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IAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro
Plant Science 153 (2000) 73 – 80 www.elsevier.com/locate/plantsci
IAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro Odile Faivre-Rampant *, Claire Kevers, Thomas Gaspar Hormonologie Fondamentale et Applique´e, Institute of Botany B 22, Uni6ersity of Lie`ge-Sart Tilman, B-4000 Lie`ge, Belgium Received 10 May 1999; received in revised form 30 November 1999; accepted 3 December 1999
Abstract The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, rac, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the rac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However, this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a Sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the rac mutant are discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Auxin oxidase; Peroxidase; Phenolic compounds; Rooting; Tobacco mutant
1. Introduction Mutants resistant, in other words more tolerant, to the exogenous application of high concentrations of natural and synthetic auxins have been isolated [1 – 4]. Such mutants might be resistant either because the level of endogenous hormone is different from the one in the wild-type or because of changes at the primary site of auxin action [5]. Shoots of the nonrooting rac mutant of tobacco [2] should belong to the latter category: according to Lund et al. [6], this rac mutant does not disrupt auxin concentrations, but involves some changes in the receptor affinity for auxin and/or in the efficiency of the transduction pathway. The problem of this mutant regarding rooting difficulties is complicated: perivascular cells in mutant stem cut-
* Corresponding author. Tel.: +32-4-3663914; fax: +32-43663859. E-mail address: [email protected] (O. Faivre-Rampant)
tings treated with IBA divide, but never form adventitious root meristems [6]. We have established in vitro shoot-forming cultures of the ‘auxin-resistant’ (nonrooting) rac mutant of tobacco and of its homologous wild-type [7]. Shoots of the rac mutant grow at a lower rate. They are characterized by higher lignin level, higher soluble peroxidase activity and higher ethylene production. These observations might be causally related to the growth inhibition as similar events have been noticed in different stress-induced growth limitations, through the cell wall rigidification and the auxin catabolism. However these relationships did not give any explanation of the rooting difficulties. In an attempt to biochemically characterize this rac mutant further, we have taken the opportunity to use its shoots in order to perform parallel analyses of auxin protection and peroxidase activities in comparison with those of its wild homologue. Gaspar et al. [8,9] indeed characterized the successive physiological phases of adventi-
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tious root formation by typical changes of peroxidase activity and polyphenolic compounds, the latter being generally active as auxin protectors. Basic isoperoxidases have been suggested to be the most effective in IAA degradation [10]. Changes in the isoperoxidase profiles were specifically observed on the cathodic side when concomitant variations of endogenous IAA levels were measured [11]. It has also been observed that the activity of certain basic isoperoxidases involved in auxin catabolism is prevented by phenolic compounds present in the extracts [12]. The activity is affected by the number of hydroxyl groups and their position relative to each other as well as to other substituents on the aromatic ring. Monophenols generally stimulate the IAA oxidation with p-substituted monophenols that are more active than o- or m-compounds, whereas o- and p-diphenols act as inhibitors (m-diphenols activate the IAA oxidation) [13]. Direct interaction and competition between the enzyme, the inhibitor or the stimulator and IAA have been discussed as the mechanism [14], as well as the functioning of the inhibitors as radical scavengers [15]. It has also been shown that different results concerning the stimulation or the inhibition can be due to different concentrations of the tested phenolic derivative [13].
2. Material and methods
2.1. Plant material The wild-type, D8, and the homozygous, rac, mutant seeds of Nicotiana tabacum cv Xanthi were surface-sterilized for 3 min in a 10% (v/v) commercial bleach solution, placed on a MS medium [16], pH 5.7, containing 0.8% (w/v) Roland agar (Brussels), 3% (w/v) sucrose and 0.13 mM BAP, as previously reported by Lund et al. [6]. Shoot apices or stem nodes were explanted for continued culture through 3 week-cycles. Cultures, in 600 ml cylindrical glass jars, containing 100 ml of MS medium (as above), covered by a glass lid maintained with a sheet of transparent plastic film, were placed under a 16 h photoperiod (Sylvania Grolux fluorescent tubes providing 15 mE m − 2 s − 1) at 28°C (day) and 25°C (night). The wild shoots showed roots at the end of each multiplication cycle, while the rac shoots did not root.
Whole shoots and basal third parts of the stems (without leaves) were sampled for the analyses. Basal parts were analyzed because they are the sites of root formation, at least in the wild-type.
