Dof Transcription Factors Are Involved in High CO 2 Induced Persimmon Fruit Deastringency

: High CO 2 treatment is a widely used deastringency technology that causes the accumulation of acetaldehyde which precipitates the astringent soluble tannins from persimmon fruit, making them more attractive to consumers. The identiﬁcation of DkADH1 and DkPDC2 (the key genes for acetaldehyde accumulation) and their regulators (e.g., ERFs), has signiﬁcantly advanced our understanding of the fruit deastringency mechanism, but other TFs are also involved in the high CO 2 response. Here, 32 DkDofs genes were identiﬁed from ‘Gongcheng-shuishi’ persimmon, with nine of them shown to differentially respond to high CO 2 treatment. Dual luciferase assay indicated that DkDof3 and DkDof6 could repress the promoters of DkADH1 and DkPDC2 , respectively. EMSA assay showed that DkDof3 and DkDof6 physically interacted with probes containing T/AAAAG elements from the DkADH1 promoter, whereas they failed to recognize similar elements from the DkPDC2 promoter. The expression of DkDof3 and DkDof6 was also found to be repressed in different persimmon cultivars in response to high CO 2 treatment. It is proposed that DkDof3 and DkDof6 were involved in fruit deastringency by regulating the expression of DkADH1 and DkPDC2 in different persimmon cultivars.


Introduction
Persimmon (Diospyros kaki L.) is a perennial deciduous fruit tree, which is native to China and has a very long cultivation history. Persimmon can be divided into four types according to the astringency of the fruit and the influence of seeds [1]: pollination-constant non-astringent (PCNA), pollination-constant astringent (PCA), pollination-variant nonastringent (PVNA) and pollination-variant astringent (PVA) types. The four types are all astringent when they are immature. The astringency of PCNA and PVNA persimmon is naturally removed on the tree before harvest, but PCA and PVA types are still astringent at harvest and need to be treated after harvest to make them edible [2]. Therefore, it is of great significance to understand the mechanism of postharvest deastringency of persimmon fruit.
The astringency of persimmon fruit is due mainly to the combination of soluble tannins and human oral mucosal proteins, which causes a dry and rough sense in the mouth [3]. Several different artificial deastringency technologies have been discovered, including ethylene treatment [4], vacuum packaging [5], high CO 2 application [6], alcohol and warm water immersion [7]. Of these, high CO 2 treatment is the most widely applied and creates a low oxygen environment [8]. Under this condition, acetaldehyde is formed and accumulates through anaerobic respiration, and combines with soluble tannins to remove fruit astringency by precipitation [9,10]. Under low oxygen conditions, acetaldehyde is produced in fruit mainly in two ways [11]. First, pyruvate is decomposed into acetaldehyde and CO 2 , catalysed by pyruvate decarboxylase (PDC, EC 4.1.1.1); Second, catalytic activity of alcohol dehydrogenase converts ethanol to generate acetaldehyde (ADH, EC 1.1.1.1). Studies have shown that the activities of ADH and PDC both increased during deastringency of persimmon fruit undergoing high CO 2 treatment, with the concomitant accumulation of acetaldehyde and ethanol [11,12]. In a previous study, Min et al. [12] isolated three DkADH genes and five DkPDC genes from persimmon. Transient overexpression experiments showed that overexpression of DkADH1 and DkPDC2 could cause a decrease in soluble tannins, indicating that DkADH1 and DkPDC2 are key targets responsible for accumulation of acetaldehyde-mediated postharvest deastringency of persimmon fruit [12,13].
Transcription factors (TFs) involved in the regulation of DkADH1 and DkPDC2 expression in persimmon fruit deastringency have been investigated. Min et al. [12] and Zhu et al. [10] found that the expression of DkADH1 and DkPDC2 was regulated by DkERF10 and DkERF9/19. Furthermore, DkERF10, DkERF9 and DkERF19 were themselves regulated by upstream DkERF20/21, DkMYB10, and DkERF18/19, respectively [10,12]. Apart from these TFs, WRKYs [6], NACs [14], ZFs [15] and other family transcription factors have also been reported to be associated with persimmon fruit deastringency caused by high CO 2 treatment. Dof genes are plant specific transcription factors [16]. Generally, their N-terminal is a highly conserved single zinc finger Dof domain, which has the dual function of interacting with DNA and protein; The Dof C-terminal is relatively variable, and encodes an important specific transcriptional regulatory domain of Dof proteins [17,18]. Studies have shown that Dof genes are involved in many processes of plant growth and development, including tissue differentiation, seed development, and stress responses among others [19]. However, few studies have reported the roles of Dof genes in the high CO 2 /hypoxia response and acetaldehyde metabolism. Additionally, the potential function of Dof genes in persimmon fruit deastringency is still unclear.
Here, we identified Dof transcription factors related to astringency removal using high CO 2 -treated persimmon fruit. The Dof family genes from persimmon fruit were analyzed and the regulatory roles of DkDofs in persimmon fruit deastringency were investigated by real-time PCR analysis, dual-luciferase assay and electrophoretic mobility shift assay (EMSA). High CO 2 reduced expression of some Dof genes. DkDof3 and DkDof6 were shown to bind to and inhibit the transcription from the DkADH1 and DkPDC2 promoters.

