Browsing by Author "Rampey, Rebekah A."

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    Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis
    (The Genetics Society of America, 2013-01) Rampey, Rebekah A.; Baldridge, Megan T.; Farrow, David C.; Bay, Sarah N.; Bartel, Bonnie
    Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.
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    Genetic analysis of IAA-conjugate sensitivity in Arabidopsis thaliana: Hydrolysis, transcriptional regulation, and metal homeostasis
    (2005) Rampey, Rebekah A.; Bartel, Bonnie
    Auxins are hormones important for numerous processes throughout plant development. Plants use several mechanisms to regulate levels of the auxin indole-3-acetic acid (IAA), including the formation and hydrolysis of amide-linked conjugates that act as storage or inactivation forms of the hormone. Certain members of an Arabidopsis amidohydrolase family hydrolyze these conjugates to free IAA in vitro. To examine the in vivo importance of auxin-conjugate hydrolysis, I generated a triple hydrolase mutant, ilr1 iar3 ill2 , which is deficient in three of these hydrolases. I compared root and hypocotyl growth of the single, double and triple hydrolase mutants on IAA-Ala, IAA-Leu, and IAA-Phe. The hydrolase mutant phenotypic profiles on different conjugates reveal the in vivo activities and relative importance of ILR1, IAR3, and ILL2 in IAA-conjugate hydrolysis. In addition to defective responses to exogenous conjugates, ilr1 iar3 ill2 roots are slightly less responsive to exogenous IAA. The triple mutant also has a shorter hypocotyl and fewer lateral roots than wild type on unsupplemented medium. As suggested by the mutant phenotypes, ilr1 iar3 ill2 imbibed seeds and seedlings have lower IAA levels than wild type and accumulate IAA-Ala and IAA-Leu, conjugates that are substrates of the absent hydrolases. These results indicate that amidohydrolases contribute free IAA to the auxin pool during germination in Arabidopsis. The regulation of IAA-conjugate hydrolase gene expression remains unknown. I have characterized the IAA-Leu-resistant mutant, ilr3-1, and cloned the defective gene, which encodes a basic helix-loop-helix leucine zipper transcription factor. ILR3 may modulate IAA-conjugate hydrolysis by regulating hydrolase expression or activity. Previous work on the IAA-Ala-resistant mutant iar1, which is defective in a predicted membrane protein similar to metal transporters, suggests a link between metal homeostasis and IAA-conjugate sensitivity. To gain insight into the function of IAR1, I conducted a genetic modifier screen to isolate second-site mutations that restore IAA-conjugate sensitivity to iar1. Here, I characterize one such mutant, mtpc2-1, and identify the gene defective as encoding a metal transport protein. The work described in this thesis suggests roles for multiple components in IAA-conjugate sensitivity in Arabidopsis, including conjugate hydrolysis, transcriptional regulation of relevant genes, and metal homeostasis.
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    Multiple Facets of Arabidopsis Seedling Development Require Indole-3-Butyric Acid–Derived Auxin
    (American Society of Plant Biologists, 2011) Strader, Lucia C.; Wheeler, Dorthea L.; Christensen, Sarah E.; Berens, John C.; Cohen, Jerry D.; Rampey, Rebekah A.; Bartel, Bonnie
    Levels of auxin, which regulates both cell division and cell elongation in plant development, are controlled by synthesis, inactivation, transport, and the use of storage forms. However, the specific contributions of various inputs to the active auxin pool are not well understood. One auxin precursor is indole-3-butyric acid (IBA), which undergoes peroxisomal β-oxidation to release free indole-3-acetic acid (IAA). We identified ENOYL-COA HYDRATASE2 (ECH2) as an enzyme required for IBA response. Combining the ech2 mutant with previously identified iba response mutants resulted in enhanced IBA resistance, diverse auxin-related developmental defects, decreased auxin-responsive reporter activity in both untreated and auxin-treated seedlings, and decreased free IAA levels. The decreased auxin levels and responsiveness, along with the associated developmental defects, uncover previously unappreciated roles for IBA-derived IAA during seedling development, establish IBA as an important auxin precursor, and suggest that IBA-to-IAA conversion contributes to the positive feedback that maintains root auxin levels.