Article pubs.acs.org/JAFC
Molecular Mechanisms Underlying γ‑Aminobutyric Acid (GABA) Accumulation in Giant Embryo Rice Seeds Guo-chao Zhao, Mi-xue Xie, Ying-cun Wang, and Jian-yue Li* Development Center of Plant Germplasm Resources, College of Life and Environmental Sciences, Shanghai Normal University, Shanghai, 200234, People’s Republic of China S Supporting Information *
ABSTRACT: To uncover the molecular mechanisms underlying GABA accumulation in giant embryo rice seeds, we analyzed the expression levels of GABA metabolism genes and contents of GABA and GABA metabolic intermediates in developing grains and germinated brown rice of giant embryo rice ‘Shangshida No. 5’ and normal embryo rice ‘Chao2-10’ respectively. In developing grains, the higher GABA contents in ‘Shangshida No. 5’ were accompanied with upregulation of gene transcripts and intermediate contents in the polyamine pathway and downregulation of GABA catabolic gene transcripts, as compared with those in ‘Chao2-10’. In germinated brown rice, the higher GABA contents in ‘Shangshida No. 5’ were parallel with upregulation of OsGAD and polyamine pathway gene transcripts and Glu and polyamine pathway intermediate contents and downregulation of GABA catabolic gene transcripts. These results are the first to indicate that polyamine pathway and GABA catabolic genes play a crucial role in GABA accumulation in giant embryo rice seeds. KEYWORDS: giant embryo rice, GABA, glutamate pathway, polyamine pathway
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INTRODUCION γ-Aminobutyric acid (also called GABA) is a nonprotein amino acid and functions as a depressive neurotransmitter in the mammalian central nervous system. It exists ubiquitously in nature in prokaryotic and eukaryotic organisms. GABA plays an important role in sleeplessness, depression, and autonomic disorder observed during the menopausal or presenile period.1 GABA can also relieve blood pressure symptom in experimental animals.2 For example, GABA-enriched food such as soybean3 and pregerminated brown rice4 can reduce blood pressure of spontaneously hypertensive rats. Meanwhile, the latest research suggested that GABA can induce α cell-mediated β-like cell neogenesis in vivo, which represents an unprecedented hope toward improved therapies for diabetes.5 GABA also plays a critical roles in plant development and stress response,6 including guidance and growth of pollen tubes in pistils as a signaling molecule.7 In plants, GABA is mainly produced from glutamate (Glu) via the catalyzation of glutamate decarboxylase (GAD).8,9 GABA derived from glutamate can be catalyzed by GABA transaminase (GABA-T) to succinic semialdehyde into the mitochondria,10−12 which can be further converted to succinate into TCA cycle by succinate semialdehyde dehydrogenase (SSADH).6 This metabolic pathway is well-known as the GABA shunt. Although it also is reported that GABA can be synthesized via the polyamines (PAs) pathway in response to abiotic stress,13−15 previous research on GABA metabolism in plants mainly focused on the Glu pathway, and the available knowledge on the relationship between the polyamine pathway and GABA accumulation is very limited. PAs include mainly putrescine (Put), spermidine (Spd), and spermine (Spm). Putrescine can be directly produced from ornithine (Orn) via ornithine decarboxylase (ODC)16 or indirectly from arginine (Arg) via arginine decarboxylase (ADC).17,18 Putrescine can be transferred to spermidine and © 2017 American Chemical Society
spermine. Catabolism of polyamines depends on oxidation. Putrescine can be oxidased by diamine oxidase (DAO) to 4aminobutyraldehyde (GABald), while polyamine oxidase (PAO) can catalyze the oxidation of spermidine or spermine to GABald, which in turn were converted to GABA by betaine aldehyde dehydrogenase (BADH).19,20 The GABA accumulation was observed after stimulation via various environmental stresses, such as anoxia, drought, and salt stress, decreasing cellular pH, temperature change, mechanical stress, and water stress.21 Stress from water-soaking is effective for stimulating production of GABA in some cereal seeds, such as brown rice,22−24 fava bean,25 barley,26 