BIOCHEMICAL AND GENETIC CHARACTERIZATION OF WHEAT (TRITICUM SPP.) KERNEL POLYPHENOL OXIDASES

Authors: Anderson, James; FUERST, PATRICK - WASHINGTON ST UNIVERSITY; Hurkman Ii, William; Vensel, William; Morris, Craig

Submitted to: Journal of Cereal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/27/2006
Publication Date: 11/9/2006
Citation: Anderson, J.V., Fuerst, E.P., Hurkman, W.J., Vensel, W.H., Morris, C.F. 2006. Biochemical and genetic characterization of wheat (Triticum spp.) kernel polyphenol oxidases. Journal of Cereal Science. 44(3):353-367.

Interpretive Summary: The data presented in this paper supports the existence of a functional multigene polyphenol oxidase (PPO) family in Triticum spp. consisting of kernel- and non-kernel sub-classes. Wheat cultivars with reduced PPO activity in kernels also had overall reduced kernel-specific PPO transcript and PPO protein levels. Results reported here are consistent with the hypothesis that PPO transcript levels in developing kernels are associated with increased PPO activity in mature kernels, which is thought to be a causative agent leading to wheat food discoloration. However, although PPO proteins were far more abundant in immature kernels, PPO activity was much greater in mature kernels; suggesting that PPO is highly latent during kernel development. Previous research has implicated discoloration of wheat foods with PPO activity originating from chromosome 2D of hexaploid wheat. Our results indicate that a wheat kernel protein identified in the 60-62 kDa range likely represents PPO resulting from chromosome 2D of hexaploid wheat. The data also indicated that, among the lines evaluated here, the 2D chromosome of hexaploid wheat had more influence on kernel PPO activity than the 2A and 2B chromosomes. Total PPO activity in mature kernels is thus a function of the specific PPO isoforms present, their abundance, and their activation levels.

Technical Abstract: Genetic variability of polyphenol oxidase (PPO) activity was investigated by evaluating PPO transcript levels, PPO activity, and protein antigenicity to a wheat PPO antibody in hexaploid, tetraploid, and aneuploid wheat (Triticum spp.) lines. DNA probe to a kernel-specific PPO (accession BT009357) hybridized to PPO clones in a cDNA library developed from immature wheat kernels but not a cDNA library developed from seedlings. Full-length coding sequence to the kernel-specific PPO accession was used to produce recombinant protein, to which wheat PPO antibody was raised. PPO transcript and antigenic proteins were most abundant during kernel development and decreased rapidly at physiological maturity. Although antigenic PPO proteins were far more abundant in immature kernels, PPO activity was much greater in mature kernels; PPO is thus highly latent during kernel development, and substantial though incomplete activation occurs during kernel maturation. Four wheat kernel antigenic protein bands of ~58, 60, 62 and 75 kD were separated by one-dimensional SDS-PAGE. Proteins of ~60 and ~62 kD were immunoprecipitated and confirmed to be PPOs by LC-MS/MS. The presence of the 2D chromosome was positively correlated with the staining intensity of antigenic ~60-62 kD proteins and with kernel PPO activity in aneuploid lines. The ~58 kD protein was not well-correlated with kernel PPO activity, while the ~75 kD protein was likely a PPO precursor. Data presented in this study confirmed that wheat PPOs consist of a functional multigene family, and indicated that, among the lines evaluated here, the 2D chromosome of hexaploid wheat had more influence on kernel PPO activity than the 2A and 2B chromosomes. Total PPO activity in mature kernels is thus a function of the specific PPO isoforms present, their abundance, and their activation levels.