The Role of Protein Post-Translational Modifications in Fruit Ripening

Abstract

Fruit ripening represents a multifaceted biological process intricately controlled by an array of plant hormones, transcription factors, and epigenetic modifications. These regulatory mechanisms are crucial in determining fruit quality and post-harvest shelf life. Recent advancements in proteomics have shifted the focus toward understanding protein post-translational modifications (PTMs), which play a crucial role in modulating protein function. PTMs enhance protein activity and stability by altering their properties after biosynthesis, thereby adding an additional layer of regulation to the ripening process. This paper provides a comprehensive review of the roles of PTMs, including ubiquitination, phosphorylation, redox modifications, and glycosylation in regulating fruit ripening. Emphasis is placed on the intricate interplay between these PTMs and key regulator factors such as plant hormones, transcriptional mechanisms, and epigenetic modifications. By exploring these interactions, this review seeks to enhance our understanding of the complex regulatory network underlying fruit ripening and to offer novel perspectives on strategies for fruit preservation.

1. Introduction

Fruit, a distinctive organ of angiosperms, is composed of the pericarp and seeds, and plays a major role in plant reproduction [1]. Fruits can be classified into two categories: dry fruits and fleshy fruits. Fleshy fruits are particularly notable for their high content of vitamins, minerals, and other essential nutrients. Additionally, they provide additional amounts of carbohydrates and dietary fiber, contributing to human health, and making them an essential part of a balanced diet [2]. Ripening fruits play a pivotal role in plant reproduction by attracting birds and other animals, thereby facilitating seed dispersal [3]. The development and ripening process of fleshy fruits involve a series of complex chemical and physiological changes. These changes include cell division and elongation, nutrient accumulation, pigment metabolism, starch degradation, cell wall modifications, and water content alterations, all of which contribute to the maturation and final quality of the fruit [2,3]. These complex changes significantly influence the color, aroma, flavor, texture, and post-harvest shelf life of fruits, thereby directly affecting their economic value [4].

Fruit can be classified into climacteric or non-climacteric types based on their physiological ripening characteristics. Climacteric fruits exhibit a respiratory peak due to a significant release of ethylene, while non-climacteric fruits do not experience this ethylene-induced respiration surge. Plant hormones, particularly ethylene, have been identified as crucial regulators of fruit ripening. Recent studies have increasingly focused on transcription factors and genetic regulatory elements that respond to these plant hormones, further elucidating the complex regulatory mechanisms underlying the ripening process [2,5]. The rapid advancement of modern biotechnology has led to increased exploration of the molecular regulatory mechanisms underlying fruit ripening. Emerging evidence has highlighted the crucial role of protein post-translational modifications (PTMs) in fruit development and ripening [2]. This paper provides a comprehensive review of the regulatory roles and mechanisms of PTMs in these processes, to enhance post-harvest fruit quality, extend post-harvest shelf life, reduce economic losses, and offer theoretical insights for fruit crop breeding.

2. Overview of Fruit Ripening

Fruit development and ripening encompass three critical stages: fruit set, growth, and ripening. Through these stages, the fruit undergoes a series of physiological and metabolic transformations. These changes include color alteration driven by chlorophyll breakdown and pigment accumulation, flavor enhancement through the synthesis and metabolism of sugars, acids, and volatile compounds, and softening, which is facilitated by cell wall remodeling and starch degradation [6,7,8]. This process is intricately regulated by a myriad of factors, including plant hormones [9], transcriptional regulatory factors [1], epigenetics, and PTMs [2]. These factors collectively constitute a complex regulatory network that dynamically coordinates fruit ripening, thereby influencing the overall fruit quality [6].

The tomato fruit, a representative climacteric fruit, possesses several advantageous characteristics including diploid genetics, self-pollination, a short life cycle, high transformation efficiency, abundant genetic resources, easy accessibility of mutant strains, and a high-quality reference genome. These attributes have established the tomato as a model organism for investigating the development and ripening of climacteric fleshy fruits [4,10]. Upon the initiation of ripening, tomato fruit experiences distinct peaks in respiration and ethylene synthesis, which are tightly regulated. The biosynthesis and response to ethylene are precisely controlled by several transcription factors, such as RIPENING INHIBITOR (RIN), COLORLESS NON-RIPENING (CNR), and NON-RIPENING (NOR) [11]. These factors are essential in regulating both ethylene synthesis and signaling pathways during ripening and in the transcriptional regulation of ripening-associated target genes [11].

