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Cited by 173Algal biology and biofuel productionLipid metabolism and biosynthesisMicrobial Metabolic Engineering and Bioproduction

Responses of Nannochloropsis oceanica IMET1 to Long-Term Nitrogen Starvation and Recovery

Open access
Dong, Hong-Po, Williams, Ernest, Wang, Da-zhi, Xie, Zhang-Xian, Hsia, Ru-ching, Jenck, Alizée, Halden, Rolf, Li, Jing +2 more·2013·Plant Physiology
DOI 10.1104/pp.113.214320PMC PMC3668043
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Abstract

The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.

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Written from the paper’s full text.

TL;DR

This study combined physiological and proteomic approaches to examine how *Nannochloropsis oceanica* IMET1 endures long-term nitrate deprivation and recovers after nitrate readdition. It reports lipid accumulation, reduced photosynthesis and protein content, and proteomic shifts toward nitrogen recycling and fatty acid synthesis, with return to steady state within 4 d after nitrogen amendment.

Key findings

  • Under nitrate depletion, lipid content increased from 24.8% dry weight on day 10 to 37.4% on day 16 and 58.7% on day 31; total fatty acids rose from 7.5% to 55.4% dry weight, with maximum fatty acid productivity of 113.9 mg L⁻¹ d⁻¹ on day 21.
  • Cellular nitrogen decreased from 6.6% to 2.1% dry weight, dissolved protein decreased from 44.4 to 14.8 µg (10⁸ cells)⁻¹, Fv/Fm decreased from 0.623 to 0.179, and Chl a decreased from 96 to 5 ng (10⁶ cells)⁻¹; zeaxanthin increased from 0.26 to 0.75 ng (10⁶ cells)⁻¹ during depletion.
  • Proteomics identified 47 proteins among 75 differentially expressed proteins during starvation, with 32 functionally annotated; Gln synthetase increased 1.8- to 3.0-fold, FabI increased 4.0-fold, trans-2-enoyl-CoA reductase increased 2.2-fold, and translation elongation factor EF-Ts decreased 4.2-fold.
  • After nitrate resupply, 150 differentially expressed proteins were detected at 24 h, 78% decreased and 22% increased; Fv/Fm rose from 0.179 on day 31 to 0.585 on day 35, carbon uptake increased 6.7-fold from day 32 to 35, and the culture returned to steady state within 4 d.

Methods

  • *N. oceanica* IMET1 was cultured in 10 L artificial seawater medium at 20°C under a 14/10-h light/dark cycle with 100 µmol photons m⁻² s⁻¹; from day 10 to 31, 2 L of culture was replaced with nitrate-free medium every 2 to 3 d, and nitrate was resupplied after day 31.
  • Physiological measurements included cell counts, in vivo Chl a fluorescence, Fv/Fm, ETR, nutrient and alkalinity analyses, elemental carbon/nitrogen, ¹⁴C-bicarbonate carbon fixation, pigment HPLC, gravimetric lipid measurement, fatty acid methyl ester GC, and transmission electron microscopy.
  • Proteomic analysis used two-dimensional gel electrophoresis (pH 4–7, 12.5% SDS-PAGE), Melanie 7.0 spot analysis, and MALDI TOF-TOF MS/MS against IMET1 transcriptome-predicted proteins; spots with greater than 1.5-fold change and P < 0.05 were selected, based on three biological replicates.

Limitations

  • Stated by the authors: many regulated proteins cannot be identified by two-dimensional gel electrophoresis, including membrane-bound proteins, faint low-abundance proteins, and poorly separated proteins; the autophagy conclusion requires further validation using autophagy markers such as Atg antibodies.
  • Stated by the authors: measured lipid content was slightly lower than previously reported for IMET1, possibly due to the cultivation system and lipid extraction method.
  • Evident from the text: proteomic conclusions rely on a limited number of identified gel spots, and the study does not report direct metabolic flux measurements to confirm the proposed pathway shifts.

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Integrity signals

Registry

Crossref · 10.1104/pp.113.214320

Cites

49 of 49 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 25 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Maximum photochemical efficiency of PSII in the dark-adapted state (Fv/Fm) in N. oceanica IMET1 was gradually reduced from 0.623 on day 10 to 0.179 on day 31.

