Bermudagrass, as a popular forage and warm season turfgrass, shows limited growth and begins to wilt when the daily mean temperature goes below 12°C, and then it enters the dormancy period when the temperature reaches 7-10°C (Munshaw et al., 2004; Huang et al., 2019). Low temperature restricts the establishment of bermudagrass in cold region. In addition, bermudagrass has advantages in the restoration of saline-alkali land due to resistance against salt stress (Xie et al., 2017). Therefore, bermudagrass often suffers from combination both of low temperature and salt stress.

Low temperature and salt stress lead to shared or specific physiological, metabolic and genes expression responses. It is well known that low temperature and salt stress restrain plant growth and reduce photosynthesis (Allen and Ort, 2001; Hasdai et al., 2006; Li et al., 2019; Zulfiqar and Ashraf, 2021b; Zulfiqar et al., 2022b). Plants experienced chloroplast structural damage, chlorophyll degradation, stomatal closure and enzyme activity decrease during both low temperature and salt stress (Meloni et al., 2003; Nowicka et al., 2018). Oxidative stress resulted from over accumulation of reactive oxygen species (ROS) and osmotic stress appear in plant response to both low temperature and salt stress (Hasanuzzaman et al., 2020a; Zulfiqar and Ashraf, 2021a; Zulfiqar and Ashraf, 2022a). Both oxidative and osmotic stress have negative effects on plant metabolism, molecular biosynthesis and cell viability (Mahajan and Tuteja, 2005; Liang et al., 2018; Zulfiqar et al., 2019; Munns et al., 2020; Zhao et al., 2021). Furthermore, both low temperature and salt stress cause alteration in ion homeostasis, which destroy the biological activity of membrane and some enzymes (Fürtauer et al., 2019; Shao et al., 2020). Differently, salt stress results in a toxic concentration of Na⁺ and inhibits absorption of K⁺ (Kim et al., 2007; Yang and Guo, 2018). Many specific ions transported genes are responsible for transportation of Na⁺ and K⁺, such as SOS (salt overly sensitive) family genes and HKT (high affinity transporter) (Li et al., 2019). Low temperature disturbs transmembrane H⁺ gradient by changing the activity of a H⁺ pumping protein, H⁺-ATPase (Ponce-Pineda et al., 2021).

Plants are equipped with fine tune resistant mechanisms to counter low temperature and salt stress (Banerjee et al., 2018). Osmoprotectants such as proline and soluble sugar are biosynthesized to remit molecular denaturation (Fürtauer et al., 2019; Zulfiqar et al., 2019; Munns et al., 2020). Plants have a complex antioxidant system, which includes enzymatic system as well as the glutathione-ascorbic acid (GSH-ASA) cycle (Bela et al., 2015). Antioxidant systems are activated to scavenge ROS. For example, antioxidant enzymes such as SOD (superoxide dismutase), POD (enzymes peroxidase) and CAT (oxidative enzyme) and their encoding genes are upregulated in the early stress response (Hasanuzzaman et al., 2020a; Hasanuzzaman et al., 2020b; Shao et al., 2020; Zulfiqar et al., 2020a; Zulfiqar and Ashraf, 2021a; Jabeen et al., 2022; Zulfiqar and Ashraf, 2022a). SOD has the ability to catalyze the conversion of superoxide anions to H₂O₂ and O₂, which is the primary substance for scavenging free radicals in living things. H₂O₂ is scavenged by enzymes CAT and POD through synergistic action to maintain a stable level of free radicals in plants (Yan et al., 2010). In addition, glyoxalase (GLY) system, closely related to GSH metabolism, was reported to participate in stress responses in the past, such as salt and heavy metal stress (Yadav et al., 2005; Singla-Pareek et al., 2006; Roy et al., 2008; Singla-Pareek et al., 2008). GLY is a type of intracellular enzyme found in both prokaryotes and eukaryotes which is mostly sublocalized in the cytoplasm and organelles. GLY system has long been known in animals and is assumed to be engaged in a variety of tasks, including cell division, proliferation, and protection against oxoaldehyde toxicity (Thornalley, 1990). In plants, salt stress increased GlLYI activity, which is accompanied by MG detoxification and decrease in GSH concentration(Parvin et al., 2019).

