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Article

Characterization of the Glutamate Decarboxylase (GAD) Gene and Functional Analysis of DlGAD3 in the Accumulation of γ-Aminobutyric Acid in Longan (Dimocarpus longan Lour.) Pulp

by
Weilin Wei
,
Tingting Zhang
,
Yongping Chen
,
Ziqi Zhou
,
Wenbing Su
,
Qizhi Xu
,
Yaling Zhang
,
Shaoquan Zheng
,
Jimou Jiang
and
Chaojun Deng
*
Fruit Research Institute, Fujian Academy of Agricultural Science, Fuzhou 350013, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(6), 686; https://doi.org/10.3390/horticulturae11060686
Submission received: 21 April 2025 / Revised: 30 May 2025 / Accepted: 10 June 2025 / Published: 15 June 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid, with many regulatory effects in humans. It aids in regulating blood glucose levels and pressure and is widely recognized for its ability to promote cognitive balance through the alleviation of stress and improvements in sleep quality. The GABA content of longan pulp is higher than that of many other fruits and vegetables; however, much is still unknown about GABA’s biosynthesis in longan. In this study, we found that the GABA content of ‘Baoshi No. 1’ (BS1) pulp was significantly higher than that of ‘Chunxiang’ (CX) pulp. The GAD activity was higher in BS1 pulp than CX pulp, while there was no significant difference in the GABA-T activity. Additionally, five GAD genes were identified in longan, and an analysis of their transcriptional levels showed that only the expression level of DlGAD3 corresponded to the GABA content and GAD activity. DlGAD3 was localized in the cytoplasm, and its transient overexpression promoted an increase in the GABA content in Nicotiana benthamiana leaves. Overall, our results show that DlGAD3 is able to promote the accumulation of GABA and may play a major role in its biosynthesis in longan pulp.

1. Introduction

Longan (Dimocarpus longan Lour.), an evergreen species within the Sapindaceae family, grows in tropical and subtropical regions and is extensively cultivated in southern China and Southeast Asian countries [1]. The longan fruit has extremely high economic and nutritional value [2] and is a famous example of “Medical Food Homology” in traditional Chinese medicine [3]. It has been recorded in the Compendium of Materia Medica (Ben Cao Gang Mu in Chinese) by Li Shizhen as an herbal medicine [4]. Several studies have shown that longan contains many bioactive products that have memory-enhancing effects and that are able to treat amnesia, insomnia, neurasthenia, heart palpitations, and fatigue [5,6].
GABA, a ubiquitous four-carbon non-proteinogenic amino acid widely found in plants and animals, is a bypass product of the tricarboxylic acid (TCA) cycle [7]. It is involved in many physiological processes in plants, including gene expression, cell wall modifications, interactions during signal transduction, responses to abiotic and biotic stress, and the regulation of plant development and carbon and nitrogen metabolism [8,9]. GABA can be used to improve the quality of fruits. For example, the use of exogenous GABA can increase the sugar (glucose, fructose, and sucrose) content of tomatoes by regulating gene expression [10]. Spraying pomegranates with GABA can increase their total phenolic and anthocyanin concentrations [11]. GABA is an inhibitory neurotransmitter in the human brain [12] and exerts several regulatory effects, such as relieving insomnia and regulating blood glucose levels and pressure, meaning that oral GABA intake can induce relaxation and diminish anxiety [13,14,15]. Enriching the GABA content in litchi juice could help to regulate the gut flora, thus ameliorating obesity [16].
The GABA shunt and polyamine metabolism regulate GABA biosynthesis [17]. In plants, GABA metabolism and biosynthesis are mainly regulated by the GABA shunt [18] (Figure 1), in which glutamate decarboxylase (GAD, EC 4.1.1.15) is a rate-limiting enzyme during [19]. GAD catalyzes the irreversible conversion of glutamate to GABA in the cytoplasm. Subsequently, GABA is transported into mitochondria and converted into succinic semialdehyde (SSA) by GABA transaminase (GABA-T). The conversion of SSA into succinate is catalyzed by semialdehyde dehydrogenase (SSADH), the enzyme activity of which depends on the presence of NAD+. Succinate then enters the TCA cycle [8,19].
The GAD gene has been cloned and studied in many species, such as Arabidopsis [20], tomatoes [21], wheat [22], and cotton [23]. Five members (GAD1–5) of the GAD gene family have been discovered in Arabidopsis thaliana [24]. The expression of GAD shows tissue specificity, and different GADs perform diverse functions and respond to different stimuli. AtGAD1 (At5G17330) is mainly expressed in the roots, while AtGAD2 (At1G65960) plays a key role in the leaves and shoots [24,25,26,27,28]. In rice, OsGAD4 shows the highest expression among the five OsGAD genes under certain stress conditions [29]. Furthermore, the overexpression of SlGAD2 and SlGAD3 has the potential to promote GABA biosynthesis in tomato fruits [30].
High-value compounds in longan, including GABA, have gradually gained attention [31]. The GABA content in longan is higher than that in many other fruits and vegetables, ranging from 51.48 mg/100 g to 180.42 mg/100 g [32,33]. Research has been conducted on the GAD gene family in longan during early somatic embryogenesis [34]; however, the key GAD gene for GABA biosynthesis in longan pulp remains unclear. In this study, we determined the differences in the GABA content and GAD and GABA-T activity between two longan cultivars. We identified five GAD genes and one GABA-T gene in longan through gene family analysis. Subsequently, we investigated the expression of the GADs and GABA-T through an RT-qPCR and determined that DlGAD3 plays a key role in GABA biosynthesis in longan pulp. The subcellular localization of DlGAD3 was found to be in the cytoplasm, and the transient transformation of DlGAD3 significantly promoted GABA production in Nicotiana benthamiana leaves. Our findings reveal a mechanism of GABA biosynthesis in longan pulp and provide a theoretical basis for further study.

