Reactive oxygen species play important effects on organisms not only in vivo but also in vitro . The atomic oxygen radical anion (O - ) has shown extremely high oxidation and reactivity towards small molecules of hydrocarbons. However, the O - effects on cells of microorganisms are scarcely investigated. This work showed the evidence that O - could react quickly with microorganisms ( Escherichia coli , Bacillus subtilis , Staphylococcus aureus , Aspergillus niger , Saccharomyces cerevisiae , and Actinomycetes ( 5046 )) and damaged the cell walls seriously as well as their intrinsic structures, arising a fast and irreversible inactivation. SEM and TEM micrographs were used to reveal the structure changes of cells before and after reacting with O - radicals. The inactivation efficiencies of the microorganisms depended on the O - intensity, the initial population of microorganisms, the exposed area, the environment, and the microorganisms’ types. Over 99% reduction of an initial1.0×107colony-forming unit (cfu), E. coli population only required less than 2 minutes while exposed to a 0.23 μ A/cm 2 O - flux under dry argon atmosphere (30°C, 1 atm). The observation of anionic intermediates (CO - , CO 2- , H 2 O - , and anionic hydrocarbons) by time-of-flight (TOF) mass spectrometry and the neutral volatile products (CO, CO 2 , and H 2 O) by quadrupole mass spectrometry (Q-MS) provided an evidence of the reactions of O - with hydrocarbon bonds of the microorganisms. The inactivation mechanism of microorganisms induced by O - was discussed.
Uncovering the underlying mechanism of salt tolerance is important to breed cotton varieties with improved salt tolerance. In this study, transcriptome and proteome sequencing were performed on upland cotton (Gossypium hirsutum L.) variety under salt stress, and integrated analysis was carried out to exploit salt-tolerance genes in cotton. Enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed on differentially expressed genes (DEGs) obtained from transcriptome and proteome sequencing. GO enrichment was carried out mainly in the cell membrane, organelle, cellular process, metabolic process, and stress response. The expression of 23,981 genes was changed in physiological and biochemical processes such as cell metabolism. The metabolic pathways obtained by KEGG enrichment included glycerolipid metabolism, sesquiterpene and triterpenoid biosynthesis, flavonoid production, and plant hormone signal transduction. Combined transcriptome and proteome analysis to screen and annotate DEGs yielded 24 candidate genes with significant differential expression. The quantitative real-time polymerase chain reaction (qRT-PCR) validation of the candidate genes showed that two genes (Gh_D11G0978 and Gh_D10G0907) responded significantly to the induction of NaCl, and these two genes were further selected as target genes for gene cloning and functional validation through virus-induced gene silencing (VIGS). The silenced plants exhibited early wilting with a greater degree of salt damage under salt treatment. Moreover, they showed higher levels of reactive oxygen species (ROS) than the control. Therefore, we can infer that these two genes have a pivotal role in the response to salt stress in upland cotton. The findings in this research will facilitate the breeding of salt tolerance cotton varieties that can be grown on saline alkaline lands.
Tall fescue (Festuca arundinacea Schreb.) is widely utilized as a major forage and turfgrass species in the temperate regions of the world and is a valuable plant material for studying molecular mechanisms of grass stress tolerance due to its superior drought and heat tolerance among cool-season species. Selection of suitable reference genes for quantification of target gene expression is important for the discovery of molecular mechanisms underlying improved growth traits and stress tolerance. The stability of nine potential reference genes (ACT, TUB, EF1a, GAPDH, SAND, CACS, F-box, PEPKR1 and TIP41) was evaluated using four programs, GeNorm, NormFinder, BestKeeper, and RefFinder. The combinations of SAND and TUB or TIP41 and TUB were most stably expressed in salt-treated roots or leaves. The combinations of GAPDH with TIP41 or TUB were stable in roots and leaves under drought stress. TIP41 and PEPKR1 exhibited stable expression in cold-treated roots, and the combination of F-box, TIP41 and TUB was also stable in cold-treated leaves. CACS and TUB were the two most stable reference genes in heat-stressed roots. TIP41 combined with TUB and ACT was stably expressed in heat-stressed leaves. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) assays of the target gene FaWRKY1 using the identified most stable reference genes confirmed the reliability of selected reference genes. The selection of suitable reference genes in tall fescue will allow for more accurate identification of stress-tolerance genes and molecular mechanisms conferring stress tolerance in this stress-tolerant species.
