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Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination
Plant Science 248 (2016) 37–44
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Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination Zhenguo Ma a,b,c , Frédéric Marsolais b,c , Mark A. Bernards c , Mark W. Sumarah b , Natalia V. Bykova d , Abir U. Igamberdiev a,∗ a
Department of Biology, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada Genomics and Biotechnology, London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON N5V 4T3, Canada c Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada d Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB R6 M 1Y5, Canada b
a r t i c l e
i n f o
Article history: Received 9 December 2015 Received in revised form 11 April 2016 Accepted 12 April 2016 Available online 13 April 2016 Keywords: Barley (Hordeum vulgare L.) Seed germination Glyoxylate cycle Succinate Citrate Malate Ascorbate Glutathione
a b s t r a c t During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2 O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction During seed germination, mobilization of stored lipids, carbohydrates and proteins takes place. In cereal seeds, the reserve of carbohydrates (starch) is found in the endosperm, while the reserve of lipids and proteins is found in the scutellum, which represents a single cotyledon of monocotyledonous plants. Starch and proteins are mobilized via the activation of corresponding amylases and proteases, while for starch breakdown an acid environment is important. Acidification of the endosperm is achieved via secretion of organic acids from the scutellum and aleurone layer into
Abbreviations: AlaAT, alanine aminotransferase; Asc, ascorbate; DHA, dehydroascorbate; DTNB, 5,5 -dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, reduced glutathione; GSO, putative glyoxysomal succinate oxidase; GSSG, oxidized glutathione; ICL, isocitrate lyase; MS, malate synthase; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; PVPP, polyvinylpolypyrrolidone; SDH, succinate dehydrogenase; TCA cycle, tricarboxylic acid cycle. ∗ Corresponding author. E-mail addresses: [email protected], a [email protected] (A.U. Igamberdiev). http://dx.doi.org/10.1016/j.plantsci.2016.04.007 0168-9452/© 2016 Published by Elsevier Ireland Ltd.
endosperm, i.e., the “digestive” process [1–3]. Organic acids are formed primarily during oxidative metabolic processes, such as the TCA cycle and the glyoxylate cycle. The latter becomes active during germination of oil-containing seeds, which includes the seeds of cereal plants, where the process of -oxidation of fatty acids is localized in the scutellum and aleurone layer [4]. Aleurone layers, according to the estimations of Newman and Briggs [5] contain approximately 2/3 of triacylglycerols of barley grain, with their concentration close to 15% of fresh weight of this tissue; the remaining 1/3 is localized in the scutellum in which lipid concentration can be even higher, up to 40% of fresh weight in maize scutellum [6]. During germination, the lipid content in these tissues decreases in correspondence with the development of -oxidation and the glyoxylate cycle. Meanwhile, the remobilization of storage reserves towards formation of carbohydrates and amino acids takes place [3,4,7]. Starchy barley seeds [8] are different from Arabidopsis oily seeds [9]. Therefore, even though the glyoxylate cycle plays a crucial role in taking advantage of acetyl-CoA from breakdown of lipids in oily seeds [10,11], it could have different functions in cereal seeds. The function of the glyoxylate cycle is usually attributed to the
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necessity of carbohydrate formation from lipids. This occurs via succinate synthesis from acetyl-CoA by the glyoxylate cycle in glyoxysomes, followed by its conversion to malate and oxaloacetate (OAA) by the TCA cycle in mitochondria, and synthesis of phosphoenolpyruvate (PEP) by PEP carboxykinase (PEPCK) in the cytosol. The latter becomes the substrate for reverse glycolysis (gluconeogenesis), in which carbohydrates are built. The glyoxylate cycle can be of main importance in the tissues of many plants, especially dicotyledons such as sunflower or castor bean, where carbohydrate reserves in seeds are low or absent. However, in cereal plants the endosperm represents a major reserve of carbohydrates, and therefore the glyoxylate cycle could serve other metabolic purposes than converting lipids to carbohydrate. Using 14 C-labelled acetate, which is easily converted to 14 C-acetylCoA, it was shown that in maize scutellum only a small amount of exogenous acetate is used for carbohydrate synthesis, while the main portion of it is detected in amino acids and organic acids [12]. This led to the conclusion that the function of the glyoxylate cycle in cereals consists mainly in providing substrates for amino acid synthesis and for acidification of the endosperm (reviewed in Ref. [13]). The function of the glyoxylate cycle to convert acetyl-CoA produced during oxidation of fatty acids of stored lipids to succinate is well established in the oily tissues of germinating seeds of dicotyledonous plants such as sunflower cotyledons and castor bean endosperm [14]. In the seeds of cereal plants the glyoxylate cycle operates in the scutellum, which represents a single cotyledon, or more precisely it has a homology to the third leaf of nymphealean seedlings [15]. The glyoxylate cycle supplies low molecular weight organic acids for the acidification of the endosperm to “digest” the stored starch, which is used as a carbohydrate supply by the developing embryo [3,13]. While the supply of carbohydrates is provided
by the endosperm, the role of the glyoxylate cycle in cereals may be expanded to include the supply of organic acids and amino acid synthesis. Operation of the glyoxylate cycle in glyoxysomes (which represents a special type of peroxisome) is accompanied by the buildup of hydrogen peroxide during the flavin-dependent oxidation of fatty acids and in other processes. While excess H2 O2 is normally scavenged by catalase, its generation affects the reduction levels of ascorbate and glutathione in the cell, which also participate in H2 O2 scavenging in the ascorbate-glutathione cycle [16]. Besides the flavin oxidation-dependent generation of H2 O2 , the formation of NADH and succinate in glyoxysomes, via transport mechanisms that bring reducing equivalents to the mitochondria, may result in the synthesis of ATP. In this study, we measured the activities of enzymes, redox levels and organic acid content in the scutellum of germinating barley seeds. While our previous study [17] was concentrated on the early germination events (first 48 h post imbibition) associated with breakage of dormancy, our current study deals with heterotrophic metabolism of germinated barley seeds that utilize storage reserves for germination and seedling growth. The results indicate that during glyoxylate cycle operation, organic acids can be used to acidify the endosperm, converted to carbohydrates or transaminated to produce amino acids. This means that the function of the glyoxylate cycle in cereals may be broad and not restricted to the conversion of storage fats to carbohydrates. 2. Material and methods 2.1. Plant material Barley (Hordeum vulgare L., cv. Harrington) seeds were soaked in ddH2 O and germinated in darkness at 25 ◦ C on two layers of filter paper in Petri dishes for 8 days. Endosperm and scutellum tissues were isolated every day and ground into fine powder in liquid nitrogen with a mortar and pestle. Aleurone layer was removed from the endosperm by a sharp blade. To prevent any contamination by endosperm, scutella were cleaned by wet paper towel, rinsed gently by ddH2 O, and dried on filter paper. The tissue powder was stored at −80 ◦ C and used within a week. For enzyme and metabolite measurements the powder was homogenized in the extraction buffer in the ratio of 50 mg tissue fresh weight to 1 mL buffer. 2.2. Measurement of protein concentration Protein concentration was measured using a standard Bradford protocol and a commercially available reagent (Sigma), according to the supplier’s protocol. Protein was extracted from tissue powder by 0.1 M HEPES (pH 7.0) containing 0.5% CHAPS and 0.1% SDS on ice. Bovine serum albumin was used as a standard. 2.3. Measurement of hydrogen peroxide
Fig. 1. Changes in H2 O2 content, ATP + ADP and ATP/ADP ratio in scutellum of barley seeds during germination. H2 O2 and ATP contents were measured by chemiluminescent methods; ADP was converted to ATP by pyruvate kinase as described in the text. The data are the means of three biological replicates ± SD.
