Metabolomic response of Euglena gracilis and its bleached mutant strain to light.

Euglena, a new superfood on the market, is a nutrient-rich, green single-celled microorganism that features the characteristics of both plant and animal. When cultivated under different conditions, Euglena produces different bioactive nutrients. Interestingly, Euglena is the only known microorganism whose chloroplasts are easy to lose under stress and become permanently bleached. We applied gas chromatography-mass spectrometry (GC-MS) to determine the metabolomes of wild-type (WT) Euglena gracilis and its bleached mutant OflB2 under light stimulation. We found a significant metabolic difference between WT and OflB2 cells in response to light. An increase of membrane components (phospholipids and acylamides) was observed in WT, while a decrease of some stimulant metabolites was detected in OflB2. These metabolomic changes after light stimulation are of great significance to the development of Euglena chloroplasts and their communications with the nucleus.

Introduction

Euglena is a single-celled eukaryotic microalga and shares characteristics of both animals and plants. It has no cell wall but contains a photosensitive eyespot structure and a flagellum. Among its bounty of nutrients are 14 types of Vitamins, 9 minerals, 18 amino acids, 11 unsaturated fatty acids, and 7 others like chlorophyll and paramylon (β-glucan). Different cultivations of Euglena result in a significant diversity of nutrients inside the cells [1]. The Euglena cells contain fully developed chloroplasts that they can grow both autotrophically under light and heterotrophically by absorbing organic matters from the environment [2]. The chloroplasts of Euglena are believed originated from the prochloron by endosymbiosis. Unlike the chloroplasts of other microalgae or higher-level plants, the chloroplasts of Euglena have poor stability and are prone to lose part of the chloroplast genome under stress such as antibiotics [2]. The Euglena cells losing part of their photosynthesis-related genes result in bleached mutants that can stably grow and multiply in heterotrophic media.

Chloroplasts are differentiated from proplastids without internal structure. In different environments or tissues, proplastids can develop into plastid structures with different functions, such as leukoplastid, amyloplast, chromoplast and chloroplast. Light is a prerequisite for Euglena chlorophyll biosynthesis, and normally chloroplasts cannot develop in the dark. In the process of chloroplast assembly, photopigments regulate the synthesis of chloroplast-related proteins under light stimulation [3]. Some of chloroplast-related proteins are encoded by nuclear genes and are transported from the cytoplasm to the protoplasts by transport peptides. These proteins help improve the structure and functional development of the chloroplasts. For example, the increases of chlorophyll, the formation of thylakoids and the development of some electron transport chain complexes all depend on these proteins [3]. In addition, cell division and chloroplast division are kept in synchronization, resulting in the metabolic state of the cell changes during the chloroplast development [4].

Metabolomics refers to the study of metabolites from the various metabolic pathways of a certain organism. Qualitative and quantitative metabolomics can be used to reveal the changes of metabolic state, detect the biomarkers, and elucidate the related metabolic pathways and mechanisms of the organism responding to environmental stimuli [5-10]. The metabolomics play an important role in systems biology, and it benefits drug development [5], toxicological study [6], and disease diagnosis [7]. The most common methods used in metabolomics include nuclear magnetic resonance spectroscopy (NMR), gas chromatography-mass spectrometry (GC-MS), liquid chromatography coupled with mass spectrometry (LC-MS). The application of metabolomics in microalgal has increased year by year. For example, the metabolome change induced by stress has been investigated in Ectocarpus siliculosu, Chlorella vulgaris and Pseudochoricystis ellipsoidea [11-13]. In Euglena, although there are countless reports to investigate the chloroplast development at the morphological levels [14-16], metabolomics studies are hardly seen [17]. GC-MS can identify hundreds of compounds at the same time, and has the advantages of high sensitivity, high resolution, and database support. We have used this separation and identification technology to detect the changes of metabolites of algae under light stimulation [17, 18].

In order to uncover the related mechanisms of chloroplast development under light stimulation in Euglena, we applied light stimulation to two strains of Euglena gracilis–wild type (WT) and bleached mutant OflB2 obtained by oxflaxicin treatment and performed GC-MS to detect the metabolome. This study reveals the physiological changes related to chloroplast development at the metabolomic level and provides a theoretical basis for further studies of the biosynthesis of chloroplast and its retrograde regulation with the nucleus.

