Fruit is an essential part of the human diet, and postharvest fungal diseases are the major cause of fruit postharvest losses worldwide. Pathogen-associated molecular patterns (PAMPs) are important elicitors from microbes, and the recognition between microbial PAMPs and plant receptors leads to PAMP-triggered immunity. Here, we identified a PAMP, PdEIX, that is an important protein elicitor with plant immunity-inducing activity, from the citrus green mold pathogen Penicillium digitatum. PdEIX showed an apoplastic location similar to that of known PAMPs, and plant receptor-like protein NbEIX2, receptor-like kinase BAK1, and other signaling components of plant immunity were required for PdEIX-triggered plant cell death in the model plant Nicotiana benthamiana. Moreover, PdEIX triggered a series of immune responses in citrus fruit and enhanced the resistance of citrus and other fruit against fungal pathogens. Our results indicate that application of a microbial PAMP as the plant immunity inducer is an effective strategy for controlling postharvest fungal diseases of fruit.

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.

Sweet potato decays easily due to its high respiration rate and reactive oxygen species (ROS) accumulation during postharvest storage. In this study, we explored the relationship between antioxidant capacity in leaves and storage properties in different sweet potato cultivars, the tuberous roots of 10 sweet potato cultivars were used as the experimental materials to analyze the storage property during storage at 11–15°C. According to the decay percentage after 290 days of storage, Xu 32 was defined as a storage-tolerant cultivar (rot percentage less than 25%); Xu 55-2, Z 15-1, Shangshu 19, Yushu, and Zhezi 3 as above-moderate storage-tolerant cultivars (rot percentage ranging from 25 to 50%); Sushu 16, Yanshu 5, and Hanzi as medium-storable cultivars (rot percentage 50–75%); and Yan 25 as a storage-sensitive cultivar (rot percentage greater than 75%). Meanwhile, analysis of the α-amylase activity in root tubers of the 10 sweet potato cultivars during storage indicated that α-amylase activity was lowest in the storage-tolerant cultivar Xu 32 and highest in the storage-sensitive cultivar Yan 25. Evaluation of antioxidant enzyme activities and ROS content in the leaves of these 10 cultivars demonstrated that cultivar Xu 32, which showed the best storage property, had higher antioxidant enzyme activity [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)] but lower lipoxygenase (LOX) activity, hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) contents, and superoxide anion radical (O 2 ⋅ – ) production rates compared with those of the storage-sensitive cultivar Yan 25 and the medium-storability cultivars Hanzi, Yanshu 5, and Sushu 16. Additionally, principal component analysis (PCA) suggested that sweet potato cultivars with different storage properties were clustered separately. Correlation and heat map analysis further indicated that CAT, APX, POD, and SOD activities were negatively correlated with α-amylase activity, while LOX activity and MDA and H 2 O 2 contents were negatively correlated with the storage property of sweet potato. Combined, our findings revealed that storage property is highly correlated with antioxidant capacity in sweet potato leaves and negatively correlated with α-amylase activity in tuberous roots, which provides a convenient means for the screening of storage-tolerant sweet potato cultivars.

As a signaling molecule, hydrogen sulfide (H2S) plays an indispensable role in the modulation of ripening and senescence in fruits and vegetables. To explore the role of H2S in regulating metabolism of postharvest tomato, ripening-related physiological parameters, activities of antioxidant enzymes and gene expression were analyzed in H2S-fumigated tomato fruits. These results show that H2S significantly delayed the color transition and softening of tomato fruit, and maintained higher level of flavonoids and lower level of anthocyanin during storage. Besides, H2S could maintain higher level of nutritional-related metabolites, such as reducing sugar, ascorbic acid during postharvest storage. Moreover, H2S decreased the rate of O2− production, inhibited the production of H2O2 and malondialdehyde (MDA), enhanced the activities of antioxidant enzymes including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (POD) in tomato fruits, while reduced the activities of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and lipoxygenase (LOX). Besides, the expression of the antioxidant-encoding genes SlCAT2, SlPOD12 was generally upregulated with H2S fumigation. Principal component analysis (PCA) suggests that H2S induced significant discrepancy mainly to the differences in firmness, anthocyanin, flavonoid and the activity of guaiacol peroxidase (POD), and the correlation analysis further shows that H2S affected pigment metabolism and nutritional quality. In conclusion, H2S could maintain better appearance and nutritional quality, and prolong the storage period of postharvest tomato fruits through activating the antioxidative system.

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.

Hydrogen sulfide (H 2 S) is a gaseous signaling molecule that plays multiple roles in plant development. However, whether endogenous H 2 S plays a role in fruit ripening in tomato is still unknown. In this study, we show that the H 2 S-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 H 2 S 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 H 2 S, suggesting a role of SlLCD1 in H 2 S 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 H 2 S, and transcriptome analysis showed that ripening-related gene expression was suppressed. Based on the results for a SlLCD1 gene-edited mutant and exogenous H 2 S application, we propose that the nuclear-localized cysteine desulfhydrase SlLCD1 is required for endogenous H 2 S generation and participates in the regulation of tomato fruit ripening.

