Molecular characterization of wheat polyphenol oxidase (PPO)

Abstract.

It is well-established that the enzyme polyphenol oxidase (PPO) is involved in undesirable browning of noodles, chapattis, middle east flat breads and steamed breads. Methods for measuring PPO activity have been developed, and the variation of PPO activity among wheat (Triticum aestivum L.) cultivars has been well documented. However, there is no report on the identification and characterization of a wheat PPO gene. PCR performed on wheat genomic DNA with oligonucleotide primers designed from conserved copper binding regions of other PPO genes resulted in amplification of a 444-bp DNA fragment. Sequence analysis identified the conserved amino acids of PPO genes indicating that the PCR product was part of the wheat PPO gene. Screening genomic and cDNA libraries using 444- and 760-bp DNA fragments as probes failed to identify a PPO gene based on conserved sequence, even though there were very strong hybridization signals for some isolates. Rapid amplification of cDNA ends (RACE) technique was used as an alternative to obtain the remaining DNA sequences in 5′ and 3′ directions based on the 444-bp partial wheat PPO gene sequence. With the use of ThermoScript Reverse Transcriptase (which functions at higher temperatures) and Advantage-GC cDNA kit, the complete DNA sequence in the 3′ direction was obtained. A similar effort in the 5′ direction resulted in amplification of a truncated 414-bp DNA sequence. Overall, 1,509-bp of putative wheat PPO DNA sequence was obtained. Alignment of deduced amino-acid sequences revealed similarity to the other PPO gene sequences, especially in the conserved copper binding regions. Southern-blot analysis performed with four different restriction enzymes revealed two to four DNA fragments, suggesting a limited number of PPO genes in wheat. Wheat genomic DNA restricted with HindIII and hybridized using a 760-bp wheat PPO probe revealed a clear distinction between wheat cultivars with high and low PPO activities. Northern-blot analysis indicated a transcript size of about 2.0-kb. PPO DNA fragment as well as RNA transcript was observed for the durum cultivar Renville which normally has very low PPO activity. Further study is needed to explain the relationship between PPO activity and the presence of PPO gene (s).