Role of GABA transaminase in the regulation of development and senescence in Arabidopsis thaliana

Current Plant Biology 19 (2019) 100119

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Role of GABA transaminase in the regulation of development and senescence in Arabidopsis thaliana Syed Uzma Jalila, M. Iqbal R. Khanb, Mohammad Israil Ansaric,

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Amity Institute of Biotechnology, Amity University, Lucknow, 226 028, Uttar Pradesh, India Department of Botany, Jamia Hamdard, New Delhi 110065, India c Department of Botany, University of Lucknow, Lucknow, 226 007, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Arabidopsis thaliana GABA shunt pathway GABA-transaminase Developmental stages pop2 mutant

GABA (gamma amino butyric acid) transaminase (GABA-T) shows significant regulatory function during plant developmental process. It is involved in the reduction of GABA to succinic semialdehyde and participates in GABA shunt pathway, which critically contributes to nitrogen metabolism during senescence stage of plant’s life cycle. Thus, to study the function of GABA-T gene in the development and senescence processes, Arabidopsis thaliana GABA-T knock out mutants (pop2-1 and pop2-3) were characterized by the assays of leaf senescence parameters (survival efficacy, total chlorophyll content, electrolyte leakage of membrane, and lipid peroxidation) and GABA shunt components in different developmental stages. GABA-T gene mutants (pop2-1 and pop2-3) impact the overall plant morphology, leaf development, flowering time and display early onset of senescence. It was observed that pop2 mutants declined the leaf survival efficacy, total chlorophyll, GABA, GABA-T as well as glutamate decarboxylase (GAD) enzymes activities. Knockout mutation in GABA-T gene adversely elevated the electrolyte leakage of membrane, lipid peroxidation at S3 stage (30 days old plant) rather than S5 stage (50 days old plant). ROS generation and cell death was also increased in pop2 mutant leaf earlier. Taken together, our results suggest a prominent role of GABA-T gene in leaf senescence and development processes in Arabidopsis thaliana.

1. Introduction In plants, GABA is a universal, non-proteinogenic, four-carbon amino acid that act as a metabolite and a signaling molecule in defence and development mechanisms [1–6].GABA accumulates in plants under the abiotic stress conditions such as exposure of hypoxia, mechanical stress, cold, darkness, heat stress, and water lodging [7–9] all these conditions in plants lead to the senescence of a leaf [10–12], and its metabolism is potentially regulated by GABA shunt pathway[13–16]. During plant development, coordination between many complex metabolic pathways catalysed by various enzymes plays a crucial role in regulating the growth, and tissue-organ differentiation [17]. GABA transaminase (GABA-T) metabolises carbon and nitrogen by GABA shunt pathway and regulates leaf senescence process in plants [18–21].GABA shunt is well-conserved metabolic pathway present in all species of eukaryotes [9] and is shown to be crucial for the developmental processes of plants [4,10,22,23]. It has been observed that nitrogen metabolism also occurs in post-harvest citrus fruit via GABA shunt pathway [11,24].

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The synthesis of GABA occurs in the cytoplasm whereas it degrades in the mitochondria. The GABA shunt pathway comprises of three important enzymes viz.GAD, GABA-T and succinic semialdehyde dehydrogenase (SSADH) [22,25]. In plants, catabolic process of proteins generates ammonia and amino acids that get metabolised into glutamine by glutamine synthetase enzyme and later this glutamine is converted into asparagine by asparagine synthetase [9,26,27]. Some amount of glutamate is also converted into GABA by the assistance of GAD enzyme. The process of GABA to glutamate transformation accelerates when protein synthesis is found to be blocked [28]. GABA changes into succinic semialdehyde with the help of enzyme GABA-T, and finally, SSADH catalyzes the conversion of succinic semialdehyde into succinate in mitochondria and enters into the Kreb’s cycle [18,22]. Earlier studies have shown that Osl2 (Gene ID 4336983) gene encoding GABA pyruvate transaminase is involved in the nitrogen metabolism in rice senescence [18]. Arabidopsis thaliana is an important plant to study development process because of its various characteristics viz. short life span, efficient reproductive phase and substantial seed production activity. In

Corresponding author. E-mail addresses: [email protected], [email protected] (M.I. Ansari).

https://doi.org/10.1016/j.cpb.2019.100119 Received 13 May 2019; Received in revised form 24 August 2019; Accepted 24 August 2019 2214-6628/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).

