NR Gene Research Articles

De Novo Transcriptome Assembly, Gene Annotations, and Characterization of Functional Profiling Reveal Key Genes for Lead Alleviation in the Pb Hyperaccumulator Greek Mustard (Hirschfeldia incana L.).

Lead (Pb) contamination is a widespread environmental problem due to its toxicity to living organisms. Hirschfeldia incana L., a member of the Brassicaceae family, commonly found in the Mediterranean regions, is characterized by its ability to tolerate and accumulate Pb in soils and hydroponic cultures. This plant has been reported as an excellent model to assess the response of plants to Pb. However, the lack of genomic data for H. incana hinders research at the molecular level. In the present study, we carried out RNA deep transcriptome sequencing (RNA-seq) of H. incana under two conditions, control without Pb(NO3)2 and treatment with 100 µM of Pb(NO3)2 for 15 days. A total of 797.83 million reads were generated using Illumina sequencing technology. We assembled 77,491 transcript sequences with an average length of 959 bp and N50 of 1330 bp. Sequence similarity analyses and annotation of these transcripts were performed against the Arabidopsis thaliana nr protein database, Gene Ontology (GO), and KEGG databases. As a result, 13,046 GO terms and 138 KEGG maps were created. Under Pb stress, 577 and 270 genes were differentially expressed in roots and aboveground parts, respectively. Detailed elucidation of regulation of metal transporters, transcription factors (TFs), and plant hormone genes described the role of actors that allow the plant to fine-tune Pb stress responses. Our study revealed that several genes related to jasmonic acid biosynthesis and alpha-linoleic acid were upregulated, suggesting these components’ implication in Hirschfeldia incana L responses to Pb stress. This study provides data for further genomic analyses of the biological and molecular mechanisms leading to Pb tolerance and accumulation in Hirschfeldia incana L.

Physiological and metabolic bases of increased growth in the tomato ethylene-insensitive mutant Never ripe: extending ethylene signaling functions.

The tomato mutant Never ripe(Nr), a loss-of-function for the ethylene receptor SlETR3, shows enhanced growth, associated with increased carbon assimilation and a rewiring of the central metabolism. Compelling evidence has demonstrated the importance of ethylene during tomato fruit development, yet its role on leaf central metabolism and plant growth remains elusive. Here, we performed a detailed characterization of Never ripe (Nr) tomato, a loss-of-function mutant for the ethylene receptor SlETR3, known for its fruits which never ripe. However, besides fruits, the Nr gene is also constitutively expressed in vegetative tissues. Nr mutant showed a growth enhancement during both the vegetative and reproductive stage, without an earlier onset of leaf senescence, with Nr plants exhibiting a higher number of leaves and an increased dry weight of leaves, stems, roots, and fruits. At metabolic level, Nr also plays a significant role with the mutant showing changes in carbon assimilation, carbohydrates turnover, and an exquisite reprogramming of a large number of metabolite levels. Notably, the expression of genes related to ethylene signaling and biosynthesis are not altered in Nr. We assess our results in the context of those previously published for tomato fruits and of current models of ethylene signal transduction, and conclude that ethylene insensitivity mediated by Nr impacts the whole central metabolism at vegetative stage, leading to increased growth rates.

De novo transcriptome of Gymnema sylvestre identified putative lncRNA and genes regulating terpenoid biosynthesis pathway

Gymnema sylvestre is a highly valuable medicinal plant in traditional Indian system of medicine and used in many polyherbal formulations especially in treating diabetes. However, the lack of genomic resources has impeded its research at molecular level. The present study investigated functional gene profile of G. sylvestre via RNA sequencing technology. The de novo assembly of 88.9 million high quality reads yielded 23,126 unigenes, of which 18116 were annotated against databases such as NCBI nr database, gene ontology (GO), KEGG, Pfam, CDD, PlantTFcat, UniProt & GreeNC. Total 808 unigenes mapped to 78 different Transcription Factor families, whereas 39 unigenes assigned to CYP450 and 111 unigenes coding for enzymes involved in the biosynthesis of terpenoids including transcripts for synthesis of important compounds like Vitamin E, beta-amyrin and squalene. Among them, presence of six important enzyme coding transcripts were validated using qRT-PCR, which showed high expression of enzymes involved in methyl-erythritol phosphate (MEP) pathway. This study also revealed 1428 simple sequence repeats (SSRs), which may aid in molecular breeding studies. Besides this, 8 putative long non-coding RNAs (lncRNAs) were predicted from un-annotated sequences, which may hold key role in regulation of essential biological processes in G. sylvestre. The study provides an opportunity for future functional genomic studies and to uncover functions of the lncRNAs in G. sylvestre.

