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A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms

2020; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.016847

1083-351X

Shungo Nakamura, Nae Fu, Kumiko Kondo, Ken‐ichi Wakabayashi, Toru Hisabori, Kazunori Sugiura,

Photoreceptor and optogenetics research

pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as "luminescent pH indicator protein" (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms. pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as "luminescent pH indicator protein" (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms. Since all enzymatic reactions are strongly affected by the pH of the reaction conditions, pH is a critical factor for most physiological phenomena in living cells. In chloroplasts, the pH of the lumenal side of the thylakoid membrane and that of the stroma are important factors in determining photosynthetic performance. For example, in addition to pH as a determinant of ATP synthase catalyzed reaction rate, the pH difference (ΔpH) across the thylakoid membrane determines the ATP synthesis rate because ΔpH is used as the driving force for ATP synthesis (1Mitchell P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism.Nature. 1961; 191: 144-148Crossref PubMed Scopus (2708) Google Scholar, 2Jagendorf A.T. Uribe E. ATP formation caused by acid-base transition of spinach chloroplasts.Proc. Natl. Acad. Sci. U. S. A. 1966; 55: 170-177Crossref PubMed Scopus (332) Google Scholar). Moreover, the enzymatic reaction steps regarding Calvin–Benson–Bassham cycle are also strongly influenced by pH in the chloroplast stroma (3Woodrow I.E. Berry J.A. Enzymatic regulation of photosynthetic Co2 fixation in C-3 plants.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1988; 39: 533-594Crossref Google Scholar, 4Buchanan B.B. Regulation of CO2 assimilation in oxygenic photosynthesis: the ferredoxin/thioredoxin system. Perspective on its discovery, present status, and future development.Arch. Biochem. Biophys. 1991; 288: 1-9Crossref PubMed Scopus (323) Google Scholar, 5Andersson I. Catalysis and regulation in Rubisco.J. Exp. Bot. 2008; 59: 1555-1568Crossref PubMed Scopus (169) Google Scholar). Despite the importance of these pH factors in photosynthetic reactions, in situ pH changes inside the chloroplast have never been determined. The photosynthetic reactions under the fluctuating light conditions are well studied in the present decades by mainly using pulse-amplitude modulation chlorophyll fluorescence measurement. The strong impacts of short-period fluctuation of the light intensity for photosynthesis performance are then described very well. Nevertheless, pH changes under these conditions cannot be determined because of the lack of the appropriate method to measure the intracellular or intraorganelle pH. Gene-encoded pH-sensitive fluorescent proteins are one of the possible solutions for this challenge. For use in plant cells, there are two major requirements. The first is that their working pH range should include the expected pH changes. The second is that the signal colors should not overlap with the absorption ranges of pigments, such as chlorophyll, xanthophyll, and carotenoids in chloroplasts. To date, several fluorescent-type pH sensor proteins have been reported, such as pHluorin (6Miesenbock G. De Angelis D.A. Rothman J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.Nature. 1998; 394: 192-195Crossref PubMed Scopus (1801) Google Scholar), pHusion (7Gjetting K.S. Ytting C.K. Schulz A. Fuglsang A.T. Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.J. Exp. Bot. 2012; 63: 3207-3218Crossref PubMed Scopus (93) Google Scholar), and GFpH (8Awaji T. Hirasawa A. Shirakawa H. Tsujimoto G. Miyazaki S. Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.Biochem. Biophys. Res. Commun. 