Hypoxia is one of the major environmental stresses caused by flooding, such as submergence and waterlogging, and has detrimental effects on plant development and growth (Zhou et al. 2020). Because of excessive water uptake, hypoxia mechanically impairs germination and seedling establishment, ultimately impacting crop production (Arguello et al. 2016; Zhang et al. 2016). Flooding also reduces seed quality in agriculturally important crops, such as soybean and cotton, by changing the distribution and accumulation of carbohydrates, proteins, and oils (Wang et al. 2018; Xu et al. 2021). Thus, hypoxia negatively regulates plant growth and development.

The phytohormone abscisic acid (ABA) mediates the response to various environmental stresses such as dehydration, salinity, and cold, and regulates water potential in plants by inducing stomata formation in submerged leaves (Iida et al. 2016) and controlling stomatal movement by guard cells (He et al. 2018). Additionally, exogenous ABA treatment can increase plant tolerance to flooding stress, most likely by compensating for the hypoxia-induced inhibition of ABA biosynthesis and promotion of ABA catabolism (De Ollas et al. 2021). In flooding-stressed soybean, ABA pretreatment increases the amount of protein through various metabolic process, which improves the survival rate and hypoxic properties of plants (Komatsu et al. 2013; Wang et al. 2018; Yin et al. 2016). In rice, ABA treatment positively regulates relative growth rate, net assimilation rate, and chlorophyll content during flooding (Saha et al. 2021).

Arabidopsis thaliana is also widely used as a model organism for molecular studies of plant adaptation to flooding-mediated hypoxic conditions. During flooding, ABA is important for the control of stomatal closure, inhibition of growth, and regulation of metabolic processes, all of which help increase plant survival under this condition (Pierik et al. 2010). In Arabidopsis, ABA-mediated stomatal closure reduces water loss, inhibits root water uptake, and adjusts metabolic activity to cope with oxygen deprivation (Voesenek and Bailey-Serres 2015). At the molecular level, ABA regulates the expression of genes involved in the flooding stress response. In Arabidopsis, ABA binds to PYR/PYL receptor proteins, inhibiting PP2C phosphatase activity, which leads to the activation of SnRK2 protein kinases. Activated SnRK2 then triggers downstream transcription factors, which in turn regulate the expression of stress-responsive genes (Cutler et al. 2010). This ABA signaling pathway plays a critical role in orchestrating the genetic response required for plant adaptation to hypoxic conditions during flooding. ABA also plays an important role in post-flooding recovery. When oxygen is reintroduced after flooding, plants overproduce reactive oxygen species (ROS), which can cause cellular damage. ABA suppresses the accumulation of ROS and facilitates cellular repair, promoting faster recovery from flooding stress (Visser et al. 2016). During this recovery process, ABA signaling pathways are essential for the regulation of cell repair mechanisms. Thus, ABA promotes plant growth under hypoxic conditions; however, the molecular mechanisms underlying the hormonal regulation of plant processes under hypoxia remain largely unknown. Therefore, additional investigation of gene expression patterns is needed to understand the basic mechanisms of ABA biosynthesis, catabolism, and signal transduction in plants under hypoxic conditions.

Sweetpotato (Ipomoea batatas [L.] Lam) is one of the food crops that could be used to address the global challenges of food and nutrition security (Kwak 2019). In the field, sweetpotato plants are highly resistant to abiotic and biotic stresses (Chen et al. 2016; Kim et al. 2013b) but are vulnerable to flooding stress (Lin et al. 2006). In previous studies, sweetpotato storage root yield was reduced by 57% during mid-season flooding (Roberts and Russo 1991), and the sweetpotato storage roots was altered under hypoxic conditions (Eguchi and Yoshida 2007). Therefore, sweetpotato cultivation may be limited in areas prone to flooding.

