Abstract
Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.
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Data availability
The data sets generated or analyzed in the current study are available from the corresponding authors upon reasonable request. Unprocessed images of luminescent flowers captured on a Sony Alpha ILCE-7M3 camera and IVIS Spectrum CT are available from Figshare (https://doi.org/10.6084/m9.figshare.11353601). Plasmid sequences are available in Genbank under the following accession numbers: pHpaB-C1, MT233533; pHpaC-C1, MT233534; pnnCPH-C1, MT233535; pnnH3H-C1, MT233536; pnnHispS-C1, MT233537; pnnLuz-C1, MT233538; pnpgA-C1, MT233539; pRcTAL-C1, MT233540; pX037, MT233541. Sanger and Illumina sequencing results are available as Supplementary Data.
Change history
04 June 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41587-020-0578-0
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Acknowledgements
This study was designed, performed and funded by Planta LLC. We thank K. Wood for assisting in manuscript development. Planta acknowledges support from the Skolkovo Innovation Centre. We thank D. Bolotin and the Milaboratory (milaboratory.com) for access to computing and storage infrastructure. We thank S. Shakhov for providing photography equipment. The Synthetic Biology Group is funded by the MRC London Institute of Medical Sciences (UKRI MC-A658-5QEA0, K.S.S.). K.S.S. is supported by an Imperial College Research Fellowship. Experiments were partially carried out using equipment provided by the Institute of Bioorganic Chemistry of the Russian Academy of Sciences Сore Facility (CKP IBCH; supported by the Russian Ministry of Education and Science Grant RFMEFI62117X0018). The F.A.K. lab is supported by ERC grant agreement 771209—CharFL. This project received funding from the European Union’s Horizon 2020 Research and Innovation Programme under Marie Skłodowska-Curie Grant Agreement 665385. K.S.S. acknowledges support by President’s Grant 075-15-2019-411. Design and assembly of some of the plasmids was supported by Russian Science Foundation grant 19-74-10102. Imaging experiments were partially supported by Russian Science Foundation grant 17-14-01169p. LC-MS/MS analyses of extracts were supported by Russian Science Foundation grant 16-14-00052p. Design and assembly of plasmids was partially supported by grant 075-15-2019-1789 from the Ministry of Science and Higher Education of the Russian Federation allocated to the Center for Precision Genome Editing and Genetic Technologies for Biomedicine. The authors would like to acknowledge the work of Genomics Core Facility of the Skolkovo Institute of Science and Technology, which performed the sequencing and bioinformatic analysis.
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Authors and Affiliations
Contributions
T.M., A.S.M., L.G.S., T.V.C., E.B.G., T.A.K., N.M.M., S.V.C., A.S.T., L.I.F., K.A.P., E.S.S., Y.V.G., V.V.N., S.A.D., P.V.S., O.A.M., V.O.S., S.M.D., A.I.B., A.S.P. and K.S.S. performed experiments. T.M., A.S.M., L.G.S., T.V.C., E.B.G., T.A.K., N.M.M., S.V.C., A.S.T., L.I.F., K.A.P., E.S.S., Y.V.G., V.V.N., S.A.D., P.V.S., O.A.M., V.O.S., S.M.D., A.I.B., A.S.P., V.V.C., S.V.D., F.A.K., I.V.Y. and K.S.S. performed data analysis. A.S.M. designed imaging setup, planned and performed experiments, analyzed data and wrote the paper. I.V.Y. and K.S.S. proposed and directed the study, planned experimentation and wrote the paper. All authors reviewed and commented on the paper draft.
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Competing interests
This work was supported by Planta LLC. I.V.Y. and K.S.S. are shareholders and employees of Planta. Planta has filed patent applications related to use of components of the fungal bioluminescent system and development of glowing transgenic organisms.
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Integrated supplementary information
Supplementary Figure 1. Engineering of autonomous luminescence in mammalian cells.
a. Overlay of ambient light (black and white) and luminescence (pseudocolor) images of a multiwell plate with HEK293T cells co-transfected with plasmids encoding genes of fungal bioluminescence pathway and caffeic acid biosynthesis: rctal, hpab, hpac, nnhisps, npga, nnh3h, nncph, nnluz. A representative luminescence image of three independent experiments. b. Light emission from autoluminescent cells, in standard МЕМ medium with 20 mM HEPES and in the same medium supplemented with caffeic acid. Data points were collected in three independent experiments.
