PIF3 is a phytochrome-interacting basic helix-loop-helix transcription factor that negatively regulates light responses, including hypocotyl elongation, cotyledon opening, and hypocotyl negative gravitropism. However, the role of PIF3 in chlorophyll biosynthesis has not been clearly defined. Here, we show that PIF3 also negatively regulates chlorophyll biosynthesis by repressing biosynthetic genes in the dark. Consistent with the gene expression patterns, the etiolated pif3 mutant accumulated a higher amount of protochlorophyllide and was bleached severely when transferred into light. The photobleaching phenotype of pif3 could be suppressed by the gun5 mutation and mimicked by overexpression of GUN5. When 4 negative phytochrome-interacting protein genes (PIF1, PIF3, PIF4, and PIF5) were mutated, the resulting quadruple mutant seedlings displayed constitutive photomorphogenic phenotypes, including short hypocotyls, open cotyledons, and disrupted hypocotyl gravitropism in the dark. Microarray analysis further confirmed that the dark-grown quadruple mutant has a gene expression pattern similar to that of red light-grown WT. Together, our data indicate that 4 phytochrome-interacting proteins are required for skotomorphogenesis and phytochromes activate photomorphogenesis by inhibiting these factors.

Edited by Ulf-Ingo Flügge Keywords:Anthocyanin biosynthesis Arabidopsis HY5 PAP1 a b s t r a c t Several positive transcription factors regulate Arabidopsis anthocyanin biosynthesis. HY5, a component of light-signaling pathways, and PAP1, an R2R3-MYB transcription factor, share common regulatory targets on anthocyanin biosynthesis genes. The epistatic interactions between the two transcription factors are currently unknown. To address this problem, we analyzed crosses between hy5 and pap1 mutants (hy5pap1) or pap1D overexpressors (hy5pap1D), performed chromatin immunoprecipitation-qPCR, and determined the PAP1 promoter region through deletion analysis. The results show that HY5 regulates PAP1 expression via direct binding to G-and ACE-boxes in the promoter region, which suggests bifurcate regulation of anthocyanin biosynthesis by HY5 via transcriptional activation of PAP1.

Phytochromes are red and far-red light photoreceptors that regulate various aspects of plant development. One of the lessunderstood roles of phytochromes is the inhibition of hypocotyl negative gravitropism, which refers to the loss of hypocotyl gravitropism and resulting random growth direction in red or farred light. This light response allows seedlings to curve toward blue light after emergence from the soil and enhances seedling establishment in the presence of mulch. Phytochromes inhibit hypocotyl negative gravitropism by inhibiting four phytochrome-interacting factors (PIF1, PIF3, PIF4, PIF5), as shown by hypocotyl agravitropism of dark-grown pif1 pif3 pif4 pif5 quadruple mutants. We show that phytochromes inhibit negative gravitropism by converting starchfilled gravity-sensing endodermal amyloplasts to other plastids with chloroplastic or etioplastic features in red or far-red light, whereas PIFs promote negative gravitropism by inhibiting the conversion of endodermal amyloplasts to etioplasts in the dark. By analyzing transgenic plants expressing PIF1 with an endodermisspecific SCARECROW promoter, we further show that endodermal PIF1 is sufficient to inhibit the conversion of endodermal amyloplasts to etioplasts and hypocotyl negative gravitropism of the pif quadruple mutant in the dark. Although the functions of phytochromes in gravitropism and chloroplast development are normally considered distinct, our results indicate that these two functions are closely related.

Seedling hypocotyls display negative gravitropism in the dark but agravitropism in the light. The Arabidopsis thaliana pif quadruple mutant (pifQ), which lacks four PHYTOCHROME-INTERACTING FACTORS (PIFs), is agravitropic in the dark. Endodermis-specific expression of PIF1 rescues gravitropism in pifQ mutant seedlings. Since phytochromes induce light responses by inhibiting PIFs and the COP1-SPA ubiquitin E3 ligase complex in the nucleus, we asked whether phyB can cell autonomously inhibit hypocotyl negative gravitropism in the endodermis. We found that while epidermis-specific expression of PHYB rescues hypocotyl negative gravitropism and all other phyB mutant phenotypes, endodermis-specific expression of PHYB does not. Epidermal phyB induces the phosphorylation and degradation of endodermal PIFs in response to red light. This induces a global gene expression pattern similar to that induced by red light treatment of seedlings expressing PHYB under the control of its own endogenous promoter. Our results imply that epidermal phyB generates an unidentified mobile signal that travels to the endodermis where it promotes PIF degradation and inhibits hypocotyl negative gravitropism.

