Melatonin Improves Salt Tolerance in Tomato Seedlings by Enhancing Photosystem II Functionality and Calvin Cycle Activity

Abstract

Salt stress severely impairs photosynthesis and development in tomato seedlings. This study investigated the regulatory role of exogenous melatonin (MT) on photosynthetic performance under salt stress by determining chlorophyll content, chlorophyll a fluorescence parameters, Calvin cycle enzyme activities, and related gene expression. Results showed that salt stress significantly reduced chlorophyll content and impaired photosystem II (PSII) functionality, as evidenced by the increased minimum fluorescence (F_o) and decreased maximum quantum efficiency of PSII (F_v/F_m) and effective PSII quantum yield (Φ_PSII). MT application mitigated these negative effects, as reflected by higher F_v/F_m, increased chlorophyll content, and lower non-photochemical quenching (NPQ). In addition, MT-treated plants exhibited improved PSII electron transport and more efficient use of absorbed light energy, as shown by elevated Φ_PSII and qP values. These changes suggest improved PSII functional stability and reduced excess thermal energy dissipation. Furthermore, MT significantly enhanced both the activity and expression of key enzymes involved in the Calvin cycle, including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), Rubisco activase (RCA), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-1,6-bisphosphatase (FBPase), fructose-bisphosphate aldolase (FBA), transketolase (TK), and sedoheptulose-1,7-bisphosphatase (SBPase), thereby promoting carbon fixation and ribulose-1,5-bisphosphate (RuBP) regeneration under salt stress. Conversely, inhibition of endogenous MT synthesis by p-CPA exacerbated salt stress damage, further confirming MT's crucial role in salt tolerance. These findings demonstrate that exogenous MT enhances salt tolerance in tomato seedlings by simultaneously improving photosynthetic electron transport efficiency and upregulating the activity and gene expression of key Calvin cycle enzymes, thereby promoting the coordination between light reactions and carbon fixation processes. This study provides valuable insights into the comprehensive regulatory role of MT in maintaining photosynthetic performance under saline conditions.

Keywords: Calvin cycle; chlorophyll a fluorescence; melatonin; salt stress; tomato seedlings.

Figure 1

Effects of exogenous melatonin on the relative growth rate of tomato seedlings under salt stress. The relative growth rates of plant height (RGR_H) (A), stem diameter (RGR_C) (B), root dry weight (RGR_R) (C), and shoot dry weight (RGR_L) (D) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 2

Effects of exogenous melatonin on photosynthetic pigment content in tomato seedlings under salt stress. The contents of chlorophyll a (Chla) (A), chlorophyll b (Chlb) (B), carotenoids (Car) (C), and total chlorophyll (Total Chl) (D), the Chla/b ratio (E), and the Car/total Chl ratio (F) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 3

Effects of exogenous melatonin on dark-adapted chlorophyll a fluorescence parameters of tomato seedlings under salt stress. The initial fluorescence (F_o) (A), maximum fluorescence (F_m) (B), maximum quantum efficiency of photosystem II (F_v/F_m) (C), and electron transport activity (F_m/F_o) (D) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 4

Effects of exogenous melatonin on light-adapted chlorophyll a fluorescence parameters of tomato seedlings under salt stress. The antenna conversion efficiency (F_v′/F_m′) (A), the photochemical quenching coefficient (qP) (B), the actual photochemical efficiency of PSII (Φ_PSII) (C), and the non-photochemical quenching coefficient (NPQ) (D) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 5

Effects of exogenous melatonin on the distribution of excitation energy between PSI and PSII of tomato seedlings under salt stress. The PSII excitation energy pressure (1 − qP) (A), PSI excitation energy allocation coefficient (α) (B), the PSII excitation energy allocation coefficient (β) (C), and the imbalance deviation coefficient of excitation energy distribution between PSI and PSII (β/α − 1) (D) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 6

Effects of exogenous melatonin on the non-cyclic electron transport rate (J_F) (A), the photochemical reaction rate (P_rate) (B), the thermal dissipation rate (D_rate) (C), and the relative limitation of photosynthetic function (L_PFD) (D) of tomato seedlings under salt stress. Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 7

Effects of exogenous melatonin on the distribution of absorbed light energy in PSII of tomato seedlings under salt stress. The proportion of absorbed light energy allocated to photochemical reactions (P) (A), the energy dissipated through non-photochemical quenching at the P680 reaction center (Ex) (B), antenna thermal dissipation (D) (C), and P + Ex + D = 1 (D) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 8

Effects of exogenous melatonin on the activity of key enzymes in the Calvin cycle of tomato seedlings under salt stress. The initial Rubisco activity (A), total Rubisco activity (B), phosphoglycerate kinase (PGK) (C), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D), fructose-1,6-bisphosphatase (FBPase) (E), fructose-1,6-bisphosphate aldolase (FBA) (F), transketolase (TK) (G), sedoheptulose-1,7-bisphosphatase (SBPase) (H), and Rubisco activase (RCA) (I) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).

Figure 9

Effects of exogenous melatonin on the gene expression of key enzymes in the Calvin cycle of tomato seedlings under salt stress. The gene expression of RbcS (A), RbcL (B), PGK (C), GAPDH (D), FBPase (E), FBA (F), TK (G), SBPase (H), and RCA (I) were measured under different treatments: Control (normal conditions without salt stress), NaCl (100 mM NaCl treatment), NM (100 mM NaCl + 100 μM melatonin), NP (100 mM NaCl + 100 μM p-CPA, a melatonin synthesis inhibitor), and NMP (100 mM NaCl + 100 μM melatonin + 100 μM p-CPA). Different letters above the bars indicate significant differences between treatments (p < 0.05) based on one-way ANOVA with LSD multiple comparisons. Values represent means ± standard errors (SE) (n = 4).