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Peroxidase and Zeatin Stability
Peroxidase and Zeatin Stability
J. VAN STADEN and C. FoRSYTH Plant Development Research Unit, Botany Department, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received August 27, 1984 ·Accepted November 13, 1984
Summary Authentic. 14C-zeatin was subjected to various enzymatic and chemical treatments in vitro. The treatments included addition of peroxidase and hydrogen peroxide separately and in combination and resulted in all cases, in breakdown of the authentic zeatin. This breakdown was increased where hydrogen peroxide was added to the reaction mixture. Oxidation of zeatin thus resulted in the detection of three new peaks of radioactivity, the most polar of which did not co-elute with any of the authentic markers tested and was not biologically active. The two less polar peaks detected co-eluted with authentic adenine and 6-{2,3,4-trihydroxy-3-methylbutylamino)-purine. Of these the latter compound stimulated the division of soybean callus.
Key words: Zeatin, oxidation, peroxidase.
Introduction While all growth responses are apparently influenced by a delicate balance between the various growth substances, the close relationship between auxins and cytokinins in a number of growth phenomena is particularly noticeable. The exact relationship between these two classes of hormones is at present not known. With respect to rooting it is generally accepted that a high auxin to cytokinin ratio is necessary to optimise this process (Skoog and Miller, 1957; Scott, 1972; Stenlid, 1982). A question which can be asked is: how is this balance achieved? From the literature it would appear that both auxins (Schneider and Wightman, 1978) and cytokinins (Whitty and Hall, 1974; McGaw and Horgan, 1983) are metabolised rapidly by oxidation processes. In cuttings the rooting process is evidenced and characterised by a rise in total peroxidase activity which is largely responsible for in vivo auxin catabolism (Gaspar, 1980). Lee {1971) has reported that exogenous cytokinin affects the distribution of indoleacetic acid oxidase isoenzymes. The enzyme N 6-(..i2-iso-pentenyl)adenosine oxidase or cytokinin oxidase, isolated by Whitty and Hall (1974) apparently has some of the properties of a mixed-function oxidase as it gave a peroxidase reaction with o-dianisidine and hydrogen peroxide. As a result it was speculated that these oxidase and peroxidase activities may play a role in the known physiological interaction of auxins and cytokinins. At present it is not known to what extent peroxidase affects cytokinins. In this investigation zeatin was subjected to peroxidase treatment and its stability to this enzyme investigated.
f. Plant. Physiol. Vol. 118. pp. 367-371 {1985)
368
J. VAN STADEN and C. FORSYTH
Materials and Methods
Cold and 8e 4C)trans-zeatin (441.1 MBq mmol- I) synthesized by Dr. J. Corse, western Regional Research Centre, Albany, U.S.A. were used in this investigation. Prior to commencing the experiments the cold (10- 5 M) and labelled zeatin were combined and the mixture then fractionated on a Sephadex LH-20 column (101x2.5cm) to establish its purity. The column was eluted with 10% methanol (Hutton and Van Staden, 1981). Fractions of 40ml each were collected and a portion of each fraction analyzed for both biological activity and radioactivity. Biological activity was established using the soybean callus bioassay (Miller, 1965). Radioactivity was determined using a Beckman LS3800 scintillation counter as described previously (Hutton and Van Staden, 1981). Only a single peak of biological- and radioactivity was detected (Fig. 1 A). This peak was subjected to HPLC analysis. It again yielded only a single UV- and radioactive peak which had a similar retention time to that of trans-zeatin (Fig. 1 C). The purified zeatin was taken to dryness and redissolved in a 0.1 M phosphate buffer at pH 6.0. The zeatin was then subjected to the treatments outlined in Table 1. All treatments were incubated at 20 °C for 3 hours whereafter 5 ml ethanol was added to each reaction flask. The respective treatments were reduced to a small volume which was filtered through a millipore filter (0.22 JLm), taken to dryness, resuspended in 0.2 ml 10% methanol and subsequently subjected to HPLC analysis. Fractions of 1 ml each were collected, half of which was used for radioassay and the remainder for the detection of biological activity.
