Effect of Different Light Quality on Photosynthetic Characteristics of Cucumber Leaves

Cucumber (Cucumis sativus L. cultivar "Changchun Mici") seedlings were cultured in Hoagland solution under irradiation with different light spectra (8 h per day) for 20 days. The red light (λmax 658 nm, λ1/2 25 nm), blue light (λmax 450 nm, λ1/2 43 nm) and white fluorescent light possessed the same fluent rate (20 μmol· m-2·s-1 ). The experimental results showed that chlorophyll content of the leaves grown under white light was 7 % and 22.4% higher than those in red and blue light, respectively. Compared with white and blue light, red light induced a lower Chl a/b ratio and a higher level of Chl b in the cucumber leaves. Measurements of the low temperature (77 K) fluorescence emission spectra and kinetics of Chl a fluorescence induction of the leaves proved that the leaves grown under red light expressed the highest PSⅡ and the lowest PSⅠactivities while the leaves under blue light had the lowest PSⅡand the highest PSⅠ activities. The O2 evolution rate of red light-grown leaves was 44.9% higher than that of the white light-grown leaves, while blue light effect was similar to that of white in respect of O2 evolution. It is concluded that light quality is an important factor in regulating the development and activities of PSⅡ and PSⅡand the O2 evolution of photosynthesis in cucumber leaves.     

Purification of the yellow fluorescent protein from Vibrio fischeri and identity of the flavin chromophore

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.     

Identification of the amino acid residues responsible for the reversible photoconversion of the monomeric red fluorescent protein TagRFP

The site-directed mutagenesis of the monomeric red fluorescent protein TagRFP and its variants was performed with the goal of generating reversibly photoactivatable fluorescent proteins. Amino acids at positions 69, 148, 165, 179, and 181 (enumeration according to the green fluorescent protein GFP) were shown to play a key role in the manifestation of the photoactivatable properties. A reversibly photoactivatable red fluorescent protein KFP-HC with excitation and emission maxima at 585 and 615 nm, respectively, was generated. The KFP-HC fluorescent intensity was decreased by 5–10 times under green light (530–560 nm) irradiation (due to the fall of the fluorescence quantum yield) and restored under irradiation with blue light (450–490 nm) or after incubation in the dark (recovery half-time of 30 min).     

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.     

金属离子对黑米花青苷色素吸收光谱的影响

以黑糯B糙米皮为实验材料 ,用 1 .5mol/L盐酸— 95 %乙醇 (V/V :1 5 / 85 )溶液提取黑米花青苷色素(BRAP) ,采用紫外可见分光光度法研究了 1 1种金属离子以及 (NH4 ) 1+ 离子对BRAP的作用。结果表明 ,未加离子条件下色素溶液可见光区λmax5 35nm ,紫外光区λmax2 80nm ,加入Al3 + 、Fe3 + 、Fe2 + 、Cu2 + 、Mn2 + 、Zn2 + 、Sn2 + 对其吸收光谱有显性影响。其中Al3 + 、Fe3 + 使 5 35nm特征吸收峰发生蓝移 ,Sn2 + 使其发生明显红移 ;Al3 + ,Fe2 + ,Mn2 + ,Zn2 +在 5 35nm附近有增加ABS值作用 ,Fe3 + 有减小ABS值作用 ;延长作用时间 ,Cu2 + 对BRAP吸收光谱的影响表现为λmax5 35nm发生蓝移 ,ABS值减小     

Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy

Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at ∼610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.     

Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent,Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment

The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA’s fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA’s fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (～70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.     

On-line green fluorescent protein sensor with LED excitation

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.     