In contrast, strawberries serve as a model for investigating non-climacteric fruit ripening. With cultivated diploid varieties and established transgenic systems, strawberries have significantly advanced our understanding of the control mechanisms governing non-climacteric fruit ripening [12]. The ripening process in strawberries is primarily regulated by abscisic acid (ABA), which orchestrates a network of downstream signaling components. These include ABA-responsive element binding protein/ABRE binding factor (AREB/ABF), ethylene response factor (ERF), MYB transcription factors, and ripening-related genes [13]. ABA influences these components through two distinct signaling pathways—ABA-FaPYR1-FaABIl-FaSnRK2 and ABA-FaABAR-FaRIPK1-FaABI4. This framework establishes a comprehensive model of ABA regulation, integrating interactions with ethylene, sugars, polyamines, coenzymes, and reactive oxygen species [13].

3. Regulation of Fruit Ripening by Protein Post-Translational Modifications (PTMs)

Following protein synthesis, proteins undergo various chemical modifications, collectively referred to as post-translational modifications (PTMs), which result in significant alterations in their structure and function [14]. These modifications are critical for the regulation of numerous cellular processes. PTMs can be broadly categorized into three types: the addition of functional groups, such as phosphorylation, methylation, and glycosylation; the covalent attachment of small peptides or proteins, including ubiquitination and SUMOylation; and the chemical modifications of amino acids, exemplified by glycation [14]. Proteins are subject to a wide array of modifications, with each distinct modification and its specific binding site imparting unique biological functions to the protein. In contrast to the limited number of unmodified proteins, potential PTMs are far more prevalent and dispersed [15]. PTMs are crucial for various life processes, including fruit development and ripening. The addition or removal of these modifications is mediated by specific enzymes commonly referred to as “writers” and “erasers”, which recognize and bind to particular sites on the protein [16]. These enzymes rely on “readers” for the recognition of PTMs, thereby facilitating signal transduction through protein–protein interactions [16]. Disrupting the “writing”, “erasing”, and “reading” processes can lead to disturbances in these modifications, consequently altering the signaling pathways and physiological activities mediated by the involved proteins. This disruption holds significant importance for investigating the molecular mechanisms underlying the regulation of PTMs during fruit ripening.

3.1. Ubiquitination and Fruit Ripening

Protein ubiquitination is a post-translational modification in which ubiquitin is covalently attached to specific substrates. This process is mediated by the coordinated actions of three enzymes: the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2), and the ubiquitin-ligating enzyme (E3), which collectively facilitate the formation of a covalent bond between ubiquitin and the target protein [15]. This process involves the formation of a high-energy thioester bond between E1 and ubiquitin in an ATP-dependent manner. Ubiquitin is then transferred to E2, which subsequently interacts with E3. E3 facilitates the attachment of ubiquitin to lysine residues on the target protein. The ubiquitinated protein is ultimately recognized and degraded by the 26S proteasome, a process that releases the ubiquitin molecules for reuse [17].

Ubiquitination plays a critical role in regulating fruit ripening by modulating protein stability and abundance. In tomato fruit, virus-induced gene silencing (VIGS) experiments have demonstrated that two E2 enzymes, SlUBC32 and SlUBC41, are involved in the regulation of pigment metabolism during tomato fruit ripening [18].

