To isolate a drought stress inducible gene, we performed RNA-seq assay and identified the pepper CaDIF1 gene from drought stress applied pepper leaves (Lim and Lee, 2016). The CaDIF1 consists of 282 amino acid, which are encoded by an 849 bp coding region ( Figure 1A ). The isoelectric point and molecular mass of CaDIF1 protein was 8.03 and 31,548 Da, respectively. The putative CaDIF1 protein contains highly conserved F-box and EID1-Like Protein (ELP) domains, including amino acid residues 30–86 and 125-252, respectively ( Figure 1A ). The amino acid sequence analysis of CaDIF1 by multiple alignment ( Figure 1A ) and phylogenetic tree analysis ( Figure 1B ) revealed that this protein has high homology with F-box proteins from other plant species ( Figures 1A, B ). In particular, CaDIF1 shares 50.3–77.0% identities and 67.1–85.1% similarities with other F-box proteins.

The CaDIF1 gene was identified in drought stress-treated pepper plants; therefore, we investigated whether the expression level of CaDIF1 was increased by drought, ABA, H₂O₂, and NaCl treatments ( Figure 2A ). First, we investigated the expression level of CaDIF1 transcripts in pepper leaves at different time points after drought stress treatment. The CaDIF1 transcripts dramatically induced at all times measured. ABA is known to function as a key modulator in the defence response to water deficit condition (Jakab et al., 2005). To examine whether ABA is also associated with CaDIF1 expression, we treated pepper plants with ABA and monitored accumulation of CaDIF1 transcripts. The CaDIF1 transcripts were strongly accumulated after ABA treatment. H₂O₂ functions as a signal component in ABA-mediated stomatal closure (Zhang et al., 2001); hence, we determined the expression level of CaDIF1 after H₂O₂ treatment. The induction of CaDIF1 transcripts were detected at 2 h after treatment and gradually increased until 24 h. High salinity treatment induced weak accumulation of CaDIF1 transcripts.

To determine the subcellular localization of CaDIF1 protein in the plant cell, GFP-tagged CaDIF1 fusion protein was transiently expressed in the epidermal cells of Nicotiana benthamiana. GFP signals were generated in the nucleus ( Figure 2B ). We also detected that the blue signals localized to the nucleus overlapped with the GFP signals by using diamidino-2-phenylindole (DAPI) staining. These results suggest that the CaDIF1 has a functional role in the nucleus.

CaDIF1 was induced by drought and ABA treatment; therefore, we postulated that this gene is involved in drought response and ABA signaling. To confirm this prediction, we conducted phenotypic analysis using loss-of function and gain-of function mutants by virus-induced gene silencing (VIGS) in pepper and generating transgenic Arabidopsis plants, respectively. We monitored CaDIF1 silencing by measurement of CaDIF1 transcripts in control (TRV2:00) and CaDIF1-knock down pepper (TRV2:CaDIF1) plants. The accumulation of CaDIF1 transcripts was low in the CaDIF1-knock down pepper leaves compared with control leaves ( Supplementary Figure S1B ). We subjected control and CaDIF1-knock down pepper plants to drought stress and compared their phenotypes ( Figure 3A ). We withheld water (11 days; middle panel) and resupply water (2 days; right panel) and assessed growth status in both plants. After treatment of drought stress and re-watering, leaf wilting phenotypes were more appeared in CaDIF1-knock down pepper plants than in control plants. The survival ratios of CaDIF1-knock down pepper plants (19.3%) were much lower than control pepper plants (71.8%) ( Figure 3B ). Previous studies suggested that water retention capacity is associated with drought tolerance; hence, the drought-sensitive phenotype of CaDIF1-knock down pepper plants is probably caused by altered water retention capacity. To asses this prediction, we monitored the fresh weight of leaves detached from control and CaDIF1-knock down pepper plants ( Figure 3C ). The leaf fresh weight of CaDIF1-knock down pepper plants was low compared with that of control plants. To examine whether the difference in water retention capacity was derived from a variation of ABA sensitivity, we measured leaf temperatures ( Figures 3D, E ). Leaf temperature shows the strength of water evaporation by transpiration, with a low left temperature reflecting high level of transpiration (Park et al., 2015). In the ABA untreated condition, leaf temperatures were not significantly different between control pepper and CaDIF1-knock down pepper plants ( Figures 3D, E ). However, after ABA treatment, the leaf temperatures of CaDIF1-knock down pepper plants were significantly low compared with control pepper plants. Consistent with leaf temperatures, the stomatal apertures were significantly larger in CaDIF1-silenced pepper plants than in control plants ( Figures 3F, G ). These results indicate that the increased transpirational water loss in CaDIF1-knock down pepper plants, which leads to reduced drought tolerance, is derived from ABA hyposensitivity.

