Fast and uniform germination is key to agricultural production and can be achieved by seed ‘priming’ techniques. Here, we characterised the responses of bread wheat (Triticum aestivum L.) seeds to a hot steam treatment (‘BioFlash’), which accelerated water uptake, resulting in faster germination and seedling growth, typical traits of primed seed. Before the completion of germination, metabolite profiling of seeds revealed advanced accumulation of several amino acids (especially cysteine and serine), sugars (ribose, glucose), and organic acids (glycerate, succinate) in hot steam-treated seeds, whereas sugar alcohols (e.g. arabitol, mannitol) and trehalose decreased in all seeds. Tocochromanols (the ‘vitamin E family’) rose independently of the hot steam treatment. We further assessed shifts in the half-cell reduction potentials of low-molecular-weight (LMW) thiol-disulfide redox couples [i.e. glutathione disulfide (GSSG)/glutathione (GSH) and cystine/cysteine], alongside the activities of the reactive oxygen species (ROS)-processing enzyme superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase. Upon the first 4 h of imbibition, a rapid conversion of LMW disulfides to thiols occurred. Completion of germination was associated with a re-oxidation of the LMW thiol-disulfide cellular redox environment, before more reducing conditions were re-established during seedling growth, accompanied by an increase in all ROS-processing enzyme activities. Furthermore, changes in the thiol-disulfide cellular redox state were associated to specific stages of wheat seed germination. In conclusion, the priming effect of the hot steam treatment advanced the onset of seed metabolism, including redox shifts associated with germination and seedling growth.

Introduction

Pre-sowing hydration, priming treatments, seed coating technologies, and seed conditioning are industrial treatments that can be grouped under the umbrella of ‘seed enhancement techniques’ [1]. Seed priming activates seed germination-related metabolism, thus accelerating and homogenising seedling emergence and establishment in the field [2,3]. Orthodox seeds (i.e. those that tolerate desiccation), produced by the majority of crops, have hardly any or no measurable metabolism at the end of maturation drying (depending on seed water contents [WCs]), but can still receive environmental cues [4,5]. Dry orthodox seeds rapidly imbibe water, accompanied by leakage of ions and solutes, before structural and oxidative damage accumulated in the desiccated state is repaired, and membrane integrity is re-established, enabling the reactivation of respiration and metabolism [6]. Co-ordinated changes in metabolism characterise germination sensu stricto, which is completed with radicle protrusion through the seed coat [7]. As the radicle protrudes, seedling growth ensues via remobilisation of storage nutrient reserves, supporting the transition from a heterotrophic seedling to a photoautotrophic plant [8,9]. The recent advances in metabolomics are leading to a better understanding of the metabolic pathways contributing to germination [1013]. However, a comprehensive overview is still lacking, and it is not known how priming influences seed metabolism.

Seed redox metabolism is closely associated with viability [14]. In the desiccated state, enzyme activity is severely restricted, and low-molecular-weight (LMW) antioxidants contribute to maintain the cellular redox state from becoming over-oxidised. During maturation drying, the synthesis of ascorbic acid (AsA) is down-regulated, and dry seeds are devoid of AsA [15,16]. Therefore, the seed cellular redox environment is governed by glutathione (γ-l-glutamyl-l-cysteinyl-glycine, GSH), the major water-soluble antioxidant and redox buffer in orthodox seeds [6,14,17]. A conversion of thiols to disulfides occurs in response to desiccation, whereby glutathione disulfide (GSSG) and S-glutathionylated proteins accumulate in the dry state, protecting protein thiol groups from irreversible oxidation [18,19]. Additionally, lipophilic antioxidants, including tocochromanols (the ‘vitamin E family’, comprising tocopherols and tocotrienols) and carotenoids, can protect membranes from oxidative damage [20,21]. During imbibition, proteins are de-glutathionylated, GSSG is converted to GSH, and later GSH and AsA are synthesised de novo, leading to the re-establishment of a reducing cellular redox environment [15,2225]. Furthermore, upon hydration, enzymes that process reactive oxygen species (ROS) are reactivated and contribute to maintain ROS at cellular concentrations at which they can act as signalling molecules, without causing oxidative stress [6,26].

Previously, we showed that tissue-specific alterations in the content of LMW thiols, and their corresponding disulfides, accompany wheat seed germination [25]. In seedlings, potentially due to the breakdown of gluten proteins in the wheat endosperm, total cysteine concentrations [i.e. cysteine (Cys) and cystine (CySS)] exceed those of total glutathione (i.e. GSH + GSSG), which is unusual because high cysteine levels can be cytotoxic [25,2729]. However, Cys is predominant in the endosperm and not in the seedling shoot or roots, but nonetheless concentrations of Cys and its half-cell reduction potential (ECySS/2Cys) are proposed to be markers of the early growth of wheat seedlings [25]. The absolute concentration of a thiol, as well as the ratio between thiol concentration and respective disulfide concentration, affect the half-cell reduction potential (Ehc), in other words the redox state, of that redox couple [30]. At the point of completion of germination, the wheat embryo possesses a much more reducing thiol-disulfide redox state than the endosperm, due to the overriding contribution of glutathione concentration and glutathione Ehc (EGSSG/2GSH) to the total LMW thiol-disulfide redox environment (Ethiol-disulfide) [25]. In proliferating cells, GSH is recruited into the nucleus as part of mitosis [31], contributing to redox regulation of the plant cell cycle from the G1 to the S phase [32,33]. Primed seeds have an advanced cell cycle, as embryo cells are synchronised at the G2 phase and ready to start mitosis [34]. This synchronisation is the basis of faster and more uniform germination of primed seeds [35]. There is general consensus that more reducing cellular conditions support proliferation in both plant and animal cells, and, in the latter, evidence of a key role for Cys is emerging [36,37]. In plants, an indication that Cys contributes to redox regulation is scarce, although previous studies showed that Cys alleviates DNA alkylation and affects seed cell cycle [3841].

Here, metabolism and redox changes associated with wheat germination were investigated, taking avantage of an industrial hot steam treatment, termed ‘BioFlash’, which resulted in a seed priming effect. The effect of this treatment on the onset of metabolism prior to radicle protrusion (i.e. completion of germination) was assessed by gas chromatography coupled to mass spectrometry (GC–MS)-based metabolite profiling. Consideration was further given to the activities of selected redox enzymes and to redox shifts in EGSSG/2GSH, ECySS/2Cys, and Ethiol-disulfide.

Materials and methods

Seed material and treatment

All experiments were conducted using bread wheat seeds (Triticum aestivum L.) of the cultivar Rebelde (Apsovsementi S.p.A., Co. Na. Se. S.r.l., Italy) produced in a field trial by the Council for Agricultural Research and Economics (CREA) in Sant'Angelo Lodigiano (Italy) in the 2013–2014 growing season. Approximately 2 kg of seeds were harvested at maturity (growth stage 90 [42]), and 1 kg was treated with ‘BioFlash’ by Hoopman equipment & engineering (Aalten, The Netherlands). Briefly, seeds were exposed to a flow of hot steam, which gradually increased internal seed temperature up to a maximum of 65 ± 0.3°C, followed by rapid cooling to room temperature. The protocol cycle was repeated twice, for an overall duration of 280 s. After the treatment, seeds were re-dried at ambient conditions then stored at 4°C in a sealed box over silica gel and used for analyses within 1 year. Seed germination was periodically tested to confirm that the advanced germination achieved by the hot steam treatment was retained. Seeds treated with ‘BioFlash’ are hereafter termed ‘hot steam-treated’ and non-treated seeds are termed ‘control’.

Seed germination, WC, conductivity, and microscopy

For all experiments, only visually intact seeds were selected, whereas damaged seeds and residual glumes were discarded. Seeds were sown in 90 mm Petri dishes (n = 7, each containing 35 seeds) between two layers of filter paper (Whatman 1, GE Healthcare, Little Chalfont, U.K.) moistened with 3 ml of distilled water and germinated at 20°C without light. A seed was considered germinated when the radicle protruded by at least 1 mm through the coleorhiza. WCs were calculated from fresh and dry weights (FWs and DWs, respectively), recorded with an XS105 analytical balance (Mettler-Toledo GmbH, Columbus, OH, U.S.A.) before and after 5 d of freeze-drying, according to the formula: WC = (FW–DW)/FW × 100. Electrical conductivity (EC) of the water used to imbibe seeds (leachate) was measured with a Cond 330i conductivity-meter (WTW Xylem Analytics Germany Sales GmbH & Co. KG, Weilheim, Germany). Control and hot steam-treated dry seeds (n = 4 replicates of 10 seeds each) were rinsed with ultrapure H2O (UPW) to remove surface-bound particles, before gently stirring in 6 ml of temperature-equilibrated UPW. The ECs of the leachates were measured after 10 min. Data were normalised to DW, after drying seeds at 103°C for 17 h. Finally, desiccated control and hot steam-treated seeds (n = 10 each) were examined for the presence of structural damage, using a VHX-S90F digital microscope for three-dimensional imaging (Keyence Corporation, Osaka, Japan).

Seedling phenotyping and analysis of photosynthetic pigments

After 48 h of imbibition, lids were removed from Petri dishes to acquire source images with a flat-bed scanner (HP Scanjet G2710, Hewlett-Packard Company, Palo Alto, CA, U.S.A.), at a resolution of 300 dpi. Images were processed using the semi-automated image-analysis toolbox SmartRoot [43], connected to ImageJ v.1.50, to compute phenotypic traits on a single-seedling basis (n = 4 replicates of 35 seedlings each); non-germinated seedlings were disregarded. Phenotyping included measuring the lengths of primary roots and the first foliage leaves, mostly sheathed by the coleoptile (for simplicity termed ‘shoot’ hereafter), and total root length, defined as the sum of the lengths of primary root and all seminal radicles.

For analysing photosynthetic pigments, the shoots were excised with a scalpel from seedlings imbibed for 72 h. Excised shoots were ground in the presence of 150 mg of analytical-grade sea sand, 1.3 mg of CaCO3 (Merck KGA, Darmstadt, Germany), and 0.5 ml of ice-cold 100% (v/v) acetone. The extract was re-suspended in 1 ml of ice-cold acetone and transferred to light-proof Eppendorf tubes, immediately before centrifugation (27 000 g, 3 min) at 4°C. One millilitre aliquots of supernatants were pipetted into Rotilabo®-single-use cells PMMA semi-micro cuvettes (Carl Roth GmbH+Co, Karlsruhe, Germany), and absorbance at 750, 663, 645, and 470 nm was measured with a Lambda 20 spectrophotometer (PerkinElmer, Inc., Walthman, MA, U.S.A.). Pure acetone was used as a blank, and values at 750 nm were subtracted from values at 663, 645, and 470 nm, before averaging the three technical replicates measured for each extract (n = 3 biological replicates of 10 seedlings each). The concentrations of total chlorophyll and carotenoids were calculated according to extinction coefficients reported by Lichtenthaler [44].

Seed preparation and metabolite profiling

Dry seeds and seeds imbibed for eight time points (n = 4 replicates of 35 seeds each) were immediately frozen in liquid nitrogen and lyophilised for 5 d, before grinding to a fine powder in 5-ml liquid-nitrogen-cooled Teflon capsules (Sartorius AG, Göttingen, Germany) with one 7-mm diameter agate bead, using a Mikro-Dismembrator S (B. Braun, Biotech International, Melsungen, Germany) at 3000 min−1 for 4 min. Ground samples were stored at −80°C in a hermetically sealed plastic box over silica gel until analysis. Unless otherwise specified, all chemicals were of analytical grade and purchased from Sigma–Aldrich.

