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The regulation of photosynthetic pigments in terrestrial Nostoc flagelliforme in response to different light colors
Algal Research 25 (2017) 128–135
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The regulation of photosynthetic pigments in terrestrial Nostoc flagelliforme in response to different light colors
MARK
Pei-pei Han, Shi-gang Shen, Rong-jun Guo, Shun-yu Yao, Ying Sun, Zhi-lei Tan, Shi-ru Jia⁎ Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Nostoc flagelliforme Light color Photosynthetic pigment Photoregulation process
Cyanobacteria have evolved a number of photosynthetic strategies by adjusting the properties of the lightharvesting pigments to cope with changes in the light environment. Nostoc flagelliforme is a terrestrial filamentous cyanobacterium that has distinct phycobilisome composition consisting of phycocyanin (PC) and allophycocyanin (APC). The photoregulation process of N. flagelliforme was explored by investigating the effects of different light colors on photosynthetic pigments, which were determined after photoautotrophic growth with red (RL), yellow (YL), green (GL), blue (BL), purple (PL) and white (WL) light of same photosynthetically active photon flux density. Compared with WL, YL, GL, BL and PL changed the amounts of pigments but did not alter the cellular ratio of PC/APC; while RL altered both the amounts and proportions of PC and APC. The observations by confocal laser scanning microscope and transmission electron microscope showed that PC, APC and chlorophyll a were distributed throughout the cells under GL, BL, PL, and WL illumination but located in the outermost layer in RL and YL-grown cells. Further study revealed that changing RL intensity would not influence the ratio of PC/APC but the distribution of pigments shifted from central cytoplasmic region to the periphery of the cells with the increase of intensity. These results implied that N. flagelliforme responded to RL by regulating the composition and location of phycobilisomes and these findings would be of importance to develop the highefficient process of N. flagelliforme culture.
1. Introduction Environmental conditions exert a strong influence upon the cell growth, morphology and pigment composition of many photoautotrophic organisms [1–3]. As photoautotrophic organism, photosynthesis and growth rates are directly affected by light environment, because it is used to drive photosynthesis and regulate cell growth and development. Moreover, light can vary in terms of light quality, light quantity and photoperiod [4–6], thus sensing and adequately responding to light is a key attribute of eco-physiological versatility of photoautotrophic organisms. Cyanobacteria, as photoautotrophic microorganisms, are one of the oldest groups of bacteria, which can date back to the Pre-Cambrian [7]. Until now, it has evolved a large variety of light-harvesting and protective pigments, and evolved many photosynthetic strategies to help them cope with changes in their light environment [8–10]. The regulation process of photosynthetic pigments to changes in light color not only affects the composition of phycobilisomes, but also has been reported to have significant influence on many cellular processes such
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as growth, photosynthesis, lipid production [1,11], nutrient utilization [12], and extracellular polysaccharides production [4]. The regulation process requires cyanobacteria to precisely measure ratios of specific light colors in its environment [13,14]. Besides the chlorophyll a (Chl a), cyanobacteria also contain several accessory pigments, including carotenoid and phycobilin, which can help them adapt to various light conditions. The phycobilins are unique among the photosynthetic pigments in that they are bonded to certain proteins, known as phycobiliproteins, which are commonly divided into three main groups: phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE). Based on the shift in PC versus PE in the subunit structure of phycobilisome in response to light colors, four types of Chromatic Acclimation (CA) processes were identified till now [15]. Nostoc flagelliforme is a terrestrial filamentous cyanobacterium with great economic value that is mainly distributed in some arid and semiarid areas of China, where they are often subjected to extreme environmental changes [16]. As a consequence, the characteristics of N. flagelliforme are quite different from the cyanobacterium in the aquatic environment, which shows strong ecological adaptability to
Corresponding author. E-mail address: [email protected] (S.-r. Jia).
http://dx.doi.org/10.1016/j.algal.2017.04.009 Received 5 December 2016; Received in revised form 25 February 2017; Accepted 18 April 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved.
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2.3. Cell observation with CLSM
extreme conditions such as alkali, desiccation and strong UV radiation [16–18]. In addition, the phycobilisome of N. flagelliforme contains only PC and APC [19], indicating the photoregulation process may be quite different from the reported CA types in cyanobacteria. Although there are more and more studies on CA in aquatic cyanobacteria [20–22], but for terrestrial N. flagelliforme, few reports about regulation process of photosynthetic pigments are available till now. Besides that, in our previous work investigating the effects of light color on N. flagelliforme culture, it was found that light color not only had strong influence on biomass accumulation, but also had obvious impact on cellular metabolism and polysaccharides biosynthesis [4,23]. Thus illustrating the responsive mechanism of photosynthetic pigments to the changes of light color is of great importance to understand the correlations between the photoregulation process and biomass accumulation and/ or cellular metabolism. Therefore, the aim of this study was to research the photoregulation process of N. flagelliforme under light with different colors. Five different colors including red, blue, green, yellow, and purple were used and compared to white light (WL) of same photosynthetically active photon flux density. The content, composition, fluorescence properties, and localizations of photosynthetic pigments were comprehensively examined to study the alterations of photosynthetic pigments dependent on the changes of light color. The ultrastructure of N. flagelliforme cells grown under different light colors was also investigated.
An adequate amount of cells were taken and placed on the slide, which was covered by cover slip. CLSM (Hitachi F-4500, Tokyo, Japan) was used to observe the fluorescence of photosynthetic pigments in cells. To determine the optimal emission wavelength, the cells were excited with three individual laser wavelengths: 488 nm, 543 nm, or 633 nm. Subsequently the emission spectrum was gathered at 10.7 nm increments as a lambda Z-series. For 488 nm excitation, the ArKr laser was set at 10% power, while for 543 nm and 633 nm excitation, the HeNe laser was set at 30% power and 10% power, respectively. The NT 80/20 filter was used to collect the images and emission scans were collected in ~10.7 nm bandwidth increments in the range from 500 to 740 nm. In order to obtain a better signal yield, scans were performed with ‘low speed’ setting (220 Hz, full scan mode) and the confocal pinhole was opened (600 mm diameter) for most measurements. The LSM FCS Zeiss 510 Meta AIM imaging software was used to obtain the acquired CLSM images. 2.4. Cell observation with TEM A total of 10 mL of sample was taken and centrifuged at 8000 × g for 15 min, and the cell pellet was collected for subsequent analysis. The methods for chemical fixation, freeze substitution, processing and embedding of the samples were used as previously reported [26]. After that, the samples were viewed using the TEM (JEM-1230, Jeol Ltd., Tokyo, Japan) operated at 80 kV and equipped with a LaB6 filament. The Gatan 1.35 K × 1.04 K × 12 bit ES500W CCD camera was used to record all the micrographs.
