Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

Authors: HANNAM, CAROL - University Of Guelph; GIDDA, SATINDER - University Of Guelph; HUMBERT, SABRINA - University Of Guelph; PENG, MINGSHENG - University Of Guelph; CUI, YUHAI - Agriculture And Agri-Food Canada; Dyer, John; ROTHSTEIN, STEVEN - University Of Guelph; MULLEN, ROBERT - University Of Guelph

Submitted to: Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2017
Publication Date: 11/16/2017
Publication URL: http://handle.nal.usda.gov/10113/5922785
Citation: Hannam, C., Gidda, S.K., Humbert, S., Peng, M., Cui, Y., Dyer, J.M., Rothstein, S.J., Mullen, R.T. 2017. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway. Botany 96:79-96.

Interpretive Summary: Nitrogen is an essential element for plant growth and development, and under low nitrogen conditions, plants undergo several physiological adaptations including reduced photosynthesis, reduced biomass production, early flowering, and mobilization of nitrogen from old to young tissues. In order to develop crops with improved nitrogen use efficiency, it is important to elucidate the molecular mechanisms involved in low nitrogen adaptation. Recently, a gene called “NITROGEN LIMITATION ADAPTATION” (NLA) was identified in the model plant Arabidopsis that was shown to play a key role in the ability of plants to adapt to low nitrogen conditions; plants harboring mutations in the NLA gene were unable to respond to low nitrogen treatments, resulting instead in early plant death. Analysis of the underlying mechanisms revealed that the NLA protein functions, at least in part, by regulating the activities of nitrogen and phosphate transporters located in the cell membrane. The protein was also localized, however, to distinct regions of the nucleus, suggesting that the protein functions within different subcellular compartments. Scientists at the University of Guelph, Agriculture and Agri-food Canada, and the ARS lab in Maricopa, Arizona, performed a detailed assessment of the structural properties of the NLA protein and determined that different parts of the protein were required for localization and function within different regions of the cell. These results shed significant light on the structure/function relationships of NLA and will guide future efforts to elucidate the role of NLA in low nitrogen adaptation. These studies will ultimately provide the knowledge base and genetic tools for improving nitrogen use efficiency in crop plants.

Technical Abstract: The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING domain that mediates degradation of phosphate transporters at the plasma membrane; ii) an SPX domain that facilitates NLA’s interaction with the phosphate transporters, and also exists in other proteins that regulate the nuclear transcription factors that control the phosphate starvation response pathway; iii) a linker domain that lies between the RING and SPX domains; and iv) a C-terminal domain, which, like the linker region, is of unknown function. Here we carried out a mutational analysis of NLA, which indicated that all the domains are not only essential for proper functioning of the protein, but also mediate its localization to the plasma membrane and/or nucleus, as well as to different subdomains within the nucleus. Overall, the results provide new insights to the distinct protein motifs within NLA and the role(s) that this protein serves at different subcellular sites with respect to the regulation of nitrogen-dependent phosphate homeostasis as well as other possible physiological activities.