At the moment, the global climate and environment are rapidly changing, and plants are being subjected to an increasing number of abiotic stresses (Zulfiqar and Hancock, 2020b; Zulfiqar et al., 2021c). However, plants are endowed with unique physiological responses under combined stress which are completely different from those under single stress (Bai et al., 2018; Henriet et al., 2019). Many studies have reported that the negative effects of stress interactions on crop productivity are much higher than when different stress components are applied alone (Mittler and Blumwald, 2010; Suzuki et al., 2014). The relative water content of tomato (Rong et al., 2017) and the biomass of Phragmites Karka (Abideen et al., 2022) were significantly decreased under compound stress. Drought and heat stress can lead to a significant reduction in Arabidopsis growth, but their combined stress has a more deleterious effect (Vile et al., 2012). However, other studies have reported the beneficial effects of the interaction of two different stresses applied simultaneously (Suzuki et al., 2014). For example, drought stress may offer protection against O₃ damage in plants (Biswas and Jiang, 2011; Zhang et al., 2019) and resulted in reduced susceptibility to powdery mildew and Botrytis cinerea (Achuo et al., 2006). Combined stress of drought and salt can reduce boron toxicity in plants (Liu et al., 2018)

Previous research has typically focused on either low temperature or salt stress, with only a few studies have been done related to the combined effect of low temperature and salt stress on plants. Bermudagrass is a valuable forage grass and warm season turfgrass with a high salt tolerance. It is critical to investigate the physiological and molecular mechanisms of bermudagrass under the combined stress of low temperature and salt, in order to improve tolerance and lengthen the growth cycle. In this study, we used physiological and molecular methods to assess the effects of low temperature and salt stress on growth traits, photosynthesis, forage quality, and stress-related gene expression in this study. As a result, the goals of this study were to: (1) investigate the interaction of low temperature and salt on bermudagrass; and (2) elucidate the synergistic mechanism of low temperature and salt.

This article looked into the defensive mechanism of bermudagrass under low temperatures and salt stress. Plants response to stress is a complex process including morphology, physiology, and biochemistry (Huang et al., 2019). Low temperature-treated bermudagrass showed reduced relative growth rate in shoots, shoot dry weight, and increased root length in previous research (Esmaili and Salehi, 2012), which matched our findings ( Figures 1B, E ). From the standpoint of plant growth phenotype, bermudagrass under the LT+S treatment is superior to that of low temperature treatment, the root dry weight reflects the same situation. Relative growth rate, shoot dry weight and root length under low temperature and low temperature + salt treatment shows no discernible differences ( Figures 1A-E ). All of this suggests that the combined stress did not produce more substantial harm to the plants; rather, a moderate amount of salt may have mitigated the damage caused by low temperatures. Maintaining proper balance of Na⁺/K⁺ and higher K⁺ concentration is considered as an important mechanism for plants to response to salt stress (Evelin et al., 2009). Lower Na⁺/K⁺ is a marker of salinity tolerance for plants (van Zelm et al., 2020). In salt-tolerant plants, K⁺ efflux can be significantly inhibited to maintain stable Na⁺/K⁺ and reduce salt stress injury (Yang and Guo, 2018). In our study, Na⁺/K⁺ remained stable and K⁺ concentration increased in root under salt stress ( Figures 3D , F ), so it can be inferred that bermudagrass A12359 has a certain salinity tolerance, which is the reason that it can alleviate the damage of low temperature to bermudagrass at the high salt concentration (2.0%).

Low temperature stress affects a variety of physiological processes in plants, the most susceptible of which is photosynthesis (Stitt and Hurry, 2002). We looked at photosynthetic indexes at the third, sixth and seventeenth d to learn more about how bermudagrass protects itself under low temperatures and different salt concentrations. The PSII reaction center becomes sensitive under stress, and the OJIP fluorescence transient curves and chlorophyll fluorescence characteristics can accurately reflect PSII’s physiological status (Chen et al., 2021). The electron transport activities (PSI and PSII) of the chloroplast thylakoid membrane were found to be dramatically reduced at low temperatures, with PSII being more vulnerable to cold pressure than PSI (Shi et al., 2016). The chlorophyll fluorescence transient response curve decreased under the three stress conditions as processing time at low temperature and salt concentration increased, compared to control, in our studies, but it decreased more noticeably under the LT treatment, and the fluorescence curve is higher under the LT+S treatment than the LT treatments when the salt concentration reaches 1.5 percent and 2.0 percent ( Figures 2B, C ).The findings revealed that an optimal salt concentration could help to relieve photosynthetic physiology in cold-stressed plants ( Figures 2A, C ). Low temperature stress causes a drop in E0, which is mostly influenced by alterations in the PSII receptor side (Van Heerden et al., 2003). φE₀ represents the reaction center of absorbed light quantum yield for electron transfer, and the higher the value, the more stressed the plants are. In this study, φE₀ significantly decreased under LT stress while improving under LT+S treatment, and the same changes were observed in area and φP₀. All of these results suggest that when LT+S was used instead of LT stress, electron transfer efficiency improved, and the effect of low temperature stress alone on plant photosynthesis was reduced.