The substrate specificity of ubiquitination is primarily determined by E3 ligases, which are categorized into three single subunit types-Homology to E6-AP C-Terminus (HECT), Really Interesting New Gene (RING), and U-Box, as well as multi-subunit complexes such as Skp-cullin-F-box (SCF) and CULLIN4-damaged-specific DNA binding protein1 (CUL4-DDB1). And the cullin-RING ubiquitin ligase (CRL) complex represents the largest family of E3 ligase in plants [19,20]. In tomatoes, the CUL4-DDB1-DET1 complex catalyzes the proteasomal degradation of GOLDEN2-LIKE (SlGLK2) by specifically targeting the Lys residues K11 and K253, thereby playing a crucial role in the regulation of chloroplast development and pigment metabolism [21]. Similarly, in apples, the U-box E3 ligase MdPUB24 interacts with MdBEL7 to promote chlorophyll degradation by enhancing the expression of Chlorophyllase (MdCLH), Pheophytinase (MdPPH2), and Red Chl Catabolite Reductase (MdRCCR2) [22]. Additionally, the INTERACTING E3 LIGASE 1 (MdMIL1) catalyzes the degradation of MYB transcription factor MdMYB308L, thereby negatively regulating anthocyanin accumulation [23]. MdPUB29, another E3 ligase, ubiquitinates basic helix-loop-helix (MdbHLH33), which in turn inhibits the expression of ethylene biosynthesis genes such as MdACO1, MdACS1, MdACS5A, ultimately slowing down the fruit ripening process [24]. In bananas, the RING-type E3 ligase MaXB3 ubiquitinates and degrades MaACS1 and MaACO1, thereby negatively regulating ethylene biosynthesis [25]. Furthermore, the E3 ligases NYC1-interacting protein 1 (MaNIP1) and RING Zinc Finger 1 (MaRZF1) are involved in high temperature-induced green ripening by degrading NON-YELLOW COLORING1 (MaNYC1), and STAY-GREEN 1 (MaSGR1), and regulating starch degradation, respectively [26,27].

Research on the regulation of fruit ripening through protein ubiquitination primarily emphasizes the catalytic role of E3 ligases. These ligases influence the levels of critical proteins involved in ripening by targeting them for degradation. This modulation of protein levels affects the expression of ripening-related genes and, consequently, the overall ripening process.

3.2. Phosphorylation and Fruit Ripening

Protein phosphorylation, the addition of a phosphate group from ATP or GTP to substrate proteins, is catalyzed by protein kinases and predominantly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues [28]. This modification plays a crucial role in regulating protein function and localization, affecting receptor protein activity, protein–protein interactions, and facilitating signal transduction within plants [29]. Phosphorylation is a dynamic and reversible biological process, with protein dephosphorylation mediated by protein phosphatases. As a fundamental regulatory mechanism, phosphorylation plays a crucial role in controlling protein activity and function [29].

Phosphorylated proteins play essential roles in various physiological activities in plants. Studies have identified 989 protein kinases and 130 protein phosphatases in Arabidopsis, while rice contains 1508 protein kinases, highlighting the extensive capacity for protein phosphorylation in plants [30].

During tomato fruit development and ripening, various processes, including ethylene biosynthesis and signaling, photosynthesis regulation, carotenoid synthesis, chlorophyll degradation, and transcription regulation are modulated by phosphorylation [28]. The protein phosphatase 2C (PP2C) family gene SlPP2C1, which is expressed in young fruit skin and flesh, accelerates fruit ripening when its expression is disrupted [31]. LeACS2 undergoes phosphorylation at the C-terminal residue Ser460, which implicates its role in ethylene signal transduction and the regulation of fruit ripening [32]. Similarly, during banana ripening, a Ser/Thr family protein kinase phosphorylates the C-terminal residues Ser476 and Ser479 of MaACS1, thereby modulating the ethylene signaling pathway [33]. MaMPK6-3 catalyzes the phosphorylation of MabZIP21 at residues T318 and S436, which enhances its transcriptional activation of ripening-related genes, including MaACO1, MaPG3, MaPL5, MaPL15, MaPE42, MaPE51, and MaEXPA15, thereby accelerating banana fruit ripening [34]. Furthermore, MaBZIP93 is phosphorylated by MaMPK2, which boosts its transcriptional activation of genes involved in cell wall modification, thus promoting banana fruit ripening [35].

The PP2C gene ABI1 in strawberries exhibits a rapid decrease in expression during fruit development. Modulating the expression of FaABI1, either silencing or overexpression, affects the expression levels of ripening-related genes, such as PG1 and PL1, thereby regulating strawberry ripening process [36]. In apples, the protein kinase MdMPK4-14G phosphorylates ETHYLENE RESPONSE FACTOR17 (MdERF17) at Thr67, which promotes chlorophyll degradation [37]. Additionally, MdSnRK2-I phosphorylates HB transcription factors MdHB1, MdHB2, and MdACS1, enhancing protein stability and thereby regulating ethylene biosynthesis and fruit ripening [38]. In litchi, LcSnRK1α-mediated phosphorylation of LcbZIP1/3 activates metabolic reprogramming genes such as DARK-INDUCIBLE 10 (LcDIN10), ASPARAGINE SYNTHASE1 (LcASN1), and ANTHOCYANIN SYNTHASE (LcANS), ensuring energy and redox homeostasis and thus regulating fruit senescence [39].