2. Materials and Methods

2.1. Plant Materials

‘Baoshi No. 1’ (BS1), a hybrid of ‘Dongbao No. 9’ × ‘Shixia’, is known for its early maturation and high quality. ‘Chunxiang’ (CX), which flowers easily and produces a high yield, is a hybrid of ‘Dongbao No. 9’ × ‘Wanxiang’. BS1 and CX fruits were obtained from the Longan Resource Nurseries, part of the National Fruit Gene Pool (Fuzhou). The pulp was cut into pieces and mixed, with three biological replicates performed for each cultivar. All the collected samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.

2.2. Samples Preparation of Metabolomics

The samples underwent vacuum freeze-drying using a lyophilizer (Scientz-100F, SCIENTZ, Ningbo, China). Before the process, the cold trap was defrosted, and the baffles and compressor filter screens were cleaned with alcohol. The samples were pretreated for freeze-drying, and the freeze-dryer was pre-cooled for three hours. Once the shelf temperature reached −50 °C, the samples, which had been stored at −80 °C, were transferred to the shelf using dry ice. Following this initial protocol, the samples were freeze-dried for 65 h under a vacuum, during which the shelf temperature was gradually increased from −45 °C to 25 °C to achieve stable, dry conditions through water sublimation. The room temperature was maintained at approximately 25 °C. The samples were ground into a fine powder (MM 400, Retsch, Haan, Germany). The grinding jar was air-dried using a blower and immersed in liquid nitrogen for 1–2 min until the bubbling ceased. Each sample was placed in a clean grinding jar to prevent direct contact and minimize heat transfer. A steel ball was added to each jar, and the jars were tightly sealed. The samples were ground at 30 Hz for 30 s, and the resulting powder was transferred to labeled Eppendorf tubes. A portion of the sample powder (50 mg) was mixed with 1200 µL of a pre-cooled 70% methanolic aqueous solution at −20 °C. The mixture was vortexed for 30 s every 30 min, and this process was repeated six times. After centrifugation at 12,000 rpm for 3 min, the supernatant was aspirated and filtered through a microporous membrane (with a 0.22 μm pore size). These samples were then subjected to metabolomic analysis.

2.3. Metabolomics Analysis

The resulting sample extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/, (accessed on 23 December 2024); MS, Applied Biosystems 4500 Q TRAP, https://sciex.com.cn/, (accessed on 23 December 2024)). The analytical conditions have been described previously by Zhu et al. (2024) [35] and Liu et al. (2023) [36]. The metabolites identified using widely targeted metabolomics were characterized using a self-built metabolic database and secondary mass spectrometry data from Metware Biotechnology, Ltd. (Wuhan, China). There were three biological replicates each for BS1 and CX: BS1-1, BS1-2, BS1-3, CX-1, CX-2, and CX-3.

