The ethylene-responsive transcription factor ERF024 is a novel regulator of climacteric fruit ripening in melon.

Fruit ripening is an essential developmental stage in Angiosperms triggered by hormonal signals such as ethylene, a major player in climacteric ripening. Melon is a unique crop showing both climacteric and non-climacteric cultivars, offering an ideal model for dissecting the genetic mechanisms underpinning this process. The major quantitative trait locus ETHQV8.1 was previously identified as a key regulator of melon fruit ripening. Here, we narrowed down ETHQV8.1 to a precise genomic region containing a single gene, the transcription factor CmERF024. Functional validation using CRISPR/Cas9 knock-out plants unequivocally identified CmERF024 as the causal gene governing ETHQV8.1. The erf024 mutants exhibited suppression of ethylene production, leading to a significant delay and attenuation of fruit ripening. Integrative multi-omic analyses encompassing RNA-seq, DAP-seq, and DNase-seq revealed the association of CmERF024 with chromatin accessibility and gene expression dynamics throughout fruit ripening. Our data suggest CmERF024 as a novel regulator of climacteric fruit ripening in melon.

Abscisic acid controls sugar accumulation essential to strawberry fruit ripening via the FaRIPK1-FaTCP7-FaSTP13/FaSPT module.

Strawberry is considered as a model plant for studying the ripening of abscisic acid (ABA)-regulated non-climacteric fruits, a process in which sugar plays a fundamental role, while how ABA regulates sugar accumulation remains unclear. This study provides a direct line of physiological, biochemical, and molecular evidence that ABA signaling regulates sugar accumulation via the FaRIPK1-FaTCP7-FaSTP13/FaSPT signaling pathway. Herein, FaRIPK1, a red-initial protein kinase 1 previously identified in strawberry fruit, not only interacted with the transcription factor FaTCP7 (TEOSINTE BRANCHEN 1, CYCLOIDEA, and PCF) but also phosphorylated the critical Ser89 and Thr93 sites of FaTCP7, which negatively regulated strawberry fruit ripening, as evidenced by the transient overexpression (OE) and virus-induced gene silencing transgenic system. Furthermore, the DAP-seq experiments revealed that FvTCP7 bound the motif "GTGGNNCCCNC" in the promoters of two sugar transporter genes, FaSTP13 (sugar transport protein 13) and FaSPT (sugar phosphate/phosphate translocator), inhibiting their transcription activities as determined by the electrophoretic mobility shift assay, yeast one-hybrid, and dual-luciferase reporter assays. The downregulated FaSTP13 and FaSPT transcripts in the FaTCP7-OE fruit resulted in a reduction in soluble sugar content. Consistently, the yeast absorption test revealed that the two transporters had hexose transport activity. Especially, the phosphorylation-inhibited binding of FaTCP7 to the promoters of FaSTP13 and FaSPT could result in the release of their transcriptional activities. In addition, the phosphomimetic form FaTCP7S89D or FaTCP7T93D could rescue the phenotype of FaTCP7-OE fruits. Importantly, exogenous ABA treatment enhanced the FaRIPK1-FaTCP7 interaction. Overall, we found direct evidence that ABA signaling controls sugar accumulation during strawberry fruit ripening via the "FaRIPK1-FaTCP7-FaSTP13/FaSPT" module.

Persulfidation and phosphorylation of transcription factor SlWRKY6 differentially regulate tomato fruit ripening.

