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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.

The electrical conductivity of LiF–KF–NaF (FLiNaK) molten system was measured in the temperature range 480–777°C. The comparison of the obtained experimental data on molten FLiNaK with the available data for individual, double and ternary fluoride melts containing KF, NaF and LiF was carried out. The resulting dependence of electrical conductivity on molar volume of the system demonstrates that at 867°C and V m larger than 23 cm 3 /mol the specific electrical conductivity is practically independent on molar volume and respectively on the molten mixture composition. The electrical conductivity of FLiNaK–CeF 3 molten systems with the cerium fluoride additions ranging from 0 to 25 mol % was measured depending on both the temperature and concentration of CeF 3 . In addition, the electrical conductivity of 0.85 FLiNaK–0.15CeF 3 –Li 2 O molten system with Li 2 O additions up to 2.3 mol % was measured. The investigation demonstrates that the addition of cerium fluoride and oxide results in a decrease of the electrical conductivity of the fluoride molten system.

—The electrochemical reduction of U(III) ions to the metal in molten LiCl–KCl eutectic in a temperature range of 673–793 K on cadmium and gallium electrodes under an inert gas atmosphere is studied by cyclic voltammetry, square-wave voltammetry, and zero current potentiometry. Reagents containing no traces of moisture, oxygen, and products of their interaction are used in the experiments. All main procedures are carried out in a dry glove box under a purified argon atmosphere. As shown by cyclic and square-wave voltammetry, the cathodic reduction of uranium(III) ions on the active cadmium and gallium electrodes in the “electrochemical window” under study occurs with a depolarization of 0.2–0.4 V and depends on the cathode material. The potential shift to the electropositive range is found to be related to the formation of intermetallic compounds of uranium with the material of the active electrodes. The potentiostatic electrolysis of the melt at the potentials of the current peaks observed on the cyclic voltammograms using the active electrodes is conducted to identify the compositions of the cathodic deposits. The X-ray diffraction (XRD) data show that the intermetallic compound UCd 11 is formed on the cadmium electrode and UGa 3 and UGa 2 are formed on the gallium electrode. SEM image and EDS analysis of the sample surface confirm the presence of fine fragments of the U–Cd and U–Ga intermetallic compounds. The conditions for the formation of alloys of a specified composition are determined by potentiostatic electrolysis. The equilibrium potentials of the U–Cd and U–Ga alloys are measured by zero current potentiometry, and the temperature dependences of the apparent standard potentials of the alloys are calculated. The electrochemical extraction of uranium from the LiCl–KCl–UCl 3 melt is studied. The determined degree of extraction of uranium from the electrolyte on the active liquid electrodes at various electrolysis times exceeds 97%.

AtATM3, an ATP-binding cassette transporter of Arabidopsis (Arabidopsis thaliana), is a mitochondrial protein involved in the biogenesis of iron-sulfur clusters and iron homeostasis in plants. Our gene expression analysis showed that AtATM3 is up-regulated in roots of plants treated with cadmium [Cd(II)] or lead (II); hence, we investigated whether this gene is involved in heavy metal tolerance. We found that AtATM3-overexpressing plants were enhanced in resistance to Cd, whereas atatm3 mutant plants were more sensitive to Cd than their wild-type controls. Moreover, atatm3 mutant plants expressing 35S promoter-driven AtATM3 were more resistant to Cd than wild-type plants. Since previous reports often showed that the cytosolic glutathione level is positively correlated with heavy metal resistance, we measured nonprotein thiols (NPSH) in these mutant plants. Surprisingly, we found that atatm3 contained more NPSH than the wild type under normal conditions. AtATM3-overexpressing plants did not differ under normal conditions, but contained less NPSH than wild-type plants when exposed to Cd(II). These results suggest a role for AtATM3 in regulating cellular NPSH level, a hypothesis that was further supported by our gene expression study. Genetic or pharmacological inhibition of glutathione biosynthesis led to the elevated expression of AtATM3, whereas expression of the glutathione synthase gene GSH1 was increased under Cd(II) stress and in the atatm3 mutant. Because the closest homolog of AtATM3 in fission yeast (Schizosaccharomyces pombe), HMT1, is a vacuolar membrane-localized phytochelatin-Cd transporter, it is tempting to speculate that glutathione-Cd(II) complexes formed in the mitochondria are exported by AtATM3. In conclusion, our data show that AtATM3 contributes to Cd resistance and suggest that it may mediate transport of glutamine synthetase-conjugated Cd(II) across the mitochondrial membrane.

The ABC-transporter superfamily is one of the largest protein families, and members can be found in bacteria, fungi, plants and animals. The first reports on plant ABC transporters showed that they are implicated in detoxification processes. The recent completion of the genomic sequencing of Arabidopsis thaliana (L.) Heynh. [Arabidopsis Genome Initiative (2000) Nature 408:796–815] showed that Arabidopsis contains more than 100 ABC-type proteins; 53 genes code for so-called full-size transporters, which are large proteins of about 150 kDa consisting of two hydrophobic and two hydrophilic domains. The large number of genes in the MDR/MRP and PDR5-like sub-clusters and the strong sequence homology found in many cases suggest functional redundancy. One reason for the high number of genes can be attributed to the duplication of large segments of Arabidopsis chromosomes. Recent results indicate that the function of this protein family is not restricted to detoxification processes. Plant ABC transporters have been demonstrated to participate in chlorophyll biosynthesis, formation of Fe/S clusters, stomatal movement, and probably ion fluxes; hence they may play a central role in plant growth and developmental processes.

Plant secondary metabolism significantly contributes to defensive measures against adverse abiotic and biotic cues. To investigate stress-induced, transcriptional alterations of underlying effector gene families, which encode enzymes acting consecutively in secondary metabolism and defense reactions, a DNA array (MetArray) harboring gene-specific probes was established. It comprised complete sets of genes encoding 109 secondary product glycosyltransferases and 63 glutathione-utilizing enzymes along with 62 cytochrome P450 monooxygenases and 26 ABC transporters. Their transcriptome was monitored in different organs of unstressed plants and in shoots in response to herbicides, UV-B radiation, endogenous stress hormones, and pathogen infection. A principal component analysis based on the transcription of these effector gene families defined distinct responses and crosstalk. Methyl jasmonate and ethylene treatments were separated from a group combining reactions towards two sulfonylurea herbicides, salicylate and an avirulent strain of Pseudomonas syringae pv. tomato. The responses to the herbicide bromoxynil and UV-B radiation were distinct from both groups. In addition, these analyses pinpointed individual effector genes indicating their role in these stress responses. A small group of genes was diagnostic in differentiating the response to two herbicide classes used. Interestingly, a subset of genes induced by P. syringae was not responsive to the applied stress hormones. Small groups of comprehensively induced effector genes indicate common defense strategies. Furthermore, homologous members within branches of these effector gene families displayed differential expression patterns either in both organs or during stress responses arguing for their non-redundant functions.

Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein (MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.

The liquidus temperatures of the BaO-BaCl2-MCl systems (with M = alkali metal) are determined by thermal analysis. The caloric effects observed during melting of the barium-containing chloride eutectic with barium oxide additions are studied. A chemical mechanism of barium oxide dissolution in the melts is confirmed. X-ray diffraction patterns taken for the melt solidified after experiment indicate the presence of barium oxychloride Ba4OCl6 in the solid phase. It is shown that the significant increase in the liquidus temperature in adding the barium oxide to barium-containing chloride molten mixtures is related to substantial changes in their composition and structure.

Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (Mrs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca2+-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca2+, Mg2+, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231—240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated 32P from (γ-32P)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts.