Korean red pepper (C. annuum L., cv. Nockwang) and tobacco (Nicotiana benthamiana) seeds were sown in loam, sand, and a compost mix of soil (vermiculite, perlite, and, peat moss, 2:3:5, v/v/v; 1:1:1, v/v/v). Plants were placed in growth room at 24±1°C with 60% relative moisture under white fluorescent light (130μmol photons·m⁻²·s⁻¹) with a 16-h light/8-h dark cycle.

To generate the CaPRR2-silenced pepper plants, a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) technique was employed, as described by Lim et al. (2018). Using the VIGS tool,¹ two 300-bp fragments of the CaPRR2 cDNA, CaPRR2 N1 (1,052–1,351), and CaPRR2 N2 (1,361–1,660) were designed to avoid off-target of silencing; each region was subsequently amplified by PCR using the primers XbaI-CaPRR2 N1 (5ʹ-TCTAGATGAAAGTAGAAGGCCTGACAA-3ʹ) and XhoI-CaPRR2 N1 (5ʹ-CTCGAGTCTCGGGTGGTTGCCAT-3ʹ) or XbaI-CaPRR2 N2 (5ʹ-TCTAGAGGAATCCTCACTCTGGACTGTAT-3ʹ) and XhoI-CaPRR2 N2 (5ʹ-CTCGAG GAGAACCGTTGATGCGTG-3ʹ). Agrobacterium tumefaciens strain GV3101 carrying pTRV1 and pTRV2:CaPRR2 N1, pTRV2:CaPRR2 N2, or pTRV2:00 as a negative control was co-infiltrated into the fully expanded cotyledons of pepper plants (OD₆₀₀=0.2 for each construct). Infected plants were placed in a growth room and maintained under the growth conditions described above for growth and spread of the virus.

Total RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed as described previously with some modifications (Lim and Lee, 2016; Lim et al., 2018). Pepper leaves at six-leaf stage were treated with ABA (100μM), H₂O₂ (100mM), mannitol (600mM), low temperature (10°C), NaCl (200mM), or drought stresses. cDNA was synthesized using harvested pepper leaves by a Transcript First Strand cDNA Synthesis kit (Roche, Indianapolis, IN, United States). The cDNA synthesized for quantitative real-time polymerase chain reaction (qRT-PCR) assay was amplified in a CFX96 Touch™ Real-Time PCR detection system (Bio-Rad, Hercules, CA, United States) using iQ™SYBR Green Supermix and specific primers (Supplementary Table S1). The relative expression level of each gene was calculated using the ∆∆Ct method as previously described (Livak and Schmittgen, 2001). The pepper Actin1 (CaACT1) gene was used for normalization (Lim and Lee, 2016).

Using the deduced amino acid sequence of CaPRR2 gene as query, its isoelectric point and a molecular weight were calculated in web tool Expasy Compute pI/MW.² Protein sequences of CaPRR2-homologous genes from other plant species were obtained through BLASTP search and were used for multiple sequence alignment analysis using web tool Clustal Omega.³ The phylogenetic tree analysis was conducted using MEGA software (version 7.0) with the neighbor-joining method.

To determine the subcellular location of CaPRR2, the full-length coding sequence (1–557) and fragments of CaPRR2 (1–151, 152–291, 292–451, 452–557, and 332–451bp) without the stop codon were inserted into the p326GFP vector. The GV3101 strain of Agrobacterium tumefaciens containing each construct was mixed with strain p19 (1:1 ratio; OD₆₀₀=0.5) and co-inoculated into fully expanded leaves of 5-week-old N. benthamiana. After 2days, the green fluorescent protein (GFP) signals were detected under a confocal microscope (510 UV/Vis Meta; Zeiss) equipped with LSM Image Browser software.

