The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

T-DNA of a crown gall tumor is organized in nucleosomes

Nucleosomes were isolated from chromatin of suspension cultured cells of Nicotiana tabacum var. White Burley, which were either habituated or transformed by Agrobacterium tumefaciens, strain T37. Chromatin repeat length in both types of tissue was identical and can be estimated to be 195 ± 10 bp. Using Southern transfer of nucleosomal DNA and hybridization with cloned nick-translated HindIII fragments of pTi C58 we show that the T-DNA originating from the Ti-plasmid of A. tumefaciens is organized in nucleosomes within the chromatin of crown gall tumor cells.     

Correlation between Auxin Resistance and the Lack of a Membrane-Bound Auxin Binding Protein and a Root-Specific Peroxidase in Nicotiana tabacum

The levels of a membrane-bound auxin binding protein (MABP) and a root-specific peroxidase (RSP) were studied in several tobacco (Nicotiana tabacum L.) cell lines including an auxin-resistant variant. Groups of cell lines were distinguished which behaved differentially with respect to MABP and RSP depending on the hormonal composition of the medium. In cell lines in which there existed a correlation between the presence or absence of MABP and that of RSP both phenotypes were expressed if kinetin (1-2 micromolar) was supplied. In contrast, neither MABP nor RSP could be detected under any hormonal conditions tested in the auxin-resistant variant which retains the ability to differentiate shoots but lacks the ability to differentiate roots. About an eightfold increase in the concentration of MABP and a dramatic increase in the activity of RSP occurred in a transformant by a mutant strain of Agrobacterium tumefaciens lacking an intact cytokinin gene when it was grown on medium containing 1 to 2 micromolar kinetin. A correlation between auxin resistance and the lack of MABP and RSP suggests that MABP might be involved in auxin-mediated root differentiation in tobacco.     

Transformation of Wall Deficient Cultured Tobacco Protoplasts by Agrobacterium tumefaciens

Attachment of virulent Agrobacterium tumefaciens to plant cells is required for transformation. To further study the components of the plant cell wall that may be involved in the attachment process, tobacco (Nicotiana tabacum L.) protoplasts were cultured in the presence of 2,6 dichlorobenzonitrile (DB), an inhibitor of cellulose biosynthesis, and then assayed for their ability to be transformed by Agrobacterium. The DB treated protoplasts were deficient in wall production. Nevertheless, they were transformable at high frequency by wild type Agrobacterium strains but not by mutant strains that lack the ability to bind to normal, walled cells. Small quantities of calcofluor white positive material present on DB treated cells were correlated with their competence to be transformed. Further, the plant:bacterial association that leads to transformation is shown to become stable within 5 hours after bacterial co-cultivation with either control or DB treated cells.     

In Vivo Modulation of T-DNA Encoded Amidohydrolase Activity in Transformed Tobacco Cells

Auxin autonomous growth of most crown gall tumor cells requires the expression of two auxin biosynthesizing genes (tms 1 and tms 2) from the T-DNA of Agrobacterium tumefaciens. The potential role of the tms 2 locus to affect auxin accumulation was studied by measuring the activity of its gene product, indoleacetamide hydrolase (AH), in cloned cells of tobacco (Nicotiana tabacum) transformed by the A6 strain of Agrobacterium tumefaciens. AH activity followed a consistent pattern over a 30 day culture cycle with a peak at 10 to 14 days. This same pattern was observed in a number of independently isolated clones as well as in uncioned tumor tissue, suggesting that AH activation is a regular process in wounded, transformed cells. Transfer of unwounded tissue to fresh media resulted in a similar pattern of AH activation, but with the peak activity only about 50 percent of the cut tissues. These results show that the tms 2 encoded AH activity is modulated over the culture cycle, and that the modulation is affected by wounding and supplying fresh nutrients in the medium. AH activity correlated closely with free indoleacetic acid levels which suggests that it can be an important determinant in controlling free IAA levels in transformed cells.     

N2-(1,3-dicarboxypropyl) ornithine in crown gall tumours

A novel acidic amino acid present in crown gall tumours induced on Nicotiana tabacum cv White Burley by Agrobacterium tumefaciens has been identified as N²-(1,3-dicarboxypropyl) ornithine.     