GC–MS-based metabolite profiling analysis was conducted according to the method described by Fiehn et al. [45,46], with minor adaptations. Metabolites were extracted in water:acetonitrile:isopropanol (2:3:3, v:v:v), supplemented with 21.26 µM 13C6-sorbitol (Campro Scientific GmbH, Berlin, Germany) and 25.00 µM 13C6,15N-valine as internal standards, and degassed with a stream of nitrogen for 5 min. During all extraction steps, samples were kept at 4°C. For quality control of contaminations in reagents and equipment, a blank was processed for each run of samples. Metabolites were extracted from 15.11 ± 0.25 mg of freeze-dried and finely ground powder, using glass beads (Carl Roth GmbH+Co, Karlsruhe, Germany) to improve extraction efficiency. After pipetting 1 ml of pre-cooled (−20°C) extraction solvent, samples were homogenised (25 Hz, 45 s) with a Tissue-Lyser (Qiagen, Hilden, Germany) and further extracted at 4°C and 1400 min−1 for 10 min, using a Compact Digital Micro plate shaker (Thermo Fisher Scientific, Waltham, MA, U.S.A.). To complete the extraction, insoluble material was pelleted by centrifugation (20 000 g, 5 min) at 4°C, and 25-µl aliquots of the supernatants were collected and dried for 3 h 15 min in a vacuum centrifuge (SpeedVac SPD111, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Thereafter, metabolites were chemically derivatised in two steps, during which all samples were exposed for the same time (15 or 40 s, for the first and second derivatisation step, respectively) to a stream of nitrogen, to avoid contaminations from condensed liquids and oxygen. Metabolites were first derivatised with 10 µl of a 20 mg ml−1 methoxyamine hydrochloride in pyridine solution at 28°C for 90 min, and then with 90 µl of N-methyl-N-trimethylsilyl-trifluoroacetamide (MTSTFA) at 37°C for 30 min. During both derivatisation steps, samples were incubated at 600 min−1 in a thermomixer (DITABIS AG, Pforzheim, Germany) and equilibrated at room temperature for at least 2 h, before 1 µl of the resulting samples was injected in splitless mode at 250°C into a Trace 1300 gas chromatograph coupled to a TSQ8000 triple quadrupole mass spectrometer (Thermo-Scientific, Waltham, MA, U.S.A.), equipped with a 30-m Rxi-5SilMS column including a 10-m integra-guard pre-column (Restek Corporation, Bellefonte, PA, U.S.A.). Metabolites were separated on the column using an oven temperature ramp starting at 70°C for 7 min, followed by an increase in 10°C min−1 up to 325°C, which was held for consecutive 4 min. Helium was used as carrier gas at a constant flow rate of 1 ml min−1, with equilibration of the system for 1 min at 70°C before each injection. Between consecutive injections, the 10-µl syringe was washed three times with hexane and three times with ethyl acetate. A mix of alkanes dissolved at 2 mg l−1 in hexane was injected in the middle of the queue to allow for the conversion of retention times into Kováts' alkane-based retention indices [47]. Mass spectra were acquired in full scan mode from m/z 50 to 600 at 5 spectra s−1, and raw data files were analysed with the ‘Automated Mass-spectral Deconvolution and Identification System’ (AMDIS) v2.71 software [48], with a minimal signal over noise ratio set at 80. Deconvoluted mass spectra and associated retention indexes were then compared against a custom-built mass spectral library and the National Institute of Standards and Technology (NIST, Gaithersburg, MD, U.S.A.), Golm, and Fiehn databases [49,50], using AMDIS and the NIST MS Search v2.0 program. Finally, peak areas for compound-specific trace ions were determined using the Xcalibur software v2.2 (Thermo-Scientific, Waltham, MA, U.S.A.), allowing for a relative quantification of identified and unknown metabolites from control and hot steam-treated seeds. Relative values of metabolites contents were obtained by normalising single metabolite peak areas to those of the internal standards and to sample DWs.

Enzyme activities

The total activities of the ROS-processing enzyme superoxide dismutase (SOD; EC 1.15.1.1, superoxide: superoxide oxidoreductase), catalase (CAT, EC 1.11.1.6), l-ascorbate peroxidase (APX, EC 1.11.1.11), and glutathione disulfide reductase (GR, EC 1.8.1.7) were measured following optimisation of the procedures by Bailly and Kranner and Sun et al. [51,52]. All extraction steps were performed by suspending finely ground seed powder in 0.5 ml of ice-cold 50 mM Sørensen's phosphate buffer, containing 1 mM ethylenediaminetetraacetic acid (EDTA), and agitating with a vortex at full speed for 30 s, before centrifuging at 20 000 g for 5 min at 4°C. The pH of the extraction buffer and reaction time were adjusted as appropriate for the individual assays, and the final volume of the reaction mixtures was set at 0.2 ml for all assays. Supernatants were kept on ice and promptly used to determine enzymes’ activities, measured at 30°C for SOD, CAT, and GR, or at 25°C for APX. Absorbance kinetics were determined using a plate reader (Synergy-HTX multi-mode reader, BioTek® Instruments, Inc., Winooski, VT, U.S.A.) and 96-well UV plates (Corning® 3635, Sigma–Aldrich, St Louis, MO, U.S.A.). Data were acquired and analysed with the Gen5™ 2.07 software (BioTek® Instruments, Inc., Winooski, VT, U.S.A.). Reaction rates at substrate saturation were calculated by changes in absorbance between 10 and 30 min, and for each biological replicate (n = 4) data from at least three technical measurements were averaged. Activity of SOD was measured by the inhibition of nitroblue tetrazolium (NBT)-formazan formation at 560 nm (ε = 8.70 mM−1 cm−1) and pH 8.0. The reaction contained 0.5 mM xanthine dissolved in 0.1 M NaOH, 0.27 mM NBT, and 0.34 U ml−1 of xanthine oxidase (EC 1.17.3.2, Calbiochem®, Merck KGA, Darmstadt, Germany). Activity of CAT was measured following hydrogen peroxide (H2O2) consumption by the decrease in absorbance at 240 nm (ε = 25.30 mM−1 cm−1) and pH 7.0, with 15 mM H2O2. Activity of APX was followed by monitoring the decrease in absorbance of AsA at 265 nm (ε = 4.06 mM−1 cm−1) and pH 7.0, in the presence of 0.22 mM AsA and 0.18 mM H2O2. Activity of GR was assessed by following the rate of NADPH oxidation at 340 nm (ε = 3.61 mM−1 cm−1) and pH 8.0, in the presence of 1 mM GSSG and 0.1 mM NADPH. The molar extinction coefficients were adjusted with path length correction.

HPLC analysis of LMW thiol-disulfide couples

The LMW thiol-disulfide redox couples of glutathione, cysteine, γ-glutamyl-cysteine (γ-Glu-Cys), and cysteinyl-glycine (Cys-Gly) were quantified by high-performance liquid chromatography (HPLC), following slightly modified protocols of Kranner and Bailly and Kranner [52,53]. For extraction, 1 ml of ice-cold 0.1 M HCl was added to 74.6 ± 0.9 mg of seed powder and homogenised (30 Hz, 1 min) with two 3 mm glass beads using a Tissue-Lyser (Qiagen, Hilden, Germany). After centrifuging at 27 000 g for 20 min at 4°C, the supernatant was split into an aliquot of 120 µl, to quantify total LMW thiols and disulfides, and into another aliquot of 400 µl, to determine LMW disulfides only. For measuring total LMW thiols and disulfides, the pH of the supernatant was adjusted to 8.0–8.3 with 180 µl of 200 mM bicine buffer, before reduction of disulfides by adding 30 µl of 3 mM dithiothreitol (DTT, Applichem GmbH, Darmstadt, Germany) for 1 h at room temperature. Then, thiols were derivatised with 20 µl of 15 mM monobromobimane (mBBr) for 15 min at room temperature in light-proof Eppendorf tubes. The derivatisation reaction was terminated with 250 µl of 0.25% (v/v) methanesulfonic acid at 4°C. For quantifying disulfides only, the pH was adjusted again to 8.0–8.3 with 200 mM bicine buffer, thiols were blocked with 30 µl of 50 mM N-ethylmaleimide (NEM) for 15 min at room temperature, before excess NEM was removed with five toluene washings. Thereafter, disulfides were reduced with DTT, and derivatisation of thiol products was conducted as for the first supernatant aliquot. Samples were centrifuged at 27 000 g for 1 h at 4°C, before separation of mBBr-derivatised cysteine, γ-Glu-Cys, Cys-Gly, and glutathione by reversed-phase HPLC, using an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, U.S.A.) on a ChromBudget 120–5-C18 column (250 × 4.6 mm, 5.0 µm particle size, BISCHOFF GmbH, Leonberg, Germany). Thiols derivatised with mBBr were separated with a gradient of 0.25% (v/v) acetic acid at pH 3.85 and methanol and detected by fluorescence (excitation at 380 nm; emission 480 nm). The column was pre-equilibrated with 12% methanol. After injection of 10 µl of analyte, the concentration of methanol was increased to 27% in 15 min at a constant flow rate of 1 ml min−1, then to 100% at 19 min to elute mBBr adducts. After 26 min, the initial methanol concentration was reset to 12% to re-equilibrate the column up to a total run time of 35 min was reached, followed by the next injection. Chromatograms and raw data were analysed using Agilent ChemStation for LC 3D systems (Agilent Technologies GmbH, Waldbronn, Germany). Data were calculated using individual calibration curves for each LMW thiol that were linear over the range of measured concentrations. Finally, the molar concentration of each LMW thiol was extrapolated by subtracting the molar concentration of the respective LMW disulfides from the measurement of total LMW thiols and disulfides.

Estimation of the Ethiol-disulfide-based cellular redox environment

The half-cell reduction potential of each LMW thiol-disulfide couple (Ei) was calculated according to the Nernst equation (eqn 1), using the WC (expressed as g H2O g−1 DW) to obtain the molar concentrations, and considering deviations in temperature and pH, compared with standard conditions.

 
formula
1

where R is the gas constant (8.314 J K−1 mol−1); T, temperature in K; n, number of transferred electrons (2 GSH → GSSG + 2 H+ + 2 e); F, Faraday constant (9.649 × 104 C mol−1); E0′, standard half-cell reduction potential of a thiol-disulfide redox couple at an assumed cellular pH of 7.3 (E0′GSSG/2GSH = −258 mV, E0′CySS/2Cys = −244 mV, E0′Cys-bis-Gly/2Cys-Gly = −244 mV, E0′bis-γ-Glu-Cys/2γ-Glu-Cys = −252 mV) [14,54]. The individual Ehc values were then mathematically combined into the LMW thiol-disulfide redox environment (Ethiol-disulfide), as previously described [54]:

 
formula
2

where Ei is the half-cell reduction potential of an individual thiol-disulfide redox couple i, and [reduced species]i is the concentration of the thiols in that redox pair.