Materials and methods

Algae cultivation and light stimulation

Wide type E. gracilis CCAP 1224/5Z was purchased from Culture Collection of Algae and Protozoa (CCAP) and maintained in our laboratory. The bleached mutant OflB2 permanently lost functional chloroplasts and was obtained by treating the WT of E. gracilis CCAP 1224/5Z with ofloxacin [2]. WT E. gracilis and the bleached mutant OflB2 were all cultured in Hetero medium (pH 3) [19] at the temperature of 23 ± 1 °C.

Absorption spectrum of the pigments

106 cells were mixed with 1 mL of 95% ethanol and incubated at 4 °C overnight. After centrifugation at 8000 g for 5 min, the absorption spectrum of supernatant was recorded by spectrometer. The detection wavelength was set to 300 nm—700 nm, and the scanning speed was 20 nm min-1.

Metabolome detection by GC-MS

To investigate the influence of light stimulation on the metabolite compositions of WT and OflB2, WT and OflB2 were first cultured under dark. After 6 days of the dark cultivation (samples entered into the platform stage), some cells were collected (used for 0 h sample under light) and the remaining cells were light-stimulated at the light intensity of 65 ± 5 μmol m-2 s-1. The light-treated samples were collected at 4 h, 12 h, 24 h, 48 h, and 72 h. Each sample had three parallels. The method for extraction of the metabolome was described in the previously published literatures [17, 18]. The metabolome was examined using the GC-7890A-MS-5975C gas chromatography-mass spectrometry system (Agilent Technologies, Inc.). The GC column was HP-5MS capillary column (Part No.: 19091S-433), which was 30.0 m (length) × 250 μm (inside diameter) × 0.25 μm (film thickness). The GC inlet temperature was set to 230 °C. Helium with purity higher than 99.999% was used as the carrier gas. 2 μL sample was injectied in splitless mode. The temperature procedure of the column oven was set as follows: 45 °C incubated for 2 min, raised to 280 °C at a rate of 5 °C min-1, 280°C incubated for 2 min. The transmission line temperature, ion source temperature and MS quadrupole were set to 290 °C, 230 °C and 150 °C, respectively. Metabolites retained in capillary column for at least 10 minutes. Full scan mode was used, and the charge-to-mass ratio detection range was set to 50–550.

The raw data from the GC-MS analysis were analyzed using the Agilent Automated Mass Spectrometry Deconvolution and Recognition System (AMDIS). For qualitative analysis of metabolites, the mass fragmentation spectrums were matched to the Fiehn database and the database of National Institute of Standards and Technology (NIST). The relative amount of each metabolite is the ratio of the peak area of the metabolite to that of the internal standard. Finally, the relative amount of metabolite was normalized by the number of cells.

Principal Component Analysis (PCA) was conducted to show the relationship between samples. In order to reveal the changes in expression patterns of metabolites, Cluster Analysis (CA) analysis was conducted and heatmap was drawn. In the heatmap, the raw data were log10-transformed to better show the differences of the expression pattern [20].

Results

Growth of WT E. gracilis and OflB2

The growths of WT E. gracilis and OflB2 under dark or light were shown in Fig 1. No matter under light or dark, the growth of WT E. gracilis was always better than that of OflB2. The cell density of WT was higher than that of OflB2 from day 3 to day 9. From day 6 to day 9, the effect of light on the growth was more obvious for WT E. gracilis than for OflB2. Under dark the highest cell density of WT was achieved at day 7 while under light it was achieve at day 9. For OflB2, there were little differences in growth between under dark and under light.

Fig 1

The growth of WT E. gracilis and OflB2 in Hetero medium under light (HL) and dark (HD) conditions.

Pigment compositions in E. gracilis and OflB2

In order to investigate the differences in pigment composition between WT E. gracilis and OflB2, the absorption spectra of pigments were recorded (Fig 2). There were absorption peaks at 433 nm and 665 nm in WT E. gracilis, but no such absorption peaks were found in OflB2, indicating that WT has pigments while OflB2 contains no pigments.

Fig 2

The absorption spectrum of the pigments in WT E. gracilis and OflB2.

GC-MS analysis

A total of 36 samples (2 strains at 6 time points with 3 biological replicates) were collected for metabolome detection. After filtering the metabolites with low quality scores (below 70), 78 and 61 metabolites were detected in WT E. gracilis and OflB2, respectively (see the S1 Table).

