The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h₂) was isolated using thioredoxin h₁ cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 by respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h₁ (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h₁ and h₂, do not have any signal peptides and are most probably cytoplasmic. Using the 3 regions of the mRNAs, two probes specific for thioredoxins h₁ and h₂ have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h₁ and a 4.5 kb fragment encodes h₂. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h₂ is present in N. sylvestris and N. tomentosiformis but that thioredoxin h₁ is absent from both putative ancestors. Thus, the thioredoxin h₁ gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells. Thioredoxin h₁ is always expressed at a lower level than h₂ in tobacco plants. In contrast, the thioredoxin hl gene is abundantly expressed in freshly isolated protoplasts, while h₂ mRNAs are not detectable.     

A strain of Pseudomonas fluorescens with two lipase-encoding genes, one of which possibly encodes cytoplasmic lipolytic activity

AIMS: A lipase-encoding gene (lipA) from a psychrotrophic strain of Pseudomonas fluorescens C9 has previously been characterized. It was also shown that when this gene was insertionally-inactivated, lipase activity was retained, suggesting that a second lipase may be present in this strain. The aim of this study was to determine whether this was the case. METHODS AND RESULTS: Using molecular cloning, chromosomal mutagenesis and enzymatic analysis, the presence of a second lipase-encoding gene (lipB) has been confirmed. The molecular weights of the putative products of lipA and lipB are 33 and 64.5 kDa, respectively, and their sequences are quite dissimilar (< 10% sequence identity). The lipB gene encodes a secreted lipase and is solely responsible for the 'lipolytic phenotype' of Ps. fluorescens C9. Expression of the lipA gene can be detected when expressed using an expression vector, but activity was only detected intracellularly in Ps. fluorescens C9, and not in the culture medium. CONCLUSION: Pseudomonas fluorescens C9 contains two dissimilar lipases. One (LipB) is secreted and responsible for the lipolytic phenotype; the evidence suggests that the other (LipA) could be intracellular, but it could be secreted and not detectable. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria may contain more than one lipase activity. Ascribing phenotypes to particular enzymes therefore requires mutational analysis. The notion of an intracellular lipase activity is novel, and, if further substantiated, begs the question as to its normal substrate and physiological role.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Seed legumains are expressed in stamens and vegetative legumains in seeds of Nicotiana tabacum L

Detailed analysis of the expression pattern of seven legumain genes from Nicotiana tabacum L. cv. SNN revealed that it showed neither coincidences with the branches of the phylogenetic tree of legumains nor with their conventional assignment to organs. This agreed well with the fact that, so far, no functional differences could be assigned to the sequence differences reflected in the branches of the tree.     

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Glycosyl-phosphatidylinositol-anchor addition signals are processed in Nicotiana tabacum

Recent studies have demonstrated the existence of glycosyl-phosphatidylinositol (GPI)-anchored proteins in higher plants. In this study we tested whether GPI-addition signals from diverse evolutionary sources would function to link a GPI-anchor to a reporter protein in plant cells. Tobacco protoplasts were transiently transfected with a truncated form of the Clostridium thermocellum endoglucanase E reporter gene (celE') fused with a tobacco secretion signal (PR-1a) at the N-terminus and either a yeast (GAS1), mammalian (Thy-1) or putative plant (LeAGP-1) GPI-anchor addition signal at the C-terminus. The yeast and plant C-terminal signals were found to be capable of directing the addition of a GPI-anchor to the endoglucanase protein (EGE') as shown by the sensitivity of the lipid component of GPI to phosphatidylinositol-specific phospholipase C (PI-PLC) digestion. In contrast, the mammalian signal was poorly processed for anchor addition. When EGE' was fused to a truncated form of the LeAGP-1 signal (missing three amino acids predicted to be critical to signal cleavage and anchor addition), a GPI-anchor was not linked to the EGE' protein indicating the necessity for the missing amino acids. Our results show the conservation of the properties of GPI-signals in plant cells and that there may be some similar preferences in GPI-addition signal sequences for yeast and plant cells.     

Six active phage-type RNA polymerase genes in Nicotiana tabacum

In higher plants, a small nuclear gene family encodes mitochondrial as well as chloroplast RNA polymerases (RNAP) homologous to the bacteriophage T7-enzyme. The Arabidopsis genome contains three such RpoT genes, while in monocotyledonous plants only two copies have been found. Analysis of Nicotiana tabacum, a natural allotetraploid, identified six different RpoT sequences. The study of the progenitor species of tobacco, N. sylvestris and N. tomentosiformis, uncovered that the sequences represent two orthologous sets each of three RpoT genes (RpoT1, RpoT2 and RpoT3). Interestingly, while the organelles are inherited exclusively from the N. sylvestris maternal parent, all six RpoT genes are expressed in N. tabacum. GFP-fusions of Nicotiana RpoT1 revealed mitochondrial targeting properties. Constructs containing the amino-terminus of RpoT2 were imported into mitochondria as well as into plastids. Thus, the dual-targeting feature, first described for Arabidopsis RpoT;2, appears to be conserved among eudicotyledonous plants. Tobacco RpoT3 is targeted to chloroplasts and the RNA is differentially expressed in plants lacking the plastid-encoded RNAP. Remarkably, translation of RpoT3 mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis, Nicotiana RpoT3 provides a second example for a non-viral plant mRNA that is exclusively translated from a non-AUG codon.     

Mapping of two white stem genes in tetraploid common tobacco (Nicotiana tabacum L.)

Leaf color is an indicator of chlorophyll (Chl) level, and isolating leaf color mutants can facilitate the understanding of Chl metabolism regulation. Here, we describe an ethyl methanesulfonate-induced light color mutant white stem 1 (ws1) in common tobacco (Nicotiana tabacum L.) that shows a phenotype highly similar to burley tobacco (Nicotiana tabacum L.), a type of air-cured tobacco that has light-colored leaves with white veins. Compared with the wild type, the light green stem of ws1 gradually became pale white along with growth, while ws1 leaves lost green color rapidly, which was positively correlated with the decline of Chl levels. A series of genetic analyses indicated that the ws1 mutant phenotype was controlled by two recessive nuclear genes ws1a and ws1b which were preliminarily mapped to the intervals of tobacco simple sequence repeat markers linkage groups 5 and 24 using the BC1F2 populations, respectively. The allelism test further revealed that the same two genes controlled the burley character in burley tobacco. Based on the Chl-deficient phenotype of ws1 and the locations of the two genes, we hypothesized that ws1a and ws1b were paralogs of each other probably originated from the ancestral species N. sylvestris and N. tomentosiformis, respectively. Both genes might share similar biological functions and expression patterns, and play key roles in the regulation of Chl biosynthesis. These results laid a solid foundation for marker-assisted selection breeding and gene function analysis of the burley character in tobacco.     

Rapidly evolving marmoset MSMB genes are differently expressed in the male genital tract

Background  

Beta-microseminoprotein, an abundant component in prostatic fluid, is encoded by the potential tumor suppressor gene MSMB. Some New World monkeys carry several copies of this gene, in contrast to most mammals, including humans, which have one only. Here we have investigated the background for the species difference by analyzing the chromosomal organization and expression of MSMB in the common marmoset (Callithrix jacchus).     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

Molecular characterization of two Brassica napus genes related to oleosins which are highly expressed in the tapetum

Two highly homologous Brassica napus flower cDNA clones, Sta 41-2 and Sta 41-9, were isolated and characterized. These clones were shown to correspond to genes expressed in the tapetum from the early uninucleate microspore stage to the dinucleate stage. The predicted Sta 41-2 and Sta 41-9 proteins possessed characteristics similar to oleosins such as a polar N-terminal domain, a large relatively conserved hydrophobic domain and a long C-terminal domain which consisted of four different groups of repeats. In addition, like oleosins, the Sta 41-2 and Sta 41-9 proteins have a basic pl, lack a signal peptide and are found in a tissue which accumulates lipids in small lipid bodies.     

Characterization of genes encoding metal tolerance proteins isolated from Nicotiana glauca and Nicotiana tabacum

We have isolated a metal tolerance protein (MTP) gene, NgMTP1, from Nicotiana glauca (a potential phytoremediator plant) and two MTP genes, NtMTP1a and NtMTP1b, from Nicotiana tabacum. These three genes shared approximately 95% homology at the amino acid level. Heterologous expression of any of these three genes complemented Zn and Co tolerance in yeast mutants to a similar extent. In yeast, these proteins were shown to be located to vacuole membrane. These results suggest that the three MTPs operate by sequestering Zn and Co into vacuoles, thereby reducing the toxicity of these metals.     

Absence of some truncated genes in the amphidiploid Nicotiana tabacum

As an initial step towards understanding how a multigene family evolves after an interspecific hybridization and subsequent chromosomal doubling, genomic Southern blots of three related species were compared: Nicotiana tabacum (the progeny), and Nicotiana sylvestris and Nicotiana tomentosiformis (the progenitors). Genomic restriction fragments generated by two endonucleases were hybridized with a cDNA of the small subunit of ribulose bisphosphate carboxylase from N. sylvestris. The restriction pattern of the DNA of the progenitors revealed considerable polymorphism of restriction-fragment lengths. All the fragments in N. tabacum, except one, have a corresponding fragment in one of the progenitors. Some fragments present in the parents were absent from the progeny: they may correspond to truncated genes consisting of only part of the 3' portion of the gene.