Results and Discussion Only one peak of biological activity and a corresponding peak of radioactivity was detected when the authentic cold and labelled zeatin mixture used in this experiment was fractionated on a Sephadex LH-20 column (Fig. 1A). When subjected to HPLC analysis only one radioactive and biologically active peak which had a similar retention time to that of trans-zeatin was detected (Fig. 1 C). That this experimental material consisted of authentic zeatin was confirmed by means of low resolution mass spectrometry. The zeatin used was not affected by the incubation conditions and a single radioactive peak which co-eluted with trans-zeatin was recovered at the end of the experiment (Table 1, treatment A). With the addition of peroxidase two polar radioactive peaks were detected, however, 92.8% of the radioactivity was still associated with trans-zeatin (Table 1, treatment B). The addition of hydrogen peroxide to the reaction medium increased the breakdown of zeatin in all cases (Table 1, treatments C, D, E). The major radioactive peak detected in all cases was polar and had a retention time of 2.1 min. The other two peaks of radioactivity had retention times very similar to those of adenine and 6-(2,3,4-trihydroxy-3-methylbutylamino)purine. Of the radioactive peaks detected following HPLC only the two less polar peaks, which co-chromatographed with 6-(2,3,4-trihydroxy-3-methylbutylamino)purine and trans-zeatin were associated with biological activity (Fig. 2). The two most polar peaks of radioactivity detected were not associated with biological activity. As one of these peaks co-chromatographed with adenine, following separation by means of HPLC, it would appear as if oxidation of zeatin did occur. The addition of hydrogen peroxide to the reaction medium accelerated the reaction and resulted in detection of a fourth
J Plant. Physiol. Vol.
118. pp. 367-371 (1985}
Peroxidase and zeatin stability
369
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RETENTION TIME min
Fig. 1: A. Biological (histogram) and radioactivity (• - • ) detected following the fractionation of a mixture of the cold and labelled zeatin used as experimental material. The mixture was fractionated on a Sephadex LH-20 column using 10% methanol as solvent. B. Separation of authentic adenine (Ade), 6-(2,3,4-trihydroxy-3-methylbutylamino)purine (THZ), and transzeatin {tZ) by reversed phase HPLC. Column Micropak MCH-5 (5 p.m, C18 bonded, 150 x 4 mm i.d.) flow rate 1 ml min -I; mobile phase, water to 4% acetonitrile over 10 min, then to 30% acetonitrile over 20 min. Absorbance was recorded with a Varian variable wavelength monitor at 265 nm fitted with a 8 p.l flow-through cell. C. HPLC separation of the biologicaland radioactive peak which co-eluted with zeatin after Sephadex fractionation. Fractions of 1 ml each were collected every minute and the radioactivity in half of each fraction recorded after 5 ml of Ready Solve EP had been added to it (• - •). The other half of each fraction was used for the detection of biological activity (histogram).
]. Plant. Physiol. Vol. 118. pp. 367-371 {1985)
370
J. VAN STADEN and C. FoRSYTH
Table 1: Distribution of radioactivity (% of total recovered) after zeatin had been subjected to various treatments. All reaction mixtures were separated by means of HPLC as described for figure 1. Ade = Adenine, THZ = 6-(2,3,4-trihydroxy-3-methylbutylamino)purine; tZ = transzeatin. Treatments as indicated in Materials and Methods
Retention time of radioactive peak (min)
B
A
2.1 17.2 (Ade) 20.1 (THZ) 25.6 (tZ)
4.6 2.6 100
92.8
c
D
E
13.8 7.3 5.7 73.2
16.8 6.0 8.4 68.8
7.9 7.7 5.0 79.4
Treatments were as follows: A= zeatin control; B = zeatin+peroxidase (Sigma, horse radish, 46 units per 2ml); C = zeatin+H202 (1ml, 3%); D = zeatin+peroxidase+H202; E = zeatin+peroxidase+H202+0.1M MnCh (1 ml).