2.4. Measurement of GABA Content

The GABA content was measured in μmol/g using a γ-Aminobutyric Acid (GABA) Content Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) following the manufacturer’s protocol.

2.5. Determination of GAD and GABA-T Activities

The GAD activity, expressed in units per gram of the sample (U/g), was quantified using a GAD Activity Assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) following the manufacturer’s instructions. The GABA-T activity, expressed in units per gram of the sample (µg/h/g), was assayed using a dedicated GABA Transaminase Activity Assay Kit (Suzhou Grace Biotechnology Co., Ltd., Suzhou, China) following the manufacturer’s protocol.

2.6. RT-qPCR Analysis

The total RNA was isolated from the pulp of BS1 and CX using an RNAprep Pure Plant Plus Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions, and cDNA was synthesized utilizing the HiScript IV RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The qPCR was performed using the Hieff UNICON® advanced qPCR SYBR Master Mix (Yeasen Biotech cat. 11185ES03) in an LC480 (Roche, Mannheim, Germany). The PCR reaction system (with a total volume of 20 µL) contained 10 µL of the PCR Master Mix, 1 µL of the cDNA template, 0.4 µL of each primer (10 μM), and 8.2 µL of ddH2O. The two-step amplification was implemented according to the manufacturer’s protocol as follows: an initial denaturation step at 95 °C for 2 min, followed by 45 cycles of 95 °C for 10 s and 60 °C for 30 s, and a final melt curve analysis (gradient dissociation) to assess the amplification specificity. DlActB was used as the reference gene [37], and the primer sequences used are provided in Supplemental Table S2. The relative gene expression was calculated using the 2−ΔΔCt method [38].

2.7. Identification of GAD in Longan and Construction of Phylogenetic Tree

The full-length protein sequences of five GAD genes (AT1G65960, AT5G17330, AT2G02010, AT2G02000, and AT3G17760) were downloaded from the TAIR database (https://www.arabidopsis.org/, (accessed on 16 December 2024)). The blast program and the five published Arabidopsis GAD sequences were used to identify all the candidate GADs in longan, and the genome of the D. longan cultivar ‘Jidanben’ was used as a reference (http://www.sapindaceae.com/, (accessed on 16 December 2024)) [39]. Simultaneously, we analyzed and downloaded the GADs of other Sapindaceae species [39], including Litchi chinensis, Nephelium lappaceum, Xanthoceras sorbifolium, Sapindus mukorossi, Acer yangbiense, and Cardiospermum halicacabum. These sequences were used to construct a phylogenetic tree using the neighbor-joining (NJ) method, and 1000 bootstrap replicates were used with the default parameters. These protein sequences are provided in Supplemental Table S3.

2.8. Transient Expression Analysis and Subcellular Localization

To verify the gene function of DlGAD3, we constructed its expression vector, pSAK227-DlGAD3-GFP, as follows: the full-length DlGAD3 gene was cloned from the cDNA templates of BS1 fruit. Subsequently, the PCR product was purified and inserted into the HindIII/EcoRI-digested pSAK277-GFP vector backbone, and pSAK227-DlGAD3-GFP was generated through homologous recombination using the ClonExpress Ultra One Step Cloning Kit (C115-01, Vazyme, Nanjing, China) following the manufacturer’s instructions. The expression vector was transformed into Agrobacterium tumefaciens (GV3101) using the heat shock method and cultured on an LB agar medium at 28 °C for 3 days. A single colony was picked and cultured overnight at 28 °C, and the agrobacterium were collected through centrifugation to infect tobacco leaves. Nicotiana benthamiana leaves were infected with the Agrobacterium using an infection buffer solution (10 mM of MgCl2, 10 mM of MES, pH 5.7, 100 μM of acetosyringone), and the GFP signals were detected using a fluorescence microscope (DM4B, Leica, Wetzlar, Germany). Subcellular localization was analyzed in infiltrated leaves at 3 days post-infiltration, and the leaf samples were collected for the measurement of GABA content. The 35S promoter-driven GFP was used as a subcellular localization positive control. The primer sequences we used are provided in Table S2.

