Given the known role of auxin in several stages of LR formation (9
) and the general use of DR5 as an auxin-signaling reporter (17
), we investigated whether prebranch site formation was related to changes in levels of this hormone. We generated different auxin-signaling reporters by fusing the promoters of INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), IAA14, and IAA19 to the luciferase coding region and tested them for response to exogenous application of auxin in the OZ. pIAA19 was expressed in the OZ and responded to exogenous auxin in a fashion similar to that of DR5:Luciferase (, and movies S3
), whereas pIAA7 and pIAA14 responded to auxin at later times. When we evaluated the expression of pIAA19 in the OZ without any hormone treatment, we did not detect an oscillatory behavior in its expression over time ( and movie S5
). Although additional transcriptional regulation of pIAA19 cannot be excluded, our data suggest that fluctuation in auxin is unlikely to be sufficient to drive the periodic pulses of DR5 expression in the OZ.
Fig. 2 Manipulation of auxin levels or signaling is not sufficient to induce prebranch site formation or ectopic branching. (A) Overlay (O) of bright-field and luciferase images, and detailed bright-field image (BF), showing expression of pIAA19 fused to the (more ...)
Induction of auxin biosynthesis in pericycle cells has been linked to acquisition of LR founder-cell identity (18
). Thus, we tested whether auxin could generate prebranch sites independently of the oscillation reported by DR5. We performed localized auxin treatments in the OZ, both when natural peaks of DR5 expression were detected and when they were not detected. Exogenous auxin treatment or auxin plus 1-N
-naphthylphthalamic acid (NPA), which should prevent auxin movement, did not lead to production of a prebranch site when the treatment was not concurrent with a DR5 oscillation (). When the DR5 oscillation was concurrent with application of auxin or auxin plus NPA, the prebranch site appeared to be shifted from the predicted position of a natural prebranch site in ~60% of the cases (). However, auxin did not seem to specify a new prebranch site, because two prebranch sites were never observed in close proximity after the auxin treatment (fig. S2
). As DR5 expression also marks LR founder cells (18
), this indicates that functional founder-cell specification normally requires the oscillation reported by DR5. However, we cannot rule out the possibility that at very high, sustained doses of auxin there may be de novo production of LRs through a mechanism that does not depend on prebranch site formation.
Additionally, it has been proposed that root bending could promote auxin accumulation preferentially at the outside of the curve, which, in turn, could induce LR formation (19
). This was particularly intriguing because we had observed that primary root bending occurred with a frequency similar to that of prebranch site production (), and prebranch sites tend to be located in the middle of the bends. Thus, we examined whether ectopic prebranch sites were induced by manual bending (generating J-hooks) near the root tip (19
). When J-hooks were formed ~5 mm from the root tip, the J-hook occurred at a position where prebranch sites were already specified (fig. S3
). We found that the first prebranch site was located at an average of 3 mm from the root tip, and we did not detect ectopic formation of prebranch sites at J-hooks (fig. S3
). Lateral roots always emerged from preexisting prebranch sites. In addition, we examined gravity-induced bends. We altered the root’s position relative to the gravity vector by rotation of either 90° or 180° in the vertical plane or by turning the plants 90° from the vertical to horizontal plane (fig. S4
). We observed that gravity-induced bends occurred in an asynchronous manner. Roots rotated 180° showed gravity-induced bending after an average of 3 hours; however, many roots took longer to bend, as seen in the skewed distribution in . Interestingly, the time between the last periodic bend prior to the 180° rotation and the subsequent gravity-induced bend followed a distribution similar to that detected for roots under normal conditions ( and ). This suggests that the root’s natural periodic bends are completed prior to gravity-induced bends. It has been reported that gravistimulation at 6-hour intervals is optimal for increasing LR density (21
). As LR development requires previous formation of a prebranch site, it is possible that the observed increase in LR density is due to the gravistimulus-promoted development of each prebranch site into a LR primordium. In addition, after rotation of the roots, we detected peaks of DR5 expression approximately every 3 hours; this is more frequent than expected for the periodic DR5 oscillation or the response to gravity alone (). It has been previously reported that auxin may accumulate following gravity-induced bending (19
). To examine this further, we observed pIAA19 expression after gravistimulation. We found that pIAA19 peaks of expression were less frequent than peaks of DR5 expression after gravistimulation (). Because pIAA19 does not oscillate, but responds to auxin, these pIAA19 peaks may report increases in auxin signaling or content due to gravity response. Under gravity response conditions, pulses of DR5 expression generated static points of DR5 expression; however, not all of these points persisted and only a few formed a prebranch site that eventually developed a LR (fig. S4
). In addition, we tested whether prebranch site formation was affected by conditions that reduced root bending by growing roots through the growth medium. We detected approximately the same number of prebranch sites for roots that grew through the agar as for roots grown along its surface (standard conditions) (). This indicates that prebranch site specification occurs in the absence of visible root bends.
Taken together, our results indicate that competence to form a LR through establishment of prebranch sites is determined by internal cues reported by peaks in DR5 expression. Auxin may contribute to this endogenous mechanism, because application of auxin appears to shift the location of a prebranch site. In addition, the root gravitropic response, which likely involves altered endogenous auxin concentrations, appears to be able to change the period of the endogenous mechanism.