Glowing plants with bioluminescent systems
Up to now, three bioluminescent systems that origin from firefly, bacteria and fungi have been reconstructed in plants. The firefly luciferase-luciferin system is a kind of D‐luciferin‐dependent systems, which is one of the most intensively studied bioluminescent systems (Fleiss & Sarkisyan, 2019). In the presence of Mg2+, oxygen and ATPs, reduced firefly luciferin can be catalyzed by firefly luciferase, and generate yellow-green luminescence. To prolong glowing, CoA could be used to deoxidize the oxidized luciferin (Gosset et al., 2020). Ow. et al. (1986) introduced Photinus pyralis luciferase gene into N. tabacum and Daucus carota for the first time. Firefly luciferase was expressed during growing process of the transgenic plant and accumulated in various plant organs, e.g., roots, stems and leaves. When watering the tobacco with solution of firefly luciferin and ATP, the substrate was transmitted to every tissues through the vasculature and react with luciferase, leading to lightening of the whole transgenic tobacco. Due to the technical difficulty to synthesize firefly luciferin in plant cells, the firefly luciferins can only be exogenously supplied. In 2017, a novel nano-technique named pressurized bath infusion of nanoparticles (PBIN) was utilized to introduce the whole firefly bioluminescent system. In PBIN, luciferin and coenzyme A (CoA) were packaged into two different nano capsules, and luciferase was linked onto the surface of silica-PEG nanocarriers. These nanoparticles could reduce luciferin cytotoxicity and diminish chemical denaturation of luciferase to some extent, causing a long light-emitting duration of up to 8 hours. To transmit the whole firefly bioluminescent system, the plant was immersed in solution containing the nanoparticles in a closed pressured aqueous chamber, in which pressurization delievered the nanoparticles into mesophyll cells through stomata. Once these nanoparticle gathered in mescophyll cells, it glowed, and produced the brightest glowing plants to date. The PBIN-based light-emitting plants were also the only glowing plants which could be modulate “on” and “off” states when CoA- and dehydroluciferin-nanoparticles were provided, respectively, thus providing viability as alternative light sources (Kwak et al., 2017).
The second bioluminescent system is discovered in three Gram-negative motile rods luminous bacteria genera, that is Vibrio ,Photobacterium , and Xenorhabdus . The lux operons (luxCDABEG ) are highly conserved through all luminous bacterial, which encode the whole bacterial bioluminescent system (Close et al., 2009). Gene luxA and luxB encode α and β subunits of bacterial luciferase, respectively, and luxCDEG encodes biosynthesis pathway of tetradecanal. Krichevsky et al. (2010) transferred the whole Photobacterium leiognathi lux operon into tobacco chloroplast and obtained the first auto-luminescent plant. Although only very dim green luminescence was observed, this auto-emission needs neither exogenous luciferin nor optical excitation. However, the cytotoxicity of the bacterial luciferin, long-chain fatty aldehyde, is ineluctable to the eukaryotic cells. It is reported that a low concentration of bacterial luciferin n-decyl aldehyde can cause toxity to many model eukaryotes, including mammalian and higher plant cells (Hollis et al., 2001), which hindered application of bacterial bioluminescent system as biomarkers or illuminants.
Recently, the third bioluminescent system originated from fungi was used to generate auto-stronge-glowing plants.The biosynthetic pathway of fungal luciferin is named caffeic acid cycle (Kotlobay et al., 2018). A gene cluster encoded four key enzymes catalyze the caffeic acid cycle inNeonothopanus gardneri , including fungal luciferase (Luz), hispidin-3-hydroxylase (H3H), hispidin synthase (Hisps), and caffeylpyruvate hydrolase (CPH). Hisps is post-translationally activated by another enzyme, NPGA (4’-phosphopantetheinyl transferase). Precursor of fungal luciferin is hispidin, which can be convert to fungal luciferin, termed 3-hydroxyhispidin. Then the luciferin is oxidized by Luz to yield photons and oxy-luciferin, named caffelpyruvic acid, followed by hydrolyzing the oxy-luciferin to pyruvic acid and caffic acid. Caffic acid can be recycled to hispidin by Hips (Purtov et al. 2015; Oba et al. 2017). Fungal caffeic acid cycle shares three intermediary metabolites with three major biosynthetic pathways of vascular plants, including shikimate, lignins and flavonoid anthocyanins condensed tannins. These common molecules make a bridge connecting the gap between fungal caffeic acid cycle and the native biosynthetic pathway in plant cells. As a result, it is reasonable to design a sophisticated fungal luciferin synthetic pathway reconstituted into tobacco metabolic routines (Mitiouchkina et al., 2019). Mitiouchkina et al. (2020) created a luminescent N. tabacum through synthetic biology, which stably displayed green luminescence. Gene of Luz , H3H ,Hisps , and CPH were transferred in the tobacco. The auto-luminescent tobacco was brighter than other reported auto-light-emitting plants. Coninsistantly, Khakhar et al. (2020) described the transiently transformation and expression of fungal bioluminescent system in both model and commercial plant species.NPGA gene was also transferred besides the four gene mentioned above. Stronge luminescence was generated from cotyledons, leaves, and petals of these genetically modified plants. The fungal bioluminescent system produces brighter light and shows no cytotoxicity and growth inhibition to plant cells, compared with that employed bacterial luminescent system (Kotlobay et al., 2018). However, drawbacks are also existed on the plant-expressed fungal bioluminescent system. For example, low pH and temperature in plant cells reduced photon yield (Reuter et al., 2020).