1. Volume 9, Issue 10, Pages 1415-1427 (October 2016)
PIF1 Regulates Plastid Development by Repressing Photosynthetic Genes in the Endodermis 
Keunhwa Kim, Jinkil Jeong, Jeongheon Kim, Nayoung Lee, Mi Eon Kim, Sangil Lee, Sun Chang Kim, Giltsu Choi 
Molecular Plant 
Volume 9, Issue 10, Pages (October 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Red Light Inhibits Hypocotyl Negative Gravitropism by Promoting the Degradation of Endodermal PIF1.
(A) Restoration of hypocotyl negative gravitropism in the pifQ by endodermal PIF1. Seedlings were grown in the dark (Dc upper panel) or continuous red light (Rc lower panel) for 3 days on a vertical plate. Scale bar, 0.5 cm.
(B) Disruption of hypocotyl negative gravitropism by endodermal expression of a dominant-negative PIF3. Seedlings were grown on a horizontal plate for 3 days in the dark. PIF3dN indicates PIF3 lacking 300 amino acids from its N terminus. Bars indicate means + SD (n = 3 biological replicates); *p < 0.05, **p < 0.01 by Student's t-test.
(C) Inhibition of endodermal PIF1 by red light determined by the expression of PIF1 target genes. Bars show the expression of two PIF1 target genes, PIL1 and PIL2, in 3-day-old dark- or continuous red-light-grown seedlings. Expression levels of PIL1 and PIL2 were normalized to those of dark-grown wild-type seedlings. Means + SD (n = 4); **p < 0.01 by Student's t-test. SCR:PIF1 indicates the SCR:PIF1/pifQ line.
(D) Degradation of endodermal PIF1 by red light. PIF1 was detected by immunoblot analysis using an α-PIF1 antibody. Empty and filled arrowheads indicate endogenous PIF1 and GFP-tagged endodermal PIF1, respectively. An asterisk indicates a non-specific band, and pS indicates the Ponceau S-stained loading control.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Endodermal PIF1 Partially Restores Wild-Type Gene Expression Patterns in Dark-Grown pifQ Mutant Seedlings.
(A) Venn diagram visualizing the overlap between DEGs from pifQ versus wild-type (WT) (pqDEGs) and SCR:PIF1/pifQ versus pifQ (epDEGs). Plants were grown in the dark for 2.5 days for these transcriptome analyses. DEGs are defined as genes with ≥2-fold changes in expression and FDR <0.05.
(B) Scatter plot comparing the expression levels of non-overlapping DEGs in the pifQ (pifQ vs Col-0) and SCR:PIF1 (SCR:PIF1/pifQ vs pifQ) transcriptomes. 307 pifQ-specific DEGs (pqsDEG) and 523 endodermal PIF1-specific DEGs (epsDEGs) are plotted. The x and y axes indicate log2 expression fold changes in the pifQ and SCR:PIF1 transcriptomes, respectively. White circles indicate genes showing statistically significant (FDR <0.05) fold change differences in the SCR:PIF1 (left) or pifQ transcriptome datasets (right). Gray circles indicate statistically insignificant genes.
(C) GSEA analysis indicating the level of enrichment for categories of non-overlapping DEGs in each transcriptome dataset. Data are presented as normalized enrichment scores mapped onto a color scale spanning blue (strong negative correlation) to yellow (strong positive correlation). Gray indicates a statistically insignificant enrichment (FDR >0.01). pifQ and SCR:PIF1 indicate the pifQ and SCR:PIF1 transcriptomes, respectively. UP and DOWN indicate gene sets comprising up- or downregulated pifQ or SCR:PIF1 DEGs.
(D) GO term enrichment using BiNGO on pqsDEGs (gray) and epsDEGs (black). White bars indicate the percentage enrichment across the whole genome. Asterisks indicate FDR <0.05 by the hypergeometric test.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 A Subset of Shared DEGs Are Direct Targets of PIF1.
(A) Heatmap visualization of the inverse patterns of expression among the DEGs shared between the pifQ and SCR:PIF1 transcriptome datasets (r = −0.82).
(B) Classification of 94 shared DEGs based on their expression patterns in the pifQ and SCR:PIF1 transcriptome datasets. The x and y axes are identical to those in Figure 2B.