The silk-spinning and Lepidopteran model insect Bombyx mori (Bombycidae) is a mulberry specialist. The BmSuc1 gene is the first β-fructofuranosidase (β-FFase) encoding gene identified in animals, and β-FFase acts as an essential sucrase for glycometabolism modulation in the silkworm larvae, involved in resistance to mulberry alkaloids. Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is an important mulberry pest leading to heavy economic loss of sericulture. However, no molecular or biochemical information is available about G. pyloalis β-FFase homologs. In this study, five β-FFase homologous genes in G. pyloalis were obtained. The genes GpSuc1a and GpSuc2c were expressed in the midgut; GpSuc2c encodes a truncated polypeptide. The expression and the localization of GpSUC1a in the midgut was characterized. Whereas recombinant GpSUC1a expressed in both Escherichia coli and BmN cells displayed little activity as compared with higher activity of BmSUC1, β-FFase activity in the larval midgut of G. pyloalis and GpSUC1a purified from the midgut were both confirmed. The data suggested that the activation of GpSUC1a is probably controlled by a more complicated post-translational regulation system in G. pyloalis larvae than that of BmSUC1 in B. mori. To study post-translational modifications (PTMs), GpSUC1a and BmSUC1 were purified from larval midguts using immunoprecipitation and subjected to LC-MS to perform PTMs analysis. Some putative N-glycosylated sites were found in GpSUC1a but none in BmSUC1, while there was more methylation in BmSUC1 than in GpSUC1a, indicating that such PTMs were supporting the differential β-FFases activities in these two mulberry feeding caterpillars.
Abstract BACKGROUND AND AIMS Copper is an essential trace element for eukaryotes and a vital cofactor for various enzymes for a multiplicity of functions. Our previous study indicated that intracellular copper overload plays an important role in renal fibrosis. However, the underlying mechanism invoked by copper overload in renal fibrosis remains largely unknown. In this study, we found copper ions in cells were mainly accumulated in mitochondria, which damage the structure and function of mitochondria. Furthermore, copper transporter 1 (CTR1), the major transporter for the copper influx, was significantly increased in fibrotic kidneys. The activity of cytochrome C oxidase (COX), a copper coenzyme mediating the final step in the electron transport chain, was decreased. Therefore, we proposed that CTR1 might be involved in mitochondrial copper overload and renal fibrosis. METHOD The expression of CTR1 was examined in ischemia-reperfusion injury (IRI 28d) mice model. The regulatory mechanisms of CTR1 in vitro were investigated by treating renal tubule epithelium cell line (NRK-52E) with TGF-β1 or copper ions with or without copper chelator tetrathiomolybdate (TM) and in vivo, we use CTR1 transgenic mice subjected to IRI operation. ICP-MS, mitoSOX Red, electron microscopy, realtime-PCR and western blot analysis were applied in the current study. RESULTS We found that stimulated by TGF-β1, COX activity was declined. Mitochondria and cytosol, especially mitochondria, accumulated a large amount of copper in fibrotic kidney tissues. Furthermore, we found Copper transporter 1 (CTR1) expression was increased in fibrotic kidneys from chronic kidney disease patients, experimental fibrosis model mice and in vitro. More importantly, compared with WT mice, CTR1+/- mice subjected to an ischemia-reperfusion injury (IRI) had reduced mitochondrial copper level and ameliorated mitochondrial function and renal fibrosis, as evidenced by improving mitochondrial structure, inhibiting mtROS production and reducing expression of α-smooth muscle actin (α-SMA), collagen I and fibronectin. In addition, after treatment with the copper chelator tetrathiomolybdate (TM), mitochondrial function and kidney fibrosis were improved. CONCLUSION Collectively, our study showed that copper overload in mitochondria could damage the mitochondrial function and lead to renal fibrosis and CTR1 is involved in the mitochondrial copper overload. Copper overload inhibits the activity of COX and impairs mitochondria, subsequently leading to renal fibrosis.
