Functional Characterization of bHLH Transcription Factors Coordinating Abiotic Stress Response, Secondary Cell Wall Biosynthesis, and Metal Homeostasis in Populus
Dimiru Tadesse1, Estella Yee1, Desigan Kumaran1, Aditi Bhat1, Crysten E. Blaby-Haas2, Timothy Paape1, and Meng Xie1* (firstname.lastname@example.org)
1Brookhaven National Laboratory; and 2Lawrence Berkeley National Laboratory
The Quantitative Plant Science Initiative (QPSI) is a capability that aims to bridge the knowledge gap between genes and their functions. A central aspect of QPSI strategy is combining genome-wide experimentation and comparative genomics with molecular-level experimentation. In this way, the team leverages the scalability of omics data and bioinformatic approaches to capture system-level information while generating sequence-specific understanding of gene and protein function. By incorporating molecular-level experimentation into the workflow, researchers are addressing the question of how a protein functions and establishing mechanistic insight into how sequence variation impacts phenotype. This knowledge serves as a touchstone for accurate genome-based computational propagation across sequenced genomes and forms the foundation for robust predictive modeling of plant productivity in diverse environments.
Populus is one of DOE’s flagship bioenergy crops as the source of renewable energy and biobased products. Gene regulatory networks (GRNs) that describe the hierarchical regulatory relationships between transcription factors (TFs), associated proteins, and their target genes are fundamental for coordinating genome-wide gene expression responses to environmental and developmental signals. The complex and dynamic behavior of plant TFs is crucial for the GRN plasticity for sensitive responses. However, it also poses a fundamental challenge in understanding molecular principles underlying GRN dynamics. Researchers previously identified two basic Helix-Loop-Helix (bHLH) TFs (PtrbHLH038 and PtrbHLH011) whose expressions were oppositely regulated in Populus leaves under iron deficiency treatment. Using the transactivation assay in protoplasts, researchers found that they are transcriptional repressors. Using their novel protoplast-based transient chromatin immunoprecipitation-sequencing (transient ChIP-seq) approach for mapping genome-wide binding targets of TFs in vivo, team members found and validated that PtrbHLH038 directly regulates PtrbHLH011 and abiotic stress-responsive genes. In contrast, PtrbHLH011 seems to have broader regulatory functions because its targets include metal transporters, growth regulators, and master regulators of secondary cell wall biosynthesis. More interestingly, protoplast-based approaches enabled the discovery that iron deficiency treatment can eliminate PtrbHLH011’s binding and repression on its target genes. By performing TurboID-based proximity labeling in protoplasts, the team identified protein cofactors that form complexes with PtrbHLH011. Based on the results described above, researchers hypothesize that PtrbHLH038 and PtrbHLH011 form a regulatory hierarchy to coordinate abiotic stress responses, secondary cell wall biosynthesis, and iron homeostasis in Populus. Transgenic plants overexpressing PtrbHLH038 and PtrbHLH011 have been generated to test the team’s hypothesis and study the biological impacts of these two transcription factors.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program as part of the Quantitative Plant Science Initiative (QPSI) science focus area.