Molecular and Hormonal Mechanisms Regulating Fleshy Fruit Ripening

Abstract

This article focuses on the molecular and hormonal mechanisms underlying the control of fleshy fruit ripening and quality. Recent research on tomato shows that ethylene, acting through transcription factors, is responsible for the initiation of tomato ripening. Several other hormones, including abscisic acid (ABA), jasmonic acid (JA) and brassinosteroids (BR), promote ripening by upregulating ethylene biosynthesis genes in different fruits. Changes to histone marks and DNA methylation are associated with the activation of ripening genes and are necessary for ripening initiation. Light, detected by different photoreceptors and operating through ELONGATED HYPOCOTYL 5(HY5), also modulates ripening. Re-evaluation of the roles of 'master regulators' indicates that MADS-RIN, NAC-NOR, Nor-like1 and other MADS and NAC genes, together with ethylene, promote the full expression of genes required for further ethylene synthesis and change in colour, flavour, texture and progression of ripening. Several different types of non-coding RNAs are involved in regulating expression of ripening genes, but further clarification of their diverse mechanisms of action is required. We discuss a model that integrates the main hormonal and genetic regulatory interactions governing the ripening of tomato fruit and consider variations in ripening regulatory circuits that operate in other fruits.

Keywords: colour; epigenetics; ethylene; flavour; fruit ripening; plant hormone; softening; transcription factor.

Figure 5

Re-evaluation of the roles of MADS-RIN and NAC-NOR. The structure of rin and nor mutant genes and their mutant phenotypes and differences compared to the corresponding CRISPR/Cas9 MADS-RIN and NAC-NOR mutants are shown. Redrawn from Li et al. [269] and Gao et al. [14]. (a) The rin mutation is the result of a partial deletion and fusion of 2 adjacent genes, RIN and MC. (b) Translated amino acids predicted for RIN and RIN-MC proteins. The colour key shows MADS-box, protein binding, I and K-region for protein structure formation, C-terminal for activation. EAR-motif indicates the repressor region. The numbers refer to amino acid positions and the colours indicate different protein domains. (c) Comparison of the phenotypes of the rin mutant and the RIN-CRISPR mutant. The ripening phenotype is much more severely inhibited in rin fruit compared to RIN-CRISPR [15,18] tomatoes. (d) Diagram of nor tomato mutation and NOR-CRISPR mutant (nor#11 and nor#19). WT tomatoes encode a full-length NAC-NOR protein of 355 aa. The nor mutant produces a truncated protein of 186 aa (NOR186). Comparison of the phenotype of the nor mutation (e) and the NOR-CRISPR mutant (f). The nor mutation produces a truncated protein (Nor186) which has an intact NAC domain. Nor186 causes a much more severe inhibition of ripening compared to the NOR-CRISPR mutant (nor#11 and nor#19), which lacks much of the NAC domain (from Gao et al. [14]).

Figure 6

The range of TFs interacting with the promoters of genes encoding fruit ripening enzymes. Redrawn and modified from Li et al. [15]. (a) Model outlining the role of ethylene and RIN in initiation and progression of climacteric ripening in tomato fruit. Ethylene can initiate ripening in a RIN-independent way leading to partial ripening. However, RIN is required for autocatalytic system-2 of ethylene production and subsequent full ripening. RIN expression is enhanced by ethylene [2,73,269]. Other factors such as NACs (NOR, NOR-like1) affect ethylene production and are also involved in the ripening genetic program [14,15,271]. We only show the involvement of ethylene, but, as discussed in the text and in Figure 7, other hormones directly regulate some ACS and ACO ethylene biosynthesis genes, as does the transcription factor RIN. (b) A model of the role of RIN and ethylene in regulating tomato fruit cell wall changes and softening. Accumulation of CEL2, XYL1, EXP1, PL, Mside1, PG and TBG4 transcripts is stimulated by ethylene [15]. RIN directly or indirectly regulates the transcription of genes involved in cell wall metabolism, such as CEL2, XYL1, EXP1, PL, Mside1 and PME1.9. RIN also inhibits expression of genes such as XTH5 and XTH8, but so far, there is no evidence that NACs including NOR and NOR-like1 inhibit transcripts of any of the cell wall metabolism genes. (c) Ethylene treatment affects expression of cell wall genes and their promoters contain ERF binding sites, which indicates that promoters of these genes might be activated by ERF TFs. RIN also targets cell wall genes and MADS proteins act in the form of protein complex. NACs also regulate softening genes, as indicated in (c). Tomato protein orthologues of FUL and ARF8 can also heterodimerise in vivo, suggesting that MADS-ARF associations may occur in diverse plant species [276]. ncRNAs appear to be involved in regulating ripening gene expression but they are omitted from the model until their diverse modes of action (Section 5) can be clarified. Subfigures (a,b) are modified from Li et al. [15].

