Abstract Detail

Genomics / Proteomics

Hu, Junjie (Grace) [1], Radovanovic, Natasa [2], Fan, Tao [3], Bykova, Natalia [2].

Redox regulation of seed dormancy release in spring wheat revealed through tissue-specific proteomics, metabolomics and antioxidant activity profiling.

Regulation of cellular redox homeostasis plays an essential role in modulating redox signaling linked to seed developmental processes and their interaction with the environment. After-ripening results in seed dormancy alleviation allowing germination to commence under favorable conditions. After-ripened seeds experience metabolic reprogramming including changes in redox homeostasis and hormonal signaling. Freshly-harvested and after-ripened imbibed seeds of spring wheat doubled haploid hybrid lines with marginal dormancy phenotypes were used for comparative analysis of biochemical and molecular redox mechanisms that regulate the transition from quiescence to highly active metabolism during germination. Tissue-specific differentially expressed total proteomes were quantitatively monitored through two-dimensional (2-D) IEF/SDS PAGE protein mapping and analysis by PDQuest software. The thiol redox-sensitive proteomes were characterized using mBBr-fluorescent Cys labelling and UV-based visualization of redox active proteins on 2D gels. Selected 2D gel spots were further analyzed by nano-LC-MS/MS for identification of proteins and their sites of modification in conjunction with MASCOT search against wheat EST database, and Scaffold bioinformatics tools for data analysis and validation. In order to gain a deeper insight into the role of tissue-specific antioxidant defense mechanisms in dormancy control, the antioxidant capacity of ascorbate–glutathione cycle enzymes, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and another antioxidant enzyme catalase was measured. Redox changes in pools of key non-protein redox metabolites in the aqueous phase ascorbate and glutathione, as well as the accumulation of potentially toxic hydrogen peroxide (H₂O₂) byproduct in response to after-ripening were examined. Redox proteomic analysis resulted in 291 high confidence protein identifications with at least two-fold change in protein expression level between dormant and after-ripened seeds, from which 49 and 101 proteins showed significant changes in expression in dormant embryos and aleurones, respectively. Moreover, 59 and 91 proteins were up-regulated in a tissue-specific manner in dormant and after-ripening treatments, respectively,. Antioxidant metabolite measurements indicated an increased level of total and reduced ascorbate in embryo from dormant seeds upon after-ripening, while the level of both reduced and total glutathione did not change sugnificantly indicating their different roles in redox regulation of dormancy. Furthermore, in contrast to a decreased dehydroascorbate reductase activity, the activities of other ascorbate recycling enzymes increased upon seeds after-ripening, with elevated activities in embryo compared to aleurone tissues. Results from enzyme and metabolite assays provide an insight into the interaction between tissue-specific antioxidant capacity to maintain redox homeostasis upon imbibition and the ability of seeds to maintain high dormancy level.

1 - Memorial University of Newfoundland, Department of Biology, 232 Elizabeth Ave., St. John's, NL, A1B 3X9, Canada
2 - Agriculture and Agri-Food Canada, Cereal research Centre, 101 Route 100, Morden, MB, R6M 1Y5, Canada
3 - Canadian Grain Commission, Government of Canada , Oilseed Section, 900-303 Main Street, Winnipeg, MB, R3C 3G8, Canada

Keywords:
Dormancy
After-ripening
Oxidative stress
Metabolomics
Tissue-specific proteomics
Redox proteomics
embryo
Aleurone layer
Spring wheat
nano-LC-MS/MS
Two dimensional (2D)IEF/SDS PAGE.

Presentation Type: Poster:Posters for Topics
Session: P
Location: Hall D/The Shaw Conference Centre
Date: Monday, July 27th, 2015
Time: 5:30 PM
Number: PGP007
Abstract ID:890
Candidate for Awards:CSPB President's Award for Best Student Presentation,CSPB Travel Bursary