Hydrogen sulfide (H2S) is a novel gaseous signaling molecule that plays a crucial role in regulating the ripening and senescence processes of plant fruits. Biaxially oriented polyethylene (BOPE) is a new type of film whose application in the preservation of fruits and vegetables has been rarely reported. This study evaluated the physical properties of BOPE and investigated whether H2S affected its characteristics. Additionally, it examined the impact of combining H2S fumigation with BOPE film packaging on the post-harvest quality retention of tomatoes. The results indicated that BOPE showed a smoother surface than polyethylene (PE) and exhibited excellent barrier properties. Furthermore, the characteristics of BOPE remained unaffected by H2S, thereby creating a more favorable environment for the post-harvest packaging and preservation of tomatoes. When the BOPE film was used in conjunction with H2S treatment, it significantly delayed the discoloration and softening processes of the fruits, while retaining higher levels of total phenolic content, total flavonoids content, and peroxidase (POD) enzyme activity, and inhibiting the increase of malondialdehyde (MDA). Additionally, principal component analysis (PCA) revealed that MDA, total phenolic content, and weight loss were the primary component factors. Correlation analysis demonstrated that MDA content was positively correlated with the water loss rate and total soluble solids, while negatively correlated with POD activity and fruit firmness. Therefore, the combination of BOPE film and H2S treatment can enhance the antioxidant capacity of postharvest tomato fruits, preserve their nutritional quality, and delay fruit senescence. This approach holds significant potential for application in postharvest fruit preservation.

Hydrogen sulfide (H2S), a gaseous signaling molecule, retards plant senescence and fruit ripening via protein persulfidation. However, the molecular mechanism underlying its coordination of fruit ripening with plant growth and development remains undefined. This study found that H2S could delay tomato fruit ripening and leaf senescence, and H2S defective mutant sllcd1 shows premature fruit ripening and stunted growth. Overexpression of SlWRKY6, which could be persulfidated by H2S, promotes fruit ripening and reduces fruit number. Molecular assays demonstrated that SlWRKY6 directly binds to the promoter of SlGIF2, a transcriptional co-activator, to repress its expression. Notably, H2S attenuates the repressive effect through persulfidation of SlWRKY6 to increase SlGIF2 transcription. Furthermore, SlGIF2 physically interacts with the GROWTH-REGULATING FACTOR 1 (SlGRF1). Functional validation showed that SlGRF1-SlGIF2 complex serves as a positive regulator of tomato plant growth and fruit yield while concomitantly delaying fruit ripening. Conversely, slgrf1 gene-edited mutants exhibit reduced plant growth, fruit yield and accelerated ripening. Mechanistically, SlGRF1-SlGIF2 complex synergistically activates two downstream target genes: SlCYCD3;1, a key cell-cycle regulator required for cell proliferation and plant growth, and SlCBSX5, which encodes an H2S biosynthesis enzyme that retards fruit ripening. This study provides a module that SlWRKY6-SlGRF1-SlGIF2 integrates H2S signal and genetically coordinates plant growth, fruit yield and fruit ripening in tomato.

Ethylene promotes fruit ripening, while hydrogen sulfide (H2S) exerts the opposite effects. However, whether H2S antagonizes ethylene-mediated regulatory mechanisms of post-harvest banana pulp ripening and senescence remains unclear. In this study, H2S alone or in combination with ethylene fumigation delayed the yellowing of banana peels by downregulating the expression of chlorophyll degradation genes (MaNYC1, MaSGR1, MaPPH and MaPAO) and prolonged the pulp softening process and reduced the release of endogenous ethylene by comparing to the control treatment. The effect on banana pulp is manifested with the preservation of nutrients, such as starch and soluble protein content, as well as the reduction of the generation of reactive oxygen species (ROS), including superoxide anion (center dot O-2) and hydrogen peroxide (H2O2), inhibiting lipid peroxidation to decrease malondialdehyde (MDA) production, while simultaneously enhancing ABTS radical scavenging rates and antioxidant enzyme activity. Bioinformatics analysis results demonstrated that H2S antagonizes ethylene by affecting the changes in fruit quality in banana pulp. Molecular analysis further revealed that the exogenous application of H2S downregulated the expression of MaACS1, MaPL, MaGWD1 and MaAMY3 genes. Collectively, these results evidence that exogenous H2S application effectively antagonized the effect of ethylene, thereby maintaining nutrient content and antioxidant enzyme activities in postharvest banana fruit.

