Ⅰ. INTRODUCTION
Italian ryegrass is a biennial or annual cool season grass, recognized forage crop due to its rapid early growth, high palatability, and digestibility. In addition, Italian ryegrass has waterlogging tolerance, makes it a major forage crop in Korea, where it is primarily cultivated in paddy fields following rice harvest (Hannaway et al., 1999;Choi et al., 2014).
Waterlogging stress in crop growth is known to occur when soil moisture content exceeds field capacity due to increased rainfall, rainy days, and poor drainage (Kaur et al., 2020). When soil is flooded, soil pores become saturated with water, and the diffusion rate of oxygen in the waterlogged environment is lower than in the air, resulting in decreased soil oxygen concentration (Olgun et al., 2008).
Plants are known to adapt to flooding stress through morphological changes, such as increasing plant growth or root aeration tissue (Hossain and Uddin, 2011). In addition, plants are known to maintain anaerobic respiration through sugar supply by increasing the utilization of soluble sugars and anaerobic respiration to adapt to energy crisis under oxygen-deficient conditions under flooding stress, and also activate metabolic adaptation mechanisms, such as the activation of antioxidant enzymes to remove various ROS, such as O2-, H2O2, and HO- equivalents, which are reactive oxygen species (ROS) that cause oxidative stress (Hossain and Uddin, 2011).
In our previous study, we compared the waterlogging stress tolerance of five Italian ryegrass varieties and reported that Florida 80 had higher tolerance than other varieties, and that Italian ryegrass may adapt to waterlogging stress by mechanisms other than the antioxidant enzyme system (Kim et al., 2022). In this study, we investigated whether Italian ryegrass adapts to waterlogging stress by mechanisms other than the antioxidant enzyme system using the Florida 80 variety.
Ⅱ. MATERIALS AND METHODS
1. Plant growth and stress treatment
The Italian ryegrass used in this study was the Florida 80 variety. Soil was prepared by mixing potting mix and sand in a 1:1 ratio and filled into pots. Ten-day old seedlings were transferred to a 40 L PVC box (58 cm × 41 cm × 25 cm), where drainage was blocked, and the water level was maintained at 1 cm above the soil surface for 21 days. The control was irrigation as needed, while the waterlogging had the water height maintained daily.
2. Measurement of ROS and total phenol contents
O2-, H2O2, and HO- were measured by the according to previously reported methods (Wu et al., 2010;Tewari et al., 2007). Total phenol content was measured according to the method of Ainsworth and Gillespie (2007). Briefly, 20 mg sample was mixed with 2 mL of 95% methanol and incubated in the dark at room temperature for 48 h. The sample was centrifuged and supernatant was mixed with 200 μL of 10% F-C reagent and 800 μL of 700 mM sodium carbonate solution, then incubated at room temperature for 2 h. The absorbance was measured at 765 nm.
3. Measurement of total soluble carbohydrate and alcohol dehydrogenase (ADH) activity
Total soluble carbohydrate concentration was measured according to the method of Iturralde Elortegui et al. (2020). Twenty milligrams of dry sample was homogenized in liquid nitrogen, extracted with distilled water at 100℃ for 10 min, and the total soluble carbohydrate concentration was calculated. Ethanol production enzyme ADH activity was measured according to the method of Liu et al. (2017). A 150 mg sample was homogenized in liquid nitrogen, crude protein was extracted, and used for ADH enzyme activity assay.
4. Root porosity
Root porosity was determined using the method described by Broughton et al. (2015). Briefly, the weight of the root samples was measured and recorded. The samples were then submerged to a depth of 1 cm in 100 mL of distilled water, and their weight was measured again. The fully submerged root samples were vacuum infiltrated at 500 mmHg for 40 sec with repeated 3 times. The samples were submerged in distilled water and weighed. The root porosity was subsequently calculated based on the weights obtained, following the method described by Thomson (1990).
5. Statistical analysis
Statistical analysis was conducted using IBM SPSS Statistics for windows, version 27.0 (Armonk, NY, USA), and an independent samples T-test was performed. Significance testing was conducted at the 95% confidence level.
