*3.4. The Responses of MDHAR during Early Flooding Treatment*

Previous studies reported that ascorbic acid (AsA) and PGB are involved in NO metabolism in plants. In vitro enzymatic assays have shown that NO scavenging is catalyzed by monodehydroascorbic acid reductase (MDHAR), which mediates binding reactions involving iron reduction in PGB in the presence of AsA and NADH [28]. AsA supports NO scavenging, directly reducing PGB in plants to produce nitrate and monodehydroascorbate (MDA). The final product of this reaction is MDA, which is efficiently reduced back to AsA in the presence of MDHAR and NADH. NO scavenging is also promoted by MDHAR [29].

We therefore investigated *MDHAR* gene expression and MDHAR-associated enzymatic activity (Figure 4). It was confirmed that the expression of different *MDHAR* genes (cytosolic, g25138; peroximal, g17489; and mitochondrial, g35177) and MDHAR enzymatic activity were higher in the flood-tolerant YJM than in the flood-sensitive JM. Changes in MDHAR expression and activity in tolerant sweet potato during flood treatment may therefore play an important role in NO regulation through interactions with PGB as well as via ROS regulation.

**Figure 4.** Changes in MDHAR activity and *MDHAR* gene expression in sweet potato cultivars treated with flooding stress for 3 d. (**A**) Relative MDHAR activity in each cultivar after 0, 0.5, or 3 d of flooding treatment. (**B**) Expression of *MDHAR* genes in each cultivar after 0, 0.5, or 3 d of flooding treatment. c*MDHAR*: cytosolic *MDHAR*; p*MDHAR*: peroxisomal *MDHAR*; m*MDHAR*: mitochondrial *MDHAR*. 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.

#### **4. Discussion**

Plants that exhibit resistance to flooding use two general survival strategies: low-O2 escape syndrome (LOES) and low-O2 quiescence syndrome (LOQS) [2,30]. Regulated anaerobic metabolism in both LOES and LOQS is equivalent to survival in low-O2 conditions. At the heart of this mechanism is an evolutionarily conserved group of TFs, ethylene response factor VIIs (ERFVIIs), which activate the genes required for anaerobic metabolism in Arabidopsis [31]. A reduction in O2 levels in plants stabilizes ERFVII TFs such as

RAP2.12 and RAP2.3, which play an important role in activating anaerobic responses at the transcriptional level [32]. Giuntoli et al. [33] also reported that the expression of a set of genes involved in the oxidative stress response is induced in flood-exposed Arabidopsis. Thus, ERFVII is a positive regulator of oxidative stress-related genes and genes involved in fermentative metabolism. The *ERFVII* TFs *RAP2.2*, *RAP2.3*, and *RAP2.12* mediate responses to oxidative stress and may act redundantly [34]. Overexpression of *RAP*-type *ERFVII* TFs also confers resistance to oxidative stress after H2O2 application. *RAP*-type *ERFVII* genes, which are involved in ROS scavenging and signaling, are themselves positively regulated by ERFVII TFs [33,34].

We investigated the expression of the representative *ERFVII* TFs *RAP2.3* and *RAP 2.12* in the sweet potato cultivars YJM and JM, which were identified as flood-resistant and flood-sensitive, respectively, in a previous study [21] (Figure 1). The expression of *ERF72/RAP2.3* and *ERF74/RAP2.12* was higher in YJM than in JM during flood stress; expression levels of these genes were increased, especially during the early period of flooding. We also measured the expression of four genes belonging to other groups within the ERF transcription factor family, *ERF1* (g31279), *ERF2* (g25395), *ERF4* (g54463), and *ERF5* (g20475). These showed either similar increases in expression in the two cultivars after flood treatment or slightly higher expression levels in JM (data not shown). A recent transcriptomic analysis of the sweet potato cultivars YJM and JM revealed changes in the expression of *ETR*, *EIN*, and *ERF*, which are all genes related to signal transduction involved in ET signaling pathways [21]. As indicated by previous studies in Arabidopsis and rice, it is highly likely that *RAP2.3* and *RAP2.12*, which are ERFVII TFs, are an important part of the mechanism enabling flood resistance in sweet potato.

