Development of auxin reporters in oilseed rape (Brassica napus)

Abstract

Auxin is a key phytohormone that regulates all aspects of plant growth, development, and environmental responses, making the precise analysis of its distribution and signaling essential for understanding plant adaptation and physiological processes. However, despite the agricultural importance of oilseed rape (Brassica napus), the lack of robust, species-specific molecular tools limits detailed studies of hormone signaling in this crop. Here, we developed and characterized reporter systems for the sensitive visualization and quantification of auxin distribution and signaling in B. napus. The DR5cc auxin signaling reporter and a novel synthetic auxin-responsive reporter, BIP3, assembled from promoter fragments of three oilseed rape Aux/IAA genes, were generated to drive GUS expression. In hairy roots, both reporters showed auxin-responsive expression in the root apical meristem that became broader after auxin treatment. In transgenic seedlings, flowers at anthesis, and 12-day-old embryos, DR5cc exhibited a more defined expression pattern than BIP3. To monitor real-time auxin dynamics under abiotic stress, DR5cc fluorescent reporters were employed in hairy roots. Mannitol and NaCl treatments induced a time-dependent increase in fluorescence, peaking at 6-12 h before returning to basal levels after 24 h. Furthermore, dual-reporter assays enabled simultaneous monitoring of auxin and cytokinin signaling, revealing distinct hormone-specific spatial responses in hairy roots. Finally, we established a quantitative DII (qDII) reporter system using degron domains from B. napus Aux/IAA proteins, providing a quantitative readout of auxin depletion. Together, these reporter systems enable spatial, temporal, and quantitative analyses of auxin dynamics during development and stress adaptation in oilseed rape.