GABA in plants (article about its mechanism)

GABA in plants (article about its mechanism)

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I am studing to plant physiology exam and i cross over the statment about GABA in plants.

It said that level of GABA increasing my thermal shock. But mechanism of effect on plants is unknown.

Can anybody give me a tipe about proper article about it?

Notes on Photobiology In Plants and Its Mechanism

Chromoprotein (photosensitive photoreceptor, blue pigment protein complex found in almost all flowering plant (angiosperms) was discovered by Borthwick et el (1952) and isolated by Butler et al (1959) from etiolated seedlings of Maize.

It exists in two interconvertible states, blue and yellow green, Pr and Pfr. Pfr is associated with cell membrane while Pr is found in diffused state in cytosol. It causes germination of seeds, inhibits flowering in SDPs and flowering in long day plants. Pr or P600 absorbs light at 660 nm and is changed to Pfr or P730.

The latter absorbs light of 730 nm and is changed back to Pr quickly. Darkness also causes this conversion but it is very slow. Phytochrome is involved in photomorphogenetic responses above 550 nm (except phototropican photonastic movements). It is required in seed germination of some plants, bud dormancy, leaf abscission, synthesis of gibberellins and ethylene, prevention of photo-oxidation, photoperiodism and morphogenesis.

Pr form is stable in dark, absorbs red light of 660 nm and changed quickly into Pfr. Pfr on absorbing far red light of 730 nm or in dark changed into Pr. This photoreversible behaviour is independent of temperature. Pr stimulates flowering in short day plants. Pfr is essential for seed germination.


At one time, phytochrome was supposed to control photomorphogenic reactions through its control on the metabolism of 2C and 3C compounds (Hendricks, 1964). Later it was thought to operate through genes (Mohar, 1966) or change in membrane permeability (Hendricks and Borthwick, 1967) by functioning as a protein enzyme for membrane structure.

Fondeville et al, (1966) observed that phytochrome controls nyctinastic (light dependent opening and closing) movements of Mimosa leaflets. Similar phytochrome controlled movements were later observed in Albizzia julibrissin and stomata. It was also found that such movements are connected with influx and efflux of K + ions. Therefore, phytochrome causes turgor changes in the cells through – change in active ion transport or membrane properties.

Phytochrome may cause photomorphogenetic response within a few seconds (Newman and Briggs, 1972) or after several hours. The former is called rapid phytochrome response. It seems that the rapid phytochrome response is caused by changes in membrane properties.

For example the excised roots of Barley present in a beaker having a solution of ATP, Mg 2+ and ascorbic acid immediately adhere to the glass surface when exposed to red light. They are released on exposure to far-red light. It shows clearly that phytochrome is located in the cell membrane (Haupt, 1972). It acts by its action on membrane bound enzymes.

Phytochrome operation may also take place through acetylcholin or related compound (Jaffe, 1970). Yamamoto and Tezuka (1972) proposed that phytochrome may act through regulation of NADP + active in the cells. It is just possible that both NADP + and acetycholine-like compounds may be related to some reactions or intermediates of phytochrome action.


In recent years, soil salinization has become an alarmingly severe problem, affecting 10% of the land surface in the world [1] as well as 42.9% of protected soil in China [2]. This issue has become the main obstacle for the sustainable production of protected agriculture. Salt stress harms crops mainly because of the presence of excess ion in soil among them, Na + and Cl − are not essential mineral but are the main ions causing salt stress injury to plants [3]. Recently, the total soil salt content in greenhouse vegetable fields increased by 69.3% (in which Na + and Cl − increased by 140 and 58% respectively) [4]. Tomato (Solanum lycopersicum L.), one of the most widely cultivated vegetable crops, is a moderately salt-sensitive crop [4, 5]. However, soil salinization often severely affects tomato fruit yield and quality by decreasing photosynthetic efficiency and disturbing physiological metabolism due to ion toxicity, osmotic stress, nutrient deficiency, etc. Apparently, compared with the slow progress of breeding [1], the regulation of salt stress tolerance by exogenous substances is a fast and effective method to relieve salt damage in crops, especially by regulating various ion transport pathways and the related metabolism.

Gamma-aminobutyric acid (GABA), a four-carbon non-proteinogenic amino acid, connects the two major metabolic pathways of carbon and nitrogen in plants and its content is significantly higher than that of other non-protein amino acids [3]. It has an important effect on plant growth and abiotic stress resistance as a signal substance or metabolic product by regulating cytoplasmic pH, acting as a temporary nitrogen pool and inducing antioxidant responses [6, 7]. Maintaining cellular ion homeostasis is an important adaptive trait of plant under salt stress [8]. Our previous study showed that exogenous GABA application influenced the absorption and inhibition of mineral elements in cucumber seedlings under NaCl stress and the addition of 5 mmol·L − 1 GABA significantly reduced the accumulation of sodium ions in cucumber roots under salt stress [9]. However, there was no strong evidence that GABA directly reduced Na + to relieve salt stress.