Plant Materials and Treatments
Astringent persimmon 'Gongcheng-shuishi' fruit were harvested from a commercial orchard at Guilin (China) in 2018. Fruit of the same size without mechanical injury were selected and treated with air or high CO 2 (95% CO 2 + 1% O 2 ) for one day in air-tight containers. Then, the control and treated fruit were transferred to 20 • C for storage in air, and nine fruit of each group were sampled every day, containing three biological replicates. The 'Jingmianshi' samples, which were collected and described by Wang et al. [20], were used for the verification of key genes expression in response to high CO 2 treatment.

Gene Isolation and Sequence Analysis
The predicted coding (CDS) sequences of persimmon Dof genes were obtained from 'Youshi' (Diospyros oleifera) genome data [21]. The full-length genes were isolated by homologous cloning, and the primers used were listed in Table S1. For phylogenetic analysis, the amino acid sequences of DkDofs were aligned with AtDofs (downloaded from the website: https://www.arabidopsis.org/ [22]) by Clustal X and the results were visualized by FigTree (v1.4.2).

Soluble Tannins Printing Assay
Soluble tannins contents were visualized by soluble tannins printing assay according to Min et al. [23]. Firstly, filter papers were soaked in FeCl 2 solution and then dried. Then, the persimmon fruit was cut lengthwise and immediately printed onto FeCl 2 -soaked filter paper. The intensity of black indicates the content of soluble tannins.

RNA Extraction and cDNA Synthesis
Total RNA was extracted by the cetyltrimethylammonium bromide (CTAB) method described in Yin et al. [4]. In short, around 1 g of pulp samples were extracted with CTAB buffer, and then washed twice with trichloromethane/isopentanol (v:v = 24:1). After precipitation with lithium chloride, it was washed twice with 75% ethanol. Then, the total RNA was obtained after dried and resolved by nuclease-free water.
The genomic DNA was then treated with DNase and synthesis of cDNA were carried out using the PrimeScript TM RT reagent Kit with gDNA Eraser (Takara, Kyoto, Japan).

Real-Time PCR Analysis
Gene-specific primers used for real-time PCR were designed using Primer3 (v.0.4.0) and double checked by melting curve and products sequencing. A CFX96 instrument (Bio-Rad) was used to perform the real-time PCR according to Yin et al. [4]. Primers used are listed in Table S2. The reaction system is 20 µL, including 10 µL ssofast evagreen Supermix (Biorad, CA, USA), 1 µL upstream primer, 1 µL downstream primer, 2 µL cDNA and 6 µL water. The PCR reaction procedure was 94 • C for 5 min, and then 94 • C 10 s, 60 • C 10 s for 45 cycles. The persimmon housekeeper gene DkACT [12] was selected as internal control to measure the expression level of other genes.

