GABA shunt and polyamine degradation pathway on γ-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia

Food Chemistry 136 (2013) 152–159

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GABA shunt and polyamine degradation pathway on c-aminobutyric acid accumulation in germinating fava bean (Vicia faba L.) under hypoxia Runqiang Yang, Qianghui Guo, Zhenxin Gu ⇑ College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s Republic of China

a r t i c l e

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Article history: Received 16 May 2012 Received in revised form 5 July 2012 Accepted 7 August 2012 Available online 16 August 2012 Keywords: Fava bean GABA accumulation GABA shunt Polyamine degradation

a b s t r a c t GABA shunt and polyamine degradation pathway on c-aminobutyric acid (GABA) accumulation in germinating fava bean under hypoxia was investigated. GABA content, GAD and DAO activity were significantly increased under hypoxia treatment. Glu and polyamine contents enhanced largely and thus supplied as sufficient substrates for GABA formation. In contrast, GABA content decreased, mainly in the embryo, after removing the hypoxia stress. DAO activity, Glu and polyamines contents decreased, while an increment of GAD activity was observed. This indicated that GAD activity can be not only regulated by hypoxia, but by the rapid growth of embryo after the recovery from hypoxia stress. When treated with AG, DAO activity was almost inhibited completely, and the GABA content decreased by 32.96% and 32.07% after treated for 3 and 5 days, respectively. Hence, it can be inferred that about 30% of GABA formed in germinating fava bean under hypoxia was supplied by polyamine degradation pathway. Ó 2012 Published by Elsevier Ltd.

1. Introduction Fava bean (Vicia faba L.) is rich in nutritive substances such as protein, carbohydrates and minerals. However, its nutritional value is limited by anti-nutritional components like vicine, convicine, tannins, (Goyoaga et al., 2008; Vilariño, Métayer, Crépon, & Duc, 2009) phytic acid, (Oomah et al., 2011) and trypsin inhibitors (Guillamon et al., 2008). Seed germination could modify the nutritional components and anti-nutrients in legumes (Azeke, Elsanhoty, Egielewa, & Eigbogbo, 2011). Furthermore, new functional components such as c-aminobutyric acid (GABA) are generated after germination (Li, Bai, Jin, Wen, & Gu, 2010). Our previous research showed that GABA content in soybean (Guo, Chen, Song, & Gu, 2011), foxtail millet (Bai et al., 2009) and fava bean (Li et al., 2010) increased significantly during socking culture with aeration. GABA exists widely in prokaryotic and eukaryotic organisms. It is a non-protein amino acid and neurotransmitter in the brain and spinal cord of mammals (Kinnersley & Turano, 2000). In recent years, GABA-enriched foods have become popular for its functional and health effects on regulating blood pressure and heart rate, and alleviation of pain and anxiety (Mody, De Koninck, Otis, & Soltesz, 1994). Glutamate decarboxylase (GAD, EC 4.1.1.15) and diamine oxidase (DAO, EC 1.4.3.6) are the rate-limiting enzymes for GABA formation. In plant cells, GABA is synthesized via the a-decarboxy1ation of glutamate (Glu) in an inreversible reaction which is cata1ysed by GAD. It is metabolised in the mitochondria to succinic ⇑ Corresponding author. Tel./fax: +86 25 84396293. E-mail address: [email protected] (Z. Gu). 0308-8146/$ - see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2012.08.008

semialdehyde and then succinate by GABA transaminase (GABA-T, EC 2.6.1.19) and succinate semialdehyde dehydrogenase (SSADH, EC 1.2.1.16), respective1y (Bown & Shelp, 1997). This metabolic pathway is known as GABA shunt. GABA can also be formed via c-aminobutyraldehyde intermediate from polyamine degradation reaction where DAO is the key enzyme (Wakte, Kad, Zanan, & Nadaf, 2011). Stressful conditions (hypoxia, salt stress, heat or cold shock, drought, mechanical injury, etc.) can strongly promote GAD and DAO activity for GABA formation (Bouche, Lacombe, & Fromm, 2003; Youn, Park, Jang, & Rhee, 2011). At present, research on GABA accumulation in plants mainly focused on the GABA shunt (Bai et al., 2009; Li et al., 2010), but very few on the polyamine degradation pathway (Xing, Jun, Hau, & Liang, 2007). Aminoguanidine (AG) is a specific inhibitor of DAO, which can block the polyamine degradation pathway (one of the pathway for GABA formation). When the polyamine degradation pathway is inhibited, it is convenient to study the contribution ratio of two concentration ways for GABA formation. Previous research showed that germinating brown rice (Komatsuzaki et al., 2007), soybean (Guo et al., 2011) and foxtail millet (Bai et al., 2009) under hypoxia could significantly increase GAD activity and GABA content. Low O2 storage of tomatoes resulted in the GAD activity increase and GABA-T activity decrease which caused the accumulation of GABA (Mae et al., 2012). All these researches mentioned above only discussed GABA shunt pathway on GABA accumulation. Limited information is available on the relationship between DAO activity and GABA accumulation, the contribution rate of GABA shunt and polyamine degradation pathway for GABA accumulation. Fava bean contains abundant protein.

