Progress 04/01/24 to 03/31/25

Outputs
Target Audience:Primary target audience: researchers in the general fields of specialty crops, specialty products, specialized metabolism, and breeding. Secondary target audience: entities involved in commercializing new specialty crop cultivars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student Jannelle Andrews (working toward Ph.D. degree) has been trained in basic techniques of molecular biology and biochemistry. How have the results been disseminated to communities of interest?Publications in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Assessing the specificity of cytochrome P450-dependent monooxygenases for the formation of different monoterpene alcohols and epoxides. We submitted a manuscript for publication (currently in revision) that documents the roles of severalcytochrome P450-dependent monooxygenases. We had initially planned to peform more work on identifying the determinants of regiospecificity in these enzymes but exciting results obtained as part of Objective 3 have led to a change of focus and we are not anticipating performing additional work on Objective 1. Objective 2. Investigating the specificity of (-)-trans-isopiperitenol dehydrogenases for diverse monoterpene alcohols as substrates. Our efforst during the next funding period will be focused on completing substrate specificity assays, including kinetic assays with selected substrates. In parallel, we will use structure-function analyses to identify amino acid residues with potential roles in conferring substrate specificity. We will then generate enzyme variants in which these residues have been exchanged to experimentalyl evaluate their roles. We are expecting to submit a manuscript reporting on the outcomes of these analyses during Q1, 2026. Objective 3. Evaluating the specificity of (+)-pulegone reductases for unsaturated monoterpene ketones as substrates. We are currently generating mutant genes of PulR that code for enzymes in which residues wit hpotentially critical roles in conferring substrate specificity have been exchanged. We are expecting to submit a manuscript reporting on our findings by the end of Q2, 2025. Objective 4. Demonstrating translational capabilities using knowledge-based approaches for breeding mints accumulating specialty oils. Building on the expansive data sets acquired for more than 70 populations from intra- and interspecific crosses between mint accessions as well as 10 F2 populations, we will begin integrating genetic sequencing and biochemical analyses to identify and chracterize candidate acetyltransferases responsible for the formation of terpinyl acetate and linalyl acetate, two important aroma volatiles. We expect to complete a manuscript reporting on the outcomes of these efforts during Q4, 2025.

Impacts
What was accomplished under these goals? Objective 1. Assessing the specificity of cytochrome P450-dependent monooxygenases for the formation of different monoterpene alcohols and epoxides. We completed a genome-wide association study of a mint population that segregates for essential oil composition. Cytochrome P450-dependent monooxygenases (CYPs) contribute critically to monoterpenoid essential oils at two branchpoints: Branchpoint 1): in the absence of a functional limonene 6-hydroxylase, C3-functionalized monoterpenoid dominate the profile, whereas in the presence of a fuinctinal limonene 6-hydroxylase, C6-functionalized monoterpenoid are predominant; Branchpoint 2): in the absence of a functional (-)-trans-isopiperitenone reductase in lines that accumulate C3-functionalized monoterpenoids, products derived from piperitenone, such as the CYP-derived piperitenone oxide, are the prinicpal constituetns in essential oils. These findings have been integrated into a manuscript that was submitted in 2024; we are currently working on the submission of a revised version. Objective 2. Investigating the specificity of (-)-trans-isopiperitenol dehydrogenases for diverse monoterpene alcohols as substrates. We have preliminary data on specific activities of peppermint (-)-trans-isopiperitenol dehydrogenase (ISPD) with 28 potential substrates (various monoterpenoid alcohols). During the next funding period, we will complete replicate analyses for these potential substrates and perform kinetic assays with selected substrates. Objective 3. Evaluating the specificity of (+)-pulegone reductases for unsaturated monoterpene ketones as substrates. The evluation of substrate specificity of peppermint (+)-pulegone reductase (PulR) has been completed. Follow-up structure-function analyses have allowed us to identify amino acid residues in the active site of PulR that might play important roles in conferring specificity. We are currently generating mutant genes of PulR that code for enzymes in which these potentially ciritical residues have been exchanged. This is a deviation from the original plan that has caused a delay in finishing up a manuscript. We are now expecting to submit a greatly enhanced manuscript by the end of Q2, 2025. Objective 4. Demonstrating translational capabilities using knowledge-based approaches for breeding mints accumulating specialty oils. A first manuscript reporting on the outcomes of our breeding efforts has been submitted and is currently under revision. The main accomplishment is the discovery of genes underlying the formation of piperitenone oxide. We are also in the process of characterizing gene candidates with elevance of teh formation of terpinyl acetate and linalyl acetate, two important aroma volatiles in mint.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Samuel C Talbot, Iovanna Pandelova, Bernd Markus Lange, Kelly J Vining, A first look at the genome structure of hexaploid Mitcham peppermint (Mentha � piperita L.), G3 Genes|Genomes|Genetics, Volume 14, Issue 12, December 2024, jkae195, https://doi.org/10.1093/g3journal/jkae195

