Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Knocking Out a Candidate Gene for Wax Production in Switchgrass Results in an Unexpected Pleiotropic Phenotype

Authors:

Katrien M. Devos1,2,4,5* (FingerMillet@uga.edu), 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 (Fig. 1). 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.

Image

Two plants side by side. The left plant is smaller with less growth and the right one is much larger.

Figure 1. Representative examples of a 3-ketoacyl-CoA synthase 5 knockout (right) and nonedited control (left).

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.