Previous studies have demonstrated that the anabolic metabolism of GABA could be activated by salt stress induction and as a result, GABA accumulation has been observed to increase rapidly in a number of plant species, such as tomato, tea, tobacco, and Arabidopsis [10,11,12,13,14]. Among these plants, the GABA content was enhanced approximately 20-fold in Arabidopsis seedlings under 150 mmol·L − 1 NaCl [13], and GABA levels increased significantly in seedlings of lentils treated with 25–100 mmol·L − 1 NaCl [15]. Furthermore, it has been demonstrated that the synthesis-related accumulation of endogenous GABA is closely related to exogenous GABA supplementation, which increased by 29% in hulless barley and by 1-fold in Caragana treated with 0.5 mmol·L − 1 and 10 mmol·L − 1 GABA, respectively, under salt stress [16, 17]. Therefore, it is believed that endogenous GABA, which is affected by salt stimulation and exogenous GABA induction, plays a vital role in improving plant resistance to salt stress via regulatory metabolic pathways [18,19,20]. However, how exogenous GABA affects endogenous GABA synthesis at transcriptional and metabolic levels remains unknown.

The improved salt tolerance due to GABA in plants is related to many physiological metabolic pathways, including the control of reactive oxygen species (ROS) accumulation in tomato [21], the regulation of redox balance and chlorophyll biosynthesis [22], the enabling of cytosolic K + retention and Na + exclusion in Arabidopsis [1] and the alteration of cell wall composition [13]. We previously reported that GABA synthesis and supplementation are crucial for enhancing salt tolerance by decreasing ROS generation and photosynthesis in tomato [23] and accelerating NO3 − reduction and assimilation in pakchoi [24]. Although there are many physiological metabolic pathways involved in the GABA-related plant salt tolerance, the mechanism by which GABA improves plant salt tolerance has not been elucidated clearly. However, these functions of GABA in plants are performed mainly via a short pathway known as the GABA shunt [7]. During this process, GABA accumulation plays a vital role due to irreversible synthesis catalysed by glutamatedecarboxylase (GAD EC 4.1.l.15) [12, 25] as well as the uptake and transport of exogenous GABA [22].

It has been shown that GAD is the most sensitive gene for GABA metabolism in response to abiotic stress. GAD enzyme activity and gene expression levels are closely related to the GABA-mediated enhancement of plant stress resistance [12]. GAD simultaneously catalyses glutamate (Glu) degradation and GABA synthesis. To date, GAD genes have been cloned and identified in various plant species, including tomato [25], citrus [26], tea [11] and other plants. Moreover, GABA levels are regulated by GAD transcriptional expression and enzyme activity regulation, which contributes approximately 61% to the accumulation of endogenous GABA in NaCl-treated soybean [27] and the GABA content decrease by approximately 50–81% in mature green fruits of SlGAD2-suppressed lines [25]. GAD gene expression significantly increases GABA accumulation in five wheat cultivars under salt and osmotic stress [28]. OsGAD2 was the most important gene for GABA accumulation in rice, exhibiting increased activity in vitro and in vivo, showing that transgenic OsGAD2 had over 40-fold higher activity than wild type (WT) [29]. SlGAD2 and SlGAD3 play key roles in regulating GABA levels in tomato fruit, showing that transgenic over-expression lines contained higher levels of GABA (2.7- to 5.2-fold) than the WT [25]. However, there were significant differences in expression sites in different plants under NaCl. CiGAD1 was expressed in the stem, leaf and seed coat of Caragana intermedia, while CiGAD2 was highly expressed in the bark [17]. The increase in expression of CiGAD1 and CiGAD2 induced GABA accumulation within 24 h of salt treatment [17]. GAD2 expression in all parts of Arabidopsis and tobacco was significantly enhanced, accompanied by increased GAD activity and increased GABA content [12, 13]. The CsGAD gene enhanced the salt and alkali tolerance of melon by increasing leaf GAD activity and GABA content [22]. However, only a limited number of studies have examined the relationship between GADs, GABA and tomato salt tolerance, and the metabolic processes and related metabolism have not been identified.

To experimentally elucidate the relationship between GABA supplementation and tomato plant salt tolerance, we investigated plant growth and changes in Na + flux and accumulation in NaCl-treated with GABA added plants. For the first time, we analysed the transcription level of four GAD genes in tomato leaves and detected the amino acid synthesis (including GABA) and metabolism of ROS in leaves to explore the physiological functions of GABA in salt-damaged tomato seedlings. The objective of the study was to elucidate the regulatory mechanism by which exogenous GABA enhances salt tolerance in tomato plants, which might provide new information regarding the molecular regulation of GAD genes and the subsequent effect of GABA on ion uptake or metabolic processes in tomato plants under high-NaCl conditions.


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How to beat the heat: Memory mechanism allows plants to adapt to heat stress

"If you can't stand the heat, get out of the kitchen," as the old saying goes. But for organisms that can't leave the proverbial kitchen when things get too hot, there's another way: researchers from Japan have discovered that plants can gain heat tolerance to better adapt to future heat stress, thanks to a particular mechanism for heat stress 'memory'.

In a study published in Nature Communications, researchers from Nara Institute of Science and Technology have revealed that a family of proteins that control small heat shock genes enables plants to 'remember' how to deal with heat stress.

Climate change, especially global warming, is a growing threat to agriculture worldwide. Because plants can't move to avoid adverse conditions, such as potentially lethal high temperatures, they need to be able to deal with factors such as heat stress effectively to survive. Therefore, improving the heat tolerance of crop plants is an important goal in agriculture.

