Plant Growth Regulation 42: 253–262, 2004. # 2004 Kluwer Academic Publishers. Printed in the Netherlands.
253
Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases Kamel Tartoura1,*, Andrea da Rocha2 and Sahar Youssef1 1
Department of Agricultural Botany, Suez-Canal University, Ismailia, Egypt; 2Department of Plant Pathology, Michigan State University, 62 Plant Biology Building, East Lansing, MI 48824, USA; *Author for correspondence (e-mail:
[email protected])
Received 17 February 2003; accepted in revised form 10 December 2003
Key words: Adventitious root formation, Basic isoperoxidases, Coumarin, Endogenous free and conjugated IAA, Indole-3-butyric acid, Mung bean cuttings
Abstract Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction. Abbreviations: ARF – Adventitious root formation; DCP – Dichlorophenol; IAA – Indole-3-acetic acid; IAA-O – IAA oxidase; IBA – Indole-3-butyric acid; iso-PER(s) – Isoperoxidase(s); PVPP – Polyvinylpolypyrolidone; PAGE – Polyacrylamide gel electrophoresis
Introduction Coumarins are naturally occurring growth active substances (Murray 1991) involved in diverse
physiological responses during plant growth and development. The role of coumarins, as plant growth regulators (Kupidlowska et al. 1994), has been reviewed by several authors (Wolf 1974;
254 Murray et al. 1982). One of their physiological effects is the stimulation of adventitious root formation (ARF). For example, exogenously applied coumarin to cuttings stimulates ARF in many plant species (Janson and Svensson 1980; Dhawan and Nanda 1982; Grosser and Chandler 1986; Tartoura 1994; Hartmann et al. 1997). Interaction between indole-3-butyric acid (IBA), commonly used to induce ARF, and coumarin in terms of ARF has not previously been studied. It is well documented that auxins play a central role in triggering the ARF process. However, reports on the endogenous indole-3-acetic acid (IAA) concentration locally determined at the potential rooting sites during the early stages of rooting have been contradictory. Many investigators reported a high level of accumulated IAA during the induction phase before the first detectable event in root initiation (Jarvis 1986; Blakesley 1994; Gaspar et al. 1994). Others have reported a decline in IAA (Berthon et al. 1989; Blakesley et al. 1991). In contrast, other investigators reported that ARF occurred without any changes in IAA level (Nordstro¨m and Eliasson 1991; Garc´ia-Go´mez et al. 1994). Basic isoperoxidases (iso-PERs) are considered as IAA oxidases (IAA-O) and are also involved in the rooting process (Gaspar and Hofinger 1989; Fet-Neto et al. 1992). Any possible correlation between endogenous free IAA, conjugated IAA and basic iso-PERs concentrations during the time-course of ARF is as yet undefined (Normanly et al. 1995; Liu et al. 1996). In this investigation, mung bean (Vigna radiata (L) Wilcz.) cuttings were used to study the interaction between applied coumarin and IBA on ARF induction. Any possible relationship to endogenous free and conjugated IAA, basic PER activity and its iso-PER analysis during the time-course of ARF was also examined. The overall aim of this work was to study the major physiological and biochemical changes that may affect mung bean V. radiata adventitious rooting.
