Plants are obligate aerobes and require molecular oxygen to enable cellular respiration and other essential processes. However, due to waterlogging, complete submergence, and poor soil drainage, plants often suffer from hypoxia and anoxia (Fukao and Bailey-Serres, 2004; Bai et al., 2013). Hypoxic conditions are characterized by anaerobic respiration responses, which facilitate energy deficits of up to 37.5% in plants because oxygen diffusion in waterlogged soil is 10,000 times lower than in well-drained soil (Gibbs and Greenway, 2003). Plants experience a series of physiological changes under hypoxia conditions, such as limited gas exchange (Voesenek and Bailey-Serres, 2015), reduced hydraulic conductivity (Tournaire-Roux et al., 2003), changes in gene expression (Xuewen et al., 2014), and increased oxidative damage (Shabala et al., 2014). Collectively, such changes redirect the energetic resources of the plant and prevent them from reaching its true genetic potential. In addition, anaerobic conditions can adversely affect leaf water potential (schildwacht, 1989), enzymatic activity (Hasanuzzaman et al., 2017), nutrient absorption and assimilation (Arduini et al., 2019), plant growth and development (Pan et al., 2021), and ultimately lead to a decline in crop yields.

Waterlogging affects over 16% of the world’s cultivated land (Ploschuk et al., 2018), and over 17 km² of the land surface is prone to flooding (Voesenek and Sasidharan, 2013). Furthermore, with the rapid climate change, heavy precipitation events are projected to increase by about 7% for every 1°C increase in global warming, leading to increased flood hazard severity (high confidence) (IPCC, 2021). Hence, there is an increasing need to understand the mechanisms of plant tolerance to waterlogging.

Cucumber (Cucumis sativus L.) is one of the world’s most important economic vegetable crops. The current estimated global area for cucumber production is approximately 2.3 million hectares, with an annual output of 87.8 million tons and an average yield of 39.3 t/ha (FAO, 2021). However, cucumbers are sensitive to waterlogging stress due to their shallow root system and high oxygen demand (Xuewen et al., 2014; Xu et al., 2016; Qi et al., 2019). The primary root system of cucumber plants quickly deteriorates under waterlogging due to a lack of oxygen, resulting in energy deficits (Gibbs and Greenway, 2003), limited mineral nutrient uptake and acquisition (Arduini et al., 2019), and a corresponding decline in growth and development (Herzog et al., 2016). Previous studies have shown that the emergence of ARs to promote gas exchange and nutrient absorption is a crucial adaptation mechanism utilized by cucumbers under waterlogging (Qi et al., 2019, 2020). In addition, due to rapidly depleted oxygen under waterlogging, root metabolism changes from aerobic respiration to anaerobic fermentation, while the concentration of CO₂ and ethylene continues to increase (Sairam et al., 2009; Pampana et al., 2016). These conditions usually result in a reduction in ATP production from 36 to 2 moles of glucose metabolized (Sousa and Sodek, 2002; Gibbs and Greenway, 2003). The lack of energy at the root system adversely affects hydraulic conductivity, photosynthetic rate, water, and mineral nutrient uptake, all of which are the result of the disruption of aquaporins function (Tournaire-Roux et al., 2003; Tan et al., 2018). Thus, the regulation of the onset and development of ARs represents a critical tool for the survival and adaptation of plants subjected to waterlogging.

