Genomic analysis of circadian clock-, light-, and growth-correlated genes reveals PHYTOCHROME-INTERACTING FACTOR5 as a modulator of auxin signaling in Arabidopsis

Abstract

Plants exhibit daily rhythms in their growth, providing an ideal system for the study of interactions between environmental stimuli such as light and internal regulators such as the circadian clock. We previously found that two basic loop-helix-loop transcription factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, integrate light and circadian clock signaling to generate rhythmic plant growth in Arabidopsis (Arabidopsis thaliana). Here, we use expression profiling and real-time growth assays to identify growth regulatory networks downstream of PIF4 and PIF5. Genome-wide analysis of light-, clock-, or growth-correlated genes showed significant overlap between the transcriptomes of clock-, light-, and growth-related pathways. Overrepresentation analysis of growth-correlated genes predicted that the auxin and gibberellic acid (GA) hormone pathways both contribute to diurnal growth control. Indeed, lesions of GA biosynthesis genes retarded rhythmic growth. Surprisingly, GA-responsive genes are not enriched among genes regulated by PIF4 and PIF5, whereas auxin pathway and response genes are. Consistent with this finding, the auxin response is more severely affected than the GA response in pif4 pif5 double mutants and in PIF5-overexpressing lines. We conclude that at least two downstream modules participate in diurnal rhythmic hypocotyl growth: PIF4 and/or PIF5 modulation of auxin-related pathways and PIF-independent regulation of the GA pathway.

Figure 1.

Phase distribution of clock- and light-regulated genes. The times of peak circadian expression are depicted. A, Genes up-regulated by light. B, Genes down-regulated by light. C, Non-light-responsive genes. D, All circadian clock-regulated genes (Covington et al., 2008). The number of genes in each column is labeled on the y axis. For circadian time (h), 0 corresponds to subjective dawn and 12 to subjective dusk.

Figure 2.

Interaction between the circadian clock and growth. The number of genes (y axis) with peak expression at a particular circadian time (x axis) is shown for all _upG or _upS genes (A), _upG and _upS genes from data excluding samples taken at subjective dusk (B), light-responsive genes in A (C), and non-light-responsive genes in A (D). Left column, _upG; right column, _upS.

Figure 3.

PIF4 and/or PIF5 affect plant sensitivity to auxin more than to GA. Plants were grown in short days for 3 d and then transferred to plates containing the indicated hormones. After an additional 7 d of growth, seedling height was measured. Dose-response curves are shown for hypocotyl length in response to treatment with IAA (A and B), picloram (C and D), or GA (E and F). B, D, and F show normalized responses; for each genotype, the values were divided by the average hypocotyl length of that genotype without added hormone. Both PIF5HA-OX3 and PIF5-OXL1 overexpress PIF5. Error bars show se; data from at least two independent experiments are shown; 166 ≤ n ≤ 264 seedlings. [See online article for color version of this figure.]

Figure 4.

Promotion of growth by high temperature is impaired in pif4 pif5. Hypocotyl lengths at low temperature (black bars) and at high temperature (gray bars) are shown. Ratios of hypocotyl length (high temperature to low temperature) are shown by the blue line and numbers. Error bars show se; data from two independent experiments are shown; 47 ≤ n ≤ 61 seedlings. [See online article for color version of this figure.]

Figure 5.

Auxin overproduction partially disrupts growth rhythms. A, Growth kinetics of auxin-overproducing yucca seedlings. Col-7 is the wild-type background for yucca. Times of light and darkness are indicated by white and black rectangles on the x axis and by white and gray areas on the plot. B, Network analysis of growth kinetics. Each node represents a genotype, and each edge indicates the similarity between their growth patterns. Degree of similarity is indicated from dotted (lower) to thick (higher) lines. Blue, yellow, magenta, and green node colors indicate wild-type, light signaling, arrhythmic clock, and auxin overproduction plants, respectively.

Figure 6.

Reduction of active GA abolishes rhythmic growth. Col is the wild-type background for the ga20ox1 ga20ox2 double mutant. The experiment was performed as described for Figure 5A.

Figure 7.

A model of PIF4- and/or PIF5-regulated auxin responses. In wild-type plants, increasing auxin levels up to an optimal concentration lead to longer hypocotyls; auxin levels higher than this cause growth inhibition. Overexpression of PIF5 sensitizes, while loss of PIF4 and/or PIF5 desensitizes, hypocotyl responses to auxin. We assume that NPA treatment reduces endogenous auxin in the hypocotyl. Note that this model fits all dose-response curves in Figure 3, A and B, and Supplemental Figure S3. [See online article for color version of this figure.]