Exploring the Role of TaERF4a in Enhancing Drought Tolerance and Regulating Dehydrin WZY1-2 Gene Expression in Wheat

TL;DR

This study shows that the TaERF4a gene helps wheat tolerate drought by boosting the expression of the WZY1-2 dehydrin gene.

Contribution

The study identifies TaERF4a as a positive regulator of the WZY1-2 gene and demonstrates its role in enhancing drought tolerance in plants.

Findings

TaERF4a overexpression in Arabidopsis improves drought tolerance by increasing SOD activity and chlorophyll content.

Silencing TaERF4a in wheat increases drought sensitivity.

TaERF4a binds to the DRE cis-element in the WZY1-2 promoter to activate its expression.

Abstract

Dehydrins (DHNs) belong to the second family of late embryogenesis abundant (LEA) proteins, which are widely distributed in plants. We cloned a SK3-type DHN gene named WZY1-2 in Zheng yin 1 cultivar of Triticum aestivum. An ERF-type transcription factor TaERF4a was found to be involved in the regulation of the dehydrin WZY1-2 gene in our last report. The stress-responsive ability and dual-luciferase assay demonstrated that TaERF4a positively regulates WZY1-2 gene transcription under stress conditions. In this study, we further characterized the role of the transcription factor TaERF4a in plant drought tolerance. Arabidopsis thaliana heterologously overexpressing TaERF4a exhibited higher survival rate, increased superoxide dismutase (SOD) activity, elevated proline and chlorophyll content, and reduced malondialdehyde (MDA) content under drought conditions. Conversely, silencing TaERF4a in Chinese spring wheat using the virus-induced gene silencing (VIGS) method increased the sensitivity of plants to drought stress. Furthermore, we identified the specific binding site of TaERF4a in the WZY1-2 promoter. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay demonstrated that TaERF4a activates the expression of the WZY1-2 dehydrin gene through binding to the DRE cis-element in its promoter. Taken together, the results of our study indicate that TaERF4a positively regulates the expression of the dehydrin WZY1-2 gene and enhances drought tolerance in plants.

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1. Introduction

As an important staple food crop worldwide, wheat often suffers from many kinds of abiotic stresses during its growth and development periods, which seriously affects yield and quality of wheat. Late embryogenesis abundant (LEA) proteins are a group of stress proteins produced by many organisms under adverse conditions. Dehydrins (DHNs) are members of the second subfamily of LEA proteins that can be classified into five subgroups—Y_n_SK_n_, Y_n_K_n_, SK_n_, K_n_, and K_n_S—based on the number and arrangement of Y-, S-, and K-segments [1]. Due to their richness in polar hydrophilic amino acids, DHNs have a wide range of biological functions, such as preventing cell dehydration, stabilizing cell membranes, binding metal ions, and scavenging hydroxyl radicals [2]. In plants, DHNs are typically found during the late embryonic development of plant seeds and vegetative tissues when subjected to various adversities, including drought, high salinity, low/high temperatures, and other abiotic stresses [3]. The wheat dehydrin WZY1-2 investigated in this study is a SK_3_-type dehydrin belonging to the SK_n_ type.

AP2/ERF proteins are a superfamily of transcription factors widely found in plants. They play important roles in regulating plant growth and development, as well as in responding to abiotic stresses. According to the differences in conserved domains and binding sequences, AP2/ERF transcription factors are divided into five subfamilies: AP2, ERF, DREB, RAV, and Soloist, all of which contain at least one AP2 domain [4,5]. The unique AP2/ERF domain, consisting of approximately 60–70 amino acids, can recognize and participate in DNA binding through two conserved amino acid sequences: the YRG element and the RAYD element [6]. These two elements form the typical three-dimensional (3D) structure within the AP2 domain, which consists of three β-sheets and one α-helix. They specifically bind to different cis-acting elements and interact with other transcription factors (TFs) to regulate gene expression. In plants, AP2/ERFs function as key regulators in multiple abiotic stress responses and hormone signaling pathways [7].