2.9. Statistical Analysis

The data were analyzed using IBM SPSS Statistics software, version 27.0 (IBM Corporation, Armonk, USA). The significance levels were tested using Student’s t-test, and *, **, and *** represent significance levels of p < 0.05, p < 0.01, and p < 0.001, respectively. The results are represented as the mean ± the standard error (SE).

3. Results

3.1. The Metabolomics Profile of the Two Longan Cultivars

Metabolome analysis was performed on the longan pulp (Figure 2A) to study the differences in metabolites between the two cultivars. The repeatability and reliability of the metabolome analysis were determined based on the overlap of the total ion current (TIC) curves for the different samples (Figure S1). A total of 1296 metabolites were detected using the UPLC-MS/MS platform (Table S1), and a category analysis revealed that the primary metabolites in the longan pulp were amino acids and derivatives (18.21%), followed by flavonoids (14.81%), phenolic acids (12.5%), lipids (11.11%), organic acids (7.71%), alkaloids (5.94%), saccharides (5.71%), nucleotides and derivatives (5.48%), terpenoids (5.09%), lignans and coumarins (4.78%), tannins (1.54%), quinones (0.39%), and others (6.71%) (Figure 2B). A hierarchical clustering heat map analysis was used to visualize the pattern of metabolite accumulation in these samples (Figure 2C). The metabolites in the pulp of the two longan cultivars were significantly different, and the biological replicates of each variety clustered together, showing the distinct metabolic characteristics of these cultivars.

3.2. Differences in GABA Content Between Two Longan Pulps

According to a metabolomics analysis, amino acids and derivatives are abundant in longan. A total of 236 amino acids and derivatives were identified, and the GABA levels differed significantly between the two longan cultivars (Figure 3A). The GABA content in BS1 was significantly higher than that in CX (Figure 3B), consistent with the results of the metabolomics analysis.

3.3. Differences in GAD and GABA-T Enzyme Activities Between Two Longan Pulps

The enzyme activities of GAD and GABA-T were measured to investigate the effect of the GABA shunt on the GABA content. The activity of GAD in BS1 was higher than that in CX (Figure 4A). However, no significant difference was observed in GABA-T activity between the two longan pulps (Figure 4B), showing that the trends in the GAD enzyme activity and GABA content were consistent.

3.4. Identification of GAD and Construction of Phylogenetic Tree for DlGADs

To confirm GAD’s function in longan pulp, five GAD genes were detected through a genome-wide analysis: D.long035428.01 (GAD1), D.long027052.01 (DlGAD3), D.long027049.01 (DlGAD3-1), D.long027050.01 (DlGAD3-2), and D.long030275.01 (DlGAD5). Using MEGA-X, a phylogenetic tree of the GAD gene family was constructed (Figure 5) comprising the GAD families of eight species: Dimocarpus longan, Arabidopsis thaliana, Litchi chinensis, Nephelium lappaceum, Xanthoceras sorbifolium, Sapindus mukorossi, Acer yangbiense, and Cardiospermum halicacabum. All the species belonged to the Sapindaceae family except for Arabidopsis. The GAD genes of longan were grouped into three groups: Group I, Group II, and Group III. DlGAD3, DlGAD3-1, and DlGAD3-2 were clustered in Group I with AtGAD2, AtGAD3, and AtGAD4. DlGAD1 was clustered in Group II with AtGAD1, and DlGAD5 was clustered in Group III with AtGAD5. This observation suggests that different members of the GAD family have different functions in longan.

3.5. Gene Expression of GAD and GABA-T in Longan Pulp

The GAD and GABA-T genes (D.long033926.01) were retrieved using the genome of ‘Jidanben’ as a reference, and we analyzed the differences in the transcriptional profiles of the two longan pulps. Notably, the transcription of DlGAD3-1 and DlGAD3-2 was not detected in either cultivar. As shown in Figure 6A, DlGAD3 exhibited significantly higher expression in the pulp of BS1 compared to CX, while the expression of DlGAD1, DlGAD5, and DlGABA-T showed no significant differences (Figure 6B–D). These findings show that the differential expression of DlGAD3 may have led to the significant differences observed in the GABA content between the two longan pulps.

