Characterization of Arabidopsis thaliana Lines with Altered Seed Storage Protein Profiles Using Synchrotron-Powered FT-IR Spectromicroscopy

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作   者：

Thushan S. Withana-Gamage;  Dwayne D. Hegedus;  Xiao Qiu; 

作者机构：

Canada;  University of Saskatchewan;  Saskatchewan S7N 0X2;  Agriculture and Agri-Food Canada;  Saskatchewan S7N 5A9;  Saskatoon;  Department of Food and Bioproduct Sciences; 

关键词：

cruciferin;  bioaccessibility;  seed storage protein;  synchrotron mid-IR;  pepsin digestion; 

期刊名称：

Journal of Agricultural and Food Chemistry

i s s n：

0021-8561

年卷期：

2013 年 61 卷 4 期

页   码：

901-912

页   码：

摘   要：

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), vC=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-helk 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.