TY - JOUR
AU - Huang, Bingru
AB - Abstract Choline, as a precursor of glycine betaine (GB) and phospholipids, is known to play roles in plant tolerance to salt stress, but the downstream metabolic pathways regulated by choline conferring salt tolerance are still unclear for non-GB-accumulating species. The objectives were to examine how choline affects salt tolerance in a non-GB-accumulating grass species and to determine major metabolic pathways of choline regulating salt tolerance involving GB or lipid metabolism. Kentucky bluegrass (Poa pratensis) plants were subjected to salt stress (100 mM NaCl) with or without foliar application of choline chloride (1 mM) in a growth chamber. Choline or GB alone and the combined application increased leaf photochemical efficiency, relative water content and osmotic adjustment and reduced leaf electrolyte leakage. Choline application had no effects on the endogenous GB content and GB synthesis genes did not show responses to choline under nonstress and salt stress conditions. GB was not detected in Kentucky bluegrass leaves. Lipidomic analysis revealed an increase in the content of monogalactosyl diacylglycerol, phosphatidylcholine and phosphatidylethanolamine and a decrease in the phosphatidic acid content by choline application in plants exposed to salt stress. Choline-mediated lipid reprogramming could function as a dominant salt tolerance mechanism in non-GB-accumulating grass species. Introduction Salinity is one of the most detrimental abiotic stress limiting plant growth and productivity in salt-affected areas (Deinlein et al. 2014, Gupta and Huang 2014). Plants adapted to saline conditions exhibit various mechanisms of salt tolerance, such as osmotic adjustment (OA), maintenance of cell membrane integrity or stability and energy storage or supply (Mansour et al. 2015, Liang et al. 2018, Tyerman et al. 2019). Understanding metabolic factors and regulatory pathways that can facilitate salt tolerance is of great significance for improving plant growth and productivity in saline areas. Glycine betaine (GB) metabolism is known to play critical roles in plant tolerance to abiotic stress, including salt stress, by facilitating OA, stabilizing membrane proteins, and protecting membranes from oxidative damages among many functions (McNeil et al. 1999, Chen and Murata 2008, Wang et al. 2016a, Tian et al. 2017). Plant species that are capable of synthesizing GB, such as spinach (Spinacia oleracea L.) (Di Martino et al. 2003) and sugar beet (Beta vulgaris) (Yamada et al. 2015), exhibit superb salt tolerance due to the accumulation of high quantity of GB in plant cells. However, not all plant species can produce GB and some plant species accumulate very low levels of GB or GB is undetectably low, including many grass species (Hitz and Hanson 1980, Peel et al. 2010, Hu et al. 2012). Exogenous application of GB can effectively improve salt tolerance in various plant species, particularly in GB non-accumulating species, such as Kentucky bluegrass in which GB was not detectable (Qian et al. 2001, Yang et al. 2012). Other metabolic pathways may become important for plant tolerance to salt stress in GB non-accumulating plants. Lipids, including phospholipids and glycolipids, are the primary components of cellular membranes controlling membrane integrity and stability, which also play key roles in plant tolerance to salt stress (Mansour 2013). Phospholipids are also involved in stress signaling and energy supply in plants exposed to abiotic stress (Rawyler et al. 2002, Hou et al. 2016). Changes in phospholipid content may take part in plant adaptation to salt stress (Chalbi et al. 2015, Singer et al. 2016, Sarabia et al. 2018). For example, in barley (Hordeum vulgare L.), the phosphatidylethanolamine (PE) content increased in tolerant cultivar while it decreased in sensitive cultivar under salinity stress (Natera et al. 2016). Also, a greater increase in the phosphatidylcholine (PC) and phosphatidylglycerol (PG) contents was found in tolerant cultivars of buffalograss (Buchloe dactyloides), compared with unchanged PG content and decreased PC content in the sensitive cultivar exposed to salt stress (Lin and Wu 1996). Glycolipids are major constituents of photosynthetic membranes (Boudière et al. 2014), and monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) are the most abundant lipids in chloroplast thylakoid membranes regulating membrane stability and fluidity (Webb and Green 1991, Narayanan et al. 2016). Studies had shown that the MGDG and DGDG contents decreased, with a lesser magnitude of changes in DGDG than in MGDG in response to abiotic stress, including salt stress, and DGDG/MGDG ratio has been associated with plant adaptation to salt stress (Liu et al. 2019). Heterologous overexpression of rice (Oryza sativa L.) MGD gene increased the MGDG content in tobacco (Nicotiana tabacum L.), which resulted in tight stacking of thylakoid membrane and high tolerance to salt stress in transgenic plants (Wang et al. 2014). Under NaCl treatment, the DGDG/MGDG ratio was found to decrease in both Arabidopsis (Arabidopsis thaliana) and Thellungiella (Thellungiella halophila), but Thellungiella showed a smaller reduction than Arabidopsis (Sui and Han 2014). In addition, changes in phospholipid and glycolipid compositions and the degree of fatty acid desaturation are also reported to play positive roles in plant tolerance to salt stress (Upchurch 2008, Guo et al. 2019). Therefore, modulation of phospholipid and glycolipid metabolism may be an effective approach to enhance plant tolerance to salt stress, particularly in plants incapable of GB synthesis. Choline is a precursor for GB and phospholipid, PC, which control both GB and PC synthesis (Su et al. 2006, Fagone and Jackowski 2013). GB is synthesized from choline, which is oxidized to betaine aldehyde by a ferredoxin-dependent choline monooxygenase (CMO), and then betaine aldehyde dehydrogenase (BADH) converts betaine aldehyde to GB in chloroplasts (Tsutsumi