Plants are often subjected to various abiotic environmental stresses, such as drought, high salt, and low temperature during their growth and development. Studies have shown that AP2/ERF transcription factors play an important role in plant resistance to abiotic stress. In particular, the DREB and ERF subfamilies are closely related to abiotic stress. For example, TaDREB, OsDREB1A, GhDBP3, and GmDREB2 genes were all induced by drought, high salt, and low temperature [8,9,10,11]. Overexpression of OsDREB1A and GmDREB2 genes in Arabidopsis improved the resistance of transgenic lines to drought and high salt stress [9,11]. The nuclear-localized protein ERF1 acts as a transcriptional activator that regulates the expression of stress-responsive genes by binding to their GCC box or DRE/CRT elements to mediate physiological responses, including proline biosynthesis and altered stomatal aperture, to enhance tolerance to different stresses [12]. Additionally, ERFs function as either positive or negative regulators of gene transcription [13]. For instance, overexpression of AtERF2 and AtERF4 in Arabidopsis thaliana revealed that AtERF2 acts as a positive regulator, while AtERF4 functions as a negative regulator of JA-responsive defense gene expression [14].

Although DHNs exhibit a variety of cell protective functions, the molecular mechanisms governing their expression have been gradually elucidated in recent years. Some studies reported that dehydrin genes can be regulated by bHLH, ABF, and DREB transcription factors. The drought stress-related bHLH transcription factor TabHLH49 positively regulated the expression of the dehydrin WZY2 gene and improved drought tolerance of wheat [15]. In grape, VviDREBA1-1 was involved in the transcriptional activation of the VviDHN2 gene by binding to the CRT cis-acting element under high CO_2_ levels to maintain table grape quality during storage at 0 °C [16]. Similarly, MsABF2 enhanced alfalfa tolerance to aluminum stress by positively regulating the expression of the MsDHN1 gene [17]. Another research in rice reported that transcription factor OsbZIP23 directly and positively regulated the expression and H3K4me3 modification levels of dehydrin genes under drought stress [18]. In a recent study, the WRKY transcription factor AmWRKY45 was reported to regulate the SK_n_-type dehydrin AmDHN4 from Ammopiptanthus mongolicus, enhancing the tolerance of Arabidopsis to low temperature and osmotic stress [19].

Therefore, we will examine other DHNs to gain a more comprehensive understanding of their regulatory mechanisms. In our previous experiment, we identified an AP2/ERF transcription factor, TaERF4a (GenBank NO.AFP49822.1), that interacts with the wheat dehydrin WZY1-2 gene promoter through a yeast one-hybrid assay [20]. Its positive regulation of the WZY1-2 gene expression was confirmed by dual-luciferase transient expression assay analysis in planta. In this study, we will further explore the role of the TaERF4a transcription factor in plants under abiotic stress and its mechanism underlying transcriptional regulation of the dehydrin WZY1-2 gene in wheat.

2. Results

2.1. Overexpression of TaERF4a Enhances Drought Tolerance in Transgenic Arabidopsis thaliana

To investigate the gene function of TaERF4a, Arabidopsis plants transformed with TaERF4a were used to identify its role in abiotic stress tolerance. The drought resistance of transgenic plants was evaluated at the vegetative growth stage. Before drought treatment, the growth phenotype of the TaERF4a-overexpressing plants at the 3-week-old seedling stage was not significantly different from that of the control plants. Next, the plants were exposed to drought stress by withholding water for 12 days. The leaves of the wild-type (WT) plants were severely wilted, whereas those of the TaERF4a-overexpressing plants were less affected. After the dehydration treatment, all plants were rewatered for 3 days. The WT plants still exhibited the leaf-wilting phenotype and eventually died, whereas the transgenic plants were less damaged, and most of them recovered growth (Figure 1A).

We also measured the root lengths of TaERF4a-overexpressing plants after osmotic stress triggered by PEG treatment. After sowing the seeds of WT and transgenic lines on 1/2 MS agar medium for 5 days, significant differences in root length were observed between the WT and transgenic lines. Moreover, after 7 days of cultivation on 1/2 MS agar medium containing 10% PEG6000, the root growth and development of both WT and transgenic seedlings were inhibited under the same treatment. The WT seedlings exhibited slower growth and shorter root lengths compared to those of the transgenic plants (Figure 2), indicating that the expression of TaERF4a improved root growth of the transformed lines under stress.

