Daily patterns of nitrogen fixation in WT vs. RCKO strains.

<p><b>A</b> and <b>C</b>, nitrogen fixation activities of the wild-type <i>R</i>. <i>palustris</i> (WT) at 30°C and 23°C. <b>B</b> and <b>D</b>, nitrogen fixation activities of the <i>kaiC</i>^<i>Rp</i>-deletion strain (RCKO) at 30°C. The three traces represent three individual cultures under anaerobic conditions that were tested at different phases of LD 12:12 after growth for ~2 weeks in LD 12:12. The black and white bars underneath represent the light conditions. Nitrogen fixation rates were calculated based on the amount of C₂H₄ (nmol) produced by 10¹⁰ cells per hour. These two-cycle LD experiments were repeated twice, each time with 3 replicate cultures; one representative experiment is shown in this figure (one-cycle LD assays were conducted in five independent experiments, each time with phasing data equivalent to those shown in this figure). The data of Fig 1 are replotted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005922#pgen.1005922.s004" target="_blank">S3 Fig</a> with all three replicate cultures averaged together (complete time series data appear in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005922#pgen.1005922.s012" target="_blank">S4 Table</a>).</p

Top-down Targeted Metabolomics Reveals a Sulfur-Containing Metabolite with Inhibitory Activity against Angiotensin-Converting Enzyme in <i>Asparagus officinalis</i>

The discovery of bioactive natural compounds containing sulfur, which is crucial for inhibitory activity against angiotensin-converting enzyme (ACE), is a challenging task in metabolomics. Herein, a new S-containing metabolite, asparaptine (<b>1</b>), was discovered in the spears of <i>Asparagus officinalis</i> by targeted metabolomics using mass spectrometry for S-containing metabolites. The contribution ratio (2.2%) to the IC₅₀ value in the crude extract showed that asparaptine (<b>1</b>) is a new ACE inhibitor

Phosphorylation patterns of KaiC^Rp in LD and LL.

<p><b>A & B</b>. Electrophoresis analyses of ³²P-labeled KaiC^Se and KaiC^Rp. Lane 1 is ³²P-labeled KaiC^Se. Lanes 2–5 are purified KaiC^Rp that was mixed with [γ-³²P]ATP. Samples for lanes 2 and 4 were immediately denatured after the addition of [γ-³²P]ATP by mixing with SDS-PAGE sample buffer (time zero samples, lanes 2 and 4). Samples for lanes 3 and 5 were incubated with [γ-³²P]ATP for 24 h at either 4°C (lane 3) or 30°C (lane 5) prior to SDS denaturation and inactivation. The samples were subjected to either regular SDS-PAGE (<b>A)</b> or phosphate-affinity SDS-PAGE with Phos-Tag (<b>B</b>). In both panels, the left portion is a Coomassie-Blue stained gel (CBB) and the right portion is the autoradiogram of P-32 radioactivity. In panel <b>B</b>, three bands of ³²P-labeled KaiC^Rp are indicated as P1, P2, P3 and an unlabeled KaiC^Rp band is indicated as NP (non-phosphorylated). <b>C & D</b>. The strain in which KaiC^Rp has been tagged with HA (RCKO + HA-kaiC^Rp) was grown under LD 12:12 and then transferred to LL. Cells were collected every 6 h. Total protein extracts were separated on SDS-polyacrylamide gels with Phos-tag and analyzed by immunoblotting using an anti-HA antibody. The specificity of the anti-HA antibody against HA-tagged KaiC^Rp was confirmed with extracts from the RCKO cells. <b>C.</b> immunoblot in which the phospho- (P1, P2, P3) and nonphospho- (NP) forms of KaiC^Rp are indicated. <b>D.</b> quantification of phosphorylation states of HA-KaiC^Rp from the immunoblot in panel C. “ZT” = Zeitgeber Time in LD, where ZT0 is lights-on and ZT12 is lights-off (complete time series data for panel D appear in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005922#pgen.1005922.s015" target="_blank">S7 Table</a>).</p

ATPase activity of KaiC^Rp and KaiC^Rp-EQ1EQ2 <i>in vitro</i> at 30°C.

<p><b>A.</b> Purified native KaiC^Rp and the KaiC^Rp-EQ1EQ2 mutant (EQ1EQ2) were incubated with1 mM ATP for 0.5, 1, 2, 4, 12, and 24 h at 30°C. The amount of ATP hydrolyzed was quantified by HPLC. Dark blue, buffer only; red, KaiC^Rp; light blue, KaiC^Rp-EQ1EQ2; green, KaiC^Se. <b>B.</b> ATPase hydrolysis rates of native KaiC^Rp at different temperatures. Q₁₀ was calculated based on these rates. These experiments were repeated twice; one representative experiment is shown in this figure.</p

Lack of robust persistence of the nitrogen fixation rhythm in LL after transfer from LD.

