The aim of this work was to study the impact of moderately long storage periods at 4 degrees C upon red orange [Citrus sinensis (L.) Osbeck] anthocyanins production and the expression of structural genes involved in their biosynthesis such as phehylalanine ammonia lyase (PAL), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and UDP-glucose flavonoid glucosyl transferase (UFGT). Our results showed that low temperature-induced anthocyanins accumulation in red orange juice vesicles after 75 days reached values eight times higher than those kept at 25 degrees C. Furthermore, real-time polymerase chain reaction showed that expression of PAL, CHS, DFR, and UFGT was strongly induced during low temperature exposure since levels of all transcripts increased at least 40-fold with respect to control samples. Interestingly, in orange fruits subjected to a brief exposure at low temperature (45 days) and subsequently kept at 25 degrees C, the anthocyanins content dropped although samples still maintained higher levels of these pigments than those registered in control oranges. Concordantly, the expression of chs, dfr, and ufgt declined upon return to control conditions, but it was always much higher in samples subjected to brief cold induction than in the control samples. On the contrary, the amount of PAL transcripts became negligible immediately after the temperature change from 4 to 25 degrees C, thus indicating that "early" and "late" genes, respectively, implicated in the first and in the last steps leading to the anthocyanins, might be affected by different regulation mechanisms.

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The health benefits associated with the consumption of anthocyanin-containing foods are extensively documented. Mature fruits of blood oranges and their hybrids are characterized by the presence of these bioactive pigments, the abundance of which can be enhanced by storing fruit at cooling nonfreezing temperature. In this work the effects of short low-temperature exposure (4 °C × 15 days) upon orange anthocyanin content and the expression of structural genes belonging to the pigment biosynthesis pathway were investigated. The results highlight that anthocyanin levels of fruit exposed to cold sharply increase, reaching, after 6 days of storage, a value 8 times higher than that observed in the time zero samples, thus suggesting that fruit with enhanced health-related attributes might be obtained at this storage stage. The analysis of gene expression shows that the amount of transcripts of all considered genes (CM1, PAL, CHS, DFR, ANS, UFGT, and GST) sharply increased after 3-6 days of cold storage, confirming previous data showing that the biosynthesis of anthocyanins is a cold-regulated pathway. By comparing the expression of selected genes (PAL, DFR, and UFGT) between blood and common oranges, it turns out that those genes strictly involved in anthocyanin biosynthesis are not cold responsive in common oranges. Moreover, the data highlight that the EST encoding the transcription factor NAC domain protein is selectively induced by cold in blood oranges but not in common oranges, thus proposing it as a candidate gene specifically involved in blood orange response to cold exposure.

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.

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