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percentage of green leaves (not indicated senescence symptoms) versus all leaves of the plants grown under optimum conditions as previously demonstrated by Chen et al. [32].

addition, the genomic data of Arabidopsis thaliana facilitates characterization of the mutants and novel genes associated with plant development. The Arabidopsis thaliana GABA-T gene (POP2; AT3G22200) associated with the class-III pyridoxal-phosphate-dependent aminotransferase family [1]. The pop2 mutant showed oversensitivity to salinity stress compared to the wild type plants [29,30]. Thus, the Arabidopsis thaliana GABA-T knockout mutants malfunctioning in GABA metabolism, i.e., ideal exemplary for understanding the role of GABA-T for regulation of phases of plant life. In the present study, we have investigated the role of GABA-T gene in plant development by characterizing Arabidopsis thaliana pop2 mutants (pop2-1 and pop2-3) containing dysfunctional GABA-T gene. We examined the different physiological processes along with the analysis of GABA shunt components of homozygous pop2 mutants and wild type plants.

2.3.2. Quantification of leaf pigment Total chlorophyll content was determined as per milligram fresh weight of the wild type and pop2 mutants leaves according to Arnon [33]. Leaf tissues were homogenized in pestle and mortar with 80 % acetone and incubated at 4 °C for 4 h prior to centrifugation at 15000 × g for 5 min. Finally, the OD of the supernatant from individual samples was estimated at 663 and 645 nm wavelength by Schimadzu’s UV-1800 UV-Vis spectrophotometer. The Arnon’s equation (below) was used to convert absorbance measurement to mg Chl g-1 leaf tissueChl a (mg g-1) = [(12.7 × A663) – (2.6 × A645)] × ml acetone/mg leaf tissue Chl b (mg g-1) = [(22.9 × A645) – (4.68 × A663)] × ml acetone/ mg leaf tissue Total Chl = Chl a + Chl b

2. Materials and methods 2.1. Plant materials and growth conditions Arabidopsis thaliana wild type (ler) and GABA-T knockout mutants (pop2-1 and pop2-3) were used in the present study. The seeds were sown in soilrite and incubated at 4 °C for 3 days and then transferred into the controlled growth chamber at 22 °C for 16 h light and 8 h dark cycle with < 50% humidity and 120–150 μmol/m2 sec light intensity. Further, homozygous plants were isolated as described in earliar study [30], the genomic DNA was isolated from the leaf tissues of plants and PCR was performed using Derived Cleaved Amplified Polymorphic Sequences (dCAPS) method for pop2-1 mutant and T-DNA flanking region primers for pop 2-3 to isolate the homozygous mutant and the homozygous plants used for experimental analysis. Leaves were collected from different developmental stages (Stages-S1, S2, S3, S4, S5 and S6) from the day of sowing (DOS), as defined in Table 1. Harvested leaves from different developmental stages were preserved in liquid nitrogen and then stored at −80 °C for further analysis.

2.4. Measurement of oxidative stress traits 2.4.1. Electrolyte leakage Electrolyte leakage of membrane was determined from the harvested leaves material of pop2 mutants and wild type plants at different developmental stages [32]. Leaves disc were dissected from different stages plants and kept in falcon tubes, added with 10 ml deionised water and the tubes containing the leaf segments were kept on rotary shaker at 100 rpm for 24 h. The electrolyte conductivity of samples was quantified by conductivity meter after 24 h (EC1). Thereafter, the leaf samples incubated at 90 °C for 1 h and cooled at room temperature for measuring the final electrolyte conductivity (EC2). The electrolyte leakage % was evaluated by the given equation: EC1/EC2 × 100. 2.4.2. Lipid peroxidation Lipid peroxidation of mutants’ and wild type plants at different stages was quantified by the amount of MDA (malondialdehyde) generated by TBA (thiobarbituric acid) as conferred by Heath and Packer [34]. Leaf tissue was grinded in 0.1 % TCA buffer and centrifuged at 12,000 rpm for 10 min. The solution of 0.5% TBA in 20% TCA was mixed with supernatant and placed at 90 °C for 30 min, thereafter samples were incubated in ice and centrifugation was done at 8000 rpm for 5 min. Further, the OD was measured at 532 nm.

