**1. Introduction**

Flooding is a major environmental stress that causes loss of crop yield in various agricultural settings. The global risk of flooding has increased dramatically in recent decades due to climate change [1]. Over the past 50 years, increasingly frequent and severe flood events have negatively impacted the lifespan of terrestrial plants. When flooding occurs, gas exchange between the plant and its environment is severely restricted, causing several internal changes in the plant. Plant organs that are submerged in water lack O2

**Citation:** Park, S.-U.; Lee, C.-J.; Park, S.-C.; Nam, K.J.; Lee, K.-L.; Kwak, S.-S.; Kim, H.S.; Kim, Y.-H. Flooding Tolerance in Sweet Potato (*Ipomoea batatas* (L.) Lam) Is Mediated by Reactive Oxygen Species and Nitric Oxide. *Antioxidants* **2022**, *11*, 878. https://doi.org/10.3390/ antiox11050878

Academic Editor: Stanley Omaye

Received: 22 March 2022 Accepted: 27 April 2022 Published: 29 April 2022

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and/or CO2 and may accumulate high levels of the gaseous hormone ethylene (ET). In addition, there are changes in the concentrations of oxygen-derived free radicals, reactive oxygen species (ROS), and nitric oxide (NO) [2]. The production, kinetics, concentration, and precise balance of these substances in flooded cells depend on the plant organ and the type of flooding. Some crop species can withstand soil inundation for only a few hours, while other flood-tolerant crops can cope with partial or complete flooding for days or months [2].

Flood-tolerant crops have anatomical, morphological, or metabolic adaptations. One of the responses to flooding is the induction of ET [3]. ET production is enhanced in crops such as rice and barley. Only flood-tolerant rice varieties, however, can withstand prolonged soil submersion or submergence phases [2]. ET is usually produced in a two-step reaction involving 1-aminocyclopropane-1 carboxylate (ACC) synthetase and ACC oxidase. ET is not only produced in response to flooding stresses but is also a modulator of stress-related morphological responses during plant growth [4,5]. In *Arabidopsis thaliana*, ET is sensed by ET receptors (ETRs), which trigger a downstream signaling cascade of transcription factors (TFs) including *APETALA2/ETHYLENE RESPONSE FACTOR* (*AP2/ERF*) [6,7].

ROS are among the key molecules mediating plant responses to flood stress [8]. Apoplastic ROS are usually generated via the respiratory burst oxidase homolog (RBOH) protein located in the plasma membrane. RBOH is a homolog of the mammalian NADPH oxidase subunit gp91phox that produces a superoxide anion (O2 −) [9]. The short-lived and highly reactive toxic chemical O2 − is converted to the nonradical hydrogen peroxide (H2O2), either spontaneously or by catalysis by superoxide dismutase (SOD) [10]. ROS might also be formed by mitochondria and/or the photosynthetic electron transport chain [8,10]. Nonenzymatic ROS-scavenging proteins, including cysteine-rich metallothionein (MT) and antioxidant enzymes such as catalase and ascorbate-glutathione cycle-related enzymes, are essential for ROS homeostasis. During flooding stress, the ROS balance of cells is disturbed due to either enhanced ROS production or a decrease in ROS scavenging capacity [8].

NO is a short-lived, highly reactive molecule that regulates several plant developmental and stress responses [11,12]. The main method of NO production is through the enzymatic and nonenzymatic reduction of nitrites [13,14]. Nitrite production is highly dependent on nitrate reductase (NR) activity. The dependence of NO production on nitrite availability means that NR plays the major role in NO production [14,15]. There are limited and contrasting data on the temporal and spatial dynamics of NO in flooded plants [15–17]. NO emissions from unsubmerged atmospheric plant tissues decreased in Arabidopsis and cotton under waterlogging, whereas NO emissions increased in three submerged deciduous tree species [15–17]. In addition, cellular and exogenous NO levels increased in various plant species during hypoxia [13,18,19]. During hypoxic conditions, this NO burst is thought to reflect an increase in NO production due to increased NR activity and nitrite accumulation. The spatiotemporal dynamics of NO and its effects may vary, therefore, depending on the flood conditions. Studying the changes in ET, ROS, and NO levels is thus important for fully understanding the mechanisms of plant flooding responses and tolerance.

We previously studied 33 sweet potato cultivars, using phenotypic and biochemical characteristics to identify flood-tolerant cultivars [20]. In addition, we recently used comparative transcriptome profiling to compare a flood-tolerant sweet potato cultivar, Yeonjami (YJM), with a flood-sensitive cultivar, Jeonmi (JM) [21]. The expression levels of several candidate genes thought to be involved in flooding tolerance correlated with the comparative transcriptomic data. However, although each sweet potato cultivar can be classified as either tolerant or sensitive to flooding stress, the molecular mechanisms of flooding resistance in ET, ROS, and NO regulation-mediated responses have not yet been reported. In this study, therefore, we conducted a transcriptome-based expression analysis of genes involved in ET, ROS, and NO regulation in two sweet potato cultivars during flooding stress.

#### **2. Materials and Methods**

### *2.1. Plant Materials*

This study involved two previously characterized cultivars of sweet potato (*Ipomoea batatas* (L.) Lam), namely, Yeonjami (YJM) and Jeonmi (JM). YJM is highly tolerant to flooding stress, whereas JM is highly sensitive [20,21]. Both cultivars were obtained from the Bioenergy Crop Research Center, National Crop Research Institute (RDA, Muan, Jeonnam, Korea). Sweet potato plants were cultivated and subjected to flooding stress as described by Park et al. [21]. Plants were grown in a growth chamber at 25 ◦C under a 16 h/8 h light/dark photocycle and 60% relative humidity. Experimental plants were grown for 2 weeks under normal conditions and then subjected to flooding stress. To induce flooding stress, water was added to the pots until approximately 65% of the aboveground tissue was submerged. Leaves were sampled 0 (control), 0.5, and 3 days after the beginning of flooding treatment. For leaf samples used in the experiment, the 3rd and 4th leaves from the top were used in four plants. The harvested leaves were above the water surface. Leaf samples were frozen immediately in liquid nitrogen and stored at −70 ◦C until further analysis.
