Resistance of Transgenic Tobacco Seedlings Expressing the ...

Plant Cell Physiol. 43(8): 939–950 (2002) JSPP © 2002

Resistance of Transgenic Tobacco Seedlings Expressing the Agrobacterium tumefaciens C58-6b Gene, to Growth-Inhibitory Levels of Cytokinin is Associated with Elevated IAA Levels and Activation of Phenylpropanoid Metabolism Ivan Gális 1, 2, Petr Šimek 3, Henri A. Van Onckelen 4, Yasutaka Kakiuchi 1 and Hiroetsu Wabiko 1, 5 1

Biotechnology Institute, Akita Prefectural University, 2-2 Minami, Ohgata, Akita, 010-0444 Japan Institute of Plant Molecular Biology AS CR, Branišovská 31, 37005 eské Bud/jovice, Czech Republic 3 Institute of Entomology AS CR, Branišovská 31, 37005 eské Bud/jovice, Czech Republic 4 Universitaire Instelling Antwerpen, Universiteitsplein 1, Antwerpen, Belgium 2

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We previously reported that the Agrobacterium tumefaciens C58-6b gene confers resistance to growth-inhibitory levels of exogenously applied N6-benzyladenine (BA, cytokinin) in transgenic tobacco (Nicotiana tabacum) seedlings. Here, we found that intracellular levels of indoleacetic acid (IAA, auxin) increased in transgenics but declined in wildtype seedlings upon BA treatment. Since exogenously supplied 1-naphthalene acetic acid (NAA), a stable synthetic auxin, counteracted the growth inhibition of wild-type seedlings by BA, we suggest that BA-induced growth inhibition in wild-type seedlings occurs, at least in part, as a result of intracellular IAA deficiency. Further HPLC analysis of cell extracts from BA-treated seedlings revealed that a fluorescent compound, later identified as the phenylpropanoid, scopolin, and the major phenolic compound, chlorogenic acid, accumulated earlier in transgenics than in wild-type seedlings. Gene transcripts encoding phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase, which are responsible for the early steps of phenylpropanoid biosynthesis, accumulated earlier and to higher levels in transgenics than in wild-type seedlings as determined by Northern hybridization analysis, thus accounting for the early accumulation of scopolin and chlorogenic acid in transgenics. As some phenolic compounds, including chlorogenic acid and scopoletin (aglycon of scopolin) are suggested to inhibit IAA catabolism, we further propose that C58-6b gene expression protects IAA from degradation by inducing the early phenylpropanoid pathway.

Introduction Tumorous crown gall disease is induced in a variety of plants upon transfer and integration of a part (T-DNA) of the Ti plasmid harbored by pathogenic Agrobacterium tumefaciens strains (Van Larebeke et al. 1974, Chilton et al. 1977). The tumors are induced by the overproduction of, and an imbalance in, the plant growth substances, cytokinin and auxin, whose biosynthesis genes are located on the T-DNA. The ipt gene encodes isopentenyl transferase, and is responsible for the early step in the synthesis of a number of active cytokinins (Akiyoshi et al. 1984). The iaaM gene codes for tryptophan monooxygenase (Van Onckelen et al. 1986, Thomashow et al. 1986), and the iaaH gene encodes indoleacetamide hydrolase (Thomashow et al. 1984, Schröder et al. 1984), both of which are required for the synthesis of indoleacetic acid (IAA). In addition to these essential genes, T-DNA contains accessory genes that control and modulate the action of cytokinin and auxin. Of these genes, the 6b gene is capable of inducing tumors on a limited number of plant hosts (Hooykaas et al. 1988) by still unknown molecular mechanisms. The 6b gene codes for a 24 kDa-polypeptide in which no functional motifs or domains have so far identified. The putative 6b protein, however, possesses a limited homology to many other proteins encoded by tumor-related genes originating from A. tumefaciens and Agrobacterium rhizogenes, the latter of which induces hairy root disease (Levesque et al. 1988). In contrast to the ipt and iaaM/iaaH genes, the 6b gene apparently possesses a variety of activities depending on the A. tumefaciens isolates from which the gene is obtained. For example, the 6b gene from the Ti plasmid, pTiAch5, reduces the cytokinin activity to induce shoot formation (Spanier et al. 1989), whereas the 6b gene from pTiS4 enhances auxin and cytokinin effects on crown gall formation (Canaday et al. 1992). Additionally, the 6b gene (AK-6b) from pTiAKE10 induces callus formation on tobacco leaf discs in the absence of exogenous cytokinin and auxin in the growth medium (Wabiko and Minemura 1996). Of the numerous chemical substances that could interact with plant growth substances, certain phenolic compounds are

Keywords: Auxin — C58-6b gene — Cytokinin — Phenylpropanoids — Tobacco. Abbreviations: BA, N6-benzyladenine; BA-7-G, N6-benzyladenine-7-glucoside; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate:CoA ligase; MS, Murashige and Skoog; NAA, 1-naphthaleneacetic acid; PAL, phenylalanine ammonia-lyase; PCR, polymerase chain reaction; RP, reversed-phase; T-DNA, transferred DNA.

