Overexpression of the Brassica rapa bZIP Transcription Factor, BrbZIP-S, Increases the Stress Tolerance in Nicotiana benthamiana

Simple Summary Energy homeostasis plays a crucial role in regulating plant defense responses. In this study, we characterized BrbZIP-S (S1-bZIP from Brassica rapa) as a key modulator of energy metabolism, including sugar and proline metabolism. In addition, plants overexpressing BrbZIP-S exhibited increased tolerance to darkness and heat stress, suggesting that BrbZIP-S regulates plant stress responses through a complex network mediated by abscisic acid, sugar, and proline. Abstract In higher plants, S1-basic region-leucine zipper (S1-bZIP) transcription factors fulfill crucial roles in the physiological homeostasis of carbon and amino acid metabolisms and stress responses. However, very little is known about the physiological role of S1-bZIP in cruciferous vegetables. Here, we analyzed the physiological function of S1-bZIP from Brassica rapa (BrbZIP-S) in modulating proline and sugar metabolism. Overexpression of BrbZIP-S in Nicotiana benthamiana resulted in delayed chlorophyll degradation during the response to dark conditions. Under heat stress or recovery conditions, the transgenic lines exhibited a lower accumulation of H2O2, malondialdehyde, and protein carbonyls compared to the levels in transgenic control plants. These results strongly indicate that BrbZIP-S regulates plant tolerance against dark and heat stress. We propose that BrbZIP-S is a modulator of proline and sugar metabolism, which are required for energy homeostasis in response to environmental stress conditions.


Introduction
Energy homeostasis in plant cells is regulated by diverse mechanisms concurring with the plant's response to environmental conditions. Energy is available in the form of carbon fixed through photosynthesis, whereas various environmental stresses affect photosynthetic carbon metabolism by affecting photosynthesis, carbon allocation, respiration, and so on [1,2]. Thus, energy management systems, including catabolic processes and anabolism repression are required for plant growth and development under energy-deprived conditions [3]. As an energy sensor, sucrose non-fermenting-1-related kinases 1 (SnRK1) assists in transcriptional and metabolic reprogramming, which regulates the carbohydrate metabolism and energy balance under low-energy stress [4,5]. Importantly, members of the basic region-leucine zipper (bZIP) transcription factor family have been found to be SnRK1-downstream mediators, which regulate the transcription of genes involved in various catabolic pathways providing alternative sources of energy and metabolites [3].
In the Arabidopsis genome, the bZIP family comprises 78 genes divided into 13 groups (A-K, M, and S). Most of these bZIP transcription factors have been found to regulate plant growth, development, senescence, seed maturation, metabolic reactions, and stress responses [6]. Among the plant bZIP family, the formation of heterodimers between S 1

Plant Growth Conditions
Seeds of Chinese cabbage (B. rapa L. ssp. pekinensis) cultivar (Chunkwang) and two lines of BrbZIP-S-overexpressing N. benthamiana (OX3 and OX7; T3 generation) were germinated and grown in a growth chamber (16 h light and 8 h dark) at 24 • C. In addition, we chose regenerated plants, which survived on the selection medium but exhibited no transcription of BrbZIP-S, as transgenic control plants (TC). Six-to eight-week-old transgenic or TC plants were incubated at 45 • C for six hours or treated with dark stress for five days. Three biological replicates (ten plants for each replicate) in each treatment were carried out.

cDNA Synthesis and Quantitative Real-Time PCR (qRT-PCR) Analysis
Total RNA was isolated from the leaves of Chinese cabbage or N. benthamiana using FavorPrep Plant Total RNA Mini Kit (Favogen, Pingtung, Taiwan) and reverse-transcribed using the Toyobo cDNA synthesis kit (TOYOBO, Co., Ltd., Osaka, Japan). qRT-PCR was performed using the Toyobo SYBR-Green Master Mix. Specific primer pairs for each gene were used (Table S1) and the transcription levels of target genes were normalized to NbEF1.

