Î³-Aminobutyric Acid (GABA) Accumulation in Tea (Camellia sinensis L

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Gamma-aminobutyric acid accumulation in tea (Camellia sinensis L.) through the GABA shunt and polyamine degradation pathways under anoxia Jieren LIAO, Xiayuan Wu, Zhiqiang Xing, Qinghui Li, Yu Duan, Wanping Fang, and Xujun Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00304 • Publication Date (Web): 29 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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Journal of Agricultural and Food Chemistry

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Manuscript for ‘Journal of Agricultural and Food Chemistry’

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Gamma-aminobutyric acid accumulation in tea (Camellia sinensis L.) through

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the GABA shunt and polyamine degradation pathways under anoxia

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Jieren Liao1, †, Xiayuan Wu2, †, Zhiqiang Xing3, Qinghui Li1, Yu Duan1, Wanping

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Fang1, Xujun Zhu1, *

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1. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, P. R.

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China

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2. College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech

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University, Nanjing 211800, P. R. China

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3. Suzhou Jiahe Foods Industry Co. Ltd, Wujiang, Suzhou 215222, China

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These authors contributed equally to this work.

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*

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(College of Horticulture, Nanjing Agricultural University, No.1 Weigang, Nanjing,

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Jiangsu Province, 210095, P. R. China)

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Telephone: +86-25-8439-5182

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Fax: +86-25-84395182

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Email: [email protected]

Corresponding author: Prof. Xujun Zhu

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ACS Paragon Plus Environment


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ABSTRACT:

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Gamma-Aminobutyric acid (GABA) is an important bioactive component of tea

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(Camellia sinensis) providing various health benefits. We studied GABA

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accumulation via the GABA shunt and the polyamine degradation pathways under

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anoxia in tea leaves. Anoxia caused a ~ 20-fold increment on GABA concentration,

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relative to fresh tea leaves. This increment was due to the increase of glutamate

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decarboxylase and diamine oxidase activities. Genes involved in GABA formation,

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such as CsGAD1, and CsGAD2, were significantly up-regulated by anoxia. The

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concentration of putrescine and spermine, two substrates for GABA production, was

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also increased by anoxia. Treating tea leaves with aminoguanidine completely

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inhibited diamine oxidase activity during anoxia, but the concentration of GABA only

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decreased by ~ 25 %. We infer that about one fourth of GABA formed in tea leaves

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under anoxia comes from the polyamine degradation pathway, opening the possibility

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of producing GABA tea based through the regulation of metabolism.

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KEYWORDS: tea; gamma-aminobutyric acid; GABA shunt; polyamine degradation;

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anoxic treatment

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INTRODUCTION

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γ-Aminobutyric acid (GABA), a non-protein amino acid, is a major inhibitory

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neurotransmitter of the central nervous system, as well as a multifunctional factor in

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neurology 1. Alterations in the concentration of GABA in the brain may be related to

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various neurological disorders including epilepsy, seizures, convulsions, Huntington’s

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disease, and Parkinsonism 2. GABA-enriched foods have been actively promoted in

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recent years due to their effects on health, on regulating blood pressure, and on the

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alleviation of pain. Various GABA-enriched foods are currently manufactured:

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GABA tea 3, 4, brown rice 5, 6, soybean 7, 8, eggs 9, sourdough bread 10, 11, and yogurt 12,

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diabetic conditions 14, and to inhibit cancer cells 15.

. The intake of GABA-enriched foods has been reported to be beneficial to prevent

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In plant cells, GABA is synthesized through the GABA shunt pathway involving

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α-decarboxylation of glutamate in an irreversible reaction. This reaction is catalyzed

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by glutamate decarboxylase (GAD). The reversible conversion of GABA to succinic

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semialdehyde by GABA transaminase (GABA-T) is followed by the irreversible

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oxidization of succinic semialdehyde (SSA) to succinate by SSA dehydrogenase

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(SSADH) (Figure 1). Succinate and α-ketoglutaric acid, which is converted from

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glutamate by glutamate dehydrogenase (GDH), enter into the tricarboxylic acid (TCA)

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cycle. Putrescine is converted to GABA by diamine oxidase (DAO), via the

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γ-aminobutyraldehyde intermediate from polyamine degradation reaction (Figure 1).

