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Changes in Peroxidase and IAA-Oxidase Activities during Adventitious bud Formation from Small Root Explants of Cichorium intybus L: Influence of Glucose
J.PlantPhysiol. Vol. 138.pp. l02-106(1991}
Changes in Peroxidase and IAA-Oxidase Activities during Adventitious bud Formation from Small Root Explants of Cichorium intybus L: Influence of Glucose BERNARD LEGRAND
and
ABDELHAMID BOUAZZA
Laboratoire de Physiologie de la differenciation et biotechnologies vegetales, Universite des Sciences et Techniques de Lille Flandres Artois, 59655 Villeneuve d'Ascq-France Received July 30, 1990 . Accepted December 17, 1990
Summary Root explants of Cichorium intybus were cultured on Heller medium supplemented with different glucose concentrations, in light or in darkness. Capacity of bud formation was strongly diminished by darkness and the average number of buds formed by explants, was not modified by glucose treatment. When explants were illuminated adventitious bud formation was strongly dependent upon the glucose concentrations. Glucose was not necessary for budding and glucose concentrations above 0.01 M decreased bud forming capacity. During differenciation of bud meristems peroxidase activity remained highest in explants wich will produce more buds, i.e. when glucose concentration was weak. Glucose effects were similar on IAA-oxidase activity, but this activity cannot be detected without PVP in the extraction buffer. The hypothesis of the regulation of bud formation by a relationship between phenolic compounds and enzymes involved in IAA catabolism, i.e. peroxidase, is discussed.
Key words: Adventitious buds in vitro, Cichorium intybus, IAA-oxidase, peroxidase, phen"ols. Abbreviations: DW = dry weight; FW = fresh weight; glc = glucose; IAA = indolylacetic acid, PVP = polyvinylpyrolidone (polyclar insoluble).
Introduction Small explants of Cichorium intybus roots are charaterized by a high regeneration and capacity through adventitious bud formation. This process can be easily controlled by exogenous nutritional factors. Among these factors, carbohydrates have been demonstrated to be very important: sucrose by Gwozdz and Szweykowska (1968), glucose by Backoula et al. (1985) and their osmotic role by Lefebvre et al. (1988). It is generally accepted that peroxidase activity and its isoenzyme patterns are altered with changes in plant development (van Huystee and Cairns, 1982). In organogenesis the role of peroxidase is often explained by the dual function of this enzyme involved in peroxidation-reactions and auxin catabolism (Hoyle, 1972). Through this last function peroxidase can modify hormonal balance in plant, leading to modulate morphogenesis. This IAA-degrading capacity is coun© 1991 by Gustav Fischer Verlag, Stuttgart
terbalanced by auxin protectors (Stonier, 1970) which are generally phenolic compounds whose synthesis can be influenced by carbohydrates (Shah and Mehta, 1978). The aim of the present work was to examine the effects of glucose on adventitious bud formation by root explants of Cichorium intybus and to determine during the first eight days of culture, thus including the induction and initiation periods of bud meristems, the variations of peroxidase and IAA oxidase activities. Because we earlier have shown (Legrand, 1978) that phenols can act on peroxidase activity, effects of glucose on the phenolic content of explant were examined too.
Material and Methods Explants (6mm diameter x 2mm height) were removed from vascular cambium of surface sterilized mature roots of Cichorium
Changes in peroxidase and IAA-oxidase: Effect of glucose
intybus L. var. Witloof cv. Flash, harvested in fields. The basal medium, solidified by agar (6 g . L - 1) consisted of macro- and microelements according to Heller (1953). The explants were individually depositate on the surface of the medium and cultured in tube containing 20 mL of basal medium supplemented or not with glucose 0.01; 0.1 or 0.3 M. Thirty explants per treatment were placed in controlled environment cabinets at constant temperature (23 °C) in continuous light (10Wm- 2) or in full darkness. Explants were harvested after 2, 4, 6 and 8 days culture, weighed and kept at -25 °C till determination of enzyme activities and phenolic content. The number of adventitious buds was determined on explants cultured during 25 days. The reported data are confirmed by the results of at least one similar experiment. For each result the standard deviation of the mean is reported. The enzymes were extracted according to the method described by Legrand and Dubois (1977). Briefly, three replicates of approximatly 3 g each of tissues were frozen in liquid nitrogen and ground to a powder with mortar and peste!. This powder was mixed with PVP (100 mg! g) and suspended in a 0.2 ionic-strength buffer to extract soluble peroxidases (El). After centrifugations (10,000 g' 10 min) and washings with the same buffer, the ionically wall bound peroxidases (E2) were extracted from the pellet with a 1.2 ionic strengh buffer by addition of NaCI 1 M in the former buffer. The pellet was washed two times in distilled water and the covalent bound peroxidases (E3) were obtained after maceration of the pellet in a pectinase (2.5 %) cellulase (0.5 %) solution in acetate buffer (pH 4.5) during 12 h at room temperature. Peroxidase activity was determined spectrophotometrically by measuring the increase in absorbance at 420 nm after 5 min in 5 mL incubation mixture containing 2.9 mL Na-K phosphate buffer (pH 6.1 0.066M), 2 mL guaiacol (1 % solution), 1 mL H 20 2 (0.2 vol solution) and 0.1 mL enzymatic extract. Peroxidase isoenzyme patterns were determined by vertical gel electrophoresis and staining with benzidine or guaiacol according to Darimont and Gaspar (1972). The wells were loaded with 75 JlL sample of the crude enzyme extracts, with 1 % (w/v) bromocresol green as a tracking dye. Measurements of IAA-oxidase activity were performed in phosphate buffer (pH 6) in the presence of Mnch (10- 3M) and 2.4dichlorophenol (10- 3M) with 0.3mL enzymatic extract and 1O- 3M IAA. The final volume was 10mL. Photocolorimetric determination of IAA destruction used Salkowski reagent after Pilet (1957). IAA-oxidase activity was expressed in Jlg IAA destroyed after 120 min. For enzymes activities the experiments were performed three times using three different extracts for each. Extraction and titration of total phenol content has been already described (Legrand, 1977). The standard curve was prepared using chlorogenic acid and all the concentrations were expressed in mg of this compound.
