Melatonin Pretreatment Confers Heat Tolerance and Repression of Heat-Induced Senescence in Tomato Through the Modulation of ABA- and GA-Mediated Pathways

Abstract

Heat stress and abscisic acid (ABA) induce leaf senescence, whereas melatonin (MT) and gibberellins (GA) play critical roles in inhibiting leaf senescence. Recent research findings confirm that plant tolerance to diverse stresses is closely associated with foliage lifespan. However, the molecular mechanism underlying the signaling interaction of MT with GA and ABA regarding heat-induced leaf senescence largely remains undetermined. Herein, we investigated putative functions of melatonin in suppressing heat-induced leaf senescence in tomato and how ABA and GA coordinate with each other in the presence of MT. Tomato seedlings were pretreated with 100 μM MT or water and exposed to high temperature (38/28°C) for 5 days (d). Heat stress significantly accelerated senescence, damage to the photosystem and upregulation of reactive oxygen species (ROS), generating RBOH gene expression. Melatonin treatment markedly attenuated heat-induced leaf senescence, as reflected by reduced leaf yellowing, an increased Fv/Fm ratio, and reduced ROS production. The Rbohs gene, chlorophyll catabolic genes, and senescence-associated gene expression levels were significantly suppressed by MT addition. Exogenous application of MT elevated the endogenous MT and GA contents but reduced the ABA content in high-temperature-exposed plants. However, the GA and ABA contents were inhibited by paclobutrazol (PCB, a GA biosynthesis inhibitor) and sodium tungstate (ST, an ABA biosynthesis inhibitor) treatment. MT-induced heat tolerance was compromised in both inhibitor-treated plants. The transcript abundance of ABA biosynthesis and signaling genes was repressed; however, the biosynthesis genes MT and GA were upregulated in MT-treated plants. Moreover, GA signaling suppressor and catabolic gene expression was inhibited, while ABA catabolic gene expression was upregulated by MT application. Taken together, MT-mediated suppression of heat-induced leaf senescence has collaborated with the activation of MT and GA biosynthesis and inhibition of ABA biosynthesis pathways in tomato.

Keywords: chlorophyll degradation; high temperature; leaf senescence; melatonin; tomato.

FIGURE 1

Exogenous application of melatonin (100 μM) delays heat induced (38/28°C for 5 days) leaf senescence in tomato. (A) Phenotypic appearance of tomato leaves during heat stress in presence or absence of melatonin treatment and (B) Chlorophyll fluorescence imaging with Fv/Fm of tomato leaves during heat stress in presence or absence of melatonin treatment.

FIGURE 2

Effects of exogenous melatonin treatment (100 μM) on senescence associated physiological attributes in tomato under heat stress (38/28°C for 5 days). (A) Changes of Fv/Fm value, (B) net photosynthetic rate (Pn), (C) stomatal conductance (Gs), (D–F) changes of Chlorophyll content, (G) relative electrolyte leakage, (H) malondialdehyde (MDA), (I) hydrogen per oxide (H₂O₂) content, (J–L) relative expression of Rbohs genes during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 3

Effects of exogenous melatonin treatment on the transcript abundance of (A–G) chlorophyll catabolic genes (RCCR, SGR1, SGR2, NYC, PAO, PPH, and NOL) and (H) senescence associated gene (SAG12) during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 4

Effects of exogenous melatonin treatment on the (A) endogenous melatonin content and (B–E) transcript abundance of melatonin biosynthesis genes (TDC, T5S, ASMT, and SNAT) during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 5

Effects of exogenous melatonin treatment on the (A) endogenous ABA content and (B–D) transcript abundance of ABA biosynthesis genes (NCED1, NCED2, and AAO3), (E,F) ABA signaling genes (ABI3 and ABI5), (G,H) ABA of catabolic genes (CYP707A1, and CYP707A2) during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 6

Effects of exogenous melatonin treatment on the (A) endogenous GA content and (B,C) transcript abundance of GA biosynthesis genes (GA20ox1 and GA20ox2), (D) GA signaling genes (GAI), (E,F) GA of catabolic genes (GA2ox1 and GA2ox2) during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 7

Effects of 1 mM sodium tungstate (ST, an ABA biosynthesis inhibitor) and 1 mM paclobutrazol (PCB, a GA biosynthesis inhibitor) on the (A) endogenous ABA and (B) endogenous GA content during heat stress with or without of melatonin treatment. Different letters denote the significant variations between the treatments and the average values were measured by Tukey’s Honestly Significant Difference (HSD) test at P < 0.05. Data represented as the mean ± standard error of triplicate biological replicates.

FIGURE 8

A probable mechanism of melatonin-mediated heat-induced leaf senescence resulting in coordination with ABA and GA biosynthesis and signaling pathway. The arrow denotes increases and bar denotes decreases.