Performing Department
(N/A)
Non Technical Summary
Thinleaf huckleberry, whose scientific name isVaccinium membranaceumDouglas ex Torr., is economically important and in increasingly high demand due to the flavonoid secondary metabolites in its berries that produce desirable dietary antioxidants and a delicious flavor profile. Although the effects of environment on flavonoid production in thinleaf huckleberry are not well understood, in closely related species it is known that light quality and elevation contribute to flavonoid production. In addition to contributing to the desirable qualities of the berries, flavonoids also defend plant leaves against damage from UV and overexposure to light. Unlike its close relatives like the blueberry and cranberry, thinleaf huckleberry has not been successfully domesticated, despite recent efforts. Identifying optimal light growing conditions will be important to reduce harvesting pressure on natural populations, which negatively impacts the animals that rely on the berries, leaves, and stems for food. In addition, understanding patterns in leaf flavonoid production in natural populations will illuminate the species' ability to adapt to the effects of climate change. This project seeks to better understand how light, latitude, and elevation affect the flavonoid profile of the leaves to assist in commercial cultivation, mediate the impact of overharvesting, assess persistence in the face of climate change, and identify correlations between geographic location and flavonoid nutrition for animals. Results from this project may impact ecological and economic aspects of forest management of thinleaf huckleberry.The proposed research is multidisciplinary and addresses three central objectives: 1) Conduct controlled treatments of different light intensities, harvest the leaves grown under the controlled conditions, analyze them for their flavonoid content and the expression levels of genes involved in flavonoid production, and compare these measurements between light intensity treatments, 2) Harvest leaves from populations at low and high elevations at three different latitudes across its geographic range, analyze the leaves for their flavonoid content and the expression levels of genes involved in flavonoid production, and compare these measurements between latitudes and elevations, and 3) Collect leaves from the same populations as Objective 2, sequence the DNA of these leaves, use the DNA sequences to construct an evolutionary tree that relates the populations to one another, and examine how evolutionary history of the populations influences the flavonoid production and gene expression levels in the leaves. Objective 1 obtains a base-line expectation for what the flavonoid production and gene expression patterns should look like at different light intensities. Objective 2 tests this pattern in the field and examines patterns of flavonoid profiles across environmental gradients. Finally, Objective 3 tests whether the patterns from Objectives 1 and 2 hold when taking into consideration evolutionary history, and also tests whether patterns of flavonoid production and gene expression can be predicted by a population's evolutionary history.Results of this project may inform optimal greenhouse growing conditions for thinleaf huckleberry potentially reducing harvesting pressure on natural populations in the future. Results may also uncover the photoprotective response of this species at higher latitudes and higher elevations, illuminating its potential ability to adapt given rapid climate change. Finally, the results may improve the understanding of ecological correlation between geographic location and leaf palatability to animals. This project combines lab and field experiments and uses cutting-edge bioinformatics tools and molecular techniques, such as metabolomics, transcriptomics, and phylogeography, to address these objectives. Data from this project will be made publicly available and results will be shared in peer-reviewed scientific journals and at national conferences.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The overarching goal of the projectis to leverage cutting edge transcriptomic, metabolomic, phylogeographic, and bioinformatic techniques to understand how light, latitude, elevation, and evolutionary history affect dietary and photoprotective flavonoid production in economically important thinleaf huckleberry. Developmental and environmental regulation of desirable and ecologically important flavonoid compounds in Vaccinium membranaceumDouglas ex Torr., thinleaf huckleberry, are not well understood, but examples in closely related species ofVacciniumshow that light quality and elevation contribute to flavonoid production and regulation. At higher light intensities, an increase in leaf flavonoid content might be attributed to the need for photoprotection against light that exceeds photosynthetic capacity. At higher latitudes, although light intensity may decrease as it passes through more atmosphere, lower temperature that can reduce photosynthetic capacity combined with longer growing season daylight length may induce flavonoid photoprotective response. Higher elevations may also increase flavonoid content due to lower temperatures and higher light intensity, particularly of ultraviolet (UV) radiation. This project will test questions in both controlled environments and the field and will uniquely incorporate phylogenetic history into the assessment of patterns of expression of these metabolites.The berry ofthinleaf huckleberry is well-known for its flavor profile and has been economically important since its establishment as a forestry industry in the United States in the 1930s. Secondary metabolites in its leaves have also been used for medicinal purposes and are an important source