soybean,27 foxtail millet,28 and buckwheat.29 In brown rice, GABA accumulation can be achieved by adjusting the soaking conditions, including water pH, temperatures, and soaking times.24 However, all of this research mentioned above mainly discussed GABA accumulation in cereal seeds under stress. The accumulation mechanism of GABA in developing grain seeds, including rice grains, is still elusive. Giant embryo rice is known for containing an enlarged embryo and more nutrients and functional components, including GABA, than those of normal embryo rice.30−32 Also, GABA content increases significantly during germination.22,32 Therefore, giant embryo rice not only is an attractive functional food but also is an excellent material to study the mechanism of GABA accumulation during the period of seeds development and germination. In this study, we investigated the molecular basis of GABA accumulation in developing giant embryo rice grains and germinated giant embryo brown rice via analyzing transcript Received: Revised: Accepted: Published: 4883
January 4, 2017 June 4, 2017 June 6, 2017 June 6, 2017 DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
Article
Journal of Agricultural and Food Chemistry
Figure 1. Expression analysis of GABA metabolic genes in developing grains of ‘Shangshida No. 5’ and ‘Chao2-10’ at 6, 10, 14, 18, 22, 26, 30 DAF. ∗∗, Significant difference (P < 0.01); ∗, significant difference (P < 0.05). were obtained from Dikma Technologies Inc. (Lake Forest, CA). All other chemicals used in this study were of analytical grade. Rice Samples. The rice cultivars used in this study, homozygous giant embryo rice (O. sativa L. japonica cv. ‘Shangshida No. 5’) and homozygous normal embryo rice (O. sativa L. japonica cv. ‘Chao2-10’), were obtained as described in a previous study.30 They were both planted in the paddy field of Shanghai Normal University. The developing rice grains of these two cultivars were harvested at 6, 10, 14, 18, 22, 26, and 30 days after flowering (DAF), respectively, frozen immediately with liquid nitrogen, and stored at −80 °C until analyses. Their mature seeds were dried in the sun and stored at 4 °C.
levels of GABA metabolism genes and contents of GABA and its metabolic intermediates. The results expanded our understanding of the GABA accumulation mechanism in developing giant embryo rice grains and germinated giant embryo brown rice, which provides a sound base for future attempts to improve rice GABA content by metabolic engineering.
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MATERIALS AND METHODS
Chemicals. GABA, glutamate, arginine, ornithine, putrescine, spermidine, spermine, and dansyl chloride were bought from SigmaAldrich Co. (St. Louis, MO). Methanol and acetonitrile of HPLC grade 4884
DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
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Journal of Agricultural and Food Chemistry
Table 1. Contents Analysis of GABA and GABA Metabolic Intermediates in the Developing Grains of Two Tested Rice Cultivars at 26 DAF and 30 DAFa 26 DAF (mg/100g, DW) cultivars
GABA
Glu
‘Shangshida No. 5’ ‘Chao2-10’
60.31 ± 0.47b 32.25 ± 0.82
3.38 ± 0.04b 6.53 ± 0.11
Arg
Orn
28.70 ± 0.61b 0.78 ± 0.02b 6.76 ± 0.22 0.33 ± 0.01 30 DAF (mg/100g, DW)
Put
Spd
Spm
6.10 ± 0.37c 3.3315 ± 0.04
3.00 ± 0.15 2.69 ± 0.03
1.65 ± 0.12 1.26 ± 0.08
cultivars
GABA
Glu
Arg
Orn
Put
Spd
Spm
‘Shangshida No. 5’ ‘Chao2-10’
63.35 ± 0.39b 36.41 ± 0.57
3.34 ± 0.11c 4.39 ± 0.01
45.11 ± 0.32b 9.11 ± 0.12
0.84 ± 0.03b 0.55 ± 0.01
5.21 ± 0.43c 2.79 ± 0.05
2.01 ± 0.06 2.00 ± 0.03
1.48 ± 0.06 1.50 ± 0.06
t-test was used to assess whether the means for two samples are different or not. Values are the mean ± SD of three analysis (n = 3). Between two type rice cultivars. bSignificant difference (P < 0.01). cSignificant difference (P < 0.05). Values without a letter have no significant difference (P > 0.05). a