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Polyamines degradation pathway leading to GABA accumulation under abiotic stress

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were reported on Arabidopsis thaliana 16, 17.

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Tea plant (Camellia sinensis) is rich in nutritive substances such as theanine,

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catechins, and minerals 18. In a seminal paper on GABA tea, it was reported that the

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GABA content in tea leaves increases 8-fold under anoxic conditions

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studies have demonstrated that GABA concentration further increases in tea shoots,

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after cycles of anaerobic, and aerobic conditions

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reduce blood pressure in Dahl salt-sensitive rats

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insomnia patients

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glutamate to GABA, which regulates GABA accumulation in tea leaves exposed to

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. Further

. GABA tea has been proved to

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, and in humans

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, and to aid

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. A recent study revealed that CsGAD1 and CsGAD2 convert

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anoxic stress and to mechanical damage 24. Although investigations have done on fava

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bean

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accumulation, as well as the contribution of the GABA shunt and polyamine

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degradation pathways for GABA accumulation in tea leaves are still unknown.

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and soybean

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, the relationship between DAO activity and GABA

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It has been shown that about 30 % of GABA formed in germinating fava bean

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(Vicia faba L.) under hypoxia is derived from the polyamine degradation pathway 25.

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The objectives of this study are to investigate the formation of GABA via the GABA

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shunt and polyamine degradation pathways in tea plant under anoxia, and to

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determine the relative contribution of these two metabolic pathways to the formation

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of GABA. Our results provide insights for the development of a GABA

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bioaccumulation strategy in tea plants based on the regulation of plant metabolism.

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MATERIALS AND METHODS

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Materials and chemicals

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One-year-old tea plants (Camellia sinensis cv. Yingshuang) were transplanted from

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a botanic garden in Nanjing Agricultural University. GABA, glutamate, putrescine,

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spermidine, spermine and aminoguanidine were purchased from Sigma-Aldrich (St.

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Louis, MO, USA). Acetonitrile and ethanol were of HPLC-grade and were purchased

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from Tedia (Fairfield, OH, USA). Other reagents were of analytical grade.

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Plant materials and treatments

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At the first incubation stage, tea plants were grown in a growth chamber with a

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photoperiod regime consisting of 12 h light (28 °C, 200 µmol·m−2·s−1, 70 % humidity)

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/ 12 h dark (22 °C, 70 % humidity). The tea plants were then grown for two weeks in

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the nutrient solution described by Wan et al.

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, and then exposed to different

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treatments as described next.

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For anoxia treatment, plucked tea leaves (one bud and three leaves) were first

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spread in a ventilated place under room temperature for 1 h, and then put in a

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Vacuum Sealer (Magic seal, MS1160) at 25 °C, 70 % humidity in the dark. After 0, 3,

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4, 7, 8, or 11 h, one bud and three leaves were collected, weighed, and stored at -

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80 °C for further analysis.

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For the water-soaking treatment, plucked tea leaves were first spread in a ventilated

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place under room temperature for 1 h, rinsed in 25 °C distilled water for 3 h, and then

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leaves were collected, weighed, and stored at - 80 °C for further analysis.

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For aminoguanidine treatment, plucked tea leaves were first sprayed by 5.0 mM

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aminoguanidine, followed by standing for 1 h under room temperature, the next

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procedure was the same as anoxia treatment.

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Determination of polyamines content

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The techniques for extraction of free polyamines, and for HPLC analysis, were

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described by Zhu et al. 28.

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Analysis of GABA and glutamate

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GABA and glutamate were extracted and purified according to Bai et al.

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then analyzed by HPLC as described by Syu et al. 30.

