Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Dhaubhadel, Sangeeta

2nd Supervisor

Kohalmi, Susanne

Co-Supervisor

Abstract

Pinto bean (Phaseolus vulgaris) is one of the leading market classes of dry beans that is affected by postharvest seed coat darkening. Bean producers and vendors encounter significant crop value loss due to the decreased consumer preference for the darker beans. Darker beans also display poor canning quality and longer cooking times. Special storage conditions can slow down bean darkening to some extent, however, maintaining such condition is expensive. The available slow darkening (SD) pinto cultivars can overcome the darkening issues of existing regular darkening (RD) cultivars, however, the SD pintos show poor agronomic performances in different environmental conditions. These sought to identify the gene/s responsible for slow darkening in pinto beans to develop improved varieties.

As part of my initial research objective, a basic-Helix-Loop-Helix (bHLH) transcription factor encoding the P (Pigment) gene was identified in pinto beans. P protein in a regulator of proanthocyanidins (PAs) biosynthesis. Using genetic complementation, transcript abundance, metabolite analysis, and inheritance study, I found a novel allele of P, Psd, whose partial loss-of-function slows down seed coat darkening by reducing the PA levels. Compared to P, Psd contains an additional glutamate residue and an arginine to histidine substitution and produces less PAs by downregulating the expression of DIHYDROFLAVONOL 4-REDUCTASE and ANTHOCYANIDIN REDUCTASE. Then, I identified that P interacts with PvMYB3A, PvMYB10B and PvWD9 proteins to form a protein complex. Additionally, the interaction study also highlighted the necessity of other plant component(s) in complex formation and its activity. Overexpression of P and PvMYB3A accumulated an elevated level of PAs in pinto bean hairy roots, that constitutively express PvWD9. Finally, a genome-wide analysis for MATE genes and their detailed characterization in common bean identified PvMATE8 as a transporter of PA monomers. PvMATE8 is able to transport epicatechin-3´-O-glucosides, not epicatechin aglycons from cytosol to the vacuole and thus ensuring substrate flow to maintain a feedback regulation for upstream epicatechin biosynthesis. Knowing the mechanism and committed steps of the PA pathway will not only help tackle postharvest darkening issues but also will make it easy to incorporate other beneficial traits in SD beans.

Summary for Lay Audience

Many colored bean varieties turn darker with time while stored after harvest, a phenomenon known as postharvest seed coat darkening. Among popular beans in North America, pinto beans are affected the most by postharvest darkening. Consumers show less preference for the darker pinto beans not only for aesthetic reasons but also because darker beans have longer cooking times. To address this issue, breeders have developed a few slow darkening (SD) cultivars that do not darken as much as the regular darkening (RD) cultivars. However, these SD cultivars are mostly developed to meet regional and consumer demands and are not adapted to diverse environmental conditions encountered during growing. This problem can be better understood if the slow darkening gene and its functional mechanism for seed coat darkening are uncovered in pinto beans.

Bean breeders have been using several genetic indicators to select SD beans, which are called markers. By analyzing those markers and the available common bean genome sequence information, I found the Pigment (P) gene. P proteins regulate the biosynthesis of a brown pigment-called, proanthocyanidins (PAs). By analyzing 21 SD and RD pinto lines, I discovered that the SD lines carry a variant of P, Psd which contains major sequence variations, leading to reduced production of PAs. Further exploration revealed that P is part of a protein complex by interacting with two other proteins, MYB and WD40. This protein complex regulates the level of PAs in the seed coat by controlling several critical steps of PA pathway. PAs accumulate in the vacuole of plant cells as polymers, whereas the subunits that form PAs are synthesized in the cytosol, a different subcellular compartment. I identified a transporter, PvMATE8, that transports PA subunits from the cytosol to the vacuole. Blocking the activity of the transporter also reduces the PA levels in the seed coat. Finally, I developed Psd-specific markers, which breeders can directly use in bean breeding to look for the SD trait. Knowing the gene responsible for the SD trait and its functional mechanism will help to save millions of dollars in the bean market.

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Available for download on Monday, December 15, 2025

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