2.2. Phenotypic analyses of wild type and pop2 mutant plant Wild type and pop2 mutants were sown in controlled growth conditions (temperature, humidity, day-night cycle) to study the phenotypic characters of wild type and mutant plants i.e. leaf size, flowering stage, silique number, silique size and seeds per silique at different developmental stages measured according to the methods described in Gaudin et al. [31]. 2.3. Assessment of physiological traits for leaf senescence

2.4.3. In vivo visualization of ROS and cell death Histochemical staining of leaves at different stages was performed with 3,3-diaminobenzidine (DAB) assay and trypan blue assay as shown by Koch and Slusarenko [35]; Thordal-Christensen et al. [36]. Leaves were submerged in the staining solutions (DAB and trypan blue) individually for 2 h and later on de-stained with acetone: acetic acid:glycerol mixture until brown or blue spots appeared. Total chlorophyll content was bleached and leaf sample were documented by visualizing leaves under a binocular [14].

The physiological traits of leaf senescence viz. leaf survival, total chlorophyll content, electrolyte leakage, lipid peroxidation, cell death, and insitu accumulation of reactive oxygen species (ROS) were measured in harvested leaves for specified days from different developmental stages. 2.3.1. Measurement of leaf survival Leaf survival was measured with the help of calculating the

2.4.4. Total protein content Total protein content of the leaves of mutant and wild type Arabidopsis thaliana plant was measured as follows. First, leaves sample were homogenised into 4 volume of extraction buffer (v/ w) [5 mM EDTA, 1.5 mM dithiothreitol (DTT), 100 mM Tris-HCl (pH 8.0), 10 % (v/v) glycerol, and 1 % (v/v) protease inhibitor cocktail] and 1 % (w/ w) polyvinylpyrrolidone (PVPP) and then centrifugation was done at 15000 × g for 20 min. at 4 °C [37]. The supernatant was transferred to new test tubes and then protein content was measured against the standard curve of Bovine Serum Albumin (BSA) as per Bradford et al. [38].

Table 1 Description of developmental stages during experimental assessments for growth and physiological traits of Arabidopsis thaliana. DOS; Day of Sowing. Stages

Description

S1 S2 S3 S4 S5 S6

Leaves of 15 days old plant from DOS Young leaf of 25 days old plant from DOS Mature leaves of 35 days old plant from DOS Mature fully expanded leaves of 45 days old plant from DOS Leaves from 55 days old plant from DOS Leaves from 65 days old plant from DOS

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Fig. 1. Variation in the flowering time and plant morphology of the wild type and pop2 mutant’s plant. (A)The variation in flowering time of wild type, pop2-1 and pop2-3 mutant.(B)Variation in leaf shape of wild type and pop2-1 and pop2-3 mutant plants(C)Variation in silique size of wild type and pop2-1 and pop2-3 mutant plants. Data represent mean ± Standard deviation of three biological replicates, *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with wild type.

