functional loss of gaba transaminase gaba t expressed early leaf senescence under various stress conditions in arabidopsis thaliana

Accepted Manuscript Title: Functional Loss of GABA Transaminase (GABA-T) Expressed Early Leaf Senescence under Various Stress Conditions in Arabidopsis thaliana Authors: Syed Uzma Jalil, Iqbal Ahmad, Mohammad Israil Ansari PII: DOI: Reference: S2214-6628(16)30077-9 http://dx.doi.org/doi:10.1016/j.cpb.2017.02.001 CPB 49 To appear in: Received date: Revised date: Accepted date: 3-12-2016 21-2-2017 22-2-2017 Please cite this article as: Syed Uzma Jalil, Iqbal Ahmad, Mohammad Israil Ansari, Functional Loss of GABA Transaminase (GABA-T) Expressed Early Leaf Senescence under Various Stress Conditions in Arabidopsis thaliana, Current Plant Biology http://dx.doi.org/10.1016/j.cpb.2017.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Functional Loss of GABA Transaminase (GABA-T) Expressed Early Leaf Senescence under Various Stress Conditions in Arabidopsis thaliana Syed Uzma Jalil1, Iqbal Ahmad2 and Mohammad Israil Ansari*1,3 Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Lucknow-226 028, India Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh-202 002, India Department of Botany, University of Lucknow, Lucknow-226 007, India *Corresponding author at: Department of Botany, University of Lucknow, Lucknow-226 007, India Tel.: +91-9839541698 Email: ansari_mi@lkouniv.ac.in, ansari_mi@hotmail.com (Dr Mohammad Israil Ansari) Abstract GABA-transaminase (GABA-T) involved in carbon and nitrogen metabolism during the plant development process via GABA shunt and GABA-T mutant, which is defective in GABA catabolism, is ideal model to examine the role of GABA-T in plant development and leaf senescence of plant We have characterized GABA transaminase knock out mutant pop 2-1 that is transition and pop2-3 which is T-DNA insertion mutant of Arabidopsis thaliana during various stress conditions In accompany with the investigation of the physiological parameters of leaf senescence during abiotic stress (dark, chilling, wounding and dehydration) and by agents (ABA, H2O2 and mannitol) induced leaf senescence conditions, our physiological evidence indicates that pop2-1 and pop2-3 mutations rapidly decreased the efficiency of leaf photosynthesis, chlorophyll content, GABA content, GABA-T, and glutamate decarboxylase (GAD) activity and on the other hand increases membrane ion leakage, malondialdehyde (MDA) level in stress induced leaves However, cell viability assay by trypan blue and insitu Hydrogen peroxidation assay by 3,3-diaminobenzidine (DAB) in stress induced leaves also display that pop2-1 and pop2-3 mutant leaves shows oversensitivity in response to different stress conditions rather than in wild type These results strongly indicate that the loss-of-function of GABA Transaminase gene induces early leaf senescence in Arabidopsis thaliana during various stress conditions Keywords- GABA- Transaminase, Leaf senescence, Abiotic stress, Osmotic stress Introduction Senescence is a genetically and developmentally synchronized event that involves a general degradation of cellular structures, followed by remobilization of the degradation of products to other growing parts of the plant This does not occur randomly, but rather includes an exceedingly regulated process of particular sequential changes in the function or structure of numerous cellular components, incorporating changes in the expression of a most of the genes[1-5].Leaf senescence occurs in an age-dependent manner, yet an unpredictable association between developmental age and factors, for example phytohormones, shading, and temperature Many Senescence-associated genes (SAGs) were isolated and identified from a variety of plant species to elucidate the molecular mechanisms underlying leaf senescence [6-12] During leaf senescence GABA shunt play an integral role in carbon/ nitrogen metabolism and it is also important for plant growth and development[13-15] GABA is one of the key component of GABA shunt and in mammalian central nervous system act as inhibitory neurotransmitter [16] In plants, GABA play role as metabolites as well as signalling molecule in many mechanism, it accumulate during various stress conditions in plant cell including leaf senescence process [13,17,18] It has been studied that GABA shunt pathway under abiotic stress can potentially regulate GABA metabolism [12] Protein degradation is the most consequential breakdown process during the leaf senescence, which