Identification and molecular characterization of two novel Arabidopsis mutants that are resistant to auxin
Auxin is a pivotal plant hormone that plays a major role in plant growth and development. While indole-3-acetic acid (IAA) is the major natural auxin found in plants, there are many synthetic chemicals with auxinic activity. 1-Naphthylacetic acid (1-NAA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and picloram are some examples of synthetic auxins. Of these, 2, 4-D and picloram are commonly used as herbicides in agriculture. While both natural and synthetic chemicals exhibit similar effects on plants, these chemicals are structurally different. Whether these structurally different chemicals function through similar molecular mechanisms is not known. Initial work in our laboratory indicates that picloram may function differently from other commonly known auxins. To identify genes that are associated with the picloram response, we used a forward genetics approach to isolate ethyl methanesulfonate (EMS) mutagenized Arabidopsis mutants (pie mutants) that were resistant to picloram. The objective of the research project was to characterize the Arabidopsis mutants pic59 and pic115 which exhibit altered response to auxin and to isolate the mutant genes by map-based cloning. Both pic59 and pic115 mutants are morphologically very similar to the wild type Col-0 plants with the exception of the short siliques in mutants. The primary root growth of pic59 exhibits differential responses to different auxins. While pic59 is highly resistant to picloram and indole butyric acid (IBA) and slightly resistant to 2, 4-D, it is sensitive to IAA. pic115 is slightly resistant to IAA but sensitive to IBA. The primary root growth of both pic59 and pic115 is resistant to the plant stress hormone abscisic acid (ABA), but is sensitive to the ethylene precursor ACC and to cytokinin (kinetin). Expression of DR5.·GFP transgene in response to different auxins is impaired in pic59 and pi115 backgrounds, suggesting that auxin-dependent gene transcription is affected in both these mutants. Degradation of HS::AXR3NT-GUS reporter gene is accelerated in both mutant backgrounds compared to wild type. This result differs from the AXR3NT-GUS degradation pattern of most previously known auxin response mutants and suggest that pic59 and pic115 may be involved in a novel auxin regulatory mechanism. By using a positional cloning technique, the pic59 mutant gene was mapped to a region of fourteen genes in the south arm of chromosome V. As there are no currently known auxin-related genes in this genetic window, pic59 represents a newly discovered gene associated with both auxin and ABA responses in Arabidopsis. The pic115 mutant gene was mapped to the north arm of chromosome II between the annotation units T23O15 and F28I8. IBR5, a gene encoding a dual specificity phosphatase (DSP) is located in this genetic window. Sequencing of this candidate gene revealed a single base pair change in the IBR5 gene, creating a missense mutation in the highly conserved dual specificity active site motif of the IBR5 gene. A PIC115:.PIC115-GUS construct revealed that the DSP enzyme is localized to areas of cell elongation. IBR5 is known to dephosphorylate mitogen activated protein kinase12 (MPK12) in Arabidopsis. These results along with results from other laboratories suggest a strong connection between auxin signaling and MAP kinase pathway in plants. It is also possible that PIC59 encodes a protein that functions as the substrate for MPK12 or a protein closely associated with the MPK12 function.
plants, arabidopsis, mutation, molecular cloning, auxin
Siriwardana, C. (2009). Identification and molecular characterization of two novel Arabidopsis mutants that are resistant to auxin (Unpublished thesis). Texas State University-San Marcos, San Marcos, Texas.