2.2. Measurement of Physiological Indices in TaERF4a-Overexpressing Plants Under Drought Stress

We further evaluated chlorophyll content, SOD activity, proline content, and MDA content in WT and TaERF4a-overexpressing plants under both normal and stress conditions (Figure 1B–E). The plants subjected to drought stress exhibited considerable increases in SOD activity and proline content in both WT and TaERF4a-overexpressing plants, but the increase was more pronounced in transgenic plants. The MDA content was significantly lower in transgenic plants compared to WT plants. Additionally, the water loss rate was measured in detached leaves from 4-week-old WT and TaERF4a-overexpressing plants. After being treated for 10 h, the water loss rates in transgenic lines were less than 72.3%, while that in the WT reached up to 84.4% (Figure 1F). This indicated that the transgenic lines had a lower water loss rate and stronger drought tolerance. Additionally, the chlorophyll content was higher in the leaves of TaERF4a-overexpressing lines than in WT plants. Therefore, the expression of TaERF4a appears to improve the tolerance of transgenic Arabidopsis to drought stress.

2.3. Silencing of the TaERF4a Gene Affects the Phenotype and Physiological Indices of Wheat Under Drought Stress Treatment

BSMV-mediated virus-induced gene silencing (VIGS) method was also employed in Chinese spring wheat to investigate the function of the TaERF4a gene. After virus inoculation, wheat leaf photobleaching was observed in both the BSMV:PDS and BSMV:TaERF4a lines, indicating that the endogenous PDS gene was silenced. After 10 days of natural drought, the leaf wilting degree of the BSMV:TaERF4a line was more severe than that of the BSMV:0 and BSMV:PDS lines (Figure 3A). qRT-PCR analysis showed that the gene expression levels of TaERF4a and WZY1-2 in the BSMV:TaERF4a lines were significantly lower than those in the other two lines (Figure 3B). Compared with the BSMV:0 and BSMV:PDS lines, the MDA content in wheat leaves of the BSMV:TaERF4a lines was significantly increased under drought stress, whereas the relative water content and chlorophyll content were significantly reduced (Figure 3C–E).

2.4. Transactivation Analysis of TaERF4a Protein

To verify the interaction between TaERF4a and the dehydrin WZY1-2 gene promoter, the bait yeast strains (Y1H Gold containing pAbAi-Pwzy1-2) transformed with the individual prey vector pGADT7-TaERF4a were able to grow on both SD/-Leu and SD/-Leu/AbA^200^ selective medium, demonstrating the interaction between TaERF4a and the WZY1-2 gene promoter [20]. Moreover, the transactivation ability of TaERF4a was analyzed using the yeast assay system (Figure 4A). Three fragments encoding the full-length TaERF4a, TaERF4a-N (1–80 aa with AP2/ERF domain), and TaERF4a-C (81–195 aa) terminal were inserted into the pGBKT7 vector, respectively. These DNA-binding domain (BD) fusion plasmids were transformed into Y2H Gold yeast cells to confirm their ability to activate the transcription of the α-galactosidase reporter gene. The yeast transformants harboring pGBKT7-TaERF4a-F or pGBKT7-TaERF4a-C grew well and displayed blue colonies on SD/-Trp medium (synthetic dropout nutrient medium) supplemented with 20 μg/mL X-α-Gal and 200 ng/mL Aureobasidin A. In contrast, yeast cells containing pGBKT7-TaERF4a-N failed to grow on SD/-Trp/X-α-Gal/AbA^200^ selective medium.

2.5. EMSA Analysis of TaERF4a Binding to the WZY1-2 Promoter

The ERF gene shares at least one AP2/ERF domain, which is composed of 60 conserved amino acid residues and is involved in DNA binding with the cis-acting elements in the promoter of stress-responsive genes, including the GCC box, dehydration-responsive elements (DREs)/C-repeat elements (CRTs) [21,22]. Therefore, the binding site of TaERF4a transcription factor to the WZY1-2 promoter was next determined. We performed the electrophoretic mobility shift assay (EMSA) to investigate the interaction and binding motif of TaERF4a to the WZY1-2 promoter in vitro. The WZY1-2 promoter was divided into three segments, P1, P2, and P3. As shown in Figure 4B, the transcription factor TaERF4a incubated with the P2 fragment showed an obvious gel migration band, indicating that the TaERF4a protein could strongly bind to the P2 fragment in vitro, whereas no significant affinity was observed after incubation with the P1 or P3 fragment. In addition, we found the DRE motif present in the P2 promoter region, which is a major target of AP2/ERF transcription factors in plant abiotic stress responses [23,24]. Next, we analyzed the binding ability of TaERF4a to the DRE cis-element. As shown in Figure 4C, TaERF4a did not bind to the mutated DRE probe or other motifs, confirming its specific binding to the DRE cis-acting element.

