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

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.

Valorization of Carrot Pomace: UVC Induced Accumulation of Antioxidant Phenolic Compounds

Carrot pomace is the main waste residue obtained during carrot juice extraction. Plant tissues respond to abiotic stresses (i.e., wounding stress and ultraviolet C (UVC) radiation) by accumulating bioactive compounds. Due to the mechanical damage occurring during juice extraction, carrot pomace undergoes extreme wounding stress. In this study, the effects of UVC light (11.8 W m−2, 0–120 min) and storage time (48 h, 25 °C) on the accumulation of phenolics compounds and the antioxidant activity (AOX) of carrot pomace were evaluated. Carrot pomace that was non-treated with UVC (control) showed a 709.5% increase in total phenolics at 48 h. A high correlation of AOX values against total phenolics (R2 = 0.87) was observed, indicating that phenolics were the main contributors to the AOX of the tissue. After UVC treatment, the pomace that was radiated for 120 min with UVC showed an increase (40.4%) in chlorogenic acid (CHA) content. At 24 h, protocatechuic acid and 3,5-dicaffeoylquinic acid, which were not detected before storage, showed accumulation by 166.5 mg/kg and 169.4 mg/kg, respectively, in UVC treated pomace. Chlorogenic acid showed the highest increase (143.6%) at 48 h in the control. Valorization of carrot pomace was achieved by increasing its concentration of antioxidant phenolics through UVC radiation.

Effects of Wounding Stress and Storage Temperature on the Accumulation of Chlorogenic Acid Isomers in Potatoes (Solanum tuberosum)

Wounding stress is an effective strategy to increase the content of bioactive compounds in horticultural crops. Potato tubers subjected to wounding stress accumulate chlorogenic acid (CGA) and CGA isomers (neo-CGA and crypto-CGA), which are phenolics that prevent and treat different chronic and degenerative diseases. In this study, the effects of wounding stress and storage temperature (10 °C and 20 °C for 168 h) on the accumulation of CGA isomers in potatoes were evaluated. Results indicated that CGA accumulation was favored when wounded potatoes were stored at 20 °C for 120 h, obtaining a 1923.1% higher concentration when compared with samples before storage. Furthermore, wounded potatoes stored at 10 °C for 120 h showed the highest neo-CGA increase in concentration (712.2%). Likewise, the highest crypto-CGA concentration (84.9% higher than control samples) was quantified in wounded potatoes stored at 20 °C for 144 h. Based on the results from both the present study and previous reports, a strategy that summarizes effective postharvest stress conditions that induce the accumulation of specific CGA isomers in potatoes is presented. The tissue with an increased content of bioactive compounds could be used as raw material to produce functional foods or could be subjected to downstream processing to produce dietary supplements.

Metabolic Profiling of Primary Metabolites and Galantamine Biosynthesis in Wounded Lycoris radiata Callus

Plants are continuously exposed to abiotic and biotic factors that lead to wounding stress. Different plants exhibit diverse defense mechanisms through which various important metabolites are synthesized. Humans can exploit these mechanisms to improve the efficacy of existing drugs and to develop new ones. Most previous studies have focused on the effects of wounding stress on the different plant parts, such as leaves, stems, and roots. To date, however, no study has investigated the accumulation of primary and galantamine content following the exposure of a callus to wounding stress. Therefore, in the present study, we exposed Lycoris radiata calli to wounding stress and assessed the expression levels of several genes involved in metabolic pathways at various time points (0, 3, 6, 12, 24, 48, 72, and 96 h of exposure). Furthermore, we quantify the primary and galantamine content using gas chromatography–time-of-flight mass spectrometry and the high-performance liquid chromatography qRT-PCR analysis of eight galantamine pathway genes (LrPAL-2, LrPAL-3, LrC4H-2, LrC3H, LrTYDC2, LrN4OMT, LrNNR, and LrCYP96T) revealed that seven genes, except LrN4OMT, were significantly expressed following exposure to wounding stress. Galantamine contents of calli after 3, 6, 12, 24, 48, 72, and 96 h of exposure were respectively 2.5, 2.5, 3.5, 3.5, 5.0, 5.0, and 8.5 times higher than that after 0 h of exposure. Furthermore, a total of 48 hydrophilic metabolites were detected in the 0 h exposed callus and 96 h exposed callus using GC-TOFMS. In particular, a strong positive correlation between galantamine and initial precursors, such as phenylalanine and tyrosine, was observed.

Modeling gene regulation in response to wounding: temporal variations, hormonal variations, and specialized metabolism pathways induced by wounding

Plants respond to wounding stress by changing gene expression patterns and inducing jasmonic acid (JA), as well as other plant hormones. This includes activating some specialized metabolism pathways, including the glucosinolate pathways, in the case of Arabidopsis thaliana. We model how these responses are regulated by using machine learning to incorporate putative cis-regulatory elements (pCREs), known transcription factor binding sites from literature, in-vitro DNA affinity purification sequencing (DAP-seq) and DNase I hypersensitive sites to predict gene expression for genes clustered by their wound response using machine learning. We found temporal patterns where regulatory sites and regions of open chromatin differed between clusters of genes up-regulated at early and late wounding time points as well as clusters where JA response was induced relative to clusters where JA response was not induced. Overall, we identified pCREs that improved model predictions of expression clusters over known binding sites. We discovered 4,255 pCREs related to wound response at different time points and 2,569 pCREs related to differences between JA-induced and non-JA induced wound response. In addition, pCREs found to be important at different wounding time points were mapped to the promoters of genes in a glucosinolate biosynthesis pathway indicating regulation of this pathway under wounding stress. Finally, we experimentally validated a predicted cis-regulatory element, CCGCGT, showing that knock-out via CRISPR-Cas9 reduces gene expression in response to wounding.