Germination of Brown Rice. ‘Shangshida No. 5’ and ‘Chao2-10’ brown rice were dried at 42 °C for 24 h to release dormancy, then were soaked in distilled water at 25 °C for 48 h, and the distilled water was replaced every 12 h. After draining the distilled water, samples were germinated in an incubator at 28 °C for 12 h. The germinated brown rice were divided into two parts, one was stored at −80 °C and the other was lyophilized in vacuum freeze-dryer, then ground into a powder, and separated through a 100 mesh sieve for analysis. Expression Analysis of GABA Metabolism Genes by Quantitative Real-Time PCR. Sequences of genes encoding OsGAD enzyme (OsGAD1, OsGAD2, OsGAD3, OsGAD4, and OsGAD5), OsADC enzyme (OsADC1, OsADC2, and OsADC3), OsODC enzyme (OsODC1, OsODC2, and OsODC3), OsDAO enzyme (OsDAO), OsPAO enzyme (OsPAO2, OsPAO6, and OsPAO7), OsBADH enzyme (OsBADH2), and OsGABA-T enzyme (OsGABA-T1, OsGABA-T2, OsGABA-T3, and OsGABA-T4) in rice were obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and Rice Genome Annotation (http://rice.plantbiology.msu.edu/) databases. Primers for PCR were designed using Primer Premier 5 software (PrimerBiosoft, Palo Alto, CA) (Supporting Information, Supplemental Table S1). Total RNA from developing grains and germinated brown rice was extracted using TRIzol reagent (Life Technologies Co., Carlsbad, CA). Reverse transcription was performed with 1 μg of total RNA using the PrimeScript RT Reagent kit (TaKaRa Bio Inc., Otsu, Shiga, Japan). The expression level was analyzed by quantitative RT-PCR analysis using a C1000 Thermal Cycler and a CFX96 Real-Time System (Bio-Rad Laboratories Inc., Hercules, CA). The cDNA samples (100 ng) were mixed with 10 μM of each primer and IQ SYBR Green Supermix (BioRad Laboratories Inc.). The amplification procedure was based on a previous description.30 Each cDNA sample was subjected to quantitative RT-PCR analysis in triplicates. The expression level of each gene was expressed as the relative value to that of Osβ-Actin. Quantification of GABA, Glu, Arg, and Orn. The developing rice grains (without husk), brown rice, and germinated brown rice were pulverized (100 mesh) using a grinder. In total, 1 g of each powdered sample were extracted with 0.1 mol/L HCl (30 mL) at 35 °C with ultrasonic wave for 60 min. A volume of 500 μL of the extraction was transferred to a 1 mL test tube and 500 μL salicylsulfonic acid (10%) was added and incubated at 4 °C for 1 h, then centrifuged at 12 000g for 45 min at 4 °C. The supernatant was filtered and diluted to a suitable concentration to measure the concentrations of GABA and amino acids by a high-speed amino acid analyzer (L-8900, Hitachi). Quantification of Polyamines. Polyamines extraction and dansylation method was adapted from SN/T 2209-2008 with modifications. Briefly, ground samples (0.2 g) were homogenized in 1.5 mL of 0.4 mol/L perchloric acid. The homogenates were incubated at 4 °C for 30 min and then centrifuged at 12 000g for 20 min. After centrifugation, the supernatant and pellet were collected separately. The pellet was further treated twice with the same procedures, and the supernatants were collected together. A volume of 1 mL of supernatant was transferred into a 10 mL test tube and 100 μL of NaOH (0.2 mol/ L), 300 μL saturated sodium carbonate, and 2 mL dansyl chloride in
acetone (10 g/mL) were added gradually. The mixture was incubated in a thermal reaction and block at 40 °C for 45 min in the dark. A volume of 100 μL of ammonia (25%−28%) was added to stop dansylation, and all of the reaction system was kept in the dark for 45 min. The mixture was diluted to 5 mL by acetonitrile. A volume of 5 μL of filtrate was taken for RP-HPLC analysis. The 5 μL samples were redissolved in methanol (60%, v/v) and injected into a fixed 20 μL loop for loading on to a 2.1 mm × 150 mm, 5 μm particle size reverse-phase (C18) column. Elution was performed at 30 °C at a flow rate of 250 μL/min using a methanol/ water stepped gradient program changing from 50% to 100% methanol over 35 min. The column was washed by 100% methanol for 15 min. Dansylated polyamines detection was performed by a Surveyor FL Plus Detector, with 365 nm excitation wavelength and an 510 nm emission wavelength. For a given treatment, three independent replication were performed. Each of the PAs produced a high correlation coefficient (r2 > 0.99) and the recovery rate of Put, Spd, and Spm were 91.42%, 104.29%, and 93.03%, respectively.
