Genome-wide regulation of light-controlled seedling morphogenesis by three families of transcription factors

Abstract

Three families of transcription factors have been reported to play key roles in light control of Arabidopsis seedling morphogenesis. Among them, bHLH protein PIFs and plant-specific protein EIN3/EIN3-LIKE 1 (EIN3/EIL1) accumulate in the dark to maintain skotomorphogenesis. On the other hand, HY5 and HY5 HOMOLOG (HYH), two related bZIP proteins, are stabilized in light and promote photomorphogenic development. To systemically investigate the transcriptional regulation of light-controlled seedling morphogenesis, we generated HY5ox/pifQein3eil1, which contained mutations of EIN3/EIL1 and four PIF genes (pifQein3eil1) and overexpression of HY5 Our results show that dark-grown HY5ox/pifQein3eil1 seedlings display a photomorphogenesis highly similar to that of wild-type seedlings grown in continuous light, with remarkably enhanced photomorphogenic phenotypes compared with the pifQ mutants. Consistent with the genetic evidence, transcriptome analysis indicated that PIFs, EIN3/EIL1, and HY5 are dominant transcription factors in collectively mediating a wide range of light-caused genome-wide transcriptional changes. Moreover, PIFs and EIN3/EIL1 independently control the expression of light-regulated genes such as HLS1 to cooperatively regulate apical hook formation, hypocotyl elongation, and cotyledon opening and expansion. This study illustrates a comprehensive regulatory network of transcription activities that correspond to specific morphological aspects in seedling skotomorphogenesis and photomorphogenesis.

Keywords: EIN3/EIL1; PIFs; ethylene signal; light signal; plant photobiology.

Fig. 1.

PIFs, EIN3/EIL1, and HY5 are the major transcription factors in directing light-controlled seedling morphogenesis. (A) Images of 4-d-old Col-0 (WT), ein3eil1 (ee), pifQ, pifQee, HY5ox/pifQee, and cop1-4 seedlings grown in the dark (D4) or in continuous red light (R4). (Scale bar, 2 mm). (B) Cotyledon photographs of 10-d-old dark-grown seedlings. (Scale bar, 0.5 mm.) (C) Hypocotyl lengths of 4-d-old seedlings grown in the dark (D4) or in R4. The WT value was set as 1, and the relative hypocotyl lengths were calculated. Error bars represent SD (n = 20). (D) Cotyledon areas of 10-d-old dark-grown etiolated seedlings. The WT value was set as 1, and the relative cotyledon areas were calculated. Error bars represent SD (n = 20).

Fig. 2.

Light-induced genome-wide transcriptional changes are largely mediated by PIFs, EIN3/EIL1, and HY5. (A) Venn diagram showing overlaps among genes regulated by light, pifQ, pifQee, and HY5ox/pifQee. The seedlings were grown for 4 d in the dark (D4) or in R4. (B) Expression levels of the PIFs/EIN3/HY5- and light-coregulated 3,267 genes in different genotypes. The FPKM values of these genes were normalized based on the Z-score method. (C) Box plot representation of the PIFs/EIN3/HY5-mediated light down-regulated (Left) or light up-regulated (Right) gene FPKM values in different genotypes. **P < 0.01, Wilcoxon test. (D and E) Gene ontology analysis of the light down-regulated (D), or light up-regulated (E) genes mediated by the PIFs/EIN3/HY5 pathway.

Fig. 3.

