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Abstract Detail



Biochemistry, metabolism, carbon flux

Freixas-Coutin, Jose A. [1], Munholland, Seth [2], Crosby, William L. [2], Pauls, K. Peter [1], Bozzo, Gale G. [1].

Identification, cloning and biochemical characterization of anthocyanidin reductase 1 isolated from the seed coat of darkening cranberry beans (Phaseolus vulgaris).

Cranberry bean (Phaseolus vulgaris) seed coat darkening occurs during postharvest storage and negatively impacts marketability of this commodity. In other legumes and Arabidopsis thaliana, seed coat darkening is associated with the biosynthesis of proanthocyanidins and their subsequent oxidation to highly reactive quinones. Anthocyanidin reductase (ANR) is a key proanthocyanidin biosynthetic enzyme known to catalyze the NADPH-dependent reduction of anthocyanidins (e.g., cyanidin) yielding flavan-3-ols. However no biochemical information exists for P. vulgaris ANR(s). Therefore, we hypothesize that expression of ANR genes is involved in cranberry bean seed coat darkening. In this study, recombinant inbred lines (RILs) from an ‘Etna’ (darkening) by ‘Wit-Rood’ (non-darkening) cranberry bean cross were greenhouse cultivated in a completely randomized design. Respective cDNA libraries were prepared from seed coat mRNA representing early, intermediate and mature developmental stages using the Illumina TruSeq™ RNA preparation protocol. Pair-end sequencing of cDNA libraries representing darkening and non-darkening RILs at each developmental stage was performed with an Illumina HiSeq 2500 platform, and transcript reads were annotated against the P. vulgaris G-19833 reference genome. Transcripts for ANR genes, PvANR1 and PvANR2 were identified, where only PvANR1 transcript abundance was significantly elevated in the seed coat of darkening relative to non-darkening cranberry beans at early and intermediate stages of development. In contrast, PvANR2 transcript abundance was marginal but similar between both RILs. Biochemical analyses were conducted using a recombinant hexahistidine (His6)-tagged PvANR1 enriched from E.coli BL21culture, following removal of the His6. The results revealed that the recombinant enzyme catalyzes the NADPH-dependent reduction of cyanidin into flavan-3-ol epimers, catechin and epicatechin in vitro, suggesting that this protein exhibits both ANR and epimerase activities. Ongoing enzyme kinetic analyses of the recombinant PvANR1 enzyme are focusing on anthocyanidin substrate specificity and the potential for ANR inhibition by the substrate cyanidin. The results may provide important information for breeding efforts aimed at preserving the visual appeal and marketability of cranberry beans.


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1 - University of Guelph, Department of Plant Agriculture, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada
2 - University of Windsor, Department of Biological Sciences, 401 Sunset Avenue, Windsor, Ontario, N9B 3P4, Canada

Keywords:
Anthocyanidin reductase
Phaseolus vulgaris
Proanthocyanidin
Seed coat darkening
Transcriptome.

Presentation Type: Oral Paper:Papers for Topics
Session: 43
Location: Hall A/The Shaw Conference Centre
Date: Tuesday, July 28th, 2015
Time: 11:00 AM
Number: 43004
Abstract ID:903
Candidate for Awards:CSPB President's Award for Best Student Presentation,CSPB Travel Bursary


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