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Influence of ethylene on cytokinin pools in tuberizing potatoes
Plant Science Letters~ 10 (1977) 19--23 @Elsevier/North-Holland Scientific Publishers, Ltd
19
INFLUENCE OF ETHYLENE ON CYTOKININ POOLS IN TUBERIZING POTATOES
J. VAN STADEN and G.G. DIMALLA
Department of Botany, University of Natal, Pietermaritzburg (South Africa) (Received March 8th, 1977) (Accepted April 22nd, 1977)
SUMMARY
Low concentrations of ethylene partly inhibit tuberization and result in the formation of "hybrid" swellings which contain limited amounts of starch. It would appear as if ethylene treatment results in an accumulation of cytokinin glucosides which may be inactive in the tuberizing process.
INTRODUCTION
Treatment of potato tubers, predisposed to the little potato disorder (premature tuberization), with high concentrations of ethrel (ethylene) results in a complete inhibition of little potato formation [1]. High concentrations of ethylene also inhibit natural tuberization [2] as well as kinetin-induced tuberization of in vitro grown stolons [3]. Mingo-Castel et al. [3] found that whenever tuberization had been triggered, the inhibitory effect of ethylene was less pronounced and that its effect was manifested as an interference with tuber development. This interference results in the formation of morphologically incomplete tubers or what is also referred to as a "hybrid" type of swelling [3]. Low concentrations of ethylene have been reported to bring about the swelling of all rapidly expanding regions of stolons, stems and axillary buds [4]. While investigating the little potato disorder it was observed that tubers predisposed to the disorder and which were treated with low concentrations of ethrel formed stolons with "hybrid" swellings at their subapical regions (Fig. 1). Development of these "hybrid" swellings soon stopped. The upper part of the stolon continued growing and developed a tightly closed apical h o o k (Fig. 1). Following this the stolons again elongated and after about 40 days a tuber was formed at the apex of the extended stolons (Fig. 2). The formation of these "hybrid" swellings seems to represent an incomplete reversal or inhibition of the tuberizing process, in which cytokinins have been implicated [5]. This paper reports on the levels of endogenous cytokinins in "hybrid" swellings that had been induced by low levels of ethylene.
20
Fig. 1. A: reversal of the little potato disorder and formation of " h y b r i d " swellings by immersing aged tubers in 100 mg/l ethrel. B: control showing little potato disorder.
Fig. 2. Development of "hybrid" swellings. A: " h y b r i d " swellings; B: elongation of apex of the " h y b r i d " swelling; C: development of small tubers at the apex of extended stolons. MATERIAL AND METHODS
Po tato tubers (Solanum tuberosum L. cv. U p - t o . l a t e ) were stored at 3°C in the dark f o r 10 months. After transferring these tubers to 20°C, p r e m a t u r e
21
tubefization occurred within I0 days (Fig. 1). Immersing the aged tubers in I00 mg/l 2-chloroethylphosphoric acid (ethrel) before transferring them to the higher temperature resulted in the formation of "hybrid" swellings (Fig. 2). Little tubers and "hybrid" swellings were collected 30 days after transferring the control and ethrel treated tubers to 20°C. The material obtained was homogenized with sufficient ethanol to give a final concentration of 80% ethanol. Cytokinins were extracted by means of a cation exchange resin [6] and assayed using the soybean bioassay [7]. Extracts were strip-loaded onto Whatman No. 1 chromatography paper and separated in isopropanol 1--25% ammonium hydroxide--water (10 : 1 : I v/v). Dried chromatograms were divided into 10 equal Rf strips and each of these individually assayed for cytokinin activity. RESULTS AND DISCUSSION
As previously reported [1] exposure to ethylene resulted in a decrease in the endogenous cytokinin levels in treated stolons that did not tuberize. "Hybrid" swellings contained lower levels of cytokinin than little tubers (Fig. 3). The activity on the paper chromatograms co~hromatographed with zeatin and zeatin riboside. No activity was detected on the chromatogram where zeatin glucoside normally occurs. This, however, does not exclude the possibility of glucosylated cytokinins being present, as impurities may inhibit the growth of soybean zG
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Fig. 3. Soybean bioMsays of 30 grof little potato tubers (A) and "hybrid" swellings (B) purified by Dowex 50 and ehromatographed in isopropanol--ammonia---water (10 : 1 : 1 v/v). Z, zeatin; ZR, zeatin riboside; ZG, zeatin glueoside. Fig. 4. Soybean biomays of 30 g little potato tubers (A) and "hybrid" swellinp (B) after fractionation on Sephadex LH-20. The broken line indicates the callus yield obtained with 10/~g/l kinetin. Z, zeatin; ZR, zeatin riboside; ZG, zeatin glucoside.
