J. Plant Physiol. Vol. 144. pp. 80-85 {1994}

Introduction

Endogenous Phytohormones in Tobacco Plants Transformed with Agrobacterium rhizogenes pRi TL-ONA Genes

E. PRINSEN1, D. CHRIQUI2, F. V ILAINE3, M. TEPFER3, and H. VAN ONCKELEN1

1 University of Antwerp (UIA), Department of Biology, Universiteitsplein 1, B-2610 Wilrijk, Belgium

2 Universite P.M. Curie, Lab. CEMV, Bk N2, 4, Place Jussieu, F-75252 Paris CEDEX OS, France

3 LN.R.A., Laboratoire de Biologie Cellulaire, F-78026 Versailles-Cedex, France

Received November 14,1993 . Accepted January 23,1994

Summary

Transgenic plants obtained from roots transformed with the T-DNA of Agrobacterium rhizogenes dis­play a modified phenotype. We analyzed endogenous indole-3-acetic acid, abscisic acid and cytokinin concentrations in apices, leaves and stems at juvenile and mature stages of normal, pRi transformed and rolA tobacco plants.

We have observed alterations in the hormonal levels in the stem apices of transformed plants. In transformed plants with a moderately altered (T) phenotype, we observed an accumulation of cytokinins at a very young age, which was not observed in transformed plants with an extremely altered phenotype (T' or rotA). All transformed plants showed reduced endogenous IAA levels in the stem apical region and consequently an attenuated basipetal auxin gradient. At the same time, the transient ABA accumula­tion observed in the apices of untransformed plants during the vegetative developmental stage was absent for all transformed plants.

The detailed kinetic hormonal analyses presented in this paper emphasize the relevance of varying hor­mone levels in the shoot apical region on the developmental pattern of both transgenic and normal plants.

Key words: Agrobacterium rhizogenes, Nicotiana tabacum cv. Xanthi, ABA, IAA, rotA, zeatin·riboside.

Abbreviations: ABA = Abscisic acid; IAA = Indole-3-acetic acid; N = Normal; ORF = open reading frame; Ri = root inducing; rotA = Ri rooting locus A; T = transformed; T' = supertransformed; ZR­equiv. = zeatin-riboside equivalents.

Morphogenetic modifications of transgenic Nicotiana taba· cum cv. Xanthi harbouring either the entire TL DNA, with either moderately (T) or extremely altered phenotype (T') or only the rotA gene of Agrobacterium rhizogenes strain A4 (Vansuyt et al., 1992), have been described previously in terms of overall morphology (Ackermann, 1977; Tepfer, 1984; Sinkar et al., 1988) as well as in terms of structural changes occurring during cell differentiation from the meris-

terns (Chriqui et al., in prep.). These studies showed an alter­ation in the coordination between the cell growth patterns of the various tissues. The major developmental abnormal­ities, i.e. shortened internodes and wrinkled leaves observed in the T' and in the rotA plants resulted from multiple changes. In particular, a precocious inhibition of cell elonga­tion was observed in the differentiating internodes, as well as an early development of internal and external phloem and a delay in xylem vessel differentiation. Plastid ultrastructure and cell wall deposition were also modified. In the pheno-