moist vermiculite and covered lightly with moist vermiculite in darkness for 48 h. After germination, the seedlings were kept in a growth chamber at 24 C with constant cool white fluorescent illumination (20 W m2 PAR), and 80% of relative humidity. Of five uniform cuttings from 7-day-old seedlings, consisting of terminal bud, one pair of primary leaves, epicotyl and 5 cm of hypocotyls, were placed in 10 mL vials containing a 4 cm solution depth of distilled water or test solutions of coumarin at 1, 10, 100, and 1000 M, IBA at 0.5, 5, and 50 M, either alone or in combination, for 24 h. The cuttings were transferred subsequently to new vials containing a 5 cm deep of distilled water for a further 6 days. Distilled water was added daily to the vials to maintain the original solution level. Incubation conditions were identical to those for seedling growth. The time-course appearance of emerging roots through epidermis was recorded and the number of these roots after 7 days was determined. The synergistic effect between coumarin and IBA on adventitious root numbers was calculated using the following formula: synergyð%Þ ¼ ½IBA/coumarin H2 O ½ðIBA H2 OÞ þ ðcoumarin H2 OÞ ½ðIBA H2 OÞ þ ðcoumarin H2 OÞ 100,
where IBA and coumarin represent the number of roots formed during individual treatments, IBA/ coumarin that formed during combined treatments, and H2O represents the number of roots formed in the control. Data are the mean values of three independent experiments, each with three replicates of 25 cuttings. Solutions of coumarin and IBA (Sigma) were prepared by dissolving the chemicals in alkaline ethanol and then diluting with distilled water. The highest concentration of ethanol in the solutions was 0.2% and thus 0.2% of ethanol was used as a control. Sampling, extraction and determination of IAA
Materials and methods Plant material and treatments Seeds of mung bean were soaked in aerated water for 3 h, then germinated in plastic trays containing
Samples of 1.5 cm length from the basal portions of the cuttings were collected at 0, 6, 12, 24, 48, 72, and 96 h during ARF. These were frozen immediately in liquid nitrogen and stored at 80 C until determination. About 5 g fresh weight (FW) samples was crushed in liquid nitrogen at 4 C
255 using a prechilled mortar and pestle and then homogenized in cold 80% methanol (MeOH) containing 200 mg L1 hydroxytoluene as an antioxidant for 24 h in darkness at 4 C. The tissues were re-extracted twice for 4 h each. The combined extracts were reduced to the aqueous phase in vacuo at 30 C using a rotary evaporator and centrifuged at 48,000 g for 30 min. Supernatants were evaporated to about 0.5 mL, then 0.5 mL of MeOH was added and the extracts were passed through an activated C18 Sep-Pak cartridge (Purves and Hollenberg 1982), followed by elution with 9 mL of 90% MeOH : water (v/v). Aliquots equal to 3 g FW were analysed by high performance liquid chromatography (HPLC) for assay of free IAA by the method of Arteca et al. (1980). The remainder of the sample was used for determination of total IAA (free + ester + amide) according to the method described by Cohen et al. (1988). Quantification of IAA by HPLC was achieved using a standard curve constructed by injection of authentic IAA (Sigma) quantities and corrected for losses of IAA during extraction and purification by adding, in a separate experiment, defined amounts of authentic IAA (Sigma) to homogenates and then quantify them by HPLC. The method used for extraction and purification was highly reproducible. The recovery rates were 80 and 74% in terms of free and conjugated IAA, respectively. The conjugated IAA (ester + amide) was calculated by subtracting the free IAA values from the total IAA amount estimated after base hydrolysis. Identification of free IAA and the free IAA released from IAA conjugates was confirmed by adding authentic IAA to a part of the sample.
Extraction and assay of basic PER activity and its iso-PER analysis Frozen tissues were crushed in liquid nitrogen at 4 C using a prechilled mortar and pestle and then homogenized in cold 0.2 M potassium phosphate buffer, pH 7.0, containing 1% polyvinylpoly pyrrolidone (PVPP; ratio: 1 fresh weight: 2 buffer, w/v). The homogenates were centrifuged at 20,000 g for 30 min at 4 C , the supernatants were decanted, the pellets were resuspended in the same amount of buffer and then centrifuged
under the same conditions as above. The two supernatants were combined and used to assay enzyme activity and the separation of basic PER isozymes by polyacrylamide gel electrophoresis (PAGE) analysis. Basic PER activity estimated by H2O2 independent oxidation of IAA was measured spectrophotometrically according to a modified method of Beffa et al. (1990). The reaction mixtures (1 ml) contained 0.5 mM IAA, 0.05 mM DCP, 0.05 mM MnCl2 and 150 mM Sodium-acetate buffer, pH 4.5. The enzymatic reaction was initiated by adding 10 L of enzyme extract. After 20 min incubation at 37 C, the non-oxidized IAA was complexed by the addition of 4 mL of a FeCl2 – H2SO4 reagent consisting of 15 mL of 0.5 M FeCl2, 50 mL H2O and 300 mL of 36 M H2SO4. The unstable red chelated dye was quantified after 15 min at 530 nm at the point of maximal dye intensity. The concentration of IAA was quantified using an IAA standard curve and the amounts of oxidized IAA were calculated by subtracting the remaining IAA from the total IAA amount. Data are the mean values of three different extracts. Proteins were assayed by the method of Bradford (1976) using bovine serum albumin as a standard. The specific activity was expressed as g of oxidized IAA h1 mg1 protein. Non-denaturing PAGE of basic iso-PER analysis was performed using 10% (w/v) polyacrylamide separating gel and 5% stacking gel. Acidic gel was buffered with 0.3 M potassium acetate buffer (pH 4.3) according to the method of Reisfield et al. (1962). The electrode buffer was 0.35 M -alanine/ acetic acid, pH 4.5. Samples were loaded in onequarter strength electrode buffer containing 10% (v/v) glycerol and 0.02% (w/v) pyronine Y as tracking dye. The gels were run at 4 C using a constant current of 75 V for 12 h. After electrophoresis, the PAGE gels were equilibrated in 50 mM sodium acetate (pH 5.0) for 20 min. The gels were then incubated at room temperature for 30–60 min in a solution containing 50 mg of 3-amino-9-ethylcarbazol, 10 mL of dimethylformamide, 200 L of 30% H2O2 and 190 mL of 100 mM sodium acetate buffer, pH 5.0 (Grahm et al. 1965). Dark brown bands appeared at the sites of PER activity. The separation of each sample using PAGE was repeated three times with identical results.