2.2. Peroxidase acti6ity and isoperoxidases Frozen explants (300 mg fresh weight taken at day 7, 14 and 21 during the growth cycle) were ground in 1 ml phosphate buffer (0.06 M, pH 6.1) and centrifuged for 10 min at 11 000×g at 4°C. The supernatant was used as the crude enzyme extract. The guaiacol-peroxidase activity and the peroxidase isoenzyme patterns by vertical starch gel electrophoresis were determined according to Darimont and Gaspar [17]; the activity was expressed in mg equivalent horseradish peroxidase (HRP) (Boehringer–Manheim) per g fresh weight. The gels were developed with benzidine. The protein concentration was determined by the Coomassie blue method [18] using bovine serum albumin as standard.
2.3. Separation and assay of auxin protectors and IAA-oxidase acti6ity The techniques to obtain auxin protectors and the determination of IAA-oxidase activity have been described elsewhere [19]. Three grams of fresh mass were ground in chilled phosphate buffer (0.06 M, pH 6.1) at a ratio of 1 g of tissue per ml of buffer. The extract was centrifuged for 20 min at 20 000×g. The resulting supernatant was filtered through a G-100 Sephadex column (2.5×35 cm2) and 2 ml samples were collected serially with a fraction collector. Their absorption was recorded at 280 nm. The assay for the inhibitors of enzymatic destruction used 4 ml reaction mixtures containing 3 ml phosphate buffer (0.06 M, pH 6.1), 0.5 ml gel fraction, 0.25 ml HRP (Boehringer–Manheim, 3 mg 10 ml − 1) and 0.25 ml IAA (4.10 − 3 M). In assays of Fig. 5, the gel fraction was replaced by 0.5 ml of a chlorogenic acid solution (10 − 3 M). The auxin protection results were expressed in percentage of inhibition of HRP used in conditions to work as IAA-oxidase. The assay for the IAA-oxidase activity used 3 ml reaction mixtures containing 2 ml phosphate buffer (0.06 M, pH 6.1), 0.25 ml gel fraction, 0.15 ml MnCl2 (10 − 3 M), 0.3 ml DCP (10 − 3 M), 0.3 ml IAA (4.10 − 3 M). The IAA-oxidase activity was
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expressed in percentage of IAA destroyed (100% IAA is in the control without enzymic fraction) calculated from the absorbance of Salkowski’s reagent at 530 nm.
2.4. Phenol content Soluble phenolic compounds were extracted from 150 mg of fresh weight by the method of Legrand [20]. The phenol content was assayed with the Folin and Ciocalteu reagent. Absorption at 750 nm is proportional to the phenolic compounds level. The results were expressed in mg of phenolic compounds (using chlorogenic acid as a reference) per mg of fresh weight. Precisely, in another work in preparation concerning the identification of the phenolics in tobacco, chlorogenic acid was found as the major compound as already shown by Vereecke et al. [21]. All the results are means of at least three separate experiments (9SD).
Fig. 2. Isoperoxidase zymograms of the different parts of the wild (D8) and mutant (rac) tobacco shoots. AS: apical stem; AL: apical leaves; BS: basal stem; BL: basal leaves. Each lane was loaded with the same amount of proteins (4.5 mg). The isoperoxidase intensity was indicated by the scale shown at the bottom of the figure.
3. Results
3.1. Peroxidase acti6ity and pattern The crude extracts of rac shoots were characterized by higher peroxidase activities during the growth cycle, with a minimal activity at day 14 for both tobacco genotypes (Fig. 1). We noted a difference at the level of the basal parts of the stems: the minimal activity occurred at day 14 for the rac mutant and at day 7 for the wild-type (Fig. 1). The isoperoxidase patterns (Fig. 2) showed quantitative and qualitative differences between the two tobacco shoots. The rac mutant had a higher number of bands (A4, C5 and C6 were not found in the wild-type) with a generally higher activity for all of them compared to the wild-type. However, two isoperoxidases (A4, A6) from the wildtype were not represented in the isoperoxidase profile of the mutant. Fig. 1. Changes in the total peroxidase activity in the whole shoots and the basal parts of the stems of the wild, D8 ( ) and the mutant, rac ( ) tobaccos during the course of the culture cycle.
3.2. IAA-oxidase acti6ity The peroxidase and IAA-oxidase activities of Sephadex-chromatographed fractions were, re-
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spectively assayed in the presence of guaı¨acol and IAA. As shown in Fig. 3, the fractions of the rac shoots exhibited two peaks of peroxidase activity contrary to data from the wild shoots: the first peak corresponding to the fractions 16–22 and the second to the fractions 30–40; the single peak for the wild shoots corresponding to the fractions 18–26. The peak of IAA-oxidase activity in the fractions 14 – 32 was qualitatively and quantitatively about the same for the two types of tobacco.