Principal Component Analysis (PCA)

PCA was conducted to see whether there were significant metabolomic differences between WT E. gracilis and OflB2 after light stimulation. The results were shown in Fig 3. The cumulative contribution of PC1 and PC2 was 76.1% for WT E. gracilis, and 73.6% for OflB2, indicating that this first two principal components account for most of the variations. For WT E. gracilis, samples with different light durations were separated from each other (Fig 3), suggesting that the metabolome of WT E. gracilis changes with light duration and WT E. gracilis cells adapt to changing light stimulation through necessary physiological and biochemical changes. In OflB2 (Fig 3), three clusters (samples with less than 24 h of light stimulation, samples with 48 h of light stimulation and samples with 72 h of light stimulation) can be found, suggesting that little changes in metabolic pathway occurred in the first 24 h of light stimulation and a certain light duration of more than 24 h can induce the metabolome change in the bleached OflB2 mutant cells. It also cannot be ruled out that the change in metabolite components after 24 h may be caused by other factors such as cell aging.

Fig 3

PCA analysis of WT E. gracilis (a) and OflB2 (b) against light treatment.

Cluster analysis

Cluster analysis was carried out to uncover the metabolomic changes after light stimulation in WT E. gracilis and OflB2. In WT E. gracilis, light stimulation results in upregulation of some metabolites (Fig 4). These metabolites includes amino acids and lipid metabolites, such as phosphonolipid (phosphoric acid, bis(trimethylsilyl) monomethyl ester), benzoic ester (phthalic acid, 6-ethyloct-3-yl 2-ethylhexyl ester), and glycine ester (glycine, N,N-bis(trimethylsilyl)-, trimethylsilyl ester), as well as the energy substance such as saccharide (d-Mannose). Light stimulation also changed the intracellular metabolism in OflB2 (Fig 4) and some stimulant metabolites decreased after light stimulation.

Fig 4

Heat map of WT E. gracilis (a) and OflB2 (b) metabolites after light stimulation.

Discussion

Since WT Euglena could grow to high cell density in either light or dark conditions, we proposed that the fully developed chloroplasts would not be helpful, even not a burden, to cells grown in the dark, where photosynthetic function is not needed. In the dark WT Euglena cannot photosynthesize and the chloroplasts inside cells may be a burden for cells. Without chloroplasts in the dark, the mutant cells were expected to grow better than WT since they have no material and energy waste for chloroplasts. Thus, the bleached mutants only growing heterotrophically may show benefit for better growth compared to WT under the same condition. However, unexpectedly, the growth of the OflB2 mutant was much slower than that of WT in the dark, probably because the OflB2 mutant has lost chloroplast and partial plastid genome, which may affect the growth and reproduction of OflB2 mutant in the dark [2].

As early as in the 1970–80s, there have been reports about the photo-induced synthesis of enzymes for fixing carbon dioxide during early chloroplast development stage [21], demonstrating that Rubisco increased its activity during the 12 h after the pre-light stimulation of the Euglena cells [22, 23]. It has also been reported that under the exposure to light, mitochondrial surface area, mitochondrial lipid levels, cytochromes were found decreased [24], and cyclophorase, such as the delta-aminolevulinic acid (pronounce) synthase (ALA synthase) [25, 26] and mitochondrial elongation factors G, was also down-regulated [27], while phosphoenol-pyruvate carboxylase slightly increased [28, 29]. Under the exposure to light, the 44 mitochondrial proteins on the whole-cell 2-D gel were detected to reduce by 31 in relative quantity, about 70% in WT Euglena [30, 31]. Visible light-induced selectivity alters the rate of specific mitochondrial protein synthesis. In addition, after exposure to light, a slightly lower cytosolic aldolase activity was found [32], similar down-regulated activity was also noticed in phosphofructokinase [32], phosphoenolpyruvate carboxykinase [33], NAD-glyceraldehyde-3-phosphate dehydrogenase [21], and malic enzyme [34]. It was concluded that, compared with OflB2, WT Euglena will experience a change in nutrition mode after light stimulation, gradually changing from heterotrophic to photoautotrophic state. This process mainly includes the synthesis of chlorophyll, chloroplast assembly, and changes in energy supplies of mitochondrial. The changes of enzyme levels caused by various physiological and biochemical reactions in the cytoplasm will eventually lead to changes in some major metabolite components.

