Understanding and Engineering Crown Root Development to Improve Water-Use Efficiency in Bioenergy Grasses
Willian Goudinho Viana1* (email@example.com), Janina Tamborski1* (firstname.lastname@example.org), Jose Sebastian2, Johannes Daniel Scharwies1, Taylor L. Clarke1, Keith E. Duncan3, Christopher N. Topp3, Hui Jiang3, Colby Starker4, Jennifer A. N. Brophy1, José R. Dinneny1,and Ivan Baxter3
1Stanford University; 2Indian Institute of Science Education and Research; 3Donald Danforth Plant Science Center; and 4University of Minnesota
This study aims to improve water-use efficiency in the bioenergy grass Sorghum bicolor by engineering crown root development. Crown roots play an important role in water and nutrient acquisition. To precisely modify crown root development, this project is engineering the location and gene expression level of a newly identified crown root regulator called Crown Root Defective using synthetic genetic circuits. Since synthetic genetic circuits have never been utilized in crop species before, researchers are currently testing and optimizing individual circuit building blocks in Setaria and Sorghum. The project goal is to enable the construction of circuits that drive gene expression in a predictable manner in these bioenergy grasses. Through the precise control of genes influencing crown root development and the identification of new root development regulators, the team aims to improve water-use efficiency.
S. bicolor is a biofuel crop that offers great potential because of its tolerance to drought, heat, and low cost of production as compared to other potential feedstocks. Despite yield gains through breeding, its productivity in suboptimal conditions is still limited. Acquisition of water and nutrients is mediated by the root, and in grasses the mature root system is primarily composed of crown roots (Viana, Scharwies, and Dinneny 2022). In the panicoid grass model Setaria viridis, soil moisture around the crown stimulates the development of crown roots, while drought conditions inhibit their growth, which facilitates the conservation of water (Sebastian et al. 2016). Despite their importance in crop productivity, the genetic mechanisms behind crown root development in response to environmental factors are not well understood. To engineer water-use efficiency in panicoid bioenergy grasses, researchers aimed to elucidate the key genetic mechanisms and subsequently alter crown root development. The team identified a mutant named crown root defective-1 (crd-1) that is specifically impaired in crown root development under well-watered conditions. Interestingly, this defect is rescued under drought stress conditions. Through Bulk Segregant Analysis, the gene was mapped to a single nucleotide polymorphism disrupting the splice site resulting in a premature stop codon of a gene encoding a WD-repeat protein. Researchers independently generated secondary alleles and complementation lines to confirm that the identified gene is in fact the causal gene for the phenotype.
Because of its promising phenotype, the team decided to leverage crd-1 to alter the number of crown roots by precisely modifying its expression level. In the past, synthetic genetic circuits were successfully used in combination with tissue-specific promoters to modify root architecture in Arabidopsis thaliana (Brophy et al. 2022). Despite success in developing tools and modifying model plants, in the past the transfer of these applications to crop plants has not always been successful. The team therefore established transient systems in Sorghum and S. viridis to test and optimize individual components of synthetic circuits for application in grass species. These circuits are built using synthetic transcription factors composed of bacterial DNA-binding proteins fused to transcriptional activation or repression domains. Through the use of transient protoplast expression, results have shown that the strength of the synthetic transcription factors is influenced by both the DNA-binding protein and activation domain. Researchers hypothesize this is due to the different conformation of each synthetic transcription factor resulting in altered accessibility of the activation domains. Moreover, research shows that the transcriptional activity of synthetic transcription factors is directly correlated to the number of binding sites utilized in the synthetic promoters. Together, the results from transient assays show that these parts can be modified in multiple aspects to drive gene expression in a predictable manner. Subsequently, genetic circuits were successfully built that implement the Boolean NOT implies logic operation in grasses.
In future research, the team aims to identify more factors influencing crown root development. WD-repeat proteins can act as scaffold proteins that organize multiprotein complexes and can also regulate gene expression. To understand the function of the WDR6 protein, researchers performed a yeast-two hybrid screen, which led to the discovery of several binding partners, including members of the Growth-Regulating Factor (GRF) family of transcription factors. The team is now working on validating these interactions and planning to conduct functional studies to further explore the role of the GRFs in crown root development. Through the identification of more players in the crown root development pathway and initial data from the genetic circuits, results indicate a promising future for engineering root architecture and other complex traits in bioenergy grasses.
Brophy, J. A. N., et al. 2022. “Synthetic Genetic Circuits as a Means of Reprogramming Plant Roots,” Science 377(6607), 747–51.
Sebastian, J., et al. 2016. “Grasses Suppress Shoot-Borne Roots to Conserve Water During Drought,” Proceedings of the National Academy of Sciences of the United States of America 113(31), 8861–66.
Viana, W. G., J. D. Scharwies, and J. R. Dinneny. 2022. “Deconstructing the Root System of Grasses Through an Exploration of Development, Anatomy and Function,” Plant, Cell and Environment 45(3), 602–19.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0018277 and DE-SC0023160.