© 1994 by Gustav Fischer Verlag, Stuttgart

typically less altered T plants the main differences were ob­served at the shoot meristem level, including a higher mitotic activity, an enlargement of the shoot apical meristem and the induction of a third foliar helix. Furthermore, different growth properties and regeneration abilities were shown by in vitro culture of root or leaf explants excised from the normal and transgenic plants and placed on hormone-free media or on media containing auxin or cytokinins (T epfer and Tempe, 1981; Tepfer, 1984; Schmulling et al., 1988, Spano et al., 1988; Capone et al., 1989). The particular growth properties of transgenic roots and the higher rooting potential of transgenic tissues were interpreted as the result of increased auxin sensitivity (Shen et al., 1988 and 1990). Spano et al. (1988) demonstrated that TE 15 (containing roLA, rolB and roLC genes) tobacco leaf explants were highly sensitive to auxin-induced root initiation, while tobacco leaf explants expressing only rolB (TH37) showed no enhanced auxin sensitivity. The combined expression of rolA, Band C enhanced auxin sensitivity of tobacco leaf protoplasts as measured by stimulation of the transmembrane electric po­tential (Shen et al., 1988 and 1990). Measuring the auxin in­duced stimulation of the transmembrane electric potential by single rol genes showed rolB to be more effective than roLA, while rolC was not active at all (Maurel et al., 1991). It was also demonstrated that rolA stimulated the auxin sensi­tivity of the proton translocation catalyzed by the plasma membrane H+-ATPase (Vansuyt et aL, 1992). It is known that plants with a modified sensitivity to auxin may exhibit abnormal growth patterns (Estelle and Sommerville, 1987; Kelly and Bradford, 1986; Mirza et al., 1984; Muller et al., 1985). However, the relations between the increased auxin sensitivity of the transgenic roL plants and their morpho­genetic modifications have not been established, and little is known about the endogenous phytohormone status in the various transgenic organs. Correlations were established be­tween endogenous IAA and lAM levels and morphological and structural changes of transgenic pea root clones, reveal­ing high IAA and lAM levels even in clones carrying only pRi TL-DNA, where no known gene for auxin synthesis is present (Prinsen et al., 1992). Recently, Schmulling et al. (1993) indicated that the expression of the different roL genes induces changed endogenous phytohormone levels and showed interrelation of various phytohormones, but the lack of kinetic analysis did not allow them to functionally correlate the endogenous hormone alterations with the ob­served morphological traits.

It is widely accepted that hormones can modify many de­velopmental processes by modulation of expression of genes that regulate pathways of growth and differentiation. Pheno­typic modifications have been described in transgenic plants overexpressing the Agrobacterium tume/aciens T-DNA genes 1,2 (tms) or 4 (ipt), respectively responsible for auxin and cy­tokinin biosynthesis (for reviews see Owens and Smigocki, 1989; Klee and Estelle, 1991). The particular phenotypes were never similar to the phenotype of the hairy root plants. However, a pertinent result was obtained by Romano et aL (1991) who described tobacco plants with reduced IAA lev­els due to expression of a 35S-iaaL gene, which displayed certain aspects of the Ri phenotype, including strongly wrinkled leaves. Although the modification in differentia-

Hormone levels in pRi transformed tobacco 81

tion patterns of the T, T' and rolA transformed plants could be due to phytohormone imbalances, no substantial altera­tion in auxin levels was found during vegetative develop­ment in leaves and stems of transformed plants (Van Oncke­len et aL, 1988). This was confirmed for floral stem segments of Brassica napus bearing pRi TL DNA Qulliard et aL, 1992). However, preliminary results on T and T' plants indicated lower IAA levels in the transgenic shoot apices at vegetative developmental stages (Van Onckelen et aL, 1988). The roLC product was shown to be a cytosolic enzyme able to hydro­lyse cytokinin-glucosides (Estruch et al., 1991 a and b), re­leasing free active hormone from cytokinin N7- and N9-glu­cosides. On the contrary, Nilsson et al. (1993 a) correlated rolC activity with a reduction of iPA concomitant with a GA19 increase. Evidence has been presented that the product of the rolB gene is a cytosolic i1-glucosidase able to hydrolyse both cytokinin-O-glucosides and indoxyl-i3-glucosides, but not IAA-glucose conjugates (Spena et al., 1993). Specific ex­pression of this gene increased the free IAA content in trans­genic anthers (Spena et aL, 1992) although expression of rolB appears not to influence the overall rate of IAA biosyn­thesis (Nilsson et aL, 1993 b). Until now no reports on the identification and biochemical function of the rolA gene product have been published.

In the present work, the endogenous contents of IAA, cy­tokinins (ZR) and abscisic acid (ABA) were determined in the apical regions, leaves and stems at juvenile and mature stages of normal, T, T' and roLA tobacco plants.

Materials and Methods

Plant material

Nicotiana tabacum L. cv. Xanthi XHFD8 (Bourgin, 1978) plants transformed with either the TL-DNA of Agrobacterium rhizogenes A4 or the rolA gene alone were described previously (Vansuyt et al., 1992). Plants were grown from homozygous seeds of the R2 generation under controlled conditions, 16/8 photoperiod, 23 ± 2°C/16 ± 2°C, and 50 Jlmollm2/s. Among Ri-transformed plants, two groups were analyzed separately, ones with a moderate (T) and extreme (T') Ri phenotype. These differences have been correlated with differences in the level of expression of rolA (Sinkar et al., 1988; Vansuyt et al., 1992). The apical region (shoot apical meristem including the first 3 young leaves), mature leaves and upper, middle and lower parts of the stem were harvested at different time inter­vals during vegetative growth, frozen at -196°C and stored at -20°C until analysis. Apices of 10 to 20 individual plants at an identical developmental stage were pooled. This pooled extract was used for further analysis. Data are expressed as means ± standard er­ror. Four separate time course experiments were performed, with similar results.