is followed by decrease in photosynthetic activities, disassembles of cellular components, degradation of macromolecules and remobilization of the nutrients to developing part of the plants [2, 4, 19-21] Ammonia released from the catabolism of proteins and nucleic acids is converted into glutamine by glutamine synthetase using glutamate as substrate During leaf senescence, the source of glutamate is likely to increase as a result of enhanced proteolytic activities and transamination reactions involving amino acids, such as alanine and aspartate, released from proteolysis Consequently, the conversion of glutamate to GABA may be enhanced due to the increased availability of the glutamate substrate for GAD Thus, the GABA shunt within mitochondria may provide carbon skeletons to replenish carboxylic acids of the TCA cycle [15, 22] It has been also investigated that Osl2 gene encoded GABA pyruvate transaminase play a key role in carbon/ nitrogen metabolism during rice leaf senescence [22] Accumulations of GABA in the senescing leaf need to be investigated to further understand the role of GABA transaminase in GABA metabolism and carbon nitrogen metabolism during leaf senescence In this study we have analysed Arabidopsis thaliana knock out mutant plants pop2-1, pop2-3 of GABA–transaminase gene Since the relation between GABA Transaminase mutant and leaf senescence is completely unknown, no attention has been given to pop2-1 and pop2-3 mutants and leaf senescence, we aim to explore the effects of GABA-T defection on leaf senescence and other stress conditions to elaborate its potential mechanism Materials and Method 2.1 Plant materials and growth conditions Arabidopsis thaliana Landsberg erecta ecotype (Ler) was use as wild type Seeds of the pop2-1 and pop2-3 mutants of GABA-Transaminase were kindly provided by Arabidopsis Biological Resource Centre (ABRC), Ohio State University, Columbia, USA Plants were grown at 22o C for long day condition (16 h light / h dark cycle) aseptically or on soil For soil growth, seeds were sown in Bio-Mix Potting Substratum, Soilrite (Keltech Energies Ltd India.) and placed at 4o C for days in dark to break seed dormancy and later transferred to normal growth conditions GABA transaminase Arabidopsis thaliana knockout mutant plants pop2-1 mutant has transition G→C, and pop 2-3 T-DNA insertion mutant The homozygous Arabidopsis thaliana T- DNA mutant pop2-3 were find out by using PCR with primer from left and right border of T-DNA and primer from flanking region pop 2-1 were screened by dCAPS method [23] Homozygous plants were used for further analysis 2.2 Treatment of detached leaves with abiotic stresses induced leaf senescence Rosette leaf (counted from bottom) was isolated from a 23-d-old Arabidopsis wild type and knock out mutant plants Under growth conditions, leaf at the time of sampling was fully expanded; these fully expanded leaves not start senescing until d later Five leaves from wild type and knock out mutant plants were used for each treatment For wounding treatment, leaves of wild type and mutants plants were wounded by needle and were floated on mm MES [2-(N-morpholino)ethane sulfonic acid] buffer (pH 5.8) and incubated for 24 h in growth chambers, without wounded leaves were used as control For dehydration treatment, we detached whole Arabidopsis rosette, we then weighed them and put on filter paper to dry for h under dim light After h, plants were weighed again to estimate water loss Leaves were put under the same conditions except for saturated humidity (to prevent leaves from losing water) were used as control For darkness treatment, leaves were floated on mm MES buffer (pH 5.8) and incubated for days in growth chambers covered with black paper or on control with proper light condition For cold treatment, leaves were floated on mm MES buffer (pH 5.8) and incubated for h at 0oC and for control kept at optimum temperature (22-25oC) in growth chamber 2.3 Treatment of detached leaves with senescence inducing agents All experiments on detached leaves were performed with the sixth rosette leaves at 23 day after leaf emergence, when the leaves were just fully developed Leaf disc were acquired by cutting leaves at the surmised centre of the petioles with a sharp tool, to minimize injuring effects The detached leaves were floated abaxial side up in a buffer (pH 5.8) containing mM MES, containing appropriate concentrations of the various senescence-inducing agents at 22°C under continuous light for days 10 mM concentration