Variation in cadmium accumulation potential and tissue distribution of cadmium in tobacco

Variation in Cd accumulation between Nicotiana species but not varieties has been observed in seedlings grown in solution culture with moderate-to-low levels of Cd. Nicotiana tabacum has been characterized as a leaf and root accumulator while Nicotiana rustica is shown to be primarily a root accumulator, having about half the leaf Cd per gram dry weight of N. tabacum. This phenotype is retained in the mature N. rustica plant. To characterize these two species which differ in their modes of Cd accumulation, tissue Cd distribution, partitioning of metal in soluble and insoluble fractions and the contribution of soluble Cd-binding proteins (peptides) to total plant Cd was assessed using mature solution cultured plants. Metal accumulation was highest in the most mature leaves and in young roots. The preponderance of young roots in N. rustica may, in part, account for low leaf/high root Cd accumulation in this species. While Cd-binding peptides appear to be a principal form of Cd in leaves and roots of seedlings and these also occur in mature leaves, Cd is equally distributed between soluble (about 80% as Cd-binding peptide) and uncharacterized insoluble forms in mature plant roots.     

Detection of the Nicotiana rustica chloroplast genome coding for the large subunit of Fraction I protein in a somatic hybrid in which only the N. tabacum chloroplast genome appeared to have been expressed

Nine plants were produced from anthers of a somatic hybrid which had been obtained by fusion of Nicotiana tabacum L. and N. rustica L. protoplants. As determined by electrofocusing, the Fraction I protein of the original somatic hybrid had largesubunit polypeptides exclusively of the N. tabacum type. Two of the plants regenerated from anthers contained Fraction-I-protein large subunits exclusively of the N. rustica type. Since each plant was regenerated from a single cell, the somatic hybrid must have had cells containing both the N. tabacum and N. rustica chloroplast genome although the latter was not expressed. Possibilities to account for this non-expression of a chloroplast genome in the somatic hybrid are discussed.     

Expression of nuclear and chloroplastic genes coding for fraction-1 protein in somatic hybrids of Nicotiana tabacum + rustica

In the sexual interspecific cross, Nicotiana rustica L.xN. tabacum L., N. rustica can serve as the female but not as the male parent. By fusion of protoplasts, the barrier to fertilization was overcome and somatic hybrids containing N. tabacum cytoplasm were produced as shown by isoelectric focusing of the Fraction-1 protein (F-1-protein). All somatic hybrids displayed polypeptides of the large subunit of F-1 protein (which is coded by the chloroplast genome) characteristic of only one or the other parental species. Two hybrids had large subunits of the N. tabacum type and two hybrids had those of the N. rustica type. Three hybrids contained three smallsubunit polypeptides (coded by the nuclear genome), one being characteristic of N. rustica, one characteristic of N. tabacum, and one with an isoelectric point common to both species. A fourth hybrid contained only two small-subunit polypeptides of the N. tabacum type but in a F-1 protein macromolecule whose large subunits were of the N. rustica type. One somatic hybrid was self-fertile and its F₂ progeny contained large- and small-subunit polypeptides indistinguishable in their isoelectric points from those in the parent F₁ hybrid. All somatic hybrids showed an aneuploid chromosome number and morphological characteristics intermediate between those of N. rustica and N. tabacum.     

Rapid flowering Nicotiana tabacum L.

 Selected Nicotiana tabacum Tobacco Introductions from the Nicotiana Germplasm Collection were crossed, back-crossed, and then selfed. When compared to parents, the new genotypes produce smaller plants with a shortened life-cycle that go from seed to seed in under 11 weeks. Received: 11 December 1998 / Revision accepted: 8 January 1999     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

Development of SCAR markers linked to three disease resistances based on AFLP within Nicotiana tabacum L

Amplified fragment length polymorphism (AFLP) was conducted on a set of 92 Nicotiana tabacum L. accessions from diverse types (flue-cured, dark air-cured, burley, oriental, and cigar wrapper) and breeding origins to identify markers associated with disease resistances. Eleven primer combinations were required to identify 33 polymorphic fragments. This allowed the identification of 92% of these accessions, and yielded sufficient information for building a neighbor joining tree. Clusters of accessions with common traits or breeding origins were observed. An important part of this polymorphism could be related to interspecific introgressions from other Nicotiana species, performed during the breeding history of N. tabacum to confer resistance to pathogens. Seven fragments were associated with three different resistances: two for the blue-mold (Peronospora tabacina Adam) resistance derived from Nicotiana debneyi Domin, two for the Va gene (Potato Virus Y susceptibility), and three for the black root rot (Chalara elegans) resistance of N. debneyi origin. Some of these markers were converted into sequence characterized amplified region markers, and validated on recombinant inbred lines or doubled-haploid lines.     

