Isolation and characterization of a Laccase gene potentially involved in proanthocyanidin polymerization in oriental persimmon (Diospyros kaki Thunb.) fruit

Proanthocyanidins (PAs, condensed tannins) are important health-promoting phytochemicals that are abundant in many plants. Oriental persimmon (Diospyros kaki Thunb.) is an excellent source of PAs because of its unique ability to accumulate large quantities of these compounds in its young fruit. There are two different spontaneous mutant phenotypes of oriental persimmons which lose their astringent taste naturally on the tree; while plants without these mutations remain rich in soluble PAs until the fruit fully ripened. The mutations are referred to as pollination-constant non-astringent genotypes named J-PCNA and C-PCNA, and are from Japan and China respectively. In this work we speculated that the loss of astringency in C-PCNA fruit is due to the soluble PAs transferred into insoluble upon polymerization, which was quite different from that of the J-PCNA. A DkLAC1 gene was isolated by the homology-based clone method. The predicted protein product of this gene showed that the DkLAC1 is a plant laccase which is phylogenetically related to the known enzyme AtLAC15 involved in the polymerization of PAs. Expression patterns of PAs biosynthetic genes associated with soluble PAs contents in three types of Oriental persimmons. Expression levels of DkLAC1 in C-PCNA type plants were linked with the reduction of soluble PAs in the flesh of the fruit. In addition the cis-elements in the DkLAC1 promoter regions indicated that the gene might also be regulated by the DkMYB4 as is seen with other well-known structural genes in Oriental persimmon. We conclude that DkLAC1 is potentially involved in PA polymerization in C-PCNA during normal ripening in C-PCNA persimmon.     

Volatile compounds associated to the loss of astringency in persimmon fruit revealed by untargeted GC–MS analysis

High resolution volatile profiling (67 compounds identified) of fruits from 12 persimmon cultivars was established and used to characterize the different astringency types of persimmon fruit before and after deastringency treatment. Analysis of the volatile profile of fruit enables us to differentiate between cultivars that at the moment of harvest produced non-astringent fruit (Pollination Constant Non Astringent—PCNA-type) from astringent ones (non-PCNA-type). Fruit failing to accumulate astringent compounds naturally (PCNA fruit) showed high levels of 3(2H)-benzofuranone, while this compound was not detected in any astringent type fruit (non-PCNA). In addition to this, PCNA cultivars also showed at harvest higher accumulation of benzeneacetaldehyde and lipid-derived aldehydes (hexanal, heptanal, octanal and decanal) than non-PCNA fruit. The application of postharvest deastringency treatment to all non-PCNA cultivars resulted on an important insolubilization of tannins. In general the CO₂-treatment enhanced the levels of acetaldehyde, however those cultivars showing high levels of dihydrobenzofuran at harvest did not present an increment of acetaldehyde. In contrast, all non-PCNA cultivars exhibited an important accumulation of lipid-derived aldehydes due to CO₂-treatment. Therefore, we propose that lipid-derived aldehydes (mainly decanal, octanal and heptanal) may be playing a role in the astringency loss. Our results suggest that 3(2H)-benzofuranone, benzeneacetaldehyde and lipid-derived aldehydes could be used as markers for both natural and artificial loss of astringency.     

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon

Proanthocyanidins(PAs) are specialized metabolites that infuence persimmon fruit quality.Normal astringent(A)-type and non-astringent(NA)-type mutants show significant variation in PA accumulation, but the infuencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by Dk DTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pa...     

Expression of ethylene response genes during persimmon fruit astringency removal

Thirteen ethylene signaling related genes were isolated and studied during ripening of non-astringent ‘Yangfeng’ and astringent ‘Mopan’ persimmon fruit. Some of these genes were characterized as ethylene responsive. Treatments, including ethylene and CO₂, had different effects on persimmon ripening, but overlapping roles in astringency removal, such as increasing the reduction in levels of soluble tannins. DkERS1, DkETR2, and DkERF8, may participate in persimmon fruit ripening and softening. The expression patterns of DkETR2, DkERF4, and DkERF5 had significant correlations with decreases in soluble tannins in ‘Mopan’ persimmon fruit, suggesting that these genes might be key components in persimmon fruit astringency removal and be the linkage between different treatments, while DkERF1 and DkERF6 may be specifically involved in CO₂ induced astringency removal. The possible roles of ethylene signaling genes in persimmon fruit astringency removal are discussed.     

<Emphasis Type="Italic">ADH</Emphasis> and <Emphasis Type="Italic">PDC</Emphasis> genes involved in tannins coagulation leading to natural de-astringency in Chinese pollination constant and non-astringency persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Thunb.)

