γ-Aminobutyric Acid Response To Water Stress And Regulation Of The Root Stem Cell Mechanism

γ-amino butyric acid (GABA) is ubiquitously found in a wide range of organisms.GABA is the inhibitory neurotransmitters in the central nervous system of animals. While, asosmolytes， GABA and proline play a crucial role in protecting plants from variousenvironmental stresses. Glutamic acid is the common precursor of these two molecules，whereglutamate decarboxylase (GAD) and⊿1-pryrroline-5-carboxylate synthetase(P5CS) isrespectively the rate-limiting enzymes in relevant biosynthesis pathway; however，thedominant metabolism pathway under water stress has not yet been established. Plantsexperience a variety of environmental stresses during development, a common response todifferent stresses is the stimulated generation of free radicals，particularly ROS，such as OH·，O2-，and H2O2. Previous studies showed that proline could remove oxygen free radicals.However whether GABA has the function of scavenging free radicals or not is unclear.Therefore this research attempts to explore the metabolic pathway of glutamate under waterstress and to compare the ability of GABA and proline scavenging reactive oxygen species. Inaddition to protecting cells from damage, GABA is also involved in the ethylene signaltransduction. Evidence showed that ethylene could be the regulator of quiescent center celldivision. But whether GABA directly involved in regulation of cell division of root quiescentcenter cells lacks of direct evidence. Thus this thesis also aims to study the regulatorymechanism of GABA involving in quiescent center cell division.To explore this，excised tobacco(SR1) leaves were used to simulate a water-stresscondition. Then we test the accumulation of proline and GABA under water dehydration andcompare the abilities of ROS scavenging. To the second issue, we use Arabidopsis wild-typeseedlings for research material. We added GABA in the medium and observed this effect ofGABA on root morphological changes including root length，the number of quiescent centercells and root cap cell layers. Then，we use PLT1genes which encode AP2transcriptionfactor and WUS gene which code homology domain transcription factor to promote stem celldynamic maitenence as entry point，through semi-quantitative technique to explore theregulatory mechanism involving γ--aminobutyric acid (GABA) signal. The final resultsshowed:1. The measured tobacco leaf RWC dramatically decreased after the leaves wereexcised from the plants, along with the time of dehydration continuation. This proved theexperimental system caused serious water stress;2. HPLC analysis of the results show that the free glutamic acid was gradually reduced with the dehydration duration in tobacco leaves, however, the content of GABA and Procontinued to increase, suggesting that water stress induces the formation of GABA and Pro;3.GABA content was much higher than that of proline in leaves under water-deficit andnon-water-deficit conditions. Specifically, the amount of GABA increased is much higherthan that of proline under a serious water loss, indicating that GABA biosynthesis is thedominant pathway from glutamic acid metabolism under these conditions;4.GAD activity demontrated an increasing trend with water stress, whereas, P5CSdemonstrated a decreasing trend. Quantitative reverse transcription polymerase chain reaction(QRT-PCR) and protein Western gel-blot analysis further confirmed this. This result provesthat GABA biosynthesis from glutamic acid is the dominant pathway under water stress;5. Colorimetric photometer assay results showed that the capacity of GABA forO2-,H2O2, and1O2quenching was significantly higher than that of proline. This indicated thatGABA acts as an effective osmolyte to reduce the production of reactive oxygen speciesunder water stress;6. The root length in medium supplemented with1μM GABA is longer than that ofcontrol, indicating that GABA can promote the growth of Arabidopsis root;7. Seedling root tips were treated with Lugol solution and transparent agent treatment,the number of root cap cell layers and the number quiescent center cells were observed undermicroscope, the results indicated that, at the root development of4d, GABA treated plantshave more1layer of root cap cell than that in control; and the number of quiescent centercells have more1in the GABA treatment than that in the control. These results indicated thatGABA could promote quiescent center cell division and differenciation; However, at8d ofroot development, there is no significant differences in root cap layer number betweenexperimental group and the control group;8. At4d of root development, the expression of PLT was significantly higher in GABAtreatment than that in the control, whereas at the8d of root development, the expression ofWUS was higher than the control. GABA may regulate the expression of these two genes tocontrol the dynamic maintenece of quiescent center cell division and differentiation.