a salicylic acid route based on the visible induction of CaSLP in response to drought (Fig. 1). Therefore, the CaSLP-silenced pepper and control plants were sprayed exogenously with 2 mM SA (Fig. 5A). No noticeable differences were found in proline, REL, and MDA contents between the CaSLP-silenced and control plants in the absence of stressful circumstances. However, after exogenous spraying SA treatment, the CaSLP-silenced pepper plants exhibited lower REL, MDA, and lower proline contents in comparison with the plants that were treated with water (Fig. 5B-D). These above-mentioned data demonstrate that exogenous application of salicylic acid drastically enhanced drought tolerance and further showed the importance of salicylic acid in CaSLP-regulated drought tolerance.

To further explore the underlying mechanism of enhanced tolerance to drought stress of CaSLP, the expression profiles of several known stomatal development-related genes were selected after drought treatment. We measured stomatal development-related genes in pepper, including SDD1, YODA, and FAMA. The qRT-PCR results showed that the expressions of SDD1, YODA, and FAMA were decreased in CaSLP-silenced cells than in control cells (Fig. 6A-C), suggesting that CaSLP acts as a positive regulator of drought signaling. We also measured the expression levels of stomatal development-related genes in Arabidopsis (AtSDD1, AtYODA, AtFAMA, AtTMM, and AtMPK3) after exposure to drought stress. The expressions of AtSDD1, AtYODA, AtFAMA, AtTMM, and AtMPK3 were greater in transgenic plants than in WT plants (Fig. 6). These results indicate that overexpression of CaSLP increases the drought tolerance of Arabidopsis.

To examine the Pst.DC3000 susceptibility, the CaSLP-silenced plants and control leaves were infected with Pst.DC3000. 10 mM MgCl₂ (control treatment) and Pst.DC3000 suspensions were injected into the plant leaves. After Pst.DC3000 treatment, the leaves of CaSLP-knockdown plants was more yellow and wilted than the control. This finding was indicated by the lower chlorophyll contents of CaSLP-knockdown plants (Fig. 7A, E, H). Additionally, to explore the accumulation of reactive oxygen species (ROS) post-Pst.DC3000 stress in the control and silenced pepper plants, the trypan blue and DAB staining of the leaf samples were performed. Figure 7B, F showed that as compared with the control, the accumulation of H₂O₂ and O₂^.− contents of CaSLP-silenced plants was markedly higher (Fig. 7I, M). These results showed that the CaSLP-silenced plants had higher ROS accumulation than WT and CaSLP-knockdown pepper plants, thereby demonstrating the decreased Pst.DC3000 resistance, as revealed by reduced cell death and bacterial numbers in pepper leaves compared with the control plants (Fig. 7C, G). To investigate the role of CaSLP-knockdown pepper plants, the bacterial numbers in CaSLP-silenced and control plants were measured. The bacterial numbers in CaSLP-silenced plants were higher than those in the control plants after Pst.DC3000 treatment (Fig. 7D). Next, we measured the expressions of stress-related genes CaNPR1, CaABR1, and CaPR1. Further analysis showed that the relative expressions of CaNPR1, CaABR1, and CaPR1 in CaSLP VIGS plants were strikingly lower than those of the control pepper plants (Fig. 7J-L). Collectively, these results illustrated that CaSLP plays a significant role in the plant’s response to Pst.DC3000.

To explore the role of CaSLP in response to disease, the CaSLP transgenic lines and WT were infected with Pst.DC3000. 10 mM MgCl₂ (control treatment) and Pst.DC3000 suspensions were injected into the plant leaves. Under untreated conditions, the color of CaSLP transgenic lines and WT was almost the same. There was no substantial difference. Whereas, after 3 days post-Pst.DC3000 injection, the WT showed more significant yellowing than the CaSLP transgenic lines. WT had lower chlorophyll content than the CaSLP transgenic lines (Fig. 8A, D, H). The bacterial counts of CaSLP transgenic lines and those treated with Pst.DC3000 were then determined. In the Pst.DC3000 treatment, the CaSLP transgenic lines showed lower bacterial numbers than WT, respectively (Fig. 8G). We also determined the DAB and Trypan blue staining. With Pst.DC3000 treatment, the WT had higher values than the CaSLP transgenic lines (Fig. 8B, E). The transgenic lines had less ROS accumulation than WT, and CaSLP transgenic lines showed increased resistance to Pst.DC3000, as revealed by reduced cell death and bacterial numbers in Arabidopsis leaves compared with WT leaves (Fig. 8C, F). To further understand the underlying molecular mechanism of CaSLP in response to disease, we measured the expression levels of SA responses and the SA biosynthesis-related genes AtNPR1, AtPAL3, and AtICS after Pst.DC3000 treatment. The expressions levels of AtNPR1, AtPAL3, and AtICS were greater in the transgenic plants than the WT plants (Fig. 8I-K). Our results indicated that CaSLP transgenic Arabidopsis improved the Pst.DC3000 resistance.