Cysteine desulfhydrase (LCD) catalyzes the generation of the signaling molecule hydrogen sulfide (H2S) in plants. In this study, we found that H2S can inhibit tomato (Solanum lycopersicum) fruit ripening and SlWRKY6 undergoes differential protein persulfidation in SlLCD1-overexpressing leaves. Then, further study indicated that SlWRKY6 could be persulfidated by H2S at Cys396. By construction of slwrky6 mutants and SlWRKY6-OE lines, we found that SlWRKY6 positively regulates leaf senescence and fruit ripening by activating the transcription of ripening-related genes STAYGREEN 1 (SlSGR1) and Senescence-Associated Gene 12 (SlSAG12). In addition, SlWRKY6 interacted with kinase SlMAPK4 and was phosphorylated at Ser33. Dual luciferase transient expression assays and electrophoretic mobility shift assays indicated that SlWRKY6 persulfidation attenuated its transcriptional regulation of target genes SlSGR1 and SlSAG12, whereas SlWRKY6 phosphorylation by SlMAPK4 activated the transcription of target genes to promote fruit ripening. Moreover, we provided evidence that SlWRKY6 persulfidation attenuated its SlMAPK4-mediated phosphorylation to inhibit tomato fruit ripening. By transient expression of SlWRKY6, SlWRKY6C396A, SlWRKY6S33A and SlWRKY6S33D in slwrky6 fruits, we found that SlWRKY6 persulfidation attenuated the expression of SlSGR1 and SlSAG12 thereby delaying tomato fruit ripening, while SlWRKY6 phosphorylation increased the expression of target genes. As tomato fruits ripened, endogenous H2S production decreased, while SlMAPK4 expression increased. Therefore, our findings reveal a model in which SlWRKY6 persulfidation due to higher endogenous H2S levels in un-ripened fruit inhibits its ability to activate SlSGR1 and SlSAG12 expression, while SlWRKY6 phosphorylation by SlMAPK4 activates its transcriptional activity, thereby promoting tomato fruit ripening.

Pepper catalase: a broad analysis of its modulation during fruit ripening and by nitric oxide.

Catalase is a major antioxidant enzyme located in plant peroxisomes that catalyzes the decomposition of H2O2. Based on our previous transcriptomic (RNA-Seq) and proteomic (iTRAQ) data at different stages of pepper (Capsicum annuum L.) fruit ripening and after exposure to nitric oxide (NO) enriched atmosphere, a broad analysis has allowed us to characterize the functioning of this enzyme. Three genes were identified, and their expression was differentially modulated during ripening and by NO gas treatment. A dissimilar behavior was observed in the protein expression of the encoded protein catalases (CaCat1-CaCat3). Total catalase activity was down-regulated by 50% in ripe (red) fruits concerning immature green fruits. This was corroborated by non-denaturing polyacrylamide gel electrophoresis, where only a single catalase isozyme was identified. In vitro analyses of the recombinant CaCat3 protein exposed to peroxynitrite (ONOO-) confirmed, by immunoblot assay, that catalase underwent a nitration process. Mass spectrometric analysis identified that Tyr348 and Tyr360 were nitrated by ONOO-, occurring near the active center of catalase. The data indicate the complex regulation at gene and protein levels of catalase during the ripening of pepper fruits, with activity significantly down-regulated in ripe fruits. Nitration seems to play a key role in this down-regulation, favoring an increase in H2O2 content during ripening. This pattern can be reversed by the exogenous NO application. While plant catalases are generally reported to be tetrameric, the analysis of the protein structure supports that pepper catalase has a favored quaternary homodimer nature. Taken together, data show that pepper catalase is down-regulated during fruit ripening, becoming a target of tyrosine nitration, which provokes its inhibition.

Abscisic acid triggers vitamin E accumulation by transient transcript activation of VTE5 and VTE6 in sweet cherry fruits.

