Transgenic tobacco (Nirotiana tabacum 1. cv Wisconsin 38) plants expressing the Agrobacferium rhizogenes r o K gene under the control of the cauliflower ...
Plant Physiol. (1993) 102: 363-371
Hormonal Characterization of Transgenic Tobacco Plants Expressing the rolC Gene of Agrobacterium rbizogenes TL-DNA’ Ove Nilsson, Thomas Moritz, Nadine Imbault’, Goran Sandberg, and Olof Olsson* Department of Plant Physiology, University of Umei, S-90187 UmeH, Sweden (O.N.); Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-90183 Ume:, Sweden (O.N., T.M., N.I., C.S., 0.0.) Subsequently, such activities have to be correlated to alterations in the hormone pools of transgenic plants expressing these activities. Thus, in iaaH/iaaM-expressing plants, which are stunted dwarfs with a strong apical dominance and excessive adventitious root formation, a correlation to increased pools of free IAA and IAA conjugates was demonstrated (Klee et al., 1987; Sitbon et al., 1992a, 1992b). Plants expressing iaaL showed, as a result of increasing the cytokinin-to-IAA ratio, cytokinin-like effects, with reduced apical dominance and reduced root growth, which were correlated to reduced levels of free IAA and increased levels of IAA conjugates (Romano et al., 1991; Spena et al., 1991). Plants expressing the ipt gene failed to develop any further than small shoots when expressing a 35s-ipt construct (Smigocki et al., 1988, 1989), but when the ipt gene was fused to a heat-shock promoter, plants expressing the gene displayed cytokinin effects in that they were dark green with reduced apical dominance and poor root formation. Accordingly, increased levels of a number of cytokinins could be detected in these plants (Medford et al., 1989; Smart et al., 1991; Smigocki, 1991). Normally, plants can actively regulate their endogenous hormone pools. Therefore, the phenotypical alterations discussed above for the transgenic plants were the results of an abnormal change in the pool size of a particular hormone. However, these changes in phenotypes and hormone pools agreed with the predicted enzymic activities of the corresponding trans-gene products when tested in vitro. Consequently, to postulate that a particular gene product affects the metabolism of one or several plant hormones in vivo, a detailed picture of the major plant hormones, including measurements of free and conjugate forms, in transgenic plants expressing these genes must be presented. When overproduced in transgenic plants, the RolC protein of Agrobacterium rhizogenes is able to cause extensive phen-
Transgenic tobacco (Nirotiana tabacum 1. cv Wisconsin 38) plants expressing the Agrobacferium rhizogenes r o K gene under the control of the cauliflower mosaic virus 35s RNA promoter were constructed. These plants displayed several morphological alterations reminiscent of changes in indole-3-acetic acid (IAA), cytokinin, and gibberellin (CA) content. However, investigationsshowed that neither the IAA pool size nor its rate of turnover were altered significantly in the rolC plants. The biggest difference between rolC and wild-type plants was in the concentrations of the cytokinin, isopentenyladenosine (iPA) and the gibberellin CA19. Radioimmunoassay and liquid chromatography-mass spectrometry measurements revealed a drastic reduction in rolC plants of iPA as well as in several other cytokinins tested, suggesting a possible reduction in the synthesis rate of cytokinins. Furthermore, gas chromatography-mass spectrometry quantifications of CAls showed a 5- to 6-fold increase in rolC plants compared with wildtype plants, indicating a reduced activity of the CA19oxidase, a proposed regulatory step in the gibberellin biosynthesis. Thus, we conclude that RolC activity in transgenic plants leads to major alterations in the metabolism of cytokinins and gibberellins. ~~
The majority of known genes encoding enzymes involved in plant hormone regulation are of bacterial origin. The iaaH and iaaM genes from Agrobacterium tumefaciens encode enzymes involved in the biosynthesis of IAA from tryptophan (Inzé et al., 1984; Schroder et al., 1984; Tomashow et al., 1984). The ipt gene product, also originating from A. tumefaciens, mediates the synthesizes of iPA-P from isopentenyl pyrophosphate and AMP (Akiyoshi et al., 1984; Bany et al., 1984), and the iaaL gene product from Pseudomonas savastanoi conjugates IAA to Lys (Glass et al., 1986; Hutzinger et al., 1986; Roberto et al., 1990). Originally, a11 of these enzymic activities were identified in cell-free systems or by expression in Escherichia coli. However, one cannot directly conclude that enzymic activities demonstrated in vitro or in E. coli cells are the same, or the most important, activities in the plant cell in vivo.
