*2.4. Identification and Functional Enrichment Analysis of Differential Expression Genes (DEGs) under Salt Stress between the AeNAC83-Overexpression Transgenic and the Wild Arabidopsis*

To understand the molecular mechanism of AeNAC83 in plant growth and salt stress response, RNA was extracted from *AeNAC83*-overexpression transgenic (OX3) and the wild (WT) *Arabidopsis* treated with 120 mM NaCl (12 samples, three replicates for each treatment) and RNA-seq was carried out using Illumina sequencing platform. The Pearson correlation coefficient heat map showed that the samples had good repeatability (Figure S1). A total of 245.54 M reads was obtained, with a total of 73.35 Gb clean data. Clean data of each sample reached 5.74 Gb, and the percentage of Q30 was at least 93.91% (Table S1). We assembled and quantified reads compared with HISAT2 using StringTie and performed fragments per kilobase of transcript per million fragments mapped (FPKM) conversion to analyze gene expression level. The screening of threshold for DEGs was Padj < 0.05 and |log2FoldChange| > 1. The hierarchical clustering analysis of DEGs under different experimental conditions was shown in Figure 5A. The volcanoplots represent the overall distribution of DEGs (Figure 5B). Compared with WT-CK, there were 1917 DEGs in OX3- CK, including 1259 up- and 658 down-regulated genes, and there were 4285 DEGs in WT-N including 2098 up- and 2187 down-regulated genes. Compared with OX3-CK, 1360 DEGs were found in OX3-N, including 692 up and 668 down-regulated genes. Compared with WT-N, there were 1537 DEGs in OX3-N, including 775 up- and 762 down-regulated genes (Figure 5C, Table S2). Venn diagram showed that the number of DEGs between "WT-CK vs. OX3-CK", "WT-CK vs. WT-N", "OX3-CK vs. OX3-N", and "WT-N vs. OX3-N" were 1917, 4285, 1360 and 1537, respectively. A total of 105 common DEGs were identified in the four comparative groups (Figure 5D).

were identified in the four comparative groups (Figure 5D).

**Figure 5.** Transcriptional variations in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment: (**A**) Expression profiles of the DEGs under NaCl treatment were shown by a heatmap; (**B**) Significance analysis of the DEGs in different comparisons by volcanoplots; (**C**) The number of up- and down-regulated genes in different comparisons; (**D**) Venn diagrams showed the proportions of the up- and down-regulated genes in four comparisons. WT-CK, OX3-CK: WT and OX3 grown under optimum conditions; WT-N, OX3-N: WT and OX3 were subjected to salt stress. Three replicates for each treatment. **Figure 5.** Transcriptional variations in *Arabidopsis* wild-type (WT) and transgenic plant (OX3) under NaCl treatment: (**A**) Expression profiles of the DEGs under NaCl treatment were shown by a heatmap; (**B**) Significance analysis of the DEGs in different comparisons by volcanoplots; (**C**) The number of upand down-regulated genes in different comparisons; (**D**) Venn diagrams showed the proportions of the up- and down-regulated genes in four comparisons. WT-CK, OX3-CK: WT and OX3 grown under optimum conditions; WT-N, OX3-N: WT and OX3 were subjected to salt stress. Three replicates for each treatment.

0.05 and |log2FoldChange| > 1. The hierarchical clustering analysis of DEGs under different experimental conditions was shown in Figure 5A. The volcanoplots represent the overall distribution of DEGs (Figure 5B). Compared with WT-CK, there were 1917 DEGs in OX3-CK, including 1259 up- and 658 down-regulated genes, and there were 4285 DEGs in WT-N including 2098 up- and 2187 down-regulated genes. Compared with OX3-CK, 1360 DEGs were found in OX3-N, including 692 up and 668 down-regulated genes. Compared with WT-N, there were 1537 DEGs in OX3-N, including 775 up- and 762 downregulated genes (Figure 5C, Table S2). Venn diagram showed that the number of DEGs between "WT-CK vs. OX3-CK", "WT-CK vs. WT-N", "OX3-CK vs. OX3-N", and "WT-N vs. OX3-N" were 1917, 4285, 1360 and 1537, respectively. A total of 105 common DEGs

#### *2.5. Identification of DEGs Involved in the Phenylpropanoid and Flavonoid Biosynthesis Path-2.5. Identification of DEGs Involved in the Phenylpropanoid and Flavonoid Biosynthesis Pathways*

