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Article

ChIP-Seq Analysis of SlAREB1 Downstream Regulatory Network during Tomato Ripening

by
Yanan He
†,
Qiong Wu
*,†,
Chunxiao Cui
,
Qisheng Tian
,
Dongdong Zhang
and
Yurong Zhang
Engineering Center of Ministry of Education, School of Food and Strategic Reserves, Henan University of Technology, Zhengzhou 450001, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Foods 2023, 12(12), 2357; https://doi.org/10.3390/foods12122357
Submission received: 19 April 2023 / Revised: 2 June 2023 / Accepted: 8 June 2023 / Published: 13 June 2023
(This article belongs to the Special Issue Research Advances of Disease Control, Preservation, Quality and Safety Control of Fruits and Vegetables)
Browse Figures
Versions Notes

Abstract

:
SlAREB1, a member of the abscisic acid (ABA) response element-binding factors (AREB/ABFs) family, was reported to play a crucial role in the expression of ABA-regulated downstream genes and affect the ripening of tomato fruit. However, the downstream genes of SlAREB1 are still unclear. Chromatin immunoprecipitation (ChIP) is a powerful tool and a standard method for studying the interactions between DNA and proteins at the genome-wide level. In the present study, SlAREB1 was proved to continually increase until the mature green stage and then decrease during the ripening period, and a total of 972 gene peaks were identified downstream of SlAREB1 by ChIP-seq analysis, mainly located in the intergenic and promoter regions. Further gene ontology (GO) annotation analysis revealed that the target sequence of SlAREB1 was the most involved in biological function. Kyoto Encylopaedia of Genes and Genomes (KEGG) pathway analysis showed that the identified genes were mainly involved in the oxidative phosphorylation and photosynthesis pathways, and some of them were associated with tomato phytohormone synthesis, the cell wall, pigment, and the antioxidant characteristic of the fruit as well. Based on these results, an initial model of SlAREB1 regulation on tomato fruit ripening was constructed, which provided a theoretical basis for further exploring the effects of the regulation mechanism of SlAREB1 and ABA on tomato fruit ripening.

1. Introduction

Fruit ripening has attracted much attention because of its particularity to plant bi-ology and its critical impact on fruit quality and shelf life, which involves changes in various appearance and flavor qualities, including color, texture, taste, and aroma [1,2]. Based on the presence or absence of respiration and ethylene transition peaks during ripening, fruit was divided into climactic and non-climactic fruit [3,4]. Tomato, a typical climacteric fruit, was widely used as a model plant for the study of fruit ripening due to its economic importance, obvious ripening period, short life cycle, extensive genome information, and significant metabolic changes [1,5,6,7,8].
Abscisic acid (ABA) is a crucial phytohormone involved in the ripening process of tomato fruit. A number of studies demonstrated that ABA has a positive impact on tomato fruit ripening [1,9,10]. Exogenous ABA treatment was shown to accelerate fruit color transition and firmness reduction and increase ethylene production during tomato ripening, as well as affect the metabolism of sugar and organic acids [11], accumulation of volatile compounds [12], and antioxidant properties [13].
The current ABA signaling model in plant can be described as follows: in the absence of ABA, PP2C inhibits SnRK2s activity through physical interactions and phosphatase activity, resulting in the inability of ABA to complete signaling. In the presence of ABA, the binding of ABA molecules to the ABA receptor PYR/PYL/RCAR induces the structural change in the receptor, which allows the ABA receptor to interact with PP2C and inhibit PP2C activity; this leads to the release of SnRK2s and, subsequently, activates the downstream ABFs/AREB/ABI5-type bZIP (basic region leucine zipper) transcription factor [14,15]. ABA response element-binding factors (AREB/ABFs), a subfamily of the basic leucine zipper (bZIP) family, are important downstream target genes of SnRK2. They can be activated by protein kinases such as SnRK2.2, SnRK2.3, and SnRK2.6 and then specifically bind to ABA response elements (ABREs and PyACGTGG/TC) in the promoter regions of downstream target genes to realize the biological regulatory function of ABA in plants [16]. In tomato, two AREB transcription factors, SlAREB1 and SlAREB2, were isolated and identified. Both of them were induced by exogenous ABA treatment, while SlAREB1 was reported as mainly involved in regulating the expression of stress-related genes and the ripening process in tomato fruit [17,18,19]. Xu et al. [20] found that SlAREB1 had a strong response to ABA and saline–alkali stress. The SlAREB1-mediated ABA signaling pathway may regulate fruit-ripening-related metabolic processes by inducing the expression of genes that encode organic acids (citric acid and malic acid), sugars (glucose and fructose), and amino acid (glutamic acid)-related synthetases in tomato fruit [17,21]. Compared with wild-type tomato fruit, the transcription levels of ethylene synthesis genes SlACS2, SlACS4, SlACO1, and SlACO3 were significantly increased in SlAREB1-overexpresseion fruit and decreased in antisense inhibition lines [21]. Yang et al. [22] found that SNAC9 interacted with SlAREB1 to affect ABA signaling and further regulate the ripening of tomato fruit and that silencing SNAC9 fruit would downregulate the expression of SlACS2 and SlACO1. Furthermore, Mou et al. [23] found that SlAREB1 interacted with NOR to promote ethylene synthesis during tomato fruit ripening. These studies suggested that SlAREB1-mediated ABA signaling may be involved in the regulation of ethylene biosynthesis and the metabolic processes associated with ripening by inducing the transcription of the corresponding genes and finally affecting tomato fruit ripening. However, the specific regulation mechanism remains to be explicated.
Chromatin immunoprecipitation (ChIP) is a powerful tool and a standard method for studying the interactions between DNA and proteins at the genome-wide level. In recent years, ChIP-Seq technology, which combines ChIP with next-generation high-throughput sequencing technology, has been widely used to identify the target gene regions that transcription factors potentially regulate in plants, owing to its advantages of having a high resolution, a low signal-to-noise ratio, and broad coverage [24,25,26,27,28]. Studies showed that SlAREB1 could regulate tomato fruit ripening, but the downstream target genes that it binds are still unclear. In this study, ChIP-Seq was performed on SlAREB1-overexpression, transgenic, mature green tomato fruit to analyze the downstream regulatory network of SlAREB1, which could provide references and research ideas for understanding the regulation mechanism of SlAREB1 and ABA on tomato fruit ripening.

2. Materials and Methods

2.1. Tomato Fruit and Exgenous ABA Treatment

Cherry tomatoes were cultivated in Aisijia Picking Garden in Linying County, Luohe city of Henan Province, China. Approximately 36 days after anthesis, mature green tomato fruit (Solanum lycopersicum L.) were manually harvested. Around 600 intact tomato fruit of uniform size were randomly collected at equal height from different plants.
The ABA treatment and storage of tomato fruit were performed per our previous study [12]. Briefly, the collected tomato fruit was sterilized and treated with 1 mM ABA (98%, HPLC, Aladdin) or sterile water (the control) under vacuum (60 kPa) for 180 s and then incubated at 20 °C and 90% relative humidity in darkness for 13 d. During the storage period, tomato fruit was sampled every 3 days, with three batches (8 fruit per batch) randomly sampled per group each time, and the pericarp tissues were frozen with liquid nitrogen and maintained at −80 °C for further use.
Tomato fruit at 6 ripening stages, including immature green (IMG1 and IMG2), mature green (MG), breaker (Br), turning (T), and red ripe (RR), were harvested, and the sampling method was the same as above.

2.2. qRT-PCR Analysis

Total RNA was extracted with RNAiso (TaKaRa, Tokyo, Japan), quantified with a BioPhotometer (D30, Eppendorf AG, Hamburg, Germany), and reverse-transcribed to cDNA using the PrimeScript® RT Reagent Kit (DRR047A, TaKaRa, Japan). Primers for selected genes were designed using the Primer 5.0 software, and the obtained sequences are shown in Table S1. The qRT-PCR experiment was performed according to the protocol of TB Green® Premix Ex Taq™ II (RR820, TaKaRa, Japan) using the QuantStudioTM 3 Real-Time PCR Instrument. Actin gene (AK328563.1) was used as the reference gene, and the results were calculated using the 2−ΔΔCT method.