2. Material and methods 2.1. Strain and culture conditions The N. flagelliforme cells (TCCC11757) cultured in BG-11 medium were obtained from the Tianjin Key Lab of Industrial Microbiology (Tianjin, China). Inoculums were prepared regularly (bi-weekly) in 500 mL shake-flask under WL at a photon flux density of 60 μmol·photons/(m2 s). Before all the experiments, the cells were incubated in a dark room for 3 days in order to reduce storage compounds, and then cells were cultured in 500 mL shake-flask containing 200 mL BG-11 medium at a photon flux density of 60 μmol·photons/(m2 s). The initial pH of the medium was adjusted to 8.0 and the shake-flask was inoculated with 10% (v/v) cell suspension i.e. the initial cell density of cell culture was 0.37 g/L dry cell weight. For the cultivation with different colors of light, WL (390–770 nm) was provided by fluorescent lamps. Red (660 nm, RL), yellow (590 nm, YL), green (520 nm, GL), blue (460 nm, BL), and purple (400 nm, PL) lights with half-band width of 5 nm were provided by different light-emitting diodes (LEDs, Model no. CDL-CF22W, Shenzhen federal heavy secco electronic Co. LTD, China). Each LED light was composed of 22 colored bulbs and the wattage and voltage of each colored bulb was 1 W and 220 V. In order to avoid any interference from external light source, all experiments were done in a dark room. A quantum sensor connected to Light Scout Dual solar quantum light meter (Spectrum Technologies, USA) was used to measure the light intensity. The cells were cultured in air conditioned environment at 25 ± 1 °C and cells grown under WL were regarded as control. According to the growth curve of N. flagelliforme under different light colors as shown in Fig.S1, samples were taken at the middle of exponential phase and beginning of stationary phase for subsequent analysis of photosynthetic pigment and cell observation with spectral confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM).
2.5. Statistical analyses Three replications from different batches of culture were performed for each experiment, and values were expressed as mean ± standard deviation. The Independent-samples t-test was used to analyze the experimental data using the SPSS statistical software (version 22.0) and the significance level was set at P < 0.05. 3. Results and discussion 3.1. The effects of light color on photosynthetic pigments In order to explore the photoregulation process of N. flagelliforme, photosynthetic pigments contents under different light conditions were measured at exponential phase and stationary phase of growth, respectively. The experiments were conducted under six different light colors: RL, BL, GL, YL, PL as well as WL at a photon flux density of 60 μmol/(m2 s). As shown in Fig. 1A, at exponential phase, the Chl a content was significantly higher under GL, WL, YL and lower under RL, than under BL and PL, while for carotenoid, it was significantly higher under PL and lower under RL, than under WL, YL, GL and BL. At stationary phase of growth (Fig. 1B), the Chl a content was higher in the order of GL, WL, YL, PL, BL and RL-grown cells with values of 4.61, 4.04, 3.97, 2.91, 2.88 and 0.98 mg/g, respectively; in comparison with exponential phase, the Chl a content remained almost unchanged, except for RL treatment, which was significantly decreased, while, the carotenoid content was slightly changed at all light conditions. The results showed that RL greatly reduced the Chl a content, and GL promoted the formation of Chl a, which corresponded with the findings of many reports [27,28]. Under PL, the highest carotenoid content was obtained with value of 1.84 mg/g, which was increased by 38.34% compared with control group. That might be explained by the fact that PL was harmful to the growth of N. flagelliforme [23], and carotenoid, as accessory pigment, could protect cell from light damage [29] besides the function of capturing light energy for use in photosynthesis.
2.2. The measurement of photosynthetic pigment The content of photosynthetic pigment was determined as previously reported [24,25]. 129
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Fig. 1. The photosynthetic pigments content of cells grown under different light colors at exponential phase (A, C) and stationary phase (B, D) of growth.
As presented in Fig. 1C, D, the changes of PC and APC contents under different light conditions presented similar patterns at both exponential and stationary phases. At exponential phase, both the PC and APC contents were significantly higher under YL, GL, BL, and lower under RL, than under WL and PL, whereas at stationary phase, they were significantly higher under GL and lower under RL, than under WL, YL, BL and PL. However, PE was not detected throughout the culture period, which was in agreement with previous study reporting only PC and APC in the isolated intact phycobilisomes [19]. The highest PC and APC contents were obtained under GL with values of 56.22 and 32.52 mg/g at stationary phase, which were respectively increased by 38.23% and 48.15%, compared to the corresponding WL control. While in RL, significant decrease (85.76% and 81.82%) in both PC and APC contents were observed compared with control. The results indicated that GL stimulated the formation of PC and APC, while RL greatly inhibited the formation of PC and APC. Interestingly, the pigmentation of RL and PL-grown cells changed from green to yellow during stationary phase, which was observed by the microscope (Fig. S2). This phenomenon might be due to an increase in the ratio of carotenoid/Chl a, as previously reported in the cyanobacteria Synechococcus and Prochlorococcus [30]. It was reported that cells would change the composition of photosynthetic pigments in response to different light environment in order to maximize the utilization of light energy during the process of photosynthesis [15]. Therefore, the composition of photosynthetic pigments at the stationary phase was subsequently calculated. As depicted in Table 1, light color had strong influence on the composition of photosynthetic pigments. In cultures incubated under RL and PL, significantly higher ratios of carotenoid/Chl a were observed compared to other light conditions. The ratio of carotenoid/ Chl a was a marker indicating cellular oxidative level [31], which
Table 1 Composition of photosynthetic pigments in the N. flagelliforme under different light colors at stationary phase of growth. Light color
WL RL YL GL BL PL
Composition of photosynthetic pigments PC/Chl a
APC/Chl a
Carotenoid/Chl a
PC/APC
6.22 4.13 5.67 7.68 8.90 6.26
3.36 2.84 3.14 4.44 5.02 3.35
0.33 0.91 0.34 0.30 0.46 0.56
1.85 1.45 1.80 1.73 1.79 1.87
± ± ± ± ± ±
0.29 0.15 0.24 0.39 0.45 0.28
± ± ± ± ± ±
0.21 0.18 0.17 0.26 0.31 0.21
± ± ± ± ± ±
0.02 0.07 0.02 0.02 0.03 0.04
± ± ± ± ± ±
0.18 0.15 0.13 0.11 0.13 0.13