The substantial decline in the rate of photosynthesis is another characteristic feature of plants subjected to waterlogging conditions. Previous studies have demonstrated the sensitivity of photosynthesis in many crops, including cucumber (Barickman et al., 2019), squash (Lin et al., 2020), tomato (Bhatt et al., 2015), and watermelon (Yetisir et al., 2006). For instance, within the first day of waterlogging treatment, cucumber plants’ CO₂ assimilation rate (A) rate declined rapidly (Kang et al., 2009; Barickman et al., 2019). Thus, even in a short period, the significant reduction in A under waterlogging conditions could lead to a decline in plant energy reserves, indicating the existence of a shared metabolic pattern. Imperatively, the factors affecting the A of plants are primarily divided into stomatal and non-stomatal limitations. Due to limited oxygen under waterlogging conditions, plants close their stomata to maintain plant water status, causing a decline in stomatal conductance (g_s) and inhibiting the exchange of CO₂ required by the plant’s basic processes (Voesenek and Bailey-Serres, 2015). Kozlowski (1984) attributed the reduction in g_s associated with high CO₂ concentration during waterlogging to a decrease in root water absorption caused by physical changes in the protoplasm and plasma membrane. Thus, waterlogging impedes the efflux of water, mineral nutrients, and related metabolites (including sucrose, amino acids, proteins, lipids, abscisic acid, etc.) from the root system, resulting in leaf dehydration and stomatal closure. Barickman et al. (2019) reported that a 10-day waterlogging caused a significant reduction in fresh and dry mass (DM) accumulation due to reduce A, g_s, and E. Consequently, a reduction in g_s eventually leads to a corresponding decrease in A and rate of transpiration (E) (Pedersen et al., 2013).

Other limitations of photosynthesis in waterlogged conditions are the decline in the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), chloroplast damage, and loss of leaf pigments (Ahmed et al., 2002; Bansal and Srivastava, 2015). These non-stomatal factors severely inhibit A under waterlogging conditions. In addition, waterlogging stress can impair the maximum quantum yield (F_v/F_m) of the photosystem (PSII), leading to photoinhibition of the PSII reaction center (Shao et al., 2013; Zhang et al., 2019). The damage of PSII also affects the photosynthetic electron transport chain and changes the amount of light energy directed to organic synthesis, thereby causing damage to the chlorophyll fluorescence parameters (Guidi et al., 2019). Previous studies have noted critical chlorophyll fluorescence parameters, including photochemical quenching (qP) and electron transport rate (ETR), as valuable evaluation tools for understanding cucumbers’ resilience to waterlogging stress.

Currently, the photosynthesis of cucumber under waterlogging has not been thoroughly studied, especially in the seedling stage. Therefore, more information about the growth, development, and physiological responses is worthy of attention, especially the differences in waterlogging tolerance between cultivars and the time-scale response during waterlogging. The present study evaluated the dynamic changes occurring in cucumber seedlings during a 10-day waterlogging period by determining physiological characteristics such as the growth rate, photosynthesis, and chlorophyll fluorescence. The relative effects of waterlogging stress on carbon assimilation and light reactions were assessed as well. The objective of this study was to explore the waterlogging tolerance of cucumber cultivars using morphological and key physiological parameters that affect carbon assimilation, such as gas exchange and chlorophyll fluorescence parameters. We hypothesized that the carbon assimilation rate of cucumber during waterlogging may be affected by stomatal and non-stomatal factors. This hypothesis was tested in two commercial cucumber cultivars exposed to 10 DOW during early developmental growth stages in a controlled environment.

Waterlogging stress imposed on the cucumber plants during the early stages of development results in dramatic changes in plant physiology and biochemistry, with detrimental impacts on morphology and yields. Our previous study on commercially cultivated cucumber plants (“Straight 8”) revealed that the seedling growth stage is extremely sensitive to waterlogging stress (Barickman et al., 2019). We hypothesized that the rapid decline of photosynthetic traits, including A, E, and g_s might contribute to the susceptibility of cucumber plants to waterlogging stress. As an adaptive response to hypoxia, the reduction in the photochemical efficiency of PSII and the formation of ARs could mitigate the potential damage to photosynthetic apparatus caused by waterlogging (Smethurst and Shabala, 2003; Olorunwa et al., 2022). With this hypothesis in mind, we evaluated plant growth, photosynthesis, and chlorophyll fluorescence parameters to identify cultivar-specific and differential responses.

