Mechanisms of the hard-to-cook phenomena in common beans: A quantitative microscopic perspective

The hard-to-cook (HTC) defect developing during adverse post-harvest storage prolongs cooking time, reducing the convenience and commercial value of beans. Since softening requires sufficient pectin solubilization to facilitate cell separation, storage-induced modifications in cell wall polysaccharides (CWPs) may limit pectin solubilization and hinder cookability. This study aimed to gain deeper mechanistic insights into HTC by simultaneously examining two main HTC hypotheses related to modifications of cotyledon CWPs, the pectin–cation–phytate and phenolic-involved hypotheses, across five bean accessions. Sequential staining of cell wall tissues with the 2F4 antibody and basic fuchsin enables concurrent visualization of the spatial distribution of Ca 2+ -crosslinked pectin along cellular junction linings and phenolic-crosslinked compounds at junction corners, indicating that both mechanisms occur concurrently. Image analysis revealed stronger fluorescence signals for both probes in aged beans than in fresh beans. Localized signals correlated more strongly with time to cook than with the cooking rate constant. No correlation was found between changes in 2F4 and basic fuchsin signals across accessions, suggesting independent development of the two mechanisms. Remarkably, phenolic crosslinking showed a greater increase upon storage than the Ca 2+ -crosslinked pectin, implying a more dominant role in HTC. This study highlights the importance of examining localized changes when investigating HTC mechanisms.