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GAD and DAO activity assay

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, and

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The tea leaves were homogenized in 70 mM potassium phosphate buffer (pH 5.8),

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containing 2 mM β-mercaptoethanol, 2 mM EDTA, 0.2 mM pyridoxal phosphate and

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100 g L−1 glycerin. Homogenates were centrifuged at 10, 000 × g for 20 min at 4 °C,

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and the supernatant was then used for enzymatic assays. The reaction mixture

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consisted of 200 µL of enzyme extract and 100 µL of substrate (10 g·L−1 glutamate,

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pH 5.8); the mixture was incubated at 40 °C for 2 h, and the reaction terminated by

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boiling in a water-bath. The suspension of the mixture was analyzed for GABA

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content. The generation of 1 µM GABA (produced from glutamate) h−1 was defined

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as one unit (1 U) of GAD activity.

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DAO activity was determined using the method described by Yang et al.

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with

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little modifications. Plant leaves (500 mg) were ground with mortar and pestle at 4 °C

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in 1.6 mL 0.1 M sodium phosphate buffer (pH 6.5) containing 5 % PVP (w/v).

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Homogenates were centrifuged at 10, 000 × g for 20 min at 4 °C. Reaction mixtures

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(3.0 mL) contained 2.5 mL 0.1 M sodium phosphate buffer (pH 6.5), 0.1 mL crude

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enzyme extracts, 0.1 mL peroxidase (250 U·mL-1), and 0.2 mL 4-aminoantipyrine/N,

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N-dimethylaniline. The reaction was initiated by the addition of 0.1 mL 20 mM

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putrescine. One activity unit of the enzyme was set as a 0.01 change of absorbance at

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555 nm.

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Gene expression analysis

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Total RNA was extracted using the Quick RNA Isolation Kit (Huayueyang, Beijing,

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China). The quality and concentration of total RNA was measured using a ONE

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DropTM OD-1000+ spectrophotometer (ONE Drop, USA); 1.0 µg total RNA was

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reverse transcribed to single-stranded cDNA using PrimeScriptTM RT Reagent Kit

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with gDNA Eraser (TaKaRa, Kyoto, Japan), according to the manufacturer’s

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instructions. Reactions for qRT-PCR analysis were performed using SYBR®Premix

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Ex TaqTM II (Tli RnaseH Plus) (TaKaRa, Kyoto, Japan), and run on an IQ5 multicolor

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real-time PCR detection system (Bio-Rad, Hercules, CA, USA). Each 20 µL of

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qRT-PCR mixture contained 10 µL of SYBR®Premix Ex TaqTM II (2×), 0.2 µM of

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each primer, and 10 ng of cDNA template. The PCR cycling program included an

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initial denaturation step (95 °C for 30 s), followed by 40 cycles of 5 s at 95 °C, and

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then 30 s at 58 °C. The primer pairs used for qRT-PCR are listed in Table S1. The

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Camellia sinensis β-actin gene was used as the reference gene

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were repeated three times with independent RNA samples, and the relative expression

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levels were calculated according to the 2-∆∆CT method 32.

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Statistical analysis

. All experiments

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Each value was expressed as means ± SE of the three independent experiments. All

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data were analyzed using SPSS 20.0 (Windows, USA). Significance was determined

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by Duncan’s test and ANOVA. A probability level of 5 % (p ≤ 0.05) was considered

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as statistically significant.

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RESULTS

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Effect of anoxia treatment on GABA accumulation and glutamate content

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GABA content in tea leaves was affected by water soaking, as well as by anoxia

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produced by vacuum in this study. The GABA content in tea leaves increased

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significantly by anoxia, and there was a concomitant decrease in the precursor

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glutamate (Figure 2). GABA content increased gradually during anoxia, reaching the

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highest concentration (0.73 mg·g−1 Freshweight) at 11 h, representing a nearly 20-fold

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increment relative to the un-treated control at 0 h (Figure 2A). As a precursor of

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GABA, glutamate is converted to GABA by GAD. We found that glutamate content

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decreased sharply in tea leaves at 3 h of anoxia; it then increased at 4 h, and decreased

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gradually afterwards. Glutamate content reached the lowest level at 11 h, representing

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an approximately 70 % decrease relative to the un-treated control at 0 h (Figure 2A).