3.6. The Subcellular Localization and Functional Analysis of the DlGAD3 Gene

DlGAD3 was cloned and fused with a GFP tag in an overexpression vector to assess its subcellular localization. GFP signals were detected in both the cytoplasm and nucleus in the positive control (GFP-tag); however, they were only detected in the cytoplasm when the GFP tag was fused with DlGAD3 (Figure 7A), indicating that DlGAD3 is localized in the cytoplasm. The GABA content was significantly higher than that in the control (empty vector) when DlGAD3 was overexpressed in Nicotiana benthamiana leaves (Figure 7B).

4. Discussion

Longan is a high-grade fruit in China and is valuable in traditional Chinese medicine [40], where it is considered an example of ‘Medical Food Homology’ [41]. Longan possesses high nutritional and medicinal value. It exhibits some medicinal properties, including anti-oxidant, immunomodulatory, anti-cancer, anti-osteoporotic, prebiotic, memory-enhancing, and anxiolytic effects [6]. In this study, we presented the results from a metabolite analysis of two longan cultivars using UPLC-MS/MS. A total of 1296 metabolites were detected and categorized into 13 groups. The top three metabolic pathways were those for amino acids and derivatives (18.21%), flavonoids (14.81%), and phenolic acids (12.5%). Longan pulp is rich in amino acids and derivatives, which may contribute to its taste and flavor [42]. Amino acids are the building blocks of protein, and functional amino acids, such as arginine, glutamate, glutamine, and glycine, have distinct properties [43,44]. GABA is a functional amino acid and possesses physiological functions, such as regulating the blood pressure, nervous system, and hormone secretion, enhancing liver and kidney function, boosting immunity, preventing cancer, and providing anti-aging effects in mammals [45,46]. It is widely recognized that GABA can promote cognitive balance by alleviating stress and improving sleep quality [47]. In plants, the physiological and biochemical functions of GABA have been widely studied and reported. GABA has metabolic roles and signaling functions [8] and is involved in regulating energy metabolism, the plant’s development and pH, the carbon/nitrogen (C/N) balance, and the defense system [8,48]. Longan produces fruit with a high GABA content, ranging from 51.48 mg/100 g to 180.42 mg/100 g [32,33]. In this study, we found that the GABA content of BS1 was higher than that of CX (Figure 3), providing a basis for studying GABA biosynthesis in longan.
Some treatments may induce changes in the enzyme activity of GAD and GABA-T to affect the synthesis of GABA; for example, CO2 treatments may cause a decline in the GABA concentration and glutamate decarboxylase activity in longan fruit [32]. In tomato fruit, UV-C treatment may increase the gene expression and activity of GAD, inhibit the expression and activity of GABA-T, and promote the biosynthesis of GABA [49]. CO2 treatment may also enhance the activity of GAD and promote the production of GABA in the postharvest fruit of some tomato varieties [50]. Ca2+ treatment may enhance the endogenous GABA content and upregulate the GAD enzyme activity in pear fruit [51], and the use of exogenous 5-aminolevulinic acid may promote an increase in the GAD enzyme activity, thereby increasing the GABA content in tomato fruit [52]. These findings suggest that GAD is a key gene involved in GABA biosynthesis.
Arabidopsis thaliana possesses five members of the GAD gene family [53]. The expression of GAD genes exhibits tissue-specific characteristics in plants: AtGAD1, AtGAD2, and AtGAD4 are mainly expressed in the roots and leaves of Arabidopsis thaliana in the vegetative stage, and AtGAD5 is expressed in the pollen. The expression of AtGAD3 has been detected in anthers and embryos [54]. The GABA content has been found to decrease when the expression of GhGAD6 is reduced [23], while the overexpression of BoGAD5 promoted an increase in the GABA content in broccoli sprouts [55]. PpGAD2 was found to be the key gene for the biosynthesis of GABA in pear flesh [56]. In this study, the GAD enzyme activity and GABA content were significantly higher in BS1 than CX (Figure 3 and Figure 4A), suggesting that the differential activity of GAD may underlie the difference in the GABA content between the two longan cultivars. Therefore, we analyzed the expression levels for the GAD gene family (Figure 5). The expression of DlGAD1 and DlGAD5 did not differ significantly, and the transcription of DlGAD3-1 and DlGAD3-2 was not detected in either cultivar. The expression of DlGAD3 in BS1 was significantly higher than that in CX (Figure 6A). We also found that the overexpression of DlGAD3 could promote GABA production in Nicotiana benthamiana leaves (Figure 7B). These results indicate that DlGAD3 may play a key role in the biosynthesis of GABA in longan pulp. However, reports have suggested that DlGAD5 cannot promote GABA production in longan embryogenic calluses, which may be due to their transient conversion efficiency [34]. This indicates that more research is needed on the role of GAD in GABA biosynthesis in longan. In one study, the expression of SlGABA-T1 led to a decrease in GABA during the tomato ripening period [57]. However, in our investigation, the GABA-T enzyme activity and the expression of DlGABA-T did not differ significantly between the two cultivars (Figure 4B and Figure 6D), indicating that GABA-T is not the cause of the difference in the GABA content between the two cultivars.

