Assessing the effects of nitrogen deposition and climate on carbon isotope discrimination and intrinsic water‐use efficiency of angiosperm and conifer trees under rising CO2 conditions

The objective of this study is to globally assess the effects of atmospheric nitrogen deposition and climate, associated with rising levels of atmospheric CO₂, on the variability of carbon isotope discrimination (Δ¹³C), and intrinsic water‐use efficiency (iWUE) of angiosperm and conifer tree species. Eighty‐nine long‐term isotope tree‐ring chronologies, representing 23 conifer and 13 angiosperm species for 53 sites worldwide, were extracted from the literature, and used to obtain long‐term time series of Δ¹³C and iWUE. Δ¹³C and iWUE were related to the increasing concentration of atmospheric CO₂ over the industrial period (1850–2000) and to the variation of simulated atmospheric nitrogen deposition and climatic variables over the period 1950–2000. We applied generalized additive models and linear mixed‐effects models to predict the effects of climatic variables and nitrogen deposition on Δ¹³C and iWUE. Results showed a declining Δ¹³C trend in the angiosperm and conifer species over the industrial period and a 16.1% increase of iWUE between 1850 and 2000, with no evidence that the increased rate was reduced at higher ambient CO₂ values. The temporal variation in Δ¹³C supported the hypothesis of an active plant mechanism that maintains a constant ratio between intercellular and ambient CO₂ concentrations. We defined linear mixed‐effects models that were effective to describe the variation of Δ¹³C and iWUE as a function of a set of environmental predictors, alternatively including annual rate (N_rate) and long‐term cumulative (N_cum) nitrogen deposition. No single climatic or atmospheric variable had a clearly predominant effect, however, Δ¹³C and iWUE showed complex dependent interactions between different covariates. A significant association of N_rate with iWUE and Δ¹³C was observed in conifers and in the angiosperms, and N_cum was the only independent term with a significant positive association with iWUE, although a multi‐factorial control was evident in conifers.     

The H3 histone chaperone NASPSIM3 escorts CenH3 in Arabidopsis

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non‐nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASP^SIM3 is co‐expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N‐terminal tail and the histone fold domain of non‐nucleosomal CenH3. Reduced NASP^SIM3 expression negatively affects CenH3 deposition, identifying NASP^SIM3 as a CenH3 histone chaperone.     

Barley HISTIDINE KINASE 1 (HvHK1) coordinates transfer cell specification in the young endosperm

Cereal endosperm represents the most important source of the world’s food; nevertheless, the molecular mechanisms underlying cell and tissue differentiation in cereal grains remain poorly understood. Endosperm cellularization commences at the maternal–filial intersection of grains and generates endosperm transfer cells (ETCs), a cell type with a prominent anatomy optimized for efficient nutrient transport. Barley HISTIDINE KINASE1 (HvHK1) was identified as a receptor component with spatially restricted expression in the syncytial endosperm where ETCs emerge. Here, we demonstrate its function in ETC fate acquisition using RNA interference‐mediated downregulation of HvHK1. Repression of HvHK1 impairs cell specification in the central ETC region and the development of transfer cell morphology, and consecutively defects differentiation of adjacent endosperm tissues. Coinciding with reduced expression of HvHK1, disturbed cell plate formation and fusion were observed at the initiation of endosperm cellularization, revealing that HvHK1 triggers initial cytokinesis of ETCs. Cell‐type‐specific RNA sequencing confirmed loss of transfer cell identity, compromised cell wall biogenesis and reduced transport capacities in aberrant cells and elucidated two‐component signaling and hormone pathways that are mediated by HvHK1. Gene regulatory network modeling was used to specify the direct targets of HvHK1; this predicted non‐canonical auxin signaling elements as the main regulatory links governing cellularization of ETCs, potentially through interaction with type‐B response regulators. This work provides clues to previously unknown molecular mechanisms directing ETC specification, a process with fundamental impact on grain yield in cereals.     

Genbank accession #: AJ131961

Plant Molecular Biology -     

Synthesis and characterization of HE-24.8: a polymeric contrast agent for magnetic resonance angiography

The physical and biological properties of a water-soluble polymeric contrast agent based on a complex of N-(2-hydroxypropyl)methacrylamide copolymer with gadolinium (HE-24.8) were investigated, and its potential for experimental magnetic resonance (MR) angiography was assessed. Relaxivities of Gd-DTPA-BMA, Gd-DTPA-HSA (human serum albumin), and HE-24.8 were determined at 1.5 T. Thermic stability and biocompatibility of HE-24.8 were assessed in vitro and by analyzing kinetics and organ distribution in rats for up to 2 weeks. For comparison, HE-24.8- and Gd-DTPA-HSA-enhanced micro-MR angiographies of brain, chest, and subcutaneous tumors in rats were performed. T1 relaxivity of HE-24.8 (21.3 +/- 1.1 mM(-1) s(-1)) was 5-fold higher than that of Gd-DTPA-BMA (4.1 +/- 0.1 mM(-1) s(-1)) and twice as high as that of Gd-DTPA-HSA (12.4 +/- 0.2 mM(-1) s(-1)). Varying the molecular weight of the polymer (15-46 kDa) did not significantly change the T1 relaxivity. In rats, 20 and 10% of the injected dose of HE-24.8 was detected at 24 and 168 h postinjection, respectively. Upon a relatively rapid initial renal clearance, no specific retention in any organ was noted, with some exception for the reticulo-endothelial system. No measurable release of gadolinium from the polymer-Gd complex or cell toxicity was observed during its incubation in aqueous environment. Excellent display of rat and tumor vascularization was achieved with Gd-DTPA-HSA and HE-24.8; however, contrast of vessels was higher in HE-24.8-enhanced scans. HE-24.8 is considered a macromolecular contrast agent highly suited for experimental MR studies.     

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells

Background

Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells.

Methods

We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro.

Results

We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus.

Conclusion

The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.     

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N- glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible to compare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.