2001; 289: 457-462Crossref PubMed Scopus (55) Google Scholar). However, there are two major problems in using them for pH measurement in photosynthetic organisms. First, the excitation light for measurement may promote light-dependent reactions in chloroplasts. These probes are of course inapplicable under dark conditions. Second, their pKa values are too low to observe pH changes in chloroplasts. The expected pH values in chloroplast stroma are reported to be between pH 7.0 (dark) and 8.0 (light) (9Heldt W.H. Werdan K. Milovancev M. Geller G. Alkalization of the chloroplast stroma caused by light-dependent proton flux into the thylakoid space.Biochim. Biophys. Acta. 1973; 314: 224-241Crossref PubMed Scopus (342) Google Scholar, 10Robinson S.P. The involvement of stromal Atp in maintaining the Ph gradient across the chloroplast envelope in the light.Biochim. Biophys. Acta. 1985; 806: 187-194Crossref Scopus (33) Google Scholar, 11Heiber T. Steinkamp T. Hinnah S. Schwarz M. Flugge U.I. Weber A. Wagner R. Ion channels in the chloroplast envelope membrane.Biochemistry. 1995; 34: 15906-15917Crossref PubMed Scopus (44) Google Scholar, 12Su P.H. Lai Y.H. A reliable and non-destructive method for monitoring the stromal pH in isolated chloroplasts using a fluorescent pH probe.Front. Plant Sci. 2017; 8: 2079Crossref PubMed Scopus (10) Google Scholar). To overcome the problems in applying the current pH sensor proteins in the chloroplast, we have developed a novel luminescent-type pH sensor protein to measure the intracellular pH of photosynthetic organisms using the bioluminescent resonance energy transfer (BRET) mechanism. In 1999, Xu et al. (13Xu Y. Piston D.W. Johnson C.H. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 151-156Crossref PubMed Scopus (474) Google Scholar) reported the first application of BRET by using Renilla luciferase (Rluc) (14Cormier M.J. Hori K. Anderson J.M. Bioluminescence in coelenterates.Biochim. Biophys. Acta. 1974; 346: 137-164Crossref PubMed Scopus (46) Google Scholar) and enhanced yellow fluorescent protein (EYFP) (15Miyawaki A. Llopis J. Heim R. McCaffery J.M. Adams J.A. Ikura M. Tsien R.Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.Nature. 1997; 388: 882-887Crossref PubMed Scopus (2460) Google Scholar) and succeeded in measuring the interaction of KaiB protein in vitro and in vivo. After this work, Hoshino et al. (16Hoshino H. Nakajima Y. Ohmiya Y. Luciferase-YFP fusion tag with enhanced emission for single-cell luminescence imaging.Nat. Methods. 2007; 4: 637-639Crossref PubMed Scopus (88) Google Scholar) reported that Rluc–EYFP fusion protein (BAF-Y) exhibited higher BRET efficiency and brighter emission and succeeded in single-cell bioluminescence imaging in 2007. Finally, Suzuki et al. (17Suzuki K. Kimura T. Shinoda H. Bai G.R. Daniels M.J. Arai Y. Nakano M. Nagai T. Five colour variants of bright luminescent protein for real-time multicolour bioimaging.Nat. Commun. 2016; 7: 13718Crossref PubMed Scopus (95) Google Scholar) reported NanoLuc–Venus fusion protein (YeNL). NanoLuc (Nluc) (18Hall M.P. Unch J. Binkowski B.F. Valley M.P. Butler B.L. Wood M.G. Otto P. Zimmerman K. Vidugiris G. Machleidt T. Robers M.B. Benink H.A. Eggers C.T. Slater M.R. Meisenheimer P.L. et al.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.Acs Chem. Biol. 2012; 7: 1848-1857Crossref PubMed Scopus (664) Google Scholar) is the brightest luciferase derived from the Oplophorus luciferase. Venus is an EYFP derivative, which is 30-fold brighter than EYFP (19Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2076) Google Scholar). YeNL was then applied to their Ca2+ indicator and successfully monitored in vivo Ca2+ dynamics (17Suzuki K. Kimura T. Shinoda H. Bai G.R. Daniels M.J. Arai Y. Nakano M. Nagai T. Five colour variants of bright luminescent protein for real-time multicolour bioimaging.Nat. Commun. 2016; 7: 13718Crossref PubMed Scopus (95) Google Scholar). Because the light intensity of the luminescent proteins is weak enough and does not influence the proton gradient formation across thylakoid membranes coupled with photosynthetic electron transfer reactions, the luminescent protein was thought to be a good tool for the GFP-based pH sensor proteins. Accordingly, we fused pH-sensitive GFP variant protein and NanoLuc (Nluc) and obtained the desired pH sensor protein Luphin (luminescent pH indicator protein) that enabled the measurement of the linear signal changes from pH 7 to 8.5. Although it is still difficult to measure pH changes in the thylakoid lumen, we have successfully used this sensor protein to observe unexpected pH changes in the cytoplasm of cyanobacteria during the light–dark transition. First, we developed a pH-sensitive GFP variant by mutating the Enhanced GFP (EGFP). EGFP is a GFP variant that emits 509 nm fluorescence by 395 nm and 488 nm excitation light, and its pKa is 6.0 (20Cormack B.P. Valdivia R.H. Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP).Gene. 1996; 173: 33-38Crossref PubMed Scopus (2413) Google Scholar). We introduced three types of mutations. The first one contains T203V and S205V mutations that delete the excited-state intramolecular proton transfer (ESIPT) pathway (21Shu X. Leiderman P. Gepshtein R. Smith N.R. Kallio K. Huppert D. Remington S.J. An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V.Protein Sci. 2007; 16: 2703-2710Crossref PubMed Scopus (61) Google Scholar). ESIPT happens when a protonated (neutral) chromophore is excited by a 395 nm light, and electron transfer occurs immediately so that the chromophore becomes anionic before emitting fluorescence. This process is called ESIPT, and because of this phenomenon, the emission wavelength from the protonated chromophore is maintained at the same wavelength of 509 nm as anionic chromophores. Therefore, the abovementioned mutations may result in the loss of 509 nm fluorescence for the 395 nm excitation light. Because protonated chromophores are dominant at lower pH conditions, the 509 nm emission caused by BRET will be weak enough at lower pH, when the GFP variant is fused with Nluc. However, the pKa of the chromophore is expected to increase due to T203 V mutation, because it is known that removing T203 increases the pKa (22Cubitt A.B. Woollenweber L.A. Heim R. Understanding structure-function relationships in the Aequorea victoria green fluorescent protein.Methods Cell Biol. 1999; 58: 19-30Crossref PubMed Google Scholar). Next, 147iG and H148S mutations were introduced, which are close to the hydroxy group of the chromophore and are expected to affect the hydrogen bond interaction and adjust pKa to be at approximately 7.5. Mutations at the same position (insertion just behind 147th amino acid and substitution at 148th amino acid) were already applied and the folding of GFP was confirmed (23Lohman J.R. Remington S.J. Development of a family of redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments.Biochemistry. 2008; 47: 8678-8688Crossref PubMed Scopus (108) Google Scholar, 24Sugiura K. Nagai T. Nakano M. Ichinose H. Nakabayashi T. Ohta N. Hisabori T. Redox sensor proteins for highly sensitive direct imaging of intracellular redox state.Biochem. Biophys. Res. Commun. 2015; 457: 242-248Crossref PubMed Scopus (27) Google Scholar). The third mutation is a part of the superfolder mutations (25Pedelacq J.D. Cabantous S. Tran T. Terwilliger T.C. Waldo G.S. Engineering and characterization of a superfolder green fluorescent protein.Nat. Biotechnol. 2006; 24: 79-88Crossref PubMed Scopus (1262) Google Scholar) that stabilize the protein structure. In a series of superfolder mutations, S30R, Y39N, F99S, N105T, V163A, and I171V were adopted, because the other mutation positions are located close to the chromophore or the mutation points included in the first and second group, and may affect the ionization process of the chromophore. Eventually, the EGFP variant displayed a fluorescence spectrum with an emission peak at 513 to 515 nm when excited at 480 nm. In the pH range of 7.0 to 8.5, the fluorescence intensity increased with increasing the ambient pH (Fig. 1). Here, we named this variant EGFPpH. Second, we fused Nanoluc (Nluc) with EGFPpH. Nluc is a bioluminescent protein that emits 450 nm luminescence by adding the substrate "Furimazine" (18Hall M.P. Unch J. Binkowski B.F. Valley M.P. Butler B.L. Wood M.G. Otto P. Zimmerman K. Vidugiris G. Machleidt T. Robers M.B. Benink H.A. Eggers C.T. Slater M.R. Meisenheimer P.L. et al.Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.Acs Chem. Biol. 2012; 7: 1848-1857Crossref PubMed Scopus (664) Google Scholar) and can be utilized as an efficient donor of BRET (17Suzuki K. Kimura T. Shinoda H. Bai G.R. Daniels M.J. Arai Y. Nakano M. Nagai T. Five colour variants of bright luminescent protein for real-time multicolour bioimaging.Nat. Commun. 