Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Plant transformation by particle bombardment of embryogenic pollen

Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 10⁴ pollen grains were GUS⁺. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS⁺ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.Abbreviations GUS ß-glucuronidase - CaMV Cauliflower Mosaic Virus - MCS multicellular structure - NPTII neomycin phosphotransferase - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl glucuronide - DAPI 4,6-diamidino-2-phenylindole - Tris Tris(hydroxymethyl)aminomethane hydrochloride - EDTA ethylenedinitrilo tetraacetic acid, disodium salt dihydrate     

Growth inhibition of suspension cultured plant cells by ribonucleases

Extracellular, stylar RNases (S-RNases) are produced by self-incompatible, solanaceous plants, such asNicotiana alata, and are thought to be involved in selfpollen rejection by acting selectively as toxins to selfpollen. In this study, the toxicity of RNases to other plant cells was tested by culturing cells ofN. alata andN. plumbaginifolia in the presence ofS-RNases fromN. alata. The growth of cultured cells ofN. plumbaginifolia was inhibited by theS-RNases, but viability was not affected. Growth of cultured cells of oneN. alata selfincompatibility genotype was inhibited by twoS-RNases, indicating that inhibition was not allele specific. Comparisons with the effects of inactivated RNase and other proteins, suggest that the inhibition of growth byS ₂-RNase was partly, but not wholly, due to RNase activity. Heat-denaturedS ₂-RNase was a very effective inhibitor of cell growth, but this inhibitory activity may be a cell surface phenomenon.     

Development of a pollen-mediated transformation method forNicotiana glutinosa

The development is described of a new procedure to genetically transform plant species using the male gametophyte as a natural transformation vector. Our system avoids the need for complicated regeneration procedures thus making it broadly applicable. Naked plasmid DNA encoding kanamycin resistance and GUS activity was introduced by particle gun bombardment into mature pollen grains ofNicotiana glutinosa. Bombarded pollen was used for pollinations and the resulting seeds were selected for kanamycin resistance. Two different kanamycin-resistant plants, designated VIP A and VIP B, were obtained in two independent experiments. In VIP A, TR2-driven GUS activity was observed in vascular bundles, trichomes and in a small number of pollen grains. DNA gel blot analysis indicated that the introduced DNA was integrated independently into the genome of VIP A and VIP B. It was shown that male and female gametophyte development and seed set were highly aberrant in both VIP A and VIP B and that the offspring of self- and cross-pollinations did not contain the transgenes. This might be caused by a recombination event during the integration of the naked DNA resulting in a deletion of part of the target chromosome. After meiosis such a deletion is lethal for the gametes. Our observation that the transgenes were detected in DNA isolated from sporophytic tissues but not in DNA from VIP A and VIP B pollen grains is in line with this explanation. Future experiments designed to increase the frequency of transformation and to transfer the transgenes to the offspring are discussed.     

Cytogenetic study of an interspecific cross of Nicotiana debneyi X N. umbratica

Summary Interspecific F₁ hybrids of Nicotiana debneyi Domin (2n=48) and N. umbratica Burbidge (2n=46), both belonging to the section Suaveolentes, showed a high degree of meiotic chromosome pairing. Two of the five F₂ plants obtained exhibited chromosome mosaicism. The first colchiploid generation (C₁) had the expected chromosome number of 2n=94 while C₂ showed 2n=88, a loss of three pairs of chromosomes. This same chromosome number continued in further colchiploid generations, followed up to C₅, except for a few plants in C₃ which showed chromosome mosaicism. The F₁ phenotype was stable through C₁ to C₅ and fertility was normal in colchiploids through all generations in spite of the loss of three pairs of chromosomes and chromosome mosaicism. This stability and fertility apparently reflect the tolerance of the genomes to the genetic adjustment of chromosome complements which is believed to be associated with the originally polyploid nature of the parental species and the chromosome doubling brought about in the amphidiploids.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