In a previous study, the phenotypic and biochemical characteristics of 33 sweetpotato cultivars were studied to identify flooding-tolerant cultivars (Park et al. 2020a). Additionally, we recently used comparative transcriptome profiling to compare a flooding-resistant sweetpotato cultivar, Yeonjami, with a flooding-sensitive cultivar, Jeonmi (Park et al. 2020b). Changes in the abundance of transcripts and proteins involved in ethylene, ROS, and nitric oxide (NO) regulation were correlated with comparative transcriptomic data collected under flooding stress (Park et al. 2022). Although sweetpotato cultivars have been classified as either sensitive or resistant to flooding stress, the role of ABA biosynthesis and signaling in the acquisition of flooding resistance has not yet been elucidated. Therefore, in this study, we performed a transcriptome-based expression analysis of genes involved in the regulation of ABA biosynthesis and signaling in two sweetpotato cultivars during flooding stress.

Plant materials and flooding stress treatment

Two sweetpotato (Ipomoea batatas [L.] Lam) cultivars were used in this study: Yeonjami (YJM; flooding resistant) and Jeonmi (JM; flooding sensitive) (Park et al. 2022). For flooding stress treatment, water was added to the pots until approximately 65% of the above-ground tissue was submerged. The 3rd and 4th leaves (from the top) of plants were harvested and were collected from four plants at each time point and frozen in liquid nitrogen.

Expression analysis

Quantitative real-time PCR (qRT-PCR) analysis was performed using Bio-Rad CFX96 thermal cycler (Bio-Rad) with EvaGreen fluorescent dye, according to the manufacturer’s instructions. Gene-specific primers used for qRT-PCR are listed in Supplementary Table 1.

Analysis of ABA levels

The levels of ABA from leaves of sweetpotato plants was measured using a Phytodetek ABA enzyme immunoassay test kit (Agdia, Inc., Elkhart, IN), as described elsewhere (Kim et al. 2013a), with slight modifications at a wavelength of 405 nm (Bio-Rad, Hercules, CA, USA).

Analysis of H₂O₂ levels

The levels of H₂O₂ from leaves of sweetpotato plants was assessed using the xylenol orange method (Bindschedler et al. 2001). Leaf tissue was ground and homogenized in a solution of 50 mM potassium phosphate buffer (pH 6.5) and the absorbance of the samples was determined at 560 nm.

Analysis of catalase activity

Total soluble protein was extracted from leaves of sweetpotato plants using extraction buffer, and the concentration of total protein was determined using the Bio-Rad Bradford assay reagent (Bradford 1976). Catalase (CAT) activity was assayed according to the method described by Aebi (1984) at 240 nm for 1 min.

Statistical analysis

Data were analyzed using one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) test. All statistical analyzes were performed using the Statistical Package for Social Sciences (SPSS 12). The level of statistical significance was set at P < 0.05.

Differential response of ABA biosynthesis- and catabolism-related genes to flooding

To investigate the response of ABA metabolism-related genes to flooding stress in sweetpotato, changes in the expressions of genes related to ABA biosynthesis and catabolism were confirmed in JM (flooding-sensitive cultivar) and YJM (flooding-resistant cultivar) (Fig. 1A). The expression-level changes in these genes were investigated by transcriptome analysis in JM and YJM in a previous study (Fig. 1B), and the differentially expressed genes were identified (Park et al. 2020b). In JM, genes encoding 9-cis-epoxycarotenoid dioxygenase (NCED) enzymes, which catalyze the final step of ABA biosynthesis, were upregulated at 0 and 0.5 days and downregulated at 3 days during the flooding treatment (Fig. 1B). On the other hand, in YJM, expression levels of NCED1 and NCED3 were increased at 0.5 and 3 days after flooding. The expression levels of selected NCED genes were confirmed by qRT-PCR. The results showed that NCED1 (g45821), NCED5 (g34763), and NCED6 (g8145) expression levels showed a decreasing trend in JM during flooding stress, whereas NCED1 (g45821), NCED3 (g54338), and NCED6 (g8145) expression levels showed an increasing trend in YJM (Fig. 1B). In addition, among the CYP707A genes, which encode cytochrome P450 monooxygenases involved in ABA catabolism, the expression levels of CYP707A1, CYP707A2, and CYP707A3 were increased at 0.5 days and that of CYP707A4 was increased at 3 days during the flooding treatment in JM (Fig. 1C). On the other hand, in YJM, CYP707A1 and CYP707A2 showed response at 0 day or 3 days during the flooding treatment, and CYP707A4 showed response at 0 day. Expression levels of selected CYP707A genes were confirmed through qRT-PCR. The results showed that the expression levels of CYP707A1 (g60684), CYP707A3 (g38495), and CYP707A4 (g60411) were increased in JM and decreased in YJM during the flooding treatment (Fig. 1C), whereas the expression level of CYP707A2 (g4939) was increased in YJM and decreased in JM during flooding.