256
Figure 1. Effect of different concentrations of coumarin and/or IBA on adventitious rooting of V. radiata cuttings. The percentage synergy is shown in parentheses. Vertical bars represent ± SD & , Control; ---*--- 500 nM IBA; --~--, 5 M IBA; , 50 M IBA.
Results Synergistic interaction of coumarin and IBA Figure 1 shows that adventitious root number increased with increasing concentration of coumarin and IBA, the maximum effect being at 1000 and 50 M, respectively, relative to the control. IBA at 100 M also significantly stimulated ARF (date not shown), but since a synergistic effect of coumarin on IBA-induced rooting could be easily estimated when using IBA at lower concentration, this treatment was excluded. Figure 1 also shows that coumarin acted synergistically with IBA in stimulating ARF compared with IBA or coumarin applied individually, with the maximum synergistic effect at 1000 M coumarin and 50 M IBA. The effect of different concentrations of one of the substances when the other is maintained constant during simultaneous application is also shown in Figure 1.
Table 1. Effect of coumarin and/or IBA on adventitious rooting of V. radiata cuttings and the time-course appearance of emerging roots through epidermis.
Treatment
Number of adventitious roots/cutting
Time of root emergence through epidermis (h) Synergy (%)
Water 6.4 (± 0.7) 72.0 (±3.6) 1000 M coumarin 34.4 (± 3.5) 78.0 (±5.6) 50 M IBA 40.5 (± 3.7) 84.0 (±5.6) 1000 M 131.8 (± 12.6) 100.0 (±6.6) coumarin + 50 M IBA
– – – 101.0
The percentage synergy is shown in parentheses. Data show the means ± SD of three replicates from two independent experiments.
at the synergistic combined treatment that significantly resulted in more roots than those in coumarin or IBA treated cuttings. Endogenous free and conjugated IAA
Time of emerging roots through epidermis Table 1 shows that time-course appearance of emerging roots at the base of the treated cuttings through epidermis was markedly delayed compared with the control. The maximum delay being
Figure 2A shows that there was a significant, transient increase in endogenous free IAA level after 6 h, in response to all treatments, with the maximum IAA concentration occurring following the combined treatment. Thereafter, endogenous
257
Figure 2. Changes in endogenous free IAA (A) and conjugated IAA (B) levels in the basal portions of V. radiata cuttings during the indicated time-course of adventitious rooting with or without treatments. Vertical bars represent ± SD. & , Control; --*--, 1000 M Coumarin; 4 , 50 M IBA; – –^––, 1000 M Coumarin +50 M IBA.
free IAA levels decreased to reach the minimum level between 24 and 48 h, with the lowest level occurring in the combined treatment. Afterwards, free IAA re-increased in both treated and untreated cuttings during the progress of ARF (Figure 2A). The different levels in free IAA during the primary events of ARF in treated and untreated cuttings (Figure 2A) were positively related to the number of roots formed (Figure 1). On the other hand, Figure 2B shows that conjugated IAA increased rapidly to reach maximum levels between 24 and 48 h in both treated and untreated cuttings. However, levels of conjugated IAA were significantly higher in treated cuttings than in untreated
ones during the early events of ARF. It was also evident that combined treatment resulted in highest conjugated IAA peak and this positively associated with the highest adventitious root production (Figures 1 and 2B). In addition, Figure 2B also shows that a gradual decrease in the conjugated IAA levels occurred during the secondary events of ARF. Total soluble basic PER activity and its iso-PER analysis An initial slight decrease in total soluble basic PER activity occurred during 6 h after application of
258
Figure 3. Changes in the specific activity of basic peroxidase levels in the basal portions of V. radiata cuttings during the time-course of ARF with or without treatments. Vertical bars represent ± SD. The symbols designations are the same as those in Figure 2.