Results On root explants callus formation began after four days and emergence of buds from the callus was observed between the twelfth and fourteenth day. The growth, under continuous light, measured by an increase in dry weight was strongly influenced by glc (Fig. 1). Without or with 0.Q1 M glc in culture medium the growth was very slow, while with 0.1 or 0.3 M glc, dry weights increased respectively two and three folds in 8 days. It could be shown that the growth increase depends on callus formation only. Bud neoformation was influenced by glucose concentration and also culture conditions (Table1). Under light the
80
103
glucose (M) --lit-
0
-
0.01
_____
0,1
-
0,3
70
60
-
50
E
Ol
Ci
x
Q)
40
Cl
E
~ 30
ci
20
10
a
4
2
6
8
Days
Fig. 1: Growth in vitro of Cichorium intybus root explants in relation to glucose concentration. Explants are cultured in continuous light.
number of buds formed by explant was the same in medium lacking glc or with 0.Q1 M, and a strongly fall was observed with 0.1 and 0.3 M glc. Under darkness the bud capacity was diminished: 70 % of explants (instead of 100 % when they were illuminated), were able to regenerate buds on basal medium or with 0.01 M glc, and only 40 % when sugar concentrations were 0.1 M and 0_3 M. The number of adventitious bud per explant was similar in all glc treatments. Effects of glc on peroxidase activity are shown in Fig. 2. For each treatment there was an increase of peroxidase activity up to a plateau beginning at day 6. This activity increased more rapidly when there was no glc in culture medium or with 0.01 M glc. For high concentrations and important decrease of peroxidase activity was found. No significant difference in isoperoxidase patterns occurred during culture whatever the treatment. Small variations in the proportions of isoperoxidases were noted, but they were not decisive. Table 1: Effects of glucose concentration on formation of adventitious buds (average number) by root explants of Cichorium intybus cultured 25 days in continuous light or in full darkness. Glucose (M.)
0
0.Q1
0.1
0.3
Continuous light Full darkness
2.20±0.38 0.78±0.41
2.69±0.43 1.25±0.30
0.95±0.31 1.17±0.22
0.57±0.43 0.73±0.40
104
BERNARD LEGRAND and ABDELHAMID BOUAZZA 20
glucose (M)
glucose (M)
--a--
18
ici 16
0
II 9 h t
IIJ
°
r3
0,01
E1
0,1
E1
0,3
0,01
~ LL
Ol
E 14
2
en
E
o
o o
o
~12
o
C\I
'
ci 10
Q. ~ .s: 13til Ql III
til "0
8
6
'x
e Ql
Q.
4 2
2
0 0
2
4
6
8
Days El
Fig. 2: Evolution of peroxidase activity in root explants of Cicho· riurn intybus cultured in continuous light with different glucose concentrations.