et al. 2015, Annunziata et al. 2019). PC is synthesized from choline chloride in the cytidyldiphosphate-choline ( CDP-Choline) pathway involving three steps catalyzed by choline kinase (CK), CTP-phosphocholine cytidylyltransferase (CCT) and cytidyltriphosphate-choline transferase (CPT) (Fagone and Jackowski 2013, Liu et al. 2018b). PC is the major phospholipid in eukaryotic cell membranes, and it has crucial roles in glycerolipid synthesis and serves as an important precursor for many signaling lipids [e.g. phosphatidic acid (PA), lysophosphatidylcholine (Lyso PC)] (Tasseva et al. 2004, Chen et al. 2019). Exogenous application of choline has been found to effectively enhance plant tolerance to abiotic stress mostly in GB-accumulating plants, such as for drought stress in Rehmannia glutinosa (Zhao et al. 2007), heat stress in bean (Phaseolus vulgaris L.) (Kreslavski et al. 2001) and salt stress in wheat (Triticum aestivum) (Salama and Mansour 2015) and seashore paspalum (Paspalum vaginatum) (Gao et al. 2020). The alleviation of drought and heat stress damages by choline has been associated with proline accumulation and protection of photosynthesis and lipids from stress damages (Zhao et al. 2007, Salama and Mansour 2015). Improved salt tolerance in GB-accumulating plant species due to choline application has been attributed to enhanced GB production, such as in wheat seeds (Salama et al. 2011) and seashore paspalum leaves (Gao et al. 2020). However, mechanisms underlying improved salt tolerance by choline are yet to completely understand and largely unknown particularly for non-GB-accumulating species. Various approaches have been taken to enhance GB accumulation in GB non-accumulating plant species, including modification of CMO enzyme activity and subcellular localization (Sakamoto and Murata 2001, Kurepin et al. 2015), choline pool (Nuccio et al. 1998) and choline transporter (Peel et al. 2010), but the increases in the GB content and effects on stress tolerance improvement are limited (Huang et al. 2000, Sakamoto and Murata 2002, Yamada et al. 2009). Therefore, we hypothesize that choline-induced salt tolerance in non-GB-accumulating plant species may mainly due to the regulation of lipid metabolism. However, specific lipids and molecular species of either phospholipids or glycolipids that may be regulated by choline contributing to salt tolerance in GB non-accumulating plants are still unclear. Such information provides insights into salt tolerance mechanisms in the CDP-Choline pathway and offers specific lipid species as potential metabolic markers for selecting salt-tolerant plants through genetic modification and breeding programs. The objectives of the present study were to examine whether exogenous choline application may enhance salt tolerance in Kentucky bluegrass and to determine how GB and major lipids metabolism affected by choline may be involved in the choline regulation of salt tolerance. The characterization of choline regulated in salt tolerance in Kentucky bluegrass provides further insights into salt regulatory mechanisms in non-GB-accumulated grass species. Results Physiological responses of Kentucky bluegrass to salt stress and choline application Leaf Fv/Fm ratio and chlorophyll content decreased in both untreated and choline-treated plants during salt stress (Fig. 1). Choline-treated plants had a significantly higher Fv/Fm than untreated plants at 14 d of salt stress while choline had no effects on Fv/Fm under nonstress conditions. Chlorophyll content in choline-treated plants maintained significantly higher level than untreated plants at 14 d (by 9.97%) and 21 d (by 17.65%) of salt treatment. Leaf EL was significantly increased during salt stress, while choline application reduced EL by 22.68%, 17.12% and 9.68% than untreated plants at 7, 14 and 21 d of salt stress, respectively. Fig. 1 Open in new tabDownload slide Effect of choline treatment and salt stress on physiological response in Kentucky bluegrass. Data were collected after 7, 14 and 21 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). The vertical bars indicate the values of LSD at P-value = 0.05. Fig. 1 Open in new tabDownload slide Effect of choline treatment and salt stress on physiological response in Kentucky bluegrass. Data were collected after 7, 14 and 21 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). The vertical bars indicate the values of LSD at P-value = 0.05. Plants under nonstress conditions exhibited higher RWC with choline application at 7 and 14 d of treatment. Leaf RWC decreased during salt stress but increased to a higher level with choline application compared to untreated plants at 21 d of salt stress treatment. Leaf OA increased during salt stress and plants with choline application had significantly higher OA compared to untreated plants during 7–21 d of salt stress. GB and choline content and associated genes of Kentucky bluegrass in response to choline treatment under salt stress To determine how choline may affect GB synthesis in Kentucky bluegrass, the content of endogenous GB and choline was measured and the expression of genes controlling GB synthesis, choline synthesis and transport was examined for plants treated with choline and exposed to 14 d of salt stress. The specific signal peak of GB and choline in HPLC chromatograms and mass spectrum is illustrated in Fig. 2a. GB was below the detectable level by HPLC–MS with extremely weak signals in all leaf samples of Kentucky bluegrass (Fig. 2b). Choline was detected and the content of endogenous choline significantly increased with salt stress (by 5.84% and 6.82% in untreated and choline-treated plants, respectively) and with choline application (by 6.80% and 7.78% in nonstress and salt-stressed plants, respectively) (Fig. 2c). Fig. 2 Open in new tabDownload slide Effect of choline treatment and salt stress on GB and choline content in Kentucky bluegrass. (a) HPLC chromatograms and mass spectrum of standards; (b) HPLC chromatograms of samples; and (c) the endogenous choline content determined by HPLC–MS analysis under different treatments. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 2 Open in new tabDownload slide Effect of choline treatment and salt stress on GB and choline content in Kentucky bluegrass. (a) HPLC chromatograms and mass spectrum of standards; (b) HPLC chromatograms of samples; and (c) the endogenous choline content determined by HPLC–MS analysis under different treatments. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. CMO controlling GB synthesis from choline was upregulated by choline while the downstream GB synthesis genes, BADH1 and BADH2, were downregulated by choline application under salt stress (Fig. 3). Choline application had no effects on the expression of those genes under nonstress conditions. Choline synthesis genes [PEAMT (phosphomethylethanolamine N-methyltransferase) and SDC (serine decarboxylase)] were all upregulated with choline application under nonstress and stress conditions. The expression level of the choline transporter gene, CTL (choline transporter-like), did not differ between choline-treated and untreated plants under nonstress conditions, while it increased with choline treatment under salt stress conditions. Fig. 3 Open in new tabDownload slide GB synthesis, choline synthesis and transport gene expression in Kentucky bluegrass in response to salt stress and exogenous choline. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 3 Open in new tabDownload slide GB synthesis, choline synthesis and transport gene expression in Kentucky bluegrass in response to salt stress and exogenous choline. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Physiological responses and gene expression of Kentucky bluegrass in response to the combination of choline and GB application under salt stress To determine whether the exogenous application of GB in the combination with choline may further improve salt tolerance, plants were treated with choline, GB and choline + GB under salt stress conditions. At 14 and 21 d of salt stress treatment, choline + GB treatment resulted in significant increases in the RWC content compared to the untreated plants (Fig. 4). Compared to the nonstress treatment, EL values in choline, GB and choline + GB treatments were significantly decreased at 14 d of salt stress, whereas at 21 d of salt stress, the significant difference was only found in GB and choline + GB treatments. Leaf OA levels in choline, GB and choline + GB treatments were significantly increased compared to the nonstress control treatment at 14 and 21 d after salt stress. Fig. 4 Open in new tabDownload slide Effect of choline, GB, choline + GB and salt stress treatments on physiological response in Kentucky bluegrass. Data were collected after 14 and 21 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 4 Open in new tabDownload slide Effect of choline, GB, choline + GB and salt stress treatments on physiological response in Kentucky bluegrass. Data were collected after 14 and 21 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. For GB synthesis genes, the CMO gene was upregulated with choline treatment and downregulated with GB and choline + GB treatments under salt stress conditions (Fig. 5). Compared to the non-choline treatment, the expression of BADH1 and BADH2 genes was significantly lower under salt stress conditions with choline, GB and choline + GB treatments, while BADH2 was upregulated in choline, GB and choline + GB treatments under nonstress conditions. Under salt stress with GB treatment, the expression of CK1 and CCT1 did not significantly change and CEPT (choline/ethanolaminephosphotransferase) gene was downregulated compared to the nonstress control treatment. CEPT was downregulated in the GB treatment and upregulated in choline + GB treatments under nonstress conditions. While choline and choline + GB application increased the expression level of CDP-choline pathway-related genes (CK1, CCT1 and CEPT) in plants exposed to salt stress. Fig. 5 Open in new tabDownload slide GB synthesis and CDP-choline pathway gene expression in Kentucky bluegrass in response to choline, GB, choline + GB and salt stress treatments. Data were collected after 14 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 5 Open in new tabDownload slide GB synthesis and CDP-choline pathway gene expression in Kentucky bluegrass in response to choline, GB, choline + GB and salt stress treatments. Data were collected after 14 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Leaf lipidomic changes in response to choline treatment under salt stress Leaf tissues were selected for lipid analysis for plants exposed to 14 d of salt stress when choline had the most pronounced positive effects on physiological parameters as described above. Lipid profiles are presented in Supplementary Fig. S1. A total of 9 lipid species and 60 lipid molecular species were detected in leaves of Kentucky bluegrass (Supplementary Table S2), including two glycolipids, MGDG and DGDG, six phospholipids, PA, PC, PE, PG, phosphatidylinositol (PI) and phosphatidylserine (PS), and one lysophospholipid, Lyso PC. The content of total lipids, glycolipids and phospholipids was reduced due to salt stress (Fig. 6). Choline application increased total lipid content by 7.85% and 8.70% in nonstress control and salt stress treatment, respectively. Similarly, total glycolipid content in choline-treated plants was significantly higher than untreated plants in the nonstress control (by 9.61%) and salt-stressed plants (by 13.04%). Total phospholipid content did not change in response to choline application under nonstress or salt stress conditions. Fig. 6 Open in new tabDownload slide Effects of choline treatment and salt stress on total lipids, glycolipids and phospholipids contents in Kentucky bluegrass. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 6 Open in new tabDownload slide Effects of choline treatment and salt stress on total lipids, glycolipids and phospholipids contents in Kentucky bluegrass. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Salt stress caused a significant decline in the total content of MGDG and DGDG in both choline-treated and untreated plants (Fig. 7). Choline treatment significantly increased total MGDG content under nonstress control (higher by 17.48 %) and salt stress (higher by 16.27 %) conditions. Total DGDG content increased in response to choline application, but it was only significantly higher than untreated plants under nonstress conditions while the increase in choline-treated plants was not statistically significant compared to untreated plants under salt stress (Fig. 7). Choline application resulted in significant increases in PC and PE contents but decreases in the PA content in plants exposed to salt stress. The content of other phospholipids, such as PG and PS, decreased under salt stress conditions in both choline-treated and untreated plants, but those phospholipids did not show significant responses to choline application exposed to nonstress control or salt stress condition (data not shown). Fig. 7 Open in new tabDownload slide Effects of choline treatment and salt stress on contents of different lipid classes in Kentucky bluegrass. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 7 Open in new tabDownload slide Effects of choline treatment and salt stress on contents of different lipid classes in Kentucky bluegrass. Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. The specific components of different lipids in response to salt stress or choline application are shown in Fig. 8. The 36:6 molecular class was the most abundant lipid species in MGDG and DGDG. For glycolipid species, compared to untreated plants, the content of MGDG 34:4, 34:2, 36:6 and 36:5 was significantly higher due to choline treatment in plants exposed to salt stress. DGDG 34:2, 36:5 and 36:4 had significantly higher content in choline-treated plants than in untreated plants under salt stress. Under nonstress conditions, the content of MGDG (34:3, 36:6) and DGDG (34:2, 36:4) was significantly increased with choline treatment. Fig. 8 Open in new tabDownload slide Effects of choline treatment and salt stress on specific molecular species of different lipid classes in Kentucky bluegrass. Data were collected after 12 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 8 Open in new tabDownload slide Effects of choline treatment and salt stress on specific molecular species of different lipid classes in Kentucky bluegrass. Data were collected after 12 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. For phospholipids species, only PC, PE and PA with significant responses to choline are further discussed. In responses to choline application, the content of PE species (34:3, 34:2, 36:6, 36:5, 36:4, 36:3 and 36:2) significantly increased under salt stress and the content of PC species, except 36:1 and 38:3, was also all increased in choline-treated plants exposed to salt stress. A significant decrease was observed in the content of all PA species (34:4, 34:3, 34:2, 34:1, 36:6, 36:5, 36:4, 36:3 and 36:2) with choline treatment, compared to untreated plants under salt stress. Under nonstress conditions, the content of PA 34:4, 34:3, 34:2 and 36:5 decreased and PC 34:3 and PE 34:3 and 36:6 contents increased in choline-treated plants compared with untreated plants. Under salt treatment, the unsaturation index of DGDG and PC increased while MGDG unsaturation index decreased with or without choline treatment (Table 1). Compared to untreated plants, the unsaturation index of PC was significantly lower in choline-treated plants under stress conditions. Table 1 The unsaturation index level of different lipid classes in Kentucky bluegrass leaves under different treatments Lipid classes . Treatments . Control . Choline treated and control . Salt stress . Choline treated and salt stress . CK . CC . S . CS . Glycolipids  DGDG 5.22 ± 0.02 b 5.21 ± 0.01 b 5.28 ± 0.02 a 5.26 ± 0.01 a  MGDG 5.74 ± 0.01 a 5.74 ±0.00 a 5.70 ± 0.01 b 5.70 ± 0.01 b Phospholipids  PA 3.24 ± 0.02 a 3.20 ± 0.02 a 3.28 ± 0.02 a 3.24 ± 0.01 a  PC 3.17 ± 0.02 c 3.18 ± 0.01 c 3.42 ± 0.02 a 3.36 ± 0.01 b  PE 3.28 ± 0.02 a 3.32 ± 0.02 a 3.34 ± 0.02 a 3.33 ± 0.02 a Lipid classes . Treatments . Control . Choline treated and control . Salt stress . Choline treated and salt stress . CK . CC . S . CS . Glycolipids  DGDG 5.22 ± 0.02 b 5.21 ± 0.01 b 5.28 ± 0.02 a 5.26 ± 0.01 a  MGDG 5.74 ± 0.01 a 5.74 ±0.00 a 5.70 ± 0.01 b 5.70 ± 0.01 b Phospholipids  PA 3.24 ± 0.02 a 3.20 ± 0.02 a 3.28 ± 0.02 a 3.24 ± 0.01 a  PC 3.17 ± 0.02 c 3.18 ± 0.01 c 3.42 ± 0.02 a 3.36 ± 0.01 b  PE 3.28 ± 0.02 a 3.32 ± 0.02 a 3.34 ± 0.02 a 3.33 ± 0.02 a Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Different lowercase letters in the same row indicate the significant difference at P ≤ 0.05 level. Open in new tab Table 1 The unsaturation index level of different lipid classes in Kentucky bluegrass leaves under different treatments Lipid classes . Treatments . Control . Choline treated and control . Salt stress . Choline treated and salt stress . CK . CC . S . CS . Glycolipids  DGDG 5.22 ± 0.02 b 5.21 ± 0.01 b 5.28 ± 0.02 a 5.26 ± 0.01 a  MGDG 5.74 ± 0.01 a 5.74 ±0.00 a 5.70 ± 0.01 b 5.70 ± 0.01 b Phospholipids  PA 3.24 ± 0.02 a 3.20 ± 0.02 a 3.28 ± 0.02 a 3.24 ± 0.01 a  PC 3.17 ± 0.02 c 3.18 ± 0.01 c 3.42 ± 0.02 a 3.36 ± 0.01 b  PE 3.28 ± 0.02 a 3.32 ± 0.02 a 3.34 ± 0.02 a 3.33 ± 0.02 a Lipid classes . Treatments . Control . Choline treated and control . Salt stress . Choline treated and salt stress . CK . CC . S . CS . Glycolipids  DGDG 5.22 ± 0.02 b 5.21 ± 0.01 b 5.28 ± 0.02 a 5.26 ± 0.01 a  MGDG 5.74 ± 0.01 a 5.74 ±0.00 a 5.70 ± 0.01 b 5.70 ± 0.01 b Phospholipids  PA 3.24 ± 0.02 a 3.20 ± 0.02 a 3.28 ± 0.02 a 3.24 ± 0.01 a  PC 3.17 ± 0.02 c 3.18 ± 0.01 c 3.42 ± 0.02 a 3.36 ± 0.01 b  PE 3.28 ± 0.02 a 3.32 ± 0.02 a 3.34 ± 0.02 a 3.33 ± 0.02 a Data were collected after 14 d of salt stress. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Data are means ± SE (n = 3). Different lowercase letters in the same row indicate the significant difference at P ≤ 0.05 level. Open in new tab Expression of lipid synthesis genes in response to salt stress and exogenous choline treatment in Kentucky bluegrass To determine major pathways of lipid synthesis that may be regulated by choline to cause differential accumulation of different lipids, as described above, gene expression analysis was performed (Fig. 9). For genes in the CPD-choline pathway (CK, CCT and CEPT), CK1, CCT1 and CEPT gene expressions were all significantly upregulated with choline treatment and increased by 2.03-, 1.5- and 2.22-fold, respectively, compared to untreated plants under salt stress conditions. Under nonstress conditions, choline application increased the CK1 gene expression level, whereas CCT1 and CEPT expression levels were not changed. Fig. 9 Open in new tabDownload slide CDP-choline pathway gene expression in Kentucky bluegrass in response to salt stress and exogenous choline. Data were collected after 14 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Fig. 9 Open in new tabDownload slide CDP-choline pathway gene expression in Kentucky bluegrass in response to salt stress and exogenous choline. Data were collected after 14 d of salt stress. Data are means ± SE (n = 3). Letters above bars indicate significant differences at the P ≤ 0.05 level. Discussion Choline plays positive role in regulating salt tolerance in GB-accumulating plants, which has been mainly attributed to the stimulation of GB synthesis (Nuccio et al. 1998, Salama et al. 2011). Our study found that choline enhanced salt tolerance in GB non-accumulating grass species, as manifested by increased photochemical efficiency and OA, and lower EL, was not due to the regulation of endogenous GB synthesis, as GB was not detected in leaves of Kentucky bluegrass regardless of choline treatment despite increases in the endogenous choline content in choline-treated plants. While CMO in the first step of GB synthesis was upregulated, BADH in the following step of GB synthesis was downregulated in choline-treated Kentucky bluegrass exposed to salt stress. In addition, choline synthesis-related genes (PEAMT and SDC) and choline transporter gene (CTL) were all upregulated with choline application under salt stress along with increased endogenous choline content. These results suggested that choline pool was not the limiting factor for GB synthesis, but the GB synthesis process could be impaired in Kentucky bluegrass. A genetic lesion may occur at the first step in GB synthesis in A. thaliana and O. sativa, leading to low or no GB synthesis (Peel et al. 2010). Therefore, choline-enhanced salt tolerance in GB non-accumulating plant species, such as Kentucky bluegrass, may go through other metabolic pathways rather than GB metabolism. Choline is also a precursor for phospholipids, as described in the Introduction. Unlike GB, lipid profiles were responsive to salt stress and choline application in Kentucky bluegrass exposed to salt stress. Lipid reprogramming by adjusting the content and composition of lipids in cellular membranes plays a key role in plant tolerance to abiotic stress (Elkahoui et al. 2004, Welti et al. 2002, Salama and Mansour 2015, Zhang et al. 2019). Along with physiological damages, as manifested by the decline in photochemical efficiency and the increase in EL, salt stress caused a significant reduction in the content of total lipids, glycolipids and phospholipids in Kentucky bluegrass in our study. Similar lipid responses to salt stress have been reported in other plant species, such as barley (H. vulgare) (Brown and DuPont 1989) and wheat (Salama and Mansour 2015). Choline application increased the content of total lipids in Kentucky bluegrass along with improvement in salt tolerance. Increases in the lipid content by choline have also been reported in halophytic seashore paspalum (Gao et al. 2020). These results indicated that choline-enhanced salt tolerance may involve modification in lipid synthesis in GB non-accumulating plant species. Phospholipids are major components of cellular membranes by forming lipid bilayers and participating in signal transduction among various functions (Namasivayam et al. 2015, Ruelland et al. 2015). PC, PE and PA are among the most abundant phospholipids in plants, with PA providing the precursors for the biosynthesis of PC and PE via the CDP-choline pathway and CDP-ethanolamine pathway, respectively (Carman and Han 2009, Arisz 2010) (https://lipidhome.co.uk/lipids/complex/pa/index.htm). These phospholipids play key roles in maintaining membrane fluidity and integrity (Tasseva et al. 2004). In our study, the content of PC decreased in salt-stressed plants and increased significantly in responses to choline treatment under salt stress. Also, PC is synthesized in the CDP-choline pathway regulated by CK, CCT and CEPT (Fagone and Jackowski 2013) and the expression level of these lipid synthesis genes was highly upregulated by choline application. The significant upregulation of CK1, CCT1 and CEPT along with the increased endogenous content of PC due to choline application indicated that the activation of CDP-choline pathway could be the dominant pathway for lipid synthesis, contributing to choline-enhanced salt tolerance in Kentucky bluegrass. The content of PE also decreased under salt stress but increased with choline treatment in Kentucky bluegrass exposed to salt stress. The PE content had been reported could affect stress signal transduction via combining with phospholipase C (PLC) as a nonspecific PLC and ultimately mediate the downstream protein kinase and regulate hyperosmotic stress (Singh et al. 2015). In addition, the ratio of PC:PE was greater in choline-treated plants than in non-choline-treated plants under salt stress in our study. The ratio of PC:PE has been positively associated with abiotic stress tolerance, including salt stress (Zhang et al. 2013), which helps to maintain stable