The yeast two-hybrid (Y2H) method was used to analyze the target protein of CaNAC035. To examine the interaction of CaNAC035 with CaSLP, the Y2H and bimolecular fluorescence complementation (BiFC) assays were used in this study. The Y2H results showed that yeast containing CaNAC035 and CaSLP grew well on the selective solid medium of SD/-Trp/-Leu/-His/-Ade, and the yeast strains turned blue on the SD/-Trp/-Leu/-His/-Ade/X-a-gal solid medium (Fig. 9A). To further identify the interaction, we performed BiFC assays, which showed that coexpression of CaNAC0-nYFP with CaSLP-cYFP displayed significant fluorescence signaling in the cell nucleus (Fig. 9B). These results showed that CaNAC035 physically interacts with CaSLP in the cell nucleus. The transcripts of CaPR1 were dramatically increased in the CaNAC035-To pepper (Fig. S2). We examined if CaNAC035 affects CaPR1 transcription directly. NAC TFs can often bind to CACG. We analyzed the promoter of CaPR1 and found that CaPR1 had an NAC core-binding site. To determine the association between CaNAC035 and the CaPR1 promoter, we carried out Y1H assays. The full length of CaNAC035 was used as the prey, the CaPR1 promoter fragment was used as bait. On medium without aureobasidin A (AbA) p53 promoter + AD-P53 (positive control), p53 promoter + AD-empty (negative control), and CaPR1 promoter + AD-CaNAC035 grew normally. However, when 200 ng/ml AbA was added, the p53 promoter + AD-P53 (positive control) and CaPR1 promoter + AD-CaNAC035 survived, the p53 promoter + AD-empty (negative control) was inhibited. Yeast cells transformed transformed and showed full-length growth well on selective media. (Fig. 9C). These results showed a direct correlation of CaNAC035 with the promoter of CaPR1. To further confirm the direct binding of CaNAC035 to the CaPR1 promoter, regulation of expression LUC/REN ratios was performed (Fig. 9D). Full-length of CaNAC035 and CaSLP were inserted into pGreenII 62-SK to get an effector, full-length of CaPR1 were inserted into pGreenII 0800-LUC to generate reporters. REN (Renilla luciferase) was used as an internal control for activity normalization. LUC:REN ratios were significantly elevated in CaNAC035-pGreen II 62-SK +  CaPR1-pGreenII 0800-LUC and CaNAC035-pGreen II 62-SK +  CaSLP-pGreen II 62-SK +  CaPR1-pGreenII 0800-LUC than the control. Dual LUC assay results showed that the CaPR1 could activate the CaNAC035 expression (Fig. 9E), indicating that CaNAC035 is a direct upstream factor of CaPR1. GUS analysis was completed to explore the activity of CaNAC035pro with the addition of CaPR1. Transient expression assays with the β-glucuronidase reporter gene (GUS) in the leaves of tobacco (Nicotiana benthamiana) showed that CaPR1 could activate the CaNAC035 expression in vivo, and CaSLP can enhances the binding of CaNAC035 to CaPR1 promoter (Fig. 9F, G, H). These results CaSLP enhances the binding of CaNAC035 to its target gene promoters.