suggested that RL and PL might induce photoinhibition and thus stimulated the formation of carotenoid to protect cells from light damage. Additionally, cells grown under BL exhibited the highest ratios of PC/Chl a and APC/Chl a, while the lowest values were obtained under RL treatment. There were some literatures reported that reducing the proportion of light harvesting pigment could not only improve the efficiency of photosynthesis, but also reduce the light damage when cells were exposed to strong light [32–35]. Although the light color altered the phycobiliproteins content, the ratios of PC/APC remained almost constant, indicating that the composition of phycobiliproteins was not influenced by the changes of light color. However, there was an exception that RL greatly reduced the phycobiliproteins content but changed the composition as well. 3.2. Observation of cellular auto-fluorescence by CLSM CLSM was used to examine the phycobiliprotein fluorescence properties and localizations, and investigate alterations in pigment 130
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Fluorescence intensity
1400
A
1200 1000
length was 633 nm, all spectra showed maximum peaks from 647 to 652 nm and 658 to 663 nm, with shoulders ranging from 675 to 680 nm, and fluorescence intensity decreased notably from 650 to 740 nm (Fig. 2C). The fluorescence in the ranges of 647–652, 658–663 and 675–680 nm can be assigned to PC, APC and Chl a, respectively [19,36]. Within the range of emission wavelength of PE (500 to 600 nm), no fluorescence was observed in all the emission spectra, which provided additional evidence that the phycobilisome of N. flagelliforme contained only PC and APC. Therefore, the 488 and 633 nm were selected as excitation wavelength of N. flagelliforme in the subsequent study. The fluorescence signal was collected at 647–652, 658–663 and 675–680 nm, and the false color green and red in Fig. 3 represented the fluorescence excited by 488 and 633 nm, respectively. At the excitation wavelength of 488 and 633 nm, both the fluorescence of PC and APC were clearly observed, while no fluorescence was emitted by Chl a at the excitation wavelength of 488 nm, which was in agreement with above result shown in Fig. 2A. CLSM observations showed that the N. flagelliforme cells were in beaded arrangement. Besides that, the fluorescence signals of the WL, GL, BL and PL-grown cells were located throughout the cells, while under RL and YL treatments, the fluorescence signals were mainly located at the periphery of the cells. These results showed that the PC, APC and Chl a in GL, BL, PL, WL-grown cells were distributed throughout the cells, while in RL and YL-grown cells, they were located in the outermost layer of cells. This response was likely correlated with effective light utilization and energy balance [27].
WL RL YL GL BL PL
800 600 400 200 0
500
550
600
650
700
750
Wavelength(nm) 600
B Fluorescence intensity
500
400
WL RL YL GL BL PL
300
200
3.3. Observation of cellular ultrastructure by TEM
100
The effects of light color on the ultrastructure of N. flagelliforme were investigated by means of TEM. After illumination with different light colors, dramatic changes occurred in the ultrastructure of N. flagelliforme. As shown in Fig. 4, at treatments of WL, GL, BL and PL illumination, the thylakoid membrane occupied almost the entire cytoplasm, whereas it was mostly located at the periphery of the cell wall after illumination by RL and YL. Under WL, BL and PL, thylakoid membrane developed a reticulate arrangement (Fig. 4A, E, F), while the arrangement of thylakoid was disorderly and the middle of the lamellae had been disintegrated in RL (Fig. 4B), which suggested that the thylakoid structure was destroyed. Since the photosynthetic pigments embedded directly in the thylakoid membrane, the photosynthetic pigments were distributed in the outermost layer of cells grown under RL. The observation was well consistent with the phenomenon observed by CLSM that strong fluorescence was distributed in the outermost layer of cells grown in RL and YL. Similarly, these results also indicated that photosynthetic pigments were distributed throughout the cells grown in WL, GL, BL and PL. The most striking feature was that the RL-grown cells contained more as well as bigger structural granule than that of cells grown in other light conditions (Fig. 4B). Structure granules, including polyglucangranules, lipid droplets, cyanophycin granules, polyphosphate granules and polyhedral bodies, were found to provide nutrients under adverse circumstances. An increase in the number and size of structural granule was probably the most common response of cyanobacterial cells under stress [37]. Therefore, it might be concluded that the cells grown in RL was under light stress.
0 500
550
600
650
700
750
700
750
Wavelength(nm) 1400
C
Fluorescence intensity
1200 1000
WL RL YL GL BL PL
800 600 400 200 0 500
550
600
650
Wavelength(nm) Fig. 2. Fluorescence emission spectra of N. flagelliforme after illumination with different light colors at the excitation wavelength of 488 (A), 543 (B), and 633 nm (C), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescence under different light colors. In order to identify the maximum emission wavelength of the N. flagelliforme, spectral scanning from 500 to 740 nm was performed at three individual excitation wavelengths: 488, 543 and 633 nm, respectively. From Fig. 2A, at the excitation wavelength of 488 nm, the main maximum peak was found between 647 and 652 nm, with shoulders ranging from 658 to 663 nm. As shown in Fig. 2B, for excitation at 543 nm, the emission spectra of cells showed distinct maximum emission peaks under different light colors, which suggested that 543 nm was unsuitable to be used as the excitation wavelength of N. flagelliforme. When the excitation wave-
3.4. The effects of RL with different light intensities on the photosynthetic pigments Based on the above results, the cells became yellow and the photosynthetic pigment content was greatly reduced under RL, suggesting that light inhibition might occur under RL of intensity at 60 μmol·photons/(m2 s). Therefore, the effects of RL with different light intensities (12, 30 and 60 μmol·photons/(m2 s)) on cell pigmentation and photosynthetic pigments were thoroughly compared. With increas131
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APC 658-663
PC 647-652
Chl a 675-680
WL
RL
YL
GL
BL
PL
Fig. 3. Images of N. flagelliforme cells under different light colors at stationary phase of growth as visualized by CLSM. (False color overlay image of the same cells obtained directly after lambda scanning by simultaneous three channel fluorescence detection. Channel 1: PC 647–652 nm; channel 2: APC 658–663 nm; channel 3: Chl a 675–680 nm; false color green and red represented the fluorescence excited by 488 and 633 nm, respectively.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
that the composition of phycobiliproteins was not influenced by the changes of RL intensity.