bilayer phase and membrane fluidity (Narayanan et al. 2018). Choline-enhanced salt tolerance in Kentucky bluegrass could be associated with the increased PC and PE contents that affect membrane stability and fluidity. Further analysis of lipid profiles for specific lipid molecular species in PC and PE revealed that PC (34:3, 34:2 and 36:5, 36:4) and PE (34:3, 34:2 and 36:5, 36:4) could contribute positively to salt tolerance in Kentucky bluegrass, as the content of these lipid species increased significantly in response to choline application under salt stress. Increased content of PC and PE that contains 34 and 36 carbons was found in plant response to Fusarium graminearum (PC 34:2, 36:4 and PE 34:2, 36:4) (Reyna et al. 2019), chilling stress (PC 34:2, 36:4 and PE 34:2, 36:4) (Li et al. 2014), and salt stress (PC 34:3, 36:5 and PE 34:2, 36:4) (Natera et al. 2016). In this study, the abundance of 16:0 acyl chains (34:3, 34:2) was higher than 18:3/18:2 acyl chains (36:5, 36:4) in PC with choline treatment and PC unsaturation index decreased with choline treatment, which suggested the positive roles of increases in the synthesis of saturated lipid species by choline for salt stress tolerance in Kentucky bluegrass. High lipid saturation helps to maintain membrane stability and integrity in plants exposed to abiotic stress, including salt stress (Hajlaoui et al. 2009, Chen et al. 2018) Therefore, choline may play positive roles in salt tolerance in association with the upregulation of PC (34:3, 34:2 and 36:5, 36:4) and PE (34:3, 34:2 and 36:5, 36:4) metabolism. PA is another abundant phospholipid serving important roles in stress responses (Wang et al. 2016b, Yao and Xue 2018). It has been reported that PA is involved in salt stress responses by affecting H+-ATPase activity and Na+/H+ exchange to maintain ion homeostasis in plants (Zhang et al. 2006, Li et al. 2019) and binding to the protein phosphatase 2C or the Gα subunit of heterotrimeric G protein to regulate the downstream ABA signal transduction and stomatal movement (Mishra et al. 2006, Darwish et al. 2009). In contrast to an increased content of PC and PE, the content of PA increased under salt stress but was significantly decreased with choline treatment under nonstress control and salt stress conditions; however, the PA content increased in response to choline application in GB-accumulating species, such as seashore paspalum (Gao et al. 2020) and wheat (Salama and Mansour 2015), under salt stress conditions. In this study, the content of PA (34:3, 34:2 and 36:5, 36:4) was significantly increased after salt treatment without choline application. In Arabidopsis, PA containing 16:0–18:2 acyl chains like 34:2, 36:4 and 36:5 had been reported to participate in coupling with MAPK cascades and regulate salt responses (Yu et al. 2010). However, the content of these PA-specific species decreased with choline application, which was opposite to the changes in PC and PE in salt stress conditions. It was reported that phospholipase D could activate by salt stress and other abiotic stress conditions, hydrolyzing phospholipids, such as PE and PC, and resulting in PA synthesis in plants (Testerink and Munnik 2005, Bargmann et al. 2009, McLoughlin and Testerink 2013). In addition, PA could be the upstream of PC and PE in lipid synthesis (Carman and Han 2009, Arisz 2010). Therefore, the reduction in the PA content and an increase in the PC and PE contents by choline suggested that choline-mediated salt tolerance involving lipid metabolism was mainly by activating the CDP-choline pathway and diverting from PA to the synthesis of PC and PE in Kentucky bluegrass. This could be a unique lipid reprogramming strategy for choline-enhanced salt tolerance in GB non-accumulating Kentucky bluegrass in this study. As important constituents of chloroplast thylakoid membranes, DGDG and MGDG are the most abundant glycolipids, which play an important role in photosynthesis, thylakoid assembly and chloroplast biosynthesis and development in plants (Awai et al. 2001, Su et al. 2009). In our study, MGDG and DGDG content decreased under salt stress. The reduction in DGDG and MGDG content were also reported in rice (O. sativa L.) (Liu et al. 2018a), Suaeda salsa L. (Sui et al. 2010) and peanut (Arachis hypogaea L.) (Liu et al. 2017) under salt stress. Choline application caused significant increases in the MGDG content in Kentucky bluegrass exposed to salt stress. MGDG is reported to function as a non-bilayer-forming lipid and indispensably participates in the thylakoid membrane formation with affecting grana stacks formation and stacking (Lee 2000). MGDG has also taken part in plant photosystem II (PSII) complex composition and plays key roles in the linear electron transport process and PSII photostability regulation (Wu et al. 2013). MGDG combined with PG is also an integral composition of photosystem I complex (Wacker et al. 2016). Furthermore, the content of specific glycolipid species, MGDG 34:4, 34:2, 36:6 and 36:5 increased due to choline treatment under salt stress while MGDG 36:6 and 36:5 were the most abundant glycolipid molecular species in Kentucky bluegrass. The increased content of MGDG, particularly MGDG 36:6 and 36:5, in choline-treated plants with improved salt tolerance suggested that those glycolipid species could be involved in choline-enhanced salt tolerance in Kentucky bluegrass. In summary, choline or GB alone and the combined application increased photochemical efficiency, leaf relative water content (RWC) and OA and reduced electrolyte leakage (EL). Choline application had no effects on the endogenous GB content and GB synthesis genes did not show responses to choline under nonstress and salt stress conditions. GB was not detected in leaves in Kentucky bluegrass with or without salt stress. Lipidomic analysis revealed significant increases in the content of total MGDG, PC and PE, and a decrease in the PA content by choline application in plants exposed to salt stress. These results