    “Maximum photochemical efficiency of PSII in the dark-adapted state (F v /F m ) was gradually reduced from 0.623 on day 10 to 0.179 on day 31 (Fig. 3A), and carbon uptake per cell as a function of C14 bicarbonate assimilation was gradually reduced following nitrogen deprivation (Fig. 4A).”
    Quote in the paperSubstantial claim
  • Findingown data

    No significant increase in C20:5ω3 content (dry weight) between day 10 and day 31 was observed in N. oceanica IMET1.

    “Meanwhile, no significant increase in C20:5 v3 content (dry weight) between day 10 and day 31 was observed (Fig. 10B).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Fv/Fm and electron transport rate of nitrogen-depleted N. oceanica IMET1 cultures were decreased significantly (P < 0.001) compared with nitrogen-replete cultures.

    “Meanwhile, F v /F m and electron transport rate (ETR) of nitrogen-depleted cultures were decreased significantly (P , 0.001) when compared with nitrogen-replete cultures (Fig. 5).”
    Quote in the paperSubstantial claim
  • Findingown data

    The majority of differentially expressed proteins in N. oceanica IMET1 were found comparing day 10 with day 31.

    “Although many proteins exhibited differential expression between day 10 and 16, the majority of differentially expressed proteins were found comparing day 10 with day 31 (Table II).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Total cells in the sampled 2 L culture fluctuated in a narrow range after day 13.

    “Nevertheless, when cells in the sampled 2 L of culture were considered, total cells fluctuated in a narrow range after day 13 (Fig. 1B).”
    Quote in the paperNot a substantial claim
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 28
  • The quote only partly states this claim. 2

  • May combine two claims.

    Findingown data

    Following nitrogen depletion, lipid content in N. oceanica IMET1 gradually increased and reached 37.4% and 58.7% of dry weight after 16- and 31-d cultures, respectively, significantly greater than nitrogen-replete cultures (24.8% of dry weight on day 10).

    “Following nitrogen depletion, the lipid content gradually increased and reached 37.4% and 58.7% of dry weight after the 16-and 31-d cultures, respectively, which were significantly greater (P , 0.05, P , 0.001) than that of nitrogen-replete cultures (24.8% of dry weight on day 10; Fig. 7A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the analyzed proteins, 47 proteins were identified, of which 32 proteins were assigned a functional annotation.

    “Among them, 47 proteins were identified, of which 32 proteins were assigned a functional annotation, including proteins involved in photosynthesis, nitrogen assimilation, protein metabolism, glycolysis, and fatty acid metabolism (Table II).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Chlorophyll a, violaxanthin, and carotenoid were decreased significantly (P < 0.05), and the ratio of chlorophyll a to carotenoid was increased significantly (P < 0.05) in nitrogen-depleted N. oceanica IMET1 cultures compared with nitrogen-replete cultures.

    “In addition, Chl a, violaxanthin, and carotenoid were decreased significantly (P , 0.05), and the ratio of Chl a to carotenoid was increased significantly (P , 0.05) in nitrogendepleted cultures when compared with nitrogen-replete cultures (Fig. 6).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When N. oceanica IMET1 was resupplied with nitrate on day 31, a total of 150 differently expressed distinct proteins were detected at 24 h, of which 78% had a greater than 1.5-fold decrease and 22% had a greater than 1.5-fold increase in relative abundance (P < 0.05).

    “When IMET1 was resupplied with nitrate on day 31, a total of 150 differently expressed distinct proteins were detected at 24 h, of which 78% had a greater than 1.5-fold decrease and 22% had a greater than 1.5-fold increase in relative abundance (P , 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Following nitrogen addition to nitrogen-starved N. oceanica IMET1, proteomic and metabolic changes occur within 24 h and the culture returns to steady state within 4 d.

    “Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Between day 10 and day 31, the levels of C16:0, C16:1Δ9, and C18:1Δ9 in percentage of total fatty acids in N. oceanica IMET1 significantly increased (P < 0.05), while no significant change for C14:0 was observed.

