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-REPSONSE 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.