We observed a significant decline in LN, LA, FM, and DM of both cultivars after 10 DOW, demonstrating the sensitivity of cucumber plants at the seedling stage to hypoxia. Previous studies revealed that waterlogging induces morphological changes in plants, especially those related to leaf photosynthesis (Zhang et al., 2019; Olorunwa et al., 2022). Analogous findings have been made in previous studies on cucurbits, including cucumber (Barickman et al., 2019), watermelon (Yetisir et al., 2006), and summer squash (Huang et al., 1995). The alterations in the morphology of cucumber plants under waterlogging can be attributed to reduced water and nutrient uptake and transportation due to energy deficits during anaerobic respiration, resulting in restricted cell expansion and leaf growth (Gibbs and Greenway, 2003; Parent et al., 2008).

The effects of waterlogging stress on leaf morphology and biomass are dependent on plant growth stage, duration, and cultivars. Zheng et al. (2021) reported a significant decline in the waterlogging sensitive “Zaojia8424” leaf biomass compared to the waterlogging tolerant cultivar “YL,” which naturally had smaller leaves. Generally, smaller leaves are more beneficial for mitigating waterlogging stress than larger leaves because of their high boundary layer conductance, which prevents heat accumulation (Leigh et al., 2017). In the current study, 10-day waterlogging reduced the leaf biomass and area in “Straight 8” and “Marketmore” cultivars, with no statistical difference between the two cultivars (Figure 1). In addition, ARs developed in the hypocotyls of both cultivars after 10 DOW. Previous studies showed a similar response for other cucurbits during their early developmental growth stages (Huang et al., 1995; Xu et al., 2016; Qi et al., 2019). The ARs of “Marketmore” was more pronounced after 10 DOW when compared to the “Straight 8” cultivar, mainly due to highly aerenchymatous cortical structures (Visser et al., 1996). In addition, after applying 1-methylcyclopropene (ethylene receptor inhibitor), 1-naphthylphthalamic acid (auxin transport inhibitor), or diphenyleneiodonium (NADPH oxidase inhibitor) to waterlogged cucumbers, Qi et al. (2019) observed the formation of ARs. This implies that auxin, ethylene, and ROS production are enhanced under excess water conditions. Also, Vidoz et al. (2010) suggested that ethylene and auxin might interact in forming ARs in waterlogged tomato hypocotyls using hormone-insensitive mutants. Since auxin and ethylene are transported polarly through ATPase-dependent carrier proteins (Visser et al., 1996), waterlogging-induced low levels of ATP cause these hormones to accumulate near the basal stem of waterlogged plants for solute transport, thereby acting as positive regulators for cucumber waterlogging tolerance.

Waterlogging induces significant changes in the photosynthetic performance of C3 crops, especially in sensitive plants like cucurbits (He et al., 2018; Barickman et al., 2019; Lin et al., 2020). This response was evident in the current study as A, g_s, and E for the two cultivars after 10 DOW treatments were significantly reduced. The results of the gas exchange analysis of cucumber plants after 10 DOW indicated that the decline in A can be due to both stomatal and non-stomatal factors according to the version of the photosynthesis model by Farquhar et al. (1980). Under waterlogging, the significant decline in A and biomass was mainly due to reduced water potential, which resulted in the rapid closure of the stomata through the effect of abscisic acid on potassium ion regulation of guard cell turgor (Sojka, 1992; Ploschuk et al., 2018). Barickman et al. (2019) opined that restricting A in cucumber plants through stomatal closure under waterlogging could limit the amount of photoassimilate available for the growth and maintenance of the cucumber vegetative and fruit tissues. In agreement with our results, Issarakraisila et al. (2007) found that the A in kale plants was associated with decreased g_s after 14 DOW treatments. Although decreased g_s is the most common response in plants to waterlogging, the reduction in C_i can also be ascribed to non-stomatal factors. The current study showed that g_s decreased in two cultivars after 10 DOW, associated with decreased A and E and increased C_i. Thus, a significant increase in C_i and a decrease in L_s in response to changes in A and decreased g_s under stressed conditions suggest an essential contribution of non-stomatal limitation to A (Farquhar and Sharkey, 1982). Previous studies (Yordanova and Popova, 2007; Liu et al., 2014; Pompeiano et al., 2019) in sensitive plants have reported similar findings. In addition, the WUE of both cultivars tested in this study decreased significantly under 10 DOW. Previous research found that waterlogging-sensitive plants have lower WUE and thus gain less carbon per unit of water lost (Meena et al., 2015; Velasco et al., 2019).