3.3. Auxin protection The auxin protection activity of the gel fractions was assayed in the presence of HRP in conditions to work as IAA-oxidase. Concerning the wildtype, the single peak of auxin protection activity in fractions 12 – 18 (Fig. 3) was of less intensity than the one exhibited by the rac mutant which appeared later, in the fractions 58–80 (Fig. 3). The larger peak of auxin protection by the rac mutant corresponded to a larger peak of absorption at 280 nm.
3.4. Phenol content The phenol levels were also determined at the
end of the growth cycle. The rac shoots contained 8.6 times more phenols (1212.6927.6 mg g − 1 fresh weight) in crude extracts than the wild shoots (140.995.8 mg g − 1 of fresh weight).
3.5. Auxin protectors The phenolic extracts of tobacco shoots were purified through a G-100 Sephadex column. The auxin protection activity was then assayed in the collected fractions (Fig. 4). The 280 nm absorption spectrum of the wild tobacco presented only two small peaks compared to two large peaks for the rac mutant. For both tobaccos, these peaks corresponded roughly to the previous peaks of absorption at 280 nm obtained after filtration of the crude enzymic extracts (Fig. 3). In addition, a large single peak of auxin protection was measured for the rac mutant and was found in the same fractions as the one resulting from the filtration of the crude enzymic extracts (Fig. 4). No IAA-oxidase activity was detectable in the fractions. As shown in Fig. 5, pure chlorogenic acid (the major phenolic compound in tobacco) was chromatographed with a single peak in fractions corresponding to compounds of low molecular
Fig. 3. G-100 Sephadex filtration of wild, D8 and mutant, rac extracts of shoots: absorbance at 280 nm ( ), peroxidase activity ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase () and IAA-oxidase activity in% of IAA destroyed ().
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Fig. 4. G-100 Sephadex filtration of wild, D8 and mutant, rac phenolic extracts of shoots: absorbance at 280 nm ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase () and IAA-oxidase activity in% of IAA destroyed ().
weight as in Fig. 3 and Fig. 4 and exhibited auxin protection properties (Fig. 5 bottom).
4. Discussion The previously shown [7] higher specific peroxidase activity (on a protein basis) of the rac mutant shoots compared to their wild counterparts was confirmed here on a fresh weight basis. The activity in both types of whole shoots reached a minimum at day 14. However, when the basal parts of the stems (where the roots are formed, at least for the wild-type) were considered separately, this minimum appeared at day 7 for the wild-type and was delayed at the 14th day for the mutant (Fig. 1). This may mean that according to previous works utilizing peroxidase as a marker of the successive rooting phases [11,24], the peroxidase minimums recorded here should correspond with the completion of the induction phases of the rooting process in both types of shoots, with a simple delay for the rac shoots. The rac shoots
Fig. 5. G-100 Sephadex filtration of 10-3 M chlorogenic acid solution: absorbance at 280 nm ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase ().
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thus should achieve a rooting inductive phase and this is in agreement with the earlier observation [6] that the rac shoots initiate cell divisions at their base even if this is not followed by the initiation of root meristems and primordia (see Section 1). The higher peroxidase activity of the tobacco mutant corresponded to additional and higher activity of both acidic and basic isoperoxidases as shown by Fig. 2. Tobacco peroxidases have been involved in lignification as well as in auxin catabolism [22,23]. It is too simple to hypothesize that an overexpression of peroxidases automatically means enhanced auxin catabolism and therefore that endogenous auxin would not be available for rooting induction. Indeed, IAA-oxidase was measured with an equivalent activity in both types of tobacco. After a Sephadex G-100 gel filtration, the peroxidases of the rac mutant divided into two peaks vs only one for the wild-type (Fig. 3) but this apparently did not influence the patterns of the IAA-oxidase activity (generally attributed to peroxidases) which were similar. However the protein elution profiles of the two tobaccos appeared quite different according to the 280 nm absorbance spectrum (with a typical peak of fractions corresponding to small molecules for the rac shoots) and to the auxin protectors. The latter showed a large peak only in the rac shoots, which corresponded, to the 280 nm peak of absorption. The phenolic content of the rac shoots was measured at a concentration 8.6 times higher than the one of the wild shoots. When purified on Sephadex, these phenols produce the typical 280 nm absorbance peak and the corresponding IAA-protectors peak. There is thus a good reason to believe that the higher auxin protection of the rac shoots is attributed to phenolic compounds. In another work in preparation, we found chlorogenic acid as the major phenolic compound in tobacco, confirming earlier results [22]. We also show here that this compound chromatographed in fractions corresponding indeed to the ones found from the crude extracts and it acted as auxin protector (Fig. 5). The effects of phenolic compounds on the enzymic oxidation of IAA in vitro are well known, but little information is available concerning their influence in vivo, because the composition, distribution and concentration of phenols in plant tissues are complex [13–15]. b-monophenolic compounds increased the rate of the IAA degradation while the 3,4-dis-