Pollination constant non-astringency (PCNA)-type persimmons are the most desirable cultivar because the fruit loses astringency naturally and does not require any treatments for edibility. The mechanism of natural astringency loss in Chinese PCNA (C-PCNA)-type persimmon is probably related to the coagulation of soluble tannins into insoluble tannins, which is quite different from that in the Japanese PCNA (J-PCNA) type. In this work, three types of persimmon cultivars were sampled: ‘Luotian-tianshi’ (C-PCNA), ‘Maekawa-jirou’ (J-PCNA), and ‘Mopanshi’ (pollination constant astringent (PCA)) were sampled. Three DkADH and four DkPDC genes were isolated from C-PCNA plants. Three candidate genes for soluble tannins coagulation identified in C-PCNA fruit (DkADH1, DkPDC1, and DkPDC2) were characterized through combined analysis of spatiotemporal expression patterns and tannin and acetaldehyde contents during fruit development. Transient over-expression in persimmon leaves showed that DkADH1 and DkPDC2 led to a significant decrease in the levels of soluble tannins in infiltrated leaves. These results indicated that DkADH and DkPDC genes should be considered key genes for natural astringency loss in C-PCNA types.     

Decreased anthocyanidin reductase expression strongly decreases silver birch (Betula pendula) growth and alters accumulation of phenolics

Phenolics, formed via a complex phenylpropanoid pathway, are important defensive agents in plants and are strongly affected by nitrogen (N) fertilization. Proanthocyanidins (PAs) are one possible endpoint of the phenylpropanoid pathway, and anthocyanidin reductase (ANR) represents a key enzyme in PA biosynthesis. In this study, the expression of silver birch (Betula pendula) anthocyanidin reductase BpANR was inhibited using the RNA interference (RNAi) method, in three consequent BpANR RNAi (ANRi birches) lines. The growth, the metabolites of the phenylpropanoid pathway, and the number of resin glands of the ANRi birches were studied when grown at two N levels. ANRi birches showed decreased growth and reduction in PA content, while the accumulation of total phenolics in both stems and leaves increased. Moreover, ANRi birches produced more resin glands than did wild‐type (WT) birches. The response of ANRi birches to N depletion varied compared with that of WT birches, and in particular, the concentrations of some phenolics in stems increased in WT birches and decreased in ANRi birches. Because the inhibition of PAs biosynthesis via ANR seriously affected birch growth and resulted in accumulation of the precursors, the native level of PAs in plant tissues is assumed to be the prerequisite for normal plant growth. This draws attention to the real plant developmental importance of PAs in plant tissues.     

Hypoxia‐responsive ERFs involved in postdeastringency softening of persimmon fruit

Removal of astringency by endogenously formed acetaldehyde, achieved by postharvest anaerobic treatment, is of critical importance for many types of persimmon fruit. Although an anaerobic environment accelerates de‐astringency, it also has the deleterious effect of promoting excessive softening, reducing shelf life and marketability. Some hypoxia‐responsive ethylene response factors (ERFs) participate in anaerobic de‐astringency, but their role in accelerated softening was unclear. Undesirable rapid softening induced by high CO₂ (95%) was ameliorated by adding the ethylene inhibitor 1‐MCP (1 μL/L), resulting in reduced astringency while maintaining firmness, suggesting that CO₂‐induced softening involves ethylene signalling. Among the hypoxia‐responsive genes, expression of eight involved in fruit cell wall metabolism (Dkβ‐gal1/4, DkEGase1, DkPE1/2, DkPG1, DkXTH9/10) and three ethylene response factor genes (DkERF8/16/19) showed significant correlations with postdeastringency fruit softening. Dual‐luciferase assay indicated that DkERF8/16/19 could trans‐activate the DkXTH9 promoter and this interaction was abolished by a mutation introduced into the C‐repeat/dehydration‐responsive element of the DkXTH9 promoter, supporting the conclusion that these DkERFs bind directly to the DkXTH9 promoter and regulate this gene, which encodes an important cell wall metabolism enzyme. Some hypoxia‐responsive ERF genes are involved in deastringency and softening, and this linkage was uncoupled by 1‐MCP. Fruit of the Japanese cultivar ‘Tonewase’ provide a model for altered anaerobic response, as they lost astringency yet maintained firmness after CO₂ treatment without 1‐MCP and changes in cell wall enzymes and ERFs did not occur.     

Carbon dioxide assimilation during postharvest removal of astringency from persimmon fruit

Application of anaerobic conditions with CO₂ or N₂ atmospheres to remove astringency from harvested persimmon fruit ( Diospryros kaki L. cv. Triumph), caused production of more acetaldehyde under CO₂ than under N₂, ¹⁴CO₂ applied in a 100% CO₂ atmosphere, for 48 h to astringent persimmon fruits was incorporated mainly into malate and very little into other metabolites, such as carbohydrate or amino acids. Application of malate or pyruvate to pulp discs of astringent persimmons caused an immediate rise in acetaldehyde production. The higher levels of acetaldehyde produced by whole fruits held in a CO₂ atmosphere, than by fruits held in a N₂ atmosphere, can be explained through fixation of atmospheric CO₂ into malate, leading to acetaldehyde production.