In the present study, the reduction in A corresponds to the reduction of V_cmax and J_max, which are considered to represent the carboxylation and regeneration of Rubisco, respectively. Previous studies have attributed the decrease in V_cmax and J_max to the decline in the amount of active Rubisco and a reduction in the contribution of ATP during photosynthesis (Flexas et al., 2014). Urban et al. (2008) considered that changes in A response to abiotic stress could be paralleled to the changes in Rubisco activity mediated by J_max to balance the modification of V_cmax when subjected to stress. In this context, the observed decrease in V_cmax and J_max for both cultivars after 10 DOW appears to be consistent with a previous study (Ma and Guo, 2014). The A/C_i further demonstrated that waterlogging stress significantly decreased A with increasing C_i. As per the fitting model of photosynthesis employed in this study (Bernacchi et al., 2001; Sharkey et al., 2007), our results indicated that waterlogging impacted the V_cmax more in “Straight 8” when compared to “Marketmore” (33% vs. 14%). Similar results were reported by Ma and Guo (2014) in waterlogging-sensitive cucumber cultivar, and they attributed the significant reduction of V_cmax under waterlogging to chlorophyll degradation. Although we did not measure changes in chlorophyll content in the present study, there is increasing evidence that decreases in A, V_cmax, and J_max are often associated with the degradation of chlorophyll content in waterlogged plants (Ma and Guo, 2014; Barickman et al., 2019). Primarily, plants subjected to waterlogging stress often experience an increase in the activities of oxidase and chlorophyllase, which are responsible for the degradation of pigments (Muhammad et al., 2021). Jans et al. (2012) suggest that the degradation of plant pigments during waterlogging can be alleviated by reducing the light level capture in a controlled environment. Thus, the downregulation of light utilization efficiency during photosynthesis in a wet environment might be a photoprotective mechanism of cucumber plants.

Moreover, the light response curves revealed that waterlogging declined A in cucumber plants, indicating that light utilization was suppressed under waterlogging. Besides the waterlogging stress, for efficient gas exchanges between cucumber leaves and the environment, the optimum PPFD for both cultivars, irrespective of the origin, was 1,500 μmol m^–2 s^–1 under the control conditions. However, above the optimum PPFD as depicted in Figure 8, revealed A photoinhibition, signifying that an increased PPFD may further hamper cucumber plants’ growth and developmental processes under waterlogging conditions (Wimalasekera, 2019).

Alterations in chlorophyll fluorescence parameters of waterlogged cucumber plants are an adaptive response to waterlogging stress, notwithstanding the photosynthetic apparatus damage. Most of the chlorophyll fluorescence parameters measured in light- and dark-adapted cucumber leaves were significantly reduced under 10-day waterlogging stress, indicating the sensitivity of cucumber plants to hypoxia stress. Previous studies demonstrated waterlogging causes a significant decline in chlorophyll fluorescence parameters, including F_o, and F_m, which reflect the PSII electron transport capacity in dark-adapted leaves (Smethurst and Shabala, 2003; Rao et al., 2021; Olorunwa et al., 2022). However, in the current study, although F_o decreased, F_m did not change significantly after 10 DOW in cucumber plants. The fact that F_m did not change significantly in response to waterlogging stress could be attributed to the fact that the degree of water stress was not severe enough to severely damage the leaves or alter metabolic pathways in cucumber plants. Moreover, Krause and Weis (1991) noted that healthy leaves had F_v/F_m values between 0.75 and 0.83, and a drop in these values indicated impaired PSII. However, the results of this study showed that the F_v/F_m of both cucumber genotypes ranged from 0.75 to 0.79 under both control and waterlogging treatments. Our current results are consistent with previous studies (Velasco et al., 2019; Lin et al., 2022), indicating that the alterations in leaf PSII at the seedling stage of cucumber were minimal under waterlogging conditions.

