*2.6. Identification of DEGs That Participate in Photosynthesis and Hormone Signal Transduction Pathways*

Thylakoid membrane photosynthetic complexes consist of photosystem II (PSII), cytochrome b6f, photosystem I (PSI), light-harvesting antenna complexes, and ATP synthase [27]. Compared with WT-CK, genes encoding key proteins involved in these complexes were almost greatly decreased in *AeNAC83*-overexpression transgenic (OX3) *Arabidopsis*, and NaCl treatment further resulted in the down-regulation of these genes, except for 1 *PsbP* gene of PSII and 1 *gamma* gene of ATP synthase (Figure 8). Chlorophyll is one of the most important pigments in higher plants, which is an important substance for photosynthesis. The total chlorophyll content was similar between OX3 and WT before salt treatment. After salt treatment, the chlorophyll content decreased dramatically in both WT and OX3 plants (Figure S2B).

**Figure 7.** Transcript abundance changes of the phenylpropanoid and flavonoid biosynthesis pathway-related DEGs in *Arabidopsis* wild type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Heat map of DEGs in phenylpropanoid biosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram. 4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4 hydroxylase; F5H, ferulate-5-hydroxylase; CCR, cinnamoyl-CoA reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; COMT, caffeic acid 3-O-methyltransferase; HCT, hydroxyl cinnamoyl transferase; CAD, cinnamyl-alcohol dehydrogenase; SGT, scopoletin glucosyltransferase; CSE, **Figure 7.** Transcript abundance changes of the phenylpropanoid and flavonoid biosynthesis pathwayrelated DEGs in *Arabidopsis* wild type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Heat map of DEGs in phenylpropanoid biosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram. 4CL, 4-coumarate-CoA ligase; C4H, cinnamate-4-hydroxylase; F5H, ferulate-5-hydroxylase; CCR, cinnamoyl-CoA reductase; CCoAOMT, caffeoyl-CoA O-methyltransferase; COMT, caffeic acid 3-O-methyltransferase; HCT, hydroxyl cinnamoyl transferase; CAD, cinnamyl-alcohol dehydrogenase; SGT, scopoletin glucosyltransferase; CSE, caffeoylshikimate esterase; CGT, coniferyl-alcohol glucosyltransferase; β-G, beta-glucosidase; POD, peroxidase. (**B**) Gene expression of DEGs in flavonoid biosynthesis pathway. DFR: dihydroflavonol 4-reductase; F3H: flavanone 3-hydroxylase; CYP73A: trans-cinnamate 4-monooxygenase; ANS: anthocyanidin synthase; FLS: flavonol synthase; CHS: chalcone synthase; PGT1: phlorizin synthase; CHI: chalcone isomerase; HCT: shikimate O-hydroxycinnamoyltransferase. \* indicate statistical significance based on two-tailed Student's *t*-test at *p*-values < 0.05.

**Figure 8.** Transcript abundance changes of photosynthesis pathway-related DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Photosynthesis pathway (ko00195). Different letters indicated the different subunits of photosynthetic complexes. (**B**) Heat map of DEGs in photosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram. **Figure 8.** Transcript abundance changes of photosynthesis pathway-related DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Photosynthesis pathway (ko00195). Different letters indicated the different subunits of photosynthetic complexes. (**B**) Heat map of DEGs in photosynthesis pathway. The log2-transformed FPKM values of DEGs were used to generate the diagram.

In addition, the abundance of hormone-related DEGs changed significantly under salt stress. A large number of genes encoding AUX1, AUX/IAA, ARF, GH3 and SAUR for the auxin signaling pathway were identified, including 2 *AUX1* genes, 8 *AUX/IAA* genes, 1 *ARF* gene, 13 *GH3* genes and 21 *SAUR* genes; 1 *CRE1* gene, 2 *AHP* genes, 11 *B*-*ARR* genes and 2 *A*-*ARR* genes for the cytokinin signaling pathway were obtained; 3 *GID1* genes, 8 *DELLA* genes and 13 *TF* genes were identified in the GA signaling pathway; DEGs involved in ABA signaling pathway were identified, including 2 *PYR* genes, 8 *PP2C* genes, 4 *SnRK2* genes and 4 *AREB/ABF* genes; 1 *ETR*, 2 *CTR1* genes, 4 *SIMKK* genes, 1 *EBF1/2* gene and 4 *ERF1/2* genes for ET signaling pathway were analyzed; 9 *BAK1* genes, 4 *BRI1* genes, 2 *BSK* genes, and 1 *TCH4* gene related to the BR signaling pathway were identified; there were 5 *JAZ* and 9 *MYC2* DEGs in the JA signaling pathway; for the SA signaling pathway, 1 *NPR1*, 4 *TGA* and 8 *PR1* were identified (Figure 9). Twelve randomly selected genes of hormone signal transduction pathways, including 1 *AUX1*, 1 *ARF*, 2 *SnRK2*, 1 *AREB/ABF*, 2 *JAZ*, 1 *MYC2*, 1 *NPR1*, 1 *TGA* and 2 *PR1*, were verified by qRT-PCR (Figure S3). The results showed that the relative expression levels were basically consistent with RNA-seq data, which supported the accuracy and reliability of transcriptome analysis results.

