1. Phytochrome Signaling in Green Arabidopsis Seedlings: Impact Assessment of a Mutually Negative phyB–PIF Feedback Loop 
Pablo Leivar, Elena Monte, Megan M. Cohn, Peter H. Quail 
Molecular Plant 
Volume 5, Issue 3, Pages (May 2012)
DOI: /mp/sss031
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
A Negative Regulatory Loop between phyB and the PIFs Regulate Growth Responses during Seedling Deetiolation. (A) Visible phenotypes of pifq, phyB, and phyBpifq pentuple mutant in the dark and under continuous R (Rc). Wild-type (WT) Col-0 and mutant seedlings were grown for 4 d in the dark or in Rc (28 μmol m−2 s−1).(B) Rc-Fluence response curves of pifq, phyB, and phyBpifq. WT and mutant seedlings were grown for 4 d under the indicated Rc fluence rates and hypocotyl lengths were measured. Data represent mean and standard error from at least 20 seedlings.(C) phyB and PIF3 protein levels (arrowheads) in WT, phyB, and pifq. Seedlings were grown for 4 d in the dark (4d-D) or in 1.26 μmol m−2 s−1 of Rc (4d-Rc). Samples were immunoblotted with antibodies against phyB and PIF3, and tubulin was used as a loading control. n.s., non-specific.(D) Quantification of the PIF3 and phyB protein levels from the blots in (C). Data were normalized to tubulin and presented as a percentage of value of WT-Dark samples.(E) Simplified model of regulation of seedling deetiolation consistent with the genetic and molecular data presented in panels (A)–(C) and elsewhere (reviewed in Leivar and Quail, 2011).
Molecular Plant 2012 5, DOI: ( /mp/sss031)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
The phyB–PIFs Negative Regulatory Loop Is Dynamically Modulated in Response to Changes in R/FR Ratio.
(A) Visible phenotypes of pifq, phyB, and phyBpifq pentuple mutant in high R/FR (Light, WL) or in low R/FR (Shade, WL+FR). Wild-type (WT) Col-0 and mutant seedlings were grown for 2 d in WLc (20 μmol m−2 s−1) from germination onward (2dWL) and for 5 additional days in the same WL conditions with (2dWL+5d[WL+FR], R/FR ratio 0.006) or without (2dWL+5dWL, R/FR ratio 6.48) supplemental FR light.
(B) Quantification of the hypocotyl elongation phenotypes of pifq, phyB, and phyBpifq. Hypocotyl length was measured from WT and mutant seedlings grown as in (A). Data represent mean and standard error from at least 20 seedlings.
(C) PIF3 protein levels (arrowheads) in WT, phyB, and pifq. Seedlings were grown for 2 d in WL (2dWL) and then transferred to WL supplemented with FR for 1 h (2dWL+1h[WL+FR]). Two-day-old dark-grown seedlings (2dD) were used as a control. Samples were immunoblotted with antibodies against PIF3 and tubulin was used as a loading control. n.s., non-specific.
(D) phyB protein levels in WT and pifq. Seedlings were grown for 2 d in WL (2dWL) and then transferred to WL supplemented with FR (2dWL+h[WL+FR]) for the indicated time (in hours). Samples were immunoblotted with antibodies against phyB and tubulin was used as a loading control.
Molecular Plant 2012 5, DOI: ( /mp/sss031)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Examination of phyB Levels in Selected pif Mutant Lines Suggests that PIF-Regulation of phyB Levels Is Not a Major Determinant of the Magnitude of SAS Responses.
(A) Quantification of the hypocotyl elongation of selected WT and pif-mutant seedlings grown as in Figure 2. Top panel: Absolute hypocotyl lengths in 2dWL, 2dWL+5dWL, and 2dWL+5d[WL+FR]. Data represent the mean and the standard error of at least 20 seedlings. Bottom panel: Differential shade-responsiveness ([2dWL+5d[WL+FR]]–[2dWL]). For each genotype, mean hypocotyl length at 2dWL was subtracted from mean hypocotyl length at 2dWL+5d[WL+FR]. Differential responsiveness in light ([2dWL+5dWL]–[2dWL]) was also plotted as reference.
