Analysis of Arabidopsis thaliana mutants defective in the oligopeptide transporter OPT3

Abstract only availableThe transport of peptides across membranes is a phenomenon found in both prokaryotes and eukaryotes as a method of obtaining amino acids, nitrogen, and carbon. Peptides can be transported by ATP-dependent transporters, as well as proton-coupled transporters. Among the latter are members of the oligopeptide transport (OPT) family, which transport tetra- and pentapeptides. Sequence comparisons led to the identification of nine OPT genes in Arabidopsis and our laboratory is investigating the role of these transporters in plant growth and development. Previous studies showed that mutations in the OPT3 gene resulted in embryo lethality. More recently, OPT3 expression was shown to increase under conditions of iron limitation, suggesting a possible role for OPT3 in transporting iron-chelates. The lethal nature of OPT3 T-DNA insertion mutation makes them difficult to study in a homozygous condition. Therefore, we sought non-lethal mutations within the OPT3 gene sequence, which can be maintained as homozygous plants. To create such mutations, we used the process of Targeted Induced Local Lesions IN Genomes (TILLING) to identify non-lethal, point mutations in the OPT3 gene. Eight mutant alleles, opt3-1 to opt3-8, were identified by TILLING. These mutants were sequenced and aligned with the other members of the OPT family to determine whether the mutations occurred within conserved regions of the protein. The mutations opt3-5 (P628S) and opt3-8 (P547L) were the first homozygous mutants identified which occurred within a highly conserved region and, therefore, were the likely candidates to disturb OPT3 function. These mutations were followed in segregating populations by CAPS (Cleaved Amplified Polymorphic Sequence) markers. Homozygous mutant lines and wild-type controls were grown on medium containing limited, moderate, or excess iron. The iron effects on the plant were determined by assaying the chlorophyll content in whole plants. These assays revealed no measurable effect of the OPT3 mutations on chlorophyll content under the conditions tested. We are now examining other opt3 alleles for a role in iron transport and other possible phenotypes displayed during plant growth and development.MU Monsanto Undergraduate Research Fellowshi

Elucidation of the functional role of oligopeptide transporters in bacterial virulence

Abstract only availableThe oligopeptide transporter (OPT) family is a relatively poorly characterized family of peptide/modified peptide transporters found in archebacteria, bacteria, fungi and plants. Plant and yeast OPTs were shown to transport tetra- and pentapeptides as well as the modified peptide glutathione. Our database analysis of sequenced bacterial genomes indicated that OPT proteins are encoded in the genomes of important human pathogens such as Pseudomonas aeruginosa, Mycobacterium tuberculosis, Neisseria meningitidis, and Haemophilus influenzae. However, functional analysis of this family of peptide transporters, especially their possible function in bacterial pathogenesis, is lacking. We obtained three P. aeruginosa strains harboring transposon insertions in the PA3934 locus, the gene predicted to encode the putative orthologous OPT protein (OptA) in P. aeruginosa PA01. Two of the optA mutant strains have in-frame fusion between PaOptA and the PhoA protein encoded within the transposon. Expression of OptA-PhoA is induced by the addition of 20 mM arginine, whereas the expression of OptA-PhoA is not affected by iron availability. The lack of iron-regulated expression of optA would indicate that it is unlikely involved in iron nutrition in P. aeruginosa. We also found that 20 mM arginine and 0.4% peptone enhanced biofilm formation by wild type PA01 strain. However, enhanced biofilm formation by arginine was not observed in the optA mutant strains. Addition of 20 mM lysine had no effect on biofilm formation. We also determined the possible function of OptA in the ability of P. aeruginosa to produce pyocyanin. We found that the optA mutant strains produced higher amounts of pyocyanin than the wild type strain. The presence or absence of arginine in the growth medium had no effect on pyocyanin production. Taken together, these results indicate that OptA is important for biofilm formation by P. aeruginosa in response to arginine and peptides, but is unlikely involved in pyocyanin production.NSF grant to G. Stace

Expression patterns of oligopeptide transporters in arabidopsis thaliana [abstract]

Abstract only availableFaculty Mentor: Dr. Gary Stacey, Plant Microbiology and Pathology & BiochemistryPeptide transport is a universally observed cellular mechanism employed by both prokaryotes and eukaryotes to transport peptides (2-6 residues) across cellular membranes in an energy-dependent manner. These peptides, upon internalization, are rapidly hydrolyzed into amino acids, which can be used for protein synthesis or as alternative sources of carbon and nitrogen. The OPT (Oligopeptide Transporter) family is involved in transport of tetra- and penta-peptides and was first identified in yeast. A distinct subfamily of nine putative Arabidopsis thaliana OPT orthologs (AtOPT1-AtOPT9) were identified by comparison to fungal OPTs. The function of OPT proteins in plant growth and development is largely unknown. One approach to help elucidate their physiological function is to determine the tissue-specific localization of each AtOPT, as well as factors that affect their expression. To accomplish this, promoter-GUS fusions of seven of the AtOPTs were utilized. The transgenic plants were stained for GUS to characterize gene expression at various stages of development, as well as under different growth conditions. Each of the AtOPT fusions exhibited similar tissue-specific expression. The AtOPTs were preferentially expressed in the vascular tissues of vegetative organs but were not expressed in root hairs and root tips. However, differential expression of the AtOPTs was observed under limiting iron conditions.Plant Genomics Internships @ M

OPT3 is a component of the iron-signaling network between leaves and roots and misregulation of OPT3 leads to an over-accumulation of cadmium in seeds.