Guaiacol-peroxidase and IAA-oxidase actlVltIes were analysed in the three fractions from six days old explants (Figs. 3 and 4). For these assays it was necessary to add PVP in buffer extraction, without PVP, no IAA-oxidase activity can be measured. About 55 % of the peroxidase activity were found in the soluble fraction (£1)' 30 % were ionically bound with cell-wall (£2)' whilst 15 % were covalent bound peroxidases (£3)' IAA-oxidase activities were correlated with these percentages. Enzyme activities were more important when explants were cultured in darkness. In each fraction maximums of peroxidase activity and IAA-oxidase activity were respectively found when culture mediums were supplemented with 0.01 M and 0.1 M of glc. Isoperoxidase patterns in different fractions are shown in Fig. 5. In fraction £1 five bands were obtained: four basic isoperoxidases, the bands C 2 and C 4 having the highest activity and one isoperoxidase AI with a low electrophoretic mobility. Ionically wall bound peroxidases were represented by three cathodic bands, among them C and C 2 showing the same electrophoretic mobility than isoperoxidases of £1' In fraction £3, three bands were detected: one basic C 1 and two anodic. Isoperoxidase activity in this fraction was essentially represented by acidic peroxidases. Since IAA-oxidase could not be detected in crude extract without PVP, it was interesting to examine the evolution of phenol content during culture (Fig. 6). In each case, phenol
E2
E3
Fig. 3: Effect of glucose concentration on peroxidase activity of different fractions. E I : Soluble peroxidase; E2 : Ionically wall bound peroxidase; E3: Covalent wall bound peroxidase from six days old root explants cultured in light or in darkness. Bars represent standard errors.
content increased with time culture, slowly when glucose was missing or with a weak concentration in culture medium, and rapidly when medium was enriched with glc. Discussion The effects of carbohydrates on adventitious bud neoformation have been investigated and our results are in contrast with those of Douglas (1985) showing that sucrose is essential for shoot formation in Populus and agreed with studies of Brown et al. (1979) on tobacco, where 4 % sucrose inhibit shoot formation. Inhibitory effects of glucose on bud neoformation in Cichonum intybus is more sensitive when explants are illuminated. The difference in budding inhibition by glc, between explants illuminated or not, gives rise to the question of variation of endogenous glucose, either by photosynthesis ability of tissues which are chlorophyllous after two days in light, or by the capacity of enzymes to hydrolyse fructosans which are essential storage carbohydrates in Cichonum intybus roots. The present results are good confirmation of earlier ones: Legrand (1977), Thorpe et aI. (1978), Thorpe and Gaspar (1978) and Kevers et al. (1981) in-
Changes in peroxidase and IAA-oxidase: Effect of glucose 50
~o
2 glucose (M)
glucose (M)
Ii 9 h t
40
II
0
EJ
0,01
105
.c N
~
30
0>
~
E
~ o
o
o
20
C>
E
o ~I
0>
2-
C>
E
III
El
'0 <=
E3
E2
Ql
..c:
c..
40
30
o
2
4
6
8
20
Days
Fig. 6: Changes in phenols contents in root explants of Cichorium in· tybus in relation to glucose concentration. Explants are cultured in continuous light.
10
El
E2
E3
Fig. 4: Effects of glucose on auxin-oxidase activity. (For explanation see Fig. 3).
PVP 5
C J
A
+
~
2~ c:-::t
::::.:)
1
1~
+
+ ]
4~
3
o~
[
~
~
~ ~
,
C:::I
CJ
c:J
- - ~ I~ '
=
=
iI
~
'F_
Fig. 5: Diagram showing isoperoxidases from the soluble (E l ), ionically wall bound (E2) and covalent wall bound (E3) fractions extracted from 6 days old root explant of Cichorium intybus. 0 ~ Increasing intensity.
dicating a variation of soluble peroxidase activity involving first an increase followed by a plateau in the course of adventitious bud formation. In light, peroxidase activity remains high in explants where bud forming capacity is important. When explants are cultured in darkness their peroxidase activity is higher than when they are illuminated, and we can wonder why bud forming capacity is low? The importance of phenolic compounds controlling growth has been demonstrated in Tobacco callus culture by their action in promoting and inhibiting bud differentiation (Lee et aI., 1982). In Cichorium intybus explant organogenesis is diminished when the phenolic content is high. Zucker (1963) had shown that synthesis of chI orogenic acid can occur in almost complete darkness but by exposing culture to light this phenolic synthesis is enhanced. Many natural phenols affect the role of enzymic oxidation of IAA (Lee et aI., 1982), and guaiacol-peroxidation (Legrand, 1974). It is interesting to note that crude extracts, obtained without PVP, are enable to destroy IAA. So in Cichorium intybus tissues exist auxin protectors which are discarded when we use PVP but in the same time peroxidase activity is diminished (data not shown). This brings us to think that among the phenols chelated by PVP, there exist not only inhibitors of IAA oxidase but also stimulators of guaiacol peroxidase. This lead us to study the nature and the evolution of phenolic compounds during adventitious bud formation. There are many studies dealing with peroxidase changes in relation to organogenesis. Their role in this process was often explained through their paricipation in auxin cat-
106
BERNARD LEGRAND and ABDELHAMID BOUAZZA
abolism and cell wall biogenesis, basic peroxidases being involved in the former and acidic peroxidases in the latter. Covalent peroxidases are represented by essentially acidic isoperoxidases and are able, as shown within, to degrade IAA. The question of specificity of basic and acidic isoperoxidases on account of this is still subject of discussion. Our data show a relationship between peroxidase activity, phenol compound content and budding capacity of the explants. During extract procedure, cell compartmentation is suppressed and peroxidase activity represents the peroxidasephenol interaction. Peroxidase activity is weaker when the amount of phenol is higher in the tissues and inversely when there is a decrease of phenol content, an increase of peroxidase activity is observed. This indirect increase of peroxidase activity leading to an enhanced IAA catabolism, so that the level of endogenous IAA is reduced and the auxin-cytokinin rate become favorable for bud formation. We can concluded only, that a low phenolic compound content and a high peroxidase activity are promoting budding. But, in fact, it is difficult to draw conclusions concerning the role in vivo of peroxidase from observations made in vitro.