The concentration of hydrogen peroxide was measured according to the method of Lu et al. [18]. The fine powder of isolated scutella was homogenized in 6% trichloroacetic acid for 30 min at 4 ◦ C, centrifuged at 15,000g for 10 min, and then insoluble polyvinylpyrrolidone (PVPP) (50 mg mL−1 ) was added. The samples were centrifuged at 15,000g for an additional 3 min. The preparation of reagents followed the method of Pérez and Rubio [19] and Lu et al. [18]. 10 mL of 6.5 mM luminol and 2 mL of 3 mM CoCl2 in 0.1 M sodium carbonate buffer (pH 10.2) were mixed, diluted to 1 L in the same buffer and stored at 4 ◦ C in the dark overnight before use. Samples, 40 L, were mixed with 10 L of the sodium carbonate buffer and the mixture was incubated at 30 ◦ C for 15 min. Catalase (EC 1.11.1.6; Sigma; 500 units) was added and incubated at the
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same condition as a control. Samples (10 L) were mixed in 5 mL SARSTEDT tubes with 200 L of reaction reagent and the chemiluminescence (CL) measure on a FB 12 Luminometer (Berthold Detection Systems GmbH, Germany). The difference of CL response between each treatment and corresponding background was considered as CL specific for H2 O2 in the samples. The amount of H2 O2 produced per gram tissue was calculated using a standard curve generated from known amounts of H2 O2 . 2.4. Measurement of ATP and ADP Extraction of ATP was conducted according to Dordas et al. [20] with modifications. The tissue powder was lysed in ice-cold 2.4 M perchloric acid (1 mL to 25 mg) for 60 min on ice and centrifuged for 5 min at 20,000g. The supernatant (0.5 mL) was neutralized with 4 M KOH, and the amount of ATP in the neutralized solution measured by chemiluminescent analysis using an ATP Detection Kit (ThermoFisher Scientific), according to supplier’s instructions. The content of ADP was determined using the EnzyLightTM ADP Assay Kit (EADP-100, BioAssay Systems), according to supplier’s instructions. Briefly, ADP was converted to ATP by pyruvate kinase (EC 2.7.1.40; Sigma) in the presence of 2 mM phosphoenolpyruvate (PEP) and 5 mM magnesium, the ATP was measured by chemiluminescence as above, and the amount of ADP was calculated by subtraction of the ATP amount before conversion.
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(Sigma). The extinction coefficient used for NADH at 340 nm was 6.22 mM−1 cm−1 . Fumarase (EC 4.2.1.2) was measured in 50 mM Tris-HCl buffer (pH 7.8) containing 50 mM l-malic acid by monitoring the formation of the double bond in fumaric acid at 240 nm using the extinction coefficient 2.44 mM−1 cm−1 [25]. 2.7. Measurement of isocitrate lyase and malate synthase Isocitrate lyase (ICL; EC 4.1.3.1) was extracted on ice with 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM MgCl2 , 1 mM EDTA and 2 mM cysteine. Samples were centrifuged at 15,000g for 10 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. ICL activity was measured in 50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl2 , 2 mM l-cysteine, 10 mM d,l-isocitrate and 4 mM phenylhydrazine hydrochloride, at 324 nm, using the extinction coefficient of glyoxylic acid phenylhydrazone (14.6 mM−1 cm−1 ) [26]. Malate synthase (MS; EC 4.1.3.2) was extracted on ice with 0.1 M HEPES, pH 7.8, containing 5 mM MgCl2 , 1 mM EDTA and 2 mM DTT. After centrifugation at 15,000g for 10 min at 4 ◦ C, and the supernatant desalted on a Sephadex G-10 column. MS activity was determined in 0.1 M HEPES, pH 7.8, containing 6 mM MgCl2 , 5 mM sodium glyoxylate, 2.5 mM acetyl-CoA and 2 mM DTNB, at 412 nm, using the extinction coefficient of 2-nitro-5-thiobenzoic acid (TNB) 13.6 mM−1 cm−1 . 2.8. Measurement of phosphoenolpyruvate carboxykinase
2.5. Measurement of ascorbate and glutathione Ascorbate and glutathione were extracted with 6% (w/v) trichloroacetic acid (1.5 mL added to 100 mg of tissue powder). The homogenate was centrifuged at 12,000g at 4 ◦ C for 20 min and the supernatant used for measurement of the reduced and oxidized ascorbate and glutathione. Ascorbate (Asc) and dehydroascorbate (DHA) were determined according to Kampfenkel et al. [21], and the absorbance was recorded at 525 nm using spectrophotometer. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined according to Zaharieva and Abadía [22]. The method is based on the reaction of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) with GSH forming 5-thionitrobenzoic acid (TNB), which can be measured spectrophotometrically at 412 nm. Oxidized glutathione (GSSG) was measured after being reduced by glutathione reductase (EC 1.8.1.7; Sigma). Calculation of reduction potential of the GSSG/2GSH couple was done using the method of Schafer and Buettner [23] using the Nernst Equation for half-cell reaction of the GSSG/2GHS couple at pH 7: Ehc (mV) = −240 − (59.1/2) log10 ([GSH]2 /[GSSG])
2.6. Measurement of succinate dehydrogenase, succinate conversion to malate, and fumarase Enzymes were extracted on ice using 50 mM Tris-HCl containing 1 mM EDTA and 2 mM dithiothreitol (DTT), pH 7.8. Samples were centrifuged at 14,000g for 10 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. Succinate dehydrogenase (SDH; EC 1.3.5.1) activity was measured in 50 mM Tris-HCl, pH 7.8, containing 5 M 2,6-dichlorophenolindophenol (DCPIP), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM KCN, and 5 mM succinate, at 600 nm [24] using the extinction coefficient 20.6 mM−1 cm−1 . Succinate conversion to malate was measured according to Igamberdiev et al. [34]. Briefly, the reaction buffer comprised 50 mM Tris-HCl, pH 7.8, 20 mM succinate, 0.6 mM NAD+ , 5 mM MgCl2 , 1 mM KCN and 5 units mL−1 NAD-malic enzyme
Determination of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) was performed according to Walker et al. [27]. The enzyme was extracted by homogenizing tissue powder with icecold 200 mM Bicine-KOH buffer, pH 9.0, containing 3 mM EDTA, 5% (w/v) polyethylene glycol (PEG) and 25 mM DTT, and centrifuged at 12,000g for 15 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. The activity of PEPCK was measured in the direction of carboxylation in 100 mM Hepes-KOH buffer, pH 6.8, containing 100 mM KCl, 0.14 mM NADH, 0.25 mM DTT, 6 mM MnCl2 , 1 mM ADP, 90 mM KHCO3 and 6 units mL−1 malate dehydrogenase (Sigma). The reaction was initiated by adding PEP (final concentration 6 mM) and the decrease in NADH was measured at 340 nm. 2.9. Measurement of alanine aminotransferase Determination of alanine aminotransferase (AlaAT; EC 2.6.1.2) activity was conducted according to Good and Muench [28]. Enzyme extraction was performed on ice using 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM DTT. The homogenate was centrifuged at 15,000g for 10 min at 4 ◦ C, and the supernatant desalted on a Sephadex G-10 column. AlaAT activity was measured in 50 mM Tris–HCl buffer, pH 7.5, containing 10 mM l-alanine, 5 mM 2-oxoglutarate, 0.1 mM NADH, and 5 units mL−1 lactate dehydrogenase (EC 1.1.1.27; Sigma) by the decrease of optical density at 340 nm due to oxidation of NADH. 2.10. Determination of pH in scutellum and endosperm Fine powder of scutellum or endosperm (10 mg) was homogenized with 1 mL of ddH2 O. Samples were centrifuged and pH was measured in supernatant using an ORION 3-Star benchtop pH meter (Thermo Scientific). 2.11. Determination of organic acids Extraction of organic acids was conducted according to the method of Jham et al. [29]. The fine powder of scutellum or
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2.12. Statistical analysis All the experiments were repeated at least three times. R was used for statistical analysis. Tukey’s test in the two-way ANOVA was used to compare the significance of difference between samples. The data in the text and figures are expressed as means ± SD of three replicates. The differences with P ≤ 0.05 were considered as statistically significant. 3. Results 3.1. Change of protein, H2 O2 and ATP concentrations in scutellum of barley seeds The content of total water-soluble proteins in scutellum tissue decreased two-fold during the first two days of germination and then stabilized at the level of about 30 mg g−1 FW (data not shown). The content of H2 O2 increased from very low values in dry seeds to more than 100 nmol g−1 FW in the first day, peaked at a maximum value of almost 250 nmol g−1 FW and then remained constant (Fig. 1). The content of ATP + ADP slightly decreased during first four days of germination and then started to increase, while the ATP/ADP ratio increased during the first four days from 2 to 5 and then decreased to values close to 3 in the next four days (Fig. 1). 3.2. Ascorbate and glutathione levels
Fig. 2. Changes in the content of reduced and oxidized species of ascorbate and glutathione, in ascorbate/dehydroascorbate ratio, and in glutathione potential in scutellum of barley seeds during germination. The potential of glutathione was calculated according to Schafer and Buettner [23]. The data are the means of three biological replicates ± SD. Abbreviations: Asc—ascorbate, DHA—dehydroascorbate.
