Seedlings Transduce the Depth and Mechanical Pressure of Covering Soil Using COP1 and Ethylene to Regulate EBF1/EBF2 for Soil Emergence

Abstract

The survival of seed plants in natural environments requires the successful emergence from the soil. In this process, the ethylene signaling pathway is utilized by plants to sense and respond to the mechanical resistance of the soil. Here, we report that constitutive photomorphogenesis 1 (COP1), a central repressor of light signaling, is a key component required for seedlings to sense the depth of soil overlay. Mutation in COP1 causes severe defects in penetrating soil, due to decreased level of EIN3, a master transcription factor in ethylene pathway that mediates seedling emergence. We show that COP1 directly targets the F box proteins EBF1 and EBF2 for ubiquitination and degradation, thus stabilizing EIN3. As seedlings grow toward the surface, the depth of soil overlay decreases, resulting in a gradual increase of light fluences. COP1 channels the light signals, while ethylene transduces the information on soil mechanical conditions, which cooperatively control EIN3 protein levels to promote seedling emergence from the soil. The COP1-EBF1/2-EIN3 module reveals a mechanism by which plants sense the depth to surface and uncovers a novel regulatory paradigm of an ubiquitin E3 ligase cascade.

Keywords: COP1; EBF1 and EBF2; EIN3; ethylene signaling; seedling emergence.

Figure 1. COP1 acts upstream of EIN3 to promote seedling emergence from the soil

(A-D) Soil emergence phenotypes (A and C) and quantitative analysis (B and D) of Col-0 (wild type) and cop1-4 mutant. Seedlings were grown under continuous white light for 7 days without (0 mm soil) or with increasing depths overlay of SiO₂ powder (A and B) and various particle size sands (C and D). Mean ±s.d., n=3. (E-F) Soil emergence phenotypes (E) and quantitative analysis (F) of Col-0, cop1-4, cop1-6 and 35S:COP1/cop1-6. Seedlings were grown under continuous white light for 7 days without (0 mm soil) or with 2.5mm soil (50-70 mesh sands) overlay. Mean ±s.d., n=3. (G) Quantitative analysis of 4-day old etiolated Col-0, cop1-4, cop1-6 and 35S:COP1/cop1-6 seedlings without (0 mm soil) or with 2.5mm soil (50-70 mesh sands) overlay. Mean ±s.d., n=3. (H-I) Soil emergence phenotypes (H) and quantitative analysis (I) of Col-0, cop1-4, EIN3ox and EIN3ox/cop1-4. Seedlings were grown under continuous white light for 7 days without (0 mm soil) or with 2.5mm soil (50-70 mesh sands) overlay. Mean ±s.d., n=3. (J) Quantitative analysis of 4-day old etiolated Col-0, cop1-4, EIN3ox and EIN3ox/cop1-4 seedlings without (0 mm soil) or with 2.5mm soil overlay. Mean ±s.d., n=3. (K-L) Images (K) and angles (L) of cotyledon opening of 3-day old etiolated Col-0, cop1-4, EIN3ox and EIN3ox/cop1-4 seedlings. Mean ±s.d., n>20. (M-N) Images (M) of cotyledons and areas (N) of single cotyledons of 7-day old etiolated Col-0, cop1-4, EIN3ox and EIN3ox/cop1-4 seedlings. Mean ±s.d., n>20.

Figure 2. EIN3 transcriptional actions are largely abolished by COP1 mutation

(A) GUS staining images of 3-day old etiolated seedlings of 5XEBS-GUS in Col-0, EIN3ox and EIN3ox/cop1-4 backgrounds. (B-F) qRT-PCR results showing the gene expressions of ERF1 (B), EBF2 (C), PIF3 (D), Lhcb1b1 (E) and Lhcb1b2 (F) in 4-day old dark-grown seedlings. The expressions were normalized to PP2A. Mean ±s.d., n=3.

Figure 3. COP1 stabilizes EIN3 protein

(A) White light (Left) and bioluminescence (Right) images of 4-day old etiolated seedlings of EIN3p-EIN3-Luciferase in ein3eil1 and cop1ein3eil1 backgrounds. The color-coded bar indicates the intensity of lucifersase activity. C.P.S stands for Counts Per Second. (B) Western blot analysis of EIN3 protein levels. Seedlings over-expressing EIN3-Myc in ein3eil1 and cop1ein3eil1 backgrounds were grown on 1/2MS medium in the dark for 4 days without (DMSO) or with MG132 pre-treatment for 12 h before harvesting. Col-0 was used as a negative control. RPT5 was used as a loading control. (C) Cell-free degradation of recombinant EIN3-His proteins in 4-day old etiolated Col-0 (top) and cop1-4 (bottom) seedlings. Equal amount of EIN3-His proteins were added into the cell extracts and incubated for the indicated periods of time, and then analyzed by immunoblots. “-” stands for the no EIN3-His protein control. Actin was used as a loading control. (D) Fluorescence microscopic analysis of the EIN3-GFP protein levels. Seedlings over-expressing EIN3-GFP in ein3eil1 and cop1ein3eil1 backgrounds were grown on 1/2MS medium for 4 days in the dark.