Recent studies have expanded our understanding of the role of phosphorylation in fruit ripening. For instance, in strawberries, the phosphorylation of the transcription factor bHLH3 by SnRK2.6 is crucial for regulating anthocyanin homeostasis during ripening [40]. SnRK2.6 negatively regulates strawberry fruit ripening by phosphorylating bHLH3, which inhibits its binding to the UFGT promoter and consequently reduces anthocyanin synthesis [40]. In tomatoes, Phytosulfokine (PSK) enhances fruit ripening and quality through the phosphorylation of the transcription factor DREB2F [41]. Conversely, in apples, the phosphorylation of MdCYTOKININ RESPONSE FACTOR4 (MdCRF4) suppresses ethylene biosynthesis, thereby slowing the fruit ripening process [42].

These findings underscore the importance of protein phosphorylation as a regulatory mechanism in fruit ripening. By modulating protein activity, stability, and interactions, phosphorylation orchestrates various physiological processes crucial for fruit development and maturation. Further research into the specific roles and interactions of phosphorylated proteins will further elucidate the molecular mechanisms underlying fruit ripening.

3.3. Redox Regulation and Fruit Ripening

Reactive oxygen species (ROS) are highly reactive oxygen-containing metabolites present in plant cells that act as signaling molecules. They mediate various signaling pathways and influence physiological activities through both signal transduction and oxidative modifications of macromolecules [43]. ROS, along with reactive nitrogen species (RNS), induce reversible oxidation of cysteine (Cys) and methionine (Met) residues in proteins [44]. Key enzymes, including methionine sulfoxide reductase (Msr), thioredoxin (Trx), and glutaredoxin (Grx), are responsible for the reduction of these oxidized proteins, thereby maintaining cellular redox homeostasis and enhancing resistance to oxidative stress [44].

Redox regulation is critical in fruit ripening and senescence. In tomatoes, fruit ripening is associated with the oxidation of methionine residues in NOR, a key ripening regulator [45]. This oxidation reduces NOR’s DNA-binding capacity and transcriptional activity, thereby inhibiting fruit ripening, however, this effect can be partially reversed by Msr [45]. Similarly, in bananas, the ethylene signaling component MaEIL9 is also regulated through redox mechanisms [46]. Methionine oxidation in MaEIL9 impairs its functional capacity, however, Msr enzymes can reduce methionine sulfoxide back to methionine, thereby restoring its DNA-binding ability and ensuring proper ethylene signaling [46]. Additionally, the transcription factor NAC42 in bananas is subject to redox regulation, particularly in response to stress-induced ripening [47]. The Msr-mediated reduction of oxidized NAC42 is vital for its functionality, indicating that redox modifications act as molecular switches to regulate protein activities during stress responses and developmental processes [47]. MaMsrB2 can also regulate the redox state of methionine in MaAPX1, thereby regulating maturation and senescence [48].

Calmodulin (CaM) is another protein regulated by redox mechanisms during fruit ripening and senescence. In litchi, methionine oxidation in CaM affects its interaction with senescence-related transcription factors, thereby influencing the aging process [49]. Methionine sulfoxide reductase (Msr) can reduce the oxidized methionine residues in CaM, preserving its functional interactions [49]. In bananas, the redox state of MaCaM influences its binding to catalase and the MaHY5-1 transcription factor, thereby modulating both fruit ripening and stress responses [50]. Additionally, in longan fruit, redox regulation of glutathione peroxidase (DlGpx) plays a crucial role in controlling senescence and quality deterioration. During senescence, there is an excessive accumulation of reactive oxygen species (ROS) and subsequent oxidation of glutathione peroxidase (DlGpx) [51]. Longan thioredoxin1 (DlTrx1) interacts with DlGpx to effectively reduce its oxidized form, thereby maintaining cellular redox homeostasis and mitigating oxidative damage throughout the senescence process [51].