2.3. Construction and Identification of SlAREB1-Overexpression Transgenic Tomato Plants

The genomic DNA of the mature green tomato fruit was extracted with high-efficiency plant genomic DNA rapid extraction kit (D200, GeneBetter, Beijing, China), the full length of the open reading frame (ORF) of SlAREB1 was amplified and cloned into pCAMBIA1301 vector via T4 ligase with NcoI and BstEII as double restriction sites, the primers used are shown in S1, and the Flag gene (3 repeats) was inserted in front of the SlAREB1 coding region during the cloning process for subsequence detection and ChIP analysis. The SlAREB1-overexpression transgenic tomato plants were obtained via the agrobacterium-mediated method with Micro-Tom plant and Hygromycin B as resistance genes [29], then the positive plants (T0 generation) were cultivated, and PCR analysis was performed both on leaves and mature green fruit of the transgenic plants to verify the successful overexpression of SlAREB1. Further ChIP-Seq analysis was carried out on the selected SlAREB1-overexpression transgenic mature green fruit.

2.4. ChIP-Seq Analysis

ChIP-Seq analysis was conducted following the method of Yang et al. [30] with modifications. Specifically, approximately 4 g of tomato peel from the SlAREB1-overexpression transgenic lines were pulverized in liquid nitrogen and cross-linked with 1% formaldehyde at room temperature for 15–30 min, and then 2.5 mL glycine (125 mM) was added to terminate the cross-linking reaction, followed by three washes to remove excess formaldehyde. Afterwards, the chromatin was extracted from the nuclei on ice using a lysis buffer containing protease inhibitors and then sonicated to obtain between 200–500 bp (20 μL of sonicated DNA were used as input sample). For the enrichment of DNA fragments bound to the target protein, 5 μL of Flag antibody (F1804, Sigma, Alexandria, VA, USA) was added to 20 μL of sonicated DNA fragment to form antibody-target protein–DNA complex, and the resulting antibody-target protein–DNA complex was immunoprecipitated using protein G beads (L00277, Sigma, Alexandria, VA, USA). The complex was eluted with elution buffer and subjected to overnight incubation at 65 °C with 20 μL of 5 M NaCl to reverse the cross-linking. Simultaneously, an input sample was mixed with 500 μL of elution buffer and 20 μL of 5M NaCl to decompose the cross-linking and was used as control. Then, the decross-linking product was mingled with 10 μL of 0.5 M EDTA, 5 μL of RNase, 20 μL of Tris-Hcl (pH 7.0), and 2 μL of proteinase K and incubated at 45 °C for 1 h. ChIP DNA Clean and Concentrator™ (Zymo Research Corp., Irvine, CA, USA) was used to purify the ChIP DNA, which was subsequently sequenced using Illumina HiSeq PE150. The quality of obtained reads was assessed with the fastqc software (version: 0.11.5) and filtered using Trimmomatic (version: 0.36). The obtained clean reads were mapped to the tomato genome (https://solgenomics, version:4.0, accessed on 11 May 2023) using BWA software (version: 0.7.15-r1140), and the peak information was analyzed with MACS software (version: 2.1.1.20160309). The entire ChIP-Seq experiment and analysis was performed with the assistance of Aijibaike Biotechnology Co., Ltd., Wuhan, China.

2.5. Statistical Analysis

Analysis of variance (ANOVA) and SPSS 22.0 software (IBM, New York, NY, USA) were used to analyze the data of SlAREB1 gene expression at a significant level of p < 0.05. The qRT-PCR results were presented as mean ± standard deviation, and the gene expression plot in this paper was created using Origin 2018 (OriginLab).

3. Results

3.1. Gene Expression of SlAREB1 during Tomato Fruit Ripening and the Effect of Exogenous ABA Treatment on It

The transcription levels of SlAREB1 in tomato fruit at different growth and development stages (immature green, mature green, breaker, turning, and red ripe) are shown in Figure 1A. The expression of SlAREB1 gradually increased during the early ripening stage, peaked at the mature green stage, declined thereafter, and exhibited a resurgence at the red ripening stage. The effect of exogenous ABA treatment on SlAREB1 gene expression is shown in Figure 1B, and, compared to the control group, ABA-treated tomato fruit had higher expression levels of SlAREB1 from the seventh day after ABA treatment. These results suggested that SlAREB1 may take part in the regulation of tomato fruit ripening, and ABA induced the expression of SlAREB1 during the ripening process.

3.2. Identification of SlAREB1-Overexpression Transgenic Tomato Plants

The results of PCR identification for SlAREB1-overexpression transgenic tomato plants are shown in Figure S1. The presence of clear and singular bands in the trans-genic tomato leaves confirmed the successful integration of SlAREB1 into the genome of transgenic tomato plants. To obtain a clearer view of the effect of SlAREB1 on tomato fruit ripening, we recorded the phenotypic changes in wild-type and transgenic tomatoes at different times after flowering. As shown in Figure 2A, the growth rates of the transgenic and wild plants were similar, and no obvious difference in phenotype was observed between them. The PCR identification results of SlAREB1-overexpression transgenic tomato fruit at the mature green stage are shown in Figure 2B; the clear and single bands in transgenic tomato fruit demonstrating that SlAREB1 was successfully overexpressed in the transgenic tomato fruit. Moreover, the qRT-PCR results showed that the expression of SlAREB1 in transgenic tomato fruit was significantly higher than that in wild-type tomato fruit (Figure 2C). Therefore, the transgenic, mature green tomato fruit was suitable for further ChIP-Seq analysis.

3.3. ChIP-Seq Analysis

3.3.1. ChIP-Seq Peak Analysis

The overview of the ChIP-Seq data of SlAREB1 is shown in Table 1, and the raw reads obtained from the two sequenced samples of SlAREB1-IP and the input were 52,720,466 and 36,263,534, respectively. The input was the control, which was the genomic DNA after ultrasound interruption. Without immunoprecipitation treatment, the DNA was directly delinked, purified, and analyzed. After quality filtration, 51,618,544 and 35,588,840 clean reads were obtained for SlAREB1-IP and the input, respectively. Subsequently, the obtained clean reads were aligned to the tomato genome, and the mapped ratios of SlAREB1-IP and the input were 75.23% and 98.14%, respectively.
A total of 972 peaks were enriched and identified in SlAREB1-overexpression transgenic tomato fruit (Table S2). These peaks on the genome were distributed in 13 chromosomes (Figure 3A). The distribution of the SlAREB1 target sequences on the gene functional elements was as follows: 48.1% in the intergenic region, 27.45% in the promoter region, 13.59% in the exon region, 8.91% in the intron region, 1.06% in the 3′-UTR end, and 0.88% in 5′-UTR end (Figure 3B).

3.3.2. Transcription Factor Prediction of Peak-Associated Genes

A total of 28 transcription factors (TFS) were identified in the enriched sequences, which were grouped into 17 TFS families, and the proportion of each TFS family to the total TFS is shown in Figure 4. The identified factors mainly focused on zf-HD-, FAR1-, and MADS-M-type transcription factor families, and the detailed gene information is listed in Table S3.

3.3.3. Gene ontology (GO) and Kyoto Encylopaedia of Genes and Genomes (KEGG) Analysis of SlAREB1 Target Sequences

GO annotation analysis showed that the target sequences of SlAREB1 were most involved in biological processes and less involved in molecular functions (Figure 5A). In terms of cell components, they were mainly related to the cell, cell part, and organelle. For the biological processes, the enrichment sequences were mainly observed in the metabolic process and cellular process. For the molecular functions, the main functional annotations were involved in binding and catalytic activity.
KEGG pathway analysis provided insights into the metabolic pathways and the specific distribution of the SlAREB1 target sequences. The top 20 metabolic pathways, as illustrated in Figure 5B, revealed that the SlAREB1 target sequence was predominantly distributed in the oxidative phosphorylation and photosynthesis pathways.

