Carboxylic acid accumulation and secretion contribute to the alkali-stress tolerance of halophyte Leymus chinensis

Leymus chinensis is a dominant halophytic grass in alkalized grasslands of Northeast China. To explore the alkali-tolerance mechanism of L. chinensis, we applied a widely targeted metabolomic approach to analyze metabolic responses of its root exudates, root tissues and leaves under alkali-stress conditions. L. chinensis extensively secreted organic acids, phenolic acids, free fatty acids and other substances having -COOH or phosphate groups when grown under alkali-stress conditions. The buffering capacity of these secreted substances promoted pH regulation in the rhizosphere during responses to alkali stress. L. chinensis leaves exhibited enhanced accumulations of free fatty acids, lipids, amino acids, organic acids, phenolic acids and alkaloids, which play important roles in maintaining cell membrane stability, regulating osmotic pressure and providing substrates for the alkali-stress responses of roots. The accumulations of numerous flavonoids, saccharides and alcohols were extensively enhanced in the roots of L. chinensis, but rarely enhanced in the leaves, under alkali-stress conditions. Enhanced accumulations of flavonoids, saccharides and alcohols increased the removal of reactive oxygen species and alleviated oxygen damage caused by alkali stress. In this study, we revealed the metabolic response mechanisms of L. chinensis under alkali-stress conditions, emphasizing important roles for the accumulation and secretion of organic acids, amino acids, fatty acids and other substances in alkali tolerance.


Introduction
Soil alkalization is a global environmental concern, and it has caused severe problems in some areas of the world, such as Northeast China.The primary causes of soil alkalization in this area are the presence of Na 2 CO 3 and NaHCO 3 , resulting in a pH level above 10 in soils (Yin et al., 2017).More than 50% of the grasslands in this region are currently at risk of soil alkalization (Ran et al., 2020), and only a few alkali-tolerant halophytes can survive under such alkaline conditions.Understanding the mechanisms of alkali-tolerant halophytes will be useful for addressing the existing issue in alkalized grasslands.Alkali stress affects plants through osmotic regulation, ion toxicity and high pH, as well as resulting in indirect oxidative stress (Wang et al., 2012;Rao et al., 2013;Cao et al., 2022).High pH levels caused by alkali stress can significantly disrupt the pH stability of plant cells and compromise the integrity of cell membranes (Fang et al., 2021).Additionally, a high pH can lead to the precipitation of mineral nutrient ions in the rhizosphere soil (Wang et al., 2012).Therefore, for plants to survive in alkalized soil, it is crucial to adjust the pH value of the rhizosphere (Li et al., 2022).
Recently, increased attention has been given to alkali stress tolerance of plants (Cao et al., 2022), especially that of glycophytes, such as Arabidopsis (Yang et al., 2019), maize (Cao et al., 2020), rice, sorghum and millet (Zhang et al., 2023).In Arabidopsis and maize, H + -ATPases play important roles in alkali tolerance (Yang et al., 2019).In rice, sorghum, millet and maize (Zhang et al., 2023), an atypical G protein gene (AT1) affects the phosphorylation of aquaporins to modulate the distribution of H 2 O 2 , a form of reactive oxygen species caused by alkali stress.Halophytes and glycophytes may employ different mechanisms to resist salt or alkali stress (Rozema and Schat, 2013;Yuan et al., 2016).For example, leaves of halophytes can generally tolerate high Na + concentrations or mitigate Na + -related damage via salt-secreting glands (Flowers and Colmer, 2008), whereas glycophytes, such as wheat, can limit influxes of Na + into aboveground organs by extracting Na + into roots (Wang and Xia, 2018).Thus, gaining a comprehensive understanding of the metabolic regulatory mechanisms responsible for alkali tolerance in halophytes will aid in improving crop alkali tolerance (Himabindu et al., 2016).
Root secretions play important roles in plant-microorganism interactions and in surviving abiotic stress conditions.The regulation of acidic substances (e.g., organic acids) is particularly crucial in this process.Root secretions containing organic acids play important roles in the detoxification of plants under heavy-metal stress (Fu et al., 2018;Awasthi et al., 2019).Moreover, plants also secrete acidic substances in response to abiotic stresses like drought (Ulrich et al., 2022), alkali stress (Yang et al., 2010;Guo et al., 2018) and low phosphorus stress (Tiziani et al., 2020).Under alkali-stress conditions, plants need to regulate the pH value outside the roots to maintain their function.The secretion of organic acids by roots is strongly associated with the alkali tolerance of plants, and it plays a crucial role in regulating cell and rhizosphere pH levels (Xiang et al., 2019;Li et al., 2022).For example, alkali stress increases secretion rates of oxalate, tartrate, malate, acetate and citrate in grape plants (Guo et al., 2018), and it also elevates secretion rates of oxalate, formate, lactate and acetate in Chloris virgata (Yang et al., 2010).
Leymus chinensis is the dominant forage grass in alkalized grasslands of Northeast China.It exhibits a remarkable ability to survive even under alkali-stress condition of 175 mM Na 2 CO 3 (pH>11) (Chen et al., 2019).L. chinensis is an important forage grass because of its high productivity, high protein content and good palatability (Lin et al., 2016;Zhao et al., 2016;Chen et al., 2019).Its wide adaptability and economic value make it an ideal species for the restoration of alkaline lands in Northeast China.Although several studies have focused on physiological characteristics, metabolism and transcriptional changes of L. chinensis under alkali stress (Jin et al., 2008;Yan et al., 2022Yan et al., , 2023)), most of them are descriptive.There is a lack of research on the metabolic regulation of different organs and the root secretions of L. chinensis grown under alkali-stress conditions.Here, a widely targeted metabolome technology, using ultra highperformance liquid chromatography/stem mass spectroscopy (UPLC-MS/MS) and real standard samples, were used to qualify and quantify metabolites in leaves, roots and root exudates of L. chinensis under alkali-stress conditions.The metabolic regulatory mechanisms and root secretion response processes of L. chinensis were investigated under alkali stress.The findings of this research will improve our understanding of plant alkali tolerance and contribute to the restoration and exploration of alkaline lands.

