Doctoral Dissertations
Date of Award
5-2022
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biochemistry and Cellular and Molecular Biology
Major Professor
Dr. Daniel Roberts
Committee Members
Dr. Brad Binder, Dr. Tessa Burch-Smith, Dr. Tarek Hewesi
Abstract
Plant nodulin 26 intrinsic proteins are categorized into three groups (NIP I, II, and III) based on pore architecture. NIP II and III participate in metalloid nutrition, whilst the function of a third (NIP I) is less understood. Here we investigate the physiological function of one NIP I protein (Arabidopsis thaliana NIP2;1) as a lactic acid channel, and also explore the structural basis for metalloid and water permeability of NIP I and NIP II proteins in general. In addition, a strategy was developed for the purification and crystallization of soybean nodulin 26 as a step towards structure determination of a NIP I protein.
NIP2;1 is specifically expressed in roots in response to low oxygen stress where it accumulates predominantly on the plasma membrane. Compared to wild type plants, nip2;1 mutant plant roots over accumulate lactic acid, and show lower lactic acid efflux and acidification of rhizosphere during hypoxia. nip2;1 plants survive hypoxia poorly compared to wild type plants, suggesting that lactic acid efflux by NIP2;1 prevents lactic acid toxicity. Nip2;1 plants show altered expression of pyruvate and lactate metabolizing enzymes, implying regulation of fermentation by lactic acid accumulation in vivo.
The structural basis for Arabidopsis NIP I and NIP II permeability was investigated by biochemical assays, plant genetics, and molecular modeling. Compared to NIP II channels, boric acid is poorly permeated by some NIP I channels (NIP4;1) but not others (NIP1;1). Over expression analysis in planta shows that NIP I proteins have some ability to transport boric acid in plants, but not as well as NIP II proteins which are bona fide physiological boric acid channels. The primary distinction between NIP I and NIP II proteins is that the latter have lost their function as aquaporin water channels. Modeling of NIP6;1 reveals a larger selectivity filter with space for five amino acids rather than the four seen in classical aquaporins and NIP I proteins. Potential models for how the NIP II pore accommodates boric acid, as well as hypotheses for gating of the NIP II pore to prevent water transport were generated by homology modeling and molecular dynamics of NIP6;1.
Recommended Citation
Beamer, Zachary, "Nodulin 26 like Intrinsic Proteins: Structurally Similar Membrane Channels with Diverse Functions in Plant Hypoxia Stress, Metalloid Nutrition & Toxicity. " PhD diss., University of Tennessee, 2022.
https://trace.tennessee.edu/utk_graddiss/7108