A previous study found that CaSLP interacts with CaNAC035 protein. To further verify the relationship between these two proteins and analyze whether CaSLP modulates drought stress in a CaNAC035-dependent manner, the CaSLP-silenced cells were inoculated into 35S:CaNAC035:GFP (Fig. 10A). qRT-qPCR showed that the expression levels of CaNAC035 and CaSLP in TRV2:00/35S:CaSLP:GFP were more significant than those in TRV2:CaNAC035/35S:CaSLP:GFP and TRV2:00/35S:GFP at 12 and 24 h (Fig. 10B, C). Under drought stress, the TRV2:00/35S:CaSLP:GFP plants exhibited higher fresh weight and survival rate than the TRV2:CaNAC035/35S:CaSLP:GFP and TRV2:00/35S:GFP plants (Fig. 10D, E). Therefore, the CaNAC035 gene was necessary for CaSLP-mediated drought stress tolerance.

Pepper (Capsicum annuum L., ‘P70’) and Arabidopsis (Columbia) seeds were obtained from the Laboratory of Vegetable Plant Biotechnology and Germplasm Innovation, Northwest A&F University, Yangling, China. The pepper plants were cultivated with a light cycle of 16 h light/8 h dark and 22 °C light /18 °C dark cycle with 75% relative humidity. The Arabidopsis seeds were sterilized before being cultivated on Murashige Skoog (MS) solid medium and vernalized for 1 day at 4 °C. The plants were restored to regular conditions after 7 days (22 °C with a photoperiod of 16 h light/8 h dark).

The total RNA was extracted according to the manufacturer’s instructions using a Tiangen RNA extraction kit (Beijing, China) and the Synthesis of cDNA was performed using a cDNA Synthesis Kit (Takara). The qPCR was conducted on the Applied Biosystems instrument using the SYBR Green Master Mix, following the manufacturer’s protocol (Ding Ning). The quantitative real-time polymerase chain reaction (qRT-PCR) primers were designed by using NCBI (https://www.ncbi.nlm.nih.gov/) and the primer was shown in Table S1. The relative expression was calculated using the 2^−ΔΔCT method (Zhuo et al. 2013). Three biological replicates were used for this study.

To generate a transient expression of CaSLP in pepper leaves, the coding region (CDS) of CaSLP was cloned and introduced into the pVBG2307::GFP vector. Then the 35S:CaSLP:GFP and 35S::GFP were transformed into A. tumefaciens strain GV3101. Transient expression in pepper leaves was performed as described by Huang et al. 2020. Leveraging the virus-induced gene silencing technique, CaSLP-silenced plants were grown. The pTRV1 and pTRV2 vectors were used for this study, and a 300-bp fragment of CaSLP was cloned and inserted into the pTRV2 vector. The pTRV1, pTRV2 (control), and pTRV2:CaSLP were separately transformed into A. tumefaciens strain GV3101. The infection method was followed as described by previous study Dai et al. 2018. The infected pepper plants were cultivated in a light/dark cycle of 22 °C/18 °C. After 28 days, we collected DNA and RNA to analyze the CaSLP transcript levels and identified positive plants using a specific primer.

For Arabidopsis transformation, the full length of CaSLP was cloned and combined into the pVBG2307:GFP vector. Then, the 35S:CaSLP:GFP fusion vector was transformed into A. tumefaciens strain GV3101. For transformation, the floral dip method was used as in previous reports (Clough and Bent 1998). Then, the transgene plants were screened on 1/2 MS solid medium, which contained 50 mg/ml kanamycin. We extracted DNA and RNA from T3 transgenic Arabidopsis lines to analyze the transcript levels of CaSLP and also confirmed the positive plants. For this experiment and future research, T3 homozygous lines were chosen.

The yeast two-hybrid assay was used for the experiment. In the pGBKT7 vector as bait plasmid, the complete length of CaNAC035 was introduced, and the full length of CaSLP was placed into the pGADT7 vector as prey plasmid. The recombinant vector was transformed into yeast Y2H. The strains were cultivated on SD (-Trp/-Leu), SD (-Trp/-Leu/-His/-Ade) and SD (-Trp/-Leu/-His/-Ade + X-α-gal) media for 3 days. Bimolecular fluorescence complementation (BiFC) was conducted as previously reported (Choi et al. 2012). To identify the proteins that can interact with a DNA sequence of interest, Y1H library screening was performed by using the Matchmaker Gold Yeast One-Hybrid Library and Screening kit (Clontech,CA, USA). The full-length CDS of CaNAC035 was placed into the pGADT7 vector to function as a prey vector. The promoter portions of CaPR1 were added to the pAbAi vectors. Recombinant plasmids were cotransformed into the Y1H yeast strain following the instructions provided by the manufacturer (Clontech, USA). For 3 days, the yeast strains were cultured on SD/Leu and SD/Leu/AbA media. The assay was similar to the widely used yeast two-hybrid assay that identifies protein protein interactions in small- or large-scale settings.

