| y-Aminobutyric acid (GABA),a four-carbon, non protein and conserved amino acid, is found in various species,from the lowest bacteria to higher organisms. In mammalian, GABA presents in high concentrations in central nervous system(CNS) as the primary inhibitory neurotransmitter. It can activate various signaling pathway via binding to seveval types of receptors anchoring in the membrane of neuro cell. And also, GABA was proved to be a significant component of the free amino acid pool in higher plants. It presents in almost all organs. The intercellular GABA content was increased significantly and quickly as a response to biotic and abiotic stress,such as cold,drought, salinity, extreme temperatures, and oxidative stress,fungus etc. Researchs have shown that such a elevation of GABA level can regulate the PH levels of cytoplasm and thus regulate the activation of several enzymes, which is PH dependent,and constituted the plant self-protected mechanisms in biochemical levels.At the same time,GABA can involve in N/C metabolism balance in plants as a significant N?C source.When the plant is infringed by insect pests,the elevated GABA content can interrupt the nervous system of insect pest,and thusresult in abnormal development and growth of insect pest. Recently,researchers demonstrated that GAGA play an important role in pollen tube elongation and orientation. Our previous work also showed that there are GABA binding sites on the tobacco mesophyll and pollen protoplasts. At the same time, some GABA related micro array data showed that exogenous GABA can mediate the expression of some genes in plants. More and more evidences support the hypothesis that some GAGA receptors or binding proteins exists to transduce GABA signal. These investigations imply that GABA is not only as a response mechanism in plants,but also as a significant signal moleculein higher plants as its role in animals,and it may playa critical regulating role during the plant development.Based on the researches described above, the main aim of our researches is to explore the possible GABA receptor or binding proteins in plants following the research methods for the discovery of GABA receptor in animals,and also to reveal the physiology and biochemistry mechanism of GABA function in plantsas a exogenous signaling molecule. For these purposes, we carrioud out following main resaech works:1. Wechose the GABA B receptor agonist: CGP64213,and coupled this molecule with Bodipy, a fluorescent group. Weused this probe to detect if its binding sites on the membrane of plant protoplast are existed. Ourresults showed that such a B type receptor agonist probe could not bind with plant cell membrane. This further support the idea that there is no homologueof a animal GABA receptors in plant. Atthe same time, this result indicates that the GABA receptors in plant may own distinct architectures compared with that in animals.2. As the GABA B type receptors was proved to act as a G protein coupled receptors, we proposed that the GABA receptors of plant were most likely a GPCR too. Wetry to purify some GPCR proteins in Arabidopsis in order to check if the plant GABA receptor is also GPCR by in vitro binding exam.Unfortunately,our experiment showed that the prokaryotic expression system is not suitable for the expression of the multi-transmembrane proteins.3. For seeking the GABA receptors in plant, we conducted the affinity chromatography as the main methods to purify GABA receptors, we coupled GABA with a new type of magnetic beads, and we plan to use it as a baitto angle for the GABA receptors or binding proteins from Arabidopsis total protein. We finally got a specific band after affinity chromatography. Massspectrometry showed the band contain18candidates. Wechose3of them forprokaryotic expression and purification. Atlast, an U-box44protein was purified.We then detected the SPR-relationship of this protein though linkong GABA on a chip by BIACORE X system,and final indicated that U-box44protein can not directely bind with GABA.4. Wedetected the effect ofexogenous GABA on the regulation of permeability of calcium permeable channels. Weused the whole cell patch clamp technology, and found that exogenous have a biphasic effect on the modulation of calcium permeable channel. It means that the high concentration of GABA have a conspicuous suppression on calcium permeable channel and low concentration of GABA can active the calcium channel. The specificity of the effect on calcium permeable channel was further confirmed by the application of the calcium channel blocker.5. Previousresearches in Arabidopsis shows that there may be a GABA gradient in pollen tube growth pathway, and this gradient might be a potential site information for regulating pollen tube orientation. We thus tested if a GABA gradient exists in the style, the main part of the pollen tube growth pathway.Take the advantage of tobacco with a long style, we dissected the tobacco style into three parts, and detected the GABA content respectively, and provided new evidence that there is really a GABA gradient in the tobacco style. This indicates the exogenous GABA existed in style may act as a signaling molecular and guide pollen tube orientation.Our work mention above shows that the GABA exist in the style on a concentration gradient, which as a signaling molecule to activate the calcium permeable channels, and it offers an example showing how style could interact with pollen tube during fertilization,and make sure the accurate growth and orientation of pollen tube. |
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