Validation of peptide MS/MS spectra using metabolic isotope labeling for spectral matching-based shotgun proteome analysis

We report an isotope labeling shotgun proteome analysis strategy to validate the spectrum-to-sequence assignments generated by using sequence-database searching for the construction of a more reliable MS/MS spectral library. This strategy is demonstrated in the analysis of the E. coli K12 proteome. In the workflow, E. coli cells were cultured in normal and (15)N-enriched media. The differentially labeled proteins from the cell extracts were subjected to trypsin digestion and two-dimensional liquid chromatography quadrupole time-of-flight tandem mass spectrometry (2D-LC QTOF MS/MS) analysis. The MS/MS spectra of the two samples were individually searched using Mascot against the E. coli proteome database to generate lists of peptide sequence matches. The two data sets were compared by overlaying the spectra of unlabeled and labeled matches of the same peptide sequence for validation. Two cutoff filters, one based on the number of common fragment ions and another one on the similarity of intensity patterns among the common ions, were developed and applied to the overlaid spectral pairs to reject the low quality or incorrectly assigned spectra. By examining 257,907 and 245,156 spectra acquired from the unlabeled and (15)N-labeled samples, respectively, an experimentally validated MS/MS spectral library of tryptic peptides was constructed for E. coli K12 that consisted of 9,302 unique spectra with unique sequence and charge state, representing 7,763 unique peptide sequences. This E. coli spectral library could be readily expanded, and the overall strategy should be applicable to other organisms. Even with this relatively small library, it was shown that more peptides could be identified with higher confidence using the spectral search method than by sequence-database searching.     

Fungicide Effectiveness during the Various Developmental Stages of Peronophythora litchii In Vitro

Litchi downy blight caused by Peronophythora litchii is one of the most destructive diseases suffered by litchi in China. This study has evaluated the activities of the fungicides dimethomorph (DMM), azoxystrobin (AZB), famoxadone (FMD), metalaxyl (MTL), cymoxanil (CYX) and mancozeb (MCB) on the mycelial growth, sporulation, zoospores release, and germination of sporangia as well as of cystospores of P. litchii . Dimethomorph and MTL inhibited mycelial growth more effectively than the other fungicides tested. Mycelial growth was affected less by MCB, and only weakly by AZB, FMD and CYX. Sporangia production was more strongly reduced by DMM and MTL than by AZB, FMD, CYX or MCB. Zoospore release from the sporangia was most sensitive to AZB and FMD, less sensitive to MCB and insensitive to DMM, MTL and CYX. Direct germination of sporangia of P. litchii was the most strongly inhibited by AZB and FMD, followed by DMM and MCB, whilst the sensitivity of this life stage to MTL and CYX was relatively low. Germination of encysted zoospores of P. litchii was the most sensitive to AZB and FMD, followed by DMM. Mancozeb was moderately active, while MTL and CYX did not inhibit this development stage. This is the first report on the in vitro response of the litchi pathogen, P. litchii, to fungicides. These findings can be valuable tools in setting up efficient disease management programmes to control litchi downy blight.     

Metabolic control analysis of developing oilseed rape (Brassica napus cv Westar) embryos shows that lipid assembly exerts significant control over oil accumulation

? Metabolic control analysis allows the study of metabolic regulation. We applied both single- and double-manipulation top-down control analysis to examine the control of lipid accumulation in developing oilseed rape (Brassica napus) embryos. ? The biosynthetic pathway was conceptually divided into two blocks of reactions (fatty acid biosynthesis (Block A), lipid assembly (Block B)) connected by a single system intermediate, the acyl-coenzyme A (acyl-CoA) pool. Single manipulation used exogenous oleate. Triclosan was used to inhibit specifically Block A, whereas diazepam selectively manipulated flux through Block B. ? Exogenous oleate inhibited the radiolabelling of fatty acids from [1-(14) C]acetate, but stimulated that from [U-(14) C]glycerol into acyl lipids. The calculation of group flux control coefficients showed that c. 70% of the metabolic control was in the lipid assembly block of reactions. Monte Carlo simulations gave an estimation of the error of the resulting group flux control coefficients as 0.27?±?0.06 for Block A and 0.73?±?0.06 for Block B. ? The two methods of control analysis gave very similar results and showed that Block B reactions were more important under our conditions. This contrasts notably with data from oil palm or olive fruit cultures and is important for efforts to increase oilseed rape lipid yields.     

Spatiotemporal pattern of gross primary productivity and its covariation with climate in China over the last thirty years

The uncertainties of China's gross primary productivity (GPP) estimates by global data‐oriented products and ecosystem models justify a development of high‐resolution data‐oriented GPP dataset over China. We applied a machine learning algorithm developing a new GPP dataset for China with 0.1° spatial resolution and monthly temporal frequency based on eddy flux measurements from 40 sites in China and surrounding countries, most of which have not been explored in previous global GPP datasets. According to our estimates, mean annual GPP over China is 6.62 ± 0.23 PgC/year during 1982–2015 with a clear gradient from southeast to northwest. The trend of GPP estimated by this study (0.020 ± 0.002 PgC/year² from 1982 to 2015) is almost two times of that estimated by the previous global dataset. The GPP increment is widely spread with 60% area showing significant increasing trend (p < .05), except for Inner Mongolia. Most ecosystem models overestimated the GPP magnitudes but underestimated the temporal trend of GPP. The monsoon affected eastern China, in particular the area surrounding Qinling Mountain, seems having larger contribution to interannual variability (IAV) of China's GPP than the semiarid northwestern China and Tibetan Plateau. At country scale, temperature is the dominant climatic driver for IAV of GPP. The area where IAV of GPP dominated by temperature is about 42%, while precipitation and solar radiation dominate 31% and 27% respectively over semiarid area and cold‐wet area. Such spatial pattern was generally consistent with global GPP dataset, except over the Tibetan Plateau and northeastern forests, but not captured by most ecosystem models, highlighting future research needs to improve the modeling of ecosystem response to climate variations.     

Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916

An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 microM, 0.087 microM, 0.055 microM and 2.74 microM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.     

Effect of vegetation on runoff-sediment yield relationship at different spatial scales in hilly areas of the Loess Plateau, North China

Whether vegetation reduces soil loss by reducing runoff volume or rather by changing runoff-sediment yield relationship has received little attention. Base on the observed data from monitoring stations and the published data from other research, this issue is addressed at different scales in hilly areas of the Loess Plateau, North China. At the plot scale, vegetation helps reduce soil loss not only by reducing runoff volume, but also by changing the runoff-sediment yield relationship, resulting that the sediment-reduction rate is higher than the runoff-reduction rate. At the watershed scale, gully erosion and mass wasting process are dominant. Vegetation measures are insufficient to control local mass movement, implying that sediment availability remains high even after vegetation is established. It is also hard for slope vegetation to change the capacity of the sediment transport system at the watershed scale. Therefore, vegetation cannot change the runoff-sediment yield relationship at the watershed scale. This implies that vegetation reduces sediment yield only by reducing runoff volume and the sediment-reduction rate approximates the runoff-reduction rate at the watershed scale. Other slope measures for soil conservation such as terraces are considered to have the same effect on the runoff-sediment yield relationship as the vegetation. Several case studies involving different spatial scales are presented and confirm this conclusion.     

基于幼化效果的山杏组培繁殖技术研究

以山杏为试验材料,选取不同部位、不同树龄、不同继代次数的组培苗,研究基于丛生芽诱导率和组培苗生根率、形态指标、生理指标3个层次的幼化效果。结果显示:(1)随着山杏外植体取材部位从树冠上部、中部、下部的依次下降,其丛生芽诱导率和组培苗生根率依次升高且部位间差异显著,其组培苗的株高、叶长、叶宽、根系数量和根系长度也随外植体取材部位下降而增加,但仅根系数量和根系长度在部位间差异显著,但其组培苗和母树叶片SOD活性、POD活性和MDA含量却逐渐降低,且组培苗叶片始终低于母树叶片。(2)随着取材母树的树龄从10a、4a、1a的不断降低,组培苗上述各项指标值均依次升高,1a树龄丛生芽诱导率及组培苗生根率、根系数量、根系长度分别为80%、96.6%、4.3条、2.40cm,极显著高于相应的10a和4a树龄的组培苗。(3)从初代培养到1次、2次继代培养,随着继代次数的增加,各类组培苗的丛生芽诱导率、株高、叶长和叶宽值不断增大,且以2次继代培养的增幅较大;同时相应组培苗叶片SOD活性、MDA含量和POD活性则呈下降趋势,但仍表现为2次继代的组培苗幼化效果较好。研究认为,各层次幼化效果指标在组培苗中表现出相同的变化规律。随着外植体在母树上取材部位的下移、取材母树年龄的降低以及组培苗继代次数的增加,山杏丛生芽诱导率和组培苗生根率、形态指标、生理指标表现出一致的变化趋势,其组培幼化效果愈来愈佳。     

The autophagy‐related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assays showed that the expression of BcATG1 was up‐regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen‐starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re‐introduction of an intact copy of the wild‐type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.     

Seasonal variation of vegetation productivity over an alpine meadow in the Qinghai–Tibet Plateau in China: modeling the interactions of vegetation productivity, phenology, and the soil freeze–thaw process

Phenology controls the seasonal activities of vegetation on land surfaces and thus plays a fundamental role in regulating photosynthesis and other ecosystem processes. Therefore, accurately simulating phenology and soil processes is critical to ecosystem and climate modeling. In this study, we present an integrated ecosystem model of plant productivity, plant phenology, and the soil freeze–thaw process to (1) improve the quality of simulations of soil thermal regimes and (2) estimate the seasonal variability of plant phenology and its effects on plant productivity in high-altitude seasonal frozen regions. We tested different model configurations and parameterizations, including a refined soil stratification scheme that included unfrozen water in frozen soil, a remotely sensed diagnostic phenology scheme, and a modified prognostic phenology scheme, to describe the seasonal variation in vegetation. After refined soil layering resolution and the inclusion of unfrozen water in frozen soil, the results show that the model adequately reproduced the soil thermal regimes and their interactions observed at the site. The inclusion of unfrozen water in frozen soil was found to have a significant effect on soil moisture simulation during the spring but only a small effect on soil temperature simulation at this site. Moreover, the performance of improved phenology schemes was good. The phenology model accurately predicted the start and end of phenology, and its precise prediction of phenology variation allows an improved simulation of vegetation production.