Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination

Abstract

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Keywords: in vivo biosensing; mitochondria; redox proteomics; redox regulation; seed germination.

Fig. 1.

Restart of respiratory energy metabolism in Arabidopsis seeds at imbibition. (A and B) Experimental setup for plate reader-based detection of genetically encoded probes in seeds at imbibition exemplified for the MgATP²⁻ FRET-sensor ATeam. (A) Representative brightfield and fluorescence (GFP-filter) images of dry seeds, either with or without expression of cytosolic ATeam (cyt-ATeam). Emission spectra of the seeds are plotted below; dashed magenta line for wildtype (Col-0) seeds, dashed green line for cyt-ATeam seeds, and green line for cyt-ATeam seeds corrected for autofluorescence of Col-0 seeds (n = 3 to 4 seed batches). (B and C) Monitoring cytosolic MgATP²⁻ levels in intact seeds through online imbibition and detection of the cyt-ATeam FRET ratio. 1: dry seed, 2: addition of water, 3: imbibition. The arrows indicate addition of water and T_1/2 equals the time point of half-maximal sensor response (n = 6 seed batches; mean normalized to last value before injection of water ± SD and corrected for Col-0 autofluorescence). (D) Total ATP, ADP, and AMP concentrations in dry seeds and after 1 and 4 h of imbibition (n = 4; mean normalized to seed dry weight + SD; 2-way ANOVA with Tukey’s multiple comparisons test with a: P > 0.05, b: P < 0.05, and e: P < 0.0001, significant differences between 1 and 4 h imbibition are indicated by a second character). Corresponding EC ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]) is shown on secondary axis (mean + SD). (E) Oxygen consumption of imbibed seeds measured with MitoXpress Xtra (fluorescence lifetime of the probe is physically quenched by oxygen). Increase of fluorescence lifetime (Δ FLT) recorded for seeds at control conditions (white circles) or supplemented with 500 µM potassium cyanide (KCN) (gray circles; n = 6 seed batches; mean normalized to amount of dry seeds ± SD). Inset shows Δ FLT rate in the timeframe of 60 to 90 min (2-sided Student’s t test with ***P < 0.001) as indicated by the green and magenta line. (F) Concentration of organic acids analyzed in dry seeds and after 1 and 4 h of imbibition (n = 4; mean normalized to seed dry weight + SD; 2-way ANOVA with Tukey’s multiple comparisons test with a: P > 0.05, b: P < 0.05, c: P < 0.01, d: P < 0.001, and e: P < 0.0001, significant differences between 1 and 4 h imbibition are indicated by a second character). Pyruvate (pyr), (iso-)citrate ((iso-)cit), 2-oxoglutarate (2OG), succinate (succ), fumarate (fum), malate (mal), and lactate (lac). (G) Working model of the restart of the respiratory energy metabolism of dry seeds early during imbibition. Width of arrow lines indicate flux rates. OXPHOS, oxidative phosphorylation.

Fig. 2.

Activation of the mitochondrial thiol redox machinery in Arabidopsis seeds at imbibition. (A) Model of glutathione and roGFP reduction via mitochondrial metabolism. TCA cycle-derived NADPH, but not NADH, acts as electron donor to the matrix thiol redox machineries, e.g., to reduce glutathione disulfide (GSSG) to glutathione (GSH) by glutathione reductase (GR), and to the thioredoxin-system (not depicted). Redox state of the matrix glutathione pool can be monitored by roGFP2 sensors. (B and C) Change in matrix glutathione redox state in imbibed seeds lacking seed coat and endosperm over time analyzed by confocal microscopy monitoring the 405/488-nm excitation ratio of roGFP2-Grx1. (B) Representative confocal images showing embryo epidermal cells imbibed for 10 and 60 min. ROIs with mitochondria are highlighted and magnified. Overlay of the 405-nm and the 488-nm channels in red and green, protein storage vacuoles appear in purple, due to an autofluorescence channel displayed in blue. (Scale bars, 10 µm.) (C) Changes in matrix roGFP2-Grx1 oxidation during seed imbibition analyzed by confocal imaging (n = 400 to 700 individual mitochondria per time point in 8 to 12 individual embryos; mean + SD; 2-sided Student’s t test with *P < 0.05 and **P < 0.01). (D) Mitochondrial roGFP2 dynamics at imbibition of intact seeds monitored by plate reader-based fluorimetry (Fig. 1 A and B). The arrow indicates addition of water and T_1/2 equals the time point of half-maximal sensor response (n = 6 seed batches; mean normalized to last value before injection of water ± SD and corrected for Col-0 autofluorescence). Inserted panel shows excitation spectra of dry seeds, dashed magenta line for Col-0 seeds, dashed green line for mt-roGFP2 seeds, and green line for mt-roGFP2 seeds corrected for autofluorescence of Col-0 seeds (n = 3 to 4 seed batches).

Fig. 3.