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RESULTS Transcript Level Analysis of GABA Metabolism Genes in Developing Rice Grains. In order to study the expression pattern of the GABA synthetic and catabolic genes in developing grains of ‘Shangshida No. 5’ and ‘Chao2-10’, genes encoding OsGAD, OsADC, OsODC, OsDAO, OsPAO, OsBADH, and OsGABA-T enzymes were analyzed at the transcript level in the developing grains of ‘Shangshida No. 5’ and ‘Chao2-10’ (obtained at 6, 10, 14, 18, 22, 26, and 30 DAF). The Osβ-Actin gene was used as a control. OsGAD isoforms transcripts showed similar expression patterns in the developing rice grains between ‘Shangshida No. 5’ and ‘Chao2-10’ in general (Figure 1A−E). Five OsGAD genes were highly expressed at the last stage of rice seeds development, and the relative expression level in ‘Shangshida No. 5’ was lower than that in ‘Chao2-10’ in general (Figure 1A−E). Interestingly, the expression levels of OsGAD1, OsGAD3, OsGAD4, and OsGAD5 genes peaked at 26 DAF both in the two tested rice cultivars (Figure 1A,C−E). However, OsGAD2 expression level in ‘Shangshida No. 5’ peaked at 10 DAF, which was much earlier than that (26 DAF) in ‘Chao2-10’ (Figure 1B). OsADC and OsODC were two types of key enzymes to synthesis putrescine, spermidine, and spermine, and expression levels of their isoform genes in ‘Shangshida No. 5’ were higher than those in ‘Chao2-10’ during seeds development (Figure 1F−K). The expression levels of OsDAO and OsPAO isoforms in ‘Shangshida No. 5’ were obviously higher than those in ‘Chao2-10’ (Figure 1L−O). OsBADH is the last step enzyme for the biosynthesis of GABA,20 and the expression level of OsBADH2 in ‘Shangshida No. 5’ was higher than that in ‘Chao210’ during seeds development in general (Figure 1P). Meanwhile, the expression levels of four OsGABA-T genes in 4885
DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
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Figure 2. Expression analysis of GABA metabolic genes in germinated brown rice of ‘Shangshida No. 5’ and ‘Chao2-10’. ∗∗, Significant difference (P < 0.01); ∗, significant difference (P < 0.05).
Table 2. Comparison Analysis of GABA and GABA Metabolic Intermediates Contents in Brown Rice and Germinated Brown Rice of Two Tested Rice Cultivarsa brown rice (mg/100 g, DW) cultivars
GABA
Glu
‘Shangshia No. 5’ ‘Chao2-10’
25.56 ± 1.90c 12.86 ± 0.37
26.75 ± 0.76c 20.14 ± 0.86
Arg
Orn
Put
50.11 ± 2.97b 0.69 ± 0.00b 4.73 ± 0.06b 12.11 ± 0.05 0.23 ± 0.00 2.74 ± 0.18 germinated brown rice (mg/100g, DW)
Spd
Spm
2.77 ± 0.06b 1.89 ± 0.06
3.58 ± 0.16 2.96 ± 0.06
cultivars
GABA
Glu
Arg
Orn
Put
Spd
Spm
‘Shangshia No. 5’ ‘Chao2-10’
50.34 ± 1.13b 22.04 ± 0.60
57.65 ± 0.10c 41.78 ± 0.20
66.17 ± 4.16c 24.56 ± 2.00
0.74 ± 0.00b 0.33 ± 0.00
20.71 ± 0.87b 11.38 ± 0.49
4.55 ± 0.11c 3.42 ± 0.21
2.13 ± 0.05c 1.80 ± 0.04
t-test was used to assess whether the means for two samples are different or not. Values are the mean ± SD of three analysis (n = 3). Between two type rice cultivars. bSignificant difference (P < 0.01). cSignificant difference (P < 0.05). Values without a lette have no significant difference (P > 0.05). a
‘Shangshida No. 5’ were generally lower than those in ‘Chao2-10’ during seeds development (Figure 1Q−T). Quantification of GABA and GABA Metabolic Intermediates Contents in Developing Rice Grains. Considering the expression levels of OsDAO and OsGABA-T1 peaked at 26 DAF, we quantified the content of GABA and GABA metabolic intermediates including Glu, Arg, Orn, Put, Spd, and Spm at 26 DAF and 30 DAF. As shown in Table 1, compared with ‘Chao210’, the contents of GABA, Arg, Orn, and Put in ‘Shangshida No. 5’ increased obviously at 26 DAF and 30 DAF grains. However, Glu content in ‘Shangshida No. 5’ was lower than that in ‘Chao210’ both at 26 DAF and 30 DAF, which was consistent with the RT-PCR results that expression level of OsGAD gene is lower in ‘Shangshida No. 5’ than that in ‘Chao2-10’ at 26 DAF and 30 DAF. Transcript Level Analysis of GABA Metabolism Genes in Germinated Brown Rice. To explore the reason for the higher GABA accumulation in the germinated giant embryo brown rice, the relative expression levels of GABA metabolism genes were determined in germinated brown rice of ‘Shangshida No. 5’ and ‘Chao2-10’. OsGAD isoform transcripts showed different expression patterns between the two tested rice cultivars (Figure 2A). OsGAD2 was detected at a low level in both of the two germinated brown rice (Figure 2A). The expression level of OsGAD3 in germinated ‘Shangshida No. 5’ brown rice was higher than that in germinated ‘Chao2-10’ brown rice (Figure 2A). However, expression levels of OsGAD1, OsGAD4, and OsGAD5 were lower in germinated ‘Shangshida No. 5’ brown rice (Figure 2A). As for OsADC isoforms, OsADC1 and OsADC3 were