suggested that choline-mediated lipid reprogramming could function as a dominant salt tolerance mechanism in non-GB-accumulating grass species (Fig. 10). Specific lipid molecular species of phospholipids (PC 34:3, 34:2 and 36:5, 36:4 and PE 34:3, 34:2 and 36:5, 36:4) and glycolipids (MGDG 36:6 and 36:5) with increased content in response to choline application could be used as biomarkers for selecting salt-tolerant germplasm. Our results provide new insights into regulatory mechanisms of salt tolerance for non-GB-accumulating species. Fig. 10 Open in new tabDownload slide An overview of GB synthesis, lipid reprogramming, and physiological responses by choline treatment and salt stress in Kentucky bluegrass. Data were collected after 14 d of salt stress. The squares represent the heat map based on the means and from left to right are CK, CC, S and CS. Blue color represents decreased content, while red color represents increased content. The red color represents increased, and the green color represents decreased. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Fig. 10 Open in new tabDownload slide An overview of GB synthesis, lipid reprogramming, and physiological responses by choline treatment and salt stress in Kentucky bluegrass. Data were collected after 14 d of salt stress. The squares represent the heat map based on the means and from left to right are CK, CC, S and CS. Blue color represents decreased content, while red color represents increased content. The red color represents increased, and the green color represents decreased. CK, control optimal condition; CC, choline treatment; S, salt stress; CS, choline treatment + salt stress. Materials and Methods Plant growth conditions Seeds of Kentucky bluegrass (Poa pratensis) (cv. ‘Baron’) were planted in plastic pots (10 cm × 7 cm × 8.5 cm) filled with fritted clay in a greenhouse. Plants were irrigated daily and watered with half‐strength Hoagland's nutrient solution (Hoagland and Arnon 1950). Uniform-size tillers were selected from 2-month-old plants established in the greenhouse and transferred to a plastic container (21-cm length × 19-cm width × 18-cm depth) filled with half-strength Hoagland's nutrient solution in a growth chamber. The nutrient solution was aerated with an aquarium pump and replaced every 3 d to provide plants with adequate aeration and nutrition. During the experimental period, the chamber was controlled at 23/20°C (day/night temperature) and 12-h photoperiod with 660 µmol m–2 s–1 photosynthetically active radiation at the canopy level. Plants were established in the hydroponic system under normal growth or nonstress conditions for 21 d prior to the exposure to experimental treatments. Experimental treatments To examine how choline application affects salt tolerance of Kentucky bluegrass and associated mechanisms, plants were subjected to choline and salt treatments. Each plant was foliar sprayed with choline chloride at 1 mM until dripping from leaves at 7 d prior to imposing salt stress and every 7 d during salt stress. The untreated control plants were sprayed with an equal volume of water. Choline chloride concentration was determined based on our preliminary test with a positive effect on plant growth under salt stress. Salt stress treatment was applied by adding NaCl daily to half-strength Hoagland's nutrient solution with the concentration increasing gradually from 30 mM to the final concentration of 100 mM. The nonstress control plants were grown in half-strength Hoagland's nutrient solution without NaCl. For the determination of potential synergistic effects of choline and GB exogenous application in Kentucky bluegrass, plants were treated with choline at 1 mM and GB at 100 mM individually or in combination when plants were exposed to salt stress. Each container was sprayed for every 7 d during the salt-treatment period. After the first 7 d of GB/choline treatment, salt treatment was applied by adding NaCl to half-strength Hoagland's nutrient solution, and the concentration increased every day by 30 mM and until the final concentration reached to 100 mM. Each of the above experiment was arranged as a split-plot design with salt treatments as main plots and choline or GB treatments as subplots. Each treatment had 4 replicates in 4 containers and 36 plants for each treatment in each container. Physiological measurements Physiological measurements were performed at 7, 14 and 21 d during salt stress treatment. For photochemical efficiency measurement expressed as the ratio of variable fluorescence to maximum fluorescence (Fv/Fm) of chlorophyll, leaves were first pretreated with dark for 30 min and then Fv/Fm reading was taken using a pulse-modulated fluorometer Fim 1500 (Analytical Development Company Ltd., Hoddesdon, UK). For chlorophyll content determination, 0.2 g of fresh leaf tissues was incubated in dimethyl sulfoxide in the dark for 48 h. The absorbance at 663 and 645 nm was measured using a spectrophotometer (Spectronic in Instruments, Rochester, NY, USA). Chlorophyll content was calculated based on the equations described by Barnes et al. (1992). For the determination of RWC, 0.5 g of fresh leaf tissues was soaked in a 50-ml centrifuge tube containing distilled water for 24 h. Leaves were blot dried with a paper towel and immediately weighed as fresh weight (Wt). Leaf tissues were then oven-dried at 80°C and weighed to determine the dry weight (Wd). Leaf RWC calculation was calculated with the equation: RWC = (Wf − Wd/Wt − Wd). EL measurement was performed following the protocol described by Jiang and Zhang (2001). Fresh leaves (0.2 g) were soaked in 35 ml of deionized water in a 50-ml plastic tube and fixed on a shaker for 24 h, conductance was then measured as initial conductivity (E0) using a conductivity meter (YSI Model 32, Yellow Spring, OH, USA). Afterward, the tubes were put into autoclave with 120°C for 20 min to kill the tissue. Final conductivity (E1) was measured after the tubes cooling to room temperature. Leaf EL was