3.3.4. Downstream Candidate Genes of SlAREB1

For the 972 peak genes obtained above, the relevant information of these genes was analyzed by matching the tomato genome. As shown in Table 2, a total of 20 and 18 genes were found associated with the oxidative phosphorylation and photosynthesis pathways, respectively. Additionally, 8 hormone-related genes (including 3 genes related to ethylene, 1 gene related to auxin, 1 gene related to gibberellin, and 1 gene related to brassinosteroid, respectively), 18 pigment-related genes, 6 cell-wall-related genes, and 3 antioxidant-related genes were identified. More detailed information for all the downstream candidate genes is listed in Table S4.

4. Discussion

ABA was proved to be a promoter of tomato fruit ripening. SlAREB1, belong-ing to the AREB/ABFs transcription factor family, played an important role in the regulation of ABA downstream gene expression. Studies demonstrated that SlAREB1 participated in the expression of stress-related genes in tomato fruit and the regulation of fruit ripening [17,18]. In this study, the expression level of SlAREB1 gradually increased in the early ripening stage of tomato fruit, peaked at the mature green stage, and decreased afterwards. After ABA treatment, the expression level of SlAREB1 commenced to rise on the seventh day and was significantly higher than that in the CK group, which was consistent with the results of Mou et al. [23], suggesting that SlAREB1 may play a pivotal role in the regulation of tomato fruit ripening and may actively participate in the ABA-mediated regulatory cascade. In addition, the growth rate and phenotype of SlAREB1-overexpression transgenic tomatoes were similar to those of wild-type tomatoes, which was in line with the findings of Bastías et al. [21], indicating that SlAREB1 is mainly involved in the regulation of metabolic programs during fruit ripening but not for the fruit phenotype.
Fruit ripening and stress were a highly intricate but coordinated process, which were predominantly regulated at the transcriptional level, and transcription factors played a pivotal role in the expression of ripening-related and stress-related genes [31,32]. Hu et al. [33] found that the zf-HD gene family primarily functions in the abiotic stress responses in tomato. In addition, the FAR1 transcription factor family was proved to be implicated in stress responses in tomato fruit, with Solyc09g057880.3 downregulated by ABA treatment and potentially serving a crucial role in stress response [34]. The MADS-box family transcription factors were reported to be involved in diverse developmental processes in plants, particularly in the specification of floral organs, fruit development, and ripening. Among them, Solyc07g017343.1 and Solyc06g034317.1 were found to exhibit distinct expression patterns in different tomato fruit development stages, and they may play a role in fruit development and ripening [35]. In the present study, 28 transcription factors were identified downstream of SlAREB1; mainly focusing on zf-HD, FAR1 and MADS-M-type transcription factor families, this indicates that SlAREB1 may be involved in the regulation of metabolic programs and fruit ripening by regulating these transcription factors.
Photosynthesis is a fundamental physiological process in plants that converts light energy into biological energy to maintain growth and development [26]. Mou et al. [36] found that ABA inhibited the expression of most photosynthesis-related genes in the process of tomato fruit ripening, indicating that ABA may modulate fruit ripening through its influence on photosynthesis. Given that SlAREB1 is an important response factor downstream of the ABA signaling pathway, it is plausible that SlAREB1 regulates photosynthesis during tomato fruit ripening. In this study, the target sequences of SlAREB1 were mainly focused on the oxidative phosphorylation and photosynthesis pathways. Notably, studies on postharvest photosynthesis of fruit are limited; therefore, our findings provide a valuable insight into the intricate interplay between photosynthesis and fruit ripening after harvest.
Oxidative phosphorylation is a complex metabolic process that occurs in mitochondria, involving the utilization of energy generated through the oxidation of sugars, lipids, and amino acids to produce adenosine triphosphate (ATP), by facilitating the combination of adenosine diphosphate (ADP) and inorganic phosphate [37], and represents a major pathway for ATP generation in plant cells [37,38]. Wang et al. [39] found that oxidative phosphorylation showed a downward trend during the red-coloring process of strawberry fruit, and the downregulation of the key genes involved in oxidative phosphorylation through Virus-Induced Gene Silencing (VIGS) inhibited respiration and ATP biosynthesis, while promoting the accumulation of sugar, ABA, ethylene, and polyamines (PA), ultimately accelerating strawberry ripening. The oxidative phosphorylation pathway, which includes the electron transport chain and phosphorylation [40], was shown to be linked to the generation of reactive oxygen species (ROS). Additionally, some enzymatic reactions involved in the biosynthesis of antioxidant compounds require energy generated from ATP decomposition [38,41]. Under unfavorable environmental conditions, plants may experience excessive accumulation of ROS, leading to oxidative stress and potential diseases or cytotoxicity from abiotic stress [42,43]. The activity of antioxidant enzymes is crucial in determining a plant’s ability to scavenge ROS. The antioxidant enzymes in plants include superoxide dismutase (SOD), catalase (CAT) [44], peroxidase (POD), polyphenol oxidase (PPO), etc. [13,45]. Glutaredoxin, a small redox protein, was also involved in the ROS scavenging pathways [46]. In the present study, three antioxidant-related genes were identified, namely the glutaredoxin family protein, peroxidase, and laccase. Laccase is a copper-containing polyphenol oxidase that also plays a role in the plant oxidation process [47]. Therefore, SlAREB1 may potentially affect the antioxidant activity of tomato fruit by interacting with these antioxidant enzymes, which influences the ROS scavenging pathways. These findings suggested that oxidative phosphorylation may play a role during tomato fruit ripening, and SlAREB1 could potentially impact tomato fruit ripening by modulating the oxidative phosphorylation pathway.
Fruit ripening is accompanied by various changes in taste (sweetness and acidity), texture (softening and firmness), and appearance (color) [48]; fruit color is one of the most important quality attributes of tomatoes that are favored by consumers and is primarily determined by pigments [49,50]. In this study, 18 pigment-related genes were identified to be SlAREB1 target genes, suggesting that SlAREB1 may potentially interact with these pigment genes to influence tomato fruit color. Furthermore, Wu et al. [51] found that ABA and ethylene synergistically regulated the accumulation of tomato fruit pigments, thereby influencing fruit color development. Since SlAREB1 played a pivotal role in the expression of ABA downstream genes, it is conceivable that it could affect these pigment-related genes, thus contributing to the modulation of tomato fruit color.
Fruit development and ripening are complex processes involved in the inter-play of multiple phytohormones, which can influence fruit quality, nutrition, and taste [52]. Mou et al. [36,53] found that exogenous ABA promoted ethylene biosynthesis and signal transduction by regulating multiple genes in ethylene synthesis and signaling pathways, thereby promoting tomato fruit ripening. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase [54] and ACC oxidase (ACO) [55] are two key enzymes in ethylene synthesis [56,57]. ACO5, a member of the ACO family, also plays a role in ethylene synthesis [58]. Ethylene response factors (AP2/ERFs) are the response factors in the ethylene signaling pathway, and they can also feedback-regulate the biosynthesis of other plant hormones such as cytokinin, gibberellin, and abscisic acid and are involved in signaling responses to hormones such as auxin, cell division, abscisic acid, and jasmonic acid [59]. In this study, the ACO5 gene and ERF genes were identified as the SlAREB1 target genes, indicating that SlAREB1 may influence ethylene biosynthesis and signaling by interacting with the ACO5 gene and ERF genes, thereby regulating tomato fruit ripening. GA-20 oxidase (Gibberellin 20-oxidase) is the key rate-limiting enzyme for gibberellin (GA) biosynthesis, and its synergistic effect is necessary for the growth of tomato fruit [60,61]. Chen et al. [62] found that exogenous gibberellic acid treatment can delay the maturation period of tomato fruit by regulating the transcriptional levels of ethylene-related genes. The auxin response factor (ARF) plays an important role in plant growth and development [63]. Brassinosteroids (BR) is a class of hormones that played an important role in plant growth and development [64]. Zhu et al. [65] found that BR positively regulated tomato fruit ripening and promoted ethylene synthesis. The brassinosteroid hydroxylase may affect the synthesis of brassinosteroid [66]. In this study, a GA-20 oxidase, an auxin response factor-ARF6, and a brassinosteroid hydroxylase were identified among the target genes of SlAREB1, but the direct impact of ARF6 on tomato fruit ripening was not demonstrated, indicating that SlAREB1 may regulate tomato fruit ripening by influencing the biosynthesis of ethylene, gibberellin, and brassinosteroids. Gibberellin and brassinosteroids potentially mediate ethylene synthesis. Further studies are needed to elucidate the exact mechanisms and interactions related to phytohormone biosynthesis and signaling involved in regulating fruit ripening by SlAREB1 and its target genes.
Fruit firmness is one of the important characteristics of tomato fruit ripening, and the metabolism of the cell wall plays a crucial role in determining the change rate of fruit firmness during ripening [67]. Several enzymes, including endogenous glucanase [68], polygalacturonase (PG) [69,70], expansin [71], pectin methylesterase [72], and PL [73], were shown to be involved in cell wall metabolism and fruit softening [74]. In addition, Zeng et al. [67] found that ABA treatment can accelerate fruit softening and promote the gene expression of β-galactosidase, SlTBG3, and SlTBG4. In this study, a total of six cell-wall-related genes were identified, including β-galactosidase and pectinesterase., indicating that SlAREB1 may regulate tomato fruit softening by modulating the expression of cell-wall-related genes, including β-galactosidase and pectinesterase, in response to ABA or other signaling pathways.
Based on the results mentioned above, a regulation model of SlAREB1 on tomato fruit ripening was drawn (Figure 6). According to the model, SlAREB1 may primarily regulate genes related to ethylene synthesis and signal transduction, gibberellin synthesis, brassinosteroid synthesis, oxidative phosphorylation, photosynthesis, antioxidants, pigment, and the cell wall, all of which play important roles during tomato fruit ripening. However, the regulatory model of SlAREB1 on tomato fruit ripening is limited as it was only determined by ChIP-Seq experiments on SlAREB1-overexpression transgenic tomatoes. Further experiments are needed to verify its interaction with related genes to clarify the regulatory model of SlAREB1 on tomato fruit ripening. In addition, it is worth noting that many of the target genes of SlAREB1 in the model have not been extensively studied, so further research is needed to fully validate and refine the proposed model. Additional experimental evidence and functional studies are necessary to elucidate the precise mechanisms by which SlAREB1 regulates these target genes and their roles in tomato fruit ripening. Further verification and improvement of the regulatory model will contribute to a more comprehensive understanding of the regulatory network governing tomato fruit ripening and the role of SlAREB1 in this process.