of nutrition for grizzly bears and American black bears, as well as ungulates and birds. Thinleaf huckleberry is not yet commercially cultivated and optimal growing conditions to maximize flavonoid content (flavor profile) in berries are unknown. Investigating how light affects thinleaf huckleberry leaves in the greenhouse can offer crucial insight into optimal commercial growing conditions, an effort that may reduce stress of over harvesting on natural populations. Illuminating the patterns of flavonoid production in leaves across gradients of light intensity via latitude and elevation may inform the evolutionary understanding of how thinleaf huckleberry adapts to abiotic stress and the ecological understanding of correlation between geography and leaf nutritional value for animals. The results of this research may aid commercial growing efforts, illuminate this species' potential ability to adapt via photoprotection to greater UV exposure or movement to higher latitudes given rapid climate change, and inform wildlife impacts due to geographic patterns of leaf nutritional value.Details of the three objectives of the proposed research are:Objective 1: Determine the expected response of secondary metabolite production in thinleaf huckleberry leaves in controlled treatments of lightintensity.H1:CHS,DFR,ANS, andUFGTwill be upregulated at higher lightexposure.H2:Quantity of anthocyanins, flavonols, and hydroxycinnamic acids will be greater in leaves matured at higher light exposure.Objective 2: Compare expression of genes in secondary metabolic pathways and secondary metabolite content of leaves between low and high elevation populations across a latitudinal gradient.H1:Higher expression ofCHS,DFR,ANS, andUFGTwill be correlated with higher latitude and higher elevation.H2:Higher quantities of anthocyanins, flavonols, and hydroxycinnamic acids will be correlated higher latitude and higher elevation.Objective 3: Examine the phylogeographic history of thinleaf huckleberry and the impact of evolutionary history on patterns of secondary metabolite production in leaves across the geographic range.H1:Expression levels ofCHS,DFR,ANS, andUFGTand quantities of anthocyanins, flavonols, and hydroxycinnamic acids have phylogenetic structure.H2:Any relationships found between latitude and elevation and secondary metabolite production hold (Aim 2: H1, H2) after accounting for phylogeny.These aims deviate from the originally proposed research by focusing on the secondary metabolites in leaves instead of berries. Due to supply constraints, Objective 1 was completed on younger (and thus non-fruiting) plants, and leaf tissue was analyzed instead of berry tissue. Due to varied fruiting time (and thus varied ripeness of fruits) during the time of field work reconnaissance, Objective 2 was completed on collected leaf tissue instead of collected berry tissue. Comparative transcriptomic analysis relies on identical environmental conditions, and collection of ripe berries from populations separated by wide temporal intervals would have introduced variation to the analysis that would be difficult to control for. These two revised objectives address the same overarching question about secondary metabolite response to light, latitude, and elevation, and they still analytically complement each other as a controlled experiment in Objective 1 and a field experiment in Objective 2. Objective 3 has only changed in that it now addresses whether the patterns of secondary metabolite production in leaves (not berries) found in Objective 1 and 2 are impacted by evolutionary history. In addition, the broader impact of informing optimal commercial growing conditions remains. However, additional broader impacts focus on photoprotective response in leaves, with implications for persistence given possible effects of climate change, and nutritional value to wildlife for whom thinleaf huckleberry is an important source of nutrition in the spring and fall.
Project Methods
Objective 1: Determine the expected response of secondary metabolite production in thinleaf huckleberry leaves in controlled treatments of light intensity.Experimental approach- Plants will be treated with different levels of lightand leaves will be harvested for RNA-Seq and metabolomics. RNA-Seq data will be used for differential gene expression (DE) analysis targetingCHS,DFR,ANS, andUFGT. Anthocyanins, flavonols, and hydroxycinnamic metabolites will be compared between treatments.Greenhouse treatments- Young plants will be purchased, grownin 6 in pots in a greenhouse, and watered with water supplemented with acidic (pH 4.5-5.5) calcium magnesium fertilizer at 100ppm N.Plants will then be placed under one of three conditions of mesh shading: high, medium, and low.Each treatment will contain eight replicates. Leaves that have budded and developedthrough the course of treatment will be harvested, placed in RNA later, and kept frozen at -80°C until RNA is extracted. Additional samples will be immediately frozen at -80°C for subsequent metabolomic analysis.RNA-Seq- Total RNA will be extracted following a modified Invitrogen Plant Reagent protocol. Leaves from one individual will be pooled. RNA quality will be confirmed with the Agilent Bioanalyzer at the PSU Genomics Core. Illumina stranded mRNA library construction and paired-end (2x150 bp) sequencing will be done at NovoGene using the NovaSeq Illumina platform.Transcriptome assembly and differential expression analysis- RNA-Seq data will be pre-processed using BBTools and assembledde novowith the Trinity pipeline. DE analysis between lighttreatments will be quantified using the DESeq Bioconductor package. To annotate transcripts, gene ontology categories and KEGG metabolic pathway IDs will be assigned using the Trinotate pipeline. In addition, HMMs will be generated to query