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.

SlERF109-like and SlNAC1 Coordinately Regulated Tomato Ripening by Inhibiting ACO1 Transcription.

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.

A D-cysteine desulfhydrase, SlDCD2, participates in tomato fruit ripening by modulating ROS homoeostasis and ethylene biosynthesis.

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 may be 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.

ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears.

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.

A Hydrogen-Sulfide-Repressed Methionine Synthase SlMS1 Acts as a Positive Regulator for Fruit Ripening in Tomato.

Ethylene is a key phytohormone that regulates the ripening of climacteric fruits, and methionine is an indirect precursor of ethylene. However, whether methionine synthase plays a role in fruit ripening in Solanum lycopersicum (tomato) is still unknown. In this study, we find that a tomato methionine synthase (named SlMS1), which could be repressed at the transcriptional level by hydrogen sulfide (H2S), acts as a positive regulator for tomato fruit ripening. By a bioinformatics analysis, it is found that SlMS1 and SlMS2 in tomato are highly homologous to methionine synthases in Arabidopsis thaliana. The expression pattern of SlMS1 and SlMS2 is analyzed in tomato, and SlMS1 expression is up-regulated during fruit ripening, suggesting its potential role in regulating fruit ripening. A potential bipartite nuclear localization signal is found in the amino acid sequence of SlMS1; thus, SlMS1 is tagged with GFP and observed in the leaves of Nicotiana benthamiana. Consistently, SlMS1-GFP shows strong nuclear localization and also cytoplasmic localization. The role of SlMS1 in regulating fruit ripening is investigated in tomato fruit by transient silencing (virus-induced gene silencing, VIGS) and transient overexpression. The results show that SlMS1 silencing causes delayed fruit ripening, evidenced by more chlorophyll and less carotenoid accumulation, while SlMS1 overexpression accelerates fruit ripening significantly compared with control. Further investigation shows that SlMS1 overexpression could up-regulate the expression of carotenoid-synthesis-related genes (PSY1, PDS, ZDS), chlorophyll-degradation-related genes (NYC1, PAO, PPH, SGR1), cell-wall-metabolism-related genes (CEL2, EXP, PG, TBG4, XTH5) and ethylene-synthesis-pathway-related genes (ACO1, ACO3, ACS2), while SlMS1 silencing causes the opposite results. The correlation analysis indicates that SlMS1 expression is negatively correlated with chlorophyll content and positively correlated with carotenoid and ripening-related gene expressions. Taken together, our data suggest that SlMS1 is a positive regulator of tomato fruit ripening and a possible target gene for the ripening-delaying effect of H2S.

Oak-inspired anti-biofouling shape-memory unidirectional scaffolds with stable solar water evaporation performance.

Biomimetic porous materials have contributed to the enhancement of solar-driven evaporation rate in interfacial desalination and clean water production. However, due to the presence of numerous microbes in water environment, biofouling should occur inside porous materials to clog the channels for water transfer, resulting in obvious inhibition of the solar-driven evaporation efficacy in long-term use. To prevent and control biofouling in porous materials for solar-driven evaporation, a facile and environment-friendly design is required in real application. Oak wood possesses vertically aligned channels for transpiration and polyphenol compounds with antimicrobial activity. In this work, inspired by the oak wood, we developed an anti-biofouling shape-memory chitosan scaffold with unidirectional channels and tannic acid coating (oak-inspired scaffold). The shape-memory property facilitated rapid decoration with oak-inspired photothermal and anti-biofouling coating inside the scaffold, respectively, which also promotes the material durability by avoiding the external force-induced permanent structure failure. More importantly, the oak-inspired tannic acid coating not only prevented bacterial adhesion and colonization, but also inhibited fungal interference. They were subjected to a microbe-rich environment, and after 3 days, the evaporation rates of the untreated chitosan scaffolds were obviously decreased to 1.24, 1.16 and 1.19 kg m-2 h-1 for C. albicans, S. aureus and E. coli, respectively, which were only 65.6, 61.4 and 63.0% of original performance (1.89 kg m-2 h-1). In comparison, the oak-inspired scaffold exhibited a high solar-driven water evaporation rate after incubation in microbial suspensions (1.80, 1.70 and 1.75 kg m-2 h-1 for C. albicans, S. aureus and E. coli after 3 days) and lake water (1.74 kg m-2 h-1 after one month). The bioinspired anti-biofouling scaffolds maintain as high as 86.7-91.8% of the solar-driven water evaporation ability after exposure to a microbe-rich environment, which is conducive to develop a biomimetic long-term durable structure in water treatment.

