Linking photoreceptor excitation to changes in plant architecture

Abstract

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.

Figure 1.

Identification of shade-responsive elements in the PIL1 promoter. (A) Induction of the PIL1 promoter after 2 h of shade treatment implicates a minimal region from −1529 to −1305 as required for shade responsiveness. A luciferase reporter was flanked with a series of 5′ deletions of PIL1 promoter and a full length of PIL1's 3′ UTR and transformed into Arabidopsis. Plotted is the fold change over white light of individual lines using a minimum of 16 independent lines. Each data point is a single measurement from an independent transformed line. (B) PIL1's 210-bp element (−1494 to −1284) confers shade responsiveness to a minimal 35S promoter. At least 16 independent lines were analyzed. (C) Two G-boxes contained within the 210-bp shade-responsive region are necessary for PIL1 induction by shade. LUC activities under the control of a wild-type or mutant G-box in the 2181-bp PIL1 native promoter are measured under the white light condition (left) or after 2-h low R:FR treatments (right). A minimum of 16 independent lines were analyzed.

Figure 2.

pif7 mutants lack an early shade avoidance response. (A) Representative seedlings of Col-0 and pif mutants grown in white light and shade. Seedlings were grown in white light for 3 d and then moved to white light or simulated shade for 5 d. (B) Quantification of hypocotyl length of Col-0, pif4pif5 (pif4-101pif5-1), pif7 mutants and sav3-2. The left Y-axis is hypocotyl lengths, and the right Y-axis is the ratio of hypocotyl lengths in shade to that in white light. (C) Quantification of cotyledon area of Col-0, pif7 mutants, and phyB-9 in response to white light and shade. (D) pif7 hypocotyls have an early growth defect that is similar to sav3. New hypocotyl growth of pif7-1 after transfer to shade was quantified using HyDE (hypocotyl-determining engine) (Cole et al. 2010). The arrow indicates the start of shade treatment. Dotted lines and roman numerals indicate phases of growth.

Figure 3.

pif7 mutants are defective in auxin biosynthesis. (A) Heat map representation of shade-regulated gene expression in Col-0 (wild type), pif7-1, and sav3-2. Red, orange, and white rows indicate RNA expression in high, medium, and low levels, respectively. The left column indicates auxin-regulated genes. Blue bars indicate auxin-induced genes, and the green bar indicates auxin-repressed genes. (B) Box plot representation of 335 auxin-induced genes between different genotypes under different light conditions. (FPKMs) Fragments per kilobase of exon per million fragments mapped. (C) Free IAA levels in Col-0 and pif7 mutants under white light and simulated shade. Col-0 and pif7 were grown in white light and moved to white light or shade for 1 h. The aerial parts were collected to measure free IAA level. (D) Direct binding of PIF7 with the G-box region of the promoters of YUC8 (−515 to −421, containing two G-boxes) and YUC9 (−1339 to −1203 bp, containing two G-boxes). After 1 h of shade treatment, ChIP assays were performed with Flag M2 antibody using 35S::PIF7-Flash transgenic plants. ChIP assays were quantified by real-time PCR after normalizing with the input DNA. PCR was performed with primers specific for the G-box region. The coding sequence (CS) region is used as a negative control.

Figure 4.

PIF7 protein abundance and phosphorylation are regulated by shade. (A) Accumulation of a fast-migrating form of PIF7 within 10 min of transfer to shade. The Western blot shows reduction of a slow-migrating form of PIF7-Flash and accumulation of a faster-migrating form of PIF7-Flash in shade. PIF7-Flash protein was visualized by anti-Myc antibody, and loading was measured by anti-actin antibody. (WL) White light; (SH) shade. (B) PIF7's post-translational modification is a dephosphorylation. Treatment of lysates from white-light-grown transgenic plants with CIP attenuated the slowly migrating band. Loading was normalized to an actin control. (C) Dephosphorylation of PIF7 is rapid and photoreversible. Seedlings were grown in continuous white light for 5 d, treated with shade (R:FR = 0.7) for the times indicated, and then returned to white light. Note the reappearance of the slowly migrating band when seedlings were returned to white light. (D) A molecular model linking shade perception by PHYB to auxin-regulated gene expression. See the text for details.