Understanding the Biosynthesis of Volatile Phenylpropenes with Multifaceted Values (2024)

Non Technical Summary
Natural products or phytochemicals made by plants have valuable functions as food ingredients and pharmaceuticals, and in industrial production. This project investigates a special class of plant natural products that occurs in carrots and in related aromatic herbs such as fennel and parsley as well as in spices such as nutmeg. These phytochemicals, also known as phenylpropenes, have been valued by humans for centuries for their flavoring, antimicrobial, insecticidal, and medicinal attributes but are also recognized for their toxic and psychoactive effects. The proposed research will unravel the formation of these compounds and unlock their vast potential for agricultural, pharmaceutical, and biotechnological applications while providing genetic information to control their levels in spices and food crops.The research project will use genomic, biochemical, and analytical techniques to identify the genes involved in the biosynthesis of phenylpropene natural products in carrot and related herbs as well as in nutmeg. Gene editing will be applied to minimize bitterness caused by the phenylpropene compounds in carrot roots, and engineering approaches will be employed to enhance the formation of the compounds in carrot leaves as natural defenses against severe carrot pests. Outcomes of the project will allow crop improvement by targeted breeding of carrot and biotechnological transfer to related crops and spices to better control phenylpropene levels. The work will further set the stage for the sustainable biotechnological production of these compounds in the development of pharmaceuticals and as critical chemical starters for high value industrial products.

Goals / Objectives
Phenylpropenes are common volatile plant natural products with diverse effects and multiple functions. They are common ingredients of aromatic herbs and spices and known to have antimicrobial and insecticidal activities. Their accumulation has been associated with bitterness and psychoactive effects; on the other hand, phenylpropenes represent natural products with pharmacological and therapeutic potential and are valuable precursors in the industrial production of fragrances and insecticide enhancers.Despite the widespread use of phenylpropenes, especially the group of methylenedioxy phenylpropenes, in crops of the Apiaceae family such as carrot and food spices such as nutmeg, the biosynthesis of these compounds remains understudied. The long term goals of this project are to a) provide essential genetic information for regulating and controlling the concentrations of methylenedioxy phenylpropenes in the roots of carrot and in spices such as nutmeg, b) (re)introduce the insecticidal activities of these compounds, particularly in the foliage of carrots and other crops, and c) enable biotechnological production of the methylenedioxy phenylpropene compounds myristicin and its precursor safrole for controlled industrial applications and potential pharmacological advancements.The specific objectives of the proposal are to:(1) Elucidate the enzymatic pathway in the biosynthesis of the methylenedioxy phenylpropene compounds myristicin and apiol in carrot and select herbs of the Apiaceae family(2) Modify myristicin accumulation in carrot roots and foliage to reduce root bitterness and potentially increase leaf resistance against carrot psylla(3) Determine key initial enzymatic steps of methylenedioxy phenylpropene biosynthesis in nutmeg as a representative spice in the Magnoliids

