EIN3/EIL1 cooperate with PIF1 to prevent photo-oxidation and to promote greening of Arabidopsis seedlings

Abstract

The ability to switch from skotomorphogenesis to photomorphogenesis is essential for seedling development and plant survival. Recent studies revealed that COP1 and phytochrome-interacting factors (PIFs) are key regulators of this transition by repressing the photomorphogenic responses and/or maintaining the skotomorphogenic state of etiolated seedlings. Here we report that the plant hormone ethylene plays a crucial role in the transition from skotomorphogenesis to photomorphogenesis by facilitating greening of etiolated seedlings upon light irradiation. Activation of EIN3/EIL1 is both necessary and sufficient for ethylene-induced enhancement of seedling greening, as well as repression of the accumulation of protochlorophyllide, a phototoxic intermediate of chlorophyll synthesis. EIN3/EIL1 were found to induce gene expression of two key enzymes in the chlorophyll synthesis pathway, protochlorophyllide oxidoreductase A and B (PORA/B). ChIP and EMSA assays demonstrated that EIN3 directly binds to the specific elements present in the PORA and PORB promoters. Genetic studies revealed that EIN3/EIL1 function in cooperation with PIF1 in preventing photo-oxidative damage and promoting cotyledon greening. Moreover, activation of EIN3 reverses the blockage of greening triggered by cop1 mutation or far-red light irradiation. Consistently, EIN3 acts downstream of COP1 and its protein accumulation is enhanced by COP1 but decreased by light. Taken together, EIN3/EIL1 represent a new class of transcriptional regulators along with PIF1 to optimize de-etiolation of Arabidopsis seedlings. Our study highlights the essential role of ethylene in enhancing seedling development and survival through protecting etiolated seedlings against photo-oxidative damage.

Fig. 1.

Ethylene facilitates greening of etiolated seedlings via an EIN3/EIL1-dependent pathway. (A) Greening rate of 3-day-old etiolated seedlings followed by 2 days of white light irradiation (WL) on MS medium supplemented with indicated concentrations of ACC. (B) Greening rate of 3- or 4-day-old etiolated seedlings followed by 2 days of WL with or without 10-μM ACC treatment. Cotyledon phenotype of 3-day-old (C) or 5-day-old (D) etiolated seedlings followed by 2 days of WL. (E and F) Greening rates of various etiolated seedlings grown in darkness for indicated number of days before exposure to WL for 2 days on MS medium with or without 10 μM ACC. Error bars represent standard error of at least three independent experiments.

Fig. 2.

EIN3/EIL1 are required for protecting seedlings from photooxidation by directly inducing the gene expression of PORA/B. (A) Fluorescence microscopy images of ROS (indicated by H₂DCFDA fluorescence) and chlorophyll fluorescence in the cotyledons of 4-day-old etiolated seedlings followed by 2 days of WL. (B) Relative fluorescence of protochlorophyllide in 3-day-old etiolated seedlings grown on MS medium with or without 10 μM ACC. (C) Relative expression levels of selected chlorophyll biosynthetic genes that are induced or repressed by EIN3/EIL1 based on microarray data. (D) Analysis of PORA/B gene expression by quantitative real-time RT-PCR of 4-day-old etiolated seedlings. (E) EMSA assays with recombinant EIN3 protein. Competition for the labeled promoter sequences was performed by adding 100-/200-/400-fold excess of unlabeled wild-type (WT) or mutant probes. (F) ChIP-PCR assays using 4-day-old etiolated seedlings. +/- Ab indicates chromatin immunoprecipitation with or without anti-EIN3 antibody. Two distinctive EIN3-binding sites were predicted in the promoter region of the PORA or PORB gene (PORA01/02, PORB01/02). Primers used for ChIP-PCR were specific to the promoter regions containing PORA01/02 or PORB01/02. Primers specific to the coding region of PORA were used as negative control. Input indicates samples before immunoprecipitation.

Fig. 3.

EIN3/EIL1 act in cooperation with PIF1 in promoting cotyledon greening. (A-D) Cotyledon phenotypes and greening rates of 4-day-old (A and B) or 5-day-old (C and D) etiolated seedlings followed by 2 days of WL. Error bars represent standard error of at least three independent experiments. (E) Relative fluorescence of protochlorophyllide in 4-day-old etiolated seedlings. (F) Fluorescence microscopy images of ROS (indicated by H₂DCFDA fluorescence) and chlorophyll fluorescence in the cotyledons of 4-day-old etiolated seedlings followed by 2 days of WL.

Fig. 4.

EIN3 protein accumulation is positively regulated by COP1 but negatively regulated by light. (A) Cotyledon phenotype of 3-day-old etiolated seedlings followed by 2 days of WL. (B) Greening rate of 2- or 3-day-old etiolated seedlings followed by 2 days of WL. (C) Relative fluorescence of protochlorophyllide in 2-day-old etiolated seedlings. (D) Immunoblot assays of EIN3 protein in 4-day-old etiolated seedlings grown on MS medium. (E) Immunoblot assays of EIN3 protein in 4-day-old etiolated seedlings grown on MS medium with indicated hours of light or dark treatment. Cotyledon phenotype (F) and greening rate (G) of seedlings that were grown in far red light (FR) for 60 h followed by 36 h of WL. Error bars represent standard error of at least three independent experiments.

Fig. 5.

A model on the action of EIN3/EIL1 and PIF1 in regulating cotyledon greening. EIN3/EIL1 and PIF1 are two classes of transcription factors that redundantly regulate chlorophyll synthesis and alleviate photooxidation of etiolated seedlings. EIN3/EIL1 and PIF1 are able to induce POR gene expression by directly binding to their promoters, and regulate a number of tetrapyrrole pathway genes to repress the accumulation of protochlorophyllide, a phototoxic intermediate in chlorophyll synthesis. Ethylene enhances seedling greening by activating EIN3/EIL1 via its canonical signal transduction pathway. Far-red light (FR) blocks seedling greening through PhyA-mediated direct inhibition of PIF1 or indirect repression of COP1, a positive regulator of EIN3/EIL1 and PIF1. Arrows and bars represent positive and negative regulation, respectively. Solid and dotted lines indicate direct and indirect regulation, respectively.