    “For changes in the percentage of fatty acids within the total fatty acids between day 10 (nitrogen replete) and day 31 (chronic nitrogen depleted; Fig. 10A), it was observed that the levels of C16:0, C16:1 D9 , and C18:1 D9 were significantly increased (P , 0.05), while no significant change for C14:0 was observed.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    About 1,500 distinct protein spots were detected on each gel in the N. oceanica IMET1 proteomic analysis.

    “Following filtering to remove speckling and background, about 1,500 distinct protein spots were detected on each gel.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    From day 1 to 7, N. oceanica IMET1 cell density increased from 4.45 × 10^6 to 25.9 × 10^6 cells mL−1 and in vivo chlorophyll a increased from 29 to 143 mg mL−1, indicating exponential growth.

    “From day 1 to 7, cell density increased from 4.45 3 10 6 to 25.9 3 10 6 cells mL 21 , and in vivo chlorophyll a (Chl a) increased from 29 to 143 mg mL 21 (Fig. 1A), indicating that the culture was in exponential growth.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Zeaxanthin in N. oceanica IMET1 decreased from 0.5 to 0.26 ng (10^6 cells)−1 during log-phase growth, followed by an increase to 0.75 ng (10^6 cells)−1 during nitrogen depletion.

    “However, zeaxanthin exhibited a different profile in that it decreased from 0.5 ng (10 6 cells) 21 to 0.26 ng (10 6 cells) 21 during log-phase growth, followed by an increase to 0.75 ng (10 6 cells) 21 during nitrogen depletion (Fig. 3B; Supplemental Table S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    From day 7 to 10, in vivo chlorophyll a increased and peaked on day 10, while cell density dropped to 22.2 × 10^6 cells mL−1.

    “From day 7 to 10, in vivo Chl a increased and peaked on day 10, but cell density dropped to 22.2 3 10 6 cells mL 21 (Fig. 1A), suggesting that cell division had ceased.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    From day 10 to 31, N. oceanica IMET1 cell density and in vivo chlorophyll a gradually decreased and were close to the starting point by day 31.

    “From day 10 to 31, 2 L of culture was replaced with medium without nitrate every 2 to 3 d.Cell density and in vivo Chl a were gradually decreased and were close to the starting point by day 31 (Fig. 1A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    During the first 5 d of growth, N. oceanica IMET1 cell size slightly reduced from 3.0 ± 0.35 μm to 2.66 ± 0.32 μm and then remained constant for the remaining experiment (2.70 ± 0.08 μm).

    “During the first 5 d of growth, there was a slight reduction in cell size (from 3.0 6 0.35 mm to 2.66 6 0.32 mm), with the size remaining constant for the full remaining period of the experiment (2.70 6 0.08 mm [n = 31]; Supplemental Fig. S1).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Only 75 differentially expressed proteins with greater than 1.5-fold increase or decrease in relative abundance (P < 0.05) and a few abundant protein spots were analyzed by MS in N. oceanica IMET1.

    “In the comparisons, when excluding different spots between replicates, those small and faint spots, only 75 differentially expressed proteins that had a greater than 1.5-fold increase or decrease in relative abundance (P , 0.05) and a few abundant protein spots were analyzed by MS.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    During long-term nitrate depletion, four isoforms of the light-harvesting complex were decreased by 1.5- to 2.5-fold, and a violaxanthin/Chl a-binding protein precursor was decreased by 1.5-fold in N. oceanica IMET1.

    “During the long-term nitrate depletion, four isoforms of the light-harvesting complex were decreased by 1.5to 2.5-fold, and a violaxanthin/Chl a-binding protein precursor was decreased by 1.5-fold (Table II).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On day 28, N. oceanica IMET1 showed poor chloroplast structure; most chloroplasts lacked outer membranes, and surface membranes of some thylakoids appeared not to be intact.

    “On day 28, IMET1 showed poor chloroplast structure.Most chloroplasts lacked outer membranes, and surface membranes of some thylakoids appeared to not be intact (Fig. 8, C and D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Enoyl-acyl carrier protein reductase (FabI) and trans-2-enoyl-CoA reductase were increased by 4.0- and 2.2-fold under nitrate depletion in N. oceanica IMET1, respectively.