Blue excitable green emitting Ce3+ doped CaS phosphor for w‐LEDs

CaS:Ce³⁺ is an efficient green‐emitting (535 nm) phosphor, excitable with blue light (450–470 nm) and was synthesized via a solid‐state reaction method by heating under a reducing atmosphere. The luminescent properties, photoluminescent (PL) excitation and emission of the phosphor were analyzed by spectrofluorophotometry. The excitation and emission peaks of the CaS:Ce³⁺ phosphor lay in the visible region, which made them relevant for light‐emitting diode (LED) application for the generation of white light. Judd‐Oflet parameters were calculated and revealed that green light emitted upon blue illumination. The prepared phosphor had strong blue absorption at 470 nm and a broad green emission band range from 490–590 nm with the peak at 537 nm. The characteristics of the CaS:Ce³⁺ phosphor make it suitable for use as a wavelength tunable green emitting phosphor for three band white LEDs pumped by a blue LED (470 nm). The Commission International de l'Eclairage co‐ordinates were calculated by a spectrophotometric method using the spectral energy distribution (0.304, 0.526) and confirm the green emission. The potential application of this phosphor is as a phosphor‐converted white light‐emitting diode. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparison of the spectral emission of lux recombinant and bioluminescent marine bacteria

The purpose of the present paper was to study the influence of bacteria harbouring the luciferase‐encoding Vibrio harveyi luxAB genes upon the spectral emission during growth in batch‐culture conditions. In vivo bioluminescence spectra were compared from several bioluminescent strains, either naturally luminescent (Vibrio fischeri and Vibrio harveyi) or in recombinant strains (two Gram‐negative Escherichia coli::luxAB strains and a Gram‐positive Bacillus subtilis::luxAB strain). Spectral emission was recorded from 400 nm to 750 nm using a highly sensitive spectrometer initially devoted to Raman scattering. Two peaks were clearly identified, one at 491–500 nm (± 5 nm) and a second peak at 585–595 (± 5 nm) with the Raman CCD. The former peak was the only one detected with traditional spectrometers with a photomultiplier detector commonly used for spectral emission measurement, due to their lack of sensitivity and low resolution in the 550–650 nm window. When spectra were compared between all the studied bacteria, no difference was observed between natural or recombinant cells, between Gram‐positive and Gram‐negative strains, and growth conditions and growth medium were not found to modify the spectrum of light emission. Copyright © 2003 John Wiley & Sons, Ltd.     

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λex=355 nm and λem=440 nm. We concluded that the optimal wavelengths for 3-AQ were λex=355 nm and λem=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λex/em values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent pro\xadperties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Geographic color variation of Phelotrupes auratus (Coleoptera,Geotrupidae) in the Kinki region,central Japan: A quantitative spectrophotometric analysis

We investigated geographic color variation of the beetle Phelotrupes auratus in the Kinki region of central Japan using a spectrophotometer. The reflectance spectrum of the dorsal surface of the elytron was measured for beetles collected from 23 sites. We focused on λmax(α), the wavelength at the peak (α peak) between 400 and 700 nm (human visual sensitivity range), for analyses of color variation. In populations distributed on the Kii Peninsula, in the southern part of the Kinki region, average λmax(α) values were lower (480–497 nm) than in populations distributed in the area west of Lake Biwa, the western part of the Kinki region (618–633 nm). For populations distributed in the areas south and southeast of Lake Biwa, a geographic cline of λmax(α) was observed approximately along an east–west transect, with average λmax values varying continuously from 624 to 557 nm. The easternmost populations along this cline had almost the same λmax(α) values as the populations distributed west of Lake Biwa. The coefficient of variation for λmax(α) tended to be larger in populations with intermediate averages than those with lower or higher average values.     

Color vision of the coelacanth (Latimeria chalumnae) and adaptive evolution of rhodopsin (RH1) and rhodopsin-like (RH2) pigments

The coelacanth, a "living fossil," lives at a depth of about 200 m near the coast of the Comoros archipelago in the Indian Ocean and receives only a narrow range of light at about 480 nm. To see the entire range of "color" the Comoran coelacanth appears to use only rod-specific RH1 and cone-specific RH2 visual pigments, with the optimum light sensitivities (lambda max) at 478 nm and 485 nm, respectively. These blue-shifted lambda max values of RH1 and RH2 pigments are fully explained by independent double amino acid replacements E122Q/A292S and E122Q/M207L, respectively. More generally, currently available mutagenesis experiments identify only 10 amino acid changes that shift the lambda max values of visual pigments more than 5 nm. Among these, D83N, E1220, M207L, and A292S are associated strongly with the adaptive blue shifts in the lambda max values of RH1 and RH2 pigments in vertebrates.     

Chromatin fluorescence after carmine staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light was 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.     