Dual Luciferase Assay
The dual luciferase assay was used to detect the regulatory effects of TFs on target genes [24]. In brief, the full-length sequences of DkDofs were inserted into pGreen II 0029 62-SK vector (SK), and the promoter fragments of DkADH1 and DkPDC2 were constructed on pGreen II 0800 Luc vector (Luc) which were completed by Min et al. [12]. Primers used were listed in Table S1. All constructed recombinant SK and Luc plasmids were transferred into Agrobacterium GV3101 and then transiently expressed on tobacco (Nicotiana benthamiana) leaves with a volume ratio of 10:1 (transcription factor: promoter). Three days after injection, the luciferase activity in leaves was detected by dual luciferase assay reagents (Promega, Madison, WI, USA).

EMSA
The full-length sequences of DkDofs were connected to the pGEX-4T-1 vector (GE), and then transformed into Rosetta (DE3) pLys bacteria (Novagen) to obtain the recombinant proteins by prokaryotic expression. Primers used are listed in Table S3. The protein induction was by adding 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37 • C. Before EMSA, the target proteins were purified by the GST-tag Protein Purification Kit (Beyotime Biotechnology). Probes used for this assay were synthesized and 3 -biotin labeled by HuaGene (Shanghai, China) and are listed in Table S4. The upstream and downstream probes were mixed and annealed to form double stranded probes before use. Finally, EMSA was performed using the LightShift Chemiluminescent EMSA kit (ThermoFisher Scientific, Waltham, MA, USA).

Statistical Analysis
The Student's t-test was performed by GraphPadPrism7. Figures were drawn with GraphPadPrism7 and Adobe Photoshop CS6.

Analysis of DkDof Genes
Based on the Diospyros oleifera genome [21], a total of 32 non-redundant Dof genes were found and their deduced amino acid sequences were used to perform phylogenetic analysis with Dof genes from Arabidopsis thaliana. These Dof family genes, both Arabidopsis and persimmon, could be divided into four major groups, and Dof genes from both plants were distributed in all four groups ( Figure 1). The distribution of the 32 DkDofs indicated that they have certain specificity in structure and different groups may play different roles and the genes clustered in the same group may have similar functions.

Analysis of DkDof Genes
Based on the Diospyros oleifera genome [21], a total of 32 non-red were found and their deduced amino acid sequences were used to perf analysis with Dof genes from Arabidopsis thaliana. These Dof family bidopsis and persimmon, could be divided into four major groups, an both plants were distributed in all four groups (Figure 1). The distr DkDofs indicated that they have certain specificity in structure and diff play different roles and the genes clustered in the same group may have Figure 1. Phylogenetic analysis of Dof family genes from Arabidopsis and pers Arabidopsis start with 'AT' and genes from persimmon start with 'EVM'. All g into four groups, highlighted in green, red, yellow and blue, respectively.

Effects of High CO2 Treatment on 'Gongcheng-Shuishi' Persimmon Fruit
The high CO2 deastringency treatment was applied to 'Gongcheng mon fruit. The results showed that after 1 d of high CO2 treatment th content has decreased significantly compared with control and this dec tinued at 2 d ( Figure 2). In short, high CO2 treatment can cause persimm gency, and the effect is more obvious at 2 d.

Effects of High CO 2 Treatment on 'Gongcheng-Shuishi' Persimmon Fruit
The high CO 2 deastringency treatment was applied to 'Gongcheng-shuishi' persimmon fruit. The results showed that after 1 d of high CO 2 treatment the soluble tannins content has decreased significantly compared with control and this decreasing trend continued at 2 d ( Figure 2). In short, high CO 2 treatment can cause persimmon fruit deastringency, and the effect is more obvious at 2 d.