Abbreviations: BAP, 6-benzylaminopurine; FAB, fast atom bombardment; HOAc, acetic acid; iP, isopentenyladenine; d-iPA, isopentenyladenosine alcohol; iPA, isopentenyladenosine; iPA-I‘, isopentenyladenosine 5’-monophosphate; LC, liquid chromatography; MeOH, methanol; NAA, naphthaleneacetic acid; PVPP, poly-Nvinylpolypyrrolidone; RIA, radioimmunoassay; SIM, selected ion monitonng; 35s promoter, cauliflower mosaic virus 35s RNA promoter; Z, zeatin; Z7G, zeatin 7-glucoside; ZR, zeatin 9-riboside.
’
This work was supported by a grant from the Swedish council for Forestry and Agricultura1 Research. * Present address: Station d’Amélioration des Arbres Forestiers, Institut National de la Recherche Agronomique, Ardon, F-45160 Olivet, France. * Corresponding author; fax 46-90-165901.
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otypical alterations reminiscent of hormone effects (Oono et al., 1987; Schmülling et al. 1988; Fladung 1990), and it has been suggested that this may be caused by a decrease in auxin biological activity or an increase in cytokinin activity (Schmiilling et al., 1988; Spena et al. 1989). Recently, it was proposed that RolC exerts its action by hydrolyzing biologically inactive cytokinin N-glucosides, thereby raising the pool of free active cytokinins (Estruch et al., 1991a). To test this hypothesis, we have investigated whether the overproduction of the RolC protein in transgenic tobacco (Nicotiana tabacum L. cv Wisconsin 38) leads to any major alterations, not only in cytokinin-to-auxin ratios, but also in ABA and GA content. In this study GAs, ABA, and free and conjugated IAA were quantified by GC-MS and cytokinins by immunoassay and LC-MS. In addition, we have established the metabolic profile and tumover rate of IAA in roZC plants. MATERIALS A N D METHODS Bacterial Strains and Plasmids
Escherichia coli DH5a was used as the recipient for plasmid amplification. Agrobacterium tumefaciens strain GV3101 (pMP90RK)(Koncz and Schell, 1986)was used as the receptor for the binary vectors and as the T-DNA donor. Agrobacterium strains were grown at 28OC in YEB medium (0.1% yeast extract, 0.5% beef extract, 0.1% peptone, 0.5% SUC,and 2 mM MgSO,). Vector Construction
Standard techniques were used for the construction of recombinant DNA plasmids (Sambrook et al., 1989). A 986bp EcoRI-HpaI fragment containing the coding region of the rolC gene was isolated from the plasmid pPCV002-ABC (Spena et al., 1987). An 8-mer BglII linker, 5'-CAGATCTG3', was blunt-end ligated to the HpaI side of the fragment, which was then recleaved with BglII. Due to the presence of an interna1 BglII site, this resulted in a 646-bp BglII fragment that was inserted into the BglII site of the vector pIC20H (Marsh et al., 1984), creating plasmid pIC20H-roK. The rolC BgZII fragment was then isolated from this plasmid and inserted into the BamHI site of the binary expression vector pPCV702 (Walden et al., 1990), resulting in a fusion of the 35s promoter to the 5' side and the nopaline synthase polyadenylation signal to the 3' side of the roIC coding region. This vector was denoted pPCV702-rolC. Plant Cultivation and Transformation
Tobacco plants (Nicotiana tabacum L. cv Wisconsin 38) were grown on fertilized peat in a greenhouse with a day temperature of about 22OC and a night temperature of 17OC. The photoperiod was 18 h, consisting of natural daylight supplemented with metal halogen lamps (Osram HQI-TS 400 W), giving a minimum quantum flux density of 150 PE m-' s-', Tobacco was transformed with A. tumefaciens GV3101 (pMP90RK) carrying the vectors pPCV702-rolC and pPVC702 (control) (Walden et al., 1990) using the leaf-disc method (Horsch et al., 1985). Transformants were selected on solid K 3 medium (Kao et al., 1974) containing 0.5 Ng/mL
