Chemical Genetics Applied to Elucidate the Physiological Role of Stress-Signaling Molecules on the Wound-Induced Accumulation of Glucosinolates in Broccoli

Abstract

Wounding stress is an effective strategy to induce glucosinolate (GS) biosynthesis in broccoli. However, there is insufficient knowledge on the physiological and molecular mechanisms underlying this stress response. Herein, a chemical-genetic approach was applied to elucidate the role of jasmonic acid (JA), ethylene (ET), and reactive oxygen species (ROS) on the wound-induced biosynthesis of GS. Broccoli was processed into chops to induce wounding stress. Broccoli chops were treated with phenidone (PHEN) and diphenyleneiodonium chloride (DPI) as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) was applied as an inhibitor of ET action. Wounding stress induced the expression of genes related to the biosynthesis of indolic and aliphatic GS, which was correlated with the accumulation of GS and modulated by the inhibitors of signaling molecules applied. Results of gene expression analysis indicated that JA played a key role in the activation of most genes, followed by ROS. Furthermore, except for the CYP79B2 gene, PHEN and 1-MCP synergistically downregulated the expression of GS biosynthetic genes evaluated, showing that the interaction between JA and ET was fundamental to modulate GS biosynthesis. Results presented herein increased our knowledge of the physiological and molecular mechanisms governing the wound-induced biosynthesis of GS in broccoli.

Cross-Talk and Physiological Role of Jasmonic Acid, Ethylene, and Reactive Oxygen Species in Wound-Induced Phenolic Biosynthesis in Broccoli

“…The concentration of inhibitors applied (PHEN, 10 mM; 1-MCP, 2000 ppb; DPI, 317 μM) and the sampling time (21 h) were determined according to previous studies [ 5 , 14 , 21 ]. Likewise, the sampling time to evaluate the expression of genes related to the stress-signaling molecules’ biosynthesis (1 h), and primary and secondary metabolite production (9 h) was selected based on a previously reported transcriptome analysis that determined the differential expression of primary and secondary metabolism-related genes as early and late wound responses in broccoli [ 21 ].…”

“…As indicated in our previous reports using a chemical-genetics approach to evaluate the role of signaling molecules on the wound response in plants, two controls were used to determine the effect of inhibitors on the expression of genes and the accumulation of phenolics: chopped broccoli exposed to air and chopped broccoli dipped in distilled water [ 10 , 12 , 14 ]. Chopped broccoli exposed to air was used as the control for 1-MCP-treated samples, whereas chopped broccoli dipped in water was used as the control for samples treated with PHEN or DPI, either applied alone or in combination with each other.…”

“…Stress signaling molecule inhibitors (1-MCP, 2000 mg L −1 ; PHEN, 10 mM; DPI, 317 μM) were applied individually or in combination in broccoli chops, as previously described [ 12 , 13 , 14 ]. To determine the effect of inhibitors’ application on the expression of genes and the accumulation of secondary metabolites, two controls were used: chopped broccoli exposed to air (control for 1-MCP treatment) and chopped broccoli dipped in distilled water (control for DPI or PHEN treatments).…”

“…Then, eATP binds to adjacent unwounded cells and triggers the wound response through the biosynthesis of reactive oxygen species (ROS). At the same time, other signaling molecules such as ethylene (ET) and jasmonic acid (JA) are produced, also playing a significant role in the wound-induced activation of the primary and secondary metabolism of plants [ 12 , 13 , 14 ]. Although the role of these signaling molecules in the postharvest wound-induced activation of the primary and secondary metabolism of the plant cells has been previously reported for crops such as carrots [ 12 , 13 ], their role in the biosynthesis of phenolics in crops such as broccoli has been overlooked.…”

“…The use of small chemical compounds that inhibit stress-signaling pathways allows for the application of chemical-genetics approaches to study stress responses [ 15 ]. For instance, phenidone (PHEN) and diphenyleneiodonium chloride (DPI) are used as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) blocks ET action [ 13 , 14 , 16 , 17 , 18 , 19 , 20 ].…”