Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

Molecular basis of the formation and removal of fruit astringency.

Astringency is a dry puckering mouthfeel mainly generated by the binding of tannins with proteins in the mouth. Tannins confer benefits such as resistance to biotic stresses and have antioxidant activity, and moderate concentrations of tannins can improve the flavor of fruits or their products. However, fruits with high contents of tannins have excessive astringency, which is undesirable. Thus, the balance of astringency formation and removal is extremely important for human consumption of fruit and fruit-based products. In recent years, the understanding of fruit astringency has moved beyond the biochemical aspects to focus on the genetic characterization of key structural genes and their transcriptional regulators that cause astringency. This article provides an overview of astringency formation and evaluation. We summarize the methods of astringency regulation and strategies and mechanisms for astringency removal, and discuss perspectives for future exploration and modulation of astringency for fruit quality improvement.

Transcriptome Analysis Revealed the Roles of Carbohydrate Metabolism on Differential Acetaldehyde Production Capacity in Persimmon Fruit in Response to High-CO2 Treatment.

Persimmon (Diospyros kaki Thunb.) fruit is unique due to the continuous accumulation of soluble tannins during fruit development in most cultivars, which causes undesired astringency. High-CO2 treatment was the most effective widely used method for astringency removal. However, differential effects of high-CO2 treatment between cultivars were observed and the molecular basis remained inclusive. Previously, one cultivar ("Luoyangfangtianshengshi," LYFTSS) showed rapid deastringency, while two cultivars ("Shijiazhuanglianhuashi," SJZLHS; "Laopige," LPG) showed slow deastringency in response to high-CO2 (95% CO2) treatment. In this study, the metabolites (acetaldehyde and ethanol) related to deastringency were further analyzed and both acetaldehyde and ethanol were higher in SJZLHS and LYFTSS than that in LPG, where acetaldehyde was undetectable. Based on the RNA-seq data, the weighted gene coexpression network analysis (WGCNA) revealed that one module, comprised of 1773 unigenes, significantly correlated with the contents of acetaldehyde and ethanol (P < 0.001). Further analysis based on the acetaldehyde metabolism pathway indicated that the differentially expressed structural genes, including previously characterized DkADH and DkPDC and also their upstream members (e.g., PFK, phosphofructokinase), showed positive correlations with acetaldehyde production. Quantitative analysis of the precursor substances indicated that sucrose, glucose, and fructose exhibited limited differences between cultivar except for malic acid. However, the content of malic acid is much less than the total soluble sugar content. To verify the correlations between these genes and acetaldehyde production, the fruit from 14 more cultivars were collected and treated with high CO2. After the treatment, acetaldehyde contents in different cultivars ranked in 30.4-255.5 µg/g FW. Real-time polymerase chain reaction (PCR) and correlation analysis indicated that the EVM0002315 (PFK) gene, belonging to carbohydrate metabolism, was significantly correlated with acetaldehyde content in fruit. Thus, it could be proposed that the differentially expressed carbohydrate metabolism related genes (especially PFK) are the basis for the variance of acetaldehyde production among different persimmon cultivars.

High CO2/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes.

Most persimmon (Diospyros kaki) cultivars are astringent and require post-harvest deastringency treatments such as 95% CO2 (high-CO2 treatment) to make them acceptable to consumers. High-CO2 treatment can, however, also induce excessive softening, which can be reduced by adding 1-methylcyclopropene (1-MCP). Previous studies have shown that genes encoding the ETHYLENE RESPONSE FACTORS (ERFs) DkERF8/16/19 can trans-activate xyloglucan endotransglycosylase/hydrolase (DkXTH9), which encodes the cell wall-degrading enzyme associated with persimmon fruit softening. In this study, RNA-seq data between three treatments were compared, namely high-CO2, high-CO2+1-MCP, and controls. A total of 227 differentially expressed genes, including 17 transcription factors, were predicted to be related to persimmon post-deastringency softening. Dual-luciferase assays indicated that DkNAC9 activated the DkEGase1 promoter 2.64-fold. Synergistic effects on transcription of DkEGase1 that involved DkNAC9 and the previously reported DkERF8/16 were identified. Electrophoretic mobility shift assay indicated that DkNAC9 could physically bind to the DkEGase1 promoter. Bimolecular fluorescence complementation and firefly luciferase complementation imaging assays indicated protein-protein interactions between DkNAC9 and DkERF8/16. Based on these findings, we conclude that DkNAC9 is a direct transcriptional activator of DkEGase1 that can co-operate with DkERF8/16 to enhance fruit post-deastringency softening.

The persimmon (Diospyros oleifera Cheng) genome provides new insights into the inheritance of astringency and ancestral evolution.

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 assembled D. oleifera genome 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 the D. oleifera genome 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 CO2 treatment to remove astringency. Using the D. oleifera genome 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.

C2H2-Type Zinc Finger Proteins (DkZF1/2) Synergistically Control Persimmon Fruit Deastringency.