S-nitrosation and persulfation are also typical reversible redox-dependent protein modifications, and the redox state they affect usually alters the structure and function of proteins, which are involved in maintaining normal cellular physiological metabolism and homeostasis [52]. NADPH is a redox compound and a key cofactor required for cell growth and proliferation. During sweet pepper ripening, hydrogen sulfide levels increased while the activity of the NADPH regenerating enzyme NADP-isocitrate dehydrogenase (NADP-ICDH) decreased. Further studies revealed that NADP-ICDH may be regulated by persulfation, S-nitrosation, and nitration through the potential targets Cys133 and Tyr450, which regulate the production of NADPH, thereby affecting the redox state of cells [52]. Similar studies have been done with respiratory burst oxidase homolog (Rboh) in sweet peppers, which can undergo S-nitrosation, Tyr-nitration, and glutathionylation, respectively, and, thus, be involved in nitro-oxidative stress associated with fruit ripening in sweet peppers [53]. And six ascorbate peroxidases (APXs) in the sweet pepper may also undergo different post-translational modifications through conserved loci Cys32 and Tyr235, including S-nitrosation, Tyr-nitration, and glutathionylation [54]. Cys396 of tomato SlWRKY6 can be persulfated by hydrogen sulfide and delays tomato fruit ripening by suppressing the expression of SlSGR1 and SlSAG12 [55]. Persulfuration at Cys206 and Cys212 of the BOI-related E3 ubiquitin-protein ligase 3 (BRG3) reduces the degradation of the ripening repressor WRKY71, while persulfuration of WRKY71 enhances its binding and transcriptional repression of the ripening positively-regulated gene CYANOALANINE SYNTHASE1 (CAS1), ultimately delaying tomato ripening [56].

It is evident that protein oxidation is prevalent during fruit ripening and senescence, and redox regulation mediated by Msr, Trx, and Grx is vital for modulating protein function. These enzymes enable the reversible oxidation of cysteine and methionine residues, acting as molecular switches that regulate protein activities. Understanding these redox processes provides valuable insights into the mechanisms of fruit ripening and senescence, offering potential strategies to enhance fruit quality and extend shelf life through targeted manipulation of redox pathways.

3.4. Glycosylation and Fruit Ripening

Glycosylation is a critical post-translational modification in eukaryotic cells, involving the covalent attachment of sugars or polysaccharides to proteins within the endoplasmic reticulum and Golgi apparatus [57]. Based on the site of sugar attachment, glycosylation can be classified into C-glycosylation, N-glycosylation, and O-glycosylation. This modification impacts protein folding, solubility, stability, biosynthesis, and activity, and is intimately linked to fruit development and ripening [57].

During strawberry fruit ripening, inhibition of N-glycosylation can retard the ripening process, highlighting the crucial role of N-glycan modification. Both Hexosaminidases FvHex1 and FvHex2 possess N-glycosylation sites [58]. Glycosidase digestion of recombinant β-Hex protein results in a reduction in molecular weight, confirming the presence of N-glycosylation [58]. The expression levels of β-Hex1 and β-Hex2 increase during ripening, and suppression of their expression or enzymatic activity can delay fruit softening and extend shelf life [58]. The activity of xyloglucan endo-transglycosylase/hydrolase (XTH) in strawberries is closely associated with fruit softening. The transcription of FcXTH1 increases as the fruit undergoes softening, and the protein contains a conserved N-glycosylation site [59]. Molecular dynamics simulations reveal that the interactions between FcXTH1 and xyloglucan are dependent on the glycosylation status of FcXTH1. Deglycosylation leads to reduced FcXTH1 activity and stability, which in turn affects the softening process [59].

Similar findings have been observed in tomatoes, where inhibition of N-glycoprotein modifying enzymes α-Man and β-Hex results in increased fruit firmness and delayed softening [60]. Glycoproteomics analysis has identified 191 differentially expressed N-glycoproteins and 252 N-glycosylation sites between green and ripe tomatoes, confirming the crucial regulatory role of N-glycosylation in fruit ripening [61].

The role of O-glycosylation in fruit ripening has been primarily investigated in tomatoes. The enzyme SPINDLY (SPY), which is involved in the ethylene signaling pathway, plays a significant role in this process [62]. Studies involving overexpression and silencing of SlSPY in transgenic tomato plants have demonstrated that SlSPY regulates ethylene signaling and interacts with ETHYLENE INSENSITIVE 2 (EIN2). SlEIN2 has two potential O-glycosylation sites, Ser771 and Thr821, where SlSPY mediates O-glycosylation. This modification enhances protein stability and promotes nuclear translocation in conjunction with phosphorylation, thereby influencing ethylene signal transduction and ripening [62].

