Increasing floods due to global climate change cause slower gas exchange on the soil surface and lack of oxygen in the soil. Accordingly, the decrease in oxygen level in the plant rhizosphere causes the carbon dioxide (CO2) concentration to increase. Additionally, when plants are flooded, they switch from aerobic to anaerobic respiration, limiting ATP production in the roots. Thus, during floods, root development in plants is limited and the absorption of water and nutrients is negatively affected due to the development of a superficial root system, thus restricting growth and development in plants (Nakamura and Noguchi, 2020). As the amount and severity of stress increases, the restriction of many factors such as the amount of chlorophyll, stomatal conductance, transport of photosynthesis products, photosynthesis rate limits vegetative growth and leads to a decrease in productivity (Reents et al., 2021; Zhang et al., 2021; Adegoye et al., 2023).
In our study on spinach, flooding stress give rise to a decrease in the leaf number (LN), leaf area (LA), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW) and root dry weight (RDW) parameters, of 40, 24, 57, 46, 49 and 20%, respectively. It has been reported by many researchers that plant development is negatively affected in different vegetable species such as spinach (Seymen, 2021), tomato (Elkelish et al., 2020), eggplant (Sarker et al., 2022) and cabbage (Seymen et al., 2022) under flooding stress situations. It has been emphasized in many studies that NO applications under abiotic stress situations contribute positively to plant growth and development by creating an important defense mechanism by reducing the negative effects of reactive oxygen species occurring during stress (Mansoor et al., 2022). It has been demonstrated that NO applications under flooding stress situations improve the plant's tolerance mechanism against flooding and oxygen deficiency (Da-Silva and Amarante., 2022). As a matter of fact, it has been reported that 50, 100, 150 µM NO applications in soybeans (Imran et al., 2022) and 100 µM NO applications in melon (Fan et al., 2014) contribute to agronomic parameters under flooding stress situations. In our study, though it was notice that the NO doses applied to spinach under flooding stress situations generally contributed to the agronomic parameters, it was notice that the application of 50 µM NO made a significant contribution.
There are many studies in which RWC and MD are unfavorable moved under stress situations (Raja et al., 2020; Korkmaz et al., 2022; Seymen et al., 2024a). Electrolyte leakage is an important index of membrane damage, which is enhanced by augmented breeding of ROS species under stress situations. It has been stated that these ROS formed cause disruptions in cellular functions in the membrane (Assaha et al., 2016; Zelinová et al., 2024). It has been reported that flooding stress applied to tomato causes cellular deterioration (Else et al., 2009). It has been reported that NO application applied under flooding situations in corn makes a significant contribution by reducing membrane damage (Jaiswal and Srivastava, 2015). Similarly, it has been reported that NO applications in barley regulate cellular events by contributing to plasma membrane electron transport (Zelinová et al., 2024). In our study, it was revealed that RWC and MD were negatively affected by flooding stress and that the applied NO doses made a significant contribution to the regulation of cellular events.
It has been reported that flooding stress causes the chlorophyll a and b contents in the leaves to change as a result of negative effects such as chlorophyll biosynthesis, membrane damage, decrease in photosynthesis activity and degradation of pigments (Seymen, 2021). The reason for these changes was interpreted as the increase in CO2 concentrations occurring during stress (Kumutha et al., 2018). In plants, stress slows down diffusion, inhibits both light absorption and carbon uptake, and prevents photooxidation, causing a decrease in plant photosynthesis (Yeung et al., 2018). It has been demonstrated in many studies that the mount of chlorophyll decreases under flooding stress situations (Seymen, 2021; Tian et al., 2021; Bharadwaj et al., 2023; Kıratlı et al., 2024; Seymen et al., 2023). It has been reported that NO applications applied under flooding stress situations significantly contribute to the pigment content in soybean (Imran et al., 2022) and wheat (Mfarrej et al., 2022) leaves. Similarly, under flooding stress situations, it was revealed that 100 µM NO dose in cucumber (Fan et al., 2014) and 500 µmol L− 1 NO dose in corn (Jaiswal and Srivastava 2015) applications contributed to the leaf pigment content. In our study, although the ten-day flooding stress applied to spinach negatively affected the pigment content, it was observed that all NO doses contributed significantly to the pigment content.
The formation of free radicals in plants under stress situations causes damage to the membrane lipid structure and proteins. Malondialdehyde (MDA) is the product of lipid peroxidation and causes damage to membrane lipids and many negative changes in ion permeability and enzyme activity (Ding et al., 2021). The causes of H2O2 accumulation in plants under stress situations are thought to be due to stress-induced negative factors such as stomatal closure, membrane lipid damage, leaf water content, oligosaccharide biosynthesis, accumulation of osmolytes, and osmotic adjustment (Akram et al., 2018). It has been observed that NO applications make a significant contribution to the reduction of MDA and H2O2 contents in spinach, which increase under flooding stress situations. In a study carried out on cotton, it was reported that SNP applied under ten-day flooding stress situations reduced the MDA content by approximately 15.2% (Zhang et al., 2022). It has been reported that NO application under flooding stress situations in Glycine max L. significantly limits H2O2 accumulation (Imran et al., 2022).
