Ribosylzeatin and zeatin in tobacco crown gall tissue

Cytokinins were extracted from two cultures of tobacco crown gall tumor tissue: an unorganized tissue and a teratoma which produced leafy shoots. On Sephadex LH-20 column chromatography, extracts of both types of tissue yielded two peaks of cytokinin activity with elution volumes similar to ribosylzeatin and zeatin. Ribosylzeatin and zeatin were detected and quantified by coupled gas chromatography — mass spectrometry selected ion monitoring (GC/MS SIM), comparable quantities being found in the two extracts. Full mass spectral evidence for the presence of ribosylzeatin in both tissues was obtained. No evidence was found for the presence of N⁶-(²-isopentenyl)adenosine (i⁶Ade) or N⁶-(²-isopentenyl)adenine (i⁶Ade) although these compounds have been reported to occur in cytokinin-habituated tobacco callus tissues.Abbreviations BAP 6-benzylaminopurine - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - i⁶ Ade N⁶-(²-isopentenyl)adenine - i⁶ Ado N⁶-(²-isopentenyl)adenosine - RFE rotary film evaporation - SIM selected ion monitoring - TLC thin-layer chromatography - TMS trimethylsilyl     

Differential expression of the potato proteinase inhibitor II promoter in transgenic tobacco

We studied temporal and spatial expression patterns of the potato proteinase inhibitor II (PI-II) promoter, using transgenic tobacco (Nkotiana tabacum L cv. Xanthi) plants that carried a fusion between the PI-II promoter and the chloramphenicol acetyltransferase (cat) gene. Pl-ll promoter activity was low when plants were young, but increased as plants grew. In 8-week-old plants, old leaves showed higher activity than young leaves. At flowering stage (ca. 15 weeks), the overall promoter activity was reduced to a lower level except in the petals. Compared with stems or petioles at the flowering stage, the roots and floral organs showed minimal activity for the Pl-ll promoter. We used several environmental stimuli to examine the induction of the Pl-ll promoter in different organs. Promoter induction was effected by wounding or methyl jasmonate in stems, petioles, sepals, and leaves. The induction was highest in leaves, as was sucrose-enhanced wound induction. These results suggest that the Pl-ll gene is temporally and spatially regulated. We also established a transient assay system in tobacco BY2 suspension cells to elucidate the upstream regulatory region of the Pl-ll promoter. A field strength of 0.75 kV/cm and 400 μF capacitance were optimal electroporation conditions for our transient assay.     

Cytokinins in tobacco crown gall tumors

Bacteria-free tobacco ($\text{Nicotiana tabacum}$ cv. Wisconsin #38) crown gall strains incited by $\text{Agrobacterium tumefaciens}$ C58, 27, B6, CGIC, and AT4 have been analyzed for cytokinin content with the tobacco callus bioassay. All tumor strains contained high total levels of cytokinins ranging from 4–810 kinetin equivalents per kg fresh weight compared to 0.5 kinetin equivalents per kg for normal callus growing on medium with 0.1 μM N6-benzyladenine. Fractionation on a column of Sephadex LH-20 separated cytokinin activity from B6 tumors into a number of components among which ribosyl-$\text{trans}$-zeatin has been purified and characterized based on uv spectrum, biological activity and mass spectrum.     

Differential expression of tomato proteinase inhibitor I and II genes during bacterial pathogen invasion and wounding

Expression of proteinase inhibitor I and II genes was investigated during infection by Pseudomonas syringae pv. tomato, the causal agent of bacterial speck disease in tomato. Inoculation of leaves with P. s. pv. tomato of two inbred tomato lines that are resistant and susceptible to the pathogen resulted in the accumulation of proteinase inhibitor I and II mRNAs in this organ. Our data showed that in the lines used in this study, proteinase inhibitor II mRNAs accumulated in leaves to higher levels than proteinase inhibitor I mRNA in response to P. s. pv. tomato infection and wounding. Proteinase inhibitor II mRNAs accumulated more rapidly in disease-resistant than in disease-susceptible plants. Proteinase inhibitor I mRNAs were first detected in the disease-susceptible line during infection and wounding. In contrast to wounding, the systemic induction of these genes during pathogen ingression was limited. These data show that the plant proteinase inhibitors constitute one of the components of the plant defense system that are induced in response to bacterial pathogen invasion.     

Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna

Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - LpDH lysopine dehydrogenase     

Isolation and characterization of proteinase inhibitor I from etiolated tobacco leaves

Proteinase Inhibitor I was induced to accumulate in tobacco (Nicotiana tabaccum) leaves by placing plants in darkness for 10 days at 27 degrees C. The inhibitor was isolated using ammonium sulfate precipitation, Sephadex G-75 chromatography, heating, and affinity chromatography with a chymotrypsin-Sepharose column. Inhibitor I was purified 232-fold with a yield of 34 mg from 2.5 kg of leaves. Affinity-purified tobacco Inhibitor I was shown to be homogeneous by gel electrophoresis in both nondissociating and dissociating buffers. The inhibitor has a molecular weight of 39,000 +/- 1000 determined by gel filtration and, like its potato and tomato counterparts, is composed of five subunits of molecular weight 8100. The tobacco Inhibitor I strongly inhibits chymotrypsin and weakly inhibits trypsin. The chemical, physical, and immunological properties of tobacco Inhibitor I indicate that it is structurally very similar to potato tuber Inhibitor I and tomato leaf Inhibitor I, although the synthesis and accumulation of the three inhibitors in their respective tissues are all under different developmental or environmental regulation.     

Regeneration of tobacco plants from crown gall tumors

Summary Tissue culture methods have been developed for regeneration of normal appearing tobacco plants from bacteria-free crown gall strains incited byAgrobacterium tumefaciens C58, IIBV7, B6, CGIC, A6NC, 27, and AT4. Regenerants fall into two categories depending on the properties of tissues from these plants. The first type of regenerant was obtained from tumors incited byA. tumefaciens C58 and it retained the potential for expression of tumor characteristics such as a nonrequirement for phytohormones (auxin and cytokinin) by explants in vitro and the presence of detectable concentrations of nopaline. Normal appearing plants obtained from C58 tumors had much lower concentrations of nopaline than the corresponding tumor tissue (130 versus 1700 μg per g dry wt) indicating a parallel repression of abnormal growth and nopaline concentrations in regenerants. The second type of regenerant was obtained from tumors incited by the otherA. tumefaciens strains and was characterized by requirements for phytohormones by explants in vitro and the apparent lack of octopine or nopaline in regenerant tissues.     

The role of pH and ion transport in oligosaccharide-induced proteinase inhibitor accumulation in tomato plants

Abstract. The induction of proteinase inhibitor (PrI) activity in young tomato plants by wounding and oligosaccharides has been shown to be prevented by pretreatment of the plants with phenolic acids such as aspirin. Aspirin applied over a wide range of concentrations is able to inhibit PrI induction by pectic fragments. The possibility that other weak acids may also inhibit PrI induction was investigated. Isobutyric acid and trimethylacetic acid were shown to be less effective as inhibitors than aspirin, and weak bases were without effect. However, it was demonstrated that various agents known to influence ATPase activity and intracellular pH were able to inhibit PrI induction, and in particular the striking effect of low concentrations of fusicoccin on PrI induction was noted.     

Hormonal control of tobacco crown gall tumor morphology

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.     

Identification of cytokinins in primary crown gall tumours of tomato

Abstract. Identification of the cytokinin complex of primary crown gall tumours of tomato ( Lycopersicon esculentum L.) by combined gas chromatography-mass spectrometry has been described. Several cytokinins have been identified which included zeatin, dihydrozeatin, isopentenyladenine, and their respective riboside and nucleotide derivatives. In addition, 6-benzylaminopurine, its riboside and the corresponding nucleotide have also been identified as major endogenous compounds in this tissue. This would appear to be the first report on the identification of cytokinins from a primary crown gall tumour tissue using unequivocal methods.     

Posttranslational modification of an isoinhibitor from the potato proteinase inhibitor II gene family in transgenic tobacco yields a peptide with homology to potato chymotrypsin inhibitor I.