"Heat stress is often repeating and changing," says lead author of the study Nobutoshi Yamaguchi. "Once plants have undergone mild heat stress, they become tolerant and can adapt to further heat stress. This is referred to as heat stress 'memory' and has been reported to be correlated to epigenetic modifications." Epigenetic modifications are inheritable changes in the way genes are expressed, and do not involve changes in the underlying DNA sequences.

"We wanted to discover how plants retain a memory of environmental changes," explains Toshiro Ito, senior author. "We examined the role of JUMONJI (JMJ) proteins in acquired temperature tolerance in response to recurring heat within a few days."

JUMONJI proteins are histone demethylases. Demethylases are enzymes that remove methyl groups from molecules such as proteins, particularly histones, which provide structural support to chromosomes. The team revealed that plants are able to maintain heat memory because of lowered H3K27me3 (histone H3 lysine 27 trimethylation) on small heat shock genes.

"We found that these proteins are necessary for heat acclimation in Arabidopsis thaliana. These results, along with future studies, will further clarify the mechanisms of plant memory and adaptation," says Yamaguchi.

This research will be relevant to genetic research in a number of fields, including biology, biochemistry, ecology, and environmental and agricultural sciences, and is applicable to the study of animals as well as plants. Understanding the epigenetic memory mechanism revealed in this study will help in working with heat tolerance to maintain the food supply in natural conditions.


Physiological and proteomic mechanisms with different temperatures and exogenous GABA through GABA shunt

Plants have evolved complex mechanisms to deal with low temperature. When plants sense low temperature, a series of protective mechanisms are triggered – including decreasing malondialdehyde (MDA) content, which is considered to be an indicator of plant oxidative stress synthesizing cryoprotectant molecules and low-molecular-weight nitrogenous compounds and improving the scavenging activity of reactive oxygen species (ROS) [40,41,42]. In addition, chlorophyll fluorescence imaging can be employed to evaluate cold tolerance in numerous plants. [43] These alterations help plants keep a metabolic balance of substance and energy in cold environments. Under low temperature, tea plants adopted a ‘survival mode’ as shown by higher free proline content, decreased MDA content, regulating the antioxidant activity and reduced photosynthesis, resulting in growth arrest (Additional file 9: Figure S1 and Additional file 10: Figure S2). GABA can be quickly accumulated and is involved in adapting to low-temperature stress [44, 45]. However, in our study, the endogenous GABA content decreased when plants were exposed to low temperature. We speculate that endogenous GABA degraded to promote synthesis of other stress response substances and feedback to the TCA cycle at low temperature, which showed that other amino acid contents and the DAPs in the TCA cycle were up-regulated at low compared to optimum temperature.

The GABA metabolism in tea plants could regulate the GABA precursor glutamate and PAs contents to relieve anoxia stress in our previous study [14]. No significant changes in GABA-T, GAD and DAO activities, Put and Spm levels for the two different temperatures compared to controls, while application of exogenous GABA. The PAO activity clearly decreased compared to control for optimum temperature at day 4, and the SPD concentration was higher at low temperature than the control (Additional file 11: Figure S3 and Additional file 12: Figure S4). The results imply that application of exogenous GABA can feed back to the TCA cycle to promote the carbon and nitrogen cycle. The amino acid contents except for alanine and lysine all declined (Additional file 7: Table S7). A reversible transformation of an amino acid from glutamic acid to pyruvic acid was catalyzed by alanine aminotransferase (AlaAT) form 2-oxoglutarate and alanine. The AlaAT-related protein (Q9S7E9) showed no significant change in treatment T2 compared to T1, which matched the report showing that AlaAT1 broke down excess alanine [46]. Lysine is synthesized by a special branch of the family pathway of aspartic acid. Carbon atoms from lysine to acetyl coenzyme A, which then enters the TCA cycle, generatingα-ketoglutaric acid [47]. These results imply that, at optimum temperature, application of exogenous GABA influenced the GABA level resulting in response of metabolism pathways to the alert in carbon and nitrogen transport.

The iTRAQ-based protein analysis was a great information-rich approach for hunting the stress-induced dynamic proteins for pinning down the key metabolic pathways, it may play important roles in the response of improving resistance to low temperature in tea plants with application of exogenous GABA. The results indicated that application of exogenous GABA at low temperature compared to control profoundly changed metabolic pathways, including amino acids biosynthesis, flavonoid biosynthesis, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms and the pentose phosphate pathway. These pathways included most of the DAPs in treatments T4/T3 however, pathways such as TCA cycle, glutathione metabolism, ascorbate and aldarate metabolism, and purine metabolism were also important. To our knowledge, this is the first report on dynamic proteomic responses to exogenous GABA application at low temperature in tea plants.

Flavonoid metabolism regulated with exogenous GABA at low temperature

Flavonoids are important secondary metabolites in plants and play important roles in many functions, which included pigment and antioxidant activities. Five DAPs related to flavonoid biosynthesis were observed in treatment T4/T3 (Fig. 7a). Anthocyanins are a major class of flavonoids, whose functions are very diverse which include antioxidant activity, UV rejection, defense of plant pathogens, leguminous nodulation, man fertility, optical signal and auxin transport govern [48]. Under salt treatments, the proline content increased while the wheat genotypes have higher anthocyanin content [49]. Low temperature induces anthocyanin synthesis in various species [50], it was also reported that overexpression of flavonol glycosides and anthocyanins in plants under abiotic stress can effectively remove ROS and improve drought tolerance of plants. [51, 52] Of the three anthocyanin-related genes selected for RT-qPCR, CsBAN was up-regulated and CsCHS and CsF3H were down-regulated (Fig. 7b), which indicating that the expression of CsCHS, CsF3H and CsBAN involved in anthocyanin biosynthesis, could be induced by cold treatment [53]. Our results also indicate that the effects of exogenous GABA for the three genes involved in anthocyanin synthesis in tea plants at low temperature differed, as did the genes in the process of mRNA expression and protein expression in the flavonoid metabolism, and should be further studied.

