Source Paper
BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth
Stella Bernardo-García, Miguel de Lucas, et al.
Source Paper
Stella Bernardo-García, Miguel de Lucas, et al.
Genes & Development • 2014
Signaling by the hormones brassinosteroid (BR) and gibberellin (GA) is critical to normal plant growth and development and is required for hypocotyl elongation in response to dark and elevated temperatures. Active BR signaling is essential for GA promotion of hypocotyl growth and suppresses the dwarf phenotype of GA mutants. Cross-talk between these hormones occurs downstream from the DELLAs, as GA-induced destabilization of these GA signaling repressors is not affected by BRs. Here we show that the light-regulated PIF4 (phytochrome-interacting factor 4) factor is a phosphorylation target of the BR signaling kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), which marks this transcriptional regulator for proteasome degradation. Expression of a mutated PIF41A protein lacking a conserved BIN2 phosphorylation consensus causes a severe elongated phenotype and strongly up-regulated expression of the gene targets. However, PIF41A is not able to suppress the dwarf phenotype of the bin2-1 mutant with constitutive activation of this kinase. PIFs were shown to be required for the constitutive BR response of bes1-D and bzr1-1D mutants, these factors acting in an interdependent manner to promote cell elongation. Here, we show that bes1-D seedlings are still repressed by the inhibitor BRZ in the light and that expression of the nonphosphorylatable PIF41A protein makes this mutant fully insensitive to brassinazole (BRZ). PIF41A is preferentially stabilized at dawn, coinciding with the diurnal time of maximal growth. These results uncover a main role of BRs in antagonizing light signaling by inhibiting BIN2-mediated destabilization of the PIF4 factor. This regulation plays a prevalent role in timing hypocotyl elongation to late night, before light activation of phytochrome B (PHYB) and accumulation of DELLAs restricts PIF4 transcriptional activity.
Objective: Analysis of plant growth patterns and protein accumulation under short day photoperiod conditions (8h light/16h dark)
This is a Diurnal Growth Analysis protocol using Arabidopsis thaliana as the model organism. The procedure involves 9 procedural steps, 2 equipment items, 3 materials. Extracted from a 2014 paper published in Genes & Development.
Model and subjects
Arabidopsis thaliana • Col-0 ecotype • unknown • Not specified • Not applicable
Study window
~1 week study window | ~24 hours hands-on
Core workflow
Generate transgenic plant lines • Generate PIF41A construct • Mobilize genomic regions and transform
Primary readouts
Key equipment and reagents
Verified items
0
Direct vendor links
0
Use this page as an execution guide, then fall back to the source paper whenever you need exact exclusions, dosing details, or assay-specific caveats.
Confirm first
Use the page like this
Start here. The step list is optimized for running the experiment, with direct vendor links available inline when you need to source a cited item.
Amplify genomic fragment including 2.4-kb promoter region and full-length PIF4 coding sequence without stop codon using PIF4promoter-f and PIF4_YFPr primers. Clone into pENTRY S/D-TOPO vector.
Note: For pPIF4::PIF4-HA pif4pif5 transgenic lines
“a genomic fragment, including a 2.4-kb promoter region and the full-length PIF4 coding sequence without stop codon, was amplified with the PIF4promoter-f and PIF4_YFPr primers and cloned into the pENTRY S/D-TOPO (Invitrogen) vector”
Substitute the NcoI–AgeI fragment of the pPIF4::PIF4 TOPO plasmid with the corresponding fragment including the PIF41A mutation.
Note: Creates pPIF4::PIF41A construct
“The pPIF4 ⊳PIF41A construct was obtained by substituting the NcoI–AgeI fragment of the pPIF4 ⊳PIF4 TOPO plasmid with the corresponding fragment, including the PIF41A mutation”
Mobilize genomic regions by LR clonase into the pGWB13-pPZP destination vector and transform via Agrobacterium into the pif4pif5 mutant.
Note: Generates transgenic lines
“These genomic regions were mobilized by LR clonase into the pGWB13-pPZP destination vector and transformed via Agrobacterium into the pif4pif5 mutant”
Cross pPIF4::PIF41A-HA plants into the bes1-D pif4pif5 mutants.
Note: Creates double mutant line
“bes1-D PIF41A plants were generated by crossing the pPIF4 ⊳PIF41A-HA plants into the bes1-D pif4pif5 mutants”
Transfer seeds to vertical MS plates supplemented with different chemical treatments as specified.
Note: Plates are vertical orientation
“Seeds were transferred to vertical MS plates supplemented with the different chemical treatments”
Grow plants in darkness or continuous red light for 5–7 days as specified.