Rice bakaenii disease (RBD) is a widespread and devastating disease mainly caused by Fusarium fujikuroi. Pydiflumetofen (Pyd) is a novel succinate dehydrogenase inhibitor (SDHI) with strong inhibitory activity against F. fujikuroi, but the mechanism of resistance to Pyd has not been well studied for this pathogen. Through fungicide adaption, a total of 12 Pyd-resistant mutants were obtained and the resistance level could be divided into three categories of high resistance (HR), moderate resistance (MR), and low resistance (LR) with resistance factors (RF) of 184.04–672.90, 12.63–42.49, and <10, respectively. Seven genotypes of point mutations in FfSdh genes (FfSdhBH248L, FfSdhBH248D, FfSdhBH248Y, FfSdhC2A83V, FfSdhC2H144Y, FfSdhDS106F, and FfSdhDE166K) were found in these mutants, among which genotype FfSdhBH248L and FfSdhC2A83V mutants showed HR, genotype FfSdhBH248D, FfSdhBH248Y, FfSdhC2H144Y, and FfSdhDE166K mutants showed MR, and genotype FfSdhDS106F mutants showed LR. Moreover, all the substitutions of amino acid point mutations including FfSdhBH248L/D/Y, FfSdhC2A83V,H144Y, and FfSdhDS106F,E166K conferring resistance to Pyd in F. fujikuroi were verified by protoplast transformation. Additionally, a positive cross-resistance was detected between Pyd and another SDHI fungicide penflufen, while no cross-resistance was detected between Pyd and phenamacril, prochloraz, azoxystrobin, carbendazim, or fludioxonil. Although pathogenicity of the mutants was increased compared with that of the wild-type parental strains, the mycelial growth rate and spore production levels of the resistant mutants were significantly decreased (P < 0.05), indicating significant fitness cost of resistance to Pyd in F. fujikuroi. Taken together, the risk of resistance to Pyd in F. fujikuroi might be moderate, and appropriate precautions against resistance development in natural populations should be taken into account when Pyd is used for the control of RBD.
FGF5 and FGF18 are key factors in the regulation of the hair follicle cycle. FGF5 is overexpressed during the late anagen phase and serves as a crucial regulatory factor that promotes the anagen-to-catagen transition in the hair follicle cycle. FGF18, which is overexpressed during the telogen phase, mainly regulates the hair follicle cycle by maintaining the telogen phase and inhibiting the entry of hair follicles into the anagen phase. The inhibition of FGF5 may prolong the anagen phase, whereas the inhibition of FGF18 may promote the transition of the hair follicles from the telogen phase to the anagen phase. In the present study, we used siRNA to suppress FGF5 or FGF18 expression as a way to inhibit the activity of these genes. Using qPCR, we showed that FGF5-targeting siRNA modified by cholesterol was more effective than the same siRNA bound to a cell-penetrating peptide at suppressing the expression of FGF5 both in vitro and in vivo . We then investigated the effects of the cholesterol-modified siRNA targeting either FGF5 or FGF18 on the hair follicle cycle in a depilated area of the skin on the back of mice. The cholesterol-modified siRNA, delivered by intradermal injection, effectively regulated the hair follicle cycle by inhibiting the expression of FGF5 and FGF18. More specifically, intradermal injection of a cholesterol-modified FGF5-targeted siRNA effectively prolonged the anagen phase of the hair follicles, whereas intradermal injection of the cholesterol-modified FGF18-targeted siRNA led to the mobilization of telogen follicles to enter the anagen phase earlier. The inhibitory effect of the cholesterol-modified FGF18-targeted siRNA on FGF18 expression was also evaluated for a topically applied siRNA. Topical application of a cream containing the cholesterol-modified FGF18-targeted siRNA on a depilated area of the skin of the back of mice revealed comparable inhibition of FGF18 expression with that observed for the same siRNA delivered by intradermal injection. These findings suggested that alopecia could be prevented and hair regrowth could be restored either through the intradermal injection of cholesterol-modified siRNA targeting FGF5 or FGF18 or the topical application of FGF18 siRNA.