Biaxially oriented polyethylene (BOPE) is a high-strength, lightweight polyethylene material with superior barrier properties. In this study, peaches were packaged using either polyethylene (PE) or BOPE film. The peaches packaged in BOPE film maintained better firmness, color retention, antioxidant content, and nutritional quality than control peaches and those packaged in PE film. Transcriptome sequencing revealed 1041 differentially expressed genes in BOPE-packaged fruit compared with that in the control, and these were enriched in the pyruvate metabolism, flavonoid biosynthesis, and glutathione metabolism pathways. Physiological analyses demonstrated that BOPE packaging reduced malondialdehyde accumulation and increased peroxidase and phenylalanine ammonia-lyase activity, potentially limiting membrane peroxidation and senescence. BOPE-packaged fruit also retained higher levels of soluble sugars, reducing sugars, free amino acids, and proline, which are critical for fruit flavor and stress adaptation. Together, these results underscore the potential of BOPE as a novel packaging material for the preservation of fruits and vegetables.

Hydrogen sulfide (H2S), as a signaling molecule, is found to delay fruit ripening and senescence by antagonizing the biosynthesis and signaling of ethylene, whereas the mechanism remains unclear. In the current work, exogenous H2S fumigation could alleviate tomato fruit ripening and an ethylene response factor SlERF.D2 was found to be persulfidated at Cys35 by mass spectrometry analysis. Meanwhile, ethylene biosynthesis related genes SlACS1 and SlACO3 were significantly downregulated at gene expression level in H2S-treated fruit. By CRISPR/Cas9 and gene overexpression, we showed that overexpression of SlERF.D2 promoted fruit ripening by accelerating chlorophyll degradation and carotenoid accumulation and upregulating the expression of ripening related genes SlPAO, SlPPH, SlSGR1, SlACS1, SlACS2, SlACS4, SlEIN2, SlACO1, and SlACO3, while the mutation of slerf.d2 delayed fruit ripening. Additionally, slerf.d2 mutant showed delayed ethylene production during tomato fruit ripening. Moreover, SlERF.D2 was found to interact with the kinase SlMAPK4 and was phosphorylated at Ser42 by yeast two-hybrid screening, pull down and LC-MS/MS. By cis-element analysis, electrophoretic mobility shift assay and dual-luciferase assay, SlERF.D2 could activate the transcription of the ethylene pathway-associated gene SlACO3 and SlEIN2. Besides, we provided evidence that SlERF.D2 persulfidation weakened the transcriptional activity of SlERF.D2 on the target gene SlACO3 and SlEIN2. In contrast, SlMAPK4-mediated phosphorylation enhanced SlERF.D2's transcriptional activation activity on SlACO3 and SlEIN2. Therefore, the present research provides insights into the mechanism of H2S in antagonizing the biosynthesis and signaling transduction of ethylene and reveals the importance of SlERF.D2 persulfidation and phosphorylation in dynamically regulating tomato fruit ripening.

Sulfur dioxide (SO2) is a potential signaling molecule, playing a crucial role in regulating multiple physiological processes in organisms. In the present study, we investigate the impact of SO2 on the germination rate of wheat seed subjected to drought stress. Drought stress was stimulated using polyethylene glycol 6000, and the findings revealed that SO2 pretreatment significantly enhanced the germination rate of wheat grain. Additionally, SO2 pretreatment resulted in increased levels of reducing sugars and soluble proteins, as well as elevated amylase activity. Furthermore, SO2 pretreatment of wheat grain significantly reduced the content of superoxide anion, hydrogen peroxide, and malondialdehyde, while increasing the activities of peroxidase (POD), ascorbate peroxidase and catalase. Additionally, SO2 pretreatment was associated with a decrease in lipoxygenase activity and an increase in the levels of endogenous hydrogen sulfide. Principal component analysis revealed that POD is the most influential factor in the seed germination process. These findings suggest that SO2 pretreatment may enhance the germination of wheat grain under drought conditions by facilitating the mobilization of storage materials and improving antioxidant capacity during the germination phase.