In summary, glycosylated proteins generally demonstrate enhanced stability compared to their non-glycosylated counterparts. Although the role of glycosylation is somewhat understood, research in this area is still limited, and the specific mechanisms remain inadequately elucidated. Further investigation using transgenic technologies, RNA interference (RNAi), and bioinformatics tools is essential to deepen our understanding of N-glycosylation enzymes and their pathways in fruit ripening.

3.5. Other Types of Modifications and Fruit Ripening

In addition to phosphorylation, ubiquitination, oxidation, and glycosylation (

Table 1. Regulation of protein post-translational modifications on fruit ripening.

Type	Fruit	Enzyme	Substrate	Site	Regulatory Effects	Reference
Ubiquitination	Tomato	SlUBC32/41			Regulates pigment metabolism	[18]
		CUL4-DDB1-DET1	SlGLK2	K11, K253	Regulates chloroplast development and pigment metabolism	[21]
	Apple	MdPUB24	MdBEL7		Promotes chlorophyll degradation	[22]
		MdMIL1	MdMYB308L		Negatively regulates anthocyanin accumulation	[23]
		MdPUB29	MdbHLH33		Inhibits ethylene biosynthesis and anthocyanin accumulation	[24]
	Banana	MaXB3	MaACS1, MaACO1		Negatively regulates ethylene biosynthesis	[25]
		MaNIP1	MaNYC1		Negatively regulates chlorophyll catabolism	[26]
		MaRZF1	MaSGR1		Negatively regulates chlorophyll catabolism	[27]
Phosphorylation	Tomato	SlPP2C1			Inhibits fruit ripening	[31]
			LeACS2	S460	Participates in ethylene signal transduction	[32]
		PSKR1	DREB2F	Y30	Increases transcription levels of ripening-related genes	[41]
	Banana	Protein kinases	MaACS1	S476, S479	Regulates ethylene biosynthesis	[33]
		MaMPK6-3	MabZIP21	T318, S436	Enhances the transcriptional activation ability of ripening-related genes and accelerates fruit ripening	[34]
		MaMPK2	MabZIP93		Enhances the transcriptional activation of cell wall modification-related genes and promotes fruit ripening	[35]
	Strawberry	FaABI1			Regulates the expression of ripening-related genes	[36]
		SnRK2.6	bHLH3		Suppresses binding to UFGT promoter and negatively regulates fruit coloring.	[40]
	Apple	MdMPK4-14G	MdERF17	T67	Promotes chlorophyll degradation	[37]
		MdSnRK2-I	MdHB1, MdHB2, MdACS1		Regulates ethylene biosynthesis	[38]
			MdCRF4		Suppresses ethylene biosynthesis	[42]
	Litchi	LcSnRK1α	LcbZIP1/3		Activates the expression of metabolic reprogramming genes and maintains energy and redox homeostasis	[39]
Redox modification	Tomato	E4, SlMsrB2	NOR	M138	Reduces the oxidized NOR and restores its function	[45]
	Banana	MaMsrB2	MaNAC42	M134, M135	Reduces the oxidized MaNAC42 and restores its function	[47]
		MaMsrB2	MaAPX1	M36	Possibly regulates the redox status of banana fruit during ripening and senescence	[48]
		MaMsrA7	MaCaM1	M77 M110	Reduces the oxidized MaCaM1 and affects its binding activity with target proteins	[50]
		MaMsrA4	MaEIL9	M129, M130 M282	Reduces the oxidized MaEIL9 and restores its function	[46]
	Longan	DlTrx1	DlGpx	C90	Regulates the redox state during fruit senescence and quality deterioration	[51]
	Sweet pepper		NADP-ICDH	C133, Y450	Regulates NADPH production, affects cell redox state	[52]
			Rboh		Involved in nitro-oxidative stress	[53]
			APXs	C32, Y235		[54]
	Tomato		SlWRKY6	C396	Suppresses the expression of SlSGR1 and SlSAG12	[55]
			BRG3	C206, C212	Reduces the degradation of the ripening repressor WRKY71	[56]
			WRKY71		Enhances binding and transcriptional repression of CAS1, delaying ripening	[56]
Glycosylation	Strawberry		FvHex1, FvHex2		Promotes fruit softening	[58]
			FcXTH1		Affects the stability of protein–ligand complexes	[59]
	Tomato	α-Man, β-Hex			Promotes fruit softening	[60]
		SlSPY	EIN2	S771, T821	Enhances protein stability and affects ethylene signal transduction	[62]

), other post-translational modifications (PTMs), such as methylation and acetylation, also play crucial roles in fruit ripening. Research on protein methylation and acetylation during fruit ripening has predominantly concentrated on histones, with comparatively limited studies addressing non-histone modifications.