the transcriptomes for gene family targets.Metabolomics- Frozen leaves will be prepared for liquid chromatography-tandem mass spectrometry (LC-MS2) to quantify anthocyanins, flavonols, and hydroxycinnamic metabolites using an untargeted approach. Samples will be prepared and analyzed following a modified protocol for the AB SCIEX 5600 Triple at the Metabolomics Facility at PSU.Objective 2: Compare expression of genes in secondary metabolic pathways and secondary metabolite content of leavesbetween low and high elevation populations across a latitudinal gradient.Experimental approach- Low and high elevation populations at each of three latitudes in western North America will be sampled. DE analysis will be conducted on the same targeted genes from Objective 1 and leaf secondary metabolite quantities will be compared between populations. Environmental co-predictors will also be collected. Generalized least squares (GLS) regression models will identify relationships between DE genes/leaf secondary metabolites and latitude/elevation/co-predictors.Population sampling- Permits will be obtained for collection of plant and soil material from all field sites in the US. During July and August when berries are ripening, seven populations of thinleaf huckleberry will be sampled across its geographic range. Thirtyindividuals from each population will be sampled. Three-fourberries and 5-8 leavesfrom each individualwill be collected, stored in RNA-later, and shipped back to PSU on dry ice. An additional set of berries and leaves will be flash frozen and shipped back to PSU on dry ice.Berries will be selected based on color.A pressed herbarium voucher specimen will be collected for each population.Environmental co-predictors- At each field site soil moisture and soil temperaturewill be measured using a Meter 5TM Sensor. Atmospheric temperature and humidity will also be measured.A soil sample will be collected at each site and submitted to the Agriculture Analytical Services Lab at PSU to measure soil pH. At each individual sampled, UV and PAR will be measured at time of collection.RNA-Seq- Total RNA will be extracted from six individuals and prepared for sequencing following methods in Objective 1. Libraries will be multiplexed on the NovaSeq Illumina platform.Transcriptome assembly and differential expression analysis- RNA-Seq data will be cleaned, assembled, and DE analysis and identification of target genes will be conducted following methods in Objective 1.Metabolomics- Following methods from Objective 1, leafsamples from six individuals per population will be send to Creative Proteomics for untargeted metabolomic analysis usingLC-MS2 to quantify anthocyanins, flavonols, and hydroxycinnamic metabolites.Generalized Least Squares- A GLS analysis will be run and relative expression of genes and metabolome measurements will be tested as a function of latitude or elevation. Environmental co-predictors will also be incorporated in these two models.Objective 3: Examine the phylogeographic history of thinleaf huckleberry and the impact of evolutionary history on patterns of secondary metabolite production in leaves across the geographic range.Experimental approach- Genomic SNP data from multiple individuals per population will be used to estimate phylogeographic history. The phylogeny will be used to test whether secondary metabolite variation has phylogenetic structure and whether latitudinal or elevational patterns of secondary metabolite variation still hold when accounting for phylogenetic bias.Plant sampling- Field sites will be the same as Objective 2, but will also include field sites in the Great Lakes region, where this species is also found. Green plant leaves will be collected from 30individuals per population and silica dried. Individuals will be spaced 5meters apart. One bilberry sample will be collected to root the phylogenetic trees with an outgroup.Genotyping-by-sequencing (GBS)- Total DNA will be extracted from leaves using the Qiagen DNeasy Plant Mini Kit. Library construction and Illumina HiSeq2500 multiplex sequencing will be completed by the University of Wisconsin Biotechnology Center. Raw data will be cleaned, assembled, and prepared for downstream analysis using cutadapt v.1.13 and Stacks.Phylogeography- Concatenated SNP data will be used to estimate a maximum likelihood tree in RAxML v.8.2.10 with the Lewis ascertainment bias correction to account for unobserved invariant sites. A multispecies coalescent tree will be estimated with SVDQuartets in PAUP v.4.0a150, which can summarize across individuals in a population to produce a tree with one tip per population.Phylogenetic signal- Relative expression data and metabolomic data from Objective 2 will be coded along the tips of the SVDQuartets summary tree. The functionphylosigin the R package 'phytools' will be run to test for phylogenetic signal. This function incorporates sampling error, or within-population variation, and will allow use of expression data from all of the 6 individuals per population sampled in Objective 2.Phylogenetic Generalized Least Squares- A phylogenetic generalized least squares analysis will be run. Under this model, which accounts for phylogenetic structure, relative expression of genes and metabolomic data from Objective 2 will be tested as a function of latitude or elevation. Environmental co-predictors will be incorporated in these two models.Results from this project will be published in peer-reviewed scientific journals and presented at national conferences to expand knowledge in the fields of botany, agriculture, and evolution. The project will be evaluated based on successful completion of outcomes. Outcomes will be considered complete if each of the aligned short term products (described under "Products") and analyses (described above) is produced.?