Analysis Of The In Vivo Interaction Of GPX3 And RRTF1 With DOS1 Using BiFC Technology In Arabidopsis

Adverse stresses can be classified as abiotic ones and biotic ones, which seriously affect plant growth and development. The abiotic stresses are diverse. They can trigger the production of reactive oxygen species (ROS), which may be the cross point of various stress signals. In recent years, scientists have analyzed the expression patterns of large numbers of genes by DNA microarray, investigated the functions of the genes and found many important cis-elements acting in ROS signal transduction.Previous results from our laboratory revealed that GPX3 (Glutathione peroxidase 3), a member of GPX family, encodes a functional protein. It can not only scavenge H₂O₂, but also regulate upstream ROS signals and activate and transfer downstream signals. AtDOS1 (Drought overly sensitive 1) has been identified to interact with GPX3 in vitro very strongly through the yeast two hybrid experiment. It localizes in nucleus, containing NLS and WWE domain, and plays a key part in plants response to drought and oxidative stress. DOS1 appears to be one of the important transcriptional regulatory factors in oxidative signal transduction in plants. Moreover, DOS1 can diffuse from the nucleus to the cytoplasm under H₂O₂ stress and transfer the redox signals. AtRRTF1 (ROS related transcription factor 1) is another gene which may interact with AtDOS1 in gene chips. It encodes a transcriptional factor containing zinc structure. The expression of AtRRTF1 is induced by H₂O₂ treatments. RRTF1 plays central roles in signal transductions of biotic and abiotic stresses. Yeast two hybrid experiment also showed that RRTF1 interacting with DOS1 in vitro. Yet, until now we still have no evidence about the in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. We still have little knowledge about the diffusion mechanisms of DOS1 in Arabidopsis under oxidative stress.In this paper, the interactions between GPX3 and DOS1 as well as RRTF1 and DOS1 in vivo were investigated using the method of splite YFP (BiFC). There was yellow fluorescence (interaction indicator) in protoplasts of WT plants transformed with GPX3-contained vector and DOS1-contained vector, as well as RRTF1-contained vector and DOS1-contained vector, indicating that DOS1 may interact with GPX3 and RRTF1 in vivo. DAPI staining analysis showed that the place in which DOS1 interacts with RRTF1 is the cell nucleus. No obvious interaction between DOS1 and RRTF1 in vivo was found in protoplasts from gpx3 plants suggesting that GPX3 may influence such interaction in vivo. To further determine if the interaction between DOS1 with GPX3 and RRTF1 affect the diffusion of DOS1 across nucleus membrane and the transferring of stress signals by DOS1 under oxidative stress, we obtained Arabidopsis gpx3 and rrtf1 T-DNA insertion mutants with DOS1-GFP tag (gpx3-DOS1::GFP, rrtf1-DOS1::GFP) by using hybridization technology between WT-DOS1::GFP and gpx3, rrtf1 mutants. Then, differences in DOS1 diffusion across nucleus membrane in the two mutants and WT were analyzed in confocal microscopy and statistical analysis. The results showed that GPX3 may influence the DOS1's diffusing movement to a certain extent. Without the GPX3, the DOS1 in guard cells would be more inclined to diffuse from the nucleus to the cytoplasm after treatments with H₂O₂ or ABA. But when the expression of RRTF1 was down-regulated, there was no obvious difference for the DOS1 diffusing movement in rrtf1 mutant with DOS1-GFP tag compared to the WT with DOS1-GFP tag. These datas indicated that RRTF1 may not affect the diffusion of DOS1 under oxidative stress.To sum up, there exists in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. GPX3 may affect the interaction between RRTF1 and DOS1. The in vivo interaction between GPX3 and DOS1 may modulate the diffusing movement of DOS1 across nucleus membrane under oxidative stress in Arabidopsis.