Histone methylation involves the addition or removal of methyl groups on lysine or arginine residues, which can significantly influence gene expression [63]. For instance, the histone lysine demethylase SlJMJ6 targets the ripening-related regulator RIN via H3K27 demethylation, leading to the upregulation of genes such as ACS4, ACO1, PL, and TBG4, thereby promoting tomato fruit ripening [63]. In contrast, SlJMJ7 reduces levels of H3K4me3, which suppresses the expression of ripening-related genes and inhibits DML2 expression, resulting in reduced DNA methylation levels. This dual suppression by SlJMJ7 affects the ripening process by both directly and indirectly repressing the expression of ripening-related genes [64].

Protein acetylation, involving the addition or removal of acetyl groups at the protein N-terminus or lysine residues, alters protein structure and function [65]. This modification is mediated by acetyltransferases and deacetylases. Histone acetylation plays a significant role in the regulation of fruit ripening [65]. In apples, MdHDA19 promotes H3K9 deacetylation, influencing ethylene production by recruiting the MdERF4-MdTPL4 complex, which directly inhibits MdACS3a expression [66]. This interaction forms an inhibitory complex comprising MdERF4, MdTPL, and MdHDA19, which regulates fruit ripening. Similarly, the MdHDA19-MdMADS6 protein complex plays a role during the early stages of fruit development [66]. In tomatoes, silencing histone deacetylases SlHDA3 [67], SlHDT1 [68], and SlHDA1 [69] via RNA interference enhances carotenoid accumulation and upregulates ethylene biosynthesis genes, such as ACS2 and ACO1, as well as ripening-related genes RIN, E4, PG, and cell wall metabolism genes HEX, MAN, and XTH5, thereby promoting fruit ripening [67,68,69].

While histone methylation and acetylation are well-established as critical regulators of fruit ripening, the roles of non-histone methylation and acetylation modifications remain relatively unexplored and warrant further investigation. Further research into these modifications, using advanced techniques such as transgenic technologies, RNA interference, and bioinformatics tools, could yield valuable insights into their functions and mechanisms in regulating fruit ripening.

4. Crosstalk Regulation of Fruit Ripening among PTMs and Other Regulations

Fruit ripening is a complex developmental process governed by the dynamic interplay of internal plant hormones, genetic regulatory factors, and external environmental cues. This intricate network encompasses plant hormones, transcriptional regulation, epigenetic modifications, post-translational modifications (PTMs), and various other regulatory factors, forming a comprehensive system that orchestrates fruit development and ripening [4]. Importantly, PTMs do not operate in isolation; one modification can trigger the addition or removal of another, thereby influencing protein recognition and cellular activities in a coordinated manner [70]. In apples, the E3 ubiquitin ligase MdPUB29, which is inhibited by elevated glucose levels, ubiquitinates MdbHLH3 to modulate ethylene biosynthesis. Simultaneously, the protein kinase MdHXK1 phosphorylates MdbHLH3 in response to glucose signals [24]. This interplay between ubiquitination and phosphorylation plays a crucial role in regulating protein stability and abundance, thereby influencing post-ripening fruit quality through the modulation of anthocyanin and ethylene production [24]. Similarly, in apples, MdNAC72 is phosphorylated by MdMAPK3, which subsequently facilitates its recognition by MdPUB24. This interaction promotes the degradation of MdNAC72 through the ubiquitin-proteasome pathway [71]. As a result, the transcriptional repression of cell wall degradation gene MdPG1 by MdNAC72 is substantially reduced. This process underscores the significance of the interplay between phosphorylation and ubiquitination in the regulation of gene expression [71].