fAA and ABA analysis

IAA and ABA were extracted and purified as described by Prin­sen et al. (1991). Frozen plant material was homogenized in 100% methanol (9 mLigfr.wt.) supplemented with 3{5(n)-lH}IAA (150Bq, 777GBq/mmol, Amersham) and DL-cis,trans-(G)H)­ABA (170Bq, 1.91 TBq/mmol, Amersham). After centrifugation (24,000 x g, 15 min, 4°C) the supernatant was diluted to 50 % meth­anol and purified on a bond-elU[ RP-C18 column. IAA and ABA were retained on a 2 mL DEAE-Sephadex column (formate condi-

82 E. PRINSEN, D. CHRIQUI, F. VlLAINE, M. TEPFER, and H. VAN ONCKELEN

tions), eluted with 6 % formic acid and bound to a bond-elut C18 column (equilibrated with 6% formic acid) coupled underneath. The retained IAA and ABA were eluted from the C18 column with 5 mL diethyl ether. Upon evaporation of the ether, the residue was dissolved in 2 x 500 ~L 100 % MeOH, dried in a speed-vac and kept at -20 °C until HPLC-analysis. IAA and ABA were separated by a preparative Ion Suppression (IS)-HPLC run (50/49.5/0.5; HzO/MeOH/HAc; O.5mLlmin; Rosil C18, 10cm, 3 ~m column, Alltech-RSL). IAA was analyzed by an analytical Ion Pairing (IP)­RP HPLC run (60/40; O.OOlM phosphate, O.OlM TBAH pH 6.61 MeOH; 0.5mL/min; same column) and measured on line with a Shimadzu RF 530 fluorescence detector (excitation at 285 nm, emis­sion at 360nm) (Horemans et al., 1984). After methylation using di­azomethane (Schlenk and Gellerman, 1960) the methylated ABA was separated from the non-methylated ABA using RP-HPLC (SOl 50; MeOH/HzO, same flow and column). ABA was analyzed on GC-ECD (argon-methane Ar/CH~; 90/10, column: OV 101 WCOT, 25m, diameter 0.25mm, Flow: through column ImLI min; through detector 30 mLI min, T: column 220 oC, Injection 280°C, Detection 280°C, solid phase injector). The endogenous IAA and ABA contents were calculated following the principles of isotope dilution.

Cytokinin analysis

Frozen plant material was homogenized in 75 % methanol (9mLlgfr.wt.) supplemented with 3H-ZR-dialcohol (± 13 ~Ci, 40 ~Cil mmol, Amersham). After centrifugation (24,000 x g, 15 min, 4°C) the supernatant was purified on a bond-elut RP-C 18 column. The column effluent was evaporated in vacuo (Biichi) and resolved in 35 % EtOH prior to RIA. Radio immuno assays were performed as described by Weiler (1980) after slight modifications using chicken egg yolk anti-Zeatin-riboside antibodies (Redig et al., 1991). Standards were tested twice and samples were tested at ten different dilutions. Standard curves were plotted after a logit trans­formation (Rodbard, 1974). The extraction yield was taken into ac­count following the principles of isotope dilution.

To compare phytohormone concentrations between the differ­ent organs, concentrations are usually expressed as pmollgfr.wt. However differences in transpiration and stomatal opening (Est­ruch et al., 1991) can influence the freshldry weight ratio. More­over, the re9uced internode length of all transformants can compli­cate isolation of the apical region during harvest. To compare the apical region of the individual plants, we have therefore chosen to express the absolute levels of phytohormones in pmol per apex. As the antibodies used in the RIA did not distinguish the riboside from the free base, concentrations and absolute levels of cytokinins were expressed as pmol ZR equivalents.

Results

Endogenous L4A levels

The endogenous IAA content in the apex, leaves and stem of tobacco plants was analyzed at different stages. Neither leaf nor stem tissue showed significant differences in free IAA concentration (data not shown). Nor did the upper, middle and lower part of the stem of seven week old plants show any differences in free IAA (Fig. 1). However, the apical region of seven week old T, T' and ralA-plants con­tained about half the endogenous IAA concentration present in the apical region of the untransformed (N) control plants. Consequently, the basipetal auxin gradient present in con­trol plants was attenuated in all transformed plants (Fig. 1).