Genomic factors responsible for abnormal morphology of pollen tubes in the interspecific cross Nicotiana tabacum ×N. rustica

Nicotiana tabacum shows unilateral pollen-pistil incongruity with N. rustica. If N. tabacum is pollinated with N. rustica, growth of the pollen tube is arrested in the middle of the style, and abundant callose deposition, tube swelling and tube winding are observed. An attempt was made to clarify the genomic factors responsible for this pollen-pistil incongruity. N. tabacum was pollinated with N. paniculata or N. undulata, progenitors of amphidiploid N. rustica. When pollinated with N. undulata, growth of the pollen tube was arrested in the middle of the style and showed abnormal morphology similar to that with N. rustica, but when pollinated with N. paniculata the pollen tube reached near the base of the style and was almost normal in appearance. These observations suggest that the factors responsible for the pollen tube abnormality of N. rustica are derived from the N. undulata genome.We also used N. sylvestris, N. glutinosa and N. otophora as pistilar parents and N. rustica or its progenitors as pollen parents to examine the genomic factors of the pistilate parents. The pollen tube features of these three species in the pistils of N. sylvestris were similar to those in the pistil of N. tabacum. Received: 25 October 1999 / Revision accepted: 2 February 2000     

The diversity of interspecific pollen-pistil incongruity in Nicotiana

Nicotiana tabacum was used as a pistillate parent and crossed with three self-compatible species, N. rustica, N. repanda and N. trigonophylla, which were previously reported to have pollen tubes unilaterally inhibited by N. tabacum pistil. Temporal and morphological observations revealed distinct differences of pollen tube behavior among these incongruous crosses. Pollen tubes of N. repanda were arrested in stigma and those of N. rustica in the middle of the style. On the other hand, pollen tubes of N. trigonophylla continued growing at a slow rate. Tubes of N. repanda and N. rustica showed morphological abnormalities such as swelling, thick wall, and irregular callose deposition. In addition, tubes of N. rustica often elongated in reverse direction and wound about in the middle of the style. Although the tubes of N. trigonophylla were apparently normal in morphology, they were distributed throughout the transmitting tissue, differing from the self-pollination of N. tabacum in which they were confined to the peripheral region of it. The diversity of pollen tube behavior indicates that physiological causes of incongruity are different among the three crosses. Bud pollination enabled pollen tubes to reach the ovary in all crosses, indicating that the N. tabacum pistil acquired its ability to inhibit foreign pollen tube elongation with its development. When interspecific hybrids between N. tabacum and the other three species were pollinated by parental species, tubes reached the ovary in all crosses, but the elongation rate of tubes slowed down and morphology was abnormal.     

Effect of X-radiation on pollen tube growth and seed setting in crosses between Nicotiana tabacum and N. rustica

Summary In vitro andin vivo studies of the effect of irradiating pollen with different doses of X-rays were carried out inNicotiana rustica andN. tabacum. Dosages upto 9600 r were found to enhance the rate of growth of pollen tubers per unit time. Advantage was taken of this observation to overcome the incompatibility normally found in the crossN. tabacum x N. rustica as a result of the inadequate growth ofrustica pollen tubes intabacum style. Thus, whenrustica pollen exposed to 4800 r and 9600 r of X-rays were used to pollinatetabacum, fertilization resulting in the formation of viable hybrid seeds occurred. In the crossN. rustica×N. tabacum there is partial seed failure as a result of the hyperplastic development of the nucellar and endothelial cells. However, when X-rayedtabacum pollen were used in the cross, seed development registered a marked improvement. Since there is now evidence for suggesting that the contents of even those pollen tubes which do not participate in fertilization have some role to play in the development of the seed, it seems likely that radiation-induced biochemical changes in pollen may prove advantageous when conditions for seed development are abnormal as in instances of somatoplastic sterility. Besides such indirect biochemical effects, irradiation of pollen may also lead to genetic changes resulting in the inactivation of zygotic lethals. Thus, there is much scope for investigating the use of radiation to overcome inter-specific and inter-generic cross-incompatibility barriers.With 2 Figures in the Text     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Mitochondrial DNA patterns are similar in gametosomatic and somatic hybrids of two Nicotiana species

Summary The segregation and recombination patterns of mitochondrial genome in the somatic hybrids of Nicotiana tabacum and N. rustica were studied by RFLP analysis using four heterologous mitochondrial DNA probes, namely cytochrome oxidase subunit I (COI), cytochrome oxidase subunit II (COII), 26s rDNA and 5s-18s rDNA. These RFLP patterns were compared with those of the gametosomatic hybrids of these two species. A preponderance of N. rustica type patterns was observed in the somatic hybrids. One of the somatic hybrids had N. rustica type pattern with COI probe, novel pattern with COII, and 26s rDNA probe and N. tabacum type pattern with 5s-18s rDNA probe. These patterns are identical to those of some of the gametosomatic hybrids and could only be due to the recombination of mitochondrial genomes of the two parents. The extent and the nature of recombination of mitochondrial genomes is similar in gametosomatic and somatic hybrids.     