    “Meanwhile, enoyl-acyl carrier protein reductase (FabI) and trans-2-enoyl-CoA reductase, involved in fatty acid synthesis, were increased by 4.0-and 2.2-fold under nitrate depletion, respectively.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    In IMET1 under nitrate starvation, the percentage of polyunsaturated fatty acids (C18:2 v6, C20:4 v6, and C20:5 v3) decreased and the percentage of saturated and monounsaturated fatty acids increased within total fatty acids.

    “In IMET1, under nitrate starvation, the percentage of polyunsaturated fatty acids (C18:2 v6 , C20:4 v6 , and C20:5 v3 ) decreased and the percentage of saturated and monounsaturated fatty acids increased within total fatty acids (Fig. 9A)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    These responses include pigment alteration, lipid accumulation, and reductions in photosynthesis, carbon fixation, and protein content.

    “These responses include pigment alteration, lipid accumulation, and reductions in photosynthesis, carbon fixation, and protein content.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    A PGK and a PK involved in glycolysis, and a succinyl-CoA synthetase involved in the TCA cycle, were up-regulated at 24 h after nitrate readdition.

    “A PGK and a PK involved in glycolysis, and a succinyl-CoA synthetase involved in the TCA cycle, were up-regulated (Table III)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In this study, one 1,3-b-glucosidase was found in IMET1 and was increased by 1.2- to 1.5-fold through chronic nitrogen depletion.

    “In this study, one 1,3-b-glucosidase was found in IMET1 and was increased by 1.2-to 1.5-fold through chronic nitrogen depletion (Table II).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    One light-harvesting complex protein showed a 1.6-fold increase, and two FNRs were up-regulated by 1.5- and 2.0-fold, respectively, at 24 h after nitrate readdition.

    “One light-harvesting complex protein showed a 1.6-fold increase, and two FNRs were up-regulated by 1.5-and 2.0-fold, respectively (Table III).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An inorganic pyrophosphatase involved in lipid degradation was increased, together with a sharp reduction of fatty acid content during the recovery.

    “Also, an inorganic pyrophosphatase involved in lipid degradation was increased, together with a sharp reduction of fatty acid content during the recovery (Figs.7B and 9; Supplemental Table S2).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Interpretationreasoning

    Lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality under chronic nitrogen starvation in N. oceanica IMET1.

    “Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Increases in zeaxanthin content relative to other pigments in N. oceanica IMET1 indicate that the light-harvesting centers were modified and that zeaxanthin was likely used for photoprotection and thermal dissipation of absorbed light.

    “In this study, increases in zeaxanthin content relative to other pigments indicate that the light-harvesting centers were modified and that zeaxanthin was likely used for photoprotection and thermal dissipation of absorbed light, since available photons exceeded the cell capacity for photosynthesis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Nitrogen starvation can lead to increased percentages of saturated and monounsaturated fatty acids as well as decreased percentages of polyunsaturated fatty acids in the total fatty acids.

    “In total, these data indicated that nitrogen starvation can lead to increased percentages of saturated and monounsaturated fatty acids as well as decreased percentages of polyunsaturated fatty acids in the total fatty acids.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Rubisco must first be carbamylated by the addition of a CO2 molecule at the active-site Lys in order to have activity.

    “Rubisco must first be carbamylated by the addition of a CO 2 molecule at the activesite Lys in order to have activity (Cleland et al., 1998).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cleland et al., 1998 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Values in the N. oceanica IMET1 proteomic analysis are based on three biological replicates.

    “Values are based on three biological replicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Reduction of Cbbx protein homolog expression may decrease Rubisco activity, which thereby leads to a reduction in photosynthetic carbon fixation in N. oceanica IMET1.

    “Since the carboxylation of ribulose-1,5-bisphosphate catalyzed by Rubisco is the primary rate-limiting factor of the Calvin cycle during the day, the reduction of Cbbx protein homolog expression may decrease Rubisco activity, which thereby leads to a reduction in photosynthetic carbon fixation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Plant autophagy is thought to play an important role in nutrient recycling under starvation.

    “Plant autophagy is thought to play an important role in nutrient recycling under starvation (Liu et al., 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Liu et al., 2009 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Autophagy was induced in N. oceanica IMET1 during nitrogen starvation, and vacuolar degradation of cytoplasmic components is an important process for N. oceanica IMET1 to cope with limited nitrogen sources.