Cyanide is produced along with ethylene biosynthesis and beta-cyanoalanine synthase (CAS) plays a crucial role in cyanide detoxification in plants, and its activity increases with fruit ripening. However, the involvement of CAS in pigment metabolism and ethylene biosynthesis during fruit ripening has not been previously reported. In this study, SlCAS1/2 were identified as highly homologous to StCAS1/2 in potato. Among them, SlCAS1 had higher expression level than SlCAS2 during tomato fruit ripening. The silencing of SlCAS1 expression by virus induced gene silencing (VIGS) increased chlorophyll content, decreased anthocyanins accumulation and affected the expression of SlNYC1, SlPPH, SlPAO and SlACO1/3 by comparing with the control. Conversely, overexpression of SlCAS1 (SlCAS1-OE) altered pigment accumulation, upregulated the expression of key anthocyanin biosynthesis genes such as SlDFR and SlANS, and increased the expression of key ethylene metabolism-related genes including SlACS2, SlACS4, SlRIN, and SlNOR, compared to the wild type. In contrast, Slcas1 mutant fruit (Delta SlCAS1) generated by CRISPR/Cas9 technology showed the opposite trend. Transcriptomic analysis comparing the wild type, SlCAS1-OE and Delta SlCAS1 fruit, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that these DEGs were mainly involved in ethylene biosynthesis, fruit ripening, and flavonoid biosynthesis. Principal component analysis (PCA) identified anthocyanin, SlACS2 and SlACO3 as the main factors. Overall, these results showed that SlCAS1 mediated tomato fruit ripening by enhancing anthocyanin and ethylene biosynthesis. These findings provide a new basis for improving tomato fruit color and ripening, offering potential strategies for improving tomato fruit quality.

The market for fresh-cut fruits and vegetables is gradually expanding and is popular among consumers, but fresh-cut fruits and vegetables are highly susceptible to browning, causing a decrease in their quality and nutrition. Although anti-browning reagents and cryopreservation methods are often used for fresh-cut fruits and vegetables, the effects are not satisfactory. In this paper, hydrogen sulfide (H2S) donor NaHS solution was used for fumigation of fresh-cut potatoes to explore the mechanism of H2S signaling on the browning of fresh-cut potatoes at the biochemical level. Fresh-cut potatoes were fumigated with H2S and it was found that H2S treatment maintained better color compared with the browning of water control. Then, total phenolic content, reactive oxygen species-related metabolites hydrogen peroxide (H2O2) and superoxide anion (·O2−), along with malondialdehyde (MDA), the activities of antioxidant enzymes, and the browning-related enzymes polyphenol oxidase (PPO), catalase (CAT), peroxidase (POD), and phenylalanine amine lyase (PAL) were determined. The results of both principal component analysis (PCA) and correlation analyses consistently indicated that CAT activity showed a strong positive correlation with the browning degree of fresh-cut potatoes. The data indicated that H2S reduced the degree of browning, increased the total phenolic content, inhibited the accumulation of reactive oxygen species (ROS) content, inhibited POD, PPO, and PAL activities, and increased CAT activity.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that extends the postharvest storage period of fruit. Biaxially oriented polyethylene (BOPE) films are widely used in fruit packaging due to their excellent properties. By analyzing the phenotypic characteristics of strawberry fruits by comparing with the control, PE and BOPE or in combination with H2S at 4 degrees C, it was shown that the H2S+BOPE treatment reduced decay and water loss, and maintained the best lustre and firmness than other groups. A total of 7,258 co-differentially expressed genes (DEGs) were identified by comparing H2S, H2S+PE and H2S+BOPE with Control by transcriptome analysis. GO, WGCNA and KEGG analyses showed that DEGs were mainly enriched in starch and sucrose metabolism, pigment metabolism and antioxidant activity from the MEyellow and MEblack modules. Moreover, these candidate genes were screened, and the expression level of CHS, PAL and DFR were significantly decreased in H2S+BOPE group than other groups by RT-qPCR. Physiological assessments indicated that H2S+BOPE treatment significantly delayed chlorophyll degradation, anthocyanin accumulation and TA content, increased reducing sugar content, carotenoid and POD activity, and maintained low MDA content in strawberries. PCA and heatmap showed that POD and pigments were main factors and the correlation analysis proved that POD activity exhibited a positive correlation with the expression of DFR (0.57) and bHLH3 (0.56), carotenoid content (0.54), and it showed a negative correlation with chlorophyll contents (-0.37). These results indicated that BOPE could effectively improve the appearance and antioxidant activity of strawberry fruits, thereby prolonging their postharvest storage period.