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The concentrations of GABA and glutamate exhibited almost the same trend during

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the water-soaking treatment as during anoxia (Figure 2B).

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Effect of anoxia treatment on DAO and GAD activity 7



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We observed that DAO activity increased gradually from 0 h to 8 h during anoxia,

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reaching the highest value after 11 h (1.56 U·g−1 FW) representing a 50-fold

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increment, relative to the control at 0 h (Figure 3A). GAD activity increased during

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the firstly 3 h of anoxia, and then decreased drastically at 7 h. GAD activity at 3 h of

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anoxia represented a 1.0-fold increment, relative to the control at 0 h. DAO and GAD

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activities during water soaking treatment of tea leaves are shown in Figure 3B. DAO

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and GAD activities exhibited the same trend during water treatment as during anoxia.

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DAO activity at 11 h represented a 1.7-fold increment, relative to the control at 0 h

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(Figure 3B).

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Anoxic treatment contributes to increased CsGADs expression related to GABA

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accumulation

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To determine whether the expression of genes related to GABA formation were

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induced by anoxia, anoxia was induced in tea leaves as described in the previous

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section. As shown in Figure 4, the expression of CsGAD1, and CsGAD2 increased

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gradually from 0 to 8 h; it further increased significantly at 11 h, reaching peaks that

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represented a 5-fold and a 10-fold increment for CsGAD1 and CsGAD2, respectively,

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relative to the control at 0 h. CsGAD3 only exhibited increased expression after 11 h

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of anoxia, but its expression levels were much lower than CsGAD1 and CsGAD2 at all

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time points.

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Effect of anoxia on polyamines content

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As additional precursors of GABA, polyamines are converted to GABA by DAO.

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After 4 h or 8 h of anoxia, the concentrations of putrescine and spermine exhibited an

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increment over 1-fold, relative to the control at 0 h. At 4 h the content of spermidine

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did not show a significant change. At 11 h of anoxia, the contents of putrescine,

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spermidine and spermine in tea leaves was much higher than that of tea leaves at 0 h

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(Figure 5).

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Effects of aminoguanidine on GABA accumulation, enzyme activity, and

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polyamine degradation

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In this study, aminoguanidine was added on top of the anoxia treatment to study the

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relative contributions of the GABA shunt and polyamine degradation pathways to

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GABA production in tea leaves under anoxia. GABA content decreased by 23.5 %

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and 29.9 % (relative to un-treated controls) after 4 and 11 h of aminoguanidine

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application, respectively (Figure 6A). Although aminoguanidine is considered as a

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specific inhibitor of DAO, GAD activity was reduced after 4 and 11 h of

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aminoguanidine application (Figure 6B). Finally, DAO activity was drastically

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inhibited, and was completely inactivated after 4 and 11 h of aminoguanidine

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application (Figure 6C). The results showed that the application of aminoguanidine

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resulted in a significant decrease of GABA content due to a decrease in DAO activity.

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However, GABA production decreased only by 29 % at 11 h of aminoguanidine

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application, when DAO was completely inactivated. These results indicated that the

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polyamine degradation pathway provided 29.9 % of GABA production, which at this

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time (11 h) was completely inhibited as a consequence of DAO inactivation.

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The concentration of putrescine increased by anoxia, and was significantly higher

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after the application of aminoguanidine for 11 h (Figure 6D). The concentration of

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spermidine (Figure 6E) increased after 4 h of aminoguanidine application

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(representing a 1.4-fold increment over the control without aminoguanidine), and then

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decreased slightly (still representing a 0.3-fold increment over the control without

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aminoguanidine). The concentration of spermine increased significantly after the

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application of aminoguanidine for 4 h, and continued to increase after 11 h (Figure

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6F). However, at 11 h there were no differences in concentration of spermine in the

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absence or presence of aminoguanidine (Figure 6F).