5. Conclusions

In this study, the expression level of SlAREB1 was examined in different stages of tomato fruit ripening, and the influence of ABA on its expression was confirmed, further supporting its role in tomato fruit ripening and its regulation by ABA. Subsequent analysis using ChIP-Seq technology identified the target genes of SlAREB1, which were found to be mainly distributed in the oxidative phosphorylation pathway and the photosynthesis pathway. Moreover, SlAREB1 was found to be involved in the regulation of tomato fruit ethylene synthesis and signal transduction, gibberellin synthesis, brassinosteroid synthesis, the cell wall, pigment, and antioxidant defense. These results provide valuable references and a theoretical basis for further investigations into the mechanism of SlAREB1 and ABA in tomato fruit ripening.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/foods12122357/s1. Table S1: Primer sequences used in this study. Table S2: Peak information statistics. Table S3: Transcription factor. Table S4: Target genes of SlAREB1. Figure S1. PCR Identification of SlAREB1 transgenic tomato plants. 1–3 were the control tomato plants, 4–5 were the transgenic tomato plants.

Author Contributions

Conceptualization, Q.W. and Y.Z.; methodology, Y.H. and Q.W.; formal analysis, Y.H., Q.W. and Q.T.; investigation, Y.H. and C.C.; resources, Q.W., Y.Z. and D.Z.; writing—original draft preparation, Y.H. and Q.W.; writing—review and editing, Y.H. and Q.W.; funding acquisition, Q.W., Y.Z. and D.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by grants from the Youth Program of the National Natural Science Foundation of China (32001753) and the High-Level Talents Research Start-Up Fund of Henan University of Technology (2020BS006).

Data Availability Statement

Sequence data are listed in this article, and some public data are noted in Supplementary Tables S1–S4.