To further investigate the in vivo function of CaDIF1, we generated transgenic Arabidopsis plants, exhibiting overexpression of the CaDIF1 gene. A real-time transcription PCR (RT-PCR) assay revealed that the accumulation of CaDIF1 transcripts was high in CaDIF1-OX plants, but not detected in the wild-type plants ( Supplementary Figure S1B ); hence, CaDIF1-OX plants were used in our subsequent phenotypic analysis. Under normal growth conditions, we observed no phenotypic differences between the wild-type and CaDIF1-OX plants ( Figures 4 and 5 ). To investigate whether altered expression of CaDIF1 affects the ABA response, we examined germination rate, primary root growth, and seedling establishment in the presence and absence of ABA ( Figure 4 ). CaDIF1-OX seeds were germinated on MS medium supplemented with 0.0 and 1.0 µM ABA. In the presence of ABA, the germination rate of CaDIF1-OX seeds at 2 days after sowing was significantly lower than that of wild-type seeds ( Figure 4A ). We measured the primary root lengths in wild-type and CaDIF1-OX plants at 8 days after sowing ( Figures 4B, C ). In the absence of ABA, the root lengths did not differ significantly between wild-type and transgenic plants; however, in the presence of 1.0 µM ABA, the roots of CaDIF1-OX plants were significantly shorter than those of wild-type plants ( Figures 4B, C ). Consistent with root growth, seedling establishment of CaDIF1-OX plants was significantly lower than that of wild-type plants ( Figures 4D, E ). These data suggest that altered CaDIF1 expression affects ABA response in germination and post-germination stages.

CaDIF1 was markedly induced by drought stress treatment ( Figure 2A ), and CaDIF1-knock down pepper plants showed an altered phenotype to drought stress ( Figure 3 ). Hence, we examined the in vivo role of CaDIF1 in response to drought stress ( Figure 5 ). First, we investigated the drought phenotype of CaDIF1-OX plants by withholding water for 14 days and then re-watering for 1 day ( Figure 5A ). Under well-watered conditions, we were not able to observe any phenotypic differences between CaDIF1-OX plants and wild-type plants ( Figure 5A , upper panel). However, under conditions of drought stress, CaDIF1-OX plants exhibited a less wilted phenotype than wild-type plants ( Figure 5A , middle panel). Furthermore, upon re-watering, the CaDIF1-OX plants resumed their growth rapidly compared with the wild-type plants ( Figure 5A , lower panel). At 1 day after re-watering, the survival ratio of CaDIF1-OX plants was 51.8–54.2%, whereas only 18.8% of wild-type plants resumed their growth ( Figure 5B ).