References BACKOULA, E., R. LEFEBVRE, and J. VASSEUR: Modalite d'action du glucose sur Ie bourgeonnement de petits explants racinaires de Cichorium intybus L. cultives in vitro C. R. Acad. Sc Paris, 301, 639-642. (1985). BROWN, D. c., D. W. LEUNG, and T. A. THORPE: Osmotic requirement for shoot formation in tobacco callus. Physiol. Plant. 46, 36-41 (1979). DARIMONT, E. and TH. GASPAR: A propos du nombre et du poids moleculaire des isoenzymes peroxydasiques de la racine de Lens culinaris Soc. Bot. Fr. Memoire Coll. Morphologie 211- 222 (1972). DOUGLAs, G. c.: Formation of adventitious buds in stem internodes of Populus hydrid TT 32 cultured in vitro: Effects of sucrose, zeatin, IAA and ABA. J. Plant Physiol. 121, 225-231 (1985). GASPAR, TH., C. PENEL, T. A. THORPE, and H. GREPPIN: Peroxidases 1970-1980. A survey of their biochemical and physiological roles in higher plants. Universite de Geneve. Centre de botanique 324 p. (1982). GASPAR, TH., C. PENEL, F. J. CASTILLO, and H. GREPPIN: A two step control of basic and acidic peroxidases and its significance for growth and development. Physiol. Plant. 64, 418-423 (1985).
GWOZDZ, E., and A. SZWEYKOWSKA: The effect of some nutritional factors on the organogenesis in root explants of Cichorium in· tybus Bull. Soc. Amis des Sciences et des Lettres de Poznan 9, 11-18 (1968). HELLER, R.: Recherches sur la nutrition minerale des tissus vegetaux cultives in vitro. Ann. Sci Nat. Bot 14,1-223 (1953). HOYLE, M. C.: Indoleacetic acid oxidase: a dual catalytic enzyme. Plant Physiol. 50, 15-18. (1972). KEVERS, c., M. COUMANS, W. DE GREFF, M. JACOBS, and TH. GASPAR: Organogenesis in habituated sugarbeet callus: Auxin content and protectors, peroxidase patterns and inhibitors. Z. Pflanzenphysioll01, 79-87 (1981). LEE, T. T., A. N. STARRAT and J. J. JEVNIKAR: Regulation of enzymic oxidation of indole-3-acetic acid by phenols : structure-activity relationships. Phytochemistry 21,517-523 (1982). LEFEBVRE, R., E. BACKOULA, and J. VASSEUR: Osmose et bourgeonnement d'explantats racinaires de Cichorium intybus L. cultives in vitro C. R. Acad. Sci. Paris 307, 385-390 (1988). LEGRAND, B.: Influence des conditions d'eclairement sur la neoformation de bourgeons par les tissus de feuilles d' endives cultives in vitro et sur I'activite peroxydasique de ces tissus. C. R. Acad. Sci Paris 278,2425-2428 (1974). - Action de la lumiere sur les peroxydases et sur la teneur en composes phenoliques de tissus de feuilles de Cichorium intybus L. cultives in vitro. Biologia Plantarum 1, 27 -33 (1977). - Les peroxydases et leur regulation au cours de la neoformation de bourgeons par des tissus de Cichorium intybus L. cultives in vitro: These. Universite Lille 219 p. (1987). LEGRAND, B. and J. DUBOIS: Evolution des peroxydases et auxinesoxydases au cours de la croissance d'une suspension cellulaire de Silene (Silene alba (Miller) E. H. L. KRAUSE). C. R. Acad. Sci. Paris 285,661-664. (1977). SHAH, R. R. and A. R. MEHTA: Effect of carbohydrates on growth, formation of phenolic compounds and related enzymes in callus cultures of Crotalaria. Indian J. of Experimental Biology 16, 768-770 (1978). STONIER, T.: The role of auxin protectors in autonomous growth. Les cultures de tissus des plantes 423 -435. Colloques internationaux CNRS. Strasbourg (1970). THORPE, T . A. and TH. GASPAR: Changes in isoperoxidases during shoot formation in tobacco callus. In vitro 14, 522-526 (1978). THORPE, T. A., M. TRAN THANH VAN, and TH. GASPAR: Isoperoxidases in epidermal layers of tobacco and changes during organ formation in vitro. Physiol. Plant. 44, 388-394 (1978). VAN HUYSTEE, R. B. and W. L. CAIRNS: Progress and prospects in the use of peroxidase to study cell development. Phytochemistry 21, 1843 -1847 (1982). ZUCKER, M.: The influence of light on synthesis of protein and chlorogenic acid in potato tuber tissue. Plant Physiol. 66, 281-285 (1963).