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of BAP (Sigma 8-9395), 0.1 Pg/mL of NAA (Sigma N-0640), 500 pg/mL of cefotaxime (Serva 16382), and 100 pg/mL of kanamycin sulfate (Serva 26898). The kanamycin-resistant shoots were induced to root on half-strength Murashige and Skoog medum (Murashige and Skoog, 1962) and then transferred to the greenhouse. The transformants displaying the strongest "RolC phenotype" were pollinated with wild-type pollen. The kanamycin-resistant offspring from these crosses a11 displayed the same phenotype as the mother plants, showing that the altered phenotype was stably inherited. Plants were harvested when the first flower bud had formed; for control plants this was after forming 27 leaves >20 mm and when reaching about 110 cm in height, and for rolC-expressing plants it was after forming 35 leaves >20 mm and reaching 70 cm in height. The plants were fractionated as specified below with leaf number 1 being the first leaf >20 mm numbered from the top of the plant, and with intemode 1 positioned immediately above it. Samples were frozen in liquid nitrogen and stored at -8OOC. Hormone measurements were in most cases done on primary rolC transformants, and on pPCV702 transformants as controls. GA measurements, LC-MS quantification of cytokinins, and studies of IAA tumover and metabolic profile were carried out using the kanamycin-resistant offspring from wild-type crosses. In these cases, wild-type Wisconsin 38 plants served as controls. Northern Blot and Hybridization
Leaves and internodes were sampled from the same plants used for hormone measurements as specified in Figure 1, and total RNA was isolated according to Logemann et al. (1987). Total RNA (25 pg) was denatured and separated on a formaldehyde-agarose gel according to Sambrook et al. (1989), and the RNA was transferred to a nylon membrane according to the manufacturer's instructions (Nytran-N, Schleicher & Schuell, Dassel, Germany). Northem hybridization was performed under stringent conditions according to Sambrook et al. (1989), using as a probe the 646-bp BglII fragment containing the entire rolC coding sequence subcloned from the plasmid pIC20H-rolC. The probe was labeled with [a-32P]dCTPby random-primed synthesis to a specific activity of 109 cpm/wg of DNA. Quantification of IAA and ABA
Tobacco plants were harvested and samples were taken from the apex, six different intemodes, and five different leaf positions as detailed in the legend to Figure 2 . Quantitative analysis of ABA, IAA, and IAA conjugates (liberated after hydrolysis for 1 h at 100°C in 7 M NaOH) was performed by GC-SIM-MS using [l3C6]IAAand ['H,]ABA as intemal standards. The method for purification and quantitative analysis by GC-MS has been described by Sundberg (1990) and Sitbon et al. (1992a). Metabolic Profile of IAA
[2'-'4C]IAA (200,000dpm) was injected in the apex of 35sroZC and wild-type tobacco. After 24 h, the apical region and the first internode of two wild-type and two rolC plants were
Hormonal Characterization of ro/C-Transgenic Tobacco
harvested and extracted individually in ice-cold methanol containing 5 mM sodium diethyldithiocarbamate. Aliquots of the methanolic extracts were dried and redissolved in 1 mL of 10% methanol in 1.0% acetic acid. Particulate matter was removed by passing the samples through a 0.22-pm filter prior to HPLC analysis. The liquid chromatograph (Waters Associates) consisted of a M680 gradient controller and two M510 pumps. Solvents were delivered at a flow rate of 1 mL/min and samples were introduced off-column via a Rheodyne model 7125 injection valve with a 200-pL loop (Rheodyne, Cotati, CA). Ionsuppressioa reversed-phase HPLC utilized a 250 X 4.6 mm i.d. 5-pm ODS Hypersil column (Capital HPLC Specialists, Bathgate Lothian, UK) that was eluted with a 25 min, 10 to 60% gradient of methanol in 0.5% aqueous acetic acid. Column eluate was mixed with liquid scintillant (10 g/L of PPO dissolved in Triton-X100:xylene:methanol[1:2:5, v/ VI), pumped at a flow rate of 3 mL/min by a Reeve Analytical (Glasgow, UK) model 9702 precision mixer, and directed to a Reeve Analytical model 9701 radioactivity monitor fitted with a 500-pL spiral glass flow cell (Reeve and Crozier, 1977).