ubstituted phenols were generally inhibitors. However Lee [25] tested a number of phenolic compounds for their effects on the IAA metabolism in maize stems, particularly on the formation of bound IAA. This author has observed that a pretreatment of the tissues with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. Mato et al. [26] have determined the changes in both the peroxidase activity and the phenolic auxin protectors during the root formation of the vine. Their study has shown that the auxin protection was due to three derivatives of tartaric acid. Different rooting abilities in Eucalyptus gunnii microcuttings were dependent on their endogenous flavonoid level [27]. Indeed, explants unable to root showed a low flavonoid concentration, whereas the same explants acquiring rooting ability increased their endogenous flavonoid level. On the contrary, rooting tests on microshoots of walnut transformed by an antisense chalcone synthase (chs) gene showed that the decreased flavonoid content in stems of transformed lines was associated with an enhanced adventitious root formation (no change in free and conjugated auxin contents was observed between control and antisense chs transformed lines) [28]. Moreover, it has also been established that some flavonoids can perturb the auxin transport by binding to a plasma membrane protein [29]. Ono [30] isolated a 2,4-D resistant variant of tobacco. It has also been shown that the auxin resistance of this variant is not due to an alteration in the mechanisms regulating intracellular auxin concentration such as uptake, export or metabolism [31]. In addition, this variant retains its ability to differentiate shoots but it lacks the ability to differentiate roots, like our rac mutant. Nakamura et al. [32] demonstrated that this 2,4-D resistant nonrooting variant has no detectable level of a membrane-bound auxin binding protein. Thus, to understand the recalcitrance to rooting of the rac mutant of tobacco, further investigations will be necessary to determine 1. the endogenous auxin contents in relation to 2. the changes in the receptor affinity for auxin, the changes in the efficiency of the transduction pathway and the H+-ATPase activity and 3. the phenolic pattern in relation with the successive rooting phases.
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Acknowledgements Odile Faivre-Rampant is gratefully indebted to the European Community for a FAIR training grant. Provision of the tobacco seeds by Dr M. Caboche (INRA, Versailles) is also gratefully acknowledged.
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[12] T. Gaspar, Integrated relationships of biochemical and physiological peroxidase activities, in: H. Greppin, C. Penel, T.H. Gaspar (Eds.), Molecular and Physiological Aspects of Plant Peroxidases, University of Gene`ve, Switzerland, 1986, pp. 455 – 468. [13] T.T. Lee, A.N. Starrat, J.J. Jevinkar, Regulation of enzymic oxidation of indole-3-acetic acid by phenols: structure-activity relationships, Phytochemistry 21 (1982) 517 – 523. [14] R. Volpert, W. Osswald, E.F. Elstner, Effects of cinnamic acid derivatives on indole acetic acid oxidation by peroxidase, Phytochemistry 38 (1995) 19 – 22. [15] S.N. Krylov, S.M. Krylova, L.B. Rubin, Threshold effect of caffeic acid on peroxidase-catalysed oxidation of indole-3-acetic acid, Phytochemistry 33 (1993) 9 – 12. [16] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant. 15 (1962) 473 – 497. [17] E. Darimont, T. Gaspar, A propos du nombre et du poids mole´culaire des isoenzymes peroxydasiques de la racine de Lens culinaris, Soc. Bot. Fr., Me´moires 1972, Coll. Morphologie, pp. 211-222. [18] M.M. Bradford, A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding, Anal. Biochem. 72 (1976) 148 – 254. [19] C. Kevers, M. Coumans, W. DeGreef, M. Hofinger, T. Gaspar, Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors, Physiol. Plant. 51 (1981) 281 – 286. [20] B. Legrand, Action de la lumie`re sur les peroxydases et sur la teneur en compose´s phe´noliques de tissus de feuilles de Cichorium intybus L. cultive´s in vitro, Biol. Plant. 19 (1977) 27 – 33. [21] D. Vereecke, E. Messens, K. Klarskov, A. De Bruyn, M. Van Montagu, K. Goethals, Patterns of phenolic compounds in leafy galls of tobacco, Planta 201 (1997) 342 – 348. [22] L.M. Lagrimini, V. Gingas, F. Finger, S. Rothstein, T.T.Y. Liu, Characterization of antisense transformed plants deficient in the tobacco anionic peroxidase, Plant Physiol. 114 (1997) 1187 – 1196. [23] I.G. Gazaryan, L.M. Lagrimini, Tobacco anionic peroxidase overexpressed in transgenic plants: aerobic oxidation of indole-3-acetic acid, Phytochemistry 42 (1996) 1271 – 1278. [24] T. Gaspar, C. Kevers, J.F. Hausman, Indissociable chief factors in the inductive phase of adventitious rooting, in: A. Altman, Y. Waisel (Eds.), Biology of Root Formation and Development, Plenum Press, New York, 1997, pp. 55 – 63. [25] T.T. Lee, Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems, Physiol. Plant. 50 (1980) 107 – 112. [26] M.C. Mato, M.L. Rua, E. Ferro, Changes in levels of peroxidases and phenolics during root formation in Vitis cultured in vitro, Physiol. Plant. 72 (1988) 84 – 88. [27] P. Curir, C.F. Van Sumere, A. Termini, P. Barthe, A. Marchesini, M. Dolci, Flavonoid accumulation is correlated with adventitious roots formation in Eucalyptus gunnii Hook micropropagated through axillary bud stimulation, Plant Physiol. 92 (1990) 1148 – 1153.
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[28] C. El Euch, C. Jay-Allemand, M. Pastuglia, P. Doumas, J.P. Charpentier, P. Capelli, L. Jouanin, Expression of antisense chalcone synthase RNA in transgenic hybrid walnut microcuttings: effect on flavonoid content and rooting ability, Plant Mol. Biol. 38 (1998) 467–479. [29] M. Jacobs, P.H. Rubery, Naturally occurring auxin transport regulators, Science 241 (1988) 346–349. [30] H. Ono, Genetical and physiological investigations of a 2,4-D resistant cell line isolated from the tissue cultures in tobacco, I: growth response to 2,4-D and IAA, Sci.
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Rep. Fac. Agri. Kobe Univ. 13 (1979) 272 – 277. [31] C. Nakamura, M. Nakata, M. Shioji, H. Ono, 2,4-D resistance in a tobacco cell culture variant: cross-resistance to auxins and uptake, efflux and metabolism of 2,4-D, Plant Cell Physiol. 26 (1985) 271 – 280. [32] C. Nakamura, H.J. Van Telgen, A.M. Mennes, H. Ono, K.R. Libbenga, Correlation between auxin resistance and the lack of a membrane-bound auxin binding protein and a root-specific peroxidase in Nicotiana tabacum, Plant Physiol. 88 (1988) 845 – 849.
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IAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro Odile Faivre-Rampant *, Claire Kevers, Thomas Gaspar Hormonologie Fondamentale et Applique´e, Institute of Botany B 22, Uni6ersity of Lie`ge-Sart Tilman, B-4000 Lie`ge, Belgium Received 10 May 1999; received in revised form 30 November 1999; accepted 3 December 1999
Abstract The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, rac, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the rac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However, this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a Sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the rac mutant are discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Auxin oxidase; Peroxidase; Phenolic compounds; Rooting; Tobacco mutant
1. Introduction Mutants resistant, in other words more tolerant, to the exogenous application of high concentrations of natural and synthetic auxins have been isolated [1 – 4]. Such mutants might be resistant either because the level of endogenous hormone is different from the one in the wild-type or because of changes at the primary site of auxin action [5]. Shoots of the nonrooting rac mutant of tobacco [2] should belong to the latter category: according to Lund et al. [6], this rac mutant does not disrupt auxin concentrations, but involves some changes in the receptor affinity for auxin and/or in the efficiency of the transduction pathway. The problem of this mutant regarding rooting difficulties is complicated: perivascular cells in mutant stem cut-
* Corresponding author. Tel.: +32-4-3663914; fax: +32-43663859. E-mail address: [email protected] (O. Faivre-Rampant)
tings treated with IBA divide, but never form adventitious root meristems [6]. We have established in vitro shoot-forming cultures of the ‘auxin-resistant’ (nonrooting) rac mutant of tobacco and of its homologous wild-type [7]. Shoots of the rac mutant grow at a lower rate. They are characterized by higher lignin level, higher soluble peroxidase activity and higher ethylene production. These observations might be causally related to the growth inhibition as similar events have been noticed in different stress-induced growth limitations, through the cell wall rigidification and the auxin catabolism. However these relationships did not give any explanation of the rooting difficulties. In an attempt to biochemically characterize this rac mutant further, we have taken the opportunity to use its shoots in order to perform parallel analyses of auxin protection and peroxidase activities in comparison with those of its wild homologue. Gaspar et al. [8,9] indeed characterized the successive physiological phases of adventi-
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tious root formation by typical changes of peroxidase activity and polyphenolic compounds, the latter being generally active as auxin protectors. Basic isoperoxidases have been suggested to be the most effective in IAA degradation [10]. Changes in the isoperoxidase profiles were specifically observed on the cathodic side when concomitant variations of endogenous IAA levels were measured [11]. It has also been observed that the activity of certain basic isoperoxidases involved in auxin catabolism is prevented by phenolic compounds present in the extracts [12]. The activity is affected by the number of hydroxyl groups and their position relative to each other as well as to other substituents on the aromatic ring. Monophenols generally stimulate the IAA oxidation with p-substituted monophenols that are more active than o- or m-compounds, whereas o- and p-diphenols act as inhibitors (m-diphenols activate the IAA oxidation) [13]. Direct interaction and competition between the enzyme, the inhibitor or the stimulator and IAA have been discussed as the mechanism [14], as well as the functioning of the inhibitors as radical scavengers [15]. It has also been shown that different results concerning the stimulation or the inhibition can be due to different concentrations of the tested phenolic derivative [13].
