Melatonin-Stimulated Triacylglycerol Breakdown and Energy Turnover under Salinity Stress Contributes to the Maintenance of Plasma Membrane H+-ATPase Activity and K+/Na+ Homeostasis in Sweet Potato

Abstract

Melatonin (MT) is a multifunctional molecule in animals and plants and is involved in defense against salinity stress in various plant species. In this study, MT pretreatment was simultaneously applied to the roots and leaves of sweet potato seedlings [Ipomoea batatas (L.) Lam.], which is an important food and industry crop worldwide, followed by treatment of 150 mM NaCl. The roles of MT in mediating K⁺/Na⁺ homeostasis and lipid metabolism in salinized sweet potato were investigated. Exogenous MT enhanced the resistance to NaCl and improved K⁺/Na⁺ homeostasis in sweet potato seedlings as indicated by the low reduced K⁺ content in tissues and low accumulation of Na⁺ content in the shoot. Electrophysiological experiments revealed that exogenous MT significantly suppressed NaCl-induced K⁺ efflux in sweet potato roots and mesophyll tissues. Further experiments showed that MT enhanced the plasma membrane (PM) H⁺-ATPase activity and intracellular adenosine triphosphate (ATP) level in the roots and leaves of salinized sweet potato. Lipidomic profiling revealed that exogenous MT completely prevented salt-induced triacylglycerol (TAG) accumulation in the leaves. In addition, MT upregulated the expression of genes related to TAG breakdown, fatty acid (FA) β-oxidation, and energy turnover. Chemical inhibition of the β-oxidation pathway led to drastic accumulation of lipid droplets in the vegetative tissues of NaCl-stressed sweet potato and simultaneously disrupted the MT-stimulated energy state, PM H⁺-ATPase activity, and K⁺/Na⁺ homeostasis. Results revealed that exogenous MT stimulated TAG breakdown, FA β-oxidation, and energy turnover under salinity conditions, thereby contributing to the maintenance of PM H⁺-ATPase activity and K⁺/Na⁺ homeostasis in sweet potato.

Keywords: K+/Na+ homeostasis; PM H+–ATPase; fatty acid β-oxidation; melatonin; sweet potato; triacylglycerol.

FIGURE 1

Effects of MT on the K⁺, Na⁺, Ca²⁺, and Mg²⁺ contents in NaCl-stressed or non-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT for 3 days before being subjected to the 150 mM NaCl stress. K⁺, Na⁺, Ca²⁺, and Mg²⁺ contents were analyzed after 7 days of salinity treatment. Columns represent the means of independent measurements of five individual plants per treatment, and bars represent the standard error of the mean. Columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 2

Effects of MT pretreatment on the salt shock-triggered transient K⁺ and H⁺ flux in the root and mesophyll tissue of Xu 32. (A,C) Transient K⁺ flux upon 150 mM NaCl shock in the root apex region (measured at 500 μm from the tip) and mature region (measured at 15 mm from the tip) of MT-pretreated (0.5 and 1.0 μM) or non-pretreated seedlings. (B,D) Transient H⁺ flux. (A–D) Each point represents the mean of eight roots at minimum collected from four individual plants. (E,F) Columns show the mean K⁺ and H⁺ flux during the period of NaCl shock (A–D, approximately 30 min). Different letters denote significant differences at P < 0.05. (G,H) Transient kinetics of K⁺ and H⁺ flux upon 150 mM NaCl shock in mesophyll tissue of MT-pretreated (100 μM) or non-pretreated seedlings. Each point represents the mean of a minimum of eight replicates. Inserted columns show the mean K⁺ and H⁺ flux during the period of NaCl shock (approximately 30 min). Different letters denote significant differences at P < 0.05.