Tocopherols are lipophilic antioxidants known as vitamin E and synthesized from the condensation of two metabolic pathways leading to the formation of homogentisate and phytyl diphosphate. While homogentisate is derived from tyrosine metabolism, phytyl diphosphate may be formed from geranylgeranyl diphosphate or phytol recycling from chlorophyll degradation. Here, we hypothesized that abscisic acid (ABA) could induce tocopherol biosynthesis in sweet cherries by modifying the expression of genes involved in vitamin E biosynthesis, including those from the phytol recycling pathway. Hence, the expression of key tocopherol biosynthesis genes was determined together with vitamin E and chlorophyll contents during the natural development of sweet cherries on the tree. Moreover, the effects of exogenously applied ABA on the expression of key tocopherol biosynthesis genes were also investigated during on-tree fruit development, and tocopherols and chlorophylls contents were analyzed. Results showed that the expression of tocopherol biosynthesis genes, including VTE5, VTE6, HPPD and HPT showed contrasting patterns of variation, but in all cases, increased by 2- and 3-fold over time during fruit de-greening. This was not the case for GGDR and VTE4, the first showing constitutive expression during fruit development and the second with marked down-regulation at ripening onset. Furthermore, exogenous ABA stimulated the production of both α- and γ-tocopherols by 60% and 30%, respectively, promoted chlorophyll degradation and significantly enhanced VTE5 and VTE6 expression, and also that of HPPD and VTE4, altogether increasing total tocopherol accumulation. In conclusion, ABA increases promote the transcription of phytol recycling enzymes, which may contribute to vitamin E biosynthesis during fruit development in stone fruits like sweet cherries.

A clathrin-related protein FaRRP1/SCD2 integrates ABA trafficking and signaling to regulate strawberry fruit ripening.

Abscisic acid (ABA) is a critical regulator for nonclimacteric fruit ripening such as in the model plant of strawberry (Fragaria × ananassa). Although FaRRP1 is proposed to participate in clathrin-mediated endocytosis of ABA, its action molecular mechanisms in ABA signaling are not fully understood. Here, using our isolated FaRRP1 (ripening-regulation protein) and candidate ABA receptor FaPYL2 and FaABAR from strawberry fruit, a series of silico and molecular interaction analyses demonstrate that they all bind to ABA, and FaRRP1 binds both FaPYL2 and FaABAR; by contrast, the binding affinity of FaRRP1 to FaPYL2 is relatively higher. Interestingly, the binding of FaRRP1 to FaPYL2 and FaABAR affects the perception affinity to ABA. Furthermore, exogenous ABA application and FaRRP1 transgenic analyses confirm that FaRRP1 participates in clathrin-mediated endocytosis and vesicle transport. Importantly, FaRRP1, FaPYL2, and FaABAR all trigger the initiation of strawberry fruit ripening at physiological and molecular levels. In conclusion, FaRRP1 not only binds to ABA but also affects the binding affinity of FaPYL2 and FaABAR to ABA, thus promoting strawberry fruit ripening. Our findings provide novel insights into the role of FaRRP1 in ABA trafficking and signaling, at least in strawberry, a model plant for nonclimacteric fruit ripening.

Transcriptome and weighted gene co-expression network analyses reveal key genes and pathways involved in early fruit ripening in Citrus sinensis.

BACKGROUND: The fruit ripening period is an important target trait in fruit tree crop breeding programs. Thus, citrus tree breeders seek to develop extreme early ripening cultivars that allow optimization of citrus maturation periods. In this study, we explored the regulatory network involved in fruit ripening in Citrus sinensis using the 'Newhall' navel orange variety and its early-ripening mutant, 'Gannanzao'. This research will provide a basis for further research on important signaling pathways, gene functions and variety breeding of Citrus sinensis related to fruit ripening period. RESULTS: Physiological analyses suggested that early fruit ripening in 'Gannanzao' is regulated by early accumulation of abscisic acid (ABA), persistently high levels of jasmonic acid (JA), and higher sucrose content in the pericarp. Pericarp samples from 'Gannanzao' and 'Newhall' navel oranges were sampled for RNA sequencing analysis at 180, 200, and 220 days after flowering; 1430 differentially expressed genes (DEGs) were identified. Functional enrichment analysis indicated that these DEGs were mainly enriched in the plant hormone signal transduction and sugar metabolism pathways, as well as other pathways related to fruit ripening. Important DEGs associated with fruit ripening in 'Gannanzao' included genes involved in ABA and JA metabolism and signal transduction, as well as sugar metabolism. Weighted gene co-expression network analysis showed that the deep pink module had the strongest correlations with ABA content, JA content, and early ripening. Based on gene functionality and gene expression analyses of 37 genes in this module, two candidate hub genes and two ethylene response factor 13 (ERF13) genes (Cs_ont_5g000690 and Cs_ont_5g000700) were identified as key genes regulated by ABA and JA signaling. These findings will help to clarify the mechanisms that underlie early citrus fruit ripening and will lead to the development of excellent genetic resources for further breeding of extreme early-ripening varieties. CONCLUSIONS: Through analyses of the 'Newhall' navel orange cultivar and its early-ripening mutant 'Gannanzao', we identified genes involved in ABA and JA metabolism, signal transduction, and sugar metabolism that were related to fruit ripening. Among these, two ERF13 genes were inferred to be key genes in the regulation of fruit ripening. These findings provide insights into the genetic architecture related to early fruit ripening in C. sinensis.