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phase consisting of a gradient starting from 92% 30 mM acetic acid, pH 3.35 (adjusted with triethylamine), and 8% acetonitrile and ending at 100% acetonitrile (10 min at 8%; 8-10% in 2 min; 10-12% in 10 min; 12-22% in 2 min; 22-25% in 21 min; 25-100% in 2 min; 8 min at 100%). The mobile phase was delivered at a flow rate of 1 mL/min by Waters model 501 pumps. One-milliliter fractions were collected, dried in vacuo, and subjected to RIA as described by Imbault et al. (1988). The radioactivity in the fractions co-chromatographing with [3H]d-iPAwas determined to estimate the final recovery (55% on average). After every second HPLC run, a cytokinin standard mixture was analyzed. The mixture contained the following cytokinins, with respective retention times: zeatin 9 -glucoside, 9 min; Z7G, 11 min; Z, 15 min; dihydrozeatin, 18 min; ZR, 22 min; dihydrozeatin 9-riboside, 24 min; isopentenyladenine 9-glucoside, 3 1 min; isopentenyladenosine, 37 min; isopentenyladenine, 42 min. Antibodies used for the RIA were polyclonals against iPA (76% cross-reactivity with ir),and ZR (52% cross-reactivity with Z) (Imbault 1989). High Resolution LC-MS-SIM of Cytokinins
Analysis of the Biological Half-life of ['3C6]IAA in the Apical Region
Tumover of [13C6]IAAin the apex and first internode of 35s-rolC and wild-type tobacco was analyzed by injection of 250 ng of [I3C6]IAAinto the apical region of the shoot of 15 wild-type and 15 RolC-expressing shoots. Three wild-type and three rolC plants were harvested individually 1, 3, 6, 9, and 24 h after application of the label, and the amount of [13C6]IAAremaining in the apex and first internode was determined by GC-SIM using 50 ng of ['H5]IAA as an intemal standard. Quantification of Cytokinins with RIA
Tobacco plants were harvested and samples were taken from upper leaves as indicated in the legend to Figure 4. The samples (500-900 mg) were lyophilized, pulverized, and extracted in 20 mL of 80% methanol/20 mM sodium phosphate buffer (pH 7.2), containing 13 mM 2,6-di-tert-butyl-4methylphenol and 36 mM /3-mercaptoethanolas antioxidants. To estimate the recovery of cytokinins, [3H]d-iPAsynthesized as described by MacDonald et al. (1981) was added as an internal standard. The extract was centrifuged and the supematant was concentrated in vacuo. The concentrated sample was diluted with 5 mL of 20 mM sodium phosphate buffer (pH 7.2) and phosphatase treated by the addition of 0.04 units/mL of acid phosphatase (Sigma P-3627). Initial purification was camed out on a 2.8-mL Varian Bond Elut quaternary amine (SAX) column (500 mg), linked to a second Sep-Pak octadecyl silica (C18)cartridge (Waters Associates AB, Partille, Sweden). The cytokinins were eluted from the C18 cartridge with 5 mL of HPLC-grade methanol. Further purification and fractionation of the individual cytokinins were performed on a reversed-phase HPLC system. The sample was introduced by a Waters 712 WISP onto a 10 cm X 8 mm i.d., 4-pm Nova-Pak CI8 cartridge (Waters Associates AB, Partille, Sweden) and eluted with a mobile
Upper leaves of 10 wild-type and 10 rolC tobacco plants were harvested and cytokinins were quantified by frit-FAB LC-MS-SIM. The samples (approximately 100 g) were lyophilized, pulverized, and extracted in 80% methanol/20 mM sodium phosphate buffer (pH 7.2) containing 13 mM 2,6-ditert-butyl-4-methylphenoland 36 mM P-mercaptoethanol as antioxidants. Prior to extraction ['H5]Z, ['H5]ZR, ['H5]Z7G, ['H6]iP, and ['H6]iPA (Apex Organics Ltd., Honiton, UK) were added as internal standards. After extraction, evaporation, and phosphatase treatment as described above, the samples were applied to a DE52 anion exchanger coupled with a CI8 cartridge. After elution of the cytokinins as described above, the samples were dissolved in 1 mL of 10 mM ammonium acetate buffer (pH 3.0) and applied to a preequilibrated 500mg SCX cartridge (Analytichem. Intemational). The cartridge was washed with buffer before eluting the cytokinins with 10 mL of 10% 2 M NH3 in MeOH. The semi-purified extract was then subjected to reversed-phase HPLC as described above, and thereafter analyzed by LC-MS-SIM as described below. The capillary frit-FAB LC-MS system (JEOL, Tokyo, Japan) has been described by Imbault et al. (1992). The mobile phase for the quantification of iPA and iP was 60% MeOH with 1% HOAc, 1%glycerol, and 0.02% PEG 300; for 2 and ZR, 40% MeOH with 1%HOAc, 1% glycerol, and 0.02% PEG 300; and for Z7G, 35% MeOH with 1%HOAc, 1%glycerol, and 0.02% PEG 300. The mass spectrometer was operated in the accelerating voltage SIM mode, recording two characteristic ions and the deuterated analogs for each cytokinin. The dwell time was 100 ms, and the resolution was 5000 with PEG 300 as reference substance. Quantification
