Hormonal Characterization of Transgenic Tobacco Plants

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.)

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, investigations showed 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. Radio- immunoassay 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 wild- type plants, indicating a reduced activity of the CA19 oxidase, 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 ip t 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.

’ 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. 363

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; Smi- gocki, 1991).

Normally, plants can actively regulate their endogenous hormone pools. Therefore, the phenotypical alterations dis- cussed 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. Conse- quently, 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-

Abbreviations: BAP, 6-benzylaminopurine; FAB, fast atom bom- bardment; 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-N- vinylpolypyrrolidone; RIA, radioimmunoassay; SIM, selected ion monitonng; 35s promoter, cauliflower mosaic virus 35s RNA promoter; Z, zeatin; Z7G, zeatin 7-glucoside; ZR, zeatin 9-riboside.

3 64 Nilsson et al. Plant Physiol. Vol. 102, 1993

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 biolog- ically 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 AND 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. Agrobacter- ium 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 986- bp 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'-CAGATCTG- 3', 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 K3 medium (Kao et al., 1974) containing 0.5 Ng/mL

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 form- aldehyde-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]dCTP by 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]IAA and ['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 (200,000 dpm) was injected in the apex of 35s- roZC 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 365

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). Ion- suppressioa 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).

Analysis of the Biological Half-life of ['3C6]IAA in the Apical Region

Tumover of [13C6]IAA in the apex and first internode of 35s-rolC and wild-type tobacco was analyzed by injection of 250 ng of [I3C6]IAA into 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]IAA remaining 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-4- methylphenol and 36 mM /3-mercaptoethanol as antioxidants. To estimate the recovery of cytokinins, [3H]d-iPA synthesized as described by MacDonald et al. (1981) was added as an internal standard. The extract was centrifuged and the su- pematant 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

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-chromato- graphing with [3H]d-iPA was 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

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-di- tert-butyl-4-methylphenol and 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 500- mg 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

366 Nilsson et al. Plant Physiol. Vol. 102, 1993

purification was essentially the same as described by Crokeret al. (1990), except for the PVPP column chromatography,which was as described by Moritz et al. (1989). The followingdeuterated 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 UniversityCanberra, Australia).

The sample was further purified by reversed-phase HPLCas described below for the quantification of cytokinins. Forpurification of free GAs, the mobile phase was a 40-minlinear gradient (10-100% methanol in 1% aqueous HOAc)delivered at a flow rate of 1 mL/min. The fractions corre-sponding to the elution volumes of the GAs were collectedand analyzed by GC-SIM-MS. After methylation and silyla-tion (Moritz et al., 1989), the GAs were injected splitless intoa Hewlett-Packard 5890 gas chromatograph equipped witha fused silica capillary column with SE-30 chemical bondedphase, 0.25 nm, 25-m long, 0.25 mm i.d. (Quadrex, NewHaven, CT). The injector temperature was 250°C and thecolumn temperature was 60°C for 2 min. The temperaturewas 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 sourceof 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 300l*A.. The acceleration voltage was 10 kV. The mass spectrom-eter was operated in the acceleration voltage SIM mode, anddata were processed by JEOL MS-MP7000D data system. Foreach GA, two characteristic ions and the deuterated analogswere recorded. The dwell time was 50 ms, and the resolutionwas 5000 with perfluorokerosene as reference substance.

RESULTS

Generation of Tobacco Plants Expressing the rolC Gene

Regeneration of ro/C-transformed plants was normal.There was no reduction in the production of shoots from theleaf discs transformed with the vector pPCV702-ro/C, nor insubsequent rooting of these shoots compared with the controlplants transformed with the vector pPCV702 alone. Fortyindependently regenerated kanamycin-resistant plants, trans-formed with the vector pPCV702-ro/C, were transferred tothe greenhouse. These plants displayed to a varying degreethe RolC phenotype, characterized by shortened internodes,light green lanceolate leaves, an increased number of inter-nodes formed before flowering, and small male-sterile flow-ers. A reduced apical dominance was evident only very earlyin development (4- to 6-leaf stage), when rolC, but not wild-type plants, produced side shoots, and late in development,after flowerbud setting, when the rolC plants again producedmore branches than the wild type. During the vegetativegrowth in the intervening period, rolC plants showed a strongapical dominance with no tendencies to side shoot formation.Similar phenotypes have been described earlier for 35S-ro/C-transformed tobacco SRI plants (Schmulling et al., 1988).