Acknowledgments

We would like to thank Li Li at Zhejiang University for checking the grammar and correcting the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kou, X.; Zhou, J.; Wu, C.E.; Yang, S.; Liu, Y.; Chai, L.; Xue, Z. The interplay between ABA/ethylene and NAC TFs in tomato fruit ripening: A review. Plant Mol. Biol. 2021, 106, 223–238. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, Y.; Wang, W.; Cai, J.; Zhang, Y.; Qin, G.; Tian, S. Tomato nuclear proteome reveals the involvement of specific E2 ubiquitin-conjugating enzymes in fruit ripening. Genome Biol. 2014, 15, 548. [Google Scholar] [CrossRef] [PubMed]
  3. Chen, Y.; Grimplet, J.; David, K.; Castellarin, S.D.; Terol, J.; Wong, D.C.J.; Luo, Z.; Schaffer, R.; Celton, J.-M.; Talon, M.; et al. Ethylene receptors and related proteins in climacteric and non-climacteric fruits. Plant Sci. 2018, 276, 63–72. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Wang, J.; Tian, S.; Yu, Y.; Ren, Y.; Guo, S.; Zhang, J.; Li, M.; Zhang, H.; Gong, G.; Wang, M.; et al. Natural variation in the NAC transcription factor NONRIPENING contributes to melon fruit ripening. J. Integr. Plant. Biol. 2022, 64, 1448–1461. [Google Scholar] [CrossRef]
  5. Li, J.; Min, D.; Li, Z.; Fu, X.; Zhao, X.; Wang, J.; Zhang, X.; Li, F.; Li, X.; Guo, Y. Regulation of Sugar Metabolism by Methyl Jasmonate to Improve the Postharvest Quality of Tomato Fruit. J. Plant Growth Regul. 2022, 41, 1615–1626. [Google Scholar] [CrossRef]
  6. Tao, X.; Wu, Q.; Fu, X.; Zhu, B.; Chen, F.; Liu, B.; Mao, L.; Luo, Z.; Li, L.; Ying, T. Understanding of exogenous auxin in regulating sucrose metabolism during postharvest tomato fruit ripening. Postharvest Biol. Technol. 2022, 189, 111913. [Google Scholar] [CrossRef]
  7. Vitale, A.; Rocco, M.; Arena, S.; Giuffrida, F.; Cassaniti, C.; Scaloni, A.; Lomaglio, T.; Guarnaccia, V.; Polizzi, G.; Marra, M.; et al. Tomato susceptibility to Fusarium crown and root rot: Effect of grafting combination and proteomic analysis of tolerance expression in the rootstock. Plant Physiol. Biochem. 2014, 83, 207–216. [Google Scholar] [CrossRef] [Green Version]
  8. Wang, D.; Yeats, T.H.; Uluisik, S.; Rose, J.K.C.; Seymour, G.B. Fruit Softening: Revisiting the Role of Pectin. Trends Plant Sci. 2018, 23, 302–310. [Google Scholar] [CrossRef]
  9. Galpaz, N.; Wang, Q.; Menda, N.; Zamir, D.; Hirschberg, J. Abscisic acid deficiency in the tomato mutant high-pigment 3 leading to increased plastid number and higher fruit lycopene content. Plant J. 2008, 53, 717–730. [Google Scholar] [CrossRef]
  10. Zhang, M.; Yuan, B.; Leng, P. The role of ABA in triggering ethylene biosynthesis and ripening of tomato fruit. J. Exp. Bot. 2009, 60, 1579–1588. [Google Scholar] [CrossRef] [Green Version]
  11. Tao, X.; Wu, Q.; Huang, S.; Zhu, B.; Chen, F.; Liu, B.; Cai, L.; Mao, L.; Luo, Z.; Li, L.; et al. Exogenous abscisic acid regulates primary metabolism in postharvest cherry tomato fruit during ripening. Sci. Hortic. 2022, 299, 111008. [Google Scholar] [CrossRef]
  12. Wu, Q.; Tao, X.; Ai, X.; Luo, Z.; Mao, L.; Ying, T.; Li, L. Contribution of abscisic acid to aromatic volatiles in cherry tomato (Solanum lycopersicum L.) fruit during postharvest ripening. Plant Physiol. Biochem. 2018, 130, 205–214. [Google Scholar] [CrossRef]
  13. Tao, X.; Wu, Q.; Aalim, H.; Li, L.; Mao, L.; Luo, Z.; Ying, T. Effects of Exogenous Abscisic Acid on Bioactive Components and Antioxidant Capacity of Postharvest Tomato during Ripening. Molecules 2020, 25, 1346. [Google Scholar] [CrossRef] [Green Version]
  14. Wang, W.; Wang, X.; Wang, Y.; Zhou, G.; Wang, C.; Hussain, S.; Adnan; Lin, R.; Wang, T.; Wang, S. SlEAD1, an EAR motif-containing ABA down-regulated novel transcription repressor regulates ABA response in tomato. GM Crops Food 2020, 11, 275–289. [Google Scholar] [CrossRef] [PubMed]
  15. Leng, P.; Yuan, B.; Guo, Y. The role of abscisic acid in fruit ripening and responses to abiotic stress. J. Exp. Bot. 2014, 65, 4577–4588. [Google Scholar] [CrossRef] [PubMed]
  16. Droege-Laser, W.; Snoek, B.L.; Snel, B.; Weiste, C. The Arabidopsis bZIP transcription factor family—An update. Curr. Opin. Plant Biol. 2018, 45, 36–49. [Google Scholar] [CrossRef] [PubMed]
  17. Bastias, A.; Lopez-Climent, M.; Valcarcel, M.; Rosello, S.; Gomez-Cadenas, A.; Casaretto, J.A. Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor. Physiol. Plant. 2011, 141, 215–226. [Google Scholar] [CrossRef] [PubMed]
  18. Orellana, S.; Yanez, M.; Espinoza, A.; Verdugo, I.; Gonzalez, E.; Ruiz-Lara, S.; Casaretto, J.A. The transcription factor SlAREB1 confers drought, salt stress tolerance and regulates biotic and abiotic stress-related genes in tomato. Plant Cell. Environ. 2010, 33, 2191–2208. [Google Scholar] [CrossRef]
  19. Yanez, M.; Caceres, S.; Orellana, S.; Bastias, A.; Verdugo, I.; Ruiz-Lara, S.; Casaretto, J.A. An abiotic stress-responsive bZIP transcription factor from wild and cultivated tomatoes regulates stress-related genes. Plant Cell. Rep. 2009, 28, 1497–1507. [Google Scholar] [CrossRef]
  20. Xu, Z.; Wang, F.; Ma, Y.; Dang, H.; Hu, X. Transcription Factor SlAREB1 Is Involved in the Antioxidant Regulation under Saline-Alkaline Stress in Tomato. Antioxidants 2022, 11, 1673. [Google Scholar] [CrossRef]
  21. Bastias, A.; Yanez, M.; Osorio, S.; Arbona, V.; Gomez-Cadenas, A.; Fernie, A.R.; Casaretto, J.A. The transcription factor AREB1 regulates primary metabolic pathways in tomato fruits. J. Exp. Bot. 2014, 65, 2351–2363. [Google Scholar] [CrossRef] [Green Version]
  22. Yang, S.; Zhou, J.; Watkins, C.B.; Wu, C.; Feng, Y.; Zhao, X.; Xue, Z.; Kou, X. NAC transcription factors SNAC4 and SNAC9 synergistically regulate tomato fruit ripening by affecting expression of genes involved in ethylene and abscisic acid metabolism and signal transduction. Postharvest Biol. Technol. 2021, 178, 111555. [Google Scholar] [CrossRef]
  23. Mou, W.; Li, D.; Luo, Z.; Li, L.; Mao, L.; Ying, T. SlAREB1 transcriptional activation of NOR is involved in abscisic acid-modulated ethylene biosynthesis during tomato fruit ripening. Plant Sci. 2018, 276, 239–249. [Google Scholar] [CrossRef] [PubMed]
  24. Small, E.C.; Maryanski, D.N.; Rodriguez, K.L.; Harvey, K.J.; Keogh, M.-C.; Johnstone, A.L. Chromatin Immunoprecipitation (ChIP) to Study DNA-Protein Interactions. Methods Mol. Biol. 2021, 2261, 323–343. [Google Scholar]
  25. GAO, S.; ZHANG, N.; LI, B. Processing and analysis of ChIP-seq data. Hereditas 2012, 34, 773–783. [Google Scholar] [CrossRef]
  26. Liu, X.; Su, L.; Li, L. Downstream target gene network regulated by AhGLK1 and AhHDA1 using ChIP-seq in peanut. Zuo Wu Xue Bao 2022, 48, 2765–2773. [Google Scholar]
  27. Wang, T.; Shen, L.; Wei, M. ChIP-seq Analysis of the Impact of Mutant IHH-GLI1 Signaling Pathway on Downstream Transcriptional Regulation. J. Nat. Sci. Hunan Norm. Univ. 2020, 43, 27–35. [Google Scholar]
  28. Zhu, L.; Xue, P.; Li, G. Genome-wide Binding Site Analysis of LAZY1 Reveals Its Downstream Regulating Network in Controlling Rice Tiller Angle. Genom. Appl. Biol. 2022, 41, 1539–1549. [Google Scholar]
