The role of gamma-aminobutyric acid in agriculture

 γ-aminobutyric acid (CAS number: 56-12-2), (GABA for short), with a relative molecular weight of 103.1, is a four-carbon, non-protein amino acid, which is widely used in vertebrates, plants and Widespread in microorganisms.

The content of GABA in plant tissues is extremely low, usually between 0.3 and 32.5 μmol/g. It has been reported in the literature that the enrichment of GABA in plants is related to the stress response experienced by the plant. When subjected to stress such as hypoxia, heat shock, cold shock, mechanical damage, and salt stress, it will lead to the rapid accumulation of GABA. It has become a research hotspot to process plant food raw materials into GABA-rich functional products after a certain stress treatment or through microbial fermentation to increase the GABA content in the body. As a novel functional factor, GABA has been widely used in the food industry. Foods developed using raw materials such as GABA-rich germinated brown rice , soybeans and broad beans are already on the market.

Metabolic pathways in plants

There are two GABA synthesis and transformation pathways in plants: one is the decarboxylation of glutamic acid to synthesize GABA by glutamic acid decarboxylase (GAD), which is called GABA shunt; One is the conversion of polyamine degradation products to form GABA, which is called polyamine degradation pathway.

GABA bypass

In higher plants, the metabolism of GABA is mainly completed by three enzymes. First, under the action of GAD, L-glutamic acid (Glu) undergoes an irreversible decarboxylation reaction at the α-position to generate GABA, and then GABA transaminase ( Under the catalysis of GABA transaminase (GABA-T), GABA reacts with pyruvate and α-ketoglutarate to generate succinic semialdehyde, which is finally catalyzed by succinic semialdehyde dehydrogenase (SSADH). Oxidative dehydrogenation to form succinic acid eventually enters the krebs circle. This metabolic pathway constitutes a branch of the TCA cycle called the GABA branch.

In plants, GAD in the cytoplasm and GABA-T and SSADH in mitochondria together regulate GABA branch metabolism, where GAD is the rate-limiting enzyme for GABA synthesis. Plant GAD contains a calmodulin (CaM) binding domain, and GAD activity is not only regulated by the concentration of Ca2+ and H+, but also by the concentration of GAD coenzyme-pyridoxal phosphate (PLP) and the substrate glutamate. This dual regulatory mechanism links the cellular accumulation of GABA with the nature and severity of environmental stress. Cold shock, heat shock, osmotic stress and mechanical injury all increase the concentration of Ca2+ in the cytosol, and Ca2+ combines with CaM to form a Ca2+/CaM complex, which can stimulate GAD gene expression and increase GAD activity under normal physiological pH conditions; while acidic pH The appearance of stimulated GAD is due to stress lowering the pH of cells, slowing down the exposure of cells to acid damage. The GABA branch in plants is considered to be the main pathway for GABA synthesis. Currently, most studies focus on how to increase GAD activity to achieve GABA enrichment.

polyamine degradation pathway

Polyamines (PAs) include putrescine (Put), spermine (Spermine, Spm) and spermidine (Spermidine, Spd), of which putrescine is the central substance in the biological metabolism of polyamines. The polyamine degradation pathway means that diamines or polyamines (PAs) are catalyzed by diamine oxidase (DAO) and polyamine oxidase (PAO), respectively, to produce 4-aminobutyraldehyde, and then 4-aminobutyraldehyde is catalyzed by diamine oxidase (DAO) and polyamine oxidase (PAO), respectively. Butyraldehyde dehydrogenase (4-amino aldehyde dehydrogenase, AMADH) dehydrogenates the process of generating GABA, and the polyamine degradation pathway finally joins the GABA branch and participates in the TCA cycle metabolism. Among them, diamine oxidase and polyamine oxidase are the key enzymes that catalyze the degradation of Put, Spd and Spm in vivo, respectively. During faba bean germination, anaerobic stress can induce the increase of key enzyme activities of polyamine synthesis and promote the accumulation of polyamines. At the same time, the activity of polyamine oxidase also increases, which promotes the synthesis and accumulation of GABA through the polyamine degradation pathway. Improve the anti-adversity ability of broad beans. Studies have shown that the content of free polyamines in soybean roots increased under salt stress, the activity of DAO increased, and the enrichment of GABA increased by 11 to 17 times. Although the polyamine degradation pathway is considered to be another important pathway for GABA synthesis, its ability to synthesize GABA in monocots is much lower than that of the GABA branch.

