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Biotechnology and Biological Transformations
ROS is a factor regulating the increased polysaccharide production by light quality in the edible cyanobacterium Nostoc flagelliforme Peipei Han, Shi-gang Shen, Rong-jun Guo, Dong-xue Zhao, Ya-hui Lin, Shiru Jia, Rong-rong Yan, and Yi-kai Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06176 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019
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Journal of Agricultural and Food Chemistry
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Original paper
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ROS is a factor regulating the increased polysaccharide production
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by light quality in the edible cyanobacterium Nostoc flagelliforme
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Pei-pei Han*, Shi-gang Shen, Rong-jun Guo, Dong-xue Zhao, Ya-Hui Lin, Shi-ru Jia*,
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Rong-rong Yan, Yi-kai Wu
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Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education,
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State Key Laboratory of Food Nutrition and Safety, College of Biotechnology,
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Tianjin University of Science and Technology, Tianjin 300457, P.R. China
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* Corresponding author: Key Laboratory of Industrial Fermentation Microbiology,
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Ministry of Education, State Key Laboratory of Food Nutrition and Safety, College of
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Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P.R.
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China. Email:
[email protected];
[email protected]; Tel: +86 22 60601598; Fax:
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+86 22 60602298 1
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ABSTRACT: To explore the regulatory factor of light quality affecting
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exopolysaccharides (EPS) production, transcriptome analysis of Nostoc flagelliforme
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cells exposed to red light (R), blue light (B), and mixed light (B:R=15:7) (BR) with
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white fluorescent light as control was performed. The differentially expressed genes
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mainly enriched in carbohydrate metabolism and energy metabolism. Significant
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enrichment in oxidation-reduction process and energy metabolism indicated the
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intracellular redox homeostasis was disrupted. Assay of reactive oxygen species (ROS)
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and malondialdehyde contents demonstrated light quality induced oxidative stress. To
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illustrate the relationship between ROS level and EPS accumulation, the effects of
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exogenous addition of ROS scavenger N-acetyl cysteine and inducer H2O2 on the
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oxidation-reduction level and EPS production were compared. The results revealed
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that light quality regulated EPS biosynthesis via the intracellular ROS level directly
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other than oxidative stress. Understanding such relationships might provide guidance
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for efficient EPS production to regulate the intracellular redox level.
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Key words: Polysaccharides, ROS, Transcriptomics, Light quality, Nostoc
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flagelliforme
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1. Introduction
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In recent years, polysaccharides have attracted a great attention from the medical
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and food industries as bioactive ingredients and food additives.1-2 Polysaccharides
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obtained from natural sources are usually considered to have low toxicity leading to
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their consumption as functional foods.3 Nostoc flagelliforme is an edible terrestrial
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cyanobacterium with great food and herbal values, which has been used as food in
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China for more than 2000 years.4 The exopolysaccharides (EPS) of N. flagelliforme
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has been proved to possess the activities of antivirus, antioxidant and anti-tumor,5-6
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which makes it as a promising resource for the development of functional foods.
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However, the low yield of EPS strongly limits the application in functional foods.
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To obtain the high EPS production yield, a lot of studies have been carried out to
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find the optimum culture condition.7-8 The control and optimization of light condition
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are regarded as one of the most important factors for the culture of photosynthetic
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microorganisms.9 Many studies reported that light quality especially red light and blue
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light could promote the production of EPS .8,10-11 However, little information is
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available on the regulatory factor of increasing EPS production and how cells respond
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to the changes of light condition.
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Singh and Montgomery reported that cells of Fremyella diplosiphon experienced
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an increased reactive oxygen species (ROS) level when cells transferred from white
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light to red light.12 Besides that, Yu et al
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acuminate were increased under blue light, but decreased under red light, compared
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with white light. ROS are known to be an important factor in cellular response and
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found that ROS contents in Camptotheca
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have been reported to be increased when microalgae are exposed to different stress
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conditions.14 ROS also act as important signaling molecules that initiate
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phosphorylation cascades with other messengers such as Ca2+, which activates major
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stress-response genes leading to cyanobacterium adapt to the adverse conditions.15
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Cells also evolve a variety of strategies in response to stress condition. This includes
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many enzymatic scavengers such as superoxide dismutase (SOD), catalase (CAT),
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and the non-enzymatic antioxidants such as polysaccharides, proline and carotenoids.