of H2O2, 500 mM concentration of Mannitol, 40µM ABA and different concentration of sucrose (0, 1, 2, 4, 6%) were used to induce oxidative stress, as indicated As a control for all treatment, the detached leaves were floated on mM MES buffer 2.4 Measurement of pigment and membrane ion leakage Determination of physiological parameters such as total chlorophyll content and membrane ion leakage of leaf senescence induced by abiotic and oxidative stresses was done For chlorophyll determination, fresh leaf material was extracted with 80% (v/v) acetone, and the absorption of the extracts was measured at 663 and 645 nm using Schimadzu UV- 1800 spectrophotometer [24] For ion leakage leaf disc are cut from each treated leaves and placed in closed vials containing 10 ml double distilled water followed by incubation on rotary shaker for 24 h, after which the electrical conductivity of solution (EC1) was determined by conductivity meter, then the samples were kept at 90°C for h, the electrical conductivity was measured again (EC2) after the solution was cooled at room temperature The electrolyte leakage (ion leakage) was defined as EC1/EC2 X 100 and expressed as percentage [25] 2.5 Lipid peroxidation The levels of lipid peroxidation was estimated with the thiobarbituric acid (TBA) assay by determining the concentration of malondialdehyde (MDA) as the end product of lipid peroxidation [26] Fresh leaf sample was homogenized in 0.1% (w/v) trichloroacetic acid (TCA) solution The homogenate was centrifuged at 15,000 g for 10 and the obtained supernatant was added to 0.5% (w/v) TBA in 20% (w/v) TCA The mixture was then incubated at 90°C for 30 min, and the reaction tubes were placed in an ice water bath Samples were centrifuged at 10,000 g for min, and the absorbance of the supernatant was read at 532 nm 2.6 Determination of GABA GABA was extracted and determined from frozen tissues of the treated leaves of wild type , pop2-1 and pop2-3 mutants [27] Harvested leaves were ground separately in microfuge tubes in liquid nitrogen until a fine powder was obtained Methanol was added to each tube and the samples were mixed for 10 Liquid from the samples was removed by vacuum drying, and 70 mM lanthanum chloride was added to each tube The tubes were shaken for 15 and centrifuged at 38, 000 g for The supernatants were transferred to new tubes, mixed with M KOH, shaken for 10 min, and centrifuged at 38, 000 g for To 550µl of the supernatant add 150µl of mM NADP+, 200µl of 0.5 M potassium pyrophosphate buffer (pH 8.6), 50µl GABase/ml (Sigma-Aldrich) prepared by dissolving required quantity in 0.1 M potassium phosphate (pH 7.2) containing 12.5% glycerol and mM 2- β-mercaptoethanol and 50 µl of 20 mM α-ketoglutarate were added, mixed well and the absorbance was measured The initial absorbance was read at 340 nm before adding α- ketoglutarate and final absorbance was read after 60 The GABA content in the sample was calculated based on the difference in absorbance The standard GABA was prepared in micromole concentration and used for constructing the calibration graph 2.7 GABA Transaminase (GABA-T) and Glutamate decarboxylase (GAD) activities Treated leaves were harvested, weighed and snap-frozen in liquid nitrogen Samples were stored at -80°C until processing Proteins extraction and enzyme assays were done as described earlier with some modifications [28] For enzyme assay, protein extraction was performed in four volumes of extraction buffer (v/w) containing 100 mM Tris-HCl (pH 8.0), 1.5 mM dithiothreitol (DTT), mM EDTA, 1% (v/v) protease inhibitor cocktail (SigmaAldrich) and 10% (v/v) glycerol and 1% (w/w) polyvinylpyrrolidone (PVP) were added to samples before homogenization Samples were then centrifuged at 20, 000 g for 20 at 4°C Supernatant was used for the enzyme assay and protein quantification Protein concentrations were calculated with bovine serum albumin as standard [29] GABA-T 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 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 to stop the reaction by incubation of samples at 97°C for GABA-T activity was determined by quantifying 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), mM b-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 UV1800 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 Lglutamate 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 GAD activity was calculated by quantifying 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+, mM 2-ketoglutarate and 0.02 units of Pseudomonas