(C) Enrichment of photosynthesis-related GO terms among class 4 genes.
(D) Identification of PIF1 targets by comparing the shared genes with genome-wide PIF1 binding sites.
(E) ChIP qPCR analysis showing direct binding of global and endodermal PIF1 to 14 PIF1 Target Genes (PTGs). ChIP was performed on 5-day-old dark-grown seedlings with an α-PIF1 antibody. Values are normalized to the 25S rDNA negative control fragment. Bars indicate means + SD (n = 4).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
PIFs Directly Induce the Expression of Repressor of Photosynthetic Genes1 (RPGE1; PTG11).
(A) Co-expression analysis between PTGs and class 4 genes using CORNET 2.0. Orange circles are PTGs and green diamonds are class 4 genes. Edges connect PTGs and class 4 genes whose expression patterns show significant correlation (p < 0.05). Edge color indicates the range of the correlation coefficient (yellow, −0.9 < r < −0.8; green, −0.8 < r < −0.7; brown, −0.7 < r < −0.5).
(B) The RPGE Family in Arabidopsis Includes RPGE1-4.
(C) Direct binding of four PIFs to the promoters of RPGE1 and RPGE2. ChIP assay was performed using an α-myc antibody on dark-grown stable transgenic plants expressing myc-tagged PIFs (PIFx-OX) or GFP (GFP-myc). RPGExp indicates RPGEx to which the promoter fragments belong. Bars indicate means + SD (n = 4) calculated relative to input DNA.
(D) Direct binding of endodermal PIF1 to the RPGE1 and RPGE2 promoters. ChIP was performed using an α-PIF1 antibody on dark-grown pifQ and SCR:PIF1/pifQ (SCR:PIF1) seedlings.
(E) PIF-dependent expression of RPGE1 and RPGE2 mRNAs. Expression levels were determined by qRT–PCR on 3-day-old dark-grown (Dc) and continuous red-light-grown (Rc) seedlings. Expression levels were normalized to PP2A levels. Bars indicate means + SD (n = 4).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5 RPGE1 Represses the Expression of Photosynthetic Genes.
(A) Pale green leaves induced by global 35S-driven RPGE1 (RPGE1-OX) and RPGE2 (RPGE2-OX). Seedlings were grown under light:dark (16 h light:8 h dark) conditions for 2 weeks.
(B) Low chlorophyll levels in RPGE1-OXs. Chlorophyll content was measured in 4-day-old Rc-grown seedlings and normalized to that of wild-type seedlings. Bars indicate means + SD (n = 4); **p < 0.01 by Student's t-test.
(C) Repression of photosynthetic genes by global RPGE1 in the dark. Expression of four nuclear genes encoding subunits of the chloroplast antenna complex was determined using 3-day-old etiolated seedlings. Bars indicate means + SD (n = 4).
(D) Repression of photosynthetic genes by SCR-driven endodermal RPGE1 (SCR:RPGE1) in the pifQ mutant background. Gene expression was determined using 2-day-old etiolated seedlings from three independent SCR:RPGE1/pifQ transgenic lines (SCR:RPGE1-x). Bars indicate means + SD (n = 4).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
Endodermal RPGE1 Partially Restores Hypocotyl Negative Gravitropism in pifQ Mutants.
(A) Partial restoration of hypocotyl negative gravitropism in three independent SCR:RPGE1/pifQ lines. Seedlings were grown on horizontal plates in the dark for 3 days. *p < 0.05, **p < 0.01 by Student's t-test. Bars indicate means + SD (n = 3).
(B) Partial restoration of endodermal amyloplast starch levels as visualized by I2-KI staining. Seedlings were grown in the dark for 3 days. Scale bar, 100 μm.
(C) A model under which endodermal PIF1 directly activates the expression of RPGE1 in the endodermis. RPGE1 then represses photosynthetic (PS) genes to suppress the conversion of amyloplasts to plastids with developed thylakoids expressing high levels of photosynthetic genes. Such suppression is partly necessary to maintain the starch-filled amyloplasts necessary for gravity sensing. Since the repression of PS genes by RPGE1 is insufficient to fully restore amyloplasts and hypocotyl negative gravitropism, there may be another factor (X) involved in this process.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions