Impact of Cytochrome P450 Enzyme on Fruit Quality.

Cytochrome P450 enzymes are monooxygenases widely diffused in nature ranging from viruses to man. They can catalyze a very wide range of reactions, including the ketonization of C-H bonds, N/O/S-dealkylation, C-C bond cleavage, N/S-oxidation, hydroxylation, and the epoxidation of C=C bonds. Their versatility makes them valuable across various fields such as medicine, chemistry, and food processing. In this review, we aim to highlight the significant contribution of P450 enzymes to fruit quality, with a specific focus on the ripening process, particularly in grapevines. Grapevines are of particular interest due to their economic importance in the fruit industry and their significance in winemaking. Understanding the role of P450 enzymes in grapevine fruit ripening can provide insights into enhancing grape quality, flavor, and aroma, which are critical factors in determining the market value of grapes and derived products like wine. Moreover, the potential of P450 enzymes extends beyond fruit ripening. They represent promising candidates for engineering crop species that are resilient to both biotic and abiotic stresses. Their involvement in metabolic engineering offers opportunities for enhancing fruit quality attributes, such as taste, nutritional content, and shelf life. Harnessing the capabilities of P450 enzymes in crop improvement holds immense promise for sustainable agriculture and food security.

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.

Methyl jasmonate advances fruit ripening, colour development, and improves antioxidant quality of 'Yoho' and 'Jiro' persimmon.

Methyl jasmonate (MJ) has potential to regulate fruit ripening and quality. 'Yoho' and 'Jiro' persimmons were sprayed with MJ (0, 2, 4, and 6 mM), four weeks before anticipated harvest to evaluate its effects on fruit colour and bioactive compounds. Preharvest MJ application significantly improved fruit colour with increased a*, b*, chroma, and colour index. The MJ 6 mM application had significantly enhanced soluble solids content (SSC), reduced total chlorophyll content in peel and pulp, and soluble and total tannins in persimmons. MJ treatments exhibited higher contents of total phenolics, flavonoids, carotenoids, and antioxidant activities. Additionally, MJ treatments enhanced the activities of shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), catalase (CAT), superoxide dismutase (SOD), peroxidase (POD) and lipoxygenase (LOX) enzymes. Overall, pre-harvest MJ application at 6 mM four weeks before anticipated harvest could be useful for advancing colour and improving bioactive compounds in 'Yoho' and 'Jiro' persimmons.

Epigenetic insights into an epimutant colorless non-ripening: from fruit ripening to stress responses.

The epigenetic machinery has received extensive attention due to its involvement in plant growth, development, and adaptation to environmental changes. Recent studies often highlight the epigenetic regulatory network by discussing various epigenetic mutants across various plant species. However, a systemic understanding of essential epigenetic regulatory mechanisms remains limited due to a lack of representative mutants involved in multiple biological processes. Colorless Non-ripening (Cnr), a spontaneous epimutant isolated from a commercial population, was initially characterized for its role in fruit ripening regulation. Cnr fruits exhibit an immature phenotype with yellow skin, attributed to hypermethylation of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-CNR (SlSPL-CNR) promoter, resulting in the repression of gene expression. In addition to DNA methylation, this process also involves histone modification and microRNA, integrating multiple epigenetic regulatory factors. Interestingly, knockout mutants of SlSPL-CNR display phenotypical distinctions from Cnr in fruit ripening, indicating complex genetic and epigenetic control over the non-ripening phenotype in Cnr fruits. Accumulating evidence suggests that Cnr epimutation is pleiotropic, participating in various biological processes such as Cd stress, Fe deficiency, vivipary, and cell death. Therefore, the Cnr epimutant serve as an excellent model for unveiling how epigenetic mechanisms are involved in diverse biological processes. This review paper focuses on recent research advances regarding the Cnr epimutant, delving into its complex genetic and epigenetic regulatory mechanisms, with the aim of enhancing our understanding and facilitating the development of high-quality, high-yield crops through epigenetic modification.

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.

Effect of SlSAHH2 on metabolites in over-expressed and wild-type tomato fruit.

Background: Tomato (Solanum lycopersicum) is an annual or perennial herb that occupies an important position in daily agricultural production. It is an essential food crop for humans and its ripening process is regulated by a number of genes. S-adenosyl-l-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) is widespread in organisms and plays an important role in regulating biological methylation reactions. Previous studies have revealed that transgenic tomato that over-express SlSAHH2 ripen earlier than the wild-type (WT). However, the differences in metabolites and the mechanisms driving how these differences affect the ripening cycle are unclear. Objective: To investigate the effects of SlSAHH2 on metabolites in over-expressed tomato and WT tomato. Methods: SlSAHH2 over-expressed tomato fruit (OE-5# and OE-6#) and WT tomato fruit at the breaker stage (Br) were selected for non-targeted metabolome analysis. Results: A total of 733 metabolites were identified by mass spectrometry using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Human Metabolome database (HMDB). The metabolites were divided into 12 categories based on the superclass results and a comparison with the HMDB. The differences between the two databases were analyzed by PLS-DA. Based on a variable important in projection value >1 and P < 0.05, 103 differential metabolites were found between tomato variety OE-5# and WT and 63 differential metabolites were found between OE-6# and WT. These included dehydrotomatine, L-serine, and gallic acid amongst others. Many metabolites are associated with fruit ripening and eight common metabolites were found between the OE-5# vs. WT and OE-6# vs. WT comparison groups. The low L-tryptophan expression in OE-5# and OE-6# is consistent with previous reports that its content decreases with fruit ripening. A KEGG pathway enrichment analysis of the significantly different metabolites revealed that in the OE-5# and WT groups, up-regulated metabolites were enriched in 23 metabolic pathways and down-regulated metabolites were enriched in 11 metabolic pathways. In the OE-6# and WT groups, up-regulated metabolites were enriched in 29 pathways and down-regulated metabolites were enriched in six metabolic pathways. In addition, the differential metabolite changes in the L-serine to flavonoid transformation metabolic pathway also provide evidence that there is a phenotypic explanation for the changes in transgenic tomato. Discussion: The metabolomic mechanism controlling SlSAHH2 promotion of tomato fruit ripening has been further elucidated.

Unraveling the key step in the aroma puzzle: Insights into alcohol acyltransferases in strawberries.

Alcohol acyltransferases (AATs) play a crucial role in catalyzing the transfer of acyl groups, contributing to the diverse aroma of fruits, including strawberries. In this research we identified nine AAT genes in strawberries through a comprehensive analysis involving phylogenetics, gene structure, conserved motifs, and structural protein model examinations. The study used the 'Camarosa' strawberry genome database, and experiments were conducted with fruits harvested at different developmental and ripening stages. The transcriptional analysis revealed differential expression patterns among the AAT genes during fruit ripening, with only four genes (SAAT, FaAAT2, FaAAT7, and FaAAT9) showing increased transcript accumulation correlated with total AAT enzyme activity. Additionally, the study employed in silico methods, including sequence alignment, phylogenetic analysis, and structural modeling, to gain insights into the AAT protein model structures with increase expression pattern during fruit ripening. The four modeled AAT proteins exhibited structural similarities, including conserved catalytic sites and solvent channels. Furthermore, the research investigated the interaction of AAT proteins with different substrates, highlighting the enzymes' promiscuity in substrate preferences. The study contributes with valuable information to unveil AAT gene family members in strawberries, providing scientific background for further exploration of their biological characteristics and their role in aroma biosynthesis during fruit ripening.

Integrative Analysis of Metabolome and Transcriptome of Carotenoid Biosynthesis Reveals the Mechanism of Fruit Color Change in Tomato (Solanum lycopersicum).

Tomato fruit ripening is accompanied by carotenoid accumulation and color changes. To elucidate the regulatory mechanisms underlying carotenoid synthesis during fruit ripening, a combined transcriptomic and metabolomic analysis was conducted on red-fruited tomato (WP190) and orange-fruited tomato (ZH108). A total of twenty-nine (29) different carotenoid compounds were identified in tomato fruits at six different stages. The abundance of the majority of the carotenoids was enhanced significantly with fruit ripening, with higher levels of lycopene; (E/Z)-lycopene; and α-, ß- and γ-carotenoids detected in the fruits of WP190 at 50 and 60 days post anthesis (DPA). Transcriptome analysis revealed that the fruits of two varieties exhibited the highest number of differentially expressed genes (DEGs) at 50 DPA, and a module of co-expressed genes related to the fruit carotenoid content was established by WGCNA. qRT-PCR analysis validated the transcriptome result with a significantly elevated transcript level of lycopene biosynthesis genes (including SlPSY2, SlZCIS, SlPDS, SlZDS and SlCRTSO2) observed in WP190 at 50 DPA in comparison to ZH108. In addition, during the ripening process, the expression of ethylene biosynthesis (SlACSs and SlACOs) and signaling (SlEIN3 and SlERF1) genes was also increased, and these mechanisms may regulate carotenoid accumulation and fruit ripening in tomato. Differential expression of several key genes in the fruit of two tomato varieties at different stages regulates the accumulation of carotenoids and leads to differences in color between the two varieties of tomato. The results of this study provide a comprehensive understanding of carotenoid accumulation and ethylene biosynthesis and signal transduction pathway regulatory mechanisms during tomato fruit development.