Changes in Peroxidase and IAA-Oxidase Activities during Adventitious bud Formation from Small Root Explants of Cichorium intybus L: Influence of Glucose BERNARD LEGRAND
and
ABDELHAMID BOUAZZA
Laboratoire de Physiologie de la differenciation et biotechnologies vegetales, Universite des Sciences et Techniques de Lille Flandres Artois, 59655 Villeneuve d'Ascq-France Received July 30, 1990 . Accepted December 17, 1990
Summary Root explants of Cichorium intybus were cultured on Heller medium supplemented with different glucose concentrations, in light or in darkness. Capacity of bud formation was strongly diminished by darkness and the average number of buds formed by explants, was not modified by glucose treatment. When explants were illuminated adventitious bud formation was strongly dependent upon the glucose concentrations. Glucose was not necessary for budding and glucose concentrations above 0.01 M decreased bud forming capacity. During differenciation of bud meristems peroxidase activity remained highest in explants wich will produce more buds, i.e. when glucose concentration was weak. Glucose effects were similar on IAA-oxidase activity, but this activity cannot be detected without PVP in the extraction buffer. The hypothesis of the regulation of bud formation by a relationship between phenolic compounds and enzymes involved in IAA catabolism, i.e. peroxidase, is discussed.
Key words: Adventitious buds in vitro, Cichorium intybus, IAA-oxidase, peroxidase, phen"ols. Abbreviations: DW = dry weight; FW = fresh weight; glc = glucose; IAA = indolylacetic acid, PVP = polyvinylpyrolidone (polyclar insoluble).
Introduction Small explants of Cichorium intybus roots are charaterized by a high regeneration and capacity through adventitious bud formation. This process can be easily controlled by exogenous nutritional factors. Among these factors, carbohydrates have been demonstrated to be very important: sucrose by Gwozdz and Szweykowska (1968), glucose by Backoula et al. (1985) and their osmotic role by Lefebvre et al. (1988). It is generally accepted that peroxidase activity and its isoenzyme patterns are altered with changes in plant development (van Huystee and Cairns, 1982). In organogenesis the role of peroxidase is often explained by the dual function of this enzyme involved in peroxidation-reactions and auxin catabolism (Hoyle, 1972). Through this last function peroxidase can modify hormonal balance in plant, leading to modulate morphogenesis. This IAA-degrading capacity is coun© 1991 by Gustav Fischer Verlag, Stuttgart
terbalanced by auxin protectors (Stonier, 1970) which are generally phenolic compounds whose synthesis can be influenced by carbohydrates (Shah and Mehta, 1978). The aim of the present work was to examine the effects of glucose on adventitious bud formation by root explants of Cichorium intybus and to determine during the first eight days of culture, thus including the induction and initiation periods of bud meristems, the variations of peroxidase and IAA oxidase activities. Because we earlier have shown (Legrand, 1978) that phenols can act on peroxidase activity, effects of glucose on the phenolic content of explant were examined too.
Material and Methods Explants (6mm diameter x 2mm height) were removed from vascular cambium of surface sterilized mature roots of Cichorium
Changes in peroxidase and IAA-oxidase: Effect of glucose
intybus L. var. Witloof cv. Flash, harvested in fields. The basal medium, solidified by agar (6 g . L - 1) consisted of macro- and microelements according to Heller (1953). The explants were individually depositate on the surface of the medium and cultured in tube containing 20 mL of basal medium supplemented or not with glucose 0.01; 0.1 or 0.3 M. Thirty explants per treatment were placed in controlled environment cabinets at constant temperature (23 °C) in continuous light (10Wm- 2) or in full darkness. Explants were harvested after 2, 4, 6 and 8 days culture, weighed and kept at -25 °C till determination of enzyme activities and phenolic content. The number of adventitious buds was determined on explants cultured during 25 days. The reported data are confirmed by the results of at least one similar experiment. For each result the standard deviation of the mean is reported. The enzymes were extracted according to the method described by Legrand and Dubois (1977). Briefly, three replicates of approximatly 3 g each of tissues were frozen in liquid nitrogen and ground to a