A more detailed kinetic analysis of the apical region was performed. Figure 2 summarizes the endogenous IAA con­tent in the apex of control, T, T' and rolA plants from an early stage of development until appearance of flower buds. The results are expressed as amount per apex. U ntrans­formed control plants at the age of 6 -12 weeks showed a clear IAA peak (40-S0pmol) (Fig. 2). In the apical region of the transformed plants, the duration of this peak was some­how shorter (6 to 10weeks) and the peak height was less ex­treme (max. 32 pmol). At the age of 13 weeks, the apical re­gion of all transformed plants contained about one third of the endogenous IAA content compared with the untrans­formed control (Fig. 2). The endogenous IAA level de-

240

200

i ~ 160

til ...... 120 '0

E .s- ao -< :!

40

0

stem fragment

Fig. I: Free IAA concentration expressed as pmollg fresh weight ± S.E. in stem apex (Apex), upper stem fragment (Su), middle stem fragment (Sm) and lower stem fragment (S,) of tobacco plants grown during 20 weeks under controlled conditions. Results are shown for untransformed control plants (.), plants transformed with TL­DNA displaying the T phenotype (_) or T' phenotype (.&.) and plants transformed with the rolA (T). At each time point, 10 to 20 individual explants were pooled for analysis.

60

50

40 '0 E .s- 30

-< :! 20

10

0 0 4 a 12 16 20

Age (weeks)

Fig. 2: Absolute levels of IAA (pmol) ± S.E. present in the apical re­gion of normal (.), T (_), T' (.&.) and rolA (T) tobacco plants grown during 20 weeks under controlled conditions, analyzed at different stages of development. At each time point, 10 to 20 indi­vidual explants were pooled for analysis.

300

200

A ~ 100 E ~

> 25

'" c::r 20 II

c: 15 N

10

5

0 0 4 8 12 16 20

Age (weeks)

Fig.3: Absolute levels of ZR-equivalents (pmol) ± S.E. present in the apical region of normal (e), T (.), T' (A) and rotA (~) to­bacco plants grown during 20 weeks under controlle? cond~tlons, analyzed at different stages of development. At ea~h time pomt, 10 to 20 individual explants were pooled for analYSIS. Samples were tested at ten different dilutions.

creased drastically in the control plants before the onset of flowering (Fig. 2, 15 weeks). The differences described were observed in 2 separate time course experiments.

Endogenous cytokinin levels

Cytokinin (zeatin-riboside-equivalent) concentrations in the apical regions, leaves and stems of the same sample were analyzed. As for IAA, no differences in zeatin-riboside con­centrations were found in leaf and stem tissue of normal and transformed plants, all varying between 2-3pmollgfr.v.:. In contrast, differences in cytokinin levels were observed 1n

shoot tips at a young stage (Fig. 3). The transient ZR-ac­cumulation shown in apices of normal plants (20 pmol) was extremely pronounced in the T-apices (80 pmol). In the apices of the rolA plants, no transient cytokinin accumulation was observed, whereas in the T' apices, as was also found in the T-plants the accumulated levels were completely diminished.

Endogenous ABA levels

Endogenous ABA levels during development are shown in figure 4. The kinetics of ABA accumulation in untransform­ed stem apical region during the growth period showed a transient ABA peak (6 nmol) at a more mature stage before flowering (10-13 weeks) (Fig. 4). This transient peak of ABA accumulation was not observed in the apical region of the transformed plants analyzed.

Discussion

In order to investigate a possible relationship between the observed morphological alterations of the transgenic plants and their endogenous phytohormone content, we analyzed endogenous IAA, ABA and cytokinin (ZR) concentrations in the apical region, leaves and stems at juvenile and mature

Hormone levels in pRi transformed tobacco 83

stages of N, T, T' and rolA tobacco plants. Although ma­ture transformed tobacco plants show morphological abnor­malities and a high expression level of rolA, rolB and rolC promoters in stems and leaves (Schmiilling et al., 1989), no differences in the endogenous IAA (Van Onckelen et al., 1988) and cytokinin levels were observed in the mature leaf or stem tissue compared to untransformed control plants. The absence of major IAA differences is also in agreement with the data from Julliard et al. (1992) on Brassica napus floral stem internodes transformed by the pRi TL-DNA and by Spano et al. (1988) on mature leaves of pRi-transformed tobacco, although the IAA values reported in the latter were about 1000 times higher compared to the endogenous auxin levels we found in Xanthi leaves (present work) as well as in leaves of SRI tobacco plants (Prinsen et al., in preparation).

