Histone deacetylase SlHDA7 impacts fruit ripening and shelf life in tomato

Abstract

Fruit ripening depends on the accurate control of ripening-related genes expression, with histone deacetylases (HDACs) playing crucial roles in transcriptional regulation. However, the functions of HDACs in fruit maturation remain largely unexplored. Here, we show that SlHDA7 acts as a suppressor of fruit ripening and functions as an H4ac HDAC in tomato. Deletion of SlHDA7 accelerated fruit ripening, while overexpression of SlHDA7 delayed the maturation process. Additionally, ethylene production and carotenoid biosynthesis significantly increased in slhda7 mutant fruits but decreased in SlHDA7-overexpressing fruits. Furthermore, SlHDA7 repress the expression of ethylene production and signaling, carotenoid metabolism, cell wall modification, and transcriptional regulation-related genes. RT-qPCR and ChIP-qPCR analyses indicated that SlHDA7 may deacetylate H4ac, leading to reduced transcript levels of ACO1, GGPPS2, Z-ISO, EXP1, and XYL1 mRNA, consequently suppressing fruit ripening. Moreover, SlHDA7 suppresses fruit ripening by targeting specific ripening-associated transcription factors (TFs) like RIN, FUL1, and ERF.E1, ultimately leading to delayed ripening and prolonged fruit shelf life. In summary, our findings indicate that SlHDA7 negatively modulates tomato fruit maturation by adjusting H4ac levels of these ripening-associated genes and key TFs.

Introduction

Fleshy fruit ripening, essential for fruit quality, is a complex genetically coordinated process. Increasing evidence indicates that fruit ripening is controlled by intricate networks that integrate internal and external factors, leading to significant alters in color, flavor, texture, and other attributes [1]. In the case of climacteric fruits like tomato, ethylene plays a vital role in regulating fruit maturation [2]. Subsequent studies indicate that ethylene-mediated regulation of fruit ripening depends on the accurate management of the expression of numerous ripening-associated genes, which are precisely regulated at the epigenetic and transcriptional levels [3, 4].

Epigenetic modifications, such as DNA methylation, noncoding RNA, and histone modifications (methylation, acetylation, ubiquitination, phosphorylation, and so on), act as a crucial regulatory mechanism that controls chromatin status and gene transcription [5–7]. DNA methylation/demethylation is vital for fruit ripening [8–11]. Knocking out DNA demethylase (SlDML2) causes a non-ripening phenotype in tomato, indicating that SlDML2 plays a crucial role in regulating tomato fruit maturation [9, 12]. Notably, SlDML2-knockout fruits exhibit defective ethylene production, highlighting the importance of SlDML2 in ripening-induced ethylene biosynthesis [8, 12]. Alongside DNA methylation/demethylation, reversible methylation/demethylation of histones appears to be a critical mechanism for regulating ripening-associated gene expression and is essential for tomato fruit ripening [13, 14]. Despite the significant roles of epigenetic modifications in fruit maturation, the molecular regulation of these ripening-related genes by epigenetic modifications still requires further elucidation.

Histone acetylation, another conserved histone modification, plays vital roles in controlling chromatin structure and gene expression [15]. Histone deacetylases (HDACs) are highly conserved enzymes in eukaryotes and are responsible for removing acetyl groups from acetylated histones at lysine residues [15–17]. Over the past decade, HDACs have garnered significant attention and have been demonstrated to play crucial roles in the plant development process, including seed [18], vegetative development [19, 20], root epidermis [21, 22], flower development [23, 24], and leaf senescence [25], as well as stress responses [26]. In fruits, HDACs have been implicated as crucial regulators of fruit development and ripening [27–31]. For instance, CsHDC1 regulates cytokinin and polyamine biosynthesis and metabolism, and its downregulation results in a short-fruit phenotype [31]. MaHDA1 and CpHDA3 have been shown to suppress fruit ripening by directly interacting with the transcriptional repressors MaERF11 and CpERF9, respectively [ 27]. Similarly, MdHDA19 inhibits apple fruit ripening via interacting with MdERF4-MdTPL4 to directly repress ripening-related gene expression [32]. In tomato, silencing of SlHDA1, SlHDA3, or SlHDT1 enhances fruit ripening, while silencing of SlHDT3 hinders fruit ripening [28, 29, 33–35]. Particularly noteworthy, SlERF.F12 directly interacts with the corepressor SlTPL2 and recruits SlHDA1/SlHDA3 to create a transcriptional complex, leading to the suppression of the initiation of tomato fruit maturation by repressing numerous ripening-associated genes’ expression [34]. Despite numerous reported roles of HDACs in regulating fruit maturation, the underlying mechanism of HDACs in fruit maturation remain largely unclear.