A nuclear-localized cysteine desulfhydrase, LCD1, plays a crucial role in mediating endogenous hydrogen sulfide production in tomatoes. However, the mechanism underlying the nuclear localization of SlLCD1 is not yet fully understood. In this study, it was found that SlLCD1 specifically interacted with nuclear import receptor importin α3 (SlIMPA3). Furthermore, it was demonstrated that silencing SlIMPA3 through virus-induced gene silencing or introducing mutations in SlIMPA3 via CRISPR/Cas9 significantly accelerated fruit ripening. Moreover, enhanced chlorophyll degradation, carotenoid accumulation, and premature upregulation of ripening-associated genes in the slimpa3 mutant indicated SlIMPA3 to be a negative regulator of fruit ripening and leaf senescence. Besides, SlIMPA3 deletion resulted in excessive hydrogen peroxide accumulation in fruits and leaves, potentially leading to premature leaf senescence and accelerated fruit ripening in the slimpa3 mutant. SlIMPA3 exhibited pronounced nuclear localization with weak distribution in the cytoplasm. SlLCD1 showed specific nuclear localization; however, after GFP tagging in slimpa3-edited tomato leaves, it migrated to the cytoplasm, suggesting that SlIMPA3 mediated the nuclear localization of SlLCD1. SlLCD1 transient expression in slimpa3 mutant fruits indicated that it did not inhibit tomato ripening following the SlIMPA3 mutation. In summary, our study revealed that SlIMPA3 interacted with SlLCD1 to facilitate its nuclear entry. Mutations in SlIMPA3 led to premature fruit ripening and leaf senescence, likely due to disrupted reactive oxygen species homeostasis resulting from SlLCD1 mislocalization in the slimpa3 mutant.

H2S is a well-known gaseous signaling molecule that plays important roles in plant response to biotic stresses. Pseudomonas syringae pv tomato (Pst) could cause enormous loss, while whether H2S could modulate plant defense against Pst is still unclear. By CRISPR/Cas9, the Sldcd1 gene editing mutant showed reduced endogenous H2S content and attenuated resistance, whereas treatment with exogenous H2S could enhance the resistance. A transcription factor, SlWRKY71, was screened and identified to promote the transcription of SlDCD1 via yeast one-hybrid, dual-luciferase reporter system, electrophoretic mobility shift assays, and transient overexpression. Here, it was found that exogenous H2S relieved the symptoms of bacterial speck disease in tomato leaves, conferring tolerance to Pst. DC3000, and the expression of the H2S-producing enzyme SlDCD1 was significantly induced. The Slwrky71 mutant also showed reduced defense in tomato leaves against Pst. DC3000, whereas SlWRKY71-OE tomato leaves showed increased tolerance. Transient overexpression of SlDCD1 in the context of Slwrky71 with exogenous H2S treatment has stronger resistance, and the overexpression of SlWRKY71 in the context of Sldcd1 showed relatively weak disease resistance, and with the addition of H2S enhanced the effect. Therefore, we concluded that SlWRKY71 could activate SlDCD1 expression and promote endogenous H2S production, thereby improving tomato leaves resistance to Pst. DC3000.

Sweet potato (Ipomoea batatas L.) is a crop native to the tropics that is weakly resistant to cold. Low temperatures are important factors limiting its storage. By analyzing the phenotypic and tuberous root color differences between Xushu 32 and Yanshu 25 under low-temperature treatment, we found that Xushu 32 showed less low-temperature damage. Transcriptome analysis was performed on the tuberous roots of the two sweet potatoes at different storage times to further investigate the molecular mechanism. There were 7039 differentially co-expressed genes in Xushu 32 and 11033 differentially co-expressed genes in Yanshu 25 after 4 and 8 days of low-temperature treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the percentage of differentially expressed genes in the glutathione metabolism pathway in Xushu 32 was significantly higher than that in Yanshu 25. Weighted correlation network analysis (WGCNA) analysis indicated that the expression modules of MElightcyan1 and MEmegenta3 were up-regulated under low-temperature stress and that glutathione S-transferases (GSTs) were hub genes with high connectivity. The candidate genes IbGST2 and IbGST4 were highly similar to a rice glutathione S-transferase (GST) (Os10g0528900) by heatmap analysis and evolutionary tree analysis and may play a role in low-temperature stress. Subsequently, gene-silenced plants of the homologous genes SlBI-GST and SlGST-T4 in tomatoes were constructed using virus-induced gene silencing (VIGS) technology, and the contents of reactive oxygen species (ROS) metabolites and expression of BI-GST, GST-T4, and the C-repeat binding transcription factors (CBFs) were determined. This suggested that BI- GST and GST- T4 genes could scavenge ROS and improve the cold tolerance of tomatoes. These results provide important insights into the roles of IbGST2 and IbGST4 in the storage characteristics of sweet potatoes under low-temperature stress.