Plasmid Construction, Plant Transformation, and Analysis of Subcellular Localization
Using Chinese cabbage cDNA as a template, the full-length BrbZIP-S (accession number: XM_009140057) was cloned into the gateway binary vector pGWB505 for the expression of BrbZIP-S. The binary vector was introduced into Agrobacterium tumefaciens GV3101 and used to transform N. benthamiana. Leaf disk explants of 3-week-old plants were inoculated into the Agrobacterium suspension, and co-cultivated in dark condition. After 48 h, leaf disks were selected with selection medium (MS medium containing 1 mg/L 6-benzylaminopurine, 0.1 mg/L indole-3-acetic acid, 300 mg/L cefotaxime, 30 mg/L hygromycin, 30 g/L sucrose, and 7.5 g/L plant agar). The transgenic lines showing hygromycin resistance were transplanted in soil and lines with a high level of BrbZIP-S-GFP protein were selected by RT-PCR.

Analysis of Proline and Pyrroline-5-Carboxylate (P5C) Contents
Leaves of N. benthamiana were homogenized in 3% sulfosalicylic acid. After centrifugation, the supernatant was used for the analysis of proline and P5C contents. The content of proline was quantified using a colorimetric assay as described by Eom et al. [14] and expressed as ng/mg of fresh weight (F.W.).
For analysis of P5C content, the supernatant of each sample was mixed with 10 mM of 2-aminobenzaldehyde, as described by La et al. [15]. After incubation at 37 • C for two hours, the absorbance at 440 nm was determined, calculated by using an extinction coefficient of 2.58 mM −1 cm −1 , and expressed as nM/mg of F.W.

Determination of Invertase Activity and Sucrose, Glucose, and Fructose Contents
To determine the activity of invertase, 0.5 g of leaf grinding material was mixed in 1 mL extraction buffer (200 mM HEPES, 3 mM MgCl 2 , 1 mM EDTA, 2% glycerol, 0.1 mM PMSF, and 1 mM benzamidine) as described by Bonfig et al. [16]. After centrifugation, the supernatant was used to analyze the vacuolar invertase (NbV-inv) activity. The pellet was washed three times with distilled water, resuspended in the extraction buffer with 1 M NaCl, and subjected to another round of centrifugation. The supernatant was used to analyze the activity of extracellular invertase (NbC-inv). The activity of NbV-inv and NbC-inv was analyzed using GOD-POD reagent [16] and the absorbance at 595 nm was determined.
The contents of glucose, fructose, and sucrose were determined using the Sucrose/D-Fructose/D-Glucose Assay Kit (Megazyme, Wicklow, Ireland). Ten milligrams of samples were extracted in 95% ethanol at 80 • C for 30 min, and an aliquot of the extracts was used to determine the chlorophyll (Chl) content. The contents of Chl a, b, and Chl a+b were calculated according to Czyczyło-Mysza et al. [17].
The accumulation of H 2 O 2 in N. benthamiana leaves was carried out using the 3,3diaminobenzidine (DAB) staining method, as described by Ji et al. [18]. In addition, MDA content was analyzed as described by Eom et al. [13] and expressed as nmol/mg of F.W. using an absorbance coefficient of 155 mM −1 cm −1 .
To determine the protein carbonyl content, protein from N. benthamiana leaves was extracted using an extraction buffer, as described by Eom et al. [13]. The same concentrations of protein were used for analyzing content of protein carbonyl using a fluorometric protein carbonyl content assay kit (BioVision, Milpitas, CA, USA).

Sucrose-Induced Repression of Translation in BrbZIP-S
Among the plant bZIP family, the S group is the largest bZIP subfamily and is divided into three subgroups (S 1 , S 2 , and S 3 ) [19]. As shown in Figure 1A, BrbZIP-S is phylogenetically closely related to the Arabidopsis S 1 -bZIP subgroup, which includes AtbZIP1, AtbZIP2, AtbZIP11, AtbZIP44, and AtbZIP53 [19]. Sucrose is a signaling molecule that negatively controls the translation of the S 1 -bZIP subgroup [20,21]. This sucrose-induced repression of translation (SIRT) is mediated by the presence of a highly conserved upstream open reading frame (uORF) found in the 5 leader region of S 1 -bZIP transcripts, called the sucrose-controlled uORF (SC-uORF) [20,21]. BrbZIP-S contained four uORFs in its 5 leader of which the third uORF showed high homology to the SC-uORF of Arabidopsis S 1 -bZIPs ( Figure 1B), indicating that BrbZIP-S retains a SIRT mechanism mediated by a conserved SC-uORF.
Similar to other plant S1-bZIPs [22], BrbZIP-S-cGFP was localized in the nucleus (Figure 1c), indicating that BrbZIP-S is a nuclear protein. Although the basic region of bZIPs serves as a nuclear localization signal, phospho-mimicking mutations can disrupt the correct localization of bZIPs, indicating that phosphorylation regulates the subcellular localization of bZIP proteins, targeting either nuclear import or cytoplasmic retention [23,24]. However, the nuclear localization of AtbZIP53-cGFP was not changed by mimicking phosphorylation of conserved serines in the DNA-binding domain, suggesting that the functionality of the nuclear localization signal in S1-bZIPs is not disrupted by phosphorylation events [24].

