Performing Department
(N/A)
Non Technical Summary
Food crops provide the essential micronutrients needed in the human diet through production of minerals and vitamins. Additionally, plants produce thousands of specialized metabolites, many of which have human health benefits. The Ericaceae family contains several economically important fruit crops that produce diverse natural products, including blueberry and cranberry. Cultivated blueberry has seen an annual increase in market size since 2000, with rising popularity due to the overall sweet fruit flavor and consumer perceived levels of known health-promoting compounds. Blueberries also produce iridoid compounds, which are potent natural products with human health benefits. Despite these known properties, little work has been done to fully understand the genetic basis of iridoid biosynthesis in cultivated blueberry. This New Investigator Seed Grant aims to obtain data to identify core iridoid biosynthetic genes in blueberry. A multi-faceted approach incorporating transcriptomic, genomic and metabolomic data will be used to elucidate genes involved in the biosynthesis of the iridoid glycoside monotropein in blueberry. The objectives of the proposal are: 1) Identify iridoid biosynthetic pathway genes in blueberry using transcriptomic, orthology, co-expression and genome sequencing analysis, and 2) Validate the computationally predicted gene targets through functional enzyme characterization. This work leverages the within-species genotypic and phenotypic diversity in blueberry iridoid production to understand the genetic basis of natural product biosynthesis in this economically important crop. Outcomes from this proposal will serve as a platform to more fully understand iridoid production in blueberry and can translate into the development of new blueberry varieties with enhanced human health benefits.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The research in this proposal addresses the genetic basis of the synthesis of plant-derived, high-value chemicals in blueberries. As part of the balanced human diet blueberries can provide essential micronutrients, vitamins, and thousands of specialized metabolites with additional health benefits. The Ericaceae plant family contains several economically important fruit crops including blueberry and cranberry. Cultivated blueberry (Vacciniumspecies) has seen an annual increase in U.S. market size since 2000. The popularity of this crop is based on their overall sweet flavor of the fruit and the consumer perceived levels of known health-promoting compounds, including vitamin C, folate, phenolic and flavonoid compounds. Blueberries also produce iridoid compounds, which are potent natural products with human health benefits. Several iridoid compounds have been identified in a subset of species within the Ericaceae, with significant inter-species diversity observed in production of the iridoid glycoside monotropein in blueberry.Despite the relevant human health properties, little work has been done to fully understand the iridoid biosynthetic pathway in cultivated blueberry.This New Investigator Seed Grant aims to address this knowledge gap by obtaining data to identify core iridoid biosynthetic genes in blueberry.Themajor hypothesisof this work is that the genes involved in iridoid biosynthesis in blueberry can be identified by comparative genomics and orthology analysis using specific blueberry varieties known to have presence/absence of this class of secondary metabolites.A multi-faceted approach incorporating transcriptomic, genomic and metabolomic data will be used to elucidate genes involved in the biosynthesis of the iridoid glycoside monotropein in cultivated blueberry. This work leverages the within-species genotypic and phenotypic diversity in blueberry iridoid production to understand the genetic basis of natural product biosynthesis in this economically important crop.The two objectives of the proposed project are:1)Identify iridoid biosynthetic pathway genes in blueberryusing transcriptomic, orthology, co-expression and genome sequence analysis; and2)Validate the computationally predicted gene targets through functional enzyme characterization.