extracted from the leaves of mutants and wild type plant at different stages as done earlier [14]. GABA-transaminase assay was performed with 15 μl of protein extract (40 μg of protein) in a reaction buffer containing 50 mM Tris-HCl (pH 8.0),0.75 mM EDTA, 1.5 mM DTT, 0.1 mM pyridoxal-5-phosphate (PLP), 10% (v/v) glycerol, 16 mM GABA and 4 mM of pyruvate in a final volume of 150 μl. Control assays were performed by boiled enzyme extract in the assay. Samples were incubated at 30 °C for 60 min, after that samples were incubated at 97 °C for 7 min. to stop the reaction. GABA-T activity was quantified by the amount of L-alanine produced by alanine dehydrogenase (AlaDH) assay. AlaDH assay was performed with 40 μl of the GABA-T assay in a reaction mix containing 50 mM sodium carbonate buffer (pH 10.0), 1 mM NAD + and 0.02 units of Bacillus subtilis AlaDH (Sigma–Aldrich) in a final volume of 200 μl. The increase of OD340 nm was recorded using Schimadzu UV- 1800 spectrophotometer. The amount of L-alanine was quantified according to external calibration curve of L-alanine. GAD assay was performed with 15 μl of protein extract (40 μg of protein) in a reaction buffer containing 150 mM potassium phosphate (pH 5.8), 0.1 mM PLP and 20 mM L-glutamate in a final volume of 150 μl. Control assays were conducted as previously described. Samples were incubated at 30 °C for 60 min, after that to stop the reaction by incubation of samples at 97 °C for 7 min. GAD activity was quantified by the amount of GABA produced by GABase assay. GABase assay was performed with 20 μl of the GAD assay in reaction mix containing 75 mM potassium pyrophosphate (pH 8.6), 3.3 mM 2-mercaptoethanol, 1.25 mM NADP+, 5 mM 2-ketoglutarate and 0.02 units of Pseudomonas

2.5. Determination of GABA shunt components 2.5.1. Quantification of GABA content GABA was isolated and quantified from frozen leaves of different developmental stages of mutants and control plants. The standard GABA was prepared in micromole concentration and then used for constructing the calibration graph. Frozen leaves were ground separately in micro centrifuge tubes in liquid nitrogen then methanol was added to each tube and the samples were incubated for 10 min at room temperature (RT). Methanol from the samples was removed by vacuum drying, and 70 mM lanthanum chloride was added to each tube. The tubes were incubated for 15 min at RT by shaken the tube and centrifuged at 38, 000g for 5 min. The supernatants were transferred to new tubes, mixed with 1 M KOH, incubated for 10 min, and centrifuged at 38, 000g for 5 min. A 550 μl of the supernatant of each sample in separate tube was taken, and then added 150 μl of 4 mM NADP+, 200 μl of 0.5 M potassium pyrophosphate buffer (pH 8.6), 50 μl GABase/ml(Sigma–Aldrich) was prepared by dissolving required quantity in 0.1 M potassium phosphate (pH 7.2) containing 12.5% glycerol and 5 mM 2- β-mercaptoethanol and 50 μl of 20 mM α-ketoglutarate, mixing them properly. The absorbance was measured. The initial absorbance was read at 340 nm before adding α- ketoglutarate and final absorbance was read after 60 min. The calibration graph of GABA was constructed as per previous study [14]. 2.5.2. Measurement of GABA-T and GAD activities Enzymes activities assay were performed from the protein that was 3

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Fig. 2. Variation in the siliques of the wild type and pop2 mutant’s plant. (A)No. of siliques per plant of wild type and pop2-1 and pop2-3 mutant plants. (B)Number of seeds per silique of wild type and pop2-1 and pop2-3 mutant’s plant. Data represent mean ± Standard deviation of three biological replicates, *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with wild type.

Fig. 3. Physiological parameter of leaf senescence at different developmental stages of pop2 mutants and wild type plant of Arabidopsis thaliana. (A) Determination of leaf survival in pop2 mutant and wild type plant.(B)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutants leaves. (C) Determination of ion leakage of wild type and mutants leaves.(D) Quantification of lipid peroxidation by the level of MDA wild type and mutant leaves. Data represent mean ± Standard deviation of three biological replicates, *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with wild type.

fluorescens GABase (Sigma–Aldrich) in a final volume of 200 μl. The increase of OD340 nm was recorded using Schimadzu UV- 1800 spectrophotometer. The amount of GABA was quantified according to external calibration curve of GABA.

