The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana

Abstract

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.

Figure 1. Generation of a lateral root transcriptomic data set.

(a) Overview of lateral root primordium stages, as described in ref. . Image adapted from ref. . (b) Gravitropic stimulation was used to induce the synchronized initiation of lateral root primordia at the site of root bending in a population of 3-day-old seedlings. Lateral root primordium stages (from I to VIII according to previous descriptions from ref. 10) were determined every 3 h from 6 to 54 h post gravity induction and are represented here as a percentage of the total number of observed lateral root primordia at each time point. (c) Root bends were microdissected for each of those 18 time points and used for RNA extraction and subsequent microarray analysis (N=450 per time point, 4 independent biological replicates). All seedlings were grown in constant light conditions, without previous entrainment. Image is adapted from ref. . (d) Principal component analysis of the lateral root data set revealed an oscillating component in the third principal component (PC3). (e) When differentially expressed genes were clustered, 17 clusters showed an oscillatory pattern. The estimate of the period length (h) and the number of genes in each cluster (N=x) is indicated above each cluster. Clusters with blue lines comprise core circadian clock genes. Expression intensities are on a log2 scale. The mean of the clusters is given by the solid line and the dotted lines show the mean±2s.d.

Figure 2. Circadian oscillations in the lateral root time course.

RNA expression patterns of the circadian clock genes CCA1, LHY, GI, TOC1, PRR7, PRR5, PRR3, ELF4 and ELF3, all of which oscillate in the lateral root data set. Expression intensities from the lateral root microarray data are on a log2 scale. Red vertical bars indicate peak times of TOC1 expression. N=4. All seedlings were grown in constant light conditions, without previous entrainment. The mean of the gene expression is given by the solid line and the error bars show the mean±2s.e.

Figure 3. Gravitropic stimulation regulates the circadian clock in the root bend.

(a) Luminescence levels of TOC1:LUC plants were continuously monitored. TOC1:LUC seedlings were grown vertically and imaged after the seeds were placed on plates. (b,c) Luminescence of control seedlings without gravitropic stimulus was monitored in: root tip and upper root (b), stem and leaf (c). (d,e) In contrast to b,c after 4 days a 90° gravitropic stimulus, as indicated by the grey section, was applied for 12 h. After that period, the plate was rotated back to original start position. (b–e) Error bars indicate s.e.; N=2. (f) From seedlings that have been exposed to a gravitropic stimulus at different ages, various tissues (root bend (g), cotyledons (h), upper root (i) and root meristem (j); indicated by circles) were dissected at the same time/age of the seedling (shown in f). (g–j) RNA was prepared and RT–qPCR was used to measure the levels of CCA1 mRNA. Only the root bends show oscillation of CCA1 expression. Error bars indicate s.d.; N=4.

Figure 4. Lateral root phenotypes of circadian clock mutants.

(a) TOC1 gene expression in the basal meristem is induced in response to auxin treatment of whole seedlings in Col-0, as well as arf7arf19 mutant plants. Expression intensities are on a log2 scale and are based on transcriptomics data with an ATH1 chip (Affymetrix), three independent biological replicates each. Error bars indicate s.d.; asterisks indicate a significant difference (P<0.05, Student’s t-test) from wildtype. (b–d) All seedlings were grown in constant light conditions, without previous entrainment. N=20 for all experiments. Error bars indicate s.d.; asterisks indicate a significant difference from wildtype. (b) Emerged lateral root density of 10-day-old C24 and toc1-1 seedlings in cm⁻¹ revealed a strong phenotype for toc1-1. (c,d) Ten-day-old seedlings were fixed and the primordium density of different developmental stages (stage I to emerged (E)) of wild-type (C24) and toc1-1 mutant seedlings in cm⁻¹ was determined. (e) Comparison of oscillating gene expression patterns from the lateral root transcriptomic time course of the circadian clock genes CCA1 and TOC1 and auxin signalling genes IAA14 and AtDAO2, demonstrate gating of auxin signalling by the circadian clock during lateral root primordium development. Expression intensities are on a log2 scale. Red vertical bars indicate peak times of TOC1 expression. All seedlings were grown in constant light conditions, without previous entrainment. The mean of the gene expression is given by the solid line and the error bars show the mean±2 s.e. N=4. (f) IAA and oxIAA accumulations exhibit circadian rhythms in roots under free-running conditions. Seedlings were germinated and entrained under 12 h light/dark cycle before transfer to constant darkness. Tissue was harvested starting at subjective dawn for 5-day seedlings. The experiment was repeated three times. White bars, light period; dark bars, dark period. Error bars indicate s.d.; N=3.