Genome-wide analysis and functional validation reveal the role of late embryogenesis abundant genes in strawberry (Fragaria × ananassa) fruit ripening.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play important roles in plant growth and development, as well as stresses responsiveness. Nowadays, it has been found that LEAs also have function in fruit ripening. However, the comprehensive analysis on a genome-wide basis of LEA family remains limited, and the role of LEA in fruit ripening has not been fully explored yet, especially in strawberry, an economic important plant and ideal material for studying fruit ripening. RESULTS: In this study, a total of 266 putative LEA proteins were identified and characterized in strawberry genome. Subcellular localization prediction indicated that they were mostly localized in chloroplast, cytoplasm and nucleus. Duplication events detection revealed that whole genome duplication or segmental was the main driver for the expansion of LEA family in strawberry. The phylogenetic analysis suggested that FaLEAs were classified into eight groups, among which, LEA2 was the largest subgroup with 179 members, followed by LEA3, dehydrin (DHN), LEA4 and SMP (seed maturation protein). The LEA1 and DHN groups were speculated to play dominant roles in strawberry fruit development and ripening, according to their larger proportion of members detected as differentially expressed genes during such process. Notably, the expression of FaLEA167 belonging to LEA1 group was altered by strawberry maturation, and inhibited by overexpression of negative regulators of ripening (a cytosolic/plastid glyceraldehyde-3-phosphate dehydrogenase, FaGAPC2 and a cytosolic pyruvate kinase, FaPKc2.2). Subsequently, overexpression of FaLEA167 significantly increased the percentage of fruit at green stage, while reduced the full red fruit proportion. In consistent, the anthocyanins content and the fruit skin color variable reflecting a range from greenness to redness (a* value) were significantly reduced. Whereas, FaLEA167 overexpression apparently up-regulated citric acid, soluble protein and malondialdehyde content, but had no obvious effects on total soluble solids, sugar, flavonoids, phenolics content and antioxidant capacity. CONCLUSIONS: These findings not only provided basic information of FaLEA family for further functional research, but also revealed the involvement of FaLEA167 in negatively regulating strawberry fruit ripening, giving new insights into understanding of FaLEA functions.

Alpha-expansins: more than three decades of wall creep and loosening in fruits.

Expansins are proteins without catalytic activity, but able to break hydrogen bonds between cell wall polysaccharides hemicellulose and cellulose. This proteins were reported for the first time in 1992, describing cell wall extension in cucumber hypocotyls caused particularly by alpha-expansins. Although these proteins have GH45 and CBM63 domains, characteristic of enzymes related with the cleavage of cell wall polysaccharides, demonstrating in vitro that they extend plant cell wall. Its participation has been associated to molecular processes such as development and growing, fruit ripening and softening, tolerance and resistance to biotic and abiotic stress and seed germination. Structural insights, facilitated by bioinformatics approaches, are highlighted, shedding light on the intricate interactions between alpha-expansins and cell wall polysaccharides. After more than thirty years of its discovery, we want to celebrate the knowledge of alpha-expansins and emphasize their importance to understand the phenomena of disassembly and loosening of the cell wall, specifically in the fruit ripening phenomena, with this state-of-the-art dedicated to them.