(B) PIF3 and PIF4/PIF5 differentially and dynamically regulate phyB levels in response to changes in the R:FR ratio. phyB protein levels were determined in wild-type Col-0 (WT), pif mutants, and PIF3-overexpressor (PIF3-OX) seedlings. Seedlings were grown under the same conditions described in Figure 2, either for 2 d in WL (2dWL, upper panel) or for 2 d in WL and then for 5 additional days in WL with (2dWL+5d[WL+FR], lower panel) or without (2dWL+5dWL, middle panel) supplemental FR. WT-1 and WT-2 refer to WT seedlings of two groups of seeds of different age used in the experiment. Samples were immunoblotted with antibodies against phyB and tubulin was used as a loading control.
(C) Quantification of phyB protein levels in WT and pif-mutant lines. phyB levels in each of the blots in (A) and in Supplemental Figure 4 were quantified, normalized to tubulin, and expressed relative to the WT value set at unity for each treatment. Mean phyB/tubulin relative to WT for each treatment was then calculated from the two biological replicates, except for pif1pif3 at 2dWL+5dWL in which only one replicate was available. Bars indicate standard deviation.
(D) PIL1 transcript levels in WT and pif-mutant seedlings grown for 2 d in WL (2dWL) or for 2 d in WL and then for 1 h in WL supplemented with FR (2dWL+1h[WL+FR]). PIL1 normalized to PP2A were determined by qPCR and the data are expressed relative to WT_2dWL set at unity. Data represent the mean and the standard deviation from two technical replicates.
Molecular Plant 2012 5, DOI: ( /mp/sss031)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
PIF3 Regulates Shade and EOD-FR Responses in Conjunction with PIF4 and PIF5.
(A) Visible phenotypes of WT, pif3, pif4, and pif5 single, double, and triple mutant seedlings in high R/FR (Light, WL) or in low R/FR (Shade, WL+FR). Wild-type (WT) Col-0 and mutant seedlings were grown as in Figure 2, for 2 d in WLc and for 5 additional days in the same WL conditions with (2dWL+5d[WL+FR]) or without (2dWL+5dWL) supplemental FR light.
(B) (Left panel) Quantification of the hypocotyl elongation of WT and pif-mutant seedlings shown in (A). Data represent the mean and the standard error from at least 20 seedlings. (Right panel) Differential shade-responsiveness. Mean hypocotyl length at [2dWL+5dWL] was subtracted from mean hypocotyl length at [2dWL+5d[WL+FR]] for each genotype.
(C) Quantification of hypocotyl elongation of WT, phyB, and phyBpif3. Seedlings were grown for 7 d in WL (2dWL+5dWL) as in Figure 2.
(D) (Left panel) Quantification of hypocotyl elongation of WT and pif-mutant seedlings in End-of-Day-Far-Red (EOD-FR) conditions. Seedlings were grown for 2 d in WL (2dWL) and then transferred for 5 additional days to either WLc (+5dWL), 14 h of WL followed by 10 h of darkness (+5d[14hW–10hD]), or 14 h of WL followed by a saturating pulse of FR (FRp) prior to the 10-h dark treatment (+5d[EOD-FR]). Data represent the mean and the standard error from at least 20 seedlings. (Right panel) Differential EOD-FR-responsiveness. Mean hypocotyl length at [2dWL+5d[14hWL–10hD]] was subtracted from mean hypocotyl length at [2dWL+5d[EOD-FR]] for each genotype.
Molecular Plant 2012 5, DOI: ( /mp/sss031)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Model Depicting a Central Role of the phyB–PIFs Negative Regulatory Loop in Regulating Growth Responses to Changes in R/FR Ratio.
In seedlings growing under continuous red light (Rc) or under high R/FR conditions (upper panel), the phy photoequilibrium is shifted towards the active Pfr state. Photoactive phy interacts with and induces the proteolytic degradation of members of the PIF quartet, and this reduction of the transcription factors inhibits the growth of the seedling. In turn, the PIFs induce the proteolytic degradation of the phyB photoreceptor, thus reducing the overall sensitivity of the seedling to light to optimize growth. In seedlings growing under low R/FR conditions (lower panel), phy photoequilibrium is shifted towards the inactive Pr form, causing a partial reversion to the etiolated state (Leivar et al., 2012). Under these conditions, the phyB–PIF negative regulatory loop is abrogated, resulting in an increase in phyB and PIF abundance. Accumulation of these PIFs induces a downstream gene-expression regulatory network that results in the induction of SAS responses (Leivar et al., 2012). A significant part of the phy-mediated growth responses to changes in R/FR is mediated by factors other than the PIF quartet, here depicted as X.
Molecular Plant 2012 5, DOI: ( /mp/sss031)
Copyright © 2012 The Authors. All rights reserved. Terms and Conditions