Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis

Abstract

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissueor cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gatewayâ-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

Spatially specific mechanisms and functions of the plant circadian clock

“…One construct, expressed under a circadian promoter, comprises the C-terminal half of luciferase fused to the C-terminus of A-Fos. A second construct consisting of the N-terminal half of luciferase fused to the bZIP domain of c-Jun is constitutively expressed under a tissue-specific promoter (either directly or indirectly through a UAS promoter transactivated by enhancer trap-driven expression of GAL4; Román et al, 2020 ). When both constructs are expressed together, the A-Fos and c-Jun domains facilitate dimerization, bringing the two halves of luciferase into close proximity to reconstitute enzymatic activity ( Endo et al, 2014 ), generating luminescence from circadian rhythms only in a target tissue (e.g.…”

“…Enhancer trap split-luciferase assays further refine this method by expressing the c-Jun-nano luciferase (nLUC) fusion under a UAS promoter, which is activated by binding of GAL4. Expressing GAL4 using enhancer trap systems, which consists of previously characterized lines that drive strong tissue-specific expression under a single enhancer ( Shima et al, 2016 ), avoids the need to use full promoters, which can introduce unwanted regulatory effects ( Román et al, 2020 ). However, spatial position of cells can affect clock gene expression even within the same tissue ( Fukuda et al, 2007 ; Wenden et al, 2012 ; Gould et al, 2018 ).…”

“…By introducing enzymes from fungi bioluminescence pathways that convert caffeic acid—an intermediate metabolite in the synthesis of lignin—into luciferin, stable auto-luminescence has been generated in several plant species ( Khakhar et al, 2020 ; Mitiouchkina et al, 2020 ). Although this approach may require ectopic expression of enzymes to produce caffeic acid in tissues where endogenous abundance is poor, auto-luminescence could be combined with tissue-specific split luciferase assays (as in Román et al, 2020 .) to study the clock in tissues with poor coverage from luciferin.…”

Arabidopsis cell suspension culture that lacks circadian rhythms can be recovered by constitutive ELF3 expression

“…We also found that calli induced from hypocotyls, leaves, and roots showed a similar expression pattern compared to callus from whole seedlings, suggesting that initial tissues did not affect the circadian rhythms in the callus (Figure 4). Several studies indicated that the circadian clock function is tissue-and cell-specific (Thain et al 2002;Takahashi et al 2015;Endo et al 2014;Román et al 2020;Gould et al 2018;James et al 2008;Greenwood et al 2019). At the tissue level, the root clock is less robust and precise than the shoot clock (Thain et al 2002;Takahashi et al 2015;Chen et al 2020) Importantly, the seedling-like rhythms obtained in callus, independent of callus source or media, indicate that freshly-derived callus can be a useful tool for studying circadian regulation.…”

An enhancer trap system to track developmental dynamics in Marchantia polymorpha

“…Enhancer trap lines can mark phenotypes that could not otherwise be easily accessible through conventional, loss‐of‐function genetic screens, for example, due to lethal effects of disrupting gene activity, or cryptic mutant phenotypes (Rojas‐Pierce & Springer, 2003 ). In such situations, locating the site of insertion within the genome can identify new gene functions and important novel promoter elements, that can then be used in further research (Radoeva et al., 2016 ; Román et al., 2020 ).…”