either coumarin or IBA to cuttings relative to the control. However, in response to combined treatment, an initial increase was observed, followed by a decrease with a minimum level between 24 and 48 h. Thereafter, a steady marked increase was noted earlier in controls compared with treated cuttings, especially those treated with coumarin plus IBA (Figure 3). Likewise, Figure 4 shows that electrophoretic separation patterns of basic iso-PERs are well correlated with its basic PER activity (Figure 3). This means that an increase or decrease in basic PER activity is accompanied by an increase or decrease in the intensity and number of enzymatic bands during the time-course of rooting. However, these results do not show an increase in the intensity or number of basic iso-PER bands even with the simultaneous combined treatment that synergistically acted in initiating more adventitious roots (Figures 1 and 4). Extending the timecourse of analysis may reveal new unique bands since this treatment delayed the emergence of roots about 28 h compared with untreated cuttings. Further research is required to fully evaluate this possibility.
Discussion Adventitious rooting process in mung bean cuttings consists of an induction phase, 0–24 h;
an early phase of root initiation, 24–48 h; a late initiation phase, 48–72 h; a root growth and development, 72 h onwards. The root induction phase is characterized by a lack of cell divisions and an accumulation of auxins. The early initiation phase is characterized by active cell division which can be inhibited by gibberellin. In this phase, auxin accumulation may be associated with both the formation of the meristematic locus and the early cell divisions. The late initiation phase, i.e., continuation of cell divisions and the beginning of differentiation into organized meristems, is associated with low levels of IAA (Jarvis 1986). Auxin and coumarin promote ARF in many different plant species including mung bean V. radiata cuttings (see Introduction section). In agreement with the results presented here, Rauscherova and Tesfa (1993), Wiesman and Riov (1994) and Pan and Gui (1997) reported that some plant growth regulators acted synergistically with IBA in stimulating more ARF in many plant species, including mung bean cuttings. The timecourse appearance of root primordia through epidermis at the bases of cuttings given simultaneously combined application of 1000 M coumarin plus 50 M IBA was significantly later than untreated and other treated cuttings. Thus, the untreated cuttings progressively entered and passed the successive phases of the adventitious rooting process, described by Jarvis (1986), 28 h
259
Figure 4. Pattern of basic isoperoxidases in the basal portions of V. radiata cuttings during the indicated time-course of adventitious rooting treated with H2O as a control (lane a), 1000 M coumarin (lane b), 50 M IBA (lane c), and their combination (lane d). Crude enzyme extracts (30 g protein) were loaded on 10% PAGE gels.
earlier than those given the simultaneous combined treatment. The delayed appearance of adventitious root primordia following treatment with a higher auxin concentration is in line with Cheng (1979) and Gronroos and Von Arnold (1987) who
reported that higher auxin concentration might delay root initiation or even inhibit the elongation of root initials. However, the combined application used in this investigation was just sufficient to stimulate ARF significantly without causing any side effects. Endogenous free auxins play a crucial role in the ARF process (Gaspar and Hofinger 1989). The data presented here are in agreement with Blakesley (1994) and Gaspar et al. (1997) who reported an early and temporary increase in the concentration of endogenous free IAA occurs during the induction phase of rooting. They suggested that the initiation phase of rooting is characterized by a decrease in free IAA to a minimum level, whereas the growth and development of root primordia is associated with a further increase in free IAA concentration. According to Laskowski et al. (1995), auxin accumulates at the potential rooting sites in the first dividing cells that form initial root primordia. Later, during adventitious root differentiation, auxin levels decrease, presumably to allow differentiation and cell elongation to proceed. However, results of other studies showed that instead of an increase in IAA concentration, a gradual decline could be observed (Berthon et al. 1989; Blakesley et al. 1991). The use of earlier sampling times in these studies might have revealed a critical IAA peak, as suggested by Blakesley (1994). Our results also indicate that there was a positive correlation between the free IAA levels measured in cuttings 6 h after application and the number of adventitious roots produced by these cuttings (Figures 1 and 2A), as also suggested by Alvarez et al. (1989) and Blakesley et al. (1991). An increase in endogenous IAA level as a result of exogenous application of other phenolic compounds was also suggested by Marigo and Boudet (1979). However, increased IAA concentration as a result of IBA-treated cuttings might be attributed to the conversion of absorbed IBA into IAA, as demonstrated in several species (Ludwig-M€ uller and Epstein 1992; Epstein and Ludwig-M€ uller 1993). Thus, the synergistic interaction between coumarin and IBA in inducing more ARF may be due to the contribution of coumarin and IBA in increasing the level of free IAA at the potential rooting sites. On the other hand, IAA conjugates are likely to be involved in regulation of auxin
