REU: Glycerol Metabolism and its Role in Biotrophy Versus Necrotrophy in an Arabidopsis/Fungal Hemibiotroph Model system.

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Description

The undergraduate intern, Mr. Jared Gabbert, for whom funds are requested will perform independent research projects that work towards the completion of a portion of the NSF-funded project (IOS 1051909) related to elucidating the role of oleic acid regulated metabolism in plant defense. The objectives of their research will be to characterize promoter domains that respond to 18:1 levels and responsible for upregulation of resistance (R) genes. Project outline Stearoyl-acyl carrier protein desaturase (SACPD) is one of the conserved enzymes among plants, which catalyzes the first desaturation step in fatty acid biosynthesis (conversion of stearate to oleate). In plants, changes in the levels of oleic acid (18:1), a major monounsaturated fatty acid (FA), results in the alteration of salicylic acid (SA)- and jasmonic acid (JA)-mediated defense responses. This is evident in the Arabidopsis ssi2/fab2 mutant, which owing to its defect in SACPD accumulates high levels of stearic acid (18:0) and reduced levels of 18:1. Consequently, replenishing 18:1 levels results in the restoration of wild-type (wt)-like signaling in the ssi2 mutant. We have identified several genes, loss-of-function of which, restore the altered defense signaling in ssi2 plants. The 18:1 content in wt plants can be lowered by glycerol application to produce ssi2-like phenotypes. The Arabidopsis mutants impaired in glycerol catabolism or generation of glycerol-3-phosphate (G3P) do not induce the SA pathway and fail to reduce their 18:1 levels after glycerol application. The plants overexpressing the ACT1 gene (encodes G3P acyltransferase) metabolize G3P rapidly and are more sensitive to glycerol application. Conversely, a mutation in act1 rescues hypersensitivity to glycerol. The 18:1 levels in plastids are regulated via acylation with G3P and a balance between G3P and 18:1 is critical for the regulation of defense signaling pathways. Moreover, loss of activity of SACPD, and thereby the reduced levels of 18:1 induce the expression of a variety of resistance (R) genes, which in turn confer broad-spectrum disease resistance to multiple pathogens. Proposed work We have previously shown that low 18:1 upregulates transcript levels of eight different R genes in Arabidopsis. Transcript profiling of ssi2 sid2 plants using microarray analyses detected 22 additional R or R-like genes that are significantly upregulated as compared to wt plants. To identify sequences mediating the low 18:1-responsive induction of these R genes, we are analyzing promoter sequences for conserved motifs. Preliminary sequence studies using the motif analysis tool at the Arabidopsis database, detected at least two 6-mer motifs that were conserved in the upstream sequences (1000 bp) of all 22 genes. In addition, three motifs were conserved in 96% and, seven motifs in 93% of the sequences analyzed. Since the promoters of R genes HRT and RPS2 also carries many of these motifs, we are carrying out deletion analysis of these promoters to identify the minimal region conferring low 18:1 responsiveness. The HRT/RPS2 promoter-GUS fusion constructs, containing various regions of the HRT/RPS2 promoters fused to the GUS gene have already been created and are currently been transformed into Arabidopsis plants. The transgenics plants will be evaluated for GUS expression under normal and low 18:1 conditions. Exogenous application of glycerol will be used as a means to lower 18:1 levels in transgenic plants. A final confirmation will be obtained by mobilizing the minimal promoter-GUS cassettes in Arabidopsis mutant backgrounds carrying low 18:1 (ssi2 and ssi2 sid2) or high 18:1 (act1). Once sequences responding to low 18:1 have been identified these will be further dissected using site-directed mutagenesis.
StatusFinished
Effective start/end date3/20/144/30/16

Funding

  • National Science Foundation

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