Here, we illustrated the function of the RPD3/HDA1 subfamily gene SlHDA7 in controlling fruit ripening in tomato. Deletion of SlHDA7 accelerated fruit ripening, while overexpression of SlHDA7 hindered fruit ripening. Through molecular and biochemical analysis, we showed that SlHDA7 suppresses the expression of ripening-associated genes by reducing histone acetylation levels, leading to decreased ethylene production and carotenoid biosynthesis during tomato fruit ripening. This research offers detailed insights into the function of SlHDA7 in fruit maturation and unveils a new understanding of the underlying mechanism of fruit maturation and carotenoid biosynthesis.

Results

Expression profile of SlHDA7 during tomato fruit maturation

Previous study has shown that the HD2 and subclass I members of RPD3/HDA1 family HDACs, including SlHDA1, SlHDA3, SlHDT1, and SlHDT3, play important roles in fruit ripening by regulating ethylene biosynthesis and carotenoid metabolism [28, 29, 33, 35, 36]. However, the function of the subclass II clade HDAC remains unexplored. According to a previous study [37], the subclass II clade HDAC genes SlHDA7 and SlHDA10 exhibited a ripening-induced expression pattern, with a particular emphasis on SlHDA7. Hence, we focused our further study on SlHDA7. SlHDA7 has an open reading frame (ORF) spanning 798 base pairs (bp), encodes a 265 amino acid protein with active sites similar to other plant HDACs and an isoelectric point predicted at 5.89. Alignment of amino acid sequence and phylogenetic analysis revealed that SlHDA7 was highly homologous to AtHDA14 (Fig. 1A), which has been implicated in regulating photosynthesis and melatonin biosynthesis [38–40].

Figure 1.

Phylogenetic analysis and expression profiles of SlHDA7. A Phylogenetic analysis of histone deacetylases (HDACs) from Arabidopsis and tomato. B Transcript levels of SlHDA7 in different tissues and at various developmental stages of tomato. BR, break stage; dpa, days post anthesis; Fw, flower; Lf, leaf; MG, mature green stage; Pi, pink stage; RR, red ripe stage; Rt, root; St, stem; Tu, turning stage. Data are means ± SD (n = 3). Different letters above bars indicate significant differences compared to fruit at MG stage by Student’s t test (*P < 0.05).

To further analyse the possible role of SlHDA7 in tomato plants, we examined its expression pattern in various tissues and fruit pericarps across development and ripening stages using RT-qPCR. Our findings revealed significantly higher expression levels of SlHDA7 in fruits and leaves compared to stems and roots (Fig. 1B). Moreover, its expression in fruits experienced a substantial upregulation during early ripening, reaching a peak at the turning stage, and then decreased in the late ripening phase (Fig. 1B), similar to previously published RNA-seq data (Fig. S1, see online supplementary material). This is slightly different from the previous report [37], which may be due to variations in the varieties. These results suggest that SlHDA7 might play a regulatory role in tomato fruit maturation.

SlHDA7 suppresses tomato fruit maturation

To explore the impact of altered SlHDA7 expression on fruit maturation, we utilized the CRISPR/Cas9 gene editing system to develop SlHDA7 knockout mutants. Two slhda7 mutants, slhda7–1 and slhda7–2, were isolated with a homozygous 1-bp deletion in the first exon, resulting in a predicted 3.1 kD truncated protein (Fig. 2A). slhda7–2 mutant also carried an addition 2-bp deletion caused by target sgRNA2 (Fig. 2A). The SlHDA7 transcript was barely detectable in these mutant lines (Fig. 2B). Additionally, we created stable transgenic plants with increased SlHDA7 levels. Two distinct transgenic lines, SlHDA7–36 and SlHDA7–47, with a C-terminal GFP fusion, showed 115- and 97-fold higher expression levels of SlHDA7 compared to the wild type (WT), and were chosen for further examination (Fig. 2B and C).

