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Abstract The tripeptide antioxidant glutathione (γ- l-glutamyl- l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat ( Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination.

In dry seeds, GSH was the predominant LMW thiol and, 15 h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48 h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E GSSG/2GSH and E CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E thiol-disulphide). The changes in E CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E GSSG/2GSH and E CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm.

We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H 2O 2) in redox regulation of bread wheat germination and early seedling growth. Seed material, germination assays, and water content measurements Bread wheat ( Triticum aestivum L.) cultivar Rebelde (Apsovsementi S.p.a., Voghera, Italy – CO.NA.SE. Consorzio Nazionale Sementi S.r.l., Italy) was grown in an open field in Sant’Angelo Lodigiano (Lodi, Italy) in 2013-2014. After harvest, seeds were immediately stored at 4 °C and. HPLC analysis of LMW thiols and disulphides At nine intervals up to 48 h after the onset of imbibition, whole seeds ( n = 4 replicates of 35 seeds), and dissected embryos and endosperms isolated after 15 h or 48 h of imbibition ( n = 4 replicates of 40 seeds) were analysed by HPLC. Freeze-dried powder from whole seeds (70 ± 6 mg), endosperms (50 ± 0.5 mg), or embryos (25 ± 5.0 mg) were extracted at 4 °C in 1 mL of 0.1 M HCl, vortexed at full speed for 1 min before centrifugation at 20,000 × g for 20 min at 4 °C.

An aliquot of 120 μL of the supernatant was used for the determination of total LMW thiols and disulphides, and 400 μL for assessing LMW disulphides according to Kranner (1998) [ Kranner I. Determination of glutathione, glutathione disulfide and two related enzymes, grutathione reductase and glucose-6-phosphate dehydrogenase, in fungal and plant cells.

In: Varma A, ed. Mychorriza manual. Berlin: Springer; 1998: 227– 241. ], and described in detail by Bailly and Kranner (2011) [ Bailly C, Kranner I. Analyses of reactive oxygen species and antioxidants in relation to seed longevity and germination. Berlin: Springer; 2011: 343– 367.

The pH of the extracts was adjusted to values between 8.0 and 8.3 with 200 mM bicine buffer. To measure both LMW thiols and disulphides, the latter were reduced by 273 μM dithiothreitol (DTT, Applichem Gmbh, Darmstadt, Germany) for 1 h at room temperature, before labelling of thiols with 857 μM monobromobimane (mBBr) for 15 min at room temperature, and stopping the reaction with 0.104% (v/v) methanesulfonic acid. To measure disulphides, thiols were blocked with 583 μM N-ethylmaleimide (NEM) for 15 min at room temperature, before excess NEM was removed five times with toluene, and disulphides were reduced with DTT, then labelled with mBBr as for measuring total LMW thiols and disulphides. Labelled LMW thiols were separated by reversed-phase HPLC using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) on a ChromBudget 120-5-C18 column (5.0 μm, BISCHOFF GmbH, Leonberg, Germany), and detected by a fluorescence detector (the excitation wavelength was set at 380 nm and the emission wavelength at 480 nm). Data were calculated using individual calibration curves for each LMW thiol that were linear over the range measured. The concentrations of LMW thiols were calculated by subtracting the concentrations of LMW disulphides from those of total LMW disulphides and thiols.

Oxalate oxidase activity measurements and quantification of extracellular H 2O 2 production Oxalate oxidase (EC 1.2.3.4, oxalate: oxygen oxidoreductase, OXO) activity of whole seeds was determined spectrophotometrically, using a modification of the method of Laker et al. (1980) [ Laker MF, Hofmann AF, Meeuse BJD. Spectrophotometric determination of urinary oxalate with oxalate oxidase prepared from moss. Clin Chem 1980;26: 827– 830., ]. OXO catalyses the oxidative breakdown of one mole of oxalate to one mole of H 2O 2 and two moles of CO 2. Briefly, H 2O 2 was indirectly quantified via the formation of indamine, catalysed by the H 2O 2-dependent horseradish peroxidase (HPOX), by following the increase in absorbance at 555 nm (A 555). Absorbance values were calibrated using a standard curve for H 2O 2 within a 2.4–120 μM range.