**Figure 9.** Transcript abundance changes of the various hormone signal transduction pathways-related DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**)

**Figure 9.** Transcript abundance changes of the various hormone signal transduction pathways-related DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) **Figure 9.** Transcript abundance changes of the various hormone signal transduction pathwaysrelated DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Plant hormone signal transduction pathways. (**B**) Heat map of DEGs in various hormone signal transduction pathways. The log2-transformed FPKM values of DEGs were used to generate the diagram.

**Figure 8.** Transcript abundance changes of photosynthesis pathway-related DEGs in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment. (**A**) Photosynthesis pathway (ko00195). Different letters indicated the different subunits of photosynthetic complexes. (**B**) Heat map of DEGs in photosynthesis pathway. The log2-transformed FPKM values of DEGs were used

#### **3. Discussion**

to generate the diagram.

Okra has important edible and medicinal value. However, due to the complexity and lack of the okra genome, it is difficult to assess the gene function of okra [28,29]. VIGS has been widely used as an effective functional genomics tool for gene function analysis [30]. NAC TFs play important roles in plant growth, development and stress responses. In this study, the role of AeNAC83 in the salt stress responses in okra was investigated. We found that the *AeNAC83*-silenced okra seedlings produced by VIGS were slightly more sensitive to salt stress (Figure 3). Further analysis found that the gene was not completely silenced, probably because VIGS sometimes silences only part of the target gene [31]. Hence, transgenic *Arabidopsis* plants over-expressing *AeNAC83* were generated to further determine the role of AeNAC83 in growth and salt stress. Over-expression of *AeNAC83* enhanced tolerance to salt stress and suppressed vegetative growth (Figure 4). Taken together, okra NAC TF AeNAC83 plays a pivotal role in mediating plant growth and defense response to salt stress.

Transcriptomic analyses showed that compared with WT-CK, there were 4285 DEGs in WT-N, while only 1360 DEGs were found in OX3-N compared with OX3-CK (Figure 5). The result showed that overexpression of *AeNAC83* resulted in the insensitivity of numerous genes to salt stress, which may be one of the reasons for enhanced resistance to salt stress. KEGG pathway enrichment analysis showed that these DEGs mainly participated in "MAPK signaling pathway-plant", "plant-pathogen interaction" and "phenylpropanoid biosynthesis", "starch and sucrose metabolism" and "circadian rhythm-plant" (Figure 6). In

plants, the rapid activation of MAPK cascades has long been observed involved in growth and development, as well as in response to drought, salinity, wounding, heat, and cold [32]. Moreover, for all comparison groups, the phenylpropanoid biosynthesis pathway was always found to contain significant enrichment of DEGs.