2016; 7: 13718Crossref PubMed Scopus (95) Google Scholar). The conjugated protein consists of EGFPpHΔC12 (EGFPpH lacking C-terminal 12 amino acids), the linker amino acid sequence of Trp-Thr (W-T), and NlucΔN4 (Nluc lacking N-terminal 4 amino acids) (Fig. 2A) and displayed the pH-dependent change of luminescent spectra (Fig. 2B). The absorption spectra under various pH values of the conjugated protein are shown in Fig. S1. As shown in Figure 2C, the luminescent signal ratio from EGFPpH to the luminescence intensity of Nluc nearly shows the linear relationship against pH in the range of 7.0 to 8.5, which is the pH range expected within the chloroplast stroma as previously described (9Heldt W.H. Werdan K. Milovancev M. Geller G. Alkalization of the chloroplast stroma caused by light-dependent proton flux into the thylakoid space.Biochim. Biophys. Acta. 1973; 314: 224-241Crossref PubMed Scopus (342) Google Scholar, 10Robinson S.P. The involvement of stromal Atp in maintaining the Ph gradient across the chloroplast envelope in the light.Biochim. Biophys. Acta. 1985; 806: 187-194Crossref Scopus (33) Google Scholar, 11Heiber T. Steinkamp T. Hinnah S. Schwarz M. Flugge U.I. Weber A. Wagner R. Ion channels in the chloroplast envelope membrane.Biochemistry. 1995; 34: 15906-15917Crossref PubMed Scopus (44) Google Scholar, 12Su P.H. Lai Y.H. A reliable and non-destructive method for monitoring the stromal pH in isolated chloroplasts using a fluorescent pH probe.Front. Plant Sci. 2017; 8: 2079Crossref PubMed Scopus (10) Google Scholar). We, therefore, named this sensor protein Luphin (luminescent pH indicator protein). To confirm the stability of the BRET signal from Luphin, we examined the performance of Luphin under varying temperatures at different pH levels. As shown in Figure 3A, the signals were mostly stable, whereas a decrease in the BRET ratio signal was observed at 30 °C. Next, we examined the influence of cations (Na+, K+, Ca2+, and Mg2+) on Luphin signals. The luminescence ratio was stable in the presence of 0 to 30 mM Na+ and K+ (Fig. 3, B–C) and 0 to 1 mM Ca2+ and Mg2+(Fig. 3, C–D). These results indicate that Luphin is suitable for measuring pH changes in living cells. To monitor the cytosolic pH changes under photosynthetic conditions, Luphin was expressed in the cytosol of the cyanobacteria Synechocystis sp. PCC 6803 (Synechocystis), one of the model phototrophs in the photosynthesis studies. The expression of the sensor protein in the cell was confirmed by using the western blotting method (Fig. S2). Before the pH change measurement, we have measured the PAM signal from the cyanobacteria containing Luphin and compared them with those from the WT cells to confirm that the luminescence from Luphin does not affect the photosynthetic parameters such as NPQ and ΦII (Fig. 4, Table S1). Then, the Luphin signals in Synechocystis cells were measured under light (white light of 40 μmol photons m−2 s−1) and dark conditions (Fig. 5A). The signals obtained from the original Synechocystis cells (WT) were shown as controls (Fig. 5, open symbols). When the cells were incubated in the dark, the Luphin signals showed that the estimated cytosolic pH was approximately 7.9. Although the pH immediately increased up to 8.0 just after turning on the light, the rapid pH decrease was observed even in the light conditions, and it reached 7.8 within 10 min. When the light was turned off, the intracellular pH unexpectedly increased to 8.0, and then, it was not maintained and finally decreased to 7.8. The estimated pH increase caused by light or dark transition was mostly suppressed (Fig. 5A, blue line) when the cells were incubated with 10 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transfer pathway inhibitor, suggesting that the observed changes of estimated pH were caused by photosynthetic electron transfer activity. To confirm that the observed signals shown in Figure 5A were caused by the proton transfer across thylakoid membranes, the uncoupling chemical carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP) was applied. The intracellular pH in the dark became 7.4 by the addition of 30 μM FCCP to the culture medium, which was at the same level as the BG11 medium used for Synechocystis culture (Fig. 5B, blue line). As expected, the Luphin signal did not respond to the light/dark transition in the presence of FCCP. Next, the light intensities of 10, 40, 70, and 100 μmol photons m−2 s−1 were used to investigate the light-intensity-dependent pH changes. The estimated pH increase within 1 min after irradiating dark-adapted Synechocystis was measured. As shown in Figure 5, C–D, the estimated pH increases after irradiation became small with increasing light intensity in the range of 10 to 100 μmol photons m−2 s−1. We could not examine the lower irradiance that was less than 10 μmol