Anti-stress and regulation

GABA has long been implicated in various stress and defense systems in plants. GABA increases as plants are stimulated and is considered to be an effective mechanism in plants in response to various external changes, internal stimuli, and ionic environment factors such as pH, temperature, and external natural enemy stimuli. GABA can also regulate the internal environment of plants, such as antioxidant, ripening, and preservation of plants. In recent years, GABA has also been found in plants as a signal molecule to transmit expanded information in plants. GABA has been found successively in soybean, Arabidopsis, jasmine, strawberry and other plants. Low concentrations of GABA help plant growth and development, while high concentrations have the opposite effect.

Response to external acidification

GABA increases rapidly in cells at low pH, and this accumulation of GABA also exists in microorganisms and animals. In plants, the intracellular H+ increases in acidic pH, which induces the increase of intracellular GABA content. The synthesis of GABA consumes H+, which alleviates intracellular acidification. This rapid response mechanism also exists in microorganisms, which increases the expression of proton respiratory chain complexes and promotes ATP synthesis while producing GABA. And up-regulated the activity of F1F0-ATP hydrolase to promote the ATP-dependent H+ excretion process under acidic conditions. In animals, cells also expel GABA and glutamate to alter the pH of the extracellular environment. More importantly, GABA is a zwitterion under physiological conditions and thus plays a role in acid-base regulation.

defense against insects

GABA contributes to the defense of plants against external natural enemies. When insects feed, cells rupture and tissue is injured due to plant injury. This mechanical cutting stimulates the increase of Ca2+ in plants, and plants secrete GABA under the stimulation of Ca2+ as a measure to resist insect feeding. There is no jasmonic acid-like signal involved in the accumulation of GABA during this process. Insects have ionotropic GABA receptors, among which the GABA-gated chloride channel subunit RDL (resistant to dieldrin) of Drosophila is the target of many insecticides. GABA induction reduces the single current of GABA receptors. Specifically, GABA acts in invertebrates through GABA receptor-gated chloride channels, and like most pesticides, through GABA receptor chloride channels, Cl- is driven downstream by an electrochemical gradient, Causes plasma membrane hyperpolarization and inhibits insect feeding. In tobacco plants overexpressing GABA, inoculated with northern nematodes, it was found that the reproductive capacity of female adult nematodes decreased overall, which can make the plants achieve the effect of defending against natural enemies. During the feeding process of the herbivorous female larvae of Ligustrum lucidum, it was found that Ligustrum lucidum would reduce its own lysine activity and make the protein nutritious, while the female larvae would secrete glycine, β-alanine, amine, etc. during this period. The molecule inhibits the reduction of lysine in plants, a process in which plants communicate with herbivorous insects that also demonstrate the function of GABA as a signaling molecule.

Protective effect on higher organisms under high temperature and freezing

Spraying GABA (200 mg/L) during the flowering period of wheat can adjust the film stability, increase the antioxidant capacity, etc., and reduce the loss of wheat under high temperature; the application of exogenous GABA also has a significant effect on the growth of cucumber seedlings. High temperature inhibits the activity of central GABAergic neurons, activates the cholinergic nervous system and causes an increase in body temperature. Long-term exposure to high temperatures increases the activity of GABAergic neurons in the hypothalamus to adapt to the environment and regulate body temperature. GABA will increase in plasma and then inhibit the concentration of catecholamines in the plasma of cold-sensitive nerve nuclei, so as to reduce the temperature of the esophagus.

Cold temperatures can reduce the biosynthetic capacity of plants, interfere with vital functions, and cause permanent damage. Low temperatures in animals can also cause injuries or even more serious injuries. The expression of biological GABA is up-regulated at low temperature, which is associated with low temperature tolerance. At low temperatures, 75% of metabolites are increased, including amino acids, sugars, ascorbates, putrescine, and some tricarboxylic acid cycle intermediates. The amino acid metabolism involved in energy metabolism and the transcriptional abundance of enzymes will increase. Can generate ATP and accumulate GHB by enhancing the GABA shunt pathway. In addition, the use of melatonin at low temperature can make spermine, spermidine and proline accumulate, and promote the expression of diamine oxidase. GABA is synthesized through the putrescine pathway, which reduces the accumulation of H2O2 and the flux of the phenylpropane pathway to achieve the effect of antiseptic and cold resistance.