The phenylpropanoid pathway that produces lignin, flavonoids, and other secondary metabolites [26], contributes to the defense and growth of plants. Fortifying cell walls by increasing their lignin content is one of the common plant defense mechanisms [33]. The 4CL, C4H and POD are core enzymes involved in the biosynthesis of flavonoids and lignin. From our analysis, the expressions of 4CL, C4H, CAD and POD-related DEGs were increased in OX3-CK and the NaCl-treated WT and OX3 samples (Figure 7A). This finding suggested that NaCl stress could cause an increase in lignin synthesis. Under normal conditions, the *AeNAC83*-overexpression plant may accumulate more lignin, which may be one of the reasons why it has stronger salt stress resistance than the wild type. In response to a variety of abiotic stress, flavonoids (including anthocyanins) play a major antioxidant role [34,35]. Flavonoids can reduce reactive oxygen species in plant tissues [36–38], which are usually produced by stress such as ultraviolet radiation, drought and salt stresses. We observed up-regulation of flavonoids genes in NaCl-treated seedlings (Figure 7B), indicating that the genes involved in stress metabolism are generally up-regulated. Some transcription factors have been identified to have regulatory functions in phenylpropanoid and flavonoid biosynthesis pathways [39]. Overexpression of *PvMYB4*, a suppress phenylpropanoid metabolism TF, caused a reduction in the lignin content and decreased recalcitrance in *Panicum virgatum* [40]. NAC is the second largest class of TFs in plants and has been shown as a key regulator of abiotic stresses [41]. In *Arabidopsis* and rice, *NAC* genes' overexpression enhanced drought and salt tolerance [20,42,43]. Additionally, overexpression of *AeNAC83* significantly induced the expression of most flavonoid biosynthesis-related DEGs under normal conditions. This result was further confirmed by the experiment of anthocyanin content determination (Figure S2A). These results showed that overexpression of *AeNAC83* improved the resistance of plants to salt stress, possibly by regulating the accumulation of lignin and flavonoids.

Chloroplast is the site of photosynthesis. High Na<sup>+</sup> levels can destroy the structure of chloroplast, damage the membrane system of plant and seriously degrade chlorophyll, resulting in the decline of photosynthesis. In the study, the expression levels of photosynthesis-related genes were significantly down-regulated in WT and transgenic *Arabidopsis* (OX-3) after NaCl treatment and the degree of decline of genes in WT was significantly higher than that of OX-3 (Figure 8). The results showed that *AeNAC83* overexpression transgenic plants had stronger photosynthetic capacity under salt stress, which could ensure more organic accumulation and improve the salt tolerance of plants. The same phenomenon has been observed in transgenic *Arabidopsis* overexpressing wheat *TaNAC67*, the chlorophyll content and Fv/Fm of which were higher than those of the control [44]. In addition, under salt stress, *AeNAC83*-overexpression transgenic plants have larger roots, suggesting that AeNAC83 may regulate roots to improve salt stress tolerance. Similarly, overexpression of soybean *GmNAC20* improved the tolerance to low temperature and salt and promoted lateral root formation [45]. Under high salt stress, *Arabidopsis thaliana AtNAC2* was highly expressed in roots, and lateral roots of transgenic plants overexpressing *AtNAC2* increased [15]. Compared with wild-type plants, *AeNAC83*-overexpression plants showed growth retardation, which may be due to the redistribution of energy between stress tolerance and normal growth and development, thereby improving the survival rate of plants under salt stress. This was consistent with transgenic rice overexpressing *ONAC022* [18].

Phytohormones, such as auxin, abscisic acid (ABA), ethylene, gibberellic acid (GA), and jasmonic acid (JA), also play central roles in salt stress response [46]. *Malus domestica* MdNAC047 induced ethylene accumulation by increasing the expression of ethylene synthesis genes *MdACS1* and *MdACO1* and TF gene *MdERF3*, enhancing tolerance to salt stress by regulating ethylene response [47]. Soybean (*Glycine Max*) GmNAC109 promoted the for-

mation of lateral roots of transgenic *Arabidopsis thaliana* and enhanced salt stress tolerance through positive regulation of auxin response gene *AtAIR3* and negative regulation of TF *AtARF2* [48]. To reveal the involvement of these hormones in the salt stress response and growth, numerous genes related to these hormones signaling pathways were identified (Figure 9). For example, in the ABA-signaling pathway, the core of salt- and drought-stress responses in plants [49], 1 *SnRK2* gene and 2 *AREB/ABF* genes were up-regulated and 1 *PYR* gene, 3 *PP2C* genes, 1 *SnRK2* gene and 1 *AREB/ABF* gene were down-regulated compared with WT-N. The expressions of JA- and SA-signaling pathways related to DEGs were increased in NaCl-treated WT and OX3 samples. These data indicated that AeNAC83 may enhance tolerance to salt stress by regulating various hormone signaling responses.

Interestingly, besides the increase in salt sensitivity in *AeNAC83*-silenced okra seedlings and salt tolerance in *AeNAC83*-overexpression transgenic *Arabidopsis*, the *AeNAC83*-silenced seedlings showed enhanced plant growth, while *AeNAC83*-overexpression lines exhibited the opposite phenotype. These data imply that AeNAC83 participates in the balance between defense responses and plant growth. In plants, the trade-off between defense and growth has attracted considerable attention, where enhanced resistance often impairs growth and development. For example, in rice, over-expression of *OsRCI*-*1* resulted in an increase in BPH resistance and a reduction in thousand-grain weight [50]. OsNAC2 negatively regulates root growth [51] and mediates abiotic stress tolerance [52].