2. Material and methods
2.1. Plant material The wild-type, D8, and the homozygous, rac, mutant seeds of Nicotiana tabacum cv Xanthi were surface-sterilized for 3 min in a 10% (v/v) commercial bleach solution, placed on a MS medium [16], pH 5.7, containing 0.8% (w/v) Roland agar (Brussels), 3% (w/v) sucrose and 0.13 mM BAP, as previously reported by Lund et al. [6]. Shoot apices or stem nodes were explanted for continued culture through 3 week-cycles. Cultures, in 600 ml cylindrical glass jars, containing 100 ml of MS medium (as above), covered by a glass lid maintained with a sheet of transparent plastic film, were placed under a 16 h photoperiod (Sylvania Grolux fluorescent tubes providing 15 mE m − 2 s − 1) at 28°C (day) and 25°C (night). The wild shoots showed roots at the end of each multiplication cycle, while the rac shoots did not root.
Whole shoots and basal third parts of the stems (without leaves) were sampled for the analyses. Basal parts were analyzed because they are the sites of root formation, at least in the wild-type.
2.2. Peroxidase acti6ity and isoperoxidases Frozen explants (300 mg fresh weight taken at day 7, 14 and 21 during the growth cycle) were ground in 1 ml phosphate buffer (0.06 M, pH 6.1) and centrifuged for 10 min at 11 000×g at 4°C. The supernatant was used as the crude enzyme extract. The guaiacol-peroxidase activity and the peroxidase isoenzyme patterns by vertical starch gel electrophoresis were determined according to Darimont and Gaspar [17]; the activity was expressed in mg equivalent horseradish peroxidase (HRP) (Boehringer–Manheim) per g fresh weight. The gels were developed with benzidine. The protein concentration was determined by the Coomassie blue method [18] using bovine serum albumin as standard.
2.3. Separation and assay of auxin protectors and IAA-oxidase acti6ity The techniques to obtain auxin protectors and the determination of IAA-oxidase activity have been described elsewhere [19]. Three grams of fresh mass were ground in chilled phosphate buffer (0.06 M, pH 6.1) at a ratio of 1 g of tissue per ml of buffer. The extract was centrifuged for 20 min at 20 000×g. The resulting supernatant was filtered through a G-100 Sephadex column (2.5×35 cm2) and 2 ml samples were collected serially with a fraction collector. Their absorption was recorded at 280 nm. The assay for the inhibitors of enzymatic destruction used 4 ml reaction mixtures containing 3 ml phosphate buffer (0.06 M, pH 6.1), 0.5 ml gel fraction, 0.25 ml HRP (Boehringer–Manheim, 3 mg 10 ml − 1) and 0.25 ml IAA (4.10 − 3 M). In assays of Fig. 5, the gel fraction was replaced by 0.5 ml of a chlorogenic acid solution (10 − 3 M). The auxin protection results were expressed in percentage of inhibition of HRP used in conditions to work as IAA-oxidase. The assay for the IAA-oxidase activity used 3 ml reaction mixtures containing 2 ml phosphate buffer (0.06 M, pH 6.1), 0.25 ml gel fraction, 0.15 ml MnCl2 (10 − 3 M), 0.3 ml DCP (10 − 3 M), 0.3 ml IAA (4.10 − 3 M). The IAA-oxidase activity was
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expressed in percentage of IAA destroyed (100% IAA is in the control without enzymic fraction) calculated from the absorbance of Salkowski’s reagent at 530 nm.
