Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes

Abstract

Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.

Figure 1.

Equipment designed and constructed for in vivo ¹³C and ¹⁵N labeling studies. A, Large tube reactor (0.5 m diameter, 1.3 m height, and 255 L volume) for ¹³CO₂ labeling of soil-grown adult rice plants. B, Small tube reactor (0.5 m diameter, 0.5 m height, and 98 L volume) for ¹³CO₂ labeling of soil-grown rice seedlings. C, Box reactor (0.5 × 0.5 × 0.5 m, 125 L volume) for simultaneous ¹³CO₂ and ¹⁵NH₄NO₃ labeling of hydroponic rice seedlings. All reactors were equipped with a temperature control, comprising a water-cooled ventilator at the bottom plate of the reactor and an external cryostat. In addition, an external CO₂ adsorption unit consisted of a high-power pump, an adsorber, and a fine dust filter. Prior to the experiments, the chosen plants were placed into the reactor, which were then closed gas tight by a rubber seal. Ambient CO₂ was removed from the reactor within 30 s. The experiments were started by injecting the desired amounts of ¹³CO₂ through an injection valve in the lid of each reactor.

Figure 2.

Impact of salt stress on carbon and nitrogen assimilation and translocation of hydroponically grown rice seedlings. The data reflect ¹³C (A) and ¹⁵N (B) enrichment in the shoot and ¹³C (C) and ¹⁵N (D) enrichment in the root. The data shown are means ± sd (n = 3) for stressed seedlings (100 mm NaCl for 6 d; white bars) and untreated controls (gray bars). At the age of 12 d, rice seedlings were simultaneously labeled with ¹³CO₂ (400 µL L⁻¹) through the reactor gas phase and with ¹⁵NH₄NO₃ (1.43 mm) supplied via the hydroponic growth medium. The labeling pulses were applied for 10 min, after which plants were either harvested directly to assess assimilation or cultivated further at ambient air and in nonlabeled medium up to 48 h to trace label translocation. The ¹³C and ¹⁵N enrichment of freeze-dried plant material was analyzed by C-IRMS coupled to EA. Asterisks indicate significant differences between mean values (P ≤ 0.05, Student’s t test). n.s., Not significant. The full data set is given in Supplemental Table S1.

Figure 3.

Relative fluxes of assimilated carbon and nitrogen in hydroponically grown rice seedlings under control conditions (A) and exposed to salt stress (100 mm NaCl for 6 d; B), calculated from ¹³C and ¹⁵N enrichment data obtained 2 h after the labeling pulse (Fig. 2). The root-to-shoot ratio for seedlings under control conditions (0.173) and seedlings exposed to high salinity (0.165) was calculated from the ¹⁵N label distribution using Equations 4 and 6. Based on this ratio, the relative reallocation of label between shoot and root was determined. The total amount of assimilated carbon and nitrogen at time zero was set to 100% carbon and nitrogen uptake, respectively, in order to provide relative data. The seedlings were analyzed at the age of 12 d. The full data sets are given in Supplemental Table S1.

Figure 4.

Carbon assimilation and translocation in adult rice plants, assessed by ¹³CO₂ isotope experiments and labeling analysis by C-IRMS coupled to EA. A, Morphology of the studied plants with sampled leaf, flag leaf, stem, and panicle. B, Tissue-specific ¹³C assimilation of rice at flowering stage (68 d), at early grain-filling stage (75 d), and at late grain-filling stage (88 d), for which soil-grown plants were labeled with 400 µL L^{−1 13}CO₂ for 10 min and harvested directly for assessment of ¹³C enrichment. C, Morphology of the studied plants with identified carbon assimilation routes. D, Time-resolved carbon assimilation and translocation of rice plants at late grain-filling stage (88 d), for which soil-grown plants were labeled with 400 µL L^{−1 13}CO₂ for 10 min and harvested directly or further cultivated at ambient air for 2, 4, 24, and 48 h prior to harvesting. In all cases, ¹³C enrichment of freeze-dried plant material is displayed as δ¹³C (‰), corrected for natural labeling. Mean values ± sd (n = 3) are shown. Different letters (a, b, or c) indicate significant differences between means (P ≤ 0.05, one-way ANOVA with Tukey’s test) of sampled organs at the individual time points. The full data sets of these experiments are given in Supplemental Tables S2 and S3.

Figure 5.