Project Methods
Experimental/Scientific Methods:Objective 1A functional genomics approach will be used to identify the unknown genes and enzymes in the biosynthetic pathway of myristicin and apiol in carrot and related species in the Apiaceae family. Since two potential pathways have been proposed for the formation of myristicin in carrot, we will initiate our studies by testing the chemical conversion of biosynthetic precursors and potential intermediates in crude protein extracts of wild carrot species in which myristicn and apiol have been detected. We will then begin the functional characterization of cytochrome P450 monooxygenase (CYP) and O-methyltransferase genes, which have been predicted as biosynthetic gene candidates based on differential gene expression analysis of wild carrot leaf and root tissues. Since CYP genes comprise large gene families, we will support our gene discovery approach with an in silico co-expression network analysis, which will be conducted together with collaborator Jennifer Wisecaver (Purdue University). To perform this analysis, additional comparative transcriptomes will be generated considering tissue-, developmental-, and hormone-induced differences in myristicin/apiol accumulation and gene expression in cultivated carrot and other wild carrot species. To identify hom*ologous biosynthetic genes in parsley and fennel in the Apiaceae family, RNA-seq will be performed with myristicin and apiol-accumulating leaf and root tissue followed by de novo transcriptome assembly. All gene candidates will be functionally characterized by heterologous expression in Nicotiana benthamiana and enzyme assays with leaf extracts or leaf disks using different proposed substrates. Alternative characterizations will be performed with recombinant enzymes expressed in yeast (for CYPs) and E. coli (for O-methyltransferases). Enzymatic products will be analyzed by gas chromatography-mass spectrometry (GC-MS).Objective 2Modification of myristicin accumulation in carrot roots and foliage will be conducted by gene knockout and overexpression experiments. Knockout of myristicin biosynthetic genes will be performed via CRISPR/Cas9 (gene editing) protocols established in the lab of Co-PD Simon. Roots of carrot plants in the T0 generation will be harvested and exposed to ethylene treatment and measured for reduced myristicin levels and the modification of other phenylpropanoid volatiles via extraction and GC-MS analysis.Gene overexpression will be conducted using a eugenol O-methyltransferase specific promoter for leaf-specific accumulation or the CaMV 35S promoter (constitutive expression). Gene expression will be performed using the Phytobrick plasmid system and Golden Gate Assembly, and Agrobacterium-mediated plant transformation will follow established protocols. Overexpression lines will be evaluated at the T0 stage between 180-300 days after planting. Plants will be tested for gene expression followed by myristicin analysis in leaves and roots.Objective 3To determine key initial enzymatic steps in eugenole and safrole biosynthesis in nutmeg, qualitative and quantitative phenylpropene analysis will first be conducted from different nutmeg tissues (leaves, flowers, maze, kernels) using GC-MS. Subsequently, RNA will be extracted from different nutmeg tissues for transcriptome and differential gene expression analysis. Candidate eugenol synthase and CYP genes that are selected in this analysis will be heterologously expressed and functionally characterized as described under Objective 1.Evaluation:Important steps as indicators of success for the outcome of the three objectives are1. Successful identification and characterization of carrot CYP and O-methyltransferase genes in the biosynthesis of myristicin and apiolMilestones:Year 1: Successful gene co-expression analysis and initial characterization of first gene candidatesYear 2: Functional characterization of additional candidate genes and identification of orthologs in parsley and fennel transcriptomesYear 3: Completion of gene functional characterization in carrot, parsley and fennel.2. Successful reduction of ethylene-induced levels of myristicin in carrot roots and accumulation of myristicin in carrot leaf tissueMilestones:Year 1: Generation of T0 eugenol synthase CRISPR/Cas9 knockout linesYear 2: Phenylpropene profiling of T0/T1 eugenol synthase KO lines; initial CRISPR/Cas9 and overexpression of CYP and O-methyltransferase pathway genesYear 3: Partial or full completion of CRISPR/Cas9 and overexpression of CYP and OMT pathway genes, phenylpropene profiling of T0/T1 KO and overexpression lines3.Successful identification and characterization of key genes in eugenol and safrole biosynthesis in nutmegMilestones:Year 1: Completed phenylpropene analysis in nutmeg tissues and transcriptome sequencingYear 2: Partially completed functional characterization of eugenol and safrole synthase gene candidates; potential completion of gene co-expression network analysisYear 3: Completion of functional characterization of eugenol and safrole synthase genesEducational Methods/Efforts:The project will provide scientific training for one postdoctoral researcher and one Ph.D. student at Virginia Tech for one year and three years, respectively. In addition, we anticipate providing an undergraduate research experience for one to two students per year at Virginia Tech. Furthermore, the project will provide training for a postdoctoral researcher at the USDA ARS Wisconsin, Madison, and another postdoctoral researcher at the Newe Yaar Research Center, Agricultural Research Organization, Israel.