Statistical analyses

Data were assessed for significance (α = 0.05) by one-way ANOVA (factor: hot-steam treatment) and two-way ANOVA analyses (main factors: hot-steam treatment and time after the onset of imbibition), using the SPSS Statistics 21 software package (IBM, New York, NY, U.S.A.) and the open source software R 3.2.1 (R Core Team, 2015) for the GC–MS-based metabolite profiling dataset. The ANOVA analyses were combined with Tukey's HSD (Honest Significant Difference) tests for post-hoc comparisons of means. The assumption of normal distribution was verified via the Shapiro–Wilk test and analysis of QQ-plots. The assumption of homoscedasticity of variances across groups was checked through Levene's test and analysis of the residuals plotted against fitted values. Whenever the latter assumption was not fulfilled, Box–Cox transformations (e.g. log10, square root, reciprocal) were applied to the data before analysis to achieve fullfilment. Total germination (TG, %) and WC (% FW) values were arcsine-transformed to simulate normal distribution. In each dataset, the cut-off value for the Cook's distance was set at 4/n (where n was the number of observations for a specific dataset), and all values with a Cook's distance greater than 4/n were considered as outliers and disregarded. Whenever the ANOVA assumptions were not fulfilled, bias-corrected accelerated bootstrap analyses were run with a sample size of 105 and two different seeds (i.e. 2000 and 200), using the Marsenne Twister random number generator. The outputs were checked with regard to the sensitivity at the decimal digits of generated 95% confidence intervals to the seeds. The association between TG and WC was assessed with Spearman's rank-order correlation in control and hot steam-treated samples, independently. For analysing the outputs of GC–MS metabolite profiling, two-way ANOVA analyses based on permutations were run with the R package ‘lmPerm’. When the interaction term was not significant (P > 0.05), an additive 2 × 2 ANOVA model was used to distinguish metabolites that were significantly different, due to either imbibition or hot-steam treatment (Supplementary Table S1D). The metabolite profiling dataset was used to conduct a Principal Component Analysis (PCA).

Results

Effects of the hot steam treatment on the physiology of wheat seeds and seedlings

Three-dimensional microscopy was used to analyse if hot-steam treatment affected the integrity of the seed surface. However, we could not detect any clear morphological differences between dry hot steam-treated and control seeds (Supplementary Figure S1). Nonetheless, between 4 and 32 h after the onset of imbibition, hot steam-treated seeds had significantly higher WCs than control seeds (Figure 1A), and imbibition led to a greater release of electrolytes by hot steam-treated seeds (inset Figure 1A). The first radicles protruded between 8 and 12 h. The time required to reach 50% of TG was significantly shorter in hot-steam-treated seeds compared with control seeds (Table 1). Between 16 and 24 h, the TG of hot steam-treated seeds was significantly higher than that of the control by ∼20%. Within 48 h, all viable hot steam-treated and control seeds had germinated, reaching 96% of TG (Figure 1B). However, when correlating WC and TG, Spearman's rank-order correlation coefficients were similar for hot steam-treated and control seeds (Table 1). Altogether, this showed that treating seeds with hot steam induced faster water uptake up to 8 h, accelerating germination (Figure 1A,B and Table 1). To confirm that seedling growth was also more advanced in hot steam-treated seeds, seedlings were phenotyped. Seedlings from hot steam-treated seeds had longer primary roots and shoots 48 h after the onset of imbibition (Figure 1C and Table 1). Likewise, the total root length was longer, and the ratio between total root and shoot lengths was higher in seedlings from hot steam-treated seeds, compared with the control (Figure 1C and Table 1). Finally, 72 h after the onset of imbibition, the contents of total chlorophylls and total carotenoids of the shoots were greater in hot steam-treated seedlings (inset Figure 1B), further indicating that seedling growth was advanced by the hot steam treatment.

Effects of hot steam treatment on germination and early seedling growth.

Figure 1.
Effects of hot steam treatment on germination and early seedling growth.

Seeds were exposed to a hot steam treatment or non-treated (control). (A) Data are means ± SEM (n = 4 replicates of 35 seeds) for WC (water content) of hot steam-treated seeds (open circles) and control seeds (closed circles), respectively. Time 0 h refers to dry seeds; error bars within symbols are not shown. Boxplots in the inset show the EC (electrical conductivity) values measured in hot steam-treated (white) and control (black) dry seeds (n = 4 replicates of 10 seeds). Whiskers indicate minima and maxima. (B) Data are means ± SEM (n = 7 replicates of 35 seeds) of total germination of hot steam-treated (open circles) and control seeds (closed circles), at 20°C in the dark. The total concentrations of photosynthetic pigments extracted from seedling shoots (white and black for hot-steam treatment and control, respectively), 72 h after the onset of imbibition, are shown as boxplots in the inset (n = 3 replicates of 10 seedlings). Asterisks denote significant differences (P < 0.05) between control and treated seeds at each time point, either using one-way ANOVA analyses followed by post-hoc Tukey's HSD test in main panels, or t-test for independent samples in insets. (C) Seedling phenotypes 48 h after the onset of imbibition. Control and hot steam-treated seedlings are presented on the left and right half of the circle, respectively.

Figure 1.
Effects of hot steam treatment on germination and early seedling growth.

Seeds were exposed to a hot steam treatment or non-treated (control). (A) Data are means ± SEM (n = 4 replicates of 35 seeds) for WC (water content) of hot steam-treated seeds (open circles) and control seeds (closed circles), respectively. Time 0 h refers to dry seeds; error bars within symbols are not shown. Boxplots in the inset show the EC (electrical conductivity) values measured in hot steam-treated (white) and control (black) dry seeds (n = 4 replicates of 10 seeds). Whiskers indicate minima and maxima. (B) Data are means ± SEM (n = 7 replicates of 35 seeds) of total germination of hot steam-treated (open circles) and control seeds (closed circles), at 20°C in the dark. The total concentrations of photosynthetic pigments extracted from seedling shoots (white and black for hot-steam treatment and control, respectively), 72 h after the onset of imbibition, are shown as boxplots in the inset (n = 3 replicates of 10 seedlings). Asterisks denote significant differences (P < 0.05) between control and treated seeds at each time point, either using one-way ANOVA analyses followed by post-hoc Tukey's HSD test in main panels, or t-test for independent samples in insets. (C) Seedling phenotypes 48 h after the onset of imbibition. Control and hot steam-treated seedlings are presented on the left and right half of the circle, respectively.

Table 1.
Effects of hot steam treatment on seed germination and phenotypic traits of seedlings from treated and control seeds 48 h after the onset of seed imbibition

Seeds were either exposed to hot steam treatment or left untreated (control).

Sample Correlation WC & TG t50 (h) Primary root (cm) Shoot (cm) Total root (cm) Root : shoot (length) 
rs P mean P mean P mean P mean P mean P 
Control 0.86 <0.001 22.4 ± 0.5 <0.001 0.86 ± 0.04 0.006 0.31 ± 0.01 <0.001 1.57 ± 0.10 0.002 4.48 ± 0.19 0.036 
HS-treated 0.87 <0.001 18.5 ± 0.6 1.03 ± 0.04 0.37 ± 0.01 2.02 ± 0.11 5.08 ± 0.22 
Sample Correlation WC & TG t50 (h) Primary root (cm) Shoot (cm) Total root (cm) Root : shoot (length) 
rs P mean P mean P mean P mean P mean P 
Control 0.86 <0.001 22.4 ± 0.5 <0.001 0.86 ± 0.04 0.006 0.31 ± 0.01 <0.001 1.57 ± 0.10 0.002 4.48 ± 0.19 0.036 
HS-treated 0.87 <0.001 18.5 ± 0.6 1.03 ± 0.04 0.37 ± 0.01 2.02 ± 0.11 5.08 ± 0.22 

rs, Spearman's rank-order correlation coefficients; t50, time to reach 50% of total germination; TG, total germination; WC, water content. The term ‘shoot’ refers to the first foliage leaves, partially sheathed by the coleoptile. Asterisks denote significant differences (t-tests for independent samples, P < 0.05; n = 4 replicates of 35 seeds).

Hot steam treatment accelerated germination-related changes in metabolites

The GC–MS-based metabolite profiling approach used led to the identification of 82 LMW metabolites, including 23 amino acids, 12 carbohydrates (seven monosaccharides, four disaccharides, and one oligosaccharide), 16 organic acids, seven sugar alcohols, six free fatty acids, phosphate and two phosphorylated compounds, five nucleic acid components, and five other apolar compounds (tocochromanols and phytosterols) (Supplementary Table S1A). A PCA score plot, based on all detected compounds, provided an overview of the metabolic changes promoted by water uptake and hot steam treatment, revealing a clear separation of dry seeds from seeds imbibed for 8 h, along principal component 1 (PC1), accounting for 36.5% of the explained variance (Figure 2). No clear clustering of the samples could be observed along PC2 and PC3, accounting respectively for 17.2 and 11.5% of the data variance. Distinctions between hot steam-treated and control seeds were not obvious in dry seeds but, 8 h after the onset of imbibition, became more pronounced along PC1 (Figure 2). With the exception of the elevated abundance of gluconate and sorbitol, the hot steam treatment did not significantly affect the contents of any metabolite prior to imbibition (Supplementary Figures S4A and S5A and Table S1D). During germination, levels of cysteine, glutamine, histidine, serine, galactose, ribose, xylose, and 2-oxoglutarate increased over 2-fold in both hot steam-treated and control seeds, whereas levels of arabitol and mannitol decreased more than 2-fold, and trehalose decreased 28-fold (Figure 3 and Supplementary Figures S2–S6). Significantly more cysteine, serine, histidine, threonine, leucine, ribose, glucose, glycerate, succinate, phosphate, and guanosine accumulated in hot steam-treated seeds, compared with control seeds, 8 h after the onset of imbibition (Figure 3 and Supplementary Figures S2–S6). Two-way ANOVA analyses revealed a significant interaction between imbibition and hot steam treatment for cysteine, serine, histidine, threonine, leucine, ribose, gluconate, and phosphate (Figure 3 and Supplementary Table S1D). Furthermore, levels of fructose, maltose, mannose, arabinose, 2-oxoglutarate, malate, α- and γ-tocopherols, and γ-tocotrienol also increased 8 h after the onset of imbibition, but less than 2-fold, and the hot steam treatment only had a limited effect (Figure 3, Supplementary Table S1D and Figures S2–S5).

PCA (Principal component analysis) of 82 metabolites identified by gas chromatography coupled to mass spectrometry (GC–MS) in hot steam-treated and control seeds, in the dry state and during germination.

Figure 2.
PCA (Principal component analysis) of 82 metabolites identified by gas chromatography coupled to mass spectrometry (GC–MS) in hot steam-treated and control seeds, in the dry state and during germination.

Squares and circles show seeds in the dry state and 8 h after the onset of imbibition, respectively. Open symbols show hot steam-treated seeds, and closed symbols show non-treated control seeds. Each data point represents a single replicate including 35 seeds (n = 4). Loading vectors are shown only for compounds with a log2 ratio of the fold-change >1, which mostly contributed to the clustering of hot steam-treated seeds compared with the control, in the dry state (left side, orange vectors) and 8 h after the onset of imbibition (right side, blue vectors). PC, principal component indicating the percentage of explained variance.

Figure 2.
PCA (Principal component analysis) of 82 metabolites identified by gas chromatography coupled to mass spectrometry (GC–MS) in hot steam-treated and control seeds, in the dry state and during germination.

Squares and circles show seeds in the dry state and 8 h after the onset of imbibition, respectively. Open symbols show hot steam-treated seeds, and closed symbols show non-treated control seeds. Each data point represents a single replicate including 35 seeds (n = 4). Loading vectors are shown only for compounds with a log2 ratio of the fold-change >1, which mostly contributed to the clustering of hot steam-treated seeds compared with the control, in the dry state (left side, orange vectors) and 8 h after the onset of imbibition (right side, blue vectors). PC, principal component indicating the percentage of explained variance.

Changes in metabolites quantified by gas chromatography coupled to mass spectrometry (GC–MS)-based metabolite profiling of dry seeds (dry) compared with seeds 8 h after the onset of imbibition (8 h).

Figure 3.
Changes in metabolites quantified by gas chromatography coupled to mass spectrometry (GC–MS)-based metabolite profiling of dry seeds (dry) compared with seeds 8 h after the onset of imbibition (8 h).

