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bioRxiv · 10.1101/2022.01.27.478073

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

Luo, M.·Hu, L.·Li, W.·Ge, L.·Qin, Y.·Zhou, Y.·Tang, W.·Wang, C.·Xu, Z.-S.·Delaplace, P.·Ma, Y.-z.·Chen, M.·Chen, J.
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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.

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Luo, M., Hu, L., Li, W., Ge, L., Qin, Y., Zhou, Y., Tang, W., Wang, C., Xu, Z.-S., Delaplace, P., Ma, Y.-z., Chen, M., Chen, J.. 2022-01-28. Heterotrimeric G protein α subunit (GPA1) regulates the response to low-nitrogen stress in Arabidopsis by interacting with AtNRT1.4 and AtATG8a. https://doi.org/10.1101/2022.01.27.478073

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