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During germination, endogenous proteases of seeds are activated and proteins are hydrolyzed into amino acids which are the essential precursors for GABA formation. Hence, fava bean seed is the good material for GABA accumulation. In this study, we investigated the fava bean germinating under hypoxia to discuss GABA shunt and polyamine degradation pathway on GABA formation, and compared the contribution rate of the two metabolic pathways.

2.2. Pretreatment of fava bean Dried seeds were sterilized using 10 ml/l sodium hypochlorite for 30 min, then washed and steeped in deionized water at 30 °C for 8 h. Thereafter, they were put into the culturing pallet (25 cm length  20 cm width  5 cm height) and incubated for 48 h in the dark (30 °C). The deionized water was given every 8 h to fulfill the metabolism starting of seeds.

2. Materials and methods 2.1. Materials and reagents

2.3. Fava bean germination

The seeds (Qi Bean 2), harvested in 2011, were supplied by Jiangsu Academy of Agricultural Sciences (Nanjing, China) and stored at 20 °C before the experiments. Horseradish peroxidase, putrescine (Put), spermidine (Spd), spermine (Spm), 4-aminoantipyrine, N,N-dimethylaniline, dimethylaminoazobenzene sulfonyl chloride (dabsyl chloride, 99%) and aminoguanidine (AG) were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other reagents were of analytical grade.

2.3.1. Non-stress germination (control) After being pretreated, the seeds were put into the culturing pallet (25 cm length  20 cm width  5 cm height) with sterilized quartz sand (2 cm in thick) and then covered with gauze and germinating in a dark incubator for 3 and 5 days, respectively. After that, the germinated fava bean seeds were rinsed with distilled water, dried on filter paper, frozen in liquid nitrogen, and stored at 20 °C until analyses.

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Fig. 1. Changes of GABA content (A and B), GAD (C and D) and DAO (E and F) activity during germination. Seeds germinating on quartz sand was defined as the control. The indexes in the two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as the mean ± SD.  Represents significant differences at p < 0.05.

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2.3.2. Hypoxia stress After pretreatment, the seeds were drained and placed in cultivated pots with lids (u 6.0 cm  9.0 cm) where they were germinating with culture solution (citrate buffer, 10 mmol/l, pH 3.5) at a constant temperature (30 °C) in a dark incubator. The culture solution was aerated by a pump (Yuyao jintai Meter Co. Ltd, Zhejiang, China). The air flow rate (1.2 l/min) was controlled by flowmeter (final dissolved oxygen concentration was 5.5 mg/l). The culture solution was replaced with the fresh one at 24 h intervals until germination was complete. The germinated fava bean seeds were rinsed with distilled water, dried on a filter paper, frozen in liquid nitrogen, and stored at 20 °C until analyses.

with 1 ml of dabsyl chloride (2 mg/ml, in acetone) and reacting at 67 °C for 10 min. After that, the reaction was stopped by putting the tubes into an ice bath and then was detected at 425 nm using UV–vis diode-array absorbance detection (DAD). The mobile phase A was acetonitrile and the mobile phase B was 0.045 mol/l CH3COONa (pH 4), the allowed time of separation of GABA and Glu was within 30 min at a constant temperature of 30 °C.

2.3.3. Stress recovery experiment The pretreated fava bean seeds were germinating under hypoxia as described above for 2 days. Then they were transferred into a culturing pallet (25 cm length  20 cm width  5 cm height) with sterilized quartz sand (2 cm thick) and then covered with gauze, germinating for 3 days as ‘‘Non-stress germination (Control)’’. The culture solution (citrate buffer, 10 mmol/l, pH 3.5) was given per 8 h to fulfill the sufficient needs for the growth of fava bean seedlings.