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.

The activities and gene expression of antioxidative enzymes and the ROS content were analyzed in two typical storage-tolerant cultivars (Xushu 32 and Shangshu 19) and another two storage-sensitive cultivars (Yanshu 25 and Sushu 16) to explore the association between the storage capacity of sweetpotato (Ipomoea batatas (L.) Lam) and ROS scavenging capability. The storage roots of the storage-tolerant cultivars maintained higher activities and expression levels of antioxidative enzymes, including ascorbate peroxidase (APX), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD); lower activity and expression of lipoxygenase (LOX); and lower accumulation of ROS metabolites compared with the storage-sensitive cultivars. The antioxidative capability and ROS parameters of leaves were positively correlated with those of storage roots. Our results provide valuable insight for evaluating the storability of sweetpotato cultivars by analyzing the capabilities of the antioxidative system and the contents of ROS metabolites.

[This corrects the article DOI: 10.1371/journal.pone.0173277.].

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.

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.

Aspergillus niger, a common saprophytic fungus, causes rot in many fruits. We studied the role of a putative catalase-peroxidase-encoding gene, cpeB, in oxidative stress and virulence in fruit. The cpeB gene was deleted in A. niger by homologous recombination, and the Δ cpeB mutant showed decreased CAT activity compared with that of the wild type. The cpeB gene deletion caused increased sensitivity to H2O2 stress, and spore germination was significantly reduced; in addition, the reactive-oxygen-species (ROS) metabolites superoxide anions (·O2-), hydrogen peroxide (H2O2), and malondialdehyde (MDA) accumulated in the Δ cpeB mutant during H2O2 stress. Furthermore, ROS metabolism in A. niger infected apples was determined, and our results showed that the Δ cpeB mutant induced an attenuated response in apple fruit during the fruit-pathogen interaction; the cpeB gene deletion significantly reduced the development of lesions, suggesting that the cpeB gene in A. niger is essential for full virulence in apples.

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

Kiwifruit ( Actinidia deliciosa ) is a typical climacteric fruit, and its ripening is closely associated with ethylene. In this study, we present evidence that H 2 S alleviated ethylene-induced ripening and senescence of kiwifruit. Kiwifruit were fumigated with ethylene released from 0.4 g·L −1 ethephon solution or H 2 S with 1 m m sodium hydrosulfide (NaHS) as the donor or in combination. Fumigation with ethylene was found to accelerate kiwifruit ripening and H 2 S treatment effectively alleviated ethylene-induced fruit softening in parallel with attenuated activity of polygalacturonase (PG) and amylase. Ethylene + H 2 S treatment also maintained higher levels of ascorbic acid, titratable acid, starch, soluble protein, and reducing sugar compared with ethylene group, whereas suppressed the increase in chlorophyll and carotenoid. Kiwifruit ripening and senescence under ethylene treatment was accompanied by elevation in reactive oxygen species (ROS) levels, including H 2 O 2 and superoxide anion and malondialdehyde (MDA), but combined treatment of ethylene plus H 2 S alleviated oxidative stress in fruit. Furthermore, the activities of antioxidative enzymes catalase (CAT) and ascorbate peroxidase (APX) were increased by ethylene + H 2 S treatment in comparison with ethylene alone, whereas the activities of lipoxygenase (LOX) and polyphenol oxidase (PPO) were attenuated by H 2 S treatment. Further investigations showed that H 2 S repressed the expression of ethylene synthesis-related genes AdSAM , AdACS1 , AdACS2 , AdACO2 , and AdACO3 and cysteine protease genes, such as AdCP1 and AdCP3 . Taken together, our findings suggest that H 2 S alleviates kiwifruit ripening and senescence by antagonizing the effect of ethylene through reduction of oxidative stress and inhibition of ethylene synthesis pathway.