®
@ AdeTHZ
tZ
24
Ade THZ tZ
30
RETENTION TIME min
Fig.2: Biological (histogram) and radioactivity (•-•) detected following the separation of enzyme treated samples of trans-zeatin by reversed phase HPLC. Conditions were as described in Figure 1. A. = Trans-zeatin + H 20 2 + peroxidase. B. = Trans-zeatin + H 20 2 + peroxidase + MnCh. Abbreviations as in Figure 1.
peak which co-chromatographed with 6-(2,3,4-trihydroxy-3-methylbutylamino)purine. This compound has been identified previously following K.Mn04 oxidation of zeatin (Van Staden et al., 1982). Cytokinin oxidase was first isolated from maize kernels (Whitty and Hall, 1974). Subsequently a similar type of enzyme with a different molecular weight was isolated from Vinca rosea (McGaw and Horgan, 1983). Specificity studies have shown that both enzymes require a !12 double bond in the N 6 side chain for activity. As a result of the enzymatic reaction side chain cleavage occurs and adenine is produced as one of the end products. Whitty and Hall (1974) indicated that the enzyme isolated had ]. Plant. PhysioL Vol. 118. pp. 367-371 {1985}
Peroxidase and zeatin stability
371
some of the properties of a mixed-function oxidase, as it gave some of the reactions associated with peroxidase. The present results indicate that peroxidase can bring about oxidation of trans-zeatin and that the reaction is accelerated when hydrogen peroxide is present in the reaction medium. The results obtained suggest that the activity of peroxidase enzymes in plant tissues may affect physiological responses related to an interaction of auxins and cytokinins to a considerable degree. Acknowledgements The financial support of the C.S.I.R. is gratefully acknowledged.
References GASPAR, T.: Rooting and flowering, two antagonistic phenomena from a hormonal point of view. Aspects and Prospects of Plant Growth Regulators - Joint DPGRG and BPGRG Symposium Monograph 6, 39-49 (1980}. HurroN, M.J. andJ. VAN STADEN: An efficient column chromatographic method for separating cytokinins. Ann. Bot. 47, 527-529 (1981}. LEE, T. T.: Cytokinin-controlled indoleacetic acid oxidase isoenzymes in tobacco callus cultures. Plant Physiol. 47, 181-185 {1971}. McGAw, B. A. and R. HoRGAN: Cytokinin oxidase from Zea mays kernels and Vinca rosea crown-gall tissue. Planta 159, 30-37 (1983}. MillER, C. 0.: Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. Natl. Acad. Sci. U.S.A. 54, 1052-1058 (1965}. ScHNEIDER, E. A. and F. WIGHTMAN: Auxins. In: Phytohormones and Related Compounds - A Comprehensive Treatise Vol. I. Eds.: LETHAM, D. S., P. B. GooDWIN, and T. J. V. HiGGINS: Elsevier/North-Holland, 1978. ScoTT, T. K.: Auxins and roots. Ann. Rev. Plant Physiol. 23, 235-258 (1972}. SKOOG, F. and C. 0. MillER: Chemical regulation of growth and organ formation in plant tissue culture in vitro. Symp. Soc. Exp. Bioi. 11, 118-131 (1957}. STENLID, G.: Cytokinins as inhibitors of root growth. Physiol. Plant. 56, 500-506 (1982}. VAN STADEN, J., S. E. DREWES, and M. J. HuTTON: Biological activity of 6-{2,3,4-trihydroxy-3methylbutylamino}purine, an oxidation product of zeatin. Physiol. Plant. 55, 143-148 (1982}. WHITTY, C. D. and R. H. HAll.: A cytokinin oxidase in Zea mays. Can. J. Bot. 52, 789-799 (1974}.