Hydrogen sulfide (H2S) is a signaling molecule that regulates plant senescence. In this study, we found that H2S delays dark-induced senescence in tomato (Solanum lycopersicum) leaves. Transcriptome and reverse transcription quantitative PCR (RT-qPCR) analyses revealed an ethylene response factor ERF.D3 is quickly induced by H2S. H2S also persulfidated ERF.D3 at amino acid residues C115 and C118. CRISPR/Cas9-mediated gene editing, and gene overexpression analyses showed that ERF.D3 negatively regulates leaf senescence and fruit ripening. Abscisic acid (ABA) levels were reduced by ERF.D3 overexpression, suggesting ERF.D3 might regulate ABA metabolism. Additionally, the ABA 8'-hydroxylase-encoding gene CYP707A2, which is required for ABA degradation, was identified as an ERF.D3 target gene through transcriptome data, RT-qPCR, dual-luciferase reporter assays, and electrophoretic mobility shift assays. ERF.D3 persulfidation enhanced its transcriptional activity toward CYP707A2. Moreover, the E3 ligase RNF217 ubiquitinated ERF.D3, which may accelerate fruit ripening during the late stage of fruit development. Overall, our study provides valuable insights into the roles of a H2S-responsive ERF.D3 and its persulfidation state in delaying leaf senescence and fruit ripening and provides a link between H2S and ABA degradation.

Hydrogen peroxide (H2O2) is relatively stable among ROS (reactive oxygen species) and could act as a signal in plant cells. In the present work, detached tomato leaves were treated with exogenous H2O2 at 10 mmol/L for 8 h to study the mechanism of how H2O2 regulates leaf senescence. The data indicated that H2O2 treatment significantly accelerated the degradation of chlorophyll and led to the upregulation of the expression of leaf senescence-related genes (NYC1, PAO, PPH, SGR1, SAG12 and SAG15) during leaf senescence. H2O2 treatment also induced the accumulation of H2O2 and malondialdehyde (MDA), decreased POD and SOD enzyme activities and inhibited H2S production by reducing the expression of LCD1/2 and DCD1/2. A correlation analysis indicated that H2O2 was significantly and negatively correlated with chlorophyll, the expression of leaf senescence−related genes, and LCD1/2 and DCD1/2. The principal component analysis (PCA) results show that H2S showed the highest load value followed by O2•−, H2O2, DCD1, SAG15, etc. Therefore, these findings provide a basis for studying the role of H2O2 in regulating detached tomato leaf senescence and demonstrated that H2O2 plays a positive role in the senescence of detached leaves by repressing antioxidant enzymes and H2S production.

Cysteine desulfhydrase catalyses 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.