Genome-wide identification of MAPK family in papaya (Carica papaya) and their involvement in fruit postharvest ripening.

BACKGROUND: Papaya (Carica papaya) is an economically important fruit cultivated in the tropical and subtropical regions of China. However, the rapid softening rate after postharvest leads to a short shelf-life and considerable economic losses. Accordingly, understanding the mechanisms underlying fruit postharvest softening will be a reasonable way to maintain fruit quality and extend its shelf-life. RESULTS: Mitogen-activated protein kinases (MAPKs) are conserved and play essential roles in response to biotic and abiotic stresses. However, the MAPK family remain poorly studied in papaya. Here, a total of nine putative CpMAPK members were identified within papaya genome, and a comprehensive genome-wide characterization of the CpMAPKs was performed, including evolutionary relationships, conserved domains, gene structures, chromosomal locations, cis-regulatory elements and expression profiles in response to phytohormone and antioxidant organic compound treatments during fruit postharvest ripening. Our findings showed that nearly all CpMAPKs harbored the conserved P-loop, C-loop and activation loop domains. Phylogenetic analysis showed that CpMAPK members could be categorized into four groups (A-D), with the members within the same groups displaying high similarity in protein domains and intron-exon organizations. Moreover, a number of cis-acting elements related to hormone signaling, circadian rhythm, or low-temperature stresses were identified in the promoters of CpMAPKs. Notably, gene expression profiles demonstrated that CpMAPKs exhibited various responses to 2-chloroethylphosphonic acid (ethephon), 1-methylcyclopropene (1-MCP) and the combined ascorbic acid (AsA) and chitosan (CTS) treatments during papaya postharvest ripening. Among them, both CpMAPK9 and CpMAPK20 displayed significant induction in papaya flesh by ethephon treatment, and were pronounced inhibition after AsA and CTS treatments at 16 d compared to those of natural ripening control, suggesting that they potentially involve in fruit postharvest ripening through ethylene signaling pathway or modulating cell wall metabolism. CONCLUSION: This study will provide some valuable insights into future functional characterization of CpMAPKs, and hold great potential for further understanding the molecular mechanisms underlying papaya fruit postharvest ripening.

Identification and virus-induced gene silencing (VIGS) analysis of methyltransferase affecting tomato (Solanum lycopersicum) fruit ripening.

MAIN CONCLUSION: Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases. Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.

Calmodulin-like protein CML15 interacts with PP2C46/65 to regulate papaya fruit ripening via integrating calcium, ABA and ethylene signals.

It is well known that calcium, ethylene and abscisic acid (ABA) can regulate fruit ripening, however, their interaction in the regulation of fruit ripening has not yet been fully clarified. The present study found that the expression of the papaya calcium sensor CpCML15 was strongly linked to fruit ripening. CpCML15 could bind Ca2+ and served as a true calcium sensor. CpCML15 interacted with CpPP2C46 and CpPP2C65, the candidate components of the ABA signalling pathways. CpPP2C46/65 expression was also related to fruit ripening and regulated by ethylene. CpCML15 was located in the nucleus and CpPP2C46/65 were located in both the nucleus and membrane. The interaction between CpCML15 and CpPP2C46/65 was calcium dependent and further repressed the activity of CpPP2C46/65 in vitro. The transient overexpression of CpCML15 and CpPP2C46/65 in papaya promoted fruit ripening and gene expression related to ripening. The reduced expression of CpCML15 and CpPP2C46/65 by virus-induced gene silencing delayed fruit colouring and softening and repressed the expression of genes related to ethylene signalling and softening. Moreover, ectopic overexpression of CpCML15 in tomato fruit also promoted fruit softening and ripening by increasing ethylene production and enhancing gene expression related to ripening. Additionally, CpPP2C46 interacted with CpABI5, and CpPP2C65 interacted with CpERF003-like, two transcriptional factors in ABA and ethylene signalling pathways that are closely related to fruit ripening. Taken together, our results showed that CpCML15 and CpPP2Cs positively regulated fruit ripening, and their interaction integrated the cross-talk of calcium, ABA and ethylene signals in fruit ripening through the CpCML15-CpPP2Cs-CpABI5/CpERF003-like pathway.

Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour-related volatiles in genome-edited tomato RIN mutants.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have inferior taste/flavour. To address this, we used genome editing to generate newer alleles of RIN (rinCR ) by targeting the K-domain. Unlike previously reported CRISPR alleles, the rinCR alleles displayed delayed onset of ripening, suggesting that the mutated K-domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and progenitor line. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, a dominant-negative phototropin1 mutant, which enhances carotenoid levels in tomato fruits. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. The metabolome of Nps1/rinCR fruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatiles in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 mutation. The Nps1 introgression into tomato provides a promising strategy for developing cultivars with extended shelf life, improved taste, and flavour.

Protein kinase FaSnRK2.6 phosphorylates transcription factor FabHLH3 to regulate anthocyanin homeostasis during strawberry fruit ripening.

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA). However, the signaling of ABA in the regulation of fruit coloration is not fully understood. Here, a transcription factor FabHLH3 key to fruit coloration is identified by yeast two hybrid library screening using FaSnRK2.6 as a bait, an ABA core signaling component negative to ripening. Indeed, this interaction is also confirmed by firefly luciferase complementation assay and pull-down assay. RT-qPCR and Western blotting analysis confirm FabHLH3 is expressed ubiquitously in strawberry and stably during fruit development. Manipulating both FabHLH3 and FaSnRK2.6 expression by overexpression and interference demonstrates that FabHLH3 and FaSnRK2.6 promote and inhibit strawberry fruit coloration, respectively, using the marker gene FaUFGT, key to anthocyanin biosynthesis. FaSnRK2.6 can phosphorylate FabHLH3, which promotes FaUFGT expression by the directly binding to its promoter. The phosphorylation inhibits the binding of FabHLH3 to FaUFGT promoter, consequently suppressing FaUFGT expression. Altogether, FaSnRK2.6, a negative kinase in ripening, interacts with and phosphorylates FabHLH3 to suppress FaUFGT expression. With the increase of ABA content in strawberry fruit ripening, the expression of FaSnRK2.6 decreased, which released FabHLH3 transcription activity and enhanced FaUFGT expression, finally promoting the coloration. Thus, our findings fill a gap how FaSnRK2.6 negatively regulates strawberry fruit coloration and ripening by FabHLH3.

Galactinol synthase 2 influences the metabolism of chlorophyll, carotenoids, and ethylene in tomato fruits.

Galactinol synthase (GolS), which catalyses the synthesis of galactinol, is the first critical enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs) and contributes to plant growth and development, and resistance mechanisms. However, its role in fruit development remains largely unknown. In this study, we used CRISPR/Cas9 gene-editing technology in tomato (Solanum lycopersicum) to create the gols2 mutant showing uniformly green fruits without dark-green shoulders, and promoting fruit ripening. Analysis indicated that galactinol was undetectable in the ovaries and fruits of the mutant, and the accumulation of chlorophyll and chloroplast development was suppressed in the fruits. RNA-sequencing analysis showed that genes related to chlorophyll accumulation and chloroplast development were down-regulated, including PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, GOLDEN 2-LIKE 2, and CHLOROPHYLL A/B-BINDING PROTEINS. In addition, early color transformation and ethylene release was prompted in the gols2 lines by regulation of the expression of genes involved in carotenoid and ethylene metabolism (e.g. PHYTOENE SYNTHASE 1, CAROTENE CIS-TRANS ISOMERASE, and 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2/4) and fruit ripening (e.g. RIPENING INHIBITOR, NON-RIPENING, and APETALA2a). Our results provide evidence for the involvement of GolS2 in pigment and ethylene metabolism of tomato fruits.

