Bioluminescence in plants: beyond as the biomarkers
During past decades, tremendous efforts have been invested for the development of optical non-invasive biomarkers in plant cells. The first bioluminescent system that was introduced into plants is firefly luciferase-luciferin system (Ow et al., 1986). Firefly luciferase can be expressed independently or fused with proteins of interst in plant cells, which provides a useful tool to study protein expression and localization, and protein-protein interactions (PPIs) (Guan et al., 2013; Sanchez et al., 2018). To detect PPIs, firefly luciferase is splited as N- and C-terminal halves and fused with two candidates of interactional proteins (Chen et al. 2008). However, firefly luciferin could not be biosynthesized in plant cells yet, so that it have to be supplied exogenously, which causes low cost-efficency.
To break those bottlenecks, several strategies were established, including using FPs as alternative biomarkers and searching novel luciferase-luciferin bioluminescent systems that can be completely biosynthesized in plants. In 1995, the green fluorescent protein (GFP) gene from Aequorea victoria was successfully expressed inArabidopsis , which brings novel revoluion in plant research (Hu & Cheng, 1995). Since then, FPs as tags are widely used in plants to monitor protein localization in vivo , recognize specific plant cells and tissues, obtain cell-specific transcriptomes and record plant cell fate selection (Ckurshumova et al., 2011). Spectral characteristics of FPs could be altered to reduce autofluorescence background from plant tissues. Various FPs covering most fluorescent ranges (i.e. yellow, blue, cyan, green, and red) have been adapted for plant cells (Blatt and Grefen, 2014). However, there are still problems remained when using FPs, including cellular damage caused by excited laser, signal weakening by plant tissues and migrating from plasmodesmata due to its smaller molecular weight (Brunkard & Zambryski 2017).
Another alternative strategy that developing novel bioluminescent systems was emerging. After elucidation of the luciferase-luciferin pairs original from bacteria and fungi, two distinguished optical reporters have been developed from these systems and now accessible in plant science. These systems have been described in their entirety and could be biosynthesized in plant cells. However, due to cytotoxicity and low photon yield of bacterial bioluminescent reactions in eukaryotes (Kotlobay et al., 2018), since autoluminescent system was firstly reported in Nicotiana tabacum , only few bacterial luciferase-luciferin pairs were reported in other transgenic plants (Cui et al., 2014; Krichevsky et al., 2010). Fungal bioluminescent system is an unique genetically encodable eukaryote luciferase-luciferin system, which was introduced into plant cells recently (Khakhar et al., 2020; Mitiouchkina et al., 2020). Less cytotoxicity and high luminescent intensity make fungal bioluminescent system as one of the most promising optical molecular tools (Reuter et al., 2020).
Nowdays, protein-directed revolution and synthetic biology boosted the development for novel bioluminescent systems. Luciferase engineering could be used to optimize the natural bioluminescent systems, such as increasing bioluminescent intensity and stability. Rational designing novel bioluminescent systems provides two representative cases, Nano Luciferase (NanoLuc) (England et al., 2016) and Nano-lantern (Doerr, 2013). NanoLuc is a small subunit (19 kDa) from shrimp Oplophorous gracilirotris luciferase (Hall et al., 2012). Split NanoLuc was developed to screen PPIs, which avoids imposing steric hindrance caused by huge firefly luciferase halves (Wang et al., 2020). Nano-lantern is an artificially designed luminescent protein fused with enhancedRenilla reniformis luciferase and yellow FP Venus. ATP production from chloroplast could be observed by Nano-lantern (Saito et al., 2012). Today, fluorescence and bioluminescence is no longer a new tool for molecular and celluar biology, but becoming a novel light sources, with which glowing plants have been obtained.