22
callus in the Rf-strips concerned (Rf 0.20--0.50). Fractionation on Sephadex LH. 20 has shown that cytokinin glucosides are indeed present in potato extracts [8]. The highest levels being recorded in old tubers and young elongating sprouts. In the stolon tips and young tubers only low levels of cytokinin glucoside could be detected [8]. Sephadex LH-20 fractionation [9] of extracts of little tubers and "hybrid" swellings which had been chromatographed on paper (Rf 0.20-0.90) revealed the presence of cell division compounds that coelute with zeatin, zeatin riboside and zeatin glucoside (Fig. 4). In the little tubers the peak co-eluting with zeatin riboside was responsible for the highest callus yield while in the "hybrid" swellings most activity was found where zeatin glucoside elutes. It is difficult to compare the activity of the different free bases with the cytokinin glucosides as their relative activity in the soybean bioassay is not yet known. Van Staden (unpublished) has found that high con-
Fig. 5. Electron micrograph showing the presence of starch (S) in "hybrid" swellings.
23
centrations of zeatin glucoside are less toxic to soybean callus than similar concentrations of zeatin and zeatin riboside. When zeatin glucoside is applied to soybean callus, free zeatin can be extracted from the dividing callus suggesting that the callus has the ability to hydrolyse zeatin glucoside as required. These findings are consistent with the view that cytokinin glucosides are storage or inactive forms of hormone [10,11]. In potatoes, it would appear as if low ethylene concentrations can bring about the conversion of free cytokinins to their glucosides. Alternatively, the higher level of cytokinin glucoside in "hybrid" swellings could have been the result of an inability of the tissue to hydrolyse the cytokinin glucoside. The resulting accumulation of inactive or storage cytokinin could explain why tuberization of ethylene treated stolons is inhibited. This may occur by eliminating the formation of a metabolic sink [12], by inhibiting cell division in the sub-apicai meristem [1], or by preventing starch synthesis [3]. Catchpole and Hillman [4] reported that swellings on potato sprouts induced by ethrel did not contain starch. An ultrastructurai investigation revealed that "hybrid" swellings formed in the presence of a low ethrel concentration did contain limited amounts of starch (Fig. 5). Lateral expansion of the procambial tissue was, however, inhibited. This may account for the development of "hybrid" swellings. If the endogenous cytokinins are indeed involved in tuberization the effect of ethylene may well be to prevent them from functioning, particularly in the cell division process. ACKNOWLEDGEMENTS The financial assistance of the C.S.I.R., Pretoria, to the senior author is grate fully acknowledged. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
G.G. Dimalla and J. van Staden, Plant Physiol., in press. A.M. Mingo-Castel, F.B. Negrn and O.E. Smith, Plant Physiol., 58 (1974) 798. A.M. Mingo-Cmztel, O.E. Smith and J. Kumamoto, Plant Physiol., 57 (1976) 480. A.H. Catchpole and J. Hillman, Nature, 223 (1969) 1387. J. van Staden and G.G. Dimalla, Ann. Bot., 40 (1976) 1117. J. van Staden, Physiol. Plant., 38 (1976) 240. C.O. Miller, Proc. Natl. Acad. Sci. (Wash.), 54 (1965) 1052. J. van Staden and G.G. Dimalla, Ann. Bot.~ in press. J. van Staden, Physiol. Plant., 36 (1976) 123. C.W. Parker and D.S. Letham, Planta, 114 (1973) 199. J. van Staden, Physiol. Plant., 38 (1976) 1. A.R. Langflle and P.L. Fomline, Plant Sci. Left., 2 (1974) 189.
