title: Characterization of Arabidopsis thaliana lines with altered seed storage protein profiles using synchrotron-powered FT-IR spectromicroscopy.
abstract: Arabidopsis thaliana  lines expressing only one cruciferin subunit type (double-knockout; CRUAbc, CRUaBc, or CRUabC) or devoid of cruciferin (triple-knockout; CRU-) or napin (napin-RNAi) were generated using combined T-DNA insertions or RNA interference approaches. Seeds of double-knockout lines accumulated homohexameric cruciferin and contained similar protein levels as the wild type (WT). Chemical imaging of WT and double-knockout seeds using synchrotron FT-IR spectromicroscopy (amide I band, 1650 cm(-1), νC═O) showed that proteins were concentrated in the cell center and protein storage vacuoles. Protein secondary structure features of the homohexameric cruciferin lines showed predominant β-sheet content. The napin-RNAi line had lower α-helix content than the WT. Lines entirely devoid of cruciferin had high α-helix and low β-sheet levels, indicating that structurally different proteins compensate for the loss of cruciferin. Lines producing homohexameric CRUC showed minimal changes in protein secondary structure after pepsin treatment, indicating low enzyme accessibility. The Synchrotron FT-IR technique provides information on protein secondary structure and changes to the structure within the cell.
authors.affiliation: Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada.
authors.forename: Thushan S
authors.initials: TS
authors.lastname: Withana-Gamage
authors.forename: Dwayne D
authors.initials: DD
authors.lastname: Hegedus
authors.forename: Xiao
authors.initials: X
authors.lastname: Qiu
authors.forename: Peiqiang
authors.initials: P
authors.lastname: Yu
authors.forename: Tim
authors.initials: T
authors.lastname: May
authors.forename: Derek
authors.initials: D
authors.lastname: Lydiate
authors.forename: Janitha P D
authors.initials: JP
authors.lastname: Wanasundara
mesh_terms: D017360:Arabidopsis / Q000737:chemistry* / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D005916:Globulins / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D055314:Seed Storage Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D012639:Seeds / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D017356:Synchrotrons
chemical_list: D029681:Arabidopsis Proteins; D005916:Globulins; D055314:Seed Storage Proteins; C571789:cruciferin A, Arabidopsis; C506890:cruciferin B, Arabidopsis; C506891:cruciferin C, Arabidopsis