]. Plant. Physiol. Vol. 118. pp. 367-371 (1985}
J. VAN STADEN and C. FoRSYTH Plant Development Research Unit, Botany Department, University of Natal, Pietermaritzburg 3200, Republic of South Africa Received August 27, 1984 ·Accepted November 13, 1984
Summary Authentic. 14C-zeatin was subjected to various enzymatic and chemical treatments in vitro. The treatments included addition of peroxidase and hydrogen peroxide separately and in combination and resulted in all cases, in breakdown of the authentic zeatin. This breakdown was increased where hydrogen peroxide was added to the reaction mixture. Oxidation of zeatin thus resulted in the detection of three new peaks of radioactivity, the most polar of which did not co-elute with any of the authentic markers tested and was not biologically active. The two less polar peaks detected co-eluted with authentic adenine and 6-{2,3,4-trihydroxy-3-methylbutylamino)-purine. Of these the latter compound stimulated the division of soybean callus.
Key words: Zeatin, oxidation, peroxidase.
Introduction While all growth responses are apparently influenced by a delicate balance between the various growth substances, the close relationship between auxins and cytokinins in a number of growth phenomena is particularly noticeable. The exact relationship between these two classes of hormones is at present not known. With respect to rooting it is generally accepted that a high auxin to cytokinin ratio is necessary to optimise this process (Skoog and Miller, 1957; Scott, 1972; Stenlid, 1982). A question which can be asked is: how is this balance achieved? From the literature it would appear that both auxins (Schneider and Wightman, 1978) and cytokinins (Whitty and Hall, 1974; McGaw and Horgan, 1983) are metabolised rapidly by oxidation processes. In cuttings the rooting process is evidenced and characterised by a rise in total peroxidase activity which is largely responsible for in vivo auxin catabolism (Gaspar, 1980). Lee {1971) has reported that exogenous cytokinin affects the distribution of indoleacetic acid oxidase isoenzymes. The enzyme N 6-(..i2-iso-pentenyl)adenosine oxidase or cytokinin oxidase, isolated by Whitty and Hall (1974) apparently has some of the properties of a mixed-function oxidase as it gave a peroxidase reaction with o-dianisidine and hydrogen peroxide. As a result it was speculated that these oxidase and peroxidase activities may play a role in the known physiological interaction of auxins and cytokinins. At present it is not known to what extent peroxidase affects cytokinins. In this investigation zeatin was subjected to peroxidase treatment and its stability to this enzyme investigated.
f. Plant. Physiol. Vol. 118. pp. 367-371 {1985)
368
J. VAN STADEN and C. FORSYTH
Materials and Methods
Cold and 8e 4C)trans-zeatin (441.1 MBq mmol- I) synthesized by Dr. J. Corse, western Regional Research Centre, Albany, U.S.A. were used in this investigation. Prior to commencing the experiments the cold (10- 5 M) and labelled zeatin were combined and the mixture then fractionated on a Sephadex LH-20 column (101x2.5cm) to establish its purity. The column was eluted with 10% methanol (Hutton and Van Staden, 1981). Fractions of 40ml each were collected and a portion of each fraction analyzed for both biological activity and radioactivity. Biological activity was established using the soybean callus bioassay (Miller, 1965). Radioactivity was determined using a Beckman LS3800 scintillation counter as described previously (Hutton and Van Staden, 1981). Only a single peak of biological- and radioactivity was detected (Fig. 1 A). This peak was subjected to HPLC analysis. It again yielded only a single UV- and radioactive peak which had a similar retention time to that of trans-zeatin (Fig. 1 C). The purified zeatin was taken to dryness and redissolved in a 0.1 M phosphate buffer at pH 6.0. The zeatin was then subjected to the treatments outlined in Table 1. All treatments were incubated at 20 °C for 3 hours whereafter 5 ml ethanol was added to each reaction flask. The respective treatments were reduced to a small volume which was filtered through a millipore filter (0.22 JLm), taken to dryness, resuspended in 0.2 ml 10% methanol and subsequently subjected to HPLC analysis. Fractions of 1 ml each were collected, half of which was used for radioassay and the remainder for the detection of biological activity.
