Marker-assisted in vivo imaging reveals prepatterning events prior to lateral root organogenesis.

Roots proliferate mainly through lateral root (LR) organogenesis events that succeed each other in a regular sequence. In the model plant Arabidopsis (Arabidopsis thaliana), live imaging of reporter lines containing the synthetic auxin-response DIRECT REPEAT5 (DR5) promoter have been instrumental for insight into prepatterning mechanisms that shape root architecture. However, depending on the reporter line in use, the course of prepatterning events leading to organogenesis may be described differently, causing confusion and misunderstandings in literature. In this study, we have setup a unique live imaging system allowing to simultaneously record the expression of bioluminescence- and fluorescence-based DR5-directed reporter lines. Based on this approach, we were able to reveal the spatio-temporal dynamics of the developmental steps taking place before and during LR organogenesis and their associated auxin response. Furthermore, manipulating primary root growth by interfering with ethylene biosynthesis in a tissue-specific manner resulted in a wild-type LR density following up mutually canceling effects by ethylene on early developmental steps. In summary, our dual live imaging has succeeded in identifying the early development steps leading to LR initiation and to place them in the correct developmental order.