Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

Secretion of trans-zeatin by Agrobacterium tumefaciens: A function determined by the nopaline Ti plasmid

Production of the plant cytokinin, trans-zeatin, by a number of strains of Agrobacterium tumefaciens was measured by a combination of traceenrichment, HPLC and radioimmunoassay and confirmed by mass spectrometry. Secretion of trans-zeatin into a culture medium is a constitutive function of those strains which harbor a nopaline Ti plasmid. Strains cured of the nopaline Ti plasmid and those which harbor octopine or agropine plasmids are non-producers. Reacquisition of nopaline plasmids by cured strains restores production.     

Agrobacterium‐produced and exogenous cytokinin‐modulated Agrobacterium‐mediated plant transformation

Agrobacterium tumefaciens is a plant pathogenic bacterium that causes neoplastic growths, called ‘crown gall’, via the transfer and integration of transferred DNA (T‐DNA) from the bacterium into the plant genome. We characterized an acetosyringone (AS)‐induced tumour‐inducing (Ti) plasmid gene, tzs (trans‐zeatin synthesizing), that is responsible for the synthesis of the plant hormone cytokinin in nopaline‐type A. tumefaciens strains. The loss of Tzs protein expression and trans‐zeatin secretions by the tzs frameshift (tzs‐fs) mutant is associated with reduced tumorigenesis efficiency on white radish stems and reduced transformation efficiencies on Arabidopsis roots. Complementation of the tzs‐fs mutant with a wild‐type tzs gene restored wild‐type levels of trans‐zeatin secretions and transformation efficiencies. Exogenous application of cytokinin during infection increased the transient transformation efficiency of Arabidopsis roots infected by strains lacking Tzs, which suggests that the lower transformation efficiency resulted from the lack of Agrobacterium‐produced cytokinin. Interestingly, although the tzs‐fs mutant displayed reduced tumorigenesis efficiency on several tested plants, the loss of Tzs enhanced tumorigenesis efficiencies on green pepper and cowpea. These data strongly suggest that Tzs, by synthesizing trans‐zeatin at early stage(s) of the infection process, modulates plant transformation efficiency by A. tumefaciens.     

The pTiC58 tzs gene promotes high-efficiency root induction by agropine strain 1855 of Agrobacterium rhizogenes

Root induction on flax (Linum usitatissimum L.) cotyledon explants by Agrobacterium rhizogenes strain 1855 is markedly increased by co-inoculation with disarmed A. tumefaciens strain LBA 4404 containing a plasmid carrying the tzs gene of pTiC58. Most of the roots (estimated to be more than 90%) were transformed. This effect is most likely due to the secretion of trans-zeatin by A. tumefaciens stimulating the division of plant cells making them more receptive to transformation by A. rhizogenes, although other explanations are possible. This observation supports the idea that the tzs gene, although not essential for transformation, may promote transformation. An obvious application for genetic engineering experiments involving transformation by A. rhizogenes, is to include a vir-induced tzs gene in the transformation system to help maximize transformation efficiency.     

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens

Summary The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid and the location, by transposon mutagenesis, of the genes involved in the production of the agrocin. The Ti plasmid was cured by the introduction of selectable plasmids carrying the origins of replication of either the nopaline Ti plasmid, pTiC58, or the octopine Ti plasmid, pTi15955. Tn5 mutagenesis indicated that the genes responsible for agrocin production and/or export are located both on the chromosome and on a plasmid, pAgJ73, which co-migrated in agarose gels with pTiJ73. As the two plasmids were separable after transposon mutagenesis, we postulate that during or after mutagenesis of the agrocin plasmid, DNA rearrangements occurred between it and pTiJ73, resulting in an increase in size of pAgJ73. We provide evidence that the rearrangements involved the duplication of nopaline catabolism genes from pTiJ73 and their insertion into pAgJ73, which facilitated the resolution of the two plasmids. As expected pTiJ73 has homology with the nopaline Ti plasmid, pTiC58.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

A functional analysis of T-DNA gene 6b: the fine tuning of cytokinin effects on shoot development

Summary The physiological function in planta of T-DNA gene 6b was studied under various experimental conditions. For this purpose the coding region of gene 6b was cloned behind the 1-promoter of the TR-DNA to enhance expression of the gene product in transformed plant cells. Expression of the recombinant gene in leaf discs of Nicotiana tabacum altered the capacity for shoot formation of the discs, induced by exogenous (i.e. BAP in the growth medium or agrobacterial trans-zeatin produced under control of gene tzs) or endogenous cytokinins (i.e. isopentenyladenosine produced under control of T-DNA gene 4). The data obtained indicate a reduction of cytokinin activity within the plant cells by the product of T-DNA gene 6b.Abbreviations AMP adenosine-5-monophosphate - BAP 6-benzylaminopurine     

Cytokinin activity of zeatin allylic phosphate, a natural compound

Zeatin allylic phosphate (ZAP) retarded chlorophyll loss in the barleyleaf senescence assay at a concentration 20 times higher than for6-benzyladenine (BA): the effective concentrations for ZAP and BA were 10 and 0.5 , respectively. Sodium molybdate,an inhibitor of phosphatases, decreased the ZAP effective concentration to 0.5 without affecting leaf senescence andtrans-zeatin activity in the control. This demonstrates theimportance of the phosphate group for ZAP activity or its penetration into leafcells. ZAP up-regulated the protein kinase activity of the barley leaf chromatinwith concentration dependence similar to that oftrans-zeatin. Conversely, ZAP was 1000 times less activethan trans-zeatin in the competition with anti-idiotypeantibodies (raised against antibody to zeatin) for binding with atrans-zeatin-binding site oftrans-zeatin-binding protein ZBP67 isolated from barleyleaves. In contrast to trans-zeatin, ZAP did not activateRNA synthesis in the presence of ZBP in the in vitro systemcontaining chromatin and RNA polymerase I isolated from barley leaves. Insummary, data presented show that ZAP possesses cytokinin activity asdemonstrated by the retardation of barley leaf senescence, but moleculartarget(s) for ZAP in barley leaf cells differs, at least partially, from thesefor trans-zeatin. It seems possible that the cytokininactivity of ZAP results from its hydrolysis while producing zeatin.     

Nucleotide sequence of the virG locus of the Agrobacterium tumefaciens plasmid pTiC58

The nucleotide sequence of the virG locus of the nopaline type plasmid pTiC58 of Agrobacterium tumefaciens has been determined. It contains an open reading frame (ORF) of 759 nucleotides and has 77% homology to the virG sequences of octopine type plasmids. Differences between the sequences of the two types of Ti plasmids in the region of virG are located predominantly outside the ORF. The amino acid sequences inferred from the two virG genes show 80% homology to each other and each shows the same moderate homologies to amino acid sequences derived from genes in a family of two-component regulatory systems. Specific differences in nucleotide and amino acid sequences as well as a structure-function model for the gene product are discussed.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Identification of cytokinins in young wheat spikes (Triticum aestivum cv. Chinese spring)

Cytokinins in young wheat (Triticum aestivum cv. Chinese spring) spikes (2–15 mm) were isolated by immunoaffinity chromatography. High-performance liquid chromatography-radioimmunoassay and gas chromatography-mass spectrometry indicated that major cytokinins present weretrans-zeatin, ribosyl-trans-zeatin, ribosyldihydrozeatintrans-zeatin-9-glucoside, and the glucosides oftrans-zeatin, ribosyl-trans-zeatin, andtrans-zeatin-9-glucoside. Dihydrozeatin,iso-pentenyladenosine, andiso-pentenyladenine were also present but at lower concentrations.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable. R. C. Durley's present address is Biological Sciences, Monsanto Company, St. Louis, Missouri 63167.     

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR limited host range - WHR wide host range - onc oncogenicity genes - iaaH indoleacetamide hydrolase gene - iaaM tryptophan monooxygenase gene - ipt isopentenyl transferase gene - tzs transzeatin secretion gene - NAA -naphthalene acetic acid - BAP 6-benzylaminopurine - Km kanamycin - Neo neomycin - Cm chloramphenicol     

Temperature-sensitive step in Ti plasmid vir-region induction and correlation with cytokinin secretion by Agrobacteria

Summary DNA transfer from Agrobacteria to plant cells requires activation of functions which are inactive under normal growth conditions. We studied two aspects with nopaline plasmid pGV3850: (1) conditions required for induction of a representative vir-region protein (virD2); for this we prepared antiserum against the protein and used the Western blot technique, and (2) correlation between vir-region induction and secretion of plant hormones. The results show that three factors are necessary and sufficient: the previously identified acetosyringone and acidic pH and, in addition, a carbon/energy source. Induction correlates with cytokinin secretion, suggesting that release of this hormone by the bacteria may play a role in tumor induction. No pronounced correlation is observed with release of indole-3-acetic acid. VirD2 induction and cytokinin secretion are temperature-dependent with similar optima. It is proposed that the thermosensitive step discovered decades ago with tumor induction in planta is in the activation of the vir functions.Abbreviations vir virulence gene - iP N⁶-(2-isopentenyl)adenine - iPA N⁶-(2-isopentenyl)adenosine - trans-Z trans-Zeatin - trans ZR, trans-Zeatinriboside - IAA indole-3-acetic acid - IPTG isopropyl--D-thiogalactoside     

Cytokinins in immature seeds of Dolichos lablab

Five cytokinins, trans-zeatin, 9-β-d-ribofuranosyl-trans-zeatin, 9-β-d-ribofuranosyl-cis-zeatin, 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)purine and 6-(trans-4-O-β-d-glucopyranosyl-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine were identified from immature seeds of Dolichos lablab.     

Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status

The effect of nitrogen on the cytokinin relations of Urtica dioica, the stinging nettle, has been investigated. The plants were grown in quartz sand and nutrient solutions providing levels of nitrate ranging from 1 to 22 mM. Nitrogen supply did not affect biomass production within the range of 3–15 mM NO ₃ ^- . However, the shoot: root ratio of biomass was significantly higher at 15 mM (standard plants) than at 3 mM (low-nitrogen plants) nitrate supply. The cytokinin patterns of the roots, stems and adult, as well as meristematic leaves of plants grown at these two levels of nitrate supply, were determined by means of high-performance liquid chromatography (HPLC) and immunoassays. Enzyme-linked immunosorbent assays (ELISAs) for zeatin riboside, dihydrozeatin riboside, isopentenyladenosine, benzyladenosine and o-hydroxybenzyladenosine enabled the quantification of 17 cytokinins, 13 of which were found in the various tissues of Urtica. trans-Zeatin and its conjugates were the predominant cytokinins in all examined samples. While the free base trans-zeatin and its O-glucoside were the major cytokinins in adult leaves, trans-zeatin riboside was prominent in the other tissues of at least the standard plants. Glucosides of the trans-zeatin type cytokinins were present only in lower amounts. However, considerable amounts of a compound, tentatively identified as cis-zeatin riboside-O-glucoside, were found, particularly in roots and meristematic leaves. Comparatively high amounts of trans-zeatin nucleotide as well as isopentenyladenosine phosphate were also demonstrated in these tissues. Analysis of the root-pressure exudates similarly showed trans-zeatin riboside and, at a lower concentration, trans-zeatin to be the only substantial components. In the low-nitrogen plants, shortage of nitrogen was manifest only in the roots; the nitrogen contents of the shoots did not respond to the nitrogen supply. Likewise, the total content of cytokinins in the shoots of the low-nitrogen plants equaled that of the standard-plant shoots, while it was lower by about 25% in the roots of the low-nitrogen plants. In the latter, the amounts of cytokinins exuded via the root-pressure fluid were also approximately 25% lower. Since the levels of only the trans-zeatin cytokinins in the roots showed a linear correlation with the shoot-to-root ratios, these cytokinins may play an important role in biomass partitioning in Urtica dioica.Abbreviations DHZ dihydrozeatin - ELISA enzyme-linked immunosorbent assay - -G glucoside - HPLC high-performance liquid chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - -N nucleotide (ribotide) - -OG O-glucoside - -R riboside - S/R shoot-to-root (ratio) - Z zeatin This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the SFB 137. The authors wish to thank Mrs. A. Fischbach for skilful technical assistence and Dr. Paul Ziegler (Lehrstuhl für Pflanzenphysiologie, University of Bayreuth, FRG) for linguistic suggestions.     

Restriction endonuclease mapping of a Rhizobium leguminosarum Sym plasmid

A circular map of the Sym plasmid pRle1001a corresponding to a genome size of 150 × 10⁶ D was established using the restriction endonucleases HpaI, SmaI, and KpnI. The map was derived from the results obtained by hybridizing individual HpaI fragments to blotted SmaI and KpnI digests of the plasmid. A complete map of the HpaI fragments was constructed in this way. Regions of homology with the structural nif-genes D and H of Klebsiella pneumoniae, with the Sym plasmid of Rhizobium trifolii 5, and with an octopine and nopaline Ti plasmid of Agrobacterium tumefaciens were mapped. A large area of plasmid pRle1001a, which carries nif structural genes, is highly conserved in the Sym plasmid of R. trifolii 5. It is assumed that this could be an area carrying genes involved in various functions that play a role in the process of symbiotic nitrogen fixation.     