ing light intensity, as shown in Fig. 5A, cell pigmentation became more and more yellow, indicating that the proportion of photosynthetic pigments might be changed. As expected, the Chl a content was significantly decreased, but the carotenoid content remained basically unchanged with the increase of light intensity (Fig. 5B). Therefore, ratios of carotenoid/Chl a were increased with the increase of light intensity, which also explained the phenomenon that cells became more and more yellow. From Fig. 5C, no significant changes in the contents of PC and APC was observed when RL intensity increased from 12 to 30 μmol·photons/(m2 s), but the contents were obviously decreased at the intensity of 60 μmol·photons/(m2 s). However the ratios of PC/APC under three light intensities remained almost unchanged, indicating
3.5. Fluorescence of cells grown under RL with different light intensities CLSM was used to compare the effects of RL with intensities of 12, 30 and 60 μmol·photons/(m2 s) on the fluorescences of Chl a, PC and APC, and all the images were collected from the signals obtained at the excitation wavelength of 488 and 633 nm. As depicted in Fig. 6, CLSM observations showed that, when light intensity was 12 μmol·photons/ (m2 s), the cells arranged in chains throughout the culture period, but when light intensity increased up to 60 μmol·photons/(m2 s), the cells 132
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Fig. 4. Effects of light color on the ultrastructure of N. flagelliforme observed by TEM. The letter T, W and SG stood for thylakoid, cell wall, and structure granule, respectively. A–F represented cells grown under WL, RL, YL, GL, BL and PL, respectively. Bar marker represents 500 nm.
Fig. 5. The changes of cell pigmentation and pigment contents of N. flagelliforme cells with different light intensities of RL at stationary phase of growth.
cated at the periphery of the cells. However, for all RL conditions, the fluorescence of the cells was located at the periphery of cells at the stationary phase of growth. The fluorescence results showed that photosynthetic pigments would not be distributed in the central cytoplasmic region but confined to the periphery of the cells with the increase of light intensity and/or culture time.
partly clumped together at the exponential phase and entirely formed clusters finally. However, when light intensity was 30 μmol·photons/ (m2 s), the cells arranged in chains at first but finally formed into a clump. This could also be explained by Masato Baba's [2] finding, which illustrated that cell gathered together to avoid high intensity of RL and to reduce the light damage by adjusting the pigment content, composition and distribution. At the exponential phase of growth, the fluorescence of cells grown in RL with intensity of 12 and 30 μmol·photons/(m2 s) was distributed throughout the cells, while under 60 μmol·photons/(m2 s) treatments, the highest fluorescence was lo-
3.6. Regulation process of photosynthetic pigments The investigation on the effects of light color on photosynthetic 133
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PC 647-652
APC 658-663
Chl a 675-680
RL-12 (exp. phase)
RL-30 (exp. phase)
RL-60 (exp. phase)
RL-12 (stat. phase)
RL-30 (stat. phase)
RL-60 (stat. phase)
Fig. 6. Cell images of N. flagelliforme grown under RL with different light intensities visualized by CLSM. False color green and red represented the fluorescence excited by 488 and 633 nm, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of phycobilisome in N. flagelliforme and the different strategy used to respond to changes of light colors from currently reported cyanobacterial species which can adapt chromatically through CA process, might be greatly due to the difference in living habitats. The regulation process in N. flagelliforme was revealed in the study; however, the regulation mechanism of the response remains to be determined in further studies. Moreover, according to the involved regulation process in response to different light colors, the corresponding light control strategy should be proposed to improve the efficiency of light utilization in order to develop the high-efficient microalgal culture process.
pigments in terrestrial N. flagelliforme showed that cells could appropriately adjust the biosynthesis of photosynthetic pigments responding to different light colors, in order to make the most use of light for photosynthesis. Compared with other light treatments, N. flagelliforme cells were more sensitive to RL treatment. Under BL, GL, YL, and PL illumination, although the amounts of pigments were changed in response to the changing light color, the cells contained fixed proportions of PC and APC, and the proportion was the same with that of WL treatment. Under RL treatment, besides the contents of photosynthetic pigments, the proportion of PC and APC was altered indicating the composition of phycobilisome was adjusted, while the composition of phycobiliproteins was not influenced by the changes of RL intensity. Therefore, the feature of photoregulation process of terrestrial N. flagelliforme is summarized that the composition as well as location of phycobilisomes is regulated in response to RL. The distinct composition
4. Conclusion In this study, the effects of five different light colors on photosynthetic pigments were comprehensively examined to investigate the 134
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photoregulation process of N. flagelliforme. The alterations in the content, composition, fluorescence properties, and localizations of photosynthetic pigments in response to different light colors showed that N. flagelliforme was responsive to RL by regulating the composition and location of phycobilisomes. Based on this study, photosynthetic efficiency can be enhanced greatly by appropriate adjusting the photosynthetic pigments in different light conditions, thus further improving the biomass and EPS production of N. flagelliforme. Therefore, these results will have significant effects on the development of high-efficient process of N. flagelliforme culture.
[12]
[13] [14]
[15] [16]
Authors' contribution
[17]
PP Han obtained the funding. PP Han and SR Jia designed the experiments. PP Han and SG Shen wrote the manuscript. SG Shen, Y Sun, and RJ Guo performed the experiments. RJ Guo and SY Yao performed the analysis. ZL Tan helped interpret the data.
[18] [19]
[20]
Conflict of interest [21]
The authors declare that they have no conflict of interest.
[22]
Acknowledgments
[23]
The authors are very grateful for the financial support from the National Natural Science Foundation of China (Grant No. 31671842 and 31201405) and Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1166).
[24]
Appendix A. Supplementary data
[25]
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2017.04.009.