BrbZIP-S Affects Sucrose Metabolism
Sucrose, the major form of carbon in higher plants, is used for internal regulation and physiological responses. As described above, S1-bZIPs, including BrbZIP-S, are known to be repressed by sucrose through translational inhibition [25,26], whereas overexpression of S1-bZIP increases the sugar content [26][27][28]. For example, fruit-specific expression of tomato S1-bZIP (SlbZIP1) or overexpression of strawberry S1-bZIP (strawberry bZIP11) resulted in tomato sweetening via the accumulation of sugar (sucrose, glucose, and fructose) [27,28]. Similarly, we found that the heterologous overexpression of BrbZIP-S induced sugar accumulation in N. benthamiana plants. As shown in Figure 2, the sucrose content was approximately 2.7-to 3-fold higher in transgenic lines (OX3 and OX7) than in TC plants. The transgenic plants also had higher levels of glucose (2.3-to 4-fold) and fructose (2.4-to 2.9-fold). In addition, overexpression of BrbZIP-S resulted in increased activity of Similar to other plant S 1 -bZIPs [22], BrbZIP-S-cGFP was localized in the nucleus ( Figure 1C), indicating that BrbZIP-S is a nuclear protein. Although the basic region of bZIPs serves as a nuclear localization signal, phospho-mimicking mutations can disrupt the correct localization of bZIPs, indicating that phosphorylation regulates the subcellular localization of bZIP proteins, targeting either nuclear import or cytoplasmic retention [23,24]. However, the nuclear localization of AtbZIP53-cGFP was not changed by mimicking phosphorylation of conserved serines in the DNA-binding domain, suggesting that the functionality of the nuclear localization signal in S 1 -bZIPs is not disrupted by phosphorylation events [24].

BrbZIP-S Affects Sucrose Metabolism
Sucrose, the major form of carbon in higher plants, is used for internal regulation and physiological responses. As described above, S 1 -bZIPs, including BrbZIP-S, are known to be repressed by sucrose through translational inhibition [25,26], whereas overexpression of S 1 -bZIP increases the sugar content [26][27][28]. For example, fruit-specific expression of tomato S 1 -bZIP (SlbZIP1) or overexpression of strawberry S 1 -bZIP (strawberry bZIP11) resulted in tomato sweetening via the accumulation of sugar (sucrose, glucose, and fructose) [27,28]. Similarly, we found that the heterologous overexpression of BrbZIP-S induced sugar accumulation in N. benthamiana plants. As shown in Figure 2, the sucrose content was approximately 2.7-to 3-fold higher in transgenic lines (OX3 and OX7) than in TC plants. The transgenic plants also had higher levels of glucose (2.3-to 4-fold) and fructose (2.4-to 2.9-fold). In addition, overexpression of BrbZIP-S resulted in increased activity of vacuolar invertase. Furthermore, N. benthamiana vacuolar invertase 1 (NbV-inv 1) was upregulated in all transgenic lines (Figure 2), suggesting that BrbZIP-S affected carbohydrate partitioning via a mechanism that includes the regulation of NbV-inv 1 expression. These results sug-gest that BrbZIP-S is a potential gene for improving sweetness via the reprogramming of sugar metabolism.
Biology 2023, 12, x 5 of 10 vacuolar invertase. Furthermore, N. benthamiana vacuolar invertase 1 (NbV-inv 1) was upregulated in all transgenic lines (Figure 2), suggesting that BrbZIP-S affected carbohydrate partitioning via a mechanism that includes the regulation of NbV-inv 1 expression. These results suggest that BrbZIP-S is a potential gene for improving sweetness via the reprogramming of sugar metabolism.