To find why CaDIF1-OX plants had drought tolerance phenotype, we measured the transpirational water loss, leaf temperature, stomatal aperture, and stress-related gene expressions. We monitored transpirational water loss by measuring the fresh weights of detached rosette leaves ( Figure 5C ). We found that the transpirational water loss was significantly lower in CaDIF1-OX plants than in the wild-type plants, suggesting that the enhanced drought tolerance of CaDIF1-OX plants was derived from a reduced transpiration rate. To determine whether low transpirational water loss in CaDIF1-OX plants was caused by increased ABA sensitivity, we measured stomatal aperture and leaf temperature after treatment with various concentrations of ABA. First, we estimated significant differences in stomatal pore sizes between wild-type and CaDIF1-OX plants after ABA treatment ( Figures 5D, E ). ABA treatment triggered a reduction in the stomatal aperture in wild-type and CaDIF1-OX plants; however, transgenic plants displayed smaller stomatal apertures than wild-type plants. These results indicate that low transpirational water loss in CaDIF1-OX plants was derived from enhanced ABA sensitivity, especially in the stomata. Next, we measured the leaf temperatures of wild-type and CaDIF1-OX plants. Consistent with the stomatal aperture, CaDIF1-OX plants exhibited significantly higher leaf temperatures than wild-type plants ( Figures 5F, G ).

In a previous studies, drought sensitivity and tolerance is related with the transcripts accumulation of stress-related genes, and that increased or decreased drought tolerance are related with altered expression of these genes (Cheong et al., 2007; Joo et al., 2019). Hence, we performed quantitative RT-PCR analysis of stress-related genes, including NCED3, DREB2A, RAB18, RD20, RD29A, and RD29B, and group A 2C type protein phosphatases (PP2Cs), such as ABI1, ABI2, and HAB1 ( Figure 6 ). The transcripts accumulation of stress-related genes were significantly high in CaDIF1-OX plants compared with wild-type plants. However, the expression level of 2C type protein phosphatases are not different between both plants. Collectively, these data indicate that enhanced expression of CaDIF1 increases drought tolerance by reducing stomatal pore size and enhancing stress-related gene expression.

To determine whether CaDIF1 is a component of SCF1 type E3 ligase complex, we performed Y2H assay using five pepper SKP proteins as prey and CaDIF1 as bait ( Figure 7A ). Yeast cell transformed with CaDIF1 and XP_016539969.1 grew on selective media, indicating that XP_016539969.1 interacts with CaDIF1 in yeast cells. We performed further analysis by designating XP_016539969.1 as CaDIS1 (Capsicum annuum DIF1-Interacting SKP 1). To confirm the interaction and localization of CaDIF1 and CaDIS1 in vivo, a bimolecular fluorescence complementation (BiFC) assay was performed ( Figure 7B ). The yellow fluorescence derived from co-expression of CaDIF1:VYNE with CaDIS1:CYCE localized constitutive strong nuclear and cytosolic localization. Consistent with these data, an in vitro pull-down assay verified that CaDIF1 interacts directly with CaDIS1 ( Figure 7C ). Taken together, these data demonstrate that CaDIF1 is able to interact with CaDIS1.

The CaDIS1 protein consists of 156 amino acids and have a conserved SKP1-like protein domain (including 4–106 amino acid residues) ( Supplementary Figure S2A ). Protein sequence assays showed that CaDIS1 has high homology with SKP1 proteins from other plants ( Supplementary Figures S2A, B ). Because the CaDIS1 is able to interact with CaDIF1, CaDIS1 plays a role as a positive regulator in ABA and drought signaling, we determined whether the transcripts accumulation of CaDIS1 is induced by ABA or abiotic stress treatments. Drought treatment induced CaDIS1 expression; however, unlike the expression patterns of CaDIF1, the CaDIS1 transcripts did not accumulate more strongly after treatment with ABA, H₂O₂, or high salinity ( Supplementary Figure S3A ). When the fusion proteins of CaDIS1-GFP was expressed in the N. benthamiana epidermal cells, this protein was also localized to the nucleus, and cytoplasm ( Supplementary Figure S3B ).

