Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Environmental Horticulture
Non Technical Summary
The chemical diversity in plant species, called the metabolome, is enormous and is the main source of human food, medicine and forms the basis for agricultural crop yield and nutritional quality. Out of the estimated 350,000 plant species on Earth, at least 31,128 plant species documented for human uses. However, only 15% are sampled thus far to study the chemical constituents, with a majority species unexplored. Medicinal plants have played a major role in drug development and certain phytochemicals continue to be widely used in their original form. The development of modern chemistry permitted the isolation and characterization of plant derived chemicals that have served as drugs or materials for the synthesis of many important drugs used today. Therefore, there exists a need and great potential for medicinal plant research to explore novel pharmaceutical compounds. By integrating multi-disciplinary approaches such as High performing liquid chromatography (HPLC), Liquid Chromatography-Mass spectrometry (LC-MS), LC-MS/MS, Gas Chromatography (GC)-MS, RNA-sequencing, gene discovery and functional characterization by utilizing CRISPR/Cas9, we want to understand the molecular mechanisms underlying the biosynthesis of specialized metabolites of agricultural/ pharmaceutical importance in the target plant species largely beloning to plant families Lamiaceae, Plantagiaceae, Rubiaceae and Amaryllidaceae with a long-term goal of metabolic engineering and production by expression in heterologous systems.
Animal Health Component
40%
Research Effort Categories
Basic
50%
Applied
40%
Developmental
10%
Goals / Objectives
Our planet Earth is home to estimated 350,000 plant species with at least 31,128 plant species documented for human uses. However, only 15% are sampled thus far to study the chemical constituents (1, 2). The chemical diversity in plant species, called the metabolome, is enormous and is the main source of human food, medicine and forms the basis for agricultural crop yield and nutritional quality. Plant metabolites are categorized as primary and secondary/specialized metabolites. Primary metabolites are structurally and functionally highly conserved in plant kingdom and are critical for growth, development and survival in plants. However, the vast chemical diversity in plant kingdom is attributed mainly to secondary/ specialized metabolites to address special needs of the plants and selected throughout the course of evolution. Specialized metabolic pathways are not well conserved, and their biosynthesis is lineage specific and is spatiotemporal (3). That means majority of specialized metabolites are only detected in defined species within specific tissues/organs or cell types at a given developmental stage under a specific environmental condition. It is imperative to understand regulatory interactions between primary and specialized metabolism, the pathway regulation including flux and further elucidate the biosynthetic pathways of specialized metabolites in order to facilitate genome engineering and synthetic biology approaches to manipulate them in planta for food and medicinal purposes.Medicinal plants have played a major role in drug development and certain phytochemicals continue to be widely used in their original form. The development of modern chemistry permitted the isolation and characterization of plant derived chemicals that have served as drugs or materials for the synthesis of many important drugs used today. Therefore, there exists a need and great potential for medicinal plant research to explore novel pharmaceutical compounds. Exploring plant derived bio-active pharmaceutical compounds from medicinally relevant species largely belonging to plant families Lamiaceae, Plantagiaceae, Rubiaceae and Amaryllidaceae in order to:I. Elucidate the molecular mechanisms underlying the biosynthesis of bio-active compounds using integrative approaches. II. Investigate the impact of controlled environments and aseptic production systems on the metabolite profiles. III. Explore micropropagation, genetic transformation and gene-editing prospects in species under study.IV. Conduct field studies to evaluate the species in FL landscape and/ other locations in the US for commercial production and involve in outreach activities to disseminate the research results. By integrating multi-disciplinary approaches such as High performing liquid chromatography (HPLC), Liquid Chromatography-Mass spectrometry (LC-MS), LC-MS/MS, Gas Chromatography (GC)-MS, RNA-sequencing, gene discovery and functional characterization by utilizing CRISPR/Cas9, we want to understand the molecular mechanisms underlying the biosynthesis of specialized metabolites in the target plant species with a long-term goal of metabolic engineering and production by expression in heterologous systems.
