Salt stress, caused by soil salinization, is an important factor for plant distribution worldwide and greatly affects plant development and productivity (Deinlein et al., 2014). Salt stress primarily causes osmotic and ionic toxicity in plant cells, which induces secondary effects of oxidative stress that greatly damage cellular components, such as membrane lipids (Zhu, 2016). Halophytes and salt-tolerant glycophyte cultivars are tolerant to saline conditions and exhibit various mechanisms, including complex physiological traits, molecular or gene networks, and metabolic pathways (Annunziata et al., 2019). The research about plant salinity stress response, such as through osmotic adjustment (OA), which maintains cell membrane integrity and stability, could provide a combination of molecular tools for developing salt-tolerant varieties in saline environments. Therefore, it is an effective method to elevate plant salinity tolerance through increasing membrane stability (Lv et al., 2021).

Salinity enhances lipid peroxidation, which, in turn, impairs cellular membrane permeability. Phospholipids and glycolipids are two basic lipids in plant cell membranes and function as maintaining membrane structure and regulating plant salt stress tolerance (Mansour, 2013; Chalbi et al., 2015). Phospholipids, like phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), could work as signaling compounds in plants exposed to abiotic stress (Hou et al., 2016; Han and Yang, 2021). In structural phospholipids, PC and PE occupy a large proportion and the remainder consists of phosphatidylglycerol (PG), phosphoinositides (PI), phosphatidylserine (PS), and PA (Furt et al., 2011). The minor lipids PA and phosphatidylinositol bisphosphate (PIP2) act as salt stress-signaling lipids that rapidly accumulate in rice (Oryza sativa) leaves, as detected using ³²P labeling (Darwish et al., 2009). Platre et al. (2018) showed that PS and PA are required to generate the electrostatic signature of the plant plasma membrane, so their function could be separated based on their varying surface charges. PC is induced by salt stress and might function in the maintenance of membrane structure and function. Liu et al. (2015) reported that PE might be indirectly converted to PC in Arabidopsis via a hypothetical methylation pathway involving the synthesis of lysophosphatidylethanolamine (LysoPE), its methylation to lysophosphatidylcholine (LysoPC), and conversion to PC. PC could be synthesized by choline from the cytidyldiphosphate‐choline (CDP‐choline) pathway (Annunziata et al., 2019). In maize roots, PC levels decreased significantly with the increased expression of some phospholipase genes under saline-alkaline stress, suggesting there was a PC mediated lipid reprogramming under stress condition (Xu et al., 2021).

Glycolipids, like monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), are major chloroplast thylakoid membranes components and primarily affect the photosystem II (PSII) characteristic (Mizusawa and Wada, 2012). In addition, a higher ratio of DGDGs/MGDGs to PCs/PEs is an indicator of the maintenance of membrane fluidity in response to abiotic stress in different plant species. It has been reported that the DGDG/MGDG ratio is response to salt stress (Guo et al., 2019). In transgenic tobacco (Nicotiana tabacum) with OsMGD gene, plants had a high salt tolerance, increased MGD activity and higher DGDG/MGDG ratios, which revealed a more stable thylakoid membrane in transgenic plant (Wang et al., 2014). In addition, the variation of membrane lipids compositions and fatty acid desaturation under salt stress condition have been identified as a common strategy for plant via affecting membranes mobility and stress signal transduction. (Magdy et al., 1993; Mansour, 2013).

Glycine betaine (GB) could synthesized from choline with two key enzyme, choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) (Annunziata et al., 2019). In halophyte Chenopodium quinoa, choline accumulation could help to increase GB to improve salt tolerance (Pottosin et al., 2014). For some species like kidney bean (Phaseolus vulgaris) and wheat (Triticum aestivum), GB is a highly abundant compatible solute during the regulation of osmotic stress (Kreslavski et al., 2001; Salama et al., 2011). In seashore paspalum, the application of choline induced salt tolerance both in different salt sensitivity cultivars with remodeling the lipid profile and increase the GB content (Gao et al., 2020). However, not all plant species has high levels of GB, such as Arabidopsis thaliana and some grass species (Sakamoto and Murata, 2002; Hu et al., 2012). The exogenous treatment of choline or GB has effectively enhanced plant tolerance to abiotic stress, particularly for these low GB level species. In the C3 Poa turfgrass, foliar application of GB alleviated the physiological injury caused by drought or salt stress by maintaining membrane stability and active SOD, POD, and APX activity (Yang et al., 2012). In rice, choline priming could mitigate the salt stress during seed germination (Huang et al., 2023). Therefore, other metabolic pathways, such as the phospholipid and glycolipid reprograming through CDP-choline pathway, might play important role in dealing with salt stress in these species.

