Chlorophyll fluorescence of Nicotiana tabacum expressing the green fluorescent protein

Evidence that the green fluorescence protein (GFP) develops a significant toxicity in plants has not been found, but it may represent a source of free radicals as a consequence of its fluorescence. In addition, green light is known to trigger the acclimatisation of the photosynthetic system towards a shady environment. Moreover, the light-harvesting system may acclimate to an increased availability of green light. Each of these effects may be induced by the GFP. Therefore, the hypothesis was tested, whether transformation of Nicotiana tabacum cv. Bursan to express the GFP could affect chlorophyll fluorescence parameters. The analysis revealed a significantly lower absorption of energy per excited cross section in GFP-transformed tobacco, a lower number of active reaction centres per excited cross section, a larger absorption and trapped energy flux leading to the reduction of the primary quinone electron acceptor of PSII per reaction centre, and a lower variable fluorescence. Additional key words: low-light stress; OJIP transient; steady-state procedure; toxicity of GFP. Received 14 July 2019, accepted 18 December 2019. +Corresponding author; e-mail: norbert.keutgen@boku.ac. at Abbreviations: ABS/CS0 – absorption of energy per excited cross section of a photosynthesizing sample (CS0) in the dark-adapted state ≈ F0 – minimum fluorescence when all reaction centres are open at the onset of fluorescence induction; ABS/CSm – absorption of energy per excited cross section of a photosynthesizing sample (CSm) at maximum fluorescence ≈ Fm – maximum fluorescence when all reaction centres are closed; ABS/RC – absorption flux of antenna Chls per reaction centre (RC); BAP – 6-benzylaminopurine; Chl – chlorophyll; CS0 – cross section of a photosynthesizing sample in the dark-adapted state; CSm – cross section of a photosynthesizing sample at maximum fluorescence; ET0/CSm – electron transport flux further than QA per CSm; ET0/RC – electron transport flux further than QA per RC; F0' – minimum chlorophyll fluorescence in the light-adapted state; Fm' – maximum fluorescence under actinic light; Ft – stationary fluorescence; Fv – variable fluorescence = F0 – Fm; Fv' – variable fluorescence in the light-adapted state = F0' – Fm'; GFP – green fluorescence protein; IAN – 3-indolylacetonitrile; MS medium – Murashige-Skoog medium; NPQ – Stern-Volmer nonphotochemical quenching parameter; PAM – pulse-amplitude modulation; PIABS – performance index for energy conservation of photons absorbed by PSII to the reduction of intersystem electron acceptors; PITotal – performance index for energy conservation from photons absorbed by PSII to the reduction of PSI end acceptors; QA – the primary quinone electron acceptor of PSII; qL – estimate of the fraction of open PSII reaction centres; qP – photochemical quenching coefficient; RC – reaction centre; RC/CSm – number of active reaction centres per excited cross section; RE0/CSm – electron flux reducing end electron acceptors at the PSI acceptor side per CSm; RE0/RC – electron flux reducing end electron acceptors at the PSI acceptor side per RC; RFd – fluorescence decrease ratio; TR0/CSm – trapped energy flux leading to QA reduction per CSm; TR0/RC – trapped energy flux leading to QA reduction per RC; δR0 – probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side; φE0 – quantum yield for electron transport; φP0 – maximum quantum efficiency of PSII; φPSII – actual photochemical quantum yield; φR0 – quantum yield for the reduction of end electron acceptors at the PSI acceptor side; ψ0 – probability (at t = 0 μs) that a trapped exciton moves an electron into the electron transport chain beyond QA. Acknowledgements: The authors gratefully acknowledge the assistance of Anna Figas for taking care of plant cultivation. Joanna Troczyńska originally developed the GFP-expressing plants and conducted preliminary Chl fluorescence tests. During the reviewing process considerable input for improvement of the manuscript was added by Hazem Kalaji and four unknown reviewers. Their contributions are gratefully acknowledged. Open access funding was supported by the BOKU Vienna Open Access Publishing Fund. range (Tsien 1998). It is characterized by a major excitation peak at 395 nm and a minor one at 475 nm. Excitation at 395 nm results in emission peaking at 509 nm, whereas excitation at 475 nm corresponds to a maximum at 503 nm (Tsien 1998, BioTek Instruments 2006). Introduction The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that results in green fluorescence when exposed to light in the blue to ultraviolet