The results concerning endogenous phytohormone con­tent in the shoot apical region showed significant differ­ences. In untransformed control apices a gradual increase in endogenous IAA levels leads to the establishment of a basi­petal IAA gradient during the mature vegetative phase. In normal plants, the prefloral phase, previously dated between 10 and 12 weeks (Chriqui et al., in prep.), is characterized by a drop in the apical IAA content, which is maintained at a low level during the beginning of the floral phase. Such very low IAA levels were also reported for flowering apices of Brassica napus (De BouilIe et al., 1989). The endogenous apical ABA levels in the untransformed controls also varied during development, showing a transient ABA accumula­tion towards the end of the vegetative growth period. Sim­ilar patterns for both IAA and ABA have been observed in Nicotiana tabacum cv. petit Havana SRI (Prinsen et al., in preparation). However, these accumulations of IAA and ABA are severely reduced in the apices of all transformed plants.

Although exogenous cytokinins were sometimes reported to stimulate flowering (Morgan et al., 1983; Carlson et al., 1987; Lejeune et al., 1988) no significant accumulation of zeatin and/or its riboside, representing the major group of

8.0 r---------------~

6.0

0 E .5 4.0 < CD <

2.0

~ 0.0

0 4 8 12 16 20

Age (weeks)

Fig. 4: Absolute levels of ABA (nmol) ± S.E. present in the apical region of normal (e), T (.), T' (A) and rotA (~) tobacco plants grown during 20 weeks under controlled conditions, analyzed at different stages of development. At each time point, 10 to 20 indi­vidual explants were pooled for analysis.

84 E. PRINSEN, D. CHRIQUI, F. VILAINE, M. TEPFER, and H. VAN ONCKELEN

cytokinins in tobacco (Riidelsheim et al., 1987), could be correlated with flower initiation. However, somewhat higher endogenous cytokinin levels were only found at the beginning of vegetative growth. The competence of the ter­minal bud for flowering in Nicotiana species changes as a function of age. In day neutral tobacco species, it was dem­onstrated that terminal buds gain competence to perceive and/or respond to an existing signal for flowering (Singer et al., 1992). The elevated IAA levels found in 6-12 weeks old normal shoot tips as well as the higher endogenous cyto­kinin content at the beginning of this period could corre­spond to a mature developmental stage where the com­petence for flowering is acquired. This period corresponds well to the finding of Vansuyt et al. (1992) who indicated that flower induction occur at 41 days for day neutral Xanthi plants. In respect to this, the absence of cytokinin ac­cumulation in the rolA apices could be related to their long delay in flowering.

The results presented here emphasize the necessity for a detailed kinetic analysis in order to evaluate the relevance of varying phytohormone levels in the shoot apical region. It is clear that from the moment alterations have taken place, triggering has already occurred and only a detailed kinetic analysis of events preceding the appearance of morpholog­ical traits can clarify ambiguities.

Compared with the untransformed controls, apices of all transgenic plants (T, T' and rolA) displayed lowered endog­enous IAA levels and consequently attenuated basipetal auxin gradient, during mature vegetative growth.

This could be correlated with the observed shortened in­ternodes, the underlying inhibition of cell elongation and the reduced apical dominance, particularly in the T' and rolA plants. Also the delay in xylem differentiation in the T' and rolA plants could result from the reduced IAA levels as it is well known that xylogenesis and lignification are IAA controlled Gacobs, 1952).

Concomitant with the reduced IAA levels, no transient ABA accumulation was observed in transgenic shoot apices. Such lowered ABA levels were also reported for transgenic Brassica napus plants harbouring the entire TL-DNA Gul­liard et al., 1993). As tobacco plants solely transgenic for rolA display all these characteristics one can expect rolA ex­pression to be mainly responsible for the observed IAA and ABA changes in T and T' plants.