CaDIF1-knock down pepper plants displayed a sensitive phenotype to drought stress ( Figure 3 ); therefore, we also postulated that CaDIS1 is involved in the defence signaling to drought stress. To confirm this prediction, we conducted phenotypic analysis by VIGS assay in pepper plants ( Figure 8 ). We examined CaDIS1 knock down by RT-PCR assay of control (TRV2:00) and CaDIS1-knock down pepper (TRV2:CaDIS1) plants. The CaDIS1 transcripts were lowly accumulated in the CaDIS1-knock down pepper leaves compared with the control leaves ( Supplementary Figure S1C ). To subject drought treatment to control and CaDIS1-knock down pepper plants, we withheld water for 11 days and re-watering for 2 days ( Figure 8A ). In comparison with control plants, CaDIS1-knock down pepper plants showed a wilted phenotype after drought stress and showed reduced drought tolerance after re-watering. The survival ratio of CaDIS1-knock down pepper plants was 36.9%, while the survival ratio of control pepper was approximately 48.7% ( Figure 8B ). Next, the transpirational water loss was compared by estimating the leaf fresh weights detached from control and CaDIS1-knock down pepper plants ( Figure 8C ). As same with the drought-sensitive phenotype, CaDIS1-knock down pepper leaves exhibited dramatically lower fresh weight than control leaves. Next, we measured leaf temperatures and stomatal apertures in the presence and absence of ABA ( Figures 8D, G ). In the absence of ABA, we were not able to detect different leaf temperatures significantly between control pepper and CaDIS1-knock down pepper plants ( Figures 8D, E ). However, after treatment with 100 µM ABA, the leaf temperatures of CaDIS1-silenced pepper were significantly lower than those of control plants. Consistent with leaf temperatures, the stomatal apertures were significantly larger in CaDIS1-silenced pepper than in control plants. ( Figures 8F, G ). These data demonstrate that the guard cells constituting stomatal pore of CaDIS1-knock down pepper plants display ABA hyposensitivity, and altered ABA sensitivity probably lead to reduced water retention in drought stress conditions.

Seeds of pepper (C. annuum L., “Nockwang”), tobacco (N. benthamiana), and A. thaliana (ecotype Col-0) were sown in a steam-sterilized compost soil mix (peat moss, perlite, and vermiculite, 5:3:2, v/v/v), sand, and loam soil (1:1:1, v/v/v). The plants were raised at 25 ± 1°C under a 16 h light/8 h dark photoperiod (130 μmol photons·m⁻²·s⁻¹). Arabidopsis [wild-type (ecotype Col-0) and CaDIF1-OX] seeds were grown on MS medium containing 1% sucrose. Details of plasmid construction and transgenic plants were described below.

VIGS analysis was performed to knockdown CaDIF1 and CaDIS1 in C. annuum (pepper) as described previously (Lim et al., 2017). A 254 bp (1–254 nucleotide sequences) fragment of the CaDIF1 or a 299 bp (2–300 nucleotide sequences) fragment of the CaDIS1 cDNA was inserted into the pTRV2 vector and introduced into Agrobacterium tumefaciens strain GV3101 via electroporation. A. tumefaciens strain GV3101 containing pTRV1, pTRV2:00, pTRV2:CaDIF1, or pTRV2:CaDIS1 was infiltrated to the fully expanded pepper cotyledons (OD₆₀₀ = 0.2, each from different construct).

Agrobacterium transformation into the A. thaliana was conducted by using the floral dip method (Clough and Bent, 1998); all mutants generated in this study were in the Col-0 background. For CaDIF1-OX, full-length cDNA sequences were integrated into the pBIN35SGFP binary vector. For selection of overexpressing plants, we used 25 μg·ml⁻¹ phosphinothricin.

