*3.1. Expression of ERFVII during Early Flooding Treatment*

To analyze the responses of sweet potato plants to flood stress, the tolerance of whole plants grown in soil was measured during early flooding treatment. Approximately 65% of the aboveground tissue of 2-month-old plants was submerged by adding excess water to the pot in a growth chamber at 25 ◦C under a 16 h/8 h light/dark photocycle. When the susceptible cultivar JM and the resistant cultivar YJM were exposed to flooding for 0, 0.5, and 3 days, the above atmospheric leaves of the JM plants showed slight wilting and curling, whereas the leaves of the YJM plants were less damaged (Figure 1A).

ET biosynthesis and signaling pathways are usually activated during flood treatment in plants [25]. TFs belonging to the *ERFVII* group, including *ERF71/HER2*, *ERF72/RAP2.3*, *ERF74/RAP2.12*, and *ERF75/RAP2.2*, are important representatives of genes that function in the ET signaling pathway activated during flood treatment at the transcriptional and post-translational levels [26,27]. Although expression analysis at the post-translational level is more important in the *ERFVIIs* [27], in this study, the expression of *ERFVII* group genes was investigated during early flood treatment at the transcriptional level (Figure 1B). The expression patterns of *ERF72/RAP2.3* (g6122), *ERF74/RAP2.12* (g55154), and *ERF74/RAP2.12* (g60549) were investigated after flood treatment using our previous transcriptomic data [21]. *ERF72/RAP2.3* was strongly induced from 0.5 d after flooding treatment in the flood-tolerant cultivar, YJM, whereas its expression decreased in the flood-sensitive JM. In contrast, *ERF74/RAP2.12* showed a weak increase in expression level in YJM, and increased expression after 3 d of flooding treatment. In JM, there was no clear pattern of changes in expression under flooding conditions, as levels increased or decreased at different times.

**Figure 1.** *Cont*.

**Figure 1.** Effects of flooding stress on the sweet potato cultivars Yeonjami (YJM) and Jeonmi (JM). (**A**) Phenotypes of aboveground portions of YJM and JM after 3 d of flooding treatment. (**B**) Expression of *ERFVII* transcription factors in each cultivar after 0, 0.5, or 3 d of flooding treatment. Different letters represent statistically significant differences between control and flooding treatment, and between flooding-treated JM and YJM, determined using two-way ANOVA with the LSD post hoc test; *p* < 0.05.

#### *3.2. The Responses of ROS during Early Flooding Treatment*

The levels of free radicals and ROS in plants usually increase during flood treatment [25]. Increases in ET biosynthesis and signaling during flood treatment modulate the amount of ROS and free radical signaling. Therefore, changes in H2O2, a representative ROS, were investigated during the early flood treatment process (Figure 2A). On day 3 of flood treatment, the H2O2 content of the flooding-tolerant cultivar, YJM, was 38% lower than that of the flooding-sensitive cultivar, JM.

We also investigated the expression levels of *RBOH*, a gene which is involved in early ROS production, during flood treatment (Figure 2B). Expression levels of *RBOHA* (g19623), *D* (g51882), and *E* (g55049) were higher in the resistant cultivar YJM than in the susceptible JM under normal untreated conditions; expression increased to higher levels in YJM than in JM within 0.5 days of flood treatment. On the other hand, *RBOHC* (g19835) showed similar changes in expression levels in both cultivars under the normal control and the early flooding treatment. After 3 days of flood treatment, the expression levels of all the

*RBOH* genes measured in the experiment were higher in JM than in YJM, due to decreases in expression in the resistant YJM cultivar between 0.5 and 3 days of flood treatment.

**Figure 2.** Expression of genes related to hydrogen peroxide (H2O2) and ROS pathways in sweet potato cultivars treated with flooding stress for 3 d. (**A**) Relative levels of H2O2 in each cultivar after 0, 0.5, or 3 d of flooding treatment. (**B**) Expression of *RBOH* genes in each cultivar after 0, 0.5, or 3 d of flooding treatment. (**C**) Expression of *MT* genes in each cultivar after 0, 0.5, or 3 d of flooding treatment. Different letters represent significant differences between control and flooding treatment, and between flooding-treated JM and YJM, determined using two-way ANOVA with the LSD post hoc test; *p* < 0.05.

Next, we measured the expression of *METALLOTHIONEIN* (*MT*) genes, which encode proteins that remove reactive oxygen species during flood treatment (Figure 2C). Expression levels of all the *MT* genes were higher in the flood-resistant YJM than in the flood-sensitive JM under normal untreated conditions. After 0.5 days of flood treatment, expression levels of *MT* genes remained higher in YJM than in JM; in particular, *MT2* (g58960 and g4909) showed a greater increase in expression in YJM than in JM. In contrast, *MT3* (g64044) showed similar expression levels in both cultivars under both control conditions and early flooding treatment. By the third day of flood treatment, however, the expression levels of *MT1* and *MT2* decreased in YJM and increased in JM.

#### *3.3. The Responses of NO during Early Flooding Treatment*

An increase in NO, together with rises in ET and ROS levels, is seen in plants at the initial stage of flood treatment; this increase in NO controls the increase in ET during flood treatment [26]. Plants usually regulate endogenous NO levels via the control of biosynthesis and scavenging. The main source of NO production is via the enzymatic and nonenzymatic reduction of nitrite (NO2 −) [13,14]. NO2 − production is highly dependent on nitrate reductase (NR). The dependency of NO production on nitrite availability makes NR the major player in NO production [14,15]. The NO generation during hypoxia is also thought to result from enhanced NR activity and NO2 − accumulation, providing a substrate for NO production [13,19].

We therefore tested the changes in nitrite (NO2 −) levels in sweet potato plants during early flood treatment. In contrast to the changes in H2O2 levels (Figure 2A), the increase in NO2 − was 31% higher in YJM than in JM (Figure 3A).

**Figure 3.** *Cont*.

**Figure 3.** Levels of nitrite (NO2 −) and expression of NO-related genes in sweet potato cultivars treated with flooding stress for 3 d. (**A**) Relative levels of NO2 − in each cultivar after 0, 0.5, or 3 d of flooding treatment. (**B**) Expression of the NO biosynthesis genes *NR* and *NIR* in each cultivar after 0, 0.5, or 3 d of flooding treatment. (**C**) Expression of *PGB* genes in each cultivar after 0, 0.5, or 3 d of flooding treatment. Different letters represent statistically significant differences between control and flooding treatment, and between flooding-treated JM and YJM, determined using two-way ANOVA with the LSD post hoc test; *p* < 0.05.

Next, we investigated changes in the expression of the NO biosynthesis genes *NI-TRATE REDUCTASE* (*NR*) and *NITRITE REDUCTASE* (*NIR*) (Figure 3B). The expression of *NR* (g1372) and *NIR* (g59680), both genes involved in early NO biosynthesis, did not differ significantly between YJM and JM under control conditions. After flood treatment, their expression gradually increased in YJM but gradually decreased in JM. We also examined the expression of the NO-scavenging *PHYTOGLOBIN* (*PGB*) genes during early flooding treatment (Figure 3C). *PGB1* (g34086), *2* (g60867), and *3* (g21239) showed higher levels of expression in YJM than in JM under control conditions and during early flood treatment (0.5 days). Their expression decreased, however, in YJM by 3 days after flooding so that, by this timepoint, the expression of *PGB*s was higher in JM than in YJM.