  29. Zhao, T.; Wu, T.; Pei, T.; Wang, Z.; Yang, H.; Jiang, J.; Zhang, H.; Chen, X.; Li, J.; Xu, X. Overexpression of SlGATA17 Promotes Drought Tolerance in Transgenic Tomato Plants by Enhancing Activation of the Phenylpropanoid Biosynthetic Pathway. Front. Plant Sci. 2021, 12, 634888. [Google Scholar] [CrossRef]
  30. Yang, B.; Liu, C.; Pan, X.; Fu, W.; Fan, Z.; Jin, Y.; Bai, F.; Cheng, Z.; Wu, W. Identification of Novel phoP-phoQ Regulated Genes that Contribute to Polymyxin B Tolerance in Pseudomonas aeruginosa. Microorganisms 2021, 9, 344. [Google Scholar] [CrossRef]
  31. Kuang, J.-F.; Wu, C.-J.; Guo, Y.-F.; Walther, D.; Shan, W.; Chen, J.-Y.; Chen, L.; Lu, W.-J. Deciphering transcriptional regulators of banana fruit ripening by regulatory network analysis. Plant Biotechnol. J. 2021, 19, 477–489. [Google Scholar] [CrossRef]
  32. Joshi, R.; Wani, S.H.; Singh, B.; Bohra, A.; Dar, Z.A.; Lone, A.A.; Pareek, A.; Singla-Pareek, S.L. Transcription Factors and Plants Response to Drought Stress: Current Un-derstanding and Future Directions. Front. Plant Sci. 2016, 7, 1029. [Google Scholar]
  33. Hu, J.; Gao, Y.; Zhao, T.; Li, J.; Yao, M.; Xu, X. Genome-wide Identification and Expression Pattern Analysis of Zinc-finger Homeodomain Transcription Factors in Tomato under Abiotic Stress. J. Am. Soc. Hortic. Sci. 2018, 143, 14–22. [Google Scholar] [CrossRef] [Green Version]
  34. Chen, Y.; Deng, J.; Chen, J.; Zeng, T.; Yu, T.; Huang, Q.; Chen, P.; Liu, Q.; Jian, W.; Yang, X. Genome-wide identification and expression analysis of FAR1/FHY3 transcription factor family in tomato. Plant Physiol. 2021, 57, 1983–1995. [Google Scholar]
  35. Wang, Y.; Zhang, J.; Hu, Z.; Guo, X.; Tian, S.; Chen, G. Genome-Wide Analysis of the MADS-Box Transcription Factor Family in Solanum lycopersicum. Int. J. Mol. Sci. 2019, 20, 2961. [Google Scholar] [CrossRef] [Green Version]
  36. Mou, W.; Li, D.; Luo, Z.; Mao, L.; Ying, T. Transcriptomic Analysis Reveals Possible Influences of ABA on Secondary Metabolism of Pigments, Flavonoids and Antioxidants in Tomato Fruit during Ripening. PLoS ONE 2015, 10, e0129598. [Google Scholar] [CrossRef] [PubMed]
  37. Kadenbach, B. Intrinsic and extrinsic uncoupling of oxidative phosphorylation. Biochim. Biophys. Acta Biomembr. 2003, 1604, 77–94. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  38. Piechowiak, T.; Sowa, P.; Tarapatskyy, M.; Balawejder, M. The Role of Mitochondrial Energy Metabolism in Shaping the Quality of Highbush Blueberry Fruit During Storage in Ozone-Enriched Atmosphere. Food Bioprocess Technol. 2021, 14, 1973–1982. [Google Scholar] [CrossRef]
  39. Wang, Q.-H.; Zhao, C.; Zhang, M.; Li, Y.-Z.; Shen, Y.-Y.; Guo, J.-X. Transcriptome analysis around the onset of strawberry fruit ripening uncovers an important role of oxidative phosphorylation in ripening. Sci. Rep. 2017, 7, 41477. [Google Scholar] [CrossRef] [Green Version]
  40. Liu, L.M.; Li, Y.; Du, G.C.; Chen, J. Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation. J. Appl. Microbiol. 2006, 100, 1043–1053. [Google Scholar] [CrossRef]
  41. Nolfi-Donegan, D.; Braganza, A.; Shiva, S. Mitochondrial electron transport chain: Oxidative phosphorylation, oxidant production, and methods of measurement. Redox. Biol. 2020, 37, 101674. [Google Scholar] [CrossRef]
  42. Tian, S.; Qin, G.; Li, B. Reactive oxygen species involved in regulating fruit senescence and fungal pathogenicity. Plant Mol. Biol. 2013, 82, 593–602. [Google Scholar] [CrossRef]
  43. Wang, Y.; Ji, D.; Chen, T.; Li, B.; Zhang, Z.; Qin, G.; Tian, S. Production, Signaling, and Scavenging Mechanisms of Reactive Oxygen Species in Fruit-Pathogen Interactions. Int. J. Mol. Sci. 2019, 20, 2994. [Google Scholar] [CrossRef] [Green Version]
  44. Merchante, C.; Alonso, J.M.; Stepanova, A.N. Ethylene signaling: Simple ligand, complex regulation. Curr. Opin. Plant Biol. 2013, 16, 554–560. [Google Scholar] [CrossRef]
  45. Zhao, W.; Ma, Z.; Liu, S.; Yang, W.; Ma, J. Transcriptome Profiling Reveals Potential Genes and Pathways Supporting Ananas comosus L. Merr’s High. Temperature Stress Tolerance. Trop. Plant Biol. 2021, 14, 132–142. [Google Scholar] [CrossRef]
  46. Zhang, S.; Wang, S.; Han, S.; Wang, D. The Research Progress of Glutaredoxin in Plants. Yuan Yi Xue Bao 2022, 34, 1135–1144. [Google Scholar]
  47. Bai, Y.; Zhou, J.-J.; Tang, Y.-L. Plant laccases and their roles in plant growth and development. Chin. Bull. Life Sci. 2022, 34, 1135–1144. [Google Scholar]
  48. Wu, S.; Wu, D.; Song, J.; Zhang, Y.; Tan, Q.; Yang, T.; Yang, J.; Wang, S.; Xu, J.; Xu, W.; et al. Metabolomic and transcriptomic analyses reveal new insights into the role of abscisic acid in modulating mango fruit ripening. Hort. Res. 2022, 9, 1136. [Google Scholar] [CrossRef] [PubMed]
  49. Kang, S.-I.; Hwang, I.; Goswami, G.; Jung, H.-J.; Nath, U.K.; Yoo, H.-J.; Lee, J.M.; Nou, I.S. Molecular Insights Reveal Psy1, SGR, and SlMYB12 Genes are Associated with Diverse Fruit Color Pigments in Tomato (Solanum lycopersicum L.). Molecules 2017, 22, 2180. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  50. Ranganath, K.G. Pigments That Colour Our Fruits: An Overview. Erwerbs-Obstbau 2022, 64, 535–547. [Google Scholar] [CrossRef]
  51. Wu, Q.; Bai, J.; Tao, X.; Mou, W.; Luo, Z.; Mao, L.; Ban, Z.; Ying, T.; Li, L. Synergistic effect of abscisic acid and ethylene on color development in tomato (Solarium lycopersicum L.) fruit. Sci. Hortic. 2018, 235, 169–180. [Google Scholar] [CrossRef]
  52. Batista-Silva, W.; Carvalho de Oliveira, A.; Oliveira Martins, A.; Siqueira, J.A.; Rodrigues-Salvador, A.; Omena-Garcia, R.P.; Medeiros, D.B.; Peres, L.E.P.; Ribeiro, D.M.; Zsogon, A.; et al. Reduced auxin signalling through the cyclophilin gene DIAGEOTROPICA impacts tomato fruit development and metabolism during ripening. J. Exp. Bot. 2022, 73, 4113–4128. [Google Scholar] [CrossRef]
  53. Mou, W.; Li, D.; Bu, J.; Jiang, Y.; Khan, Z.U.; Luo, Z.; Mao, L.; Ying, T. Comprehensive Analysis of ABA Effects on Ethylene Biosynthesis and Signaling during Tomato Fruit Ripening. PLoS ONE 2016, 11, e0154072. [Google Scholar]
  54. Isaacson, T.; Ronen, G.; Zamir, D.; Hirschberg, J. Cloning of tangerine from tomato reveals a carotenoid isomerase essential for the production of beta-carotene and xanthophylls in plants. Plant Cell 2002, 14, 333–342. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Kou, X.; Zhao, Y.; Wu, C.; Jiang, B.; Zhang, Z.; Rathbun, J.R.; He, Y.; Xue, Z. SNAC4 and SNAC9 transcription factors show contrasting effects on tomato carotenoids biosynthesis and softening. Postharvest Biol. Technol. 2018, 144, 9–19. [Google Scholar] [CrossRef]
  56. Chen, H.; Bai, S.; Kusano, M.; Ezura, H.; Wang, N. Increased ACS Enzyme Dosage Causes Initiation of Climacteric Ethylene Production in Tomato. Int. J. Mol. Sci. 2022, 23, 10788. [Google Scholar] [CrossRef]
  57. Yu, W.; Sheng, J.; Zhao, R.; Wang, Q.; Ma, P.; Shen, L. Ethylene biosynthesis is involved in regulating chilling tolerance and SlCBF1 gene expression in tomato fruit. Postharvest Biol. Technol. 2019, 149, 139–147. [Google Scholar] [CrossRef]