[26]
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The regulation of photosynthetic pigments in terrestrial Nostoc flagelliforme in response to different light colors
MARK
Pei-pei Han, Shi-gang Shen, Rong-jun Guo, Shun-yu Yao, Ying Sun, Zhi-lei Tan, Shi-ru Jia⁎ Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Nostoc flagelliforme Light color Photosynthetic pigment Photoregulation process
Cyanobacteria have evolved a number of photosynthetic strategies by adjusting the properties of the lightharvesting pigments to cope with changes in the light environment. Nostoc flagelliforme is a terrestrial filamentous cyanobacterium that has distinct phycobilisome composition consisting of phycocyanin (PC) and allophycocyanin (APC). The photoregulation process of N. flagelliforme was explored by investigating the effects of different light colors on photosynthetic pigments, which were determined after photoautotrophic growth with red (RL), yellow (YL), green (GL), blue (BL), purple (PL) and white (WL) light of same photosynthetically active photon flux density. Compared with WL, YL, GL, BL and PL changed the amounts of pigments but did not alter the cellular ratio of PC/APC; while RL altered both the amounts and proportions of PC and APC. The observations by confocal laser scanning microscope and transmission electron microscope showed that PC, APC and chlorophyll a were distributed throughout the cells under GL, BL, PL, and WL illumination but located in the outermost layer in RL and YL-grown cells. Further study revealed that changing RL intensity would not influence the ratio of PC/APC but the distribution of pigments shifted from central cytoplasmic region to the periphery of the cells with the increase of intensity. These results implied that N. flagelliforme responded to RL by regulating the composition and location of phycobilisomes and these findings would be of importance to develop the highefficient process of N. flagelliforme culture.
1. Introduction Environmental conditions exert a strong influence upon the cell growth, morphology and pigment composition of many photoautotrophic organisms [1–3]. As photoautotrophic organism, photosynthesis and growth rates are directly affected by light environment, because it is used to drive photosynthesis and regulate cell growth and development. Moreover, light can vary in terms of light quality, light quantity and photoperiod [4–6], thus sensing and adequately responding to light is a key attribute of eco-physiological versatility of photoautotrophic organisms. Cyanobacteria, as photoautotrophic microorganisms, are one of the oldest groups of bacteria, which can date back to the Pre-Cambrian [7]. Until now, it has evolved a large variety of light-harvesting and protective pigments, and evolved many photosynthetic strategies to help them cope with changes in their light environment [8–10]. The regulation process of photosynthetic pigments to changes in light color not only affects the composition of phycobilisomes, but also has been reported to have significant influence on many cellular processes such
⁎
as growth, photosynthesis, lipid production [1,11], nutrient utilization [12], and extracellular polysaccharides production [4]. The regulation process requires cyanobacteria to precisely measure ratios of specific light colors in its environment [13,14]. Besides the chlorophyll a (Chl a), cyanobacteria also contain several accessory pigments, including carotenoid and phycobilin, which can help them adapt to various light conditions. The phycobilins are unique among the photosynthetic pigments in that they are bonded to certain proteins, known as phycobiliproteins, which are commonly divided into three main groups: phycocyanin (PC), allophycocyanin (APC) and phycoerythrin (PE). Based on the shift in PC versus PE in the subunit structure of phycobilisome in response to light colors, four types of Chromatic Acclimation (CA) processes were identified till now [15]. Nostoc flagelliforme is a terrestrial filamentous cyanobacterium with great economic value that is mainly distributed in some arid and semiarid areas of China, where they are often subjected to extreme environmental changes [16]. As a consequence, the characteristics of N. flagelliforme are quite different from the cyanobacterium in the aquatic environment, which shows strong ecological adaptability to
Corresponding author. E-mail address: [email protected] (S.-r. Jia).
http://dx.doi.org/10.1016/j.algal.2017.04.009 Received 5 December 2016; Received in revised form 25 February 2017; Accepted 18 April 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved.
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2.3. Cell observation with CLSM
extreme conditions such as alkali, desiccation and strong UV radiation [16–18]. In addition, the phycobilisome of N. flagelliforme contains only PC and APC [19], indicating the photoregulation process may be quite different from the reported CA types in cyanobacteria. Although there are more and more studies on CA in aquatic cyanobacteria [20–22], but for terrestrial N. flagelliforme, few reports about regulation process of photosynthetic pigments are available till now. Besides that, in our previous work investigating the effects of light color on N. flagelliforme culture, it was found that light color not only had strong influence on biomass accumulation, but also had obvious impact on cellular metabolism and polysaccharides biosynthesis [4,23]. Thus illustrating the responsive mechanism of photosynthetic pigments to the changes of light color is of great importance to understand the correlations between the photoregulation process and biomass accumulation and/ or cellular metabolism. Therefore, the aim of this study was to research the photoregulation process of N. flagelliforme under light with different colors. Five different colors including red, blue, green, yellow, and purple were used and compared to white light (WL) of same photosynthetically active photon flux density. The content, composition, fluorescence properties, and localizations of photosynthetic pigments were comprehensively examined to study the alterations of photosynthetic pigments dependent on the changes of light color. The ultrastructure of N. flagelliforme cells grown under different light colors was also investigated.
An adequate amount of cells were taken and placed on the slide, which was covered by cover slip. CLSM (Hitachi F-4500, Tokyo, Japan) was used to observe the fluorescence of photosynthetic pigments in cells. To determine the optimal emission wavelength, the cells were excited with three individual laser wavelengths: 488 nm, 543 nm, or 633 nm. Subsequently the emission spectrum was gathered at 10.7 nm increments as a lambda Z-series. For 488 nm excitation, the ArKr laser was set at 10% power, while for 543 nm and 633 nm excitation, the HeNe laser was set at 30% power and 10% power, respectively. The NT 80/20 filter was used to collect the images and emission scans were collected in ~10.7 nm bandwidth increments in the range from 500 to 740 nm. In order to obtain a better signal yield, scans were performed with ‘low speed’ setting (220 Hz, full scan mode) and the confocal pinhole was opened (600 mm diameter) for most measurements. The LSM FCS Zeiss 510 Meta AIM imaging software was used to obtain the acquired CLSM images. 2.4. Cell observation with TEM A total of 10 mL of sample was taken and centrifuged at 8000 × g for 15 min, and the cell pellet was collected for subsequent analysis. The methods for chemical fixation, freeze substitution, processing and embedding of the samples were used as previously reported [26]. After that, the samples were viewed using the TEM (JEM-1230, Jeol Ltd., Tokyo, Japan) operated at 80 kV and equipped with a LaB6 filament. The Gatan 1.35 K × 1.04 K × 12 bit ES500W CCD camera was used to record all the micrographs.