5. Conclusions

In summary, the DlGAD3 gene may promote GABA biosynthesis in longan pulp. Through a metabolomics analysis, we found that the GABA content in BS1 was significantly higher than that in CX. Subsequently, we found that the enzyme activity of GAD and the expression of DlGAD3 in BS1 were significantly higher than those in CX. A functional analysis of DlGAD3 showed its localization in the cytoplasm and its ability to increase the GABA content in Nicotiana benthamiana leaves through transient overexpression. Collectively, these findings demonstrate that DlGAD3 is able to promote GABA accumulation and underlies the differences in the GABA content between BS1 and CX.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/horticulturae11060686/s1: Figure S1: The TIC maps from QC samples mass spectrometry; Table S1: UPLC-MS/MS-based metabolite identification and quantification in longan pulp; Table S2: Primers used for quantitative real-time PCR and vector construction; Table S3: Proteins sequences of GADs used in this study for phylogenetic tree.

Author Contributions

Conceptualization, C.D., J.J. and S.Z.; formal analysis, W.S. and Z.Z.; funding acquisition, C.D., J.J. and S.Z.; investigation, W.W., T.Z. and Q.X.; methodology, Y.C. and T.Z.; project administration, J.J. and S.Z.; resources, Q.X. and Y.Z.; software, Y.C., Z.Z. and Y.Z.; supervision, J.J. and S.Z.; visualization, W.W.; writing—original draft, C.D. and W.W.; writing—review and editing, W.S. and J.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Funding information: Fujian Basic Research Project of Provincial Public Welfare Research Institutes: 2022R1028002; China Agriculture Research System-Litchi and Longan (No.CARS-32-24); the Financial Special Project of the Fujian Academy of Agricultural Sciences (2023), and the Technology Innovation Team Program of the Fujian Academy of Agricultural Sciences (CXTD2021004-1).

Data Availability Statement

The original contributions presented in this study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
GADglutamate decarboxylase
BS1Baoshi No. 1
CXChunxiang
GABAγ-aminobutyric acid
TCAtricarboxylic acid
SSAsuccinic semialdehyde
GABA-TGABA transaminase
SSADHsemialdehyde dehydrogenase