powder with mortar and peste!. This powder was mixed with PVP (100 mg! g) and suspended in a 0.2 ionic-strength buffer to extract soluble peroxidases (El). After centrifugations (10,000 g' 10 min) and washings with the same buffer, the ionically wall bound peroxidases (E2) were extracted from the pellet with a 1.2 ionic strengh buffer by addition of NaCI 1 M in the former buffer. The pellet was washed two times in distilled water and the covalent bound peroxidases (E3) were obtained after maceration of the pellet in a pectinase (2.5 %) cellulase (0.5 %) solution in acetate buffer (pH 4.5) during 12 h at room temperature. Peroxidase activity was determined spectrophotometrically by measuring the increase in absorbance at 420 nm after 5 min in 5 mL incubation mixture containing 2.9 mL Na-K phosphate buffer (pH 6.1 0.066M), 2 mL guaiacol (1 % solution), 1 mL H 20 2 (0.2 vol solution) and 0.1 mL enzymatic extract. Peroxidase isoenzyme patterns were determined by vertical gel electrophoresis and staining with benzidine or guaiacol according to Darimont and Gaspar (1972). The wells were loaded with 75 JlL sample of the crude enzyme extracts, with 1 % (w/v) bromocresol green as a tracking dye. Measurements of IAA-oxidase activity were performed in phosphate buffer (pH 6) in the presence of Mnch (10- 3M) and 2.4dichlorophenol (10- 3M) with 0.3mL enzymatic extract and 1O- 3M IAA. The final volume was 10mL. Photocolorimetric determination of IAA destruction used Salkowski reagent after Pilet (1957). IAA-oxidase activity was expressed in Jlg IAA destroyed after 120 min. For enzymes activities the experiments were performed three times using three different extracts for each. Extraction and titration of total phenol content has been already described (Legrand, 1977). The standard curve was prepared using chlorogenic acid and all the concentrations were expressed in mg of this compound.
Results On root explants callus formation began after four days and emergence of buds from the callus was observed between the twelfth and fourteenth day. The growth, under continuous light, measured by an increase in dry weight was strongly influenced by glc (Fig. 1). Without or with 0.Q1 M glc in culture medium the growth was very slow, while with 0.1 or 0.3 M glc, dry weights increased respectively two and three folds in 8 days. It could be shown that the growth increase depends on callus formation only. Bud neoformation was influenced by glucose concentration and also culture conditions (Table1). Under light the
80
103
glucose (M) --lit-
0
-
0.01
_____
0,1
-
0,3
70
60
-
50
E
Ol
Ci
x
Q)
40
Cl
E
~ 30
ci
20
10
a
4
2
6
8
Days
Fig. 1: Growth in vitro of Cichorium intybus root explants in relation to glucose concentration. Explants are cultured in continuous light.
number of buds formed by explant was the same in medium lacking glc or with 0.Q1 M, and a strongly fall was observed with 0.1 and 0.3 M glc. Under darkness the bud capacity was diminished: 70 % of explants (instead of 100 % when they were illuminated), were able to regenerate buds on basal medium or with 0.01 M glc, and only 40 % when sugar concentrations were 0.1 M and 0_3 M. The number of adventitious bud per explant was similar in all glc treatments. Effects of glc on peroxidase activity are shown in Fig. 2. For each treatment there was an increase of peroxidase activity up to a plateau beginning at day 6. This activity increased more rapidly when there was no glc in culture medium or with 0.01 M glc. For high concentrations and important decrease of peroxidase activity was found. No significant difference in isoperoxidase patterns occurred during culture whatever the treatment. Small variations in the proportions of isoperoxidases were noted, but they were not decisive. Table 1: Effects of glucose concentration on formation of adventitious buds (average number) by root explants of Cichorium intybus cultured 25 days in continuous light or in full darkness. Glucose (M.)
0
0.Q1
0.1
0.3
Continuous light Full darkness
2.20±0.38 0.78±0.41
2.69±0.43 1.25±0.30
0.95±0.31 1.17±0.22
0.57±0.43 0.73±0.40
104
BERNARD LEGRAND and ABDELHAMID BOUAZZA 20
glucose (M)
glucose (M)
--a--
18
ici 16
0
II 9 h t
IIJ
°
r3
0,01
E1
0,1
E1
0,3
0,01
~ LL
Ol
E 14
2
en
E
o
o o
o
~12
o
C\I
'
ci 10
Q. ~ .s: 13til Ql III
til "0
8
6
'x
e Ql
Q.
4 2
2
0 0
2
4
6
8
Days El
Fig. 2: Evolution of peroxidase activity in root explants of Cicho· riurn intybus cultured in continuous light with different glucose concentrations.