    “These results may suggest that autophagy was induced in IMET during nitrogen starvation and that the vacuolar degradation of cytoplasmic components is an important process for N. oceanica IMET1 to cope with limited nitrogen sources.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    In the marine diatom T. pseudonana, proteins for nitrate and nitrite reduction were decreased during nitrogen starvation, while Glu synthase and Gln synthetase were increased.

    “In the marine diatom T. pseudonana, proteins for nitrate and nitrite reduction were decreased during nitrogen starvation, while Glu synthase and Gln synthetase were increased (Hockin et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hockin et al., 2012 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The N. oceanica IMET1 nitrogen starvation and recovery experiment was repeated two times.

    “The experiment was repeated two times.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Autophagy is the major pathway by which proteins and organelles are targeted for degradation in the vacuoles of plants.

    “Autophagy is the major pathway by which proteins and organelles are targeted for degradation in the vacuoles of plants (Liu et al., 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Liu et al., 2009 source

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  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Carbonic anhydrase catalyzes the reversible conversion of CO2 to HCO3− and produces CO2 in the vicinity of Rubisco from actively transported HCO3−.

    “This enzyme catalyzes the reversible conversion of CO 2 to HCO 3 2 and produces CO 2 in the vicinity of Rubisco from actively transported HCO 3 2 (Huertas and Lubian, 1998).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Huertas and Lubian, 1998

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fatty acids synthesized in the chloroplast are transferred to the endoplasmic reticulum for fatty acid elongation.

    “Subsequently, the fatty acids synthesized in the chloroplast are transferred to the endoplasmic reticulum for fatty acid elongation (Poulos, 1995).”
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    • cites Poulos, 1995 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The oxygen-evolving complex is capable of splitting water into oxygen and hydrogen.

    “The oxygen-evolving complex is capable of splitting water into oxygen and hydrogen (Mayfield et al., 1987).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Mayfield et al., 1987 source

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    Premisecitation

    The acyl carrier protein-dependent system in chloroplasts usually synthesizes fatty acids with chain lengths of C16 and C18.

    “In addition, it is noteworthy that the ACP-dependent system in the chloroplasts usually synthesizes fatty acids with chain lengths of C16 and C18 (Hendren and Bloch, 1980).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Hendren and Bloch, 1980

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  • May combine two claims.

    Interpretationown data

    N. oceanica IMET1 may reduce protein synthesis and increase protein degradation in response to long-term nitrogen starvation.

    “These results suggest that IMET1 may reduce protein synthesis and increase protein degradation in response to the longterm nitrogen starvation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In 18:3-type plants, all unsaturated fatty acids are synthesized by the pathway that begins with stearoyl-ACP desaturase.

    “In 18:3-type plants, all unsaturated fatty acids are synthesized by the pathway that begins with stearoyl-ACP desaturase (Cheesbrough, 1990).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Cheesbrough, 1990 source

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  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Nitrate concentration was measured two times, and data from the second experiment are presented.

    “Nitrate concentration was measured two times, and data from the second experiment are presented here.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In microalgae, fatty acid de novo synthesis is performed in the chloroplast, where acetyl-CoA produced from photosynthesis and glycolysis is converted to malonyl-CoA, which then serves as a building block for fatty acid chain extension in the acyl carrier protein enzyme complex.

    “In microalgae, fatty acid de novo synthesis is performed in the chloroplast, where acetyl-CoA produced from photosynthesis and glycolysis is converted to malonyl-CoA, which then serves as a building block for fatty acid chain extension in the acyl carrier protein (ACP) enzyme complex (Ohlrogge and Browse, 1995).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ohlrogge and Browse, 1995

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  • Classified as probably not a substantial claim.

    Premisecitation

    Triacylglycerol accumulation in cells of C. reinhardtii can mitigate reactive oxygen species formation at PSI following nitrogen deprivation by sequestering excess electrons moving through the photosynthetic electron transport chain.

    “However, it has been reported that triacylglycerol accumulation in cells of C. reinhardtii can mitigate reactive oxygen species formation at PSI following nitrogen deprivation by sequestering excess electrons moving through the photosynthetic electron transport chain (Li et al., 2012).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Li et al., 2012

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In N. oceanica IMET1, 2-Cys peroxiredoxin increased more than other peroxidases, suggesting that it is a key antioxidant involved in the nitrogen stress response.