3. Results 3.1. The pop2 mutants shows alteration in flowering time, leaf development, and morphology To analyse the phenotypic characters, leaf size, flowering stage, silique number, silique size and seeds per silique were measured at different stages (Fig. 1). In the current study, we found that the two GABAT mutants flower earlier than the wild type plants. Flowering in the mutant pop2-3 began at18.6 ± 1.15 days and in pop2-1 began 25.3 ± 2.6 days from the DOS, whereas in wild type plants, flowering began by 36.3 ± 1.5 days (Fig. 1A). Mutation in GABA-T strongly affected leaf morphology and a reduction in leaf size in pop2-1 mutant in comparison to wild type plants (Fig. 1B).The pop2-1mutant plants were fertile, but they produced smaller and fewer siliques. The siliques of wild type plant were larger

2.6. Statistical analysis The values are mean ± SD for three independent replicates of individual experiment. Statistical significance was evaluated by One-way ANOVA with Dunnett’s multiple comparison test using a p value < 0.05 by Graph Pad prism.

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Fig. 4. Insitu localization of hydrogen peroxide by DAB and cell viability by Trypan blue assay of pop2 mutants and wild type plant leaves at different developmental stages.(A) Determination of ROS and cell viability in wild type leaves. (B) Determination of ROS and cell viability in pop2-1 leaves. (C) Determination of ROS and cell viability in pop2-3 leaves.

3.2. Physiological changes in pop2 mutants leaves To study the physiological changes in the wild type and pop2 mutants’ plant, we measured leaf survival, chlorophyll content, electrolyte leakage, and total protein content of the both type of plants (mutants and wild types) leaves at different developmental stages. First, we have investigated the consequence of leaf longevity (expressing the leaf survival) in the GABA-T mutants and wild type (Fig. 3). It was observed that the leaves survival % of both mutants were stable up to S2 stage, whereas, in wild type almost at S3 stage. Most of the leaf population to survive was at S3 stage in the pop2-1(S1-100 ± 0.0%, S2-100 ± 0.0%, S3-71.6 ± 4.4%) and pop2-3(S1-100 ± 0.0%, S2-100 ± 0.0%, S376.7 ± 8.2%) mutants, while it was longer at S5 stage, in the wild type (S1-100 ± 0.0%, S2-100 ± 0.0%, S3-98.13 ± 3.2%, S490.56 ± 6.3%, S5-61.06 ± 5.1%) plants (Fig. 3 A). The leaf survival percentage was decreased from S4 stage in pop2-1(42.7 ± 3.1 %) and pop2-3 (47.23 ± 6.1%) mutants, whereas, S5 stage in wild type plant (61.06 ± 5.1%). Total chlorophyll content of pop2-1 and pop2-3 mutants (1.20 ± 0.06 and 1.29 ± 0.02 mg g-1 FW respectively) at S1 stage was lesser as compared to the wild type plants 1.97 ± 0.01 mg g-1 FW (Fig. 3B). Chlorophyll content in wild type plant was stable up to S3 stage (S1- 1.97 ± 0.19 mg g-1 FW, S2-1.9 ± 0.085 mg g-1 FW, S31.79 ± 0.107 mg g-1 FW) while, at S2 stage was stable in pop2-1 (S11.20 ± 0.067 mg g-1 FW, S2-1.19 ± 0.097 mg g-1 FW) and pop2-3 (S11.29 ± 0.093 mg g-1 FW, S2-1.28 ± 0.041 mg g-1 FW) mutants.

Fig. 5. Determination of GABA content of pop2 mutant and wild type leaves of Arabidopsis thaliana at different developmental stages. Data represent mean ± Standard deviation of three biological replicates, *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with wild type.

(1.76 ± 0.20 cm) compared to pop2-3 (0.9 ± 0.1 cm) and pop2-1 (0.36 ± 0.05 cm) (Fig. 1C). Number of siliques per plant were also decreased in mutants compared to wild type plants (WT:184.06 ± 10.69, pop2-1:160.3 ± 17.06, and pop2-3:171 ± 18.5) as shown in Fig. 2A.Number of seeds per silique was maximum in wild type plants (44.66 ± 14.57) and minimum in pop2-1 mutant plant (3.66 ± 0.57) and in medium range in pop2-3 mutant (43.33 ± 5.68) (Fig. 2B).