Role in antioxidant and oxidative processes

GABA shunting, as an intermediate in the branch pathway of the tricarboxylic acid cycle, is closely related to the energy cycle. At the same time, GABA functions as a regulator of oxidative metabolites. When Arabidopsis SSADH mutants were exposed to high temperature, it was found that their reactive oxygen intermediates (ROI) accumulated, resulting in plant death [7], which proved that there is a relationship between ROI and GABA. Similarly, the mutant strains of SSADH and GABA-T genes have a large number of ROIs at high temperature, and the use of the ROI-eliminating agent N-tert-butyl-α-phenylnitrone (PBN) can make a large amount of GABA accumulate, thereby improving the survival rate of yeast. Therefore, the GABA shunt pathway is considered to have a role in suppressing ROI at high temperature. In the process of GABA shunt, SSA can be converted into GHB via GLYR/SSAR, and GHB is closely related to ROI. GHB and ROI were abundantly accumulated in SSADH deletion mutants, and guabatelin inhibited the accumulation of GHB and ROI and inhibited peroxidative death. The GABA shunt process can reduce the accumulation of ROI and protect the organisms from oxidative damage and peroxidative decay caused by high temperature.

Maintain carbon and nitrogen balance

The balance of carbon and nitrogen metabolism involves many physiological processes, including energy metabolism, amino acid metabolism and so on. Since GABA synthesis and shunting pathways involve nitrogen metabolism, GABA is also an important part of the tricarboxylic acid cycle in the energy cycle, and the GABA shunting pathway competes with the respiratory chain for SSADH, so GABA has been considered an important part of carbon and nitrogen metabolism for a long time. The branched glutamate synthesis GABA pathway of the tricarboxylic acid cycle is one of the key factors for plants to rapidly respond to external stimuli. The vast majority of NH3+ is synthesized through the glutamine synthetase/glutamate synthetase pathway (glutamine synthetase/gluta-mate synthetase, GS/GOGAT), which is considered to be the main synthetic pathway of amino acids. Most of the free amino molecules are fixed by glutamine. Glutamate is considered to be the main accumulation form of nitrogen in the old roots of plants. Nitrogen is stored in amino acids such as arginine. nitrogen requirements. Similarly, amino acids are also involved in energy metabolism by being converted into precursors or intermediates of the tricarboxylic acid cycle. In the study of spinach, it was found that proline accounted for 8.1% to 36.% of total free amino acids, GABA accounted for 12.8% to 22.2%, and glutamic acid accounted for 5.6% to 21.5%. Glutamate is the precursor of GABA and proline. At low temperature, plants will shun glutamate nitrogen into GABA and proline to regulate nitrogen metabolism. In addition, except for NADP+-dependent citrate dehydrogenase, glutamine synthase in roots and shoots, and phosphoenolpyruvate carboxylase in shoots, almost all primary nitrogen metabolism in Arabidopsis thaliana cultured under 50 mmol/L GABA Enzyme activities related to nitrate uptake were affected. In Arabidopsis thaliana cultured under NaCl conditions, it was found that the accumulation of GABA led to the increase of overall amino acids in Arabidopsis thaliana. GAD activity and protein levels in Arabidopsis leaves cultured with different nitrogen compounds (10mmol/L NH4Cl, 5mmol/L NH4NO3, 5mmol/L glutamate and 5mmol/L glutamine) as the sole nitrogen source, respectively different, indicating that GAD plays a role in nitrogen metabolism.

Increased GAD activity and increased GABA and banana dopamine were also found in NO-stressed bananas. Glutamate dehydrogenase activity and GAD expression increased transiently under salt stress, which in turn increased the flux of GABA shunt and other related pathways to regulate carbon and nitrogen balance. The ratios of NADH:NAD+ and ADP:ATP under stress can also affect GABA-T, resulting in GABA accumulation. Under salt stress, plants use the C/N balance pathway more to relieve stress.