Supplementary Figure 2.
Structure of the plasmid used to create plant lines with self-sustained luminescence.
Supplementary Figure 3. Phenotype of glowing and wild type Nicotiana tabacum plants.
Potted glowing plants (line NT001) and wild type (line NT000) plants of the same age in ambient light (a, b) and in the dark (c). The 4-week old plants on panel a are representative of 100 wild-type and 100 glowing plants grown from in-vitro in four separate experiments. d. Comparison of chlorophyll a, chlorophyll b and carotenoid content in leaves, leaf dimensions and plant height of 8-week old plants of the transgenic line NT001 and the wild type line NT000.The boxes extend from the lower to upper quartile values of the data, orange line represents the median. Whiskers represent a full data range. The exact sample sizes are denoted above each box plot, the statistics (U) and p-values of two-tailored Mann-Whitney U test are noted in the lower right part of each plot.
Supplementary Figure 4. Bioluminescent Nicotiana tabacum and Nicotiana benthamiana plants.
a and b are photos of potted plants in ambient light and in the dark, respectively, captured on Sony Alpha ILCE-7M3 camera. Leaves marked with pink triangles on a, as well as control leaves from wild type plants, were then cut off and imaged in IVIS Spectrum CT in ambient light and in the dark separately for Nicotiana tabacum (c, d) and Nicotiana benthamiana (e, f). Black paper box around the control leaves visible on images c and e was used to prevent light emitted by transgenic plants from illuminating leaves of the control plants. All images correspond to a single experiment performed on three glowing and one wild-type Nicotiana tabacum, and three glowing and one wild-type Nicotiana benthamiana plants.
Supplementary Figure 5. Flowers collected from the wild type and bioluminescent Nicotiana tabacum and Nicotiana benthamiana lines, imaged on IVIS Spectrum CT.
Nicotiana tabacum (a, b) and Nicotiana benthamiana (c, d) lines. a and c are photos in ambient light, b and d are photos in the dark. The black paper box around the control flowers visible on images a and c was used to prevent light emitted by transgenic plants from illuminating flowers of the control plants. All flowers depicted were randomly collected from different plants in a single imaging experiment (6 glowing and 3 wild-type Nicotiana tabacum, 6 glowing and 4 wild-type Nicotiana benthamiana plants).
Supplementary Figure 6. Quantitative comparison of light emission from flowers of Nicotiana tabacum (right side of each photo) and Nicotiana benthamiana (left side of each photo).
a and b are images in ambient light and in the dark captured on IVIS Spectrum CT. c and d are images of the same flowers in ambient light and in the dark captured on Sony Alpha ILCE-7M3. On c and d, the flowers were imaged along with the calibrated light source XLS-4 that emits 1.6*109 photons/sec at 525 nm. This calibrated light source was separately imaged on IVIS Spectrum CT, see Figshare (doi: 10.6084/m9.figshare.11871888) for raw files. All the flowers were collected from different plants in a single imaging experiment (14 glowing Nicotiana tabacum, and 13 glowing Nicotiana benthamiana plants).
Supplementary Figure 7. ROI selection for the analysis of light emission from flowers used to generate brightness values in the Supplementary Table 1 (note that the numbers on the image represent the total photon flux per second for each ROI and not the average radiance).
Analysis of normalised total flux in the designated ROIs is available at Figshare (doi: 10.6084/m9.figshare.11871888). The flowers demonstrated on this figure are the same as in the Supplementary Figure 6.
Supplementary Figure 8. Photos of glowing Nicotiana benthamiana plant taken on a smartphone in ambient light and in the dark with 30-second exposure.
(A) Taken on a smartphone in ambient light. (B) Taken in the dark with 30-second exposure. Images are the result of a single experiment.
Supplementary Figure 9. Infiltration of Nicotiana benthamiana leaves with solutions of hispidin precursors.