<p><b>A</b>, nitrogen fixation activities of the WT (red) and RCKO (green) strains at 30°C. <b>B</b>, nitrogen fixation activities of the WT (red) and RCKO (green) strains at 23°C. Data is plotted as the mean of three individual cultures. Data points are mean +/- S.D. Black-white bars indicate the light/dark conditions: black is dark, white is light. These experiments were repeated five times (five times for WT, and five times for RCKO), each time with three replicate cultures; one representative experiment is shown in this figure (the three replicate cultures of this experiment are plotted separately in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005922#pgen.1005922.s005" target="_blank">S4 Fig</a>, and complete time series data appear in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005922#pgen.1005922.s014" target="_blank">S6 Table</a>).</p

Table_2_Metabolomic Evaluation of the Quality of Leaf Lettuce Grown in Practical Plant Factory to Capture Metabolite Signature.XLSX

<p>Vegetables produce metabolites that affect their taste and nutritional value and compounds that contribute to human health. The quality of vegetables grown in plant factories under hydroponic cultivation, e.g., their sweetness and softness, can be improved by controlling growth factors including the temperature, humidity, light source, and fertilizer. However, soil is cheaper than hydroponic cultivation and the visual phenotype of vegetables grown under the two conditions is different. As it is not clear whether their metabolite composition is also different, we studied leaf lettuce raised under the hydroponic condition in practical plant factory and strictly controlled soil condition. We chose two representative cultivars, “black rose” (BR) and “red fire” (RF) because they are of high economic value. Metabolite profiling by comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) resulted in the annotation of 101 metabolites from 223 peaks detected by GC-MS; LC-MS yielded 95 peaks. The principal component analysis (PCA) scatter plot showed that the most distinct separation patterns on the first principal component (PC1) coincided with differences in the cultivation methods. There were no clear separations related to cultivar differences in the plot. PC1 loading revealed the discriminant metabolites for each cultivation method. The level of amino acids such as lysine, phenylalanine, tryptophan, and valine was significantly increased in hydroponically grown leaf lettuce, while soil-cultivation derived leaf lettuce samples contained significantly higher levels of fatty-acid derived alcohols (tetracosanol and hexacosanol) and lettuce-specific sesquiterpene lactones (lactucopicrin-15-oxalate and 15-deoxylactucin-8-sulfate). These findings suggest that the metabolite composition of leaf lettuce is primarily affected by its cultivation condition. As the discriminant metabolites reveal important factors that contribute to the nutritional value and taste characteristics of leaf lettuce, we performed comprehensive metabolite profiling to identify metabolite compositions, i.e., metabolite signature, that directly improve its quality and value.</p

Table_3_Metabolomic Evaluation of the Quality of Leaf Lettuce Grown in Practical Plant Factory to Capture Metabolite Signature.XLSX

<p>Vegetables produce metabolites that affect their taste and nutritional value and compounds that contribute to human health. The quality of vegetables grown in plant factories under hydroponic cultivation, e.g., their sweetness and softness, can be improved by controlling growth factors including the temperature, humidity, light source, and fertilizer. However, soil is cheaper than hydroponic cultivation and the visual phenotype of vegetables grown under the two conditions is different. As it is not clear whether their metabolite composition is also different, we studied leaf lettuce raised under the hydroponic condition in practical plant factory and strictly controlled soil condition. We chose two representative cultivars, “black rose” (BR) and “red fire” (RF) because they are of high economic value. Metabolite profiling by comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) resulted in the annotation of 101 metabolites from 223 peaks detected by GC-MS; LC-MS yielded 95 peaks. The principal component analysis (PCA) scatter plot showed that the most distinct separation patterns on the first principal component (PC1) coincided with differences in the cultivation methods. There were no clear separations related to cultivar differences in the plot. PC1 loading revealed the discriminant metabolites for each cultivation method. The level of amino acids such as lysine, phenylalanine, tryptophan, and valine was significantly increased in hydroponically grown leaf lettuce, while soil-cultivation derived leaf lettuce samples contained significantly higher levels of fatty-acid derived alcohols (tetracosanol and hexacosanol) and lettuce-specific sesquiterpene lactones (lactucopicrin-15-oxalate and 15-deoxylactucin-8-sulfate). These findings suggest that the metabolite composition of leaf lettuce is primarily affected by its cultivation condition. As the discriminant metabolites reveal important factors that contribute to the nutritional value and taste characteristics of leaf lettuce, we performed comprehensive metabolite profiling to identify metabolite compositions, i.e., metabolite signature, that directly improve its quality and value.</p

Data_Sheet_1_Metabolomic Evaluation of the Quality of Leaf Lettuce Grown in Practical Plant Factory to Capture Metabolite Signature.DOCX