Plants and seeds are the main dietary sources of zinc, iron, manganese, and copper, but are also the main entry point for toxic elements such as cadmium into the food chain. We report here that an Arabidopsis oligopeptide transporter mutant, opt3-2, over-accumulates cadmium (Cd) in seeds and roots but, unexpectedly, under-accumulates Cd in leaves. The cadmium distribution in opt3-2 differs from iron, zinc, and manganese, suggesting a metal-specific mechanism for metal partitioning within the plant. The opt3-2 mutant constitutively up-regulates the Fe/Zn/Cd transporter IRT1 and FRO2 in roots, indicative of an iron-deficiency response. No genetic mutants that impair the shoot-to-root signaling of iron status in leaves have been identified. Interestingly, shoot-specific expression of OPT3 rescues the Cd sensitivity and complements the aberrant expression of IRT1 in opt3-2 roots, suggesting that OPT3 is required to relay the iron status from leaves to roots. OPT3 expression was found in the vasculature with preferential expression in the phloem at the plasma membrane. Using radioisotope experiments, we found that mobilization of Fe from leaves is severely affected in opt3-2, suggesting that Fe mobilization out of leaves is required for proper trace-metal homeostasis. When expressed in yeast, OPT3 does not localize to the plasma membrane, precluding the identification of the OPT3 substrate. Our in planta results show that OPT3 is important for leaf phloem-loading of iron and plays a key role regulating Fe, Zn, and Cd distribution within the plant. Furthermore, ferric chelate reductase activity analyses provide evidence that iron is not the sole signal transferred from leaves to roots in leaf iron status signaling

Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics

Insertional mutagenesis is a powerful tool for determining gene function in both model and crop plant species. Tnt1, the transposable element of tobacco (Nicotiana tabacum) cell type 1, is a retrotransposon that replicates via an RNA copy that is reverse transcribed and integrated elsewhere in the plant genome. Based on studies in a variety of plants, Tnt1 appears to be inactive in normal plant tissue but can be reactivated by tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean (Glycine max). Experiments showed that the Tnt1 element was stably transformed into soybean plants by Agrobacterium tumefaciens-mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern-blot analysis revealed that the copy number of transposed Tnt1 elements ranged from four to 19 insertions, with an average of approximately eight copies per line. These insertions showed Mendelian segregation and did not transpose under normal growth conditions. Analysis of 99 Tnt1 flanking sequences revealed insertions into 62 (62%) annotated genes, indicating that the element preferentially inserts into protein-coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed throughout the soybean genome. Furthermore, fluorescence in situ hybridization experiments validated that Tnt1 inserted into multiple chromosomes. Passage of transgenic lines through two different tissue culture treatments resulted in Tnt1 transposition, significantly increasing the number of insertions per line. Thus, our data demonstrate the Tnt1 retrotransposon to be a powerful system that can be used for effective large-scale insertional mutagenesis in soybean

Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds

Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits

Critical role for uricase and xanthine dehydrogenase in soybean nitrogen fixation and nodule development

Abstract De novo purine biosynthesis is required for the incorporation of fixed nitrogen in ureide exporting nodules, as formed on soybean [Glycine max (L.) Merr.] roots. However, in many cases, the enzymes involved in this pathway have been deduced strictly from genome annotations with little direct genetic evidence, such as mutant studies, to confirm their biochemical function or importance to nodule development. While efforts to develop large mutant collections of soybean are underway, research on this plant is still hampered by the inability to obtain mutations in any specific gene of interest. Using a forward genetic approach, as well as CRISPR/Cas9 gene editing via Agrobacterium rhizogenes‐mediated hairy root transformation, we identified and characterized the role of GmUOX (Uricase) and GmXDH (Xanthine Dehydrogenase) in nitrogen fixation and nodule development in soybean. The gmuox knockout soybean mutants displayed nitrogen deficiency chlorosis and early nodule senescence, as exemplified by the reduced nitrogenase (acetylene reduction) activity in nodules, the internal greenish‐white internal appearance of nodules, and diminished leghemoglobin production. In addition, gmuox1 nodules showed collapsed infected cells with degraded cytoplasm, aggregated bacteroids with no discernable symbiosome membranes, and increased formation of poly‐β‐hydroxybutyrate granules. Similarly, knockout gmxdh mutant nodules, generated with the CRISPR/Cas9 system, also exhibited early nodule senescence. These genetic studies confirm the critical role of the de novo purine metabolisms pathway not only in the incorporation of fixed nitrogen but also in the successful development of a functional, nitrogen‐fixing nodule. Furthermore, these studies demonstrate the great utility of the CRISPR/Cas9 system for studying root‐associated gene traits when coupled with hairy root transformation