The relative electric conductivity(REC),semilethal temperature(LT50) of florets under low temperature below 0℃,and SOD activity,content of MDA,soluble sugar and soluble protein,and pollen viability of florets from room temperature(14.4℃～16.2℃) to 3℃ were investigated in two winter cut chrysanthemum cultivars 'Hanzi' and 'Hanhuang',respectively.The results showed that the REC of two cultivars increased significantly below-6℃,and the REC in ray and tubular florets of 'Hanhuang' were lower than that in 'Hanzi'.The LT50 of two cultivars were-9℃ or much lower.LT50 of 'Hanhuang' was lower than that of 'Hanzi',while LT50 of ray florets was higher than that in tubular florets of both cultivars.With a decrease in temperature from room temperature to 3℃,the activity of SOD,content of soluble protein and viability of pollen increased at the beginning then decreased,while the content of MDA was opposite.And soluble sugar content gradually increased with the temperature drop,'Hanhuang' showed higher ability in enhancing the SOD activity,soluble protein and soluble sugar content than 'Hanzi',where the ability in tubular florets was higher than that in ray florets.The pollen viability of 'Hanzi' decreased earlier and more quickly than that of 'Hanhuang' under low temperature.All of these showed that 'Hanhuang' was more cold tolerant than 'Hanzi',and the limited low temperature of freezing injury was below-6℃,while 9℃/or 6℃ were critical temperature point of physiological barrier.
Wetlands act as persistent natural carbon sinks over long time scales. Understanding the response of these carbon reservoirs to climate change is critical to assessing potential climate feedbacks. We conducted a study of an 860‐cm‐long sediment core in Dahu Swamp in south China to determine how the carbon accumulation rate ( CAR ) has varied as a function of palaeohydrology and palaeoclimate over the past 47 000 years. From an orbital time scale, our results show that the CAR in Dahu Swamp is relatively low in the wet periods of Marine Isotope Stage 3 (MIS 3) (mean: 46.7 gC m −2 a −1 ) and MIS 1 (mean: 28.2 gC m −2 a −1 ), compared to the dry periods of MIS 2 (mean: 59.9 gC m −2 a −1 ). At centennial and millennial scales, the highest CARs of Dahu Swamp mainly occur in organic‐rich silt or clay (gyttja) layers, which correspond to the relatively dry climate (e.g. c . 48 000–41 000, c. 33 000–32 000, c. 15 800–14 900 and c. 4400–4250 cal. a BP ). The CAR of Dahu Swamp is mainly controlled by local hydrological variations that are closely related to the East Asian summer monsoon ( EASM ) intensity, which may be co‐influenced by orbitally induced summer insolation forcing and internal feedback processes (e.g. Atlantic Meridional Overturning Circulation and El Niño/Southern Oscillation). Based on comparison with the CARs in monsoonal regions of China, we consider that precipitation may be the key factor for wetland CAR in EASM areas, whereas temperature is more important in Qinghai–Tibetan Plateau regions under Indian summer monsoon influence. The CAR of Dahu Swamp provides valuable records of wetland carbon accumulation dynamics in subtropical monsoon regions, which contradict the traditional patterns in global northern wetlands.
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