Results and Discussion Only one peak of biological activity and a corresponding peak of radioactivity was detected when the authentic cold and labelled zeatin mixture used in this experiment was fractionated on a Sephadex LH-20 column (Fig. 1A). When subjected to HPLC analysis only one radioactive and biologically active peak which had a similar retention time to that of trans-zeatin was detected (Fig. 1 C). That this experimental material consisted of authentic zeatin was confirmed by means of low resolution mass spectrometry. The zeatin used was not affected by the incubation conditions and a single radioactive peak which co-eluted with trans-zeatin was recovered at the end of the experiment (Table 1, treatment A). With the addition of peroxidase two polar radioactive peaks were detected, however, 92.8% of the radioactivity was still associated with trans-zeatin (Table 1, treatment B). The addition of hydrogen peroxide to the reaction medium increased the breakdown of zeatin in all cases (Table 1, treatments C, D, E). The major radioactive peak detected in all cases was polar and had a retention time of 2.1 min. The other two peaks of radioactivity had retention times very similar to those of adenine and 6-(2,3,4-trihydroxy-3-methylbutylamino)purine. Of the radioactive peaks detected following HPLC only the two less polar peaks, which co-chromatographed with 6-(2,3,4-trihydroxy-3-methylbutylamino)purine and trans-zeatin were associated with biological activity (Fig. 2). The two most polar peaks of radioactivity detected were not associated with biological activity. As one of these peaks co-chromatographed with adenine, following separation by means of HPLC, it would appear as if oxidation of zeatin did occur. The addition of hydrogen peroxide to the reaction medium accelerated the reaction and resulted in detection of a fourth
J Plant. Physiol. Vol.
118. pp. 367-371 (1985}
Peroxidase and zeatin stability
369
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>-
1-
Q:
~<
2
THZ 20.4
u
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24
30
0
6
RETENTION TIME min
Fig. 1: A. Biological (histogram) and radioactivity (• - • ) detected following the fractionation of a mixture of the cold and labelled zeatin used as experimental material. The mixture was fractionated on a Sephadex LH-20 column using 10% methanol as solvent. B. Separation of authentic adenine (Ade), 6-(2,3,4-trihydroxy-3-methylbutylamino)purine (THZ), and transzeatin {tZ) by reversed phase HPLC. Column Micropak MCH-5 (5 p.m, C18 bonded, 150 x 4 mm i.d.) flow rate 1 ml min -I; mobile phase, water to 4% acetonitrile over 10 min, then to 30% acetonitrile over 20 min. Absorbance was recorded with a Varian variable wavelength monitor at 265 nm fitted with a 8 p.l flow-through cell. C. HPLC separation of the biologicaland radioactive peak which co-eluted with zeatin after Sephadex fractionation. Fractions of 1 ml each were collected every minute and the radioactivity in half of each fraction recorded after 5 ml of Ready Solve EP had been added to it (• - •). The other half of each fraction was used for the detection of biological activity (histogram).
]. Plant. Physiol. Vol. 118. pp. 367-371 {1985)
370
J. VAN STADEN and C. FoRSYTH
Table 1: Distribution of radioactivity (% of total recovered) after zeatin had been subjected to various treatments. All reaction mixtures were separated by means of HPLC as described for figure 1. Ade = Adenine, THZ = 6-(2,3,4-trihydroxy-3-methylbutylamino)purine; tZ = transzeatin. Treatments as indicated in Materials and Methods
Retention time of radioactive peak (min)
B
A
2.1 17.2 (Ade) 20.1 (THZ) 25.6 (tZ)
4.6 2.6 100
92.8
c
D
E
13.8 7.3 5.7 73.2
16.8 6.0 8.4 68.8
7.9 7.7 5.0 79.4
Treatments were as follows: A= zeatin control; B = zeatin+peroxidase (Sigma, horse radish, 46 units per 2ml); C = zeatin+H202 (1ml, 3%); D = zeatin+peroxidase+H202; E = zeatin+peroxidase+H202+0.1M MnCh (1 ml).