Aspergillus niger is a common pathogenic fungus causing postharvest rot of fruit and vegetable, whereas the knowledge on virulence factors is very limited. Superoxide dismutase [SOD (EC 1.15.1.10)] is an important metal enzyme in fungal defense against oxidative damage. Thus, we try to study whether Cu/Zn-SOD is a virulence factor in A. niger . Cu/Zn-SOD encoding gene sodC was deleted in A. niger [MA70.15 (wild type)] by homologous recombination. The deletion of sodC led to decreased SOD activity in A. niger , suggesting that sodC did contribute to full enzyme activity. Δ sodC strain showed normal mycelia growth and sporulation compared with wild type. However, sodC deletion markedly increased the cell’s sensitivity to intracellular superoxide anion generator menadione. Besides, spore germination under menadione and H 2 O 2 stresses were significantly retarded in Δ sodC mutant compared with wild type. Further results showed that sodC deletion induced higher superoxide anion production and higher content of H 2 O 2 and malondialdehyde (MDA) compared with wild type, supporting the role of SOD in metabolism of reactive oxygen species (ROS). Furthermore, Δ sodC mutant had a reduced virulence on chinese white pear ( Pyrus bretschneideri ) as lesion development by Δ sodC was significantly less than wild type. The determination of superoxide anion, H 2 O 2 , and MDA in A. niger -infected pear showed that chinese white pear infected with Δ sodC accumulated less superoxide anion, H 2 O 2 , and MDA compared with that of wild type A. niger , implying that Δ sodC induced an attenuated response in chinese white pear during fruit–pathogen interaction. Our results indicate that sodC gene contributes to the full virulence of A. niger during infection on fruit. Aspergillus niger is one of the most common species found in fungal communities. It is an important fermentation industrial strain and is also known to cause the most severe symptoms in fruit during long-term storage (Pel et al., 2007). Meanwhile, plants activate their signaling pathways to trigger defense responses to limit pathogen expansion. One of the earliest host responses after pathogen attack is oxidative burst, during which large quantities of ROS are generated by different host enzyme systems, such as glucose oxidase (Govrin and Levine, 2000). ROS such as singlet oxygen, superoxide anion, hydroxyl (OH − ), and H 2 O 2 are released to hinder the advance of pathogens (Gara et al., 2003). ROS can react with and damage cellular molecules, such as DNA, protein, and lipids, which will limit fungal propagation in the host plant (Apel and Hirt, 2004).

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.

New anthocyanins from purple sweet potato (Solanum tuberosum) were purified and the probiotic properties were investigated in this study. After pretreatment of purple sweet potato, the anthocyanins were extracted using the water extraction method, and the extraction conditions were studied. Subsequently, the resulting solution was filtered with a membrane of 3 kDa molecular weight cut-off; and the filtrate was subjected to three kinds of macroporous adsorption resins (AB-8, DM301 and X-5) for further purification. Meanwhile, the proliferation effect of anthocyanins on the bifidobacteria was also investigated. The results demonstrated that the optimal extraction condition for anthocyanins was temperature 60 degrees C, liquid solid ratio 10:1, time 2 h and pH3.0. X-5 presented the best absorption capability (89.00 +/- 0.24%) as well as desorption capability (85.11 +/- 0.14%), indicating that X-5 was the suitable potential resin for purification. The yield of the anthocyanins was 0.095%, and the purity of anthocyanins was confirmed by high performance liquid chromatography (HPLC). It was found that the new anthocyanins could stimulate bifidobacteria growth and four phenolic acids (protocatechuic acid, ferulic acid, caffeic acid and vanillic acid) were produced; suggesting that the anthocyanins could be utilized as prebiotics.

本文研究了黑米花青素对小白鼠的抗氧化及免疫调节作用.以生理盐水为载体,维生素C作为阳性对照,分别采用高、中、低三种剂量的黑米花青素粗提物及其纯化物,灌胃喂养小白鼠30d后,测定小白鼠的脾和胸腺指数,血清、肝脏、心脏和肾脏中总抗氧化力(T-AOC)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化酶(GSH-Px)的活力,丙二醛(MDA)和羟自由基含量.研究结果表明:该花青素能增加小白鼠的脾和胸腺指数,显著提高其体内T-AOC水平和SOD、GSH-Px的活性,降低MDA和羟自由基的含量.因此,黑米花青素具有提高小白鼠的体内抗氧化及免疫调节作用.

[Objective]To study the volatile constituents from the leaves of Rubus chingii Hu.[Methods]The volatile constituents of R.chingii leaves were analyzed by HS-GC/MS.The constituents were identified by mass spectra using NIST.The relative percentage content of volatile constituents was calculated by the relative peak area method.[Results]A total of 37 constituents were isolated and identified.Among them,hexadecanoic acid had the maximum content.[Conclusions]The volatile constituents of R.chingii leaves were isolated and analyzed by HS-GC/MS,which provided scientific references for the development and utilization of R.chingii leaves.

The cultivation mode based on the enterprise's scientific research project can make the gradu-ates directly involve in research work under the combined direction of the tutor and the enterprise's re-search personnel .In this paper ,by using theoretical and empirical methods ,the basic condition of the cultivation of the graduates'scientific research ability during the process of enterprise-university coop-eration is investigated .The research subject ,research experience and research cognition are the im-portant parts of the cultivation .The cultivation strategy is also put forward ,i .e .to explore the culti-vation mode of graduates according to the requirement of the enterprise and the rules of graduates'cul-tivation during the process of the enterprise-university cooperation .