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to flavonoid biosynthesis. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). BAN: NAD(P)-binding Rossmann-fold superfamily protein CHI1: chalcone flavanone isomerase 1 CHS: chalcone synthase F3H: flavanone 3-hydroxylase FLS1: flavonol synthase 1

Amino acid metabolism and ascorbate (AsA)/glutathione cycle and exogenous GABA at low temperature

The levels of GABA, proline, tryptophan, histidine, asparagine and alanine showed the greatest changes in abundance in response to GABA application at low temperature compared to control (Additional file 7: Table S7). Combining with the metabolome and proteome, we found no significant relation between them. There are few published studies of amino acid export in the xylem under saline conditions [54]. After being treated with exogenous GABA at low temperature for 7 days, except for glycine, contents of all amino acids in leaves rose greatly.

Glutamine synthetase (GS) is an enzyme in the process of plant primary nitrogen assimilation, since glutamine and other related nitrogenous compounds are converted by it catalyzing ammonium, which is also a substrate for protein synthesis. The reabsorption of ammonia in photorespiration is the main role of chloroplast GS, and chloroplastic GS aided nitrogen assimilation in chloroplasts of YL [55]. GS was down-regulated here, and most amino acid contents increased, indicating that GS played an important role in the process that GABA works as a signal molecule when plants exposed to cold stress. Cysteine synthase catalyzes the synthesis of cysteine from O-acetylserine and disulfides, which serves as the only amino acid containing disulfide bonds (S–S) that protecting cellular environments from oxidative stress [55]. Cysteine synthase and spermidine synthase might be associated to the rise molecular chaperone activity and molecular chaperone and the decline in oxidative stress [56]. In the present study, cysteine and spermidine contents increased significantly but no differences between treatment T4 and T3, suggesting regulatory mechanisms occurring in response to cysteine and spermidine and involving both transcriptional and post-translational levels (Fig. 8a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to amino acid metabolism and AsA/glutathione cycle. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: ASA1: anthranilate synthase alpha subunit 1 CM2: chorismate mutase 2 GGAT2: glutamate--glyoxylate aminotransferase 2-like GLN1–4: glutamine synthetase 1–4 GPX7: glutathione peroxidase 7 GSTF10: glutathione S-transferase PHI 10 GSTF9: glutathione S-transferase PHI 9 LAP2: Cytosol aminopeptidase family protein LGALDH: L-galactose dehydrogenase MDAR5: monodehydroascorbate reductase 5, chloroplastic-like METK4: S-adenosylmethionine synthetase 4 PGK3: phosphoglycerate kinase precursor RPE: ribulose-5-phosphate-3-epimerase SK1: shikimate kinase 1 TKL-2: transketolase 2

Cell process and in vitro stress produce AsA, a step in the biosynthetic pathway of which is the catalysis of GDP-mannose-3′, 5′-diisomerase (GME) between GDP-galactose and GDP-mannose [57]. Spinach L-galactose dehydrogenase (L-GalDH) show invertible inhibition by AsA, the final product of biosynthetic pathway [58]. AsA peroxidase (APX) plays a main role in cellular H2O2 metabolism. GME, L-GalDH and leucyl amino peptidase had lower expression in tea plants, but APX, glutathione S-transferase and glutathione exhibited an opposite trend (Fig. 8a), which implied that exogenous GABA application might regulate AsA and glutathione metabolism by different mechanisms. Sensitive genes in amino acid metabolism and the AsA/glutathione cycle had their relative expressions determined using RT-qPCR (Fig. 8b) and these were consistent with the related DAPs.

TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organs and GABA shunt and exogenous GABA at low temperature

The TCA cycle includes important approaches to plant synthetic amino acid, energy supply and different biological movement course [59]. Malate dehydrogenase (MDH) catalyzes the effect of NAD+ on NADH oxidation of malic acid was reproduced in mitochondrial matrix during TCA cycle [60]. Environmental stresses including drought, heat, salinity and aluminum can decrease the MDH level in various plant species [61,62,63]. Exogenous GABA application at low temperature resulted in lower MDH level compared with the control, suggesting that exogenous GABA improved the low-temperature-induced reduction in MDH to catalyze the inhibited malate to oxaloacetate during malate metabolism. All of the DAPs in the TCA cycle showed greater down-regulation in treatment T4 compared to T3 (Fig. 9a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organisms. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: AGT1: alanine-glyoxylate transaminase FBA2: fructose-bisphosphate aldolase 2 GAPB: glyceraldehyde-3-phosphate dehydrogenase B subunit GDH3: glutamate dehydrogenase 3 GGAT2: glutamate--glyoxylate aminotransferase 2-like GLDP2: glycine decarboxylase P-protein 2 GLO1: glyoxalase NADP-ME4: NADP-malic enzyme 4 PDH-E1: pyruvate dehydrogenase E1 PMDH1: peroxisomal NAD-malate dehydrogenase 1 RBCL: ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit RBCS-2B: Ribulose bisphosphate carboxylase small chain 2B SDH1–1: succinate dehydrogenase 1–1