endosperm was homogenized with 80% methanol for 30 min at 40 ◦ C and centrifuged at 15,000g for 10 min. The supernatant was used for determination of organic acids. Each sample solution (100 L) was transferred to a glass vial and dried under nitrogen using a Reacti-Vap Evaporator (Thermo Scientific). Sample derivatization was performed according to the method of Roessner et al. [30]. Dry residues of organic acids were derivatized by adding 50 L of MOX Reagent (2% methoxyamine hydrochloride in pyridine, Thermo Scientific), vortexed and incubated for 2 h at 37 ◦ C. Next, 50 L of 50% BSTFA in pyridine was added into each vial. Vials were vortexed and incubated for another 40 min at 70 ◦ C. Organic acids were analyzed by GC–MS essentially according to Roessner et al. [30]. Briefly, trimethylsilyl (TMS) derivatives of organic acids were separated in a Varian CP-3800 (USA) gas chromatograph (GC) equipped with a flame ionization detector (FID) and an ion trap mass spectrometer (Varian 220-MS, USA) (GC–MS). The GC was equipped with a pair of CP-Sil 5CB low bleed MS columns (WCOT silica 30 m × 0.25 mm ID), one in line with the FID and the other with the MS. Temperature in the injector oven and in the FID oven was set at 230 ◦ C and 300 ◦ C, respectively. Samples (1 L) were injected twice (once to each column) in splitless mode and simultaneously eluted with the following oven temperature program: the initial temperature of 70 ◦ C was held for 5 min, followed by a temperature increase (7.5 ◦ C min−1 ) to 300 ◦ C, where the final temperature was held for 2 min. High purity helium was used as a carrier gas and constant flow rate was 1 mL min−1 . Compounds were identified on the basis of their co-elution with authentic standards and their EI-mass spectra (50–650 amu). Quantification of the organic acids in samples was based on FID peak areas and independently derived calibration curves for citrate, malate and succinate.
The total pool of ascorbate (and dehydroascorbate) decreased in scutellum during the first four days of germination, then started to stabilize and slightly increased by day 8. The contents of reduced and oxidized glutathione dropped sharply during the first day of germination and then stabilized. The reduction level of ascorbate (ascorbate to dehydroascorbate ratio) decreased in the first day, then started to increase before stabilizing between days 3–6 and then increasing again (Fig. 2). The glutathione potential was lower during the first three days of germination, then increased to a higher level between days 4–6, and again decreased by day 8. 3.3. Content of organic acids in the scutellum and endosperm of barley seeds The GC–MS analysis of the main organic acids produced in the glyoxylate and tricarboxylic acid cycles (succinic, malic and citric acid) in the scutellum and endosperm of barley seeds (Fig. 3) revealed that the most abundant organic acid in scutellum is citrate, at a concentration of 6–8 mol g−1 FW during the whole study period of germination and seedling establishment (from 0 to 8 days). The concentration of succinate (0.1–0.3 mol g−1 FW) was 30–60 times lower as compared with citrate. The concentration of malate was almost as high as citrate during the first two days and then decreased by 3–4 times. By contrast, the concentration of citrate in the endosperm increased during the first four days of germination, while that of malate decreased from very high levels in the first 2–3 days to two-three orders of magnitude lower levels by days 4–5. The concentration of succinate was negligible during the whole period of germination (Fig. 3). The pH value of homogenized tissue water extracts decreased for both the scutellum and endosperm during germination. In the scutellum the decrease was moderate, from 5.8 to 5.9 at the first day, down to 5.2–5.3 by days 5–6. In endosperm, however, acidification was significant during first two days of germination, as the pH value in dry seeds dropped from 4.4 to 3.5 by the fourth day of germination, before slightly increasing (to 3.7) at the days 6–8.
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Fig. 3. Changes in the content of succinate, malate and citrate and in the value of pH in scutellum and endosperm during germination of barley seeds. Organic acids were measured by GC–MS, pH was determined in the tissue total extract. The data are the means of three biological replicates ± SD. The details are presented in the text.
3.4. Activities of glyoxylate cycle enzymes and of succinate oxidation The activity of ICL increased gradually from very low values until reaching 30 nmol min−1 mg−1 protein on day 4, where it remained stable for the next three days, and then started to decline. The activity of MS increased quickly during first two days, then stabilized at the level of 50–60 nmol min−1 mg−1 protein, and from day 4 started to decrease (Fig. 4). The peak of MS (the enzyme producing malate) activity appeared earlier than that of ICL, which produces succinate. The pattern of succinate oxidation more closely followed the activity profile of ICL, with SDH activity being several times lower than the activity of succinate oxidation measured via the detection of malate formation (Fig. 4). The intensity of succinate oxidation measured by malate formation from succinate reached 15–16 nmol min−1 mg−1 protein, while SDH activity measured by reduction of DCPIP did not exceed 5 nmol min−1 mg−1 protein. 3.5. Activities of fumarase, phosphoenolpyruvate kinase and alanine aminotransferase The activity of fumarase was approximately seven fold higher than the rate of succinate oxidation (measured by malate formation). It increased during germination, being at the highest level between days 3 and 7, before declining (Fig. 5). PEPCK and AlaAT revealed similar profiles of activity, with a gradual increase from the start of germination, reaching a maximum activity at days 4–5 and then declining. The measurable rate of transamination catalyzed by AlaAT was one order of magnitude higher than the rate of PEP synthesis in the PEPCK reaction, which was on the same level as fumarase activity (Fig. 5).
Fig. 4. Changes in activities of isocitrate lyase, malate synthase, succinate dehydrogenase and succinate oxidation to malate in scutellum of barley seeds during germination. These activities reflect operation of the glyoxylate cycle and conversion of its product succinate. The details of spectrophotometric measurements are described in the text. The data are the means of three biological replicates ± SD.
4. Discussion In the current study we used germinating barley seeds, which develop the glyoxylate cycle in scutellar tissues during the first few days of germination, to further explore the role of the glyoxylate cycle in stored starch mobilization. It has been reported that barley embryos do not contain the enzymes of the glyoxylate cycle [4] and that this cycle only operates in the aleurone layer; however, this result is not supported by the present study. The increase of key enzymes of the glyoxylate cycle, ICL and MS, was observed immediately after imbibition of seeds, with ICL peaking at the 4th day, while the highest activity of MS appeared even earlier (Fig. 4). In the study of Holtman et al. [4] ICL and MS were not detected in scutella of barley seeds, while in aleurone layers the activities of both enzymes peaked at the 4th day of germination exhibiting the activities of 80 and 110 nmol min−1 per 10 aleurone layers correspondingly. These values approximately match the activities per mg protein, considering aleurone layer as 5% of total weight of 40 mg barley seed and 50 mg protein per g of aleurone fresh weight, the values corresponding to those estimated in Briggs et al. [7]. In our study ICL peaked after 4–7 days of germination with maximum activity of 30 nmol min−1 mg−1 protein while MS in scutellum peaked on the days 2 and 3 with the activity of 60 nmol min−1 mg−1 protein. Apparently lower activities of MS and ICL as compared to those reported in aleurone layers by Holtman et al. [4] could explain their conclusion about localization of the glyoxylate cycle only in aleurone layers in germinating barley seeds, also gluconeogenesis and
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Fig. 5. Changes in activities of PEP carboxykinase, fumarase, and alanine aminotransferase in scutellum of barley seeds during germination. These enzymes catalyze reactions that are involved in determining metabolic directions of the glyoxylate cycle product succinate conversion either to carbohydrate or amino acid synthesis. The details of spectrophotometric measurements are described in the text. The data are the means of three biological replicates ± SD.
its hormonal regulation in barley are strongly cultivar-dependent [31], which can also result in discrepancies in different studies. While both the aleurone layer and the scutellum contain triacylglycerols, the functions of these two tissues are not the same. The aleurone layer is a part of the endosperm, and it excretes organic acids for starch breakdown [3]. Although Drozdowicz and Jones [3] detected much lower excreting capacity by the scutellum as compared to the aleurone, other studies [1,2] reported very significant excretion of organic acids by the scutellum and established the role of plasma membrane ATPase in this process. On the other hand, the scutellum can be a rapid and immediate source of soluble carbohydrates for developing embryos, and, therefore, the products of its metabolism are also used for growth of the radicle, coleoptile and first leaves. The uptake of carbohydrates by the embryo occurs not only via gluconeogenesis in the scutellum but also via their transport to the scutellum from the endosperm [3], and may take place in exchange of organic acids. Further studies are needed to elucidate the mechanism(s) of transport between the scutellum and endosperm. The profiles of activities of ICL and MS (Fig. 4) indicate that the formation of succinate and malate increases respectively in the scutellum with the development of activities of these enzymes in glyoxysomes, while citrate is formed in the reaction catalyzed by citrate synthase that operates both in glyoxysomes and mitochondria [32]. Malate can be also formed from OAA by the malate dehydrogenase (MDH) reaction and from fumarate by fumarase.