Incorporation of ¹³C into protein amino acids upon ¹³CO₂ labeling. Enrichment of extracted amino acids was analyzed by GC-C-IRMS. A, Time-resolved pattern of a rice seedling (top, age of 12 d) and an adult rice plant at late grain filling (bottom, age of 88 d), for which soil-grown plants were labeled with 400 µL L^{−1 13}CO₂ for 10 min and then harvested directly or further cultivated at ambient air for 2, 4, 24, and 48 h prior to harvesting. Statistical analysis was conducted using Student’s t test, whereby significant differences (P ≤ 0.05) between seedling and adult leaves are marked with asterisks. B, Tissue-resolved pattern of an adult rice plant at late grain filling, for which plants were labeled with 400 µL L^{−1 13}CO₂ for 10 min followed by 24 h of cultivation at ambient air prior to harvesting of leaf, flag leaf, stem, and panicle. Statistical analysis was done by one-way ANOVA with Tukey’s test, whereby different letters (a, b, or c) indicate significant differences between means of the different tissues. C, Tissue-specific amino acid metabolism, visualized as Venn diagrams, which display the relation of amino acids based on the statistical significance between measured ¹³C enrichments. Significant differences between means of amino acid ¹³C enrichments (P ≤ 0.05) were determined by one-way ANOVA with Tukey’s test. Amino acids that do not show significantly different ¹³C enrichment are located in equally colored ellipses. The enrichment data are provided as mean values (n = 3) and reflect atomic percent excess corrected for natural isotopes. The full data sets are given in S4 to S6, respectively. To facilitate comparison, the data are normalized for the highest enrichment of each data set, which was set to 100%, and visualized by a color code between yellow (0%) and red (100%). CIT, Citrate; E4P, erythrose 4-phosphate; F6P, Fru-6-P; FUM, fumarate; GAP, glyceraldehyde 3-phosphate; G6P, Glc-6-P; MAL, malate; OAA, oxaloacetate; 2OG, 2-oxoglutarate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate; PYR, pyruvate; R5P, ribose 5-phosphate; RU5P, ribulose 1,5-bisphosphate; SDL, seedling shoot; SUC, succinate.

Figure 6.

Integrated analysis of carbon and nitrogen metabolism by combined labeling of hydroponically grown rice seedlings with ¹³CO₂ and ¹⁵NH₄NO₃. Tissue-specific enrichment of extracted amino acids was quantified by GC-C-IRMS. Integrated views on transport and biosynthetic routes of Ala, Gly, Ser, Glu/Gln, Asp/Asn, and Pro regarding assimilated ¹³C (light gray) and ¹⁵N (dark gray) labeling (A), assessed in shoot (B) and root (C), are represented by bar graphs over the first 4 h of tracing. The data reflect means ± sd (n = 3). At the age of 12 d, rice seedlings were simultaneously labeled with 400 µL L^{−1 13}CO₂ and 1.43 mm ¹⁵NH₄NO₃ for 10 min and then either harvested directly for assessment of label assimilation or cultivated further at ambient air for 2 to 4 h for assessment of label translocation. Asterisks indicate significant differences between mean values at P ≤ 0.05 (Student’s t test). APE, Atomic percent excess; n.s., not significant. The full data set is given in Supplemental Table S7.

Figure 7.

A and B, Mode-of-action analysis of the effect of the herbicide imazapyr on rice seedlings using ¹³CO₂ labeling in combination with EA-C-IRMS to assess carbon assimilation (A) and with GC-C-IRMS to assess ¹³C enrichment of extracted proteinogenic amino acids (B). The experimental setup contained seedlings at the age of 12 d subjected to imazapyr (62.5 g ha⁻¹ active ingredient) or control treatment (62.5 g ha⁻¹ control solution). Four hours after the treatment, seedlings were labeled for 10 min with 400 µL L^{−1 13}CO₂. The assimilation of ¹³C, determined immediately after the labeling pulse from freeze-dried plant material, is expressed as δ¹³C (‰), corrected for natural isotopes. The enrichment of extracted amino acids was determined after 2 h of further cultivation at ambient air. The δ¹³C values of the amino acids of imazapyr-treated rice seedlings were normalized to those of the control seedlings. C, Phenotypes of rice seedlings 7 d after treatment with the control solution (left plant) and the imazapyr solution (right plant). Asterisks indicate significant differences between mean values of imazapyr-treated and control plants at P ≤ 0.05 (Student’s t test). n.s., Not significant. Mean values ± sd (n = 3) are shown. The full data sets are given in Supplemental Tables S8 and S9.