Ethylene is crucial for cotyledon greening and seedling survival during de-etiolation

Abstract

The most remarkable change of de-etiolation in seedling is chlorophyll synthesis and greening. This transition is achieved by photoreduction of dark-accumulated protochlorophyllide (Pchlide) in light. However, overaccumulation of Pchlide results in phototoxicity to plants, so appropriate accumulation and quick reduction of Pchlide are crucial for survival of seedlings during the transition from dark to light. We found that this vital process is tightly regulated by the plant gaseous hormone ethylene. Transgenic analysis using a promoter-GUS reporter system showed that the ethylene signaling was able to activate the expression of PORA (protochlorophyllide oxidoreductase A) gene in seedling cotyledons. We further found that application of ethylene rescued the greening defect of the flu mutant, which over-accumulated Pchlide in the dark. Additionally, genetic studies revealed that Ethylene Insensitive 3 (EIN3) and EIN3-like 1 (EIL1）regulate Pchlide accumulation and cotyledon greening largely independent of Phytochrome-Interacting Factor 1 (PIF1) but partly dependent on PIF3. Therefore, the ethylene signaling via EIN3/EIL1 presents a new pathway to constrain phototoxic Pchlide accumulation in darkness, and simultaneously facilitate Pchlide reduction to synthesize chlorophyll upon light exposure. Our results thus uncover an essential role of ethylene in protecting seedlings from photo-oxidative damage during the process of de-etiolation.

Figure 1

Ethylene activates PORA gene expression in cotyledons of etiolated seedlings. The imaging of GUS staining of 4-day-old dark-grown seedlings (#6, #3 represent two independent transgenic lines containing PORA-promoter-GUS) was shown. The seedlings were grown on MS medium supplemented with or without 10 µM ACC or 100 µM AgNO₃ (Ag⁺).

Figure 2

Ethylene application greatly represses Pchlide overaccumulation in the flu mutant and improves its greening rate. (A) Greening rate quantification of 5-day-old etiolated seedlings transferred to white light (WL) for 1 day. Error bars represent standard error of at least three independent experiments, and more than 50 seedlings were used for calculation each time. (B) Relative fluorescence of Pchlide of 3-day-old etiolated seedlings grown on MS medium supplemented with or without 10 µM ACC.

Figure 3

Ethylene application represses Pchlide overaccumulation and improves greening rates of the pif3 mutant via the action of EIN3/EIL1. (A and C) Greening rate quantification of 4-day-old etiolated seedlings transferred to white light (WL) for 1 day. Error bars represent standard error of at least three independent experiments, and more than 50 seedlings were used for calculation each time. (B and D) Relative fluorescence of Pchlide of 3-day-old etiolated seedlings grown on MS medium supplemented with or without 10 µM ACC.