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(0.028 ± 0.004 μM mg-1) and pop2-3 (0.023 ± 0.004 μM mg-1) mutants, and the increase was considerably higher in both mutants than wild type plants (Fig. 3D). When leaves of wild type and both mutants were assayed by DAB and trypan blue at different developmental stages, the density of brown patches for hydrogen peroxide was higher in both mutants’ leaves from S3 stage as compared with S5 stage in wild type plant. A large number of blue spots were detected in leaves of mutants from S3 stage as compared with wild type plants and also appeared in S5 stage (Fig. 4), indicating that ROS generation and cell death rate were elevated in pop2-1 and pop2-3 mutants’ leaf earlier, rather than wild type. The wild type and mutants were grown-up in sterile condition on MS medium and early symptoms of leaf senescence were observed in pop2 mutants, but not in the wild type plants at S3 stage. 3.3. Determination of GABA content at different developmental stages In the present study, we examined whether GABA-T mutant plants can cause any alteration in GABA content or not. The evaluation of GABA content in wild type as well as pop2 mutants shows that GABA content of mutants’ leaves was elevated from S3 stage whereas in wild type it is stable at S3 stage and start increasing from S4 stage (Fig. 5). Maximum GABA content was found in S4 stage in pop2-1 and pop2-3 (0.71 ± 0.05 and 0.91 ± 0.07 μmoles g-1 FW, respectively) and S5 stage in wild type plant (0.93 ± 0.06 μmoles g-1 FW). After reaching to the maximum level of GABA content, it was declined at S5 stage in mutant plants and at S6 stage in wild type plants (Fig. 5).

Fig. 6. Determination of enzymatic activities of leaves of pop2 mutant and wild type plant of Arabidopsis thaliana at different developmental stages. (A) Enzymatic activity of GABA-T in wild type and pop2-1 and pop2-3 mutants. (B) GAD activity in wild type and pop2-1 and pop2-3 mutants leaves.

3.4. Determination of enzyme activity (GABA-T and GAD) during different leaf developmental stages

Chlorophyll content sharply declined in pop2-1(0.77 ± 0.063 mg-1 FW) and pop2-3(0.63 ± 0.073 mg g-1 FW) in S3 stage, as compare in S4 stage in wild type plant (0.91 ± 0.094 mg g-1 FW) (Fig. 3B). Electrolyte leakage of membrane began to increase at S3 stage in pop2-1(48.38 ± 0.04 %) and pop2-3 (54.37 ± 0.06%) mutants, and constantly elevating during the growth period up to S6. Nevertheless, electrolyte leakage in wild type leaves initiated to escalate until S4 stage (32.55 ± 0.07 %) and there was no any escalation during the growth period (Fig. 3C). The MDA content began to escalate at S4 stage in wild type plants (0.021 ± 0.009 μM mg-1), but already at S3 in the pop2-1

GABA-T and GAD enzyme activities were assayed and compared during different leaf developmental stages of wild type and pop2 mutants (Fig. 6A, B).The enzymatic activities of GABA-T as well as GAD were considerably higher in control plants (GABA-T 12.28 ± 0.76 nmoles min-1 mg-1protein and GAD 20.97 ± 2.8 nmoles min.-1 mg-1 protein) than in pop2-1 and pop2-3 mutants (GABA-T 0.89 ± 0.29 nmoles min-1 mg-1 protein, 2.34 ± 0.54, respectively; GAD 8.01 ± 1.4, 10.09 ± 1.6 nmoles min-1 mg-1 protein respectively) in S1 stage. The enzymes (GABA-T and GADA) activity of pop2-1 and pop2-3 Fig. 7. Involvement of GABA transaminase in the mechanism leaf senescence process via GABA shunt pathway. GDH: Glutamate dehydrogenase, GAD: Glutamate decarboxylase, αKGDH: α-ketoglutarate dehydrogenase, GABAT: GABA-transaminase, SSADH: Succinic semialdehyde dehydrogenase, SCS: Succinyl CoA synthetase, TCA: Tricarboxylic Acid, CaM: Calmodulin (Calcium binding protein) GS: Glutamine synthetase, AS: Asparagine synthetase. The dotted arrow indicates the degradation of macromolecules from senescence leaves /transfer of nutrients to the developing parts of the plants and the dark arrows indicates the direction of the reaction during senescence.