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DISCUSSION High accumulation of GABA in tea leaves under anoxic conditions has been

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19, 20

; the cause of this accumulation has been identified

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reported

. However,

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information on the relationship of GABA accumulation, glutamate content, and

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related enzyme activity during anoxic treatment is still limited. GABA enriching

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methods mainly focus on vacuum treatment and water soaking during tea manufacture

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22

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anoxia. This reduction may be a response of plants to anoxia stress, as glutamate was

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used by GAD to form more GABA (Figure 2A). This idea is confirmed by previous

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reports indicating that anoxia stress contributes to GABA formation, while reducing

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GABA transamination via decreasing the pH in the cytoplasm 25. A previous study

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indicated that, after 6 h of anoxia, the GABA content of tea leaves increased nearly

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80-fold relative to the control 24. In the present study, the tea leaves were first spread

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under room temperature for 1 h before anoxia treatment, producing only

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approximately 20-fold increment over the control. We inferred that the spreading

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treatment modified GABA accumulation in tea leaves, as the free amino acid content

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increased from 1.94 % to 2.18 % by 1 h-spreading reported by Xing et al 34.

. Glutamate is an important amino acid playing a key role in amino acid metabolism . We observed that glutamate content in tea leaves decreased from 7 to 11 h under

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Although DAO activity and GABA content increased continuously in tea leaves

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under anoxia from 0 to 11 h, GAD activity did not increase after 3 h of treatment. This

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observation may indicate that DAO activity plays an important role for GABA

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accumulation in tea leaves

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. Another possibility may be that GABA contributed to

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increase GAD activity at the first 3 h of anoxia, and that GABA was being converted

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to succinic acid by GABA-T and SSADH simultaneously.

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Assessment of tea quality by sensory evaluation is commonly used after tea

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processing. Taste and aroma are key factors that affect tea quality. We compared the

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taste components (Table S2) and aroma components (Table S3) of tea leaves that were

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used to manufacture tea, after anoxia treatment, water-soaking treatment, and control

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treatment. The results showed that, both the taste and aroma components of tea

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manufactured after the anoxia treatment tea were relatively higher than those of tea

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manufactured after water soaking. The taste and aroma components of tea

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manufactured after the anoxia treatment were slightly lower than those of tea

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manufactured from control leaves. Anoxia treatment was selected for further testing,

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because it resulted in high GABA accumulation, and relatively high quality of tea.

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In keeping with a recent report

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, the mRNA levels of CsSSADH1, CsSSADH2,

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CsGABA-T1, CsGABA-T2, CsGDH1, CsGDH2, and CsP5CS (data not shown) did not

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increase after anoxia treatment in tea leaves, as they did after the aerobic treatment.

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These results indicate that, increased expression of CsGAD1 and CsGAD2 may play

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an important role in the accumulation of GABA in tea leaves after anoxia.

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Environmental stress increases GABA accumulation through two different

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mechanisms 37. In the first mechanism, metabolic or mechanical disruptions caused by

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stress can lead to cytosolic acidification, which induces pH-dependent activation of

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GAD and GABA synthesis. In the second mechanism, stress initiates a signal

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transduction

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Ca2+/calmodulin-dependent GAD activity and GABA synthesis. In Arabidopsis

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thaliana, genes involved in the GABA shunt pathway (except AtGAD3 and AtGAD4,

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which are highly homologous to CsGAD2) exhibited little expression under anoxia 24,

pathway

in

which

increased

cytosolic

11


Ca2+

stimulates


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38

.

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Putrescine, spermidine and spermine can be catalyzed by DAO or PAO (polyamine

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oxidase), directly or indirectly, and then converted into GABA via formation of

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the γ-aminobutyraldehyde intermediate

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spermidine and spermine accumulated in tea leaves after anoxia (Figure 5), but the

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accumulation of spermine was considerably lower than that of putrescine and

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spermidine. Polyamine metabolism is a complex process regulated by various signal

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transduction pathways

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GABA, as well as DAO activity increased under other abiotic stresses, which were

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also considered as polyamine derived GABA functions in response to the abiotic

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stress

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identifying and characterizing GABA receptor channels, providing a decisive

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argument in the long-standing dispute between the metabolic and signaling functions

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of GABA 42.