Altered Proline Metabolism in BrbZIP-S Transgenic N. benthamiana Plants
One of the most important multifunctional amino acids in plants is proline, which is a proteinogenic amino acid synthesized from glutamate and ornithine [29]. Proline biosynthesis is induced by various stressors, and its catabolism is activated in darkness and during stress relief via TOR-and SnRK1-dependent signaling [30,31]. Overexpression of AtbZIP11 has been shown to lead to reduced proline content [32], indicating that S1-bZIP plays a role in coordinating sucrose and proline metabolism. We showed that OX plants contained a much lower proline level than the TC plants ( Figure 3). In addition, P5C was slightly increased in OX plants, indicating that proline catabolism was activated by BrbZIP-S.

Altered Proline Metabolism in BrbZIP-S Transgenic N. benthamiana Plants
One of the most important multifunctional amino acids in plants is proline, which is a proteinogenic amino acid synthesized from glutamate and ornithine [29]. Proline biosynthesis is induced by various stressors, and its catabolism is activated in darkness and during stress relief via TOR-and SnRK1-dependent signaling [30,31]. Overexpression of AtbZIP11 has been shown to lead to reduced proline content [32], indicating that S 1 -bZIP plays a role in coordinating sucrose and proline metabolism. We showed that OX plants contained a much lower proline level than the TC plants ( Figure 3). In addition, P5C was slightly increased in OX plants, indicating that proline catabolism was activated by BrbZIP-S.
Biology 2023, 12, x 6 of 10 plants (Figure 3). In contrast, the expression levels of NbP5CS 1 and 2 were down-regulated ( Figure 3). These results suggest that BrbZIP-S induces metabolic changes in proline metabolism.

BrbZIP-S Controls Darkness-Induced Senescence in Transgenic Plants
Various investigations have shown that unfavorable environmental stressors, including light deprivation, lead to rapid leaf senescence [34]. The understanding of darknessor age-induced senescence is of high economic relevance as senescence can significantly decrease the post-harvest shelf-life of a plant as well as lead to yield loss in agriculture [35]. In Arabidopsis, the low content of trehalose 6-phosphate, a non-competitive inhibitor of SnRK1 [36], was shown to lead to delayed senescence [37]. During darkness-induced senescence, it was found that ProDH-mediated proline catabolism provides energy and glutamate, which play a crucial role in nitrogen remobilization [38]. Therefore, we presumed that the increasing proline catabolism by BrbZIP-S delays darkness-induced senescence. To test this hypothesis, we transferred six-week-old plants to extended darkness for five days and then analyzed the Chl levels, an integral part of leaf senescence [39]. As shown in Figure 4, the levels of Chl a, Chl b, and Chl a+b decreased by 58.2% (1.29 μg/mg F.W. to 0.54 μg/mg F.W.), 74.7% (0.57 g/mg F.W. to 0.14 g/mg F.W.), and 63.3% (1.87 μg/mg F.W. to 0.68 μg/mg F.W.), respectively, in plants kept in darkness, compared to those in normal growth conditions. However, the contents of Chl a, Chl b, and Chl a + b were significantly higher in OX3 and OX7 lines than in TC plants under dark conditions, indicating that BrbZIP-S overexpression delayed Chl degradation during darkness-induced senescence. It was previously shown that nutrient-deprivation-induced senescence and agedependent senescence were delayed in plants overexpressing KIN10 (the main component of Arabidopsis SnRK1) and the rice SnRK1, respectively [3,40], suggesting the essential

BrbZIP-S Controls Darkness-Induced Senescence in Transgenic Plants
Various investigations have shown that unfavorable environmental stressors, including light deprivation, lead to rapid leaf senescence [34]. The understanding of darknessor age-induced senescence is of high economic relevance as senescence can significantly decrease the post-harvest shelf-life of a plant as well as lead to yield loss in agriculture [35]. In Arabidopsis, the low content of trehalose 6-phosphate, a non-competitive inhibitor of SnRK1 [36], was shown to lead to delayed senescence [37]. During darkness-induced senescence, it was found that ProDH-mediated proline catabolism provides energy and glutamate, which play a crucial role in nitrogen remobilization [38]. Therefore, we presumed that the increasing proline catabolism by BrbZIP-S delays darkness-induced senescence. To test this hypothesis, we transferred six-week-old plants to extended darkness for five days and then analyzed the Chl levels, an integral part of leaf senescence [39]. As shown in Figure 4, the levels of Chl a, Chl b, and Chl a+b decreased by 58.2% (1.29 µg/mg F.W. to 0.54 µg/mg F.W.), 74.7% (0.57 g/mg F.W. to 0.14 g/mg F.W.), and 63.3% (1.87 µg/mg F.W. to 0.68 µg/mg F.W.), respectively, in plants kept in darkness, compared to those in normal growth conditions. However, the contents of Chl a, Chl b, and Chl a + b were significantly higher in OX3 and OX7 lines than in TC plants under dark conditions, indicating that BrbZIP-S overexpression delayed Chl degradation during darkness-induced senescence. It was previously shown that nutrient-deprivation-induced senescence and age-dependent senescence were delayed in plants overexpressing KIN10 (the main component of Arabidopsis SnRK1) and the rice SnRK1, respectively [3,40], suggesting the essential role of S 1 -bZIPs, including BrbZIP-S, in plant survival is through inducing remobilization of proline under darkness and nutrient deprivation conditions. Biology 2023, 12, x 7 of 10 role of S1-bZIPs, including BrbZIP-S, in plant survival is through inducing remobilization of proline under darkness and nutrient deprivation conditions.

