GABA mediates phenolic compounds accumulation and the antioxidant system enhancement in germinated hulless barley under NaCl stress
Introduction
Barley grains are widely consumed due to their positive dietary, processing characteristics and bioactive compounds such as β-glucans and tocols (Marconi, Graziano, & Cubadda, 2007). Furthermore, it has been found that a wide range of antioxidant compounds with a phenolic structure exist in barley (Hernanz et al., 2001). Phenolic compounds are secondary metabolites of plants derived from the shikimic acid metabolic pathway. Phenylalanine ammonia lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), coumaric acid 3-hdroxylase (C3H), caffeic acid Omethyltransferase (COMT) and ferulic acid 5-hydroxylase (F5H) are the key enzymes available participating in the biosynthesis of phenolics. The biosynthesis of phenolics is a complex network of chemical reactions, which is an endogenously regulated process during plant growth and development (Crozier, Jaganath, & Clifford, 2007), or which can be stimulated by exogenous factors such as salinity (Navarro, Flores, Garrido, & Martinez, 2006). Positive changes of phenolic components content under salt stress have been confirmed in plants such as buckwheat (Lim, Park, Kim, Jeong, & Kim, 2012) and barley (Ghafoor, Al-Juhaimi, Ozcan, & Jahurul, 2014). Phenolic compounds have strong antioxidant capacity and play an important role in lower occurrence and lower mortality rates of cardiovascular diseases and certain cancers (Anderson et al., 2001). Therefore, the metabolism and accumulation of phenolic compounds have been extensively studied.
In addition to phenolic compounds, γ-aminobutyric acid (GABA) in germinated hulless barley was also induced under adversity (Mazzucotelli, Tartari, Cattivelli, & Forlani, 2006). As is well known, GABA is a four-carbon non-protein amino acid and is widely found in bacteria, fungi, algae and higher plants. GABA is metabolized via decarboxylation of glutamate or degradation of polyamine (Wang et al., 2014). The former, known as GABA shunt, is the main metabolic pathway in hulless barley seedlings. Glutamate decarboxylase (GAD) is the key enzyme involved in GABA biosynthesis. The activity of GAD could be inhibited by 3-mercaplopropionic acid (3-MP), a GABA synthesis inhibitor, which has been frequently used in neurological studies (Ding & Tsunekawa, 2004) and might lead to decreased level of GABA (Ling et al., 2013). Previous studies have shown that GABA acts as a neurotransmitter in mammalian brain tissues. Recently, researchers found that GABA accumulated rapidly in response to variety of stresses such as acidosis, anoxia, cold, heat, draught and salt, which inferred that GABA might function as signaling molecules in plants (Kinnersley and Turano, 2000, Roberts, 2007). Further evidence for GABA signaling in plants came from the fact that a GABA concentration gradient is necessary for pollen tube growth and guidance in Arabidopsis (Palanivelu, Edlund, Preuss, & Brass, 2003). GABA has also been shown to act as a signaling molecule for nitrate uptake modulation (Beuve et al., 2004) and 14-3-3 gene regulation (Lancien & Roberts, 2006).
Our previous research showed that phenolic compounds and GABA content increased simultaneously in germinated barley with or without NaCl stress. Besides, exogenous GABA significantly increased the content of phenolic compounds and GABA (Fig. S1). This implied a close relationship between GABA and phenolics. Whether the induction of GABA represented a consequence of NaCl stress without any functional roles in plant protection or was involved in NaCl induced phenolics production was still unclear. Based on the fact that the phenolics and GABA metabolisms are not interrelated, and GABA has been suggested as a signaling molecule in plants, we speculate that GABA acts as a signaling molecule and functions in phenolic compounds enrichment under NaCl stress. To our best knowledge, reports on the mediation of GABA in NaCl stress-induced phenolics biosynthesis of germinated hulless barley are not available in the literature.
The aims of the current study were to systematically investigate the effects of GABA on phenolic components and antioxidant system under NaCl stress in germinated hulless barley, and to assess a possible relationship between GABA accumulation and phenolics production. This would provide new ideas for the enrichment of phenolic compounds and bring practical implications for the commercialization of functional foods in food industries.
Section snippets
Material and experimental design
The hulless barley seeds were purchased from Institute of Agricultural Sciences of the Yangtze River Bank (Jiangsu, China).
Barley seeds were soaked in 0.5% sodium hypochlorite for 15 min and then washed with deionized water. After that, they were immersed in deionized water at 25 °C for 6 h with a ratio 1:5 (w/v), and then, all the seeds were sown evenly on trays with an automatic spraying system provided a 2 min mist every 1 h which were placed in a growth chamber at 25 °C in dark for 2 days
Changes of GABA and total phenolic content during germination
Results showed that NaCl stress induced an increment of GABA content by 53% and 15% at day 4 and day 6 of germination compared with the control (Fig. 1A). The hulless barley treated with GABA under NaCl stress had higher endogenous GABA content, which was 1.28 and 1.18 times higher than that under only NaCl stress. The Fig. 1B and C showed that the total phenolic content increased by 18% under NaCl stress at the 6th day of germination compared with the control (p < 0.05). Exogenous application
Discussion
In the present study, NaCl stress increased the total phenolic content and phenolic acid content (Fig. 1B and C and Table 2) in germinated hulless barley. These results came from the enhancement of activity (Fig. 2A, C, E, G, I and K) and gene expression (Fig. 2B, D, F, H, J and L) of key enzymes participating in phenolics biosynthesis. PAL is involved in the first committed step in the phenylpropanoid pathway. Indeed, we noticed that PAL was relatively highly related to the accumulation of
Conclusion
GABA acting as an important signal molecule induced phenolic components accumulation by regulating the gene expressions of PAL, C4H, 4CL, C3H, COMT and F5H that participating in phenylpropanoid metabolism as well as the enzymes activity and the enhanced antioxidant system by regulating levels of POD, CAT, SOD, APX, GR and GST activity.
Acknowledgements
Financial support was provided by the Fundamental Research Fund of the Central University (KYZ201744), China and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Notes
The authors declare no competing financial interest.
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