Figure 1. Expression of genes related to abscisic acid (ABA) biosynthesis and catabolism in two sweetpotato cultivars, JM (flooding sensitive) and YJM (flooding resistant), subjected to flooding stress treatment. (A) Simple schematic of ABA biosynthesis and catabolism pathways. (B) Results of transcriptome analysis (shown in the heat map) and quantitative reverse-transcription (qRT)-PCR analysis of NCED genes. (C) Results of transcriptome analysis (shown in the heat map) and qRT-PCR analysis of CYP707A genes. Statistically significant differences between JM and YJM were determined by one-way analysis of variance, followed by the LSD post-hoc test (*P < 0.05)

Differential response of ABA signaling-related genes to flooding

To investigate the response of ABA signaling genes to flooding stress in sweetpotato, we monitored the expression of Pyrabactin Resistance (PYR) genes, encoding intracellular ABA receptors, in YJM and JM plants during the flooding treatment. Most of the PYR genes were upregulated 3 days after the flooding treatment in JM and at 0 and 0.5 days after flooding in YJM (Fig. 2A). Validation of the expression levels of selected PYR genes through qRT-PCR showed that the expression of PYR9 (g9406) was increased at 0 day of flooding in both JM and YJM (Fig. 2B), while the expression of PYR1 (g55788), PYR4 (g25480), and PYR8 (g39321) decreased or remained unchanged in JM but decreased in YJM during the flooding treatment. Next, the expression of ABSCISIC ACID INSENSITIVE 5 (ABI5) genes, encoding ABA signaling transcription factors, was monitored in YJM and JM during the flooding treatment (Fig. 2A). The expression of ABI5-4 and ABI5-5 was increased at 0 day of flooding in JM. On the other hand, in YJM, the expression of most ABI5 genes was increased at 0 day of flooding, while that of ABI5-5 (g38687) and ABI5-7 (g34305) was increased after 3 days of flooding (Fig. 2C). The results of qRT-PCR showed that the expression of ABI5-2 (g58697) and ABI5-7 (g34305) was increased during the flood treatment in JM and YJM, respectively, whereas the expression of ABI5-4 (g59676) and ABI5-5 (g43242) was decreased during the flooding treatment in both JM and YJM.

Figure 2. Expression of genes related to abscisic acid (ABA) signaling in sweetpotato cultivars JM and YJM subjected to flooding stress. (A) Simple schematic of ABA signaling pathways. (B) Results of transcriptome analysis (shown in the heat map) and quantitative reverse-transcription (qRT)-PCR analysis of PYR genes. (C) Results of transcriptome analysis (shown in the heat map) and qRT-PCR analysis of ABI5 genes. Statistically significant differences between JM and YJM were determined by one-way analysis of variance, followed by the LSD post-hoc test (*P < 0.05)