Transcription factors are pivotal in the regulation of gene expression, functioning as modulators that either activate or inhibit target genes. They act as intermediaries between transcriptional regulation and PTMs [72]. PTM-related enzymes can directly interact with transcription factors, mediating their modifications and thereby influencing their stability, DNA binding ability, and transcriptional control of downstream genes. For instance, E4/SlMsrB2-mediated redox modification of NOR in tomatoes [45], MaMsrA4-mediated redox modification of MaEIL9, and MaMPK6-3-mediated phosphorylation of MabZIP21 in bananas all affect hormone signaling pathways and the expression of ripening-related genes [34]. These modifications influence the DNA binding capacity and transcriptional activity of the respective transcription factors, thereby modulating the fruit ripening process.

The role of epigenetic modifications in fruit development and ripening has increasingly garnered attention in research. Epigenetic modifications, including DNA methylation, histone PTMs, chromatin remodeling, and the action of non-coding RNA, respond to external environmental signals and regulate chromatin states, thereby modulating gene expression [72]. Research has demonstrated that the interplay between epigenetics mechanisms and transcriptional regulation, as well as the interactions among various epigenetic modifications, plays a crucial role in the regulation of fruit ripening. In the case of tomatoes, for example, the histone H3K4 demethylase SlJMJ7 has been identified as a negative regulator of key genes involved in ethylene biosynthesis genes (ACS2, ACS4, ACO6), transcriptional regulators (RIN, NOR), and DNA demethylation gene DML2 through the demethylation of H3K4me3 marks [64]. The loss of the SlJMJ7 function results in elevated expression of DML2, which in turn causes genome-wide DNA hypomethylation, indirectly facilitating the expression of ripening-related genes. This indicates that SlJMJ7, via histone demethylation, engages in a complex interplay with transcriptional regulation and DNA methylation to coordinate the regulation of fruit ripening [64]. Nevertheless, further detailed investigations into the interactions and mutual influences of various post-translational modifications (PTMs) in the regulation of fruit ripening are necessary to gain a more comprehensive understanding of the underlying molecular mechanisms.

5. Conclusions and Future Perspectives

Fruit ripening is a multifaceted developmental process governed by a dynamic interplay of plant hormones, genetic factors, and environmental cues. This intricate regulatory network integrates transcriptional regulation, epigenetic modifications, and a range of post-translational modifications (PTMs) including ubiquitination, phosphorylation, redox modifications, and glycosylation (Figure 1). Ubiquitination, in particular, plays a crucial role by involving E3 ubiquitin ligases that target key regulatory proteins for degradation, thereby modulating the progression of fruit ripening. Phosphorylation, orchestrated by protein kinases and phosphatases, exerts a dynamic influence on the activity and stability of proteins involved in fruit ripening. Redox modifications, mediated by enzymes such as Msr, Trx, and Grx, act as molecular switches that modulate protein functions. Glycosylation, including both N- and O-glycosylation, plays a critical role in stabilizing proteins and regulating their activity, thereby significantly affecting fruit softening and ethylene signaling. Additionally, methylation and acetylation, particularly of histones, play essential roles in gene expression regulation during fruit ripening.

Despite considerable advancements, our understanding of post-translational modifications (PTMs) in fruit ripening remains incomplete. Research on non-histone methylation and acetylation is still limited, leaving many potential regulatory mechanisms unexamined. The crosstalk between different PTMs and their integrated impact on fruit ripening is not yet well characterized, constraining our comprehension of how these modifications interact to regulate protein functions and gene expression. Moreover, existing studies have predominantly focused on model fruit species such as tomatoes, thereby limiting our ability to discern the conservation and divergence of PTM-mediated regulatory networks across different fruit types.

Future research should broaden its focus to include investigations of non-histone methylation and acetylation in fruit ripening, to uncover new regulatory layers and mechanisms. In-depth studies on the crosstalk between different PTMs are essential to elucidate their coordinated roles in the regulation of fruit ripening. To achieve this, advanced technologies such as high-throughput sequencing, VIGS, RNAi, and proteomic sequencing should be employed to identify and characterize PTM-related enzymes and their substrates. Furthermore, extending research to non-model fruit species will be instrumental in uncovering both conserved and unique regulatory mechanisms involved in fruit ripening. Integrating multi-omics approaches, including genomics, proteomics, and epigenomics, will offer a more comprehensive understanding of the complex regulatory networks governing this process. Such insights have the potential to significantly enhance post-harvest storage and preservation strategies, as well as drive advancements in fruit quality and overall agricultural productivity.