Open AccessAn efficient protocol for functional studies of apple transcription factors using a glucocorticoid receptor fusion system
Author(s) -
Estevan Joan,
GómezJiménez Sara,
Falavigna Vítor da Silveira,
Camuel Alicia,
Planel Lisa,
Costes Evelyne,
Andrés Fernando
Publication year - 2020
Publication title -
applications in plant sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.64
H-Index - 23
ISSN - 2168-0450
DOI - 10.1002/aps3.11396
Subject(s) - biology , transcription factor , cycloheximide , glucocorticoid receptor , gene , transcription (linguistics) , microbiology and biotechnology , genetics , protein biosynthesis , linguistics , philosophy
Premise We report a protocol for studying the function of apple ( Malus × domestica ) transcription factors based on the glucocorticoid receptor (GR) system, which allows the dexamethasone (DEX)‐mediated activation of plant transcription factors to monitor the expression levels of their potential target genes. Methods and Results Apple leaves are transformed with a vector that allows the expression of the studied transcription factor (i.e., FLOWERING LOCUS C [MdFLC]) fused to GR. Calli derived from the transformed leaves are treated with DEX and cycloheximide, a protein synthesis inhibitor. Compared with other methods, combining the GR system with cycloheximide treatments enables the differentiation between direct and indirect transcription factor target genes. Finally, the expression levels of putative MdFLC target genes are quantified using quantitative reverse transcription PCR. Conclusions We demonstrate the efficiency of our GR system to study the function of apple transcription factors. This method is accessible to any laboratory familiar with basic molecular cloning procedures and the apple leaf–mediated agro‐transformation technique.