Transcriptomics Reveals the ERF2-bHLH2-CML5 Module Responses to H2S and ROS in Postharvest Calcium Deficiency Apples.

Calcium deficiency usually causes accelerated quality deterioration in postharvest fruit, whereas the underlining mechanism is still unclear. Here, we report that calcium deficiency induced the development of bitter pit on the surface of apple peels compared with the healthy appearance in control apples during postharvest storage. Physiological analysis indicates that calcium-deficient peels contained higher levels of superoxide anion (O2•-), malondialdehyde (MDA), total phenol, flavonoid contents and polyphenol oxidase (PPO) activity, and reduced calcium, H2S production, anthocyanin, soluble protein content, and peroxidase (POD) activity compared with those in calcium-sufficient peels. The principal component analysis (PCA) results show that calcium content, ROS, and H2S production were the main factors between calcium-deficient and calcium-sufficient apple peels. Transcriptome data indicated that four calmodulin-like proteins (CMLs), seven AP2/ERFs, and three bHLHs transcripts were significantly differentially expressed in calcium-deficient apple peels. RT-qPCR and correlation analyses further revealed that CML5 expression was significantly positively correlated with the expression of ERF2/17, bHLH2, and H2S production related genes. In addition, transcriptional co-activation of CML5 by ERF2 and bHLH2 was demonstrated by apple transient expression assays and dual-luciferase reporter system experiments. Therefore, these findings provide a basis for studying the molecular mechanism of postharvest quality decline in calcium-deficient apples and the potential interaction between Ca2+ and endogenous H2S.

Roles of a Cysteine Desulfhydrase LCD1 in Regulating Leaf Senescence in Tomato.

Hydrogen sulfide (H2S), a novel gasotransmitter in both mammals and plants, plays important roles in plant development and stress responses. Leaf senescence represents the final stage of leaf development. The role of H2S-producing enzyme L-cysteine desulfhydrase in regulating tomato leaf senescence is still unknown. In the present study, the effect of an L-cysteine desulfhydrase LCD1 on leaf senescence in tomato was explored by physiological analysis. LCD1 mutation caused earlier leaf senescence, whereas LCD1 overexpression significantly delayed leaf senescence compared with the wild type in 10-week tomato seedlings. Moreover, LCD1 overexpression was found to delay dark-induced senescence in detached tomato leaves, and the lcd1 mutant showed accelerated senescence. An increasing trend of H2S production was observed in leaves during storage in darkness, while LCD1 deletion reduced H2S production and LCD1 overexpression produced more H2S compared with the wild-type control. Further investigations showed that LCD1 overexpression delayed dark-triggered chlorophyll degradation and reactive oxygen species (ROS) accumulation in detached tomato leaves, and the increase in the expression of chlorophyll degradation genes NYC1, PAO, PPH, SGR1, and senescence-associated genes (SAGs) during senescence was attenuated by LCD1 overexpression, whereas lcd1 mutants showed enhanced senescence-related parameters. Moreover, a correlation analysis indicated that chlorophyll content was negatively correlated with H2O2 and malondialdehyde (MDA) content, and also negatively correlated with the expression of chlorophyll degradation-related genes and SAGs. Therefore, these findings increase our understanding of the physiological functions of the H2S-generating enzyme LCD1 in regulating leaf senescence in tomato.

A single amino acid mutant in the EAR motif of IbMYB44.2 reduced the inhibition of anthocyanin accumulation in the purple-fleshed sweetpotato.