Seeds treated with hot steam are referred to as ‘HST’, and non-treated seeds used as control are abbreviated ‘Ctr’. Only metabolites that showed significant changes during germination are indicated. Detected metabolites (rows) are shown in groups, and differential abundance is indicated by the colour scale on the bottom. Numbers on the scale indicate relative values of metabolite contents determined by normalising the peak areas of each metabolite to that of the internal standards and to the sample dry weight. Maximum average values were set to 1 for each metabolite. Arrows indicate the trends of metabolites that increased or decreased in abundance during germination. Asterisks on the left side indicate a significant interaction (P< 0.05) between ‘time after the onset of imbibition’ and ‘treatment’ in the 2 × 2 ANOVA analyses of identified metabolites.

Figure 3.
Changes in metabolites quantified by gas chromatography coupled to mass spectrometry (GC–MS)-based metabolite profiling of dry seeds (dry) compared with seeds 8 h after the onset of imbibition (8 h).

Seeds treated with hot steam are referred to as ‘HST’, and non-treated seeds used as control are abbreviated ‘Ctr’. Only metabolites that showed significant changes during germination are indicated. Detected metabolites (rows) are shown in groups, and differential abundance is indicated by the colour scale on the bottom. Numbers on the scale indicate relative values of metabolite contents determined by normalising the peak areas of each metabolite to that of the internal standards and to the sample dry weight. Maximum average values were set to 1 for each metabolite. Arrows indicate the trends of metabolites that increased or decreased in abundance during germination. Asterisks on the left side indicate a significant interaction (P< 0.05) between ‘time after the onset of imbibition’ and ‘treatment’ in the 2 × 2 ANOVA analyses of identified metabolites.

Germination-induced changes in the activities of ROS-processing enzymes were affected by hot steam treatment

Prior to seed imbibition, the hot steam treatment only affected SOD activity, which was significantly higher in control seeds and, after 4 h of imbibition, declined to the level of hot steam-treated seeds (Figure 4A). Maximal SOD activities were found between 24 and 32 h after the onset of imbibition in hot steam-treated samples, and only between 32 and 48 h in the control. Activities of CAT rose steadily throughout imbibition and, in both hot steam-treated and control seeds after 48 h, were 3.9-fold higher than in dry seeds (Figure 4B). In contrast, APX activity significantly increased only 32 h after imbibition had been initiated (Figure 4C) and, after 48 h, was 1.2-fold higher in hot steam-treated seeds compared with control (Figure 4C). In both hot steam-treated and control seeds, GR activities doubled within 8 h after the onset of imbibition. After 20 h, GR activities reached maximal values and were 1.2-fold higher in hot steam-treated seeds (Figure 4D), which at that time interval also showed 25% higher TG than control seeds (Figure 1B). A significant 1.8-fold decrease in GR activity occurred between 20 and 24 h after the onset of imbibition, before activity increased again during seedling growth, whereby 48 h after the onset imbibition hot steam-treated seedlings had significantly higher GR activity than control seedlings (Figure 4D).

Impact of hot steam treatment on the activities of ROS-processing enzymes, during germination and early seedling growth.

Figure 4.
Impact of hot steam treatment on the activities of ROS-processing enzymes, during germination and early seedling growth.

Data are means ± SEM (n = 4 replicates of 35 seeds) of hot steam-treated seeds (open circles) and control (closed circles) seeds. (AD) The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione disulfide reductase (GR). Data were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test (P < 0.05), but only significant differences at the same time intervals are shown (asterisks).

Figure 4.
Impact of hot steam treatment on the activities of ROS-processing enzymes, during germination and early seedling growth.

Data are means ± SEM (n = 4 replicates of 35 seeds) of hot steam-treated seeds (open circles) and control (closed circles) seeds. (AD) The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione disulfide reductase (GR). Data were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test (P < 0.05), but only significant differences at the same time intervals are shown (asterisks).

Hot-steam treatment advanced changes in the thiol-disulfide cellular redox environment

Among the metabolite groups identified, amino acids showed the most pronounced responses to the hot steam treatment, whereby 8 h after the onset of imbibition serine, histidine, threonine, leucine, and the redox-active cysteine accumulated more in hot steam-treated seeds than control (Figure 3, Supplementary Table S1A–D and Figure S2). The cysteine residue is responsible for nucleophilicity of the tripeptide antioxidant GSH. However, GSH is too labile for reliable detection by the untargeted GC–MS-based metabolite profiling analysis adopted and was therefore measured by a targeted HPLC approach. Previously, we reported that total glutathione levels (i.e. GSH + GSSG), on a DW basis, hardly changed up to 48 h after the onset of imbibition of wheat seeds, but levels of GSH and GSSG fluctuated [25]. The influence that these fluctuations had on the EGSSG/2GSH can be seen in Figure 5A, together with the influence of hot-steam treatment. Between 4 and 8 h after the onset of imbibition (before any completion of germination), EGSSG/2GSH first shifted towards more negative values, indicative of more reducing conditions. Between 8 and 32 h, when TG ranged between 0 and 88%, EGSSG/2GSH progressively increased towards more oxidising conditions (i.e. less negative values), which sharply shifted towards more reducing conditions by the time all viable seeds had germinated (Figures 1B and 5A). The overall pattern was that EGSSG/2GSH redox shifts were more advanced in hot steam-treated seeds.

Effects of hot steam treatment on the cellular LMW thiol-disulfide redox environment (Ethiol-disulfide), during germination and early seedling growth.

Figure 5.
Effects of hot steam treatment on the cellular LMW thiol-disulfide redox environment (Ethiol-disulfide), during germination and early seedling growth.

Data are means ± SEM (n = 4 replicates of 35 seeds) of hot steam-treated seeds (open circles) and non-treated control (closed circles) seeds. (A) Redox shifts in the half-cell reduction potentials of glutathione (EGSSG/2GSH), (B) in the half-cell reduction potentials of cysteine (ECySS/2Cys), and (C) in the mathematically combined Ethiol-disulfide, upon imbibition. Data were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test (P < 0.05), but only significant differences at the same time intervals are shown (asterisks).

Figure 5.
Effects of hot steam treatment on the cellular LMW thiol-disulfide redox environment (Ethiol-disulfide), during germination and early seedling growth.

Data are means ± SEM (n = 4 replicates of 35 seeds) of hot steam-treated seeds (open circles) and non-treated control (closed circles) seeds. (A) Redox shifts in the half-cell reduction potentials of glutathione (EGSSG/2GSH), (B) in the half-cell reduction potentials of cysteine (ECySS/2Cys), and (C) in the mathematically combined Ethiol-disulfide, upon imbibition. Data were tested for significance using one-way ANOVA analyses followed by post-hoc Tukey's HSD test (P < 0.05), but only significant differences at the same time intervals are shown (asterisks).

In control seeds, ECySS/2Cys did not change until 24 h, but then sharply decreased towards more reducing conditions by ∼60 mV until 48 h, as EGSSG/2GSH did after 32 h (Figure 5B). In hot steam-treated seeds, ECySS/2Cys changed as in control seeds, although values were significantly more reducing after 8, 20, and 24 h (Figure 5B).

The Ehcs of the four most abundant LMW thiol-disulfide redox couples, including (bis)-γ-glutamyl-cyst(e)ine and cyst(e)inyl-(bis)-glycine (together accounting for ∼8% of total LMW thiols, Supplementary Table S2), were mathematically combined into the Ethiol-disulfide. In dry seeds, Ethiol-disulfide values were around −0.62 mV M and did not significantly differ between hot-steam-treated and control seeds. However, 8 h after the onset of imbibition, a significantly larger oxidative shift occurred in hot steam-treated seeds (Figure 5C), which took up more water, diluting cellular thiols, and disulfides [NB: the WC was used to calculate the molar concentrations of LMW thiol-disulfide couples, and, in the Nernst equation, the Ehc of each LMW thiol-disulfide couple depends on both the thiol-disulfide ratio and the thiol molar concentration, as the concentration of a LMW thiol is a squared term (eqn 1)]. Between 32 and 48 h, when TG reached a maximum, Ethiol-disulfide values started shifting back towards more reducing conditions, as a consequence of LMW disulfides being reduced to thiols. Notably, 48 h after the onset of imbibition, the Ethiol-disulfide values were significantly more negative by 1.6-fold in the more developed seedlings from hot steam-treated seeds (Figure 5C). In dry seeds, the most influential Ehc to Ethiol-disulfide was EGSSG/2GSH (Supplementary Table S2), with values around −192 mV in both hot steam-treated and control seeds. However, the contribution of ECySS/2Cys to Ethiol-disulfide increased during germination and early seedling growth. After 8 and 48 h, the contribution of ECySS/2Cys to Ethiol-disulfide exceeded that of EGSSG/2GSH in hot steam-treated samples (Supplementary Table S2).

Discussion

Hot-steam treatment had a seed priming effect, advancing germination and seedling growth

Hot steam treatment has been used at least since the 1990s to surface sterilise seeds [5557]. Here, it was shown that hot steam-treated wheat seeds also germinated faster and more uniformly than control seeds (Figure 1B and Table 1). The seedlings from hot steam-treated seeds showed advanced growth with longer primary roots and shoots, and higher ratios between total root lengths and shoot lengths, suggesting that radicles were growing at a faster rate (Figure 1C and Table 1). In addition, the shoots of 3-day-old seedlings from hot steam-treated seeds had higher contents of photosynthetic pigments, in agreement with advanced seedling development (inset Figure 1B). Therefore, the hot steam treatment, besides surface sterilisation, also entailed a seed priming effect.

Seed imbibition is typically accompanied by solute leakage (e.g. amino acids, sugars, organic acids, and inorganic salts), increasing the EC of the water in which seeds are imbibed [58]. Within the first 10 min of imbibition, electrolyte leakage was higher in hot steam-treated seeds (inset in Figure 1A). Interestingly, although the hot steam treatment only lasted ∼4 min, it enabled treated seeds to take up water faster (Figure 1A), a trait also found in response to other priming treatments [3,59].

Hot steam treatment advanced seed metabolism during germination

The few recent studies available on metabolite profiling of wheat seeds describe metabolite changes in response to different genetic backgrounds, environmental factors, and dormancy [6063]. A comparative metabolomic analysis, using GC–MS, recently reported distinct metabolic changes in the wheat embryo and endosperm during seed germination [64]. Here, we provide evidence for an advancement of seed metabolism by hot steam treatment, allowing us to pinpoint metabolic changes occurring during wheat seed germination.

Accelerated accumulation of amino acids supported faster seed germination and seedling growth

Germination is accompanied by the remobilisation of seed storage proteins and de novo synthesis of proteins, requiring an increased turnover of amino acids, which are also involved in various metabolic pathways, including energy production [12]. Previous studies on wheat showed that proteins in embryos are remobilised earlier and independently of the subsequent remobilisation of storage proteins in the endosperm that support seedling growth. Furthermore, not all amino acids released from early proteolysis of storage reserves in the embryo are used to synthesise new proteins, but are maintained in the cytoplasm, where they could enter energetic pathways [12,65,66]. During the first 8 h of germination, 12 of the identified amino acids accumulated significantly. Of these Cys, serine, histidine, threonine, and leucine accumulated more in response to hot steam treatment (Figure 3 and Supplementary Figure S2). Alongside its role as proteinogenic amino acid, Cys is the end product of sulfur assimilation and an important building block for many molecules involved in redox metabolism, including LMW thiols (e.g. GSH) and iron-sulfur clusters. Furthermore, Cys is required for synthesis of enzyme cofactors (biotin, vitamin B7, and thiamine, vitamin B1), secondary metabolites for plant defence (e.g. some phytoalexins), and the proteinogenic amino acid methionine, which contributes to the synthesis of the phytohormone ethylene [67,68]. In wheat, we recently showed that Cys rapidly accumulates during seed germination and early seedling growth [25]. The content of serine, a precursor of Cys [67,69], rose within 8 h after the onset of imbibition in response to the hot steam treatment (Figure 3 and Supplementary Figure S2A). This increase provides the possibility that enhanced serine biosynthesis supported the rapid increase in Cys, potentially complementing Cys accumulating from storage protein remobilisation and GSH metabolism [25]. Furthermore, germinating hot steam-treated seeds also contained more histidine (Figure 3 and Supplementary Figure S2C), an amino acid that is essential for embryo development in Arabidopsis [70] and is implicated in recycling nitrogen from glutamine, whose level increased in hot steam-treated and control seeds alike (Figure 3 and Supplementary Figure S2F). The hot steam treatment also advanced the accumulation of threonine, an aspartate derivative, and methionine (Figure 3 and Supplementary Figure S2D,J). These amino acids are substrates for the synthesis of isoleucine, which, with leucine and valine, forms the interconnected pathways to synthesise branched-chain amino acids [71,72]. These pathways were apparently advanced in hot steam-treated seeds, in which branched-chain amino acids accumulated more than in control seeds during germination (Figure 3 and Supplementary Figure S2E,I,K). Summarising, during 8 h of germination, an accumulation of all detected amino acids occurred, which was accelerated in hot steam-treated seeds.

