Molecular and biochemical characterization of wheat (Triticum aestivum. L) polyphenol oxidases (PPOs)

Abstract

Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in the undesirable brown discoloration of food products. Wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function for two important reasons, namely (1) the large size an complexity of its (allohexaploid) genome, and (2) its economic importance. Prior to this study, the molecular and biochemical properties of wheat PPOs were largely unknown. To remedy this situation, we have performed several BLAST searches of expressed sequence tag (EST) databases, using a known wheat PPO sequence as a search tool. This study suggested the presence of at least six PPO genes in hexaploid wheat, falling into two different phylogenetic clusters of three genes each. Presence of a wheat PPO multigene family was confirmed by Southern blotting. A combination of biochemical (enzyme purification and mass spectrometric analysis) and molecular (Northern) approaches confirmed that members of one cluster are not expressed in the developing kernels and senescing flag leaves, while regulation of one or several members of the other gene cluster controls PPO activity in these tissues. Our data, including immunoblotting and enzyme activity studies, further indicated that wheat PPOs are synthesized as inactive precursor (early kernel development) which are proteolytically processed and activated as the kernels mature. Activation of PPO precursor proteins was also demonstrated in vitro, in presence of purified trypsin. In these experiments, PPO activity increased during the first four hours and remained stable thereafter, indicating that the protein domains responsible for catalytic activity are quite stable. Research performed as a part of this dissertation has also demonstrated that wheat PPO activity is influenced by strong anionic detergents such as SDS and N-lauroylsarcosine. The corresponding experiments indicated that these detergents influenced both enzyme extraction and activity, at least in high-PPO wheat varieties. This work also has practical aspects, making PPO assays (as used by breeders for germplasm screening) more robust. In conclusion, as a result of this dissertation, wheat PPOs have emerged as a fascinating example of a plant multigene family with complex transcriptional and posttranscriptional regulation.