The 110 seedlings depicted on panel (i) are representative of three independent experiments. Photos were captured on Sony Alpha ILCE-7M3 ( Online Methods). tabacum plants at germination ( i), vegetative ( ii) and flowering ( iii) stages light emission from roots ( iv) and cross section of flowers (v).

The brightness allowed us to capture detailed images on consumer-grade cameras with exposure times of 0.5–30 seconds, providing comparable quality to that of more expensive luminescence imaging equipment ( Figure 2, Supplementary Figures 3–8).īioluminescent plants at various stages of development.Ī. Light emission at all developmental stages was visible to the naked eye, with intensity from the flowers reaching 10 10 photons/min ( Supplementary Table 1).

This suggests that unlike bacterial bioluminescence, expression of the caffeic acid cycle is not toxic and does not impose an obvious burden on plant growth. The overall phenotype, chlorophyll and carotenoid content, flowering time, and seed germination did not differ from the wild-type tobacco, with the exception of a 12% increase in median height of transgenic plants ( Supplementary Figure 3 Supplementary Note 2). DNA cassettes were used containing codon-optimised versions of the four previously reported Neonothopanus nambi bioluminescence genes: nnluz (luciferase), nnhisps (hispidin synthase), nnh3h (hispidin-3-hydroxylase), and nncph (caffeoyl pyruvate hydrolase) ( Figure 1, Online Methods, Supplementary Figure 2, Supplementary Note 1).įifteen plant lines were independently obtained with confirmed genome integrations. We engineered the autonomously glowing tobacco plants via random-site genome integration using Agrobacterium-mediated transformation. Luminescence spectrum is rendered from dataset published in Ref. Absorbance spectrum of leave is representative of experiment performed on three leaves. Abbreviations: 4CL - 4-coumarate:CoA ligase C3H - p-coumaric acid 3-hydroxylase C4H - cinnamic acid 4-hydroxylase CCOMT - caffeoyl-CoA 3-O-methyltransferase CCR - cinnamoyl-CoA reductase CHI - chalcone isomerase CHS - chalcone synthase CPH - putative caffeoyl pyruvate hydrolase H3H - hispidin-3-hydroxylase HispS - hispidin synthase Luz - luciferase PAL - phenylalanine ammonia-lyase. The fungal or plant origin of enzymes is indicated with mushroom and plantlet symbols, respectively. The caffeic acid cycle shares metabolites with some of the major plant biosynthetic pathways. Spectrum of fungal bioluminescence ( Neonothopanus nambi, in green) overlaid onto the absorbance spectrum of plant leaves ( Nicotiana tabacum, in dark gray). Although caffeic acid is not native to animals, autonomous luminescence can be enabled by including two additional enzymes needed for its biosynthesis from tyrosine - tyrosine ammonia lyase and coumarate 3-hydroxylase, or their functional equivalents ( Figure 1b, Supplementary Figure 1) 3.įeatures of the fungal bioluminescence system.Ī. Moreover, the green luminescence fits within an optical transparency window of pigmented plant tissues ( Figure 1a).

Thus, the caffeic acid cycle in luminous fungi is well suited for integration into plant metabolism. Here, we report light emission in Nicotiana tabacum and benthamiana plants without the need for exogenous substrate, by integrating fungal bioluminescence genes into the nuclear genome.Ĭaffeic acid is a key component of the phenylpropanoid pathway in vascular plants, leading to lignin and a host of other secondary metabolites. Although the metabolic pathway underlying fungal bioluminescence (the caffeic acid cycle) was recently reported 2, applying this to luminescent imaging in multicellular organisms had only been demonstrated with externally-supplied substrate. Bioluminescent reporters have not been broadly applied in plants because exogenous addition of luciferin can be toxic, technically cumbersome and expensive.

Luminous plants