Hypoxic environments are generally undesirable for most plants, but for astringent persimmon, high CO2 treatment (CO2 > 90%), also termed artificial high-CO2 atmosphere (AHCA), causes acetaldehyde accumulation and precipitation of soluble tannins and could remove astringency. The multiple transcriptional regulatory linkages involved in persimmon fruit deastringency have been advanced significantly by characterizing the ethylene response factors (ERFs), WRKY and MYB; however, the involvement of zinc finger proteins for deastringency has not been investigated. In this study, five genes encoding C2H2-type zinc finger proteins were isolated and designed as DkZF1-5. Phylogenetic and sequence analyses suggested the five DkZFs could be clustered into two different subgroups. qPCR analysis indicated that transcript abundances of DkZF1/4 were significantly upregulated during AHCA treatment (1% O2 and 95% CO2) at day 1, DkZF2/5 at both day 1 and 2, while DkZF3 at day 2. Dual-luciferase assay indicated DkZF1 and DkZF2 as the activators of deastringency-related structural genes (DkPDC2 and DkADH1) and transcription factors (DkERF9/10). Moreover, combinative effects between various transcription factors were investigated, indicating that DkZF1 and DkZF2 synergistically showed significantly stronger activations on the DkPDC2 promoter. Further, both bimolecular fluorescence complementation (BiFC) and yeast two hybrid (Y2H) assays confirmed that DkZF2 had protein-protein interactions with DkZF1. Thus, these findings illustrate the regulatory mechanisms of zinc finger proteins for persimmon fruit deastringency under AHCA.

High-CO2/Hypoxia-Responsive Transcription Factors DkERF24 and DkWRKY1 Interact and Activate DkPDC2 Promoter.

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.

DkNAC7, a novel high-CO2/hypoxia-induced NAC transcription factor, regulates persimmon fruit de-astringency.

Artificial high-CO2 atmosphere (AHCA, 95% CO2 and 1% O2) has been widely applied as a postharvest de-astringency treatment for persimmon fruit. AHCA increases expression of transcription factors, including ethylene response factors (DkERF), that target de-astringency genes. Here, the promoter of DkERF9, a previously characterized AHCA-inducible and de-astringency regulator, was utilized to screen a cDNA library by yeast one hybrid assay. A novel NAC transcription factor, named DkNAC7, was identified. Dual-luciferase assay indicated that DkNAC7 could not only trans-activate the promoter of DkERF9, but also activated the previously identified deastringency-related gene DkPDC2. Real-time PCR analysis showed that DkNAC7 was up-regulated by AHCA treatment, in concert with the removal of astringency from persimmon fruit and subcellular localization showed DkNAC7 was located in the nucleus. Thus, these results indicate that DkNAC7 is a putative transcriptional activator involved in regulating persimmon fruit deastringency by trans-activition on both DkERF9 and DkPDC2, which encodes pyruvate decarboxylase.

A transcription factor network responsive to high CO2/hypoxia is involved in deastringency in persimmon fruit.

Plant responses to anaerobic environments are regulated by ethylene-response factors (ERFs) in both vegetative and productive organs, but the roles of other transcription factors (TFs) in hypoxia responses are poorly understood. In this study, eight TFs (DkbHLH1, DkMYB9/10/11, DkRH2-1, DkGT3-1, DkAN1-1, DkHSF1) were shown to be strongly up-regulated by an artificial high-CO2 atmosphere (1% O2 and 95% CO2). Dual-luciferase assays indicated that some TFs were activators of previously characterized DkERFs, including DkMYB10 for the DkERF9 promoter, DkERF18/19 and DkMYB6 for the DkERF19 promoter, and DkERF21/22 for the DkERF10 promoter. Yeast one-hybrid and cis-element mutagenesis confirmed these physical interactions with one exception. The potential roles of these TFs in persimmon fruit deastringency were analysed by investigating their transient over-expression (TOX) in persimmon fruit discs, which indicated that DkMYB6TOX, DkMYB10TOX, DkERF18TOX, and DkERF19TOX were all effective in causing insolubilization of tannins, concomitantly with the up-regulation of the corresponding genes. These results indicated that multiple TFs of different classes are responsive to high-CO2/hypoxia in fruit tissues, and that a TF-TF regulatory cascade is involved in the hypoxia responses involving the Group VII DkERF10, and DkERFs and DkMYBs.

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 (Dkß-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.

Involvement of DkTGA1 Transcription Factor in Anaerobic Response Leading to Persimmon Fruit Postharvest De-Astringency.

Persimmon fruit are unique in accumulating proanthocyanidins (tannins) during development, which cause astringency in mature fruit. In 'Mopanshi' persimmon, astringency can be removed by treatment with 95% CO2, which increases the concentrations of ethanol and acetaldehyde by glycolysis, and precipitates the soluble tannin. A TGA transcription factor, DkTGA1, belonging to the bZIP super family, was isolated from an RNA-seq database and real-time quantitative PCR indicated that DkTGA1 was up-regulated by CO2 treatment, in concert with the removal of astringency from persimmon fruit. Dual-luciferase assay revealed that DkTGA1 had a small (less than 2-fold), but significant effect on the promoters of de-astringency-related genes DkADH1, DkPDC2 and DkPDC3, which encode enzymes catalyzing formation of acetaldehyde and ethanol. A combination of DkTGA1 and a second transcription factor, DkERF9, shown previously to be related to de-astringency, showed additive effects on the activation of the DkPDC2 promoter. Yeast one-hybrid assay showed that DkERF9, but not DkTGA1, could bind to the DkPDC2 promoter. Thus, although DkTGA1 expression is positively associated with persimmon fruit de-astringency, trans-activation analyses with DkPDC2 indicates it is likely to act by binding indirectly DkPDC2 promoter, might with helps of DkERF9.