expressed in a relative lower level compared with OsADC2 in both tested rice, and the OsADC2 was higher expressed in germinated ‘Chao2-10’ brown rice compared with germinated ‘Shangshida No. 5’ brown rice (Figure 2B). The expression patterns of three OsODC isoforms were similar, which were expressed at a higher level in germinated ‘Shangshida No. 5’ brown rice compared with germinated ‘Chao2-10’ brown rice (Figure 2C). OsDAO gene presented a higher expression level in germinated ‘Shangshida No. 5’ brown rice, while OsPAO and OsBADH2 presented a lower expression level (Figure 2D). In addition, the expression levels of the four OsGABA-T genes in germinated ‘Chao2-10’ brown rice were higher than those in germinated ‘Shangshida No. 5’ brown rice, indicating that the degradation of GABA in germinated ‘Chao2-10’ brown rice is more active than that in ‘Shangshida No. 5’ (Figure 2E). Quantification of GABA and GABA Metabolic Intermediates Contents in Brown Rice and Germinated Brown Rice. We quantified GABA and GABA metabolic intermediates in brown rice and germinated brown rice. As shown in Table 2, the GABA content in ‘Shangshida No. 5’ brown rice (25.56 mg/ 100 g) was higher than that in ‘Chao2-10’ brown rice (12.86 mg/ 100 g), and the Glu and PAs pathway metabolic intermediates Arg, Orn, Put, Spd, and Spm contents in ‘Shangshida No. 5’ brown rice were also higher than those in ‘Chao2-10’ brown rice. Compared with brown rice of ‘Shangshida No. 5’ and ‘Chao2-10’, germinated brown rice contained higher GABA contents, 50.34 mg/100 g and 22.04 mg/100 g, respectively. Interestingly, the contents of Glu and PAs pathway metabolic intermediates Arg, 4886
DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
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Journal of Agricultural and Food Chemistry
‘Shangshida No. 5’ brown rice compared with germinated ‘Chao2-10’ brown rice. In this study, we also found that the contents of GABA, Glu, Arg, and Put were all significantly higher in germinated brown rice than that in brown rice of two tested rice cultivars (Table 2), indicating that higher GABA accumulation in germinated brown rice is not the only result from Glu derived pathway but also results from PAs derived pathways. It is different from a previous study, which only suggested GABA accumulation resulted from higher OsGAD activity in the brown rice after water soaking.22 In the germinated brown rice, OsGAD2 transcript was nearly undetectable, then, compared with OsGAD2, OsGAD1 and OsGAD3 transcripts were relative high, which is consistent with previous reports,22 in which OsGAD3 and OsGAD1 were the major transcripts with an overlapping but slightly distinct expressing pattern but OsGAD2 transcripts only appeared upon coleoptile emergence. Notably, expressions of OsGAD4 and OsGAD5 have not been analyzed in the previous reports.22 This study analyzed for the first time the expression pattern of OsGAD4 and OsGAD5 in the germinated brown rice and found the expression level of OsGAD4 is highest among the five OsGAD genes. Moreover, in developing rice grains, the transcripts of OsGAD1, OsGAD3, OsGAD4, and OsGAD5 genes peaked at 26 DAF both in the two test rice cultivars. However, the OsGAD2 expression level peaked at 26 DAF in ‘Chao2-10’, in marked contrast, which peaked at 10 DAF in ‘Shangshida No. 5’ (Figure 1B). These analyses suggested that OsGAD2 might have a unique function during seeds development and germination. Yang et al. reported that Put, Spd, and Spm content increased in leaves under drought stress.38 However, Do et al. reported that Put and Spd content decreased and Spm content increased in rice leaves