Involvement of the GH38 Family Exoglycosidase α-Mannosidase in Strawberry Fruit Ripening.

Exoglycosidase enzymes hydrolyze the N-glycosylations of cell wall enzymes, releasing N-glycans that act as signal molecules and promote fruit ripening. Vesicular exoglycosidase α-mannosidase enzymes of the GH38 family (EC 3.2.1.24; α-man) hydrolyze N-glycans in non-reduced termini. Strawberry fruit (Fragaria × ananassa) is characterized by rapid softening as a result of cell wall modifications during the fruit ripening process. Enzymes acting on cell wall polysaccharides explain the changes in fruit firmness, but α-man has not yet been described in F. × ananassa, meaning that the indirect effects of N-glycan removal on its fruit ripening process are unknown. The present study identified 10 GH38 α-man sequences in the F. × ananassa genome with characteristic conserved domains and key residues. A phylogenetic tree built with the neighbor-joining method and three groups of α-man established, of which group I was classified into three subgroups and group III contained only Poaceae spp. sequences. The real-time qPCR results demonstrated that FaMAN genes decreased during fruit ripening, a trend mirrored by the total enzyme activity from the white to ripe stages. The analysis of the promoter regions of these FaMAN genes was enriched with ripening and phytohormone response elements, and contained cis-regulatory elements related to stress responses to low temperature, drought, defense, and salt stress. This study discusses the relevance of α-man in fruit ripening and how it can be a useful target to prolong fruit shelf life.

Persulfidome of Sweet Pepper Fruits during Ripening: The Case Study of Leucine Aminopeptidase That Is Positively Modulated by H2S.

Protein persulfidation is a thiol-based oxidative posttranslational modification (oxiPTM) that involves the modification of susceptible cysteine thiol groups present in peptides and proteins through hydrogen sulfide (H2S), thus affecting their function. Using sweet pepper (Capsicum annuum L.) fruits as a model material at different stages of ripening (immature green and ripe red), endogenous persulfidated proteins (persulfidome) were labeled using the dimedone switch method and identified using liquid chromatography and mass spectrometry analysis (LC-MS/MS). A total of 891 persulfidated proteins were found in pepper fruits, either immature green or ripe red. Among these, 370 proteins were exclusively present in green pepper, 237 proteins were exclusively present in red pepper, and 284 proteins were shared between both stages of ripening. A comparative analysis of the pepper persulfidome with that described in Arabidopsis leaves allowed the identification of 25% of common proteins. Among these proteins, glutathione reductase (GR) and leucine aminopeptidase (LAP) were selected to evaluate the effect of persulfidation using an in vitro approach. GR activity was unaffected, whereas LAP activity increased by 3-fold after persulfidation. Furthermore, this effect was reverted through treatment with dithiothreitol (DTT). To our knowledge, this is the first persulfidome described in fruits, which opens new avenues to study H2S metabolism. Additionally, the results obtained lead us to hypothesize that LAP could be involved in glutathione (GSH) recycling in pepper fruits.

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.

Class I TCP in fruit development: much more than growth.

Fruit development can be viewed as the succession of three main steps consisting of the fruit initiation, growth and ripening. These processes are orchestrated by different factors, notably the successful fertilization of flowers, the environmental conditions and the hormones whose action is coordinated by a large variety of transcription factors. Among the different transcription factor families, TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR (TCP) family has received little attention in the frame of fruit biology despite its large effects on several developmental processes and its action as modulator of different hormonal pathways. In this respect, the comprehension of TCP functions in fruit development remains an incomplete puzzle that needs to be assembled. Building on the abundance of genomic and transcriptomic data, this review aims at collecting available TCP expression data to allow their integration in the light of the different functional genetic studies reported so far. This reveals that several Class I TCP genes, already known for their involvement in the cell proliferation and growth, display significant expression levels in developing fruit, although clear evidence supporting their functional significance in this process remains scarce. The extensive expression data compiled in our study provide convincing elements that shed light on the specific involvement of Class I TCP genes in fruit ripening, once these reproductive organs acquire their mature size. They also emphasize their putative role in the control of specific biological processes such as fruit metabolism and hormonal dialogue.

Evidence Supporting a Role of Alternative Splicing Participates in Melon (Cucumis melo L.) Fruit Ripening.