Tobacco plants transformed with the Agrobacterium T-DNA gene 1 contain high amounts of indole-3-acetamide

The T-DNA genes 1 and 2 of the Ti plasmid of Agrobacterium tumefaciens are involved in the biosynthesis of IAA in transformed plant cells. Previously, it has been shown that gene 2 codes for an amidohydrolase able to convert IAM into IAA. We have isolated Nicotiana tabacum regenerates transformed with either gene 1 or genes 6a and 6b of the T-DNA. The tobacco plants transformed with gene 1 contain 500–1000-times more IAM as compared to plants transformed with genes 6a and 6b, and as compared to untransformed control plants. No drastic differences in endogenous IAA concentrations were observed between the three plant types analyzed.     

Inhibition by Ethylene of Auxin-Promotion of Flower Bud Formation in Tobacco Explants Is Absent in Plants Transformed by Agrobacterium rhizogenes

The in vitro regeneration of flower buds was studied in pedicel explants from tobacco (Nicotiana tabacum L., cv Petit Havana) transformed with Agrobacterium rhizogenes, pRi 1855 (agropine type). At a low concentration (0.1 micromolar) of 1-naphthalene-acetic acid, pedicel strips from phenotypically aberrant plants regenerated two to three times more flower buds than explants from untransformed tobacco. Intermediate bud numbers were observed in transformants with a less extreme phenotype. The results can be explained by an increased sensitivity of the transformed explants to auxin with respect to flower bud regeneration. The effect of transformation on the auxin response is fully accounted for by the absence of a negative interaction of endogenous ethylene with 1-naphthaleneacetic acid, a phenomenon normally encountered in untransformed tissues. Three observations led to this conclusion. Application of 1 micromolar AgNO₃ to untransformed explants increased the number of flower buds to the level observed in transformed tissues but had no effect on transformed pedicel strips; exposure to 10 microliters per liter ethylene strongly reduced the response to auxin at all concentrations in untransformed explants but was almost ineffective in the transformed tissues; and endogenous ethylene synthesis occurred at the same rate in both types of explants.     

Influence of plant cultivar and plasmid-DNA on transformation rates in tobacco and moth bean

Incubation of protoplasts with polyethylene glycol (PEG) and plasmid DNA containing the coding region for aminoglycoside phosphotransferase gene (NPT II) proved to be a simple transformation method for Nicotiana tabacum and Vigna aconitifolia, a drought-tolerant grain legume. In both plant species examined, the plant cultivar was an important factor, which clearly influenced transformation rates. The use of different expression signals derived from gene VI of the cauliflower mosaic virus (plasmid pABD1) or from the nopaline synthase gene of Agrobacterium tumefaciens (plasmid pLGV neo2103) also resulted in different frequencies of stably transformed colonies. Plants could be regenerated from kanamycin-resistant line of tobacco and moth bean.     

Pathogenesis-related proteins in plants and tissues ofNicotiana tabacum transformed byAgrobacterium tumefaciens

Large amounts of pathogenesis-related (PR) proteins were found inNicotiana tabacum crown gall tissue, following transformation of normal tobacco cells withAgrobacterium tumefaciens. In contrast, PR proteins were not detected in leaves of grafted plants that had been recovered from crown gall tissue even though these plants were still transformed as shown by their inability to form roots and ability to produce octopine. No difference was observed in susceptibility to virus infection between untransformed and transformed plants grafted onto identical rootstocks. The results are discussed in relation to physiological factors controlling PR protein induction and virus resistance.     

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression

Background  

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation.     

Host and T-DNA determinants of cytokinin autonomy in tobacco cells transformed byAgrobacterium tumefaciens

The hormone autonomy of tobacco (Nicotiana tabacum L.) cells transformed byAgrobacterium tumefaciens containing mutations attmr (the “rooty” locus) of the pTiT37 plasmid has been examined. These cells require cytokinin, but not auxin for continuous growth in culture, indicating that the function of thetmr locus is to specify or induce cytokinin autonomy. Examination of tissues from plants regenerated from cells transformed by the mutant bacteria showed that the auxin independent phenotype is suppressed, but can be reinitiated in culture by exposure to an exogenous supply of auxin. In addition the developmental state of the cells from such regenerated plants can exert a profound influence on their cytokinin autonomy phenotype.     

Effects of hornonal balance during leaf-disc infection of Nicotiana plumbaginifolia with Agrobacterium tumefaciens nopaline strain C58

Abstract

The technique of leaf-disc transformation has been used in order to study the influence of different hormonal balances on the process of transformation of Nicotiana plumbaginifolia tissues with Agrobacterium tumefaciens nopaline-strain C58.