2.6. Dual-Luciferase Assay of TaERF4a in Plants

We demonstrated that TaERF4a can positively regulate the expression of the WZY1-2 dehydrin gene by using the dual-luciferase transient expression assay [20]. To test whether TaERF4a depends on the DRE cis-element to activate the WZY1-2 gene expression, a luciferase assay was also performed in planta. After mixing A. tumefaciens harboring the p35S::TaERF4a (effector vector) with those containing the 2 × DRE-minimal 35S promoter::Rluc (reporter vector 2) or the mutant DRE cis-element 2 × DREm-minimal 35S promoter::Rluc (reporter vector 3), the mixture was instantaneously transformed into N. benthamiana leaves by agro-infiltration and then subjected to luciferase activity analysis (Figure 5). We found that, in the plants transformed with reporter vector 2, the expression of TaERF4a resulted in a more than 4.9-fold increase in luciferase activity (Rluc/Fluc ratio) compared with the GUS control. In contrast, in the plants transformed with reporter vector 3 containing the mutant DRE element, luciferase activity showed a similar level to that of the control.

3. Discussion

Plants evolved a variety of defense mechanisms to counter adverse environmental factors, in which dehydrins play an important role in the plant stress response. We previously isolated and characterized the full-length cDNA, genomic, and promoter sequences of the SK_3_-type dehydrin WZY1-2 in the Zheng yin 1 cultivar of Triticum aestivum [23]. Moreover, TaERF4a was identified as a potential upstream regulator of the WZY1-2 gene through yeast one-hybrid assay. TaERF4a responds to abiotic stress and positively regulates the expression of WZY1-2 dehydrin.

Transcription factors have multiple functions not only in plant growth and development, but also in abiotic stress responses. Studies have shown that the AP2/ERF family of transcription factors is widely involved in regulating plant responses to various abiotic stresses. Kumar et al. found that rice plants overexpressing Os-AP2/ERF-N22 showed higher relative water content, membrane stability index, osmotic potential, stomatal conductance, wax content, and radical scavenging activity [25]. Huang et al. reported that OsERF19-overexpressing lines increased the tolerance of rice plants to salt stress and upregulated the expression of stress-responsive genes, including OsLEA3, OsNHX1, OsHKT6, and OsOTS1 [26]. Under heat stress, AP2/ERF transcription factor ERF1 positively regulates heat tolerance in plants by binding to DRE cis-elements in heat-responsive genes, such as HSFA3 and HSPs, and activating their expression [12]. In addition, studies have shown that the TaERF1 gene can participate in JA, ET, and ABA signal transduction pathways and activate the expression of stress resistance genes. Furthermore, TaERF1-overexpressing Arabidopsis significantly increased its resistance to drought, high salt, and high temperature, while reducing leaf water loss by decreasing stomatal aperture [27].