Expression of DkDofs in Response to High CO2 Treatment
In order to explore the effects of DkDofs on persimmon fruit deastringency, their ex pression patterns in control and high CO2-treated samples were analyzed. Real-time PCR experiments were performed to measure the expression of most DkDofs. Although w were unable to identify specific primers for genes EVM0007955, EVM0009058 EVM0011394, EVM0021836 and EVM0022952. EVM0025419 and EVM0024362 were up regulated and EVM0003933, EVM0019694, EVM0017946, EVM0024406, EVM0011284 EVM0012621 and EVM0031008 were down-regulated after 1 d in response to high CO ( Figure 3). There were also some genes, such as EVM0018852 and EVM0023131, differen tially expressed at 2 d ( Figure 3). Since the soluble tannins of fruit have decreased at 1 d (Figure 2), the nine DkDofs differentially expressed at 1 d were selected as candidate gene for further investigations into the mechanism of deastringency.

Expression of DkDofs in Response to High CO 2 Treatment
In order to explore the effects of DkDofs on persimmon fruit deastringency, their expression patterns in control and high CO 2 -treated samples were analyzed. Real-time PCR experiments were performed to measure the expression of most DkDofs. Although we were unable to identify specific primers for genes EVM0007955, EVM0009058, EVM0011394, EVM0021836 and EVM0022952. EVM0025419 and EVM0024362 were up-regulated and EVM0003933, EVM0019694, EVM0017946, EVM0024406, EVM0011284, EVM0012621 and EVM0031008 were down-regulated after 1 d in response to high CO 2 ( Figure 3). There were also some genes, such as EVM0018852 and EVM0023131, differentially expressed at 2 d ( Figure 3). Since the soluble tannins of fruit have decreased at 1 d (Figure 2), the nine DkDofs differentially expressed at 1 d were selected as candidate genes for further investigations into the mechanism of deastringency. EER REVIEW 6 of 11

Regulatory Effects of DkDof1-7 on DkADH1 and DkPDC2 Promoters
Full-length sequences of seven DkDofs were isolated (designated as DkDof1-7, corresponding to EVM0031008, EVM0019694, EVM0011284, EVM0025419, EVM0003933, EVM0024406 and EVM0012621). Since DkADH1 and DkPDC2 have been identified as the key targets involved in the high CO2-induced deastringency process, the regulatory effects of DkDof1-7 on their promoters were analyzed. Dual luciferase assay found no significant trans-activation effect, but DkDof3 and DkDof6 were found to be repressors of the DkADH1 and DkPDC2 promoters with a 0.5-fold reduction in expression (Figure 4). The transcriptional effects of DkDof2 and DkDof7 on transcription from the DkADH1 promoter and DkDof5 on the DkPDC2 promoter also reached statistical significance, but the . Expression of DkDofs in response to high CO 2 treatment in 'Gongcheng-shuishi' fruit. EVM0031008, EVM0019694, EVM0011284, EVM0025419, EVM0003933, EVM0024406 and EVM0012621 were designated as DkDof1-7. DkACT was used as internal control. Error bars represent standard errors for three replicates (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Regulatory Effects of DkDof1-7 on DkADH1 and DkPDC2 Promoters
Full-length sequences of seven DkDofs were isolated (designated as DkDof1-7, corresponding to EVM0031008, EVM0019694, EVM0011284, EVM0025419, EVM0003933, EVM0024406 and EVM0012621). Since DkADH1 and DkPDC2 have been identified as the key targets involved in the high CO 2 -induced deastringency process, the regulatory effects of DkDof1-7 on their promoters were analyzed. Dual luciferase assay found no significant trans-activation effect, but DkDof3 and DkDof6 were found to be repressors of the DkADH1 and DkPDC2 promoters with a 0.5-fold reduction in expression (Figure 4). The transcriptional effects of DkDof2 and DkDof7 on transcription from the DkADH1 promoter and DkDof5 on the DkPDC2 promoter also reached statistical significance, but the differences did not reach the selected thresholds (more than 2-fold or less than 0.5-fold) (Figure 4). differences did not reach the selected thresholds (more than 2-fold or less th (Figure 4).

