Saline-alkali tolerant plants show excellent potentials for preventing soil salinization, improving the ecological environment, and providing live conditions for the other plants that have lower tolerance to saline-alkali stress . Emerging studies have focused on biological responses of saline-alkali tolerant plant, aiming to decipher saline-alkali tolerant mechanisms. For example, Chenopodium quinoa Wild. has been used to investigate the genotype-dependent variability in salinity responses from morphological, physiological, cellular, and molecular aspects . However, these plant tolerance or defense studies are mainly conducted by indoor control [16, 22, 23]. The application of indoor control cannot fully reflect the real responses of plants to saline-alkali stress as plants survive from saline-alkali soil usually undergo a long-term adaptation and evolution with habitat. In the current study, we measured the expressions of metabolites in S. salsa and P. tenuiflora, which survive in saline-alkali soil, using GC-MS and LC-qTOF-MS and demonstrated diverse metabolites with varied intensities in S. salsa and P. tenuiflora.
Many sugars have been identified as regulatory components in the control of glycolytic flux in a variety of stress survival strategies . They not only act as readily available energy source for plant growth under stress, but function as osmoprotectants to maintain osmotic balance and stabilize macromolecules . Soluble sugars provide adaptive buffer for plants under saline-alkali stress and play an important role in regulating osmotic pressure [25, 26]. In our studies, many soluble sugars, including sorbose, fucose, and D-talose, are highly expressed. They have the ability to balance osmotic pressure and protect the biological structures of plants from desiccation damage [27, 28]. Notably, many metabolites in glycolysis/gluconeogenesis pathways were found to be significantly accumulated in P. tenuiflora, indicating that the production of downstream products through metabolic flux from these pathways is essential for salt-alkali tolerance. Therefore, it is likely that P. tenuiflora can regulate the central metabolism by effectively utilizing carbon, accumulating carbon assimilation production, and providing more material and energy to promote the tolerance against salt-alkali stress.
Nitrogen metabolism has been reported to be strongly interconnected with carbon metabolism . Sufficient carbon skeleton source and energy supply are important for the assimilation of nitrogen and the synthesis of amino acids . Plants respond to high salinity by limiting protein synthesis, promoting protein degradation, and changing amino acid compositions [31, 32]. Here, significant enrichment of amino acids was discovered in S. salsa. Therefore, we speculate that S. salsa uses different salt-alkali response strategy as P. tenuiflora. In P. tenuiflora, a large portion of carbon influx to sugars while only amino acids isoleucine, norleucine, and aspartatic acid were highly accumulated (Figure. 3a). Isoleucine and norleucine can improve salt resistance and maintain the metabolic and osmotic homeostasis under stress . Aspartic acid can act as an immediate donor of amino groups for the synthesis of other amino acids . Many other amino acids, including glutamine, proline, alanine, tyrosine, ornithine, and 3-hydroxynorvaline, were identified to be significantly accumulated in S. salsa. Glutamine has an elevated nitrogen-to-carbon ratio and can use limited carbon skeletons to response to environmental stresses [33, 34]. Proline is generally considered as an osmotic regulator and an active oxygen scavenger in response to high salinity . Like molecular chaperone, proline can form a protective film . Proline are mainly produced by glutamate synthesis pathway and ornithine synthesis pathway . Activation of ornithine synthesis pathway also plays a vital role in improving plant salt tolerance. Noteworthy, aminobutyric acid, which is involved in various stress response and defense mechanisms, is also considerably accumulated in S. salsa. Aminobutyric acid can maintain carbon and nitrogen balance, protect plants from oxidative stress, and regulate the pH value of cytoplasmic. These differentially regulated amino acids help S. salsa to survive under saline-alkali stress.
Alcohols help to reserve available water in plants and thus are considered as essential osmotic regulators. In our current study, accumulated alcohols in the aboveground part of S. salsa were discovered (Fig. 5). These alcohols benefit the maintenance of osmotic pressure balance in cytoplasm and contribute to the regulation of water loss . In addition, alcohols work as natural scavengers of salinity-induced reactive oxygen species and protect the biomolecules against oxidative damage .
The roles of soluble sugars, alcohols, and amino acids in resisting saline-alkali stress have been well acknowledged. Our current study revealed that a large proportion of differently expressed metabolizes were acids, implying the potential involvement of acids in plant protection. Acids can enhance plant stress resistance and stabilize intracellular pH . We found that many acids were accumulated in the aboveground part of S. salsa. These acids may help to maintain ionic balance by neutralizing alkali and excess toxic ions. They can also affect the fluidity and hydrophobicity of cell membrane, which is crucial for cell membrane activity maintenance and saline-alkali stress defense . Notably, some acids, such as nonanoic acid methyl ester, methyl hexadecanoate, and phenylacetic acid, have obvious flavors and are volatile. Secretion of these acids may affect the surrounding environment and influence soil composition (Table 2). Volatile substances are also communication factors that contribute to plant defense and reproduction . Therefore, these volatile substances may improve soil properties via signal transmission and communication. The pioneer role of S. salsa may be partially attributed to the successful secretion of these allelopathic compounds under saline-alkali stress.
Our GC-MS results showed that gallic acid, vanillic acid, protocatechuic acid, and catechol, acids subordinated to phenolic compounds, were obviously accumulated. These compounds are secondary metabolites that originate from phenylalanine metabolism [20, 42, 43]. Gallic acid and protocatechuic acid are the precursors of tannin, which can affect plant thickness and reduce water evaporation . Moreover, a larger number of other phenolic compounds were investigated. Bioactive phenolic compounds are important biofactories of plants under stress [44, 45]. These compounds are mainly divided into the benzoid acid derivatives with C6C1 carbon skeleton (C6C1-compounds), the hydroxycinnamic acid derivatives with C6C3 carbon skeleton (C6C3-compounds), and the flavonoids with C6C3C6 carbon skeleton (C6C3C6-compounds). Our results demonstrated the enrichment of C6C1-compounds in S. salsa as well as enrichment of C6C3- and C6C3C6-compounds in P. tenuiflora (Figure. 5). C6C1-compounds, usually induced by biotic elicitors, are signaling molecules that defense stress. C6C3C6-compounds are flavonoids that can directly enhance the chemical defense of plants and help plants adapting to their environments . Significantly accumulation of phenolic compounds in S. salsa and P. tenuiflora may thus benefit their saline-alkali tolerance.