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Corresponding author: E-mail, [email protected]; Fax, +81-185-45-2678. 939

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Phenolics and auxin in cytokinin-treated tobacco

scopolin and chlorogenic acid (CGA), preferentially accumulate to higher levels than in wild-type control seedlings. We propose that the C58-6b gene modulates the production of phenolic compounds and these compounds protect IAA from degradation and thus counteract the growth inhibitory effect of cytokinins.

Results

Fig. 1 Growth of tobacco seedlings in response to BA treatment. Seeds from wild-type (WT, panels A, C) and the C58-6b-transgenic, 3026-1-2 (B, D) plant lines, were placed on hormone-free (A, B) or 2.2 mM BA-containing (C, D) MS medium, and cultivated under a 16-h photoperiod at 25°C for 2 weeks. Horizontal bar represents 1 cm.

known to influence cytokinin/auxin metabolism and action, and may thus play a considerable physiological role in plant growth and development. For example, dehydrodiconiferyl alcohol glucosides promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins (Teutonico et al. 1991) and a number of flavonoids have been demonstrated to be efficient inhibitors of auxin transport (Jacobs and Rubery 1988, Mathesius et al. 1998, Murphy et al. 2000). Furthermore, it has been demonstrated that, in vitro, some hydroxycinnamic acids, such as caffeic acid and ferulic acid, can inhibit IAA oxidase activity (Lee et al. 1982), which is responsible for decarboxylation of IAA, although such an IAA oxidasemediated pathway is considered to represent the minor part of IAA catabolism in vivo (Normanly et al. 1995). In contrast, caffeic acid has been shown to promote the activity of cytokinin oxidase (Wang and Letham 1995). These latter two observations suggest the possibility that some phenylpropanoids may participate in controlling the endogenous cytokinin/auxin balance, and thus may be of a great importance, as the cytokinin/auxin ratio is believed to play a fundamental role in determining plant growth and development. Previously, we reported that transgenic tobacco seedlings expressing the C58-6b gene from pTiC58 showed resistance to growth inhibitory levels of exogenous cytokinin (N6-benzyladenine, BA) without affecting the efficiency of cytokinin inactivation (Gális et al. 1999). We now show that, in these transgenic seedlings, the IAA levels transiently increase and that the phenolic compounds,

Resistance of transgenic seedlings to growth inhibitory levels of BA We transformed tobacco plants with the C58-6b gene, and obtained two independent lines, 3026-1-2, and 3026-32. The line 3026-1-2, which has been described previously, is a T1 generation carrying a single C58-6b gene insertion in a homozygous constitution. Another line used in this study, 3026-32, is a T0 generation, containing a single chromosome insertion in a heterozygous constitution. Seeds obtained from each transgenic plant were plated onto hormone-free MS medium or the same medium containing growth inhibitory levels of 2.2 mM BA. Transgenics grew slightly faster than wildtype seedlings on hormone-fee medium (Gális et al. 1999, data not shown). In contrast, transgenic seedlings showed typical resistance and growth promotion, whereas wild-type seedlings were inhibited (Fig. 1; Gális et al. 1999). Stem elongation and swelling, root enlargement, leaf expansion, and callus formation in hypocotyl region were typical morphological characteristics of transgenic seedlings in the presence of BA (Gális et al. 1999). The 3026-32-seedlings showed notably earlier and higher rates of growth than the 3026-1-2-seedlings (Fig. 2A). The ratio of the growth on the medium with BA to that without BA also demonstrated the preferential growth of 3026-1-2seedlings compared with wild-type seedlings (Fig. 2B). It should be noted that 3026-32-seedlings segregated in a 3 : 1 ratio with respect to the C58-6b gene (see Materials and Methods), and hence the weight measurements and subsequent molecular analysis of 3026-32-seedlings are underestimated due to the presence of slowly growing untransformed seedlings. Northern hybridization analysis indicated that the differential growth between wild-type and transgenic seedlings was accompanied by concomitant induction of C58-6b gene transcript accumulation, reaching maximum levels at 13 d of cultivation (Gális et al. 1999, data not shown). Intracellular IAA accumulation We have previously shown that the accumulation of glucosylated-BA (BA-7-G) is comparable between transgenic and wild-type seedlings upon BA treatment, and thus cytokinin inactivation does not seem to explain the cytokinin resistance of the transgenic plants (Gális et al. 1999). In many cases, cytokinins manifest their physiological effects on plant growth in association with auxin. To examine the possible involvement of auxin in BA-resistance, we determined intracellular


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Fig. 2 Growth of seedlings. Wild-type (triangle), 3026-1-2 (open square), and 3026-32 (closed square) derived seedlings were grown on hormone-free MS medium or the same medium supplemented with 2.2 mM BA as shown in Fig. 1, and the fresh weight (FW) of entire seedlings was measured. (A) Growth profiles of seedlings in the presence of BA. The mean ± SE fresh weight of a single seedling, obtained from three independent experiments are shown. (B) Ratio of the growth denotes that the fresh weight of wild-type (WT, open square) and transgenic (302612, closed diamond) seedlings grown with BA was divided by that of wild-type and transgenic seedlings grown without BA respectively.