Figure 7

Model of tomato fruit ripening regulation. ERFs, ethylene response factors; ARFs, auxin response factors; SA, salicylic acid; ABA, ABA response element binding factors; JA, MYC Family bHLH TFs, etc.; BR, brassinosteroid response factors; MADSs include MADS-box TFs such as RIN, FUL1/2, TAGL1, etc.; NACs include NAC family TFs such as NOR, NOR-like1, etc.

Figure 1

The carotenoid biosynthesis pathway and variations in different fruits (redrawn and modified from Lado et al. [33] and Luan et al. [30]). Each fruit labelled with a number in blue is positioned in the pathway at the level of the predominant carotenoid responsible for its coloration: 1, Lemon (Citrus limon); 2, Sweet orange Pinalate mutant; 3, Tomato tangerine mutant; 4, Grape (Vitis vinifera); Immature green pepper (Capsicum annuum); Kiwifruit (Actinidia chinensis); Peel of immature mandarin (Citrus reticulata, Citrus clementina, Citrus unshiu); Avocado (Persea americana); 5, Tomato (Solanum lycopersicum); Red watermelon (Citrullus lanatus); Pulp of red grapefruit (Citrus paradisi); Red papaya (Carica papaya); Gac (Momordica cochinchinensis); 6, Orange-flesh melon (Cucumis melo); Orange-flesh apricot (Prunus armeniaca); Orange-flesh pumpkin (Cucurbita maxima); 7, Yellow papaya (Carica papaya); Loquat (Eriobotyra japonica); Pulp of mandarin (Citrus reticulata, Citrus clementina, Citrus unshiu); 8, Yellow-flesh peach (Prunus persica); Orange pepper (Capsicum annuum); 9, Mango. PSY, phytoene synthase; PDS, phytoene desaturase; ZISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, lycopene isomerase; LCY, lycopene cyclase; CYCB, chromoplast specific lycopene β-cyclase; CHX, carotene hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin de-epoxidase; NSY, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase. Note that the hormone ABA, which is discussed in Section 3.3, is produced from this pathway. Some of these metabolites are also utilised in the biosynthesis of hormones, such as GA, strigolactones and β-cyclocital, but these pathways are omitted from this figure because they are not discussed in this review.

Figure 2

The anthocyanins biosynthetic pathway and variations in different fruits. This figure is redrawn and modified from Gayomba et al. [44]. The flavonol biosynthetic pathway is illustrated, showing enzymatic steps, and the responsible enzyme is indicated in red. CHI, chalcone isomerase; CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol reductase; OMT1, flavone 3′-O-methyltransferase 1.

Figure 3

Biochemical origin of volatile compounds in fruits (redrawn from Aragüez and Valpuesta [86]; Klee and Tieman [88]). Key aspects of primary metabolism, which provides the substrates for secondary metabolism, are shown on the left. Solid lines indicate a validated step in a pathway, with the responsible enzyme indicated in red. Volatiles are indicated in blue. Broadly, volatiles are derived from the fatty acid (for example, Z-3-hexenol), carotenoid cleavage (for example, geranylacetone) or phenylpropanoid (the C6–C3 compounds) pathways. In addition, volatile alcohols can be reversibly converted to esters by the action of an alcohol acetyltransferase (AAT1) and a carboxymethylesterase (carboxylesterase 1 (CXE1)). LoxC, lipoxygenase C; HPL, fatty acid hydroperoxide lyase; ADH2, alcohol dehydrogenase 2; BCAT, branched-chain amino acid aminotransferases; AADC, aromatic amino acid decarboxylase; CCD1, carotenoid cleavage deoxygenase 1.

Figure 4

Ethylene response of WT and RIN-deficient tomato fruits (reproduced with the permission from Li et al. [15]). Effect of exogenous ethylene and ethylene perception inhibitor 1-MCP treatment on ripening progression of RIN-CRISPR tomato fruit. WT and RIN-CRISPR tomato fruits were picked at MG stages and treated and replenished daily with ethylene (100 ppm) and 1-MCP (10 ppm) or air continually for up to 15 days. Fruits in horizontal rows are biological replicates. Enlarged photos of representative samples are shown compared to WT fruits on the right. The red scale bar represents 2 cm.