All extraction steps were conducted at 4 °C (on ice). 50 ± 0.5 mg of seed powder were homogenised in 0.4 mL of 50 mM succinate buffer (pH 3.8) containing 1 mM EDTA and 9.6 mM oxalic acid in 2 mL Eppendorf tubes with two 3 mm glass beads (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), using a TissueLyser II (Qiagen, Hilden, Germany) for 1 min at 30 Hz.

After centrifugation at 20,000 × g for 5 min at 4 °C, the supernatant was collected and centrifuged again at 20,000 × g for 2 min. OXO activity was measured in 96-well BRANDplates ® (pureGRADE™ S-clear, Sterile R, BRAND GmbH + CO KG, Wertheim, Germany), using a Synergy-HTX plate reader (BioTek ® Instruments, Inc., Winooski, VT) at 35 °C after pre-incubating 51.2 μL of protein extract and oxalic acid for 15 min. The increase in A 555 was followed for 10 min after adding 185 μL of a chromogenic solution (790 μM of N,N-dimethylaniline and 110 μM of 3-methyl-2-benzothiazolinone hydrazone dissolved in 50 mM succinate buffer, pH 3.8) and 2.5 U/mL of HPOX in a total volume of 240 μL, with a final concentration of oxalic acid of 2 mM.

A reaction mixture without protein extract and oxalic acid was used as a blank. Three technical replicates for each biological replicate ( n = 4) were measured. H 2O 2 was quantified using a “Red Hydrogen Peroxide Assay Kit”, following optimisation of instructions by the manufacturer (Enzo Life Sciences Inc., Farmingdale, NY). Whole seeds and seedlings were incubated in 1 mL of two times concentrated reaction mixture containing 10 μL of red peroxidase substrate stock solution in dimethyl sulphoxide (DMSO), 40 μL of 20 U/mL HPOX stock solution, and 950 μL of assay buffer in darkness at room temperature for 30 min, then diluted 1:2 with the reaction buffer. Fluorescence of the HPOX- and H 2O 2-catalysed formation of Resorufin (excitation wavelength =540 ± 35 nm, emission wavelength =590 ± 20 nm) was measured with a Synergy-HTX plate reader (BioTek ® Instruments, Inc., Winooski, VT). Data were corrected for background fluorescence occurring in the presence of pure assay buffer.

Three technical replicates were measured for each biological replicate ( n = 4, each containing 25 seeds). The content of extracellular H 2O 2 produced was calculated from a standard curve for H 2O 2 (0–3 μM range), and data are expressed as nmol g −1 DW s −1. Statistical analysis Data were analysed for significance (α =.05) by one-way ANOVA in combination with Tukey's HSD (Honest Significant Difference) test for post-hoc comparisons of means ( p-value ≤.05), using the IBM SPSS Statistics 21 software package. Arcsine transformation was applied to germination and WC values to simulate normal distribution of data.

The assumption of normal distribution was assessed via the Shapiro–Wilk test, and further verified with QQ-plots. The assumption of homoscedasticity of variances across groups was checked through Levene's test, and, whenever not respected, the appropriate mathematical transformations were applied to the data. To compare different seed structures at the same time interval a one-sample t-test was applied ( p-value ≤.05). Seed germination and water uptake The first radicle protruded 12 h after the onset of imbibition, and 50% of total germination (TG) was reached after approximately 22 h (). Within 48 h, 96% of seeds had germinated. The radicle and two seminal roots developed (in wheat this is indicative of healthy seedlings), and the coleoptile (a protective sheath covering the emerging shoot in monocotyledon plants) could be clearly distinguished in most seedlings.

After 8 h of imbibition, the water uptake slowed down (), typical for Poaceae seeds [ Clarke JM. Measurement of relative water-uptake rates of wheat seeds using agar media. Can J Plant Sci 1980;60: 1035– 1038.,, Rathjen JR, Strounina EV, Mares DJ. Water movement into dormant and non-dormant wheat ( Triticum aestivum L.) grains. J Exp Bot 2009;60: 1619– 1631.,, ].

Changes in WC during imbibition of Triticum aestivum seeds. After 15 and 48 h from the onset of imbibition, NG seeds were sorted from G seeds.