The observed difference in endogenous ZR concentrations (Fig. 3, 6 weeks) could be correlated with the varying root­ing capacities of the different phenotypes. T plants showed an overdeveloped rooting system as compared with the con­trol plants, whereas the solely expression of rolA (rolA plants) or the overexpression of rolA (T' plants) results in a drastically reduced rooting system (Chriqui et al., in prep.). However, it can not be excluded that the enhanced cytoki­nin concentration in the T phenotype is directly related to the expression of rolC, coding for a cytokinin-N-glucosidase (Estruch et al., 1991 a and b).

Taking into account these results together with the pre­viously reported shift in polyamine content (Martin-Tanguy et al., 1990; Mengoli et al., 1992), in polyamine oxidation (Mengoli et al., 1992), and the increase in plastid peroxidase and polyphenol oxidase activities (Chriqui et al., in prep.)

that occurred in the T, T' and rolA plants, it appears that rolA gene expression is correlated with an overall increase in oxidative processes. A more detailed study of the metabo­lism of each hormone in relation to the specific expression of rolA remains necessary.

Acknowledgements

This work was supported by a Belgian State Prime Minister's of­fice, Research Program IVAP 38 and a Bridge program nr BIOT­CT90-0179-(SMA). HVO is a research director of the Belgian N.F.W.O.

References

ACKERMANN, c.: Pflanzen aus Agrobacterium rhizogenes Tumoren an Nicotiana tabacum. Plant Sci. Lett. 8, 23-30 (1977).

BOURGIN, j.: Valine-resistant plants from in vitro selected tobacco cells. Mol. Gen. Genet. 161, 225-230 (1978).

CAPONE, 1., L. SPANO, M. CARDARELLI, D. BELINCAMPI, A. PETIT, and P. CONSTANTINO: Induction and growth roots properties of car­rot with different complements of Agrobacterium rhizogenes T-DNA. Plant Mol. BioI. 13,43-52 (1989).

CARLSON, D. R., D. j. DYER, C. D. COTTERMAN, and R. C. DURLEY: The physiological basis for cytokinin induced increases in pot set in IX93-100 soybeans. Plant Physiol. 84, 233 - 239 (1987).

CHRIQUI, D., A. GUIVARC'H, C. BESNARD, M. NOIN, F. VILAINE, M. TEPFER, E. PRINSEN, and H. VAN ONCKELEN: Comparative mor­phogenesis of tobacco plants transformed with the TL-DNA or the rolA gene of Agrobacterium rhizogenes. J. Plant Physiol. in prep.

DE BOUILLe, P., B. SOTTA, E. MIGINIAC, and A. MERRIEN: Hormones and inflorescence development in oilseed rape. Plant Physiol. Biochem. 27 (3),443 -450 (1989).

ESTELLE, M. A. and C. SOMMERVILLE: Auxin resistant mutants of Arabidopsis thaliana with an altered morphology. Mol. Gen. Ge­net. 206, 200-206 (1987).

ESTRUCH, j. j., D. CHRIQUI, K. GROSSMANN, J. SCHELL, and A. SPENA: The plant oncogene rolC is responsible for the release of cyto­kinins from glucoside conjugates. EMBO journ. 10 (10),2889-2895 (1991 a).

ESTRUCH, J. J., A. PARETS-SOLER, T. SCHMULLING, and A. SPENA: Cy­tosolic localization in transgenic plants of the rolC peptide from Agrobacterium rhizogenes. Plant Mol. BioI. 17, 547 -550 (1991 b).

HOREMANS, S., H. VAN ONCKELEN, P. RUDELSHEIM, and J. DE GREEF: Study of parameters involved in the determination of IAA and ABA in plant materials. j. of Exp. Bot. 35 (161), 1832-1845 (1984).

JACOBS, W.: The role of auxin in differentiation of xylem around a wound. Amer. J. Bot. 39,301-309 (1952).

jULLIARD, J., B. SOTTA, G. PELLETIER, and E. MIGINIAC: Enhancement of naphthaleneacetic acid-induced rhizogenesis in TL-DNA­transformed Brassica napus without significant modification of auxin levels and auxin sensitivity. Plant Physiol. 100, 1277 -1282 (1992).

jULLIARD, j., F. PELeSE, B. SOTTA, R. MALDINEY, C. PRIMARD-BRISSET, L. jouANIN, G. PELLETIER, and E. MIGINIAC: TL-DNA transfor­mation decreases ABA level. Physiol. Plant. 88, 654-660 (1993).