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EPS could effectively scavenge ROS, and demonstrated protective effects against the
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inhibition of the fusion between the membrane vesicles in cyanobacteria.16 A previous
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study showed that varying light conditions resulted in the co-occurrence of ROS and
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EPS accumulation in cyanobacteria.17 However, there is limited information about
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this phenomenon. Understanding the mechanisms behind increased EPS accumulation
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in response to different stress conditions, especially different light qualities, is crucial
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to enable key manipulations at the biochemical and physiological level for increasing
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EPS production.
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Transcriptomics, as one of the rapidly developing systems biology approaches,
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have become essential for understanding how microorganisms respond and adapt to
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changes in their physical environment.18 In this study, transcriptional analysis of N.
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flagelliforme cells exposed to three typical light qualities including red light (R), blue
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light (B), and mixed light (B:R=15:7) (BR) with white light (W) as control was
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performed by RNA sequencing (RNA-seq) technology, and differentially expressed
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genes (DEGs) were analyzed to reveal the effect of light qualities on N. flagelliforme 4
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cells and clarify the mechanisms involved in the EPS production. Subsequently, the
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relationship between ROS level and EPS accumulation was investigated by
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comparing the effects of exogenous addition of ROS scavenger N-acetyl cysteine
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(NAC) and ROS inducer H2O2 on the intracellular oxidation-reduction level and EPS
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production of N. flagelliforme. The study aimed to reveal the possible mechanism of
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light quality affecting EPS production and explore the factor of regulating EPS
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production in N. flagelliforme.
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2. Materials and methods
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2.1 Strain and culture conditions
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Tianjin Key Lab of Industrial Microbiology (Tianjin, China) provided the N.
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flagelliforme cells (TCCC11757), which were cultured in BG-11 medium. Inocula
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were regularly (bi-weekly) prepared in 500 mL Erlenmeyer flasks at 25°C under
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continuous cool-white fluorescent light illumination of 60 μmol/(m2 s). Before all the
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experiments, cells were firstly incubated in the dark room for 3 days, in order to
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reduce the storage compounds such as cyanophycin granules and polyphosphate
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granules, which would provide nutrients under adverse circumstances, and avoid the
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influence of white light from pre-culture.
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Under R, B, and BR treatments, the cells were cultured in 500 mL shake-flasks
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containing 200 mL BG-11 medium19 for 16 days. The temperature, pH and light
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intensity were controlled at 25°C, 9 and 60 μmol/(m2 s), respectively. The cell culture
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was shaken three times a day manually. Each light-emitting diode light (Shenzhen
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federal heavy secco electronic Co. LTD, China) consisted of 22 colored bulbs which 5
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emitted at specific wavelengths. The half-band widths are 5 nm for red bulb (660 nm)
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and blue bulb (460 nm). The wattage and voltage of each colored bulb was 1 W and
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220 V. R and B were composed of 22 red and blue bulbs, respectively, and BR was
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composed of 15 blue and 7 red bulbs. The light intensity was measured using a
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quantum sensor connected to Light Scout Dual solar quantum light meter (Spectrum
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Technologies, USA). The cells grown under W were treated as control.
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2.2 RNA extraction and high-throughput sequencing
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TRIzol Reagent (Invitrogen) was used to extract the total RNA of each sample, and
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Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop
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(Thermo Fisher Scientific Inc.) and 1% agarose gel were used to quantify and qualify
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the extracted RNA . A total of 1 μg total RNA with the value of RNA integrity
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number above 7 was used for construction of complementary DNA (cDNA) library
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based on the manufacturer’s instructions (NEBNext Ultra RNA Library Prep Kit for
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Illumina). AxyPrep Mag PCR Clean-up (Axygen) was used to clean up the PCR
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products. Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA)
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Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) were used to validate and
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quantify the PCR products, respectively.
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Then based on the manufacturer’s protocol (Illumina, San Diego, CA, USA), the
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libraries with different indices were multiplexed and loaded on an Illumina HiSeq
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instrument. A 2×150 bp paired-end (PE) configuration was used to perform the
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sequencing. Image analysis and base calling were performed by the HiSeq Control
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Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. 6
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2.3 De novo transcriptome assembly and annotation
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The raw reads generated were filtered using Trimmomatic (v0.30) with quality
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value>20 and other contaminants, such as adapters were also trimmed. Parameters
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such as adapter trimming, sliding window, leading and trailing were considered for
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filtration. Minimum length was taken as 75 bp. The quality of the RNA-seq reads was
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assessed with FastQC (v0.10.1). QC passed reads were subjected to de novo assembly
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with Trinity and final assembled transcripts were generated. These contigs were then
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further processed with sequence clustering software TGICL to form longer sequences
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defined as unigenes.
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The generated unigenes were used for BLASTX alignment (E