of GAs
Ten wild-type and 10 rolC tobacco plants were divided and pooled into upper leaves, lower leaves, upper internodes, and lower internodes (approximately 150 g). The method for
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purification was essentially the same as described by Croker et al. (1990), except for the PVPP column chromatography, which was as described by Moritz et al. (1989). The following deuterated GAs were added as internal standards: 17,17[2H2]GA,, 17,17-[2H2]GA19, 17,17-[2H2]GA20, and 17,17[2H2]GA53 (Lewis Mander, Australian National University Canberra, Australia). The sample was further purified by reversed-phase HPLC as described below for the quantification of cytokinins. For purification of free GAs, the mobile phase was a 40-min linear gradient (10-100% methanol in 1% aqueous HOAc) delivered at a flow rate of 1 mL/min. The fractions corresponding to the elution volumes of the GAs were collected and analyzed by GC-SIM-MS. After methylation and silylation (Moritz et al., 1989), the GAs were injected splitless into a Hewlett-Packard 5890 gas chromatograph equipped with a fused silica capillary column with SE-30 chemical bonded phase, 0.25 nm, 25-m long, 0.25 mm i.d. (Quadrex, New Haven, CT). The injector temperature was 250°C and the column temperature was 60°C for 2 min. The temperature was thereafter increased by 20°C/min to 200°C and by 4°C/ min to 260°C. The column effluent was led to the ion source of a double focusing JEOL JMS-SX102 mass spectrometer (JEOL, Tokyo, Japan). The interface temperature was 250°C, the electron energy was 70 eV at an ionization current of 300 l*A.. The acceleration voltage was 10 kV. The mass spectrometer was operated in the acceleration voltage SIM mode, and data were processed by JEOL MS-MP7000D data system. For each GA, two characteristic ions and the deuterated analogs were recorded. The dwell time was 50 ms, and the resolution was 5000 with perfluorokerosene as reference substance. RESULTS
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the plants used for hormone analysis. Leaves and internodes were sampled as detailed in the figure. The plants used were wild-type (WT) and ro/C-transformed (C4, C7, C9, C10) plants. Total RNA (25 Mg) was loaded in each lane. The rolC transcript was compared with a DMA standard run in parallel and was estimated to be about 600 bases.
different plant tissues. As seen in Figure 1, all plant parts showed high expression of the rolC gene with only small differences between organs and between plants. This general expression pattern fits well with what has been described earlier for various 35S promoter-reporter gene fusions in a number of different plants (Jefferson et al., 1987; An et al., 1988; Williamson et al. 1989; Nilsson et al., 1992). Thus, no selection for plants expressing the RolC protein to a lesser degree or in a different organ-specific manner has taken place during the regeneration procedure. Importantly, all plants strongly expressed the rolC gene in those parts of the plant used for hormone measurements.
Generation of Tobacco Plants Expressing the rolC Gene
IAA Quantification
Regeneration of ro/C-transformed plants was normal. There was no reduction in the production of shoots from the leaf discs transformed with the vector pPCV702-ro/C, nor in subsequent rooting of these shoots compared with the control plants transformed with the vector pPCV702 alone. Forty independently regenerated kanamycin-resistant plants, transformed with the vector pPCV702-ro/C, were transferred to the greenhouse. These plants displayed to a varying degree the RolC phenotype, characterized by shortened internodes, light green lanceolate leaves, an increased number of internodes formed before flowering, and small male-sterile flowers. A reduced apical dominance was evident only very early in development (4- to 6-leaf stage), when rolC, but not wildtype plants, produced side shoots, and late in development, after flowerbud setting, when the rolC plants again produced more branches than the wild type. During the vegetative growth in the intervening period, rolC plants showed a strong apical dominance with no tendencies to side shoot formation. Similar phenotypes have been described earlier for 35S-ro/Ctransformed tobacco SRI plants (Schmulling et al., 1988).