Binding Abilities of DkDof3 and DkDof6 on DkADH1 and DkPDC2 Promoters
In order to explore whether the regulatory effects of DkDof3 and DkD DkADH1 and DkPDC2 promoters were direct or indirect, their abilities to bin promoters were tested. The specific DNA binding sequence for Dof family T/AAAAG [25]. There were five and three T/AAAAG motifs in the DkADH1 a promoters, respectively, which were designed as probes with nearby sequen 30 bp ( Figure 5A) (Sequences of probes were listed in Table S4). EMSA resu that DkDof3 could bind to the P1, P2, P4 and P5 probes from DkADH1 pr DkDof6 could bind to the P2, P3, P4 and P5 probes from DkADH1 promoter The unlabeled DkADH1-P4 probe was designed to verify the specificity of the the results show that adding the unlabeled DkADH1-P4 probe could reduce of labeled DkADH1-P4 probe to DkDof3 and DkDof6 proteins ( Figure 5D). T indicate that both DkDof3 and DkDof6 directly bind to the DkADH1 promote neither of them was found to bind to the probes from the DkPDC2 promoter, gested that their regulatory effects on DkPDC2 promoter might be indirect (F Figure 4. The regulatory effects of DkDof1-7 on the promoters of DkADH1 and DkPDC2. Empty SK and TF-SK means empty SK plasmid and recombinant SK construct containing the TF gene, respectively. The LUC/REN value of empty SK plus corresponding promoter was set as 1. Error bars represent standard errors from three replicates (**, p < 0.01; ***, p < 0.001). DkDof2, 3, 6 and 7 inhibit the activity of the promoter of DkADH1 whereas DkDof3 and 6 inhibit activity of the DkPDC2 promoter.

Binding Abilities of DkDof3 and DkDof6 on DkADH1 and DkPDC2 Promoters
In order to explore whether the regulatory effects of DkDof3 and DkDof6 on the DkADH1 and DkPDC2 promoters were direct or indirect, their abilities to bind these two promoters were tested. The specific DNA binding sequence for Dof family proteins is T/AAAAG [25]. There were five and three T/AAAAG motifs in the DkADH1 and DkPDC2 promoters, respectively, which were designed as probes with nearby sequences of about 30 bp ( Figure 5A) (Sequences of probes were listed in Table S4). EMSA results indicate that DkDof3 could bind to the P1, P2, P4 and P5 probes from DkADH1 promoter and DkDof6 could bind to the P2, P3, P4 and P5 probes from DkADH1 promoter ( Figure 5B). The unlabeled DkADH1-P4 probe was designed to verify the specificity of the binding and the results show that adding the unlabeled DkADH1-P4 probe could reduce the binding of labeled DkADH1-P4 probe to DkDof3 and DkDof6 proteins ( Figure 5D). These results indicate that both DkDof3 and DkDof6 directly bind to the DkADH1 promoter. However, neither of them was found to bind to the probes from the DkPDC2 promoter, which suggested that their regulatory effects on DkPDC2 promoter might be indirect ( Figure 5C).

Expression of DkDof3 and DkDof6 in 'Jingmianshi' Fruit
In order to further verify the relations between DkDof3 and 6 and persimmon fruit deastringency, the changes in their expression were analyzed in another persimmon cultivar ('Jingmianshi'). As found for 'Gongcheng-shuishi', high CO2 treatment also effectively accelerated deastringency in 'Jingmianshi' fruit [20], and inhibited the expression of DkDof3 and DkDof6 (Figure 6), which confirmed that these two Dof genes were negatively correlated with persimmon fruit deastringency.

Expression of DkDof3 and DkDof6 in 'Jingmianshi' Fruit
In order to further verify the relations between DkDof3 and 6 and persimmon fruit deastringency, the changes in their expression were analyzed in another persimmon cultivar ('Jingmianshi'). As found for 'Gongcheng-shuishi', high CO 2 treatment also effectively accelerated deastringency in 'Jingmianshi' fruit [20], and inhibited the expression of DkDof3 and DkDof6 (Figure 6), which confirmed that these two Dof genes were negatively correlated with persimmon fruit deastringency.