To analyze CaDIF1 and CaDIS1 expression in pepper plants, we treated with ABA (100 μM), H₂O₂ (100 μM), drought, or NaCl (200 mM) as described previously (Lim et al., 2015a; Lim et al., 2015b). Pepper leaves were harvested at 0, 2, 6, 12, and 24 h after each treatment. For the determination of ABA sensitivity, we measured the seed germination rates, cotyledon development, and primary root lengths on ABA containing MS plants. For drought sensitivity analysis, we used 4-week-old pepper and 3-week-old Arabidopsis plants and subjected to drought stress by withholding water for 11 and 14 days, respectively. After re-watering for 2 days and 1 day, respectively, we calculated the survival. To determine drought sensitivity or tolerance, transpirational water loss was measured. Pepper and Arabidopsis rosette leaves were detached, and the reduced fresh weight was measured for each time points. For quantitative RT-PCR assay, 4-week-old CaDIF1-OX transgenic Arabidopsis plants were subjected to drought stress. All the experiments were conducted three times.

Leaf temperatures and stomatal apertures were measured as described previously (Lim and Lee, 2016). Briefly, A. thaliana and pepper leaf peels were floated in buffer with 10 μM CaCl₂, 10 mM MES–KOH (pH 6.15), and 50 mM KCl, in day condition. After incubation for 3 h, stomata begins to close by replacing the buffer with 10 and 20 μM of ABA. Then after 2.5, 100 stomata apertures per 10 leaves were measured under a Nikon Eclipse 80i microscope. To analyze the thermal imaging, we used full expanded first and second leaves of pepper plants and Arabidopsis were treated with 100 μM ABA. For measurement of leaf temperatures, we used infrared camera (T420; FLIR) equipped with FLIR ver 5.2 software. All the experiments were conducted three times.

To investigate subcellular localization and BiFC assay of CaDIF1 and CaDIS1, we used the open reading frame of CaDIF1, CaDIS1, and pBIN35SGFP, Pro-35S:VYNE, and Pro-35S:CYCE vectors, which express the proteins driven by the 35S promoter. The different constructs were introduced into the A. tumefaciens strain GV3101 and selected by 50 μg·ml⁻¹ kanamycin. These cells were combined with the silencing suppressor p19 and introduced into the N. benthamiana epidermal cells by using agroinfiltration method. At 3 days after infiltration, leaf discs were cut, and the lower epidermal cells were examined under a confocal microscope (510 UV/Vis Meta; Zeiss) equipped with LSM Image Browser software.

Yeast-two hybrid (Y2H) assays were performed as described previously (Lim et al., 2017). The cDNA fragments of CaDIF1 or CaDIS1 were subcloned into the pGBKT7 or pGADT7 vectors, respectively, and co-transformed into yeast strain AH109 using the lithium acetate-mediated transformation method (Ito et al., 1983). To evaluate the interaction between bait and prey proteins, transformant candidates were selected on SC-Leu-Trp medium. Next, 10-fold serial dilutions were prepared from each yeast cell culture (OD₆₀₀ = 0.5), and 5 μl of each sample was spotted onto SC-Leu-Trp medium or SC-Ade-His-Leu-Trp medium. Yeast colonies were incubated at 30°C for 5 days.

For expression of the maltose‐binding protein (MBP) and glutathione‐S‐transferase (GST) recombinant proteins, the pMAL‐c2X (New England Biolabs) and pGEX‐4T‐3 (GE Healthcare) vectors were respectively used. Each vector, harboring the full‐length cDNA sequence of CaDIS or CaDIF1, was introduced into Escherichia coli strain BL21 cells. The fusion proteins were induced and purified according to the manufacturer’s instructions.

For the pull‐down assay, 5 μg of GST or GST‐CaDIF1 were incubated with 5 μg of MBP or MBP‐CaDIS1 for 2 h at 4°C on constant rocking in 0.5 ml of binding buffer (20 mM Tris–HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5% Tween‐20) containing GST resin. After boiling at 97°C for 5 min, eluted proteins were analyzed using SDS‐PAGE, followed by western blotting and immunodetection with anti‐MBP and anti‐GST antibodies.