Functional Characterization Of MAG2 In Arabidopsis Thaliana

One of the major vesicle trafficking pathways in plants is the transport of vesicles from ER to the central lytic vacuole or the protein storage vacuole. Vesicle trafficking requires membrane fusion, mediated by SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins. Recently, it was shown that multisubunit tether complexes (MTCs) are essential for intracellular transport and have been proposed to function in the initial attachment of transport vesicles to their target membranes. Arabidopsis MAG2, which is related to yeast TIP20 in Dsl1p complex and mammalian RINT-1 in syntaxin 18 complex, functions in the transpost of storage protein precursors between the ER and Golgi apparatus.It has been reported that vesicle trafficking plays an important role in abiotic stress tolerance. The mag2 mutation causes hypersensitivity to salt stress, osmotic stress, ABA, sugar, and PAC, an inhibitor of GA biosynthesis, which is characterized by reduced root elongation and chlorophyll content, and impaired ionic and osmotic responses during seed germination and early seedling establishment. Interestingly, mutation in mag2l, a homolog of mag2 in Arabidopsis, increases osmotic stress tolerance in germination and seedling development. The mag2l mutation causes hyposensitivity to ABA and PAC treatments in germination and seedling establishment. Many stress-responsive genes, ABA biosynthesis and ABA signal genes are downregulated or upregulated in mag2 plants. In addition, the mag2 mutation causes impaired basal thermotolerance in seed germination and seedling development. However, no significant phenotype is observed in mag2l plant after heat shock treatment.Seed germination and seedling establishment of mag2 and zw10, which codes a homologous protein of yeast Dsllp and mammalian ZW10, are sensitive to DTT treatment. BiP2;GUS expression results show that BiP2 expression in mag2 and zw10 plants is higer than that of wt and mag2l plants with or without DTT treatment. Our yeast two hybrid assay (Y2H) shows that MAG2 interacts with ZW10CR(304-540). The expression pattern of mag2, mag2l, Atzw10, AtSec20, AtUfel, and AtSec39 in different tissues or ABA, GA, and ethylene mutants are very similar. Combined the previous protein interaction results (Li et al.,2006,2009), we propose that a similar MTC, which is related to yeast Dsllp complex or mammalian syntaxin 18 complex, is also existed in Arabidopsis.Transient expression results show that MAG2 and MAG2L localize in ER and punctate structures. The TIP20 domain in MAG2 and MAG2L proteins is essential for these two proteins subcellular localization. Sec20 localizes in ER structure. Transient expression of ER markers in mag2, mag2l, zw10, and sec20 protoplasts shows that the morphology of ER is not affected in these mutants with or without NaCl, Mannitol and BFA treatments. Transient expression of ER to vacuole markers in mag2, mag2l, zw10, and sec20 protoplasts shows that the anterograde trafficking pathway from ER to vacuole is not affected in these mutants. Confocal imaging of transgenic plants with ER to cell outer space marker shows that mutation of mag2 does not affect ER-to-apoplast anterograde trafficking pathway. Our results indirectly suggest that mag2 mutation impairs the Golgi-to-ER retrograde trafficking in Arabidopsis.In short, the results in this article imply that MAG2 plays an important role in plant adaptation to abiotic stresses and ER stress, beyond the function in intracellular vesicle trafficking. The involvement of MAG2 in abiotic stress and ER stress responses may shed light on membrane fusion or vesicle transport in stress adaptation.