Endosperms (dark grey bars) and their embryos or seedlings (light grey bars) were dissected and analysed separately. Data are means ± SE ( n = 4 replicates of 40 endosperms and embryos). After the onset of imbibition, bars labelled with the same letter do not differ significantly (one-way ANOVA analyses followed by post-hoc Tukey's HSD test, p-value ≤.05). Asterisks indicate significant differences (one-sample t-test, p-value ≤.05) between seed structures at the same time interval. Changes in WC during imbibition of Triticum aestivum seeds.

After 15 and 48 h from the onset of imbibition, NG seeds were sorted from G seeds. Endosperms (dark grey bars) and their embryos or seedlings (light grey bars) were dissected and analysed separately. Data are means ± SE ( n = 4 replicates of 40 endosperms and embryos). After the onset of imbibition, bars labelled with the same letter do not differ significantly (one-way ANOVA analyses followed by post-hoc Tukey's HSD test, p-value ≤.05).

Asterisks indicate significant differences (one-sample t-test, p-value ≤.05) between seed structures at the same time interval. Changes in LMW thiols and disulphides in whole seeds during germination and early seedling growth GSH + GSSG [hereafter termed “total glutathione”] and Cys + CySS [hereafter referred to as “cyst(e)ine”] dominated the LMW thiol-disulphide redox pool in dry whole seeds, whereas the concentrations of Cys-Gly + Cys-bis-Gly [hereafter termed as “cyst(e)inyl-(bis)-glycine”] and γ-Glu-Cys + bis-γ-Glu-Cys [hereafter referred to as “(bis)-γ-glutamyl-cyst(e)ine”] were lower by one order of magnitude (). Dry wheat seeds contained 4.7 times higher concentrations of total glutathione than cyst(e)ine; total glutathione was 7.4 and 67.8 times more abundant than cyst(e)inyl-(bis)-glycine and (bis)-γ-glutamyl-cyst(e)ine, respectively (). Disulphide contents in dry whole seeds, expressed as a percentage of total thiols and disulphides, were 30% for bis-γ-Glu-Cys, 36% for GSSG, 40% for Cys, and 41% for Cys-bis-Gly (). Species of the Poaceae may also contain a GSH homologue termed hydroxy-methyl-glutathione (hGSH, γ- l-glutamyl- l-cysteinyl-β-serine) [ Klapheck S, Chrost B, Starke J, Zimmermann H. Γ-Glutamylcysteinylserine: a new homolog of glutathione in plants of the family Poaceae. Bot Acta 1992;105: 174– 179.

], but no corresponding peak was detected in seeds or seedlings during the first 48 h after the onset of imbibition. Concentrations of LMW thiols and corresponding disulphides during germination and early seedling growth of Triticum aestivum. The abscissa shows the time after the onset of imbibition, and time 0 indicates dry seeds.

(A) GSH and GSSG; (B) Cys and CySS; (C) Cys-Gly and Cys-bis-Gly; (D) γ-Glu-Cys and bis-γ-Glu-Cys. Data are means ± SE for the four LMW thiols (white bars) and corresponding disulphides (black bars). Data labelled with the same letters do not differ significantly (one-way ANOVA analyses followed by post-hoc Tukey's HSD test, p-value ≤.05). For ease of comparison, germination curve is indicated by the grey line. Concentrations of LMW thiols and corresponding disulphides during germination and early seedling growth of Triticum aestivum. The abscissa shows the time after the onset of imbibition, and time 0 indicates dry seeds. (A) GSH and GSSG; (B) Cys and CySS; (C) Cys-Gly and Cys-bis-Gly; (D) γ-Glu-Cys and bis-γ-Glu-Cys.

Data are means ± SE for the four LMW thiols (white bars) and corresponding disulphides (black bars). Data labelled with the same letters do not differ significantly (one-way ANOVA analyses followed by post-hoc Tukey's HSD test, p-value ≤.05). For ease of comparison, germination curve is indicated by the grey line. Total glutathione concentrations rose 1.3-fold within the first 8 h of imbibition, GSH increased by 43%, and GSSG decreased by 42%.

A further significant increase in total glutathione, mostly due to GSH, was recorded during early seedling growth (i.e. Between 24 h and 48 h after the onset of imbibition) (). Cyst(e)ine concentrations steadily increased within 24 h, after which concentrations greatly increased ().