*ways* KEGG pathway enrichment analysis was performed to interpret the functions of these DEGs. The top three DEGs participated in "MAPK signaling pathway-plant", "plant-pathogen interaction" and "phenylpropanoid biosynthesis" in WT-CK vs. OX3-CK and WT-CK vs. WT-N comparison groups (Figure 6A,B). The top three DEGs participated in "phenylpropanoid biosynthesis", "MAPK signaling pathway", and "starch and sucrose metabolism" in OX3-CK vs. OX3-N comparison group (Figure 6C). The top three DEGs participated in "phenylpropanoid biosynthesis", "circadian rhythm-plant", and "starch KEGG pathway enrichment analysis was performed to interpret the functions of these DEGs. The top three DEGs participated in "MAPK signaling pathway-plant", "plantpathogen interaction" and "phenylpropanoid biosynthesis" in WT-CK vs. OX3-CK and WT-CK vs. WT-N comparison groups (Figure 6A,B). The top three DEGs participated in "phenylpropanoid biosynthesis", "MAPK signaling pathway", and "starch and sucrose metabolism" in OX3-CK vs. OX3-N comparison group (Figure 6C). The top three DEGs participated in "phenylpropanoid biosynthesis", "circadian rhythm-plant", and "starch and sucrose metabolism" in WT-N vs. OX3-N comparison group (Figure 6D).

and sucrose metabolism" in WT-N vs. OX3-N comparison group (Figure 6D). The phenylpropanoid biosynthesis pathway was always found to contain significant enrichment of DEGs for all comparisons (Figure 6). Through integration, 13 gene families The phenylpropanoid biosynthesis pathway was always found to contain significant enrichment of DEGs for all comparisons (Figure 6). Through integration, 13 gene families involved in phenylpropanoid biosynthesis were identified (Figure 7A), including 129 DEGs. The POD family was the most represented (49 DEGs) among these 13 gene families, whereas the C4H, F5H, and CSE family were the least represented, with only 1 DEG (Figure 7A). The phenylpropanoid biosynthesis pathway provides the precursors for the flavonoid biosynthesis pathway [26]. Hence, we analyzed the DEGs involved in flavonoid biosynthesis and 10 gene families were identified, among which the HCT family was the most represented (7 DEGs). The expression levels of most DEGs were up-regulated by NaCl treatment, except for 2 *CHI* genes and 1 *HCT* gene (Figure 7B). In addition, under normal conditions, nearly half of the flavonoid biosynthesis-related DEGs were significantly induced by overexpression of *AeNAC83*. *CHI*, *F3H*, *DFR*, and *ANS* are crucial structural genes involved in the anthocyanin synthesis pathway. Anthocyanins, a class of flavonoids distributed ubiquitously in the plant, play important roles in the growth and development of plants and stress response. The content of anthocyanins in OX3 was significantly higher

than that of WT before salt treatment. Salt stress induced the accumulation of anthocyanins in WT, while there was no significant change in anthocyanin content in OX3 (Figure S2A). OX3 (Figure S2A).

involved in phenylpropanoid biosynthesis were identified (Figure 7A), including 129 DEGs. The POD family was the most represented (49 DEGs) among these 13 gene families, whereas the C4H, F5H, and CSE family were the least represented, with only 1 DEG (Figure 7A). The phenylpropanoid biosynthesis pathway provides the precursors for the flavonoid biosynthesis pathway [26]. Hence, we analyzed the DEGs involved in flavonoid biosynthesis and 10 gene families were identified, among which the HCT family was the most represented (7 DEGs). The expression levels of most DEGs were up-regulated by NaCl treatment, except for 2 *CHI* genes and 1 *HCT* gene (Figure 7B). In addition, under normal conditions, nearly half of the flavonoid biosynthesis-related DEGs were significantly induced by overexpression of *AeNAC83*. *CHI*, *F3H*, *DFR*, and *ANS* are crucial structural genes involved in the anthocyanin synthesis pathway. Anthocyanins, a class of flavonoids distributed ubiquitously in the plant, play important roles in the growth and development of plants and stress response. The content of anthocyanins in OX3 was significantly higher than that of WT before salt treatment. Salt stress induced the accumulation of anthocyanins in WT, while there was no significant change in anthocyanin content in

*Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 9 of 19

**Figure 6.** The top 20 enriched KEGG pathways enrichment analysis of DEGs in the four comparison groups. (**A**–**D**) KEGG pathway enrichment analysis of DEGs in the WT-CK vs. OX3-CK, WT-CK vs. WT-N, OX3-CK vs. OX3-N, and WT-N vs. OX3-N comparisons, respectively. **Figure 6.** The top 20 enriched KEGG pathways enrichment analysis of DEGs in the four comparison groups. (**A**–**D**) KEGG pathway enrichment analysis of DEGs in the WT-CK vs. OX3-CK, WT-CK vs. WT-N, OX3-CK vs. OX3-N, and WT-N vs. OX3-N comparisons, respectively.