We reported the relatively significant differences in either response patterns or metabolite components between WT and OflB2 under light stimulation in this study. The individual significantly changed components are somehow involved in the accumulation of chlorophyll, formation of thylakoid structures [35], and synthesis of photosynthesis-related proteins and others in the process of chloroplast development. It was once reported that the biosynthesis of phosphatidylglycerol (PG) is the basis of embryonic development as well as normal membrane structure of chloroplasts and mitochondria [36]. It was stated that galactolipids, monogalactosyl-diacylglycerol (MGDG), and digalactosyl-diacylglycerol (DGDG), major lipids in the thylakoid membrane required for its development, could be essential for maintaining optimal efficiency in photosynthesis [37, 38]. X-ray crystallographic studies of photosystem I (PS I) and photosystem II (PS II) also indicated that thylakoid lipids were contained in the crystal structures of both compounds, and that PG and MGDG were both found near the reaction center of PS I and PS II [39, 40]. In addition, we found that compared with the control group WT-0h, WT-4h had a different metabolite composition, further suggesting that cell metabolism of WT E. gracilis changes very quickly, 4 h as a short period of time after light stimulation. This change is significantly represented by the increase of some important metabolites. For instance, the increase of phosphonolipid may be related to the increase of membrane components; the formation of thylakoids in chloroplasts will be accelerated after light stimulation; and the increase of other amino acids and amides is associated with the synthesis of some photosynthetic proteins. Therefore, it is safe to conclude that the changes in the metabolite components and contents of WT Euglena after light treatment are mostly due to the accumulation of substances and energy for photosynthesis.

Since WT E. gracilis performs photosynthesis, it can be understood that the enhancement of metabolic activity is to prepare some substances for photosynthesis. However, bleached mutant OflB2 did not contain functional chloroplasts and thus performed no photosynthesis, and we infer that light stimulation may reduce the transcription of certain genes and result in the decrease of the levels of major metabolites in the cells.

Combined with other Omic data analyses, such as genomic, transcriptomic, and proteomic data (unpublished) conducted in WT E. gracilis and bleached mutants, we propose that the regulation of light induced photoreactivity in Euglena is more likely to occur at the post-transcriptional level. Metabolomic analysis showed that metabolome of WT E. gracilis under light stimulation is majorly represented by its relation with the increase of membrane components. For example, light promotes the formation of thylakoids in chloroplasts; the increase of other amino acids and amides is associated with the synthesis of some photosynthetic proteins. However, under light stimulation, metabolome of the bleached OflB2 is characterized by the down-regulation of some metabolite components. It is proposed that light stimulation slows down the metabolism of the bleached Euglena cells to a certain extent, making it resemble a semi-dormant state in a short period of time, thereby reducing the damage of light stress against cells without any functional chloroplasts. In summary, the metabolomic analyses of WT E. gracilis and bleached mutant OflB2 showed that metabolite component changes occur in both strains after light stimulation. In the WT E. gracilis, it is mainly represented by the increase of some metabolites such as phosphoric ester involved in membrane synthesis and various amino acids in protein synthesis for photosynthesis. In contrast, in the bleached OflB2, metabolomes were mainly characterized by the decrease of some secondary metabolites. In addition, cells of the two strains showed different response patterns to light stimulation: WT E. gracilis changed more rapidly than the bleached cells. Therefore, there is a significant difference in the response mechanism between the two strains to light stimulation. We propose that the main cause in WT E. gracilis is to switch from heterotrophy to autotrophy, so as to prepare itself for chloroplast development and photosynthesis. However, the bleached mutant OflB2 cells are at the heterotrophic state, and light stimulation may work as a stress instead of a chloroplast development. As a result, its cells adapt to the new environment by changing the metabolomic status.

Intracellular metabolites detected with GC-MS in WT E. gracilis and OflB2.

(XLSX)

Click here for additional data file.

15 Jul 2019

PONE-D-19-15939

Metabolomic response of Euglena gracilis and its bleached mutant strain to light

PLOS ONE

Dear Dr. Wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Aug 29 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Haitao Shi

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

1. Thank you for including the following funding information within the acknowledgements section of your manuscript; "This work was supported by National Natural Science Foundation of China (31670116), the Guangdong Innovation Research Team Fund (2014ZT05S078), and the Shenzhen Grant Plan for Science & Technology (JCYJ20160308095910917, JCYJ20170818100339597), used for the design of the study, data collection, data analysis and manuscript writing. The authors gratefully acknowledge the supports from the Instrumental Analysis Center of Shenzhen University (Xili Campus)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

"NO - Include this sentence at the end of your statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

2. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This is an interesting study of a unique type of algae. The experimental design is sound and the results are useful. There are a few points needs to be amended or addressed.