The onset of anaerobic conditions triggers a burst of ROS in Arabidopsis due to changes in NADPH oxidase activity in membranes and an imbalance in the mitochondrial electron transfer system [35]. Arabidopsis *RBOHD* mutants are less tolerant of anaerobic conditions and show negative effects on *ALCOHOL DEHYDROGENASE1* (*ADH1*) expression, compared to wild-type seedlings immersed in water [35–37]. This suggests that ROS generated by *RBOHD* under these conditions may be a positive signal necessary for plant tolerance of flooding-mediated hypoxia. Yamauchi et al. [38] observed high levels of induction of *RBOH* expression in parallel with repression of *MT*, which encodes an ROS scavenger, in maize roots that did not show tissue reduction following treatment with the NADPH oxidase inhibitor diphenyleneiodonium (DPI). High expression of *RBOH* can therefore induce cell death in plants during flood treatment through oxidative burst. High *RBOH* expression appears to act either by suppressing the expression of ROS-scavenging genes, including the *MT*s, or, during flooding, through signal transduction pathways that increase the expression of a downstream gene that causes ROS to be removed and activates an appropriate defense mechanism. We observed that the expression of *RBOHA*, *D*, and *E* increased during the initial flood treatment in the flood-resistant cultivar YJM, and the expression of *MT2* significantly increased in the flooding treatment after 12 h (Figure 2). Therefore, flood tolerance in sweet potato appears to involve activation of the pathway involving the ROS scavenger protein MT.

Like ROS, NO is detrimental to plant cells, but it is also a key component of plant response-related signaling pathways [39]. In plants, NO is involved in the degradation of transcriptional regulators, which leads to the activation of key hypoxia-responsive genes [40]. Indeed, in anaerobic plants, NO availability negatively modulates the activation of responses induced by ERFVII TFs. Arabidopsis NR mutants such as *nitrate reductase 1* (*nia1*) and *nia2* exhibit changes in anaerobic gene transcription due to the impaired production of NO, which alters the activation of genes downstream of *ERFVII* in reaction to anaerobic conditions [40]. Therefore, NO destabilizes *ERFVII* and inhibits downstream signaling pathways. Hartman et al. [41] reported that early ET capture from flooded Arabidopsis increases transcription of the NO scavenger nonsymbiotic *PGB1*, thereby reducing the solubility of NO and promoting the stability of ERFVII. This phenomenon occurs prior to severe hypoxia in plants and acts as a priming event, enhancing the plant's tolerance to upcoming stressful conditions. In the current study, a greater increase in NO2 − levels during flood treatment was observed in the flood-resistant cultivar YJM than in JM, a flood-sensitive cultivar (Figure 3A); although there was no significant change during the initial flood treatment, a difference was observed after 3 days of treatment. During the flood treatment, the expression of NR and NIR, genes involved in NO2 − levels, gradually increased in YJM but decreased gradually in JM (Figure 3B). It therefore appeared that exposure to flooding activated response mechanisms regulated by NO2 − generation in YJM, a flood-resistant sweet potato cultivar.

PGB, which scavenges NO, potentially promotes the interaction between NO and ET. Levels of *PGB* mRNA increase during submergence and hypoxic conditions in several plant species [42,43]. NO also increases the levels of *PGB* mRNA in rice, cotton, and spinach, indicating a feedback mechanism [44–46]. Interactions and feedback regulation between NO and ET have also been shown at the level of ERFVII, as ERFVII stability and action are highly dependent on NO and oxygen levels [40,47]. When NO or oxygen concentrations are reduced, ERFVII TFs accumulate, thereby promoting transcription of downstream target genes. Interestingly, several genes regulating ERFVII TFs contain hypoxia-responsive promoter elements, including several ET signaling genes and the NO-scavenging gene *PGB* [31]. Therefore, ERFVII TFs are upregulated by NO, oxygen, and ET, whereas ERFVII action can, in turn, induce ET biosynthesis and NO scavenging, creating a positive feedback loop [31]. We observed that the expression of *PGB*s increased strongly within 0.5 days of flood treatment in the flood-resistant cultivar YJM, i.e., during the initial period of flooding (Figure 3); in contrast, the expression of *PGB*s increased slowly and gradually in JM. In this study, therefore, the expression of NO2 − generation-related genes gradually increased in response to flooding, but the expression of *PGBs* increased immediately, suggesting that the overall level of NO2 − increased gradually in the flooding-tolerant YJM. It is possible that NO2 − generated through the nonenzymatic pathway via mitochondria may have influenced the increase in NO2 − levels, in addition to NO2 − generated by the enzymatic pathway acting through NR and NIR. These results suggest that increases in NO2 − generation and elimination affected the ROS signaling mechanism through the expression of *ERFVII*, thereby activating the flood resistance mechanism.