Mutations in Phytochrome Interacting Factors (PIFs) induce a conversion of the endodermal amyloplasts necessary for gravity sensing to plastids with developed thylakoids accompanied by abnormal activation of photosynthetic genes in the dark. In this study, we investigated how PIFs regulate endodermal plastid development by performing comparative transcriptome analysis. We show that both endodermal expression of PIF1 and global expression of the PIF quartet induce transcriptional changes in genes enriched for nuclear-encoded photosynthetic genes such as LHCA and LHCB. Among the 94 shared differentially expressed genes identified from the comparative transcriptome analysis, only 14 genes are demonstrated to be direct targets of PIF1, and most photosynthetic genes are not. Using a co-expression analysis, we identified a direct target of PIF, whose expression pattern shows a strong negative correlation with many photosynthetic genes. We have named this gene REPRESSOR OF PHOTOSYNTHETIC GENES1 (RPGE1). Endodermal expression of RPGE1 rescued the elevated expression of photosynthetic genes found in the pif quadruple (pifQ) mutant and partly restored amyloplast development and hypocotyl negative gravitropism. Taken together, our results indicate that RPGE1 acts downstream of PIF1 in the endodermis to repress photosynthetic genes and regulate plastid development.

Plant seedlings germinating under the soil are challenged by rough soil grains that can induce physical damage and sudden exposure to light, which can induce photobleaching. Seedlings overcome these challenges by developing apical hooks and by suppressing chlorophyll precursor biosynthesis. These adaptive responses are, respectively, regulated by the phytochrome and ethylene signaling pathways via the PHYTOCHROME-INTERACTING FACTORs (PIFs) and the ETHYLENE INSENSITIVE 3 (EIN3)/EIN3-LIKE transcription factors. Although many processes downstream of phytochrome and ethylene signaling are similar, it remains unclear if and where these pathways converge. Here, we show PIFs and EIN3 induce similar changes in the transcriptome without robustly regulating each other’s signaling pathways. PIFs and EIN3 target highly overlapped gene promoters and activate subsets of the co-target genes either interdependently or additively to induce plant responses. For chlorophyll biosynthesis, PIFs and EIN3 target and interdependently activate the expression of HOOKLESS1. HOOKLESS1, in turn, represses chlorophyll synthesis genes to prevent photobleaching. Thus, our results indicate an integration of the phytochrome and ethylene signaling pathways at the level of transcriptional gene regulation by two core groups of transcription factors, PIFs and EIN3.

PHYTOCHROME-INTERACTING FACTOR1 (PIF1) is a basic helix-loop-helix transcription factor that inhibits light-dependent seed germination in Arabidopsis thaliana. However, it remains unclear whether PIF1 requires other factors to regulate its direct targets. Here, we demonstrate that LEUNIG_HOMOLOG (LUH), a Groucho family transcriptional corepressor, binds to PIF1 and coregulates its targets. Not only are the transcriptional profiles of the luh and pif1 mutants remarkably similar, more than 80% of the seeds of both genotypes germinate in the dark. We show by chromatin immunoprecipitation that LUH binds a subset of PIF1 targets in a partially PIF1-dependent manner. Unexpectedly, we found LUH binds and coregulates not only PIF1-activated targets but also PIF1-repressed targets. Together, our results indicate LUH functions with PIF1 as a transcriptional coregulator to inhibit seed germination.

Starch granules in the endodermis of plant hypocotyls act as statoliths that promote hypocotyl negative gravitropism—the directional growth of hypocotyls against gravity—in the dark. To identify the molecular components that regulate hypocotyl negative gravitropism, we performed a mutagenesis screen and isolated reduced gravitropic 1 (rgv1) mutants that lack starch granules in their hypocotyl endodermis and show reduced hypocotyl negative gravitropism in the dark. Using whole genome sequencing, we identified three different rgv1 mutants that are allelic to the previously reported early starvation 1 mutant, which is rapidly depleted of starch just before the dawn. ESV1 orthologs are present in starch-producing green organisms, suggesting ESV1 is a functionally conserved protein necessary for the formation of starch granules. Consistent with this, we found that liverwort and rice ESV1 can complement the Arabidopsis ESV1 mutant phenotype for both starch granules and hypocotyl negative gravitropism. To further investigate the function of ESV1 in other plants, we isolated rice ESV1 mutants and found that they show reduced levels of starch in their leaves and loosely packed starch granules in their grains. Both Arabidopsis and rice ESV1 mutants also lack starch granules in root columella and show reduced root gravitropism. Together, these results indicate ESV1 is a functionally conserved protein that promotes gravitropic responses in plants via its role in starch granule formation.