Hot steam-treated seeds showed larger ribose pool and advanced starch breakdown during germination

Carbohydrates are the main organic compounds in wheat seeds, where they represent up to 85% of the global pool of stored reserves, mainly as starch [73]. During germination, most of the LMW carbohydrates detected accumulated, probably due to starch remobilisation in the endosperm to support embryo growth (Figure 3). Starch breakdown is key to cereal germination and seedling growth, whereby α-amylases and α-glucosidases synergistically catabolise starch granules to maltose and glucose in the amylolytic pathway [74,75]. Fructose can be a further intermediate of starch breakdown, generated by invertases [76]. Levels of glucose, fructose, and maltose increased within 8 h of germination. Since glucose rose significantly more in hot steam-treated than in control seeds, hot steam treatment probably advanced the amylolytic pathway (Figure 3 and Supplementary Figure S3B,G,I). The degradation of trehalose by trehalase could also account for glucose accumulation [61]. In contrast with all other detected carbohydrates, the non-reducing disaccharide trehalose sharply dropped during germination (Figure 3 and Supplementary Figure S3D). Trehalose participates in cell division and cell wall biosynthesis during Arabidopsis embryo development and maturation [7779]. In dry seeds, trehalose is a particularly abundant osmoprotectant that essentially contributes to seed desiccation tolerance, presumably by replacing water, stabilising membrane lipids, and preventing unfolding of enzymes and proteins [61,80,81]. During germination, ribose, a component of ATP and RNAs, also accumulated more in hot steam-treated than in control seeds (Figure 3 and Supplementary Figure S3A), suggesting its utilisation for the transcription of new RNAs, especially rRNAs. This is essential for assembling polysomes and commencing protein synthesis during germination [65,8284]. Both cell division in the embryo and protein remobilisation in the endosperm requires continuous remodelling of cell walls, through a turnover of arabinoxylans [85]. Xylose and arabinose, in the form of arabinoxylans, are constituents of the hemicellulose fraction of cell walls in seed embryo and endosperm [86] and, during germination, accumulated in both hot steam-treated and control seeds (Figure 3 and Supplementary Figure S3E,F). In summary, further to advancing the accumulation of amino acids, the hot steam treatment also advanced carbohydrate metabolism upon germination.

Mitochondrial metabolites accumulated during seed germination

Mitochondria, formed during seed maturation, enlarge and differentiate their inner membranes in response to seed imbibition, and aerobic respiration is reactivated [87]. Three detected tricarboxylic acid (TCA) cycle intermediates significantly increased upon imbibition. However, only levels of succinate were higher in hot steam-treated seeds than control seeds (Figure 3 and Supplementary Figure S4C,D,F), providing no clear evidence that resumption of TCA cycle and aerobic metabolism were advanced by hot steam treatment.

Threonate can originate from ascorbate degradation in dry orthodox seeds, which down-regulate ascorbate synthesis and mainly rely on GSH as water-soluble antioxidant [14,17,88]. Therefore, during germination, the significant drop of threonate contents could be reminiscent of the down-regulated ascorbate synthesis (Supplementary Figure S4E). In agreement with this assumption, de novo synthesis of ascorbate is detected only 10 h after the onset of imbibition in durum wheat (Triticum durum Desf.) [15], the tetraploid relative of bread wheat.

Sugar alcohols decreased and tocochromanols increased during seed germination

Seed germination was accompanied by a clear decrease in sugar alcohols (Figure 3 and Supplementary Figure S5). Exposing wheat seeds to osmotic stress during imbibition induces an increase in endogenous sugar alcohols, which are commonly used molecules for seed osmopriming [59,89]. Reserve polyols are also known to promote fungal spore germination, whereas their metabolism in seed germination remains species dependent [90,91]. Nonetheless, the production of sugar alcohols is typical of seed maturation drying and often accompanies the acquisition of desiccation tolerance, with a putative role in protecting protein and enzyme thiol groups from oxidative modification [19,80]. Interestingly, compared with the control, re-dried hot steam-treated seeds contained more sorbitol (Figure 3 and Supplementary Figure S5A). Furthermore, levels of arabitol, mannitol, ribitol, and xylitol decreased during germination (Figure 3 and Supplementary Figure S5B–E). Contrarily, the cyclic polyol quinate (Figure 3 and Supplementary Figure S5F), an intermediate at a branch point of the shikimate pathway [92], accumulated in both hot steam-treated and control seeds, suggesting the activation of this pathway during germination. Finally, tocochromanols, lipid-soluble antioxidants of the ‘vitamin E family’, increased during germination, as found in other species [9397], indicating that tocochromanols are an important part of the metabolic reshuffle that accompanies seed germination (Figure 3 and Supplementary Figure S6C–E).

SOD, CAT, and APX activities increased upon seed imbibition, and the increase in APX activity was advanced by the hot steam treatment

Seed priming typically leads to an increase in the activities of ROS-processing enzymes [98]. A distinct ROS burst also usually accompanies seed imbibition [99], potentially associated with the resumption of aerobic respiration and electron leakage from the mitochondrial electron transport chain [100]. The activities of SOD and CAT rose upon germination and early seedling growth (Figure 4A,B) and were likely related to radicle protrusion and elongation, as previously observed in maize and wheat embryos [101,102]. The activities of GR and APX, which support embryo growth in conjunction with other peroxidases to modify cell walls [99,102,103], also rose during germination in both hot steam-treated and control seeds (Figure 4C,D). Notably, de novo ascorbate synthesis is initiated during germination and early seedling growth, in parallel to increasing APX activity [15], which was advanced by the hot steam treatment.

Fluctuations in EGSSG/2GSH and GR activity accompanying germination and early seedling growth were advanced by the hot steam treatment

Increased GR activity paralleled a shift of EGSSG/2GSH values that became more negative (i.e. more reducing conditions) within 8 h after the onset of imbibition (Figures 4D and 5A). Reducing cellular conditions are required for division of root apical cells, as required to complete germination, and are generally essential for plant growth [5,37,104106]. We showed that after 15 h of imbibition, when only a certain proportion of wheat seeds had germinated, embryos of non-germinated seeds were characterised by more negative EGSSG/2GSH values (i.e. more reducing conditions) than those of seeds that had completed germination [25]. A large abundance of GSH is critical to establish the highly reducing EGSSG/2GSH values in the nuclei necessary to progress the cell cycle into the G1/S transition [33,107]. In contrast, less negative EGSSG/2GSH values are required for tissue differentiation [30,108,109] and were found to be indicative of progress towards the seed to seedling transition. Between 12 and 24 h after the onset of imbibition, the relationships between EGSSG/2GSH values and GR activities were less clear, and the proportion between germinated and non-germinated seeds varied considerably. However, between 32 and 48 h, when most seeds had developed into seedlings, a clear trend between high GR activities and more reducing conditions, indicated by more negative EGSSG/2GSH values, became evident again (Figures 4D and 5A). In summary, less negative EGSSG/2GSH values (i.e. more oxidising conditions) accompanied the completion of germination, and more negative EGSSG/2GSH values accompanied seedling growth. This is in agreement with previous results on tissue and cell cultures, where shifts in EGSSG/2GSH towards more reducing conditions were associated with cell proliferation and shifts towards more oxidising conditions with tissue differentiation [25,32,109,110]. In hot steam-treated seeds, the pattern of changes in EGSSG/2GSH was advanced. Therefore, we suggest that redox changes in EGSSG/2GSH are associated with the transition from seed to seedling.

The ECySS/2Cys as independent redox node in wheat germination and early seedling growth

The physiological relevance of redox shifts in ECySS/2Cys values has been almost neglected in plants. To date, only one study indicated that the Cys/CySS redox couple may have a regulatory function independent of the GSH/GSSG redox couple [67]. However, in animal cells, the Cys/CySS redox couple is regarded as one of the core nodes of thiol-dependent redox signalling. Other nodes are GSH/GSSG and thioredoxin redox couples which, together with Cys/CySS, are maintained at independent Ehcs in different cellular compartments [36,111]. As ECySS/2Cys values are typically less negative than EGSSG/2GSH, the Cys/CySS couple in mammalian plasma is envisaged to reflect redox events occurring in the more oxidising environment of the extracellular space [111115]. In animal cells, shifts in ECySS/2Cys values may affect cell function, leading to cell proliferation, differentiation, adhesion, and apoptosis [115]. With few exceptions, and independently from EGSSG/2GSH, more reducing ECySS/2Cys values enhance cell proliferation, whereas more oxidising ECySS/2Cys values induce cell differentiation [36,115]. In plants, oxidative shifts in ECySS/2Cys occur during seed ageing, after-ripening, and exposure to salt stress [54,109,116], whereas reducing shifts are suggested to accompany the remobilisation of storage proteins in wheat endosperm [25]. In this study, ECySS/2Cys was always less negative than EGSSG/2GSH, in both control and hot steam-treated seeds, up to 32 h after the onset of imbibition (Figure 5A,B). After 48 h, when 96% of seed had germinated and Cys concentrations sharply increased, ECySS/2Cys values shifted below those of EGSSG/2GSH (compare Figures 1A and 5B). Therefore, we suggest that ECySS/2Cys represents a thiol-based node in redox circuitry, which is regulated independently of EGSSG/2GSH in wheat seed germination and early seedling growth. The ECySS/2Cys node could involve additional circuits for redox signalling, probably mediated by S-cysteinylation, to tune cellular proliferation and differentiation at distinct stages of the seed to seedling transition. Similarly to barley seeds, where Cys affects the cell cycle [41], reducing shifts in ECySS/2Cys values may be indicative of cell cycle progression in wheat. Such shifts were significantly larger in hot steam-treated seeds before the completion of germination and at the time intervals when their TG was significantly greater (Figures 1A and 5B). Hence, ECySS/2Cys shifts towards more reducing conditions were associated with the completion of germination.

Redox shifts in the thiol-disulfide redox environment related to advanced germination and early seedling growth

It is commonly recognised that cellular redox balance cannot be described by a single redox couple [30]. Consequently, we combined the LMW thiol-disulfide redox couples into the Ethiol-disulfide. Shifts in Ethiol-disulfide towards more oxidising conditions were correlated with seed ageing and dormancy, and reducing shifts with increased germination and viability [54,116,117]. After 8 h of water uptake, more oxidising cellular conditions were associated with differential contents of metabolites, indicative of the advanced metabolism of hot steam-treated seeds (Figure 5C). This is also the stage when wheat embryos synthesise new DNA; hence, the embryonic cells of hot steam-treated seeds were possibly more synchronised at the G2 phase, as typical of priming treatments [35,83]. Furthermore, more oxidising conditions are key for progressing the cell cycle from the G1 and the G2 checkpoints, which are responsive to redox switches [37]. In faster germinating hot steam-treated seeds, Ethiol-disulfide values showed an advanced shift towards more reducing conditions, which widened after 48 h when ECySS/2Cys became the main contributor to the Ethiol-disulfide (Supplementary Table S2).