2. Material and methods 2.1. Strain and culture conditions The N. flagelliforme cells (TCCC11757) cultured in BG-11 medium were obtained from the Tianjin Key Lab of Industrial Microbiology (Tianjin, China). Inoculums were prepared regularly (bi-weekly) in 500 mL shake-flask under WL at a photon flux density of 60 μmol·photons/(m2 s). Before all the experiments, the cells were incubated in a dark room for 3 days in order to reduce storage compounds, and then cells were cultured in 500 mL shake-flask containing 200 mL BG-11 medium at a photon flux density of 60 μmol·photons/(m2 s). The initial pH of the medium was adjusted to 8.0 and the shake-flask was inoculated with 10% (v/v) cell suspension i.e. the initial cell density of cell culture was 0.37 g/L dry cell weight. For the cultivation with different colors of light, WL (390–770 nm) was provided by fluorescent lamps. Red (660 nm, RL), yellow (590 nm, YL), green (520 nm, GL), blue (460 nm, BL), and purple (400 nm, PL) lights with half-band width of 5 nm were provided by different light-emitting diodes (LEDs, Model no. CDL-CF22W, Shenzhen federal heavy secco electronic Co. LTD, China). Each LED light was composed of 22 colored bulbs and the wattage and voltage of each colored bulb was 1 W and 220 V. In order to avoid any interference from external light source, all experiments were done in a dark room. A quantum sensor connected to Light Scout Dual solar quantum light meter (Spectrum Technologies, USA) was used to measure the light intensity. The cells were cultured in air conditioned environment at 25 ± 1 °C and cells grown under WL were regarded as control. According to the growth curve of N. flagelliforme under different light colors as shown in Fig.S1, samples were taken at the middle of exponential phase and beginning of stationary phase for subsequent analysis of photosynthetic pigment and cell observation with spectral confocal laser scanning microscope (CLSM) and transmission electron microscopy (TEM).
2.5. Statistical analyses Three replications from different batches of culture were performed for each experiment, and values were expressed as mean ± standard deviation. The Independent-samples t-test was used to analyze the experimental data using the SPSS statistical software (version 22.0) and the significance level was set at P < 0.05. 3. Results and discussion 3.1. The effects of light color on photosynthetic pigments In order to explore the photoregulation process of N. flagelliforme, photosynthetic pigments contents under different light conditions were measured at exponential phase and stationary phase of growth, respectively. The experiments were conducted under six different light colors: RL, BL, GL, YL, PL as well as WL at a photon flux density of 60 μmol/(m2 s). As shown in Fig. 1A, at exponential phase, the Chl a content was significantly higher under GL, WL, YL and lower under RL, than under BL and PL, while for carotenoid, it was significantly higher under PL and lower under RL, than under WL, YL, GL and BL. At stationary phase of growth (Fig. 1B), the Chl a content was higher in the order of GL, WL, YL, PL, BL and RL-grown cells with values of 4.61, 4.04, 3.97, 2.91, 2.88 and 0.98 mg/g, respectively; in comparison with exponential phase, the Chl a content remained almost unchanged, except for RL treatment, which was significantly decreased, while, the carotenoid content was slightly changed at all light conditions. The results showed that RL greatly reduced the Chl a content, and GL promoted the formation of Chl a, which corresponded with the findings of many reports [27,28]. Under PL, the highest carotenoid content was obtained with value of 1.84 mg/g, which was increased by 38.34% compared with control group. That might be explained by the fact that PL was harmful to the growth of N. flagelliforme [23], and carotenoid, as accessory pigment, could protect cell from light damage [29] besides the function of capturing light energy for use in photosynthesis.
2.2. The measurement of photosynthetic pigment The content of photosynthetic pigment was determined as previously reported [24,25]. 129
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Fig. 1. The photosynthetic pigments content of cells grown under different light colors at exponential phase (A, C) and stationary phase (B, D) of growth.
As presented in Fig. 1C, D, the changes of PC and APC contents under different light conditions presented similar patterns at both exponential and stationary phases. At exponential phase, both the PC and APC contents were significantly higher under YL, GL, BL, and lower under RL, than under WL and PL, whereas at stationary phase, they were significantly higher under GL and lower under RL, than under WL, YL, BL and PL. However, PE was not detected throughout the culture period, which was in agreement with previous study reporting only PC and APC in the isolated intact phycobilisomes [19]. The highest PC and APC contents were obtained under GL with values of 56.22 and 32.52 mg/g at stationary phase, which were respectively increased by 38.23% and 48.15%, compared to the corresponding WL control. While in RL, significant decrease (85.76% and 81.82%) in both PC and APC contents were observed compared with control. The results indicated that GL stimulated the formation of PC and APC, while RL greatly inhibited the formation of PC and APC. Interestingly, the pigmentation of RL and PL-grown cells changed from green to yellow during stationary phase, which was observed by the microscope (Fig. S2). This phenomenon might be due to an increase in the ratio of carotenoid/Chl a, as previously reported in the cyanobacteria Synechococcus and Prochlorococcus [30]. It was reported that cells would change the composition of photosynthetic pigments in response to different light environment in order to maximize the utilization of light energy during the process of photosynthesis [15]. Therefore, the composition of photosynthetic pigments at the stationary phase was subsequently calculated. As depicted in Table 1, light color had strong influence on the composition of photosynthetic pigments. In cultures incubated under RL and PL, significantly higher ratios of carotenoid/Chl a were observed compared to other light conditions. The ratio of carotenoid/ Chl a was a marker indicating cellular oxidative level [31], which
Table 1 Composition of photosynthetic pigments in the N. flagelliforme under different light colors at stationary phase of growth. Light color
WL RL YL GL BL PL
Composition of photosynthetic pigments PC/Chl a
APC/Chl a
Carotenoid/Chl a
PC/APC
6.22 4.13 5.67 7.68 8.90 6.26
3.36 2.84 3.14 4.44 5.02 3.35
0.33 0.91 0.34 0.30 0.46 0.56
1.85 1.45 1.80 1.73 1.79 1.87
± ± ± ± ± ±
0.29 0.15 0.24 0.39 0.45 0.28
± ± ± ± ± ±
0.21 0.18 0.17 0.26 0.31 0.21
± ± ± ± ± ±
0.02 0.07 0.02 0.02 0.03 0.04
± ± ± ± ± ±
0.18 0.15 0.13 0.11 0.13 0.13