2.4. Phenol content Soluble phenolic compounds were extracted from 150 mg of fresh weight by the method of Legrand [20]. The phenol content was assayed with the Folin and Ciocalteu reagent. Absorption at 750 nm is proportional to the phenolic compounds level. The results were expressed in mg of phenolic compounds (using chlorogenic acid as a reference) per mg of fresh weight. Precisely, in another work in preparation concerning the identification of the phenolics in tobacco, chlorogenic acid was found as the major compound as already shown by Vereecke et al. [21]. All the results are means of at least three separate experiments (9SD).
Fig. 2. Isoperoxidase zymograms of the different parts of the wild (D8) and mutant (rac) tobacco shoots. AS: apical stem; AL: apical leaves; BS: basal stem; BL: basal leaves. Each lane was loaded with the same amount of proteins (4.5 mg). The isoperoxidase intensity was indicated by the scale shown at the bottom of the figure.
3. Results
3.1. Peroxidase acti6ity and pattern The crude extracts of rac shoots were characterized by higher peroxidase activities during the growth cycle, with a minimal activity at day 14 for both tobacco genotypes (Fig. 1). We noted a difference at the level of the basal parts of the stems: the minimal activity occurred at day 14 for the rac mutant and at day 7 for the wild-type (Fig. 1). The isoperoxidase patterns (Fig. 2) showed quantitative and qualitative differences between the two tobacco shoots. The rac mutant had a higher number of bands (A4, C5 and C6 were not found in the wild-type) with a generally higher activity for all of them compared to the wild-type. However, two isoperoxidases (A4, A6) from the wildtype were not represented in the isoperoxidase profile of the mutant. Fig. 1. Changes in the total peroxidase activity in the whole shoots and the basal parts of the stems of the wild, D8 ( ) and the mutant, rac ( ) tobaccos during the course of the culture cycle.
3.2. IAA-oxidase acti6ity The peroxidase and IAA-oxidase activities of Sephadex-chromatographed fractions were, re-
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spectively assayed in the presence of guaı¨acol and IAA. As shown in Fig. 3, the fractions of the rac shoots exhibited two peaks of peroxidase activity contrary to data from the wild shoots: the first peak corresponding to the fractions 16–22 and the second to the fractions 30–40; the single peak for the wild shoots corresponding to the fractions 18–26. The peak of IAA-oxidase activity in the fractions 14 – 32 was qualitatively and quantitatively about the same for the two types of tobacco.
3.3. Auxin protection The auxin protection activity of the gel fractions was assayed in the presence of HRP in conditions to work as IAA-oxidase. Concerning the wildtype, the single peak of auxin protection activity in fractions 12 – 18 (Fig. 3) was of less intensity than the one exhibited by the rac mutant which appeared later, in the fractions 58–80 (Fig. 3). The larger peak of auxin protection by the rac mutant corresponded to a larger peak of absorption at 280 nm.
3.4. Phenol content The phenol levels were also determined at the
end of the growth cycle. The rac shoots contained 8.6 times more phenols (1212.6927.6 mg g − 1 fresh weight) in crude extracts than the wild shoots (140.995.8 mg g − 1 of fresh weight).
3.5. Auxin protectors The phenolic extracts of tobacco shoots were purified through a G-100 Sephadex column. The auxin protection activity was then assayed in the collected fractions (Fig. 4). The 280 nm absorption spectrum of the wild tobacco presented only two small peaks compared to two large peaks for the rac mutant. For both tobaccos, these peaks corresponded roughly to the previous peaks of absorption at 280 nm obtained after filtration of the crude enzymic extracts (Fig. 3). In addition, a large single peak of auxin protection was measured for the rac mutant and was found in the same fractions as the one resulting from the filtration of the crude enzymic extracts (Fig. 4). No IAA-oxidase activity was detectable in the fractions. As shown in Fig. 5, pure chlorogenic acid (the major phenolic compound in tobacco) was chromatographed with a single peak in fractions corresponding to compounds of low molecular
Fig. 3. G-100 Sephadex filtration of wild, D8 and mutant, rac extracts of shoots: absorbance at 280 nm ( ), peroxidase activity ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase () and IAA-oxidase activity in% of IAA destroyed ().
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Fig. 4. G-100 Sephadex filtration of wild, D8 and mutant, rac phenolic extracts of shoots: absorbance at 280 nm ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase () and IAA-oxidase activity in% of IAA destroyed ().
weight as in Fig. 3 and Fig. 4 and exhibited auxin protection properties (Fig. 5 bottom).
