Browsing by Author "Dirk, Lynnette M.A."

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    A misannotated locus positively influencing Arabidopsis seed germination is deconvoluted using multiple methods, including surrogate splicing
    (Elsevier B.V., 2017) Majee, Manoj; Wu, Shuiqin; Salaita, Louai; Gingerich, Derek; Dirk, Lynnette M.A.; Chappell, Joseph; Hunt, Art G.; Vierstra, Richard; Downie, A. Bruce
    A screen of activation tagged lines of Arabidopsis thaliana retrieved COLD TEMPERATURE GERMINATING10-D(tag) (CTG10-D(tag)) seeds, capable of radicle protrusion in advance of wild type (WT) at suboptimal- and optimal-temperatures. Genomic walking revealed T-DNA in the intragenic region that upregulates expression of the At4g19330 locus previously predicted to encode an F-BOX protein. A combination of surrogate splicing, primer scanning, RACE, and Illumina PolyAdenylation Tag (PAT) sequencing in petunia (Petunia X hybrida) and Arabidopsis were required to demonstrate that the region around At4g19330 was misannotated and is actually compromised of two separate genes. Even though homologous regions nearby and elsewhere on chromosome 4 are confounding elements in the molecular characterization of the locus, we could determine that the 5′ entity encodes a ribonucleoprotein of unknown function whereas the 3′ gene includes the promoter and full coding region of an F-BOX protein. Although both genes were upregulated, only independently-transformed lines over-expressing the F-Box exhibited enhanced completion of seed germination. We named it CTG10, and a single, poorly penetrant, mutant line of ctg10 manifested the expected reduced completion of seed germination. Whereas CTG10-OE lines are hyposensitive to the gibberellin biosynthetic inhibitor paclobutrazol, the ctg10 mutant line is hypersensitive; a phenotype which could be alleviated when transgenically rescued with CTG10. The F-Box moiety promoted association of CTG10 with ASK proteins in yeast two hybrid assays, indicating that it likely assembles into an SCF-type ubiquitin ligase to promote the ubiquitination of one or more substrates. In this capacity CTG10 might target a protein repressing seed germination for polyubiquitination and subsequent proteasomal degradation.