Plant materials
The test organism used in this study was L. chinensis variety 'Jingmu-3', which was provided by the Heilongjiang Academy of Agricultural Sciences.It is not a nationally protected plant, and therefore, no special permission was required for its use in this research.

Plant cultivation
The experiment was conducted at Jilin Agricultural University (125.42°E,43.81°N), Changchun, China.L. chinensis seeds (8 per pot) were sown in pots filled with clean sand and cultured using Hoagland nutrient solution for 30 days before being exposed to alkali-stress treatments.The control group was cultured with Hoagland nutrient solution, whereas the alkali-stress group (8 plants/pot) was irrigated thoroughly once a day with Hoagland nutrient solution containing 180 mM alkaline salt (molar ratio NaHCO 3 : Na 2 CO 3 = 9:1, pH 8.7).All the pots were placed in a greenhouse (23-25°C day and 17-20°C night, 16 h light).After 2 days of treatment, the mature leaves and roots of L. chinensis (tillering stage) were harvested and stored at −80°C for a metabolomics analysis.The root exudates were collected using Li et al.'s method in which root exudates were collected by rinsing sand with distilled water (Li et al., 2022).The collected exudates were then stored at −80°C for a metabolomics analysis.Each treatment had three biological replicates, with each biological replicate being a pool of all the plants or root exudates from a pot.