photons m−2 s−1 due to the limit of the irradiation facility in the laboratory. The gradual decrease of the estimated pH during light irradiation and the increase just after turning the light off were unpredicted, although we successfully observed pH changes associated with photosynthetic electron transfer activities. To elucidate the cause of the gradual pH decrease during light irradiation, we examined the effect of far-red (FR) light (40 μmol photons m−2 s−1 with peak emission at 730 nm) in the presence of 10 μM DCMU (Fig. 6A). Only the cyclic electron transfer pathway will work under these conditions because FR light only excites PSI, and DCMU inhibits plastoquinone (PQ) reduction at the PSII site. The PQ pool may be therefore rather oxidized than the situation with the white light irradiation. Interestingly, the increase of the estimated pH caused by turning on the FR light was maintained during the FR light irradiation for 10 min (Fig. 6A); however, it was then decreased in the dark. The next white light irradiation in the presence of DCMU did not induce the pH shift (Fig. 6A) as indicated in Figure 5A. These results suggest that the gradual pH decrease during white light irradiation may have been induced by PQ pool reduction, and ATP synthase must first dissipate the formed proton gradient across thylakoid membranes to synthesize ATP. In contrast, the estimated pH increase observed after turning off the light was mostly suppressed by the addition of 10 μM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) (Fig. 6B). This result suggests that the estimated pH increase after turning off the light is related to the oxidation of plastoquinol (PQH2) since DBMIB inhibits PQH2 oxidation at the cytochrome b6f (Cyt b6f) site, which is accumulated during irradiation. In this study, we developed a novel luminescent-type pH sensor protein "Luphin." By adding the substrate "Furimazine," Luphin emitted two luminescence peaks at 450 nm and 513 nm. The ambient pH can be measured as a ratio of these two luminescence intensities as shown in Figure 2B. Unlike other fluorescent-type sensors, Luphin does not require excitation light for measurement, because it is a BRET sensor that fuses pH-sensitive GFP with Nluc. This working mechanism has significant advantages when applied to photosynthetic organisms because photosystems are hardly excited by luminescence from Nluc, and autofluorescence from chlorophyll molecules is hardly induced during measurements. It is also suitable for use in weakly alkaline spaces, such as chloroplast stroma and cyanobacterial cytoplasm, as the emission ratio varies dynamically and linearly over a pH range of 7.0 to 8.5. For measuring pH in plant cells, Luphin has several advantages over the previously reported BRET-type pH sensor, pHlash (26Zhang Y. Xie Q. Robertson J.B. Johnson C.H. pHlash: a new genetically encoded and ratiometric luminescence sensor of intracellular pH.PLoS One. 2012; 7e43072Crossref PubMed Scopus (17) Google Scholar). In this sensor protein, Renilla luciferase "Rluc8" (27Loening A.M. Fenn T.D. Wu A.M. Gambhir S.S. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output.Protein Eng. Des. Sel. 2006; 19: 391-400Crossref PubMed Scopus (302) Google Scholar) was fused with a YFP variant "cpVenus" (28Nagai T. Yamada S. Tominaga T. Ichikawa M. Miyawaki A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10554-10559Crossref PubMed Scopus (805) Google Scholar). The Nluc used in this study was about 25 times brighter than Rluc8, and the dynamic range calculated as a luminescence ratio of Luphin in the range of pH 7.0 to 8.5 was greater than that of pHlash, suggesting that Luphin is more appropriate for use in photosynthetic organisms. On another note, a disadvantage of sensor proteins utilizing Rluc or Nluc is that the luminescence emitting reaction is accompanied by O2 consumption and CO2 production. For example, under light conditions, increased O2 production from PSII may promote the luciferase reaction. Therefore, the luminescence intensity from Luphin that is expressed in Synechocystis may be enhanced. In addition, since photosynthetic and respiratory electron transfers involve O2 production and consumption, the electron transfer rate may be affected by the O2 consumption during the luciferase reaction. Luphin can be localized to subcellular organelles by fusing the targeting signal sequence because it is a gene-encoded sensor protein. For example, the pH dynamics in the chloroplast stroma is a great interest in the research field of photosynthesis, and Luphin must be a good tool for this purpose. Indeed, there is a report that the luminescence from the luciferase introduced into the chloroplast was successfully observed with keeping the shape of a leaf by using EM-CCD (29Saito K. Chang Y.F. Horikawa K. Hatsugai N. Higuchi Y. Hashida M. Yoshida Y. Matsuda T. Arai Y. Nagai T. Luminescent proteins for high-speed single-cell and whole-body imaging.Nat. Commun. 