Heterotrimeric G protein α subunit (GPA1) regulates the response to low-nitrogen stress in Arabidopsis by interacting with AtNRT1.4 and AtATG8a

Abstract

Efficient nitrogen absorption and utilization are important factors for higher plants to increase yield and reduce eutrophication (caused by excessive use of nitrogen fertilizers). Heterotrimeric G proteins, including three subunits of , {beta}, and {gamma}, participate in the pathway regulating nitrogen absorption and utilization in plants. However, the regulatory mechanism remains largely obscured. In this study, our results revealed that the G protein subunit (AtGPA1) mutant gpa1-4 was tolerant to low-nitrogen stress in Arabidopsis. AtGPA1 was shown to directly interact with a nitrate transporter (AtNRT1.4) and a key autophagy-related protein (AtATG8a) on the plasma membrane using the yeast hybrid system and pull-down assay (in vitro) and BiFC assay (in vivo). GUS staining and subcellular localization showed that AtGPA1 and AtNRT1.4 were co-expressed in roots and leaf veins and on the plasma membrane. Under low-nitrate conditions, the single mutant gpa1-4 and NRT1.4RNAi plants (AtNRT1.4RNA interference plants) and the double mutant NRT1.4RNAi/gpa1-4 plants (AtNRT1.4RNA interference plants on a gpa1-4 background) were healthier than the wild type plants. Moreover, the phenotype of the double mutant NRT1.4RNAi/gpa1-4 plants was closer to that of the NRT1.4RNAi plants compared to that of the gpa1-4 mutants. The results of the nitrate efflux rate assay in roots were consistent with the phenotypic changes under low-nitrogen conditions. These results indicated that AtGPA1 is an upstream factor that regulated the response to low-nitrogen stress through interaction with AtNRT1.4. In addition, we found that transgenic plants overexpressing AtATG8a were more tolerant to low-nitrogen stress, and their phenotype was similar to that of gpa1-4 mutants and double mutant ATG8aOX/gpa1-4 plants (AtATG8a overexpressing plants on a gpa1-4 mutant background). Further, autophagosome observations were consistent with the phenotypes in mutant plants, indicating that AtGPA1 regulated the response to low-nitrogen stress in Arabidopsis plants by affecting the autophagosome assembly. Our findings may provide a new model for improving nitrogen-use efficiency through genetical modification to boost crop yields. One sentence summaryAtGPA1 negative regulates low nitrogen stress response by interaction with a nitrate transporter, AtNRT1.4 and an autophagy-related protein, AtATG8a in Arabidopsis.