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

Engineering Continuous Trait Variation in Bioenergy Feedstocks to Optimize Growth on Marginal Lands

Authors:

Jennifer A. N. Brophy* (jbrophy@stanford.edu), Janina Tamborski, Isabel Goldaracena Aguirre

Institutions:

Department of Bioengineering, Stanford University

Goals

As climate change progresses, bioenergy crops will need to withstand increasingly formidable water, nutrient, and temperature stresses. Though yields of C4 grasses, such as Sorghum bicolor, have increased through breeding and improved agronomy, annual yield gains will be hard hit by impending abiotic stressors (Prasad et al. 2021). Thus, new germplasm must be developed to maintain, or continue to enhance, yields. Unfortunately, relatively little is known about the traits that contribute to abiotic stress tolerance in sorghum or other related next-generation C4 feedstock species. The project aims to develop a novel synthetic biology–based approach to determine the contribution of individual root features to abiotic stress tolerance. Synthetic genetic circuits will be employed to generate continuous variation in root depth and root branch density, so their contribution(s) to stress tolerance can be studied with the ultimate goal of identifying optimums and generating more resilient plants.

Abstract

Trait variation is key to understanding the contribution of specific plant features to environmental stress resilience. By measuring the fitness of plants with changes in a specific trait under different stress conditions, researchers can identify traits that are associated with greater resilience (Upadhyaya et al. 2016). The project is engineering sorghum plants to have variation in two key root traits—depth and branch density. The goal is to develop lines that dramatically reduce the number of plants that need to be phenotyped in gene-environment experiments in order to increase the number of abiotic stress conditions that can be tested simultaneously. The project’s approach utilizes synthetic genetic circuits to tissue—specifically titrate—the expression of genes that control root development. Researchers have engineered Buffer gate components that can be used to vary gene expression over several orders of magnitude in C4 grass protoplasts and are in the process of validating them in stably transformed lines. To tune root branch density, the team plans to express mutant Auxin/Indole-3-Acetic Acids (Aux/IAAs) at varying levels in specific root layers. The mutant Aux/IAAs should inhibit auxin response and prevent the development of lateral roots in a concentration dependent manner (Brophy et al. 2022). Researchers designed a library of mutant SbAux/IAAs with disruptions to their “degron” regions and have begun testing them in sorghum protoplasts (Moss et al. 2015). Initial results suggest that these mutant proteins are resistant to auxin-mediated degradation and can constitutively suppress the auxin transcriptional reporter DR5 (Yang et al. 2017). To modify root depth, the team is using CRISPR-Cas9 to knock out homologs of DEEPER ROOTING 1 (DRO1)—a gene identified in rice that alters root growth angles (Uga et al. 2013). Researchers are testing CRISPR guide RNA activity in sorghum protoplasts and plan to use the most efficacious guides for stable transformation. Once knocked out, DRO1 will be reintroduced at a variety of expression levels using Buffer gates. This work is building toward a new approach for understanding the contribution of root architecture features to plant fitness.

References

Brophy, J. A. N., et al. 2022. “Synthetic Genetic Circuits as a Means of Reprogramming Plant Roots,” Science 377, 747–51.

Moss, B. L., et al. 2015. “Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics,” Plant Physiology 169, 803–13.

Prasad, V. B. R., et al. 2021. “Drought and High Temperature Stress in Sorghum: Physiological, Genetic, and Molecular Insights and Breeding Approaches,” International Journal of Molecular Sciences 22, 9826.

Uga, Y., et al. 2013. “Control of Root System Architecture by DEEPER ROOTING 1 Increases Rice Yield Under Drought Conditions,” Nature Genetics 45, 1097–102.

Upadhyaya, H. D., et al. 2016. “Sorghum Germplasm Resources Characterization and Trait Mapping.” In The Sorghum Genome, 77–94. Eds. Rakshit, S., and Y-H. Wang. Springer International Publishing, Cham. DOI:10.1007/978-3-319-47789-3_4.

Yang, J., et al. 2017. “Dynamic Regulation of Auxin Response during Rice Development Revealed by Newly Established Hormone Biosensor Markers,” Frontiers in Plant Science 8.

Funding Information

This research was supported by the DOE Office of Science, BER program, grant no. DE-SC0024057.