Northern Analysis of Transformed Plants

The same plants that were used for hormone measure-ments were analyzed for their expression of the rolC gene in

UJT C4 C7 C9 C I O

— too in o in

— vO

o in10 . K) .i co i co o inW >»- V. V- V- ^ _ ^ - ^ ^ ^(0 M O *^ (0 03 ® ** «^ *̂ O CD 03 «^ ^ ^^a > c a ) c u u a ) c e c a > a u c c c

fillll !••§••••Figure 1. Northern analysis of the transcription of the rolC gene inthe plants used for hormone analysis. Leaves and internodes weresampled as detailed in the figure. The plants used were wild-type(WT) and ro/C-transformed (C4, C7, C9, C10) plants. Total RNA (25Mg) was loaded in each lane. The rolC transcript was compared witha DMA standard run in parallel and was estimated to be about 600bases.

different plant tissues. As seen in Figure 1, all plant partsshowed high expression of the rolC gene with only smalldifferences between organs and between plants. This generalexpression pattern fits well with what has been describedearlier for various 35S promoter-reporter gene fusions in anumber of different plants (Jefferson et al., 1987; An et al.,1988; Williamson et al. 1989; Nilsson et al., 1992). Thus, noselection for plants expressing the RolC protein to a lesserdegree or in a different organ-specific manner has taken placeduring the regeneration procedure. Importantly, all plantsstrongly expressed the rolC gene in those parts of the plantused for hormone measurements.

IAA Quantification

The differences in free IAA concentrations were very smallbetween rolC transformants and control plants. The inter-nodes showed a gradient of free IAA concentrations alongthe plant, with concentrations decreasing toward the base ofthe plant (Fig. 2, A and B). In contrast, there was a lowerconcentration of conjugated IAA in the internodes of rolCtransformants compared with controls (Fig. 2C). This differ-ence was even more pronounced in leaves, which showedonly 50% of the control concentration of conjugated IAA(Fig. 2D).

Metabolic Profiles of IAA

After 24 h, no quantitative or qualitative differences wereobserved between wild-type and 355-ro/C plants in metabolicprofiles of applied 2-[14C]IAA, indicating a similar IAA me-tabolism (data not shown).

Turnover Rate of IAA

Figure 3 shows an experiment comparing the combinedeffects of turnover and transport of IAA from the apex andfirst internode of rolC transformants and wild-type plants.We could not detect any differences, indicating a similar rateof IAA turnover and transport.

k

.Wl

w r 2 A Free IAA

M M C \

v

c. E o, E O V

-

Hormonal Characterization of roK-Transgenic Tobacco

C9 RolC

fl CIO RolC 2o

40

10 20

O z 9 0 I Conjugated IAA M fi 40 60

30 40

20

20 10

o n Apex 1-3 5 9 13 17 25

Internode number

B Free IAA wr1 w r 2

0 C9 RolC

D Conjugated IAA

1-3 5 9 13 17 Leaf number

Figure 2. Free (A and B) and conjugated (C and D) IAA content in apex, internodes 1-3, 5, 9, 13, 17, and 25 (A and C), and in leaves 1-3, 5, 9, 13, and 17 (B and D) in two control plants transformed with the vector pPCV702 alone (WT 1, WT 2) and two rolC-expressing plants (C9, CIO) transformed with the vector pPCV702-rolC. Note the different scales for IAA content.

367

Biological half life time of IAA 250 r I

WT RolC

Cytokinin Quantification

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.

O 2 4 6 8 1 0 12 11 16 1 8 2 0 2 2 2 4

Time (hrs)

Figure 3. Rate of inactivation and transport of ['3C6]lAA applied to the apex and first leaves of wild-type and rolC tobacco plants. ['3C6]lAA (250 ng/plant) was injected into the apical region of the shoot and tissue harvested after a O-, 3-, 6-, 9-, or 24-h period of metabolism. The amount of ["C6]IAA remaining in the apex and first leaves of individual plants was determined by GC-SIM.

GA Quantification

The internodes of rolC transformants showed generally lower concentrations of the tested GAs compared with wild- type (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.

368 Nilsson et al. Plant Physiol. Vol. 102, 1993

8

RIA T A

LC-MS 40

E 30

$ & 20

10

O Z ZR iPA iP Z7G

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 1 O wild-type (WT) and 1 O 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

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).