The differences in free IAA concentrations were very small between rolC transformants and control plants. The internodes showed a gradient of free IAA concentrations along the plant, with concentrations decreasing toward the base of the plant (Fig. 2, A and B). In contrast, there was a lower concentration of conjugated IAA in the internodes of rolC transformants compared with controls (Fig. 2C). This difference was even more pronounced in leaves, which showed only 50% of the control concentration of conjugated IAA (Fig. 2D).
Northern Analysis of Transformed Plants
The same plants that were used for hormone measurements were analyzed for their expression of the rolC gene in
Metabolic Profiles of IAA
After 24 h, no quantitative or qualitative differences were observed between wild-type and 355-ro/C plants in metabolic profiles of applied 2-[14C]IAA, indicating a similar IAA metabolism (data not shown). Turnover Rate of IAA
Figure 3 shows an experiment comparing the combined effects of turnover and transport of IAA from the apex and first internode of rolC transformants and wild-type plants. We could not detect any differences, indicating a similar rate of IAA turnover and transport.
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Hormonal Characterization of roK-Transgenic Tobacco
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Cytokinin Quantification
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Cytokinins were quantified in young leaves, both with RIA on 2 rolC and 2 wild-type plants (Fig. 4A) and with LC-MS on 10 pooled rolC and 10 pooled wild-type plants (Fig. 4B). Both of these methods revealed lower cytokinin concentrations in the role plants, although the RIA method gave lower absolute values. These kinds of discrepancies are often seen when comparing RIA and GC-MS data, and are probably due to the interna1 standardization procedure utilized in the RIA (Roger Horgan, personal communication). The LC-MS quantifications showed that the concentration of iPA in rolC plants was reduced to approximately 20% of the wild-type values and these plants also displayed lowered amounts of a11 other tested cytokinins, except for Z, which was slightly increased in the rolC plants (wild-type approximately 0.73 and rolC approximately 1.1 pmol/g fresh weight). It should be noted that the nucleoside estimates in this investigation reflect the sum of the nucleoside and nucleotide pools obtained by phosphatase treatment of the extracts. GA Quantification
The internodes of rolC transformants showed generally lower concentrations of the tested GAs compared with wildtype (Fig. 5, A and B). In leaves of the rolC plants, the most dramatic change, compared with the wild type, was a 5 to 6 times increase in the concentration of GA19. In upper leaves, this was also coupled to a decrease in GA1 to about 30% of the wild-type concentration.
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out that some aspects of the RolC phenotype are indicative of an increased cytokinin-to-auxin ratio (Schmülling et al., 1988; Spena et al., 1989). Such changes in hormone activity could be achieved by the RolC protein modifying either the plant pool of, or sensitivity to, free active hormones. Therefore, a detailed comparison of free and conjugated hormone pools in rolC and wild-type plants is crucial to determine any effects of the RolC protein on systems regulating plant hormones. In this investigation, the levels of free and conjugated IAA, cytokinins, GAs, and ABA in transgenic tobacco plants expressing the rolC gene from the 3 5 s promoter were determined. In addition, the metabolic profile and tumover rate of IAA was studied. To find out if RolC somehow alters the ratio between auxin and cytokinins (Schmülling et al., 1988; Spena et al., 1989),
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Figure 4. Content of Z, ZR, iPA, iP, and Z7C as measured by RIA (A) and LC-MS (6).In A, the samples consist of pooled leaves 5 to 8 from two control plants transformed with the vector pPCV702 alone (WT) and two rolC-expressing plants (C4, C7) transformed with the vector pPCV702-rolC (RolC). In B, the samples consist of pooled upper leaves from 1O wild-type (WT) and 1O rolC-expressing plants (RolC). Note the different scales for cytokinin content and that the nucleoside quantifications reflect the sum of nucleosides and nucleotides. The cytokinin content is expressed as pmol of ZR and iPA equivalents/g fresh weight in the RIA, and as pmol of cytokininslg fresh weight in the LC-MS quantifications. Z7C was not determined in the RIA quantifications.