260 levels and the plant tissues could produce active IAA by hydrolysing these IAA conjugates (Bandurski et al. 1995). According to Wiesman et al. (1989), IBA, which initiates and stimulates the development of a larger number of roots, also increases significantly the level of IAA conjugates. Therefore, it seems that the initiation and development of roots caused by coumarin and/or IBA requires, or is associated with, increased conjugated IAA. Our data further show that the levels of IAA conjugates started to decrease after reaching maximum levels (Figure 2B). However, the rate of decrease in conjugated IAA was not proportional to the rise in the free IAA levels (Figure 2A). This may be explained by the fact that some conjugated IAA might be hydrolysed in the later stages of the rooting process and the released IAA is rapidly metabolized when high auxin activity is required for rooting, as suggested by Nordstro¨m et al. (1991); and/or conjugated IAA might be subjected to a direct PER-mediated conjugated IAA oxidation during the progress of ARF, as suggested by Garcia-Go´mez et al. (1995). Indeed, basic PERs (Figures 3 and 4), which are maintained at relatively high levels of activity, might be involved in free IAA oxidation, as reported by Gazaryan et al. (1996) and Lagrimini et al. (1997) or conjugated IAA oxidation, as reported by Tsurumi and Wada (1990), Pl€ uss et al. (1989) and Tuominen et al. (1994). Thus, the principal metabolic pathway of IAA in vivo involves conjugation of IAA followed by oxidation of the indole ring system. Basic PERs are thought to play a crucial role in regulating endogenous IAA levels (Gazaryan et al. 1996; Lagrimini et al. 1997). The minimum basic PER activity found in all treatments at the early phases of adventitious rooting (Figure 3) was also noted previously in other works (Moncousin et al. 1988; Gaspar et al. 1994). Interestingly, this minimum level was observed following application of the cytokinin benzylaminopurine to V. radiata cuttings that inhibits significantly adventitious rooting (data not shown). This indicates that there was not an inverse relation between the accumulation of IAA (Figure 2) and the initial decrease in basic PER (Figure 3). Thus, the direct relation between accumulated IAA and this minimum PER has to be re-examined, as suggested by Gaspar et al. (1997). However, The initial increase in
basic PER activity in cuttings given the combined treatment (Figure 3A) may be attributed to catabolism of the excess supraoptimal amounts of the auxin and phenolic compounds that were applied to the cuttings to induce rooting. In terms of ARF, many authors reported a sharp initial increase in basic PER activity during the primary events of different plant species followed by a sharp decrease during the secondary events of ARF (see references in Gaspar and Hofinger 1989). In contrast, the results presented here indicate that the maximum increase in basic PER activity occurred during the secondary events of ARF concomitantly with the initiation and development of adventitious roots. Further, the values of this enzymatic activity were also related to the number of roots formed (Figures 1, 3, and 4), as also found by De Klerk et al. (1990). According to Quiroga et al. (2000), basic PER was mainly involved in the biosynthesis of lignin that may take place during growth and emergence of the root primordia and the formation of vascular tissues between the root primordia and the vascular tissues of the cuttings. Comparison of the dynamics of conjugated IAA accumulation (Figure 2A and B) and the activity of basic iso-PERs (Figures 3 and 4) led us to conclusion that the former but not the latter is responsible for downregulation of endogenous free IAA levels following root induction phase. Our results fully support the previous studies conducted by Gus’Kov et al. (1980) and Nordstro¨m and Eliasson (1991) who reported that auxin applied to stem cuttings does not undergo rapid basic PER-mediated IAA oxidation, but passes from the free into the conjugated state. Our results are also in accordance with Normanly et al. (1995) who disagreed that catabolism of IAA via basic PER-mediated IAA oxidation is a mechanism of IAA degradation in vivo whereby (a) products of IAA oxidase, noted by the loss of the carboxyl carbon, do not appear to be present in plants in significant amounts (Ernstsen et al. 1987), and (b) experiments with transgenic plants showed that there was no change in IAA levels associated with over-expression or a 90% decrease in PER levels (Lagrimini 1991). Thus, the formation of IAA conjugation is most likely the main mechanism involved in IAA oxidation during the primary events of ARF.
