Bermudagrass [Cynodondactylon (L) Pers.] is widely used in golf courses, sports fields and lawns globally (Fan et al., 2014). As a representative warm-season grass, its optimal growth temperature ranges from 26°C to 35°C (Fan et al., 2014). Cold stress is considered to be a key environmental factor that limits its growth and distribution (Fan et al., 2014).

When two Bermudagrass [Cynodon dactylon (L.) Pers. var. dactylon] cultivars, Riviera (cold tolerant) and Princess-77 (cold sensitive) were exposed to cold acclimation at 8/4°C (day/night), relative EL values were remarkably increased, resulting in the damage to cell membrane (Zhang et al., 2006). Previous results showed that H₂O₂ content increased in plants tissues after low temperature treatment (Fadzillah et al., 1996; O’Kane et al., 1996). To protect themselves from cold-induced damage, plants have evolved multiple mechanisms to enhance their cold tolerance, which include alterations of membrane fluidity, metabolism homeostasis, enzyme activity and the C-repeat-Binding Factor/DRE-Binding Factor (CBF/DREB) pathway (Thomashow, 1999; Zhu et al., 2004; Chinnusamy et al., 2010; Knight and Knight, 2012).

Photosynthesis plays a vital role in photochemical and biochemical process, which transforms solar energy into chemical energy of biomass production, but it is highly sensitive to low temperature. Previous studies suggested that when rice were exposed to cold conditions of 5, 10, 15, 20, and 25°C for 16 h, there was a steady decline in photosynthesis of cold-sensitive rice cultivars ‘Milyyang23,’ compared to that of cold-resistant ‘Stejaree45’(Jeong et al., 2002). Krause (1994) reported that under cold stress, the efficiency of photosynthesis electron transport decreased notably in plant, which resulted in excessive energy generation. Generally, when plants are subjected to photo inhibitory conditions, reactive oxygen species (ROS) are formed, which leads to severe injury of PS II components (Osmond, 1994). The extent of inactivation relies on the balance between inactivation and re-synthesize of PS II components during the low temperature stress (Allakhverdiev et al., 2008; Chen et al., 2013).

As we all know that plants will change the way of carbon metabolism if photosynthesis is affected. There are two naturally stable isotopes of carbon, ¹²C and ¹³C. Majority of the carbon is ¹²C (98.9%), with 1.1% being ¹³C (O’Leary, 1988). The distribution of isotopes among different compounds can reveal information about the physical, chemical and metabolic processes involved in carbon transformations (Farquhar et al., 1989). Recently, stable carbon isotope analysis is a rapidly developing technique which may reveal many processes of carbon dynamics in plants. Sustaining this technological trend will enable researchers to discover how plants respond to abiotic and biotic stress (Dawson et al., 2002). Previous studies reported that conditions triggering stomatal closure, such as salinity, water stress and decrease the CO₂ supply to carboxylation sites increased the δ13C of plants (Rivelli et al., 2002; Araus et al., 2003; Yousfi et al., 2010), but the evaluation of whether variation in δ13C is the result of changes in intrinsic photosynthetic capacity or stomatal conductance remains unknown (Scheidegger et al., 2000; Farquhar et al., 2007).

ABA is a vital phytohormone that regulates many essential physiological and biochemical processes, and it has a key role in stress resistance during plant growth and development (Verslues and Zhu, 2005; Fujii et al., 2009; Kim et al., 2016). Previous study suggested that cold stress is accompanied by increased levels of endogenous ABA in many plants (Mantyla et al., 1995), and exogenous ABA treatment could enhance plant cold resistance (Kumar et al., 2008; Kim et al., 2016). Under low temperature condition, plants activate downstream gene expression through both ABA-dependent and ABA-independent pathways. In Arabidopsis, the expression levels of ABA responsive transcription factor, ABF1 and ABF4, were induced under cold condition (Choi et al., 2000). Furthermore, exogenous application ABA also increased the content of soluble sugar and proline, improved water retention (Deng et al., 2005; Huang et al., 2015), reduced membrane lipid peroxidation, alleviated cell membrane injury effectively (Zhou and Guo, 2005; Huang et al., 2015), and enhanced photosynthesis (He et al., 2008).

Recently, profound progress has been made in investigating ABA participation in abiotic stress response, while studies about the effects of exogenous ABA treatment under cold conditions in Bermudagrass species are still very limited. Here, we aim at investigating the possible cold adaptive role of ABA in the cold-sensitive and cold- resistance Bermudagrass genotypes, particularly focusing on cell membrane stability, photosynthesis and stable isotope signatures. This study provides some insights into the possible physiological and molecular mechanisms underlying Bermudagrass response to cold stress.

