László Márton

Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3

Silver nitrate effectively promoted shoot regeneration in wheat (Triticum aestivum L.) callus cultures derived from immature embryos. This effect could be observed in both weakly and strongly regenerating cultivars, and in using material from both field and greenhouse grown plants. The role of silver ions as an inhibitor of ethylene action was supported by a reversal of the inhibitory effects of 2,4-D and ethylene on morphogenesis in wheat callus cultures.Enhancement of shoot regeneration by silver nitrate was also observed in callus cultures of non-regenerating or weakly regenerating mutants of Nicotiana plumbaginifolia Viv. derived from cell cultures.

Transmission of paternal chloroplasts in Nicotiana

SummaryTransmission of paternal chloroplasts was observed in Nicotiana, considered to inherit organelles in a strictly maternal way. Plants carrying streptomycin resistant plastids were used as pollen donors. Cell lines with paternal plastids in the offspring were selected as green (resistant) sectors on calli induced from the seedlings on streptomycin-containing media. The presence of paternal plastids in the regenerated plants was confirmed by restriction analysis. In the Nicotiana plumbaginifolia ♀xN. plumbaginifolia Np(SR1)3 ♂ and the N. plumbaginifolia Np(gos)29 ♀xN. tabacum SR1 ♂ crosses 2.5% and 0.07% of the offspring were found to contain paternal (tabacum) plastids, respectively. These plants, however, carried maternal mitochondria exclusively. This sexual cybridization method offers a simple way to transfer chloroplasts solely, a goal not accessible by protoplast fusion.

Respiratory control over photosynthetic electron transport in chloroplasts of higher-plant cells: evidence for chlororespiration

Flash-induced primary charge separation, detected as electrochromic absorbance change, the operation of the cytochrome b/f complex and the redox state of the plastoquinone pool were measured in leaves, protoplasts and open-cell preparations of tobacco (Nicotiana tabacum L.), and in isolated intact chloroplasts of peas (Pisum sativum L.). Addition of 0.5–5 mM KCN to these samples resulted in a large increase in the slow electrochromic rise originating from the electrogenic activity of the cytochrome b/f complex. The enhancement was also demonstrated by monitoring the absorbance transients of cytochrome f and b6 between 540 and 572 nm. In isolated, intact chloroplasts with an inhibited photosystem (PS) II, low concentrations of dithionite or ascorbate rendered turnover of only 60% of the PSI reaction centers, KCN being required to reactivate the remainder. “Silent” PSI reaction centers which could be reactivated by KCN were shown to occur in protoplasts both in the absence and presence of a PSII inhibitor. Contrasting spectroscopic data obtained for chloroplasts before and after isolation indicated the existence of a continuous supply of reducing equivalents from the cytosol.Our data indicate that: (i) A respiratory electron-transport pathway involving a cyanide-sensitive component is located in chloroplasts and competes with photosynthetic electron transport for reducing equivalents from the plastoquinone pool. This chlororespiratory pathway appears to be similar to that found in photosynthetic prokaryotes and green algae. (ii) There is an influx of reducing equivalents from the cytosol to the plastoquinone pool. These may be indicative of a complex respiratory control of photosynthetic electron transport in higher-plant cells.

Nitrate reductase deficient cell lines from haploid protoplast cultures ofNicotiana plumbaginifolia

SummaryNitrate reductase deficient (NR-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.Out of 136 chlorate resistant clones 29 were fully deficient in nitrate reductase. The rest of the clones contained decreased or normal levels of NR activity (91 and 16 clones, respectively).Further characterization was carried out in 9 clones which were fully deficient in NR and in 2 clones containing resisdual (0–5%) NR activity. The clones were tentatively classified as defective in the apoenzyme (7 clones including the 2 with residual NR activity) or the cofactor (4 clones) of NR by the xanthine dehydrogenase activity and in vitro enzyme complementation. The cofactor defectives could be further classified into two groups. In one of these (2 clones) the NR activity could be partially restored by unphysiologically high (0.2–1 mM) molybdate in the culture medium. The other two are new types which have not been described in flowering plants.Plant regeneration was obtained only in the clones which contained residual NR activity.

Complementation in somatic hybrids indicates four types of nitrate reductase deficient lines in Nicotiana plumbaginifolia

SummaryAllelism of nine nitrate reductase deficient (NR−) Nicotiana plumbaginifolia cell lines was tested by complementation after protoplast fusion. Complementation was recognized by the appearance of somatic hybrid colonies growing on a selective NH4+/NO3− medium which cannot support the growth of NR− lines. All five apoenzyme defective (NA) lines were non-complementing and therefore allelic. The apoenzyme and the cofactor defective (NX) lines were complementing, as expected, and gave somatic hybrids with restored nitrate reductase activity. The four cofactor defective lines were found to belong to three complementation groups (NX1 and NX9; NX21; NX24). Two of these (NX21 and NX24) are of new types which have not been previously described in flowering plants. In the somatic hybrids restoration of NR activity was accompanied by the restoration of plant regeneration ability.

