A misannotated locus positively influencing Arabidopsis seed germination is deconvoluted using multiple methods, including surrogate splicing

Manoj Majee, Shuiqin Wu, Louai Salaita, Derek Gingerich, Lynnette M.A. Dirk, Joseph Chappell, Art G. Hunt, Richard Vierstra, A. Bruce Downie

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)74-85
Number of pages12
JournalPlant Gene
Volume10
DOIs
StatePublished - Jun 2017

Bibliographical note

Funding Information:
This work was supported by a pilot project and a full research grant to ABD from the Kentucky Tobacco Research and Development Center (University of Kentucky, Lexington, KY, 40546-0312, USA) and a National Science Foundation IOS Grant (0849230, ABD). The activation-tagged lines were acquired from The Arabidopsis Biological Resource Center (The Ohio State University, Columbus, OH, USA). The T-DNA insertional mutant was identified in the SALK SIGnAL resource (SALK Institute, San Diego, CA, USA) and obtained from ABRC. A modified pRTL2 vector (NotI sites introduced 5′ and 3′ to the cassette) was the kind gift of Ms. Gulvadee Chaiyaprasithi. Mr. David Martin provided excellent technical assistance in aspects of the project. The authors declare no conflict of interest with the publication of this paper.

Publisher Copyright:
© 2017 The Authors

Keywords

  • Activation tagging
  • F-Box
  • Germination
  • Proteasome
  • Seed
  • Surrogate splicing

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Genetics
  • Plant Science

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