FUNCTIONAL ANALYSIS AND BIOCHEMICAL CHARACTERIZATION OF HETEROTRIMERIC G PROTEIN IN ARABIDOPSIS

Open Access
- Author:
- Wang, Shiyu
- Graduate Program:
- Biochemistry, Microbiology, and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 18, 2007
- Committee Members:
- Ming Tien, Committee Member
Teh Hui Kao, Committee Member
David Scott Gilmour, Committee Member
Sarah Mary Assmann, Committee Member
Nina Fedoroff, Committee Member
Alan G Jones, Committee Member - Keywords:
- HETEROTRIMERIC G PROTEIN IN ARABIDOPSIS
- Abstract:
- Heterotrimeric G proteins are recognized as important signal transduction molecules in all eukaryotic organisms from yeasts to humans. Compared to mammals and invertebrates, simplicity of G protein gene family in Arabidopsis provides a unique advantage for investigating its functions in various aspects of different physiological processes. Extensive studies in the last 10 years have shown that the Arabidopsis heterotrimeric G protein is involved in a transducing a variety of signals, including light, hormones, biotic and abiotic stressors. First, we analyzed the functional roles of heterotrimeric G protein in Arabidopsis oxidative stress responses to ozone (O3). Arabidopsis thaliana plants of homozygous gpa1-4 (Gα null mutation) are less sensitive to O3 damage than wild-type Columbia-0 plants, whereas agb1-2 (Gβ null mutation) plants are more sensitive to O3 damage than wild-type Columbia-0 plants. The genes encoding the α and β subunits of the Arabidopsis heterotrimeric G protein are differentially expressed in the course of the oxidative stress response to O3, suggesting that they play different roles in the plant’s O3 responses. The first peak of the biphasic oxidative burst elicited by O3 in wild-type plants is absent in both mutant plants, suggesting that the first peak of the oxidative burst requires both α and β subunits or the intact heterotrimeric G protein. The second peak is missing in gpa1-4 mutant plants, but is normal in agb1-2 mutant plants, suggesting that the second peak of the oxidative burst requires only the α subunit but not the β subunit. Thus, the opposing O3 phenotypes and the differential ROS production patterns between gpa1-4 and agb1-2 mutant plants indicate that the α and β subunits play separable roles in cell death associated oxidative stress response to O3. Second, we analyzed the functional role of heterotrimeric G protein of Arabidopsis unfolded protein responses to Tm. Seedlings of agb1-2 mutant plants with a null mutation in the gene coding for the β subunit of the heterotrimeric G protein are more resistant to growth inhibition by the protein glycosylation inhibitor tunicamycin (Tm) than wildtype plants and gpa1-4 plants with a null mutation in the gene encoding the α subunit of the heterotrimeric G protein. Leaves of agb1-2 mutant plants exhibit markedly less cell death after Tm treatment than those of wildtype plants. The transcriptional response of agb1-2 mutant plants to Tm is less pronounced than that of wildtype plants. On the other hand, AtrbohD (AtrbohD null mutation) and AtrbohD/F (AtrbohD and AtrbohF null mutation) mutant plants show more cell death in response to treatment with Tm than wildtype plants and AtrbohF (AtrbohF null mutation) mutant plants. The protective role of the endogenous ROS elicited by Tm treatment against Tm-induced cell death is also seen using ROS scavengers. Moreover, the transcriptional responses are delayed by the pretreatment with ROS scavengers. A majority of the Arabidopsis Gβ protein is associated with the endoplasmic reticulum (ER) and is degraded after Tm treatment, while Gα protein is not. Collectively, these observations indicate that the Gβγ complex, not Gα, plays an important role in cell death associated with the unfolded protein response in Arabidopsis. Moreover these observations imply a direct role for both ROS and G protein signaling in the UPR. To elucidate the mechanism of G protein signaling transduction, we characterized the Arabidopsis heterotrimeric G protein complex. Using fluorescence resonance energy transfer (FRET) and co-immunoprecipitation, we show interaction between the β1 and γ subunits and between the α1 and β1γ subunits of the Arabidopsis heterotrimeric G proteins in plant protoplasts. We report that both the Gα and the Gβ subunits are associated with macromolecular complexes in plasma membrane fractions. We estimate the size of the G-protein-containing plasma membrane complex to be approximately 669 kD based on blue native polyacrylamide gel electrophoresis, suggesting that the heterotrimeric G protein is present in a complex with other proteins. We find that Gα1 is present in both the large ca. 669 kD complex and in smaller complexes in plants homozygous for the agb1-2 Gβ1 null allele. Deletion of the Gβ1 interacting domain in Gα1, ahGα1, abolishes normal function of Gα1 in the oxidative stress response to ozone, suggesting the interaction between Gα1 and Gβ1γ complex may be required for transmitting the O3 signal in plant cells. Treatment of the plasma membrane fraction with hydrogen peroxide (H2O2), an important signaling molecule, results in the partial dissociation of the Gα1 complex. This observation suggests direct or indirect activation of G protein signaling by ROS in the oxidative stress response.