Optimization of “planterns”
One of the problems is low luminescent intensity, which hinders the population of planterns. Though some glowing plants generated a bright emission of 1.44 × 1012 photons/s (Kwak et al. 2017), many other auto-glowing plants produce a much dimmer luminescence of 1.3 × 106 photons/s (Krichevsky et al., 2010) and 1.6 × 109 photons/s (Mitiouchkina et al., 2020), which is only bright enough to see in entire darkness with naked eyes. Enhancement of whole-plant glowing intensity could be considered as follow. Bioluminescent intensity is determined by both characteristics and accumulation of luciferase-luciferin pairs. Plant intracellular conditions also interfere bioluminescent intensity. Once the luminescence is produced, transparency window (TW) effects transmission efficiency (Strack, 2019).TW can be measured and sketched by spectrophotometer (Mitiouchkina et al., 2020). Because the TW is effected by pigments, plant species, apparatus and growth status, the bioluminescent spectrum should be altered according to TW.
As a result, the improvement of glowing intensity could be achieved from two aspects, i.e., the optimization of bioluminescent systems and the transgenic strategies. By engineering of luciferase, promotes feasibility to enhance bioluminescent intensity, spectral characteristics, pH and thermal stability by random mutagenesis, site-specific mutagenesis and in scilicon design (Branchini et al., 2019; Fujii et al., 2007; Modestova & Ugarova, 2016). Protein fusion or spilt could also alter bioluminescent characteristics. A chimeric firefly luciferase of North American firefly (Photinus pyralis ) and Japanese firefly (Luciola italica ) luciferase exhibited 1.4-fold enhanced bioluminescence quantum yield compared with the wide-type firefly luciferases (Branchini et al., 2014). Nano-lantern and NanoLuc are shown to improve luminescent intensity significantly, though exogenously supplied luciferin is necessary. Similar nano-lantern or NanoLuc might be developed based on the other bioluminescent systems whose luciferin could be biosynthesized, e.g., fungal bioluminescent system. Recently, as structural basis for the spectral difference was gradually clarified, bioluminescent color could also be changed by designing luciferin. It was reported that firefly luciferins with elongated π-system, such as phenyl or phenol, exhibit an obvious red-shift of emission spectrum (Yao et al., 2020). It was reported that firefly luciferin analog with less conjugated π-system present blue-shift in absorption and fluorescence (Pirrung et al., 2019). The designed luminescent pairs provide more selections, and these luciferin analogs might be able to be biosynthesized in plant cells. Given that the potentially cytotoxicity and growth inhibition to higher plants caused by bioluminescent systems, protecting groups were introduced into luciferin is reported possible, which can decrease cytotoxicity and enhance the accumulation (Yuan et al., 2017).
To optimize transgenic methodologies, substanial accumulation of luciferase-luciferin pairs can be achieved via common genetic engineering technique, such as codon optimization, inserting translational enhancer and several tandemly ranging the gene cluster (Sasaki et al., 2014). Engineering subcellular organelles (e.g. chloroplast and vacuole) into specified “scintillon” is another promising strategy. Increasing the accumulation of the whole luciferin pathway can be achieved through the development of chloroplast transformation technique (Kwak et al., 2019; Jin & Daniell, 2015). Bacterial bioluminescent system has been stably expressed in chloroplast (Krichevsky et al., 2010). Maternal inheritance of the chloroplast genome prevents genes from escaping into the environment through pollen grains (Yu et al., 2020), which may helps popularize planterns as alternative light sources in future (Callaway, 2013). Meanwhile, vacuoles could be employed to accumulate FPs or luciferase-luciferin pairs. Proteins associatd to bioluminescence can be fused with sequence specific vacuolar sorting signals (Viegas et al., 2017). The potential strategy to enhance the suboptimized glowing plants would be helpful for designing better glowing plants in future.