Oscillating gene expression determines competence for periodic Arabidopsis root branching

Abstract

Plants and animals produce modular developmental units in a periodic fashion. In plants, lateral roots form as repeating units along the root primary axis; however, the developmental mechanism regulating this process is unknown. We found that cyclic expression pulses of a reporter gene mark the position of future lateral roots by establishing prebranch sites and that prebranch site production and root bending are periodic. Microarray and promoter-luciferase studies revealed two sets of genes oscillating in opposite phases at the root tip. Genetic studies show that some oscillating transcriptional regulators are required for periodicity in one or both developmental processes. This molecular mechanism has characteristics that resemble molecular clock-driven activities in animal species.

Fig. 1

Periodic branching and bending are marked by repeated pulses of DR5 reporter expression. (A) LRs develop from the primary root (PR), and younger LRs are located closer to the PR tip. (B) DR5 fused to the luciferase reporter gene is expressed in the oscillation zone (OZ) and prebranch site(s). (C) Quantification of DR5 expression in the OZ over time. Arrows mark the time when the primary root bends. (D) Overlay of luciferase and bright-field images of a DR5-expressing root. Bright-field image was taken 5 days after the luciferase picture. Asterisks indicate LR primordia that did not emerge. (E and F) Graph of the distribution of prebranch site production and bending over time (n = 15 and 30, respectively). (Inset) Root bends with time interval (t_i) between bends. (G) Prebranch site production and root length under different growth conditions (n = 40). Scale bars, 1 mm.

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 luciferase reporter. (B) Quantification of pIAA19 and DR5 expression in the OZ after localized auxin treatments (n = 10). (C) Quantification of pIAA19 expression in the OZ over time. (D) Time series of a DR5:Luciferase-expressing root after OZ-localized auxin treatment. Yellow arrows indicate natural DR5 oscillations. Red arrow shows the location of the auxin treatment. (E) Percentage of plants showing misplaced prebranch sites in response to OZ-localized auxin and NPA treatments. (F) Distribution of the time to bend in response to gravity after a 180° turn (n = 39). (G) Time between consecutive pulses of DR5 expression over a 20-hour period under normal conditions and after changing the relative position of roots to gravity by turning. (H) Number of peaks of DR5 or pIAA19 expression detected over a 20-hour period after a 180° turn (n = 10). (I) Prebranch site production and root length for roots grown on the surface (control) or through the growth medium (n = 15 for each). Scale bars, 250 µm. Error bars represent SD.

Fig. 3

Identification of genes with periodic expression behavior in the oscillation zone. (A) Venn diagram showing the number of genes exhibiting periodic behavior as identified by two different methods. (B) Heat maps of the two sets of genes found to oscillate in the OZ. Yellow indicates higher and blue indicates lower relative expression. (C and D) Overlay (O) of bright-field and luciferase images showing expression of the promoter of (C) ZAT11 in phase with DR5, and (D) ARF7, in antiphase, fused to the luciferase gene. Bright-field images (BF) detailing the OZ are shown to the right (C and D). Scale bars, 250 µm. (E) Quantification of pZAT11:Luciferase and (F) pARF7:Luciferase expression in the OZ over time. (G) Expression profiles for oscillating genes in cluster A3 as expressed in the RootMap. Red line represents average gene expression. (H) Time series of pARF7:Luciferase (top) and pATAF1:Luciferase (bottom) expression (cluster A3). Dotted lines mark a fixed spatial position. (I) Gene Ontology (GO) term enrichment in the phase (P) and antiphase (A) gene clusters and (J) their enrichment in data sets profiling LR initiation and in transcription factor families.

Fig. 4

Oscillating genes encoding transcription factors are necessary for regular prebranch site formation and root bending. (A) Bright-field images of seedlings showing root bending and emerged LRs of wild type (Col-0) and mutants. (B) Quantification of the number of prebranch sites and LRs for a variety of genotypes (n = 25). Error bars, SD. (C) Distribution of bending over time in Col-0 compared with mutants (n = 25). Note the increased distribution of time between bends in the mutants. (D and E) Expression of pSHP1:Luciferase in the OZ. (D) Bright-field and Luciferase images overlaid. (E) Time series showing SHP1 expression pulses over time. White and yellow arrows mark first and second expression pulses, respectively; asterisk marks position of a future LR primordium. (F) Quantification of DR5:Luciferase expression in the OZ in arf7-1 mutants. Arrows indicate the times when primary root bending occurred. (G) Luciferase images of Col-0 (wt) and arf7-1 expressing the DR5:Luciferase reporter. White arrows mark prebranch sites. (H) Quantification of the frequency of bending in fez-3 mutants (n = 15). Bending of fez-3 and wild type is similar [compare with (C)]. Scale bars: (A and G), 1 mm; (D and E), 250 µm.