The role of auxin-mediated gene activation in the bryophyte, Physcomitrium patens

Perception and response to the hormone auxin are critical to plant growth and development. Expression of auxin-response genes is tightly regulated via known mechanisms of both activation and repression. Across the plant lineage, auxin-response gene induction is performed by AUXIN-RESPONSE FACTOR (ARF), activating transcription factors. Conversely, AUXIN/INDOLE ACETIC ACID proteins repress expression. Studies of gain-of-function constitutive-repression lines and ARF loss-of-function mutants have advanced the field. Yet, there is a need for a comparative study of aberrant auxin-signaling mutants to understand the developmental consequences of constitutive repression versus the absence of auxin-mediated gene induction. Using CRISPR/Cas9 gene-editing tools, we mutated each activating ARF gene in the model bryophyte, Physcomitrium patens. The resulting septuple loss-of-function mutant line (arfasept) has severe developmental phenotypes and a diminished ability to respond to exogenous auxin. However, phenotypic analysis revealed that the arfasept line is not as severely affected as the constitutive-repression lines. Expression analysis of several auxin-response genes demonstrates that auxin-mediated gene induction is abolished in both arfasept and constitutive-repression lines but that basal expression levels are higher in the arfasept lines. Our results suggest that the expression of auxin-regulated genes important for developmental progression is maintained, albeit at reduced levels, in the absence of ARFs.