PIFs and EIN3/EIL1 independently regulate the expression of light-responsive genes. (A) Representative images of GUS staining in 4-d-old dark-grown seedlings. 5XEBS-GUS transgenic lines harbor five tandem repeats of EBS fused with the GUS reporter gene. C₂H₄ indicates that the seedlings were treated with C₂H₄ gas for 4 h before staining. PIF1p- to PIF5p-GUS indicates that the GUS reporter gene is driven, respectively, by the promoter of PIF1 to PIF5 genes. (Scale bar, 0.2 mm.) (B and C) qRT-PCR analysis of the expression levels of light-repressed (B) or light-activated (C) genes in 4-d-old etiolated seedlings grown on half-strength Murashige and Skoog (1/2 MS) medium supplemented with 10 µM β-estradiol. iPIF1/pifQee to iPIF5/pifQee are the inducible transgenic plants in which the respective PIF1 to PIF5 genes driven by β-estradiol–induced pER8 promoters are transformed into pifQee background. EIN3/pifQee presents the transgenic plants with EIN3 native promoter-driven EIN3 gene in the pifQee background. The gene expression levels were normalized to that of two internal control genes (PP2A and SAND) and relative to the WT sample. Error bars indicate the SD of three technical repeats. All experiments were repeated independently three times.

Fig. 4.

Apical hook formation is promoted by PIFs and EIN3/EIL1 through directly activating HLS1 transcription. (A and B) Apical hook images of 4-d-old etiolated seedlings grown on half-strength Murashige and Skoog (1/2 MS) medium without (MS) or with (ACC) 10 µM ACC supplementation (A) or on 1/2 MS medium supplemented with 10 µM β-estradiol (B). (C and D) qRT-PCR analysis of HLS1 gene expression levels in 4-d-old etiolated seedlings grown on 1/2 MS medium supplemented without or with 10 µM ACC (C) or on 1/2 MS medium supplemented with 10 µM β-estradiol (D). (E) Visualization of EIN3 and PIFs ChIP-seq data in the genomic regions encompassing the promoter region of HLS1 gene. (F) ChIP-qPCR analyzing the associations of PIFs and EIN3 with HLS1 promoter in 4-d-old etiolated seedlings grown on 1/2 MS medium supplemented with 10 µM β-estradiol (Left) and without or with 10 µM ACC (Right). iPIF1 to iPIF5 are the respective iPIF1/pifQee to iPIF5/pifQee transgenic plants, and EIN3 presents the EIN3/pifQee transgenic plants. WT and PP2A promoter (PP2Ap) were used as controls. **P < 0.01, Student’s t test. Error bars indicate the SD of three technical repeats. All experiments were repeated independently three times.

Fig. 5.

PIFs and EIN3/EIL1 cooperatively regulate hypocotyl elongation, cotyledon opening, and cotyledon expansion. (A and B) Representative images (A) and hypocotyl lengths (B) of 4-d-old etiolated seedlings grown on half-strength Murashige and Skoog (1/2 MS) medium supplemented with 10 µM β-estradiol. (Scale bar, 2 mm.) The WT value was set as 1, and the relative hypocotyl lengths were calculated. Error bars represent SD (n = 20). *P < 0.05, Student’s t test. (C–F) Representative cotyledon images (C and E) and cotyledon areas (D and F) of 4-d-old etiolated seedlings grown on 1/2 MS medium supplemented with 10 µM β-estradiol (C and D) and without (MS) or with (ACC) 10 µM ACC (E and F). (Scale bar, 0.5 mm.) The WT value was set as 1, and the relative cotyledon areas were calculated. Error bars represent SD (n = 20).

Fig. 6.

Model of the transcriptional regulation of light-controlled seedling morphogenesis by PIFs, EIN3/EIL1, and HY5. (A) PIFs, EIN3/EIL1, and HY5 direct the transcriptional network underlying light responses in seedlings. PIFs and EIN3/EIL1 cooperatively repress light-induced morphological transition, including apical hook unfolding, cotyledon opening and expansion, and inhibition of hypocotyl elongation, whereas HY5 inhibits hypocotyl elongation to promote light responses. (B) Light oppositely regulates the protein levels of PIFs, EIN3/EIL1, and HY5 to initiate seedling morphological transition. When grown in the dark, photoreceptor phyB is inactive, and the central repressor COP1 is activated to control the protein abundance of downstream factors. PIFs and EIN3/EIL1 accumulate and function collectively in sustaining skotomorphogenesis. Upon light activation, phyB directly induces rapid degradation of PIFs and EIN3/EIL1, and indirectly stabilizes HY5 by repressing COP1’s action to promote the photomorphogenic development.