Hence, after 48 h, cyst(e)ine was the most abundant LMW thiol-disulphide redox couple, and its concentration became 1.8 times higher than that of total glutathione (). Within the first 12 h of imbibition, cyst(e)inyl-(bis)-glycine decreased 2.3-fold, (bis)-γ-glutamyl-cyst(e)ine increased 2.8-fold, then both plateaued for the subsequent 12 h (). From 24 h of imbibition, when approximately 62% of seeds had germinated (), cyst(e)inyl-(bis)-glycine and (bis)-γ-glutamyl-cyst(e)ine both increased progressively.

Changes in LMW thiol-disulphide redox couples in endosperm and embryo on a DW and on a seed basis After 15 and 48 h from the onset of imbibition, seeds were sorted into two lots, G and NG, and embryos and seedlings were separated from the endosperms. At these intervals, we characterised the concentrations of LMW thiols and disulphides, expressed on a DW basis (), and their corresponding E hcs in the two seed structures (). As in whole seeds, total glutathione and cyst(e)ine were the predominating LMW thiol-disulphide redox couples in both endosperm and embryo. Fifteen hours after the onset of imbibition, the endosperms isolated from NG seeds (termed “NG15_endosperms”) contained the same concentrations of LMW thiols and disulphides as the endosperms isolated from germinated seeds (termed “G15_endosperms”), and had the same thiol-disulphide ratios ( p-value.05; ).

By contrast, embryos isolated from NG seeds 15 h after the onset of imbibition (termed “NG15_embryos”) contained 3–19 times higher concentrations of all LMW thiols than whole dry seeds. Embryos with radicles protruding at least 1 mm and isolated from germinated seeds 15 h after the onset of imbibition (termed “G15_embryos”) had about one third more total glutathione, one forth more cyst(e)inyl-(bis)-glycine, and five times more (bis)-γ-glutamyl-cyst(e)ine than NG15_embryos. In particular, G15_embryos contained more disulphides than NG15_embryos ( p-value ≤.05), except for CySS (). Concentration, on a DW basis, and redox state of four low-molecular-weight (LMW) redox couples in structures isolated from Triticum aestivum seeds upon imbibition. After 15 h from the onset of imbibition, white bars for LMW thiols and black bars for their corresponding disulphides show dry weight-based concentrations in endosperms and embryos isolated from NG seeds or G seeds. After 48 h from the onset of imbibition, bars show thiol and disulphide DW-based concentrations for endosperms and seedlings isolated from germinated seeds (panels A--D). Bars on the right side show the half-cell reduction potentials of the four LMW thiol-disulphide redox couples in the endosperm (dark grey bars) and embryo or seedling (light grey bars), respectively (panels E--H).

Data for endosperms and embryos or seedlings were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test for thiols (lower case letters) and disulphides (upper case letters). Data points labelled with the same letter do not differ significantly ( p-value ≤.05; italics is used for comparing the endosperms). Data are means ± SE ( n = 4 replicates of 40 endosperms and embryos or seedlings per condition). NG: non-germinated; G: germinated; 15 and 48 indicate the hours after the onset of imbibition. Concentration, on a DW basis, and redox state of four low-molecular-weight (LMW) redox couples in structures isolated from Triticum aestivum seeds upon imbibition.

After 15 h from the onset of imbibition, white bars for LMW thiols and black bars for their corresponding disulphides show dry weight-based concentrations in endosperms and embryos isolated from NG seeds or G seeds. After 48 h from the onset of imbibition, bars show thiol and disulphide DW-based concentrations for endosperms and seedlings isolated from germinated seeds (panels A--D).

Bars on the right side show the half-cell reduction potentials of the four LMW thiol-disulphide redox couples in the endosperm (dark grey bars) and embryo or seedling (light grey bars), respectively (panels E--H). Data for endosperms and embryos or seedlings were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test for thiols (lower case letters) and disulphides (upper case letters). Data points labelled with the same letter do not differ significantly ( p-value ≤.05; italics is used for comparing the endosperms).

Data are means ± SE ( n = 4 replicates of 40 endosperms and embryos or seedlings per condition). NG: non-germinated; G: germinated; 15 and 48 indicate the hours after the onset of imbibition. After 48 h from the-onset of imbibition, 96% of seeds had germinated (). Only germinated seeds were characterised for their concentrations of LMW thiols and disulphides in isolated seed structures 48 h after the onset of imbibition. Total glutathione in the endosperms isolated from germinated seeds 48 h after the onset of imbibition (termed “G48_endosperms”) dropped by 16% compared to G15_endosperms ( p-value ≤.05).

Conversely, cyst(e)ine, cyst(e)inyl-(bis)-glycine, and (bis)-γ-glutamyl-cyst(e)ine increased about five-, two-, and four-fold, with high disulphide percentages (52, 44, and 40%, here arranged from the highest to the lowest contribution to each thiol-disulphide redox pool). In seedlings with coleoptile, radicle, and two seminal roots isolated from germinated seeds 48 h after the onset of imbibition (termed “G48_embryos”), total glutathione, cyst(e)inyl-(bis)-glycine, and (bis)-γ-glutamyl-cyst(e)ine concentrations dropped two-, three-, and two-fold compared to G15_embryos, whereas cyst(e)ine almost doubled. Low disulphide percentages were found for total glutathione (0.1%), cyst(e)inyl-(bis)-glycine (1.5%), and (bis)-γ-glutamyl-cyst(e)ine (1.8%), but not for (bis)-γ-glutamyl-cyst(e)ine, which was found as bis-γ-Glu-Cys for 20.4% (). In summary, G15_embryos showed markedly higher concentrations of LMW thiols and disulphides than NG15_embryos, but this trend was not evident in NG15_endosperms and G15_endosperms. NG15_ embryos, G15_embryos and G48_embryos had far higher thiol-disulphide ratios than NG15_endosperms, G15_endosperms and G48_endosperms (except for the redox couple γ-Glu-Cys/bis-γ-Glu-CySS in G15_embryos) (). Thereafter, a phase of early seedling growth was identified by comparing G15_embryos and G48_embryos. In both G48_embryos and G48_endo-sperms, total glutathione declined, while cyst(e)ine increased.

In contrast, cyst(e)inyl-(bis)-glycine and (bis)-γ-glutamyl-cyst(e)ine increased in G48_endosperms, but decreased in G48_embryos (). When the total contents of LMW thiol-disulphide redox couples were expressed on a seed basis, their concentrations and proportions in the overall LMW thiol-disulphide redox pool changed distinctly (). In particular, NG15_endosperms and G15_endosperms had the same concentrations and proportional distributions of the four LMW thiol-disulphide redox couples. In G48_endosperms, the total LMW thiol-disulphide redox pool increased 1.5-fold, and cyst(e)ine became the dominant LMW thiol-disulphide redox couple (). By contrast, after 15 h, the total LMW thiol-disulphide redox pool in G15_embryos was greater than in NG15_ embryos, with a larger proportion represented by (bis)-γ-glutamyl-cyst(e)ine (). The total LMW thiol-disulphide redox pool further increased in G48_embryos compared to G15_embryos, with a decline in cyst(e)inyl-(bis)-glycine, and an increase in cyst(e)ine proportions, but total glutathione remained the dominant LMW thiol-disulphide redox couple (). Changes in LMW thiol-disulphide half-cell reduction potentials in endosperm and embryo The tissue WCs (g H 2O g −1 DW; ) were used to calculate the molar concentrations of all thiols and disulphides (), which are required for the individual E hcs of the four thiol-disulphide redox couples ().

The E GSSG/2GSH, the half-cell reduction potential of the cysteinyl-glycine/cystinyl-bis-glycine redox couple (E Cys-bis-Gly/2Cys-Gly), and the half-cell reduction potential of the γ-Glu-Cys/bis-γ-Glu-Cys redox couple (E bis-γ-Glu-Cys/2γ-Glu-Cys) did not differ between NG15_endosperms and G15_endosperms. However, values of the half-cell reduction potential of the CySS/2Cys redox couple (E CySS/2Cys) were more oxidising in NG15_endosperms. E CySS/2Cys shifted back to more negative values in G48_endosperms compared to G15_endosperms (), and E GSSG/2GSH and E Cys-bis-Gly/2Cys-Gly slightly shifted towards more positive values (). In G15_embryos, E GSSG/2GSH and E Cys-bis-Gly/2Cys-Gly values were by 19 mV and 15 mV more positive than in NG15_embryos. These values shifted back to more negative values (by 41 mV and 6 mV) in G48_embryos (). Values of E CySS/2Cys did not differ significantly between NG15_embryos and G15_embryos or G15_embryos and G48_embryos. The E bis-γ-Glu-Cys/2γ-Glu-Cys did not differ between NG15_embryos and G15_embryos, but in G48_embryos, it shifted to a more negative value than that of NG15_embryos.