In this study, we examined the physiological response of Kentucky bluegrass with exogenous choline chloride under salt stress. Characterizing the GB and lipids (mainly phospholipids and glycolipids) metabolism that may be involved in the choline regulation of salt tolerance could provide theoretical basis for the important role of lipid metabolism in CDP-choline pathway during plant under salt stress condition. The selected lipid species in our study could be molecular markers for further salt stress tolerance improvement in salt-sensitive grass species.

Soil salinity is an important factor that affecting turfgrass growth and utilization. Kentucky bluegrass is sensitive to salt stress, and mitigation of the detrimental effects of salt stress and improvement in its tolerance is urgently required. Choline has been identified play role in plant salt stress tolerance (Incharoensakdi and Karnchanatat, 2003). In the present study, Fv/Fm, relative water content, chlorophyll content, and OA were increased, and EL were decreased in choline chloride -treated plants in salt treated plants. This illustrated that exogenous choline chloride could improve the salt tolerance of Kentucky bluegrass. Membrane remodeling is an important metabolic regulatory mechanism in plants exposed to salt stress (Guo et al., 2022). In soybeans, a short-term salt stress treatment could trigger a dynamic reprogramming in both galactolipids and phospholipids (Liu et al., 2021). Similar results have been reported in potato (Solanum tuberosum) (Yu et al., 2019), cotton (Gossypium herbaceum) (Liu et al., 2022), and Carex rigescens (Hu et al., 2021). As a precursor, choline is participated in two key pathways including GB metabolism pathway and lipid metabolism pathway in plant (Annunziata et al., 2019). In our study, endogenous GB metabolism pathway had not obviously response to choline application, while lipid (galactolipids and phospholipids) remodeling, were occurred with choline treatment, especially under salt stress. These results show that choline-mediated lipid metabolism is the central salt tolerance mechanism in Kentucky bluegrass.

Our data showed that the glycolipid and phospholipid contents were increased with choline chloride treatment. Choline alters the reorganization of lipid composition to protect membrane from the injury caused by salt stress, and similar results have been reported for the seashore paspalum (Gao et al., 2020). Plant glycolipids contribute to the formation of stacked thylakoid membranes and DGDG and MGDG content have been reported to decrease after salt stress in rice (Wang et al., 2014) and Suaeda salsa (Sui et al., 2010). In our study, an increase in the content of DGDG and MGDG was observed under salt stress with choline chloride treatment. DGDG and MDGD are likely to act as stabilizers in the organization of thylakoid membranes and are indispensable components of the light-harvesting complex II in plant photosynthesis (Guo et al., 2019). Furthermore, plants can alter the thylakoid membrane lipids component to resist abiotic stress. The content of MGDG 36:6, 36:5, and DGDG 34:3, 36:6, and 36:5 was significantly higher in choline chloride treated plants under salt stress, suggesting that these glycolipid species make a difference in choline mediated salt tolerance improvement. Similarly, the levels of 18:2-containing glycolipids (like MGDG-36:5 and DGDG-36:5) were upregulated by different abiotic stresses in tall fescue (Festuca arundinacea) (Zhang et al., 2019), C. rigescens (Hu et al., 2021), and rice (Wang et al., 2019). These results suggest that unsaturated 18:2-containing glycolipids might be significant in abiotic stress responses in plants.

Under salt stress condition, the PC content was significantly higher in choline chloride treated plants. PC participate in abiotic stress tolerance in plants (Tasseva et al., 2004). Studies have confirmed membrane PC has positive role in plant salt tolerance in Arabidopsis thaliana (Qiao et al., 2018), cordgrass (Spartina patens) (Wu et al., 2005) and tomato (Lycopersicon esculentum) (Kerkeb et al., 2001). In addition, in Catharanthus roseus and Mesembryanthemum crystallinum which had high salt tolerence (Elkahoui et al., 2004; Barkla et al., 2018), PC species (36:5 and 36:4) increased after salt stress compared to those in salt-sensitive plants, suggesting that they are involved in salt stress adaptation (Magdy et al., 1994). In the present study, the PC unsaturation index decreased with exogenous choline chloride. In PC-specific molecular species, the abundance of 16:0 acyl chains were higher than that of 18:3/18:2 acyl chains after choline chloride treatment. High lipid saturation contributes to plant cell membrane stability and integrity under abiotic stress (Li et al., 2020), indicating that these saturated lipid species may play an important role in choline-mediated salt stress tolerance in Kentucky bluegrass.