Purple-fleshed sweetpotato (Ipomoea batatas（L.）Lam.) is rich in anthocyanins. R2R3-type MYB transcription factors（TFs）with EAR motifs inhibiting anthocyanin biosynthesis have been reported, and there is still a lack of information on how mutations in the EAR motifs of MYBs affect anthocyanin accumulation. In this study, we obtained three IbMYB44 TFs by bioinformatics. Among these TFs, IbMYB44.1, IbMYB44.3 with a complete EAR motif and IbMYB44.2 with a single amino acid mutant in the EAR motif caused an amino acid substitution from leucine to valine. RT-qPCR analysis showed that IbMYB44s was expressed at lower levels in the purple-fleshed sweetpotato than in nonpurple-fleshed sweetpotato (P < 0.01). Transient expression assays showed that the inhibitory effect of IbMYB44.1/3 was stronger than IbMYB44.2 in tobacco leaves and red-skinned pears. RT-qPCR analysis further proved that IbMYB44.1/3 significantly inhibited the expression of anthocyanin biosynthesis-related genes compared with IbMYB44.2 in tobacco leaves and red-skinned pears. A dual luciferase reporter assay showed that IbMYB44s cannot directly activate the IbANS promoter, and the result was also verified by yeast one-hybrid (Y1H) experiments. Moreover, we identified the interaction of IbMYB340 with IbMYB44.1, IbMYB44.2 and IbMYB44.3 via yeast two-hybrid (Y2H) assays. Thus, IbMYB44.1/3 could interact with IbMYB340 to negatively regulate anthocyanin biosynthesis. This study enriched the regulatory network of anthocyanins and also provided a theoretical basis for a single amino acid mutant from leucine to valine in the EAR motif of IbMYB44.2 affecting anthocyanin biosynthesis in the purple-fleshed sweetpotato.

IbERF71, with IbMYB340 and IbbHLH2, coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potato (Ipomoea batatas L.).

KEY MESSAGE: The transcription factor (TF) IbERF71 forms a novel complex, IbERF71-IbMYB340-IbbHLH2, to coregulate anthocyanin biosynthesis by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Purple-fleshed sweet potato (Ipomoea batatas L.) is very popular because of its abundant anthocyanins, which are natural pigments with multiple physiological functions. TFs involved in regulating anthocyanin biosynthesis have been identified in many plants. However, the molecular mechanism of anthocyanin biosynthesis in purple-fleshed sweet potatoes has rarely been examined. In this study, TF IbERF71 and its partners were screened by bioinformatics and RT-qPCR analysis. The results showed that the expression levels of IbERF71 and partners IbMYB340 and IbbHLH2 were higher in purple-fleshed sweet potatoes than in other colors and that the expression levels positively correlated with anthocyanin contents. Moreover, transient expression assays showed that cotransformation of IbMYB340+IbbHLH2 resulted in anthocyanin accumulation in tobacco leaves and strawberry receptacles, and additional IbERF71 significantly increased visual aspects. Furthermore, the combination of the three TFs significantly increased the expression levels of FvANS and FvGST, which are involved in anthocyanin biosynthesis and transport of strawberry receptacles. The dual-luciferase reporter system verified that cotransformation of the three TFs enhanced the transcription activity of IbANS1. In addition, yeast two-hybrid and firefly luciferase complementation assays revealed that IbMYB340 interacted with IbbHLH2 and IbERF71 but IbERF71 could not interact with IbbHLH2 in vitro. In summary, our findings provide novel evidence that IbERF71 and IbMYB340-IbbHLH2 form the regulatory complex IbERF71-IbMYB340-IbbHLH2 that coregulates anthocyanin accumulation by binding to the IbANS1 promoter in purple-fleshed sweet potatoes. Thus, the present study provides a new regulatory network of anthocyanin biosynthesis and strong insight into the color development of purple-fleshed sweet potatoes.

A nuclear-localized cysteine desulfhydrase plays a role in fruit ripening in tomato.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays multiple roles in plant development. However, whether endogenous H2S plays a role in fruit ripening in tomato is still unknown. In this study, we show that the H2S-producing enzyme L-cysteine desulfhydrase SlLCD1 localizes to the nucleus. By constructing mutated forms of SlLCD1, we show that the amino acid residue K24 of SlLCD1 is the key amino acid that determines nuclear localization. Silencing of SlLCD1 by TRV-SlLCD1 accelerated fruit ripening and reduced H2S production compared with the control. A SlLCD1 gene-edited mutant obtained through CRISPR/Cas9 modification displayed a slightly dwarfed phenotype and accelerated fruit ripening. This mutant also showed increased cysteine content and produced less H2S, suggesting a role of SlLCD1 in H2S generation. Chlorophyll degradation and carotenoid accumulation were enhanced in the SlLCD1 mutant. Other ripening-related genes that play roles in chlorophyll degradation, carotenoid biosynthesis, cell wall degradation, ethylene biosynthesis, and the ethylene signaling pathway were enhanced at the transcriptional level in the lcd1 mutant. Total RNA was sequenced from unripe tomato fruit treated with exogenous H2S, and transcriptome analysis showed that ripening-related gene expression was suppressed. Based on the results for a SlLCD1 gene-edited mutant and exogenous H2S application, we propose that the nuclear-localized cysteine desulfhydrase SlLCD1 is required for endogenous H2S generation and participates in the regulation of tomato fruit ripening.

