Polyphenol oxidase plays a critical role in melanin formation in the fruit skin of persimmon (Diospyros kaki cv. 'Heishi').

In this study, the melanin in persimmon and its formation were investigated. Melanin was found to be deposited on the cell walls of the upper epidermis and subepidermal cells in persimmon skin and the isolated pigment appears to have lamellar structures. Diagnostic analysis of the isolated pigment showed results that were similar to those of melanin from other sources. Ultraviolet-visible spectroscopy revealed that the extracted skin pigment displayed a broadband, structureless absorption profile that increased progressively towards shorter wavelengths. The Fourier transform infrared spectroscopy assay revealed that melanin in persimmon skin exhibits many characteristic absorption peaks. The phenolic profile analysis suggested that the precursors of this pigment may include gallic acid, procyanidin B1, procyanidin B2, ferulic acid and epigallocatechin gallate. The PPO activity and DkPPO expression significantly increased during melanin formation, and transient overexpression of DkPPO promoted melanin synthesis. These results indicate that the isolated pigment was a type of melanin and that PPO plays a critical role in its formation.

Identification of xyloglucan endotransglucosylase/hydrolase genes (XTHs) and their expression in persimmon fruit as influenced by 1-methylcyclopropene and gibberellic acid during storage at ambient temperature.

Xyloglucan endotransglucosylase/hydrolase (XTH) is thought to contribute to fruit softening by degrading xyloglucan that is a predominant hemicellulose in the cell wall. In this study, two full-length XTH genes (DKXTH1 and DKXTH2) were identified from 'Fupingjianshi' persimmon fruit, and the expression level of both XTH genes was investigated during softening for 18-24 d using RT-qPCR. Sequence analysis showed that DKXTH1 and DKXTH2 contained a putative open reading frame of 861 and 876 bp encoding polypeptides of 287 and 292 amino acid residues, respectively, which contained the conserved DEIDFEFLG motif of XTH, a potential N-linked glycosylation signal site. RT-qPCR analysis showed that DKXTH1 and DKXTH2 in untreated fruit had different expression patterns during fruit softening, in which maximum expression occurred on days 3 and 12 of ripening, respectively. 1-Methylcyclopropene (1-MCP) and gibberellic acid (GA(3)) treatments delayed the softening and ethylene peak of persimmon fruit, as well as suppressed the expression of both XTH genes, especially DKXTH1. These results indicated that the expression of both XTH genes might be ethylene dependent action, and closely related to softening of persimmon in the early (DKXTH1) and later (DKXTH2) ripening stages.

Purification and chemical characterisation of a cell wall-associated ß-galactosidase from mature sweet cherry (Prunus avium L.) fruit.

Using four different chromatographic steps, ß-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium ß-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and ß-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing ß-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry ß-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple ß-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry ß-galactosidase exhibited a strict specificity towards p-nitrophenyl ß-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

Involvement of negative feedback regulation in wound-induced ethylene synthesis in 'Saijo' persimmon.

Wounding is one of the most effective stress signals to induce ethylene synthesis in persimmon (Diospyros kaki Thunb.). We found that wound-induced ethylene biosynthesis is subjected to negative feedback regulation in mature 'Saijo' persimmon fruit since ethylene production was enhanced by 1-methylcyclopropene (1-MCP) (an inhibitor of ethylene perception) pretreatment, which was approximately 1.8 fold of that in control tissues (without 1-MCP pretreatment). Wound-induced 1-aminocyclopropane-1-carboxylate (ACC) synthase activity and DK-ACS2 gene expression were substantially increased by 1-MCP pretreatment after 12 h, which resulted in much higher ACC content in 1-MCP pretreated tissues than that in a control after 24 h. These results indicated that wound-induced DK-ACS2 gene expression was negatively regulated by ethylene in mature persimmon fruit. However, 1-MCP pretreatment had no effect on DK-ACO1 gene expression, suggesting the independence of wound-induced DK-ACO1 on ethylene. Out of accord with DK-ACO1 gene expression, ACC oxidase activity was enhanced 48 h after wounding in 1-MCP pretreated tissues, reaching a peak 1.5-fold higher than that in control tissues at 60 h.

Partial purification of latent persimmon fruit polyphenol oxidase.

Persimmon fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning with Triton X-114 and ammonium sulfate fractionation between 50 and 75%. The enzyme, which showed both monophenolase and diphenolase activities, was partially purified in a latent form and could be optimally activated by the presence of 1 mM sodium dodecyl sulfate (SDS) with an optimum pH of 5.5. In the absence of SDS, the enzyme showed maximum activity at acid pH. SDS-PAGE showed the presence of a single band when L-DOPA was used as substrate. The apparent kinetic parameters of the latent enzyme were determined at pH 5.5, the V(m) value being 15 times higher in the presence of SDS than in its absence, whereas the K(M) was the same in both cases, with a value of 0.68 mM. The effect of several inhibitors was studied, tropolone being the most active with a K(i) value of 0.45 microM. In addition, the effect of cyclodextrins (CDs) was studied, and the complexation constant (K(c)) between 4-tert-butylcatechol (TBC) and CDs was calculated using an enzymatic method. The value obtained for K(c) was 15580 M(-1).