®
@ AdeTHZ
tZ
24
Ade THZ tZ
30
RETENTION TIME min
Fig.2: Biological (histogram) and radioactivity (•-•) detected following the separation of enzyme treated samples of trans-zeatin by reversed phase HPLC. Conditions were as described in Figure 1. A. = Trans-zeatin + H 20 2 + peroxidase. B. = Trans-zeatin + H 20 2 + peroxidase + MnCh. Abbreviations as in Figure 1.
peak which co-chromatographed with 6-(2,3,4-trihydroxy-3-methylbutylamino)purine. This compound has been identified previously following K.Mn04 oxidation of zeatin (Van Staden et al., 1982). Cytokinin oxidase was first isolated from maize kernels (Whitty and Hall, 1974). Subsequently a similar type of enzyme with a different molecular weight was isolated from Vinca rosea (McGaw and Horgan, 1983). Specificity studies have shown that both enzymes require a !12 double bond in the N 6 side chain for activity. As a result of the enzymatic reaction side chain cleavage occurs and adenine is produced as one of the end products. Whitty and Hall (1974) indicated that the enzyme isolated had ]. Plant. PhysioL Vol. 118. pp. 367-371 {1985}
Peroxidase and zeatin stability
371
some of the properties of a mixed-function oxidase, as it gave some of the reactions associated with peroxidase. The present results indicate that peroxidase can bring about oxidation of trans-zeatin and that the reaction is accelerated when hydrogen peroxide is present in the reaction medium. The results obtained suggest that the activity of peroxidase enzymes in plant tissues may affect physiological responses related to an interaction of auxins and cytokinins to a considerable degree. Acknowledgements The financial support of the C.S.I.R. is gratefully acknowledged.
References GASPAR, T.: Rooting and flowering, two antagonistic phenomena from a hormonal point of view. Aspects and Prospects of Plant Growth Regulators - Joint DPGRG and BPGRG Symposium Monograph 6, 39-49 (1980}. HurroN, M.J. andJ. VAN STADEN: An efficient column chromatographic method for separating cytokinins. Ann. Bot. 47, 527-529 (1981}. LEE, T. T.: Cytokinin-controlled indoleacetic acid oxidase isoenzymes in tobacco callus cultures. Plant Physiol. 47, 181-185 {1971}. McGAw, B. A. and R. HoRGAN: Cytokinin oxidase from Zea mays kernels and Vinca rosea crown-gall tissue. Planta 159, 30-37 (1983}. MillER, C. 0.: Evidence for the natural occurrence of zeatin and derivatives: Compounds from maize which promote cell division. Proc. Natl. Acad. Sci. U.S.A. 54, 1052-1058 (1965}. ScHNEIDER, E. A. and F. WIGHTMAN: Auxins. In: Phytohormones and Related Compounds - A Comprehensive Treatise Vol. I. Eds.: LETHAM, D. S., P. B. GooDWIN, and T. J. V. HiGGINS: Elsevier/North-Holland, 1978. ScoTT, T. K.: Auxins and roots. Ann. Rev. Plant Physiol. 23, 235-258 (1972}. SKOOG, F. and C. 0. MillER: Chemical regulation of growth and organ formation in plant tissue culture in vitro. Symp. Soc. Exp. Bioi. 11, 118-131 (1957}. STENLID, G.: Cytokinins as inhibitors of root growth. Physiol. Plant. 56, 500-506 (1982}. VAN STADEN, J., S. E. DREWES, and M. J. HuTTON: Biological activity of 6-{2,3,4-trihydroxy-3methylbutylamino}purine, an oxidation product of zeatin. Physiol. Plant. 55, 143-148 (1982}. WHITTY, C. D. and R. H. HAll.: A cytokinin oxidase in Zea mays. Can. J. Bot. 52, 789-799 (1974}.
]. Plant. Physiol. Vol. 118. pp. 367-371 (1985}