In this study, we investigated the role of TaERF4a transcription factor in response to plant abiotic stress. Proline, MDA, SOD, and chlorophyll content were measured in WT and transgenic lines under drought stress. These physiological indices in transgenic plants were superior to those in WT plants. In contrast, VIGS-mediated TaERF4a-silencing plants exhibited a drought-sensitive phenotype and lower biochemical indices after exposure to drought stress. These results indicate that TaERF4a has a positive effect on plant drought tolerance. As is well known, drought is one of the most common abiotic stresses for plants, negatively affecting water and nutrient uptake and impeding root growth and development. Plant roots grown in soil can perceive different environmental stresses and adapt their architecture accordingly [28]. Notably, TaERF4a-overexpressing Arabidopsis produced longer roots than WT plants under drought stress conditions. Furthermore, studies reported that the AP2/ERF transcription factors improve stress resistance by regulating the osmotic potential and reactive oxygen species scavenging capacity. For example, BpERF13 upregulates superoxide dismutase (SOD) and peroxidase (POD) genes, thereby enhancing the tolerance of transgenic plants to cold stress [29]. Meanwhile, AP2/ERF transcription factors (TFs) are involved in stress responses by regulating the expression of stress-related genes. For instance, AgDREB1 and AgDREB2 act as transcriptional activators to regulate downstream genes by binding to the corresponding DRE/CRT element, thus enhancing the stress resistance of celery [30]. To elucidate the putative mechanism of TaERF4a on drought stress tolerance, qRT-PCR analyses were performed to examine the expression patterns of stress-responsive genes involved in the ROS-scavenging system, proline biosynthesis, and other pathways in both WT and TaERF4a-overexpressing plants before and after drought stress treatment (Figure 6). The expression levels of drought stress-related and ROS scavenging genes, including copper/zinc superoxide dismutase 1 (CSD1), peroxidase (POD1), Δ-1-pyrroline-5-carboxylate synthase1 (P5CS1), responsive to dehydration 29A (RD29A), and responsive to ABA 18 (RAB18), were upregulated to improve ROS scavenging ability and decrease stress-induced damage. Moreover, the expression levels of these genes were markedly higher in transgenic plants than in WT plants. The expression of these stress resistance genes is consistent with the stress tolerance phenotype of transgenic plants under drought conditions. Therefore, these results suggest that TaERF4a may function positively to activate the expression of stress signaling-related genes in response to drought stress.

Transcription factors participate in plant stress responses by regulating gene expression through binding to downstream stress response gene promoters [31]. Yeast one-hybrid and EMSA assays demonstrated that the TaERF4a protein binds and interacts with the WZY1-2 gene promoter. A luciferase assay showed that TaERF4a positively regulates the expression of the dehydrin WZY1-2 gene, as reported in our previous study. In VIGS-mediated transient expression analysis, the WZY1-2 gene expression level was significantly reduced in BSMV:TaERF4a lines under drought stress compared to the control (Figure 3B). Furthermore, TaERF4a transcription factor binds the DRE cis-acting element in the WZY1-2 promoter to regulate its expression. Thus, these results indicate that TaERF4a is a potential positive regulator of WZY1-2 expression.

As dehydrins are localized to the nucleus, they can also protect nucleic acids and proteins by binding to them against damage from ROS under stress conditions [32]. In our last report, GFP fusion and BiFC assays demonstrated that the dehydrin WZY1-2 localizes to the nucleus as a homodimer [33]. The ability of dehydrin to form dimers at specific subcellular locations in planta can help strengthen its biological function [34]. Additionally, our earlier research revealed that the expression of WZY1-2 dehydrin in wheat increases with drought severity at the seedling stage, suggesting its role in drought stress response [35].

Taken together, we propose a potential model to explain the regulatory mechanism underlying the stress resistance of the WZY1-2 dehydrin in wheat. The transcript level of TaERF4a gene is upregulated under abiotic stress. TaERF4a activates WZY1-2 gene transcription by directly binding to the DRE cis-acting element in the WZY1-2 promoter. The nucleus-localized WZY1-2 then forms a homodimer to mediate the response to abiotic stress in wheat (Figure 7).

4. Materials and Methods

4.1. Plant Growth Conditions

Wheat (Triticum aestivum cv. Chinese spring), Arabidopsis thaliana (ecotype Col-0), and tobacco (Nicotiana benthamiana) were grown in a growth chamber at 22 °C and 60% humidity under 16 h/8 h light/dark cycles with a constant white light intensity of 250 μmol photons·m^−2^·s^−1^.

4.2. Acquisition of TaERF4a-Overexpressing Transgenic Arabidopsis thaliana

The amplified TaERF4a coding sequence was cloned into the Kpn I/Hind III digested plant expression vector pCAMBIA1302. The recombinant plasmid pCAMBIA1302:TaERF4a was then transformed into Agrobacterium tumefaciens strain GV3101 using the electric shock method. Agrobacterium-mediated transformation of Arabidopsis was performed via the floral dipping method to generate TaERF4a overexpression in transgenic plants. Homozygous T3 progeny of the transgenic Arabidopsis plants was selected for further analysis.

4.3. VIGS-Mediated TaERF4a Gene Silencing in Wheat

In vitro transcripts of the Barley stripe mosaic virus vectors BSMV-α and BSMV-β were mixed with the transcripts of BSMV-γ (negative control), BSMV-γ-PDS (positive control), or BSMV-γ-PDS-TaERF4a (experimental group) at an equal ratio of 3 μL each. The transcript mixture was then mixed with 91 μL FES buffer and used for viral inoculation on tobacco leaves. The transcription method followed the protocol of the T7 High Yield RNA Transcription kit (Novoprotein, Suzhou, China), and plant inoculations were performed as previously described [15]. The experiment was repeated three times, and the results were averaged.