fluorescens GABase (Sigma-Aldrich) in a final volume of 200 μl The increase of OD at 340 nm was recorded using Schimadzu UV- 1800 spectrophotometer The amount of GABA was quantified according to external calibration curve of GABA 2.8 Detection of ROS and cell death in leaves Trypan blue (TB) stain was used to visualize dead cells [30] H2O2 was detected in situ of stress induced leaves using 3,3-diaminobenzidine (DAB) (Sigma –Aldrich) [31] Results 3.1 The influence of abiotic stress induced leaf senescence on pop 2-1, pop2-3 mutants and wild type plants Abiotic stresses leads to enhance damage that occurs in plants exposed to a various stressful conditions and provoke leaf senescence due to disruption of cellular homeostasis and osmoregulation [32-34, 7, 9] In order to study the relationship between leaf longevity and abiotic stress tolerance, we first examined the responses to various abiotic stress conditions in two mutant pop2-1 and pop 2-3 and wild type plants To minimize any developmental effects, full-grown fifth-sixth rosette leaves were used in all experiments The fifth-sixth rosette leaves of the wild type and mutants were detached and treated with days dark, h cold, h wounding and h dehydration stress condition which are all known to induce stress The response to abiotic stress was monitored by measuring the chlorophyll content, Ion leakage percentage, lipid peroxidation, GABA content and enzyme assay (GABA-T and GAD) We first evaluated the effect of dark stress for days on detached leaves in the presence of MES buffer (Fig 1) The chlorophyll content of wild type, pop2-1, and pop2-3 leaves dropped to 1.152±0.178, 0.104 ± 0.103, and 0.304±0.242 mg-1respectively (Fig 1A) The ion leakage percentage of pop2-1 and pop2-3 mutants increases 97.67% and 95.30% respectively as compared to wild type plants 68.70% after dark treatment (Fig 1B) The MDA content were significantly increased to 0.083±0.008 and 0.093±0.009 in pop2-1 and pop2-3 mutants leaves respectively in comparison to wild type 0.044±0.006 µM mg-1 as in (Fig 1C) GABA content, GABA- T and GAD activity were increased in wild type leaves in comparison to mutant plants (Fig D and E) respectively DAB assay for hydrogen peroxide and trypan blue assay for cell death shown in (Fig 5A) In order to examine the impact of cold stress (0oC) for h on pop2-1, pop2-3 mutant and wild type plants detached leaves in the presence of MES buffer The chlorophyll content of pop2-1 and pop2-3 leaves dropped to 1.082±0.013, 0.928 ± 0.096 respectively than wild type leaves 1.685±0.054 mg-1 (Fig 2A) The ion leakage percentage of pop2-1and pop2-3 mutants increases 86.50% and 79.67% respectively as compared to wild type plants 53.10% after cold treatment as shown in (Fig.2B).The MDA content were significantly increased to 0.057±0.006 and 0.072±0.015 in pop2-1 and pop2-3 mutant leaves respectively in comparison to wild type 0.035±0.009 µM mg-1 as in (Fig 2C) GABA content, GABA- T and GAD activity increased in wild type leaves than mutant plants (Fig 2D and E) respectively DAB assay for hydrogen peroxide and trypan blue assay for cell death shown in (Fig.5B) We investigate the effect of wounding stress on pop2-1, pop2-3 mutant and wild type plants detached leaves for 24 h in the presence of MES buffer The chlorophyll content of pop21and pop2-3 leaves decreased to 0.689±0.143, 0.763 ± 0.101 respectively than wild type leaves 1.429±0.102 mg-1 (Fig 3A) The ion leakage percentage of pop2-1 and pop2-3 mutants increases 97.74% and 95.20% respectively as compared to wild type plants 68.36% after wounding stress as shown in (Fig 3B) The MDA content were significantly increased to 0.027±0.001 and 0.036±0.002 in pop2-1 and pop2-3 mutant leaves respectively in comparison to wild type 0.009±0.001 µM mg-1 as shown in (Fig 3C) GABA content, GABA- T and GAD activity were also increased in wild type leaves rather than mutant plants (Fig 3D and E) respectively DAB assay for hydrogen peroxide and trypan blue assay for cell death shown in (Fig 5C) The impact of dehydration stress was observed on pop2-1, pop2-3 mutant and wild type plants detached leaves for h The chlorophyll content of pop2-1 and pop2-3 leaves decreased to 0.397±0.032, 0.731 ± 0.001 respectively than wild type leaves 1.693±0.021 mg-1 (Fig 4A) The ion leakage percentage of pop2-1 and pop2-3 mutant increases 94.38% and 90.04% respectively as compared to wild type plants 63.90% after wounding stress as shown in (Fig 4B) The MDA content were significantly increased to 0.079±0.002 