Antisense Expression of the Peptide Transport Gene AtPTR2-B Delays Flowering and Arrests Seed Development in Transgenic Arabidopsis Plants

Previously, we identified a peptide transport gene, AtPTR2-B, from Arabidopsis thaliana that was constitutively expressed in all plant organs, suggesting an important physiological role in plant growth and development. To evaluate the function of this transporter, transgenic Arabidopsis plants were constructed expressing antisense or sense AtPTR2-B. Genomic Southern analysis indicated that four independent antisense and three independent sense AtPTR2-B transgenic lines were obtained, which was confirmed by analysis of the segregation of the kanamycin resistance gene carried on the T-DNA. RNA blot data showed that the endogenous AtPTR2-B mRNA levels were significantly reduced in transgenic leaves and flowers, but not in transgenic roots. Consistent with this reduction in endogenous AtPTR2-B mRNA levels, all four antisense lines and one sense line exhibited significant phenotypic changes, including late flowering and arrested seed development. These phenotypic changes could be explained by a defect in nitrogen nutrition due to the reduced peptide transport activity conferred by AtPTR2-B. These results suggest that AtPTR2-B may play a general role in plant nutrition. The AtPTR2-B gene was mapped to chromosome 2, which is closely linked to the restriction fragment length polymorphism marker m246.

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

SummaryChloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F1 and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.

Triazine-resistant Nicotiana mutants from photomixotrophic cell cultures

SummaryTriazine-resistant mutants have been isolated in photomixotrophic cell cultures of Nicotiana plumbaginifolia. Triazine herbicides inhibit photosynthesis and cause extensive photodestruction of chloroplasts (bleaching) in sensitive plants. Selection was based on the greening ability of the resistant cells in the presence of 10-4 M terbutryn, under normal culture conditions, but in a medium containing a low sugar concentration. In the mutant plants, as compared to wild type, two to three orders of magnitude higher concentrations of triazines resulted in inhibition of photosynthetic electron transport and greening. The resistance was inherited maternally.

Differential mercury volatilization by tobacco organs expressing a modified bacterial merA gene.

ABSTRACTMercury pollution is a major environmental problem accompanying industrial activities. Most of the mercury released ends up and retained in the soil as complexes of the toxic ionic mercury (Hg2+), which then can be converted by microbes into the even more toxic methylmercury which tends to bioaccumulate. Mercury detoxification of the soil can also occur by microbes converting the ionic mercury into the least toxic metallic mercury (Hg0) form, which then evaporates. The remediation potential of transgenic plants carrying the MerA gene from E. coli encoding mercuric ion reductase could be evaluated. A modified version of the gene, optimized for plant codon preferences (merApe9, Rugh et al. 1996), was introduced into tobacco by Agrobacterium-mediated leaf disk transformation. Transgenic seeds were resistant to HgCl2 at 50 μM, and some of them (10-20% ) could germinate on media containing as much as 350 μM HgCl2, while the control plants were fully inhibited or died on 50 μM HgCl2. The rate of elemental mercury evolution from Hg2+ (added as HgCl2) was 5-8 times higher for transgenic plants than the control. Mercury volatilization by isolated organs standardized for fresh weight was higher (up to 5 times) in the roots than in shoots or the leaves. The data suggest that it is the root system of the transgenic plants that volatilizes most of the reduced mercury (Hg0). It also suggests that much of the mercury need not enter the vascular system to be transported to the leaves for volatilization. Transgenic plants with the merApe9 gene may be used to mercury detoxification for environmental improvement in mercury-contaminated regions more efficiently than it had been predicted based on data on volatilization of whole plants via the upper parts only (Rugh et al. 1996).Communicated by László Márton, Department of Biological Sciences, University of South Carolina, 700 Sumter Street, Columbia, SC29208, USA

Comparative biochemical characterization of mutants at the nitrate reductase/molybdenum cofactor loci cnxA, cnxB, and cnxC of Nicotiana plumbaginifolia

Abstract Mutations in any of the three gene loci cnxA, cnxB, cnxC can lead to a total loss of nitrate reductase activity in Nicotiana species. The cnx loci are involved in synthesis and processing of the molybdenum cofactor, which is an essential structural constituent of nitrate reductase. The biochemical properties of cnxA, cnxB and cnxC mutant cell lines of Nicotiana plumbaginifolia were examined further. The cnxA line (N×9) was found to possess a catalytically defective but dimerization-active and under in vivo/in vitro-conditions repairable molybdenum cofactor, thus, resembling the properties of N. tabacum cnxA lines. The cnxB (N×24) and cnxC (N×21) mutants. however, show a phenotype very different from cnxA. This new phenotype is characterised by an irreversible loss of both the catalytic function and dimerization ability of the molybdenum cofactor which makes it likely that the molybdopterin moiety of the cofactor is defective or lacking in these mutants. In this report we summarize and compare the phenotypic data presently available for the Nicotiana loci cnxA, cnxB and cnxC. Possible functions of the gene products of these loci will be discussed.