Ⅲ. RESULTS AND DISCUSSION
To determine the effect of waterlogging stress on the formation of adventitious roots in Italian ryegrass, the number of adventitious roots was checked after stress treatment. In Florida 80, the number of adventitious roots increased compared to the control as the waterlogging stress treatment period increased (Fig. 1A-C). When soil is flooded, the oxygen concentration in the soil decreases, reducing root respiration, and adventitious roots are formed from other tissues to alleviate waterlogging stress (Miyashita and Shiono, 2023). These results indicate that Italian ryegrass forms adventitious roots to absorb oxygen from the water surface during waterlogging treatment.
To determine the degree of aerenchyma formation in the root cortex of Italian ryegrass under waterlogging stress treatment, root porosity was measured. Waterlogging treatment significantly increased root porosity compared to the control (Fig. 2). Florida 80 in the control showed a root porosity of about 20%, which is similar to the results of aerenchyma and root porosity in rice and sea barley (Xu et al., 2022). By contrast, after waterlogging treatment, Italian ryegrass showed a root porosity of about 70%. These results suggest that Italian ryegrass has developed mechanisms to alleviate waterlogging stress by delivering oxygen absorbed from the leaves or adventitious roots to the root tips (Zhang et al., 2015).
The changes in the contents of different types of ROS in response to waterlogging stress were investigated in Italian ryegrass leaves and roots. While only the H2O2 content increased in the leaves under waterlogging stress compared to the control, the contents of O2-, H2O2 and HO- increased in the roots (Fig. 3). These results are similar to findings in pigeon pea and cutton, where ROS levels in the roots increased following waterlogging stress (Pan et al., 2022). This suggests that while the shoot are exposed to the air, the roots being submerged in water, accumulate ROS that act as signaling molecules to promote the formation of aerenchyma in the root tissues.
To mitigate oxidative stress induced by waterlogging stress, both enzymatic antioxidant responses and non-enzymatic antioxidant responses, such as those involving total phenolic compounds, play important roles (Herken et al., 2001). Florida 80 exhibited increased levels of phenolic compounds in both leaf and root tissues compared to the control under waterlogging stress. One of these phenolic compounds, vitamin E, protects cell membranes from lipid peroxidation caused by HO- (Herken et al., 2001). According to the study by Kim et al. (2022), Italian ryegrass subjected to waterlogging stress showed a decrease in the activity of antioxidant enzymes, suggesting that nonenzymatic antioxidants, specifically phenolic compounds, may play a more significant role in the antioxidant mechanisms.
To investigate the changes in ethanol fermentation metabolism, the contents of soluble carbohydrates and the activity of the ethanol fermentation enzyme ADH were measured. Florida 80 subjected to waterlogging stress showed an increase in soluble carbohydrate concentration in both leaves and roots compared to the control (Fig. 5). However, the activity of ADH increased only in the roots (Fig. 6). It is known that plants that escape waterlogging stress, the increase in soluble carbohydrate concentration under waterlogging stress is reported to be a result of starch being broken down into soluble carbohydrates by a-amylase (Cannarozzi et al., 2018). The increase in ADH activity only in the roots suggests that ethanol fermentation metabolism primarily occurred in the roots because the root tissues could not absorb oxygen due to waterlogging (Liu et al., 2017). These results indicate that Italian ryegrass has an excellent ability to activate ethanol fermentation metabolism to alleviate energy deficiency caused by reduced root respiration.
Ⅳ. CONCLUSION
When Italian ryegrass is exposed to waterlogging stress, it generates energy through ethanol fermentation, which induces the formation of adventitious roots. Additionally, ROS signals in the roots induce the formation of aerenchyma, thereby increasing oxygen availability. To mitigate lipid peroxidation caused by ROS, phenolic mechanisms are employed to suppress it. These changes suggest that Italian ryegrass has developed mechanisms to escape waterlogging stress and enhance oxygen absorption from both the air and water surface. Results will aid in the development of waterlogging tolerant varieties in the future.