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

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. There- fore, 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),

400

Mo

O

e004 B Lower Internodes

400

Mo

O Ic Uowr

600

400

200

O

ID Lower

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 369

400-

A w r 1 II wI-2 c9 Role

Apex 1-3 5 9 13 17 25

Internode number

600-

500-

400.

a" 300.

200.

100.

O.

9

1-3 5 9 13 17

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, C1 O) 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 RolC- expressing 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

the RolC protein exerts its action in vivo by releasing active cytokinins from their inactive N-glucoside conjugates. In- deed, 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 improb- able 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 GAS3 by 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, GAS3 is 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 mon- ocotyledons 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

3 70 Nilsson et al. Plant Physiol. Vol. 102, 1993

interesting that GAI is reduced almost 3 times, which correlates well with the seemingly smaller cells in the leaves of rolC plants (Estruch e t al., 1991a). Taken together, these results indicate that parts of the developmental alterations caused by the rolC expression could be explained by alterations in GA metabolism.

The ABA measurements show a decrease of ABA in rolC leaves compared with controls. However, such differences are normal in plants with varying water status (Zeevaart, 1980). The rolC leaves have a lower dry weight-to-fresh weight ratio than control leaves (wild-type, 16.4 * 1.4% SD; rolC, 10.6 -+ 1.2% SD; n = 9), and also appear to have a higher turgor, a s described previously for rolABC plants (Ooms et al., 1986), indicating a condition of reduced water stress that correlates well with the lowered ABA levels.

In this study, we have measured bulk hormone concentrations in whole leaves and stems, and, therefore, it can be argued that changes in hormone levels occurring a t a subcellular, cellular, or tissue leve1 are masked when estimating bulk hormone concentrations. However, the RolC protein has been shown to be localized in the cytosol (Estruch et al., 1991b), and when expressed from the 35S-promoter, effects of the RolC protein can be detected in tobacco mesophyll cells, both in membrane potential assays (Maurel et al., 1991) and in anatomical studies (Spena et al., 1989; Estruch et al., 1991a). Thus, the RolC protein is expressed to high levels in a dominant cell type of tobacco leaves and 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 the RolC protein in transgenic plants clearly influences the metabolism of cytokinins and 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 the RolC enzymic activity. Further insight into the primary effect of the 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

Akiyoshi DE, Klee HJ, Amasino R, Nester EW, Gordon MP (1984) T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis. Proc Natl Acad Sci USA 81: 5994-5998

An G, Costa MA, Mitra A, Ha SB, Marton L (1988) Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants. Plant Physiol88: 547-552

Barry GF, Rogers SG, Fraley RT, Brand L (1984) Identification of a cloned cytokinin biosynthetic gene. Proc Natl Acad Sci USA 81:

Beinsberger SEI, Valcke RLM, Deblaere RY, Clijsters HMM, De Greef JA, Van Onckelen HA (1991) Effects of the introduction of Agrobacterium tumefaciens T-DNA ipt gene in Nicotiana tabacum L. cv. Petit Havana SRl plant cells. Plant Cell Physiol32: 489-496

4776-4780

Croker SJ, Hedden P, Lenton JR, Stoddart JL (1990) Comparison of gibberellins in normal and slender barley seedlings. Plant Phys- i0194 194-200

Estruch JJ, Chriqui D, Grossmann K, Schell J, Spena A (1991a) The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates. EMBO J 10: 2889-2895

Estruch JJ, Parets-Soler A, Schmidling T, Spena A (1991b) Cyto- solic localization in transgenic plants of the rolC peptide from Agrobacterium rhizogenes. Plant Mo1 Biol17: 547-550

Fladung M (1990) Transformation of diploid and tetraploid potato clones with the rolC gene of Agrobacterium rhizogenes and characterization of transgenic plants. Plant Breeding 104 295-304

Glass NL, Kosuge T (1986) Cloning the gene for indoleacetic acid- lysine synthetase from Pseudomonas syringae subsp. savastanoi. J Bacterioll66: 598-603

Grossmann K, Kwiatkowski J, Hauser C (1991) Phytohormonal changes in greening and senecing intact cotyledons of oilseed rape and pumpkin: influence of the growth retardant BAS 111..W. Physiol Plant 83: 544-550

