*2.3. Overexpression of AeNAC83 in Arabidopsis Inhibits Plant Growth and Improves Salt Tolerance*

To further investigate the role of AeNAC83 in salt stress and growth, over-expression of *AeNAC83* gene transgenic Arabidopsis plants were produced. The lines OX3 and OX7 with high expression of *AeNAC83* were selected for further phenotypic analysis from at least 10 homozygous transgenic lines (Figure 4A). *Int. J. Mol. Sci.* **2022**, *23*, x FOR PEER REVIEW 7 of 19

**Figure 4.** Roles of AeNAC83 in salt tolerance and in growth in *Arabidopsis* transgenic plants. Fourday-old seedlings of wild-type (WT) and two transgenic lines (OX3 and OX7) were transplanted on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl for 10 days. (**A**) Semi-quantitative analysis of *AeNAC83* gene expression in wild-type (WT) and *AeNAC83*-overexpression transgenic plants. The first bands show *AeNAC83* gene expression (30 cycles) and the bands below show *AtActin* gene expression (26 cycles) used as internal control. (**B**) Phenotypes of seedlings on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl, respectively. (**C**,**D**) Fresh weight and primary root length of seedlings at the end of the experiment in (**B**). Data are presented as mean ± SD (*n* = 10). Different letters denote significant differences at *p* < 0.05, using ANOVA and Duncan's multiple tests. (**E**) Root phenotype of seedlings on 1/2 MS medium without NaCl treatment. The red arrows indicated the crown root. **Figure 4.** Roles of AeNAC83 in salt tolerance and in growth in *Arabidopsis* transgenic plants. Fourday-old seedlings of wild-type (WT) and two transgenic lines (OX3 and OX7) were transplanted on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl for 10 days. (**A**) Semi-quantitative analysis of *AeNAC83* gene expression in wild-type (WT) and *AeNAC83*-overexpression transgenic plants. The first bands show *AeNAC83* gene expression (30 cycles) and the bands below show *AtActin* gene expression (26 cycles) used as internal control. (**B**) Phenotypes of seedlings on 1/2 MS medium supplemented with 0, 120, or 150 mM NaCl, respectively. (**C**,**D**) Fresh weight and primary root length of seedlings at the end of the experiment in (**B**). Data are presented as mean ± SD (*n* = 10). Different letters denote significant differences at *p* < 0.05, using ANOVA and Duncan's multiple tests. (**E**) Root phenotype of seedlings on 1/2 MS medium without NaCl treatment. The red arrows indicated the crown root.

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

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 <

The *Arabidopsis thaliana* ecotype Columbia (wild-type, WT) and transgenic seedlings were grown in 1/2 MS medium for 4 days and then transplanted to medium with 0, 120 or 150 mM NaCl for 10 days to observe the phenotypic difference. Under normal conditions, significant differences existed in the growth between the WT and transgenic plants. Compared with WT, *AeNAC83*-overexpression transgenic plants exhibited many altered phenotypes (Figure 4B), including small rosette, short primary roots and promoted crown roots and root hairs (Figure 4E). The fresh weight (Figure 4C) and primary root length (Figure 4D) of the transgenic plants were significantly declined than those of wild-type plants. After NaCl treatment for 10 days, better performance was observed for transgenic lines than WT. Most WT leaves showed albinistic symptoms, and the number of crown roots of transgenic lines was greater than in control (Figure 4D). The fresh weight of the transgenic lines was greater than that of WT exposed to salt stress, and there was no obvious difference in primary root length between WT and OX7 transgenic lines. Together, these results suggest that over-expression of *AeNAC83* inhibited plant growth and enhanced tolerance to salt stress.