PE and PC are the major phospholipid components of extraplastidial membranes, and PE can regulate stress signal transduction under hyperosmotic stress via phospholipase C (Pokotylo et al., 2014; Singh et al., 2015). Under salt stress, the PE content was significantly increased in choline chloride treated plants. Studies have reported that PE increases in association with PC, resulting in abiotic stress due to increased membrane fluidity (Higashi and Saito, 2019). The choline group of PC is a bilayer-forming lipid, whereas the ethanolamine group of PE is a non-lamellar-forming lipid, which affects the formation of the bilayer phase and preserves membrane fluidity (Toumi et al., 2008; Narayanan et al., 2018). Therefore, the PC/PE ratio may reflect a cellular response to maintain membrane stability and balance between the membrane and storage lipids (Narayanan et al., 2018). In the present study, the PC/PE ratio increased in response to salt stress with exogenous choline chloride, illustrating that the elevated abundance of PC rather than PE with choline chloride treatment could help maintain the membrane integrity and fluidity in Kentucky bluegrass.

PA has been reported to crucial for salt stress responses by helping maintain ion homeostasis (Yao and Xue, 2018; Liu et al., 2019). In addition, PA is a major intermediate that guiding the PC and PE biosynthesis from the CDP-choline and CDP-ethanolamine pathways, respectively (Carman and Han, 2009). The PA content and specific molecular species decreased under salt stress in Kentucky bluegrass, and the result was the opposite of the PC and PE variation. Under abiotic stress conditions, phospholipase D could hydrolyze of PE and PC and produce PA in plants (Jiang et al., 2019). Therefore, these results suggest that choline can improve salt tolerance in Kentucky bluegrass by promoting PA downstream of PC and PE synthesis through the CDP-choline pathway.

The phosphatidylserine (PS) content can be regulated by abiotic stress in plants (Higashi and Saito, 2019). PS includes a large amount of lipid species, such as PS (40:3), which contains very-long-chain fatty acids and has been reported to increase in heat stress-susceptible wheat genotype plants (Narayanan et al., 2016). In the present study, choline chloride treatment increased the PS (40:3 and 40:2) content under salt stress conditions. However, the function of the increased PS content under salt stress is unknown. A previous study reported that PS decarboxylase (PSD) converts PS to PE through endoplasmic reticulum (Yamaoka et al., 2011). Therefore, the changes in PS in plants exposed to salinity stress with choline chloride treatment may rely on the PSD-mediated metabolic pathway and, together with PE, may play a role in membrane stability.

LysoPC is a lipid class with only one fatty acyl chain on the glycerol moiety of glycerolipids, which is surface-active, alters membrane fluidity, and affects membrane receptor functions (Zhang et al., 2018). In the present study, choline chloride treatment increased LysoPC content under salt stress conditions. In Schizochytrium sp., LysoPC lipid species were significantly enriched in the choline metabolism pathway, contributing to improved salt stress tolerance (Jiang et al., 2019). However, the role of LysoPC in stress tolerance in plants remains unclear. Plant Ca²⁺ plays a significant role in cell membrane integrity, and endogenous Ca²⁺ levels may decrease during salt stress (Hashemipetroudi et al., 2022). A previous study found that the content of LysoPC increased in response to Ca²⁺ deprivation (IsHizuKA et al., 2000), demonstrating that LysoPC might play an essential role in Ca²⁺ maintaining plasma membrane stability.

In summary, choline chloride increased the salt tolerance of Kentucky bluegrass by increasing Fv/Fm, relative water content, chlorophyll content, and OA and reducing leaf EL. Lipidomic results showed that the content of total MGDG, DGDG, PC, PE, PS, and LysoPC were increased, and PA content was decreased with exogenous choline chloride when exposed to salt stress. The leaf GB content and synthesis related genes had no response to exogenous choline chloride, indicating that choline triggered lipid metabolism was a primary mechanism in Kentucky bluegrass response to salt stress condition The specific lipid molecular species of glycolipids (MGDG 36:6, 36:5, and DGDG 34:3, 36:6, 36:5), phospholipids (PC 36:5, 36:4, PE 34:3, 34:2, 36:5, 36:4, PI 34:2, and PS 40:3, 40:2), and lysophospholipids (LysoPC 16:0, 18:3, 18:2) were increased exposed to exogenous choline chloride. Our results provide lipid biomarkers for further improvement of salt-sensitive grass species.

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

ZZ: Writing – original draft, Writing – review and editing, Software, Data curation, Formal Analysis, Validation. YL: Validation, Writing – review and editing, Data curation, Formal Analysis, Software. XM: Data curation, Software, Writing – review and editing, Formal Analysis. YL: Writing – review and editing, Investigation, Validation. CZ: Validation, Writing – review and editing, Data curation. GY: Writing – review and editing, Resources, Supervision. ZW: Supervision, Writing – review and editing, Funding acquisition. KZ: Funding acquisition, Supervision, Writing – review and editing, Software, Writing – original draft.