2.6. GAD and DAO activity assay

2.5. Polyamines determination Free polyamines were analysed by HPLC as described by Xing et al. (2007).

GAD activity was determined according to Bai et al. (2009), the GABA content in reaction solution was analysed by the standard method. One unit of GAD activity was defined as release of 1 lmol of GABA produced from glutamate per 60 min at 40 °C. The GAD activity of plant tissues is defined as units of GAD activity of 1 lg protein. DAO activity was determined according to Yang, Chen, and Gu (2011).

2.3.4. Aminoguanidine (AG) treatment The germination procedure was the same to ‘‘Hypoxia stress’’ except that the culture solution containing 7.5 mmol/l of AG.

2.7. Protein determination

2.4. GABA and Glu assay

Protein content was measured from the supernatant by using a protein–dye reagent (Coomassie blue G-250) with bovine serum albumin as a standard according to Yang et al. (2011).

GABA and Glu were extracted and purified according to Bai et al. (2009) The residues were dissolved with 2 ml of 1 mol/l NaHCO3 (pH 9.0) and centrifuged at 6000g for 10 min. GABA and Glu were determined by high performance liquid chromatography (HPLC, Agilent 1200, USA) with a ZORBAX Eclipse AAA reversed-phase column (3.5 lm), 4.6  150 mm i.d. as described by Syu, Lin, Huang, and Lin (2008) The amino acid solution (1 ml, pH 9.0) was mixed

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Fig. 2. Changes of Glu (A and B) and polyamines (C–H) content during germination. Seeds germinated on quartz sand with spraying cultural solution was defined as the control. The indexes in the two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as mean ± SD.  Represents significant differences at p < 0.05.

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3. Results and discussion

Hypoxia treatment is an efficient method for GABA accumulation in germinating seeds (Bai et al., 2009). However, limited information on the relationship among GABA accumulation, precursors and enzymes activities has been reported. We investigated the relationship of them during fava bean germination as well as the distribution in cotyledon and embryo of fava bean seeds.

GABA content (mg/g DW)

3.1. Hypoxia stress on GABA accumulation

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3.1.2. Glu and polyamines content Glu and polyamines, precursors of GABA, are converted to GABA by GAD and DAO, respectively. Glu content increased significantly in non-stress seeds (Control). However, it increased initially and decreased afterwards in sprouts under hypoxia, showing the highest level in 3-day sprouts, which was 1.51-fold of the control (Fig. 2A). Glu content in cotyledon of 5-day sprouts under hypoxia

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3.1.1. GABA content, GAD and DAO activity GABA content increased significantly with the germination time under hypoxia stress (p < 0.05). GABA content of 5-day fava bean sprout under hypoxia was 2.21-fold higher than that of the control (Fig. 1A), and was 1.57- and 8.26-fold higher than the control in cotyledon and embryo, respectively (Fig. 1B). GAD activity increased firstly and then decreased, exhibiting the highest value of 0.89 U/lg protein after germinating for 3 days, which was 9.90-fold of the control. The GAD activity of 5-day sprout was 1.20-fold of the control (Fig. 1C). No significant (p > 0.05) change of GAD activity was detected in cotyledon while there was a 1.73-fold increment in GAD activity of embryo compared to the control (Fig. 1D). In addition, it was noticed that GAD activity in embryo was 7.21-fold of that in cotyledon under hypoxia treatment (Fig. 1D). DAO activity increased gradually during the monitored germination period, which was 4.09- and 4.93-fold of the control after germinating under hypoxia for 3 and 5 days, respectively (Fig. 1E). It was 1.12 U/mg protein in cotyledon of hypoxia treated seeds while was not detectable in cotyledon of untreated seeds. In embryo, the DAO activity was 2.06-fold of the control. It can also be seen that DAO activity in embryo was 7.21-fold of that in cotyledon under hypoxia (Fig. 1F). When fava bean was germinating under non-stressful condition (Control), DAO activity had no significant changes during germination while GAD activity increased rapidly in 5-day sprouts and was only 17.28% less than that under hypoxia. Although GAD activity of sprouts under control increased, no significant increment was observed in GABA content from 3- to 5-day of germination. The reason may be when GABA was being formed, it was also being converted to succinic acid by GABA transaminase (GABA-T) and succinate semialdehyde (SSADH) simultaneously to participate in tricarboxylic acid (TCA) cycle or as materials for synthesis of other substances (Bown & Shelp, 1997). Compared to the sprouts grew under hypoxia with higher GABA content, it was noticed that GABA could not be accumulated under non-stress condition. The optimum reaction pH of GAD is about 5.8 (Zhang, Yao, Chen, & Wang, 2007) and that of DAO is 6.5 (Yang, Chen, Han, & Gu, 2012). Under hypoxia stress, pH in cytoplasm decreased by 0.4–0.8 (Kurkdjian & Guern, 1989) to create a acidic condition where GAD and DAO present high activity, which is helpful for GABA formation. In addition, it was reported that the optimum reaction pH of other enzymes for GABA degradation are between 8.6 and 9.5 (Busch & Fromm, 1999; Narayan & Nair, 1990), so the enzymes activity are relatively inhibited in hypoxia condition. Consequently, hypoxia environment benefits for GABA synthesis while it is not helpful for GABA transamination (Renault et al., 2010).

