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Abstract Members of the Metal Tolerance Protein (MTP) family are critical in mediating the transport and tolerance of divalent metal cations. Despite their significance, the understanding of MTP genes in mustard (Brassica juncea) remains limited, especially regarding their response to heavy metal (HM) stress. In our study, we identified MTP gene sets in Brassica rapa (17 genes), Brassica nigra (18 genes), and B. juncea (33 genes) using the HMMER (Cation_efflux; PF01545) and BLAST analysis. For the 33 BjMTPs, a comprehensive bioinformatics analysis covering the physicochemical properties, phylogenetic relationships, conserved motifs, protein structures, collinearity, spatiotemporal RNA-seq expression, GO enrichment, and expression profiling under six HM stresses (Mn2+, Fe2+, Zn2+, Cd2+, Sb3+, and Pb2+) were carried out. According to the findings of physicochemical characteristics, phylogenetic tree, and collinearity, the allopolyploid B. juncea’s MTP genes were inherited from its progenitors, B. rapa and B. nigra, with minimal gene loss during polyploidization. Members of the BjMTP family exhibited conserved motifs, promoter elements, and expression patterns across subgroups, consistent with the seven evolutionary branches (G1, G4–G9, and G12) of the MTPs. Further, spatiotemporal expression profiling under HM stresses successfully identified specific genes and crucial cis-regulatory elements associated with the response of BjMTPs to HM stresses. These findings may contribute to the genetic improvement of B. juncea for enhanced HM tolerance, facilitating the remediation of HM-contaminated areas.

Ascorbate peroxidases (APXs) are key components of the ascorbate–glytathione cycle, which plays an important role in removing excess reactive oxygen species (ROS) in plants. Herein, MaAPX1 was verified as being involved in the ripening and senescence of banana fruit, exhibiting responsiveness to the accumulation of ROS and the oxidation of proteins. Site-directed mutation was applied to explore the mechanism of MaAPX1 activity changes. We found that the 32-site cysteine (Cys, C) served as a potential S-nitrosylation site. The mutant MaAPX1C32S activity was decreased significantly when Cys32 was mutated to serine (Ser, S). Intriguingly, the neighboring conserved 36-site methionine (Met, M), which is adjacent to Cys32, displayed an enzyme activity that was approximately five times higher than that of the wild-type MaAPX1 when mutated to lysine (Lys, K). Utilizing LC-MS/MS spectroscopy coupled with stopped-flow analysis showed that the enhanced MaAPX1M36K activity might be due to the increased S-nitrosylation level of Cys32 and the promotion of intermediate (compound I, the first intermediate product of the reaction of APX with H2O2) production. Molecular docking simulations showed that the S-N bond between Cys32 and Lys36 in MaAPX1M36K might have a function in protecting the thiol of Cys32 from oxidation. MaAPX1M36K, a promising mutant, possesses immense potential for improving the antioxidant capabilities of APX in the realm of bioengineering technology research.

Ascorbate peroxidase (APX) is a key antioxidant enzyme that is involved in diverse developmental and physiological process and stress responses by scavenging H2O2 in plants. APX itself is also subjected to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of APX in plants remains poorly understood. Here, we identified and confirmed that MaAPX1 interacts with methionine sulfoxide reductase B2 (MsrB2) in bananas. Ectopic overexpression of MaAPX1 delays the detached leaf senescence induced by darkness in Arabidopsis. Sulfoxidation of MaAPX1, i.e., methionine oxidation, leads to loss of the activity, which is repaired partially by MaMsrB2. Moreover, mimicking sulfoxidation by mutating Met36 to Gln also decreases its activity in vitro and in vivo, whereas substitution of Met36 with Val36 to mimic the blocking of sulfoxidation has little effect on APX activity. Spectral analysis showed that mimicking sulfoxidation of Met36 hinders the formation of compound I, the first intermediate between APX and H2O2. Our findings demonstrate that the redox state of methionine in MaAPX1 is critical to its activity, and MaMsrB2 can regulate the redox state and activity of MaAPX1. Our results revealed a novel post-translational redox modification of APX.

Litchi (Litchi chinensis Sonn.) is a well-known traditional medicine since ancient times. The crude extract of litchi leaf exhibited good bioactivity against cancer cells. In this work, three sesquilignans, ehletianol C (1), sesquipinsapol B (2) and sesquimarocanol B (3), and one lignan glycoside schizandriside (4) were isolated from the ethyl acetate-soluble extract of litchi leaf by column chromatography. Their structures were further elucidated by ESI-MS and NMR spectral studies. Ehletianol C, sesquipinsapol B and sesquimarocanol B were detected from this species for the first time. Sesquipinsapol B (EC50 values less than 2.0 μg/mL) and sesquimarocanol B (EC50 values less than 2.4 μg/mL) showed very high cytotoxicities against nasopharynx carcinoma cells CNE1 and CNE2 cells, respectively. Thus, they could be utilized as good drug candidates against nasopharynx cancer. Moreover, three sesquilignans showed effective in vitro cytotoxicities against human cancer cells HepG2 and Hela. Four lignans showed better antioxidant activities than butylated hydroxytoluene (BHT).

