Source Paper
BR-dependent phosphorylation modulates PIF4 transcriptional activity and shapes diurnal hypocotyl growth
Stella Bernardo-García, et al.
Source Paper
Stella Bernardo-García, 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: Assessment of seedling development and hypocotyl length in darkness or continuous red light under various chemical treatments
This is a Light-Dependent Seedling Development protocol using Arabidopsis thaliana as the model organism. The procedure involves 10 procedural steps, 2 equipment items, 4 materials. Extracted from a 2014 paper published in Genes & Development.
Model and subjects
Arabidopsis thaliana • Col-0 ecotype
Study window
~1 week study window | ~24 hours hands-on
Core workflow
Generate transgenic lines • Create PIF41A construct • Mobilize genomic regions into destination vector
Primary readouts
Key equipment and reagents
Verified items
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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
Note: Fragment cloned into pENTRY S/D-TOPO vector
“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”
Substitute NcoI–AgeI fragment of pPIF4::PIF4 TOPO plasmid with corresponding fragment including PIF41A mutation
Note: Generates 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”
Use LR clonase to mobilize genomic regions into pGWB13-pPZP destination vector
Note: Prepares construct for Agrobacterium transformation
“These genomic regions were mobilized by LR clonase into the pGWB13-pPZP destination vector”
Transform destination vector via Agrobacterium into pif4pif5 mutant plants
Note: Generates pPIF4::PIF4-HA pif4pif5 transgenic lines
“transformed via Agrobacterium into the pif4pif5 mutant”
Cross pPIF4::PIF41A-HA plants into bes1-D pif4pif5 mutants
Note: Creates double mutant transgenic 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 oriented vertically for growth measurement
“Seeds were transferred to vertical MS plates supplemented with the different chemical treatments”
Grow seedlings in darkness or continuous red light for 5–7 days as specified
Note: Light condition depends on experimental group
“grown in darkness or continuous red light for 5–7 d, as specified”
Photograph seedling plates for subsequent analysis
Note: Images used for hypocotyl length measurement
“Plates were photographed, and the ImageJ software was used to measure”
Use ImageJ software to measure seedlings' hypocotyl length from plate photographs
Note: Primary outcome measure for seedling development
“the ImageJ software was used to measure the seedlings' hypocotyl length”
Conduct diurnal growth and protein accumulation studies in plants grown in short days (8 h light/16 h dark)
Note: Separate experimental condition for temporal analysis
“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.
Assessment of seedling development and hypocotyl length in darkness or continuous red light under various chemical treatments
Objective
Assessment of seedling development and hypocotyl length in darkness or continuous red light under various chemical treatments
Subjects
From paperArabidopsis thaliana • Col-0 ecotype
Cohort notes
From paperTransgenic lines: pPIF4::PIF4-HA pif4pif5, pPIF4::PIF41A-HA pif4pif5, and bes1-D PIF41A plants
Generate transgenic lines
Create PIF41A construct
Mobilize genomic regions into destination vector
Transform into pif4pif5 mutant
Seedling hypocotyl length
From paperHypocotyl length measured using ImageJ software from plate photographs
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Diurnal growth patterns
From paperHypocotyl length measured using ImageJ software from plate photographs
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Protein accumulation
From paperHypocotyl length measured using ImageJ software from plate photographs
Artifact type
Endpoint measurements summarized by group or timepoint
Comparison focus
Compare endpoint magnitude between groups, timepoints, or both
Seedling hypocotyl length
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Diurnal growth patterns
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Protein accumulation
From paperRaw artifact
Per-sample or per-animal endpoint measurements collected during the experiment
Processed artifact
Structured table with cleaned measurements ready for comparison
Final reported form
Summary statistics and between-group or across-timepoint comparisons
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
Preprocessing / cleaning
Hypocotyl length measured using ImageJ software from plate photographs
Scoring or quantification
Quantify the primary readouts for this experiment: Seedling hypocotyl length; Diurnal growth patterns; Protein accumulation.
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.
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
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