Abstract
Bioluminescent detection has become a powerful method that is used extensively in numerous areas in life science research. Given that fluorescence detection in plant cells is difficult owing to the autofluorescence of chlorophyll, the use of a luciferin–luciferase system should be effective in plant biology. However, the suitable optical window for a luminescence system in plants remains unexplored. In this study, we sought to determine the optical window and optimal luciferase reporter system for terrestrial plant analyses using Arabidopsis thaliana as a model organism. We compared six different luciferase systems and found the green enhanced Nano-lantern (GeNL)–furimazine combination to be the optimal luciferase reporter. Spectral measurements of GeNL–furimazine showed that its luminescence peak falls within the range of optical transparency for chlorophyll and, therefore, enables greater penetration through a layer of cultured A. thaliana cells. Moreover, A. thaliana plants expressing GeNL with furimazine emitted strong luminescence, which could be detected even with the naked eye. Thus, the GeNL–furimazine combination should facilitate biological analyses of genes and cellular functions in A. thaliana and all other terrestrial plants.
Highlights
Luciferase has been used as a useful reporter gene for both in vitro and in vivo bioluminescent imaging to assess biological events in different organisms [1,2,3,4,5]
For luminescent imaging in A. thaliana cells, we examined Photinus pyralis firefly luciferase (FLuc) and NLuc, which is among the brightest luciferases
These results indicated that green enhanced Nano-lantern (GeNL) is a suitable luciferase that can be used in luciferase reporter systems in A. thaliana cells and that by applying weak basic pH conditions, the luciferase activity of secreted GeNL can be further enhanced
Summary
Luciferase has been used as a useful reporter gene for both in vitro and in vivo bioluminescent imaging to assess biological events in different organisms [1,2,3,4,5]. Various types of luciferase genes have been discovered or developed, including firefly, Renilla, and Vargula luciferases [1,3,4,5,6] These systems can be classified according to the type of luciferin employed [1,3,4,5,6]. Coelenterazine is found in many marine organisms, including Renilla, Vargula, ctenophores, and shrimps, unlike firefly luciferin, it does not require ATP for the luminescence reaction. Among these systems, NanoLuc (NLuc, known as NanoKAZ [7]), a luciferase utilizing coelenterazine originating from Oplophorus, is one of the most widely used luciferases for in vivo bioimaging due to its small size, brightness, and ATP independence [8]
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