One key post-transcriptional modification mechanism that dynamically controls a number of physiological processes in plants is alternative splicing (AS). However, the functional impacts of AS on fruit ripening remain unclear. In this research, we used RNA-seq data from climacteric (VED, Harukei 3) and non-climacteric (PI, PS) melon cultivars to explore alternative splicing (AS) in immature and mature fruit. The results revealed dramatic changes in differential AS genes (DAG) between the young and mature fruit stages, particularly in genes involved in fruit development/ripening, carotenoid and capsaicinoid biosynthesis, and starch and sucrose metabolism. Serine/arginine-rich (SR) family proteins are known as important splicing factors in AS events. From the melon genome, a total of 17 SR members were discovered in this study. These genes could be classified into eight distinct subfamilies based on gene structure and conserved motifs. Promoter analysis detected various cis-acting regulatory elements involved in hormone pathways and fruit development. Interestingly, these SR genes exhibited specific expression patterns in reproductive organs such as flowers and ovaries. Additionally, concurrent with the increase in AS levels in ripening fruit, the transcripts of these SR genes were activated during fruit maturation in both climacteric and non-climacteric melon varieties. We also found that most SR genes were under selection during domestication. These results represent a novel finding of increased AS levels and SR gene expression during fruit ripening, indicating that alternative splicing may play a role in fruit maturation.

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.

Movement of ACC oxidase 3 mRNA from seeds to flesh promotes fruit ripening in apple.

Xenia, the phenomenon in which the pollen genotype directly affects the phenotypic characteristics of maternal tissues (i.e., fruit ripening), has applications in crop production and breeding. However, the underlying molecular mechanism has yet to be elucidated. Here, we investigated whether mobile mRNAs from the pollen affect the ripening and quality-related characteristics of the fruit using cross-pollination between distinct Malus domestica (apple) cultivars. We demonstrated that hundreds of mobile mRNAs originating from the seeds are delivered to the fruit. We found that the movement of one of these mRNAs, ACC oxidase 3 (MdACO3), is coordinated with fruit ripening. Salicylic acid treatment, which can cause plasmodesmal closure, blocks MdACO3 movement, indicating that MdACO3 transcripts may move through the plasmodesmata. To assess the role of mobile MdACO3 transcripts in apple fruit, we created MdACO3-GFP-expressing apple seeds using MdACO3-GFP-overexpressing pollen for pollination and showed that MdACO3 transcripts in the transgenic seeds move to the flesh, where they promote fruit ripening. Furthermore, we demonstrated that MdACO3 can be transported from the seeds to fruit in the fleshy-fruited species tomato and strawberry. These results underscore the potential of mobile mRNAs from seeds to influence fruit characteristics, providing an explanation for the xenia phenomenon. Notably, our findings highlight the feasibility of leveraging diverse pollen genomic resources, without resorting to genome editing, to improve fruit quality.

The Transcription Factor MiMYB8 Suppresses Peel Coloration in Postharvest 'Guifei' Mango in Response to High Concentration of Exogenous Ethylene by Negatively Modulating MiPAL1.

Anthocyanin accumulation is regulated by specific genes during fruit ripening. Currently, peel coloration of mango fruit in response to exogenous ethylene and the underlying molecular mechanism remain largely unknown. The role of MiMYB8 on suppressing peel coloration in postharvest 'Guifei' mango was investigated by physiology detection, RNA-seq, qRT-PCR, bioinformatics analysis, yeast one-hybrid, dual-luciferase reporter assay, and transient overexpression. Results showed that compared with the control, low concentration of exogenous ethylene (ETH, 500 mg·L-1) significantly promoted peel coloration of mango fruit (cv. Guifei). However, a higher concentration of ETH (1000 mg·L-1) suppressed color transformation, which is associated with higher chlorophyll content, lower a* value, anthocyanin content, and phenylalanine ammonia-lyase (PAL) activity of mango fruit. M. indica myeloblastosis8 MiMYB8 and MiPAL1 were differentially expressed during storage. MiMYB8 was highly similar to those found in other plant species related to anthocyanin biosynthesis and was located in the nucleus. MiMYB8 suppressed the transcription of MiPAL1 by binding directly to its promoter. Transient overexpression of MiMYB8 in tobacco leaves and mango fruit inhibited anthocyanin accumulation by decreasing PAL activity and down-regulating the gene expression. Our observations suggest that MiMYB8 may act as repressor of anthocyanin synthesis by negatively modulating the MiPAL gene during ripening of mango fruit, which provides us with a theoretical basis for the scientific use of exogenous ethylene in practice.

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.

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.

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.