Three-week-old control and CaPRR2-silenced pepper plants were subjected to drought stress by withholding water for 14days. The survival rate was measured by counting the plant number resumed their growth 2days after re-watering. To determine the water loss, the first and second leaves were detached from both pepper plants. The fresh weights of the detached leaves were measured hourly. For the analysis of the relative water content (RWC), the first leaves were detached from 3-week-old pepper plants of each line, and the fresh weight (FW) was measured. The leaves were incubated under turgid conditions for 8h, and the turgid weight (TW) was measured. The dry weight (DW) was measured after drying in a 60°C dry oven for 4days. The RWC of each plant line was calculated using the formula [RWC=(FW−DW)/(TW−DW)×100]. For qRT-PCR analysis, 3-week-old TRV2:CaPRR2 and TRV2:00 plants were carefully removed from the soil and subjected to drought stress. After 6h, the first and second leaves of each plant line were harvested, and total RNA was isolated. The experiments were repeated three times.

Measurements of the leaf temperature and stomatal pore size were performed as previously described (Lim and Lee, 2016). Six-leaf stage pepper plants were used for the thermal image analysis. Thermal images were taken before and after treatment with 0 and 100μM ABA for 6h using an infrared camera (FLIR system; T420), and leaf temperature was measured with FLIR Tools + version 5.2 software.

TRV2:CaPRR2 and TRV2:00 leaf peels of the first and second leaves were floated on stomatal opening solution (SOS: 10mM CaCl₂, 10mM MES-KOH, pH 6.15, and 50mM KCl) under light conditions for 2.5h. After 2.5h, the leaf peels were transferred to fresh SOS containing 0 and 20μM ABA and incubated for an additional 2.5h. For measurement of the stomatal apertures, 100 stomata per sample were observed under a Nikon Eclipse 80i microscope. Each experiment was performed independently three times.

Three-week-old CaPRR2-silenced and control pepper plants were hydroponically subjected to salt stress using water containing 200mM NaCl. The survival rate was calculated by counting the number of plants 4days after salt stress treatment. To determine the chlorophyll content, various concentrations of NaCl solution were applied to the leaf discs of TRV2:CaPRR2 and TRV2:00 plants. Five days after salt stress treatment, the leaf disc chlorophyll content was measured spectrophotometrically, as described previously (Lim et al., 2015). For qRT-PCR analysis, 3-week-old TRV2:CaPRR2 and TRV2:00 plants were hydroponically subjected to salt stress using water containing 200mM NaCl. After 6h, the first and second leaves of each plant line were harvested, and total RNA was isolated. All experiments were repeated three times.

Three-week-old CaPRR2-silenced and control pepper plants were subjected to salt stress by hydroponically incubating them to various concentrations of NaCl solution. Proline contents of harvested leaf samples were determined by a ninhydrin-based colorimetric assay at 520nm as described by Abrahám et al. (2010). Malondialdehyde (MDA) contents were measured by thiobarbituric acid (TBA) assay as previously described (Heath and Packer, 1968; Lim et al., 2015).

Statistically significant differences between genotypes were determined using Student’s t test. Results were considered significant at p<0.05.

To isolate PRR genes from pepper plants, we conducted BLASTP search using amino acid sequences of nine Arabidopsis PRR (APRR) genes as queries and found 12 gene loci for putative CaPRR in pepper plants. To determine their genetic relation, we performed a phylogenetic tree analysis with the deduced amino acid sequences of 12 CaPRR genes and 9 APRR genes and found that these genes were simply clustered into two groups (Figure 1A). There were one or two candidate genes of PRR homolog, corresponding to APRR1, APRR2, APRR3, APRR5, APRR7, and APRR9, in the pepper genome. Of the APRRs, it has been suggested that APRR2 may be associated with plant responses to abiotic stress, such as salt and drought, based on the interaction with CML9 (Perochon et al., 2010). Hence, we selected CA06g13040 as APRR2 homolog for further study and named CaPRR2; in protein sequence, CA06g13040 shares 48.2% identity/59.5 similarity with APRR2, but CA00g25810 shares 44% identity/55.7% similarity. The CaPRR2 gene consists of a 1,674-bp open reading frame, encoding 557 amino acid residues with an isoelectric point of 6.17 and a molecular weight of 61.81kDa. Multiple sequence alignment analysis revealed high amino acid sequence identity (48.2–85.1%) and similarity (59.4–89.3%) between CaPRR2 and other plant PRR2 proteins (Supplementary Figure S1). CaPRR2 contains a highly conserved cheY-homologous receiver domain and an MYB-like DNA-binding domain.

