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Transcriptomic and Biochemical Analyses of Proanthocyanidin Metabolism in Seed Coats of Cranberry Bean (Phaseolus vulgaris L.)

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Title: Transcriptomic and Biochemical Analyses of Proanthocyanidin Metabolism in Seed Coats of Cranberry Bean (Phaseolus vulgaris L.)
Author: Freixas Coutin, Jose
Department: Department of Plant Agriculture
Program: Plant Agriculture
Advisor: Bozzo, GalePauls, K. Peter
Abstract: Postharvest-related seed coat darkening is a major problem in common bean (Phaseolus vulgaris L.), including cranberry bean, as these seeds are graded lower in the commercial market. Proanthocyanidins accumulate in the seed coats of dicotyledonous species, including legumes and their oxidation to quinones promotes seed coat darkening. The upregulated expression of genes encoding late steps in proanthocyanidin biosynthesis (e.g., ANTHOCYANIDIN REDUCTASE, ANR), are associated with seed coat darkening in Medicago truncatula and Arabidopsis thaliana. In this thesis, total proanthocyanidin levels in seed coats increased with seed maturation in a darkening-susceptible cranberry bean recombinant inbred line (RIL), whereas these compounds were undetectable in non-darkening RIL seed coats. RNA sequencing was implemented to investigate transcript changes in darkening and non-darkening RIL seed coats as a function of bean development. The transcript levels for 1336 genes were varied between darkening and non-darkening RIL seed coats at one or more stages of seed development. A model clustering analysis revealed the differentially expressed genes formed 14 gene clusters based on their mode of expression. Structural and regulatory genes of the proanthocyanidin biosynthesis pathway were prevalent in cluster 2 and upregulated in seed coats of the darkening RIL. This included myeloblastosis proto-oncogene (MYB) transcription factors with phylogenetic similarity to proanthocyanidin activating MYBs from other plant species. Moreover, regions upstream of the transcription start site in cluster 2 genes were enriched in MYB and basic helix-loop-helix consensus binding sites as revealed by a transcription factor binding site enrichment analysis. A principal component analysis revealed that transcript abundance of two genes of unknown function and three proanthocyanidin biosynthesis genes, FLAVANONE 3-HYDROXYLASE 1, DIHYDROFLAVONOL 4-REDUCTASE 1 and PvANR1 were highly associated with seed coat proanthocyanidin accumulation in the darkening cranberry bean RIL. Biochemical characterization of a recombinant PvANR1 was performed following the removal of the hexahistidine-tag. PvANR1 catalyzed the NADPH-dependent reduction of cyanidin to catechin and epicatechin in vitro; moreover, high cyanidin concentrations inhibited the formation of both of these products. This study provides biochemical and molecular information for traditional breeding strategies aimed at preserving the aesthetic quality of edible dry beans during postharvest storage.
Date: 2017-06
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