PIF3 is a repressor of chloroplast development

Abstract

The phytochrome-interacting factor PIF3 has been proposed to act as a positive regulator of chloroplast development. Here, we show that the pif3 mutant has a phenotype that is similar to the pif1 mutant, lacking the repressor of chloroplast development PIF1, and that a pif1pif3 double mutant has an additive phenotype in all respects. The pif mutants showed elevated protochlorophyllide levels in the dark, and etioplasts of pif mutants contained smaller prolamellar bodies and more prothylakoid membranes than corresponding wild-type seedlings, similar to previous reports of constitutive photomorphogenic mutants. Consistent with this observation, pif1, pif3, and pif1pif3 showed reduced hypocotyl elongation and increased cotyledon opening in the dark. Transfer of 4-d-old dark-grown seedlings to white light resulted in more chlorophyll synthesis in pif mutants over the first 2 h, and analysis of gene expression in dark-grown pif mutants indicated that key tetrapyrrole regulatory genes such as HEMA1 encoding the rate-limiting step in tetrapyrrole synthesis were already elevated 2 d after germination. Circadian regulation of HEMA1 in the dark also showed reduced amplitude and a shorter, variable period in the pif mutants, whereas expression of the core clock components TOC1, CCA1, and LHY was largely unaffected. Expression of both PIF1 and PIF3 was circadian regulated in dark-grown seedlings. PIF1 and PIF3 are proposed to be negative regulators that function to integrate light and circadian control in the regulation of chloroplast development.

Fig. 1.

Dark-grown phenotype of pif mutant seedlings. (A) Protochlorophyllide accumulation in WT and pif mutant seedlings in darkness. (B) Cotyledons of WT and pif mutant seedlings after 4 d in the dark. (C) Hypocotyl growth of WT and pif mutant seedlings in darkness. Values shown in A and C are the mean ± SE of 4 and 3 independent experiments, respectively. Photographs shown in B are representative and at the same scale.

Fig. 2.

Plastid ultrastructure in pif mutant seedlings. Transmission electron micrographs of plastids from WT (A, E, and I), pif1 (B, F, and J), pif3 (C, G, and K), and pif1pif3 (D, H, and L) seedlings. Seedlings were grown for 4 d in the dark (A–D), 2 d in the dark followed by 1 d of WL (110 μmol·m⁻²·s⁻¹) (E–H), or 4 d in the dark followed by 1 d of WL (I–L). [Scale bars: 500 nm (A–D) and 1 μm (E–L).]

Fig. 3.

Light-grown phenotype of pif mutant seedlings. (A) Chlorophyll accumulation in WT and pif mutant seedlings after transfer to WL (110 μmol·m⁻²·s⁻¹) after 4 d in the dark. (B) Chlorophyll levels in WT and pif mutant seedlings after 4 d in the dark and either 2 or 4 h of WL. (C and D) Chlorophyll levels in WT and pif mutant seedlings after 8 h of WL after different dark periods (C) or after 4 d in the dark and transfer to 1 d of WL of different fluence rates (D). Values shown are the mean ± SE of 4 independent experiments.

Fig. 4.

Expression of tetrapyrrole synthesis genes in pif mutant seedlings. (A) Real-time PCR data showing expression of HEMA1 in dark-grown pif mutant seedlings. Data are presented as the fold difference from WT after normalizing to the control gene YLS8. (B) GUN4 and CHLH expression as for (A). (C) Glu-TR protein levels in WT and pif mutant seedlings after 2 d in the dark. One of 2 repeat experiments with similar results is shown, and equal protein loading was confirmed by staining duplicate gels. (D) Expression of HEMA1 in dark-grown WT and pif mutant seedlings replotted from (A). (E) Expression of PIF1, PIF3, and HEMA1 in dark-grown WT seedlings. (F) Expression of HEMA1 in WT and pif mutants after either 3 d in the dark (filled symbols) or 2 d in the dark + 1 d of WL (110 μmol·m⁻²·s⁻¹; open symbols). Vertical bars indicate the level of light induction. Values shown are the mean ± SE of ≥3 independent experiments.

Fig. 5.

Model for regulation of tetrapyrrole synthesis genes and chloroplast development in pif mutant seedlings.