Progress 09/15/03 to 09/14/06
Outputs
Auxins have number of agricultural applications including induction of adventitious or lateral root formation. Only recently have some of the primary molecular events been uncovered, largely due to advances based on genetic approaches. The finding that protein products of Aux/IAA gene family a The objectives of this proposal were to 1) Create and introduce into plants promoter-reporter gene constructs for IAA5, IAA6, and IAA19 2) Characterize the spatial and temporal expression patterns of this group of closely related auxin-inducible genes. In addition, we accomplished a third objective that used loss-of-function alleles of these three genes to identify phenotypic effects in the single, double, and triple mutants and the correlation of those phenotypes with the expression patterns. Objective 1: Greater than a dozen transgenic lines for each promoter-reporter gene construct were identified. Based on the segregation of the antibiotic resistance, 10 lines with single
sites of T-DNA integration were selected and used for preliminary characterization. Based on the preliminary observations, two lines for each construct was selected for detailed analysis. Objective 2: The data indicate that IAA5, IAA6, and IAA19 are expressed in unique, as well as overlapping tissues during Arabidopsis development. These findings support the notion that closely related gene family members might evolve differential expression patterns. The differences in expression patterns, in turn, allow the plant to respond or accommodate responses to a wide range of developmental and environmental cues. Objective 3: Previously, loss-of-function alleles for IAA5, IAA6, and IAA19 were described (Overvoorde et al., 2005). Double and triple knockout combinations of these alleles were created and used to search for phenotypic effects. While the roots of these lines showed wild-type responses to growth on media containing increasing concentrations of IAA, 2,4-D, NAA, ACC, BA, GA, and
ABA, the triple knockout had reduced capacity to form adventitious roots in an assay that we developed. The double knockouts indicated that loss of two genes were responsible for this change in phenotype. Preliminary data indicate that the promoters of these two genes also show activity at the site of emerging adventitious roots. Collectively, these observations provide new insight into the expression patterns and functional roles of these closely related genes. The functional and reporter-based assays provide new tools for determining the mechanism of auxin-mediated changes in gene expression
Impacts
The plant hormone auxin controls a number of important processes. The results of this project describe the tissue-specific expression of three closely related auxin-inducible genes. The unique and overlapping patterns of expression correlate well with the phenotypes associated with lines that lack two or three of these genes. Specifically, two the gene products are required for the formation of adventitious roots. In addition to the scientific contributions, the funding of this project has served as a recruiting tool for the field of plant biology. Ten undergraduate students participated in aspects of this project. Of the seven that have already graduated, four are in graduate school (two in areas related to plant biology and the other two in pharmacology), one is working as a lab technician and will be applying for graduate school, one is in a Teach for America program, and the last is working in a biotech company an will be applying for graduate school next year.
Finally, a pair of projects has been spun off from this work. One of these has been used to obtain external funding and the second will form the basis of a future application.
Publications
- Bush, S. 2004. Defining the Function and Expression of the Aux/IAA Genes IAA5, IAA6, and IAA19 in Arabidopsis thaliana. Honors Thesis; Department of Biology; Macalester College.
- Nalle, S. 2005. Spatial and Temporal Expression of the Early Auxin Response Genes IAA5, IAA6, and IAA19/MSG2 in Arabidopsis. Honors Thesis; Department of Biology; Macalester College.
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Progress 10/01/03 to 09/30/04
Outputs
The goal of this proposal is to define the temporal and spatial expression patterns of three genes (IAA5, IAA6, and IAA19) that belong to the 29-member Aux/IAA gene family. The proposal has three objectives, and significant progress has been made on the all three. Based on suggestions from reviewers and the development of the Gateway cloning (Invitrogen), Objective 1 was modified to incorporate this new approach. A binary vector was created that allows the recombination-mediated cloning of promoter elements upstream of a nuclear-localized, binary reporter gene, mGFP-GUS. This approach has been used to successfully create and confirm by sequencing constructs for the double CaMV 35S promoter and the promoters of IAA5, IAA6, and IAA19. Objective 2: Generate transgenic plants containing the mGFP-GUS reporter gene under the control of the IAA5, IAA6, or IAA19 promoter elements. These binary vector constructs have been transferred into Agrobacterium strains and used to
transform wild-type plants. We are currently screening for transgenic seedlings and will use these plant materials to pursue Objective 3: Determine the spatial and temporal expression patterns of IAA5, IAA6, and IAA19. In addition to progressing with the creation of transgenic reporter lines, we have identified and optimized a set of primers for quantitative real-time PCR analysis of these three genes and other members of the Aux/IAA gene family. Analysis of the hormone responsive and tissue expression patterns will be conducted in the coming year.
Impacts
We are contributing to an understanding of phytohormone-regualted gene expression by studying a closely-related set of proteins that are involved mediating the effects of the plant hormone auxin. In addition to better understanding a biological process, this work could provide insights into the regulation of multigene families, which could be beneficial for modification of agriculturally important plant species.
Publications
- Bush, Susan. B.S. Honors thesis, 2004. Defining the Function and Expression of the Aux/IAA Genes IAA5, IAA6, and IAA19 in Arabidopsis thaliana
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