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concentration as 57 mM of IAA (data not shown) did not counteract growth inhibition possibly because of the rapid catabolism of the exogenously administered IAA (see Discussion). Therefore, we employed the synthetic auxin, NAA, which is known to be metabolized at a slower rate than IAA in vivo. Indeed, addition of NAA, optimally at 0.27 mM, promoted the growth of wild-type seedlings in the presence of inhibitory BA levels, so that the growth rate and morphological appearance of these wild-type seedlings were comparable to those of BAtreated transgenic seedlings without NAA addition except that addition of both NAA and BA led to a slight undifferentiated callus growth (Fig. 4, upper panel). The BA-treated transgenic seedlings, however, showed no further growth increases when supplemented with NAA at the same concentration examined for wild-type seedlings (data not shown). From these results, we suggest that previously observed preferential accumulation of intracellular auxin in transgenic seedlings might have counteracted, at least in part, the growth inhibitory effect of cytokinins.

Fig. 3 Accumulation of IAA in BA-treated seedlings. Wild-type (WT, triangle) and transgenic (3026-1-2, open square) seedlings were treated with 2.2 mM BA, extracted and separated by two rounds of HPLC, and detected by fluorescence. Values correspond to the means ± SE of three independent measurements.

levels of the naturally occurring auxin, IAA, during seedling growth in the presence of BA. The IAA levels in wild-type seedlings continuously decreased, whereas in the transgenic 3026-1-2-seedlings, the IAA levels started to increase at day 8. Maximum levels, which were approximately twice those in the wild type, occurred at day 12, when the accelerated growth of transgenic seedlings was already apparent, and then gradually declined (Fig. 3). The decline of free IAA after day 12 in the transgenic seedlings possibly reflects the fact that the majority of IAA is typically maintained in small, localized tissues, for example shoot apices, while tissues with lower IAA content, such as leaves, expand greatly during this stage of seedling development. Thus, IAA accumulation in transgenic seedlings correlated with their preferential growth together with C58-6b transcript accumulation in the presence of BA. Effects of exogenous auxin on BA-induced growth inhibition We further examined whether exogenously added auxin could influence the growth of seedlings in the presence of BA. Seeds of the wild-type plant were plated onto MS medium containing increasing amounts of IAA together with growthinhibitory levels of BA. IAA was unable to support the growth of wild-type seedlings (Fig. 4, lower panel), and even as high a

Identification of scopolin as a differentially accumulating product in BA-treated seedlings To explore possible chemical compounds associated with IAA accumulation and hence BA-resistance, we prepared cell extracts from 13-day-old, BA-treated seedlings, and analyzed the products by HPLC according to the method which was described previously (Gális et al. 1999). A comparison of the UV270 HPLC chromatographic profiles revealed an early and efficient accumulation of a predominant compound, with a retention time of 35 min (and thus termed a compound, RT35) in the extracts prepared from young transgenic plants (Fig. 5). To identify the structure of the RT35 compound, the peak fraction was isolated, treated with b-glucosidase, and subsequently separated by HPLC. A shift in the retention time from 35 to 57 min (the corresponding compound, RT57) suggested the presence of a glucosylation pattern in RT35 molecule. We also detected a strong blue fluorescence of both RT35 and its aglycon (RT57) under UV (254 and 366 nm), and the UV absorbance spectrum of RT35 in water showed two maxima, at 292 and 340 nm. The molecular weights of the metabolite, 354, and its aglycon 192 (M minus 162, M minus glucose residue), were obtained from electrospray ionization (ESI) mass spectra (data not shown). To determine the structure of RT35 compound in more detail, the electron impact (EI) mass spectrum of the metabolite with a very weak molecular peak and aglycon fragments (Fig. 6). These data suggested a possible coumarin structure, and we therefore tested scopoletin as one of the potential structures of RT57 (RT35 minus glucose residue). HPLC and mass spectrometry data revealed that authentic scopoletin standard and RT57 were identical, confirming that the unknown RT35 structure is scopoletin glucoside, scopolin. An early and large-scale increase in scopolin content was observed in transgenic plants at 9–11 d, whereas scopolin in the controls began to accumulate only after 16 d of cultivation (Fig. 7). Furthermore, the 3026-32-seedlings, which showed


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Fig. 4 Effect of auxins on the growth of BA-treated wild-type seedlings. Wild-type seedlings were cultivated on MS media containing the indicated amounts of either IAA or NAA, together with 2.2 mM BA under a 16-h photoperiod at 25°C for 2 weeks.