KELLY, M. and K. BRADFORD: Insensitivity of the diageotrophica to­mato mutant to auxin. Plant Physiol. 82, 713 -717 (1986).

KLEE, H. and M. ESTELLE: Molecular genetic approaches to plant hormone biology. Annu. Rev. Plant Mol. BioI. 42, 529-551 (1991).

LEJEUNE, P., J. M. KINET, and G. BERNIER: Cytokinin fluxes during floral induction in the long day plant Sinapis alba L. Plant Phys­iol. 86, 1095-1098 (1988).

MARTIN-TANGUY, J., D. TEPFER, M. PAYNOT, D. BURTIN, L. HEISLER, and C. MARTIN: Inverse relationship between polyamine levels and the degree of phenotypic alteration induced by the root-in­ducing, left-hand transferred DNA from Agrobacterium rhizoge· nes. Plant Physiol. 92, 912-918 (1990).

MAUREL, c., H. BARBIER-BRYGOO, A. SPENA, J. TEMPE, and J. GUERN: Single rol genes from the Agrobacterium rhizogenes TL-DNA al­ter some of the cellular responses to auxin in Nicotiana tabacum. Plant Physiol. 97, 212-216 (1991).

MENGOLI, M., D. CHRIQUI, and N. BAGNI: Protein, free amino acid and polyamine content during development of hairy root Nico· tiana tabacum plants. J. Plant Physiol. 139, 697 -702 (1992).

MIRZA, J., G. OLSEN, T. H. IVERSEN, and E. P. MAHER: The growth and gravitropic responses of wild type and auxin resistant mu­tants of Arabidopsis thaliana. Physiol. Plant. 60, 516-522 (1984).

MORGAN, D. G., J. M. KEILLER, and A. o. PRYNNE: Nitrogen growth regulators and pod development in oilseed rape. In: Interactions between nitrogen and growth regulators in the control of plant development. British Plant Growth Regulator Group mono­graph 9, 75-86 (1983).

MULLER, J. F., J. GOUJAUD, and M. CABOCHE: Isolation in vitro of naphthaleneaceitic acid-tolerant mutants of Nicotiana tabacum which are impaired in root morphogenesis. Mol. Gen. Genet. 199,194-200 (1985).

NILSSON, 0., A. CROZIER, T. SCHMDLLING, G. SANDBERG, and O. OLSSON: Indole-3-acetic acid homeostasis in transgenic tobacco plants expressing the Agrobacterium rhizogenes rolB gene. The PlantJ. 3,681-689 (1993).

NILSSON, 0., T. MORITZ, N. 1MBAULT, G. SANDBERG, and O. OLS­SON: Hormonal characterization of transgenic tobacco plants ex­pressing the rolC gene of Agrobacterium rhizogenes TL-DNA. Plant Physiol. 102, 363-371 (1993).

OWENS, L. D. and A. C. SMIGOCKI: Transfer of phytohormone genes to induce morphogenesis in plants. Adv. Cell Culture 7, 183-199 (1989).

PRINSEN, E., J. BERCETCHE, D. CHRIQUI, and H. VAN ONCKELEN: Pi· sum sativum epicotyls inoculated with Agrobacterium rhizogenes agropine strains harbouring various T-DNA fragments: Mor­phology, histology and Endogenous Indole-3-acetic acid and in­dole-3-acetamide. J. of Plant Physiol. 140, 75 - 83 (1992).

PRINSEN, E., P. RDDELSHEIM, and H. VAN ONCKELEN: Extraction, Pu­rification and Analysis of Endogenous Indole-3-acetic acid and Abscisic acid. In: NEGRUTlU, I. and G. GHARTI-CHHETRI (eds.): A laboratory guide for Cellular and Molecular Plant Biology, pp. 175-185 and pp. 323-324. Birkhauser Verlag, Basel/Boston/ Berlin (1991).

REDIG, P., E. PRINSEN, W. VAN DONGEN, and H. VAN ONCKELEN: Immunoaffinity Chromatography and LC-MS for ABA analysis. Physiol. Plant. 85 (3) II, pA 29 (1991).

RODBARD, D.: Statistical quality control and routine data processing for radioimmunoassays and immunoradiometric assays. Clin. chern. 20 (10),1255-1270 (1974).

ROMANO, C. P., M. B. HEIN, and H. KLEE: Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi. Genes Dev. 5, 438-446 (1991).