  58. Sell, S.; Hehl, R. A fifth member of the tomato 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene family harbours a leucine zipper and is anaerobically induced. DNA Seq. 2005, 16, 80–82. [Google Scholar] [CrossRef] [PubMed]
  59. Gu, C.; Guo, Z.H.; Hao, P.P.; Wang, G.M.; Jin, Z.M.; Zhang, S.L. Multiple regulatory roles of AP2/ERF transcription factor in angiosperm. Bot. Stud. 2017, 58, 10788. [Google Scholar] [CrossRef] [Green Version]
  60. Mignolli, F.; Mariotti, L.; Laura Vidoz, M. Reduced gibberellin biosynthesis and response in fruits of the auxin insensitive diageotropica tomato mutant. Plant Growth Regul. 2022, 99, 505–513. [Google Scholar] [CrossRef]
  61. Wu, J.-M.; Chen, R.-F.; Huang, X.; Qiu, L.-H.; Li, Y.-R. Studies on the Gene of Key Component GA20-oxidase for Gibberellin Biosynthesis in Plant. Biol. Bull. 2016, 32, 1–12. [Google Scholar]
  62. Chen, S.; Wang, X.-J.; Tan, G.-F.; Zhou, W.-Q.; Wang, G.-L. Gibberellin and the plant growth retardant Paclobutrazol altered fruit shape and ripening in tomato. Protoplasma 2020, 257, 853–861. [Google Scholar] [CrossRef] [PubMed]
  63. Li, Y.; Han, S.; Qi, Y. Advances in structure and function of auxin response factor in plants. J. Integr. Plant Biol. 2023, 65, 617–632. [Google Scholar] [CrossRef]
  64. Zhu, Z.; Liang, H.; Chen, G.; Tang, B.; Tian, S.; Hu, Z. Isolation of the brassinosteroid receptor genes and recharacterization of dwarf plants by silencing of SlBRI1 in tomato. Plant Growth Regul. 2019, 89, 59–71. [Google Scholar] [CrossRef]
  65. Zhu, T.; Tan, W.-R.; Deng, X.-G.; Zheng, T.; Zhang, D.-W.; Lin, H.-H. Effects of brassinosteroids on quality attributes and ethylene synthesis in postharvest tomato fruit. Postharvest Biol. Technol. 2015, 100, 196–204. [Google Scholar] [CrossRef]
  66. Zhao, B.; Li, J. Regulation of Brassinosteroid Biosynthesis and Inactivation. J. Integr. Plant Biol. 2012, 54, 746–759. [Google Scholar] [CrossRef]
  67. Zeng, W.-J.; Yang, J.; Chen, S.-M.-J.; Wei, X.-Y.; Wen, G.-Q.; He, Z.-Y.; Wang, Q.; Chen, X.-T.; Zhou, J.-Y.; Zou, J. Expression of Cell Wall Metabolism Genes During Tomato (Solanum lycopersicum) Maturation and the Effects of Treatment with Exogenous C2H4 and ABA on Their Expression. J. Biotechnol. 2021, 29, 1040–1049. [Google Scholar]
  68. He, Y.; Li, J.; Ban, Q.; Han, S.; Rao, J. Role of Brassinosteroids in Persimmon (Diospyros kaki L.) Fruit Ripening. J. Agric. Food Chem. 2018, 66, 2637–2644. [Google Scholar] [CrossRef] [PubMed]
  69. Jiang, F.; Lopez, A.; Jeon, S.; de Freitas, S.T.; Yu, Q.; Wu, Z.; Labavitch, J.M.; Tian, S.; Powell, A.L.T.; Mitcham, E. Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking. Hort. Res. 2019, 6, 17. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  70. Yang, Y.; Lu, L.; Sun, D.; Wang, J.; Wang, N.; Qiao, L.; Guo, Q.; Wang, C. Fungus Polygalacturonase-Generated Oligogalacturonide Restrains Fruit Softening in Ripening Tomato. J. Agric. Food Chem. 2022, 70, 759–769. [Google Scholar] [CrossRef]
  71. Perini, M.A.; Sin, I.N.; Villarreal, N.M.; Marina, M.; Powell, A.L.T.; Martinez, G.A.; Civello, P.M. Overexpression of the carbohydrate binding module from Solanum lycopersicum expansin 1 (Sl-EXP1) modifies tomato fruit firmness and Botrytis cinerea susceptibility. Plant Physiol. Biochem. 2017, 113, 122–132. [Google Scholar] [CrossRef] [PubMed]
  72. Micheli, F. Pectin methylesterases: Cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 2001, 6, 414–419. [Google Scholar] [CrossRef] [PubMed]
  73. Shi, L.; Liu, Q.; Qiao, Q.; Zhu, Y.; Huang, W.; Wang, X.; Ren, Z. Exploring the effects of pectate and pectate lyase on the fruit softening and transcription profiling of Solanum lycopersicum. Food Control 2022, 133, 108636. [Google Scholar] [CrossRef]
  74. Ao, Y.; Yang, M.-Y.; Zhang, C.; Wu, Q. Research Progress on the Mechanisms of Cell Wall Actions during Ripening and Softening Processes of Tomato Fruits. Storage Process 2021, 21, 118–125. [Google Scholar]
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Figure 1. Expression of SlAREB1 during tomato fruit ripening (A) and the effect of exogenous ABA treatment on it (B). Error bars represent the standard error (SE) of three biological replicates. In (A), the gene expression of SlAREB1 at the immature green stage is normalized to one, and the asterisk (*) indicates the expression level of the gene in other stage is significantly different from the expression level at IMG1 (p < 0.05); in (B), the expression of SlAREB1 in the control group on day 1 is normalized to one, and the asterisk (*) indicates the expression level of the gene in the ABA treatment group is significantly different from that in the control group at the same time (p < 0.05).
Figure 1. Expression of SlAREB1 during tomato fruit ripening (A) and the effect of exogenous ABA treatment on it (B). Error bars represent the standard error (SE) of three biological replicates. In (A), the gene expression of SlAREB1 at the immature green stage is normalized to one, and the asterisk (*) indicates the expression level of the gene in other stage is significantly different from the expression level at IMG1 (p < 0.05); in (B), the expression of SlAREB1 in the control group on day 1 is normalized to one, and the asterisk (*) indicates the expression level of the gene in the ABA treatment group is significantly different from that in the control group at the same time (p < 0.05).
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Figure 2. Construction of SlAREB1-overexpression transgenic tomato plants. (A) Phenotypes between wild-type and SlAREB1-overexpression transgenic tomato fruit; (B) PCR products’ identification of SlAREB1-overexpression transgenic tomato fruit at mature green stage; 1–3 are the control tomato plants, and 4–6 are the transgenic tomato plants; (C) qRT-PCR validation of SlAREB1 overexpression in transgenic tomato fruit. The samples of wild-type tomato fruit at the mature green stage are normalized to one, and the asterisk (*) indicates the SlAREB1 expression level between the transgenic and wild-type tomato fruit at the mature green stage is significantly different at the significance level of p < 0.05.
Figure 2. Construction of SlAREB1-overexpression transgenic tomato plants. (A) Phenotypes between wild-type and SlAREB1-overexpression transgenic tomato fruit; (B) PCR products’ identification of SlAREB1-overexpression transgenic tomato fruit at mature green stage; 1–3 are the control tomato plants, and 4–6 are the transgenic tomato plants; (C) qRT-PCR validation of SlAREB1 overexpression in transgenic tomato fruit. The samples of wild-type tomato fruit at the mature green stage are normalized to one, and the asterisk (*) indicates the SlAREB1 expression level between the transgenic and wild-type tomato fruit at the mature green stage is significantly different at the significance level of p < 0.05.
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Figure 3. Peak distribution of SlAREB1 enriched genes. (A) Distribution of peaks on the genome. (B) Distribution of peaks on gene functional elements.
