Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive‐stranded RNAs. Here, we have established a BNYVV full‐length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV‐based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co‐localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV‐based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV‐based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.     

Determination of oxytetracycline hydrochloride in milk and egg white samples using Ru(bipy)32+–Ce(SO4)2 chemiluminescence

A novel, rapid and sensitive chemiluminescence (CL) method for the determination of oxytetracycline hydrochloride (OTCH) is described in this paper. The presented method was based on the fact that OTCH could immensely enhance the CL of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium (II) in acidic medium. Under optimal experimental conditions, CL intensity was favorably linear for OTCH in the range 5.0 × 10^?7 to 5.0 × 10^?5 g/ml, with a detection limit of 1.5 × 10^?7 g/ml (S/N = 3). The relative standard detection was 4.76% for 5.0 × 10^?6 g/ml (n = 11). This method was successfully applied to the analysis of OTCH in milk and egg white samples. According to the results of the kinetic curves for OTCH in the Ru(bipy)₃²⁺–Ce(SO₄)₂ CL system, together with CL and ultraviolet (UV)–visible spectra, the possible mechanism of the CL reaction is discussed briefly.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

黄壤稻田土壤微生物生物量碳磷对长期不同施肥的响应

本研究以进行了22年的黄壤稻田长期定位试验为依托,分析了不同施肥模式下土壤碳(C)、磷(P)与微生物生物量C(MBC)、P(MBP)的变化及其耦合特征,旨在探讨黄壤地区合理培肥模式以提高土壤磷素有效性.试验包括10个处理,分别为不施肥(CK)、单施氮肥(N)、磷钾配合(PK)、氮钾配合(NK)、氮磷配合(NP)、氮磷钾配合(NPK)、单施有机肥(M)、3种有机无机肥配施(1/4M+3/4NP、0.5MNP、MNPK).结果表明:与不施肥相比,N和NK处理土壤有机碳(TOC)、全磷(TP)、MBC、MBP均有不同程度的降低,施磷处理(PK、NP、NPK)则有不同程度的提升;与不施肥和施用无机肥相比,施用有机肥各处理TOC、MBC、MBP及MBP/TP均显著增加,其中M和MNPK处理增幅最大.MBC/MBP、TOC/MBP、MBC/TP以施用有机肥处理最低,N处理最高.土壤MBC、MBP及其耦合关系与土壤TOC和有效磷均呈极显著相关,TOC是影响MBC、MBP、MBP/TP的直接因素,而有效磷则是影响MBC/MBP、TOC/MBP、MBC/TP的直接因素.土壤MBP及碳、磷耦合关系各指标可以有效区分单施化肥和施用有机肥的不同施肥方式,可作为评价黄壤稻田磷素肥力的生物学指标.配施有机肥是增加黄壤稻田磷有效性、提高土壤供磷潜力和保持土壤生物健康的有效途径.     

From canopy to seed: Loss of snow drives directional changes in forest composition

Climate change is altering the conditions for tree recruitment, growth, and survival, and impacting forest community composition. Across southeast Alaska, USA, and British Columbia, Canada, Callitropsis nootkatensis (Alaska yellow‐cedar) is experiencing extensive climate change‐induced canopy mortality due to fine‐root death during soil freezing events following warmer winters and the loss of insulating snowpack. Here, we examine the effects of ongoing, climate‐driven canopy mortality on forest community composition and identify potential shifts in stand trajectories due to the loss of a single canopy species. We sampled canopy and regenerating forest communities across the extent of C. nootkatensis decline in southeast Alaska to quantify the effects of climate, community, and stand‐level drivers on C. nootkatensis canopy mortality and regeneration as well as postdecline regenerating community composition. Across the plot network, C. nootkatensis exhibited significantly higher mortality than co‐occurring conifers across all size classes and locations. Regenerating community composition was highly variable but closely related to the severity of C. nootkatensis mortality. Callitropsis nootkatensis canopy mortality was correlated with winter temperatures and precipitation as well as local soil drainage, with regenerating community composition and C. nootkatensis regeneration abundances best explained by available seed source. In areas of high C. nootkatensis mortality, C. nootkatensis regeneration was low and replaced by Tsuga. Our study suggests that climate‐induced forest mortality is driving alternate successional pathways in forests where C. nootkatensis was once a major component. These pathways are likely to lead to long‐term shifts in forest community composition and stand dynamics. Our analysis fills a critical knowledge gap on forest ecosystem response and rearrangement following the climate‐driven decline of a single species, providing new insight into stand dynamics in a changing climate. As tree species across the globe are increasingly stressed by climate change‐induced alteration of suitable habitat, identifying the autecological factors contributing to successful regeneration, or lack thereof, will provide key insight into forest resilience and persistence on the landscape.     

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.     

The Arabidopsis thaliana trehalase is a plasma membrane-bound enzyme with extracellular activity

The lack of trehalose accumulation in most plant species has been partly attributed to the presence of an active trehalase. Although trehalose synthesis enzymes are thought to be cytosolic, and previous studies have indicated that trehalase activity is extracellular, the exact location of the enzyme has not yet been established in plant cell. We present evidence that the yet uncharacterised full-length Arabidopsis trehalase is a plasma membrane-bound protein, probably anchored to the membrane through a predicted N-terminal membrane spanning domain. The full-length AtTRE1, when expressed in yeast can functionally substitute for the extracellularly active trehalase Ath1p, by sustaining the growth of an ath1 null mutant strain on trehalose and at pH 4.8. We further demonstrate that AtTRE1 expressed in yeast is plasma membrane-bound as in plant cell. In light of these findings, the regulation of plant cell endogenous trehalose by trehalase is discussed.     

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.     

Automated work-flow for processing high-resolution direct infusion electrospray ionization mass spectral fingerprints

The use of mass spectrometry (MS) is pivotal in analyses of the metabolome and presents a major challenge for subsequent data processing. While the last few years have given new high performance instruments, there has not been a comparable development in data processing. In this paper we discuss an automated data processing pipeline to compare large numbers of fingerprint spectra from direct infusion experiments analyzed by high resolution MS. We describe some of the intriguing problems that have to be addressed, starting with the conversion and pre-processing of the raw data to the final data analysis. Illustrated on the direct infusion analysis (ESI-TOF-MS) of complex mixtures the method exploits the full quality of the high-resolution present in the mass spectra. Although the method is illustrated as a new library search method for high resolution MS, we demonstrate that the output of the preprocessing is applicable to cluster-, discriminant analysis, and related multivariate methods applied directly to mass spectra from direct infusion analysis of crude extracts. This is done to find the relationship between several terverticillate Penicillium species and identify the ions responsible for the segregation.