In conclusion, we showed that the hot-steam treatment advanced seed water uptake, impacting on metabolism and resulting in faster germination and seedling growth. Furthermore, LMW thiol-disulfide redox switches were shown to be associated with germination and the transition from seed to seedling. In particular, besides EGSSG/2SGH, we propose ECySS/2Cys as key node in redox signalling during wheat seed germination and seedling growth.

Abbreviations

     
  • AMDIS

    automated mass-spectral deconvolution and identification system

  •  
  • ANOVA

    analysis of variance

  •  
  • APX

    ascorbate peroxidase

  •  
  • AsA

    ascorbic acid

  •  
  • CAT

    catalase

  •  
  • Cys

    cysteine

  •  
  • Cys-Gly

    cysteinyl-glycine

  •  
  • CySS

    cystine

  •  
  • DTT

    dithiothreitol

  •  
  • DW

    dry weight

  •  
  • Ebis−γ-Glu-Cys/2γ-Glu-Cys

    half-cell reduction potential of the γ-glutamyl-cysteine/bis-γ-glutamyl-cystine redox couple

  •  
  • EC

    electrical conductivity

  •  
  • ECys-bis-Gly/2Cys-Gly

    half-cell reduction potential of the cysteinyl-glycine/cystinyl-bis-glycine redox couple

  •  
  • ECySS/2Cys

    half-cell reduction potential of the cysteine/cystine redox couple

  •  
  • EDTA

    ethylenediaminetetraacetic acid

  •  
  • EGSSG/2GSH

    half-cell reduction potential of the glutathione/glutathione disulfide redox couple

  •  
  • Ehc

    half-cell reduction potential

  •  
  • Ei

    half-cell reduction potential of an individual thiol-disulfide redox couple

  •  
  • Ethiol-disulfide

    LMW thiol-disulfide redox environment

  •  
  • FW

    fresh weight

  •  
  • GC–MS

    gas chromatography coupled to mass spectrometry

  •  
  • GR

    glutathione disulfide reductase

  •  
  • GSH

    glutathione

  •  
  • GSSG

    glutathione disulfide

  •  
  • H2O2

    hydrogen peroxide

  •  
  • HPLC

    high-performance liquid chromatography

  •  
  • LMW

    low-molecular-weight

  •  
  • mBBr

    monobromobimane

  •  
  • MTSTFA

    N-methyl-N-trimethylsilyl-trifluoroacetamide

  •  
  • NBT

    nitroblue tetrazolium

  •  
  • NEM

    N-ethylmaleimide

  •  
  • PC

    principal component

  •  
  • PCA

    principal component analysis

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase

  •  
  • t50

    time to reach 50% of total germination

  •  
  • TCA

    tricarboxylic acid

  •  
  • TG

    total germination

  •  
  • UPW

    ultrapure water

  •  
  • WC

    water content

  •  
  • γ-Glu-Cys

    γ-glutamyl-cysteine

Author Contribution

D.G., T.R., and P.V. conceived the study. All co-authors contributed to the experimental design. D.G., T.R., and M.L. conducted the experiments. D.G. and E.A. analysed the data. D.G., T.R., and I.K. wrote the manuscript, which was revised and approved by all remaining co-authors.

Funding

The Italian Lombardy Region granted D.G. and M.L. with scholarships in the framework of the Project ID 46547514 ‘Advanced Priming Technologies for the Lombardy Agro-Seed Industry — PRIMTECH’ (Action 2). D.G. received financial support by the University of Innsbruck's PhD programme ‘Doktoratsstipendium NEU aus der Nachwuchsförderung 2015 der Universität Innsbruck, 2. Tranche’ and by the Seventh Framework Programme of the European Union ‘EcoSeed’ (Impacts of Environmental Conditions on Seed Quality), grant 311840.

Acknowledgments

We are grateful to Werner Kofler (Department of Botany, University of Innsbruck, Austria) for conducting three-dimensional microscopy of seeds and capturing images. We thank Jan Willem Hoopman and Jan Eenink from Hoopman equipment & engineering (Aalten, The Netherlands) for conducting the hot steam seed treatment.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