RDDELSHEIM, P., E. PRINSEN, M. VAN LUSEBETTENS, D. INZE, M. VAN MONTAGU, J. DE GREEF, and H. VAN ONCKELEN: The effect of

Hormone levels in pRi transformed tobacco 85

mutations in the T-DNA encoded auxin pathway on the endoge­nous phytohormone content in cloned Nicotiana tabacum Crown Gall tissues. Plant Cell Physiol. 28 (3),475-484 (1987).

SCHLENK, H. and J. L. GELLERMAN: Esterification of fatty acids with diazo methane on a small scale. Anal. Chern. 32, 1412 -1414 (1960).

SCHMDLLING, T., J. SCHELL, and A. SPENA: Single genes from Agro· bacterium rhizogenes influence plant development. EMBO J. 7 (9),2621- 2629 (1988).

SCHMDLLING, T., S. BEINSBERGER, J. DE GREEF, J. SCHELL, H. VAN ONCKELEN, and A. SPENA: Construction of a heat-inducible chi­meric gene to increase the cytokinin content in transgenic plant tissue. FEBS Lett. 249 (2),401-406 (1989).

SCHMDLLING, T., M. FLADUNG, K. GROSSMANN, and J. SCHELL: Hor­monal content and sensitivity of transgenic tobacco and potato plants expressing singe rol genes of Agrobacterium rhizogenes T-DNA. The Plant Journal 3 (3), 371-383 (1993).

SHEN, W. H., A. PETIT, J. GUERN, and J. TEMPE: Hairy roots are more sensitive to auxin than normal roots. Proc. Natl. Acad. Sci. USA 85,3417 -3421 (1988).

SHEN, W. H., E. DAVIOUD, C. DAVID, BARBIER-BRYGOO, J. TEMPE, and J. GUERN: High sensitivity to auxin is a common feature of hairy root. Plant Physiol. 94, 554-560 (1990).

SINGER, S. R., C. H. HANNON, and S. C. HUBER: Acquisition of com­petence for floral development in Nicotiana buds. Planta 188, 546-550 (1992).

SINKAR, V., F. PYTHOUD, F. WHITE, E. NESTER, and M. GORDON: rolA locus of the Ri-plasmid directs developmental abnormal­ities in transgenic plants. Genes Dev. 2, 688-698 (1988).

SPANO, L., D. MARIOTTI, M. CARDARELLI, C. BKANCA, and P. CON­STANTINO: Morphogenesis and auxin sensitivity of transgenic to­bacco with different complements of Ri T-DNA. Plant Physiol. 87, 479-483 (1988).

SPENA, A., J. J. ESTRUCH, G. HANSEN, K. LANGENKEMPER, S. BERGER, and J. SCHELL: The Rhizogenes tale: modification of plant growth and physiology by an enzymatic system of hydrolysis of phytohormone conjugates. In: NESTER, E. W. and D. P. S. VERMA (eds.): Advances in Molecular Genetics of Plant Microbe Interac­tions. Vol. II., pp. 109-124. Kluwer Acad. Pub I. (1993).

SPENA, A., J. J. ESTRUCH, E. PRINSEN, W. NACKEN, H. VAN ONCKE­LEN, and H. SOMER: Anther-specific expression of the rolB gene of Agrobacterium rhizogenes alters anther development and whole flower growth. Theor. Appl. Genet. 84, 520-527 (1992).

TEPFER, D.: Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the trans­formed genotype and phenotype. Cell 37, 959-967 (1984).

TEPFER, D. and J. TEMPE: Production d'agropine par des racines for­mees sous l'action d'Agrobacterium rhizogenes, souche A4. C.R. Acad. Sc. 292, 153 -156 (1981).

VAN ONCKELEN, H., E. PRINSEN, D. CHRIQUI, M. TEPFER, and J. DE GREEF: Endogenous Auxin Levels in Normal and Hairy Root Tobacco Plants, Med. Fac. Landbouww. Rijksuniv. Gent 53 (4 a), 1685 -1693 (1988).

VANSUYT, D., F. VILAINE, M. TEPFER, and M. ROSSIGNOL: rolA mod­ulates the sensitivity to auxin of the proton translocation cata­lyzed by the plasma membrane H+ -ATPase in transformed to­bacco. FEBS lett. 298, 89-92 (1992).

WEILER, E. W.: Radioimmunoassay for trans-zeatin and related cy­tokinins. Planta 149, 155-162 (1980).