Figure 4. COP1 is responsible for the 26S proteasome-mediated EBF1 and EBF2 protein degradation

Western blots showing the EBF1(A, left panel) or EBF2 (B, left panel) protein levels. Seedlings over-expressing EBF1-TAP of EBF2-TAP in Col-0 (WT) and cop1-4 mutant backgrounds were grown on 1/2MS medium in the dark for 4 days without (DMSO) or with MG132 pre-treatment for 12 h before harvesting. Col-0 was used as a negative control. RPT5 was used as a loading control. Right panel shows the quantification analysis of the three biological replicates of EBF1 (A, right panel) or EBF2 (B, right panel) protein levels after normalizing to RPT5. The protein levels EBF1-TAP/EBF2-TAP in Col-0 backgrounds without MG132 treatment (DMSO) was set as 1. Mean ±s.d., n=3.

Figure 5. EBF1 and EBF2 physically interact with COP1 in vitro and in vivo

(A) N-terminal fragments of COP1 directly interact with EBF1 and EBF2 in yeast two-hybrid assays. Left diagrams indicate the various fragments of COP1 fused with the activation domain. Full length EBF1 and EBF2 fused with LexA DNA binding domain were the prey constructs in the assay. (B) COP1 and EBF1/2 directly interact in pull-down assays. Purified COP1-His was used as prey and was pulled down by the baits EBF1-MBP, EBF2-MBP and MBP, respectively. Anti-MBP and anti-His were used for the immunoblot analysis. (C) BiFC assay reveals that COP1 interacts with EBF1 and EBF2 in the nucleus of Nicotiana benthamiana leaf cells. Full length COP1 or EBF1 and EBF2 were fused to the split N-terminal or C-terminal (YFPⁿ or YFP^c) fragments of YFP. GST fused to YFPⁿ or YFP^c fragments were used as negative controls. Red arrow indicates the position of YFP speckles. Bar = 20 μm. (D) Semi-in vivo co-immunoprecipitation assay of COP1 with EBF1 and EBF2 proteins. EBF1-GFP and EBF2-GFP overexpression transgenic plants and Col-0 control were grown in the dark for 4 days. Equal amount of COP1-His proteins were added into the cell extracts and immunoprecipitated using anti-GFP antibody and immunoblotted using indicated antibodies.

Figure 6. COP1 directly ubiquitinates both EBF1 and EBF2 proteins

(A-B) In vitro ubiquitination assays of EBF1 (A) or EBF2 (B) by COP1. Purified recombinant COP1-His and EBF1-MBP or EBF2-MBP were used in the assays. The ubiquitination of EBF1 or EBF2 was analyzed by western blot using an anti-MBP antibody.

Figure 7. COP1 modulates the level of EIN3 proteins in response to the increased light fluences as seedlings grow towards the surface

(A-B) Western blots showing EIN3 protein levels in the 4-day old seedlings grown in the dark (A) or white light (B). Seedlings over-expressing EIN3-Myc in ein3eil1 (E3Mee) and cop1ein3eil1 (E3Meec-4) backgrounds were grown on 1/2MS medium with or without 2.5mm soil (50-70 mesh Sand) covering. Bottom panels show the quantification analysis of the three biological replicates of EIN3-Myc protein levels after normalizing to Actin. The protein levels of EIN3-Myc/ein3eil1 without soil covering was set as 1. Mean ±s.d., n=3. (C-D) Western blots showing the EIN3-Myc protein levels in ein3eil1 (C) or cop1ein3eil1 (D) background seedlings upon mechanical stresses. Seedlings were grown on 1/2MS medium for 4 days in the dark, then were pressed by a glass plate with the pressure of about 150Pa for the indicated periods of time. Bottom panels show the quantification analysis of the three biological replicates of EIN3-Myc protein levels after normalizing to Actin. The protein level of EIN3-Myc/ein3eil1 without mechanical stress was set as 1. Mean ±s.d., n=3. (E) Light fluence in the soil increases as seedlings grow towards the soil surface. The light fluence was measured under the water-saturated soils with indicated particle sizes and depths. (F) Western blots showing the EIN3 and HY5 protein levels in ein3eil1 and cop1ein3eil1 seedlings grown under indicated light fluences (μmolm²s^-1) for 4 days. Dark-grown 4 day-old Col-0 was used as a negative control. Bottom panel shows the quantification analysis of the three biological replicates of EIN3-Myc protein levels after normalizing to Actin. The protein level of EIN3-Myc/ein3eil1 in the dark was set as 1. Mean ±s.d., n=3.