Figure 2.

SlHDA7 represses tomato fruit maturation. A Generation of SlHDA7 knockout mutants. B Analysis of the gene expression level of SlHDA7 in the SlHDA7 overexpression and knockout mutant lines by RT-qPCR. ACTIN was used as the internal control. Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05). C Western blot analysis of the protein level of SlHDA7 in the SlHDA7 overexpression plants. An anti-GFP antibody was used for detection, and an anti-H3 antibody was used for internal control. D Phenotypes of the WT, SlHDA7 overexpression lines SlHDA7–36 and SlHDA7–47, and the SlHDA7 knockout mutant lines slhda7–1 and slhda7–2 during fruit maturation. Bar = 2 cm.

Phenotypic analysis indicated that the SlHDA7-OE lines exhibited delayed fruit maturation compared to WT fruits, while the absence of SlHDA7 function led to early ripening (Fig. 2D). Corresponding to the ripening phenotype, slhda7–1 and slhda7–2 fruits displayed higher ethylene production rates, along with increased carotenoid and soluble pectin contents, but lower chlorophyll and protopectin levels compared to WT fruits. Conversely, the SlHDA7-OE fruits demonstrated contrasting patterns for these physiological characteristics (Fig. 3B–F). These findings indicate that SlHDA7 functions as a suppressor of tomato fruit maturation.

Figure 3.

Expression level of SlHDA7 and ripening-related physiological properties of the WT, SlHDA7-OE, and slhda7 mutant at 36, 39, and 42 dpa. A Relative expression levels of SlHDA7 in the slhda7–1, slhda7–2, SlHDA7–36, and SlHDA7–47 lines by RT-qPCR. ACTIN was used as the reference control. B–F Ethylene production rate (B), total chlorophyll content (C), carotenoid content (D), propectin content (E), and soluble pectin content (F) in the fruits of the WT, SlHDA7-OE lines, and slhda7 mutant fruits at 36, 39, and 42 dpa. Data are means ± SD (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

SlHDA7 deacetylates H4ac in tomato

Given the correlation between HDACs function and their deacetylase activity, we verified SlHDA7’s histone deacetylation activity by comparing histone lysine acetylation levels in the WT, slhda7 mutant, and SlHDA7-OE lines using specific antibodies. The H4ac levels in the SlHDA7-OE lines were lower than these in the WT (Fig. 4A and B), while there were no significant differences in H4ac levels in the slhda7 mutants (Fig. 4C and D). Additionally, global levels of H3K9ac and H3K14ac showed no significant variance among the WT, slhda7 mutants, and SlHDA7-OE lines (Fig. 4A–D). These findings suggest that SlHDA7 may be responsible for H4ac deacetylation during tomato fruit ripening.

Figure 4.

SlHDA7 functions as a histone H4ac deacetylase. A Assessment of global acetylation levels of H3K9, H3K14, and H4 in the WT and SlHDA7-OE lines. B Statistical evaluation of Fig. 4A. C Assessment of global acetylation levels of H3K9, H3K14, and H4 in the WT and slhda7 mutant lines. D Statistical evaluation of Fig. 4C. The H3 and H4 were utilized as a loading controls. Data are means ± SE (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

SlHDA7 regulates the expression of ripening-related genes

To identify genes influenced by SlHDA7 during tomato fruit ripening, we compared the transcriptomes of WT and slhda7–1 fruits at 36 dpa by RNA-seq analysis. A total of 6451 genes exhibited significant differential expression (fold change >2.5, FDR < 0.05) between WT and slhda7–1 fruits. Among the 6451 genes regulated by SlHDA7, 1914 were suppressed, and 4537 were activated (Fig. 5A; Table S2, see online supplementary material). Gene Ontology (GO) enrichment analysis indicated that the upregulated genes in slhda7–1 fruits were notably involved in secondary metabolite biosynthesis and oxidoreductase activity, whereas the downregulated genes were associated with protein kinase activity and plant hormone signal transduction pathways (Fig. 5B). Moreover, numerous genes associated with ripening-related transcription regulation, the abscisic acid-activated signalling pathway, the ethylene biosynthesis process, flavonoid biosynthesis, and carotenoid biosynthesis were notably upregulated in the slhda7–1 fruits (Fig. 5B; Table 1; Table S2, see online supplementary material).