MDH is located in glyoxysomes, cytosol and mitochondria; therefore it is difficult to estimate the contribution of different isoforms to malate production, while the intensity of TCA cycle could be estimated from activities of succinate dehydrogenase, which is lower than isocitrate lyase at the peak of the glyoxylate cycle. Also, the maximal capacities of these enzymes measured in vitro may not fully reflect the situation taking place in vivo. The concentration of citrate was always high in the scutellum (Fig. 3), while in the endosperm it was much lower, slightly increasing to the 4th day of germination. Malate concentration was very high in the first three days in the endosperm and scutellum and then decreased. This indicates that the scutellum may export malate and citrate for the acidification of the endosperm, which was supported by measured changes in pH values in the scutellum and endosperm during germination. That is, the pH value of total water extract of the scutellum gradually decreased during germination, while in the endosperm we observe a significant acidification during first three days post imbibition before the pH level stabilized (Fig. 3). Contrary to malate and citrate, the level of succinate was low both in the scutellum and endosperm (Fig. 3) despite the fact that this acid represents the main exported product from the glyoxylate cycle. This suggests that the enzymes utilizing succinate are not limiting and succinate is efficiently metabolized further in the scutellum of germinating seeds. The common point of view is that succinate is transported from glyoxysomes to the mitochondria, where it is converted to fumarate by SDH and then to malate by fumarase. While the activity of fumarase in the scutellum is quite high, peaking at 4–5 day of germination (Fig. 5), the activity of SDH measured in our study was lower than the activity of ICL (Fig. 4). The succinate oxidation rate, however, measured as malate formation from succinate, was several times higher, which may indicate that mitochondrial SDH is not the only engine for the oxidation of succinate. For example, there was a report of a plasma membrane protein oxidizing succinate and using nitrate as electron acceptor [33]. More important could be a flavin-containing oxidase located in glyoxysomal membranes that is capable of converting succinate to malate, forming hydrogen peroxide in the reaction [34]. This oxidase has a low affinity for succinate (Km ∼ 18 mM) but taking into account the intensive formation of succinate in the glyoxylate cycle, it may be important for succinate conversion to malate without leaving glyoxysomes in germinating cereal seeds when succinate concentration reaches high levels. In dicotyledonous plants the activity of this enzymatic system was not detected [34]. The development of lipid mobilization, the glyoxylate cycle and other metabolic processes in the scutellum is accompanied by the buildup of hydrogen peroxide (Fig. 1). This can be explained by participation of flavin-containing oxidases in -oxidation, the oxidation of succinate and other oxidative processes occurring at this time. On the other hand, the formation of NADH during -oxidation of fatty acids or via the glyoxylate cycle can result in redox transfer to mitochondria, where the oxidation of reducing equivalents is coupled to the formation of ATP. The increase in the ATP/ADP ratio, starting from germination and peaking at the 4th day, indicates that the mobilization of lipids is connected with ATP synthesis. Some decrease in the total ATP + ADP level by this time may also be related to biosynthetic processes as ATP is used in nucleic acid and coenzyme synthesis. In contrast to the cotyledons of dicotyledonous plants, the scutellum exhibits some activity of the glycine decarboxylase complex [35], which may serve for the provision of outflow of glyoxylate from the glyoxylate cycle to glycine and serine synthesis. Cellular or tissue changes in redox level can be monitored via measurement of reduced and oxidized species of ascorbate and glutathione [16]. It is seen from Fig. 2 that the reduction level of ascorbate (measured as the ratio of ascorbate to dehydroascorbate) increases from day 1 to day 3 of germination. Even more
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Fig. 6. Scheme of metabolic processes in barley seeds during germination. Abbreviations: AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; FUM, fumarase; GSO, putative glyoxysomal succinate oxidase; ICL, isocitrate lyase; MDH, malate dehydrogenase; MS, malate synthase; PEPCK, phosphoenolpyruvate carboxykinase; SDH, succinate dehydrogenase; Explanation is given in the text.
pronounced was the increased value of glutathione potential, reflecting the portion of reduced glutathione in the total glutathione pool. This means that during germination, scutellar tissue is characterized by an increase of redox level together with the accumulation of H2 O2 and synthesis of ATP. The role of ascorbate and glutathione in keeping H2 O2 levels under control is particularly important, since glyoxysomal catalase is only capable of maintaining H2 O2 at millimolar levels due to its low affinity [13,16]. GSH and GSSG can play a role in activating quiescent cells [36] and initiating cell division and differentiation [23,37]. Gluconeogenesis (reverse glycolysis) is linked to the glyoxylate cycle through the synthesis of carbohydrates via the formation of PEP in the PEPCK reaction [38]. The activity of this enzyme increased during germination, peaking at the 4–5th day. Also at that time, peak AlaAT activity occurred, and the transamination rate of pyruvate was several times higher than PEPCK activity (Fig. 5). In agreement with the previous data [12], this may indicate the importance of glyoxysome-like organelles in the scutellum in the formation of amino acids and synthesis of new proteins for the developing embryo. Thus the function of -oxidation of fatty acids followed by the glyoxylate cycle and the reactions of the TCA cycle in cereals may not be limited to synthesis of carbohydrates but also be related to synthesis of amino acids and to acidification of the endosperm for starch mobilization. It can also provide efficient operation of the TCA cycle by supplying substrate to it when the main oxidation process is the mobilization of fatty acids (the anaplerotic role for TCA cycle). On the other hand, the scutellum serves as a nutrient transporter, carrying sugars [39] and peptides [40] from the endosperm to the embryo to support seed germination and seedling growth, which is achieved in particular due to the difference of pH value between endosperm and scutellum [41]. Fig. 6 provides an overview of the proposed importance of the glyoxylate cycle in the scutellum of germinating cereal seeds for the maintenance of several metabolic and physiological processes during germination. Mobilization of stored lipids occurs via -oxidation of fatty acids in glyoxysomes followed by the glyoxylate cycle, with its key enzymes MS and ICL, in which succinate is formed. Succinate can be transported to mitochondria where it is converted to malate by SDH and fumarase, or may be directly
oxidized in glyoxysomes by a putative glyoxysomal succinate oxidase (GSO). Malate is converted by MDH to OAA, which, in turn, is converted to PEP by PEPCK. This pathway leads to carbohydrate synthesis in reverse glycolysis or to amino acid synthesis via aspartate and alanine aminotransferases. The main source of carbohydrates for the embryo is the endosperm where starch breakdown is facilitated by acidification caused by malate and citrate and possibly other acids. The study reveals new aspects of the organization of metabolic processes in early germination of cereal seeds. It unambiguously shows the presence of the glyoxylate cycle in barley scutellum, and the prevalence of citrate and malate over succinate in the organic acids formed, and demonstrates that the operation of the glyoxylate cycle in cereal seeds may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis. These processes determine the germination potential of seeds and their viability at early stages of plant development, which has also a long-term practical importance for barley agronomy. Acknowledgement This work was supported by the grants from the Natural Sciences and Engineering Research Council of Canada (to A.U.I. and F.M.). References [1] S.M. Shchiparev, G.V. Chuprova, V.V. Polevoi, Secretion of acids by isolated scuttella of corn, Vestnik Leningradskogo Universiteta Biologiya (4) (1976) 130–133. [2] S.M. Shchiparev, E.A. Alieva, A.N. Shinkarev, The effect of the inhibitors of the plasma membrane ATPase on the acidification of medium by scutellums of Zea mays, Vestnik Sankt-Peterburgskogo Universiteta Biologiya (2) (1993) 86–88. [3] Y.M. Drozdowicz, R.L. Jones, Hormonal regulation of organic and phosphoric acid release by barley aleurone layers and scutella, Plant Physiol. 108 (1995) 769–776. [4] W.L. Holtman, J.C. Heistek, K.A. Mattern, R. Bakhuizen, A.C. Douma, -oxidation of fatty acids is linked to the glyoxylate cycle in the aleurone but not in the embryo of germinating barley, Plant Sci. 99 (1994) 43–53. [5] J.C. Newman, D.E. Briggs, Glyceride metabolism and gluconeogenesis in barley endosperm, Phytochemistry 15 (1976) 1453–1458.
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Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination Zhenguo Ma a,b,c , Frédéric Marsolais b,c , Mark A. Bernards c , Mark W. Sumarah b , Natalia V. Bykova d , Abir U. Igamberdiev a,∗ a
Department of Biology, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada Genomics and Biotechnology, London Research and Development Centre, Agriculture and Agri-Food Canada, London, ON N5V 4T3, Canada c Department of Biology, University of Western Ontario, London, ON N6A 5B7, Canada d Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, MB R6 M 1Y5, Canada b
a r t i c l e
i n f o
Article history: Received 9 December 2015 Received in revised form 11 April 2016 Accepted 12 April 2016 Available online 13 April 2016 Keywords: Barley (Hordeum vulgare L.) Seed germination Glyoxylate cycle Succinate Citrate Malate Ascorbate Glutathione
a b s t r a c t During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2 O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis. © 2016 Published by Elsevier Ireland Ltd.
1. Introduction During seed germination, mobilization of stored lipids, carbohydrates and proteins takes place. In cereal seeds, the reserve of carbohydrates (starch) is found in the endosperm, while the reserve of lipids and proteins is found in the scutellum, which represents a single cotyledon of monocotyledonous plants. Starch and proteins are mobilized via the activation of corresponding amylases and proteases, while for starch breakdown an acid environment is important. Acidification of the endosperm is achieved via secretion of organic acids from the scutellum and aleurone layer into
Abbreviations: AlaAT, alanine aminotransferase; Asc, ascorbate; DHA, dehydroascorbate; DTNB, 5,5 -dithiobis-(2-nitrobenzoic acid); DTT, dithiothreitol; GSH, reduced glutathione; GSO, putative glyoxysomal succinate oxidase; GSSG, oxidized glutathione; ICL, isocitrate lyase; MS, malate synthase; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PEPCK, PEP carboxykinase; PVPP, polyvinylpolypyrrolidone; SDH, succinate dehydrogenase; TCA cycle, tricarboxylic acid cycle. ∗ Corresponding author. E-mail addresses: [email protected], a [email protected] (A.U. Igamberdiev). http://dx.doi.org/10.1016/j.plantsci.2016.04.007 0168-9452/© 2016 Published by Elsevier Ireland Ltd.
endosperm, i.e., the “digestive” process [1–3]. Organic acids are formed primarily during oxidative metabolic processes, such as the TCA cycle and the glyoxylate cycle. The latter becomes active during germination of oil-containing seeds, which includes the seeds of cereal plants, where the process of -oxidation of fatty acids is localized in the scutellum and aleurone layer [4]. Aleurone layers, according to the estimations of Newman and Briggs [5] contain approximately 2/3 of triacylglycerols of barley grain, with their concentration close to 15% of fresh weight of this tissue; the remaining 1/3 is localized in the scutellum in which lipid concentration can be even higher, up to 40% of fresh weight in maize scutellum [6]. During germination, the lipid content in these tissues decreases in correspondence with the development of -oxidation and the glyoxylate cycle. Meanwhile, the remobilization of storage reserves towards formation of carbohydrates and amino acids takes place [3,4,7]. Starchy barley seeds [8] are different from Arabidopsis oily seeds [9]. Therefore, even though the glyoxylate cycle plays a crucial role in taking advantage of acetyl-CoA from breakdown of lipids in oily seeds [10,11], it could have different functions in cereal seeds. The function of the glyoxylate cycle is usually attributed to the
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necessity of carbohydrate formation from lipids. This occurs via succinate synthesis from acetyl-CoA by the glyoxylate cycle in glyoxysomes, followed by its conversion to malate and oxaloacetate (OAA) by the TCA cycle in mitochondria, and synthesis of phosphoenolpyruvate (PEP) by PEP carboxykinase (PEPCK) in the cytosol. The latter becomes the substrate for reverse glycolysis (gluconeogenesis), in which carbohydrates are built. The glyoxylate cycle can be of main importance in the tissues of many plants, especially dicotyledons such as sunflower or castor bean, where carbohydrate reserves in seeds are low or absent. However, in cereal plants the endosperm represents a major reserve of carbohydrates, and therefore the glyoxylate cycle could serve other metabolic purposes than converting lipids to carbohydrate. Using 14 C-labelled acetate, which is easily converted to 14 C-acetylCoA, it was shown that in maize scutellum only a small amount of exogenous acetate is used for carbohydrate synthesis, while the main portion of it is detected in amino acids and organic acids [12]. This led to the conclusion that the function of the glyoxylate cycle in cereals consists mainly in providing substrates for amino acid synthesis and for acidification of the endosperm (reviewed in Ref. [13]). The function of the glyoxylate cycle to convert acetyl-CoA produced during oxidation of fatty acids of stored lipids to succinate is well established in the oily tissues of germinating seeds of dicotyledonous plants such as sunflower cotyledons and castor bean endosperm [14]. In the seeds of cereal plants the glyoxylate cycle operates in the scutellum, which represents a single cotyledon, or more precisely it has a homology to the third leaf of nymphealean seedlings [15]. The glyoxylate cycle supplies low molecular weight organic acids for the acidification of the endosperm to “digest” the stored starch, which is used as a carbohydrate supply by the developing embryo [3,13]. While the supply of carbohydrates is provided
by the endosperm, the role of the glyoxylate cycle in cereals may be expanded to include the supply of organic acids and amino acid synthesis. Operation of the glyoxylate cycle in glyoxysomes (which represents a special type of peroxisome) is accompanied by the buildup of hydrogen peroxide during the flavin-dependent oxidation of fatty acids and in other processes. While excess H2 O2 is normally scavenged by catalase, its generation affects the reduction levels of ascorbate and glutathione in the cell, which also participate in H2 O2 scavenging in the ascorbate-glutathione cycle [16]. Besides the flavin oxidation-dependent generation of H2 O2 , the formation of NADH and succinate in glyoxysomes, via transport mechanisms that bring reducing equivalents to the mitochondria, may result in the synthesis of ATP. In this study, we measured the activities of enzymes, redox levels and organic acid content in the scutellum of germinating barley seeds. While our previous study [17] was concentrated on the early germination events (first 48 h post imbibition) associated with breakage of dormancy, our current study deals with heterotrophic metabolism of germinated barley seeds that utilize storage reserves for germination and seedling growth. The results indicate that during glyoxylate cycle operation, organic acids can be used to acidify the endosperm, converted to carbohydrates or transaminated to produce amino acids. This means that the function of the glyoxylate cycle in cereals may be broad and not restricted to the conversion of storage fats to carbohydrates. 2. Material and methods 2.1. Plant material Barley (Hordeum vulgare L., cv. Harrington) seeds were soaked in ddH2 O and germinated in darkness at 25 ◦ C on two layers of filter paper in Petri dishes for 8 days. Endosperm and scutellum tissues were isolated every day and ground into fine powder in liquid nitrogen with a mortar and pestle. Aleurone layer was removed from the endosperm by a sharp blade. To prevent any contamination by endosperm, scutella were cleaned by wet paper towel, rinsed gently by ddH2 O, and dried on filter paper. The tissue powder was stored at −80 ◦ C and used within a week. For enzyme and metabolite measurements the powder was homogenized in the extraction buffer in the ratio of 50 mg tissue fresh weight to 1 mL buffer. 2.2. Measurement of protein concentration Protein concentration was measured using a standard Bradford protocol and a commercially available reagent (Sigma), according to the supplier’s protocol. Protein was extracted from tissue powder by 0.1 M HEPES (pH 7.0) containing 0.5% CHAPS and 0.1% SDS on ice. Bovine serum albumin was used as a standard. 2.3. Measurement of hydrogen peroxide
Fig. 1. Changes in H2 O2 content, ATP + ADP and ATP/ADP ratio in scutellum of barley seeds during germination. H2 O2 and ATP contents were measured by chemiluminescent methods; ADP was converted to ATP by pyruvate kinase as described in the text. The data are the means of three biological replicates ± SD.