RNA sequencing and de novo assembly of Solanum trilobatum leaf transcriptome to identify putative transcripts for major metabolic pathways

Solanum trilobatum L. is an important medicinal plant in traditional Indian system of medicine belonging to Solanaceae family. However, non-availability of genomic resources hinders its research at the molecular level. We have analyzed the S. trilobatum leaf transcriptome using high throughput RNA sequencing. The de novo assembly of 136,220,612 reads produced 128,934 non-redundant unigenes with N50 value of 1347 bp. Annotation of unigenes was performed against databases such as NCBI nr database, Gene Ontology, KEGG, Uniprot, Pfam, and plnTFDB. A total of 60,097 unigenes were annotated including 48 Transcription Factor families and 14,490 unigenes were assigned to 138 pathways using KEGG database. The pathway analysis revealed the transcripts involved in the biosynthesis of important secondary metabolites contributing for its medicinal value such as Flavonoids. Further, the transcripts were quantified using RSEM to identify the highly regulated genes for secondary metabolism. Reverse-Transcription PCR was performed to validate the de novo assembled unigenes. The expression profile of selected unigenes from flavonoid biosynthesis pathway was analyzed using qRT-PCR. We have also identified 13,262 Simple Sequence Repeats, which could help in molecular breeding. This is the first report of comprehensive transcriptome analysis in S. trilobatum and this will be an invaluable resource to understand the molecular basis related to the medicinal attributes of S. trilobatum in further studies.

WAFs lead molting retardation of naupliar stages with down-regulated expression profiles of chitin metabolic pathway and related genes in the copepod Tigriopus japonicus

Oil pollution is considered being disastrous to marine organisms and ecosystems. As molting is critical in the developmental process of arthropods in general and copepods, in particular, the impact will be adverse if the target of spilled oil is on molting. Thus, we investigated the harmful effects of water accommodated fractions (WAFs) of crude oil with an emphasis on inhibition of chitin metabolic pathways related genes and developmental retardation in the copepod Tigriopus japonicus. Also, we analysed the ontology and domain of chitin metabolic pathway genes and mRNA expression patterns of developmental stage-specific genes. Further, the developmental retardation followed by transcriptional modulations in nuclear receptor genes (NR) and chitin metabolic pathway-related genes were observed in the WAFs-exposed T. japonicus. As a result, the developmental time was found significantly (P<0.05) delayed in response to 40% WAFs in comparison with that of control. Moreover, the NR gene, HR3 and chitinases (CHT9 and CHT10) were up-regulated in N4–5 stages, while chitin synthase genes (CHS-1, CHS-2-1, and CHS-2-2) down-regulated in response to WAFs. In brief, a high concentration of WAFs repressed nuclear receptor genes but elicited activation of some of the transcription factors at low concentration of WAFs, resulting in suppression of chitin synthesis.Thus, we suggest that WAF can lead molting retardation of naupliar stages in T. japonicus through down-regulations of chitin metabolism. These findings will provide a better understanding of the mode of action of chitin biosynthesis associated with molting mechanism in WAF-exposed T. japonicus.

Effects of salt stress on the expression of key genes related to nitrogen assimilation and transport in the roots of the cultivated tomato and its wild salt-tolerant relative