A. Representative photo of transgenic bioluminescent Nicotiana benthamiana leaves injected with solution of individual hispidin precursors. The images are representative of four experiments repeated independently with similar results B, C. Injections of hispidin and luciferin in leaves of bioluminescent and wild-type N. benthamiana plants. The experiment was repeated twice independently with similar results. D. Box plots showing luminescence response at sites of injections of mixtures of hispidin precursors of various compositions. Data points are shown, Luminescence change is absolute change of signal (ΔL = luminescence after injection – initial luminescence). Strikethrough text highlights the absence of a compound in the mixture. Notably, only mixtures containing caffeic acid induced the increase of luminescence. The box extends from the lower to upper quartile values of the data, orange line represents the median. Whiskers represent a full data range. Groups containing multiple reagens (Kruskal-Wallis H Test, H-statistic=48.59, p=7.11e-10) and single potential reagents (Kruskal-Wallis H Test, H-statistic: 68.65, p=8.3e-15) were analyzed separately. P-values of post-hoc pairwise Mann-Whitney U-tests highlighting the effect of caffeic acid on the luminescence response are indicated below the brackets between the box plots E. Effect of injections of three successive luciferin precursors on the intensity of luminescence of N. benthamiana leaves.
Supplementary Figure 10.
Photos of cross-section of flowers from a glowing Nicotiana tabacum plant in ambient light (A) and in the dark (B). The images of two flowers from different plants depicted on the figure are representative of six cut flowers from three plants.
Supplementary Figure 11. Dynamics of bioluminescence following injuries in leaves.
A. A representative image of a leaf before injury. B. Close-up photo of the cut site. C. Dynamics of bioluminescence at the cut site following injury (exposure time — 5 seconds). Scale bar corresponds to 1 cm. The video version of the panel C is available as Supplementary Video 7. The experiment with leaf injury was repeated three times with similar results.
Supplementary Figure 12. Pruning-induced sustained increase in bioluminescence in lateral shoots.
A. Photo under ambient light. Lateral shoots are marked with yellow arrows. The cut is highlighted by a dashed yellow line on the enlarged photo (bottom). B. Photo captured in the dark. The images are representative of two imaging experiments performed on six plants with similar results.
Supplementary Figure 13. Dynamics of luminescence in whole plants.
a. Photo of transgenic Nicotiana tabacum plant sprayed with 5 mM methyl jasmonate solution or buffer (control) before and after treatment. Two plants depicted are representative of six plants analyzed in three independent imaging experiments. b. Photo of transgenic Nicotiana tabacum plant incubated in a closed vial with ripe banana skin. Two plants depicted are representative of four plants analyzed in two independent imaging experiments. c. Circadian oscillations of luminescence. Photos of three plants were being captured constantly for ten days, in normal light conditions (17.5 h day; days 1-3 and 8-10) or in constant darkness (days 4-7). Each image of Plant 1 was compressed into a single vertical line of pixels to create the kymogram. The mean brightness of plants 1-3 is displayed on the graph. The graph on days 1-3 is representative of nine plants in three independent experiments. The full graph (days 1-10) shows the luminescence of three plants in a single experiment. Pseudocolor is used for visual clarity.
Supplementary Figure 14. Southern blot of DNA extracted from various glowing transgenic tobacco lines, with the probe annealing to nnluz gene.
a. Southern blot of DNA extracted from various glowing transgenic tobacco lines, with the probe annealing to nnluz gene. The results of the blot indicate the presence of two copies of nnluz gene in the genome of the line NT001 (used for all N. tabacum experiments in this study). The Southern blot is from a single experiment performed on DNA samples extracted from five transgenic plant lines and one control line. b. Unprocessed full scan image of the same blot.
Supplementary Figure 15. Patterns of bioluminescence dynamics in leaves of young glowing plants.
Overlay of photos in ambient light and in the dark is shown on A. Dynamics of luminescence from the two plants marked with a square on A is displayed on B. The video version of panel B is available as Supplementary Video 9. The images on panel B are representative of six plants in this experiment, and one more time-lapse experiment with three plants was performed with similar results.
