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Background We have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function. Results When grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase. Conclusions Our data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.

Background Nicotiana rustica (Aztec tobacco), like common tobacco ( Nicotiana tabacum ), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3–3 Gb; 2n = 2x =24), Nicotiana undulata , Nicotiana paniculata and Nicotiana knightiana . Results In total, 41% of N. rustica genome originated from the paternal donor ( N. undulata ), while 59% originated from the maternal donor ( N. paniculata / N. knightiana ). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata . Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica . Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata , and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot. Conclusions The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applied.

Land-sea climatic proxies have been obtained from the Last Glacial section of IMAGES core MD95-2043 (western Mediterranean Sea). Vegetation and alkenone derived SST curves indicate rapid (~150 years) and synchronous terrestrial and marine climatic changes, paralleling the Dansgaard-Oeschger (D-O) climatic variability over Greenland. This frequency of climate change can be related to shifts between the two modes of operation of the North Atlantic Oscillation (NAO). Transfer functions applied to the pollen data indicate that there was an amplification of the climatic signal during Heinrich events (HEs) in comparison with other D-O stadials. The development and persistence of both Scandinavian and Atlantic Mobile Polar Highs over southwestern Europe may explain the extreme cooling (~10 degree C) and dryness (400 mm) during Heinrich events 5 and 4 in the Mediterranean region. Comparison of the results of core MD95-2043 with similar climatic data from IMAGES core MD95-2042, located off Portugal, indicates that thermal and precipitation gradients occurred between the Mediterranean and the Atlantic sides of Iberia within HEs. HEs 4 and 5 are associated with more humid conditions in the Atlantic (by 200 mm) than in the Mediterranean site, as is the case at the present time. This comparison also illustrates the different behaviour of these areas during the D-O stadials. In contrast with the Mediterranean site, the Atlantic site shows similar precipitation and temperature drops for all the D-O stadials, including those related to the HEs. Here we propose the operation of different Mobile Polar Highs (MPH) as the driving mechanism for this difference in behaviour between the Atlantic and Mediterranean sides of Iberia. HEs are related to a stronger influence of the Scandinavian MPH, forcing a severe aridification and cooling of the full Iberian Peninsula. The Atlantic MPH may have been dominant during the other stadials, which would preferentially affect Southwestern Iberia.

The Mediterranean Sea sustains a rich and fragile ecosystem currently threatened by multiple anthropogenic impacts that include, among others, warming, pollution, and changes in seawater carbonate speciation associated to increasing uptake of atmospheric CO2. This environmental change represents a major risk for marine calcifiers such as planktonic foraminifera, key components of pelagic Mediterranean ecosystems and major exporters of calcium carbonate to the sea floor, thereby playing a major role in the marine carbon cycle. In this study, we investigate the response of planktic foraminifera calcification in the northwestern Mediterranean Sea on different timescales across the industrial era. This study is based on data from a 12-year-long sediment trap record retrieved in the in the Gulf of Lions and seabed sediment samples from the Gulf of Lions and the promontory of Menorca. Three different planktic foraminifera species were selected based on their different ecology and abundance: Globigerina bulloides, Neogloboquadrina incompta, and Globorotalia truncatulinoides. A total of 273 samples were weighted in both sediment trap and seabed samples. The results of our study suggest substantial different seasonal calcification patterns across species: G. bulloides shows a slight calcification increase during the high productivity period, while both N. incompta and G. truncatulinoides display a higher calcification during the low productivity period. The comparison of these patterns with environmental parameters indicate that controls on seasonal calcification are species-specific. Interannual analysis suggests that both G. bulloides and N. incompta did not significantly reduce their calcification between 1994 and 2005, while G. truncatulinoides exhibited a constant and pronounced increase in its calcification that translated in an increase of 20 % of its shell weight. The comparison of these patterns with environmental data reveals that optimum growth conditions affect positively and negatively G. bulloides and G. truncatulinoides calcification, respectively. Sea surface temperatures (SSTs) have a positive influence on N. incompta and G. truncatulinoides calcification, while carbonate system parameters appear to affect positively the calcification of three species in the Gulf of Lions throughout the 12-year time series. Finally, comparison between sediment trap data and seabed sediments allowed us to assess the changes of planktic foraminifera calcification during the late Holocene, including the pre-industrial era. Several lines of evidence indicate that selective dissolution did not bias the results in any of our data sets. Our results showed a weight reduction between pre-industrial and post-industrial Holocene and recent data, with G. truncatulinoides experiencing the largest weight loss (32 %–40 %) followed by G. bulloides (18 %–24 %) and N. incompta (9 %–18 %). Overall, our results provide evidence of a decrease in planktic foraminifera calcification in the western Mediterranean, most likely associated with ongoing ocean acidification and regional SST trends, a feature consistent with previous observations in other settings of the world's oceans.