Project Methods
Scutellaria Project 1: 1. Production trials with Scutellaria arenicola, S. integrifolia, S. lateriflora and S. baicalensisSelected germplasm of Scutellaria will be grown, sourced from FL nurseries and seeds collected from native environment. Replicated field experiments will be conducted in a randomized block design for each species at multiple locations. 2. Manipulate controlled environments in order to maximize bio-active metabolitesControlled environments ensure safety and efficacy for medicinal plant production and can help maximize bioactive compound synthesis by optimizing environmental factors such as CO2, light (quality and quantity) among others. In Scutellaria, CO2 enrichment has resulted in higher concentration of wogonin, baicalin and baicalein in plant tissues compared with non-enriched system . In addition, studies have demonstrated that different light conditions impact phenylpropanoid pathway, specifically plants grown under light-emitting diodes (LED) have increased accumulation of phenylpropanoids. All species will be evaluated for growth and bioactive compounds in growth chambers equipped with LEDs and CO2 injection control capabilities. 3. Quantify price and quality of existing Scutellaria products and develop enterprise budget based on FL dataCurrently there are more than 50 Scutellaria products sold by various sources including Amazon, ebay, Walmart, herbal medicine stores etc., throughout the country. However, no data is available on the quality of the active metabolites present in the products or the dollars returned from commercial production of Scutellaria in FL. Pharmaceutically active compounds will be quantified from various products available in the market compared to airdried and freeze-dried samples from our experimental results to account for any differences that might be due to sample preparation process.Pharmaceutical metabolite analyses: We will analyze flavonoids by HPLC by using standards available to quantify the compounds. Targeted metabolites from leaves, shoots and roots will be analyzed and quantified for samples from the field experiments, controlled environment studies and commercial products. The active flavone concentrations will be used as biomarkers to evaluate variations within various commercial products.Bacopa Project 2: 1. Selection of germplasm and metabolite profilingB. monnieri, B. caroliniana, B. innominate, B. rotundifolia, B. floribunda are available in the U.S. in nurseries and natural habitats. Targeted metabolites from leaves and shoots will be analyzed and quantified from the germplasm collected using HPLC and LC-MS/MS methods. 2. RNA-sequencing in order to identify the differentially expressed candidate genes between species and tissue typesRNA from multiple species and tissue types will be sequenced to identify the candidate genes involved in the biosynthesis of bio-active compounds by comparison of expression profiles of species that have varied bio-active compound profiles and by co-expression analyses of genes. RNA will be isolated from leaf, stem and root tissues and RNA-sequencing (RNA-seq) libraries will be constructed and sequenced in single-end mode on the Illumina HiSeq 4000 platform. Three biological replicates will be performed to address any variation attributable to biological variation. De-novo transcriptome assembly will be performed, and differential gene expression analysis will be performed relative to B. monnieri species and various tissue types in each species to identify candidates involved in the biosynthetic pathway of bio-active metabolites.3. Establish tissue culture micropropagation, regeneration and gene-editing technologies to functionally characterize the candidate genesTissue culture micropropagation techniques will be established using MS media (MS salts, 3% sucrose, 5 g/L phytoagar) using nodes and apical meristems. Plant regeneration will be performed using stem and leaf segments from four-week old tissue culture plants on step I (MS salts, 3% sucrose, 5 g/L phytoagar, 1 mg/L thiamine-HCl, 0.8 mg/L zeatin-riboside and 2 mg/L 2,4-D) for 5 days. Explants will then be transferred to Step II media (MS salts, 3% sucrose, 5 g/L phytoagar, 1 mg/L thiamine-HCl, 0.8 mg/L zeatin- riboside, 2 mg/L gibberellic acid) and will be regularly transferred to new step II plates once in every 10 days. The number of explants forming calli and shoots will be considered for calculating the regeneration rate. In order to establish gene-editing methodologies, Phytoene Desaturase (PDS) gene will be targeted for knock-out using CRISPR/Cas9. Single guide RNAs will be designed targeting two potential 5' regions of the PDS gene and CRISPR/Cas9 constructs will be generated. Genetic transformation protocols will be established using Agrobacterium mediated transformation. Since the phenotype of PDS knock-out is a visible albino phenotype, determination of gene silencing can be evaluated during callus phase. Eventually, candidate genes identified in the transcriptomic studies will be evaluated functionally using CRISPR/Cas9.Mitragyna Project 3: 1. Authentication of genetic identity of Mitragyna collection through DNA analysesMitragyna speciosa ecotypes including Speciosa, Mengda, Bumblebee, R, RB, Red Indonesia, Pink Indonesia, Rifat, MS Indo, MS Malay, MS Thai, MS Vietnam along with other species including Mitragyna hirsuta, M. diversifolia are available in our germplasm collection. Metabolite profiling of these species for the bioactive alkaloids revealed that some of these ecotypes do not possess Mitragynine but have other analogous bioactive compounds and furthermore there are qualitative and quantitative differences in the chemical profile between the samples. In order to identify the genetic basis of the collection and authenticate the origin of the germplasm, DNA barcoding of the existing germplasm will be performed. Genomic DNA will be isolated from meristematic leaves of all the ecotypes and species in our germplasm to perform DNA barcoding. Chloroplast markers including rbcL, matK, trnL-trnF will be amplified and sequenced by sanger sequencing. Multiple sequence alignments will be done to compare the DNA sequences and will be further correlated with the chemical profile of bioactive actives compounds. This correlation between genetics and biochemistry of species not only helps in categorizing the species and ecotypes but also allows us to hypothesize on the role of plant age/maturity on the chemical profile of individual species. All the species that share a similar DNA barcode, but not the chemical profile will be further examined for age related bio active accumulation.2. Gene-editing through de novo induction of meristems in Mitragyna speciosaTissue culture remains one of the biggest bottle necks in developing transgenic plants and gene-editing especially in tree species where the in vitro propagation and regeneration could be time consuming. Concomitantly expressing developmental regulators and gene-editing reagents was recently demonstrated to create transgenic and gene-edited shoots through de novo meristem induction in various plant species (24). A similar strategy will be applied to test the induction of genetically modified meristems in Mitragyna speciosa. Developmental regulators Wuschel (WUS) and Shoot Meristemless (STM) along with a luciferase reporter on the T-DNA region of a binary vector will be delivered to young seedlings of M. speciosa via Agrobacterium tumefacians to develop transgenic lines by circumventing tissue culture based genetic transformation method. Based on the callus induction and luciferase activity, co-expressing CRISPR/Cas9 cassette expressing Cas9 and guide RNA targeting PDS will be verified based on photo bleaching phenotype in order to establish gene-editing methodology for functional characterization of genes involved in the biosynthetic pathway of bioactive alkaloid production in this medicinal plant.