Inorganic nitrogen is a key element for plant growth under salt stress. A comparative study including physiological responses, ion content, transcript regulation of ammonium/nitrate transporters (AMTs/NRTs) as well as key enzymes for nitrogen assimilation was undertaken in wild salt-tolerant tomatoes (Solanum pennellii) and cultivated tomatoes (Solanum lycopersicon) exposed to 100mM NaCl for 1 and 7days. In comparison to S. lycopersicon, S. pennellii was more salt tolerant as evidenced by its higher survival rate, lower biomass reduction, and less salt injury (reduced electrolyte leakage and proline accumulation). In root tissues of both species, salt exposure (7days) reduced the mRNA expression levels of low affinity nitrate transporters (NRT1.1 and NRT1.2). This was associated with a decline in both nitrate content and expression level of the nitrate reductase gene (NR). Salt-stressed root tissues of S. pennellii showed relatively higher mRNA expression of the high affinity ammonium transporters (AMT1.1 and AMT1.2) compared to S. lycopersicon. The root ammonium content was increased only in S. lycopersicon going hand in hand with a reduction in mRNA level of cytosolic glutamine synthetase (GS1) after 7days of salt stress, whereas the expression level of GS1 was unchanged in S. pennellii, suggesting a lower salt-induced inhibition in ammonium assimilation in this species. Our comparative study demonstrated that the salt-tolerant and salt-sensitive tomato species show differential contribution of the nitrogen transporters and key genes associated with nitrogen assimilation under salt stress. While the reduction in the expression of key components of NO3− uptake (NRT1.1, NRT1.2) and assimilation (NR gene) in both species, likely contributed to the reduction in plant growth under salt stress, the observed salt tolerance for S. pennellii was associated with relative higher mRNA expression of ammonium uptake and assimilation genes. These results provide crucial knowledge for tomato breeding employing salt-tolerant wild species in salt-induced nitrogen- deficient environments.

Feeding behavior and performance of Nasonovia ribisnigri on grafts, detached leaves, and leaf disks of resistant and susceptible lettuce

AbstractAphids are dependent on the phloem sap of plants as their only source of nutrients. Host‐plant resistance in lettuce, Lactuca sativa L. (Asteraceae), mediated by the Nr gene is used to control the lettuce aphid Nasonovia ribisnigri (Mosely) (Hemiptera: Aphididae). The resistance is located in the phloem; however, the exact mechanism of resistance is unknown. In this study, we investigated whether the resistance factor (or factors) is synthesized in the root or in the shoot. The feeding behavior and performance of avirulent N. ribisnigri were studied on grafts of resistant and susceptible lettuce. In addition, the persistence of resistance in excised lettuce tissue was measured, by studying the feeding behavior and performance of N. ribisnigri on detached leaves and leaf disks of resistant lettuce. It appears that the resistance factor encoded by the Nr gene is produced in the shoots: aphid feeding was reduced on resistant shoots grafted on susceptible roots, whereas aphids were able to feed on grafts of susceptible shoots on resistant roots. Partial loss of resistance was observed after detachment of leaves and excision of leaf disks from resistant plants. Aphids fed longer on excised resistant plant tissue compared with intact resistant plants; however, compared with excised plant tissue of the susceptible cultivar, the time spent on feeding was shorter, indicating resistance was not completely lost. Our findings caution against the use of excised leaf material for aphid resistance bioassays.

Involvement of nitrate reductase in the ameliorating effect of 5-aminolevulinic acid on NaCl-stressed barley seedlings

Exogenous application of 5-aminolevulinic acid (ALA) is widely used to decrease the effect of different abiotic stresses in various crops while the nitrogen metabolism is a possible target of ALA in plants. We investigated the effect of exogenous ALA on growth, activity of nitrate reductase (NR; EC 1.6.6.1), synthesis of proline and ROS generation in seedlings of the salt-sensitive barley cultivar (Hordeum vulgare L. cv. Gonar). Obtained data indicated that the ALA (20, 40 and 80 mg L−1) increased elongation and leaf blade expansion of barley seedlings exposed to 150 mM NaCl as compared to control plants, which were not treated by ALA. NR gene (Nar1) expression, NR protein content and enzymatic activities increased in ALA-treated plants. ALA also induced accumulation of free proline and decreased generation of superoxide anion radicals. Overall, this study demonstrates that ALA effects on salt-stressed barley involves changes of NR activities both at transcriptional and translational levels and leads to complex physiological modifications, such as improvement of growth and antiradical defense.

To bind or not to bind: the taxonomic scope of nuclear receptor mediated endocrine disruption in invertebrate phyla.