To survive extreme environmental conditions, botanical species have developed some complex adaptive strategies. Recently, ABA and ABA-mimicking ligand, AM1, have been reported to play a crucial role in plants defense against cold stress (Verslues and Zhu, 2005; Cheng et al., 2016; Kim et al., 2016). However, the mechanisms of ABA response to cold stress in Bermudagrass are still largely unknown. In the current study, different physiological mechanisms of exogenous ABA were investigated between cold-sensitive and cold-resistance Bermudagrass genotypes response to cold stress, including cell damage, the process of photosystem II and the alterations of δ13C value.

Cell membrane stability is considered to be a reliable indicator of biotic and abiotic stress- induced cellular damage (Premachandra et al., 1992). The results of this study showed that the leaf EL increased significantly under cold stress, and this increasing rate was higher in the S than the R genotype. Exogenous application of ABA plays an effective role in lowering EL level in the both Bermudagrass genotypes. Interestingly, EL decrease was more in S genotype than R genotype, while the level in the R genotype was still lower than S genotype (Figure 2B). This observation resonates with the results of Kumar et al. (2008), who reported that ABA-treated plants had lower EL than those of water-treated plants. The MDA content can be used to evaluate the extent of lipid peroxidation, which further reflects the extent of oxidation injure to the cellular damage (Södergren et al., 2001), and lipid peroxidation was induced by cold stress in many plants (Zhang et al., 2006). Similar to the EL, cold stress significantly increased the MDA content in leaves, which was dramatically decreased by exogenous ABA application in the both genotypes (Figure 2A). This observation suggests that cold stress leads to lipid peroxidation, and exogenous ABA played a positive role in maintaining cell membrane stability and normal function, which is consistent with Guschina et al. (2002), who reported that ABA may decrease membrane damage in mosses by diminishing the lipid change.

Reactive oxygen species induced oxidative damage and cell membranes injury (Fridovich, 1978; Sharma et al., 2012). Under cold stress, there is a cellular increase in the superoxide and hydroxyl free radicals which increase membrane lipid peroxidation, leading to cellular damage and destruction (Mittler, 2002). H₂O₂ is one of the ROS, and previous research indicated an increase in the amount of leaf H₂O₂ when plants were subjected to low temperature (Wise and Naylor, 1987; Hodgson and Raison, 1991; Prasad et al., 1994). In the present study, cold stress dramatically induced the H₂O₂ accumulation compared to the control under normal condition, and it was lower in R genotype than S genotype. However, after exogenous application of ABA in the plants of two genotypes, the H₂O₂ accumulation significantly decreased (Figure 3). This observation suggests that ABA played a crucial role in alleviating oxidation damage during cold stress, which is consistent previous reports from Kumar et al. (2008) and Guo et al. (2012), who reported that exogenous ABA eased oxidative damage of cell membrane by increasing the activity of ROS enzymes under cold stress.

Chlorophyll a fluorescence has been widely studied under biotic and abiotic stress (Chen et al., 2013). Cold stress has been reported to decrease photosynthetic electron transport efficiency in plant (Hodgson and Raison, 1989). In this study, alteration of OJIP transient curves suggested that exogenous ABA treatment could enhance cold stress resistance in both genotypes, particularly for the R genotype (Figure 4). As an indicator of plant vitality, PI is a sensitive parameter of JIP-test in evaluating plant photochemical activities under stress condition. It combines several parameters that are evaluated through the fluorescence OJIP (Yusuf et al., 2010), and a strong correlation was found between PI and physiological parameters in several studies on stress (Strasser et al., 2007; Zubek et al., 2009). In the study, PI_ABS and PI_total decreased significantly under cold stress, and they were strongly improved by exogenous ABA in both genotypes, suggesting that exogenous ABA plays a shielded role in cold resistance of two Bermudagrass. Furthermore, cold stress significantly influenced the photosynthetic efficiency, such as decreased the values of φP0, φE0 and , and they were partly recovered by ABA in Bermudagrass (Table 2). These data implied that ABA enhances the quantum yield of the electron transport flux on the sides of donor and acceptor of PS II in both Bermudagrass, which is favorable for maintenance high-level photosynthetic property in response to cold stress.