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:Primary target audience: researchers in the general fields of specialty crops, specialty products, specialized metabolism, and breeding. Secondary target audience: entities involved in commercializing new specialty crop cultivars. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A visiting student (Summeyye Yilmaz) from the University of Bonn (Germany) wrote an M.Sc. thesis based on the work she had done as part of this project. She defended her thesis successfully in March, 2024. Summeyye is now applying for graduate school. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Assessing the specificity of cytochrome P450-dependent monooxygenases for the formation of different monoterpene alcohols and epoxides. Identify and characterize the gene coding for piperitenone monooxygenase and publish this finding. Continue experiments to assess the regiospecificity of different cytochrome P450-dependent monooxygenases (CYPs) by generating mutant genes that code for enzyme variants with altered active site residues and assaying these variants for changes in regiospecificity. The longer-term goal is to define theactive site residues that determine the regiospecificity of hydroxylation reactions with monoterpene substrates. Objective 2. Investigating the specificity of (-)-trans-isopiperitenol dehydrogenases for diverse monoterpene alcohols as substrates. Complete assays of (-)-trans-isopiperitenol dehydrogenase (ISPD) from peppermint with various monoterpene alcohols as potential substrates. Perform kinetic assays with selected substrates. Submit manuscript reporting on the outcomes of this sub-project. Continue crystallization of ISPD, with and without bound ligands, to hopefully obtain a crystal structure. Objective 3. Evaluating the specificity of (+)-pulegone reductases for unsaturated monoterpene ketones as substrates. Complete kinetic assays with selected substrates. Submit manuscript reporting on the outcomes of this sub-project. Begin assessing the functions of medium chain dehydrogenases/reductases (MDRs) with homology to pulegone reductase of peppermint across plant lineages. The longer-term goal is to assess the evolution of a pulegone reductase activity from ancestral MDRs. Objective 4. Demonstrating translational capabilities using knowledge-based approaches for breeding mints accumulating specialty oils. Continue the characterization of genes underlying the formation of terpinyl acetate. Begin with characterizing the genes underlying the formaiton of linalyl acetate. The aroma has been described as "floral, sweet and citric, and additionally as minty and slightly caraway-like". Continue the development of mint lines with novelty oils as specialty crops.

Impacts
What was accomplished under these goals? Objective 1. Assessing the specificity of cytochrome P450-dependent monooxygenases for the formation of different monoterpene alcohols and epoxides. Leaf glandular trichomes were isolated from several mint accessions and cultivars that were expected to express different cytochrome P450-dependent monooxygenase (CYP) genes, ultimately leading to the accumulation of different monoterpenoid products. Isolated glandular trichome cells were further processed to obtain microsomal membrane preparations. These were assayed for CYP activity with various substrates. Peppermint (Black Mitcham cultivar) was shown to exert limonene 3-hydroxylase and menthofuran synthase activity, while an activity for the conversion of piperitenone to piperitenone oxide was detected in horsemint accession CMEN 585. We are currently testing the function of candidate genes for the piperitenone oxidation activity using an E. coli-based heterologous expression system. Objective 2. Investigating the specificity of (-)-trans-isopiperitenol dehydrogenases for diverse monoterpene alcohols as substrates. We just started assaying (-)-trans-isopiperitenol dehydrogenase (ISPD) from peppermint with various monoterpene alcohols as potential substrates. We also initiated the crystallization of ISPD, with and without bound ligands, to hopefully obtain a crystal structure. Objective 3. Evaluating the specificity of (+)-pulegone reductases for unsaturated monoterpene ketones as substrates. We assayed (+)-pulegone reductase (PulR) from peppermint with > 40 unsaturated monoterpene ketones and structurally similar compounds as potential substrates. We are currently performing kinetic assays with selected substrates. A manuscript reporting on these efforts should be ready for submission in fall 2024. We also initiated the crystallization of PulR, with and without bound ligands, to hopefully obtain a crystal structure that is substantially improved over the currently available sturcture. Objective 4. Demonstrating translational capabilities using knowledge-based approaches for breeding mints accumulating specialty oils. We have performed more than 70 intra- and inter-species crosses with a diverse set of mint accessions. We are now in the process of generating F2 populations of progeny from selected crosses to assess the genetics underlying the accumulation of different monoterpenoids across accessions. The current focus is on characterizing the genes underlying the formation of terpinyl acetate. The aroma of this volatile has been described as "mildly herbaceous, sweet and refreshing, reminiscent of spicy bergamot-lavender type oils". We are working toward developing mint lines with high terpinyl acetate content as specialty crops.

Publications