Plants respond to salt, severe temperatures, and other factors through the GLY system, which consists of GlyI, GlyII, and GSH (coenzyme). The detoxification of methylglyoxal, a by-product of carbohydrate metabolism, is its primary physiological role (Roy et al., 2008). In this study, since the expression levels of GLYI and antioxidant oxidases related genes change early under stress conditions (Wang et al., 2020; Zhang et al., 2021), the gene expression levels were measured once after 6 h of treatment, and then again at the end of the experiment (17d), so as to observe the gene expression changes. Under salt, mannitol and heavy metal stress, GLYI expression in mustard was dramatically increased (Veena et al., 1999). GLYI was considerably elevated in both roots and leaves after 6 h of salt treatment in our experiment ( Figures 4A, B ), which is consistent with earlier research. Low temperature causes mechanical constraints-membrane damage, whereas salinity causes malignancy by disrupting the ion and osmotic balance of cells (Mahajan and Tuteja, 2005). Osmotic stress can particularly promote the production of GLYI, at which point the GLY system is triggered to repair the damage (Inoue et al., 1998). Low temperature stress was found to increase the levels of reactive oxygen species (ROS) and lower enzyme antioxidant activity in plants (Aroca et al., 2005; Sasaki et al., 2007), affecting the antioxidant system. In our study, antioxidant enzyme genes (Cu-Zn/SOD, POD2, and CAT1) in roots were significantly down-regulated under low temperature stress ( Figures 4D , F , H ). When exposed to low temperature stress alone, the expression of GLYI in leaves does not rise and even decreases dramatically in roots, and several indicators reveal that the injury to plants is highest at this time. However, when compared to low temperature treatment, the expression level of GLYI was higher during LT+S stress, indicating that the GLY system was activated again, reducing the damage caused by low temperature. Furthermore, the GLY system raises the level of free reduced glutathione, which is necessary for the removal of harmful reactive oxygen species (e.g., H₂O₂) and organic peroxides that accumulate in stressed plants, as well as the maintenance of other antioxidants (Frendo et al., 2013). Despite the roles of antioxidant enzymes and GLY in stress are clearly, we indicated that these genes may involve in stress acclimation to several different environmental adversity.

Crude protein and crude fiber are two important indexes for measuring the nutritional value of herbage, as well as important contents for improving herbage quality. The first selection factors for high quality forage are high crude protein content and low crude fiber content (Barth, 2012). Previous research has shown that plants in low temperature environments reduce structural carbohydrate content while increasing soluble carbohydrate and protein content to minimize plant harm (Vo and Johnson, 2001). In our experiment, compared to the control, bermudagrass protein content increased significantly under LT and LT+S treatment ( Figure 5B ). This phenomenon could be explained by the fact that the LT treatment was set at 10 -15°C and the cells were subjected to low temperature stress but did not reach the point where the fluidity of the cell membrane becomes weak and rigidity increases. At this point, the cell membrane’s phase transition reduces the ability of the membrane protein to bind to phospholipids and causes it to become free protein, resulting in an increase in protein content in the plant. Crude fat is a nutrient with a high calorific value that can enhance feed palatability (Li et al., 2019). According to the test results, the crude fat content was lowest under low temperature treatment and significantly higher under low temperature + salt treatment ( Figure 5A ), proving once again that appropriate salt can mitigate the damage caused by low temperature.

At present, plants are subjected to more and more abiotic stresses (Zulfiqar and Hancock, 2020b; Zulfiqar et al., 2021c), and the physiological responses under combined stress and single stress are completely different. Therefore, the changes of phenotype, photosynthesis, Na⁺, K⁺, gene expression and forage quality under single salt, low temperature stress and combined stress were analyzed in this study to explore the interaction between low temperature and salinity on bermudagrass. It was found that low temperatures cause more damage to bermudagrass, but moderate salt addition can mitigate the damage by enhancing photosynthesis, improving the expression of antioxidant system genes (Cu-Zn/SOD, POD2 and CAT1) and glyoxalase system GLYI gene in roots and thus improves forage quality. This provides putative pathways improving turfgrass and forage tolerance to combination stress.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

All authors contributed largely to the work presented in this article. Conceived and designed the experiments: JF. Performed the experiments XZ, GW, XL, YX. Analyzed the data: XZ, YY. Language modification: EA. Wrote the paper: XZ, YY. All authors contributed to the article and approved the submitted version.

This work was supported by National Key R&D Program of China (2019YFD0900702) and Agricultural Variety Improvement Project of Shandong Province (2019LZGC010).

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.

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