Overexpression of BrbZIP-S Increases the Heat Tolerance of N. benthamiana
Among unfavorable environmental conditions, higher temperatures above critical thresholds affect crop growth and development, leading to significant yield loss. Therefore, improving heat tolerance is of profound importance to the production yields of crops and will greatly secure food security [41]. Plant S1-bZIPs play a key role in plant innate immunity and the response to unfavorable environmental conditions [11]. In our previous study, BrbZIP-S was induced under abiotic stress conditions [13], indicating the possible involvement of BrbZIP-S in the stress response. To analyze the function of BrbZIP-S in heat stress responses, OX and TC plants were treated at 45 °C for six hours. As shown in Figure 5a, the leaves of the OX3 and OX7 lines were less wilted than those of the control plants. In addition, H2O2 was less accumulated in the OX3 and OX7 lines than in the TC plants (Figure 5b). After the subsequent recovery incubation at 25 °C, severe damage was observed in the TC plants, whereas only a slight wilting was seen in the OX3 and OX7 lines (Figure 5a). The levels of MDA and protein carbonyls were significantly lower in OX lines than in TC plants (Figure 5c,d), indicating that BrbZIP-S increased heat tolerance in N. benthamiana plants.
Soluble sugars are osmoprotectants, which can protect cell membranes by scavenging toxic reactive oxygen species generated under various stress conditions, including heat stress [42,43]. In addition, SlbZIP1 and OsbZIP71 exert important roles in abiotic stress tolerance via modulating abscisic acid (ABA)-mediated pathways [12,44]. Under heat stress conditions, the transcription levels of genes involved in sucrose metabolism including sucrose synthase and invertase, were increased by ABA [45]. Therefore, one possible explanation should be that the BrbZIP-S-induced sugar metabolism reprogramming seen above ( Figure 2) contributes to improved heat tolerance.

Overexpression of BrbZIP-S Increases the Heat Tolerance of N. benthamiana
Among unfavorable environmental conditions, higher temperatures above critical thresholds affect crop growth and development, leading to significant yield loss. Therefore, improving heat tolerance is of profound importance to the production yields of crops and will greatly secure food security [41]. Plant S 1 -bZIPs play a key role in plant innate immunity and the response to unfavorable environmental conditions [11]. In our previous study, BrbZIP-S was induced under abiotic stress conditions [13], indicating the possible involvement of BrbZIP-S in the stress response. To analyze the function of BrbZIP-S in heat stress responses, OX and TC plants were treated at 45 • C for six hours. As shown in Figure 5A, the leaves of the OX3 and OX7 lines were less wilted than those of the control plants. In addition, H 2 O 2 was less accumulated in the OX3 and OX7 lines than in the TC plants ( Figure 5B). After the subsequent recovery incubation at 25 • C, severe damage was observed in the TC plants, whereas only a slight wilting was seen in the OX3 and OX7 lines ( Figure 5A). The levels of MDA and protein carbonyls were significantly lower in OX lines than in TC plants ( Figure 5C,D), indicating that BrbZIP-S increased heat tolerance in N. benthamiana plants.

Conclusions
Energy homeostasis under energy-deprived conditions is very common and important in regulating plant defense responses. In this study, we analyzed the physiological function BrbZIP-S, and suggested that BrbZIP-S acts as a positive factor in stress tolerance against darkness and heat stress. BrbZIP-S may be regulated by stress response pathways via a complex network mediated by ABA, sugar, and proline. Further functional dissec-