Six flavonoids, namely luteolin (1), epicatechin (2), kaempferol 3-O-β-glucoside (3), kaempferol 3-O-α-rhamnoside (4), procyanidin A2 (5) and rutin (6) were purified from the EtOAc-soluble extract of litchi leaf by column chromatography. Their structures were elucidated by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) evidences. Luteolin and kaempferol 3-O-α-rhamnoside were found from this plant for the first time. Procyanidin A2 exhibited high anticancer activities against human hepatoma HepG2 and human cervical carcinoma Hela cells. However, it had poor anticancer activities against human lung cancer A549 and human breast cancer MCF-7 cells. Luteolin, epicatechin, procyanidin A2 and rutin showed good antioxidant activities than butylated hydroxytoluene (BHT). The antimicrobial activity assay indicated that luteolin possessed the strongest antimicrobial activity against Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Salmonella and Bacillus thuringiensis. Epicatechin, procyanidin A2 and rutin showed relatively weak antimicrobial activities.

β-aminobutyric acid (BABA) is a new environmentally friendly agent to induce disease resistance by priming of defense in plants. However, molecular mechanisms underlying BABA-induced priming defense are not fully understood. Here, comprehensive analysis of priming mechanism of BABA-induced resistance was investigated based on mango-Colletotrichum gloeosporioides interaction system using iTRAQ-based proteome approach. Results showed that BABA treatments effectively inhibited the expansion of anthracnose caused by C. gleosporioides in mango fruit. Proteomic results revealed that stronger response to pathogen in BABA-primed mango fruit after C. gleosporioides inoculation might be attributed to differentially accumulated proteins involved in secondary metabolism, defense signaling and response, transcriptional regulation, protein post-translational modification, etc. Additionally, we testified the involvement of non-specific lipid-transfer protein (nsLTP) in the priming acquisition at early priming stage and memory in BABA-primed mango fruit. Meanwhile, spring effect was found in the primed mango fruit, indicated by inhibition of defense-related proteins at priming phase but stronger activation of defense response when exposure to pathogen compared with non-primed fruit. As an energy-saving strategy, BABA-induced priming might also alter sugar metabolism to provide more backbone for secondary metabolites biosynthesis. In sum, this study provided new clues to elucidate the mechanism of BABA-induced priming defense in harvested fruit.

Anthocyanin accumulation is crucial for the development of quality for most fruit. The mechanism underlying the regulation of anthocyanin biosynthesis by transcription factors in litchi fruit remains largely unknown. In this study, we isolated one NAC (NAM, ATAF1/2 and CUC2) TF gene, LcNAC13. Expression of LcNAC13 was upregulated as ripening proceeded, followed by the accumulation of anthocyanins. Electrophoretic mobility shift assay (EMSA) and transient expression assay showed that LcNAC13 could negatively regulate the expression of anthocyanin biosynthesis-related genes, including LcCHS1/2, LcCHI, LcF3H, LcF3’H, LcDFR, and LcMYB1. Furthermore, LcR1MYB1, as one R1-MYB type MYB, was identified to physically interact with LcNAC13 and reverse the effect of LcNAC13. Taken together, these results suggested that LcNAC13 and LcR1MYB1 may act together to antagonistically regulate anthocyanin biosynthesis during litchi fruit ripening, which helps to provide new insights into the regulatory networks of anthocyanin biosynthesis.

As the average life expectancy continues to increase, so does the need for resorbable materials designed to treat, augment, or replace components and functions of the body. Naturally occurring biopolymers such as silks are already attractive candidates due to natural abundance and high biocompatibility accompanied by physical properties which are easily modulated through blending with another polymer. In this paper, the authors report on the fabrication of biocomposite materials made from binary blends of Bombyx mori silk fibroin (SF) protein and renewably sourced low molecular weight nylon 610 and high molecular weight nylon 1010. Films were characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Results of this study demonstrated that enhanced structural and thermal properties were achievable in composite films SF-N610/N1010 due to their chemical similarity and the possible formation of hydrogen bonds between nylon and silk molecular chains. This study provides useful insight into the sustainable design of functional composite materials for biomedical and green technologies.