Transmission of organelles in triploid hybrids produced by gametosomatic fusions of two Nicotiana species

Summary Gametosomatic hybrids produced by the fusion of microspore protoplasts of Nicotiana tabacum Km⁺Sr⁺ with somatic cell protoplasts of N. rustica were analysed for their organelle composition. For the analysis of mitochondrial (mt)DNA, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA and mtDNA with four DNA probes of mitochondrial origin: cytochrome oxidase subunit I, cytochrome oxidase subunit II, 26s rDNA and 5s-18s rDNA. Of the 22 hybrids analyzed, some had parental-type pattern for some probes and novel-type for the others, indicating interaction between mtDNA of the two parent species. For chloroplast (cp)DNA analysis, species-specific patterns were generated by Southern hybridization of restriction endonuclease digests of total DNA with large subunits of ribulose bisphosphate carboxylase and cpDNA as probes. All the hybrids had N. rustica-specific patterns. Hybrids were not resistant to streptomycin, a trait encoded by the chloroplast genome of N. tabacum. In gametosomatic fusions of the two Nicotiana species, mitochondria but not the chloroplasts are transmitted from the parent contributing microspore protoplasts.     

Nucleo-cytoplasmic incompatibility in cybrid plants possessing an Atropa genome and a Nicotiana plastome.

Summary Twenty-nine cybrids possessing an Atropa belladonna nuclear genome and a Nicotiana tabacum plastome were selected from two independent protoplast fusion experiments. In contrast to the previously described reciprocal, green and fertile cybrids with a Nicotiana nuclear genome and an Atropa plastome (Kushnir et al. 1987), the plants obtained were totally chlorophyll-deficient. An Atropa nuclear genome and a Nicotiana plastome from these chlorophyll-deficient cybrids were combined with an Atropa or a Scopolia plastome and a Nicotiana nuclear genome, respectively, in control fusion experiments. All of these nuclear genome/plastome combinations gave rise to normal, green plants. Therefore, we conclude that an N. tabacum plastome is incompatible with an A. belladonna nuclear genome.     

Comparison of the mitochondrial genome of Nicotiana tabacum with its progenitor species

Summary Mitochondrial DNAs from Nicotiana tabacum, an amphiploid, and its putative progenitor species, N. sylvestris and N. tomentosiformis were compared in structure and organization. By using DNA transfer techniques and cloned fragments of known genes from maize and N. sylvestris as labeled probes, the positions of homologous sequences in restriction digests of the Nicotiana species were analyzed. Results indicate that the mitochondrial DNA of N. tabacum was inherited from N. sylvestris. Conservation in organization and sequence homology between mtDNAs of N. tabacum and the maternal progenitor, N. sylvestris, provide evidence that the mitochondrial genome in these species is evolutionarily stable. Approximately one-third of the probed restriction fragments of N. tomentosiformis mtDNA showed conservation of position with the other two species. Pattern variations indicate that extensive rearrangement of mtDNA has occurred in the evolution of these Nicotiana species.     

Deep sequencing of the ancestral tobacco species Nicotiana tomentosiformis reveals multiple T‐DNA inserts and a complex evolutionary history of natural transformation in the genus Nicotiana

Nicotiana species carry cellular T‐DNA sequences (cT‐DNAs), acquired by Agrobacterium‐mediated transformation. We characterized the cT‐DNA sequences of the ancestral Nicotiana tabacum species Nicotiana tomentosiformis by deep sequencing. N. tomentosiformis contains four cT‐DNA inserts derived from different Agrobacterium strains. Each has an incomplete inverted‐repeat structure. TA is similar to part of the Agrobacterium rhizogenes 1724 mikimopine‐type T‐DNA, but has unusual orf14 and mis genes. TB carries a 1724 mikimopine‐type orf14‐mis fragment and a mannopine‐agropine synthesis region (mas2‐mas1‐ags). The mas2′ gene codes for an active enzyme. TC is similar to the left part of the A. rhizogenes A4 T‐DNA, but also carries octopine synthase‐like (ocl) and c‐like genes normally found in A. tumefaciens. TD shows a complex rearrangement of T‐DNA fragments similar to the right end of the A4 TL‐DNA, and including an orf14‐like gene and a gene with unknown function, orf511. The TA, TB, TC and TD insertion sites were identified by alignment with N. tabacum and Nicotiana sylvestris sequences. The divergence values for the TA, TB, TC and TD repeats provide an estimate for their relative introduction times. A large deletion has occurred in the central part of the N. tabacum cv. Basma/Xanthi TA region, and another deletion removed the complete TC region in N. tabacum. Nicotiana otophora lacks TA, TB and TD, but contains TC and another cT‐DNA, TE. This analysis, together with that of Nicotiana glauca and other Nicotiana species, indicates multiple sequential insertions of cT‐DNAs during the evolution of the genus Nicotiana.     