Metabolomics analysis
Relative concentrations of metabolites were quantified using a widely targeted metabolomics approach based on local MS/MS data library constructed with authentic standards (Chen et al., 2013(Chen et al., , 2020)).A mixed sample of equal volume of all extracts was loaded into the QTRAP system to construct a MS2 spectral tag library.Retention time, m/z ratio and fragmentation information were used to identify each metabolite by matching to the in-house database MWDB (https://www.metware.cn),and all the identified metabolites were quantified using a MRM method (Chen et al., 2013(Chen et al., , 2020)).The full scan investigation of MS/MS data was continuously evaluated using Analyst 1.6.3software (AB Science, Waltham, MA, USA) to collect and trigger the acquisition of MS/ MS spectra.
A KEGG enrichment analysis was performed on 183 upregulated metabolites in the leaves.A total of 75 metabolic pathways were enriched, which included 14 biosynthesis of other secondary metabolites, 13 amino acid metabolism pathways, 12 carbohydrate metabolism pathways (e.g., fructose and mannose metabolism), 7 lipid metabolism pathways (e.g., fatty acid degradation) and 3 energy metabolism pathways (nitrogen metabolism, sulfur metabolism and carbon fixation in photosynthetic organisms).The upregulated metabolites in the leaves were significantly enriched in 2-oxocarboxylic acid metabolism, D-amino acid metabolism, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, tropane, piperidine and pyridine, alkaloid bio-synthesis, glucosinolate biosynthesis, valine, leucine and isoleucine biosynthesis, arginine and proline metabolism, cyanoamino acid metabolism, vitamin B6 metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, FIGURE 3 KEGG enrichment of upregulated metabolites in leaves of L. chinensis after expose to alkali stress.

Metabolic pathway analyses of roots and leaves
Leaves and roots enhance the biosynthesis of amino acids, other secondary metabolites and carbohydrates to support growth, and root secretions are increased to adapt to alkali stress.In the energy metabolism pathway, L. chinensis roots and leaves had increased levels of fumaric, L-malic and D-erythrose-4-phosphate, with the enhancement being greater in roots than in leaves.After expose to alkali stress, the levels of 2-aminoethanesulfonic acid and D-glucose were increased only in the roots, whereas L-cysteine, citric acid and glycerone phosphate showed increased levels only in the leaves (Supplementary Figure 5).In the amino acid metabolism pathway, levels of L-cysteine, L-ornithine, L-leucine, L-tyrosine, Ltryptophan, L-phenylalanine and L-isoleucine were increased in the leaves but were decreased in the roots.Additionally, the level of L-aspartate was lower in the leaves but higher in the roots, indicating greater accumulations of amino acids in the leaves (Supplementary Figure 6).In the carbohydrate metabolism pathway, levels of D-glucose, D-mannose, D-glucoronic acid, Dsorbitol, D-galactose, D-arabitol, ribitol, D-arabinose and D-xylonic acid were increased in the roots but decreased in the leaves.Additionally, sorbitol-6-phosphate was found to be decreased in the roots but increased in the leaves, indicating that the roots tended to accumulate more carbohydrates (Supplementary Figure 7).For the biosynthesis of secondary metabolites pathway, roots exhibited higher levels of phlorizin, apigenin, chrysin and syringetin compared with leaves.Levels of piperidine and phenylpyruvate, but not cadaverine, were lower in roots, whereas other secondary metabolites showed enhanced accumulations in roots (Supplementary Figure 8).Effects of alkali stress on the secretion rates of free fatty acids in L. chinensis.The levels of the metabolites were upregulated only in root exudates, not in roots or leaves.The asterisks (*) indicate significant differences between treatments.

FIGURE 6
Effects of alkali stress on secretion rates of organic acids in L. chinensis.The levels of the metabolites were upregulated only in root exudates, not in roots or leaves.The asterisks (*) indicate significant differences between treatments.

FIGURE 7
Effects of alkali stress on secretion rates of phenolic acids containing -COOH in L. chinensis.The levels of the metabolites were upregulated only in root exudates, not in roots or leaves.The asterisks (*) indicate significant differences between treatments.

FIGURE 8
Effects of alkali stress on accumulations of flavonoids in roots of L. chinensis.The levels of the metabolites were upregulated only in roots, not in root exudates or leaves.The asterisks (*) indicate significant differences between treatments.