In addition, MDH participates in amino acid synthesis because of the reactions of malic acid, acetoacetic acid and aspartic acid [64]. With the significant decrease in MDH and succinate dehydrogenase and increases in aspartic acid, the contents of threonine, isoleucine, lysine, alanine, valine and serine were elevated with exogenous GABA application at low temperature. It was reported that the levels of alanine, valine and serine were affected by the stimulation of elevated endogenous GABA under abiotic stresses: Bermuda grass under heat stress [65] and tobacco under salt [66]. The GABA shunt was considered to be a part of the TCA cycle during respiration [67,68,69], which is also important in primary carbon and nitrogen metabolism [70]. Increased GABA promoted the alanine substance accumulation, which entered the proline metabolism and TCA cycle. All amino acid contents except for glycine were increased by elevated GABA at low temperature, which suggested that GABA increased the carbon and nitrogen metabolism. However, the proteins related to the GABA shunt showed no significant changes, but the GABA precursors and the enzyme activity of GABA metabolism clearly changed.

Glyoxylate cycle was firstly discovered in microorganisms, which provided substrates for biosynthetic processes and respiration [71]. In addition to lipids, glyoxylate cycle is another carbon source necessary for growth after germination [72]. Most of the proteins involved in the glyoxylate cycle were down-regulated in response to low temperature with exogenous GABA compared to the control. Furthermore, the exogenous GABA could relieve the cold damage to photosystem II, which was crucial in carbon metabolic [73]. There are many signaling molecules, such as nitrate, ammonium, sugar, amino acids and organic acids, also contain interactions between carbon and nitrogen metabolism [74]. Nitrogen uptake and metabolism is contributed by carbon metabolism since it requires carbon skeletons, reducing power, ATP, reductants and co-transporters. [73, 75,76,77]. Their control may be closely linked and coordinated at the level of gene expression [78]. However, only aminomethyl transferase and fructose-bisphosphate aldolases (FBAs) were up-regulated in the TCA cycle, glyoxylate and dicarboxylate metabolism, and carbon fixation in photosynthetic organisms in treatment T4 compared to T3. Fructose 1,6-bisphosphate aldolases, which are important in the Calvin-Benson cycle (CBC), were dramatically altered when tomato seedlings suffer from heat/cold stresses [79]. This might imply that exogenous GABA application could improve photosynthesis to regulate low-temperature response through FBA expression and enzyme activity.

The above evidence suggests that, as a signal molecule, GABA regulates the glyoxylate cycle, the TCA cycle and carbon fixation in photosynthetic organ metabolism in a complex manner, especially in the carbon and nitrogen cycle. It will be interesting to establish how tea plants respond to low temperature via different mechanisms and how the signal molecule GABA influences the operating mechanisms. Several genes in the TCA cycle, glyoxylate cycle and carbon fixation in photosynthetic organisms had their relative expression determined by RT-qPCR (Fig. 9b) and the expression mode of genes was similar to the response of related DAPs.

Oxidative pentose phosphate pathway and purine metabolism and exogenous GABA at low temperature

The energy and metabolic intermediates in the biosynthesis are mainly derived from the pentose oxidation pathway. However, further details are needed on how the pathway and its effects on other processes in plants. Nitrate is the major source of nitrogen that plants need, and the location of nitrate assimilation significantly affect the plant energy budget [73]. This implies that exogenous GABA application could affect the energy budget in tea plants. Only FBA in the oxidative pentose phosphate pathway was up-regulated in treatment T4 compared to T3 here. Lu et al. [80] reported that all FBA genes in Arabidopsis thaliana, except AtFBA6, were up-regulated in response to cold stress. The carbon skeleton of the amino acid synthesis pathway arises from different sectors of the respiratory pathway. Some root in the oxidative pentose phosphate pathway (or, in the light, the CBC) and glycolysis (Ery4P and PEP for the synthesis of aromatic amino acids), some stem from the final product of glycolysis (pyruvate for alanine) and other organic acids from the TCA cycle [73]. For treatment T4 compared to T3, FBA was not only in glycolysis cycle but also in CBC, FBA2 and FBA3 genes were up-regulated, but the DAPs in the TCA cycle were down-regulated. Testing how the application of exogenous GABA in tea plants affects the respiration pathway of amino acid biosynthesis through reverse genetic pathway under low temperature, is further needed.

Purine nucleotides are produced by two distinct routes in plants: de novo and salvage pathways. The de novo synthesis employs 5-phosphoribosyl-1-pyrophosphate, aspartate, glycine, glutamine, HCO 3− and 10-formyltetrahydrofolateas for building blocks, which could be found in all plants, such as tea-leaves however, the salvage pathways are more diverse and less well understood [81, 82]. Tea plants engender special nitrogen compounds including theanine and caffeine and their effects on human health have been studied in detail [83]. In order to against herbivores and pathogens, young shoots will accumulate caffeine and leaves may be treated as a chemical defense of young soft tissue [84]. All the DAPs in purine metabolism, except ureidoglycolate amidohydrolase, were up-regulated (Fig. 10a).