Effects of potassium levels over the range occurring in host plants on the growth of an insect herbivore (Manduca sexta)

Third instar tobacco hornworms (Manduca sexta L.: Sphingidae) on low dietary potassium had a lower relative growth rate than individuals on diets with potassium concentrations reflecting those in host-plants, due to decreased consumption rate, lower efficiencies of conversion of ingested and digested food (ECI and ECD), and a prolonged growth/feeding phase. Furthermore, these larvae, when placed on a diet with a moderate potassium concentration through the fourth stadium, ended up being smaller due to lower ECI and less biomass gained, and had a prolonged growth phase, which suggest an irreversible cost of the previous low potassium diet. Third instar hornworms on high potassium diets had lower ECI and ECD, and they had a prolonged growth phase. These individuals, when placed on a moderate potassium diet in the fourth stadium, gained less biomass, than those previously offered hostplant-like-potassium diets. Body potassium concentrations (% dw) at the end of the third stadium were similar among treatment groups. With increasing potassium concentrations in the diet, utilization efficiencies of potassium decreased and potassium concentrations in the frass increased. Correspondingly, water content (% fw) of the newly-molted fourth instar larvae declined with increasing potassium, indicating a passive loss of water during potassium excretion. Low and high dietary potassium reduced survivorship of third instar larvae; fourth instar caterpillars previously fed the low potassium diet also had poor survivorship. We conclude that, within the normal range of potassium concentrations in the hostplants, caterpillar performance is largely unaffected by potassium concentration, but that potassium-poor and potassium-rich diets, such as those hornworms may sometimes experience, can reduce growth and survivorship.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

Effect of aluminium on the growth of 34 plant species: A summary of results obtained in low ionic strength solution culture

The results from many experiments conducted over 5 years to determine the tolerance of 34 plant species (87 cultivars) to aluminium (Al) are summarised. All experiments were conducted in a temperature-controlled glasshouse using a low-ionic-strength solution culture technique. The activity of Al³⁺ (M) at which top yields were reduced by 50% (Al_RY50) was determined for each cultivar.The species Bromus wildenowii, Cynosurus cristatus, Hordeum vulgare, Triticum aestivum (cvs Warigal, Scout, Sonora-63), Avena byzantina, Arabidopsis thaliana, Lycopersicon esculentum and Nicotiana plumbaginifolia were all very sensitive to Al (Al_RY50<1). The species Poa pratense, Lolium perenne (NZ-derived cultivars), Lotus corniculatus, Avena sativa (cvs West, Carbeen, Camellia and Coolabah), Triticum aestivum (cvs Cardinal and Waalt), Allium cepa and Asparagus officinalis were sensitive to Al (Al_RY50 1–2).The pasture grass species Lolium perenne (Australian and European and derived cultivars), Lolium hybridum and Lolium multiflorum, Dactylis glomerata (Apanui and Kara), Phalaris aquatica, Festuca arundinacea and the pasture legumes species Trifolium pratense, Trifolium repens and Trifolium subterraneum were all moderately sensitive to Al (Al_RY50 2–5). Other species that were also moderately sensitive included Triticum aestivum (cvs Atlas-66, BH146, and Carazinho), Avena sativa (cvs Swan and Blackbutt), Avena Strigosa, Petunia x and Phaseolus vulgaris (cvs Red Kidney, Black Turtle and Haricot).The most tolerant species (Al_RY50>5) were (in order of increasing tolerance) Phaseolus vulgaris (cvs Tendergreen, The Prince and Yatescrop), Cucurbita maxima, Dactylis glomerata (cv Wana), Paspalum dilatatum, Lotus pedunculatus, Ehrharta calycina, Medicago sativa, Holcus lanatus, Festuca rubra, Phaseolus lunatus and Agrostis tenuis. Agrostis tenuis was at least twice as tolerant as the next most tolerant species (Al_RY50>30 compared to 15.6).     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate