Diethyl aminoethyl hexanoate ameliorates salt tolerance associated with ion transport, osmotic adjustment, and metabolite reprograming in white clover

Abstract

Background: Soil salinization is a serious environmental hazard, limiting plant growth and production in different agro-ecological zones worldwide. Diethyl aminoethyl hexanoate (DA-6) as an essential plant growth regulator (PGR) exhibits a beneficial role in improving crop growth and stress tolerance. However, the DA-6-regulated effect and mechanism of salt tolerance in plants are still not fully understood. The objective of current study was to disclose salt tolerance induced by DA-6 in relation to changes in water and redox balance, photosynthetic function, ionic homeostasis, and organic metabolites reprogramming in white clover (Trifolium repens).

Results: A prolonged duration of salt stress caused water loss, impaired photosynthetic function, and oxidative injury to plants. However, foliar application of DA-6 significantly improved osmotic adjustment (OA), photochemical efficiency, and cell membrane stability under salt stress. In addition, high salinity induced massive accumulation of sodium (Na), but decreased accumulation of potassium (K) in leaves and roots of all plants. DA-6-treated plants demonstrated significantly higher transcript levels of genes involved in uptake and transport of Na and K such as VP1, HKT8, SOS1, NHX2, NHX6, and SKOR in leaves as well as VP1, HKT1, HKT8, H⁺-ATPase, TPK5, SOS1, NHX2, and SKOR in roots. Metabolomics analysis further illustrated that DA-6 primarily induced the accumulation of glucuronic acid, hexanoic acid, linolenic acid, arachidonic acid, inosose, erythrulose, galactopyranose, talopyranose, urea, 1-monopalmitin, glycerol monostearate, campesterol, stigmasterol, and alanine.

Conclusions: The DA-6 significantly up-regulated transcript levels of multiple genes associated with increased Na⁺ compartmentalization in vacuoles and Na⁺ sequestration in roots to reduce Na⁺ transport to photosynthetic organs, thereby maintaining Na⁺ homeostasis under salt stress. The accumulation of many organic metabolites induced by the DA-6 could be attributed to enhanced cell wall and membrane structural stability and functionality, OA, antioxidant defense, and downstream signal transduction in leaves under salt stress. The present study provides a deep insight about the synergistic role of DA-6 in salt tolerance of white clover.

Keywords: Gene expression; Ionic homeostasis; Metabolomics; Organic acids; SOS pathway; Sugars.

Fig. 1

Changes in (A) relative water content (RWC) and (B) osmotic potential (OP) in leaves of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 2

Changes in (A) total chlorophyll (Chl) content, (B) Chl a content, (C) Chl b content, (D) Chl a/b ratio, (E) the ratio of variable to maximal fluorescence (Fv/Fm), and (F) performance index on an absorption basis (PIABS) in leaves of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 3

Changes in (A) electrolyte leakage (EL), (B) superoxide anion (O₂^.−) content, (C) hydrogen peroxide (H₂O₂) content, and (D) malondialdehyde (MDA) content in leaves of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 4

Changes in (A) sodium ion (Na⁺) content, (B) potassium ion (K⁺) content, and (C) K⁺/Na⁺ ratio in leaves and roots of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 5

Changes in relative expression levels of genes in (A) leaves and (B) roots of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 6

Effects of exogenous application of DA-6 on changes in (A) heat map of 58 metabolites, (B) percentage of down-regulated, up-regulated, and unchanged metabolites, and (C) relative contents of organic acids, sugars, alcohols, and other metabolites in leaves of white clover plants under normal and saline conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 7

Changes in (A) and (B) relative quantity of sugars and (C) sugar alcohols in leaves of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 8

Changes in (A) and (B) relative quantity of organic acids and (C) other metabolites in leaves of white clover influenced by DA-6 application under normal and salt stress conditions. Different letters represent significant difference among different treatments (n = 4, and p ≤ 0.05). C, control; C + DA-6, control pretreated with DA-6; S, salt stress; S + DA-6, salt-affected plants pretreated with DA-6

Fig. 9

Integrative pathways were mediated by foliar DA-6 application in white clover plants under salt stress. Red, green, or white words indicate significant increases, decreases, or no changes, respectively