(PPARγ). 2 The fact that most research on NR-mediated disruption has focused on vertebrate models significantly hinders our understanding on the wider biological and phylogenetic impact of EDCs and emerging pollutants. This is particularly relevant as invertebrates represent more than 95% of the known animal species, and have key roles in ecosystem functions. With the exception of the imposex phenomenon in prosobranch gastropods, that has indisputably been associated with tributyltin binding to RXR, 3 very few studies have linked NRs and endocrine perturbation in invertebrate phyla. This is most likely related with the lack of knowledge of invertebrate endocrinology and genomic NR collection. In contrast to vertebrates, the NR gene family diversity outside chordates was assumed to be comparatively smaller for a considerable time. However, the full genome sequences from a few invertebrate species suggest that this is a problem of biased taxonomic

Application of exogenous substances reduces tobacco-specific nitrosamines content by regulating biosynthesis of nicotine and nitrite in burley tobacco

Tobacco-specific nitrosamines (TSNAs) are carcinogenic chemicals found in tobacco plants. The increasing health consciousness of individuals had led to an increased interest in research on reducing TSNAs content. The aim of this study was to use a pot experiment in which exogenous substances were applied to burley tobacco to dissect the mechanism of TSNAs production. The results indicated that spraying the exogenous substances IAA, NAA, SA and combination thereof on burley tobacco after topping decreased TSNAs content by 2.69–29.4 % in upper leaves and 0.23–39.3 % in middle leaves without affecting total sugar, total nitrogen, potassium and chlorine contents. The application of exogenous substances could down-regulate expression of the NR gene and the activity of the NR enzyme, resulting in less accumulation of the TSNAs precursor nitrite. The exogenous substances significantly reduced nicotine accumulation, which was consistent with low enzyme activities and the down-regulated expressions of genes involved in nicotine biosynthesis, especially significant in the case of quinolinate phosphoribosyltransferase. These results suggested that the application of exogenous substances on burley tobacco after topping could reduce TSNAs content which may be attributed to the regulation of exogenous substances on nitrite and nicotine. This also implies one potential improvement to agronomic practices aimed at controlling the accumulation of TSNAs in burley tobacco.

Performance and feeding behaviour of two biotypes of the black currant-lettuce aphid, Nasonovia ribisnigri, on resistant and susceptible Lactuca sativa near-isogenic lines.

The black currant-lettuce aphid, Nasonovia ribisnigri, is an important pest of cultivated lettuce, Lactuca sativa. Since 1982, the control of this aphid on lettuce is largely based on host plant resistance, conferred by the Nr gene, introgressed from Lactuca virosa. The resistance mechanism remains to be identified. N. ribisnigri populations virulent on the Nr-based resistance in lettuce have emerged in several locations in Europe since 2007. The objective of this study was to investigate the resistance mechanism mediated by the Nr gene in lettuce by detailed studies of aphid feeding behaviour and performance. Both avirulent (Nr:0) and virulent (Nr:1)biotypes of N. ribisnigri were studied on five resistant and two susceptible near isogenic lines (NILs). In addition, survival and colony development were quantified.Nr:0 aphids showed a strong decrease in sieve element ingestion and took longer to accept a sieve element on resistant NILs compared with susceptible NILs, and no aphids survived on the resistant NIL. Nr:1 aphids fed and performed equally well on the resistant and susceptible NILs. The resistance mechanism against Nr:0 aphids encoded by the Nr gene seems to be located in the phloem, although we also observed differences in feeding behaviour during the pathway phase to the phloem. Nr:1 aphids were highly virulent to the resistance conferred by the Nr gene. The consequences of the appearance of Nr:1 aphids for control of N. ribisnigri are discussed.

New sources of resistance to lettuce aphids in Lactuca spp.

