No abstract

Polyphenol oxidase (PPO) activity was found to be low in the leaves, roots, inflorescence tissues and developing and mature fruit of pineapple (Ananas comosus L.). In fruit affected by the chill-induced internal browning disorder known as Blackheart, PPO activity was 10-fold higher than in unaffected fruit, and there was a direct correlation between PPO activity and the severity of Blackheart symptoms. Degenerate oligonucleotide primers were designed to conserved regions of plant PPO genes, and used to amplify two distinct pineapple PPO cDNAs, designated PINPPO1 (2181 bp) and PINPPO2 (1319 bp), which share 81% sequence identity at the DNA level and show a high degree of homology to other plant PPO genes. PINPPO1 encodes a peptide of 604 amino acids, including a putative transit peptide of 95 amino acids and two copper-binding regions, CuA and CuB, which are highly conserved in plant PPOs. Southern analysis suggested the presence of at least four PPO genes in pineapple. Expression of PINPPO1 and PINPPO2 was low in roots, leaves, inflorescence tissues and developing fruit, but was strongly up-regulated in response to chilling and wounding. These results indicate that PPO is synthesised de novo in response to chilling of pineapple fruit, and implicate a role for the enzyme in the development of Blackheart disorder.

The contribution of polyphenol oxidase (PPO) and peroxidase (POD) to enzymic browning in sugarcane juice was investigated. Inactivation of these enzymes with heat resulted in juice of lower color (absorbance measured at 420 nm), but POD was found to be more heat stable than PPO. Salicylhydroxamic acid (SHAM) completely inhibited PPO activity and markedly reduced juice color but had no effect on POD activity. Removal of oxygen in the presence of the substrate chlorogenic acid also stopped color formation. Upon subsequent addition of oxygen, browning continued, indicating that the process was oxygen dependent. Color development in juice was complete after 20-30 min even though PPO was still active. Addition of chlorogenic acid at this point restarted browning, suggesting that color development was limited by the availability of phenolic substrates. Varietal differences were observed in levels of PPO activity, phenolics, and color. There was a correlation between juice color and phenolic content but not between juice color and PPO activity. The sugarcane PPO enzyme was most active with chlorogenic acid. It was not active with p-diphenols and was inhibited by SHAM, suggesting that it is a catechol oxidase-type enzyme (EC 1.10.3.1) and not a lacease (EC 1.10.3.2). It is atypical in that it was inhibited by SDS. The results suggest that enzymic browning contributes significantly to color formation in sugarcane juice and that PPO is the major enzyme involved.

Polyphenol oxidase (PPO) activity in sugarcane (a C4 grass) was highest in the growing point and declined down the stalk. Sugarcane PPO with an apparent molecular mass of 45 kDa was purified to homogeneity from immature stem tissue. Western analysis of sugarcane extracts with a polyclonal antibody raised to this protein suggested it resulted from cleavage of a 60 kDa protein during purification. The antibody was used to screen a sugarcane stem cDNA library. A full-length PPO clone (sugppo 1) was characterised and shown to encode a 67 kDa precursor protein comprising a plastid transit sequence of 8 kDa and a mature PPO protein of 59 kDa. High levels of expression of sugppo 1 were detected in the growing point of the stalk and in the immature tissue immediately below it, but no message was detected in RNA from mature stem or leaf. Comparison with other PPO sequences indicated that sugppo 1 was significantly different to PPO genes in C3 dicotyledonous plants.