FIGURE 9
Effects of alkali stress on accumulations of saccharides and alcohols in roots of L. chinensis.The levels of the metabolites were upregulated only in roots, not in root exudates or leaves.The asterisks (*) indicate significant differences between treatments.

Discussion
4.1 Alkali stress induced the secretion of phenolic acids, organic acids and fatty acids in L. chinensis High soil pH can lead to the precipitation of various mineral element ions surrounding roots, reducing their bioavailability (Turner et al., 2020).High pH caused by alkali stress also affects the root morphology and functions of plants, consequently impacting above-ground symptoms (Turner et al., 2020).Therefore, plants that survive under alkali-stress conditions must adjust the pH of their rhizospheres.The application of organic acids immediately lowers the soil pH (Rukshana et al., 2014), and plants, such as grape hybrid rootstocks, seabuckthorn, Chloris virgata, wheat and grapevine, can mitigate the damage caused by alkali stress through the secretion of organic acids (Chen et al., 2009;Yang et al., 2010;Guo et al., 2018;Xiang et al., 2019;Li et al., 2020).Our previous study revealed that alkali stress strongly inhibits photosynthesis and growth of L. chinensis, and it disturbed the ion balance (Wang et al., 2020).Here, our results indicated that L. chinensis extensively secreted organic acids, phenolic acids, free fatty acids and other substances containing a -COOH or phosphate group after expose to alkali stress.The extensive secretion of these Effects of alkali stress on accumulations of glycerol phospholipids (lipids) in leaves of L. chinensis.The levels of the metabolites were upregulated only in leaves, not in root exudates or roots.The asterisks (*) indicate significant differences between treatments.
substances having a buffering capacity should promote pH regulation in the rhizosphere during the response of L. chinensis to alkali stress, allowing L. chinensis to exhibit a strong resistance to alkali stress.Additionally, during responses to alkali stress, L. chinensis had enhanced secretion rates for many metabolites without a -COOH or H 2 PO 4 group, and this may play a crucial role in the interactions between L. chinensis roots and rhizosphere microorganisms that facilitate nutrient uptake.Alkali stress enhances the glycolysis level of L. chinensis roots by upregulating the expression of rate-limiting enzyme-encoding (6-phosphofructokinase and pyruvate kinase) genes (Wang et al., 2020).Glycolysis provides ATP, NADH, phosphoenolpyruvate and acetyl-CoA for metabolic processes and stress responses.Thus, enhanced glycolysis should support more energy and carbon sources for the alkali-stress responses of L. chinensis roots, including the accumulation and secretion of organic solutes.Li et al. also reported that alkali stress enhances the glycolysis level of halophyte Puccinellia tenuiflora roots by upregulating the expression of a rate-limiting enzyme-encoding (6phosphofructokinase) gene to relieve the damage caused by alkali stress (Li et al., 2022).This suggests that enhanced glycolysis by upregulating rate-limiting enzyme-encoding genes may be a common response of halophytes to alkali stress.Genetic modifications of the rate-limiting glycolysis-related enzymeencoding genes may be a promising strategy for improving the alkali tolerance of crop and forage grasses, and this should be investigated in depth.