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as well as hys1 (hypersenescence1) early-senescence mutants of Arabidopsis [32,47,49]. Our result revealed the increased the level of MDA and cell damage in mutant plants in earlier stage of leaf development as compared to wild type (Fig. 3D). Similarly, the MDA content elevated on 40 days in control plants, on the other hand at 30 days in (Glutathione reductase 2) igr2-9 as well asigr2-14 mutant’s plant of Arabidopsis [50]. Taken together, this physiological data suggested that pop2 mutants promptly declined the efficacy of photosynthesis and instigated precocious foliar senescence. Our data provide evidence of crucial role of GABA-T in foliar senescence. The measurement of GABA content in pop2 mutant and wild type revealed that GABA content of pop2 mutants leaves were elevated from S3, whereas, in wild type it stable at S3 stage and start increasing from S4 stage (Fig. 5), similar result was found in case of rice leaf senescence [18]. In response to various abiotic stresses, the detached leaves of pop2 mutants showed greater sensitivity and prior senescence than the wild type [14,51]. This anticipated that GABA-T regulating both cellular fortification as well as senescence activities. The pop2 mutants behave similarly to the wild type until S2 stage. Similar experiment was performed for EAS1(early-senescence) mutant of Arabidopsis thaliana and found that EAS1 mutants hastily declined photosynthesis efficacy, which resulted precocious senescence after 35 days in optimum growth conditions [52]. Furthermore, the pat14 (palmitoyl transferase) mutants of Arabidopsis thaliana showed similar to wild type up to flowering stage [53]. GABA-T and GAD activities were assayed at different leaf developmental stages in wild type and mutant plant. The enzymes activity was reached at peak level at S4 stage in pop2 mutants plant, whereas, in wild type plant enzymatic activity reached at peak level at S5 stage (Fig. 6A,B). Similar investigation was done on the rice Osl2 gene that encode GABA-T, that expression is specific leaf senescence of rice, which validate the up-regulation of GABA-T activities in foliar senescence, maximum activity showed at the S3 stage (senescence leaves with 45–60% Chl) of rice [18].