Figure 5.

Transcriptome analysis of DEGs modulated by SlHDA7 in tomato fruits. A Volcano plots of the RNA-seq data. B GO enrichment analysis of SlHDA7-upregulated and downregulated genes by DAVID. C Validation the results of RNA-seq by RT-qPCR. ACTIN was used as the reference control. Data are means ± SD (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

Table 1.

Ripening-associated genes expression changes were compared between slhda7–1 mutant and WT fruit at 36 dpa.

Gene name	Gene ID	Log2(fold change)	Annotation
Ethylene biosynthesis and signaling
ACS2	Solyc01g095080	4.533	1-aminocyclopropane-1-carboxylate synthase 2
ACS4	Solyc05g050010	3.533	1-aminocyclopropane 1-carboxylate synthase 4
ACO1	Solyc07g049530	3.065	1-aminocyclopropane-1-carboxylate oxidase 1
ACO3	Solyc07g049550	1.598	1-aminocyclopropane-1-carboxylate oxidase 3
ETR3	Solyc09g075440	1.591	Ethylene receptor never ripe
ETR4	Solyc06g053710	1.381	Ethylene receptor homolog precursor
ERF.E1	Solyc09g075420	2.457	Ethylene response factor 1
ERF.E3	Solyc03g123500	1.069	Ethylene response factor 3
ERF.E4	Solyc01g065980	1.010	Ethylene response factor 4
ERF.H1	Solyc06g065820	4.285	Ethylene response factor 3-like
Carotenoid biosynthesis
DXS1	Solyc01g067890	1.821	1-D-deoxyxylulose 5-phosphate synthase
IDI1	Solyc04g056390	1.103	Isopentenyl diphosphate isomerase
GGPPS1	Solyc11g011240	2.534	Geranylgeranyl pyrophosphate synthase 1
GGPPS2	Solyc04g079960	5.113	Geranylgeranyl pyrophosphate synthase 2
PSY1	Solyc03g031860	3.969	Phytoene synthase 1
CrtISO	Solyc10g081650	1.728	Prolycopene isomerase
ZISO	Solyc12g098710	5.890	15-cis-zeta-carotene isomerase
CrtR-b2	Solyc03g007960	1.639	Beta-carotene hydroxylase
Cell wall degradation
MAN4	Solyc01g008710	6.567	Mannan endo-1,4-beta-mannosidase 4 precursor
PL	Solyc03g111690	2.764	Pectate lyase 18
PME1	Solyc03g123620	1.065	Pectinesterase
EXP1	Solyc06g051800	2.574	Expansin precursor 1
CEL8	Solyc08g082250	1.643	Endo-beta-1,4-D-glucanase precursor 8
CEL2	Solyc09g010210	3.678	Endo-1,4-beta-glucanase precursor 2
PL8	Solyc09g091430	6.352	PREDICTED: probable pectate lyase 8
XYL1	Solyc10g047030	3.010	Beta-D-xylosidase 1 precursor
PG2a	Solyc10g080210	8.518	Polygalacturonase-2 precursor
TBG4	Solyc12g008840	4.448	Galactosidase precursor
Maturation regulator and chlorophyll metabolism
AP2a	Solyc03g044300	1.165	APETALA2-like protein
NAC9	Solyc04g005610	1.582	PREDICTED: NAC transcription factor 29
RIN	Solyc05g012020	3.712	MADS-box transcription factor isoform 1
FUL1	Solyc06g069430	2.697	AGAMOUS-like MADS-box protein AGL8 homolog
E4	Solyc03g111720	4.582	Methionine sulfoxide reductase
E8	Solyc09g089580	4.477	1-aminocyclopropane-1-carboxylate oxidase -like
SGR1	Solyc08g080090	1.714	Senescence-inducible chloroplast stay-green protein 1
CAO	Solyc11g012850	1.279	Chlorophyllide a oxygenase