The role of drought and flooding

At the end of the 20th century, it was found that drought can reduce the nitrogen fixation of roots and the diffusion of O2, which makes plants hypoxia and leads to the accumulation of GABA. Glutamate and aspartate content increased under hypoxic conditions. Under drought, GAD activity increased and GABA-T accumulated rapidly. Under drought conditions, root and stem growth and leaf area extension were inhibited, reactive oxygen species increased, and the production of low molecular osmotic regulators such as GABA and other amino acids, polyols, and organic acids increased, and the expression of enzymes against oxidative damage was up-regulated. Studies have shown that under drought conditions, the expression of genes related to cellular homeostasis, scavenging of reactive oxygen species, stable protection of structural proteins, osmotic regulators, and transporters is up-regulated. Exogenous GABA enables plants to maintain higher relative water content, reduces electrolyte leakage, lipid, peroxide, carbon metabolism and can improve membrane stability. In addition, exogenous GABA can also induce the increase of GABA-T and α-valerate dehydrogenase activities, and inhibit GAD activity to increase GABA and glutamate. At the same time, GABA accelerates the synthesis of polyamines, inhibits the decomposition of polyamines, and further activates the activities of σ-1-pyrroline-5-carboxylic acid synthase, proline dehydrogenase and ornithine-σ-aminotransferase, resulting in GABA pre- High accumulation and metabolism of the enrichment. GABA can also increase the activity of catalase (CAT) and peroxidase (POX) by promoting the expression of chlorophyll, increase the content of proline and sugar, regulate osmosis and reduce oxidation. Plants experience a drop in pH under waterlogging. Long-term waterlogging will cause soil hypoxia and short-term waterlogging will increase GABA. There is a direct relationship between stomatal closure under waterlogging and abscisic acid. GABA increases due to H+ rise and hypoxia. At the same time, the accumulation of alanine can improve the viability of plants under hypoxic conditions. Under hypoxic conditions, GABA can enhance photosynthesis through indirect regulation, reduce stomatal limitation, and increase oxygen flux. The activity of GAD increased under hypoxia, while GABA could alleviate the damage to plant seedlings caused by hypoxia, and exogenous GABA could alleviate root growth inhibition under hypoxia and rapidly grow adventitious roots. Adventitious root growth can also alleviate hypoxia in plants. 

In addition, the levels of other amino acids related to the TCA cycle except GABA, glutamate and alanine decreased under waterlogging and hypoxia conditions. GABA and glutamate can be used as direct synthetic substrates of alanine, and through this anaerobic pathway, ATP can be generated twice as much as glycolysis to ensure energy supply. GABA also has the ability to eliminate reactive oxygen species intermediates and detoxify plants and prevent programmed cell eath (PCD) indirectly through H2O2 signaling, among other roles. 

other physiological effects

50mmol/L GABA and different salt concentrations have different effects on plant seedlings. When NO3- ion is lower than 40mmol/L, GABA will stimulate root elongation, and when NO3- ion is greater than 40mmol/L, GABA will inhibit root elongation . Moreover, GABA stimulates the absorption of low concentrations of NO3- and inhibits the uptake of high concentrations of NO3-, while enzymes such as GS are regulated by nitrogen. The above studies suggest that nitrogen has a certain role in regulating plant growth. Under the stimulation of NaCl (50mmol/L), the glycosylation metabolism of plants will initiate changes, and the effects include changes in the tricarboxylic acid cycle, GABA metabolism, amino acid synthesis and shikimate-mediated secondary metabolism. Higher salt ions lead to the oxidative degradation of soybean polyamines to GABA. Plant GABA receptors have root tolerance that modulates pH and Al3+.

Plant GAD expression and γ-hydroxybutyrate transcript abundance increased during bacterial infection, resulting in increased GABA. Tobacco plants with high GABA synthesis levels were less susceptible to A. tumefaciens C58 infection. GABA can induce the expression of the Agrobacterium ATTKLM operon, which reduces the concentration of N-(3-oxooctanoyl) homoserine lactone and down-regulates the quorum sensing signal (or hormone), which affects its toxicity to plants. GABA also plays a role in the signal communication between plants and bacteria. GABA can inhibit the expression of Hrpl gene in bacteria (the Hrpl gene encodes a protein that sensitizes plants or causes tissue diseases), and at the same time inhibits the expression of hrp genes in plants, so that plants are free from allergies Response (hrp: Controls the pathogenic ability of plant pathogens and causes allergic reactions).

In addition, GABA also has a ripening effect. GABA can stimulate ethylene biosynthesis by stimulating the transcriptional abundance of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Under waterlogging, ethylene can provide oxygen to plants by promoting the growth of adventitious roots. High concentrations of GABA can inhibit the growth of plant and bacterial GABA transaminase (GABA-T, GABT) mutants, and high concentrations can inhibit bacterial reproduction in plants. Inhibition of GABA-T in tomato leads to the accumulation of GABA, causing dwarfism in tomato.

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