and 0.087±0.008 in pop2-1 and pop2-3 mutants leaves respectively in comparison to wild type 0.031±0.002 µM mg-1 as in (Fig 4C) GABA content, GABA- T and GAD activity in wild type leaves increased and dropped in mutant plants (Fig 4D and E) respectively DAB assay for hydrogen peroxide and trypan blue assay for cell death shown in (Fig 5D) 3.2 Leaves of mutant plants display early-senescence during oxidative stress treatment Phytoharmones not only play key role in growth and development but also involved in senescence process of plants [4, 19, 21] Many researchers have analysed that exogenous abscisic acid (ABA) are known to accelerates the leaf senescence In response to ABA treatment, ABA were given to detached leaves of wild type and knockout mutants of Arabidopsis thaliana plants for days in the normal climate chambers to induce leaf senescence After exogenous treatment of ABA, the results showed that ABA could promotes leaf senescence of pop2-1, pop2-3 mutants and wild type plants but the senescence symptoms of pop2-1 mutant leaves appeared earlier than pop2-3 mutant and were more pronounced than those of wild type leaves For leaf senescence observation we have done chlorophyll content determination The leaf senescence after ABA treatment, chlorophyll content in pop2-1 was significantly decreased from second day of treatment in contrast pop2-3 and late in wild type, results are presented in (Fig 6A) The ion leakage percentage was also observed (Fig 6B) the membrane ion leakage percentage in pop 2-1 mutant leaves increased significantly from the second day of treatment than pop2-3 mutant and in last wild type The MDA accumulation in the pop2-1 mutants were shown elevated after second day of treatment and in pop 2-3 mutants it increases after third day whereas in wild type MDA level increased after fourth day of treatment (Fig 6C) The GABA content of pop 2-3 mutant leaves were high than pop2-1 mutant and wild type plant and increased from third day of treatment but in pop 2-1 it increases from second day of treatment on the other hand in wild type GABA content increases from fourth day of treatment(Fig 6D) The enzyme activity (GABA –T and GAD) were much more in wild type plants than pop2-1 and pop2-3 mutant During the treatment the enzyme activity of wild type plant leaves were increases from 4th day whereas in pop2-1 and pop 2-3 mutant it increases from second day of treatment (Fig 6E and F) Trypan blue (TB) staining was used for the investigation of cell viability When treated leaves were stained with TB, a large number of blue patches were observed in leaves of pop2-1 from second day of treatment than pop2-3, but were rarely present in wild-type plants (Fig 9), indicating that cell death is increased in pop2 mutants leaf H2O2 treatment was given to the detached leaves of wild type and pop2-1 and pop2-3 mutants of Arabidopsis thaliana plants for days in the normal climate chambers to stress induced leaf senescence The results showed that H2O2 could accelerates leaf senescence of pop2-1, pop2-3 mutants and wild type plants but the senescence symptoms of pop2-1 mutant leaves appeared earlier than pop2-3 mutant and were more pronounced than those of wild type leaves as shown in (Fig 7C) Chlorophyll content in pop2-1 was significantly decreased under H2O2 treatment from third day of treatment in contrast pop2-3 and late in wild type as shown in (Fig 7A) The ion leakage percentage was presented in (Fig 7B) and observed that ion leakage percentage in pop2-1 mutant leaves increased significantly from the third day of treatment than pop2-3 mutant and in last wild type plant The MDA accumulation in the pop2-1and pop 2-3 mutants were shown elevated after 3rd day of treatment whereas in wild type MDA level increased after fourth day of treatment (Fig 7C) The GABA content of pop 2-1 and pop 2-3 mutant leaves were increased from third day of treatment on the other hand in wild type GABA content increases from fourth day of treatment (Fig 7D) The activity (GABA–T and GAD) were much more in wild type plants than pop2-1 and pop2-3 mutant During the treatment, the GABA- T activity of wild type plant leaves were slightly increases from fourth day whereas in pop2-1 and pop 2-3 mutant from third day and then decreased from fourth day of treatment (Fig 7E) GAD activity increased in pop 2-1 and pop 2-3 from third day and in wild type it increases from fourth day of treatment (Fig 7F) Trypan blue (TB) staining was used for the investigation of cell viability When treated