Furthermore, we found that the qP of cucumber plants (representing the proportion of excitation energy dissipated at the expense of photochemical utilization) was significantly reduced under waterlogging stress. Corresponding results have been reported by He et al. (2018) in cucumber plants and Zheng et al. (2021) in watermelon at the seedling stage. These studies attributed the decline in qP to either a slower rate of consumption of reductant and ATP produced by non-cyclic electron transport compared to the rate of excitation of open PSII or damage to PSII reaction centers (Urban et al., 2008). Taken together, the evidence from the current and previous studies further supports the fact that waterlogging leads to a decrease in photorespiration and corresponding damage to the PSII reaction centers of sensitive plants, including cucurbits. Conversely, the NPQ of cucumber plants revealed that the excess excitation energy not used during photochemistry increased in response to waterlogging conditions. Increased NPQ is considered a protective mechanism of the photosynthetic apparatus (Demmig-Adams and Adams, 2018). This result is consistent with previous reports (Ma and Guo, 2014; Bhatt et al., 2015; He et al., 2018) that cucurbit and other fruit crops have increased NPQ and decreased ETR under waterlogged conditions.

According to Kramer et al. (2004), the energy absorbed in PSII is divided into three partitions, including Φ_PSII, Φ_NPQ, and Φ_NO. The current study revealed a decline in Φ_PSII, and increments in Φ_NPQ, and Φ_NO in cucumber plants under waterlogging, which corroborates with previous studies (Zhang et al., 2019; Zheng et al., 2021). Since the Φ_PSII is a product of qP and F_v′/F_m′ (Genty et al., 1989), the decrease in Φ_PSII under waterlogging may be due to qP and F_v′/F_m′ reduction. Moreover, the correlation analysis revealed a positive relationship between Φ_PSII, qP, and F_v′/F_m′. Increases in Φ_NO in response to waterlogging indicate the lack of photochemical energy conversion and photoprotective regulatory mechanisms, and the excess electron can be used to form singlet oxygen in the chloroplast (Zheng et al., 2021). In both cultivars, an increase in the 1-qL was also demonstrated in response to waterlogging, suggesting that a more significant proportion of the PSII reaction centers were closed during actinic irradiance, thus resulting in a decline in Φ_PSII, as denoted by Kramer et al. (2004). The alterations in the qL under waterlogging further indicate that the greater percentage of the energy absorbed in PSII was converted into chemically fixed energy whereas the remaining quanta were dissipated into heat and fluorescence.

Using morphological and physiological processes, we explored the response of cucumber cultivars to waterlogging stress. The light- and carbon-dependent reactions of photosynthesis were shown to be distinct at the photosynthesis level, with the light-dependent reactions increasing, whereas carbon assimilation was considerably impeded. Decreased stomatal conductance, increased CO₂ concentration, down-regulated Rubisco carboxylation and regeneration, and absorbed energy dissipated in the form of heat were the main factors that reduced carbon assimilation rate in cucumber plants under waterlogging stress. In addition, the results of this study demonstrated that waterlogging impairs the photoreactions of photosynthesis of cucumber cultivars and that damage could be exacerbated with increasing light intensity.

Overall, our results support the hypothesis that cucumber plants are sensitive to waterlogged conditions, and most effects of waterlogging stress are similar in the two commercial cultivars we analyzed. Notably, the “Marketmore” cultivar appeared more tolerant to waterlogging stress with respect to morphological performance. These observations suggest that cucumber plants can be adapted to waterlogging conditions since they rapidly generate ARs to facilitate gas diffusion and increase plant survival when oxygen concentration is low. However, the newly developed ARs might not contribute to the waterlogging tolerance of cucumbers due to waterlogging-caused inhibition in the carbon metabolism, and PSII reaction centers.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

OO, BA, SB, and TB designed the experiments. SB collected physiological and morphological data. OO and BA did the statistical analysis using SAS. OO wrote the first draft of the manuscript. SP, GP, and TB made significant comments and inputs to successive versions of the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.