ABA Quantification
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As can be deduced from Figure 6A, the content of ABA in intemodes were comparable between control and rolC transformants, decreasing toward the base of the plant. In contrast, ABA levels in leaves 5 to 25 were markedly lower in rolC transformants, being about 50% of the concentrations in the control leaves (Fig. 68).
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DISCUSSION
To date, the single rolC gene from A. rhizogenes TL-DNA has been used to transform tobacco and potato plants, in which it has been expressed from both its endogenous promoter and from the heterologous 3 5 s promoter (Oono et al., 1987; Schmülling et al., 1988; Fladung, 1990). Different transgenic 35S-rolC plant species show many common phenotypical features such as reduced internodal length, reduced apical dominance leading to increased branching, smaller light green leaves, and reduced flower size (Schmdling et al., 1988; Fladung, 1990). However, little is known about the specific biochemical activity of the RolC protein and how this relates to the typical RolC phenotype. It has been pointed
Figure 5. Content of GA53, GAI9, G A z ~and , CAI in pooled upper internodes (A), lower internodes (B), upper leaves (C), and lower leaves (D) in 10 wild-type and 10 transformed plants expressing the rolC gene. Note the different scales for C A content.
Hormonal Characterization of ro/C-Transgenic Tobacco
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Leaf number Figure 6 . ABA content in apex, internodes 1-3, 5, 9, 13, 17, and 25 (A), and leaves 1-3, 5, 9, 13, and 17 (B), in two control plants transformed with the vector pPCV702 alone (WT 1, WT 2) and two roK-expressing plants (C9, C1O) transformed with the vector pPCV702-rolC. Note the different scales for ABA content.
we measured the pools of IAA and cytokinins as well as the metabolism of IAA. The measurements of IAA showed no differences in the content of free IAA between rolC plants and controls, but there was a reduction of conjugated IAA in the rolC plants. To elucidate this further, the metabolic profile of apically injected 2-[14C]IAA, as well as the tumover of apically injected ['3Cs]IAA, was studied. These experiments did not reveal any significant difference in either the rate of IAA conjugation/catabolism or the qualitative nature of the conjugates and catabolites formed in the rolcplant. The lower content of IAA conjugates is not reflected in a change of IAA metabolism, as revealed by the short-term turnover studies. This is probably due to a small change in IAA conjugation accumulated over time. In summary, it is unlikely that the primary action of the RolC enzyme is on IAA metabolism, and thus the reduced IAA conjugate levels in rolC leaves are presumably a secondary effect of the RolC activity. The content of the cytokinin riboside iPA was decreased to about 20% of the control in the leaves of the RolCexpressing plants. This is in contrast with the data presented by Estruch et al. (1991a), who detected increased levels of iPA in the RolC-producing patches of tobacco somatic mosaics carrying a 35s-Ac-rolC gene construct. The same authors also demonstrated that the RolC protein is able to hydrolyze a variety of cytokinin glucosides in vitro and proposed that
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the RolC protein exerts its action in vivo by releasing active cytokinins from their inactive N-glucoside conjugates. Indeed, our data show that Z7G is slightly reduced and Z slightly increased in rolC tobacco compared with wild type. However, the most dramatic change is the strong reduction in the concentration of iPA, one of the first compounds in the cytokinin biosynthesis pathway, and the general trend in our investigation is not an increase but rather a decrease in cytokinins. Consequently, our data indicate that rolC plants may exhibit reduced cytokinin biosynthesis. Therefore, even if the RolC protein hydrolyzes Z7G in vivo, it seems improbable that the resulting small increase in Z could account for the profound changes in growth and development displayed by rolC-expressing plants. It is well known that increased cytokinin concentrations lead to a dark green color of both leaves and calli, directly caused by an increase in the Chl content (Beinsberger et al., 1991; Grossman et al., 1991). The leaves of both 35s-rolC plants (Schmiilling et al., 1988) and the rolC-expressing patches of the somatic mosaics (Spena et al., 1989; Estruch et al., 1991a) are pale green in color due to a reduced Chl content (Fladung, 1990), which indicates decreased cytokinin levels in these plants, giving support to the cytokinin measurements presented in