leaves were stained with TB, a large number of blue patches were observed in leaves of pop2-1 from third day of treatment than pop2-3, but were rarely present in wild-type plants (Fig.9), indicating that cell death is increased in pop2 mutant leaf The oxidative damage phenotype and the production of the ROS in pop2-1, and pop2-3 as result of the mannitol treatments were in agreement with the sensitivity of the same mutants under the same treatments In response to mannitol treatment chlorophyll content in pop2-1 was significantly decreased from second day of treatment in contrast pop2-3 and late in wild type (Fig 8A) Membrane ion leakage percentage in pop2-1 mutant leaves increased significantly from the second day of treatment than pop2-3 mutant and in last wild type (Fig 8B) The MDA accumulation in the pop2-1 and pop 2-3 mutants were shown elevated after third day of treatment whereas in wild type MDA level increased after fourth day of treatment (Fig 8C) The GABA content of pop 2-1 and pop 2-3 mutant leaves were increased from third day of treatment on the other hand in wild type GABA content increases from fourth day of treatment (Fig 8D) The activity (GABA–T and GAD) were much more in wild type plants than pop2-1 and pop2-3 mutant During the treatment, the GABA- T activity of wild type plant leaves were slightly increases from fourth day whereas in pop2-1 and pop 2-3 mutant from third day and then decreased from fourth day of treatment (Fig 8E) GAD activity increased in pop 2-1 and pop 2-3 from 3rd day and in wild type it increases from 4th day of treatment (Fig 8F) Trypan blue (TB) staining was used for the investigation of cell viability When treated leaves were stained with TB, a large number of blue patches were observed in leaves of pop2-1 from third day of treatment than pop2-3, but were rarely present in wild-type plants (Fig 9), indicating that cell death is increased in pop2 mutants leaf Discussion A crucial link between GABA-T and leaf senescence has yet to be discovered via genetic analysis It has been reported that senescence is last step leaf development process GABAtransaminase play important role in nitrogen recycling during leaf senescence process by GABA shunt [15, 18] Different function of GABA shunt has been reported by several scientists [12, 35, 36] To determine whether abiotic and oxidative stress tolerance and GABA-T are linked in plants during leaf senescence, we have compared various responses to abiotic and oxidative stress in GABA-T mutants and in wild-type Arabidopsis thaliana plants We have established a leaf senescence screening system based on chlorophyll fluorescence and observed chlorophyll degradation in pop2-1 and pop2-3 mutant of Arabidopsis thaliana and elevation of ion leakage, lipid peroxidation and cell damage in mutant plants (Fig and 5) Our physiological evidence indicates that pop2-1 and pop2-3 mutations rapidly decreased the efficiency of leaf photosynthesis and caused early leaf senescence In response to osmotic or oxidative stress, the detached leaves of pop 2-1 and pop2-3 mutant also displayed phenotypes of higher sensitivity and earlier senescence than those of the wild type It appears that GABA-T controls both cellular protection activities and senescence activities (Fig 1) The oversensitivity of the pop2-1 and pop2-3 mutants to the abiotic and oxidative stress treatment indicates that a GABA-T defect impaired GABA accumulation that might be required for growth and implicates that GABA is involved in intracellular signalling especially during leaf senescence under stress conditions (Fig 1D) Similarly, GABA enhanced growth in Rhizobium leguminosarum, as a key source of carbon and nitrogen, which reveal the importance of GABA shunt in growth and metabolism of plants [37] on the other hand ataldh10A8 and ataldh10A9 T-DNA-insertion mutants reduced 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leaves (E) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after dark treatment Data are mean ± standard deviation (SD) of three biological replicates Fig.2 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in cold stress treatment (A) Determination of chlorophyll content of wild type, pop2-1 and pop2-3 leaves (B) Determination of ion leakage of wild type and mutants leaves in cold stress (C) Quantification of lipid peroxidation by the level of MDA in cold treated wild type and mutants leaves (D) Quantification of GABA content in cold stress of wild type and mutants leaves (E) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after cold treatment Data are mean ± standard deviation (SD) of three biological replicates Fig.3 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in wounding stress treatment (A)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutants leaves (B) Determination of ion leakage of wild type and mutants leaves in wounding stress (C) Quantification of lipid peroxidation by the level of MDA in wounding treated wild type and mutants leaves (D) Quantification of GABA content in wounding stress of wild type and mutants leaves (E) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after wounding treatment.Data are mean ± standard deviation (SD) of three biological replicates Fig.4 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in dehydration stress treatment (A)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutants leaves (B) Determination of ion leakage of wild type and mutants leaves in dehydration stress (C) Quantification of lipid peroxidation by the level of MDA in dehydration treated wild type and mutant leaves (D) Quantification of GABA content in dehydration stress of wild type and mutants leaves (E) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after dehydration treatment Data are mean ± standard deviation (SD) of three biological replicates Fig.5 GABA-T mutation accelerates ROS activity and cell death in Arabidopsis thaliana under abiotic stresses (A) DAB and trypan blue assay shows elevation of hydrogen peroxide and cell death in detached leaves of pop 2-1 and pop2-3 mutants rather than wild type under dark stress condition (B) DAB and trypan blue assay on detached leaves of wild type, pop2-1 and pop2-3 mutants leaves under cold stress (C) DAB and trypan blue assay on detached leaves of wildtype, pop2-1 and pop2-3 mutants leaves under wounding stress (D) DAB and trypan blue assay on detached leaves of wildtype, pop2-1 and pop2-3 mutants leaves under dehydration stress Fig.6 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in ABA stress treatment (A)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutants leaves (B) Determination of ion leakage of wild type and mutants leaves in ABA stress (C) Quantification of lipid peroxidation by the level of MDA in ABA treated wild type and mutant leaves (D) Quantification of GABA content in ABA stress of wild type and mutants leaves (E-F) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after ABA treatment Fig.7 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in hydrogen peroxide (H2O2) stress treatment (A)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutant leaves (B) Determination of ion leakage of wild type and mutants leaves in H2O2 stress (C) Quantification of lipid peroxidation by the level of MDA in H2O2 treated wild type and mutant leaves (D) Quantification of GABA content in H2O2 stress of wild type and mutants leaves (E-F) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after H2O2 treatment Fig.8 GABA-T mutation accelerates leaf senescence in Arabidopsis thaliana in mannitol stress treatment (A)Determination of chlorophyll content of wild type, pop2-1 and pop2-3 mutants leaves (B) Determination of ion leakage of wild type and mutants leaves in mannitol stress (C) Quantification of lipid peroxidation by the level of MDA in mannitol treated wild type and mutant leaves (D) Quantification of GABA content in mannitol stress of wild type and mutants leaves (E-F) Enzymatic activity of GABA-T and GAD in wild type and mutants leaves after mannitol treatment Fig.9 Effect of Oxidative stress on cell viability of knockout mutants and wildtype plant leaves.by Trypan blue staining (A) Before Trypan blue staining (B) After Trypan blue Table Evaluation of physiological parameters of leaf senescence of control of pop2-1, pop2-3 mutants and wild type plants of A thaliana Data are mean ± standard deviation (SD) of three biological replicates ... results strongly indicate that the loss- of- function of GABA Transaminase gene induces early leaf senescence in Arabidopsis thaliana during various stress conditions Keywords- GABA- Transaminase, Leaf. .. In this study, for the investigation of the role of GABA- T in leaf senescence during various stress conditions , we have characterize GABA transaminase knock out mutant pop 2-1 that is transition... Accumulations of GABA in the senescing leaf need to be investigated to further understand the role of GABA transaminase in GABA metabolism and carbon nitrogen metabolism during leaf senescence In this