Ascorbate peroxidase in fruits and modulation of its activity by reactive species.

Ascorbate peroxidase (APX) is one of the enzymes of the ascorbate-glutathione cycle and is the key enzyme that breaks down H2O2 with the aid of ascorbate as an electron source. APX is present in all photosynthetic eukaryotes from algae to higher plants and, at the cellular level, it is localized in all subcellular compartments where H2O2 is generated, including the apoplast, cytosol, plastids, mitochondria, and peroxisomes, either in soluble form or attached to the organelle membranes. APX activity can be modulated by various post-translational modifications including tyrosine nitration, S-nitrosation, persulfidation, and S-sulfenylation. This allows the connection of H2O2 metabolism with other relevant signaling molecules such as NO and H2S, thus building a complex coordination system. In both climacteric and non-climacteric fruits, APX plays a key role during the ripening process and during post-harvest, since it participates in the regulation of both H2O2 and ascorbate levels affecting fruit quality. Currently, the exogenous application of molecules such as NO, H2S, H2O2, and, more recently, melatonin is seen as a new alternative to maintain and extend the shelf life and quality of fruits because they can modulate APX activity as well as other antioxidant systems. Therefore, these molecules are being considered as new biotechnological tools to improve crop quality in the horticultural industry.

Auxin responsive factor MdARF17 promotes ethylene synthesis in apple fruits by activating MdERF003 expression.

KEY MESSAGE: Auxin (AUX) promotion of apple fruit ripening is ethylene-dependent, and AUX-MdARF17-MdERF003 plays a role in AUX-promoted ethylene synthesis in apple. Phytohormones play important roles in plant growth and fleshy fruit ripening, and the phytohormone auxin (AUX) can either promote or inhibit the ripening of fleshy fruits. Although AUX can influence ethylene (ETH) synthesis in apple (Malus domestica) fruits by affecting ETH system II, this mechanism remains to be explored. Here, we identified an ETH response factor (ERF) family transcription factor, MdERF003, whose expression could be activated by naphthalene acetic acid. The transient silencing of MdERF003 inhibited ETH synthesis in fruits, and MdERF003 could bind to the MdACS1 promoter. To explore the upstream target genes of MdERF003, we screened the MdARF family members by yeast one-hybrid assays of the MdERF003 promoter, and found that the transcription factor MdARF17, which showed AUX-promoted expression, could bind to the MdERF003 promoter and promote its expression. Finally, we silenced MdERF003 in apple fruits overexpressing MdARF17 and found that MdERF003 plays a role in MdARF17-promoted ETH synthesis in apple. Thus, AUX-MdARF17-MdERF003 promotes ETH synthesis in apple fruits.

Characterization of leucine aminopeptidase (LAP) activity in sweet pepper fruits during ripening and its inhibition by nitration and reducing events.

KEY MESSAGE: Pepper fruits contain two leucine aminopeptidase (LAP) genes which are differentially modulated during ripening and by nitric oxide. The LAP activity increases during ripening but is negatively modulated by nitration. Leucine aminopeptidase (LAP) is an essential metalloenzyme that cleaves N-terminal leucine residues from proteins but also metabolizes dipeptides and tripeptides. LAPs play a fundamental role in cell protein turnover and participate in physiological processes such as defense mechanisms against biotic and abiotic stresses, but little is known about their involvement in fruit physiology. This study aims to identify and characterize genes encoding LAP and evaluate their role during the ripening of pepper (Capsicum annuum L.) fruits and under a nitric oxide (NO)-enriched environment. Using a data-mining approach of the pepper plant genome and fruit transcriptome (RNA-seq), two LAP genes, designated CaLAP1 and CaLAP2, were identified. The time course expression analysis of these genes during different fruit ripening stages showed that whereas CaLAP1 decreased, CaLAP2 was upregulated. However, under an exogenous NO treatment of fruits, both genes were downregulated. On the contrary, it was shown that during fruit ripening LAP activity increased by 81%. An in vitro assay of the LAP activity in the presence of different modulating compounds including peroxynitrite (ONOO-), NO donors (S-nitrosoglutathione and nitrosocyteine), reducing agents such as reduced glutathione (GSH), L-cysteine (L-Cys), and cyanide triggered a differential response. Thus, peroxynitrite and reducing compounds provoked around 50% inhibition of the LAP activity in green immature fruits, whereas cyanide upregulated it 1.5 folds. To our knowledge, this is the first characterization of LAP in pepper fruits as well as of its regulation by diverse modulating compounds. Based on the capacity of LAP to metabolize dipeptides and tripeptides, it could be hypothesized that the LAP might be involved in the GSH recycling during the ripening process.

