Inhibition of the shade avoidance response by formation of non-DNA binding bHLH heterodimers

Abstract

In shade-intolerant plants such as Arabidopsis, a reduction in the red/far-red (R/FR) ratio, indicative of competition from other plants, triggers a suite of responses known as the shade avoidance syndrome (SAS). The phytochrome photoreceptors measure the R/FR ratio and control the SAS. The phytochrome-interacting factors 4 and 5 (PIF4 and PIF5) are stabilized in the shade and are required for a full SAS, whereas the related bHLH factor HFR1 (long hypocotyl in FR light) is transcriptionally induced by shade and inhibits this response. Here we show that HFR1 interacts with PIF4 and PIF5 and limits their capacity to induce the expression of shade marker genes and to promote elongation growth. HFR1 directly inhibits these PIFs by forming non-DNA-binding heterodimers with PIF4 and PIF5. Our data indicate that PIF4 and PIF5 promote SAS by directly binding to G-boxes present in the promoter of shade marker genes, but their action is limited later in the shade when HFR1 accumulates and forms non-DNA-binding heterodimers. This negative feedback loop is important to limit the response of plants to shade.

Figure 1

The pif4pif5 mutations are largely epistatic over hfr1 in long-term shade conditions. Seedlings were grown for 8.5 days in high R/FR (white bars) or for 4 days in high R/FR followed by 4.5 days in low R/FR (black bars). (A) Hypocotyl length measurements, data are represented as the mean, error bars represent 2 × s.e. values, n=15. (B) Gene expression of PIL1 and XTR7 was determined by Q–PCR analysis. Biological triplicates were performed with technical triplicates for each sample. Values were normalized with EF1α and GAPC-2. Relative expressions to Col-0 in high R/FR are shown. Error bars represent s.e. values of biological triplicates.

Figure 2

The pif4pif5 mutations are epistatic over hfr1 in early responses to shade. Seedlings were grown for 6 days in high R/FR (12 h light–12 h dark) and then either kept in high R/FR ratios or shifted to low R/FR ratios. The expression of PIL1 and XTR7 was analysed by Q–PCR. Three technical replicas were performed for each sample. Values were normalized with EF1α and GAPC-2. Relative expressions to Col-0 (point 0) are shown. Error bars represent s.e. values of technical triplicates.

Figure 3

HFR1 interacts with PIF4 and PIF5. (A, B) Co-immunoprecipitation of in vitro-transcribed and -translated proteins (³⁵S-Met labelled). The HA tag was used for immunoprecipitation of PIF4 (A) or PIF5 (B) using HA-antibodies coupled to agarose beads. Proteins were separated by SDS–PAGE and visualized by autoradiography (immunoprecipitation,IP). The lanes come from the same gel and intervening lanes have been removed (indicated by a dividing line) (C) Bimolecular fluorescence complementation (BiFC) with HFR1/HFR1^* with PIF4 or PIF5 in plant cells. Onion cells were co-bombarded with N– and C–YFP fusion proteins. 1/3/5/7 dsRED signal of transfected cells; 2/4/6/8 YFP channel; Scale bar=100 μm. (D) Co-immunoprecipitation of HFR1–Flag and PIF5–HA. 35S∷HFR1–3 × Flag (HFR1–Flag), 35S∷PIF5–3 × HA (PIF5–HA) and seedlings expressing both transgenes (HFR1–Flag and PIF5–HA) were grown for 3 days in the dark. After 2 h 30 min in low R/FR condition, proteins were extracted and co-immunoprecipitated using anti-Flag antibodies. Proteins were separated by SDS–PAGE, western blotted and detected using antibodies raised against HA and Flag.

Figure 4

HFR1 inhibits PIF5 transactivation activity in Arabidopsis cells. (A) Schematic presentation of the constructs including the positions of the 3 G-boxes present in the PIL1 promoter. (B) Arabidopsis cells were co-bombarded with the pPIL1∷GUS or pPIL1*∷GUS (PIL1 promoter in which the 3 G-boxes are mutated) and either a vector control or PIF5. The transactivation activity of the effectors is given with the GUS values normalized to luciferase activity (the internal transfection control). Values are represented as mean of three different transfections±s.e. (C) Arabidopsis cells were co-bombarded with the pPIL1∷GUS construct and combinations of the different effector constructs as indicated in the figure. The transactivation activity is calculated as in (B).

Figure 5

HFR1 prevents PIF4 and PIF5 from binding to the G-box DNA sequence. Electromobility shift assays (EMSA) in (A–D) were performed using in vitro-transcribed and -translated proteins, and a ³²P-radiolabelled DNA probe of the PIL1 promoter sequence containing a double G-box. (A and C) The DNA probe (lane 1–9) was incubated with TNT master mix (lane 1) or PIF4 (A)/PIF5 (C) with increasing amounts of unlabelled probe (lane 3–5) or mutated unlabelled probe (lane 6–8). Lane 9 contains HFR1. (B, D) Lane 1: PIF4 or PIF5 alone; Lane 2: PIF4 or PIF5 with HFR1; Lane 3: PIF4 or PIF5 with HFR1^*. The arrow indicates the specific PIF–DNA complex. FP, free probe.

Figure 6

PIF5–HA, but not HFR1–HA, binds to the promoter of shade-induced genes in vivo. Chromatin immunoprecipitation (ChIP) from 12-day-old Col, 35S∷HFR1–3 × HA (HFR1) and 35S∷PIF5–3 × HA (PIF5) seedlings. (A) Schematic representation of the PIL1, XTR7 and HFR1 genes, including the regions amplified after ChIP and the position of G-boxes. (B) Immunoprecipitated DNA was quantified by Q–PCR using primers in the promoter region containing G-boxes (region 1, 3 and 5) or control regions without G-boxes (region 2, 4 and 6). Data are average of technical triplicates of the Q–PCR (values±s.d.). Data from one representative ChIP experiment are shown.