To assess drought tolerance in pepper plants, the leaves of CaSLP-silenced and control pepper plants were subjected to a 15-day drought. Leaves of pepper were collected at different stages after drought treatment. To further identify the role of CaSLP in transgenic Arabidopsis drought stress tolerance, 3-week-old T3 transgenic and WT lines were used in the experiments. The T3 transgenic and WT lines were treated with drought treatment for 10 d. Samples for gene expression analysis were collected, and phenotypic changes were observed and photographed. Following the drought treatments, samples were collected to investigate stress-related gene expression and physiological indicators. Well-watered plants were used as the positive control. The bacterium Pst.DC3000 was incubated overnight in Kings B solid medium containing 25 mg mL⁻¹ rifampicin at 28℃ and then resuspended in 10 mM MgCl₂. The whole plant was inoculated with a bacterial suspension of 10⁷ colony- forming units (CFU) mL⁻¹ and 0.02% Silwet L-77 (Li et al. 2015). Trypan blue staining and bacterial population counts (CFU) were performed 3 days after inoculation to evaluate disease symptoms (Bai et al. 2012; Wolfe et al. 2000).

The malondialdehyde (MDA) content was performed essentially as previously described (Liu et al. 2006), the relative electrolyte leakage (REL) was determined based on an earlier study (Dahro et al. 2016), the water loss rate was examined according to the protocol of Zhang et al. (2022), the accumulation of H₂O₂ and O₂^.− contents were conducted by histochemical staining of 3,3’-diaminobenzidine (DAB) and nitro-blue tetrazolium (NBT), respectively, using the method of Wang et al. (2011) O₂^.− the content was examined based on the method of Ma et al. (2016), the H₂O₂ measurement was determined as described by Geng and Liu (2018). The stomatal aperture assay was performed as described by Jiang et al. (2014). The stomatal index was determined as the number of stomata divided by the total number of epidermal cells. For the application of an salicylic acid spray, CaSLP-silenced and control plants were exogenously given 2 mM salicylic acid, and the plants were exposed to drought to determine the effects of exogenous salicylic acid on CaSLP’s response to drought. Exogenous treatment with 2 mM salicylic acid was performed for 2 days. We measured the growth performance and calculated the physiological index. We added Tween-20 (0.05%) for exogenous spraying of salicylic acid on CaSLP-silenced and control plants.

To rapidly and accurately determine the activity of a given promoter, the CaPR1 promoter elements were introduced into a pGreen62-SK vector. After being placed into a pGreen0800-luciferase (LUC) vector, CaNAC035’s coding sequences were obtained. Four-week-old tobacco leaves were injected with the recombinant vectors after being cotransformed into Agrobacterium Gv3101 bacteria (Nicotiana benthamiana). Using the Dual-Luciferase® machine, the ratio of luciferase (LUC) and Renilla (REN) was computed to calculate transient expression (Promega, WI, USA). Transient GUS activity was determined with tobacco leaves. The CaPR1 promoter was inserted into pCAMBIA1300-GUS to activate the GUS reporter gene. The ORFs of CaSLP and CaNAC035 were cloned into the pVBG2307:GFP plasmid to obtain 35S:CaSLP and 35S:CaNAC035 recombinant plasmids, respectively. Different combinations were injected into tobacco leaves (5 weeks old) for Agrobacterium-mediated transformation. The injected tobacco seedlings grew for approximately 2 days under normal conditions. The GUS operations were also carried out as indicated by (Ma et al. 2021).

One-way analysis of variance (ANOVA) tests with significant differences of P 0.05 (*) and P 0.01 (**) were used for statistical studies in the statistical software SPSS (version 21.0, USA).

Not applicable.

All authors approve the manuscript and consent to publication of the work.

The authors declare no conflict of interest.