Rebooting the thiol redox machinery of isolated mitochondria to define the proteomic landscape of mitochondrial proteinaceous Cys redox switches. (A) Schematic model of quiescent mitochondria and their metabolic reactivation by substrate addition. Substrates are metabolized reducing NAD⁺ and NADP⁺. Both NADH and NADPH can be oxidized by the electron transport chain (ETC), but only NADPH can act as reductant for the matrix thiol redox machineries. Matrix-targeted roGFP2-Grx1 acts as artificial target of the glutathione redox machinery. (B) Representative roGFP2-Grx1 reduction kinetics of quiescent mitochondria, either supplemented with 10 mM 2OG or 10 mM citrate (n = 4 technical replicates, mean ± SD; dashed line indicates addition of substrates and red box highlights the oxidation state after 25 min). The mt-roGFP2-Grx1 redox state calculated for each metabolic condition after 25 min on the Right; for calibration of the sensor, mitochondria were incubated with 5 mM dipyridyldisulfide (DPS, oxidant) or 20 mM DTT (DTT, reductant) for 20 min (2-sided Student’s t test with **P < 0.01 and ****P < 0.0001). Inset shows state II oxygen consumption of isolated mitochondria supplemented either with 2OG or citrate (n = 5, mean + SD). (C and D) Abundance profiles of the redox states of Cys peptides in quiescent and respiring mitochondria. Isolated mitochondria supplemented with citrate (C) or 2OG (D) and incubated for 25 min before differential thiol labeling with iodoTMTs. Redox states of individual peptides quantified for isolated mitochondria with and without respiratory substrate (blue and red bars, respectively). The distribution of cysteine peptide oxidation levels is shown by the proportion of the total number of peptides in each 2% quantile of percentage oxidation (n = 3, mean + SD). In Insets, percentage of Cys oxidation without substrate addition is plotted against percentage of Cys oxidation after substrate addition for individual Cys peptides. Cys peptides with significant change in their Cys-oxidation state are displayed in blue (t test corrected for multiple comparisons by Benjamini, Krieger, and Yekutieli with <2% false discovery rate [FDR]). (E) The 5 most enriched GO terms of the “cellular component” and “biological process” categories of a PANTHER-overrepresentation analysis of all significantly redox-switched Cys peptides. (F) All identified Cys peptides of the individual subunits of the mitochondrial complex I mapped on the schematic structure according to Braun et al. (32) (Left); all Cys peptides identified for enzymes of the TCA cycle and closely associated (Right). Blue: significantly redox-switched Cys peptide; red: not significantly redox-switched Cys peptide. (G) Redox shift analysis through immunodetection of aconitase proteins (ACO) in seed protein extracts alkylated with methylpolyethylene glycolmaleimide reagent (MM-PEG24). Protein extracts from dry seeds (dry), seeds imbibed for 1 h (1 h), and dry seeds and treated with Tris(2-carboxyethyl)phosphine (TCEP) for reduction (n = 3 biological replicates, see SI Appendix, Fig. S8A). Note that the 3 Arabidopsis ACO proteins are localized in the mitochondrial matrix and the cytosol. (H) Relative activities of alternative oxidase (AOX, Left panels, see also SI Appendix, Fig. S8B), NAD-malic enzyme (NAD-ME), and aconitase (ACO) in isolated mitochondria assayed at different redox potentials adjusted by a DTT/dithiane buffer. RoGFP2 to validate the redox conditions in the assay media (Lower panels). Calculated redox potentials are adjusted for assay pH. AOX: 7.5, NAD-ME: 6.7, ACO: 7.8 (n = 3 biological replicates, mean + SD, 1-way ANOVA with Dunnett’s multiple comparisons test, *P < 0.05, ***P < 0.001, and ****P < 0.0001).

Fig. 4.

The effect of impairing the mitochondrial thiol redox machinery on energy metabolism and germination. Synchronized seeds of Col-0, gr2, trx-o1, and ntr a/b lines were analyzed for their performance during germination. (A) Oxygen consumption of imbibed seeds measured with MitoXpress Xtra. (i) Oxygen uptake as indicated by increase in fluorescence lifetime (Δ FLT) over time (n = 5 seed batches; mean normalized to amount of dry seeds + SD). (ii) Δ FLT rate in the 60- to 90-min time window (2-sided Student’s t test with **P < 0.01 and ***P < 0.001 if compared with Col-0). (B) Total ATP, ADP, and AMP concentration (i) in dry seeds and (ii) after 1 h of imbibition (n = 4; mean normalized to seed dry weight + SD; 2-way ANOVA with Dunnett’s multiple comparisons test with b: P < 0.05, c: P < 0.01, d: P < 0.001, e: P < 0.0001 if compared with Col-0). Corresponding EC ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]) is shown on secondary axis (mean + SD). (C) Concentration of organic acids analyzed (i) in dry seeds and (ii) after 1 h of imbibition (n = 4; mean normalized to seed dry weight + SD; 2-way ANOVA with Dunnett’s multiple comparisons test with b: P < 0.05, c: P < 0.01, d: P < 0.001 and e: P < 0.0001 if compared with Col-0). Pyruvate (pyr), (iso-)citrate [(iso-)cit], 2-oxoglutarate (2OG), succinate (succ), fumarate (fum), malate (mal), and lactate (lac). (D) Germination over 5 d (i) for freshly harvested seeds and (ii) after a CDT by storage at 37 °C and 75% relative humidity for 19 d to mimic seed aging. The statistical analysis of germination rates of gr2, trx-o1, and ntr a/b as compared to Col-0 seeds is shown in SI Appendix, Table S1.