BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth methods
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BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
To estimate the stabilization of this mutant protein, we treated PIF4 and PIF41A seedlings with cycloheximide (CHX) and with CHX and BL ( ). In these studies, PIF4 is destabilized by 30 min of CHX application, while PIF41A is still detectable after 90 min of CHX treatment. Also, upon BL application, PIF4 half-life was similar to that of the PIF41A protein, whereas this hormone did not have any effect on PIF41A protein levels ( ). Consistent with these results, when PIF4 and PIF41A lines were crossed to the bin2.1 mutant, we observed that constitutive activation of BIN2 in these seedlings leads to a strong reduction in PIF4 protein levels but has only a minor effect on PIF41A protein stability ( ).
BIN2 phosphorylation plays a main role in destabilizing the PIF4 protein at dawn
Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treated for 12 h with 0.5 µM BRZ before starting the assay. Three hours before lights on, seedlings were changed to 0.5 µM BRZ (mock) or 1 µM BL (BL) medium, and 50 µM CHX was added 1 h later. Seedlings were kept in darkness and harvested at different times after CHX application, as indicated. Western blot analyses of these samples showed that the wild-type PIF4 protein is destabilized after 30 min of CHX treatment, in contrast to PIF41A, which is still detected by 90 min of CHX application. BL treatment increased PIF4 stability but did not have any effect on...
The concerted action of PIFs and BES1/BZR1 modulates rhythmic hypocotyl growth
Although it is widely accepted that PIFs accumulate only in darkness, we show that the PIF4 protein follows a pattern of accumulation similar to that of its transcript. We generated pif4pif5 lines expressing the PIF4-HA or PIF41A-HA proteins under the control of its native promoter and selected lines with wild-type levels of these transgenes. pPIF4 ∷PIF4-HA lines had a hypocotyl length and expressed the PIL1 and PRE5 genes to levels similar to those of the Col-0 plants (; Supplemental Figs. S7, S12). pPIF4 ∷PIF41A-HA lines, in contrast, were much taller and showed increased levels of expression of these genes ( ). pPIF4 ∷PIF41A plants are actually taller than the bes1-D mutant and, in the presence of BRZ, show increased levels of expression of the PIL1 and PRE5 transcripts, suggesting that PIF4 destabilization plays an important role in BRZ repression of hypocotyl g...
Materials and methods
Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark).
In vitro kinase and phosphorylation assays
PIF4-His, PIF41A-His, MBP-BES1, and BIN2-GST fusion proteins were purified using glutathione agarose (Clontech), amylose agarose (New England Biolabs), or Ni-NTA agarose (Qiagen) beads. For in vitro kinase assays, PIF4-His, MBP-BES1, and GST-BIN2 proteins were coincubated with 32 Pγ-ATP for 40 min at 37°C. Two micromolar and 20 µM of the BIN2 kinase inhibitor bikinin (Calbiochem) were added to the incubation mix for specific inhibition. Extracts of mock-, BL-, and BRZ-treated seedlings were incubated for 1 h at 37°C with or without CIP (New England Biolabs) to assay for in vivo protein phosphorylation.
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To estimate the stabilization of this mutant protein, we treated PIF4 and PIF41A seedlings with cycloheximide (CHX) and with CHX and BL ( ). In these studies, PIF4 is destabilized by 30 min of CHX application, while PIF41A is still detectable after 90 min of CHX treatment. Also, upon BL application, PIF4 half-life was similar to that of the PIF41A protein, whereas this hormone did not have any effect on PIF41A protein levels ( ). Consistent with these results, when PIF4 and PIF41A lines were crossed to the bin2.1 mutant, we observed that constitutive activation of BIN2 in these seedlings leads to a strong reduction in PIF4 protein levels but has only a minor effect on PIF41A protein stability ( ).
Mutation of the conserved BIN2 phosphorylation motif increases the half-life of the PIF41A protein and leads to continuous growth. ( A ) Analysis of the half-life of the PIF4 and PIF41A proteins. Transgenic pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 seedlings were grown in SD for 5 d and treated for 12 h with 0.5 µM BRZ before starting the assay. Three hours before lights on, seedlings were changed to 0.5 µM BRZ (mock) or 1 µM BL (BL) medium, and 50 µM CHX was added 1 h later. Seedlings were kept in darkness and harvested at different times after CHX application, as indicated. Western blot analyses of these samples showed that the wild-type PIF4 protein is destabilized after 30 min of CHX treatment, in contrast to PIF41A, which is still detected by 90 min of CHX application. BL treatment increased PIF4 stability but did not have any effect on the PIF1A protein. Note that in BL-treated seedlings, the half-life of PIF4 becomes similar to that of PIF41A. ( B ) Stability of the PIF4 and PIF41A protein in the bin2.1 mutant background. pPIF4 ∷PIF4-HA pif4pif5 and pPIF4 ∷PIF41A-HA pif4pif5 lines were crossed with the bin2.1 mutant,...
Although it is widely accepted that PIFs accumulate only in darkness, we show that the PIF4 protein follows a pattern of accumulation similar to that of its transcript. We generated pif4pif5 lines expressing the PIF4-HA or PIF41A-HA proteins under the control of its native promoter and selected lines with wild-type levels of these transgenes. pPIF4 ∷PIF4-HA lines had a hypocotyl length and expressed the PIL1 and PRE5 genes to levels similar to those of the Col-0 plants (; Supplemental Figs. S7, S12). pPIF4 ∷PIF41A-HA lines, in contrast, were much taller and showed increased levels of expression of these genes ( ). pPIF4 ∷PIF41A plants are actually taller than the bes1-D mutant and, in the presence of BRZ, show increased levels of expression of the PIL1 and PRE5 transcripts, suggesting that PIF4 destabilization plays an important role in BRZ repression of hypocotyl growth. Time-course studies to analyze diurnal levels of accumulation of the PIF4 and PIF41A proteins showed that the wild-type PIF4 protein starts to accumulate at the end of the night, peaks during the day, and is reduced after transition to dark. PIF41A protein levels were higher than those of the...
Seeds were transferred to vertical MS plates supplemented with the different chemical treatments and grown in darkness or continuous red light for 5-7 d, as specified. Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length. Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark).
PIF4-His, PIF41A-His, MBP-BES1, and BIN2-GST fusion proteins were purified using glutathione agarose (Clontech), amylose agarose (New England Biolabs), or Ni-NTA agarose (Qiagen) beads. For in vitro kinase assays, PIF4-His, MBP-BES1, and GST-BIN2 proteins were coincubated with 32 Pγ-ATP for 40 min at 37°C. Two micromolar and 20 µM of the BIN2 kinase inhibitor bikinin (Calbiochem) were added to the incubation mix for specific inhibition. Extracts of mock-, BL-, and BRZ-treated seedlings were incubated for 1 h at 37°C with or without CIP (New England Biolabs) to assay for in vivo protein phosphorylation.
Machine-readable layer
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