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Fig. 3. Effect of recovery on GABA content (A and B), GAD (C and D) and DAO (E and F) activity. Seeds germinating under hypoxia was defined as the control. The indexes inthe two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as mean ± SD.  Represents significant differences at p < 0.05.

was less than that of the control, while in embryo it was 2.02-fold of the control (Fig. 2B). After germinating for 3 days, Put content was less than that of the control, but Spd and Spm content had no significant changes. However, the contents of Put, Spd and Spm in 5-day sprouts under hypoxia were all higher than that of the control (Fig. 2C, E and G). Put content in cotyledon increased (Fig. 2D), while Spd and Spm content decreased compared to the control (Fig. 2F and H). In embryo, the three polyamines content all increased significantly (Fig. 2D, F and H). During seed germination, endogenous enzymes are activated and hydrolyze insoluble stored substances into small molecular substances to provide materials and energy for tissues expansion. Free amino acids are also produced in this procedure. Glu is a very important amino acid and plays a key role during the metabolism of other amino acids (Wu, 2009). Hence, its content would increase during seeds germination. In the present study, Glu content of fava bean sprouts increased significantly during germination under hypoxia and non-stress condition, providing huge amounts of materials for GABA formation. However, Glu content of sprouts decreased from 3- to 5-day (Fig. 2A) under hypoxia which may be a plant response to hypoxia stress as Glu was catalysed by GAD to form more GABA (Fig. 1A and B). This was further confirmed by the fact that hypoxia stress is helpful for GABA formation

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and unbeneficial for GABA transamination by decreasing pH value of cytoplasm (Kurkdjian & Guern, 1989). Furthermore, the GAD activity of 3- and 5-day sprouts under hypoxia exhibited a higher level than non-stress counterparts (Fig.1C). Therefore, Glu was highly underutilised by GAD for the synthesis of GABA during germination under hypoxia. Put is a kind of diamine, Spd and Spm are tripropanolamine and fouramine, respectively. They can be catalysed by DAO directly or indirectly and then converted into GABA via c-aminobutyraldehyde intermediate formation (Wakte et al., 2011). In the present study, the results showed that Put, Spd and Spm could be accumulated in fava bean during germination under hypoxia, and they mainly existed in embryo (Fig. 2D, F and H). Compared with nonstress seeds (Control), Put was accumulated significantly either in cotyledon or embryo, while Spd and Spm mainly existed in embryo, and decreased significantly in cotyledon. Polyamine metabolism is a very complicated process (Xu, Shi, Ding, & Xu, 2011), and it was assumed to be related to signal transduction (Groppa & Benavides, 2008) which needs further investigations to give precise explanations. 3.2. Effects of the recovery from hypoxia on GABA accumulation After hypoxia stress for 2 days, fava bean seeds were transferred into a culturing pallet (25 cm length  20 cm width  5 cm height) with sterilized quartz sand (2 cm thick) continuing germination for another 3 days. Then, the GABA content, its precursors contents (Glu and polyamine levels) and related enzymes (GAD and DAO) activities were analysed to explore the impact of recovery on GABA accumulation. 3.2.1. GABA content, GAD and DAO activity From Fig. 3A, after the removal of hypoxia stress for 3 days, GABA content decreased (from 2.29 mg/g DW to 1.25 mg/g DW)