The allotetraploid plant Nicotiana tabacum (common tobacco) is a major crop species and a model organism, for which only very fragmented genomic sequences are currently available. Here we report high-quality draft genomes for three main tobacco varieties. These genomes show both the low divergence of tobacco from its ancestors and microsynteny with other Solanaceae species. We identify over 90,000 gene models and determine the ancestral origin of tobacco mosaic virus and potyvirus disease resistance in tobacco. We anticipate that the draft genomes will strengthen the use of N. tabacum as a versatile model organism for functional genomics and biotechnology applications. Common tobacco ( Nicotiana tabacum ) is a widely cultivated and economically important non-food crop. Here, the authors report the draft genome sequences for three of the most common tobacco varieties and provide insights into the evolution of tobacco through a comparative analysis with closely related species.

Background Selection of optimal computational strategies for analyzing metagenomics data is a decisive step in determining the microbial composition of a sample, and this procedure is complex because of the numerous tools currently available. The aim of this research was to summarize the results of crowdsourced sbv IMPROVER Microbiomics Challenge designed to evaluate the performance of off-the-shelf metagenomics software as well as to investigate the robustness of these results by the extended post-challenge analysis. In total 21 off-the-shelf taxonomic metagenome profiling pipelines were benchmarked for their capacity to identify the microbiome composition at various taxon levels across 104 shotgun metagenomics datasets of bacterial genomes (representative of various microbiome samples) from public databases. Performance was determined by comparing predicted taxonomy profiles with the gold standard. Results Most taxonomic profilers performed homogeneously well at the phylum level but generated intermediate and heterogeneous scores at the genus and species levels, respectively. kmer-based pipelines using Kraken with and without Bracken or using CLARK-S performed best overall, but they exhibited lower precision than the two marker-gene-based methods MetaPhlAn and mOTU. Filtering out the 1% least abundance species—which were not reliably predicted—helped increase the performance of most profilers by increasing precision but at the cost of recall. However, the use of adaptive filtering thresholds determined from the sample’s Shannon index increased the performance of most kmer-based profilers while mitigating the tradeoff between precision and recall. Conclusions kmer-based metagenomic pipelines using Kraken/Bracken or CLARK-S performed most robustly across a large variety of microbiome datasets. Removing non-reliably predicted low-abundance species by using diversity-dependent adaptive filtering thresholds further enhanced the performance of these tools. This work demonstrates the applicability of computational pipelines for accurately determining taxonomic profiles in clinical and environmental contexts and exemplifies the power of crowdsourcing for unbiased evaluation.