Hedden P, Croker SJ (1992) Regulation of gibberellin biosynthesis in maize seedlings. In CM Karssen, LC Van Loon, D Vreugdenhil, eds, Progress in Plant Growth Regulation. Kluwer Academic Pub- lishers, Dordrecht, The Netherlands, pp 534-544

Horsch RB, Fry JE, Hoffman N, Wallroth M, Eichholtz D, Rogers S, Fraley RT (1985) A simple and general method for transfemng genes into plants. Science 227: 1129-1132

Hutzinger O, Kosuge T (1986) Microbial synthesis and degradation of indole-3-acetic acid. 111. The isolation and characterization of indole-3-acetyl-c-~-lysine. Biochemistry 7: 601-605

Imbault N (1989) Analyse des cytokinines dans les pousses en cours de differenciation florale et dans les bourgeons sexues du sapin de Douglas (Pseudotsuga menziesii [Mirb.] Franco). PhD thesis. L'U- niversité dOrleans

Imbault N, Moritz T, Nilsson O, Chen HJ, Bollmark M, Sandberg G (1993) Separation and identification of cytokinins using combined capillary liquid chromatography-mass spectrometry. Biol Mass Spectrom 22: 201-210

Imbault N, Tardieu 1, Joseph C, Zaerr JB, Bonnet-Masimbert M (1988) Possible role of isopentenyladenine and isopentenyladenosine in flowering of Pseudotsuga menziesii: endogenous variations and exogenous applications. Plant Physiol Biochem 26: 289-295

Inze D, Follin A, Van Lijsebettens M, Simoens C, Genetello C, Van Montagu M, Schell J (1984) Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens: further evidence that two genes are involved in indole-3-acetic acid synthesis. Mo1 Gen Genet 194 265-274

Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: 6- glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907

Kao KN, Constabel F, Michayluk MR, Gamborg OL (1974) Plant protoplast fusion and growth of intergeneric hybrid cells. Planta

Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffmann NL (1987) The effects of overproduction of two Agrobacterium tumefaciens auxin biosynthetic gene products in transgenic petunia plants. Genes Dev 1: 86-96

Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes camed by a new type of Agrobacterium binary vector. Mo1 Gen Genet 204: 383-396

Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant tissues. Ana1 Biochem 163 16-20

MacDonald EMS, Akiyoshi DE, Morris RO (1981) Combined high- performance liquid chromatography-radioimmunoassay for cytokinins. J Chromatogr 214 101-109

MacMillan J (1987) Gibberellin deficient mutants of maize and pea and the molecular basis of gibberellin action. In GV Hoad, JR Lenton, MB Jackson, RK Atkin, eds, Hormone Action in Plant Development: A Critica1 Appraisal Butterworths, London, pp

Marsh JL, Erfle M, Wykes EJ (1984) The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 3 2 481-485

Maurel C, Barbier-Brygoo H, Spena A, Tempé J, Guern J (1991)

120 215-227

73-87

Hormonal Characterization of rolC-Transgenic Tobacco 371

Single ro l genes from Agrobacterium rhizogenes TL-DNA alter some of the cellular responses to auxin in Nicotiana tabacum Plant Physiol97: 212-216

Medford JI, Horgan R, El-Sawi S, Klee HJ (1989) Alterations of endogenous cytokinins in transgenic plants using a chimeric isopentenyl transferase gene. Plant Cell 1: 403-413

Moritz T, PhilipsonJJ, Oden PC (1989) Detection and identification of gibberellins in Sitka spruce (Picea sitchensis) of different ages and coning ability by bioassay, radioimmunoassay and gas chromatography-mass spectrometry. Physiol Plant 7 5 325-332

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assay with tobacco tissue cultures. Physiol Plant 1 5 473-479

Nilsson O, Aldén T, Sitbon F, Little CHA, Chalupa V, Sandberg G, Olsson O (1992) Spatial pattem of CaMV 35s promoter- luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging. Transgenic Re- search 1: 209-220

Ooms G, Atkinson J, Bossen ME, Leigh RA (1986) TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro. Planta 168

Oono Y, Handa T, Kanaya K, Uchimiya H (1987) The TL-DNA gene of Ri plasmids responsible for dwarfness of tobacco plants. Jpn J Genet 6 2 501-505

