Analysis of Membrane Transporter Systems Expressed During Symbiotic Nitrogen Fixation in the Model Legume Medicago Truncatula

Abstract

As the second largest crop family, legumes provide a significant component to the human diet as well as livestock nutrition through forage species. Unique from most other plants groups, legumes are able to form a symbiosis with naturally occurring soil rhizobia, diazotrophs, which are able to reduce atmospheric nitrogen to usable forms of nitrogen for the plant. In return for providing reduced nitrogen, differentiated rhizobia are sustained by dicarboxylates, branched chain amino acids, and other nutrients from the plant. In order for the movement of nutrients to be translocated between symbionts and from the cytosol to cell organelles, other cells, and plant tissues, membrane transporters must be present to move substrates across lipid bilayers. Secondary transporters supply the necessary amino acids and additional nutrients to and from the nitrogen fixation zone of nodules to support symbiotic nitrogen fixation. While the biosynthesis pathways of amino acids to feed symbiosis is well understood, the involved transporters, including importers and exporters, of amino acids, as well as transporters of other nutrients to support infected cells are largely unknown and relatively few transporters have been functionally characterized in legumes. In order to assess potential amino acid transporters, I performed a genomic inventory of putative transporters belonging to the Amino Acid Polyamine-Organocation superfamily by compiling a list of transporters classified under the superfamily in the Transporter Classification Database, along with their protein size, transmembrane domains (TMDs), and compared them to characterized members of transporter families to determine confidence of functionality. I also compiled expression data to determine expression patterns to manually annotate candidate nodule specific transporters of interest for further study. Using the model species Medicago truncatula to study symbiotic nitrogen fixation, I genetically characterized the nodule specific membrane transporter, MtAPC1 through in silico analysis of expression patterns, followed by transcriptional analysis of inoculation with rhizobial mutants to determine essential steps of infection necessary for gene expression. Transcriptional activity of MtAPC1 was also fine-tuned to identify cell-type within the nodule and the impact of the MtAPC1 knockdown in nodule development during symbiotic nitrogen fixation. I also characterized expression patterns of a copper transporter, MtCOPT1, regarding its transcriptional activity in nodules and its capability of importing copper into the cell cytosol. In addition, I preliminarily analyzed of two putative nodule-specific transporters, MtSEN1 and MtOPT1 for iron and short oligopeptides (3-8 amino acids in length) though in silico analysis with publicly available datasets, as well as determined expression patterns by RT-qPCR of nodulating root inoculated with rhizobial mutants and wild-type rhizobia. v

Hydrolytic Enzymes, Proteases, Permeases, and Transporters as Cryptococcus neoformans Weapons in Human Infections

“…For example, nitrogen starvation induces Trp biosynthetic enzymes encoded by Trp2,Trp3,Trp4,and Trp5 [112]; also, under repressive NCR, Aap2 and Aap3 are induced in the presence of Lys-containing medium [106]. However, amino acid supplements in SD do repress the general amino acid control (GAAC) but promote the APC-superfamily membrane transporters/permeases (APC, AAAP, AGCS, CCC, HAAAP, BCCT, SSS, NSS, NCS1, NCS2, and SulP families [114][115][116]).…”

Transcription Factors Control the Virulence-Associated Metabolic Enzymes and Membrane Proteins in Response to Nutrient Bioavailability in Cryptococcal Infection

“…For example, nitrogen starvation induces Trp biosynthetic enzymes encoded by Trp2, Trp3, Trp4, and Trp5 [92]; also, under repressive NCR, Aap2 and Aap3 are induced in the presence of Lys-containing media [52]. However, amino acid supplements in SD do repress the general amino acid control (GAAC) but promote the APC-superfamily membrane transporters/permeases, such as APC, AAAP, AGCS, CCC, HAAAP, BCCT, SSS, NSS, NCS1, NCS2, and SulP families [94][95][96].…”