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significantly (p < 0.05). In cotyledon, GABA content had no significant changes (p > 0.05), but it decreased by 65.75% in embryo compared to the control (Fig. 3B). When stressed continuously for 5 days, GAD activity decreased by 33.33% compared with that stressed for 2 days. While after recovery, it increased by 101.76% compared to that in the stressed seeds (Fig. 3C). GAD activities in cotyledon and embryo were higher than that of the control (Fig. 3D). The reason may be continuous hypoxia treatment brought down the cell viability of fava bean seeds, so that GAD activity was inhibited simultaneously. After 3 days recovery, the life activity of the seeds increased, and the GAD synthesis was stimulated at the same time which resulted in the increment of GAD activity. On the contrary, an opposite trend was observed on DAO activity. After recovery, DAO activity decreased by 39.05% (Fig. 3E), and it decreased in cotyledon while increased significantly in embryo (Fig. 3F). Compared to hypoxia stress, a decrease in DAO activity coincided with the reduction in GABA accumulation of germinating fava bean seeds after recovery. GABA content had no significant changes in cotyledon while decreased significantly in embryo, DAO activity decreased significantly in cotyledon while increased in embryo. Hence, it can be found that GABA content and DAO activity were all sensitive to hypoxia stress. Interestingly, GAD activity increased significantly after recovery, but GABA content decreased. The reason may be under stress, plant produces some substances such as proline (Pro) and GABA to resist stress condition (Xu et al., 2011). While recovery induced the decrease of GABA content, indicating that GABA is a molecule or signal vector for plant fight against to stress. Glu, a important metabolism intermediate of those substances (Wu, 2009), can be catalysed by GAD and converted to GABA, and also can be converted into Pro (Delauney & Verma, 1993) or other substances via a D1-pyrroline-5-carboxylic acid (P5C) intermediate formation under the catalysis of D1-pyrroline-5-carboxylate synthetase (P5CS). After

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Fig. 4. Effect of recovery on Glu (A and B) and polyamines (C–H) content. Seeds germinating under hypoxia was defined as the control. The indexes in the two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as mean ± SD.  Represents significant differences at p < 0.05.

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needs cotyledon to supply nutrients and energy. Hence, after removing hypoxia treatment, Glu was generated substantially and transported into embryo. Under GAD catalysis, it was converted to GABA and go into TCA circle (Bown & Shelp, 1997) to provide carbon skeleton and nitrogen source for the formation of new organs, but not to be accumulated. Under hypoxia stress, a great amount of polyamines were accumulated. When stress was removed, three kinds of polyamines content (Put, Spm and Spd) decreased significantly as well as GABA content, indicating that polyamine and GABA are related to stress resistance of plant (Xu et al., 2011).

recovery, an enhancement of life activity, accelerated metabolism and radicle expansion occurred. The formation of new organs needs the degradation of storage protein or amino acids to provide sufficient nitrogen. GAD is an essential enzyme for amino acids metabolism, and the increase of GAD activity is helpful for enhancing amino acids metabolic rate. Hence, it can be concluded that the change of GAD activity not only can be regulated by stress condition, but also by rapid growth of plant after the removal of the stress treatment.

GABA content (mg/g DW)

3.2.2. Glu and polyamines content Although recovery had no significant effects on Glu content in germinating fava bean (Fig. 4A), Glu content slightly increased in cotyledon while decreased significantly in embryo (Fig. 4B). The content sequence of polyamines in germinated fava bean was Put > Spd > Spm and their content decreased significantly after recovery (Fig. 4C, E and G). Put, Spd and Spm content decreased by 63.37%, 75.11% and 62.63% in cotyledon, respectively, and by 76.14%, 59.46% and 68.51% in embryo, respectively (Fig. 4D, F and H). After the removal of hypoxia stress, Glu content in germinating fava bean had no significant changes, because it was produced in cotyledon and consumed in embryo at the same time. Embryo is an important organ in which the life activities are more vigorous. Before the occurrence of fibrous root, the development of seedling

3.3. Effects of AG on GABA accumulation under hypoxia AG is a specific inhibitor of DAO, addition of which during fava bean seeds germination could significantly inhibit DAO activity (Yang et al., 2012), cutting off the polyamine degradation pathway of GABA metabolism. Here, AG was applied to study the contribution rate of GABA shunt and polyamine degradation pathway for GABA formation in germinating fava bean under hypoxia. 3.3.1. GABA content, GAD and DAO activity Fig. 5 showed the effects of AG on GABA accumulation and the key enzymes activities for GABA formation of germinating fava bean under hypoxia. AG application led to a decrease of GABA con-

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Fig. 5. Effects of AG on GABA content (A and B), GAD (C and D) and DAO (E and F) activity. Seeds germinating under hypoxia and without the addition of AG was defined as the control. The indexes in the two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as mean ± SD.  Represents significant differences at p < 0.05.