suggested that RL and PL might induce photoinhibition and thus stimulated the formation of carotenoid to protect cells from light damage. Additionally, cells grown under BL exhibited the highest ratios of PC/Chl a and APC/Chl a, while the lowest values were obtained under RL treatment. There were some literatures reported that reducing the proportion of light harvesting pigment could not only improve the efficiency of photosynthesis, but also reduce the light damage when cells were exposed to strong light [32–35]. Although the light color altered the phycobiliproteins content, the ratios of PC/APC remained almost constant, indicating that the composition of phycobiliproteins was not influenced by the changes of light color. However, there was an exception that RL greatly reduced the phycobiliproteins content but changed the composition as well. 3.2. Observation of cellular auto-fluorescence by CLSM CLSM was used to examine the phycobiliprotein fluorescence properties and localizations, and investigate alterations in pigment 130
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Fluorescence intensity
1400
A
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length was 633 nm, all spectra showed maximum peaks from 647 to 652 nm and 658 to 663 nm, with shoulders ranging from 675 to 680 nm, and fluorescence intensity decreased notably from 650 to 740 nm (Fig. 2C). The fluorescence in the ranges of 647–652, 658–663 and 675–680 nm can be assigned to PC, APC and Chl a, respectively [19,36]. Within the range of emission wavelength of PE (500 to 600 nm), no fluorescence was observed in all the emission spectra, which provided additional evidence that the phycobilisome of N. flagelliforme contained only PC and APC. Therefore, the 488 and 633 nm were selected as excitation wavelength of N. flagelliforme in the subsequent study. The fluorescence signal was collected at 647–652, 658–663 and 675–680 nm, and the false color green and red in Fig. 3 represented the fluorescence excited by 488 and 633 nm, respectively. At the excitation wavelength of 488 and 633 nm, both the fluorescence of PC and APC were clearly observed, while no fluorescence was emitted by Chl a at the excitation wavelength of 488 nm, which was in agreement with above result shown in Fig. 2A. CLSM observations showed that the N. flagelliforme cells were in beaded arrangement. Besides that, the fluorescence signals of the WL, GL, BL and PL-grown cells were located throughout the cells, while under RL and YL treatments, the fluorescence signals were mainly located at the periphery of the cells. These results showed that the PC, APC and Chl a in GL, BL, PL, WL-grown cells were distributed throughout the cells, while in RL and YL-grown cells, they were located in the outermost layer of cells. This response was likely correlated with effective light utilization and energy balance [27].
WL RL YL GL BL PL
800 600 400 200 0
500
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650
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750
Wavelength(nm) 600
B Fluorescence intensity
500
400
WL RL YL GL BL PL
300
200
3.3. Observation of cellular ultrastructure by TEM
100
The effects of light color on the ultrastructure of N. flagelliforme were investigated by means of TEM. After illumination with different light colors, dramatic changes occurred in the ultrastructure of N. flagelliforme. As shown in Fig. 4, at treatments of WL, GL, BL and PL illumination, the thylakoid membrane occupied almost the entire cytoplasm, whereas it was mostly located at the periphery of the cell wall after illumination by RL and YL. Under WL, BL and PL, thylakoid membrane developed a reticulate arrangement (Fig. 4A, E, F), while the arrangement of thylakoid was disorderly and the middle of the lamellae had been disintegrated in RL (Fig. 4B), which suggested that the thylakoid structure was destroyed. Since the photosynthetic pigments embedded directly in the thylakoid membrane, the photosynthetic pigments were distributed in the outermost layer of cells grown under RL. The observation was well consistent with the phenomenon observed by CLSM that strong fluorescence was distributed in the outermost layer of cells grown in RL and YL. Similarly, these results also indicated that photosynthetic pigments were distributed throughout the cells grown in WL, GL, BL and PL. The most striking feature was that the RL-grown cells contained more as well as bigger structural granule than that of cells grown in other light conditions (Fig. 4B). Structure granules, including polyglucangranules, lipid droplets, cyanophycin granules, polyphosphate granules and polyhedral bodies, were found to provide nutrients under adverse circumstances. An increase in the number and size of structural granule was probably the most common response of cyanobacterial cells under stress [37]. Therefore, it might be concluded that the cells grown in RL was under light stress.
0 500
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600
650
700
750
700
750
Wavelength(nm) 1400
C
Fluorescence intensity
1200 1000
WL RL YL GL BL PL
800 600 400 200 0 500
550
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Wavelength(nm) Fig. 2. Fluorescence emission spectra of N. flagelliforme after illumination with different light colors at the excitation wavelength of 488 (A), 543 (B), and 633 nm (C), respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescence under different light colors. In order to identify the maximum emission wavelength of the N. flagelliforme, spectral scanning from 500 to 740 nm was performed at three individual excitation wavelengths: 488, 543 and 633 nm, respectively. From Fig. 2A, at the excitation wavelength of 488 nm, the main maximum peak was found between 647 and 652 nm, with shoulders ranging from 658 to 663 nm. As shown in Fig. 2B, for excitation at 543 nm, the emission spectra of cells showed distinct maximum emission peaks under different light colors, which suggested that 543 nm was unsuitable to be used as the excitation wavelength of N. flagelliforme. When the excitation wave-
3.4. The effects of RL with different light intensities on the photosynthetic pigments Based on the above results, the cells became yellow and the photosynthetic pigment content was greatly reduced under RL, suggesting that light inhibition might occur under RL of intensity at 60 μmol·photons/(m2 s). Therefore, the effects of RL with different light intensities (12, 30 and 60 μmol·photons/(m2 s)) on cell pigmentation and photosynthetic pigments were thoroughly compared. With increas131
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APC 658-663
PC 647-652
Chl a 675-680
WL
RL
YL
GL
BL
PL
Fig. 3. Images of N. flagelliforme cells under different light colors at stationary phase of growth as visualized by CLSM. (False color overlay image of the same cells obtained directly after lambda scanning by simultaneous three channel fluorescence detection. Channel 1: PC 647–652 nm; channel 2: APC 658–663 nm; channel 3: Chl a 675–680 nm; false color green and red represented the fluorescence excited by 488 and 633 nm, respectively.) (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
that the composition of phycobiliproteins was not influenced by the changes of RL intensity.