Citrinin is a toxic secondary metabolite of Penicillium citrinum and its contamination in many food items has been widely reported. However, research on the citrinin biosynthesis pathway and its regulation mechanism in P. citrinum is rarely reported. In this study, we investigated the effect of different carbon sources on citrinin production by P. citrinum and used transcriptome analysis to study the underlying molecular mechanism. Our results indicated that glucose, used as the sole carbon source, could significantly promote citrinin production by P. citrinum in Czapek’s broth medium compared with sucrose. A total of 19,967 unigenes were annotated by BLAST in Nr, Nt, Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Transcriptome comparison between P. citrinum cultured with sucrose and glucose revealed 1085 differentially expressed unigenes. Among them, 610 were upregulated while 475 were downregulated under glucose as compared to sucrose. KEGG pathway and Gene ontology (GO) analysis indicated that many metabolic processes (e.g., carbohydrate, secondary metabolism, fatty acid and amino acid metabolism) were affected, and potentially interesting genes that encoded putative components of signal transduction, stress response and transcription factor were identified. These genes obviously had important impacts on their regulation in citrinin biosynthesis, which provides a better understanding of the molecular mechanism of citrinin biosynthesis by P. citrinum.

Thioredoxins (Trxs) are small proteins with a conserved redox active site WCGPC and are involved in a wide range of cellular redox processes. However, little information on the role of Trx in regulating low-temperature stress of harvested fruit is available. In this study, three full-length Trx cDNAs, designated MaTrx6, MaTrx9 and MaTrx12, were cloned from banana (Musa acuminata) fruit. Phylogenetic analysis and protein sequence alignments showed that MaTrx6 was grouped to h2 type with a typical active site of WCGPC, whereas MaTrx9 and MaTrx12 were assigned to atypical cys his-rich Trxs (ACHT) and h3 type with atypical active sites of GCAGC and WCSPC, respectively. Subcellular localization indicated that MaTrx6 and MaTrx12 were located in the plasma membrane and cytoplasm, respectively, whereas MaTrx9 showed a dual cytoplasmic and chloroplast localization. Application of ethylene induced chilling tolerance of harvested banana fruit, whereas 1-MCP, an inhibitor of ethylene perception, aggravated the development of chilling injury. RT-qPCR analysis showed that expression of MaTrx12 was up-regulated and down-regulated in ethylene- and 1-MCP-treated banana fruit at low temperature, respectively. Furthermore, heterologous expression of MaTrx12 in cytoplasmic Trx-deficient Saccharomyces cerevisiae strain increased the viability of the strain under H2O2. These results suggest that MaTrx12 plays an important role in the chilling tolerance of harvested banana fruit, possibly by regulating redox homeostasis.

Transcription factors (TFs) are important regulators of plant growth and development and responses to stresses. TFs themselves are also prone to multiple posttranslational modifications (PTMs). However, redox-mediated PTM of TFs in plants remains poorly understood. Here, we established that NON-RIPENING (NOR), a master TF regulating tomato (Solanum lycopersicum) fruit ripening, is a target of the Met sulfoxide reductases A and B, namely E4 and SlMsrB2, respectively, in tomato. Met oxidation in NOR, i.e. sulfoxidation, or mimicking sulfoxidation by mutating Met-138 to Gln, reduces its DNA-binding capacity and transcriptional regulatory activity in vitro. E4 and SlMsrB2 partially repair oxidized NOR and restore its DNA-binding capacity. Transgenic complementation of the nor mutant with NOR partially rescues the ripening defects. However, transformation of nor with NOR-M138Q, containing mimicked Met sulfoxidation, inhibits restoration of the fruit ripening phenotype, and this is associated with the decreased DNA-binding and transcriptional activation of a number of ripening-related genes. Taken together, these observations reveal a PTM mechanism by which Msr-mediated redox modification of NOR regulates the expression of ripening-related genes, thereby influencing tomato fruit ripening. Our report describes how sulfoxidation of TFs regulates developmental processes in plants. [ABSTRACT FROM AUTHOR]

Senescence is a key factor resulting in deterioration of nonclimacteric fruit. NAC transcription factors are important regulators in plant development and abiotic stress responses, yet little information regarding the role of NACs in regulating non-climacteric fruit senescence is available. In this study, we cloned 13 NAC genes from litchi (Litchi chinensis) fruit, and analyzed subcellular localization and expression profiles of these genes during post-harvest natural and low-temperature- delayed senescence. Of the 13 NAC genes, expression of LcNAC1 was up-regulated in the pericarp and pulp as senescence progressed, and was significantly higher in senescencedelayed fruit than that in naturally senescent fruit. LcNAC1 was induced by exogenous ABA and hydrogen peroxide. Yeast one-hybrid analysis and transient dual-luciferase reporter assay showed that LcNAC1 was positively regulated by the LcMYC2 transcription factor. LcNAC1 activated the expression of LcAOX1a, a gene associated with reactive oxygen species regulation and energy metabolism, whereas LcWRKY1 repressed LcAOX1a expression. In addition, LcNAC1 interacted with LcWRKY1 in vitro and in vivo. These results indicated that LcNAC1 and LcWRKY1 form a complex to regulate the expression of LcAOX1a antagonistically. Taken together, the results reveal a hierarchical and co-ordinated regulatory network in senescence of harvested litchi fruit. [ABSTRACT FROM AUTHOR]

