A Luciferase Reporter Assay to Identify Chemical Activators of ABA Signaling
Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especi
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Introduction Chemical genomics has arisen as an alternative to overcome the limitations of classical genetics, including redundancy and lethality. This recent discipline uses small molecules to study biological systems and to understand protein function, helping to elucidate a biological process in a specific organism [1, 2]. Chemical genomics often involves high-throughput screening (HTS) of large chemical libraries for compounds that cause the desired effect, i.e., phenotype of interest, regulation of the target activity, or reporter activation [2]. Phenotypic screenings are aimed to discover small molecules able to control a particular trait. However, the
Glenn R. Hicks and Chunhua Zhang (eds.), Plant Chemical Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 2213, https://doi.org/10.1007/978-1-0716-0954-5_10, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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identification of the target modulated by the chemical compound is not trivial and represents a bottleneck for this approach [3]. Also, phenotypic-driven chemical screenings make it more laborious to carry out structure–activity relationship (SAR) studies and to use them to rationally design improved chemical analogs. Alternatively, target-based approaches, with the purpose of identifying new small molecules that interact with a specific target and modulate its activity, are also frequently used [4, 5]. In this case, it is preferable to have previous knowledge about the target of choice and about its involvement into the cellular process of interest. In any case, an easy and informative assay needs to be developed in order to study the impact of chemical probes on the target’s activity. In addition to phenotypic and targeted approaches, reporter-based screenings have been widely used to evaluate the effects of compounds on a pathway or target of interest. For instance, direct quantification of β-glucuronidase activity (GUS activity) in Arabidopsis thaliana (Arabidopsis) has been used in HTS of chemical libraries for modulators of salicylic acid signaling [6] and for inducers of plant defense responses [7–9]. Luciferase (LUC) has several advantages over GUS or GFP reporters. It has a relatively short half-life (3 h) compared to GFP (24 h) or GUS (days) [10] and also allows for nondestructive signal detection with virtually no background issues [10]. Additionally, reporter-based screens can provide either quantitative or semiquantitative data, allowing for compound ranking and prioritization. Understanding the processes of adaptation and tolerance to water stress, especially in plants with agronomic interest, is very important nowadays. Under adverse environmental conditions, such as drought, salinity, and extreme temperatures, plant growth and productivity are severely affected [11]. The plant hormone abscisic acid (ABA) is crucial for plant stress adaptation, with a major relevance in drought resistance [12, 13]. ABA regulates guard cell closure, limiting transpiration under w
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