Project Methods
Objective 1:Identify iridoid biosynthetic pathway genes in blueberryHypothesis: The combination of analyses using transcriptomics, orthology, co-expression and genome sequence analysis will reveal the candidate iridoid biosynthesis genesObjective 1, Task 1.A new cultivar and tissue panel will be generated for select cultivars to expand the genetic and phenotypic diversity needed for identification of monotropein biosynthetic genes. Both Ornablue and Concord cultivars have been previously analyzed for monotropein content in several tissues. We already havede novotranscriptome assemblies completed for tissues from Ornablue (+) and Concord (-) which can be used for candidate gene identification using orthology analysis to previously characterized iridoid biosynthetic genes inC. roseus. Three additional cultivars 1) Summit, a previously identified monotropein-positive cultivar that is a southern highbush ecotype (Leisner et al., 2017) 2) Cara's choice (monotropein-negative), a half-sibling of Summit (Ehlenfeldt & Stretch, 2005) and northern highbush ecotype, and 3) Tifblue, a rabbiteye cultivar (V. virgatum)have been selected for analysis to expand the panel diversity. Tifblue is a late season blueberry variety that is one of the most important cultivars in rabbiteye breeding (Hancock et al., 2008).Rabbiteye blueberries are native to the southeastern U.S. and a major ecotype grown in Alabama and southern Georgia (Coneva, 2013). In this new expanded panel, root tissue will be analyzed for all cultivars as other plant species has shown significant production of iridoids in plant roots (e.g., Kellner et al., 2015). As the origin and/or transport of monotropein production in blueberry are unknown, analysis of root tissue is invaluable. This panel will also include young leaves sprayed with methyl jasmonate (MeJa).Previous work has demonstrated that application of MeJa can increase the level of monoterpenes in cranberry (Rodriguez-Saona et al., 2013),C. roseus(Liscombe et al., 2011), and other species (Wang et al., 2010; Nagatoshi et al., 2011; Góngora-Castillo et al., 2012; Semiz et al., 2012). It is hypothesized that increasing monotropein production will increase expression and/or abundance of genes related to iridoid biosynthesis and be a valuable asset in gene discovery and co-expression analysis.All samples for the blueberry tissue diversity panel will be collected from verified cultivars.Monotropein content of ground tissues will be analyzed and quantified by targeted LC-MS using an authentic standard as described in Leisner et al. (2017). For transcriptomic analysis, RNA will be isolated and cDNA libraries will be constructed and sequenced with the Illumina HiSeq4000 sequencer. In addition to short-read sequencing,transcriptome assemblies for all cultivars will be reconstructed using isoform sequencing (Iso-Seq) from Pacific Biosciences (PacBio). Short-read Illumina data generated from the HiSeq4000 will be used to error correct the reconstructed transcriptomes and generate gene expression data.From the Iso-Seq analysis a high-quality, full-length transcripts will be generated with no assembly required. Gene expression analysis will be completed by mapping each RNA-Seq library to the transcriptome for each cultivar followed by determination of transcript abundance values.Objective 1, Task 2.Enzymes in threekey steps in the early iridoid biochemical pathway will be selected for targeted orthology analysis - GES, G8H and GOR. These genes represent the first committed steps in iridoid biosynthesis (Miettinen et al., 2014) and should share sufficient homology withC. roseusto utilize comparative genomic and orthology analysis. We will also use the new data to validate our previous work with ISY.Orthology analysis will be completed and potential orthologs will be analyzed for presence/absence variants and transcript abundance by mapping the transcript sequences to theV. corymbosumgenome (cv. Draper, Colle et al., 2019) and to the transcriptome assembly for each cultivar.Co-expression analyses, including hierarchical clustering, principal component analyses, mutual rank, and self-organizing maps will be used to identify the candidate genes involved in the early iridoid biosynthetic pathway (Obayashi & Kinoshita, 2009; Saeed et al., 2003; Wehrens & Buydens, 2007).Objective 2: Validate the computationally predicted gene targets through functional enzyme characterizationHypothesis:The functionality of the gene products can be confirmed by biochemical analysis.Objective 2, Task 1.In order to validate the computationally predicted gene targets, cloning of genes encoding three key enzymes (GES, G8H, GOR) will be completed for two blueberry genotypes, with the long-term goal of confirming enzyme sequences for all cultivars (outside the scope of this grant). The entire coding sequence will be amplified and cloned into pOPINF expression vector using In-Fusion cloning (Takara Bio). Plasmids containing the cloned gene will be transformed into competentE. colior yeast expression strain cells to produce protein in quantities suitable for enzymatic analysis. After lysing the cells by sonication, the soluble portion will be loaded onto Ni-NTA agarose resin (Qiagen), and the recombinant His-tagged protein eluted. The purified recombinant protein will then be used in Task 2.Objective 2, Task 2.To validate enzyme function, end-point enzyme assays for the enzymes of interest will be done according to Lichman et al., 2020. This is key to confirming the cloned gene sequence produces a protein that has the assigned function in the iridoid biosynthetic pathway. To obtain anin vitroprofile of reaction products for the enzyme of interest GC-MS will be performed(Yang et al., 2009; Geu-Flores et al., 2012). The early biosynthetic intermediates, such as geranyl diphosphate, geraniol, and 8-hydroxygeraniol required as a substrate for these enzymes are commercially available. Completion of this objective will be done in collaboration with Dr. Sarah O'Connor, Director at the Max Planck Institute for Chemical Ecology.