### **4. Materials and Methods**

#### *4.1. Multiple Sequence Alignment and Phylogenetic Analysis*

The amino acid sequences of NAC proteins were obtained from the TAIR (https: //www.arabidopsis.org/) and NCBI. The accession number and references for other NACs: *Arabidopsis thaliana*, ATAF1 (AT1G01720), ATAF2 (AT5G08790) [4], CUC1 (AT3G15170), CUC2 (AT5G53950) [5], CUC3 (AT1G76420) [53], AtNAC1 (AF198054.1) [14], AtNAC2 (AT5G39610) [15], AtNAP (AJ222713) [54]; rice, OsNAC2 (Os04g0460600) [51,55]; soybean, GmNAC20 (EU440353.1) [45]; petunia, PhNAM (X92204) [3]. Multiple sequence alignment of AeNAC83 with other 11 NAC members was carried out using ClustalX 2.0 and modified with GeneDoc. The phylogenetic tree was generated using the MEGA7 software with the neighbor-joining (NJ) method, Poisson-corrected distances and 1000 replicates.

### *4.2. RNA Isolation and qRT-PCR*

Mature seeds of okra (*Abelmoschus esculentus* L.) Cultivar "xian zhi" was used in this study. Plant growth conditions and treatment were performed according to our previous research [25]. Okra seedlings with only two true leaves under normal conditions were selected for salt treatment, irrigated with 300 mM NaCl solution, and the control was irrigated with water. At 1 d, 3 d, 5 d and 7 d after treatment, RNA was extracted from the second true leaf, and the expression of the *AeNAC83* gene was analyzed by qRT-PCR. The primers were listed in Table S3.

#### *4.3. Subcellular Localization of GFP-AeNAC83 Fusion Protein*

The coding sequence of AeNAC83 was amplified by RT-PCR with primers 50 -GAGCTC ATGGAGAAGCTTAGTTTTGT-30 and 50 -TCTAGAAGGTTTTCTTCTGAAGTAAG-30 , and cloned into the SacI/XbaI sites of pCAMBIA1300-sGFP under the control of the CaMV 35S promoter, resulting in 35Sp:: AeNAC83:GFP construct. The constructed 35Sp:: AeNAC83:GFP vector was introduced into *Agrobacterium* strain GV3101, and transiently transformed in tobacco epidermal cells. The empty vector was used as a negative control. The distribution of fluorescence was imaged 48 h after the inoculation by a confocal laser-scanning microscope (Zeiss LSM 710).

#### *4.4. Virus-Induced Gene Silencing (VIGS) of AeNAC83 and NaCl Treatment*

A specific sequence of about 300bp from AeNAC83 was amplified by RT-PCR with primers 50 -GAATTCATGGAGAAGCTTAGTTTT-30 and 50 -GGTACCTTTCCTGTCGATTCC GGT-30 , and cloned into the EcoRI/KpnI sites of pTRV2, resulting in TRV-NAC83 construct. The construct was transferred into *Agrobacterium tumefaciens* GV3101.

A single colony of the *Agrobacterium tumefaciens* containing TRV1, TRV-*NAC83* or empty TRV2 was inoculated into 3ml YEP liquid medium with 50 mg/L rifampicin and 50 mg/L kanamycin, and cultured at 28 ◦C for 12 h at 200 rpm. For secondary activation, 1 mL culture was added to 50 mL of YEP and grown until OD600 reached 1–1.5. Agrobacteria were resuspended in a monomethylamine (MMA) solution (20 µM acetylsyringone, 10 mM MgCl<sup>2</sup> and 10 mM MES, PH 5.6) to a final concentration of OD600 = 1 and placed without shaking at 28 ◦C for 3 h in dark. Then the cotyledons of okra seedlings that cotyledons were fully stretched and the true leaves had not yet grown were used to inoculate with the suspension of TRV1 and TRV-*NAC83* mixed at a ratio of 1:1 (v/v). pTRV2 and pTRV1 empty vectors were used as negative controls. The inoculated seedlings were maintained for 12 h in the dark and employed to a 16-h light/8 h dark cycle at 25 ◦C in a growth chamber. After 25 days, the *AeNAC83* mRNA level was measured and the phenotype of leaves was observed. Then the *AeNAC83*-silenced okra seedlings and the negative control were irrigated with 300 mM NaCl solution for 7 days. The leaf fresh weight and total chlorophyll content was measured [25].