    “In IMET1, 2-Cys peroxiredoxin increased more than other peroxidases, suggesting that it is a key antioxidant involved in the nitrogen stress response.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    2-Cys peroxiredoxin is active with various thioredoxin isoforms and functions as an antioxidant toward reactive oxygen species in C. reinhardtii.

    “It has been shown that 2-Cys peroxiredoxin is active with various thioredoxin isoforms and functions as an antioxidant toward reactive oxygen species in C. reinhardtii (Goyer et al., 2002).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Goyer et al., 2002 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The growth experiment was repeated two times.

    “The experiment was repeated two times.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    The increase of succinyl-CoA synthetase might facilitate the use of various carbon skeletons produced by the catabolism of some amino acids into the TCA cycle via succinyl-CoA.

    “The increase of succinyl-CoA synthetase might facilitate the use of various carbon skeletons produced by the catabolism of some amino acids into the TCA cycle via succinyl-CoA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Different responses of the TCA cycle in C. reinhardtii and T. pseudonana to nitrogen starvation have been observed, showing that the flux of the TCA cycle may be adaptable.

    “Different responses of the TCA cycle in C. reinhardtii and T. pseudonana to nitrogen starvation have been observed (Miller et al., 2010;Hockin et al., 2012), showing that the flux of the TCA cycle may be adaptable.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Miller et al., 2010 source

      Not checked in the free run.

    • cites Hockin et al., 2012 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Our results further support the hypothesis that under nitrate starvation, chrysolaminaran might be hydrolyzed into Glc, which then enters cytosolic glycolysis and provides acetyl-CoA precursors for fatty acid biosynthesis.

    “Our results further support the hypothesis that under nitrate starvation, chrysolaminaran might be hydrolyzed into Glc, which then enters cytosolic glycolysis and provides acetyl-CoA precursors for fatty acid biosynthesis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Gel annotations and matching fidelity were checked manually to eliminate matching errors caused by the software.

    “Gel annotations and matching fidelity were checked manually to eliminate matching errors caused by the software.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Three gels representing independent samples were analyzed for each time point.

    “Three gels representing independent samples were analyzed for each time point.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Based on sequencing of Nannochloropsis spp. genomes, the form of carbohydrate reserve in Nannochloropsis spp. is not starch but chrysolaminaran, similar to the diatom Phaeodactylum tricornutum.

    “Based on the sequencing of Nannochloropsis spp.genomes, it is proposed that the form of carbohydrate reserve in Nannochloropsis spp. is not starch but chrysolaminaran (J.Xu, personal communication), similar to the diatom Phaeodactylum triconum (Kroth et al., 2008).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites J.Xu, personal communication

      Not checked in the free run.

    • cites Kroth et al., 2008 source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All the procedures for two-dimensional gel electrophoresis and gel analysis were repeated independently.

    “All the procedures for two-dimensional gel electrophoresis and gel analysis were repeated independently.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    The retention of low expression levels of the majority of proteins in IMET1 after 24 h might be due to a delay in the synthesis of free amino acids after nitrogen provision.

    “This might be due to a delay in the synthesis of free amino acids after nitrogen provision.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Fatty acid oxidation might provide the energy for this metabolic shift during nitrogen recovery, whereas acetyl-CoA could be shifted to the TCA cycle.

    “These results suggest that fatty acid oxidation might provide the energy for this metabolic shift during nitrogen recovery, whereas acetyl-CoA could be shifted to the TCA cycle.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Plumley and Schmidt (1989) also showed an increased mRNA expression of light-harvesting complex II in C. reinhardtii following a nitrogen pulse.

    “Plumley and Schmidt (1989) also showed an increased mRNA expression of light-harvesting complex II in C. reinhardtii following a nitrogen pulse.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Plumley and Schmidt (1989)

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In C. reinhardtii, protein synthesis in nitrogen-limited cells is immediately stimulated by the nitrogen pulse, increasing 10-fold within 30 min in both soluble and membrane fractions.