The concentration of hydrogen peroxide was measured according to the method of Lu et al. [18]. The fine powder of isolated scutella was homogenized in 6% trichloroacetic acid for 30 min at 4 ◦ C, centrifuged at 15,000g for 10 min, and then insoluble polyvinylpyrrolidone (PVPP) (50 mg mL−1 ) was added. The samples were centrifuged at 15,000g for an additional 3 min. The preparation of reagents followed the method of Pérez and Rubio [19] and Lu et al. [18]. 10 mL of 6.5 mM luminol and 2 mL of 3 mM CoCl2 in 0.1 M sodium carbonate buffer (pH 10.2) were mixed, diluted to 1 L in the same buffer and stored at 4 ◦ C in the dark overnight before use. Samples, 40 L, were mixed with 10 L of the sodium carbonate buffer and the mixture was incubated at 30 ◦ C for 15 min. Catalase (EC 1.11.1.6; Sigma; 500 units) was added and incubated at the
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same condition as a control. Samples (10 L) were mixed in 5 mL SARSTEDT tubes with 200 L of reaction reagent and the chemiluminescence (CL) measure on a FB 12 Luminometer (Berthold Detection Systems GmbH, Germany). The difference of CL response between each treatment and corresponding background was considered as CL specific for H2 O2 in the samples. The amount of H2 O2 produced per gram tissue was calculated using a standard curve generated from known amounts of H2 O2 . 2.4. Measurement of ATP and ADP Extraction of ATP was conducted according to Dordas et al. [20] with modifications. The tissue powder was lysed in ice-cold 2.4 M perchloric acid (1 mL to 25 mg) for 60 min on ice and centrifuged for 5 min at 20,000g. The supernatant (0.5 mL) was neutralized with 4 M KOH, and the amount of ATP in the neutralized solution measured by chemiluminescent analysis using an ATP Detection Kit (ThermoFisher Scientific), according to supplier’s instructions. The content of ADP was determined using the EnzyLightTM ADP Assay Kit (EADP-100, BioAssay Systems), according to supplier’s instructions. Briefly, ADP was converted to ATP by pyruvate kinase (EC 2.7.1.40; Sigma) in the presence of 2 mM phosphoenolpyruvate (PEP) and 5 mM magnesium, the ATP was measured by chemiluminescence as above, and the amount of ADP was calculated by subtraction of the ATP amount before conversion.
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(Sigma). The extinction coefficient used for NADH at 340 nm was 6.22 mM−1 cm−1 . Fumarase (EC 4.2.1.2) was measured in 50 mM Tris-HCl buffer (pH 7.8) containing 50 mM l-malic acid by monitoring the formation of the double bond in fumaric acid at 240 nm using the extinction coefficient 2.44 mM−1 cm−1 [25]. 2.7. Measurement of isocitrate lyase and malate synthase Isocitrate lyase (ICL; EC 4.1.3.1) was extracted on ice with 50 mM Tris-HCl buffer, pH 7.5, containing 5 mM MgCl2 , 1 mM EDTA and 2 mM cysteine. Samples were centrifuged at 15,000g for 10 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. ICL activity was measured in 50 mM Tris-HCl, pH 7.5, containing 5 mM MgCl2 , 2 mM l-cysteine, 10 mM d,l-isocitrate and 4 mM phenylhydrazine hydrochloride, at 324 nm, using the extinction coefficient of glyoxylic acid phenylhydrazone (14.6 mM−1 cm−1 ) [26]. Malate synthase (MS; EC 4.1.3.2) was extracted on ice with 0.1 M HEPES, pH 7.8, containing 5 mM MgCl2 , 1 mM EDTA and 2 mM DTT. After centrifugation at 15,000g for 10 min at 4 ◦ C, and the supernatant desalted on a Sephadex G-10 column. MS activity was determined in 0.1 M HEPES, pH 7.8, containing 6 mM MgCl2 , 5 mM sodium glyoxylate, 2.5 mM acetyl-CoA and 2 mM DTNB, at 412 nm, using the extinction coefficient of 2-nitro-5-thiobenzoic acid (TNB) 13.6 mM−1 cm−1 . 2.8. Measurement of phosphoenolpyruvate carboxykinase
2.5. Measurement of ascorbate and glutathione Ascorbate and glutathione were extracted with 6% (w/v) trichloroacetic acid (1.5 mL added to 100 mg of tissue powder). The homogenate was centrifuged at 12,000g at 4 ◦ C for 20 min and the supernatant used for measurement of the reduced and oxidized ascorbate and glutathione. Ascorbate (Asc) and dehydroascorbate (DHA) were determined according to Kampfenkel et al. [21], and the absorbance was recorded at 525 nm using spectrophotometer. Reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined according to Zaharieva and Abadía [22]. The method is based on the reaction of 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) with GSH forming 5-thionitrobenzoic acid (TNB), which can be measured spectrophotometrically at 412 nm. Oxidized glutathione (GSSG) was measured after being reduced by glutathione reductase (EC 1.8.1.7; Sigma). Calculation of reduction potential of the GSSG/2GSH couple was done using the method of Schafer and Buettner [23] using the Nernst Equation for half-cell reaction of the GSSG/2GHS couple at pH 7: Ehc (mV) = −240 − (59.1/2) log10 ([GSH]2 /[GSSG])
2.6. Measurement of succinate dehydrogenase, succinate conversion to malate, and fumarase Enzymes were extracted on ice using 50 mM Tris-HCl containing 1 mM EDTA and 2 mM dithiothreitol (DTT), pH 7.8. Samples were centrifuged at 14,000g for 10 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. Succinate dehydrogenase (SDH; EC 1.3.5.1) activity was measured in 50 mM Tris-HCl, pH 7.8, containing 5 M 2,6-dichlorophenolindophenol (DCPIP), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM KCN, and 5 mM succinate, at 600 nm [24] using the extinction coefficient 20.6 mM−1 cm−1 . Succinate conversion to malate was measured according to Igamberdiev et al. [34]. Briefly, the reaction buffer comprised 50 mM Tris-HCl, pH 7.8, 20 mM succinate, 0.6 mM NAD+ , 5 mM MgCl2 , 1 mM KCN and 5 units mL−1 NAD-malic enzyme
Determination of phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) was performed according to Walker et al. [27]. The enzyme was extracted by homogenizing tissue powder with icecold 200 mM Bicine-KOH buffer, pH 9.0, containing 3 mM EDTA, 5% (w/v) polyethylene glycol (PEG) and 25 mM DTT, and centrifuged at 12,000g for 15 min at 4 ◦ C and the supernatant desalted on a Sephadex G-10 column. The activity of PEPCK was measured in the direction of carboxylation in 100 mM Hepes-KOH buffer, pH 6.8, containing 100 mM KCl, 0.14 mM NADH, 0.25 mM DTT, 6 mM MnCl2 , 1 mM ADP, 90 mM KHCO3 and 6 units mL−1 malate dehydrogenase (Sigma). The reaction was initiated by adding PEP (final concentration 6 mM) and the decrease in NADH was measured at 340 nm. 2.9. Measurement of alanine aminotransferase Determination of alanine aminotransferase (AlaAT; EC 2.6.1.2) activity was conducted according to Good and Muench [28]. Enzyme extraction was performed on ice using 50 mM Tris-HCl buffer, pH 7.5, containing 1 mM DTT. The homogenate was centrifuged at 15,000g for 10 min at 4 ◦ C, and the supernatant desalted on a Sephadex G-10 column. AlaAT activity was measured in 50 mM Tris–HCl buffer, pH 7.5, containing 10 mM l-alanine, 5 mM 2-oxoglutarate, 0.1 mM NADH, and 5 units mL−1 lactate dehydrogenase (EC 1.1.1.27; Sigma) by the decrease of optical density at 340 nm due to oxidation of NADH. 2.10. Determination of pH in scutellum and endosperm Fine powder of scutellum or endosperm (10 mg) was homogenized with 1 mL of ddH2 O. Samples were centrifuged and pH was measured in supernatant using an ORION 3-Star benchtop pH meter (Thermo Scientific). 2.11. Determination of organic acids Extraction of organic acids was conducted according to the method of Jham et al. [29]. The fine powder of scutellum or
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2.12. Statistical analysis All the experiments were repeated at least three times. R was used for statistical analysis. Tukey’s test in the two-way ANOVA was used to compare the significance of difference between samples. The data in the text and figures are expressed as means ± SD of three replicates. The differences with P ≤ 0.05 were considered as statistically significant. 3. Results 3.1. Change of protein, H2 O2 and ATP concentrations in scutellum of barley seeds The content of total water-soluble proteins in scutellum tissue decreased two-fold during the first two days of germination and then stabilized at the level of about 30 mg g−1 FW (data not shown). The content of H2 O2 increased from very low values in dry seeds to more than 100 nmol g−1 FW in the first day, peaked at a maximum value of almost 250 nmol g−1 FW and then remained constant (Fig. 1). The content of ATP + ADP slightly decreased during first four days of germination and then started to increase, while the ATP/ADP ratio increased during the first four days from 2 to 5 and then decreased to values close to 3 in the next four days (Fig. 1). 3.2. Ascorbate and glutathione levels
Fig. 2. Changes in the content of reduced and oxidized species of ascorbate and glutathione, in ascorbate/dehydroascorbate ratio, and in glutathione potential in scutellum of barley seeds during germination. The potential of glutathione was calculated according to Schafer and Buettner [23]. The data are the means of three biological replicates ± SD. Abbreviations: Asc—ascorbate, DHA—dehydroascorbate.