Hydrogen Sulfide Maintained the Good Appearance and Nutrition in Post-harvest Tomato Fruits by Antagonizing the Effect of Ethylene.

Hydrogen sulfide (H2S) could act as a versatile signaling molecule in delaying fruit ripening and senescence. Ethylene (C2H4) also plays a key role in climacteric fruit ripening, but little attention has been given to its interaction with H2S in modulating fruit ripening and senescence. To study the role of H2S treatment on the fruit quality and nutrient metabolism, tomato fruits at white mature stage were treated with ethylene and ethylene plus H2S. By comparing to C2H4 treatment, we found that additional H2S significantly delayed the color change of tomato fruit, and maintained higher chlorophyll and lower flavonoids during storage. Moreover, H2S could inhibit the activity of protease, maintained higher levels of nutritional-related metabolites, such as anthocyanin, starch, soluble protein, ascorbic acid by comparing to C2H4 treatment. Gene expression analysis showed that additional H2S attenuated the expression of beta-amylase encoding gene BAM3, UDP-glycosyltransferase encoding genes, ethylene-responsive transcription factor ERF003 and DOF22. Furthermore, principal component analysis suggested that starch, titratable acids, and ascorbic acid were important factors for affecting the tomato storage quality, and the correlation analysis further showed that H2S affected pigments metabolism and the transformation of macromolecular to small molecular metabolites. These results showed that additional H2S could maintain the better appearance and nutritional quality than C2H4 treatment alone, and prolong the storage period of post-harvest tomato fruits.

MYB44 competitively inhibits the formation of the MYB340-bHLH2-NAC56 complex to regulate anthocyanin biosynthesis in purple-fleshed sweet potato.

BACKGROUND: Anthocyanins, which have important biological functions and have a beneficial effect on human health, notably account for pigmentation in purple-fleshed sweet potato tuberous roots. Individual regulatory factors of anthocyanin biosynthesis have been identified; however, the regulatory network of anthocyanin biosynthesis in purple-fleshed sweet potato is unclear. RESULTS: We functionally determined that IbMYB340 cotransformed with IbbHLH2 in tobacco and strawberry receptacles induced anthocyanin accumulation, and the addition of IbNAC56a or IbNAC56b caused increased pigmentation. Furthermore, we confirmed the interaction of IbMYB340 with IbbHLH2 and IbNAC56a or IbNAC56b via yeast two-hybrid and firefly luciferase complementation assays; these proteins could form a MYB340-bHLH2-NAC56a or MYB340-bHLH2-NAC56b transcriptional complex to regulate anthocyanin biosynthesis by binding to the IbANS promoter rather than the IbUFGT promoter. Furthermore, it was found by a transient expression system in tobacco leaves that IbMYB44 could decrease anthocyanin accumulation. Moreover, the interaction of IbMYB44 with IbMYB340 and IbNAC56a or IbNAC56b was verified. This result suggested that IbMYB44 acts as a repressor of anthocyanin in sweet potato. CONCLUSIONS: The repressor IbMYB44 affected anthocyanin biosynthesis by competitively inhibiting the IbMYB340-IbbHLH2-IbNAC56a or IbMYB340-IbbHLH2-IbNAC56b regulatory complex formation. Overall, the present study proposed a novel regulatory network whereby several vital TFs play key roles in regulating anthocyanin biosynthesis, and it provides strong insight into the potential mechanism underlying anthocyanin biosynthesis in sweet potato tuberous roots with purple color.

PyWRKY26 and PybHLH3 cotargeted the PyMYB114 promoter to regulate anthocyanin biosynthesis and transport in red-skinned pears.