4. Discussion The previously shown [7] higher specific peroxidase activity (on a protein basis) of the rac mutant shoots compared to their wild counterparts was confirmed here on a fresh weight basis. The activity in both types of whole shoots reached a minimum at day 14. However, when the basal parts of the stems (where the roots are formed, at least for the wild-type) were considered separately, this minimum appeared at day 7 for the wild-type and was delayed at the 14th day for the mutant (Fig. 1). This may mean that according to previous works utilizing peroxidase as a marker of the successive rooting phases [11,24], the peroxidase minimums recorded here should correspond with the completion of the induction phases of the rooting process in both types of shoots, with a simple delay for the rac shoots. The rac shoots
Fig. 5. G-100 Sephadex filtration of 10-3 M chlorogenic acid solution: absorbance at 280 nm ( ), auxin protection in% of inhibition of HRP used in conditions to work as IAA-oxidase ().
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thus should achieve a rooting inductive phase and this is in agreement with the earlier observation [6] that the rac shoots initiate cell divisions at their base even if this is not followed by the initiation of root meristems and primordia (see Section 1). The higher peroxidase activity of the tobacco mutant corresponded to additional and higher activity of both acidic and basic isoperoxidases as shown by Fig. 2. Tobacco peroxidases have been involved in lignification as well as in auxin catabolism [22,23]. It is too simple to hypothesize that an overexpression of peroxidases automatically means enhanced auxin catabolism and therefore that endogenous auxin would not be available for rooting induction. Indeed, IAA-oxidase was measured with an equivalent activity in both types of tobacco. After a Sephadex G-100 gel filtration, the peroxidases of the rac mutant divided into two peaks vs only one for the wild-type (Fig. 3) but this apparently did not influence the patterns of the IAA-oxidase activity (generally attributed to peroxidases) which were similar. However the protein elution profiles of the two tobaccos appeared quite different according to the 280 nm absorbance spectrum (with a typical peak of fractions corresponding to small molecules for the rac shoots) and to the auxin protectors. The latter showed a large peak only in the rac shoots, which corresponded, to the 280 nm peak of absorption. The phenolic content of the rac shoots was measured at a concentration 8.6 times higher than the one of the wild shoots. When purified on Sephadex, these phenols produce the typical 280 nm absorbance peak and the corresponding IAA-protectors peak. There is thus a good reason to believe that the higher auxin protection of the rac shoots is attributed to phenolic compounds. In another work in preparation, we found chlorogenic acid as the major phenolic compound in tobacco, confirming earlier results [22]. We also show here that this compound chromatographed in fractions corresponding indeed to the ones found from the crude extracts and it acted as auxin protector (Fig. 5). The effects of phenolic compounds on the enzymic oxidation of IAA in vitro are well known, but little information is available concerning their influence in vivo, because the composition, distribution and concentration of phenols in plant tissues are complex [13–15]. b-monophenolic compounds increased the rate of the IAA degradation while the 3,4-dis-
ubstituted phenols were generally inhibitors. However Lee [25] tested a number of phenolic compounds for their effects on the IAA metabolism in maize stems, particularly on the formation of bound IAA. This author has observed that a pretreatment of the tissues with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. Mato et al. [26] have determined the changes in both the peroxidase activity and the phenolic auxin protectors during the root formation of the vine. Their study has shown that the auxin protection was due to three derivatives of tartaric acid. Different rooting abilities in Eucalyptus gunnii microcuttings were dependent on their endogenous flavonoid level [27]. Indeed, explants unable to root showed a low flavonoid concentration, whereas the same explants acquiring rooting ability increased their endogenous flavonoid level. On the contrary, rooting tests on microshoots of walnut transformed by an antisense chalcone synthase (chs) gene showed that the decreased flavonoid content in stems of transformed lines was associated with an enhanced adventitious root formation (no change in free and conjugated auxin contents was observed between control and antisense chs transformed lines) [28]. Moreover, it has also been established that some flavonoids can perturb the auxin transport by binding to a plasma membrane protein [29]. Ono [30] isolated a 2,4-D resistant variant of tobacco. It has also been shown that the auxin resistance of this variant is not due to an alteration in the mechanisms regulating intracellular auxin concentration such as uptake, export or metabolism [31]. In addition, this variant retains its ability to differentiate shoots but it lacks the ability to differentiate roots, like our rac mutant. Nakamura et al. [32] demonstrated that this 2,4-D resistant nonrooting variant has no detectable level of a membrane-bound auxin binding protein. Thus, to understand the recalcitrance to rooting of the rac mutant of tobacco, further investigations will be necessary to determine 1. the endogenous auxin contents in relation to 2. the changes in the receptor affinity for auxin, the changes in the efficiency of the transduction pathway and the H+-ATPase activity and 3. the phenolic pattern in relation with the successive rooting phases.
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Acknowledgements Odile Faivre-Rampant is gratefully indebted to the European Community for a FAIR training grant. Provision of the tobacco seeds by Dr M. Caboche (INRA, Versailles) is also gratefully acknowledged.
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