Misexpression approaches for the manipulation of flower development

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1110; p. 383
• Main Authors: Xu, Yifeng, Gan, Eng-Seng, Ito, Toshiro
• Format: Journal Article
• Language: English
• Published: United States 2014
• Subjects: Agrobacterium - genetics; Arabidopsis - genetics; Cloning, Molecular; Dexamethasone - pharmacology; Flowers - drug effects; Flowers - genetics; Flowers - growth & development; Gene Expression - drug effects; Genes, Plant - genetics; Genetic Engineering - methods; Organ Specificity; Phenotype; Promoter Regions, Genetic - genetics; Protein Biosynthesis - drug effects; Transcription, Genetic - drug effects; Transformation, Genetic
• DOI: 10.1007/978-1-4614-9408-9_22

The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that complements the screening of loss-of-function mutants and that has been successfully applied to studying the mechanisms of flower development. In addition, the functions of genes of interest can be further analyzed through reverse genetics. A commonly used method is gene overexpression, where strong, often ectopic expression can result in an opposite phenotype to that caused by a loss-of-function mutation. When overexpression is detrimental, the misexpression of a gene using tissue-specific promoters can be useful to study spatial-specific function. As flower development is a multistep process, it can be advantageous to control gene expression, or its protein product activity, in a temporal and/or spatial manner. This has been made possible through several inducible promoter systems, as well as by constructing chimeric fusions between the ligand binding domain of the glucocorticoid receptor (GR) and the protein of interest. Upon treatment with a steroid hormone at a specific time point, the fusion protein can enter the nucleus and activate downstream target genes. All these methods allow us to genetically manipulate gene expression during flower development. In this chapter, we describe methods to produce the expression constructs, method of screening, and more general applications of the techniques.