under drought stress, and GABA was undetectable in their studies.41 Legocka and Kluk found that in Lupinus luteus seedlings, Put content increased significantly in response to drought and osmotic stress.42 A higher level of Put can result in a higher level of GABA in giant embryo brown rice. Similarly, a decreased Put content may cause a lower content of GABA, which can explain why Do et al. did not detect GABA.41 It is known that giant embryo rice resulted from the dysfunction of GE gene, which encodes a cytochrome P450 monooxygenases.33−36 Current evidence points to the occurrence of intricate cross-talks between PAs, hormones, and other metabolic pathways in the plant.43,44 Our results, together with other previous studies, rendered us to speculate that the GE gene might affect polyamine metabolism through its regulation of levels of hormones in giant embryo rice seeds, which merits further investigations. The content of GABA in ‘Shangshida No. 5’ grains at 26 DAF and 30 DAF was 60.31 mg/100 g and 63.35 mg/100 g respectively (Table 1), while in brown rice, GABA content was 25.56 mg/100 g (Table 2). The GABA concentration in “Chao210” grains at 26 DAF and 30 DAF was 32.25 mg/100 g and 36.41 mg/100 g respectively (Table 1); however, in brown rice, it was 12.86 mg/100 g (Table 2). These results suggested that the GABA concentration in brown rice was much lower than that in developing grains at 26 DAF and 30 DAF of the two tested cultivars. However, the similar difference was not observed in the concentration of Put, Arg, and Orn. It suggested that GABA might be more easily lost than Put, Arg, and Orn during the drying process of seeds. Similar cases have also been reported,30 suggesting a decrease of γ-tocopherol and γ-tocotrienols during the sunshine or hot-air-based drying of the giant embryo seeds. In order to prevent nutrient lost in daily rice production,
Orn, Put, and Spd also were higher in germinated brown rice than those in brown rice, especially Glu and Put contents.
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DISCUSSION ‘Shangshida No. 5’ is a new giant embryo rice derived from giant embryo gene (GE) mutation in ‘Chao2-10’ japonica rice. GE encodes an a cytochrome P450 monooxygenases.33−36 The giant embryo brown rice and germinated brown rice usually contain higher GABA content.22,23,31 Our study verified that GABA content in the giant embryo brown rice was higher than that in the normal embryo brown rice, and germinated giant embryo brown rice also had higher levels of GABA than that in giant embryo brown rice before germination (Table 2). These results indicated that the new giant embryo rice ‘Shangshida No. 5’ possesses more healthy benefits and could be used to produce healthy foods. Previous studies on GABA anabolism mainly focused on the Glu derived pathway in rice.9,22,37 Liu et al. measured the role of OsGAD activity and the expression of three OsGAD homologous genes to accumulation of GABA in water-soaked rice grains and suggested that the accumulation of GABA in rice grains could be attributed to the increase of OsGAD transcripts.22 Khwanchai et al. showed that variation in the GABA amount in germinated brown rice was due to both the OsGAD activity and the amount of Glu, which affected accumulation of GABA in tested five cultivars.37 Akama et al. reported that seed-specific expression of truncated OsGAD2 produces GABA-enriched rice grains.9 PAs metabolism was usually studied under drought38,39 and salt stress40 in rice. However, the relationship between PAs metabolism and GABA accumulation in rice seeds has not been reported yet. For the first time, this study took the Glu derived pathway, PAs derived pathway, and GABA catabolism pathway together into consideration to investigate the GABA accumulation mechanism in developing giant embryo rice grains and germinated giant embryo brown rice. In the developing normal embryo rice ‘Chao2-10’ grains, the expression levels of GABA synthetic genes, including Glu derived and PAs derived pathways genes were relative low at 6−14 DAF and slowly up-regulated from 18 DAF. Those results indicated that the accumulation of GABA in developing grains result mainly from Glu derived and PAs derived pathways. When compared the expression levels of GABA metabolism genes in developing grains between ‘Shangshida No. 5’ and ‘Chao2-10’, it is clear that the expression levels of OsGAD genes and OsGABA-T genes were downregulated but the expression levels of OsADC, OsODC, OsDAO, and OsPAO genes were up-regulated in ‘Shangshida No. 5’ at almost all tested stages (Figure 1). That was coincident with the relatively lower content of Glu but higher contents of Arg, Orn, Put in ‘Shangshida No. 5’ at 26 DAF and 30 DAF (Table 1), indicating that the higher accumulation of GABA in developing giant embryo grains was the result from the higher activation of PAs metabolism and the more reduction of OsGABA-T expression but not the Glu pathway. The result is completely different from previous research that GABA in water-soaked rice grains is synthesized from Glu.22,37 According to GABA metabolic intermediates contents and GABA metabolism pathway genes expression data in germinated ‘Shangshida No. 5’ brown rice and ‘Chao2-10’ brown rice, we found the upregulated expressions of OsGAD3, OsODC1, OsODC2, OsODC3, OsDAO, and down-regulated expression of four OsGABA-T (Figure 2) coincident with the higher content of Glu and Put in germinated ‘Shangshida No. 5’ brown rice (Table 2). That might be the reason for higher accumulation of GABA in germinated 4887
DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
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Journal of Agricultural and Food Chemistry ORCID
cryopreservation without drying might may be a recommended method. In conclusion, our study suggested that GABA accumulation in developing normal embryo rice grains was mainly originated from Glu derived and PAs derived pathways, while the higher GABA content in developing giant embryo rice grains was mainly from the up-regulated activity of PAs derived pathway and downregulated activity on GABA catabolism but not the Glu pathway (Figure 3). This study reported for the first time the molecular
Jian-yue Li: 0000-0003-1334-8084 Funding
We are grateful to the Shanghai Science and Technology Commission for providing grants (Grant 063919141) in support of our study. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are indebted to Ms. Juan Gui of the Instrumental Analysis Center of Shanghai Jiao Tong University for providing technical support of the high-performance liquid chromatograph and highspeed amino acid analyzer.
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Figure 3. GABA metabolic pathways in developing rice grains of ‘Shangshida No. 5’. The metabolites and genes detected in this study are indicated in black underline. Green ↓: Decline in metabolite content or gene expression level in ‘Shangshida No. 5’ compared with those of ‘Chao2-10’. Green ↑: Increase in metabolite content or gene expression level in ‘Shangshida No. 5’ compared with those of ‘Chao2-10’. Compared with those in ‘Chao2-10’, in developing ‘Shangshida No. 5’ grains, the upregulation of gene transcripts and intermediate contents in polyamine pathway and the downregulation of GABA catabolic gene transcripts were observed, which result in higher GABA accumulation. Meanwhile, and downregulation of OsGAD transcripts and Glu contents in developing ‘Shangshida No. 5’ grains were also observed, as compared with those in ‘Chao2-10’. Abbreviations: ADC, arginine decarboxylase; AGIH, agmatine iminohydrolase; CPAH, N-carbamoylputrescine amidohydrolase; BADH2, 4-aminobutyraldehyde dehydrogenase 2; DAO, diamine oxidase; GABA, 4-aminobutyrate; GABA-T, GABA transaminase; GAD, glutamate decarboxylase; ODC, ornithine decarboxylase; PAO, polyamine oxidase.
mechanisms underlying GABA accumulation in developing giant embryo rice grains, which provide a scientific basis for the attempts in increasing GABA content in rice grains via metabolic engineering.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b00013. Supplemental Table 1, detailed primer sequences for quantitative RT-PCR of GABA metabolic genes (PDF)
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DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
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DOI: 10.1021/acs.jafc.7b00013 J. Agric. Food Chem. 2017, 65, 4883−4889