endosperm was homogenized with 80% methanol for 30 min at 40 ◦ C and centrifuged at 15,000g for 10 min. The supernatant was used for determination of organic acids. Each sample solution (100 L) was transferred to a glass vial and dried under nitrogen using a Reacti-Vap Evaporator (Thermo Scientific). Sample derivatization was performed according to the method of Roessner et al. [30]. Dry residues of organic acids were derivatized by adding 50 L of MOX Reagent (2% methoxyamine hydrochloride in pyridine, Thermo Scientific), vortexed and incubated for 2 h at 37 ◦ C. Next, 50 L of 50% BSTFA in pyridine was added into each vial. Vials were vortexed and incubated for another 40 min at 70 ◦ C. Organic acids were analyzed by GC–MS essentially according to Roessner et al. [30]. Briefly, trimethylsilyl (TMS) derivatives of organic acids were separated in a Varian CP-3800 (USA) gas chromatograph (GC) equipped with a flame ionization detector (FID) and an ion trap mass spectrometer (Varian 220-MS, USA) (GC–MS). The GC was equipped with a pair of CP-Sil 5CB low bleed MS columns (WCOT silica 30 m × 0.25 mm ID), one in line with the FID and the other with the MS. Temperature in the injector oven and in the FID oven was set at 230 ◦ C and 300 ◦ C, respectively. Samples (1 L) were injected twice (once to each column) in splitless mode and simultaneously eluted with the following oven temperature program: the initial temperature of 70 ◦ C was held for 5 min, followed by a temperature increase (7.5 ◦ C min−1 ) to 300 ◦ C, where the final temperature was held for 2 min. High purity helium was used as a carrier gas and constant flow rate was 1 mL min−1 . Compounds were identified on the basis of their co-elution with authentic standards and their EI-mass spectra (50–650 amu). Quantification of the organic acids in samples was based on FID peak areas and independently derived calibration curves for citrate, malate and succinate.
The total pool of ascorbate (and dehydroascorbate) decreased in scutellum during the first four days of germination, then started to stabilize and slightly increased by day 8. The contents of reduced and oxidized glutathione dropped sharply during the first day of germination and then stabilized. The reduction level of ascorbate (ascorbate to dehydroascorbate ratio) decreased in the first day, then started to increase before stabilizing between days 3–6 and then increasing again (Fig. 2). The glutathione potential was lower during the first three days of germination, then increased to a higher level between days 4–6, and again decreased by day 8. 3.3. Content of organic acids in the scutellum and endosperm of barley seeds The GC–MS analysis of the main organic acids produced in the glyoxylate and tricarboxylic acid cycles (succinic, malic and citric acid) in the scutellum and endosperm of barley seeds (Fig. 3) revealed that the most abundant organic acid in scutellum is citrate, at a concentration of 6–8 mol g−1 FW during the whole study period of germination and seedling establishment (from 0 to 8 days). The concentration of succinate (0.1–0.3 mol g−1 FW) was 30–60 times lower as compared with citrate. The concentration of malate was almost as high as citrate during the first two days and then decreased by 3–4 times. By contrast, the concentration of citrate in the endosperm increased during the first four days of germination, while that of malate decreased from very high levels in the first 2–3 days to two-three orders of magnitude lower levels by days 4–5. The concentration of succinate was negligible during the whole period of germination (Fig. 3). The pH value of homogenized tissue water extracts decreased for both the scutellum and endosperm during germination. In the scutellum the decrease was moderate, from 5.8 to 5.9 at the first day, down to 5.2–5.3 by days 5–6. In endosperm, however, acidification was significant during first two days of germination, as the pH value in dry seeds dropped from 4.4 to 3.5 by the fourth day of germination, before slightly increasing (to 3.7) at the days 6–8.
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Fig. 3. Changes in the content of succinate, malate and citrate and in the value of pH in scutellum and endosperm during germination of barley seeds. Organic acids were measured by GC–MS, pH was determined in the tissue total extract. The data are the means of three biological replicates ± SD. The details are presented in the text.
3.4. Activities of glyoxylate cycle enzymes and of succinate oxidation The activity of ICL increased gradually from very low values until reaching 30 nmol min−1 mg−1 protein on day 4, where it remained stable for the next three days, and then started to decline. The activity of MS increased quickly during first two days, then stabilized at the level of 50–60 nmol min−1 mg−1 protein, and from day 4 started to decrease (Fig. 4). The peak of MS (the enzyme producing malate) activity appeared earlier than that of ICL, which produces succinate. The pattern of succinate oxidation more closely followed the activity profile of ICL, with SDH activity being several times lower than the activity of succinate oxidation measured via the detection of malate formation (Fig. 4). The intensity of succinate oxidation measured by malate formation from succinate reached 15–16 nmol min−1 mg−1 protein, while SDH activity measured by reduction of DCPIP did not exceed 5 nmol min−1 mg−1 protein. 3.5. Activities of fumarase, phosphoenolpyruvate kinase and alanine aminotransferase The activity of fumarase was approximately seven fold higher than the rate of succinate oxidation (measured by malate formation). It increased during germination, being at the highest level between days 3 and 7, before declining (Fig. 5). PEPCK and AlaAT revealed similar profiles of activity, with a gradual increase from the start of germination, reaching a maximum activity at days 4–5 and then declining. The measurable rate of transamination catalyzed by AlaAT was one order of magnitude higher than the rate of PEP synthesis in the PEPCK reaction, which was on the same level as fumarase activity (Fig. 5).
Fig. 4. Changes in activities of isocitrate lyase, malate synthase, succinate dehydrogenase and succinate oxidation to malate in scutellum of barley seeds during germination. These activities reflect operation of the glyoxylate cycle and conversion of its product succinate. The details of spectrophotometric measurements are described in the text. The data are the means of three biological replicates ± SD.
4. Discussion In the current study we used germinating barley seeds, which develop the glyoxylate cycle in scutellar tissues during the first few days of germination, to further explore the role of the glyoxylate cycle in stored starch mobilization. It has been reported that barley embryos do not contain the enzymes of the glyoxylate cycle [4] and that this cycle only operates in the aleurone layer; however, this result is not supported by the present study. The increase of key enzymes of the glyoxylate cycle, ICL and MS, was observed immediately after imbibition of seeds, with ICL peaking at the 4th day, while the highest activity of MS appeared even earlier (Fig. 4). In the study of Holtman et al. [4] ICL and MS were not detected in scutella of barley seeds, while in aleurone layers the activities of both enzymes peaked at the 4th day of germination exhibiting the activities of 80 and 110 nmol min−1 per 10 aleurone layers correspondingly. These values approximately match the activities per mg protein, considering aleurone layer as 5% of total weight of 40 mg barley seed and 50 mg protein per g of aleurone fresh weight, the values corresponding to those estimated in Briggs et al. [7]. In our study ICL peaked after 4–7 days of germination with maximum activity of 30 nmol min−1 mg−1 protein while MS in scutellum peaked on the days 2 and 3 with the activity of 60 nmol min−1 mg−1 protein. Apparently lower activities of MS and ICL as compared to those reported in aleurone layers by Holtman et al. [4] could explain their conclusion about localization of the glyoxylate cycle only in aleurone layers in germinating barley seeds, also gluconeogenesis and
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Fig. 5. Changes in activities of PEP carboxykinase, fumarase, and alanine aminotransferase in scutellum of barley seeds during germination. These enzymes catalyze reactions that are involved in determining metabolic directions of the glyoxylate cycle product succinate conversion either to carbohydrate or amino acid synthesis. The details of spectrophotometric measurements are described in the text. The data are the means of three biological replicates ± SD.
its hormonal regulation in barley are strongly cultivar-dependent [31], which can also result in discrepancies in different studies. While both the aleurone layer and the scutellum contain triacylglycerols, the functions of these two tissues are not the same. The aleurone layer is a part of the endosperm, and it excretes organic acids for starch breakdown [3]. Although Drozdowicz and Jones [3] detected much lower excreting capacity by the scutellum as compared to the aleurone, other studies [1,2] reported very significant excretion of organic acids by the scutellum and established the role of plasma membrane ATPase in this process. On the other hand, the scutellum can be a rapid and immediate source of soluble carbohydrates for developing embryos, and, therefore, the products of its metabolism are also used for growth of the radicle, coleoptile and first leaves. The uptake of carbohydrates by the embryo occurs not only via gluconeogenesis in the scutellum but also via their transport to the scutellum from the endosperm [3], and may take place in exchange of organic acids. Further studies are needed to elucidate the mechanism(s) of transport between the scutellum and endosperm. The profiles of activities of ICL and MS (Fig. 4) indicate that the formation of succinate and malate increases respectively in the scutellum with the development of activities of these enzymes in glyoxysomes, while citrate is formed in the reaction catalyzed by citrate synthase that operates both in glyoxysomes and mitochondria [32]. Malate can be also formed from OAA by the malate dehydrogenase (MDH) reaction and from fumarate by fumarase.