The use of resistant cultivars is one of the best ways to protect lettuce from aphid pests. At present, there are cultivars available with nearly complete resistance to Nasonovia ribisnigri biotype Nr:0 (based on the Nr gene) and partial resistance to Macrosiphum euphorbiae. Nevertheless, a new biotype of N. ribisnigri (Nr:1) able to overcome the resistance based on the Nr gene is expanding around Europe and has become a major threat of lettuce. In the present work, we report the presence of this new biotype in southeastern Spain, a major lettuce-producing region. Furthermore, a pool of 264 germplasm accessions belonging to Lactuca genus was tested in a greenhouse assay to search for new resistance sources to N. ribisnigri. The most promising accessions were retested in the laboratory to characterize the resistance by means of free-choice and antibiosis assays against biotypes Nr:0 and Nr:1 of N. ribisnigri and against a clone of M. euphorbiae. Three accessions of L. virosa showed resistance against the target aphid species and could be of interest for ongoing breeding programs. The accessions CGN16272 and CGN13361 were both partially resistant to the Nr:1 biotype of N. ribisnigri and to M. euphorbiae, and CGN13355, in spite of not being resistant to N. ribisnigri, showed a near complete resistance to M. euphorbiae. The study of the feeding behavior of N. ribisnigri biotypes showed that the Nr:1 biotype is able to maintain a similar phloem feeding ingestion pattern on genotypes bearing the Nr gene and on N. ribisnigri-susceptible lettuce genotypes. Moreover, as aphids rejected L. virosa as a feeding source due to superficial factors (high level of antixenosis), no differences in the level of antibiosis between such genotypes were detected. A second set of screening assays were conducted on 40 accessions of L. virosa in order to select for resistance against the Nr:1 biotype. The results showed three accessions with high levels of resistance (CGN05148, CGN21399 and CGN16274) against Nr:1 that could be of interest in lettuce breeding programs.

Hybridization, polyploidy, and evolutionary transitions between monoecy and dioecy in Bryonia (Cucurbitaceae)

Correns's 1903 (Berichte der Deutschen Botanischen Gesellschaft 21: 133-147) crosses between a monoecious and a dioecious species of Bryonia revealed the simple Mendelian inheritance of dioecy and provided the first instance of an XY sex determination system in any organism. Bryonia ranges from the Canary Islands to Central Asia and comprises seven dioecious and three monoecious species; its closest relative, Ecballium elaterium, has dioecious and monoecious populations. We used chloroplast (cp) and nuclear (nr) gene phylogenies to infer sexual system evolution in Bryonia. We also tested for associations between sexual system and ploidy level, based on published and original chromosome counts. Conflicts between cp and nr topologies imply that the dioecious hexaploid B. cretica arose from hybridization(s), probably involving the dioecious diploids B. dioica, B. syriaca, and/or B. multiflora. A tetraploid dioecious endemic on Corsica and Sardinia probably originated from B. dioica via autopolyploidy. While the cp phylogeny resolves few species relationships, the nr tree implies at least two evolutionary changes in sexual system. There is no correlation between sexual system and ploidy level. Molecular clocks suggest that the deepest divergence, between a species on the Canary Islands and the ancestor of all remaining species, occurred ca. 10 million years ago.

Potential Sources of Genetic Resistance in Lactuca spp. to the Lettuce Aphid, Nasanovia ribisnigri (Mosely) (Homoptera: Aphididae)

Lettuce aphid, Nasanovia ribisnigri (Mosely) (Homoptera: Aphididae), is an economically important pest of lettuce (Lactuca sativa L.). High-level resistance found in a wild relative, Lactuca virosa L. accession PIVT 280, is conditioned by the Nr gene, which has been transferred to European cultivars and is being commercially transferred to U.S.-adapted cultivars. New sources of resistance to guard against possible resistance-breaking strains of lettuce aphid were sought in a greenhouse screening of 1203 accessions of lettuce, which included 1047 accessions of L. sativa L., seven accessions of Lactuca perennis L., 18 accessions of Lactuca saligna L., 125 accessions of L. serriola L., and six accessions of L. virosa L. Two new and potentially unique sources of resistance to lettuce aphid were found in L. serriola accession PI 491093 and L. virosa accession PI 274378. The genetic relationship of resistance in PI 491093 and PI 274378 with that in PIVT 280 remains to be determined.

Lipoteichoic acid modulates hepatic drug metabolizing enzymes by activating the Toll‐like receptor 2 signaling pathway

Background:Altered expression of drug metabolizing enzymes (DMEs) in inflammation can affect the metabolism and toxicity of drugs. It is known that inflammatory responses are induced in immune cells by Lipopolysaccahride (LPS) and Lipoteichoic acid (LTA), derived from gram −ve and gram +ve bacteria, respectively. We reported that LPS modulates gene expression of hepatic DMEs and their regulatory nuclear receptors (NRs: PXR, CAR and RXRα) by activating Toll‐like receptor (TLR) 4 signaling pathway. We hypothesize that LTA can modulate NR and DME gene expression by activating the TLR2 signaling pathway. Methods: Adult male C57BL/6 mice were i.p. injected with 6 μg/g bw LTA or saline. Livers were harvested and RNA and proteins were analyzed by real‐time PCR and western blotting, respectively.Results:LTA treatment for 2h resulted in ∼80% suppression of CAR and target DME genes, Cyp2b10, Cyp2a4 and Sultn. The NR, PXR, and the main phase 1 DME, Cyp3a11 were reduced ∼70% by 4–8 h of LTA treatment. Nuclear protein levels of RXRα were reduced in LTA‐treated mice. LTA injection induced cytokine production and cell‐signaling components in the liver. The effects of LTA on NRs, DMEs, cytokines and cell‐signaling pathways were blocked in TLR2−/− mice, indicating that the effects of LTA on hepatic genes were mediated by TLR2.Conclusions:TLR2 is involved in regulating DME gene expression during induction of inflammation by LTA.

Evolution of a novel subfamily of nuclear receptors with members that each contain two DNA binding domains

BackgroundNuclear receptors (NRs) are important transcriptional modulators in metazoans which regulate transcription through binding to the promoter region of their target gene by the DNA binding domain (DBD) and activation or repression of mRNA synthesis through co-regulators bound to the ligand binding domain (LBD). NRs typically have a single DBD with a LBD.ResultsThree nuclear receptors named 2DBD-NRs, were identified from the flatworm Schistosoma mansoni that each possess a novel set of two DBDs in tandem with a LBD. They represent a novel NR modular structure: A/B-DBD-DBD-hinge-LBD. The 2DBD-NRs form a new subfamily of NRs, VII. By database mining, 2DBD-NR genes from other flatworm species (Schmidtea mediterranea and Dugesia japonica), from Mollusks (Lottia gigantean) and from arthropods (Daphnia pulex) were also identified. All 2DBD-NRs possess a P-box sequence of CEACKK in the first DBD, which is unique to 2DBD-NRs, and a P-box sequence of CEGCKG in the second DBD. Phylogenetic analyses of both DBD and ligand binding domain sequences showed that 2DBD-NR genes originate from a common two DBD-containing ancestor gene. A single 2DBD-NR orthologue was found in Arthropoda, Platyhelminths and Mollusca. Subsequent 2DBD-NR gene evolution in Mollusks and Platyhelminths involved gene duplication. Chromosome localization of S. mansoni 2DBD-NR genes by Fluorescent in situ hybridization (FISH) suggests that 2DBD-NR genes duplicated on different chromosomes in the Platyhelminths. Dimerization of Sm2DBDα indicates that 2DBD-NRs may act as homodimers, suggesting either that two repeats of a half-site are necessary for each DBD of 2DBD-NRs to bind to its target gene, or that each 2DBD-NR can recognize multiple sites.Conclusion2DBD-NRs share a common ancestor gene which possessed an extra DBD that likely resulted from a recombination event. After the split of the Arthropods, Mollusks and Platyhelminths, 2DBD-NR underwent a recent duplication in a common ancestor of Mollusks, while two rounds of duplication occurred in a common ancestor of the Platyhelminths. This demonstrates that certain NR gene underwent recent duplication in Prostostome lineages after the split of the Prostostomia and Deuterostomia.

Receptor‐Mediated Targeting of Toxin Results in Selection of Genetically Protected Hepatocytes Without Bystander Toxicity

AIM To target G418 specifically to liver cells via asialoglycoprotein receptors (AsGR) for selection of stably transfected neomycin resistant hepatocytes (NR+) without collateral damage. METHODS: A novel G418 conjugate was synthesized by peptide coupling to a galactose-terminal asialoorosomucoid (AsOR). Uptake studies were done by labeled-ligand assays, and toxicity by MTT assays on Huh7 (AsGR+, NR−), GFP-Huh7 (AsGR+, NR+), and 3T3 (AsGR−, NR−) cell lines. RESULTS: Uptake of conjugate (7–8 moles) of G418 per AsOR was the same as for AsOR alone. Conjugate inhibited growth of Huh7 cells by 84.3%. This inhibition was blocked by excess free AsOR. Incubation of 3T3 cells with free G418, but not conjugated G418 resulted in toxicity. GFP-Huh cells were not susceptible either to free or conjugated G418. Co-cultures of GFP-Huh7 and 3T3 in G418 alone resulted in only inhibition of 3T3 cells. In contrast, co-cultures with AsOR-G418 resulted in no significant toxicity to either cell type. CONCLUSIONS: G418-protein conjugates retained inhibitory activity targeted against AsGR (+), NR (−) cells. G418 modification to form a conjugate did not block its activity. Lack of effect of G418 conjugate on ASGR (−) cells suggests that the effects were specific, and not due to release of free G418 in the medium. The presence of NR gene in AsGR (+) cells protected those cells, and the effect without collateral damage to co-cultured AsGR (−) cells. Supported by Lopata Chair in Hepatitis Research and R01-DK042182.

Responses of Nasonovia ribisnigri (Homoptera: Aphididae) to susceptible and resistant lettuce.

Nymphs and alates of aphid Nasonovia ribisnigri (Mosley) (Homoptera: Aphididae) were tested on 10 lettuce cultivars with N. ribisnigri resistance gene Nr and 18 cultivars without the resistance gene in various bioassays. Bioassays used whole plants, leaf discs, or leaf cages to determine susceptibility of commercial lettuce cultivars to N. ribisnigri infestation and to evaluate screening methods for breeding lettuce resistance to N. ribisnigri. Resistant and susceptible plants were separated in 3 d when using whole plant bioassays. Long-term (> or =7 d) no-choice tests using leaf cages or whole plants resulted in no survival of N. ribisnigri on resistant plants, indicating great promise of the Nr gene for management of N. ribisnigri. Effective screening was achieved in both no-choice tests where resistant or susceptible intact plants were tested separately in groups or individually and in choice tests where susceptible and resistant plants were intermixed. Leaf discs bioassays were not suitable for resistance screening. All lettuce cultivars without the resistance gene were suitable hosts for N. ribisnigri, indicating the great importance of this pest to lettuce production and the urgency in developing resistant lettuce cultivars to manage N. ribisnigri.

The main origin of endogenous NO in higher non-leguminous plants

The main origin of nitric oxide (NO) production in higher non-leguminous plants is studied in this paper. NO content in the leaf of wheat ( Triticum aestivum L.), orchid ( Malaxis monophyllos L.) and aloe ( Aloe vera L.) was decreased over 90% by the treatment of heat or microwave. It indicates that NO is primarily enzymically produced in higher non-leguminous plants. Nitrate reductase (NR; EC 1.6.6.1) activity and NO content were about 74% and 56%, respectively, in wheat seedling grown in a medium lacking molybdenum. Sodium tungstate almost completely inhibited NR activity and decreased NO content near 90% in the wheat seedling extraction. Taking out inducing factors such as KNO 3 and light of NR gene expression, the NR activity was less than 30% and the NO content was less than 20% of the control in the wheat seedling. These results suggest that NO is mainly produced through NR pathway in wheat seedlings. When NaNO 2 was applied to wheat seedlings for 10 h in dark before harvest, the NO content vastly increased but dramatically decreased after treatment by heat or microwave. The provision of NaNO 2 as above and added exogenous Cu 2+ decreased both NR and nitrite reductase (NiR; EC 1.7.7.1) activity, but increased NO content. These results suggest that the conversion from NO 2 – to NO in wheat seedling is also enzymic and this process is independent of NR and NiR.

Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris.

In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed from most other higher-plant NRs by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099-1105]. In order to elucidate these deviating properties in more detail, the NR gene from R. communis was cloned, expressed heterologously and characterized. The deduced protein sequence showed that Ricinus NR has a serine phosphorylation site and a 14-3-3 binding motif, a common characteristic of NRs. Functional Ricinus NR protein was expressed in the yeast Pichia pastoris and compared with the features of Arabidopsis thaliana NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was not inactivated by incubation with MgATP. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3 proteins were prepared from 4-day-darkened (and therefore NR-free) leaves of Ricinus, and added to the assay of RcNR to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, its unusually high Mg2+-sensitivity was restored, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixture and ATP. Thus, insensitivity to ATP appears to be an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and appeared to interact specifically with recombinant Ricinus NR to provide the Mg2+-sensitivity of the authentic leaf enzyme.