Non-random chloroplast segregation inNicotiana tabacum (+)N. rustica somatic hybrids selected by dual nuclear-encoded resistance

Nicotiana tabacum (+)N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistantNicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistantN. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessedN. rustica chloroplasts and only one hadN. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar toN. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns ofcoxll hybridization identical withN. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.Contribution No. 1439 Plant Research CentreCurrent address: Plant Biotechnology Institute, National Research Council, 110 Gymmasium Road, Saskatoon, Saskatchewan S7N OW9, Canada     

Root exudates from sunflower (Helianthus annuus L.) show a strong adsorption ability toward Cd(II)

Abstract

Cd(II) adsorption of root exudates from sunflower (Helianthus annuus L.) seedling was investigated by Cd ion-selective electrode, Fourier Transform Infrared spectroscopy, and fluorescence spectroscopy. Root exudates from Helianthus annuus L. had strong adsorption ability toward Cd(II). The adsorption process was pH-dependent and the maximum adsorption capacity, 150.8 mg g^?1, was observed at pH 7.0. Root exudates had pK _a1 at 4.7 for carboxyl and pK _a2 at 9.2 for phenolic, and amino groups. The aliphatic and aromatic (C?H) groups, amide III group, and the C (=O)?O and sulfonate groups were responsible for Cd(II) adsorption. The excitation emission matrix fluorescence spectroscopy showed protein-like substances participated in Cd adsorption and formed strong complexes, with conditional stability constants of 4.70 and 4.32, which is a little lower than that determined by potentiometric methods, 5.13. The strong Cd complexing ability of root exudates implies that root exudates may significantly affect mobility, toxicity, and phytoavailability of Cd. Cd binding of root exudates may be attributed to its interaction with the proteins, polysaccharides, and phenolic compounds in root exudates.     

Effects of RNases on rejection of pollen from Nicotiana tabacum and N. plumbaginifolia

S-RNases are implicated in both inter- and intra-specific pollen rejection in Nicotiana. At least two mechanisms contribute to S-RNase dependent rejection of pollen from self compatilble species such as Nicotiana plumbaginifolia and N. tabacum. Cloned S-RNases from self incompatible N. alata expressed in styles of self compatible N. tabacum cause rejection of N. tabacum pollen through a factor-independent mechanism. However, rejection of N. plumbaginifolia pollen occurs only when S-RNases are expressed in conjunction with non-S-RNase factors from N. alata (factor-dependent pollen rejection). Here, we compared the pollen rejection activity of four RNases in these two systems. Recombinant RNase expression levels in the factor-dependent N. plumbaginifolia system were insufficient to cause pollen rejection. However, three S-RNases were active in the factor-independent N. tabacum pollen rejection system. This system shows the broadest specificity of any system so far examined. However, RNaseI from E. coli could not cause N. tabacum pollen rejection. Thus, RNase activity alone is not sufficient for pollen rejection. Our results suggest that S-RNases are specially adapted to function in pollen rejection. Received: 15 December 2000 / Accepted: 1 May 2001     

Parental origin of the allotetraploid tobacco Nicotiana benthamiana

Nicotiana section Suaveolentes is an almost all‐Australian clade of allopolyploid tobacco species including the important plant model Nicotiana benthamiana. The homology relationships of this clade and its formation are not completely understood. To address this gap, we assessed phylogenies of all individual genes of N. benthamiana and the well studied N. tabacum (section Nicotiana) and their homologues in six diploid Nicotiana species. We generated sets of 44 424 and 65 457 phylogenetic trees of N. benthamiana and N. tabacum genes, respectively, each collectively called a phylome. Members of Nicotiana sections Noctiflorae and Sylvestres were represented as the species closest to N. benthamiana in most of the gene trees. Analyzing the gene trees of the phylome we: (i) dated the hybridization event giving rise to N. benthamiana to 4–5 MyA, and (ii) separated the subgenomes. We assigned 1.42 Gbp of the genome sequence to section Noctiflorae and 0.97 Gbp to section Sylvestres based on phylome analysis. In contrast, read mapping of the donor species did not succeed in separating the subgenomes of N. benthamiana. We show that the maternal progenitor of N. benthamiana was a member of section Noctiflorae, and confirm a member of section Sylvestres as paternal subgenome donor. We also demonstrate that the advanced stage of long‐term genome diploidization in N. benthamiana is reflected in its subgenome organization. Taken together, our results underscore the usefulness of phylome analysis for subgenome characterization in hybrid species.