Abstract Background Several studies have highlighted the role of host–microbiome interactions in the pathogenesis of inflammatory bowel disease (IBD), resulting in an increasing amount of data mainly focusing on Western patients. Because of the increasing prevalence of IBD in newly industrialized countries such as those in Asia, the Middle East, and South America, there is mounting interest in elucidating the gut microbiota of these populations. We present a comprehensive analysis of several IBD-related biomarkers and gut microbiota profiles and functions of a unique population of patients with IBD and healthy patients from Kazan (Republic of Tatarstan, Russia). Methods Blood and fecal IBD biomarkers, serum cytokines, and fecal short-chain fatty acid (SCFA) content were profiled. Finally, fecal microbiota composition was analyzed by 16S and whole-genome shotgun sequencing. Results Fecal microbiota whole-genome sequencing confirmed the presence of classic IBD dysbiotic features at the phylum level, with increased abundance of Proteobacteria, Actinobacteria, and Fusobacteria and decreased abundance of Firmicutes, Bacteroidetes, and Verrucomicrobia. At the genus level, the abundance of both fermentative (SCFA-producing and hydrogen (H2)-releasing) and hydrogenotrophic (H2-consuming) microbes was affected in patients with IBD. This imbalance was confirmed by the decreased abundance of SCFA species in the feces of patients with IBD and the change in anaerobic index, which mirrors the redox status of the intestine. Conclusions Our analyses highlighted how IBD-related dysbiotic microbiota—which are generally mainly linked to SCFA imbalance—may affect other important metabolic pathways, such as H2 metabolism, that are critical for host physiology and disease development.

The Messinian salinity crisis and its precursor events have been the greatest environmental perturbation of the Mediterranean Sea to date, offering an opportunity to study the response of marine ecosystems to extreme hydrological change and a large-scale biological invasion. The restriction of the marine connection between the Mediterranean and the Atlantic Ocean resulted in stratification of the water column and high-amplitude variations in seawater temperature and salinity already from the early Messinian. Here, we present a unified and revised marine fossil record of the Mediterranean (https://doi.org/10.5281/zenodo.13358435, Agiadi et al., 2024) that covers the Tortonian stage, the pre-evaporitic Messinian stage, and the Zanclean stage and encompasses 23 032 occurrences of calcareous nannoplankton, dinoflagellates, foraminifera, corals, ostracods, bryozoans, echinoids, mollusks, fishes, and marine mammals. This record adheres to the FAIR principles, is updated in terms of taxonomy, and follows the currently accepted stratigraphic framework. Based on this record, knowledge gaps are identified, which are due to spatiotemporal inconsistencies in sampling effort and the distribution of sedimentary facies, as well as the inherent differences in the preservation potential between the groups. Additionally, sampling bias in old records may have distorted the record in favor of larger, more impressive taxa within groups. This record is now ready to be used to answer both geological and biological questions about the Mediterranean Sea and beyond and is amendable when new fossil data are brought to light.

Agrobacterium (Rhizobium)-mediated transformation leads to the formation of crown galls or hairy roots on infected plants. These effects develop due to the activity of T-DNA genes, gathered on a big plasmid, acquired from agrobacteria during horizontal gene transfer. However, a lot of plant species are known to contain such sequences, called cellular T-DNAs (cT-DNAs), and maintain normal phenotypes. Some of the genes remain intact, which leads to the conclusion of their functional role in plants. In this study, we present a comprehensive analysis of the cT-DNAs in the Nicotiana noctiflora Hook. genome, including gene expression and opine identification. Deep sequencing of the Nicotiana noctiflora genome revealed the presence of two different cT-DNAs, NnT-DNA1 and NnT-DNA2, which contain the intact genes iaaM, iaaH, acs, orf13, orf13a, and orf14. According to the expression analysis results, all these genes are most active in roots in comparison with other organs, which is consistent with data on cT-DNA gene expression in other plant species. We also used genetic engineering approaches and HPTLC and HPLC-MS methods to investigate the product of the acs gene (agrocinopine synthase), which turned out to be similar to agrocinopine A. Overall, this study expands our knowledge of cT-DNAs in plants and brings us closer to understanding their possible functions. Further research of cT-DNAs in different species and their functional implications could contribute to advancements in plant genetics and potentially unveil novel traits with practical applications in agriculture and other fields.