Guaiacol-peroxidase and IAA-oxidase actlVltIes were analysed in the three fractions from six days old explants (Figs. 3 and 4). For these assays it was necessary to add PVP in buffer extraction, without PVP, no IAA-oxidase activity can be measured. About 55 % of the peroxidase activity were found in the soluble fraction (£1)' 30 % were ionically bound with cell-wall (£2)' whilst 15 % were covalent bound peroxidases (£3)' IAA-oxidase activities were correlated with these percentages. Enzyme activities were more important when explants were cultured in darkness. In each fraction maximums of peroxidase activity and IAA-oxidase activity were respectively found when culture mediums were supplemented with 0.01 M and 0.1 M of glc. Isoperoxidase patterns in different fractions are shown in Fig. 5. In fraction £1 five bands were obtained: four basic isoperoxidases, the bands C 2 and C 4 having the highest activity and one isoperoxidase AI with a low electrophoretic mobility. Ionically wall bound peroxidases were represented by three cathodic bands, among them C and C 2 showing the same electrophoretic mobility than isoperoxidases of £1' In fraction £3, three bands were detected: one basic C 1 and two anodic. Isoperoxidase activity in this fraction was essentially represented by acidic peroxidases. Since IAA-oxidase could not be detected in crude extract without PVP, it was interesting to examine the evolution of phenol content during culture (Fig. 6). In each case, phenol
E2
E3
Fig. 3: Effect of glucose concentration on peroxidase activity of different fractions. E I : Soluble peroxidase; E2 : Ionically wall bound peroxidase; E3: Covalent wall bound peroxidase from six days old root explants cultured in light or in darkness. Bars represent standard errors.
content increased with time culture, slowly when glucose was missing or with a weak concentration in culture medium, and rapidly when medium was enriched with glc. Discussion The effects of carbohydrates on adventitious bud neoformation have been investigated and our results are in contrast with those of Douglas (1985) showing that sucrose is essential for shoot formation in Populus and agreed with studies of Brown et al. (1979) on tobacco, where 4 % sucrose inhibit shoot formation. Inhibitory effects of glucose on bud neoformation in Cichonum intybus is more sensitive when explants are illuminated. The difference in budding inhibition by glc, between explants illuminated or not, gives rise to the question of variation of endogenous glucose, either by photosynthesis ability of tissues which are chlorophyllous after two days in light, or by the capacity of enzymes to hydrolyse fructosans which are essential storage carbohydrates in Cichonum intybus roots. The present results are good confirmation of earlier ones: Legrand (1977), Thorpe et aI. (1978), Thorpe and Gaspar (1978) and Kevers et al. (1981) in-
Changes in peroxidase and IAA-oxidase: Effect of glucose 50
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Fig. 6: Changes in phenols contents in root explants of Cichorium in· tybus in relation to glucose concentration. Explants are cultured in continuous light.
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Fig. 4: Effects of glucose on auxin-oxidase activity. (For explanation see Fig. 3).
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Fig. 5: Diagram showing isoperoxidases from the soluble (E l ), ionically wall bound (E2) and covalent wall bound (E3) fractions extracted from 6 days old root explant of Cichorium intybus. 0 ~ Increasing intensity.
dicating a variation of soluble peroxidase activity involving first an increase followed by a plateau in the course of adventitious bud formation. In light, peroxidase activity remains high in explants where bud forming capacity is important. When explants are cultured in darkness their peroxidase activity is higher than when they are illuminated, and we can wonder why bud forming capacity is low? The importance of phenolic compounds controlling growth has been demonstrated in Tobacco callus culture by their action in promoting and inhibiting bud differentiation (Lee et aI., 1982). In Cichorium intybus explant organogenesis is diminished when the phenolic content is high. Zucker (1963) had shown that synthesis of chI orogenic acid can occur in almost complete darkness but by exposing culture to light this phenolic synthesis is enhanced. Many natural phenols affect the role of enzymic oxidation of IAA (Lee et aI., 1982), and guaiacol-peroxidation (Legrand, 1974). It is interesting to note that crude extracts, obtained without PVP, are enable to destroy IAA. So in Cichorium intybus tissues exist auxin protectors which are discarded when we use PVP but in the same time peroxidase activity is diminished (data not shown). This brings us to think that among the phenols chelated by PVP, there exist not only inhibitors of IAA oxidase but also stimulators of guaiacol peroxidase. This lead us to study the nature and the evolution of phenolic compounds during adventitious bud formation. There are many studies dealing with peroxidase changes in relation to organogenesis. Their role in this process was often explained through their paricipation in auxin cat-
106
BERNARD LEGRAND and ABDELHAMID BOUAZZA
abolism and cell wall biogenesis, basic peroxidases being involved in the former and acidic peroxidases in the latter. Covalent peroxidases are represented by essentially acidic isoperoxidases and are able, as shown within, to degrade IAA. The question of specificity of basic and acidic isoperoxidases on account of this is still subject of discussion. Our data show a relationship between peroxidase activity, phenol compound content and budding capacity of the explants. During extract procedure, cell compartmentation is suppressed and peroxidase activity represents the peroxidasephenol interaction. Peroxidase activity is weaker when the amount of phenol is higher in the tissues and inversely when there is a decrease of phenol content, an increase of peroxidase activity is observed. This indirect increase of peroxidase activity leading to an enhanced IAA catabolism, so that the level of endogenous IAA is reduced and the auxin-cytokinin rate become favorable for bud formation. We can concluded only, that a low phenolic compound content and a high peroxidase activity are promoting budding. But, in fact, it is difficult to draw conclusions concerning the role in vivo of peroxidase from observations made in vitro.