#### *4.5. Generation of AeNAC83 Transgenic Arabidopsis Plants*

The *Agrobacterium* GV3101 containing the 35Sp:: *AeNAC83*:GFP construct was transformed into *Arabidopsis thaliana* ecotype Columbia (wild-type, WT) via the inflorescence infiltration method. Transformed lines were selected on 1/2 MS medium with 30 mg/L hygromycin and then confirmed by PCR. Two *AeNAC83* homozygous transgenic lines (OX-3 and OX-7) of the T4 generations with high *AeNAC83* expression were selected for further phenotypic analysis. The primers were listed in Table S3.

#### *4.6. Performance of Transgenic Lines under Salt Stress Treatment*

Seeds of WT and OX-3 and OX-7 were kept at 4 ◦C for 3 days and then plated on 1/2 MS medium under a 16-h light/8-h dark cycle at 23 ◦C in a growth chamber. Four-day-old seedlings were transplanted on 1/2 MS medium containing 0, 120 or 150 mM NaCl. After 10 days, the total fresh weight and primary root length were measured. Samples of whole plants of OX3 and WT treated with 0 or 120 mM NaCl were harvested and frozen in liquid nitrogen for Illumina sequencing assay. Seedlings were sown on 1/2 MS medium without NaCl treatment for two weeks, and the roots were imaged using a stereo microscope.

#### *4.7. RNA Isolation and Illumina Sequencing*

Total RNA was extracted from the leaves and roots of 14-day *AeNAC83*-overexpression transgenic (OX3) and the wild (WT) *Arabidopsis* treated with 120 mM NaCl. RNase-free DNase I (Takara, Dalian, China) was used to remove the DNA. cDNA libraries were constructed as described by our previous study [25]. The constructed libraries were sequenced on the Illumina platform. The resulting reads (clean reads) were mapped to the Arabidopsis reference genome using HISAT2. FPKM was used to estimate gene expression levels. Differential expression analysis of two groups was performed using the DESeq2 with an adjusted *p*-value < 0.05.

#### *4.8. Gene Annotation and Enrichment Analysis*

Based on a Wallenius non-central hyper-geometric distribution [56], GOseq R packages were used to analyze GO enrichment of DEGs. KOBAS software [57] was used to test whether DEGs were statistically enriched in KEGG pathways.

#### *4.9. Measurement of Anthocyanin Content*

Anthocyanins were extracted from seedlings of WT and OX3 grown in 1/2 MS medium supplemented with 0 or 120 mM NaCl. Leaf material from each treatment was weighed W (g), and then 1 mL of acidic methanol containing 1% HCl (*v*/*v*) was

added and kept in the dark at 4 ◦C for 24 h. The leaching solution was centrifuged at 13,000 rpm for 10 min, and the absorbance was measured at the wavelength of 530 and 657 nm, respectively. The relative content of anthocyanins was calculated by the formula: Qanthocyanins = (A<sup>530</sup> − 0.25 × A657)·g <sup>−</sup><sup>1</sup> FW [58].

#### **5. Conclusions**

In this study, we demonstrate that the nucleus-located AeNAC83 participates in salt stress tolerance in okra. Additionally, AeNAC83 negatively regulates plant growth, indicating a possible node of trade-off between okra resistance and growth. Transcriptome analysis revealed that most of the genes involved in flavonoid biosynthesis and photosynthesis were up-regulated and down-regulated, respectively. Various plant hormone signaling pathway-related DEGs were also identified. Our study provides a comprehensive understanding of the molecular mechanism of AeNAC83 involved in plant growth and salt tolerance.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3390/ ijms231710182/s1.

**Author Contributions:** Y.Z. conceived and designed the research, and drafted the manuscript; X.Z. and T.W. performed the experiments; S.G. and J.H. analyzed the data. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of Zhejiang Province (LQ22C130003) and Scientific Research Fund of Zhejiang A&F University (2020FR057).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The transcriptional data was deposited in the NCBI Sequence ReadArchive (BioProject: PRJNA857503). All data generated or analysed during this study are included in this published article and its Supplementary Materials files.

**Conflicts of Interest:** The authors declare no conflict of interest.

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