this paper. Furthermore, a cross between a dark-green cytokinin-overproducing 35s-ipt plant and a pale-green 35s-rolC plant produces hybrids with a general RolC phenotype, except for normally colored leaves (T. Schmiilling, personal communication). In other words, the pale-green color of rolC leaves is complemented by cytokinin-mediated Chl stabilization from the ipt plants, but in a11 other aspects the RolC phenotype is dominant over the Ipt phenotype. Thus, the pale-green color of RolC leaves is probably due to reduced cytokinin levels, but other aspects of the RolC phenotype are not directly caused by altered cytokinin pools. Taken together, these results indicate that the observed developmental alterations must be explained in other ways than in a mere alteration of the IAAIcytokinin balance. Two of the most striking differences between rolC and wild-type plants are the shortened internodes and reduced leaf size of the rolC plants. These changes could reflect a lower GA activity. Accordingly, we quantified GA,, GA19, GA20,and GAS3by GC-MS in rolC and wild-type plants. The GA measurements revealed different GA pattems in internodes and leaves (Fig. 5), which probably reflects the balance between sites of synthesis and transport of the GAs. In our plant material, the sample with the most rapidly expanding tissue is the upper leaves (Fig. 5C), and consequently this is the place where GAs probably play an important role. In upper leaves of rolC plants, GAS3is increased 3 times and GAlg is increased almost 6 times, suggesting that the enzyme GA19 oxidase has a lower activity in rolC plants compared with wild type. This is particularly interesting, since in monocotyledons the GA19-to-GA20 conversion is suggested to be a regulatory step in GA synthesis (Croker et al., 1990; Hedden and Croker, 1992), and results from Pisum (Ross et al., 1992) also support this hypothesis for dicots. The higher GA19 concentration in the rolC plants does not cause phenotypic alterations by itself, since only GAl is regarded to be the active GA per se ( e g Phinney, 1984; MacMillan, 1987). It is
Nilsson et al.
3 70
interesting that GAI is reduced almost 3 times, which correlates well with t h e seemingly smaller cells in the leaves of rolC plants (Estruch e t al., 1991a). Taken together, these results indicate that parts of t h e developmental alterations caused by the rolC expression could be explained by alterations in GA metabolism. The ABA measurements show a decrease of ABA i n rolC leaves compared with controls. However, such differences are normal i n plants with varying water status (Zeevaart, 1980). The rolC leaves have a lower dry weight-to-fresh 1.4% SD; weight ratio t h a n control leaves (wild-type, 16.4 rolC, 10.6 -+ 1.2% SD; n = 9), and also appear t o have a higher turgor, a s described previously for rolABC plants (Ooms e t al., 1986), indicating a condition of reduced water stress that correlates well with t h e lowered ABA levels. In this study, we h a v e measured bulk hormone concentrations i n whole leaves a n d stems, and, therefore, it can be argued that changes i n hormone levels occurring a t a subcellular, cellular, or tissue leve1 are masked when estimating bulk hormone concentrations. However, t h e RolC protein h a s been shown to be localized i n the cytosol (Estruch et al., 1991b), a n d when expressed from t h e 35S-promoter, effects of the RolC protein can b e detected i n tobacco mesophyll cells, both in membrane potential assays (Maurel et al., 1991) and in anatomical studies (Spena et al., 1989; Estruch e t al., 1991a). Thus, t h e RolC protein is expressed to high levels in a dominant cell type of tobacco leaves a n d is not confined to a small subcellular compartment. Therefore, it should be possible, even when analyzing whole leaves, to detect significant changes in the hormone pools. In summary, our data show that expression of t h e RolC protein i n transgenic plants clearly influences the metabolism of cytokinins a n d GAs, and in both cases RolC seems to reduce the rate of synthesis of these compounds. However, a t this point it is not possible to say if these differences are primary or secondary effects of t h e RolC enzymic activity. Further insight into t h e primary effect of t h e RolC activity might be gained from more direct studies of the metabolism of different GAs and cytokinins in rolC plants.
*
ACKNOWLEDCMENTS
We wish to thank Monica Burstrom and Gun Lovdahl for technical assistance and Dr. Angelo Spena for the gift of the pPCV002-ABC plasmid. Received December 4, 1992; accepted December 10, 1992. Copyright Clearance Center: 0032-0889/93/102/0363/09. LITERATURE CITED
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