To investigate the organ-specific expression of CaPRR2, we examined CaPRR2 transcript levels in various pepper plant tissues using quantitative RT-PCR analysis. At the seedling and mature stages, CaPRR2 was strongly expressed in young leaf and stem, and compared to these organs, the others, such as root, flower, and green fruit, had <50% expression levels (Figure 1B). To investigate whether CaPRR2 is associated with the environmental stress response, we examined the induction of CaPRR2 transcripts after exposure to ABA, drought, H₂O₂, mannitol, NaCl, and low temperature (Figure 1C). The CaPRR2 transcripts tended to decrease 3h after exposure to ABA, drought, and mannitol by low than 50%. In response to H₂O₂ and NaCl treatment, the CaPRR2 expression levels were peaked (1.6 to 2-fold) at 12h. Low-temperature exposure led to gradual induction of CaPRR2, particularly at 12h, with 5-fold increase. These results suggest that CaPRR2 may be involved in the abiotic stress signaling.

To investigate the subcellular localization of CaPRR2 in plant cells, the GFP reporter gene was fused to the C-terminal region of CaPRR2 under the control of the 35S promoter (Pro35S:CaPRR2-GFP), and the GFP-fused proteins were transiently expressed in the epidermal cells of N. benthamiana. Using the nuclear marker, diamidino-2-phenylindole, we showed that the GFP signals were localized in the nucleus (Figure 2A). To investigate the domain that determines CaPRR2 localization in the nucleus, we fractionated CaPRR2 and observed the localization of the resulting constructs (Figure 2B). The deletion construct carrying the MYB domain (292–451 amino acid residues) was localized only in the nucleus. To verify whether the MYB domain is associated with CaPRR2 localization in the nucleus, we fractionated this construct, except for the MYB domain (322–451 amino acid residues; Figure 2C). The resulting construct was localized in the nucleus and the cytoplasm. These results suggest that CaPRR2 is localized in the nucleus by the MYB domain and functions in the nucleus.

The expression levels of CaPRR2 varied according to different stress treatments; hence, CaPRR2 is likely involved in abiotic stress signaling. We performed a phenotypic analysis of pepper plants using VIGS assays (Figures 3, 4). First, we generated two VIGS constructs – CaPRR2 N1 (1,052–1,351bp) and CaPRR2 N2 (1,361–1,660bp) – in the CaPRR2 gene. Using RT-PCR analysis, we verified that the expression levels of CaPRR2 were significantly lower in the CaPRR2-silenced pepper plants (TRV2:CaPRR2 N1 and TRV2:CaPRR2 N2) than in the control pepper plants (TRV2:00; Supplementary Figure S2). To examine the drought response of CaPRR2-silenced pepper plants, we withheld the watering of TRV2:CaPRR2 and TRV2:00 plants for 14days and then re-watered the plants for 2days (Figure 3A). Under well-watered and drought stress conditions, we observed no phenotypic differences between both plant lines (Figure 3A, left panel). However, after re-watering for 2days, TRV2:CaPRR2 pepper plants showed a less wilted phenotype than TRV2:00 plants (Figure 3A, right panel). The survival rates of TRV2:CaPRR2 and TRV2:00 pepper plants were 58.3–61.1 and 30.5%, respectively (Figure 3B). We wondered whether this drought-tolerant phenotype of CaPRR2-silenced pepper plants is derived from alteration of water status. Since leaf relative water content is widely used as an indicator of water stress in plants, we monitored a change in the relative water content from TRV2:CaPRR2 and TRV2:00 plants in response to drought stress. As shown in Figure 3C, TRV2:CaPRR2 plant lines displayed significantly higher relative water content at all time points after drought treatment than TRV2:00 plants. We also evaluated the transpirational water loss by measuring the leaf fresh weights of TRV2:CaPRR2 and TRV2:00 pepper plants. At various time points after detachment, the transpirational water loss was significantly lower in TRV2:CaPRR2 than in TRV2:00 plants, consistent with a decrease in the relative water content (Figure 3D).