We previously reported comparative transcriptome profiling to compare a floodtolerant sweet potato cultivar with a flood-sensitive cultivar [21]. A higher number of some of the ROS signaling-related genes such as mitogen-activated protein kinase (MAPK) and ET signaling-related genes were upregulated in the tolerant cultivars than in the susceptible cultivar. Recently, another study also reported comparative transcriptome profiling using different soybean cultivars [48]. A higher number of some of the ROS-related genes such as glutathione S-transferase and lipoxygenase were upregulated in the tolerant cultivars than in the susceptible cultivar. The number of some phytohormone ABA-related TFs of the basic leucine zipper domain was also higher in the tolerant cultivars than in the susceptible cultivar. Similar to our previous study, the expression levels of several candidate genesrelated to ROS and phytohormones thought to be involved in flooding tolerance correlated with the comparative transcriptomic data.

This study addressed the transmission of the plant signaling molecules ET, ROS, and NO and showed how these signals were correlated in flood-tolerant and flood-sensitive cultivars of sweet potato (Figure 5). Consistent with previous research, a signaling pathway acting through ERFVII was activated in flood-resistant plants. In addition, our results suggest that the expression of *RBOH* and *MT* genes, which encode components of an ROS signaling pathway involving ERFVII, played an important role in enabling the flood resistance of sweet potato. This study also suggests that activation of the NO biosynthesis and scavenging cycle and an increase in MDHAR activity likely regulated ERFVII expression and levels of ROS via other pathways.

**Figure 5.** A suggested model of the ET-, ROS-, and NO-related biological processes and genes involved in influencing the flooding response that occurs in sweet potato leaves under early flooding. Flooding conditions result in the induction of ERFVII transcription factors and the expression of RBOHs, which produce O2 − radicals from oxygen in the apoplast. RBOH expression may enhance RBOH activity and increase ROS levels in the apoplast as well as the cytosol. Expression of genes encoding ROS scavengers, such as MTs, increases, leading to an increase in ROS signaling and scavenging responses and inducing the response in flooding-tolerant sweet potato. The NO synthesis-scavenging cycle regulates ERFVII-mediated signaling. MDHARs are also potentially involved in NO/ROS-mediated responses in sweet potato under early flooding conditions. ERF: ethylene response factor; ET: ethylene; ROS: reactive oxygen species; RBOH: respiratory burst oxidase homolog; MT: metallothionein; NO: nitric oxide; NR: nitrate reductase; NIR: nitrite reductase; PGB: phytoglobulin; MDHAR: monodehydroascorbate reductase.

#### **5. Conclusions**

In conclusion, we characterized changes in the expression of flooding response-related genes in flood-tolerant and flood-sensitive sweet potato cultivars that were associated with response mechanisms mediated by ET, ROS, and NO. This approach identified candidate sweet potato genes whose expression was associated with changes in specific responses involved in metabolic and signaling pathways related to ET, ROS, and NO. The use of marker-assisted selection to identify genes linked to flood tolerance offers several advantages over flooding control in an integrated management system. Further investigation, based on large-scale genomic and transcriptomic analysis, is required to elucidate the exact role played by each candidate gene in regulating the signaling pathways involved in flooding tolerance responses of sweet potato during flooding stress. Transgenic plants with an enhanced or reduced expression of candidate genes will be generated to determine their role in the mechanisms conferring flooding tolerance. Overall, our results provide valuable information for enabling the development of crops with enhanced tolerance to hypoxic stress induced by flooding.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/xxx/s1. Table S1: List of primers used for quantitative real-time PCR (qRT-PCR) analysis of sweet potato genes.

**Author Contributions:** Conceptualization, S.-U.P., C.-J.L., H.S.K. and Y.-H.K.; data curation, S.-C.P.; formal analysis, K.J.N., K.-L.L. and S.-S.K.; funding acquisition, S.-S.K.; methodology, S.-U.P., C.-J.L., H.S.K. and Y.-H.K.; resources, S.-S.K., H.S.K. and Y.-H.K.; validation, K.J.N., K.-L.L. and S.-S.K.; visualization, K.J.N., K.-L.L. and S.-S.K.; writing—original draft, S.-U.P., C.-J.L., H.S.K. and Y.-H.K.; writing—review and editing, S.-U.P., C.-J.L., H.S.K. and Y.-H.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (2020R1A2C1004560, 2021R1A2C400188711) and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM5372113).

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

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

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

#### **Abbreviations**



#### **References**

