**1. Introduction**

Salinity is one of the most detrimental abiotic stresses which limits plant growth and reduces biomass and grain yield severely worldwide. Salt stress impairs the productivity of plants by affecting cell growth and metabolic process, causing irreversible damage to seed germination, seedling growth and crop yield [1]. To counter the adverse effects of environmental stress, plants have evolved complex mechanisms to cope with salt stress at both physiological and biochemical levels [2]. The improvement of morphological structure and physiological metabolism level is controlled by the expression of stress-response genes, and transcription factors (TFs) play a prominent part in regulating these genes. Numerous TFs, including NAC TFs, have been identified to be involved in plant growth and salt stress response.

The plant-specific NAC TF family is one of the largest TF families in plants, named after the initials of the *NAM* (no apical meristem) gene from *Petunia hybrida* [3], the *ATAF1/2* [4] and *CUC2* (cup-shaped cotyledon) [5] from *Arabidopsis thaliana*. In 1996, Souer et al. [3] cloned the first NAC transcription factor gene *NAM* from *Petunia*, which affects the formation and differentiation of *Petunia* apical meristem. Subsequently, NAC TFs were successively identified in *Arabidopsis* [6], rice [7], soybean [8], tomato [9] and other species. The N-terminal of all NAC TFs contains a conserved NAC domain composed of about

**Citation:** Zhao, X.; Wu, T.; Guo, S.; Hu, J.; Zhan, Y. Ectopic Expression of *AeNAC83*, a NAC Transcription Factor from *Abelmoschus esculentus*, Inhibits Growth and Confers Tolerance to Salt Stress in *Arabidopsis*. *Int. J. Mol. Sci.* **2022**, *23*, 10182. https://doi.org/10.3390/ ijms231710182

Academic Editors: Jian Zhang and Zhiyong Li

Received: 15 July 2022 Accepted: 30 August 2022 Published: 5 September 2022

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150 amino acid residues, and the C-terminal contains a highly variable transcriptional activation region (TAR) [6].

Many studies have shown that NAC TFs not only play an important role in plant growth and development, such as secondary wall formation [10–12], leaf senescence [13], and lateral root development [14,15], but also participate in response to abiotic stresses [16,17]. NAC TFs respond to salt stress mainly through maintaining intracellular Na<sup>+</sup> and K<sup>+</sup> concentrations and the relative homeostasis of the intracellular environment by activating the expression of stress response genes and protecting the stability of cell structure by enhancing the accumulation of osmoregulation substances such as soluble sugar, proline, and betaine. For example, compared with WT, transgenic rice overexpressing *ONAC022* with lower Na<sup>+</sup> accumulation and transpiration rate, and increased free proline and soluble sugar content, enhanced salt tolerance significantly [18], which were also observed in transgenic rice overexpressing *ONAC009* and *ONAC058* [19,20]. Wheat TaNAC29 improved plant salt tolerance by inducing the expression of stress-related genes, enhancing the antioxidant system, and scavenging reactive oxygen species [21]. Under salt stress, *NAC13*-overexpressing transgenic poplar showed enhanced salt tolerance and the *NAC13*-suppressing plants increased sensitivity to salt stress [22].

Okra (*Abelmoschus esculentus* L.), a medicinal and edible plant, has attracted increasing attention worldwide [23,24]. However, no molecular characterization of any NAC family member's impact on okra growth and salt stress response has been reported. In our previous study, an NAC TF gene *AeNAC83* from okra was up-regulated and exposed to salt stress [25]. In the present work, we cloned the coding sequence of AeNAC83 by using full-length isoforms obtained in okra and silenced the gene by the whole plant virus-induced gene silencing (VIGS). The *AeNAC83*-silenced okra seedlings exhibited a significantly higher biomass accumulation under normal conditions and were more sensitive to salt stress than that in Mock plants. Moreover, we further investigated its functions in transgenic *Arabidopsis* overexpressing *AeNAC83*. The overexpression of *AeNAC83* improved salt resistance and inhibited plant growth. According to the transcriptome analysis, the improved resistance and inhibited growth were related to the up-regulation of flavonoid biosynthesis-related genes and the down-regulation of photosynthesis-related genes in transgenic plants. The above work will provide important resources for molecular breeding of plant stress resistance.