This study was conducted to increase the bioactivity of litchi pericarp polysaccharides (LPPs) biotransformed by Aspergillus awamori. Compared to the non-A. awamori-fermented LPP, the growth effects of A. awamori-fermented LPP on Lactobacillus bulgaricus and Streptococcus thermophilus were four and two times higher after 3 days of fermentation, respectively. Increased 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and DNA protection activity of litchi pericarp polysaccharides were also achieved after A. awamori fermentation. Moreover, the relative content of glucose and arabinose in LPP after fermentation decreased from 58.82% to 22.60% and from 18.82% to 10.09%, respectively, with a concomitant increase in the relative contents of galactose, rhamnose, xylose, and mannose. Furthermore, lower molecular weight polysaccharides were obtained after A. awamori fermentation. It can be concluded that A. awamori was effective in biotransforming LPP into a bioactive mixture with lower molecular weight polysaccharides and higher antioxidant activity and relative galactose content. [ABSTRACT FROM AUTHOR]

Background: Litchi (Litchi chinensis Sonn.) pericarp is a major byproduct which contains a significant amount of polyphenol. This study was designed to biotransformation litchi pericarp extract (LPE) by Aspergillus awamori to produce more bioactive compounds with stronger antioxidant activities. Results: The study exhibited that the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities significantly (p < 0.05) increased from 15.53% to 18.23% in the water-extracted fraction and from 25.41% to 36.82% in the ethyl acetate-extracted fraction. Application of DNA cleavage assay further demonstrated the enhanced protection effect of the fermented phenolics on DNA damage. It is also noted that the water-extracted fraction of the fermented LPE possessed a much stronger capacity than the ethyl acetate-extracted fraction to prevent from damage of supercoiled DNA. Interestingly, it was found that some new compounds such as catechin and quercetin appeared after of A. awamori fermentation of LPE, which could account for the enhanced antioxidant activity. Conclusion: The DPPH radical scavenging activity and DNA protection effect of LPE were increased by Aspergillus awamori bioconversion while some compounds responsible for the enhanced antioxidant activity were identified. This study provided an effective way of utilizing fruit pericarp as a readily accessible source of the natural antioxidants in food industry and, thus, extended the application area such as fruit by-products. [ABSTRACT FROM AUTHOR]

Rapid and efficient methods to assess nanoparticle toxicity are desired in current research. Here we showed that Escherichia colilabeled by green fluorescent protein can be a good model bacterium for assessing acute toxicity of TiO2(about 50% inhibition ratios after 135 min exposure). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that TiO2nanoparticles (NPs) can influence certain protein expression in the recombinant bacterium, and the obvious effects in repressed expression and elevated expression were observed in 30/40, 10/20 μg mL−1treated cells, respectively. However, the GFP expression (27 kD) was not influenced by introduced TiO2NPs. The change of the fluorescence intensity may be caused by the damage in folding and chromophore formation of the GFP post-translational modification due to generated reactive oxygen species. Furthermore, TiO2NPs at higher concentrations decreased their toxicity because of aggregation. 20 μg mL−1humic acid (HA) introduced to the medium can decrease the fluorescent inhibition owing to the barrier of steric hindrance it provides between NPs and cells. [ABSTRACT FROM AUTHOR]

Pericarp browning is the major cause of deterioration of harvested litchi fruit. Water loss plays a role in pericarp browning of litchi fruit. This study investigated the effects of humidification with dry fog on pericarp browning and quality of litchi fruit stored at low temperature. Litchi fruit were stored in a non-humidified cold chamber (control) or in a humidified cold room using Tabor atomizer system that generated 95% relative humidity (RH) without depositing water on the fruit surface at 4°C. Control fruit stored in cold room without added humidity underwent rapid weight loss, accompanied by severe pericarp browning after 25 d of storage. However, slight weight loss and no obvious pericarp browning were found in humidified-fruit. Moreover, humidification maintained well the integrity of cell membrane and inhibited polyphenol oxidase activity during early storage. In addition, respiration rate was obviously inhibited in humidified-fruit compared with control fruit. This study might provide a convenient approach to reduce pericarp browning of harvested litchi fruit by humidifying the fruit using the Tabor atomizer at low temperature instead of packaging with film. [ABSTRACT FROM AUTHOR]