As a typical climacteric fruit, tomato (Solanum lycopersicum) is widely used for studying the ripening process. The negative regulation of tomato fruits by transcription factor SlNAC1 has been reported, but its regulatory network was unclear. In the present study, we screened a transcription factor, SlERF109-like, and found it had a stronger relationship with SlNAC1 at the early stage of tomato fruit development through the use of transcriptome data, RT-qPCR, and correlation analysis. We inferred that SlERF109-like could interact with SlNAC1 to become a regulatory complex that co-regulates the tomato fruit ripening process. Results of transient silencing (VIGS) and transient overexpression showed that SlERF109-like and SlNAC1 could regulate chlorophyll degradation-related genes (NYC1, PAO, PPH, SGR1), carotenoids accumulation-related genes (PSY1, PDS, ZDS), ETH-related genes (ACO1, E4, E8), and cell wall metabolism-related genes expression levels (CEL2, EXP, PG, TBG4, XTH5) to inhibit tomato fruit ripening. A dual-luciferase reporter and yeast one-hybrid (Y1H) showed that SlNAC1 could bind to the SlACO1 promoter, but SlERF109-like could not. Furthermore, SlERF109-like could interact with SlNAC1 to increase the transcription for ACO1 by a yeast two-hybrid (Y2H) assay, a luciferase complementation assay, and a dual-luciferase reporter. A correlation analysis showed that SlERF109-like and SlNAC1 were positively correlated with chlorophyll contents, and negatively correlated with carotenoid content and ripening-related genes. Thus, we provide a model in which SlERF109-like could interact with SlNAC1 to become a regulatory complex that negatively regulates the tomato ripening process by inhibiting SlACO1 expression. Our study provided a new regulatory network of tomato fruit ripening and effectively reduced the waste of resources.

Red-skinned pears (Pyrus L.) are preferred to consumers for their attractive color and abundant anthocyanins. Pyrus ETHYLENE RESPONSE FACTOR 3 (PyERF3) positively regulates anthocyanin biosynthesis through interacting with Pyrus myeloblastosis family 114 (PyMYB114) and Pyrus basic helix-loop-helix 3 (PybHLH3) in red-skinned pears. However, the role of APETALA2/ethylene response factors (AP2/ERFs), which negatively regulate anthocyanin biosynthesis, remains unclear in red-skinned pears. Here, we validated that 2 AP2/ERFs, PyERF4.1 and PyERF4.2, screened from the transcriptome data of 'Starkrimson' pear (Pyrus communis L.) and its green mutant, inhibit anthocyanin biosynthesis in transgenic pear calli, as well as in overexpression and gene-edited tomato (Solanum lycopersicum) fruits. Meanwhile, the co-transformation of PyERF4.1/PyERF4.2 with PyERF3-PyMYB114-PybHLH3 inhibited anthocyanin biosynthesis in pear fruits and strawberry (Fragaria vesca) receptacles. Further assays showed that PyMYB114 activated the transcription of PyERF4.1/PyERF4.2; PyERF4.1/PyERF4.2 then interacted with PyERF3 to affect the stability of the PyERF3-PyMYB114-PybHLH3 complex, thereby inhibiting the transcription of the anthocyanin biosynthesis gene Pyrus anthocyanidin synthase (PyANS). Furthermore, deletion of the ERF-associated-amphiphilic repression (EAR) motif eliminated the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis, and a mutation of the PyERF4.2-EAR motif (LxLxM to LxLxL) strengthened the inhibitory effect, demonstrating that the EAR motif is indispensable for the inhibitory effect of PyERF4.1/PyERF4.2 on anthocyanin biosynthesis in pears. Our study has shed light on a feedback regulatory loop mechanism that balances the excessive accumulation of anthocyanins in red-skinned pears, providing insights into the regulatory mechanism of anthocyanin biosynthesis and the regulatory network of coloration in red-skinned pears.

Hydrogen sulfide(H2S)is involved in multiple processes during plant growth and development.D-cysteine desulfhydrase(DCD)can produce H2S with D-cysteine as the substrate;however,the potential developmental roles of DCD have not been explored during the tomato lifecycle.In the present study,SlDCD2 showed increasing expression during fruit ripening.Compared with the control fruits,the silencing of SlDCD2 by pTRV2-SlDCD2 accelerated fruit ripening.A SlDCD2 gene-edited mutant was constructed by CRISPR/Cas9 transformation,and the mutant exhibited accelerated fruit ripening,decreased H2S release,higher total cysteine and ethylene contents,enhanced chlorophyll degradation and increased carotenoid accumulation.Additionally,the expression of multiple ripening-related genes,including NYC1,PAO,SGR1,PDS,PSY1,ACO1,ACS2,E4,CEL2,and EXP was enhanced during the dcd2 mutant tomato fruit ripening.Compared with the wild-type fruits,SlDCD2 mutation induced H2O2 and malondialdehyde(MDA)accumulation in fruits,which led to an imbalance in reactive oxygen species(ROS)metabolism.A correlation analysis indicated that H2O2 content was strongly positively correlated with carotenoids content,ethylene content and ripening-related gene expression and negatively correlated with the chlorophyll content.Additionally,the dcd2 mutant showed earlier leaf senescence,which maybe due to disturbed ROS homeostasis.In short,our findings show that SlDCD2 is involved in H2S generation and that the reduction in endogenous H2S production in the dcd2 mutant causes accelerated fruit ripening and premature leaf senescence.Additionally,decreased H2S in the dcd2 mutant causes excessive H2O2 accumulation and increased ethylene release,suggesting a role of H2S and SlDCD2 in modulating ROS homeostasis and ethylene biosynthesis.