stronger resistance to BA than 3026-1-2-seedlings, began to accumulate scopolin earlier than the 3026-1-2-seedlings, indicating that differential accumulation of scopolin is correlated with the degree of resistance of transgenic seedlings to BA (Fig. 7). We identified only low levels of scopolin (maximum about 0.5 mg per g seedling) in both wild-type and transgenic seedlings without treatment of cytokinin. The scopoletin levels were low and usually represented less than 1–2% of the total scopolin content in both transgenic and wild-type seedlings upon BA treatment. Intracellular CGA accumulation Since scopolin was preferentially accumulated in transgenic seedlings by BA treatment, the levels of other phenolic compounds could also be influenced. Among the diverse molecular species of the soluble phenolic fraction, CGA has been reported to comprise the major phenolic constituent of tobacco plants (Elkind et al. 1990). In order to examine changes in CGA levels, we separated cell extracts from BAtreated seedlings by HPLC and examined the absorbancy at 270 nm. A major UV270-absorbing peak of the separated phenolics was found to be CGA as determined by ESI mass spectra (data not shown). Intracellular CGA levels declined between 7 and 9 d of cultivation in all transgenic and wild-type seedlings (Fig. 8), during which time the differential growth patterns were not yet evident. However, after 8 d, the CGA began to accumulate differentially; accumulation was rapid and continu-

Fig. 5 HPLC analysis of the BA (2.2 mM)-treated extracts from 13day-old seedlings. (A) wild-type control plants; (B) C58-6b transgenic line, 3026-1-2. The main peak at 35 min (RT35) is present predominantly in the transgenic plants.

ous in the transgenic seedlings, but remaining at steady-state levels in wild-type seedlings until 16 d (Fig. 8). The rate of CGA accumulation was more rapid in the 3026-32 seedlings with stronger resistance to BA, than in the less resistant 3026-

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Fig. 6


EI-mass spectrum of the HPLC isolated peak fraction at 34–35 min (RT35).

Fig. 7 Accumulation of scopolin in BA-treated seedlings. Wild-type (WT, triangle) and transgenic (3026-1-2, open square; 3026-32, closed square) seedlings were treated with 2.2 mM BA, and extracts were analyzed by HPLC/fluorescence detection. Values correspond to the means ± SE of three independent experiments.

Fig. 8 Accumulation of CGA in BA-treated seedlings. Wild-type (WT, triangle) and transgenic (3026-1-2, open square; 3026-32, closed square) seedlings were treated with 2.2 mM BA, extracted and separated by HPLC, and detected by UV absorbance detector at 280 nm. Values correspond to the means ± SE of three independent experiments.

1-2 seedlings. After 20 d, all plants accumulated CGA at comparable levels. These results demonstrate that CGA levels correlate well with the extent of BA-resistant growth during early seedling development.

Phenylpropanoid biosynthesis gene expression Scopolin and CGA are one of the stable end-products of the branching pathway of phenylpropanoid metabolism as depicted in Fig. 9. The phenylpropanoid biosynthesis pathway


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Fig. 9 The biosynthesis pathway of phenolic compounds according to Harborne (1980). The steps in which PAL, C4H, and 4CL enzymatic activities are involved are shown. The thick arrow implies that cytokinin induces PAL gene expression. CGA and scopolin are double-underlined.

is initiated by phenylalanine ammonia-lyase (PAL), which converts L-phenylalanine to trans-cinnamic acid. This compound is further metabolized to p-coumaric acid by cinnamate 4-hydroxylase (C4H), and to other derivatives by steps catalyzed by 4-coumarate:CoA ligase (4CL) and other enzymes. To address the possibility that the induction of gene expression encoding these enzymes could be responsible for the increased accumulation of scopolin, transcript levels of these genes at different stages of seedling development in the presence of BA were examined by Northern hybridization analysis. PAL transcripts in both transgenic and wild-type seedlings without BA treatment showed comparable levels throughout the cultivation period (Fig. 10). Upon BA treatment, PAL transcripts increasingly accumulated with time in both wild-type and transgenic seedlings, however, the transcripts in transgenic seedlings were induced earlier and to higher levels than those in wild-type seedlings throughout development (Fig. 10), notably even at 9 d when differential growth among tobacco lines could not yet be observed (Fig. 2). The accumulation of both C4H and 4CL transcripts was induced by BA in both transgenic seedlings and wild-type seedlings. As with PAL gene induction, C4H and 4CL transcripts accumulated earlier in transgenic seedlings than in wild-type controls (Fig. 10). These results indicate that the PAL, C4H, 4CL genes are simultaneously induced by BA treatment and that the higher transcript levels in transgenics strongly correlate with the preferential accumulation of scopolin and CGA.