The virB operon of Agrobacterium tumefaciens pTiC58 encodes 11 open reading frames

Summary Agrobacterium tumefaciens genetically transforms plant cells by transferring a copy of its T-DNA to the plant where it is integrated and stably maintained. In the presence of wounded plant cells this process is activated and mediated by the products of the vir genes which are grouped into six distinct loci. The largest is the virB locus spanning 9.5 kb. Transposon mutagenesis studies have shown that virB gene products are required for virulence but their functions remain largely unknown. To provide information relevant to understanding the function of VirB polypeptides, the nucleotide sequence of the virB operon from a nopaline plasmid, pTiC58, is presented here. Eleven open reading frames (ORFs) are predicted from this sequence. The predicted sizes of 10 of the 11 VirB polypeptides are verified by specific expression in Escherichia coli. Only the product of the smallest ORF potentially encoding a 5.8 kDa polypeptide has not been detected. The initiation of translation of five virB ORFs occurs at codons that overlap the termination codons of the ORF immediately upstream; thus, translational coupling may be an important mechanism for efficient translation of the large virB polycistronic mRNA. Based on hydropathy plot analysis nine of the virB ORFs encode proteins that may interact with membranes; these data support the earlier hypothesis (Engstromm et al. 1987) that virB gene products may form a membrane pore or channel to mediate exit of the T-DNA copy (T-strands) from Agrobacterium into the plant cell. A comparison of the two published octopine virB sequences with the nopaline sequence presented here is made.     

Agrobacterium vitis nopaline Ti plasmid pTiAB4: relationship to other Ti plasmids and T-DNA structure

The Ti plasmid of the Agrobacterium vitis nopaline-type strain AB4 was subcloned and mapped. Several regions of the 157 kb Ti plasmid are similar or identical to parts of the A. vitis octopine/cucumopine (o/c)-type Ti plasmids, and other regions are homologous to the nopaline-type Ti plasmid pTiC58. The T-DNA of pTiAB4 is a chimaeric structure of recent origin: the left part is 99.2% homologous to the left part of the TA-DNA of the o/c-type Ti plasmids, while the right part is 97.1 % homologous to the right part of an unusual nopaline T-DNA recently identified in strain 82.139, a biotype Il strain from wild cherry. The 3 non-coding regions of the ipt genes from pTiAB4 and pTi82.139 are different from those of other ipt genes and contain a 62 by fragment derived from the coding sequence of an ipt gene of unknown origin. A comparison of different ipt gene sequences indicates that the corresponding 62 by sequence within the coding region of the AB4 ipt gene has been modified during the course of its evolution, apparently by sequence transfer from the 62 by sequence in the 3 non-coding region. In pTi82.139 the original coding region of the ipt gene has remained largely unmodified. The pTiAB4 6b gene differs from its pTi82.139 counterpart by the lack of a 12 by repeat in the 3 part of the coding sequence. This leads to the loss of four glutamic acid residues from a series of ten. In spite of these differences, the ipt and 6b genes of pTiAB4 are functional. Our results provide new insight into the evolution of Agrobacterium Ti plasmids and confirm the remarkable plasticity of these genetic elements. Possible implications for the study of bacterial phylogeny are discussed.     

Hormonal regulation of zeatin-riboside accumulation by cultured tobacco cells

Auxin (11 M -naphthaleneacetic acid) and cytokinin (1.4 M kinetin) regulate cytokinin accumulation by cytokinin-requiring (C^-) and cytokinin-autotrophic (C⁺) lines of Havana 425 tobacco (Nicotiana tabacum L.) tissues. No trans-zeatin riboside (ZR) (<0.5 pmol·g^-1 fresh weight) was detected in six C^- and nine C⁺ lines grown for 14 d on auxin + cytokinin and auxin medium, respectively. C⁺ lines, but not C^- lines accumulated ZR (1.9–5.1 pmol·g^-1 fresh weight) when incubated on hormone-free medium but both lines accumulated ZR when incubated on kinetin medium. Therefore, it appears that kinetin treatment can induce ZR accumulation and that this accumulation is blocked by auxin treatment. Similar effects were obtained with some lines of cells autotrophic for both auxin and cytokinin. Tobacco plants carrying the dominant Habituated leaf-1 allele (Hl-1) differ from wild-type plants in that leaf-derived tissues in culture exhibit a C⁺ phenotype. No differences in ZR content were found in C⁺ leaf tissues from Hl-1/Hl-1 plants and C⁺ tissues that arise epigenetically in wild-type plants. This indicates that the H-1 allele does not act to induce overproduction of ZR. The Hl-1 allele is known to have oncogenic functions similar to the isopentenyl transferase (ipt) locus of the Ti plasmid. Although Hl-1/Hl-1 cells transformed with Ti plasmids defective at the ipt locus are tumorigenic and hormone-autotrophic in culture, they contain low levels of ZR typical of non-transformed Hl-1/Hl-1 cells. Therefore, the high levels of ZR characteristics of cells transformed with wild-type Ti plasmids are not necessary for expression of the tumor phenotype.Abbreviations C^- cytokinin-requiring phenotype - C⁺ cytokinin-autotrophic phenotype - Hl-1 habituated leaf-1 locus - IPA isopentenyladenosine - ipt isopentenyltransferase gene - ZR trans-zeatin riboside