40

39

. Our results showed that putrescine,

that still need further investigation. Levels of putrescine,

16, 17, 41

. In wheat, the role of GABA as a signaling effector was revealed by

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Aminoguanidine is a specific inhibitor of DAO. Reduced DAO activity causes the

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polyamine degradation pathway of GABA metabolism to be blocked 25, 31. As GABA

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precursors, the concentrations of glutamate and polyamines changed accordingly with

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GABA formation in tea leaves during anoxia; this is also reflected by GAD and DAO

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activities. Glutamate decarboxylation (catalyzed by GAD). As well as polyamine

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degradation (catalyzed by DAO), can produce GABA. Polyamines (especially

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spermidine and spermine) accumulated when DAO activity was blocked by

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aminoguanidine, which indicated that DAO is the key enzyme for polyamine

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metabolism 43.

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ACKNOWLEDGMENTS

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This work was supported by Natural Science Fund of Jiangsu Province

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(BK20140714), National Natural Science Foundation of China (31400584), China

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Postdoctoral Science Foundation funded project (2015M581810), and Research Fund

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for the Post Doctoral Project of Jiangsu Province (1501134C).

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SUPPORTING INFORMATION

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Table S1. Primers used in this study.

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Table S2. Taste components in tea from different treatments.

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Table S3. Aroma components in tea from different treatments.

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FIGURE CAPTIONS

451

Figure 1. Biosynthesis and metabolism pathway of GABA in plants in relation to

452

polyamines, and TCA cycle. ADC, arginine decarboxylase; DAO, D-amino acid

453

oxidase; GABA, γ-aminobutyric acid; GABA-T, GABA transaminase; GAD,

454

glutamate

455

decarboxylase; PAO, polyamine oxidase; P5CS, pyrroline-5-carboxylate synthase;

456

SPDS, spermidine synthase; SPMS, spermine synthase; SSADH, succinic

457

semialdehyde dehydrogenase; TCA, Tricarboxylic acid.

decarboxylase;

GDH,

glutamate

dehydrogenase;

ODC,

ornithine

458 459

Figure 2. Changes in GABA and glutamate concentration during water soaking (A)

460

and anoxia treatments (B). All experiments were performed in triplicate. Data

461

represent the mean value ± standard deviation. Means with different letters are

462

significantly different from each other (p ≤ 0.05). Glu, glutamate.

463 464

Figure 3. Changes in DAO and GAD activity during water soaking (A) and anoxia

465

treatment (B). Data represent the mean value ± standard deviation. Means with

466

different letters are significantly different from each other (p ≤ 0.05).

467 468

Figure 4. Expression of CsGAD1, CsGAD2, and CsGAD3 during anoxia treatment.

469

Transcript abundance was calculated according to the difference in cycle threshold

470

values between the target gene and β-actin transcripts normalized by the 2-∆∆CT

471

method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data

472


473

significantly different from each other (p ≤ 0.05).

474

19



475

Figure 5. Changes in the concentration of polyamines during anoxia treatment. Data

476


477

significantly different from each other (p ≤ 0.05). Put, putrescine; Spd, spermidine;

478

Spm, spermine.

479 480

Figure 6. Effects of aminoguanidine on the concentrations of GABA and polyamines,

481

and on the activity of DAO and GAD during anoxia treatment. Data represent the

482

mean value ± standard deviation. Means with different letters are significantly

483

different from each other (p ≤ 0.05). AG, aminoguanidine; Put, putrescine; Spd,

484

spermidine; Spm, spermine.

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Figure 1

21



Figure 2

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Figure 3

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Figure 4

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Figure 5

25



Figure 6

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TOC Graphic

a-ketoglutaric acid

GDH

Glutamate

Ornithine

GAD

TCA cycle

DAO Putrescine

GABA

GABA-T Succinic acid

SSADH

Succinic semialdehyde


Î³-Aminobutyric Acid (GABA) Accumulation in Tea (Camellia sinensis L

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