#### **2. Results**

#### *2.1. Isolation and Characterization of AeNAC83*

We first cloned the coding sequence of AeNAC83 from okra, which encoded a protein of 255 amino acids. Then, multiple sequence alignment was performed with other NAC TFs from *Arabidopsis* and other species. AeNAC83 has a high homology with other NAC members, containing one NAC domain with five conserved regions (Figure 1A). In order to reveal the evolutionary relationship between AeNAC83 and other NAC TFs, a phylogenetic tree was constructed. All the selected NAC members were clustered into two distinct subgroups, and AeNAC83 belonged to the ATAF subgroup (Figure 1B).

To determine the subcellular location of AeNAC83, the 35Sp:: *AeNAC83*:GFP fusion protein was constructed and transiently expressed in tobacco epidermal cells. Fluorescence analysis showed that the control empty vector was expressed in both the nucleus and cytoplasm, while the protein was only localized in the nucleus (Figure 2A), indicating that AeNAC83 was localized in the nucleus.


after treatment, and then the transcription level of the *AeNAC83* gene in the second true leaf of the seedlings was determined by qRT-PCR. We found that compared with the control, the expression of *AeNAC83* was up-regulated at three time points after salt treatment, and the expression was highest on the first day, then gradually decreased (Figure 2B). The

result suggests that AeNAC83 may play a regulatory role in salt stress in okra.

**Figure 1.** Characterization of AeNAC83: (**A**) Multiple sequence alignment of AeNAC83 and its homologous NAC proteins. The NAC domain with five conserved regions (a–e) are indicated by red lines; (**B**) Phylogenetic analysis of AeNAC83 with its homologous NAC proteins. **Figure 1.** Characterization of AeNAC83: (**A**) Multiple sequence alignment of AeNAC83 and its homologous NAC proteins. The NAC domain with five conserved regions (a–e) are indicated by red lines; (**B**) Phylogenetic analysis of AeNAC83 with its homologous NAC proteins.

**Figure 2.** Subcellular localization and expression pattern of AeNAC83: (**A**) Subcellular localization of AeNAC83 in *N. benthamiana*. *N. benthamiana* cells were transformed with 35Sp:: *AeNAC83*:GFP or pCAMBIA1300-GFP. After incubating for 48 h, the transformed cells were observed under a confocal microscope. The photographs were taken under detecting GFP fluorescence, bright field, and in combination (merge), respectively. Empty vector (pCAMBIA1300-GFP) was used as a control; (**B**) Expression of *AeNAC83* in okra seedlings after 300 mM NaCl treatment by qRT-PCR. Total RNA for **Figure 2.** Subcellular localization and expression pattern of AeNAC83: (**A**) Subcellular localization of AeNAC83 in *N. benthamiana*. *N. benthamiana* cells were transformed with 35Sp:: *AeNAC83*:GFP or pCAMBIA1300-GFP. After incubating for 48 h, the transformed cells were observed under a confocal microscope. The photographs were taken under detecting GFP fluorescence, bright field, and in combination (merge), respectively. Empty vector (pCAMBIA1300-GFP) was used as a control; (**B**) Expression of *AeNAC83* in okra seedlings after 300 mM NaCl treatment by qRT-PCR. Total RNA for expression analysis was isolated from leaves of two-week-old seedlings after 300 mM NaCl treatment for 1, 3, 5, and 7 days. Data are presented as mean ± SD (*n* = 3). Different letters denote significant differences at *p* < 0.05, using ANOVA and Duncan's multiple tests.

Our previous analysis showed that salt stress induced the expression of *AeNAC83*. To further confirm whether AeNAC83 participates in the salt resistance, the two-week-old okra seedlings were irrigated with 300 mM NaCl and sampled at 1, 3, 5 and 7 days after treatment, and then the transcription level of the *AeNAC83* gene in the second true leaf of the seedlings was determined by qRT-PCR. We found that compared with the control, the expression of *AeNAC83* was up-regulated at three time points after salt treatment, and the expression was highest on the first day, then gradually decreased (Figure 2B). The result suggests that AeNAC83 may play a regulatory role in salt stress in okra. mixture containing the pTRV2-*NAC83* vector and pTRV1 into the cotyledons of okra. Negative control was performed by using empty vectors pTRV1 and pTRV2. The Ae*NAC83* mRNA levels of newly grown young true leaves at 25-days post-inoculation were detected to assess the efficiency of VIGS. As shown in Figure 3A, the mRNA levels of AeNAC83 in silenced plants decreased dramatically compared with the control, accounting for about 60% of the control, indicating that the gene was partially silenced. To determine the roles of AeNAC83 in salt stress and plant growth, the *AeNAC83* silenced okra seedlings produced by VIGS were irrigated with water or 300 mM NaCl solution for 7 days. Under normal conditions, the total leaf fresh weight of *AeNAC83*-si-

expression analysis was isolated from leaves of two-week-old seedlings after 300 mM NaCl treatment for 1, 3, 5, and 7 days. Data are presented as mean ± SD (*n* = 3). Different letters denote signif-