2012; 3: 1262Crossref PubMed Scopus (186) Google Scholar). In addition, the internal pH change caused by photosynthetic reactions in chloroplasts can be measured simultaneously with excitation of photosystems by a red light with a wavelength longer than 600 nm, because the emission wavelength of Luphin is shorter than 600 nm. Accordingly, Luphin must be an excellent tool for in situ measurements of pH dynamics in photosynthetic organisms. In this study, we succeeded in the real-time monitoring of changes in the estimated pH in the cytoplasm by using Luphin that was expressed in Synechocystis cells. The observed changes should be mainly caused by changes in photosynthetic electron transfer activities (Fig. 5). The process of photosynthetic electron transfer and proton transfer at the thylakoid membrane of Synechocystis is briefly described as follows. First, the electrons, produced by the water-splitting reaction on the PSII, are transferred to PQ, and PQ is then reduced to PQH2. One electron of PQH2 is transferred to plastocyanin (PC) when PQH2 is oxidized at the Qo-site on cytochrome b6f (Cyt b6f). Simultaneously, the other electron is transferred to the Qi-site on Cyt b6f. The electron is used for the rereduction of PQ at the Qi-site. This electron circulation between the PQ pool and Cyt b6f is called the Q cycle (30Mitchell P. Possible molecular mechanisms of protonmotive function of cytochrome systems.J. Theor. Biol. 1976; 62: 327-367Crossref PubMed Scopus (898) Google Scholar). The electron of PC is transferred to Fd via PSI and eventually produces NADPH from NADP+. Proton influx is coupled with the electron transfer at both PSII and Cyt b6f and then generates ΔpH across thylakoid membranes. In addition, unlike green algae and green plants, cyanobacteria possess respiratory chain complexes in the thylakoid membranes. Therefore, respiratory electron transfer mainly occurs on thylakoid membranes under dark conditions. In a series of observed changes in the estimated pH in the cytoplasm of Synechocystis (Fig. 5A), the transient pH decrease during the irradiation of white light and the pH increase after turning off the light were unpredicted results. The observed changes were possibly due to the reduction of the PQ pool (Fig. 6A) and PQH2 oxidation at Cyt b6f (Fig. 6B), which are suggested by the inhibition experiments. Based on these observed changes, we here propose the mechanism of electron transfer promotion and suppression in the thylakoid membranes as follows.1.Within 1 min of irradiation, photosynthetic electron transfer is promoted and pH increase in the cytoplasm is induced (Fig. 7A).2.The PQ pool is reduced a few minutes later, and PQ, which is required for electron transfer in the Q cycle, is deprived at the Qi-site on Cyt b6f. The photosynthetic electron transfer is then suppressed. Consequently, the formation rate of ΔpH becomes slower than that of ΔpH dissipation by ATP synthase, and therefore the pH decrease in the cytoplasm is initiated (Fig. 7B).3.After turning off the light, PQ reduction at PSII is terminated and the PQ pool is rather oxidized. Therefore, the shortage of PQ at the Qi-site is relieved and the electron transfer at Cyt b6f is activated. Finally, the pH increase in the cytoplasm that accompanies PQH2 oxidation is observed, which is accumulated during irradiation. Under dark conditions, the electrons of PC must be consumed at the respiratory chain pathway, such as cytochrome c oxidase (COX) (Fig. 7C). In chloroplast, the acidification of the lumen is thought to be one of the major factors regulating linear electron transport (see the review by Tikhonov (31Tikhonov A.N. pH-Dependent regulation of electron transport and ATP synthesis in chloroplasts.Photosynth. Res. 2013; 116: 511-534Crossref PubMed Scopus (113)