MDH is located in glyoxysomes, cytosol and mitochondria; therefore it is difficult to estimate the contribution of different isoforms to malate production, while the intensity of TCA cycle could be estimated from activities of succinate dehydrogenase, which is lower than isocitrate lyase at the peak of the glyoxylate cycle. Also, the maximal capacities of these enzymes measured in vitro may not fully reflect the situation taking place in vivo. The concentration of citrate was always high in the scutellum (Fig. 3), while in the endosperm it was much lower, slightly increasing to the 4th day of germination. Malate concentration was very high in the first three days in the endosperm and scutellum and then decreased. This indicates that the scutellum may export malate and citrate for the acidification of the endosperm, which was supported by measured changes in pH values in the scutellum and endosperm during germination. That is, the pH value of total water extract of the scutellum gradually decreased during germination, while in the endosperm we observe a significant acidification during first three days post imbibition before the pH level stabilized (Fig. 3). Contrary to malate and citrate, the level of succinate was low both in the scutellum and endosperm (Fig. 3) despite the fact that this acid represents the main exported product from the glyoxylate cycle. This suggests that the enzymes utilizing succinate are not limiting and succinate is efficiently metabolized further in the scutellum of germinating seeds. The common point of view is that succinate is transported from glyoxysomes to the mitochondria, where it is converted to fumarate by SDH and then to malate by fumarase. While the activity of fumarase in the scutellum is quite high, peaking at 4–5 day of germination (Fig. 5), the activity of SDH measured in our study was lower than the activity of ICL (Fig. 4). The succinate oxidation rate, however, measured as malate formation from succinate, was several times higher, which may indicate that mitochondrial SDH is not the only engine for the oxidation of succinate. For example, there was a report of a plasma membrane protein oxidizing succinate and using nitrate as electron acceptor [33]. More important could be a flavin-containing oxidase located in glyoxysomal membranes that is capable of converting succinate to malate, forming hydrogen peroxide in the reaction [34]. This oxidase has a low affinity for succinate (Km ∼ 18 mM) but taking into account the intensive formation of succinate in the glyoxylate cycle, it may be important for succinate conversion to malate without leaving glyoxysomes in germinating cereal seeds when succinate concentration reaches high levels. In dicotyledonous plants the activity of this enzymatic system was not detected [34]. The development of lipid mobilization, the glyoxylate cycle and other metabolic processes in the scutellum is accompanied by the buildup of hydrogen peroxide (Fig. 1). This can be explained by participation of flavin-containing oxidases in -oxidation, the oxidation of succinate and other oxidative processes occurring at this time. On the other hand, the formation of NADH during -oxidation of fatty acids or via the glyoxylate cycle can result in redox transfer to mitochondria, where the oxidation of reducing equivalents is coupled to the formation of ATP. The increase in the ATP/ADP ratio, starting from germination and peaking at the 4th day, indicates that the mobilization of lipids is connected with ATP synthesis. Some decrease in the total ATP + ADP level by this time may also be related to biosynthetic processes as ATP is used in nucleic acid and coenzyme synthesis. In contrast to the cotyledons of dicotyledonous plants, the scutellum exhibits some activity of the glycine decarboxylase complex [35], which may serve for the provision of outflow of glyoxylate from the glyoxylate cycle to glycine and serine synthesis. Cellular or tissue changes in redox level can be monitored via measurement of reduced and oxidized species of ascorbate and glutathione [16]. It is seen from Fig. 2 that the reduction level of ascorbate (measured as the ratio of ascorbate to dehydroascorbate) increases from day 1 to day 3 of germination. Even more
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Fig. 6. Scheme of metabolic processes in barley seeds during germination. Abbreviations: AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; FUM, fumarase; GSO, putative glyoxysomal succinate oxidase; ICL, isocitrate lyase; MDH, malate dehydrogenase; MS, malate synthase; PEPCK, phosphoenolpyruvate carboxykinase; SDH, succinate dehydrogenase; Explanation is given in the text.
pronounced was the increased value of glutathione potential, reflecting the portion of reduced glutathione in the total glutathione pool. This means that during germination, scutellar tissue is characterized by an increase of redox level together with the accumulation of H2 O2 and synthesis of ATP. The role of ascorbate and glutathione in keeping H2 O2 levels under control is particularly important, since glyoxysomal catalase is only capable of maintaining H2 O2 at millimolar levels due to its low affinity [13,16]. GSH and GSSG can play a role in activating quiescent cells [36] and initiating cell division and differentiation [23,37]. Gluconeogenesis (reverse glycolysis) is linked to the glyoxylate cycle through the synthesis of carbohydrates via the formation of PEP in the PEPCK reaction [38]. The activity of this enzyme increased during germination, peaking at the 4–5th day. Also at that time, peak AlaAT activity occurred, and the transamination rate of pyruvate was several times higher than PEPCK activity (Fig. 5). In agreement with the previous data [12], this may indicate the importance of glyoxysome-like organelles in the scutellum in the formation of amino acids and synthesis of new proteins for the developing embryo. Thus the function of -oxidation of fatty acids followed by the glyoxylate cycle and the reactions of the TCA cycle in cereals may not be limited to synthesis of carbohydrates but also be related to synthesis of amino acids and to acidification of the endosperm for starch mobilization. It can also provide efficient operation of the TCA cycle by supplying substrate to it when the main oxidation process is the mobilization of fatty acids (the anaplerotic role for TCA cycle). On the other hand, the scutellum serves as a nutrient transporter, carrying sugars [39] and peptides [40] from the endosperm to the embryo to support seed germination and seedling growth, which is achieved in particular due to the difference of pH value between endosperm and scutellum [41]. Fig. 6 provides an overview of the proposed importance of the glyoxylate cycle in the scutellum of germinating cereal seeds for the maintenance of several metabolic and physiological processes during germination. Mobilization of stored lipids occurs via -oxidation of fatty acids in glyoxysomes followed by the glyoxylate cycle, with its key enzymes MS and ICL, in which succinate is formed. Succinate can be transported to mitochondria where it is converted to malate by SDH and fumarase, or may be directly
oxidized in glyoxysomes by a putative glyoxysomal succinate oxidase (GSO). Malate is converted by MDH to OAA, which, in turn, is converted to PEP by PEPCK. This pathway leads to carbohydrate synthesis in reverse glycolysis or to amino acid synthesis via aspartate and alanine aminotransferases. The main source of carbohydrates for the embryo is the endosperm where starch breakdown is facilitated by acidification caused by malate and citrate and possibly other acids. The study reveals new aspects of the organization of metabolic processes in early germination of cereal seeds. It unambiguously shows the presence of the glyoxylate cycle in barley scutellum, and the prevalence of citrate and malate over succinate in the organic acids formed, and demonstrates that the operation of the glyoxylate cycle in cereal seeds may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis. These processes determine the germination potential of seeds and their viability at early stages of plant development, which has also a long-term practical importance for barley agronomy. Acknowledgement This work was supported by the grants from the Natural Sciences and Engineering Research Council of Canada (to A.U.I. and F.M.). References [1] S.M. Shchiparev, G.V. Chuprova, V.V. Polevoi, Secretion of acids by isolated scuttella of corn, Vestnik Leningradskogo Universiteta Biologiya (4) (1976) 130–133. [2] S.M. Shchiparev, E.A. Alieva, A.N. Shinkarev, The effect of the inhibitors of the plasma membrane ATPase on the acidification of medium by scutellums of Zea mays, Vestnik Sankt-Peterburgskogo Universiteta Biologiya (2) (1993) 86–88. [3] Y.M. Drozdowicz, R.L. Jones, Hormonal regulation of organic and phosphoric acid release by barley aleurone layers and scutella, Plant Physiol. 108 (1995) 769–776. [4] W.L. Holtman, J.C. Heistek, K.A. Mattern, R. Bakhuizen, A.C. Douma, -oxidation of fatty acids is linked to the glyoxylate cycle in the aleurone but not in the embryo of germinating barley, Plant Sci. 99 (1994) 43–53. [5] J.C. Newman, D.E. Briggs, Glyceride metabolism and gluconeogenesis in barley endosperm, Phytochemistry 15 (1976) 1453–1458.
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