Effect of application of exogenous GABA at low temperature on (a) DAPs and (b) RT-qPCR analysis of genes related to oxidative pentose phosphate pathway and purine metabolism. Transcript abundance was calculated according to the difference in cycle threshold values between the target gene and β-actin transcripts normalized by the 2 −ΔΔCT method. The mRNA levels of the genes in tea leaves at 0 h were set as 1.0. Data represent the mean value ± standard deviation. Means with different letters significantly differ from each other (p ≤ 0.05). Abbreviations: ALDH1: Aldehyde dehydrogenase CYFBP: Fructose-1,6-bisphosphatase FBA3: fructose-1, 6-bisphosphate aldolase

Thus, the evidence suggests that exogenous GABA application either directly or indirectly stimulated flux into amino acid and caffeine biosynthesis and regulated the plant energy budget. This affected the resistance to cold in tea plants and the quality of tea flavor through the oxidative pentose phosphate pathway and purine metabolism. Key genes in the oxidative pentose phosphate pathway and purine metabolism were selected for RT-qPCR, which showed that expression mode of genes were similar to that of related DAPs (Fig. 10b). Above all, the effects of exogenous GABA at low temperature on physiological index and DAPs in metabolism pathways, were summarized on Fig. 11.

Effect of exogenous GABA at low temperature on physiological index and DAPs in metabolism pathways. Models of physiological index and possible metabolism pathways by exogenous GABA compared to that without GABA under low temperature in the tea plants. Antioxidant activities had no significant difference (green), Polyamine contents increased slightly (blue), while other index and the metabolism pathways above were affected by exogenous GABA (red)


The identification of phytochemicals in herbal materials is a critical step during the process of system biology analysis. Herbal materials are often subjected to extraction, concentration, and/or purification, resulting in the phytochemical compositions alteration. The phytochemical data from current databases (e.g., TCMSP, TCMID, may not be used directly for system biology investigation. Additional methods for phytochemical identification, such as UPLC-Q-TOF/MS, should be a complementary tool to obtain more accurate results of phytochemical compositions (Shen et al., 2013). In current work, a series of flavonoid glycosides and saponins were identified from SZJ extract, in which the spinosin derivatives including 6’’’-vanilloylspinosin, 6’’’-para-hydroxylbenzoylspinosin, 6’’’-sinapoylspinosin, 6’’’-para-coumaroylspinosin, 6’’’-feruloyspinosin, 6’’’-(-)-phaseoylspinosin, and 6’’-O-feruloylspinosin are rare in other plant species. The studies on their bioactivities and effective targets have been so poorly reported that there is not enough data for system biology analysis. In addition, their chemical structures are complicated and contain multi-chiral centers that bring a great challenge to obtain the potential targets through reverse virtual fishing technique. Traditional system biology analysis would take ADME (absorption, distribution, metabolism, and excretion) screening strategy that may exclude these glycosides with low oral bioavailability and low drug-likeness (Yue et al., 2017a Yue et al.,�). For instance, ginsenosides are the dominant phytochemicals in ginseng and are thought to be contributed to its multiple bioactivities (Ru et al., 2015 Kim et al., 2017). However, the above analysis strategy, i.e., ADME screening, would exclude the ginsenosides along with their contributions on efficacy when performing systematic research of ginseng. Therefore, such an analytical strategy is incomplete and not systematic. In fact, it has been well demonstrated that the metabolites of the ginsenosides are responsible for the specific bioactivities (Chen et al., 2018 Kim, 2018). Similarly, the chemoinformatics and pharmacoinformatics approach indicated that jujubogenin was the effective GABAA agonist, neither jujuboside A nor jujuboside B (Chen, 2009). Gut microbes play an important role in favoring phytochemicals transformation into metabolites endowed with biological activity (Dey, 2019). As a result, the strategy that involves the metabolites of glycosides in gastrointestinal environment (e.g., gut microbes) will be a more reasonable approach to understand the actual efficacy and mechanism of herbal materials in which the glycosides are considered as the main active components.