261 Conclusions The results of the present investigation suggest that coumarin acted synergistically with IBA in stimulating more adventitious root production in mung bean V. radiata cuttings. In addition, there was a positive relationship between the level of IAA accumulation and the number of adventitious roots formed. The synergistic interaction between coumarin and IBA in inducing more ARF may be due to the contribution of coumarin and IBA in increasing the level of free IAA at the potential rooting sites. Furthermore, basic isoPER activity was not a major participant in the oxidation of endogenous free IAA that increases transiently during the early phases of adventitious rooting process. Rather, IAA conjugation is the major mechanism involved in IAA oxidation pathway. The modes of synergistic interaction between coumarin and IBA in inducing more adventitious roots required further work to fully elucidate this mechanism. The interaction between coumarin and IBA with other biological substances, may also participate in the synergistic effect in terms of ARF in V. radiata mung bean cuttings. The interaction between these two substances in terms of certain biological substances is in progress. References Alvarez R., Nissen S.J. and Sutter E.G. 1989. Relationship between indole-3-acetic acid levels in apple (Malus pumila Mill) rootstocks cultured in vitro and adventitious root formation in the presence of indole-3-butyric acid. Plant Physiol. 89: 439–443. Arteca R.N., Poovaiah B.W. and Smith O.E. 1980. Use of high performance liquid chromatography for the determination of endogenous hormone levels in Solanum tuberosum L. subjected to carbon dioxide enrichment of the root zone. Plant Physiol. 65: 1216–1219. Bandurski R.S., Cohen J.D., Slovin J.P. and Reinecke D.M. 1995. Auxin biosynthesis and metabolism. In: Davies P.J. (ed.), Plant Hormones: Physiology, Biochemistry and Molecular Biology, Kluwer Academic Publishers, Kluwer Academic Press, Dordrecht, pp. 39–65. Beffa R., Martin H.V. and Pilet P.-E. 1990. In vitro oxidation of indoleacetic acid by soluble auxin-oxidases and peroxidases from maize roots. Plant Physiol. 94: 485–491. Berthon J.Y., Maldiney R., Sotta B., Gaspar T. and Boyer N. 1989. Endogenous levels of plant hormones during the course of adventitious rooting in cuttings of Sequoiadendron giganteum in vitro. Biochem. Physiol. Pflanzen 184: 405–412.
Blakesley D. 1994. Auxin metabolism and adventitious root initiation. In: Davis T.D. and Haissig B.E. (eds), Biology of Adventitious Root Formation, Plenum Press, New York, pp. 143–154. Blakesley D., Weston G.D. and Elliott M.C. 1991. Endogenous level of indole-3-acetic acid and abscisic acid during the rooting of Cotinus coggygria taken at different times of the year. Plant Growth Regul. 10: 1–2. Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254. Cheng T.Y. 1979. Recent advances in development of in vitro techniques for Douglas fir. In: Sharp W.R., Larsen P.O., Paddock E.F. and Raghavan V. (eds), Plant Cell and Tissue Culture: Principles and Applications, Ohio State University press, Columbus, pp. 493–508. Cohen J.D., Bialek K., Slovin J.P., Baldi B.G. and Chen K.-H. 1988. Development of genetic and analytical systems for studies of auxin metabolism. In: Pharis R.P. and Rood S.B. (eds), Plant Growth Substances 1988, Springer-Verlag, Berlin, pp. 45–56. De Klerk G.J., Smulders R. and Benschop M. 1990. Basic peroxidases and rooting in microcuttings of Malus. Acta Hort. 280: 29–36. Dhawan R.S. and Nanda K.K. 1982. Stimulation of root formation on Impatiens balsamina L. cuttings by coumarin and the associated biochemical changes. Biol. Plant. 24: 177–182. Epstein E. and Ludwig-M€ uller J. 1993. Indole-3-butyric acid in plants: occurrence, synthesis, metabolism and transport. Physiol. Plant. 