We have recently discovered a novel mechanism, the lateral root clock, which converts temporal information into a spatial pattern, somewhat analogous to the vertebrate segmentation clock. The lateral root clock determines the root physical network through positioning of lateral roots. These features are critical for effective exploration of the soil environment. To characterize the lateral root clock we generated a reporter with the DR5 promoter driving firefly luciferase. Imaging of this reporter construct revealed oscillations with a period of about 6 hours as well as expression that appeared after each oscillation and remained as a series of illuminated spots along the length of the root. These points were precisely where lateral roots form and thus were named “prebranch sites”. This oscillatory process appeared to be buffered against changes in the rate of root growth, suggesting that it had features of a biological clock.
To identify the genes oscillating in this region of the root we isolated sections from the region we called the oscillation zone (OZ) from individual seedlings containing a DR5-GUS construct. We determined the level of GUS mRNA in each section and used this to order the roots along the oscillation wave. We then performed a microarray analysis on the individual sections from 20 roots. We identified approximately 2,000 genes appearing to oscillate in phase with DR5 and another 1400 genes appearing to oscillate in anti-phase with DR5. Several promoters from genes that encoded transcription factors were able to drive oscillation in transgenic plants. To determine if these genes are involved in lateral root formation, insertional mutations were phenotyped. Single mutations had fairly subtle effects, but double and triple mutants exhibited dramatic reductions in the number of lateral roots and prebranch sites. Our working hypothesis is that networks of transcription factors are configured in such a way as to generate oscillations in expression and that this emergent behavior establishes the periodic positioning of lateral roots.