Red pear is favored because of its bright appearance and abundant anthocyanins. Anthocyanin biosynthesis is controlled by transcription factors (TFs) forming regulatory complexes. In red-skinned pears, the WRKY TFs have a significant relationship with anthocyanin biosynthesis, but the molecular mechanism of the WRKY TFs involved in regulating color formation in red-skinned pear is unclear. In this study, the TFs PyWRKY31 and PyWRKY26 were screened as candidate genes for controlling anthocyanin biosynthesis by transcriptome data and bioinformatics analysis. The effect of anthocyanin accumulations after cotransformation of PyWRKY31 or PyWRKY26 with its partners PyMYB10, PyMYB114, and PybHLH3 was verified in tobacco leaves and strawberry receptacles by a transient expression system. RT-qPCR analysis and a dual-luciferase reporter system further confirmed that this cotransformation activated the expression of PyDFR, PyANS, and PyUFGT in anthocyanin biosynthesis and PyGST in anthocyanin transport instead of the PyABC transporter and PyAVP. Furthermore, the cotransformed PyWRKY26 and PybHLH3 could bind to the PyMYB114 promoter, and PyWRKY26 directly activated the transcription of PyMYB114. In addition, the TF PyWRKY26 could interact with PybHLH3, as confirmed by firefly luciferase complementation and yeast two-hybrid (Y2H) assays. These results showed that the interaction of PyWRKY26 and PybHLH3 could cotarget the PyMYB114 promoter, which resulted in anthocyanin accumulation in red-skinned pear. This study further strengthened the understanding of the regulatory mechanism of anthocyanin accumulation and contributed to improving the appearance of red-skinned pears.

Functional Characterization of a Cystathionine ß-Synthase Gene in Sulfur Metabolism and Pathogenicity of Aspergillus niger in Pear Fruit.

Aspergillus niger, which is a fungal pathogen, causes rot in a variety of fruits. In this study, the cystathionine ß-synthase cbsA gene was deleted by homologous recombination to study its role in sulfur metabolism and pathogenicity of A. niger. The results showed that Δ cbsA strain maintained normal mycelia growth and sporulation compared with the control strain A. niger MA 70.15, whereas the contents of cysteine and glutathione (GSH) increased significantly after cbsA deletion. However, Δ cbsA strain showed reduced endogenous H2S production. Further results showed that cbsA gene deletion induced higher resistance to cadmium stress and stronger infectivity to pears. It was also found that a stronger response of reactive oxygen species (ROS) production was induced in Δ cbsA mutant-infected pear compared with the control strain. In all, the present research suggested the important role of cbsA in sulfur metabolism and pathogenicity of A. niger in pear fruit.

Central Role of Adenosine 5'-Phosphosulfate Reductase in the Control of Plant Hydrogen Sulfide Metabolism.

Hydrogen sulfide (H2S) has been postulated to be the third gasotransmitter in both animals and plants after nitric oxide (NO) and carbon monoxide (CO). In this review, the physiological roles of H2S in plant growth, development and responses to biotic, and abiotic stresses are summarized. The enzymes which generate H2S are subjected to tight regulation to produce H2S when needed, contributing to delicate responses of H2S to environmental stimuli. H2S occupies a central position in plant sulfur metabolism as it is the link of inorganic sulfur to the first organic sulfur-containing compound cysteine which is the starting point for the synthesis of methionine, coenzyme A, vitamins, etc. In sulfur assimilation, adenosine 5'-phosphosulfate reductase (APR) is the rate-limiting enzyme with the greatest control over the pathway and probably the generation of H2S which is an essential component in this process. APR is an evolutionarily conserved protein among plants, and two conserved domains PAPS_reductase and Thioredoxin are found in APR. Sulfate reduction including the APR-catalyzing step is carried out in chloroplasts. APR, the key enzyme in sulfur assimilation, is mainly regulated at transcription level by transcription factors in response to sulfur availability and environmental stimuli. The cis-acting elements in the promoter region of all the three APR genes in Solanum lycopersicum suggest that multiple factors such as sulfur starvation, cytokinins, CO2, and pathogens may regulate the expression of SlAPRs. In conclusion, as a critical enzyme in regulating sulfur assimilation, APR is probably critical for H2S generation during plants' response to diverse environmental factors.