Note: Light conditions vary by experimental condition
“grown in darkness or continuous red light for 5–7 d, as specified”
Photograph plates containing seedlings for subsequent analysis.
Note: Images used for hypocotyl length measurement
“Plates were photographed, and the ImageJ software was used to measure the seedlings' hypocotyl length”
Use ImageJ software to measure seedlings' hypocotyl length from photographs.
Note: Quantitative measurement of growth
“the ImageJ software was used to measure the seedlings' hypocotyl length”
Conduct diurnal growth and protein accumulation studies in plants grown under short day photoperiod conditions.
Note: Short day conditions are 8 hours light/16 hours dark
“Diurnal growth and protein accumulation studies were performed in plants grown in short days (8 h light/16 h dark)”
This section explains what the experiment is doing, which readouts matter, what the data artifacts usually look like, and how the analysis should flow from raw capture to reported result.
Analysis of plant growth patterns and protein accumulation under short day photoperiod conditions (8h light/16h dark)
Objective
Analysis of plant growth patterns and protein accumulation under short day photoperiod conditions (8h light/16h dark)
Subjects
From paperArabidopsis thaliana • Col-0 ecotype • unknown • Not specified • Not applicable
Cohort notes
From paperTransgenic lines including pPIF4::PIF4-HA pif4pif5, pPIF4::PIF41A, and bes1-D PIF41A plants
Generate transgenic plant lines (Not specified)
Generate PIF41A construct (Not specified)
Mobilize genomic regions and transform (Not specified)
Generate bes1-D PIF41A plants (Not specified)
Seedling hypocotyl length
From paperImageJ software used to measure hypocotyl length from plate photographs
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Diurnal growth patterns
From paperImageJ software used to measure hypocotyl length from plate photographs
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Protein accumulation levels
From paperImageJ software used to measure hypocotyl length from plate photographs
Artifact type
Representative image panels with region or marker comparisons
Comparison focus
Compare staining intensity, structure, or cell counts across matched conditions
Seedling hypocotyl length
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Diurnal growth patterns
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Protein accumulation levels
From paperRaw artifact
Field or section images captured from matched samples
Processed artifact
Selected representative panels with quantified intensity, counts, or area measurements
Final reported form
Per-group imaging summaries with representative figures and quantified endpoints
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
Preprocessing / cleaning
ImageJ software used to measure hypocotyl length from plate photographs
Scoring or quantification
Quantify the primary readouts for this experiment: Seedling hypocotyl length; Diurnal growth patterns; Protein accumulation levels.
Normalization
Normalize image-derived measurements against the matched acquisition or segmentation rules before comparing groups.
Statistical comparison
Statistical method not yet structured for this page.
Reporting output
Report representative outputs alongside summary comparisons for Seedling hypocotyl length, Diurnal growth patterns, Protein accumulation levels.
Source links and direct wording from the methods section for validation and deeper review.
Citation
Stella Bernardo-García et al. (2014). BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth. Genes & Development
“”
“”
“”
“”
Direct vendor pages are linked from the protocol above. This section stays focused on the full comparison view and the prep checklist.
Gather these items before starting the experiment. Check off items as you prepare.
Not specified • Not mentioned • Not mentioned • Not mentioned
Not specified • Not mentioned • Not mentioned • Not mentioned
Not specified • Not mentioned • Not mentioned • Not mentioned
Invitrogen • Not mentioned • Not mentioned • Not mentioned
Not specified • Not mentioned • Not mentioned • Not mentioned
Not specified • Not mentioned
Use this section as the page quality checkpoint. It keeps section navigation, evidence access, readiness, and verification meaning in one place.
Current status surfaces were computed from experiment data updated Feb 28, 2026.
Source access
Jump back into the original paper or the methods evidence section when you need exact wording, exclusions, or method-specific caveats.
This protocol has structured steps plus evidence quotes, and is ready for canonical sync.
Steps
9
Evidence Quotes
14
Protocol Items
5
Linked Products
0
Canonical Sync
Pending
What this means
The completeness score reflects how much structured protocol data is present: steps, methods evidence, listed materials, linked products, and paper provenance.
Computed from the current experiment record updated Feb 28, 2026.
Canonical Sync shows whether a ConductGraph-backed protocol is available for this experiment route right now. It is a sync-status signal, not a claim that every downstream vendor link or step detail is perfect.
Steps
9
Evidence
14
Specific Products
0/0
Canonical Sync
Pending
What this score means
The verification score reflects evidence coverage, subject detail, paper provenance, step depth, and whether linked products resolve to specific item pages instead of generic searches.
Computed from the current experiment record updated Feb 28, 2026.
A page can have structured steps and still need review when evidence is thin, product links are generic, or canonical protocol coverage is still pending.
What still needs work