The VIGS method was used to assess the *AeNAC83* gene function. We constructed the pTRV2-*NAC83* vector with a 336-bp fragment and then inoculated the *Agrobacterium*

#### *2.2. Performance of AeNAC83-Silenced Plants under Salt Stress* lenced seedlings was significantly higher than Mock plants, indicating that AeNAC83

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 5 of 19

icant differences at *p* < 0.05, using ANOVA and Duncan's multiple tests.

*2.2. Performance of AeNAC83-silenced Plants under Salt Stress*

The VIGS method was used to assess the *AeNAC83* gene function. We constructed the pTRV2-*NAC83* vector with a 336-bp fragment and then inoculated the *Agrobacterium* mixture containing the pTRV2-*NAC83* vector and pTRV1 into the cotyledons of okra. Negative control was performed by using empty vectors pTRV1 and pTRV2. The *AeNAC83* mRNA levels of newly grown young true leaves at 25-days post-inoculation were detected to assess the efficiency of VIGS. As shown in Figure 3A, the mRNA levels of AeNAC83 in silenced plants decreased dramatically compared with the control, accounting for about 60% of the control, indicating that the gene was partially silenced. may be involved in plant growth. After the 7-day treatment, the seedling growth was inhibited obviously (Figure 3B), and leaf fresh weight of *AeNAC83*-silenced plants decreased significantly than that of Mock plant (Figure 3C). The total chlorophyll content in *AeNAC83*-silenced plants was not significantly different from that in the control before salt treatment, whereas after salt treatment the chlorophyll content increased in the Mock plants (Figure 3D). The results showed that *AeNAC83*-silenced plants were more sensitive to NaCl treatment, indicating that AeNAC83 may play a positive regulatory role in salt stress.

**Figure 3.** Phenotype analysis of *AeNAC83*-silenced okra plants produced by VIGS under salt stress. The *Agrobacterium tumefaciens* GV3101 cell culture harboring the pTRV2 or *AeNAC83*-pTRV2 together with pTRV1 were mixed with a ratio of 1:1 and syringe-infiltrated into okra cotyledons. At 25 d post-inoculation (dpi), the leaves were used for gene expression assay and 300 mM NaCl treatment for 7 days. (**A**) Expression analysis of *AeNAC83* in *AeNAC83*-silenced okra seedlings by qRT-PCR. (**B**) Images of plant phenotype. (**C**) The leaf fresh weight. (**D**) Total chlorophyll content. Data **Figure 3.** Phenotype analysis of *AeNAC83*-silenced okra plants produced by VIGS under salt stress. The *Agrobacterium tumefaciens* GV3101 cell culture harboring the pTRV2 or *AeNAC83*-pTRV2 together with pTRV1 were mixed with a ratio of 1:1 and syringe-infiltrated into okra cotyledons. At 25 d post-inoculation (dpi), the leaves were used for gene expression assay and 300 mM NaCl treatment for 7 days. (**A**) Expression analysis of *AeNAC83* in *AeNAC83*-silenced okra seedlings by qRT-PCR. (**B**) Images of plant phenotype. (**C**) The leaf fresh weight. (**D**) Total chlorophyll content. Data are presented as mean ± SD (*n* = 10). Different letters denote significant differences at *p* < 0.05, using ANOVA and Duncan's multiple tests.

To determine the roles of AeNAC83 in salt stress and plant growth, the *AeNAC83* silenced okra seedlings produced by VIGS were irrigated with water or 300 mM NaCl solution for 7 days. Under normal conditions, the total leaf fresh weight of *AeNAC83* silenced seedlings was significantly higher than Mock plants, indicating that AeNAC83 may be involved in plant growth. After the 7-day treatment, the seedling growth was

inhibited obviously (Figure 3B), and leaf fresh weight of *AeNAC83*-silenced plants decreased significantly than that of Mock plant (Figure 3C). The total chlorophyll content in *AeNAC83*-silenced plants was not significantly different from that in the control before salt treatment, whereas after salt treatment the chlorophyll content increased in the Mock plants (Figure 3D). The results showed that *AeNAC83*-silenced plants were more sensitive to NaCl treatment, indicating that AeNAC83 may play a positive regulatory role in salt stress.