When the rate of energy absorption by chlorophyll exceeds utilization capacity of the organism, the tapped energy is used in photosynthesis or dissipated as heat and ROS are formed (Huner et al., 1998; Pospíšil, 2012). When plants were exposed to cold stress, high PS II excitation pressure caused photosynthetic acclimatization due to the decline in PS II antenna size, which in return reduces photosynthetic efficiency (Huner et al., 1998). Our study showed that exogenous ABA application reduced the values of ABS/RC, RE0/RC and DI0/RC for two genotypes, indicating ABA restrained the cold-triggered energy absorption, trapping and dissipation hence reduced the excessive energy production. Interesting, ET0/RC significantly increased in the R genotype under low temperature, while the values decreased in the S genotype (Table 2). This suggested that exogenous ABA reduced the energy flow for electron transport in the R genotype to weaken photosynthesis in response to cold stress. This could be associated with the inherent photosynthesis intensity of the R genotype, which may confer stronger resistance to low temperature.

The inhibition of photosynthesis under cold stress may be attributable to damage to enzyme, thus affecting carbon isotopic fractionation of plants with the photosynthesis (Berry and Bjorkman, 1980; Farquhar, 1983). Bermudagrass is a C₄ perennial species, and previous studies have showed that C₄ plants have a δ13C value of -9‰ to -14‰ (Bender, 1971; Smith and Epstein, 1971). However, in this study, the values of δ13C are about -19‰ in normal condition for both genotypes (Figure 5), which probably due to growth of the plants in a glasshouse for a long time, where CO₂ concentration was higher than the that of the air. The finding corresponds to Smith and Boutton (1981), who reported that δ13C values decreased as CO₂ concentration increased. Conditions that induced stomatal closure, confined the CO₂ supplement to carboxylation sites, which increased the δ13C values of plants (Farquhar and Richards, 1984; Rivelli et al., 2002; Araus et al., 2003; Yousfi et al., 2012). Low temperature induced stomatal closure, and ABA combined with receptors on the outside of the stomatal guard cell membrane, which further induced stomatal clusore (Cornic and Ghashghaie, 1991; Assmann and Shimazaki, 1999). Therefore in theory, exogenous ABA application may weaken the carbon isotopic fractionation of plants. However, in our study, cold stress increased δ13C values in both genotypes, which were alleviated by ABA. Prior research has identified that the activities of several photosynthetic enzymes, such as PEP-carboxylase, RuDP-carboxylase, were found to be higher in the presence of ABA (Sankhla and Huber, 1979), which could reduce δ13C values via enhanced carbon isotopic fractionation. Moreover, Farquhar et al. (1982) identified that the fractionation caused by diffusion was only 4.4‰, however, the fractionation caused by carboxylation reactions peaked to 30‰. Therefore, the effect of diffusion on stoma could almost be ignored compared to the carboxylation reactions. These results illustrated that ABA heightened the carbon isotope fractionation in both Bermudagrass under cold stress, which may be related to the interaction of stomatal closure and enzyme activities of photosynthesis. However, the regulation of carbon isotope fractionation by ABA is extremely complex under cold stress, therefore, the detailed mechanism still needs further investigation.

As an important plant hormone, ABA has been reported to regulate the expression of many genes. In our study, both cold condition and ABA treatment directly triggered expression level of ABF1, and the level in the R genotype was higher than that of S genotype, suggesting that ABF1 plays a positive role of ABA pathway, especially for the R Bermudagrass (Figure 6A). This result is consistent with previous reports from Cheng et al. (2016), who reported that ABF1was up-regulated by AM1 pretreatment and cold stress. Thomashow (1999) and Chinnusamy et al. (2007) has reported that CBF genes play vital roles in cold stress. As expected, our findings showed that CBF1 expression was up-regulated in response to cold stress, which was similar to the study of Shi et al. (2014). In addition, the cold-induced up-regulatiod was generally enhanced by ABA treatment, and the R genotype showed higher levels than the S genotype, indicating that exogenous ABA accelerate CBF1 gene expression, thus promoting COR genes transcription and enhancing cold tolerance of Bermudagrass, especially for the R genotype (Figure 6B).

Previous studies have suggested that LEA class proteins are highly hydrophilic, which may function to maintain water and protect macromolecules in dehydrated cells (Chakrabortee et al., 2007). In addition, the LEA protein can function as an antioxidant under water stress, and ABA promotes the synthesis of LEA class proteins. In the present study, under cold stress, the LEA gene was generally up-regulated, and the cold-induced up-regulation was enhanced by ABA application, suggesting that exogenous ABA enhanced the transcription of LEA and increased accumulation of the LEA protein (Figure 6C).

In summary, exogenous ABA alleviated cold caused oxidative damage in both Bermudagrass genotypes, and more prominently in the R genotype. This may be related to maintenance of cell membrane stability, improvement of the process of photosystem II, increase of carbon isotopic fractionation.

LC and JF conceived and designed the experiments. XH, LC, ZH, and AL analyzed the data. XH performed the experiments and wrote the manuscript. LC, HS, ZH, and EA helped to revise the manuscript. All authors read and approved the final manuscript.

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.