    “In C. reinhardtii, protein synthesis in nitrogen-limited cells is immediately stimulated by the nitrogen pulse, increasing 10-fold within 30 min in both soluble and membrane fractions (Plumley and Schmidt, 1989).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Plumley and Schmidt, 1989

      Not checked in the free run.

  • May combine two claims.

    Interpretationnone given

    Lipid accumulation and autophagy may play a key role in maintaining vitality and ensuring the positive incorporation of recycled nitrogen.

    “In addition, lipid accumulation and autophagy may play a key role in maintaining vitality and ensuring the positive incorporation of recycled nitrogen.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Each carbon fixation experiment was performed in triplicate.

    “each experiment was performed in triplicate.”
    Quote in the paperNot a substantial claim
58 of 58

  • The paper relies on this without support.

    The effect of long-term nitrogen deprivation on Nannochloropsis species was largely unknown before this study.

    “Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown.”

  • The paper relies on this without support.

    The sampled culture and selected time points are representative of the physiological state of the whole N. oceanica IMET1 culture.

    “These sampling points were chosen based on the changes of in vivo Chl a concentrations and the nitrate concentration in medium as benchmarks.”

  • The paper relies on this without support.

    The annotated functions of the identified proteins correspond to their actual roles in the indicated metabolic pathways.

    “which play an important role in the elongation step of protein synthesis”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Day 10 is described as both logarithmic growth and ceased cell division

    Minor

    The methods say the culture was in logarithmic growth on day 10, but the results say cell density fell from day 7 to day 10 and infer that cell division had ceased.

  • Listed glycolysis enzyme did not increase during nitrate depletion

    Minor

    The text includes PPH among enzymes that increased during nitrate depletion, but Table II reports PPH ratios of 0.7 on day 16 and 1.0 on day 31 relative to day 10.

2 of 2

Cites

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1 of the 50 references couldn’t be matched to a known paper.

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Authors
Dong, H.-P., Williams, E., Wang, D.-Z., Xie, Z.-X., Hsia, R.-C., Jenck, A., Halden, R., Li, J., Chen, F., & Place, A. R.
Affiliations
  • Research Center for Harmful Algae and Marine Biology, Jinan University, Guangzhou 510632, China (H.-P.D.)
  • University of Maryland, Baltimore (US)
  • Xiamen University (CN)
  • Center for Environmental Security Biodesign Institute/Security and Defense Systems Initiative, Arizona State University, Tempe, Arizona 85287 (A.J., R.H.); and
  • Arizona State University (US)
  • Chinese Academy of Sciences (CN)
ORCID
Venue
Plant Physiology
Publisher
Oxford University Press (OUP)
Published
2013-05-01
Volume
162
Issue
2
Pages
1110-1126
ISSN
  • 0032-0889 (ISSN-L)
  • 1532-2548
DOI
10.1104/pp.113.214320
PMC
PMC3668043
OpenAlex
W2147709159
Semantic Scholar
4efdd9263e56b74afb1ceabd7103b2abe4c990ab
Cited by
173
References
50
Citations by year
2026: 5 · 2025: 11 · 2024: 10 · 2023: 9 · 2022: 4 · 2021: 15 · 2020: 23 · 2019: 14 · 2018: 12 · 2017: 14 · 2016: 21 · 2015: 19 · 2014: 15 · 2013: 1
FWCI
6.78
Type
journal article
Language
en
Open access
Licence
Locations
  • PLANT PHYSIOLOGY (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Institutional knowledge base (Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences) (submittedVersion)
Funders
  • Xiamen University
  • National Institute of Environmental Health Sciences (R01ES021949)
Topics
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Algal biology and biofuel production
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
Keywords
Nannochloropsis oceanica, nitrogen starvation, nitrate deprivation, nitrogen recovery, proteome, oleaginous microalgae, lipid accumulation, nitrogen recycling, autophagy, nitrogen scavenging, fatty acid synthesis, glycolysis, protein turnover, biodiesel feedstock, proteomic biomarkers
MeSH terms
Autophagy, Enzymes, Fatty Acids, Glycolysis, Nitrates, Nitrogen, Proteins, Down-Regulation, Plastids, Proteomics, Lipid Metabolism, Stramenopiles, Microalgae

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

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