This review is concerned with the mechanisms controlling fruit softening. Master genetic regulators switch on the ripening programme and the regulatory pathway branches downstream, with separate controls for distinct quality attributes such as colour, flavour, texture, and aroma. Ethylene plays a critical role as a ripening hormone and is implicated in controlling different facets of ripening, including texture change, acting through a range of transcriptional regulators, and this signalling can be blocked using 1-methylcyclopropene. A battery of at least seven cell-wall-modifying enzymes, most of which are synthesized de novo during ripening, cause major alterations in the structure and composition of the cell wall components and contribute to the softening process. Significant differences between fruits may be related to the precise structure and composition of their cell walls and the enzymes recruited to the ripening programme during evolution. Attempts to slow texture change and reduce fruit spoilage by delaying the entire ripening process can often affect negatively other aspects of quality, and low temperatures, in particular, can have deleterious effects on texture change. Gene silencing has been used to probe the function of individual genes involved in different aspects of ripening, including colour, flavour, ethylene synthesis, and particularly texture change. The picture that emerges is that softening is a multi-genic trait, with some genes making a more important contribution than others. In future, it may be possible to control texture genetically to produce fruits more suitable for our needs.

Summary: 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.

Identification and functional characterization of hypoxia-responsive transcription factors is important for understanding plant responses to natural anaerobic environments and during storage and transport of fresh horticultural products. In this study, yeast one-hybrid library screening using the persimmon (Diospyros kaki) pyruvate decarboxylase (DkPDC2) promoter identified three ethylene response factor (ERF) genes (DkERF23/DkERF24/DkERF25) and four WRKY transcription factor genes (DkWRKY/DdkWRKY5/DkWRKY6/DkWRKY7) that were differentially expressed in response to high CO₂ (95%, with 4% N₂ and 1% oxygen) and high N₂ (99% N₂ and 1% oxygen). Yeast one-hybrid assays and electrophoretic mobility shift assays indicated that DkERF23, DkERF24, DkERF25, DkWRKY6, and DkWRKY7 could directly bind to the DkPDC2 promoter. Dual-luciferase assays confirmed that these transcription factors were capable of transactivating the DkPDC2 promoter. DkERF24 and DkWRKY1 in combination synergistically transactivated the DkPDC2 promoter, and yeast two-hybrid and bimolecular fluorescence complementation assays confirmed protein–protein interaction between DkERF24 and DkWRKY1. Transient overexpression of DkERF24 and DkWRKY1 separately and in combination in persimmon fruit discs was effective in maintaining insolubilization of tannins, concomitantly with the accumulation of DkPDC2 transcripts. Studies with Arabidopsis (Arabidopsis thaliana) homologs AtERF1 and AtWRKY53 indicated that similar protein–protein interactions and synergistic regulatory effects also occur with the DkPDC2 promoter. We propose that an ERF and WRKY transcription factor complex contributes to responses to hypoxia in both persimmon fruit and Arabidopsis, and the possibility that this is a general plant response requires further investigation.

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit.

Persimmon (Diospyros kaki) is an oriental perennial woody fruit tree whose popular fruit is produced and consumed worldwide. The persimmon fruit is unique because of the hyperaccumulation of proanthocyanidins during fruit development, causing the mature fruit of most cultivars to have an astringent taste. In this study, we obtained a chromosome-scale genome assembly for ‘Youshi’ (Diospyros oleifera, 2n = 2x = 30), the diploid species of persimmon, by integrating Illumina sequencing, single-molecule real-time sequencing, and high-throughput chromosome conformation capture techniques. The assembledD. oleiferagenome consisted of 849.53 Mb, 94.14% (799.71 Mb) of which was assigned to 15 pseudochromosomes, and is the first assembled genome for any member of the Ebenaceae. Comparative genomic analysis revealed that theD. oleiferagenome underwent an ancient γ whole-genome duplication event. We studied the potential genetic basis for astringency development (proanthocyanidin biosynthesis) and removal (proanthocyanidin insolublization). Proanthocyanidin biosynthesis genes were mainly distributed on chromosome 1, and the clustering of these genes is responsible for the genetic stability of astringency heredity. Genome-based RNA-seq identified deastringency genes, and promoter analysis showed that most of their promoters contained large numbers of low oxygen-responsive motifs, which is consistent with the efficient industrial application of high CO₂treatment to remove astringency. Using theD. oleiferagenome as the reference, SLAF-seq indicated that ‘Youshi’ is one of the ancestors of the cultivated persimmon (2n = 6x = 90). Our study provides significant insights into the genetic basis of persimmon evolution and the development and removal astringency, and it will facilitate the improvement of the breeding of persimmon fruit.