The last paragraph of the introduction, no result should be presented or discussed in the introduction.

Line 103, what does it mean "The solvent was delayed by 10 min"?

Line 191, why the low growth of growth of the OflB2 mutant is unexpected? this needs better explanation.

It is hard for readers to understand when conclusion is made from unpublished data. Suggest to delete or modify the 2nd paragraph of the summary.

Reviewer #2: The manuscript applied gas chromatography-mass spectrometry (GC-MS) to determine the metabolomes of wild-type (WT) Euglena gracilis and its bleached mutant OflB2 under light stimulation. These metabolomic changes after light stimulation are of great significance to the development of Euglena chloroplasts and their communications with the nucleus. However, The manuscript is relatively simple, the authors should consider the following points:

1. Figure 1 cannot be found.

2. Morphological changes of chlorophyll and other physiological indicators should be observed

3. Add GC-MS Analysis of Raw Data Graph.

3. It is suggested that genomics, transcriptome and proteomics data be published together to better reveal the mechanism of light effects on them.

4.There are some grammatical errors in the manuscript,please modify the language further.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

22 Oct 2019

Reviewer #1

1.Questions: The last paragraph of the introduction, no result should be presented or discussed in the introduction.

Response: Thanks for your constructive advices. We have moved the sentence “the relatively significant differences in either response time or metabolite components between WT and OflB2 under light stimulation” from introduction into discussion (lines 189-190).

2.Questions: Line 103, what does it mean "The solvent was delayed by 10 min"?

Response: Sorry for the confusion caused by the unclear description. We changed the "The solvent was delayed by 10 min" to "Metabolites retained in capillary column for at least 10 minutes"(lines 95-96).

3.Questions: Line 191, why the low growth of growth of the OflB2 mutant is unexpected? this needs better explanation.

Response: Since WT Euglena could grow heterotrophically and autotrophically under different culture conditions, and in the dark WT Euglena cannot photosynthesize and the chloroplasts inside cells may be a burden for cells. Without chloroplasts in the dark, the mutant cells were expected to grow better than WT since they have no material and energy waste for chloroplasts. We have added this paragraph into the revised manuscript (Line 159-162).

4.Questions: It is hard for readers to understand when conclusion is made from unpublished data. Suggest to delete or modify the 2nd paragraph of the summary.

Response: Thanks for useful suggestions. We have deleted the conclusion based on unpublished data.

Reviewer #2

1.Questions: Figure 1 cannot be found.

Response: Sorry for low mistake by the first time submissioner. We have added figures in the revised version and this time we promise every figure are included.

2.Questions: Morphological changes of chlorophyll and other physiological indicators should be observed

Response: in the new version we added and analyzed the differences of pigments between wild type and the bleached mutant in lines 121-125

3.Questions: Add GC-MS Analysis of Raw Data Graph.

Response: we have added a supplementary material containing the raw analysis data of GC-MS in the newly revised version.

4.Questions: It is suggested that genomics, transcriptome and proteomics data be published together to better reveal the mechanism of light effects on them.

Response: thanks for constructive advice. We agree completely with you. We have the RNA-seq and proteomics data but still wait for the ongoing Euglena genome assemble, to make a better annotation and analysis.

5.Questions: There are some grammatical errors in the manuscript, please modify the language further.

Response: we have corrected the grammatical errors as possible as we can. Please check for the details in the revised manuscript.

Academic Editor：

We will only publish funding information present in the Funding Statement section of the online submission form.

Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation.

Response: we have deleted the funding information from the manuscript text, as request. We amended the list of authors and ensure that each author has only one major affiliation.

Submitted filename: Responses to editor and reviewers.docx

Click here for additional data file.

25 Oct 2019

Metabolomic re​sponse of Euglena gracilis and its bleached mutant strain to light

PONE-D-19-15939R1

Dear Dr. Wang,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Haitao Shi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

31 Oct 2019

PONE-D-19-15939R1

Metabolomic re​sponse of Euglena gracilis and its bleached mutant strain to light

Dear Dr. Wang:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Haitao Shi

Academic Editor

PLOS ONE