References BACKOULA, E., R. LEFEBVRE, and J. VASSEUR: Modalite d'action du glucose sur Ie bourgeonnement de petits explants racinaires de Cichorium intybus L. cultives in vitro C. R. Acad. Sc Paris, 301, 639-642. (1985). BROWN, D. c., D. W. LEUNG, and T. A. THORPE: Osmotic requirement for shoot formation in tobacco callus. Physiol. Plant. 46, 36-41 (1979). DARIMONT, E. and TH. GASPAR: A propos du nombre et du poids moleculaire des isoenzymes peroxydasiques de la racine de Lens culinaris Soc. Bot. Fr. Memoire Coll. Morphologie 211- 222 (1972). DOUGLAs, G. c.: Formation of adventitious buds in stem internodes of Populus hydrid TT 32 cultured in vitro: Effects of sucrose, zeatin, IAA and ABA. J. Plant Physiol. 121, 225-231 (1985). GASPAR, TH., C. PENEL, T. A. THORPE, and H. GREPPIN: Peroxidases 1970-1980. A survey of their biochemical and physiological roles in higher plants. Universite de Geneve. Centre de botanique 324 p. (1982). GASPAR, TH., C. PENEL, F. J. CASTILLO, and H. GREPPIN: A two step control of basic and acidic peroxidases and its significance for growth and development. Physiol. Plant. 64, 418-423 (1985).
GWOZDZ, E., and A. SZWEYKOWSKA: The effect of some nutritional factors on the organogenesis in root explants of Cichorium in· tybus Bull. Soc. Amis des Sciences et des Lettres de Poznan 9, 11-18 (1968). HELLER, R.: Recherches sur la nutrition minerale des tissus vegetaux cultives in vitro. Ann. Sci Nat. Bot 14,1-223 (1953). HOYLE, M. C.: Indoleacetic acid oxidase: a dual catalytic enzyme. Plant Physiol. 50, 15-18. (1972). KEVERS, c., M. COUMANS, W. DE GREFF, M. JACOBS, and TH. GASPAR: Organogenesis in habituated sugarbeet callus: Auxin content and protectors, peroxidase patterns and inhibitors. Z. Pflanzenphysioll01, 79-87 (1981). LEE, T. T., A. N. STARRAT and J. J. JEVNIKAR: Regulation of enzymic oxidation of indole-3-acetic acid by phenols : structure-activity relationships. Phytochemistry 21,517-523 (1982). LEFEBVRE, R., E. BACKOULA, and J. VASSEUR: Osmose et bourgeonnement d'explantats racinaires de Cichorium intybus L. cultives in vitro C. R. Acad. Sci. Paris 307, 385-390 (1988). LEGRAND, B.: Influence des conditions d'eclairement sur la neoformation de bourgeons par les tissus de feuilles d' endives cultives in vitro et sur I'activite peroxydasique de ces tissus. C. R. Acad. Sci Paris 278,2425-2428 (1974). - Action de la lumiere sur les peroxydases et sur la teneur en composes phenoliques de tissus de feuilles de Cichorium intybus L. cultives in vitro. Biologia Plantarum 1, 27 -33 (1977). - Les peroxydases et leur regulation au cours de la neoformation de bourgeons par des tissus de Cichorium intybus L. cultives in vitro: These. Universite Lille 219 p. (1987). LEGRAND, B. and J. DUBOIS: Evolution des peroxydases et auxinesoxydases au cours de la croissance d'une suspension cellulaire de Silene (Silene alba (Miller) E. H. L. KRAUSE). C. R. Acad. Sci. Paris 285,661-664. (1977). SHAH, R. R. and A. R. MEHTA: Effect of carbohydrates on growth, formation of phenolic compounds and related enzymes in callus cultures of Crotalaria. Indian J. of Experimental Biology 16, 768-770 (1978). STONIER, T.: The role of auxin protectors in autonomous growth. Les cultures de tissus des plantes 423 -435. Colloques internationaux CNRS. Strasbourg (1970). THORPE, T . A. and TH. GASPAR: Changes in isoperoxidases during shoot formation in tobacco callus. In vitro 14, 522-526 (1978). THORPE, T. A., M. TRAN THANH VAN, and TH. GASPAR: Isoperoxidases in epidermal layers of tobacco and changes during organ formation in vitro. Physiol. Plant. 44, 388-394 (1978). VAN HUYSTEE, R. B. and W. L. CAIRNS: Progress and prospects in the use of peroxidase to study cell development. Phytochemistry 21, 1843 -1847 (1982). ZUCKER, M.: The influence of light on synthesis of protein and chlorogenic acid in potato tuber tissue. Plant Physiol. 66, 281-285 (1963).