Discussion Expression of the A. tumefaciens C58-6b gene, as induced by BA in transgenic seedlings, antagonizes the growthinhibitory effect of BA. We have identified specific metabolic alterations in the transgenic seedlings, which we propose are

Fig. 10 Northern blot analysis of PAL, C4H and 4CL gene expression. Seedlings from wild type (WT), transgenic lines, 3026-1-2 and 3026-32 were cultivated either with (+) or without (–) BA (2.2 mM). Northern hybridization was performed, using 32P-labeled PAL, C4H and 4CL gene fragments as probes. The same filters were rehybridized with a probe encoding the 18S rRNA of A. thaliana to standardize loading. Numbers above individual lanes represent days of cultivation of the seedlings.

responsible for their resistance phenotype. First, a naturally occurring auxin, IAA, while found to decline in wild-type plants, began to increasingly accumulate in transgenic seedlings in response to BA. Second, the phenolic compounds, scopolin and CGA, were found to accumulate more rapidly and to greater extents in transgenic than in wild-type seedlings. Auxin counteracts the growth inhibition by cytokinin One notable early phenotypic trait of BA-treated seed-

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lings was an enhanced hypocotyl elongation of C58-6b transgenics compared to that of wild-type controls (Gális et al. 1999). This elongation could have reflected the enhanced auxin levels in transgenic seedlings, since auxin is known to promote stem elongation (reviewed by Evans 1985). The conclusion that the increased intracellular IAA levels in transgenic seedlings may counteract the growth-inhibitory effects of BA is reinforced by the finding that exogenous application of the synthetic auxin, NAA, in the medium, together with BA, alleviates the growth inhibition in wild-type seedlings. The optimal NAA concentration for the growth alleviation was approximately 0.3 mM, which suggests that relatively small amounts of NAA are sufficient to counteract the BAinduced growth inhibition. In contrast to NAA, when various concentrations of IAA were added to the medium with BA, virtually no growth enhancement in wild-type seedlings could be observed. This discrepancy could be related to the differential metabolism of NAA and IAA, particularly when the hormones are supplied exogenously. Ribnicky et al. (1996) demonstrated that the intracellular levels of free NAA in NAA-treated carrot hypocotyls remained high, whereas most of the externallysupplied IAA was readily converted to conjugated forms that are considered to be physiologically less-active or inactive. Furthermore, both the free and conjugated forms of IAA were degraded in carrot upon extended culturing (Ribnicky et al. 1996). Therefore, the absence of growth enhancement of BAtreated wild-type seedlings upon addition of IAA could be due to the rapid conversion of the active, free IAA to biologically inactive (or less active) conjugated forms and subsequent catabolism by oxidization (Bandurski et al. 1995). From these observations, we consider it most likely that IAA is rapidly metabolized in BA-treated wild-type tobacco seedlings, whereas in C58-6b-transgenic seedlings, free, active IAA is maintained at levels that are sufficient to counteract the BAinduced growth inhibition. Tinland et al. showed that the 6b gene from pTiTm4 (T6b) increased tumor formation induced by the iaa (auxin biosynthesis) genes (Tinland et al. 1989), and further suggested that the plant growth response could have resulted from reduction of inhibitory effects of high auxin levels (Tinland et al. 1990). However, no cytokinin was required to reveal such effects. The T-6b gene conferred increased sensitivity of N. rustica protoplast to exogenously applied NAA and BA. This is consistent with the hypothesis that the 6b gene could protect auxin from inactivation (Tinland et al. 1992). C58-6b gene-mediated activation of phenylpropanoid synthesis In both wild-type and transgenic tobacco seedlings, BA treatment activated transcript levels of PAL, C4H, and 4CL genes, and led to a higher accumulation of both scopolin and CGA in the tissues. These results are consistent with the previous reports of the co-ordinated induction of PAL, C4H, and 4CL gene expression (Logemann et al. 1995, Reinold and Hahlbrock 1996, Koopmann et al. 1999), and also in good

agreement with the fact that PAL gene expression is induced by cytokinins (Nagai et al. 1994), and that the resulting increase in PAL activity enhances scopolin accumulation in tobacco tissue cultures (Hino et al. 1982, Sharan et al. 1998). In addition, tobacco plants expressing the ipt gene accumulated more scopolin as compared with untransformed plants (Hamdi et al. 1995). Elevated PAL levels in tobacco resulted in high levels of a number of phenolics, including CGA, supporting evidence that PAL controls the influx into the phenylpropanoid biosynthesis pathway (Bate et al. 1994). Furthermore, Tamagnone et al. (1998) reported that tobacco plants transgenic for the Myb308 transcription factor gene, which specifically suppresses C4H and 4CL transcript levels, contained fewer phenolic compounds including CGA, providing evidence that the early steps of phenylpropanoid synthesis are important in determining the amount of end products of the pathway, such as scopolin and CGA. While co-activation of PAL/C4H/4CL gene expression was similar in both transgenic and wild-type seedlings, the difference appeared to be the earlier up-regulation of the PAL/C4H/4CL gene expression in transgenics compared to wild-type seedlings in response to BA, resulting in an earlier and more pronounced accumulation of the phenolic compounds, scopolin and CGA. It could be that increased C58-6b gene product directly promotes PAL/C4H/4CL transcript accumulation. This hypothesis is consistent with the finding that the AK-6b gene might be directly involved in the transcriptional control of cell division (Kitakura et al. 2002). Alternatively, cytokinin levels or sensitivity to cytokinin could be increased in C58-6b-transgenics, and so induce the onset of accumulation of PAL/C4H/4CL transcripts. This possibility, however, seems less likely, since no observable changes in cytokinin metabolism were found in transgenic seedlings (Gális et al. 1999). Furthermore, if sensitivity to cytokinins would be increased, we would expect the C58-6b gene expression to enhance the growth-inhibitory effects of BA on transgenic seedlings. In fact, the C58-6b gene confers the reverse effect, cytokinin resistance. Spanier et al. (1989) reported that the 6b gene from pTiAch5 reduced cytokinin activity (namely, reduced shoot formation) under moderate concentration of BA application, whereas the same gene stimulated shoot formation at excess concentration of BA. The 6b genes from pTiAch5, pTiC58 and pTiTm4 have been reported to stimulate cytokinin-induced (upon ipt gene expression) tumor growth (Tinland et al. 1989). It may be that when the cytokinin levels are toxic, the 6b gene could confer cytokinin resistance. It has been reported that PAL transcripts accumulation and enzymatic activity were inhibited by the product, t-cinnamic acid (Bolwell et al. 1988, Blount et al. 2000). Similar feedback inhibition was observed in enzymatic activities of C4H, 4CL (Lamb and Rubery 1976, Knobloch and Harlbroch 1977). The C58-6b gene may act to release such product-associated feedback inhibition of PAL/C4H/4CL gene expression or enzymatic activity, which may lead to preferential PAL/C4H/4CL gene