Phinney BO (1984) Gibberellin AI, dwarfism and the control of shoot elongation in higher plants. In A Crozier, JR Hillman, eds, The Biosynthesis and Metabolism of Plant Hormones, Vol 23. Cambridge University Press, London, pp 17-4 1

Reeve DR, Crozier A (1977) Radioactivity monitors for high-performance liquid chromatography. J Chromatogr 137: 271-282

Roberto FF, Klee H, White F, Nordeen R, Kosuge T (1990) Expres- sion and fine structure of the gene encoding N'-(indole-3-acetyl)- L-lysine synthetase from Pseudomonas savastanoi. Proc Natl Acad Sci USA 87: 5797-5801

Romano CP, Hein MB, Klee HJ (1991) Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi. Genes Dev 5: 438-446

Ross JJ, Willis CL, Gaskin P, Reid JB (1992) Shoot elongation in Lathyrus odoratus L.: gibberellin levels in light- and dark-grown tal1 and dwarf seedlings. Planta 187: 10-13

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning, A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Schmiilling T, Schell J, Spena A (1988) Single genes from Agrobac- terium rhizogenes influence plant development. EMBO J 7:

Schroder G, Waffenschmidt S, Weiler E, Schroder J (1984) The T-

106-112

2621-2629

region of Ti plasmids codes for an enzyme synthesizing indole-3- acetic acid. Eur J Biochem 138 378-391

Sitbon F, Hennion S, Sundberg B, Little CHA, Olsson O, Sandberg G (1992a) Transgenic tobacco plants co-expressing the Agrobacter- ium tumefuciens iaaM and iaaH genes display altered growth and indoleacetic acid metabolism. Plant Physiol99 1062-1069

Sitbon F, Little CHA, Olsson O, Sandberg G (199211) Correlation between the expression of T-DNA IAA biosynthetic genes from developmentally regulated promoters and the distribution of IAA in different organs of transgenic tobacco. Physiol Plant 8 5

Smart CM, Scofield SR, Bevan MW, Dyer TA (1991) Delayed leaf senesence in tobacco plants transformed with tmr, a gene for cytokinin production in Agrobacterium. Plant Cell 3: 647-656

Smigocki AC (1991) Cytokinin content and tissue distribution in plants transformed by a reconstructed isopentenyl transferase gene. Plant Mo1 Bioll6 105-115

Smigocki AC, Owens LD. (1988) Cytokinin gene fused with a strong promoter enhances shoot organogenesis and zeatin levels in transformed plant cells. Proc Natl Acad Sci USA 8 5 5131-5135

Smigocki AC, Owens LD (1989) Cytokinin-to-auxin ratios and morphology of shoots and tissues transformed by a chimeric isopentenyl transferase gene. Plant Physiol 91: 808-81 1

Spena A, Aalen RB, Schulze S (1989) Cell-autonomous behavior of the rolC gene of Agrobacterium rhizogenes during leaf development: a visual assav for transposon excision in transgenic plants. Plant

679-688

- . Cell 1: 1157-'1164

Suena A. Prinsen E. Fladunz M. Schulze SC. Van Onckelen H '(1991) The indoleacetic acid-yysine synthetase gene of Pseudomonas syringae subsp. savastanoi induces developmental alterations in transgenic tobacco and potato plants. Mo1 Gen Genet 227:

Spena A, Schell J (1987) The expression of a heat-inducible chimeric gene in transgenic tobacco plants. Mo1 Gen Genet 206 436-440

Sundberg B (1990) Influence of extraction solvent (buffer, methanol, acetone) and time on the quantification of indole-3-acetic acid in plants. Physiol Plant 78: 293-297

Thomashow LS, Reeves S, Thomashow MF (1984) Crown gall oncogenesis: evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyses synthesis of indoleacetic acid. Proc Natl Acad Sci USA 81: 5071-5075

Walden R, Koncz C, Schell J (1990) The use of gene vectors in plant molecular biology. Methods Mo1 Cell Biol 1: 175-194

Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB (1989) Differential accumulation of a transcript driven by the CaMV 35s promoter in transgenic tobacco. Plant Physiol90 1570-1576

Zeevart JAD (1980) Changes in the levels of abscisic acid and its metabolites in excised leaf blades of Xanthium strumarium during and after water stress. Plant Physiol66: 672-678

205-212

Documents

Hormonal Characterization of Transgenic Tobacco Plants