ing light intensity, as shown in Fig. 5A, cell pigmentation became more and more yellow, indicating that the proportion of photosynthetic pigments might be changed. As expected, the Chl a content was significantly decreased, but the carotenoid content remained basically unchanged with the increase of light intensity (Fig. 5B). Therefore, ratios of carotenoid/Chl a were increased with the increase of light intensity, which also explained the phenomenon that cells became more and more yellow. From Fig. 5C, no significant changes in the contents of PC and APC was observed when RL intensity increased from 12 to 30 μmol·photons/(m2 s), but the contents were obviously decreased at the intensity of 60 μmol·photons/(m2 s). However the ratios of PC/APC under three light intensities remained almost unchanged, indicating
3.5. Fluorescence of cells grown under RL with different light intensities CLSM was used to compare the effects of RL with intensities of 12, 30 and 60 μmol·photons/(m2 s) on the fluorescences of Chl a, PC and APC, and all the images were collected from the signals obtained at the excitation wavelength of 488 and 633 nm. As depicted in Fig. 6, CLSM observations showed that, when light intensity was 12 μmol·photons/ (m2 s), the cells arranged in chains throughout the culture period, but when light intensity increased up to 60 μmol·photons/(m2 s), the cells 132
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Fig. 4. Effects of light color on the ultrastructure of N. flagelliforme observed by TEM. The letter T, W and SG stood for thylakoid, cell wall, and structure granule, respectively. A–F represented cells grown under WL, RL, YL, GL, BL and PL, respectively. Bar marker represents 500 nm.
Fig. 5. The changes of cell pigmentation and pigment contents of N. flagelliforme cells with different light intensities of RL at stationary phase of growth.
cated at the periphery of the cells. However, for all RL conditions, the fluorescence of the cells was located at the periphery of cells at the stationary phase of growth. The fluorescence results showed that photosynthetic pigments would not be distributed in the central cytoplasmic region but confined to the periphery of the cells with the increase of light intensity and/or culture time.
partly clumped together at the exponential phase and entirely formed clusters finally. However, when light intensity was 30 μmol·photons/ (m2 s), the cells arranged in chains at first but finally formed into a clump. This could also be explained by Masato Baba's [2] finding, which illustrated that cell gathered together to avoid high intensity of RL and to reduce the light damage by adjusting the pigment content, composition and distribution. At the exponential phase of growth, the fluorescence of cells grown in RL with intensity of 12 and 30 μmol·photons/(m2 s) was distributed throughout the cells, while under 60 μmol·photons/(m2 s) treatments, the highest fluorescence was lo-
3.6. Regulation process of photosynthetic pigments The investigation on the effects of light color on photosynthetic 133
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PC 647-652
APC 658-663
Chl a 675-680
RL-12 (exp. phase)
RL-30 (exp. phase)
RL-60 (exp. phase)
RL-12 (stat. phase)
RL-30 (stat. phase)
RL-60 (stat. phase)
Fig. 6. Cell images of N. flagelliforme grown under RL with different light intensities visualized by CLSM. False color green and red represented the fluorescence excited by 488 and 633 nm, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of phycobilisome in N. flagelliforme and the different strategy used to respond to changes of light colors from currently reported cyanobacterial species which can adapt chromatically through CA process, might be greatly due to the difference in living habitats. The regulation process in N. flagelliforme was revealed in the study; however, the regulation mechanism of the response remains to be determined in further studies. Moreover, according to the involved regulation process in response to different light colors, the corresponding light control strategy should be proposed to improve the efficiency of light utilization in order to develop the high-efficient microalgal culture process.
pigments in terrestrial N. flagelliforme showed that cells could appropriately adjust the biosynthesis of photosynthetic pigments responding to different light colors, in order to make the most use of light for photosynthesis. Compared with other light treatments, N. flagelliforme cells were more sensitive to RL treatment. Under BL, GL, YL, and PL illumination, although the amounts of pigments were changed in response to the changing light color, the cells contained fixed proportions of PC and APC, and the proportion was the same with that of WL treatment. Under RL treatment, besides the contents of photosynthetic pigments, the proportion of PC and APC was altered indicating the composition of phycobilisome was adjusted, while the composition of phycobiliproteins was not influenced by the changes of RL intensity. Therefore, the feature of photoregulation process of terrestrial N. flagelliforme is summarized that the composition as well as location of phycobilisomes is regulated in response to RL. The distinct composition
4. Conclusion In this study, the effects of five different light colors on photosynthetic pigments were comprehensively examined to investigate the 134
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photoregulation process of N. flagelliforme. The alterations in the content, composition, fluorescence properties, and localizations of photosynthetic pigments in response to different light colors showed that N. flagelliforme was responsive to RL by regulating the composition and location of phycobilisomes. Based on this study, photosynthetic efficiency can be enhanced greatly by appropriate adjusting the photosynthetic pigments in different light conditions, thus further improving the biomass and EPS production of N. flagelliforme. Therefore, these results will have significant effects on the development of high-efficient process of N. flagelliforme culture.
[12]
[13] [14]
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Authors' contribution
[17]
PP Han obtained the funding. PP Han and SR Jia designed the experiments. PP Han and SG Shen wrote the manuscript. SG Shen, Y Sun, and RJ Guo performed the experiments. RJ Guo and SY Yao performed the analysis. ZL Tan helped interpret the data.
[18] [19]
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Conflict of interest [21]
The authors declare that they have no conflict of interest.
[22]
Acknowledgments
[23]
The authors are very grateful for the financial support from the National Natural Science Foundation of China (Grant No. 31671842 and 31201405) and Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1166).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.algal.2017.04.009.
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References
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