expression. To validate such product inhibition, measurement of phenolic intermediates, such as t-cinnamic acid or pcoumaric acid, is necessary. However, so far we are unable to determine the predominant intermediates presumably due to rapid turnover. Relationship between IAA accumulation and phenylpropanoid metabolism in BA-treated transgenic seedlings Cellular levels of IAA are maintained by a complex homeostatic regulatory mechanisms including biosynthesis, transport, conjugation, and catabolism. Eklöf et al. (1997) reported that transgenic tobacco expressing the ipt gene and hence continuously generating cytokinins at high levels, accumulated less free IAA than wild-type plants as a result of reduced IAA synthesis. However, in our case, if the continuous decline of IAA in BA-treated wild-type seedlings was caused by the defective de novo biosynthesis of IAA, exogenously supplied IAA in excess should have resulted in elevated intracellular accumulation of free IAA, which ultimately could counteract the growth inhibition of the wild-type seedlings by BA. However, as we noted above, exogenous IAA did not restore the growth of BA-treated wild-type seedlings. In addition to free forms, IAA also exists as conjugates with sugars and amino acids (Östin et al. 1998). To examine the possibility that the C58-6b gene mediates release of free IAA from its conjugates in BA-treated seedlings, the measurement of total IAA is needed. Nevertheless, the feeding experiment with IAA and its stable, oxidase-resistant analog NAA suggests that the susceptibility to degradation, rather than the conjugation rate per se, is responsible for auxin-mediated growth restoration in BA-treated seedlings. Ribnicky et al. (1996) has shown that even though the proportions of conjugated IAA and NAA in carrot tissues are similar, the IAA and its conjugates are rapidly degraded, while free and conjugated NAA levels stay high during the course of the experiment. Thus, stability of IAA could be responsible for higher levels of IAA accumulation and hence resistance to BA in transgenic in comparison with wild-type seedlings. Inactivation of IAA is suggested to take place through decarboxylative and non-decarboxylative oxidative reactions. Several phenolic compounds have been suggested to protect IAA from decarboxylation (Lee et al. 1982), whereas diphenols, such as caffeic acid and CGA, inhibited IAA oxidation catalyzed by the tobacco anionic peroxidase (Gazaryan and Lagrimini 1996). The peroxidase is reported to be less active in the presence of scopoletin (scopolin aglycon) (Sirois and Miller 1972). Despite such numerous studies, the physiological importance of the peroxidase-catalyzed oxidation of IAA is limited (Normanly et al. 1995), as exemplified in wound response (Catalá et al. 1994). In the present study, however, we demonstrate that the resistance to cytokinins in C58-6b transgenic tissues is accompanied by the increased levels of IAA in transgenic plants. The increased IAA accumulation in transgenic 3026-1-2 line started at day 8 of cultivation (Fig. 3),

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almost coincident with the increased accumulation of CGA (Fig. 8). The result suggests that CGA and/or metabolic intermediates leading to CGA synthesis act as possible protectants of IAA. By contrast, increase of IAA preceded the increased scopolin accumulation which occurred after 10 d (Fig. 7). It is possible that metabolic intermediates of scopolin synthesis (rather than scopolin itself, Fig. 9) protected IAA from inactivation at the early stages of seedling development. At later times, scopolin, together with the phenolic compounds described above, might be synergistically involved in IAA protection. Preliminary experiments showed that exogenous application of CGA or p-coumaric acid together with BA conferred resistance to BA to wild-type seedlings, and thus supporting the conclusion that IAA is stabilized by phenolic compounds. The coordinate accumulation of IAA and phenolic compounds, together with the finding that oxidase-resistant auxin NAA induces growth of the cytokinin inhibited wild-type seedlings, suggests the possibility that under specific conditions, such as cytokinin stress, the IAA decarboxylation pathway could participate in the control of IAA levels in plant tissues and that activation of phenylpropanoids by C58-6b gene may counteract IAA inactivation. Overall, our observations suggest the interesting possibility that in some cases the phenylpropanoid pathway may serve as an intrinsic regulatory system that controls the endogenous cytokinin/auxin balance which is believed to play a fundamental role in plant growth and development.