4.4. Quantitative Real-Time PCR

Trizol was used to isolate total RNA from the sample, and first-strand cDNA was synthesized from total RNA (1 μg) by using TransScript One-step cDNA Synthesis SuperMix (TransGen, Beijing, China) according to the manufacturer’s instructions. The reaction system was as follows: 2 × TransStart Tip Green qPCR SuperMix 10 μL, Forward Primer (10 μmol/L) 0.4 μL, Reverse Primer (10 μmol/L) 0.4 μL, cDNA template (20 ng/μL) 1 μL, ddH_2_O 8.2 μL. The qPCR reaction was pre-denatured at 94 °C for 4 min, 40 cycles of 94 °C for 30 s, and 60 °C for 30 s, followed by a melting curve analysis. All reactions were performed in triplicate to ensure repeatability of the results, and data analysis was conducted using the 2^−ΔΔCt^ method. Primer sequences are listed in Table S1.

4.5. Performance of TaERF4a-Overexpressing Arabidopsis thaliana Plants Under Drought Conditions

Seeds of the wild-type (WT) and T3 homozygous TaERF4a-overexpressing lines were sown on 1/2 MS agar medium and incubated at 22 °C under 16 h light/8 h dark cycles for 3 days. The germinated seedlings were then transferred to the same medium supplemented with 10% PEG 6000 and grown for another week. Additionally, to investigate the drought tolerance of the transgenic lines, both WT and transgenic seedlings grown under normal conditions for 3 weeks were subjected to water deficit by withholding water for 12 days. The rehydration assay was carried out over the following three days.

4.6. Measurements of Malondialdehyde (MDA), Water Loss Rate, and Chlorophyll Content Physiological Indices

MDA content, SOD activity, and proline content were quantified using commercial detection kits (Jiancheng Bioengineering Institute, Nanjing, China). The water loss rate, relative water content, and chlorophyll content of wheat leaves were measured as previously described [36,37].

4.7. Transactivation Analysis of TaERF4a

The transactivation ability of the TaERF4a protein was analyzed using the Y2H Gold yeast strain. The full-length and two truncated fragments of TaERF4a fused with the GAL4 DNA-binding domain of the pGBKT7 plasmid were transformed into Y2H Gold cells, respectively, and then grown on SD-Trp/X-α-Gal/AbA medium to confirm their transactivation ability.

4.8. Electrophoretic Mobility Shift Assay

The binding of transcription factor TaERF4a with the fragment of WZY1-2 promoter was analyzed by electrophoretic mobility shift assay (EMSA). The WZY1-2 promoter sequence was divided into three segments: P1 (−718 to −462 bp), P2 (−472 to −218 bp), and P3 (−220 to +15 bp). The EMSA was performed using the LightShift^®^ Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. The WZY1-2 promoter fragment was incubated with purified TaERF4a protein at room temperature for 30 min and then separated by 6% native polyacrylamide gel electrophoresis.

4.9. Dual-Luciferase Transient Expression Assay

To investigate whether TaERF4a relies on the DRE element to activate WZY1-2 gene expression, a dual-luciferase assay was conducted in N. benthamiana. The effector vector pCAMBIA1301:TaERF4a was transformed into Agrobacterium tumefaciens strain GV3101. Reporter vector 2 was constructed by inserting two copies of the DRE cis-element upstream of the minimal 35S promoter and ligating it to the dual-luciferase reporter vector pC0390-RUC. Reporter vector 3 was constructed similarly but with two copies of a mutant DRE cis-element. An equal mixture of Agrobacterium transformants containing the effector vector and either reporter vector 2 or vector 3 was injected into tobacco leaves through agroinfiltration. The plants were cultured under normal light conditions for 2 days after being incubated in darkness for 8 h. The infiltrated leaves were collected and frozen in liquid nitrogen for subsequent luciferase activity (RLuc/Fluc) analysis using the Dual-Luciferase^®^ Reporter Assay System (Promega, Fitchburg, WI, USA).