Figure 3. Peak distribution of SlAREB1 enriched genes. (A) Distribution of peaks on the genome. (B) Distribution of peaks on gene functional elements.
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Figure 4. Transcription factor prediction of peak-associated genes.
Figure 4. Transcription factor prediction of peak-associated genes.
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Figure 5. GO (A) and KEGG (B) analysis of SlAREB1 target sequences. (A) Red represents molecular functions (MF); green represents cellular components (CC); blue represents biological processes (BP).
Figure 5. GO (A) and KEGG (B) analysis of SlAREB1 target sequences. (A) Red represents molecular functions (MF); green represents cellular components (CC); blue represents biological processes (BP).
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Figure 6. SlAREB1 regulates tomato fruit ripening model. Different colors represent different anabolic pathways.
Figure 6. SlAREB1 regulates tomato fruit ripening model. Different colors represent different anabolic pathways.
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Table
Table 1. Statistical analysis of raw data.
Table 1. Statistical analysis of raw data.
SampleRaw ReadsClean ReadsClean RatioMapped ReadsMap Rate
SlAREB1-IP52,720,46651,618,54497.91%38,834,54575.23%
Input36,263,53435,588,84098.14%34,927,95198.14%
Raw reads: the number of original sequencing reads; clean reads: the number of reads obtained by filtering raw reads; mapped reads: the total number of reads on the alignment; mapped rate: the proportion of the total number of reads on the alignment.
Table
Table 2. Target genes of SlAREB1 (partial).
Table 2. Target genes of SlAREB1 (partial).
Gene IDSubject LengthSubject StartSubject EndSubject Annotation
Oxidative phosphorylation
Solyc00g013180.139019,741,9229,745,822NADH-ubiquinone oxidoreductase chain 4
Solyc00g014830.3224110,120,36710,122,607NADH dehydrogenase subunit 7
Solyc00g019730.2111210,827,17110,828,282Cytochrome c oxidase subunit 3
Solyc00g019950.1141510,844,93510,846,349NADH dehydrogenase subunit 9
Solyc00g117655.119515,444,46715,444,661NADH-ubiquinone oxidoreductase chain 1
Solyc01g020470.219930,837,74330,837,941NADH dehydrogenase subunit 9
Solyc01g056670.149355,571,58155,572,073NADH dehydrogenase subunit 4L
Solyc03g013460.124745,900,76245,901,008Cytochrome c oxidase subunit 3
Solyc03g043610.21467,121,3827,121,527ATP synthase subunit a
Solyc05g016220.113815,092,14915,092,286Ycf1
Solyc05g023920.131830,101,77330,102,090NADH-ubiquinone oxidoreductase chain 1
Solyc07g019510.333811,845,80411,846,141Cytochrome c oxidase subunit 1
Solyc08g029260.1109637,265,40837,266,503NADH dehydrogenase subunit 2
Solyc10g045750.121235,863,08835,863,299NADH-ubiquinone oxidoreductase chain 4
Solyc10g049470.115745,831,44145,831,597Ycf1
Solyc11g021240.214913,415,63913,415,787Ycf1
Solyc11g021300.115413,419,57713,419,730Ycf1
Solyc11g030570.133922,056,50222,056,840NADH-ubiquinone oxidoreductase chain 4
Solyc12g035550.133041,911,35041,911,679Ycf1
Solyc12g035930.115344,564,59844,564,750DNA-directed RNA polymerase subunit beta
Photosynthesis
Solyc00g230070.1241318,659,72818,662,140Photosystem II CP43 chlorophyll apoprotein
Solyc01g017090.324723,795,39023,795,636NADH-quinone oxidoreductase subunit L
Solyc01g017440.114323,869,85123,869,993DNA-directed RNA polymerase subunit alpha
Solyc01g017740.114625,041,92625,042,071Cytochrome b6
Solyc01g056870.235657,175,86457,176,219Ycf2
Solyc02g011755.115914,135,56214,135,720Photosystem I iron-sulfur center
Solyc02g080635.129145,373,84845,374,138Photosystem II CP43 reaction center protein
Solyc03g122000.343771,495,01771,495,453Cytochrome b6-f complex subunit 4
Solyc04g049003.117438,943,03338,943,206Cytochrome c biogenesis protein CcsA
Solyc05g016220.113815,092,14915,092,286Ycf1
Solyc10g012230.11464,687,5024,687,647Ycf2
Solyc10g047410.121440,732,14740,732,360Photosystem II CP43 chlorophyll apoprotein
Solyc10g049470.115745,831,44145,831,597Ycf1
Solyc11g018700.21729,107,4189,107,589Ycf15
Solyc11g021210.115213,408,19413,408,345Cytochrome c biogenesis protein ccsA
Solyc11g021240.214913,415,63913,415,787Hypothetical chloroplast RF1
Solyc11g021300.115413,419,57713,419,730Hypothetical chloroplast RF1
Solyc12g035550.13304191135041,911,679Ycf1
Phytohormones
Solyc07g026650.319630,146,53730,146,732ACO5
Solyc09g059510.344554,883,43554,883,879ERF
Solyc00g179240.217117,318,51417,318,684MADS-box
Solyc10g045690.114335,000,92535,001,067Gibberellin 20-oxidase
Solyc12g006350.2344870,483870,826Auxin response factor 6
Solyc12g006860.21871,281,5241,281,710Brassinosteroid hydroxylase
Pigment
Solyc00g019730.2111210,827,17110,828,282Cytochrome c oxidase subunit 3
Solyc00g049210.137812,649,89512,650,272Cytochrome c-type biogenesis protein CcmF
Solyc01g017740.114625,041,92625,042,071Cytochrome b6
Solyc02g021770.118624,252,66924,252,854Cytochrome c oxidase subunit 1
Solyc03g013460.124745,900,76245,901,008Cytochrome c oxidase subunit 3
Solyc03g013390.129646,389,35446,389,649Cytochrome c oxidase subunit 3
Solyc03g122000.343771,495,01771,495,453Cytochrome b6-f complex subunit 4
Solyc05g023720.124829,081,74629,081,993Apo cytochrome f
Solyc05g025700.131435,921,16935,921,482Cytochrome c biogenesis FC
Solyc07g019510.327411,845,10911,845,382Cytochrome c oxidase subunit 1
Solyc07g032450.116739,127,89539,128,061Cytochrome b6
Solyc09g015880.317011,297,88911,298,058Cytochrome c oxidase subunit 2
Solyc09g050020.285135,489,75635,490,606Cytochrome b
Solyc11g021210.115213,408,19413,408,345Cytochrome c biogenesis protein ccsA
Solyc11g028160.143220,552,58020,553,011Cytochrome c biogenesis
Solyc11g039360.144545,467,50945,467,953Cytochrome c biogenesis FC
Solyc11g056410.227745,648,87145,649,147Cytochrome c oxidase subunit 2
Solyc11g063620.226249,936,63549,936,896Cytochrome c biogenesis FN
Cell wall
Solyc07g042220.216155,403,94855,404,108Beta-galactosidase
Solyc10g076430.115259,507,91959,508,070Pectinesterase
Solyc00g011890.31719,408,5689,408,738Galactokinase-like protein
Solyc03g071520.119119,361,0381,9361,228Galactosyltransferase family
Solyc05g025500.319232,902,29832,902,489Glucan endo-1 3-beta-glucosidase 6
Solyc07g017730.32027,823,0237,823,224Glucan endo-1 3-beta-glucosidase 5
Antioxidant
Solyc01g067460.220775,956,53675,956,742Glutaredoxin family protein
Solyc02g092580.313754,267,59154,267,727Peroxidase
Solyc06g050530.319133,316,52433,316,714Laccase
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MDPI and ACS Style

He, Y.; Wu, Q.; Cui, C.; Tian, Q.; Zhang, D.; Zhang, Y. ChIP-Seq Analysis of SlAREB1 Downstream Regulatory Network during Tomato Ripening. Foods 2023, 12, 2357. https://doi.org/10.3390/foods12122357

AMA Style

He Y, Wu Q, Cui C, Tian Q, Zhang D, Zhang Y. ChIP-Seq Analysis of SlAREB1 Downstream Regulatory Network during Tomato Ripening. Foods. 2023; 12(12):2357. https://doi.org/10.3390/foods12122357

Chicago/Turabian Style

He, Yanan, Qiong Wu, Chunxiao Cui, Qisheng Tian, Dongdong Zhang, and Yurong Zhang. 2023. "ChIP-Seq Analysis of SlAREB1 Downstream Regulatory Network during Tomato Ripening" Foods 12, no. 12: 2357. https://doi.org/10.3390/foods12122357

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