The GABAA receptors are chloride channels and are composed of several subunit classes (α, β, γ, δ, and ϵ) (Olsen and Sieghart, 2008). GABAergic neurotransmission plays an important role in anxiety status. Previous studies have shown that deficit of GABAA receptors and reduction of GABA transmission were observed in people with anxiety-like symptoms (Horowski and Dorow, 2002 Nutt and Malizia, 2004 Hasler et al., 2008). In contrast, positive modulation of GABAA receptors and enhancement of GABA transmission have shown anxiolytic effects. Classic benzodiazepines reduce anxiety by interacting with the GABAA receptors via the benzodiazepine binding site, which is present at the interface of 㬑, 㬒, 㬓, or 㬕 subunits and γ subunit of GABAA receptors (Möhler, 2012). Other classes of compounds, GABA, barbiturates, and alcohol also could act at different benzodiazepine binding sites to increase the opening of the chloride channel resulting in enhancement of inhibitory synaptic transmission (Harris, 1990 Schousboe and Redburn, 1995). The results of our system biology study suggested the GABAA receptors signaling is a significant pathway involving in anxiolytic effect of SZJ. In fact, pharmacologic study has found that spinosin, a major C-glycoside flavonoid in SZJ, exerted anxiolytic-like effects via modulation of GABAA and 5-HT receptors (Liu et al., 2015). Similarly, 6′′′-feruloylspinosin and spinosin have been reported to significantly enhance the expression of GABRA1 and GABRA5 mRNA in rat hippocampal neurons (Qiao et al., 2016). In addition, it has been found that stimulation of jujuboside A at 50 µg/mL could increase the mRNA transcription levels of GABRA1, GABRA5, GABRB1, and GABRB2 in hippocampal neurons (You et al., 2010 Wang et al., 2015) however, long time stimulation of jujuboside A at a high dose of 100 µg/mL result in the decrease of GABRA1 and GABRB2 mRNAs expression (You et al., 2010). These results suggested a two-way modulatory effect of SZJ on GABRA1 and other GABAA receptors. Such benefits were similar to what we found in this work, that is, SZJ extract enhanced mRNA level of GABRA1 in non-H2O2 treated SH-SY5Y cells, but inhibited the H2O2-induced overexpression of GABRA1. Therefore, combining with the results from literatures and our results, it was suggested that SZJ exhibited anxiolytic effects through modulating GABAA receptors, in which a two-way modulation of GABRA1 may play an important role.

It was well established that the alteration of various behaviors in anxiety disorders including appetite, mood, sleep, and cognitive function have been linked to the serotonergic system (Liu Y. et al., 2018 Liu et al., 2019). Serotonin receptors are prevalent throughout the nervous system and the periphery, and they potentially control the serotonergic neurotransmission throughout the brain and neuronal activity to alleviate neuropsychiatric disorders (Okazawa et al., 1999). Generally, the activation of HTR1A, HTR2A receptors can produce anxiolytic effects, whereas inactivation of them increases anxiety-like behaviors (Clinard et al., 2015 Spiacci et al., 2016). Involvement of other 5-HT receptors including HTR1B, HTR1B, and HTR2C in the mechanisms of anxiety have also been recognized (Graeff et al., 1996 Griebel et al., 1997 McCorvy and Roth, 2015). Similarly, our system biology analysis found that the serotonergic synapse pathway was dominant in anxiolytic effects mechanism of SZJ extract, in which different subtypes of 5-HT receptors were involved. Notably, as the same effect on GABRA1, SZJ extract also showed a two-way modulation on HTR1A and HTR2A in our RT-qPCR test. Genetic studies in animal models have suggested that anxiety-like behavior can increase when the HTR1A function is eliminated or overexpressed (Overstreet et al., 2003). Hence, these results suggested the involvement of modulating serotonergic synapse pathway, specifically two-way modulation of HTR1A and HTR2A in anxiolytic effects of SZJ.

In addition, the cannabinoid receptors (CNR) are extensively expressed in areas of the nervous system and have been found closely associated with anxiety behavior (Akirav, 2011). It has been well illustrated that endocannabinoid (eCB) reduces the serotonin release in the central nervous system and increases the expression and function of HTR1A in the hippocampus via the activation of CNR1 (Haj-Dahmane and Shen, 2011 Patel et al., 2017). Beyond CNR1, eCB system could exert actions on other targets including CNR2, transient receptor potential vanilloid receptor type 1 (TRPV1), or cyclooxygenase-2 (COX2) to participate in improvement of anxiety (Patel et al., 2017). In addition, cyclic AMP-responsive element-binding protein (CREB) has been suggested to be crucial for the role of HTR1A in modulating anxiety-related behaviors via mediating hippoacampus structural plasticity (Zhang J. et al., 2016). Intriguingly, this systematic analysis work showed phytochemicals in SZJ extracts potentially act on the above mentioned targets including CNR1, CNR2, TRPV1, COX2, and CREB. These results, to some extent, suggested that the mechanism of action of SZJ in anti-anxiety may also involve those pathways/targets that indirectly modulate eCB and serotoninergic systems. More attention needs to be paid to those targets/pathways in further experimental studies on anxiolytic effects of SZJ.

The phytochemicals in herbal medicines are the substantial basis for their pharmacologic actions. Those phytochemicals with good bioactivity and high content are considered to be the chemical markers in quality control of the herbal medicines. Jujuboside A and spinosin are used to quality markers of SZJ crude drug in Chinese Pharmacopoeia (Edition 2020). Combining the results from literatures reports (Han et al., 2009 Abdoul-Azize, 2016) and our results, the modulations of GABAergic and serotoninergic systems seem the major mechanisms of SZJ exerting anxiolytic effects, as well as the traditional efficacy of nourishing heart and calming mind. Based on that, we abstracted the phytochemicals-targets-pathway sub-networks of GABAergic and serotoninergic synapse pathways. As shown in Figure 7, it demonstrated that metabolites of C-glycosides (spinosin, etc.) and jujubosides (jujuboside A, etc.) including apigenin, kaempferol, naringenin, genkwanin, and jujubogenin were involved in modulation of GABAergic and serotoninergic synapse pathways. The result provides the extra evidence to support that C-glycosides and jujubosides are responsible for the anxiolytic effects of SZJ, and they support jujuboside A and spinosin as chemical markers for quality control of SZJ and its preparations. Beyond the C-glycosides and jujubosides, the involvements of triterpenic acid (e.g., betulinic acid) and alkaloid (zizyphusine) were also observed in modulation of GABAergic and serotoninergic synapse pathways. Specifically, betulinic acid is a function of the modulating GABAergic system via multiple subtypes of GABAA receptors, whereas zizyphusine is a function of the modulating serotoninergic system via multiple subtypes of 5-HT receptors. Notably, it has been reported that zizyphusine was identified as one of the principal components in SZJ by principal component analysis (Sun et al., 2014). And according to records in TCMIP, zizyphusine is exclusively derived from Ziziphus jujube (fruit or seeds), and its bioavailability and druglikeness is much better than C-glycosides and jujubosides. These findings recommend that the involvement of zizyphusine in quality control of SZJ extract and pharmacologic actions in anxiolytic effect is worth investigating in the future. Because the pharmacologic study of zizyphusine is poor at present, more attention could be paid to research and development of zizyphusine as a potential natural anti-anxiety drug.