Changes in contents of ABA, H₂O₂ and CAT activity to flooding

The changes in ABA content in resistant and susceptible sweetpotato cultivars during flooding treatment were investigated (Fig. 3A). During flooding treatment, ABA content increased over time in the resistant cultivar YJM, while it decreased over time in sensitive cultivar JM. The changes in ABA levels were similar to the changes in the expression pattern of the NCED1 gene shown in Fig. 1. ABA has also been reported to regulate defense mechanisms against ROS, including hydrogen peroxide (H₂O₂), through ROS signaling (Li et al. 2022; Ye et al. 2011). Therefore, in this study, the levels of H₂O₂, a representative ROS, and activity of catalase (CAT), a representative antioxidant enzyme that removes H₂O₂, were determined during flooding treatment. During flooding treatment, the level of H₂O₂ increased more strongly in the susceptible cultivar JM than in resistant cultivar YJM (Fig. 3B). On the other hand, CAT activity, which removes H₂O₂, increased more strongly in resistant cultivar YJM than in the sensitive cultivar JM (Fig. 3C), and this difference in CAT activity between the two cultivars mirrored the difference in ABA levels (Fig. 3A).

Figure 3. Changes in the abscisic acid (ABA) and H₂O₂ content and catalase (CAT) activity in sweetpotato cultivars JM and YJM subjected to flooding stress. (A) ABA content, (B) H₂O₂ content, (C) CAT activity. Statistically significant differences between JM and YJM were determined by one-way analysis of variance, followed by the LSD post-hoc test (*P < 0.05)

Being a gaseous plant hormone, ethylene has difficulty exiting the plant cells under flooding conditions, which leads to its rapid accumulation within cells and consequently physiological changes in plants (Alpuerto et al. 2016; Hattori et al. 2009). Therefore, ethylene acts as a key signal for plant adaptation during flooding and regulates the growth and development of plants under hypoxic conditions through interactions with other plant hormones including ABA (Steffens et al. 2006; Vidoz et al. 2010).

In our previous study, we analyzed the physiological and biochemical characteristics of sweetpotato cultivars to select those exhibiting flooding resistance and sensitivity traits (Park et al. 2020a). In addition, transcriptome analysis was also performed on YJM, a cultivar selected as flood-resistant, and JM, a cultivar selected as flood-vulnerable, and genes involved in flooding resistance at the transcriptional level were identified (Park et al. 2020b). As reported in many studies, ethylene-mediated ROS and NO metabolism play a very important role during flood stress. Indeed, a recent study confirmed changes in ethylene-mediated ROS metabolism and its interaction with NO-related metabolism in flooding-resistant YJM and sensitive JM (Park et al. 2022). However, the previous study was restricted to characterizing the physiology, transcriptome, and ROS and NO metabolism of flooding-resistant and flood-sensitive sweetpotato cultivars. ABA metabolism and signaling were not investigated. The present study addresses this gap in our knowledge by investigating ABA metabolism and signaling changes in flood-resistant and flood-sensitive sweetpotato cultivars.

Many studies have reported that ethylene inhibits ABA biosynthesis during flooding. Ethylene accumulated to flooding stress induces seedling elongation in Rumex palustris by inhibition of ABA biosynthesis (Benschop et al. 2006). Mechanistically, the ethylene accumulation inhibits the NCED expression, which leads to the degradation of ABA into phaseic acid (PA) and consequently a decline in ABA level (Benschop et al. 2006; Saika et al. 2007). Ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) cause a rapid increase in the expression of CYP707A (ABA catabolism) genes, encoding ABA 8'-hydroxylases, and their expression is inhibited by pretreatment with 1-methylcyclopropene (1-MCP), an ethylene perception inhibitor (Saika et al. 2007). Another study confirmed that the rapid decrease in ABA level in rice under flooding conditions is controlled by the OsABA8ox1 expression (Pan et al. 2021; Saika et al. 2007). The endogenous ABA level of R. palustris plants was reduced in petioles after flooding (Cox et al. 2004). Recently, flooding was also shown to decrease ABA levels in tomato roots (De Ollas et al. 2021). The expression of OsNCED1, OsNCED2, and OsNCED3 in rice was reduced during flooding treatment (Saika et al. 2007). In Arabidopsis thaliana, expression of AtNCED3 was reduced in roots upon submergence, and the endogenous ABA content in roots was reduced (Hsu et al. 2011). In addition, the transcription of two NCED genes in Solanum dulcamara was significantly downregulated upon submergence, resulting in a significant decrease in ABA level (Dawood et al. 2016). However, another study reported that AtNCED4 expression was upregulated in Arabidopsis seedlings upon submergence (Hsu et al. 2011). These results suggest that the regulation of ABA biosynthesis in both aerial and underground plant parts is distinct and warrants further exploration. In this study, we investigated the expression levels of NCED genes during flooding treatment in JM, a flooding-sensitive sweetpotato cultivar, and YJM, a flooding-resistant sweetpotato cultivar (Fig. 1). Most NCED genes showed higher expression levels in JM than in YJM early in the flooding treatment (0.5 days). Although the expression of some NCED genes was increased in YJM after the flooding treatment, NCED expression levels in JM were higher at 0 and 0.5 days of the flooding treatment. Therefore, it is thought that expression-level changes in NCED genes during the flooding treatment are important, but differences in ABA levels due to the differences in NCED expression levels are also important.