References

References
1
Taylor
,
A.G.
,
Allen
,
P.S.
,
Bennett
,
M.A.
,
Bradford
,
K.J.
,
Burris
,
J.S.
and
Misra
,
M.K.
(
1998
)
Seed enhancements
.
Seed Sci. Res.
8
,
245
256
2
Bradford
,
K.J.
(
1995
)
Seed Development and Germination
,
Marcel Dekker
,
New York
3
Jisha
,
K.C.
,
Vijayakumari
,
K.
and
Puthur
,
J.T.
(
2013
)
Seed priming for abiotic stress tolerance: an overview
.
Acta Physiol. Plant.
35
,
1381
1396
4
Footitt
,
S.
,
Douterelo-Soler
,
I.
,
Clay
,
H.
and
Finch-Savage
,
W.E.
(
2011
)
Dormancy cycling in Arabidopsis seeds is controlled by seasonally distinct hormone-signaling pathways
.
Proc. Natl Acad. Sci. U.S.A.
108
,
20236
20241
5
Long
,
R.L.
,
Kranner
,
I.
,
Panetta
,
F.D.
,
Birtić
,
S.
,
Adkins
,
S.W.
and
Steadman
,
K.J.
(
2011
)
Wet-dry cycling extends seed persistence by re-instating antioxidant capacity
.
Plant Soil
338
,
511
519
6
Kranner
,
I.
,
Minibayeva
,
F.V.
,
Beckett
,
R.P.
and
Seal
,
C.E.
(
2010
)
What is stress? Concepts, definitions and applications in seed science
.
New Phytol.
188
,
655
673
7
Bewley
,
J.D.
,
Bradford
,
K.J.
,
Hilhorst
,
H.W.M.
and
Nonogaki
,
H.
(
2013
)
Seeds: Physiology of Development, Germination and Dormancy
,
Springer
,
New York
8
Bewley
,
J.D.
(
1997
)
Seed germination and dormancy
.
Plant Cell
9
,
1055
1066
9
Bassel
,
G.W.
(
2016
)
To grow or not to grow?
Trends Plant Sci.
21
,
498
505
10
Fait
,
A.
,
Angelovici
,
R.
,
Less
,
H.
,
Ohad
,
I.
,
Urbanczyk-Wochniak
,
E.
,
Fernie
,
A.R.
et al. 
(
2006
)
Arabidopsis seed development and germination is associated with temporally distinct metabolic switches
.
Plant Physiol.
142
,
839
854
11
Shu
,
X.-L.
,
Frank
,
T.
,
Shu
,
Q.-Y.
and
Engel
,
K.R.
(
2008
)
Metabolite profiling of germinating rice seeds
.
J. Agric. Food Chem.
56
,
11612
11620
12
Rosental
,
L.
,
Nonogaki
,
H.
and
Fait
,
A.
(
2014
)
Activation and regulation of primary metabolism during seed germination
.
Seed Sci. Res.
24
,
1
15
13
Kazmi
,
R.H.
,
Willems
,
L.A.J.
,
Joosen
,
R.V.L.
,
Khan
,
N.
,
Ligterink
,
W.
and
Hilhorst
,
H.W.M.
(
2017
)
Metabolomic analysis of tomato seed germination
.
Metabolomics
13
,
145
14
Kranner
,
I.
,
Birtić
,
S.
,
Anderson
,
K.M.
and
Pritchard
,
H.W.
(
2006
)
Glutathione half-cell reduction potential: a universal stress marker and modulator of programmed cell death?
Free Radical. Biol. Med.
40
,
2155
2165
15
De Gara
,
L.
,
de Pinto
,
M.C.
and
Arrigoni
,
O.
(
1997
)
Ascorbate synthesis and ascorbate peroxidase activity during the early stage of wheat germination
.
Physiol. Plant.
100
,
894
900
16
De Gara
,
L.
,
de Pinto
,
M.C.
,
Moliterni
,
V.M.C.
and
D'Egidio
,
M.G.
(
2003
)
Redox regulation and storage processes during maturation in kernels of Triticum durum
.
J. Exp. Bot.
54
,
249
258
17
Foyer
,
C.H.
and
Noctor
,
G.
(
2011
)
Ascorbate and glutathione: the heart of the redox hub
.
Plant Physiol.
155
,
2
18
18
Kranner
,
I.
and
Grill
,
D.
(
1996
)
Significance of thiol-disulfide exchange in resting stages of plant development
.
Bot. Acta
109
,
8
14
19
Colville
,
L.
and
Kranner
,
I.
(
2010
)
Desiccation tolerant plants as model systems to study redox regulation of protein thiols
.
Plant Growth Regul.
62
,
241
255
20
Menè-Saffrané
,
L.
,
Jones
,
A.D.
and
DellaPenna
,
D.
(
2010
)
Plastochromanol-8 and tocopherols are essential lipid-soluble antioxidants during seed desiccation and quiescence in Arabidopsis
.
Proc. Natl Acad. Sci. U.S.A.
107
,
17815
17820
21
Fernández-Marín
,
B.
,
Miguez
,
F.
,
Méndez-Fernández
,
L.
,
Agut
,
A.
,
Becerril
,
J.M.
,
Garcia-Plazaola
,
J.I.
et al. 
(
2017
)
Seed carotenoid and tocochromanol composition of wild Fabaceae species is shaped by phylogeny and ecological factors
.
Front. Plant Sci.
8
,
1428
22
Fahey
,
R.C.
,
Di Stefano
,
D.L.
,
Meier
,
G.P.
and
Bryan
,
R.N.
(
1980
)
Role of hydration state and thiol-disulfide status in the control of thermal stability and protein synthesis in wheat embryo
.
Plant Physiol.
65
,
1062
1066
23
Kranner
,
I.
and
Grill
,
D.
(
1993
)
Content of low-molecular-weight thiols during the imbibition of pea seeds
.
Physiol. Plant.
88
,
557
562
24
Tommasi
,
F.
,
Paciolla
,
C.
,
de Pinto
,
M.C.
and
De Gara
,
L.
(
2001
)
A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds
.
J. Exp. Bot.
52
,
1647
1654
25
Gerna
,
D.
,
Roach
,
T.
,
Stöggl
,
W.
,
Wagner
,
J.
,
Vaccino
,
P.
,
Limonta
,
M.
et al. 
(
2017
)
Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth
.
Free Radic. Res.
51
,
568
581
26
Bailly
,
C.
,
El-Maarouf-Bouteau
,
H.
and
Corbineau
,
F.
(
2008
)
From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology
.
C. R. Biol.
331
,
806
814
27
Anderson
,
M.E.
and
Meister
,
A.
(
1987
)
Intracellular delivery of cysteine
.
Method Enzymol.
143
,
313
325
28
Osman
,
L.P.
,
Mitchell
,
S.C.
and
Waring
,
R.H.
(
1997
)
Cysteine, its metabolism and toxicity
.
Sulfur Rep.
20
,
155
172
29
Ohtsu
,
I.
,
Wiriyathanawudhiwong
,
N.
,
Morigasaki
,
S.
,
Nakatani
,
T.
,
Kadokura
,
H.
and
Takagi
,
H.
(
2010
)
The L-cysteine/L-cystine shuttle system provides reducing equivalents to the periplasm in Escherichia coli
.
J. Biol. Chem.
285
,
17479
17487
30
Schafer
,
F.Q.
and
Buettner
,
G.R.
(
2001
)
Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple
.
Free Radic. Biol. Med.
30
,
1191
1212
31
Markovic
,
J.
,
Borrás
,
C.
,
Ortega
,
Á.
,
Sastre
,
J.
,
Viña
,
J.
and
Pallardó
,
F.V.
(
2007
)
Glutathione is recruited into the nucleus in early phases of cell proliferation
.
J. Biol. Chem.
282
,
20416
20424
32
Potters
,
G.
,
De Gara
,
L.
,
Asard
,
H.
and
Horemans
,
N.
(
2002
)
Ascorbate and glutathione: guardians of the cell cycle, partners in crime?
Plant Physiol. Biochem.
40
,
537
548
33
Diaz-Vivancos
,
P.
,
Wolff
,
T.
,
Markovic
,
J.
,
Pallardó
,
F.V.
and
Foyer
,
C.H.
(
2010
)
A nuclear glutathione cycle within the cell cycle
.
Biochem. J.
431
,
169
178
34
Black
,
M.
,
Bewley
,
J.D.
and
Halmer
,
P.
(
2006
)
The Encyclopedia of Seeds: Science, Technology and Uses
,
CABI
,
Wallingford
35
Varier
,
A.
,
Vari
,
A.K.
and
Dadlani
,
M.
(
2010
)
The subcellular basis of seed priming
.
Curr. Sci. India
99
,
450
456
36
Go
,
Y.-M.
and
Jones
,
D.P.
(
2008
)
Redox compartmentalization in eukaryotic cells
.
Biochim. Biophys. Acta, Gen. Subj.
1780
,
1273
1290
37
Diaz-Vivancos
,
P.
,
de Simone
,
A.
,
Kiddle
,
G.
and
Foyer
,
C.H.
(
2015
)
Glutathione - linking cell proliferation to oxidative stress
.
Free Radic. Biol. Med.
89
,
1154
1164
38
Mikaelsen
,
K.
and
Pedersen
,
K.
(
1968
)
Protective effectiveness of cysteine, cysteamine and cystamine against X-ray induced chromosome aberrations at different stages of mitosis
.
Hereditas
60
,
399
406
39
Kaul
,
B.L.
(
1969
)
Modification of the radiomimetic effects of nitrosoguanidine by cysteine in plants
.
Experientia
25
,
74
75
40
Kaul
,
B.L.
and
Choudhary
,
D.K.
(
1972
)
Cystein protection against ethylenimine damage in barley seeds
.
Radiat. Bot.
12
,
57
61
41
Rubluo
,
A.
(
1982
)
The effect of L-cysteine on pre-soaked barley seeds treated with methyl methanesulfonate
.
Experientia
38
,
326
327
42
Zadoks
,
J.C.
,
Chang
,
T.T.
and
Konzak
,
C.F.
(
1974
)
A decimal code for the growth stages of cereals
.
Weed Res.
14
,
415
421
43
Lobet
,
G.
,
Pagès
,
L.
and
Draye
,
X.
(
2011
)
A novel image-analysis toolbox enabling quantitative analysis of root system architecture
.
Plant Physiol.
157
,
29
39
44
Lichtenthaler
,
H.K.
(
1987
)
Chlorophylls and carotenoids: pigments of photosynthetic biomembranes
.
Method Enzymol.
148
,
350
382
45
Fiehn
,
O.
,
Kristal
,
B.
,
Van Ommen
,
B.
,
Sumner
,
L.W.
,
Sansone
,
S.-A.
,
Taylor
,
C.
et al. 
(
2006
)
Establishing reporting standards for metabolomic and metabonomic studies: a call for participation
.
OMICS
10
,
158
163
46
Fiehn
,
O.
,
Wohlgemuth
,
G.
,
Scholz
,
M.
,
Kind
,
T.
,
Lee
,
D.Y.
,
Lu
,
Y.
et al. 
(
2008
)
Quality control for plant metabolomics: reporting MSI-compliant studies
.
Plant J.
53
,
691
704
47
Kováts
,
E.
(
1958
)
Gas-chromatographische Charakterisierung organischer Verbindungen .1. Retentionsindices aliphatischer Halogenide, Alkohole, Aldehyde und Ketone
.
Helv. Chim. Acta
41
,
1915
1932
48
Stein
,
S.E.
(
1999
)
An integrated method for spectrum extraction and compound identification from gas chromatography/mass spectrometry data
.
J. Am. Soc. Mass Spectr.
10
,
770
781
49
Kopka
,
J.
,
Schauer
,
N.
,
Krueger
,
S.
,
Birkemeyer
,
C.
,
Usadel
,
B.
,
Bergmuller
,
E.
et al. 
(
2005
)
GMD@CSB.DB: the Golm Metabolome Database
.
Bioinformatics
21
,
1635
1638
50
Kind
,
T.
,
Wohlgemuth
,
G.
,
Lee
,
D.Y.
,
Lu
,
Y.
,
Palazoglu
,
M.
,
Shahbaz
,
S.
et al. 
(
2009
)
Fiehnlib: mass spectral and retention index libraries for metabolomics based on quadrupole and time-of-flight gas chromatography/mass spectrometry
.
Anal. Chem.
81
,
10038
10048
51
Sun
,
Y.
,
Oberley
,
L.W.
and
Li
,
Y.
(
1988
)
A simple method for clinical assay of superoxide dismutase
.
Clin Chem.
34
,
497
500
PMID:
[PubMed]
52
Bailly
,
C.
and
Kranner
,
I.
(
2011
)
Analyses of Reactive Oxygen Species and Antioxidants in Relation to Seed Longevity and Germination
,
Springer
,
Berlin
53
Kranner
,
I.
(
1998
)
Determination of Glutathione, Glutathione Disulfide and two Related Enzymes, Glutathione Reductase and Glucose-6-Phosphate Dehydrogenase, in Fungal and Plant Cells
,
Springer
,
Berlin
54
Birtić
,
S.
,
Colville
,
L.
,
Pritchard
,
H.W.
,
Pearce
,
S.R.
and
Kranner
,
I.
(
2011
)
Mathematically combined half-cell reduction potentials of low-molecular-weight thiols as markers of seed ageing
.
Free Radic. Res.
45
,
1093
1102
55
Watts
,
J.E.
,
de Villiers
,
O.T.
and
Watts
,
L.
(
1993
)
Sterilization of wheat seeds for tissue culture purposes
.
S. Afr. J. Bot.
59
,
641
642
56
Forsberg
,
G.
,
Andersson
,
S.
and
Johnsson
,
L.
(
2002
)
Evaluation of hot, humid air seed treatment in thin layers and fluidized beds for seed pathogen sanitation
.
Z. Pflanzenk. Pflanzen.
109
,
357
370
57
Studer
,
P.
,
Heller
,
W.E.
,
Hummerjohann
,
J.
and
Drissner
,
D.
(
2013
)
Evaluation of aerated steam treatment of alfalfa and mung bean seeds to eliminate high levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica, and Listeria monocytogenes
.
Appl. Environ. Microb.
79
,
4613
4619
58
Shephard
,
H.L.
and
Naylor
,
R.E.L.
(
1996
)
Effect of the seed coat on water uptake and electrolyte leakage of sorghum (Sorghum bicolor (L.) Moench) seeds
.
Ann. Appl. Biol.
129
,
125
136
59
Paparella
,
S.
,
Araújo
,
S.S.
,
Rossi
,
G.
,
Wijayasinghe
,
M.
,
Carbonera
,
D.
and
Balestrazzi
,
A.
(
2015
)
Seed priming: state of the art and new perspectives
.
Plant Cell Rep.
34
,
1281
1293
60
Matthews
,
S.B.
,
Santra
,
M.
,
Mensack
,
M.M.
,
Wolfe
,
P.
,
Byrne
,
P.F.
and
Thompson
,
H.J.
(
2012
)
Metabolite profiling of a diverse collection of wheat lines using ultraperformance liquid chromatography coupled with time-of-flight mass spectrometry
.
PLoS ONE
7
,
e44179
61
Francki
,
M.G.
,
Hayton
,
S.
,
Gummer
,
J.P.A.
,
Rawlinson
,
C.
and
Trengove
,
R.D.
(
2016
)
Metabolomic profiling and genomic analysis of wheat aneuploid lines to identify genes controlling biochemical pathways in mature grain
.
Plant Biotechnol. J.
14
,
649
660
62
Liu
,
C.
,
Ding
,
F.
,
Hao
,
F.
,
Yu
,