Reassessing the contribution of TOMATO AGAMOUS-LIKE1 to fruit ripening by CRISPR/Cas9 mutagenesis.

KEY MESSAGE: CRISPR/Cas9-edited TOMATO AGAMOUS-LIKE1 (TAGL1) provided new insights into fruit ripening. TOMATO AGAMOUS LIKE 1 (TAGL1) has been identified as playing a key role in the process of tomato fruit development and ripening. We have re-evaluated the functions of TAGL1 using CRISPR/Cas9 mutagenesis. Three KO mutants contained frameshift mutations resulting in premature termination codons due to a 1 bp insertion. TAGL1-KO mutants exhibited dark immature fruits and orange ripening fruits. The fruit shape was characterized by a prominent pointed tip at the end and the pericarp thickness was significantly thinner. TAGL1-KO mutants showed reduced ethylene biosynthesis, increased firmness, and delayed onset of ripening. The chlorophyll content of TAGL1-KO mutants was higher in the mature green stage and the lycopene content of TAGL1-KO mutants in the ripening stage was lower compared to the WT. ACS2, ACS4, ACO1, ACO3, PG2a, PL, PME, EXP1, and PSY1 in the mutants were significantly down-regulated during ripening. Ripening fruits in the double mutant of rin and tagl1 showed a more extreme phenotype than the rin mutant suggesting that the double mutation acts synergistically during ripening. TAGL1-targeted mutagenesis by CRISPR/Cas9 strengthens its regulatory functions controlling ripening parameters and provides new insights into fruit ripening.

A tomato NAC transcription factor, SlNAP1, directly regulates gibberellin-dependent fruit ripening.

In tomato (Solanum lycopersicum), the ripening of fruit is regulated by the selective expression of ripening-related genes, and this procedure is controlled by transcription factors (TFs). In the various plant-specific TF families, the no apical meristem (NAM), Arabidopsis thaliana activating factor 1/2 (ATAF1/2), and cup-shaped cotyledon 2 (CUC2; NAC) TF family stands out and plays a significant function in plant physiological activities, such as fruit ripening (FR). Despite the numerous genes of NAC found in the tomato genome, limited information is available on the effects of NAC members on FR, and there is also a lack of studies on their target genes. In this research, we focus on SlNAP1, which is a NAC TF that positively influences the FR of tomato. By employing CRISPR/Cas9 technology, compared with the wild type (WT), we generated slnap1 mutants and observed a delay in the ethylene production and color change of fruits. We employed the yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) assays to confirm that SlNAP1 directly binds to the promoters of two crucial genes involved in gibberellin (GA) degradation, namely SlGA2ox1 and SlGA2ox5, thus activating their expression. Furthermore, through a yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BIFC) and luciferase (LUC) assays, we established an interaction between SlNAP1 and SlGID1. Hence, our findings suggest that SlNAP1 regulates FR positively by activating the GA degradation genes directly. Additionally, the interaction between SlNAP1 and SlGID1 may play a role in SlNAP1-induced FR. Overall, our study provides important insights into the molecular mechanisms through which NAC TFs regulate tomato FR via the GA pathway.