FIGURE 3

Effects of MT on steady-state fluxes of K⁺, H⁺, and Na⁺ in root different regions of NaCl-stressed or non-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT or 1.0 μM (root) +100 μM (leaf) MT for 3 days and then subjected to the 150 mM NaCl stress. The steady-state fluxes at the root apex (500–3000 μm from the tip) and mature region (10–15 mm from the tip) were measured after 1, 3, and 5 days of NaCl treatment. Each column is the mean of a minimum of 10 roots collected from five individual seedlings, and bars represent the standard errors of the mean. Columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 4

Effects of MT on the PM H⁺–ATPase activity and ATP level in the root and leaf tissues of NaCl-stressed or non-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT for 3 days and then subjected to the 150 mM NaCl stress. (A,B) H⁺ pumping activity in PM vesicles purified from the root and leaf tissues of Xu 32 after 5 and 15 days of NaCl treatment. (C,D) ATP hydrolysis activity. (A–D) Each column is the mean of three independent experiments. (E,F) Relative ATP level. Each column is the mean of six individual plants. Columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 5

Effects of MT on the leaf lipidome in NaCl-stressed or non-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT for 3 days and then subjected to 150 mM NaCl stress. The mature leaves were collected at the same position after 7 days of NaCl treatment for lipidomic profiling. (A) Total amount of lipids in various head group classes. (B–J) Abundance of lipid molecular species of nine main lipid classes. Each column is the mean of five repetitions, and columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 6

Effects of MT on the expression level of genes related to the TAG synthesis and breakdown, FA β-oxidation, and energy turnover in the leaves of NaCl-stressed or non-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT for 3 days and then subjected to 150 mM NaCl stress. The mature leaves were collected at the same position after 1, 4, and 7 days of NaCl treatment. Each column is the mean of four replications, and columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 7

DMP reversed the effects of MT on the TAG accumulation, K⁺ and Na⁺ content, PM H⁺–ATPase activity, and cellular ATP level in NaCl-stressed Xu 32. Xu 32 seedlings were pretreated with 0.5 μM (root) + 100 μM (leaf) MT for 3 days and then subjected to NaCl (150 mM) plus DMP (25 μM) treatment. (A,B) Mature leaves and fine roots were collected from various treatment groups (5 days after treatment), stained with BODIPY 493/503 and then observed under a fluorescence microscope. For each treatment, a minimum of 10 root segments and leaf disks from four individual plants was observed. Representative images showing the alteration of lipid droplet accumulation. (C–F) Na⁺ and K⁺ content in the leaves (C,E) and roots (D,F) of Xu 32 under various treatment conditions (7 days after treatment). Columns represent the means of five repetitions, whereas bars represent the standard error of the mean. (G–J) H⁺ pumping and ATP hydrolysis activity in PM vesicles purified from the leaves (G,I) and roots (H,J) of Xu 32 under different conditions (5 days after treatment). Each column is the mean of three independent experiments. (K,L) Relative ATP level in the leaves (K) and roots (L) of Xu 32 under different conditions (5 days after treatment). Each column is the mean of six repetitions. Columns labeled with different letters indicate significant difference at P < 0.05.

FIGURE 8

Schematic model showing the mediation of MT on salt tolerance in sweet potato. In this model, MT recovers the inhibitory effects of salt stress on the expression of SDP1 and lipase activity and accelerates FA release from TAG. Thus, several free FAs with appropriate acyl chain are transported to the peroxisome for β-oxidation. The resulting acetyl-CoA is used for catalyzing the production of succinates and citrates by isocitrate lyase and citrate synthase. Then, succinate and citrate enter the mitochondria for the TCA cycle. Thus, abundant ATPs were generated for driving the activity of H⁺–ATPase across the PM and tonoplast. Therefore, the increased PM H⁺–ATPase activity blocks the K⁺ efflux via depolarization-activated K⁺ outward channels. In addition, the Na⁺/H⁺ antiport activity across the PM and tonoplast is fuelled by the generated proton motive force. Finally, the K⁺/Na⁺ homeostasis is maintained under salinity stress.