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Fig. 6. Effect of AG on Glu (A and B) and polyamines (C–H) content. Seeds germinating under hypoxia and without the addition of AG was defined as the control. The indexes in the two organs (cotyledon and embryo) of 5-day sprouts were determined. Each point was expressed as mean ± SD.  Represents significant differences at p < 0.05.

tent of 3- and 5-day sprouts by 32.96% and 32.07%, respectively (Fig. 5A), where GABA content in cotyledon and embryo of 5-day sprout were 83.47% and 45.60% of the control (without AG), respectively (Fig. 5B). Although AG is the specific inhibitor of DAO, the application of which also inhibited GAD activity (Fig. 5C and D), which was assumed to be the inhibition of physiological activities of fava bean sprouts caused by AG. It was found that the elongation of radicle was significantly inhibited when AG was applied (data not shown). DAO activity was significantly inhibited by AG (Figs. 5E, 3F) and was completely inactivated in 3-day sprout. However, DAO activity occurred in 5-day sprout (Fig. 5E), which may be a result of the adaptation of sprouts from 3- to 5day towards AG. DAO activity was inhibited completely in cotyledon while showed a higher level (69.80% of the control) in embryo (Fig. 5F). AG is the specific inhibitor of DAO, but it could also inhibit the growth of fava bean sprouts, and thus GAD activity decreased, slightly. The addition of AG resulted in the significant decrease of DAO activity resulting in a significant decrease in GABA content. In 3-day sprout where DAO activity was totally inhibited, a 32.96% decrease in GABA formation was detected compared to the control. In other words, GABA formation from polyamine degradation pathway was completely inhibited as a consequence of the inactivation of DAO. Hence, it could be inferred that the polyamine degradation pathway provided 32.96% of GABA formation, which agreed with our previous research that polyamine degradation pathway accounts for about 30% of GABA formation in germinating fava bean under non-stress condition (Yang et al., 2011). 3.3.2. Glu and polyamines content The conversion from Glu to GABA is catalysed by GAD, and the role of DAO is to convert polyamine to GABA. Glu content of sprouts increased firstly and then decreased significantly with the germination time (Fig. 6A), while it was lower than that of the control when AG was applied. Glu content of 5-day sprout was 45.51% of the control, and it was lower than the control in cot-

yledon and embryo. From Fig. 6C, E and G, it was showed that Put, Spd and Spm contents in the control group increased significantly with the germination time. While they showed various trends in AG treated group. The changing trend of Put was similar to that of the control, but Put content was increased by AG application compared with the control. Spd and Spm contents increased firstly and then decreased, which were 231.48% and 486.47% of the controls in 3-day sprouts, respectively. In cotyledon of AG treated group, Put content was higher than that in the control, while in embryo it was lower (Fig. 6D). The content of Spd and Spm showed an opposite trend (Figs. 6F, 4E). GABA can not only be formed by Glu decarboxylation which is catalysed by GAD, but also produced from polyamine degradation by DAO. Being the precursors, Glu and polyamines showed corresponding changes with GABA formation in germinating fava beans. As a key intermediate product of amino acids metabolism, Glu level increased sharply during germination to meet the growth requirements of sprouts. However, Glu content decreased with the time prolongation of AG treatment since Glu was converted to small molecule substance including Pro and GABA to resist abiotic stress (Alvarez, Tomaro, & Benavides, 2003; Maldonado, Sanchez-Ballesta, Alique, Escribano, & Merodio, 2004). When AG was applied, Glu content was significantly lower than that of the control, which may be attributed to the degradation of Glu to meet the needs of fighting against the adverse condition induced by the inhibition of DAO activity. When DAO activity was inhibited by AG, polyamines (especially Put) were accumulated as DAO is the key enzyme for polyamine metabolism (Bouchereau, Aziz, Larher, & Martin-Tanguy, 1999). 4. Conclusions Hypoxia treatment is an efficient method for accumulating GABA in germinating seeds. The levels of Glu and polyamines (Put, Spd and Spm) which are the precursors of GABA were regulated by seed germination and hypoxia treatment. GAD activity

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