Expression of DkDof3 and DkDof6 in 'Jingmianshi' Fruit
In order to further verify the relations between DkDof3 and 6 and persimmon fruit deastringency, the changes in their expression were analyzed in another persimmon cultivar ('Jingmianshi'). As found for 'Gongcheng-shuishi', high CO2 treatment also effectively accelerated deastringency in 'Jingmianshi' fruit [20], and inhibited the expression of DkDof3 and DkDof6 (Figure 6), which confirmed that these two Dof genes were negatively correlated with persimmon fruit deastringency.

Discussion
It is very important for the development of the persimmon industry to study fruit deastringency technologies. In recent years, research to precisely control fruit astringency without affecting other quality attributes has evolved from simple technology development [11,26] to investigations into the molecular basis of the mechanisms of deastringency [10,12]. Previous studies have identified DkADH1 and DkPDC2 as key target genes of high CO 2 -induced persimmon fruit deastringency [12]. Moreover, several TFs have been reported to regulate high CO 2 -induced persimmon fruit deastringency, such as ERFs [12], MYBs [10] and WRKYs [6]. However, these TFs cannot fully explain the whole process of fruit deastringency. Here, the involvement of Dof families in deastringency regulation was studied which was little reported before. A total of 32 Dof genes were identified, based on the Diospyros oleifera genome [21], and nine DkDofs were probably involved in deastringency regulation as they were differentially expressed in response to high CO 2 treatment (Figure 3). Among them, DkDof3 (EVM0011284) and DkDof6 (EVM0024406) could trans-repress the promoters of DkADH1 and DkPDC2 (Figure 4), which enriches the regulation targets of Dof family. Besides, DkDof3 and DkDof6 were clustered together (blue group) and were homologous to Arabidopsis AT2G37590.1 (Figure 1), which has been shown to be involved in vascular development [27]. As ADH and PDC are also the key enzymes involved in hypoxia response, whether this blue group of Dof genes including AT2G37590.1 have flood resistance function is worth exploring.
TFs can be divided into activators and repressors according to their regulatory effects on the target genes [28]. Many TFs have been reported to regulate hypoxia responses or fruit deastringency. Some of these TFs have been found to form complexes, such as DkERF24 and DkWRKY1 [6]. Some are involved in a regulatory cascade, for example, DkMYB10 was identified as the upstream activator of DkERF9 [10]. Some TFs individually regulate the expression of ADH or PDC, such as DkERF23 [6]. However, these reported that TFs are all activators. It has been reported previously that Dof family genes could play roles as repressors. For example, ZmDof1 inhibited pollen formation by binding to and suppressing the promoter of Zm401, which was the key gene for pollen formation [29]. AtDAG1 (DOF AFFECTING GERMINATION1) controls seed dormancy and germination by inhibiting the expression of abscisic acid degradation gene CYP707A2 and gibberellin synthesis gene AtGA3ox1 [30]. Here, DkDof3 and DkDof6 were verified to be involved in fruit deastringency by suppressing transcription of DkADH1 and DkPDC2 (Figure 4), which showed that the deastringency process is jointly regulated by transcriptional activators and repressors. Besides, DkDof3 and DkDof6 could regulate both DkADH1 and DkPDC2 promoters and might play a greater role in fruit deastringency, as most previously identified TFs only regulated one of them ( Figure 7).   In conclusion, 32 DkDofs were identified from persimmon fruit, with nine of them differentially expressed in response to high CO 2 treatment, which were considered to be involved in persimmon fruit deastringency. Of these, DkDof3 and DkDof6 were identified as repressors that could directly and indirectly trans-repress the promoters of DkADH1 and DkPDC2, respectively. Moreover, the expression of DkDof3 and DkDof6 was repressed in another persimmon cultivar in response to high CO 2 treatment, which suggested that the regulatory roles of them may be conserved in different cultivars. These findings add to our understanding of persimmon fruit deastringency and the function of Dof family genes.
Author Contributions: Conceptualization, X.Y.; methodology, W.W. and X.L.; validation, W.W. and X.Y.; investigation, R.J.; writing-original draft preparation, R.J. and W.W.; writing-review and editing, K.C. and X.Y. All authors have read and agreed to the published version of the manuscript.