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Figure 1. Overview of the metabolic pathway of GABA in plants.
Figure 1. Overview of the metabolic pathway of GABA in plants.
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Figure 2. Analysis of metabolic characteristics of pulp from two longan cultivars using pie chart and heat map. (A) BS1 and CX longan fruit. (B) Classes of metabolites identified in longan pulp. (C) Clustering heat map of all metabolites.
Figure 2. Analysis of metabolic characteristics of pulp from two longan cultivars using pie chart and heat map. (A) BS1 and CX longan fruit. (B) Classes of metabolites identified in longan pulp. (C) Clustering heat map of all metabolites.
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Figure 3. Results for GABA from metabolomics analysis (A) and determination of GABA content (B) in longan pulp. Data are represented as mean ± standard error (SE) from three biological replicate assays, and significance levels were determined using Student’s t-test. ** p < 0.01; *** p < 0.001.
Figure 3. Results for GABA from metabolomics analysis (A) and determination of GABA content (B) in longan pulp. Data are represented as mean ± standard error (SE) from three biological replicate assays, and significance levels were determined using Student’s t-test. ** p < 0.01; *** p < 0.001.
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Figure 4. Activities of GAD (A) and GABA-T (B) in longan pulp. Data are presented as mean ± standard error (SE) from three biological replicate assays, and significance levels were determined using Student’s t-test. * p < 0.05.
Figure 4. Activities of GAD (A) and GABA-T (B) in longan pulp. Data are presented as mean ± standard error (SE) from three biological replicate assays, and significance levels were determined using Student’s t-test. * p < 0.05.
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Figure 5. Phylogenetic tree of the GAD gene families. Different branch colors represent different groups.
Figure 5. Phylogenetic tree of the GAD gene families. Different branch colors represent different groups.
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Figure 6. Analysis of GADs and GABA-T expression in longan pulp. (A) Relative expression of DlGAD3; (B) Relative expression of DlGAD1; (C) Relative expression of DlGAD5; (D) Relative expression of GABA-T. Data are presented as mean ± standard error (SE) from three biological replicates, and significance levels were determined using Student’s t-test. *** p < 0.001. All gene expression data were normalized to BS1, with its expression level set as 1.
Figure 6. Analysis of GADs and GABA-T expression in longan pulp. (A) Relative expression of DlGAD3; (B) Relative expression of DlGAD1; (C) Relative expression of DlGAD5; (D) Relative expression of GABA-T. Data are presented as mean ± standard error (SE) from three biological replicates, and significance levels were determined using Student’s t-test. *** p < 0.001. All gene expression data were normalized to BS1, with its expression level set as 1.
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Figure 7. Functional analysis of DlGAD3. (A). Subcellular localization of DlGAD3 in Nicotiana benthamiana leaves. Scale = 50 μm. (B). GABA content. Data are presented as mean ± standard error (SE) from four biological replicates, and significance levels were determined using Student’s t-test. ** p < 0.01.
Figure 7. Functional analysis of DlGAD3. (A). Subcellular localization of DlGAD3 in Nicotiana benthamiana leaves. Scale = 50 μm. (B). GABA content. Data are presented as mean ± standard error (SE) from four biological replicates, and significance levels were determined using Student’s t-test. ** p < 0.01.
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MDPI and ACS Style

Wei, W.; Zhang, T.; Chen, Y.; Zhou, Z.; Su, W.; Xu, Q.; Zhang, Y.; Zheng, S.; Jiang, J.; Deng, C. Characterization of the Glutamate Decarboxylase (GAD) Gene and Functional Analysis of DlGAD3 in the Accumulation of γ-Aminobutyric Acid in Longan (Dimocarpus longan Lour.) Pulp. Horticulturae 2025, 11, 686. https://doi.org/10.3390/horticulturae11060686

AMA Style

Wei W, Zhang T, Chen Y, Zhou Z, Su W, Xu Q, Zhang Y, Zheng S, Jiang J, Deng C. Characterization of the Glutamate Decarboxylase (GAD) Gene and Functional Analysis of DlGAD3 in the Accumulation of γ-Aminobutyric Acid in Longan (Dimocarpus longan Lour.) Pulp. Horticulturae. 2025; 11(6):686. https://doi.org/10.3390/horticulturae11060686

Chicago/Turabian Style

Wei, Weilin, Tingting Zhang, Yongping Chen, Ziqi Zhou, Wenbing Su, Qizhi Xu, Yaling Zhang, Shaoquan Zheng, Jimou Jiang, and Chaojun Deng. 2025. "Characterization of the Glutamate Decarboxylase (GAD) Gene and Functional Analysis of DlGAD3 in the Accumulation of γ-Aminobutyric Acid in Longan (Dimocarpus longan Lour.) Pulp" Horticulturae 11, no. 6: 686. https://doi.org/10.3390/horticulturae11060686

APA Style

Wei, W., Zhang, T., Chen, Y., Zhou, Z., Su, W., Xu, Q., Zhang, Y., Zheng, S., Jiang, J., & Deng, C. (2025). Characterization of the Glutamate Decarboxylase (GAD) Gene and Functional Analysis of DlGAD3 in the Accumulation of γ-Aminobutyric Acid in Longan (Dimocarpus longan Lour.) Pulp. Horticulturae, 11(6), 686. https://doi.org/10.3390/horticulturae11060686

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