Fruit quality is defined by attributes that give value to a commodity.Flavor,texture,nutrition,and shelf life are key quality traits that ensure market value and consumer acceptance.In pear fruit,soluble sugars,organic acids,amino acids,and total flavonoids contribute to flavor and overall quality.Transcription factors(TFs)regulate the accumulation of these metabolites during development or in response to the environment.Here,we report a novel TF,PpbZIP44,as a positive regulator of primary and secondary metabolism in pear fruit.Analysis of the transient overexpression or RNAi-transformed pear fruits and stable transgenic tomato fruits under the control of the fruit-specific E8 promoter demonstrated that PpZIP44 substantially affected the contents of soluble sugar,organic acids,amino acids,and flavonoids.In E8::PpbZIP44 tomato fruit,genes involved in carbohydrate metabolism,amino acid,and flavonoids biosynthesis were significantly induced.Furthermore,in PpbZIP44 overexpression or antisense pear fruits,the expression of genes in the related pathways was significantly impacted.PpbZIP44 directly interacted with the promoter of PpSDH9 and PpProDH1 to induce their expression,thereby depleting sorbitol and proline,decreasing citrate and malate,and enhancing fructose contents.PpbZIP44 also directly bound to the PpADT and PpF3H promoters,which led to the carbon flux toward phenylalanine metabolites and enhanced phenylalanine and flavonoid contents.These findings demonstrate that PpbZIP44 mediates multimetabolism reprogramming by regulating the gene expression related to fruit quality compounds.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that delays color change during fruit ripening. Whether H2S affects anthocyanin biosynthesis in red-skinned pears (Pyrus L.) remains unclear. Here, we found that H2S substantially inhibits anthocyanin accumulation in red-skinned pears and the expression of several genes encoding transcription factors is affected in response to H2S signaling. For example, PyMYB10 and PyMYB73 were down-regulated, whereas PyMYB114 and PyMYB6 were up-regulated. Bioinformatics analysis showed that PyMYB73 and PyMYB6, each containing an EAR motif, may negatively regulate anthocyanin accumulation. Transient expression analysis showed that PyMYB73 substantially promotes anthocyanin biosynthesis by co-transforming with PyMYB10/PyMYB114 + PybHLH3; however, PyMYB6 inhibited anthocyanin biosynthesis in strawberry (Fragaria vesca) receptacles and pear fruits, and PyMYB73 interacted with PyMYB10 and PyMYB6 but not PyMYB114 or PybHLH3. Further investigation showed that Cys194 and Cys218 of PyMYB10 were modified by persulfidation and that PyMYB10Cys218Ala substantially increased anthocyanin accumulation by a transient transformation system. Co-transformation of PyMYB10Cys218Ala + PyMYB73/PyMYB6 also promoted anthocyanin accumulation in pear fruits. Yeast two-hybrid assays showed that the mutation of PyMYB10 did not affect the interaction between PyMYB10 and PyMYB73, but it inhibited interaction with PyMYB6. Moreover, H2S weakened the interaction between PyMYB10 and PyMYB73 but enhanced the interaction with PyMYB6. Thus, we provided a model in which PyMYB10 undergoes persulfidation at Cys218, enhancing the interaction with PyMYB6 and reducing the interaction with PyMYB73. These subsequently results in lower expression of the anthocyanin biosynthesis-related genes Pyrus dihydroflavonol 4-reductase (PyDFR), Pyrus anthocyanidin synthase (PyANS), Pyrus UDP-glucose: flavonoid 3-glucosyl transferase (PyUFGT) and Pyrus glutathione S-transferase (PyGST), thereby inhibiting anthocyanin accumulation in red-skinned pears. Our findings provided a molecular mechanism for H2S-mediated anthocyanin biosynthesis in red-skinned pears.