Metabolic responses in roots and leaves
Alkali stress can strongly influence the metabolism of plants.For instance, grapevines subjected to alkali stress accumulate phenolic acids, flavonoids and alkaloids (Lu et al., 2023).Similarly, Cinnamomum bodinieri roots respond to alkali stress by accumulating alkaloids, flavonoids, N-acetyl-L-phenylalanine, lauric acid, palmitic acid, oxaloacetic acid and other substances to alleviate the negative effects of alkali stress (Han et al., 2023).Alkali stress triggers a noteworthy accumulation of nearly all free amino acids and ribose in mature wheat leaves, thereby enhancing tissue tolerance (Xiao et al., 2020).Similarly, the concentrations of amino acids, alkaloids, and organic acids in Lycium barbarum fruits are enhanced under salt-alkali stress (Liang et al., 2022).Hence, organic acids, amino acids and secondary metabolites significantly contribute to plant alkali-stress tolerance.Alkali stress specifically enhanced the accumulations of numerous flavonoids, saccharides and alcohols in the roots, but few in the leaves, of L. chinensis.Enhanced accumulations of flavonoids, saccharides and alcohols enables the more efficient removal of reactive oxygen species and the alleviation of oxygen damage caused by alkali stress.In addition, under alkali stress, L. chinensis leaves exhibited enhanced accumulations of free fatty acids, lipids, amino acids, organic acids, phenolic acids, and alkaloids, which should play important roles in maintaining cell membrane stability and osmotic regulation, as well as supporting the production of substrates for alkali-stress responses of the roots through long-distance transport.Additionally, there are 18 shared upregulated substances with a -COOH in the leaves and root exudates, but only 5 shared upregulated substances with a -COOH in the roots and root exudates.This indicated that leaves may be the main organs that provide carboxylic acid substances that are secreted by L. chinensis roots under alkali-stress conditions.The KEGG enrichment analysis revealed that the upregulated metabolites in both roots and leaves were associated with amino acid, secondary, carbohydrate and energy metabolism pathways.The enhancement Effects of alkali stress on accumulations of amino acids and amino acid derivatives in leaves of L. chinensis.The levels of the metabolites were upregulated only in leaves, not in root exudates or roots.The asterisks (*) indicate significant differences between treatments.
of amino acid, carbohydrate and energy metabolism pathways in the leaves and roots facilitates the alkali-stress responses of L. chinensis.Alkali stress upregulates the expression of many key genes, including phenylalanine ammonia-lyase (rate-limiting enzyme for phenol synthesis) gene, 4-coumarate-CoA ligase gene, flavonoid 3'-monooxygenase gene, flavonoid 3'-monooxygenase gene, cinnamoyl-CoA reductase gene, and flavanone 3dioxygenase gene involved in phenol (including phenolic acids and flavonoids) biosynthesis in leaves and roots of L. chinensis (Wang et al., 2020).These upregulated genes caused by alkali stress may enhance the synthesis of phenolic acids and flavonoids during responses of L. chinensis to alkali stress.Our results suggest that responses of plants to alkali stress require a complex network that coordinates numerous metabolic pathways, involving numerous genes.The genes (e.g., phenylalanine ammonia-lyase gene and 6phosphofructokinase gene) involved in the synthesis of key metabolites and energy metabolism may form key nodes of the alkali-stress response network, but upstream regulatory genes may be effective targets for the genetic improvement of plant alkali tolerance.Molecular regulatory mechanisms of alkali tolerance should be explored in crops or model plants, with an emphasis on the upstream regulatory genes for key metabolites and energy metabolism.Our study provides useful information for further research on the molecular regulatory mechanisms of plant alkali tolerance.

Conclusions
The extensive secretion of substances having a buffering capacity promoted pH regulation of the rhizosphere during responses of L. chinensis to alkali stress.In the roots of L. chinensis, the enhanced accumulations of flavonoids, saccharides and alcohols enabled the more effective removal of reactive oxygen species and the alleviation of oxygen damage caused by alkali stress.L. chinensis leaves exhibited enhanced accumulations of free fatty acids, lipids, amino acids, organic acids, phenolic acids and alkaloids, which should play important roles in maintaining cell membrane stability and osmotic regulation, as well as supporting the production of substrates for alkali-stress responses in the roots.
FIGURE 1 Venn diagram showing the number of detected metabolites in root exudates, root tissues and leaf tissues of L. chinensis.(A) Comparison of all the identified root exudates from plants grown under control and alkali-stress conditions.(B) Comparison of the numbers of the detected metabolites in root exudates, root tissues and leaf tissues.(C) Comparison of the numbers of upregulated metabolites in root exudates, root tissues and leaf tissues.

FIGURE 11
FIGURE 11Effects of alkali stress on accumulations of alkaloids in leaves of L. chinensis.The levels of the metabolites were upregulated only in leaves, not in root exudates or roots.The asterisks (*) indicate significant differences between treatments.