The Function Of MaGAD1 In Ethylene Biosynthesis And Signal Transduction Pathways

Glutamate decarboxylase (GAD; EC 4.1.1.15)-a kind of intracellular enzymes is a pyridoxalphosphate(PLP)-dependent enzyme that catalyses the irreversible a-decarboxylation of L-glutamate to y-aminobutyrate (GABA),which exists widely in animal and plant,It is a kind of intracellular enzymes.The role played by GABA in plants, however,remains to be precisely delineated, whereas its function as an inhibitory neurotransmitter in animals is fairly well understood (Lee et al.,2001).Genes encoding GAD proteins have been identified in a number of different plants such as Arabidopsis thaliana (Bouche et al.,2004; Turano and Fang 1998), rice (Akama et al.,2001; Ohs et al.,2005), cowpea (Johanson et al.,1997), tomato (Gallego et al.,1995), and tobacco (Yuns and Ohs 1998).Sequence analysis showed that this protein has two conserved domains of glutamate decarboxylase, a pyridoxal-5’-phosphate binding domain in the middle region and a calmodulin (CaM)-binding domain at the carboxyl terminus, which is a unique feature in plants (Arazi et al.,1995; Baum et al.,1993; Gallego et al.,1995).The GAD activity can be regulated in vivo by Ca2+/CaM and pH(Snedden et al.,1995; Snedden et al.,1996; Turano and Fang 1998; Zik et al.,1998).Abundant amounts of GABA rapidly accumulates in a variety of plant organs under several environmental stress conditions(e.g. Mechanical damage, chilling, salt stress, plant hormones, hypoxia),and GAD expression,GAD activity are also induced in response to these stress. Therefore,GAD play role in regulation of cytosolic pH, carbon effluxes into the tricarboxylic acid cycle, nitrogen metabolism,transport,storage, and deterrence of insects (Yunlong Zhuang et al.,2010; Juan et al.,2010; Bouche and Fromm 2004; Shelp et al.,1999; Alan WB et al.,1997; Pedro PG et al.,1995).The GAD is isolated from banana fruit(Mum acuminata L.AAA group cv.Brazilian) and designated as MaGAD1.RT-PCR analysis shows that MaGAD1 expresses in roots, stems, leaves, flowers and fruits.The expression lever is higher in fruits than in other organs. So, MaGAD1 probably plays a certain role in different physiological course, and is closely related with fruit mature.In order to further study the function and molecular mechanism of MaGADl under various stresses, banana seedings(Musa acuminata L.AAA group cv. Brazilian) were treated with ethephon, waterlogging stress or wound stress. And then the expression of MaGAD1, GAD activity and the content of GABA were investigated. The results showed that the MaGAD1 expression increased observably when the banana seedings were treated with ethephon, GAD activity and GABA levels closely matched MaGAD1 expression. The MaGAD1 expression was not markedly induced under waterlogging stress or wound stress. However, GAD activity and GABA levels had a strong response to stress. These results suggested that the expression of MaGADl was induced by ethephon and MaGAD1 may mainly participate ethylene responsive pathway rather than waterlogging and wood stress response pathway which may be regulated by some other GAD family numbers.Based on the above research, in order to further study the close relationship between MaGAD1 and ethylene, by means of agrobacterium mediated inflorescence soaking method, we used the constructed vector named PC 1302-MaGAD1 to transfer extraneous MaGAD1 to Arabidopsis thaliana. We got 21 positive plants by resistance selection and PCR. Moreover, at the T3 generation,11 homozygous genotypes were screened. After southern blot detecting, we selected C-4,C-6 homozygous genotypes to be researched in follow-up experiment.During the stage of seeds germination, OE(MaGAD1 overexpressing lines’) germination rate were higher than wild type’s, as well as stronger sensitivity to exogenous ACC.When treated adult plants of all the lines with 0.2%o ethephon, we monitored the ethylene responsive genes. The results revealed that all these genes exhibited much higher level in OE lines, suggested that MaGAD1 transgenic lines indeed had higher sensitivity to ethylene signal.All the evidence mentioned above demonstrate that, MaGAD1 could affect plant mature and aging by regulating key genes expression of ethylene metabolic pathways.