Proline is an amino acid that accumulates in plants in response to adverse environmental situations and forms a defense in various stress responses. Proline plays an important role in regulating structures such as osmatic pressure settings, membranes, and proteins, and in tolerating the negative effects of stress by clearing free O2 radicals (Barber and Müller 2021). Additionally, plants attempt to survive by synthesizing proline internally in reply to tolerating a wide range of stress reaction (Barickman et al., 2019). The formation of ROS under stress situations damages protein structures in plants, causing their decrease (Santos et al., 2020). NO, on the other hand, interacts with ROS in adverse environmental situations and plays a defensive role in the physiological and biochemical responses of plants to stress (Piacentini et al., 2020). When the data we obtained were examined, it was seen that 50, 100 and 150 µM NO applications conduce to the increased proline content under flooding stress situations, while 100, 150 and 200 µM NO applications conduce remarkably to the decreasing protein content under stress situations. As a matter of fact, when the studies were examined, it was reported that NO applied to cucumber (Fan et al., 2014) and wheat (Mfarrej et al., 2022) under flooding stress situations made a remarkably contribution to the proline and protein contents.
Plants cause an increase in the production of reactive oxygen species (ROS) under adverse environmental situations. Plants develop a defense mechanism by synthesizing many enzymatic antioxidant enzymes such as POD (peroxidase), CAT (catalase) and SOD (superoxide dismutase) to suppress ROS produced under adverse environmental situations (Seymen et al., 2024b). During stress, SOD enables the conversion of H2O2 and O2 and reduces accumulation, while POD ensures the clearance of ROS by clearing H2O2 in the extracellular space. CAT, on the other hand, takes part in cleaning ROS by converting H2O2 into O2 (Rajput et al., 2021). In many studies, it has been reported that NO applications play a role in preventing ROS accumulation under stress situations, changing gene expression as a signaling molecule, and thus protecting against stress by mitigating oxidative damage occurring in plant cells (Fan et al., 2014; Ekinci et al., 2020). It has been reported that NO applied to soybeans under flooding stress situations for 3 and 7 days caused a significant increase in SOD, POD, CAT activities (Imran et al., 2022). It has been shown that NO applied to cucumber under flooding situations prevented ROS accumulation by significantly increasing SOD, POD, CAT contents (Fan et al., 2014). The results in our study were determined to be compatible with other studies. 100 µM NO application under stress situations increased SOD and POD activity, while 200 µM NO application made a significant contribution by increasing the CAT content.
One of the main effects of oxygen deficiency during flooding stress is the inhibition of photosynthesis. The main reasons for this effect have been reported to be due to many reasons such as decrease in leaf area and chlorophyll content, increase in leaf temperature, leaf senescence, and wilting (Parent et al., 2008; Zhang et al., 2019). Chlorophyll fluorescence is a widely used method to determine the characteristics of the plant that occur during stress (Jiang et al., 2017). In other words, when plants are exposed to stress situations, decreases in Fv/Fm values are stated as an indicator of stress. It has been stated that these diminish affect the photosynthetic efficiency of the leaves (Baker, 2008). On the other hand, it is stated that under stress situations, stomatal closure restricts carbon absorption and uptake, causing a diminish in the transport and production of photosynthesis products. Therefore, stomatal conductance is directly related to plant yield (Liao et al., 2022). In our current study, flooding stress caused a decrease in photosynthetic efficiency, stomatal conductance, and chlorophyll fluorescence, while causing an increase in leaf temperature. It was observed that 50 µM NO application made a significant contribution to both QPSII and Fv/Fm. In a study carried out on soybeans, it was reported that flooding stress limited photosynthetic efficiency and stomatal conductance, while applied NO increased stomatal conductance and photosynthetic efficiency (Imran et al., 2022). It has been determined that NO applications under stress situations play an active role in photosynthesis and stomatal conductance and increase tolerance to stress situations by regulating cell death (Hasanuzzaman, et al., 2018).
PCA is an important analysis method in definition substantial parameters in stress studies and determining the effectiveness of the applications (Kıratlı et al., 2024; Seymen et al., 2024b). In our study, the fact that the first two components work over 75% means that the reliability of the analysis method is high. However, the first component explained at a rate as high as 55% of the study emerged as the component defining flooding stress. In many stress studies, it has been explained that the first component explains stress and is the application that creates the most important variables in the study (Mozafari et al., 2019; Seymen et al., 2019; Yavuz et al., 2021; Seymen, 2021; Kal et al., 2023; Seymen et al., 2023; Yavuz et al., 2023; Kıratlı et al., 2024; Seymen et al., 2024b). The application of 50 µM nitric oxide, located in the positive region of the first component, has emerged as an application that is closer to full irrigation and contributes to agronomic and physiological parameters compared to other applications and provides tolerance under flood stress situations.