不同波长蓝光LED对两个品种水稻秧苗生长的影响

传统的大田育秧方式容易受到自然条件的影响,如在连续的阴雨天容易导致烂秧,而采用人工可控的工厂化育秧则有助于解决此类问题。LED光源具有光质纯、光效高以及低能耗等优势,是一种有望在工厂化育秧中应用的人工光源。该研究以生长特性差异明显的水稻天优998(籼稻)和圣稻14(粳稻)为材料,在光强为(60±5)μmol·m~-2·s~-1、光周期为12 h·d~-1的条件下,以450、470和490 nm 3个波长的蓝光LED照射天优998和圣稻14秧苗,考察了不同波长蓝光对两个品种的三叶期和五叶期水稻秧苗生长的影响。结果表明:蓝光处理下两个品种、不同秧龄幼苗的壮苗指数、根系活力和根数增加,体内碳、氮代谢活性增强,抗氧化物酶活性也会增加。3个波长的蓝光对两个品种、不同秧龄水稻秧苗生长的影响存在差异,450 nm照射下水稻秧苗的各项生长指标较优,生产上育秧可根据秧龄选择450 nm或490 nm作为独立和组合光源。     

Fluorescence studies of phosphoribulokinase, a light-regulated, chloroplastic enzyme

Recent characterization of spinach phosphoribulokinase has revealed that the homodimeric molecule contains only two tryptophans per 44-kDa subunit. We have performed steady-state and frequency domain studies of the intrinsic fluorescence of this protein. The fluorescence properties reflect contributions from both types of tryptophan residues. One of these appears to be relatively exposed to solvent and the quencher, acrylamide; fluoresce with a lambda max of 345 nm; decay with a fluorescence lifetime of 6.3 ns; have a relatively red-shifted absorption spectrum; and have a certain degree of independent motional freedom, with respect to the protein. The other tryptophan residue appears to be more buried; fluoresce with lambda max of 325 nm; have a lifetime of 1.7 ns; have a relatively blue-shifted absorption spectrum; and not to enjoy independent motional freedom. On comparison of phase-resolved spectral data and solute quenching data, we suggest that resonance energy transfer between the blue and red tryptophan residues may occur. We also describe the strategy of simultaneously fitting Stern-Volmer quenching data collected at two emission wavelengths.     

PHOTOSYNTHETIC LIGHT-HARVESTING FUNCTION OF VIOLAXANTHIN IN NANNOCHLOROPSIS SPP. (EUSTIGMATOPHYCEAE)1

Whole cell absorption spectra of the Eustigmatophycean algae Nannochloropsis salina Bourrelly and Nannochloropsis sp. reveal the presence of a distinct absorption peak at 490 nm. The lack of chlorophylls b and c in these species indicates that this peak must be attributed to carotenoid absorption. In vivo fluorescence excitation spectra for chlorophyll a emission show a corresponding maximum at 490 nm. This peak is more clearly resolved than carotenoid maxima in other algal classes due to the absence of accessory chlorophylls. The carotenoid composition of the two Nannochloropsis species shows that violaxanthin and vaucheriaxanthin are the main contributors to 490 nm absorption. Violaxanthin accounts for approximately 60% of the total carotenoid in both clones. We conclude that light absorption by violaxanthin, and possibly by vaucheriaxanthin, is coupled in energy transfer to chlorophyll a and that violaxanthin is the major light-harvesting pigment in the Eustigmatophyceae. This is the first report of the photosynthetic light-harvesting function of this carotenoid.     

Chromatin Fluorescence after Carmine Staining

After staining with dilute solutions (0.1 mg/ml in distilled water) of commercial carmine, a strong reddish orange fluorescence was observed in nuclei from cell smears and frozen and paraffin tissue sections. Optimal exciting light ws 436 nm (violet-blue) or 450-490 nm (blue). Compact chromatin from interphase nuclei, mitotic and meiotic chromosomes and the kinetoplast of Trypanosoma cruzi showed the highest fluorescence, while the basophilic cytoplasm appeared weakly fluorescent. No emission was observed in cartilage matrix, mast cell granules or goblet cell mucin. This selective method could be valuable in microscopic and cytochemical studies on chromatin because the carmine fluorescence is stable and preparations can be dehydrated and mounted permanently without changes in the fluorescence pattern.