To clarify whether SlHDA7 indirectly regulates tomato fruit ripening through other HDACs, we analysed the expression of previously reported SlHDACs in RNA-seq data. The result revealed that the expression of SlHDACs did not show significant difference between the WT and the slhda7 mutant fruits except for SlHDA7, SlHDA5, and SlHDT3 (Fig. S2, see online supplementary material). Observing this, we first consider whether the sequences similarity among HDACs results in CRISPR/Cas9 off target effects, leading to reduced expression of SlHDA5 and SlHDT3. However, a multiple sequence alignment of sgRNA1 and sgRNA2 among tomato SlHDACs revealed that both sgRNAs were specific to SlHDA7 and did not cause off-target effects (Fig. S3, see online supplementary material). Additionally, SlHDT3 is known to positively regulate tomato fruit ripening [35], while SlHDA5 responds to abiotic stress [41]. Because the expression of SlHDT3 is significantly downregulated during tomato fruit ripening, particularly from IMG to BR stage [35, 37], the unexpected down-regulation expression of SlHDT3 in slhda7 mutant may be related to the WT fruit at the mature green stage, whereas the slhda7 fruit is at the break stage. SlDML2 mediates DNA demethylation of ripening-related genes, playing a crucial regulatory role in tomato fruit ripening. Our study revealed that the expression of SlDML2 did not show significant difference between the WT and the slhda7–1 fruits. Moreover, DNA methylation in the SlHDA7 promoter did not differ significantly between WT and the sldml2 fruits [12]. Taken together, these findings indicated that the ripening changes in the slhda7 fruits may primarily due to the absence of SlHDA7.

In addition, we conducted an RT-qPCR assay to confirm the accuracy of the RNA-seq results. The expression of the 16 fruit ripening-associated genes, such as ACO3, ERF.E4, ETR3, ERF.H1, IDI1, CrtISO, CrtR-b2, DXS1, MAN4, PME1, CEL8, PL, NAC9, AP2a, CAO, and SGR1 were confirmed to be upregulated in the slhda7 fruits compared with the WT fruits (Fig. 5C). Overall, these findings indicate that SlHDA7 delays fruit ripening by influencing the expression of ripening-related genes associated with ethylene biosynthesis and response, carotenoid metabolism, cell wall modification, and transcriptional regulation.

SlHDA7 inhibits the expression of ripening-related genes via deacetylation of H4ac during tomato fruit maturation

Previous studies have revealed that fruit ripening is dependent on the precise regulation of numerous ripening-related genes, which are tightly controlled at the epigenetic and transcriptional levels [3, 4]. Based on our previous findings, we postulated that SlHDA7 retards fruit ripening through its H4ac deacetylase activity. To validate this hypothesis, we initially investigated the expression of key maturation-related genes, such as ACO1, ACS2, ACS4, ERF.E1, GGPPS2, GGPPS3, PSY1, ZISO, PG2a, EXP1, XYL1, CEL2, RIN, E4, E8, and FUL1 in WT, SlHDA7-OE, and slhda7 mutant fruits. As expected, RT-qPCR analysis revealed up-regulation of these genes in slhda7 mutant fruits, while down-regulation in SlHDA7-OE fruits at 36 and 39 dpa (Fig. 6).

Figure 6.

Ripening-associated genes expression profiles in WT, slhda7, and SlHDA7-OE fruits at 36 and 39 dpa. ACTIN was used as the reference control. Data are means ± SD (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

To further validate this hypothesis, we conducted ChIP-qPCR assays to evaluate the H4ac levels of these ripening-related genes in SlHDA7-OE, slhda7, and WT fruits at 36 dpa. In comparison to WT fruits, the H4ac levels at the promoter regions of ACO1, ERF.E1, GGPPS2, ZISO, EXP1, XYL1, RIN, and FUL1 were decreased in SlHDA7-OE fruits but notably increased in slhda7 fruits (Fig. 7). Because H4ac is an active marker, these results suggest that SlHDA7 might function as a suppressor of fruit maturation by inhibiting the transcription of certain ripening-related genes through the elimination of H4ac.

Figure 7.

