Knocking Out a Candidate Gene for Wax Production in Switchgrass Results in an Unexpected Pleiotropic Phenotype
Authors:
Katrien M. Devos1,2,4,5* ([email protected]), Gurjot S. Sidhu1,2, Eudald Illa-Berenguer1,2, Bochra A. Bahri1,2,3, Wayne Parrott1,2,4, Thomas H. Pendergast IV1,2,4,5, Gerald A. Tuskan2
Institutions:
1Institute of Plant Breeding, Genetics, and Genomics, University of Georgia–Athens; 2Center for Bioenergy Innovation; 3Department of Plant Pathology, University of Georgia–Griffin; 4Department of Crop and Soil Sciences, University of Georgia–Athens; 5Department of Plant Biology, University of Georgia–Athens
URLs:
Goals
The Center for Bioenergy Innovation’s (CBI) vision is to accelerate domestication of bioenergy-relevant, nonmodel plants and microbes to enable high-impact innovations along the bioenergy and bioproduct supply chain while focusing on sustainable aviation fuels (SAF). CBI has four overarching innovation targets: (1) develop sustainable, process-advantaged biomass feedstocks; (2) refine consolidated bioprocessing with co-treatment to create fermentation intermediates; (3) advance lignin valorization for biobased products and aviation fuel feedstocks; and (4) improve catalytic upgrading for SAF blendstocks certification.
Abstract
Switchgrass, Panicum virgatum, a grass native to North America, is of intense interest as a dedicated feedstock for the production of SAF. Switchgrass ecotypes differ by a number of characteristics, including the presence of wax on leaves and stems. Lowland ecotypes generally contain high levels of C33 β-diketones and hydroxy-β-diketones, which are associated with the formation of crystalline wax tubes on the abaxial leaf side and a blueish plant color (Bragg et al. 2020; Weaver et al. 2018). In contrast, β-diketones are largely lacking from upland accessions, which therefore have glossy green leaves. Researchers previously identified a cluster of genes as strong candidates for the quantitative trait locus that was identified for wax variation in an F2 population from a cross between the lowland genotype AP13 and the upland genotype VS16 (Qi et al. 2021). One of the candidate genes, a likely 3-ketoacyl-CoA synthase 5 (KCS-5), was knocked out in Performer7, a transformable lowland accession, using CRISPR-Cas9. Interestingly, while edited plants had the expected glossy green color, they were also shorter in stature and had more tillers compared to the controls. To determine the effect of KCS-5 knockout on transcription, an RNA-seq analysis was conducted on two independent KCS-5 knockout plants and two nonedited control plants. A total of 1,781 and 415 genes were differentially expressed (DE) in leaves and stems, respectively, between the KCS-5 knockout lines and nonedited controls (p-value≤0.05, log2-fold difference≥1). 64% of the genes DE in stems were also DE in leaves. Work is ongoing to determine the affected pathways as well as the effect of the KCS-5 knockout on sustainability.
References
Bragg, J., et al. 2020. “Environmentally Responsive QTL Controlling Surface Wax Load in Switchgrass,” Theoretical and Applied Genetics 133, 3119–37. DOI:10.1007/s00122-020-03659-0.
Qi, P., et al. 2021. “Quantitative Trait Locus Mapping Combined with Variant and Transcriptome Analyses Identifies a Cluster of Gene Candidates Underlying the Variation in Leaf Wax Between Upland and Lowland Switchgrass Ecotypes,” Theoretical and Applied Genetics 134, 1957–75. DOI:10.1007/s00122-021-03798-y.
Weaver, J. M., et al. 2018. “Cuticular Wax Variants in a Population of Switchgrass (Panicum virgatum L.),” Industrial Crops and Products 117, 310–16. DOI:10.1016/j.indcrop.2018.02.081.
Funding Information
Funding was provided by the CBI led by Oak Ridge National Laboratory. CBI is funded as a U.S. DOE Bioenergy Research Center supported by the BER program in the DOE Office of Science under FWP ERKP886. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. DOE under contract no. DE-AC05-00OR22725.