M.
,
Lei
,
H.
,
Wu
,
X.
et al. 
(
2016
)
Reprogramming of seed metabolism facilitates pre-harvest sprouting resistance of wheat
.
Sci. Rep.
6
,
20593
63
Das
,
A.
,
Kim
,
D.-W.
,
Khadka
,
P.
,
Rakwal
,
R.
and
Rohila
,
J.S.
(
2017
)
Unraveling key metabolomic alterations in wheat embryos derived from freshly harvested and water-imbibed seeds of two wheat cultivars with contrasting dormancy status
.
Front. Plant Sci.
8
,
1203
64
Han
,
C.
,
Zhen
,
S.
,
Zhu
,
G.
,
Bian
,
Y.
and
Yan
,
Y.
(
2017
)
Comparative metabolome analysis of wheat embryo and endosperm reveals the dynamic changes of metabolites during seed germination
.
Plant Physiol. Bioch.
115
,
320
327
65
Marcus
,
A.
(
1969
)
Seed germination and the capacity for protein synthesis
.
Symp. Soc. Exp. Biol.
23
,
143
160
PMID:
[PubMed]
66
Marsh
,
L.
,
Datta
,
K.
and
Marcus
,
A.
(
1982
)
Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool
.
Plant Physiol.
70
,
67
73
67
López Martin
,
M.C.
,
Becana
,
M.
,
Romero
,
L.C.
and
Gotor
,
C.
(
2008
)
Knocking out cytosolic cysteine synthesis compromises the antioxidant capacity of the cytosol to maintain discrete concentrations of hydrogen peroxide in Arabidopsis
.
Plant Physiol.
147
,
562
572
68
Romero
,
L.C.
,
Aroca
,
M.A.
,
Laureano-Marin
,
A.M.
,
Moreno
,
I.
,
García
,
I.
and
Gotor
,
C.
(
2014
)
Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana
.
Mol. Plant.
7
,
264
276
69
Wirtz
,
M.
and
Hell
,
R.
(
2006
)
Functional analysis of the cysteine synthase protein complex from plants: structural, biochemical and regulatory properties
.
J. Plant Physiol.
163
,
273
286
70
Ingle
,
R.A.
(
2011
)
Histidine Biosynthesis
,
The American Society of Plant Biologists
,
Rockville, U.S.A.
71
Binder
,
S.
(
2010
)
Branched-chain Amino Acid Metabolism in Arabidopsis Thaliana
,
The American Society of Plant Biologists
,
Rockville, U.S.A.
72
Joshi
,
V.
,
Joung
,
J.-G.
,
Fei
,
Z.J.
and
Jander
,
G.
(
2010
)
Interdependence of threonine, methionine and isoleucine metabolism in plants: accumulation and transcriptional regulation under abiotic stress
.
Amino Acids
39
,
933
947
73
Shewry
,
P.R.
and
Hey
,
S.J.
(
2015
)
The contribution of wheat to human diet and health
.
Food Energy Secur.
4
,
178
202
74
Goldstein
,
L.D.
and
Jennings
,
P.H.
(
1975
)
The occurrence and development of amylase enzymes in incubated, de-embryonated maize kernels
.
Plant Physiol.
55
,
893
898
75
Sun
,
Z.
and
Henson
,
C.A.
(
1990
)
Degradation of native starch granules by barley α-glucosidases
.
Plant Physiol.
94
,
320
327
76
Bogdan
,
J.
and
Zagdańska
,
B.
(
2006
)
Changes in the pool of soluble sugars induced by dehydration at the heterotrophic phase of growth of wheat seedlings
.
Plant Physiol. Bioch.
44
,
787
794
77
Eastmond
,
P.J.
,
van Dijken
,
A.J.H.
,
Spielman
,
M.
,
Kerr
,
A.
,
Tissier
,
A.F.
,
Dickinson
,
H.G.
et al. 
(
2002
)
Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation
.
Plant J.
29
,
225
235
78
Gómez
,
L.D.
,
Baud
,
S.
,
Gilday
,
A.
,
Li
,
Y.
and
Graham
,
I.A.
(
2006
)
Delayed embryo development in the ARABIDOPSIS TREHALOSE-6-PHOSPHATE SYNTHASE 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation
.
Plant J.
46
,
69
84
79
Iturriaga
,
G.
,
Suárez
,
R.
and
Nova-Franco
,
B.
(
2009
)
Trehalose metabolism: from osmoprotection to signaling
.
Int. J. Mol. Sci.
10
,
3793
3810
80
Crowe
,
J.H.
,
Carpenter
,
J.F.
and
Crowe
,
L.M.
(
1998
)
The role of vitrification in anhydrobiosis
.
Annu. Rev. Physiol.
60
,
73
103
81
Oliver
,
A.E.
,
Crowe
,
L.M.
and
Crowe
,
J.H.
(
1998
)
Methods for dehydration-tolerance: depression of the phase transition temperature in dry membranes and carbohydrate vitrification
.
Seed Sci. Res.
8
,
211
221
82
Chen
,
D.
,
Schultz
,
G.
and
Katchalski
,
E.
(
1971
)
Early ribosomal RNA transcription and appearance of cytoplasmic ribosomes during germination of the wheat embryo
.
Nat. New Biol.
231
,
69
72
83
Rejman
,
E.
and
Buchowicz
,
J.
(
1971
)
The sequence of initiation of RNA, DNA and protein synthesis in the wheat grains during germination
.
Phytochemistry
10
,
2951
2957
84
Brooker
,
J.D.
,
Tomaszewski
,
M.
and
Marcus
,
A.
(
1978
)
Preformed messenger RNAs and early wheat embryo germination
.
Plant Physiol.
61
,
145
149
85
Kuroyama
,
H.
and
Tsumuraya
,
Y.
(
2001
)
A xylosyltransferase that synthesizes β-(1 → 4)-xylans in wheat (Triticum aestivum L.) seedlings
.
Planta
213
,
231
240
86
Barron
,
C.
,
Surget
,
A.
and
Rouau
,
X.
(
2007
)
Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition
.
J. Cereal Sci.
45
,
88
96
87
Botha
,
F.C.
,
Potgieter
,
G.P.
and
Botha
,
A.M.
(
1992
)
Respiratory metabolism and gene expression during seed germination
.
Plant Growth Regul.
11
,
211
224
88
Parsons
,
H.T.
and
Fry
,
S.C.
(
2012
)
Oxidation of dehydroascorbic acid and 2,3-diketogulonate under plant apoplastic conditions
.
Phytochemistry
75
,
41
49
89
Thind
,
S.K.
(
1991
)
Effects of a long chain aliphatic alcohol mixture on growth and solute accumulation in water stressed wheat seedlings under laboratory conditions
.
Plant Growth Regul.
10
,
223
234
90
Aitken
,
W.B.
and
Niederpruem
,
D.J.
(
1972
)
Isotopic studies of carbohydrate metabolism during basidiospore germination in Schizophyllum commune. 1. Uptake of radioactive glucose and sugar alcohols
.
Arch. Mikrobiol.
82
,
173
183
91
Gupta
,
A.K.
and
Kaur
,
N.
(
2005
)
Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants
.
J. Biosci.
30
,
761
776
92
Guo
,
J.
,
Carrington
,
Y.
,
Alber
,
A.
and
Ehlting
,
J.
(
2014
)
Molecular characterization of quinate and shikimate metabolism in Populus trichocarpa
.
J. Biol. Chem.
289
,
23846
23858
93
Simontacchi
,
M.
,
Caro
,
A.
,
Fraga
,
C.G.
and
Puntarulo
,
S.
(
1993
)
Oxidative stress affects α-tocopherol content in soybean embryonic axes upon imbibition and following germination
.
Plant Physiol.
103
,
949
953
94
Carciochi
,
R.A.
,
Galván-D'Alessandro
,
L.
,
Vandendriessche
,
P.
and
Chollet
,
S.
(
2016
)
Effect of germination and fermentation process on the antioxidant compounds of quinoa seeds
.
Plant Food Hum. Nutr.
71
,
361
367
95
Wang
,
Y.
,
Li
,
M.
,
Xu
,
F.
,
Chai
,
L.
,
Bao
,
J.
and
Shen
,
S.
(
2016
)
Variation in polyphenols, tocols, γ-aminobutyric acid, and antioxidant properties in whole grain Rice (Oryza sativa L.) as affected by different germination time
.
Cereal Chem. J.
93
,
268
274
96
Jiang
,
J.S.
,
Chen
,
Z.H.
,
Ban
,
L.P.
,
Wu
,
Y.D.
,
Huang
,
J.P.
,
Chu
,
J.F.
et al. 
(
2017
)
P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid mediated seed germination
.
Sci. Rep.
7
,
40625
97
Roach
,
T.
,
Nagel
,
M.
,
Börner
,
A.
,
Eberle
,
C.
and
Kranner
,
I.
(
2018
)
Changes in tocochromanols and glutathione reveal differences in the mechanisms of seed ageing under seedbank conditions and controlled deterioration in barley
.
Environ. Exp. Bot.
156
,
8
15
98
Bailly
,
C.
,
Benamar
,
A.
,
Corbineau
,
F.
and
Côme
,
D.
(
2000
)
Antioxidant systems in sunflower (Helianthus annuus L.) seeds as affected by priming
.
Seed Sci. Res.
10
,
35
42
99
Kranner
,
I.
,
Roach
,
T.
,
Beckett
,
R.P.
,
Whitaker
,
C.
and
Minibayeva
,
F.V.
(
2010
)
Extracellular production of reactive oxygen species during seed germination and early seedling growth in Pisum sativum
.
J. Plant Physiol.
167
,
805
811
100
Wojtyla
,
L.
,
Lechowska
,
K.
,
Kubala
,
S.
and
Garnczarska
,
M.
(
2016
)
Different modes of hydrogen peroxide action during seed germination
.
Front. Plant Sci.
7
,
66
101
Leprince
,
O.
,
Deltour
,
R.
,
Thorpe
,
P.C.
,
Atherton
,
N.M.
and
Hendry
,
G.A.F.
(
1990
)
The role of free radicals and radical processing systems in loss of desiccation tolerance in germinating maize (Zea mays L)
.
New Phytol.
116
,
573
580
102
Cakmak
,
I.
,
Strbac
,
D.
and
Marschner
,
H.
(
1993
)
Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds
.
J. Exp. Bot.
44
,
127
132
103
Roach
,
T.
,
Beckett
,
R.P.
,
Minibayeva
,
F.V.
,
Colville
,
L.
,
Whitaker
,
C.
,
Chen
,
H.
et al. 
(
2010
)
Extracellular superoxide production, viability and redox poise in response to desiccation in recalcitrant Castanea sativa seeds
.
Plant Cell Environ.
33
,
59
75
104
Seal
,
C.E.
,
Zammit
,
R.
,
Scott
,
P.
,
Nyamongo
,
D.O.
,
Daws
,
M.I.
and
Kranner
,
I.
(
2010
)
Glutathione half-cell reduction potential as a seed viability marker of the potential oilseed crop Vernonia galamensis
.
Ind. Crop. Prod.
32
,
687
691
105
Rouhier
,
N.
,
Cerveau
,
D.
,
Couturier
,
J.
,
Reichheld
,
J.P.
and
Rey
,
P.
(
2015
)
Involvement of thiol-based mechanisms in plant development
.
Bba-Gen Subjects.
1850
,
1479
1496
106
Calvi
,
G.P.
,
Aud
,
F.F.
,
Ferraz
,
I.D.K.
,
Pritchard
,
H.W.
and
Kranner
,
I.
(
2017
)
Analyses of several seed viability markers in individual recalcitrant seeds of Eugenia stipitata McVaugh with totipotent germination
.
Plant Biol.
19
,
6
13
107
Schnaubelt
,
D.
,
Queval
,
G.
,
Dong
,
Y.
,
Diaz-Vivancos
,
P.
,
Makgopa
,
M.E.
,
Howell
,
G.
et al. 
(
2015
)
Low glutathione regulates gene expression and the redox potentials of the nucleus and cytosol in Arabidopsis thaliana
.
Plant Cell Environ.
38
,
266
279
108
Stasolla
,
C.
(
2010
)
Glutathione redox regulation of in vitro embryogenesis
.
Plant Physiol. Bioch.
48
,
319
327
109
Zagorchev
,
L.
,
Seal
,
C.E.
,
Kranner
,
I.
and
Odjakova
,
M.
(
2012
)
Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L
.
Free Radic. Res.
46
,
656
664
110
Belmonte
,
M.F.
,
Donald
,
G.
,
Reid
,
D.M.
,
Yeung
,
E.C.
and
Stasolla
,
C.
(
2005
)
Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca)
.
J. Exp. Bot.
56
,
2355
2364
111
Jones
,
D.P.
(
2006
)
Redefining oxidative stress
.
Antioxid. Redox Signal.
8
,
1865
1879
112
Jones
,
D.P.
,
Mody
,
V.C.
,
Carlson
,
J.L.
,
Lynn
,
M.J.
and
Sternberg
,
P.
(
2002
)
Redox analysis of human plasma allows separation of pro-oxidant events of aging from decline in antioxidant defenses
.
Free Radic. Biol. Med.
33
,
1290
1300
113
Roede
,
J.R.
,
Uppal
,
K.
,
Liang
,
Y.L.
,
Promislow
,
D.E.L.
,
Wachtman
,
L.M.
and
Jones
,
D.P.
(
2013
)
Characterization of plasma thiol redox potential in a common marmoset model of aging
.
Redox Biol.
1
,
387
393
114
Zheng
,
Y.X.
,
Ritzenthaler
,
J.D.
,
Burke
,
T.J.
,
Otero
,
J.
,
Roman
,
J.
and
Watson
,
W.H.
(
2018
)
Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression
.
Free Radic. Biol. Med.
118
,
13
22
115
Jones
,
D.P.
,
Go
,
Y.-M.
,
Anderson
,
C.L.
,
Ziegler
,
T.R.
,
Kinkade
,
J.M.
and
Kirlin
,
W.G.
(
2004
)
Cysteine/cystine couple is a newly recognized node in the circuitry for biologic redox signaling and control
.
FASEB J.
18
,
1246
1248
116
Morscher
,
F.
,
Kranner
,
I.
,
Arc
,
E.
,
Bailly
,
C.
and
Roach
,
T.
(
2015
)
Glutathione redox state, tocochromanols, fatty acids, antioxidant enzymes and protein carbonylation in sunflower seed embryos associated with after-ripening and ageing
.
Ann. Bot.
116
,
669
678
117
Nagel
,
M.
,
Kranner
,
I.
,
Neumann
,
K.
,
Rolletschek
,
H.
,
Seal
,
C.E.
,
Colville
,
L.
et al. 
(
2015
)
Genome-wide association mapping and biochemical markers reveal that seed ageing and longevity are intricately affected by genetic background and developmental and environmental conditions in barley
.
Plant Cell Environ.
38
,
1011
1022