A member of the potato proteinase inhibitor II (PPI-II) gene family under the control of the cauliflower mosaic virus 35S promoter has been introduced into tobacco (Nicotiana tabacum). Purification of the PPI-II protein that accumulates in transgenic tobacco has confirmed that the N-terminal signal sequence is removed and that the inhibitor accumulates as a protein of the expected size (21 kD). However, a smaller peptide of approximately 5.4 kD has also been identified as a foreign gene product in transgenic tobacco plants. This peptide is recognized by an anti-PPI-II antibody, inhibits the serine proteinase chymotrypsin, and is not observed in nontransgenic tobacco. Furthermore, amino acid sequencing demonstrates that the peptide is identical to a lower molecular weight chymotrypsin inhibitor found in potato tubers and designated as potato chymotrypsin inhibitor I (PCI-I). Together, these data confirm that, as postulated to occur in potato, PCI-I does arise from the full-length PPI-II protein by posttranslational processing. The use of transgenic tobacco represents an ideal system with which to determine the precise mechanism by which this protein modification occurs.     

Auxin and cytokinin levels in selected and temperature-induced morphologically distinct tissue lines of tobacco crown gall tumors

Two morphologically distinct tissue culture lines were selected from a tobacco (Nicotiana tabacum L. ‘Wisconsin 38’) crown gall tumor: (1) a green teratomous line producing numerous abnormal shoots, designated leafy; and (2) a white, hard, spherical tissue, designated white. These were originally isolated at 27°C, but temperature changes were also found to control these morphologies, with 33°C favoring the development of the leafy form and 21°C favoring the white form. The higher temperature was also correlated with a decrease in the endogenous levels of both auxin and cytokinin, whereas the lower temperature was correlated with an increase in the levels of these growth regulators. There was no significant difference, however, in the auxin and cytokinin levels in the two forms maintained at 27°C, suggesting that the basis for morphological changes at this temperature resides either in very subtle differences in auxin and cytokinin or in factors other than these two growth regulators.     

Cytokinin metabolism in autonomous and crown gall tissue cultures

When tissues ofCatharanthus roseus A6 crown gall were incubated on medium supplemented with 50 (μM N6-isopentenyladenine (i⁶Ade), endogenous i⁶Ade, N6-isopentenyladenosine (i⁶A) and i⁶A nucleotide (i⁶AXP) increased to. 6, 5 and 12 nmol g^-1, respectively, during 100 h. Whereas i⁶Ade and i⁶AXP increased rapidly during the initial 4 h and then remained relatively constant, the level of i⁶A continued to increase to 25 nmol g^-1 by 16 h and then decreased; Ribosylzeatin (io⁶A) and its nucleotide (io⁶AXP) remained constant at 1.5 and 1.7 nmol g^-1, respectively. Upon transfer to cytokininless medium, i⁶Ade and i⁶AXP declined rapidly but i⁶A increased to 10 nmol g^-1 after 4 h and then declined. Again, io⁶A and io⁶AXP were unchanged. Prolonged incubation of crown gall tissue on i⁶Ade completely inhibited growth. By contrast, nonrtransformed, autonomous tissue lines fromCalycanthus fertilis andActinidia chinensis Xarguta continued to proliferate on this medium. TheActinidia Une was shown to metabolize i⁶Ade to zeatin and to accumulate this cytokinin to levels in excess of 70 nmol g^-1.     

Sugar response element enhances wound response of potato proteinase inhibitor II promoter in transgenic tobacco

The promoter region of the potato proteinase inhibitor II (PI-II) gene was studied to identifycis-acting regulatory sequences involved in sugar response using transgenic tobacco plants. The 5 control region covering an 892 nucleotide sequence upstream from the cap site and a 32 nucleotide untranslated region of the PI-II promoter was able to activate a reporter chloramphenicol acetyltransferase (cat) gene by wounding or by incubating in a sugar-free medium. This wound response was further enhanced by sugar. Hexoses, disaccharides, and some trisaccharides were strong inducers whereas pentoses, deoxy sugars, sugar acids, TCA cycle intermediates, amino acids, and other carbohydrates had little effect on the promoter activity. Deletion of the sequence between-892 and-573 abolished the wound response but not the sugar response. An additional 5 deletion to-453 removed the sugar inducibility. Locations of thecis-acting regulatory elements were further elucidated by 3 deletion analysis. Deletion of the downstream region from-520 did not affect the wound of sugar response of the promoter. However, 3 deletion mutant-574 was unable to respond to sugar but did respond weakly to wounding. Further deletion to-624 abolished both responses. Therefore, it can be concluded that a wound response element is located in between-624 and-574 and that the response is further enhanced by a sugar response element located in the sequence between-573 and-520.