Materials and Methods Recombinant clones The recombinant plasmid, pCB3026 containing the C58-6b gene from the pTiC58 of A. tumefaciens C58 strain, has been described previously (Gális et al. 1999). Intragenic regions of the genes encoding PAL, C4H, and 4CL were amplified by PCR as follows. PCR was performed as described previously (Gális et al. 1999), except that an annealing temperature of 58°C was employed to isolate the 4CL gene segment. Primers, 5¢-CCTTGCTCTTGTGAATGGTA-3¢ and 5¢-AGGTTCTCTTAGCGACTTGG-3¢, and genomic DNA of Nicotiana tabacum cv. Samsun as a template, were used to isolate the 879-bp PAL gene intragenic segment (Accession no. X78269). To isolate the C4H gene segment, primers were designed based on the sequences reviewed by Nedelkina et al. (1999). Nested PCR was performed with the genomic DNA used above, first with the degenerate primers, 5¢-GA(C/ T)(A/C)TCTTCCTCCT(T/C)CG(C/T)ATG-3¢ and 5¢-GCNGG(A/G) TT(A/G)TTNGCNA(A/G)CCACCA-3¢, and subsequently with the nested primers, 5¢-AT(A/C/T)TT(C/T)ACNGGNAA(A/G)GGNGA(A/ G)GA(C/T)ATG-3¢ and 5¢-C(T/A)CCATA(G/A)TTATA(C/A)TCAAA (G/A)CTCTG-3¢, generating a 350-bp DNA fragment. To amplify the 610 bp-4CL gene segment, primers 5¢-GCGATTTTGATTATGCAG-3¢ and 5¢-GTTGGGATGGTTGAGAAG-3¢ were used together with cDNA isolated from N. tabacum cv. Xanthi NC callus tissues to obtain the mixture of both isoforms of the structurally-similar 4CL genes of tobacco (Accession no. U50845, U50846). Amplified DNA segments were cloned into the vector, pGEM-T, and the identity of the amplified DNAs was verified by DNA sequence determination (PAL, C4H) or extensive restriction analysis (4CL).

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Transgenic plants and growth conditions The C58-6b gene was introduced into tobacco plants (N. tabacum L. cv. Samsun) by Agrobacterium-mediated gene transfer as described previously (Gális et al. 1999). The transgenic and wild-type plants were transferred to soil and allowed to grow under 16 h-light/ 8 h-dark conditions at 27°C. Seeds obtained from two independently isolated transgenic plant lines were used in this study. One line, 30261-2, which has been described previously, is one of the T1 progenies of the primary transformant 3026-1, and carries a single insertion of the C58-6b gene in a homozygous constitution. Another line, 3026-32, which was used in this study, is a primary transformant (T0 generation), containing a single chromosome insertion as evidenced by a Mendelian segregation (3 : 1) for kanamycin resistance in the T1 generation. The seeds obtained from the line 3026-1-2 (T2 generation), and those from 3026-32 (T1 generation), were surface-sterilized with 1% (v/v) sodium hypochlorite for 5 min and placed onto modified MS medium (Gális et al. 1999) supplemented with BA, IAA, and NAA at various concentrations. Basically it takes several days for seeds to germinate and green tissues appeared in a further 2–3 d. Growth measurement was initiated at 7 d after seeds were placed onto the media, when green tissues were fully established. IAA measurements The method employed for purification and analysis of IAA was that described by Prinsen et al. (1995). In brief, whole seedlings were homogenized in liquid nitrogen and extracted in 80% (v/v) methanol at 4°C. A tritium-labeled internal standard 3-[5(n)-3H]indolylacetic acid (specific activity 777 GBq mmol–1, 0.5 kBq per sample, Amersham), was included with the methanol solution to estimate the recovery efficiency of the purification procedure. Supernatants, obtained by centrifugation of the extracts, were further pre-purified over reversed-phase (RP)-C18 columns (Sep-Pak cartridges, C18, Waters, Milford, U.S.A.), evaporated to the water phase and diluted with 0.04 M (pH 6.5) ammonium acetate buffer. Samples were passed through a DEAE Sephadex A25 (Pharmacia Biotech, Uppsala, Sweden) column, and the IAA retained on the column was eluted with 4% formic acid, followed by concentration on an underneath-coupled RP-C18 cartridge. IAA was eluted from the cartridge with 100% ether and evaporated to dryness. Extracts were purified by preparative ion suppression RP-HPLC (Waters 600 Multisolvent Delivery System; Puresil C18 column, 150´4.6 i.d. mm, Waters): samples were run isocraticaly in 40/0.5/59.5 (v/v/v; methanol/acetic acid/water) at a flow rate 0.5 ml min–1, relevant fractions were collected and evaporated to dryness. IAA was further separated using ion pairing RP-HPLC in a buffer consisting of 40/60 (v/v); methanol/10 mM tetra butyl ammonium hydroxide, 1 mM potassium phosphate pH 6.6 at a flow rate of 0.5 ml min–1 and determined with fluorescent detector (Waters 474, excitation at 285 nm and emission at 360 nm). These two successive rounds of HPLC under different pH conditions allowed us to separate and measure IAA in a pure form. Mass spectrometric analysis ESI mass spectra were recorded on a LCQ ion trap mass spectrometer (Thermoquest/Finnigan, San Jose, U.S.A.). Samples, dissolved in 50% methanol, were continuously infused into the electrospray ion source held at 3.5 or 4 kV. EI mass spectra of the unfused samples were recorded on a double focusing profile mass spectrometer (Kratos, Manchester, U.K.). Samples, placed in glass capillaries, were introduced into the mass spectrometer via a direct insertion probe. The mass spectrometer operating conditions were set at 70 eV energy, 200°C ion source temperature, 2000 resolution (static, 10% valley), 100 mA ion current, and a 50–500 mass range scanned at 3 s

per decade. Molecular weight of the fragments was estimated by the peak matching technique (resolution 7000, static, 10% valley) with perfluorokerosene as a reference in the ion source. Scopolin and CGA measurements Initially, to detect scopolin, plant materials (1–2 g) were extracted in 80% (v/v) methanol, purified by DEAE Sephadex, and analyzed by RP-HPLC with a UV absorbance detector at 270 nm as described previously (Gális et al. 1999). Subsequently, we simplified the procedure as follows. Entire plant tissues (100 mg), including shoots and roots, were homogenized in 400 ml of 80% (v/v) methanol, incubated for 60 min on ice and centrifuged for 20 min at 18,500´g in a micro centrifuge at 4°C. Ten-ml aliquots of the supernatant were separated by HPLC (column 5mSymmetry Shield RP8, 4.6´250 mm and Waters 600E multisolvent delivery system, Waters Co., Milford, U.S.A.) using a gradient of 100% methanol/20 mM sodium acetate pH 5 buffer (starting at a ratio of 25/75; in 4.5–15 min linearly to 70/ 30; in 25–30 min, linearly from 70/30 to 100/0) at a flow rate of 1 ml min–1. The scopolin peak fraction was detected using a fluorescence detector (Waters 474 Scanning Fluorescence Detector), with excitation and emission parameters of 360 nm and 460 nm, respectively. Since an authentic scopolin standard is not commercially available, we used scopoletin for quantification of scopolin. To this end, the isolated scopolin peak was treated with almond b-glucosidase (1 unit per sample, Sigma) to release scopoletin which, under our experimental conditions, possessed 4.9 times higher fluorescence intensity than scopolin. This value was used as a recalculation factor for scopolin content. For CGA measurement, extracts were separated similarly by RPHPLC and detected with a UV absorbance detector at 280 nm. The UV spectrum of the corresponding peak was determined and compared with that of CGA standard and its identity was further confirmed by ESI mass spectrometric analysis. Northern hybridization analysis To isolate total RNA, plant tissues (1 g) were ground in liquid nitrogen and mixed with 5 ml of extraction buffer (50 mM Tris-HCl pH 8, 300 mM NaCl, 5 mM EDTA, 2% (w/v) SDS, 10 mM bmercaptoethanol, and 1 mM aurintricarboxylic acid). The samples were heated for 2 min at 50°C, followed by the addition of KCl to a final concentration of 0.3 M, and then centrifuged (20,000´g) for 10 min at 4°C. RNA was precipitated from the supernatant by the addition of 8 M LiCl to give 2 M. The precipitates were dissolved in 0.5 ml of water, treated with phenol, and the RNA was precipitated with 50 ml of 3 M sodium acetate pH 5.2 and 550 ml of isopropanol. Aliquots of RNA were separated by 1% agarose-formaldehyde gel electrophoresis and blotted onto Hybond-N+ nylon membranes (Amersham, U.K.). Northern hybridization was performed at 42°C in the presence of 50% formamide with 32P-radioactively-labeled probes as described previously (Wabiko et al. 1990). Membranes were washed under low stringency washing conditions (2´ SSC, 0.5% SDS at 65°C) to ensure identification of mRNAs of various PAL, C4H and 4CL isoforms encoded by similar nucleotide sequences.

Acknowledgments We thank Drs. S. Youssefian and J. L. Smith for critical reading of the manuscript, Dr. W. Van Dongen for the ES-MS/MS analysis, and Ms. E. Kudo for helpful technical assistance. This research was financially supported by the Sasagawa Scientific Research Grant (No. 9-264) from the Japan Science Society, and by The Naito Foundation to I.G.


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(Received January 2001; Accepted June 5, 2002)