Modulation of Enhanced Antioxidant Activity by Hydrogen Sulfide Antagonization of Ethylene in Tomato Fruit Ripening.

Ethylene (C2H4) and hydrogen sulfide (H2S) play important physiological roles in regulating fruit ripening and senescence. The mechanism of H2S in ethylene-induced tomato fruit ripening and senescence is still unknown. Here, we show that exogenous H2S reduced the production of superoxide anion (·O2-), malondialdehyde (MDA), and H2O2 in tomato fruit. Further, additional H2S was found to induce the activities of guaiacol peroxidase, catalase, ascorbate peroxidase, and superoxide dismutase compared with C2H4 treatment alone, whereas the activities of lipoxygenase, polyphenol oxidase, and phenylalanine ammonia lyase were adversely affected. Moreover, the expression of the antioxidant-encoding genes SlAPX2, SlCAT1, SlPOD12, and SlCuZnSOD was generally up-regulated with C2H4-H2S cotreatment, compared with their expression after ethylene treatment. Thus, the present results suggest that exogenous H2S acts as a fruit-ripening regulator by antagonizing the effect of ethylene, thereby providing a potential application for H2S in the postharvest storage of fruit.

Hydrogen sulfide inhibits the growth of Escherichia coli through oxidative damage.

Many studies have shown that hydrogen sulfide (H2S) is both detrimental and beneficial to animals and plants, whereas its effect on bacteria is not fully understood. Here, we report that H2S, released by sodium hydrosulfide (NaHS), significantly inhibits the growth of Escherichia coli in a dose-dependent manner. Further studies have shown that H2S treatment stimulates the production of reactive oxygen species (ROS) and decreases glutathione (GSH) levels in E. coli, resulting in lipid peroxidation and DNA damage. H2S also inhibits the antioxidative enzyme activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) and induces the response of the SoxRS and OxyR regulons in E. coli. Moreover, pretreatment with the antioxidant ascorbic acid (AsA) could effectively prevent H2S-induced toxicity in E. coli. Taken together, our results indicate that H2S exhibits an antibacterial effect on E. coli through oxidative damage and suggest a possible application for H2S in water and food processing.

Correction: Hydrogen sulfide alleviates postharvest ripening and senescence of banana by antagonizing the effect of ethylene.

[This corrects the article DOI: 10.1371/journal.pone.0180113.].

Hydrogen sulfide alleviates postharvest ripening and senescence of banana by antagonizing the effect of ethylene.

Accumulating evidence shows that hydrogen sulfide (H2S) acts as a multifunctional signaling molecule in plants, whereas the interaction between H2S and ethylene is still unclear. In the present study we investigated the role of H2S in ethylene-promoted banana ripening and senescence by the application of ethylene released from 1.0 g·L-1 ethephon solution or H2S with 1 mM sodium hydrosulfide (NaHS) as the donor or in combination. Fumigation with ethylene was found to accelerate banana ripening and H2S treatment effectively alleviated ethylene-induced banana peel yellowing and fruit softening in parallel with decreased activity of polygalacturonase (PG). Ethylene+H2S treatment also delayed the decreases in chlorophyll and total phenolics, and increased the accumulation of flavonoid, whereas decreased the contents of carotenoid, soluble protein in banana peel and reducing sugar in pulp compared with ethylene treatment alone. Besides, ethylene+H2S treatment suppressed the accumulation of superoxide radicals (·O2-), hydrogen peroxide (H2O2) and malondialdehyde (MDA) which accumulated highly in ethylene-treated banana peels. Furthermore H2S enhanced total antioxidant capacity in ethylene-treated banana peels with the 2,2'-azobis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) assay. The result of quantitative real-time PCR showed that the combined treatment of ethylene with H2S down-regulated the expression of ethylene synthesis genes MaACS1, MaACS2 and MaACO1 and pectate lyase MaPL compared with ethylene treatment, while the expression of ethylene receptor genes MaETR, MaERS1 and MaERS2 was enhanced in combination treatment compared with ethylene alone. In all, it can be concluded that H2S alleviates banana fruit ripening and senescence by antagonizing the effect of ethylene through reduction of oxidative stress and inhibition of ethylene signaling pathway.