88: 382–389. Ernstsen A., Sandberg G. and Lundstro¨m K. 1987. Identification of oxindole-3-acetic acid, and metabolic conversion of indole-3-acetic acid to oxindole-3-acetic acid in Pinus sylvestris seeds. Planta 172: 47–52. Fet-Neto A.G., Teixeira S.L., Dasilva E.A.M. and Anna R.S. 1992. Biochemical and morphological changes during in vitro rhizogenesis in cuttings of Sequoia sempervirens (D. Don) EndI. J. Plant Physiol. 140: 720–728. Garcia-Go´mez M.L., Sa´nchez-Romero C., Barcelo´-Mu noz A., Heredia A. and Pliego-Alfaro F. 1994. Levels of endogenous indole-3-acetic acid and indole-3-acetylasparatic acid during adventitious rooting in avocado microcuttings. J. Exp. Bot. 45: 865–870. Garcia-Go´mez M.L., Sa´nchez-Romero C., Barcelo´-Mu noz A., Heredia A. and Pliego-Alfaro F. 1995. Peroxidase activity during adventitious root formation in avocado microcuttings. Can. J. Bot. 73: 1522–1526. Gaspar T. and Hofinger M. 1989. Auxin metabolism during rooting. In: Davis T.K., Haissig B.E. and Sankhla N. (eds), Adventitious Root Formation in Cuttings, Dioscorides Press, Portland, OR, USA, pp. 117–131. Gaspar T., Kevers C. and Hausman J.F. 1997. Indissociable chief factors in the inductive phase of adventitious rooting. In: Altman A. and Waisel Y. (eds), Biology of Root Formation and Development, Plenum Press, New York, pp. 55–63. Gaspar T., Kevers C., Hausman J.F. and Ripetti V. 1994. Peroxidase activity and endogenous free auxin during adventitious root formation. In: Lumsden P.J., Nicholas J.R. and Davies W.J. (eds), Physiology, Growth and Development of
262 Plants in Culture, Kluwer Academic Publishers, Dordrecht, pp. 289–298. Gazaryan I.G., Lagrimini L.M., Ashby G.A. and Thorneley R.N.F. 1996. Mechanism of indole-3-acetic acid oxidation by plant peroxidase: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases. Biochem. J. 313: 841–847. Grahm R.C., Lundholm U. and Karnovsky M.J. 1965. Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbazole. J. Histochem. Cytochem. 13: 150–152. Gronroos R. and Von Arnold S. 1987. Initiation of roots on hypocotyls cuttings of Pinus contorta in vitro. Physiol. Plant. 69: 227–236. Grosser J.W. and Chandler J.L. 1986. In vitro multiplication of Swingle citrumelo rootstock with coumarin. Hort. Sci. 21: 518–520. Gus’Kov A.V., Turetskaya R.Kh., Grin N.Ya. and Kefeli V.I. 1980. Effect of auxin on auxin oxidase activity in bean stem and leaf cuttings during the process of rhizogenesis. K. A. Timiryazev Institute of Plant Physiology, Academy of Sciences of the USSR, Moscow, pp. 431–435. Hartmann H.T., Kester D.E., Davies F.T. Jr and Geneve R.L. 1997. Propagation: Principles and Practices 6th edn. Plant Prentice Hall, Upper Saddle River, NJ. Janson E. and Svensson S. 1980. Coumarin effects on Glycine max hypocotyl explants. Physiol. Plant. 48: 486–490. Jarvis B.C. 1986. Endogenous control of adventitious rooting in non-woody cuttings. In: Jackson M.B. (ed.), New Root Formation in Plants and Cuttings, Martinus Nijhoff Publisher, Dordrecht, 191–122. Kupidlowska E., Kowalec M., Sulkowski G. and Zobel A.M. 1994. The effect of coumarins on root elongation and ultrastructure of meristematic cell protoplast. Ann. Bot. 73: 525–530. Lagrimini L.M. 1991. Peroxidase, IAA oxidase and auxin metabolism in transformed tobacco plants (Abstract No. 497). Plant Physiol. 96: S-77. Lagrimini L.M., Gingas V., Finger F., Rothstein S. and Liu T.T. 1997. Characterization of antisense transformed plants deficient in the tobacco anionic peroxidase. Plant Physiol. 114: 1187–1196. Laskowski M.J., Williams M.E., Nusbaum H.C. and Sussex I.M. 1995. Formation of lateral root meristems is a two-stage process. Development 121: 3303–3310. Liu Z.-H., Hsiao I.-C. and Pan Y.