were stable till S2 stage, whereas it was stable till S3 stage in wild type plant (Fig. 6).These activities goes up peaking at S4 stage in pop21(GABA-T 4.32 ± 0.98 nmoles min-1 mg-1 protein, GAD 19.36 ± 1.91 nmoles min-1 mg-1protein) and pop2-3 (GABA-T 5.07 ± 1.27 nmoles min-1 mg-1 protein, GAD 23.09 ± 1.86nmoles min-1. mg-1protein) mutant plants and S5 stage in wild type (GABA-T 17.85 ± 1.03 nmoles min-1 mg-1protein, GAD 36.08 ± 3.81 nmoles min-1 mg-1protein) plant. The enzymes activity was decline at S5 stage in mutant plants and at S6 stage in wild type plants (Fig. 6A,B). 4. Discussion Leaf senescence is a vital stage in the life span of plants and well-coordinated process involving retardation in photosynthesis, degradation of molecules and mobilization of nutrient [39]. The senescence process is directed by several internal and environmental influences [17,40,41]. SAGs (senescence associated genes) have been recognized in different plants, among these genes, GABA-T has been also identified as leaf senescence gene [11,18]. Additionally, GABA is also involved in the signaling process of plants [10,42]. The expression of GABA-T is reflected to play a prominent function in regulation of nitrogen metabolism mechanism of plant development and foliar senescence via GABA shunt [22] (Fig. 7). Several investigators [15,29,34] have studied different function of GABA shunt. Since the disruption of GABA-T genes in Arabidopsis thaliana, pop2 mutants’ phenotypes display morphological alteration than wild type plant, which provides a direct clue to gene function. By analysing pop2 mutants, we reported that pop2 mutants have an overall reduction organ size, modification in flowering time (Fig. 1A) defects in leaf morphology (Fig. 1B) and silique size (Fig.2C) and have shortened life cycle compared to wild type Arabidopsis plants. It has been also reported that the pop2 mutants roots were more sensitive to salinity stress and morphology of pop2 mutant might differ from control plants [29,30,43,44]. The mutation of GABA-T gene may silence various genes that regulating development and thus defects the plant morphology. It has been reported that SSADH phenotype suppressed by Arabidopsis thaliana mutant of GABA-T gene [44]. The pop2 mutants display abridged seed development (Fig. 3B), suggesting that involvement of GABA-T activity is limited to flower parts in Arabidopsis thaliana [1]. Foliar senescence is reliant on plant age in optimum conditions or under adverse environmental condition, and the pigment degradation in senescence is generally perceived in matured leaves. In present study, pop2 mutant display more yellow leaves as compared to wild type plants at S3 stage, proposing that pop2 mutants have a precocious-senescence phenotype. Additionally, we have determined alteration in various physiological features in pop2 mutants viz. leaf survival, chlorophyll content, ion leakage, MDA content, insitu hydrogen peroxidation assay and cell death [45] at different leaf developmental stages in response to leaf senescence. Leaf survival was lesser in pop2 mutants as compared to wild type plants (Fig. 3A). Similar studies were reported in egy1 mutant encoding a plastid metalloprotease [32] and apg7-1 mutant encoding the ATP-dependent activating enzyme in Arabidopsis thaliana that indicated precocious senescence [46]. We have observed chlorophyll degradation in pop2 mutants in earlier stage rather than wild type plant as shown in (Fig. 3B),that similarly reported in egy1 mutants [32], in porB-1porC-1(NADPH:Pchlide oxidoreductases) mutant [48], and in mex1 (maltose excess 1) mutants of Arabidopsis thaliana [47]. Our result also showed that elevation of electrolyte leakage that is also similarly found in egy1 mutants’ leaf of Arabidopsis thaliana [32] to escalate 15 days after leaf formation and is constantly increasing during the growth period. Conversely, electrolyte leakage in wild type leaves commenced to elevate from 30 days (Fig. 3C). Proteins are essential constituents of living cells as well as crucial for the appropriate functioning of an entity, and could be declined through foliar senescence (Fig. 7). The pop2 mutants had reduced total protein contents, comparable results was also perceived in egy1, apg7-1

5. Conclusion In conclusion, GABA shunt plays important role in regulating growth and development of plants. GABA transamination is an enzymatic reaction of GABA shunt pathway that is catalyzed by GABA-T which potentially regulates the metabolism of GABA in plants. We have characterized the GABA-T knockout pop2 mutants at different developmental stages of Arabidopsis thaliana. We have seen dramatic changes in flowering time, leaf development, morphology as well as pod size and shape in mutant plants. Our results revealed that the GABA-T implicated in the regulation of senescence process in addition to its involvement in nitrogen and carbon metabolism. Thus, the GABA-T is significant regulator of plant development in Arabidopsis thaliana and plays key role in biosynthesis and catabolism of GABA. Conflict of interest Authors have no conflict of interest. Acknowledgements The authors acknowledge SERB, DST New Delhi for their financial support to Dr. Mohammad Israil Ansari for this work and grateful to the Arabidopsis Biological Resource Centre (ABRC), USA for providing the mutants seeds of Arabidopsis thaliana. We are thankful to Professor Nazneen Khan, Department of English, University of Lucknow, India and Dr. Arif Siddiquie, Amity University Uttar Pradesh, Lucknow, India for editing the manuscript critically. References [1] R. Palanivelu, L. Brass, A.F. Edlund, et al., Pollen tube growth and guidance is

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