ChIP-qPCR analysis of the H4ac levels of ripening-associated genes in SlHDA7-OE, slhda7, and WT fruits. The black arrows indicate the positions of the different primers used for ChIP-qPCR. Chromatin was isolated from the WT, slhda7–1, and SlHDA7–36 fruits at 36 dpa with an anti-H4ac antibody. ACTIN was used as the reference control. Data are means ± SD (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

Knockout SlHDA7 shortens the fruit shelf life

To investigate the impact of SlHDA7 overexpression and knockout on the postharvest shelf life, fruits of WT, SlHDA7-OE, and slhda7 were harvested at the Br + 7 stage (the 7th day after the break stage) and stored under the same conditions at room temperature. The skin of the WT fruits began to wrinkle after 7 days of storage, while the SlHDA7-OE fruits only showed slight shrinkage after 14 days of storage (Fig. 8A). In contrast, the slhda7 fruits experienced accelerated shrinkage, with severe shrinkage and deformation after 49 days of storage (Fig. 8A). To explore the potential mechanism linked to the fruit shelf life of the WT, SlHDA7-OE, and slhda7 fruits, we monitored the weight loss rate during storage. As depicted in Fig. 8B, the weight loss rate of the slhda7 fruits was notably higher than that of WT fruits after 35 days of storage, but significantly lower for SlHDA7-OE fruits (Fig. 8B). These findings suggest that SlHDA7 prolongs fruit shelf life by reducing water loss rate and delaying ripening.

Figure 8.

SlHDA7 extends the fruit shelf life of tomato. A Phenotypes of WT, SlHDA7-OE, and slhda7 mutant fruits stored for 49 days. B The weight loss rate of WT, SlHDA7-OE, and slhda7 mutant fruits during 49 days post-harvest storage. Data are means ± SD (n = 3). Asterisks indicate significant differences compared to WT by Student’s t test (^*P < 0.05).

Discussion

Fruit ripening, the final stage of fruit development, is synergistically controlled by both internal developmental factors and external environmental factors. It depends on the accurate control of numerous ripening-related genes to remodel biochemical and physiological processes that determine fruit quality [42]. Despite the identification of numerous epigenetic factors associated with fruit ripening [14, 34, 36], the functions and impacts of most HDACs in controlling this process remain ambiguous. In this study, we demonstrate that SlHDA7, a subclass II clade histone deacetylase, might function as a suppressor of tomato fruit maturation by deacetylating H4ac to inhibit the transcription of crucial ripening-related genes.

SlHDA7 functions as a suppressor of tomato fruit maturation

Histone acetylation is an essential epigenetic marker closely linked to gene activation or inhibition. Recent research indicates that histone acetylation and deacetylation, regulated by histone acetyltransferases and histone deacetylases, play crucial functions in plant development, stress response, and fruit ripening [13, 36, 43]. For instance, AtHDA9 depressingly affects salt and drought stress by modulating histone acetylation levels of numerous stress-responsive genes in Arabidopsis [44]. MaHDA1 and MdHDA19 negatively regulate banana fruit ripening via directly interacting with transcriptional repressors to form a repressor complex to suppresses the expression of many ripening-related genes [27, 32]. In tomato, SlHDA1, SlHDA3, or SlHDT1 delay fruit ripening, while SlHDT3 promotes it [28, 29, 33, 35]. Despite the reported essential roles of HDACs in modulating fruit maturation, the regulatory mechanisms of HDACs in this process remain largely unclear.

Here, we present evidence supporting that SlHDA7 functions as a suppressor of fruit maturation in tomato. Firstly, the expression of SlHDA7 significantly increased during tomato fruit maturation (Fig. 1C). Additionally, knockout of SlHDA7 was found to accelerate tomato fruit ripening, whereas overexpression of SlHDA7 led to delayed ripening (Fig. 2D). Moreover, a multitude of ripening-associated genes participated in ethylene production, carotenoid synthesis, cell wall metabolism, and key ripening-related transcription factors were upregulated in the slhda7 mutant fruits, while downregulated in SlHDA7 overexpression fruits (Fig. 6). These findings collectively suggest that SlHDA7 exerts a negative influence on tomato fruit ripening.

