The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

A proteome-scale analysis of vertebrate protein amino acid occurrence: Thermoadaptation shows a correlation with protein solvation but less so with dynamics

Despite differences in behaviors and living conditions, vertebrate organisms share the great majority of proteins, often with subtle differences in amino acid sequence. Here, we present a simple way to analyze the difference in amino acid occurrence by comparing highly homologous proteins on a subproteome level between several vertebrate model organisms. Specifically, we use this method to identify a pattern of amino acid conservation as well as a shift in amino acid occurrence between homeotherms (warm-blooded species) and poikilotherms (cold-blooded species). Importantly, this general analysis and a specific example further establish a broad correlation, if not likely connection between the thermal adaptation of protein sequences and two of their physical features: on average a change in their protein dynamics and, even more strongly, in their solvation. For poikilotherms, such as frog and fish, the lower body temperature is expected to increase the protein–protein interaction due to a decrease in protein internal dynamics. In order to counteract the tendency for enhanced binding caused by low temperatures, poikilotherms enhance the solvation of their proteins by favoring polar amino acids. This feature appears to dominate over possible changes in dynamics for some proteins. The results suggest that a general trend for amino acid choice is part of the mechanism for thermoadaptation of vertebrate organisms at the molecular level.     

Acclimation response of spring wheat in a free-air CO2 enrichment (FACE) atmosphere with variable soil nitrogen regimes. 1. Leaf position and phenology determine acclimation response

We have examined the photosynthetic acclimation of wheat leaves grown at an elevated CO₂ concentration, and ample and limiting N supplies, within a field experiment using free-air CO₂ enrichment (FACE). To understand how leaf age and developmental stage affected any acclimation response, measurements were made on a vertical profile of leaves every week from tillering until maturity. The response of assimilation (A) to internal CO₂ concentration (C_i) was used to estimate the in vivo carboxylation capacity (Vc_max) and maximum rate of ribulose-1,5-bisphosphate limited photosynthesis (A _sat). The total activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and leaf content of Rubisco and the Light Harvesting Chlorophyll a/b protein associated with Photosystem II (LHC II), were determined. Elevated CO₂ did not alter Vc_max in the flag leaf at either low or high N. In the older shaded leaves lower in the canopy, acclimatory decline in Vc_max and A _sat was observed, and was found to correlate with reduced Rubisco activity and content. The dependency of acclimation on N supply was different at each developmental stage. With adequate N supply, acclimation to elevated CO₂ was also accompanied by an increased LHC II/Rubisco ratio. At low N supply, contents of Rubisco and LHC II were reduced in all leaves, although an increased LHC II/Rubisco ratio under elevated CO₂ was still observed. These results underscore the importance of leaf position, leaf age and crop developmental stage in understanding the acclimation of photosynthesis to elevated CO₂ and nutrient stress. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosynthesis of the sesquiterpene germacrene D in Solidago canadensis: 13C and (2)H labeling studies

The biogenetic origin of the isoprenoid building blocks of the sesquiterpene germacrene D was studied in Solidago canadensis. Feeding experiments were carried out with 1-[5,5-D(2)]deoxy-D-xylulose-5-phosphate (D(2)-DOXP), [5-13C]mevalonolactone (13C-MVL) and [1-13C]-D-glucose. The hydrodistillate of a cut shoot fed with D(2)-DOXP was investigated by enantio-MDGC-MS and the volatile fraction of a shoot supplied with 13C-MVL was examined by GC-C-IRMS. The incorporation of [1-13C]-D-glucose was analyzed by quantitative 13C NMR spectroscopy after isolation of germacrene D from the essential oil. Our labeling studies revealed that the biosynthesis of the C-15 skeleton of sesquiterpene germacrene D in Solidago canadensis proceeds predominantly via the methylerythritol phosphate pathway.     

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

Background and Aims

Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.

Methods

Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.

Key Results

A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.

Conclusions

Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.     

Involvement of two type 2C protein phosphatases BcPtc1 and BcPtc3 in the regulation of multiple stress tolerance and virulence of Botrytis cinerea

Type 2C Ser/Thr phosphatases (PP2Cs) are involved in various cellular processes in many eukaryotes, but little has been known about their functions in filamentous fungi. Botrytis cinerea contains four putative PP2C genes, named BcPTC1, ‐3, ‐5, and ‐6. Biological functions of these genes were analysed by gene deletion and complementation. While no phenotypes aberrant from the wild type were observed with mutants of BcPTC5 and BcPTC6, mutants of BcPTC1 and BcPTC3 had reduced hyphal growth, increased conidiation, and impaired sclerotium development. Additionally, BcPTC1 and BcPTC3 mutants exhibited increased sensitivity to osmotic and oxidative stresses, and to cell wall degrading enzymes. Both mutants exhibited dramatically decreased virulence on host plant tissues. All of the defects were restored by genetic complementation of the mutants with wild‐type BcPTC1 and BcPTC3 respectively. Different from what is known in Saccharomyces cerevisiae, BcPtc3, but not BcPtc1, negatively regulates phosphorylation of BcSak1 (the homologue of S. cerevisiae Hog1) in B. cinerea, although both BcPTC1 and BcPTC3 were able to rescue the growth defects of a yeast PTC1 deletion mutant under various stress conditions. These results demonstrated that BcPtc1 and BcPtc3 play important roles in the regulation of multiple stress tolerance and virulence of B. cinerea.     

Stable carbon and nitrogen isotope signatures of decomposing tropical macrophytes

The Pantanal of Mato Grosso, Brazil, is a large, seasonal wetland, which exhibits high macrophyte productivity at the beginning of the rainy season, when the floodplain becomes flooded. During inundation, from December through May, there is rapid turnover of decomposing macrophyte litter, which is subsequently colonized and consumed by various organisms. In this paper, the variation in the carbon and nitrogen isotope signatures of decomposing macrophytes and detritus was determined to provide an isotopic baseline for the elucidation of higher trophic levels. Seven abundant macrophyte species, Cyperaceae sp., Pontederia lanceolata, Cabomba furcata, Salvinia auriculata, Eichhornia crassipes, Nymphaea amazonum and Paspalum repens, were exposed in mesocosm decomposition experiments lasting 21 or 100 days. Stable isotope ratios of carbon and nitrogen and the atomic C/N ratios were determined for decomposing plant material, particulate organic matter (POM), the microbial film, and aquatic invertebrate larvae. The ¹³C values for the macrophytes did not change during decomposition. However, the variability of ¹⁵N was high (range of ± 6 ) due to microbial activity. There was no consistent difference in the isotopic signatures of macrophytes and POM. C/N ratios decreased from 17 to 50 in macrophytes, to 7 to 12 in POM. The isotopic signatures and C/N ratios of the microbial film were the same as those of POM. We concluded that heterotrophic processes did not fractionate stable carbon isotopes but caused an increase in the variability of stable nitrogen ratios and a change in the C/N ratios in our experimental system. Therefore, it was not possible to distinguish fresh and senescent material or even POM when used as a food source. The ¹³C values of the aquatic larvae were closely coupled to those of the carbon source provided.