Context and background

Bioluminescent reporters such as the firefly luciferase have been widely used in eukaryotic systems. They are particularly appealing for use in plants as they do not require excitation and hence autofluorescence that is inherent to plant tissue can be avoided. A major disadvantage however is the need to deliver the luciferase substrate exogenously – causing issues with homogenous substrate delivery and tissue penetration. It would thus be highly desirable to generate plants that already produce the luciferase substrate. Kotlobay et al. (1) identified a novel luciferase/luciferin biosynthesis system, referred to as the caffeic acid cycle, in the eukaryotic fungus Neonothopanus nambi (Fig. 1). The fungal luciferin 3-hydroxyhispidin is produced from caffeic acid through the activity of two biosynthetic enzymes, hispidin synthase (HispS) and hispidin-3-hydroxylase (H3H). The luciferase (luz) subsequently generates a bioluminescent signal by oxidising 3-hydroxyhispidin; the resulting product can be recycled to caffeic acid by caffeoylpyruvate hydrolase (CPH). In their preprints, Mitiouchkina et al. and Khakhar et al. deploy this system in plants and show that it can be harnessed to monitor gene expression.

 

Figure 1. Fungal bioluminescent pathway (caffeic acid cycle) used to create bioluminescent plants. Reproduced from Figure 1 of the preprint (Khakhar et al.) under a CC BY-NC-ND 4.0 license.

Key findings

1. The fungal caffeic acid cycle effectively labels multiple plant species without the need for lasers or other light sources

Caffeic acid is an intermediate of the plant’s phenylpropanoid biosynthesis pathway and present in virtually all plant species. The authors of both preprints introduced genes encoding the enzymes of the fungal caffeic acid cycle into plants to test whether the pathway can be reconstituted in planta, thereby bypassing the need to externally supply luciferin. Transient expression of the enzymes in multiple plant species including tobacco, Arabidopsis and tomato resulted in auto-luminescence of the transformed organs. When the pathway was stably integrated into the tobacco genome, auto-luminescence was observed across the whole plant and throughout development. Mitiouchkina et al. further observed that luminescence varied across organs – over the time of day and strongly increased upon wounding – likely correlating with the activity of the phenylpropanoid pathway that generates caffeic acid.

2. This system can be used to visualise spatio-temporal changes in gene expression

The luminescence pathway developed in these preprints potentially represents a powerful reporter system. Khakhar et al. adapted this system to observe spatio-temporal changes in gene expression by expressing the fungal luciferase from selected promoters. Expression from the Petunia ODORANT1 promoter lead to exclusive auto-luminescence in flowers with a diurnal rhythm that peaked at dusk, in agreement with previous observations. Expression from the abscisic acid (ABA)-responsive AtRAB18 promoter on the other hand conferred strong increase in luminescence when plants were treated with ABA or dessicated, a stress known to boost ABA levels. None of these specific responses were observed when the luciferase was expressed from the constitutive 35S promoter, providing proof of concept that the fungal bioluminescence pathway (FBP) can be employed as a reporter system for gene expression.

 

 

Why we chose this paper

Many model organisms or cell lines are highly amenable to bioluminescence reporter systems such as the firefly luciferase. However, a major disadvantage of these systems is the requirement of exogenous substrate, particularly in plants where tissue permeability varies greatly. The preprints presented here represent an excellent solution to this problem by negating the need for exogenous substrate whilst providing a powerful and adaptable reporter system.

 

Open questions

1. The authors of both preprints highlight the excellent use of this system as a tool within plant biology. How applicable is this reporter system in different plant groups (monocots, ferns, bryophytes)? Is this also applicable to other organisms and do the authors envisage adoption in different systems?
2. To what extent may the observed variations in the phenylpropanoid pathway activity affect FBP-based reporter systems? Are there tissues/conditions that might be more suitable for analyses with FBP than others?
3. How stable is the expression system across plant generations? Has silencing of these genes in subsequent generations been observed?

 

References

1. Kotlobay AA, Sarkisyan KS, Mokrushina YA, Marcet-Houben M, Serebrovskaya EO, Markina NM, et al. Genetically encodable bioluminescent system from fungi. Proc Natl Acad Sci. 2018 Dec 11;115(50):12728–32.

 

doi: https://doi.org/10.1242/prelights.15189

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