<p>Vegetables produce metabolites that affect their taste and nutritional value and compounds that contribute to human health. The quality of vegetables grown in plant factories under hydroponic cultivation, e.g., their sweetness and softness, can be improved by controlling growth factors including the temperature, humidity, light source, and fertilizer. However, soil is cheaper than hydroponic cultivation and the visual phenotype of vegetables grown under the two conditions is different. As it is not clear whether their metabolite composition is also different, we studied leaf lettuce raised under the hydroponic condition in practical plant factory and strictly controlled soil condition. We chose two representative cultivars, “black rose” (BR) and “red fire” (RF) because they are of high economic value. Metabolite profiling by comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) resulted in the annotation of 101 metabolites from 223 peaks detected by GC-MS; LC-MS yielded 95 peaks. The principal component analysis (PCA) scatter plot showed that the most distinct separation patterns on the first principal component (PC1) coincided with differences in the cultivation methods. There were no clear separations related to cultivar differences in the plot. PC1 loading revealed the discriminant metabolites for each cultivation method. The level of amino acids such as lysine, phenylalanine, tryptophan, and valine was significantly increased in hydroponically grown leaf lettuce, while soil-cultivation derived leaf lettuce samples contained significantly higher levels of fatty-acid derived alcohols (tetracosanol and hexacosanol) and lettuce-specific sesquiterpene lactones (lactucopicrin-15-oxalate and 15-deoxylactucin-8-sulfate). These findings suggest that the metabolite composition of leaf lettuce is primarily affected by its cultivation condition. As the discriminant metabolites reveal important factors that contribute to the nutritional value and taste characteristics of leaf lettuce, we performed comprehensive metabolite profiling to identify metabolite compositions, i.e., metabolite signature, that directly improve its quality and value.</p

Table_4_Metabolomic Evaluation of the Quality of Leaf Lettuce Grown in Practical Plant Factory to Capture Metabolite Signature.XLSX

<p>Vegetables produce metabolites that affect their taste and nutritional value and compounds that contribute to human health. The quality of vegetables grown in plant factories under hydroponic cultivation, e.g., their sweetness and softness, can be improved by controlling growth factors including the temperature, humidity, light source, and fertilizer. However, soil is cheaper than hydroponic cultivation and the visual phenotype of vegetables grown under the two conditions is different. As it is not clear whether their metabolite composition is also different, we studied leaf lettuce raised under the hydroponic condition in practical plant factory and strictly controlled soil condition. We chose two representative cultivars, “black rose” (BR) and “red fire” (RF) because they are of high economic value. Metabolite profiling by comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) resulted in the annotation of 101 metabolites from 223 peaks detected by GC-MS; LC-MS yielded 95 peaks. The principal component analysis (PCA) scatter plot showed that the most distinct separation patterns on the first principal component (PC1) coincided with differences in the cultivation methods. There were no clear separations related to cultivar differences in the plot. PC1 loading revealed the discriminant metabolites for each cultivation method. The level of amino acids such as lysine, phenylalanine, tryptophan, and valine was significantly increased in hydroponically grown leaf lettuce, while soil-cultivation derived leaf lettuce samples contained significantly higher levels of fatty-acid derived alcohols (tetracosanol and hexacosanol) and lettuce-specific sesquiterpene lactones (lactucopicrin-15-oxalate and 15-deoxylactucin-8-sulfate). These findings suggest that the metabolite composition of leaf lettuce is primarily affected by its cultivation condition. As the discriminant metabolites reveal important factors that contribute to the nutritional value and taste characteristics of leaf lettuce, we performed comprehensive metabolite profiling to identify metabolite compositions, i.e., metabolite signature, that directly improve its quality and value.</p

Table_1_Metabolomic Evaluation of the Quality of Leaf Lettuce Grown in Practical Plant Factory to Capture Metabolite Signature.XLSX

<p>Vegetables produce metabolites that affect their taste and nutritional value and compounds that contribute to human health. The quality of vegetables grown in plant factories under hydroponic cultivation, e.g., their sweetness and softness, can be improved by controlling growth factors including the temperature, humidity, light source, and fertilizer. However, soil is cheaper than hydroponic cultivation and the visual phenotype of vegetables grown under the two conditions is different. As it is not clear whether their metabolite composition is also different, we studied leaf lettuce raised under the hydroponic condition in practical plant factory and strictly controlled soil condition. We chose two representative cultivars, “black rose” (BR) and “red fire” (RF) because they are of high economic value. Metabolite profiling by comprehensive gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) resulted in the annotation of 101 metabolites from 223 peaks detected by GC-MS; LC-MS yielded 95 peaks. The principal component analysis (PCA) scatter plot showed that the most distinct separation patterns on the first principal component (PC1) coincided with differences in the cultivation methods. There were no clear separations related to cultivar differences in the plot. PC1 loading revealed the discriminant metabolites for each cultivation method. The level of amino acids such as lysine, phenylalanine, tryptophan, and valine was significantly increased in hydroponically grown leaf lettuce, while soil-cultivation derived leaf lettuce samples contained significantly higher levels of fatty-acid derived alcohols (tetracosanol and hexacosanol) and lettuce-specific sesquiterpene lactones (lactucopicrin-15-oxalate and 15-deoxylactucin-8-sulfate). These findings suggest that the metabolite composition of leaf lettuce is primarily affected by its cultivation condition. As the discriminant metabolites reveal important factors that contribute to the nutritional value and taste characteristics of leaf lettuce, we performed comprehensive metabolite profiling to identify metabolite compositions, i.e., metabolite signature, that directly improve its quality and value.</p