Supplementary Figure 16. Photos of cross-section of stem of a glowing Nicotiana tabacum plant in ambient light and in the dark.
(A) In ambient light. (B) in the dark.The robust light emission belongs to the cells of shoot pith parenchyma (in the center of the stem). Weaker bioluminescence is observed from core parenchyma cells (periphery of the stem). Maximal light intensity corresponds to the region of the axillary bud — newly forming meristem — and surrounding parenchymatous cells. Completely lignified tissues, xylem and phloem, with cambium in between, lack luminescence (visible at the right side of the stem as a dark band). The images are representative of two experiments with similar results.
Supplementary Figure 17. Expression of nnluz in N. tabacum throughout the day measured by qPCR on 9 cDNA samples obtained from plants grown in natural light conditions.
All data points, including the technical replicates are shown as dots, each colour represents a different biological sample. The blue dots represent the mean Ct values at each time point. Experiment was performed once.
Supplementary information
Supplementary Information
Supplementary Figs. 1–17, Tables 1–3 and Notes 1–7.
Supplementary Video 1
Injection of hispidin in leaves of autonomously luminescent N. tabacum. Time-lapse luminescence imaging after injection of hispidin (into the central area of the upper part of the blade) and caffeic acid (into the apical part) of leaves of autonomously luminescent N. tabacum. Luminescence intensity is shown in pseudocolor. The video is representative of two imaging experiments performed on six plants.
Supplementary Video 2
Germinating T1 seeds. Time-lapse imaging of transgenic N. tabacum T1 seeds germinating in transparent agar. Overlay of photos in ambient light (gray) and luminescence (pseudocolor) is shown. The video of five germinating seeds is representative of two imaging experiments with similar results (in total, 32 seedlings).
Supplementary Video 3
Regenerating roots. Time-lapse luminescence imaging of regenerating roots of cut shoots of transgenic N. tabacum plants (photos every 5 min). Luminescence intensity is shown in pseudocolor. The video of roots of two individual plants is representative of three independent imaging experiments on six plants.
Supplementary Video 4
Root microscopy. Time-lapse microscopy luminescence imaging of regenerating roots of cut shoots of transgenic N. tabacum plants (photos every 5 min). Luminescence intensity is shown in pseudocolor. The video shows the roots of an individual plant and is representative of two independent experiments of imaging with high magnification with similar results.
Supplementary Video 5
Long time-lapse throughout lifespan of plants. Time-lapse imaging of transgenic N. tabacum plants from vegetation to flowering. Luminescence intensity is shown in pseudocolor. Yellow asterisks indicate daytime. This long-term time-lapse is a result of a single experiment
Supplementary Video 6
Time-lapse imaging of flowers. Time-lapse luminescence of flowers of autonomously luminescent N. tabacum. Luminescence intensity is shown in pseudocolor. The depicted flowers from a single plant are representative of imaging experiments with similar results.
Supplementary Video 7
Leaf injuries in N. tabacum. Time-lapse luminescence imaging after injury of leaf blade of autonomously luminescent N. tabacum. Luminescence intensity is shown in pseudocolor. The experiment with leaf injury was repeated three times with similar results.
Supplementary Video 8
Dynamic luminescence after pruning. Time-lapse luminescence imaging of pruning-induced lateral shoots of autonomously luminescent N. tabacum. Luminescence intensity is shown in pseudocolor. The video is representative of two time-lapse imaging experiments performed on six plants with similar results.
Supplementary Video 9
Dynamic patterns of luminescence in leaves of young plants. Dynamics patterns of bioluminescence in leaves of young glowing plants. Time relative to the start of the day (+HH format) as well as absolute time (HH:MM format) is displayed in the lower left corner of the video. The video is representative of two imaging experiments with simila results.
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Mitiouchkina, T., Mishin, A.S., Somermeyer, L.G. et al. Plants with genetically encoded autoluminescence. Nat Biotechnol 38, 944–946 (2020). https://doi.org/10.1038/s41587-020-0500-9
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DOI: https://doi.org/10.1038/s41587-020-0500-9
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