Fluorescence Properties of a Novel Cyanobacteriochrome GAF Domain from Spirulina that Exhibits Moderate Dark Reversion.

Cyanobacteriochromes (CBCRs) are biliproteins for photoreception that are present in cyanobacteria. These proteins possess one or more unique cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that can covalently bind the linear tetrapyrrole (bilin). Light absorption triggers the photoisomerization of bilin between the 15Z and 15E photostates. The 15E photoproduct of some CBCR GAF domains can revert to the stable 15Z state in the absence of light. In some cases, this property makes these domains function as sensors of light intensity or as red/dark optogenetic switches. However, there have been few reports regarding the applicability of these fluorescent properties. Here, we report a red/green cyanobacteriochrome GAF domain from Spirulina subsalsa, designated SPI1085g3, which exhibited photoconversion from the red-absorbing dark state (Pr, λmax = 642 nm) to the orange-absorbing photoproduct state (Po, λmax = 590 nm), and exhibited moderate dark reversion (t1/2 = 3.3 min) from the Po state to the Pr state. The SPI1085g3 Pr state exhibited intense red fluorescence (λmax = 662 nm), with a quantum yield of 0.14. The fluorescence was switched off by red light irradiation and increased in the dark. Replacement of Cys448 of SPI1085g3 with Ser resulted in a slightly improved fluorescence quantum yield and nearly 13-fold faster dark reversion (t1/2 = 15.2 s) than that of the wild type. This novel red/dark-switchable fluorescent biliprotein expands the present repertoire and diversity of photoswitchable fluorescent protein candidates.

Chromophorylation of a Novel Cyanobacteriochrome GAF Domain from Spirulina and Its Response to Copper Ions.

Cyanobacteriochromes (CBCRs) are phytochrome-related photoreceptor proteins in cyanobacteria and cover a wide spectral range from ultraviolet to far-red. A single GAF domain that they contain can bind bilin(s) autocatalytically via heterologous recombination and then fluoresce, with potential applications as biomarkers and biosensors. Here, we report that a novel red/green CBCR GAF domain, SPI1085g2 from Spirulina subsalsa, covalently binds both phycocyanobilin (PCB) and phycoerythrobilin (PEB). The PCB-binding GAF domain exhibited canonical red/green photoconversion with weak fluorescence emission. However, the PEB-binding GAF domain, SPI1085g2-PEB, exhibited an intense orange fluorescence (λabs.max = 520 nm, λfluor.max = 555 nm), with a fluorescence quantum yield close to 1.0. The fluorescence of SPI1085g2-PEB was selectively and instantaneously quenched by copper ions in a concentration-dependent manner and exhibited reversibility upon treatment with the metal chelator EDTA. This study identified a novel PEB-binding cyanobacteriochrome-based fluorescent protein with the highest quantum yield reported to date and suggests its potential as a biosensor for the rapid detection of copper ions.

[Responses of CO2 fluxes to light intensity and temperature in rice paddy field].

CO2 fluxes in rice paddy ecosystem in subtropical hilly region were measured continuously using eddy covariance technique. The objectives were to investigate the responses of CO2 fluxes to light intensity and temperature in the paddy ecosystem. Results showed a rectangular hyperbolic light-response function could be used to describe the relationship of CO2 flux and photosynthetic photon flux density (PPFD). The absolute values of CO2 fluxes increased with the increment of PPFD. When PPFD was higher than 1000 micromol/(m2 x s), the maximum was observed. CO2 fluxes responded differently to light between early and late rice. Values of quantum yield of late rice (0.0465-0.0999 micromol/micromol) were general higher than that of early rice (0.0176-0.0541 micromol/micromol). Moreover, the quantum yield and the maximum rate of photosynthesis assimilation in the blooming stage were higher than that in tillering and ripening stages. In nighttime, respiration from soil and plants (ecosystem respiration, Reco) changed exponentially with the increase of soil temperature at the depth of 5 cm (T5), 10 cm (T10), and 20 cm (T20), respectively. Whereas, T5 was more feasible than others to be considered as the temperature parameter for Reco calculation. During early rice growing season, Reco was more sensitive to temperature change than that during late rice growing season.

[Seasonal characteristics of CO2 fluxes from the paddy ecosystem in subtropical region].

CO2 fluxes from paddy ecosystem in subtropical hilly region were measured continuously using eddy covariance technique. Based on data rejecting, correcting and filling, the daily and annual CO2 fluxes were calculated from the instantaneous values, respectively. The objectives were to investigate the variation of CO2 fluxes on seasonal temporal scale, analyze the relationship between CO2 fluxes and environmental factors, and to quantify the annual net ecosystem exchange (NEE) from the paddy ecosystem. Results show the values of GPP, R(eco) and NEE are higher from Jun. to Sep. and lower in the other months. The NEE from May to Sep. accounted for above 80% of the annual value and is crucial to the whole annual value. Photosynthetically active radiation (PAR) and mean daily air temperature (T(a)) were two main influential factors for controlling the seasonal trend of GPP and NEE and could be described by binary linear functions, respectively. The annual NEE in paddy ecosystem was 2 475.6 g/(m2 x a). This is showed that paddy ecosystem was a carbon sink for the atmosphere in subtropical region.

[Estimation of CO2 fluxes from rice paddies based on transparent chamber measurement].

Closed chamber provide a valuable tool for measuring CO2 exchange fluxes. In general, the change rate of CO2 concentration is assumed to be constant in the short measurement time and a linear regression method is used to estimate the CO2 fluxes. However, due to the physical and physiological effects, the change rate of CO2 is not always constant. A linear regression method and an exponential regression method are compared in this study. Results show that during the growing stages except for the ripening, nonlinear relationship of CO2 concentration versus time was estimated in the sunny daytime. Absolute values of CO2 fluxes calculated by linear regression were lower than that by exponential regression. Whereas, CO2 concentration changed linearly with time in cloudy days or in nighttime. And no significant difference was found between the results calculated with these two methods. Accumulative CO2-C fluxes with exponential and linear regressions were compared with the values of net ecosystem exchange of CO2-C (NEE), which were calculated by net primary production (NPP) minus soil respiration. The values with the exponential regression method were closer to NEE than those with linear regression. Therefore, the linear regression method could result in underestimation of carbon budget of ecosystem. Based on transparent chamber measurement, the exponential regression is more feasible to calculate CO2 fluxes than the linear regression.