C58 Agrobacterium strain incites withish, compact undifferentiated calli on Nicotiana tabacum, whilst allows the recover of shoots in N. plumbaginifolia. Shooting can be modulated through the hormonal balance of the culture medium mostly during the tissue culture phase (phase 2) subsequent to the period of infection (phase 1). Complete absence of hormones in phase 1 and 2 does not result in any callus formation or shooting on the edges of the infected leaf-discs, thus indicating that in this case the transformed cells need hormones at least in initial phase of development. In both the phases, addition of 2,4-D, NAA and kinetin resulted necessary in the stimulation of callus and shooting, namely in phase 2, and kinetin played a determinant role. Direct shooting also leads to an higher frequency of NOS⁺ regenerated shoots.     

Expression of the acidic-subunit of amarantin, carrying the antihypertensive biopeptides VY, in cell suspension cultures of Nicotiana tabacum NT1

A modified version of amarantin, main seed storage protein of Amaranthus hypochondriacus, carrying four antihypertensive biopeptides Val-Tyr into the acidic-subunit of the protein, was expressed in cell suspension cultures of Nicotiana tabacum L. NT1. Cell growth and viability kinetics were assessed to determine optimal conditions for genetic transformation via Agrobacterium tumefaciens. Selection of putative transgenic calli was conducted using 25 μg ml^?1 hygromycin. Presence of the transgene was confirmed using histological glucuronidase assay and PCR analysis. Accumulation and expression of the recombinant protein was detected using Western blot analysis. Protein hydrolysate of transgenic calli showed high levels of inhibition of the angiotensin converting enzyme, with an IC₅₀ value of 3.5 μg ml^?1. This was 10-fold lower than that of protein extracts of wild-type cells, with an IC₅₀ of 29.0 μg ml^?1.     

Free and Conjugated Indoleacetic Acid (IAA) Contents in Transgenic Tobacco Plants Expressing the iaaM and iaaH IAA Biosynthesis Genes from Agrobacterium tumefaciens

The Agrobacterium tumefaciens T-DNA gene iaaM was introduced by leaf-disc transformation into transgenic tobacco (Nicotiana tabacum) plants expressing the iaaH gene. Regenerated calli were screened for the presence of indole-3-acetamide (IAM), by gas chromatography-multiple ion monitoring-mass spectrometry, and IAM-containing calli were further analyzed for free and conjugated indoleacetic acid (IAA). It was found that transgenic calli on average contained twice as much free IAA and three times more conjugated IAA than calli from wild-type plants. About 40% of the transformed calli could be regenerated to plants. The distribution of free and conjugated IAA was measured in transformed plants with a normal phenotype and compared with equivalent wild-type plants. The IAA content of transgenic plants was only slightly increased, whereas IAA-conjugate levels were enhanced significantly. These data suggest that conjugation of IAA may serve as a regulatory mechanism, contributing to maintenance of steady-state IAA pool sizes during tobacco growth and development.     

Transgenic Tobacco (Nicotiana tabacum SR1) Plants Expressing the Gene Coding for Serratia marcescens Nuclease

The gene coding for the secreted Serratia marcescens endonuclease was fused with the mannopine synthase promoter of Agrobacterium tumefaciens Ti plasmid and transferred to Nicotiana tabacum SR1 plants. The promoter is leaf- and root-specific. The resulting transgenic plants demonstrated elevated nuclease activity. The level of the transgene product was determined in the transgenic lines.     

Tropane alkaloids production in transgenic Hyoscyamus niger hairy root cultures over-expressing Putrescine N-methyltransferase is methyl jasmonate-dependent

The cDNA from Nicotiana tabacum encoding Putrescine N-methyltransferase (PMT), which catalyzes the first committed step in the biosynthesis of tropane alkaloids, has been introduced into the genome of a scopolamine-producing Hyoscyamus niger mediated by the disarmed Agrobacterium tumefaciens strain C58C1, which also carries Agrobacterium rhizogenes Ri plasmid pRiA4, and expressed under the control of the CaMV 35S promoter. Hairy root lines transformed with pmt presented fivefold higher PMT activity than the control, and the methylputrescine (MPUT) levels of the resulting engineered hairy roots increased four to fivefold compared to the control and wild-type roots, but there was no significant increase in tropane alkaloids. However, after methyl jasmonate (MeJA) treatment, a considerable increase of PMTase and endogenous H6Hase as well as an increase in scopolamine content was found either in the transgenic hairy roots or the control. The results indicate that hairy root lines over-expressing pmt have a high capacity to synthesize MPUT, whereas their ability to convert hyoscyamine into scopolamine is very limited. Exposure to MeJA strongly stimulated both polyamine and tropane biosynthesis pathways and elicitation led to more or less enhanced production simultaneously.