Pheophytinase activity and gene expression of chlorophyll-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets

Pheophytinase (PPH) activity and gene expression of chlorophyll (Chl)-degrading enzymes relating to UV-B treatment in postharvest broccoli (Brassica oleracea L. Italica Group) florets were determined. PPH is involved in the dephytylation of Mg-free Chl a, pheophytin (Phy) a. However, in vitro chlorophyllase (Chlase, EC.3.1.1.14) also uses Phy a as a substrate to produce pheophorbide (Pheide) a by dephytylation. For an accurate determination of PPH activity, the PPH protein fraction was separated from Chlase protein by ammonium sulfate precipitation. The protein precipitated by 45–60% saturated ammonium sulfate included a little bit of Chlase activity and was suitable for PPH determination. PPH activity in broccoli florets treated with a UV-B dose of 19 kJ m−2 was repressed for the first 2 d of storage at 15 °C, whereas it increased gradually with senescence of control broccoli florets. The expression level of BoCLH1 was reduced in broccoli florets on day 4 of storage, while BoCLH2 and BoCLH3 were up-regulated with UV-B treatment. A high BoPAO expression level was found in senescent broccoli florets, and the up-regulation of this gene was delayed by UV-B treatment. The highest expression level of BoPPH was found in the control, and its expression was clearly repressed by UV-B treatment on day 2 of storage. We suggest that the up-regulation of Chl-degrading enzyme genes could be delayed by UV-B treatment, resulting in the suppression of floret yellowing in stored broccoli.

► An accurate method for pheophytinase (PPH) activity determination was established.
► PPH was separated from chlorophyllase by ammonium sulfate precipitation.
► PPH activity in broccoli florets treated with UV-B was slightly repressed.
► UV-B delayed up-regulation of chlorophyll (Chl)-degrading enzyme genes including PPH.
► UV-B could delay Chl degradation by repressing enzyme activity and gene expression.