calculated as EL = E0/E1. For leave OA measurement, 0.2 g of fresh leaf tissues was fully hydrated in deionized water for 24 h at 4°C. After achieving complete turgor, leaf tissues were frozen in liquid nitrogen and subsequently stored at −20°C. A 10-μl sap expressed from leaf tissues was placed in the chambers of an osmometer for the measurement of osmolarity (A) (Wescor, Inc., Logan, UT, USA). The osmotic potential was determined based on the equation: Ψ(MPa) = −A × 0.001 × 2.58. OA value was then calculated according to Ψ(nonstress control) and Ψ(salt stress) plants (Gao et al. 2020). Lipidomic profiling analysis Fresh leaf tissues were harvested at 14 d of salt stress treatment for lipid profiling analysis. Lipid extraction, electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis and quantification were performed based on the method described in previous studies (Zhang et al. 2019). Briefly, lipids were extracted from 0.3 g of fresh leaves with a 1.5 ml of chloroform and 0.6 ml of water mixed solution and shaken for 1 h and then treated with 4 ml of chloroform/methanol (2:1, V/V) with 0.01% butylated hydroxytoluene mix solution and shaken for 0.5 h at room temperature. The step of chloroform and methanol extraction was then repeated six times, and the last repeat was gone through overnight until the leaves turned into white color. All the extracted solution was mixed together and evaporated with nitrogen gas. Finally, the dried lipid extract was redissolved in 1 ml of chloroform and stored at 80°C for further ESI-MS/MS analysis. ESI-MS/MS data analysis was performed as described previously (Zhang et al. 2019, Gao et al. 2020). The remaining leaf tissues were dried in an oven with 105°C, and dry weight was recorded for further lipid content calculation. The unsaturation index calculation of each lipid head group was carried out as the equation: [(N × mol% lipid)]/100 where N is the number of double bonds in the lipid molecular species and mol% is the composition percentage of individual lipid molecular species (Su et al. 2009). Quantitation of GB and choline content by HPLC–MS GB and choline contents were measured according to the method in Grieve and Grattan (1983), Koc et al. (2002) and Nuccio et al. (1998) with modification. The standard stock solution of GB (Sigma, No. 61962) and choline chloride (Sigma, No. C7017) was prepared by dissolving in distilled water in a 25-ml flask. The working standard solutions of 400–0.39 µmol/l choline chloride and betaine derivative were made by appropriate dilution of the stock solution with distilled water. Frozen leaf tissues (0.2 g of fresh weight) were extracted in 5 ml of a mixture of methanol/chloroform/water (12:5:3, v/v/v) for overnight in dark. All samples were centrifuged at 12,000 rpm for 10 min, and the supernatant was diluted 100× with distilled water and a portion (10 μl) of this final supernatant was injected into HPLC. HPLC–UV/Vis–MS (Agilent 1100 series LC/MSD trap, Waldbronn, Germany) was used to separate and quantify betaine and choline in leaf extracts. For HPLC, the mobile phase A was 15 mmol/l acetic acid and the mobile phase B was 100% acetonitrile. The flow rate was 0.6 ml/min. The gradient were 5–7% B from 0 to 10 min, 7–15% B from 10 to 15 min, and 15–25% B from 15 to 20 min. The column was equilibrated with 5% B for 5 min between injections. The column was thermostatic at 25°C. The injection volume was 5 μl. The diode-array detector (DAD) was set at 210, 254 and 280 nm, with a spectrum scan range from 200 to 600 nm. For the MS part, the nebulizer was set at 40 psi and drying gas at 350°C with its flow rate at 12 l/min. Nitrogen was used as nebulizing and drying gas. Positive ionization mode was used, the collision energy termed as compound stability was set at 80% and the scan range was 50–800 m/z. The data were collected and calculated with software Agilent ChemStation A.08.03 and LC/MSD Trap Control 5.1. RNA extraction, cDNA synthesis and qRT-PCR analysis Total RNA was extracted from leaves using the TRIzol regent method and treated with Turbo DNA-free Kit (Ambion, Austin, TX, USA) to remove DNA. The cDNA was synthesized using 2 μg of total RNA by High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Grand Island, NY, USA). The cDNAs were diluted with RNase-free water to a concentration of 1:20 prior to the qRT-PCR analyses. qRT-PCRs were performed with the Step-One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the SYBR PCR Master Mix (Life Technologies, Grand Island, NY, USA). Relative gene expression levels were determined based on the 2−△△CT method (Livak and Schmittgen 2001). Primer sequences are presented in Supplementary Table S1. Each qRT-PCR analysis was performed in three biological replicates. Statistical analysis Data were subjected to the analysis of variances using a statistical program SPSS v20.0 (SPSS Inc., Chicago, IL, USA). Significant differences between treatments were determined using Fisher’s protected least significant difference (LSD) test at the 0.05 probability level. A P-value of <0.05 was considered a statistically significant difference. Supplementary Data Supplementary data are available at PCP online. Acknowledgments This work is supported by Rutgers Center of Turfgrass Science. The lipid analyses were performed at the Kansas Lipidomic Research Center Analytical Laboratory. We thank the China Scholarship Council (CSC) for scholarship (File No. 201806350077). We thank Stephanie Rossi for her assistance with plant management. Disclosures The authors have no conflicts of interest to declare. 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TI - Choline-Mediated Lipid Reprogramming as a Dominant Salt Tolerance Mechanism in Grass Species Lacking Glycine Betaine
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcaa116
DA - 2021-02-04
UR - https://www.deepdyve.com/lp/oxford-university-press/choline-mediated-lipid-reprogramming-as-a-dominant-salt-tolerance-tJUC3FjwYY
SP - 2018
EP - 2030
VL - 61
IS - 12
DP - DeepDyve
ER -