SlHDA7 inhibits tomato fruit maturation by removing H4ac at ripening-related gene loci

In eukaryotes, histone acetylation and deacetylation are directly connected with chromatin status and genes expression, while HDACs control gene expression via removing acetyl groups from histone tails, resulting in chromatin condensation and transcriptional repression. Our RNA-seq data revealed that SlHDA7 likely modulate the expression of various maturation-related genes participating in ethylene production and signaling, carotenoid synthesis, cell wall metabolism, and ripening-related transcription factors, underscoring the significance of SlHDA7 in coordinating tomato fruit ripening transcription (Fig. 5). To uncover the function of SlHDA7 in tomato fruit maturation, we performed the RT-qPCR and ChIP-qPCR assays to identify putative target genes of SlHDA7.

Numerous studies have revealed that ethylene is the main trigger of climacteric fruit ripening [2, 45, 46]_. In the present study, knockout of SlHDA7 promoted ethylene biosynthesis, whereas overexpression of SlHDA7 suppressed this process (Fig. 3C). Furthermore, key genes involved in ethylene synthesis and signaling, such as ACO1, ACS2, ACS4, and ERF.E1 [46, 47] were significantly upregulated in slhda7 fruits but downregulated in SlHDA7-OE fruits (Fig. 6). Additionally, ChIP-qPCR assays indicated that SlHDA7 suppresses the expression of ACO1 and ERF.E1 by reducing H4ac levels at their gene loci during tomato fruit ripening (Fig. 7). Carotenoid accumulation and cell wall degradation lead to the red softened fruit, which are the two major characteristics of tomato ripening. Our findings revealed that SlHDA7-mediated removal of H4ac represses the expression of crucial carotenoid biosynthesis (GGPPS2 and ZISO) and cell wall metabolism (EXP1and XYL1) related genes (Fig. 7). Collectively, these findings indicate that SlHDA7 delays tomato fruit ripening via inhibiting ripening-related structural genes expression.

In addition to ripening-related structural genes, transcription factors orchestrate fruit ripening via the integration of internal signals with environmental cues [48, 49]. In tomato, RIN and FUL1 are crucial for fruit ripening based on mutant or knockout analyses [50, 51]. Notably, RIN and FUL1 positively regulate the tomato fruit maturation by controlling various maturation-related genes participating in ethylene synthesis and signaling, chlorophyll degradation, carotenoid metabolism, and cell wall degradation, and transcription regulation [52–54]. In this work, we revealed that SlHDA7 eliminates the acetyl group from the RIN and FUL1 chromatin, leading to their downregulation (Fig. 7). Because HDACs lack DNA binding ability, they are typically recruited by various transcription factors or interacting proteins to form complexes that regulate target gene expression. It is probable that SlHDA7 identifies its target genes through interactions with transcription factors or other proteins.

In summary, our results indicate that SlHDA7 might function as a suppressor of fruit maturation by deacetylating H4ac to inhibit the transcription of genes participating in ethylene synthesis and signaling, carotenoid biosynthesis, cell wall metabolism, and crucial ripening-related transcription factors in tomato, providing new insights into the impact of epigenetic regulation on fruit maturation.

Materials and methods

Plant materials and growth conditions

Solanum lycopersicum (cv. Ailsa Craig (AC)) wild-type (WT) and transgenic plants were cultivated in a greenhouse with natural light conditions (23°C with a 16 h/8 h photoperiod). The fruits’ development and ripening stages were accurately determined by tagging at anthesis, which was recorded as days post-anthesis (dpa). The pericarp of harvested fruits were promptly frozen in liquid nitrogen and preserved at −80°C for additional analysis.

Generation of transgenic plants

The coding sequence of SlHDA7 (Solyc01g009110) was cloned and inserted into the pBI121-GFP vector to create the pBI121-SlHDA7-GFP construct. To produce the slhda7 mutants, two specific sgRNA sequences were inserted into the pPTG-sgRNA-Cas9-AtU6–1 vector to target SlHDA7 simultaneously [55]. The resulting vectors were confirmed through sequencing, then transformed into the cotyledon explants of the tomato cultivar AC by Agrobacterium tumefaciens GV3101 mediated transformation [56]. Kanamycin-resistant, PCR and Sanger sequencing were used for identifying positive transformants. All primers used in this study were listed in Table S1 (see online supplementary material).