Figure 7 Abstracted phytochemicals-targets-pathway sub-networks of serotoninergic synapse (A) and GABAergic pathways (B). Cytoscape (Version 3.6.1) was applied to construct the sub-networks.

There are some limitations of our current research. First, we did not conduct the behavioral test to confirm the anxiolytic-like effect of SZJ. Such benefit of SZJ was concluded on the base of previous pharmacodynamic studies, as well as clinical practice experience of traditional medicine. Our in vitro mRNA expression evaluation of GABAA and 5-HT receptors only used a single concentration of SZJ extract, which corresponds to approximately 90% cell viability in CellTiter-Glo assay. Leveraging the integrated approach of system biology, UPLC-Q-TOF/MS and RT-qPCR, present work contributed to the illustration of potential mechanism of action involved in the anxiolytic-like effect of SZJ. However, further in-depth preclinical studies are warranted to verify the results obtained from the current analysis.

GABA Supplements

Currently gamma aminobutyric acid is commercially available as a dietary supplement, both natural and synthetic. Natural GABA is created by a fermentation process that uses a bacteria called Lactobacillus hilgardii.

Many people consume it to sleep better and decrease anxiety. It is also famous in athletes, as it seems to contribute to fat loss and to the development of muscle mass.

This is because it produces an intense increase in growth hormone, which is critical for muscle. In addition, it allows better sleep, something that those who do bodybuilding need.

However, the use of this supplement is subject to controversy. Many believe there is a lack of scientific evidence about its benefits.

In addition, it appears that it is difficult for GABA in blood to cross the blood-brain barrier to reach the brain. Therefore, it could not act on the neurons of our nervous system.

GABA-mimetic effect on neurodegeneration and neuroregenerative potential

Behavioral experiments have lent support to the GABA-mimetic activity of Ashwagandha root extract. GABAergic neurodegeneration due to neuroleptic-induced excitotoxicity and oxidative stress is one of the etiopathological mechanisms in the pathophysiology of tardive dyskinesia (Gunne et al., 1993) and GABA agonists are shown to be effective in ameliorating the symptoms of tardive dyskinesia. The beneficial effect of Ashwagandha root extract might be due to its GABA mimetic activity. Ashwagandha, its constituents and the metabolites of its constituents promote the growth of nerves after taking it for 7 days.

An intriguing study demonstrated that chronic oral administration of withanoside IV attenuated the axonal, dendritic and synaptic losses and memory deficits induced by amyloid peptide Aβ(25�) in mice (Kuboyama et al, 2006). After oral administration in mice, withanoside IV was metabolized into sominone, which induced marked recovery in neurites and synapses and also enhanced axonal and dendritic outgrowth and synaptogenesis. These effects were maintained for at least 7 days after discontinuing withanoside IV administration. These data suggest that withanoside IV, and its metabolite, sominone, may have clinical usefulness as antidementia drugs.

Another team found that the methanol extract of Ashwagandha (5 mg/ml) significantly increased the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. The effect of the extract was dose-and time-dependent. mRNA levels of the dendritic markers MAP2 and PSD-95 by RT-PCR were found to be markedly increased by treatment with the extract. Immunocytochemistry demonstrated the specific expression of MAP2 in neurites extended by the extract. These results suggest that the methanol extract of Ashwagandha promotes the formation of dendrites (Kulkarni et al., 1993).

Aluminium Toxicity and Its Tolerance in Plant: A Review

Aluminium (Al) toxicity is one of the major abiotic stress problems around the globe where acidic soil is present. Al shows a toxic effect between the soil pH 4.5 and 5.5. Root growth inhibition is the most prodigious symptom of Al toxicity in plants. Aluminium toxicity adversely affects the plant growth and development which ultimately reduces the yield. However, the extent of toxicity depends on the genotype of the plant, that is the plant is either the Al-sensitive or Al-tolerant type. Plants have several mechanisms to cope with the toxic effects of aluminium which include exclusion mechanism and internal tolerance mechanism. This review discusses the harmful impacts of aluminium on morphological, anatomical, physio-biochemical, and molecular aspects of the plant. This review also discusses the strategies to reduce the toxic effects of aluminium in plant and various aluminium-responsive genes which can be used in genetic manipulation for better crop development.

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Watch the video: Δημήτρης Κούβελας Γιατί με διώκει ο εισαγγελέας Ο Βασιλακόπουλος ούτε 10 για διαδικασίες φαρμάκων 48 (May 2022).


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