Due to their highest molar concentrations (), E GSSG/2GSH and E CySS/2Cys were the most influential contributors to the mathematically combined E thiol-disulphide of dry whole seeds, NG15_endosperms, G15_endosperms, G48_endosperms, and NG15_embryos, G15_embryos and G48_embryos (i.e. All measured samples), whereas E Cys-bis-Gly/2Cys-Gly and E bis-γ-Glu-Cys/2γ-Glu-Cys together contributed less than 8% (). E CySS/2Cys contributed more to E thiol-disulphide in endosperms than in embryos or seedlings, and the contributions of E CySS/2Cys increased in both seed structures during early seedling growth. Relative contribution of the individual half-cell reduction potentials (E hcs) of the four LMW thiol-disulphide redox couples to the LMW thiol-disulphide redox environment (E thiol-disulphide) in dry whole seeds and isolated Triticum aestivum seed structures upon imbibition.

Values for the individual contributions of the thiol-disulphide redox couples are expressed as percentages of E thiol-disulphide calculated for dry whole seeds and seed structures isolated after 15 or 48 h from the onset of imbibition. Data show means ± SE ( n = 4). G: germinated; NG: non-germinated.

Changes in the H 2O 2 production during germination and early seedling growth The H 2O 2 production rate decreased three-fold from the first imbibition interval to 16 h after the onset of imbibition (). Between 16 and 32 h after the onset of imbibition, when the majority of seeds germinated (), the rate of H 2O 2 production increased by five-fold and further increased after 48 h ().

Similar to the pattern of H 2O 2 production, the activity of OXO decreased in the first 16 h of imbibition and then increased, but the trend was much less pronounced (). Changes in H 2O 2 production and OXO activity during Triticum aestivum seed germination and early seedling growth. White bars show the rates of H 2O 2 production (left axis) and black bars show OXO activity (right axis). Both variables were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test. Bars labelled with the same letter do not differ significantly ( p-value ≤.05). Upper case letters are used for H 2O 2 production rates, and lower case letters for OXO activity.

Data show means ± SE ( n = 3 replicates of 25 seeds for H 2O 2 production; n = 4 replicates of 35 seeds for OXO activity). Changes in H 2O 2 production and OXO activity during Triticum aestivum seed germination and early seedling growth. White bars show the rates of H 2O 2 production (left axis) and black bars show OXO activity (right axis). Both variables were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test. Bars labelled with the same letter do not differ significantly ( p-value ≤.05). Upper case letters are used for H 2O 2 production rates, and lower case letters for OXO activity.

Data show means ± SE ( n = 3 replicates of 25 seeds for H 2O 2 production; n = 4 replicates of 35 seeds for OXO activity). Overview of the changes in H 2O 2 and LMW thiols and disulphides during bread wheat germination and early seedling growth. From left to right, changes in LMW thiol-disulphides and H 2O 2 production rates in Triticum aestivum are schematically represented for a whole dry seed, seed structures isolated from non-germinated seeds after 15 h from the onset of imbibition, and seed structures isolated from germinated seeds after 15 and 48 h. Whole dry seed, endosperm including aleurone (large oval), and embryo or seedling (small oval) are divided by vertical lines.

These lines delimit areas proportional to the concentrations of total glutathione (i.e. GSH (glutathione) + GSSG (glutathione disulphide), area left of line) and cyst(e)ine (i.e. Cys (cysteine)+ CySS, (cysteine) area right of line), in the respective seed structure. The redox states (E hcs in mV) of total glutathione and cyst(e)ine are indicated by the blue-to-red (reducing-to-oxidising) shadings of each area, as shown by the bottom right scale. Yellow background shadings indicate the rates of H 2O 2 production (nmol g −1 DW s −1), as shown by the bottom left scale. The dashed vertical line separates seeds from seedlings.

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