Fruit ripening characteristics

The ethylene production rate was determined following a previously published protocol [57, 58]. Chlorophyll and carotenoids were extracted and measured as previously described [59] and are expressed as μg g⁻¹ FW. The propectin and soluble pectin contents were measured as described previously [60]. Three biological replicates were conducted, with one replicate consisting of six fruits.

RT-qPCR assays

Total RNA of roots, stems, leaves, flowers, and tomato pericarp at 10, 20, 30, 36, 39, and 40 dpa was extracted with the HiPure Plant RNA Mini Kit (Magen, Guangzhou, China). First-strand cDNA was synthesized with a PrimeScript TM Reagent Kit (RR036A, Takara Bio Inc., Shiga, Japan) following the method described previously [56]. RT-qPCR was performed with an ABI7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) via using SYBR® Premix Ex TaqTM II (RR420A, TakaraBio Inc., Shiga, Japan). The gene expression level was calculated using the 2^−ΔΔCt method. The analysis included three independent biological replicates. ACTIN (Solyc03g078400) was chosen as the reference control.

RNA-seq assay

RNA was extracted from three independent WT and slhda7 fruits at 36 dpa. The RNA extraction, library preparation, and Illumina sequencing were carried out at the Gene Denovo Company (Guangzhou, China) using the Illumina NovaSeq 6000 platform. The clean reads were compared with HISAT2 to the reference genome SL3.0. Gene expression levels were quantified using an RPKM value. Differentially expressed genes (DEGs) were identified based on the fold changes >2.5 with FDR <0.05 using DESEQ2. The gene functional enrichment analysis of the DEGs was conducted using the DAVID database (https://david.ncifcrf.gov/gene2gene.jsp).

Analysis of global histone acetylation

The levels of global histone acetylation in the leaves were assessed following previously established protocols [61]. Specific anti-acetylated histone antibodies were utilized for Western blot analysis. The quantification of immunoblotting signals was carried out using ImageJ. The specific anti-acetylated antibodies used in western blot assay were listed as: anti-H3 (ab1791; Abcam), anti-H3ac (17–615; Millipore), anti-H3K9ac (ab32129; Abcam), anti-H3K14ac (ab52946; Abcam), anti-H4 (ab177840; Abcam), and anti-H4ac (06–866; Millipore).

ChIP-qPCR analysis

ChIP-qPCR assays were conducted following the method described previously [13]. Fruits from the WT, slhda7–1, and SlHDA7–36 lines at 36 dpa were collected and promptly fixed with 1% formaldehyde under vacuum for 15 min, then quenched by adding glycine. After crosslinking, the chromatin complex was sonicated to approximately 500 bp fragments. Immunoprecipitation was carried out using an anti-acetyl-histone H4ac antibody (06–866; Millipore). The enrichment was assessed through qPCR and was normalized to the relative enrichment compared to the input. All primers utilized in ChIP-qPCR are listed in Table S1 (see online supplementary material).

Postharvest storage test

The fruits of the WT, SlHDA7 overexpression lines, and SlHDA7 knockout mutants were collected at the breaking stage and placed on a clean plate under greenhouse conditions for 16 hours during the day (25°C) and 8 hours during the night (22°C). The phenotype and fruit weight were monitored every 7 days. Each postharvest storage test utilized 20 fruits.

Statistical analysis

We used SPSS version 7.5 for statistical analysis. Variations among different sample groups were conducted with either Student’s t-test or ANOVA.

Author contributions

G.J. designed the experiments. Y.Z., Z.L., and H.H. performed the experiments. Y.Z. and G.J. wrote the paper. X.D., Y.J., X.S., and H.Q. edited the manuscript. All authors discussed and approved the final manuscript.

Data availability

The RNA-Seq data have been deposited in the Dryad in the following repository (https://datadryad.org/stash/share/dFeS6NvPQK_xhouVJUpnPQYRN2R5VkjHNAvbqiel2R8).

Conflict of interests

The authors declare no competing interests.

Supplementary data

Supplementary data is available at Horticulture Research online.