More the 99% of total water loss from leaf occurs through stomata (Kane et al., 2020). Plants close stomata in response to water deficit, and it is well-known that phytohormone ABA is involved in this process (Cutler et al., 2010). Based on these, we hypothesized that the drought-tolerant phenotype displayed by CaPRR2-silenced pepper plants may be caused by altered ABA sensitivity. To prove this, we measured leaf temperatures and stomatal apertures before and after ABA treatment. At 6h after ABA treatment, leaf temperatures were significantly higher in TRV2:CaPRR2 than in TRV2:00 plants (Figures 3E,F). Moreover, at 3h after ABA treatment, stomatal apertures of TRV2:CaPRR2 were significantly smaller than those of TRV2:00 plants (Figures 3G,H). Taken together, these results revealed that CaPRR2 functions as a negative regulator of drought stress by regulating ABA-induced stomatal closing.

Next, we explored whether the biological role of CaPRR2 is associated with the response to salt stress. To conduct phenotypic analyses under salt stress conditions, 3-week-old TRV2:CaPRR2 and TRV2:00 plants were subjected to salt stress by hydroponically growing them in 200mM NaCl solution. In the absence of NaCl, we observed no phenotypic differences between TRV2:CaPRR2 and TRV2:00 plants (Figure 4A, left panel). However, when both plant lines were subjected to salt stress for 5days, TRV2:CaPRR2 exhibited a less wilted phenotype than TRV2:00 plants (Figure 4A, right panel). The survival rates of TRV2:CaPRR2 were 66.6–75%, whereas only 33.3% of TRV2:00 plants resumed their growth (Figure 4B). As shown in Figure 4A, salt stress triggered leaf senescence; the leaves of both plant lines treated with salt stress turned to pale yellow, compared to the non-treated plants. To evaluate this difference quantitatively, we compared the chlorophyll content between TRV2:CaPRR2 and TRV2:00 plants using leaf discs from plants exposed to salt stress (Figure 4C). Consistent with the salt-tolerant phenotype, TRV2:CaPRR2 plants exhibited significantly higher chlorophyll contents than TRV2:00 plants (Figure 4D). Plants resist osmotic stress by induced salt stress through production of various osmoprotectants, including proline (Nuccio et al., 1999). Measurement of proline content from TRV2:CaPRR2 and TRV2:00 plants treated with salt stress revealed that proline content after treatment was significantly higher in TRV2:CaPRR2 than in TRV2:00 (Figure 4E). Salt stress can induce membrane lipid peroxidation (Katsuhara et al., 2005); hence, we also measured MDA contents from TRV2:CaPRR2 and TRV2:00 plants under salt stress conditions. MDA was highly accumulated in both plant lines by salt stress, but TRV2:CaPRR2 had lower MDA contents than TRV2:00 (Figure 4F). These results indicate that CaPRR2 plays a negative role in salt tolerance.

Several previous studies have suggested that stress-related genes regulate osmotic tolerance (Zhang et al., 2006; Tran et al., 2007; Aubert et al., 2010). When the expression levels of stress-related genes are altered, osmotic tolerance increases or decreases. CaPRR2 expression was negatively correlated with drought and salt tolerance. We wondered how silencing of CaPRR2 gene affects the expression levels of stress-related genes in response to drought and salt stress. To test this, we performed qRT-PCR analysis of stress-related genes – including CaNCED3, CaOSR1, and CaRAB18 – in the leaves of pepper plants that had been subjected to drought and salt stress (Figure 5). After drought stress treatment, CaOSR1 and CaRAB18 genes in TRV2:CaPRR2 leaves showed approximately 1.7- to 2.6-fold and 1.5- to 1.8-fold increase, respectively, compared to those in TRV2:00 leaves. Similarly, salt stress significantly induced these genes; its levels were >2.5-fold higher in TRV2:CaPRR2 than in TRV2:00. In contrast, CaNCED3 expression was significantly induced by the drought treatment but not by the NaCl treatment. Drought stress induced expression level of CaNCED3 to 1.5- to 2-fold in TRV2:CaPRR2 compared to in TRV2:00. These data suggest that a reduced expression of CaPRR2 affects the expression levels of stress-related genes, and this likely affects the osmotic stress response.