In general, there are two ABA catabolism pathways in plants: 1) oxidative inactivation pathway, in which ABA is converted to PA, and 2) combined inactivation pathway, in which ABA combines with glucose to form ABA glucose ester. Studies have shown that the transcription of CYP707A5 genes is upregulated during submergence in rice, promoting ABA degradation (Yang and Choi 2006). CYP707A5 genes have been shown to encode ABA 8’-hydroxylase enzymes, which are involved in ABA degradation (Krochko et al. 1998). The expression of CYP707A1-1 is upregulated while that of CYP707A1-2 is downregulated in both partial and complete flooding in S. dulcamara (Yang et al. 2018). Therefore, hypoxia caused by flooding promotes ABA catabolism. In this study, we investigated the expressions of CYP707A genes in JM and YJM plants during the flooding treatment (Fig. 1). Most CYP707A genes showed higher expression levels in the flooding-sensitive cultivar JM than in the flooding-resistant cultivar YJM at 0.5 and 3 days of flooding. Although the expressions of some CYP707A genes was upregulated in YJM at 3 days, the CYP707A genes that exhibited higher expressions in YJM than in those of JM under untreated conditions (0 day) showed decreased expression levels during the flooding treatment. Therefore, it is thought that changes in ABA biosynthesis and catabolism levels, regulated by NCED and CYP707A genes, respectively, during flooding treatment affect the physiological characteristics of sweetpotato plants.

Interestingly, the expression of PYR and Pyrabactin Resistance-Like (PYL) genes, encoding ABA receptors, has been reported to upregulation after flooding stress (Arbona et al. 2017; De Ollas et al. 2021). This serves as a feedback for maintaining a certain ABA signaling in plants in flooded soil. On the other hand, the expression of ABA signaling genes, including ABI5, LATE EMBRYOGENESIS ABUNDANT 5 (LEA5) and LEA14, has been reported to decrease upon flooding (Yang et al. 2018). In this study, the expression levels of PYR and ABI5 genes were investigated in sweetpotato cultivars JM (flooding sensitive) and YJM (flooding resistant) during flooding (Fig. 3). The expression of PYR genes was higher in YJM than in JM and showed a decreasing trend during the flooding treatment. Consistently, the expression of ABI genes was also higher in YJM than in JM and showed a decreasing trend during the flooding treatment.

ABA has been reported to regulate the hypoxic stress response of plants downstream of ethylene. A previous study showed that ethylene regulates the physiological response of sweetpotato plants to flooding stress through ROS and nitric oxide signaling (Park et al. 2022). In this study, we investigated the expression levels of ABA metabolism- and signaling-related genes in the sweetpotato cultivars JM and YJM, based on the transcriptome analysis of these cultivars conducted previously, and it could be inferred that the flooding response mechanism regulated by ethylene is related to ABA. Thus, the regulation of plant growth and development under flooding stress is highly complex, and a single phytohormone may not fully reflect the adaptation strategy of plants under hypoxic conditions. ABA is thought to be a representative plant hormone regulated by the ethylene signaling mechanism during waterlogging stress and is assumed to perform an important function.

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 (2021R1A2C400188711).