-W. 1996. Effect of naphthaleneacetic acid on endogenous indole-3-acetic acid, peroxidase and auxin oxidase in hypocotyls cuttings of soybean during root formation. Bot. Bull. Acad. Sin. 37: 247–253. Ludwig-M€ uller J. and Epstein E. 1992. Indole-3-acetic acid is converted to indole-3-butyric acid by seedlings of Zea mays. In: Karssen C.M., Van Loon L.C. and Vreugdenhil D. (eds), Progress in Plant Growth Regulation, Kluwer, Dordrecht, pp. 188–193. Marigo G. and Boudet A. 1979. Effects of an increase in level of phenolic compounds on the auxin content and growth of Lycopersicum esculentum. Z. Phlanzenphysiol. 92: 33–38. Moncousin C.H., Favre J.M. and Gaspar T. 1988. Changes in peroxidases activity and endogenous IAA levels during adventitious rooting in vine cuttings. In: Kutacek M., Bandurski R.S. and Krekule J. (eds), Physiology and
Biochemistry of Auxins in Plants, Dioscorides Press, Portland, OR, pp. 331–337. Murray R.D.H. 1991. Naturally occurring plant coumarins. Prog. Chem. Org. Nat. Prod. 58: 83–343. Murray R.D.H., Mendez J. and Brown S.A. 1982. The Natural Coumarins: Occurrence, Chemistry and Biochemistry. J. Wiley and Sons, Chichester. Nordstro¨m A.C. and Eliasson L. 1991. Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in cuttings. Physiol. Plant. 82: 599–605. Nordstro¨m A.C., Jacops F.A. and Eliasson L. 1991. Effect of exogenous indole-3-acetic acid and indole-3-butyric acid on internal levels of the respective auxins and their conjugation with aspartic acid during adventitious root formation in pea cuttings. Plant Physiol. 96: 856–861. Normanly J., Slovin J.P. and Cohen J.D. 1995. Rethinking auxin biosynthesis and metabolism. Plant Physiol. 107: 323–329. Pan R.C. and Gui H.Y. 1997. Physiological basis of synergistic effects of IBA and triadimefon on rooting of mung bean. Plant Growth Regul. 22: 91–98. Pl€ uss R., Jenny T. and Meier H. 1989. IAA-induced adventitious root formation in greenwood of Populus tremula and formation of 2-indolone-3-acetylasparatic acid, a new metabolite of exogenously applied indole-3-acetic acid in cuttings of Populus tremula L. Physiol. Plant. 75: 89–96. Purves W.K. and Hollenberg S.M. 1982. Metabolism of exogenous indoleacetic acid to its amide conjugate in Cucumis sativus L. Plant Physiol. 70: 283–286. Quiroga M., Guerrero C., Botella M.A., Botella A., Barcelo A., Amaya I., Medina M.I., Alonso F.J., Forchetti S.M., Tigier H. and Valpuesta V. 2000. A tomato peroxidase involved in the synthesis of lignin and suberin. Plant Physiol. 122: 1119–1127. Rauscherova L. and Tesfa L. 1993. Interaction of paclobutrazol and other growth regulators in the process of rhizogenesis in Ligustrum vulgare. Sci. Hort. 167: 167–173. Reisfield R.A., Lewis V.J. and Williams D.E. 1962. Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature 195: 281–283. Tartoura K.A.H. 1994. Physiological studies on propagation of apple trees. PhD dissertation, Faculty of Agriculture, Suez Canal University, Ismailia, Egypt. Tsurumi S. and Wada S. 1990. Oxidation of indole-3acetylasparatic acid in Vicia. In: Pharis R.P. and Rood S.P. (eds), Plant Growth Substances 1988, Springer-Verlag, pp. 353–359. € stin A., Sandberg B. and Sundberg G. 1994. A Tuominen H., O novel metabolic pathway for indole-3-acetic acid in apical shoots of Populus tremuloides (Michx). Plant Physiol. 106: 1511–1520. Wiesman Z. and Riov J. 1994. Interaction of paclobutrazol and indole-3-butyric acid in relation to rooting mung bean (Vigna radiata) cuttings. Physiol. Plant. 92: 608–612. Wiesman Z., Riov J. and Epstein E. 1989. Characterization and rooting ability of indole-3-butyric acid conjugates formed during rooting of mung bean cuttings. Plant Physiol. 91: 1080–1084. Wolf F.T. 1974. Effects of coumarin upon plant growth and development. J. Tenn. Acad. Sci. 49: 27–33.
