The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that can be applied to study the mechanisms of flower development, complementing the screening of loss-of-function mutants. In addition, the functions of genes of interest can be further analyzed through reverse genetics. A commonly used method is gene overexpression, where 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 inducible proteins by constructing chimeric fusions between the ligand-binding domain of the glucocorticoid receptor (GR) and the protein of interest. The recently introduced CRISPR-Cas9-based platform provides a new way of bioengineering transcriptional regulators in plants. By fusing a catalytically inactive dCas9 with functional activation or repression domains, the CRISPR-Cas9 module can achieve transcriptional activation or repression of endogenous 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.
Keywords: Activation tagging; CRISPR-Cas9; Glucocorticoid receptor; Misexpression; Overexpression; Promoter.
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