19
INFLUENCE OF ETHYLENE ON CYTOKININ POOLS IN TUBERIZING POTATOES
J. VAN STADEN and G.G. DIMALLA
Department of Botany, University of Natal, Pietermaritzburg (South Africa) (Received March 8th, 1977) (Accepted April 22nd, 1977)
SUMMARY
Low concentrations of ethylene partly inhibit tuberization and result in the formation of "hybrid" swellings which contain limited amounts of starch. It would appear as if ethylene treatment results in an accumulation of cytokinin glucosides which may be inactive in the tuberizing process.
INTRODUCTION
Treatment of potato tubers, predisposed to the little potato disorder (premature tuberization), with high concentrations of ethrel (ethylene) results in a complete inhibition of little potato formation [1]. High concentrations of ethylene also inhibit natural tuberization [2] as well as kinetin-induced tuberization of in vitro grown stolons [3]. Mingo-Castel et al. [3] found that whenever tuberization had been triggered, the inhibitory effect of ethylene was less pronounced and that its effect was manifested as an interference with tuber development. This interference results in the formation of morphologically incomplete tubers or what is also referred to as a "hybrid" type of swelling [3]. Low concentrations of ethylene have been reported to bring about the swelling of all rapidly expanding regions of stolons, stems and axillary buds [4]. While investigating the little potato disorder it was observed that tubers predisposed to the disorder and which were treated with low concentrations of ethrel formed stolons with "hybrid" swellings at their subapical regions (Fig. 1). Development of these "hybrid" swellings soon stopped. The upper part of the stolon continued growing and developed a tightly closed apical h o o k (Fig. 1). Following this the stolons again elongated and after about 40 days a tuber was formed at the apex of the extended stolons (Fig. 2). The formation of these "hybrid" swellings seems to represent an incomplete reversal or inhibition of the tuberizing process, in which cytokinins have been implicated [5]. This paper reports on the levels of endogenous cytokinins in "hybrid" swellings that had been induced by low levels of ethylene.
20
Fig. 1. A: reversal of the little potato disorder and formation of " h y b r i d " swellings by immersing aged tubers in 100 mg/l ethrel. B: control showing little potato disorder.
Fig. 2. Development of "hybrid" swellings. A: " h y b r i d " swellings; B: elongation of apex of the " h y b r i d " swelling; C: development of small tubers at the apex of extended stolons. MATERIAL AND METHODS
Po tato tubers (Solanum tuberosum L. cv. U p - t o . l a t e ) were stored at 3°C in the dark f o r 10 months. After transferring these tubers to 20°C, p r e m a t u r e
21
tubefization occurred within I0 days (Fig. 1). Immersing the aged tubers in I00 mg/l 2-chloroethylphosphoric acid (ethrel) before transferring them to the higher temperature resulted in the formation of "hybrid" swellings (Fig. 2). Little tubers and "hybrid" swellings were collected 30 days after transferring the control and ethrel treated tubers to 20°C. The material obtained was homogenized with sufficient ethanol to give a final concentration of 80% ethanol. Cytokinins were extracted by means of a cation exchange resin [6] and assayed using the soybean bioassay [7]. Extracts were strip-loaded onto Whatman No. 1 chromatography paper and separated in isopropanol 1--25% ammonium hydroxide--water (10 : 1 : I v/v). Dried chromatograms were divided into 10 equal Rf strips and each of these individually assayed for cytokinin activity. RESULTS AND DISCUSSION
As previously reported [1] exposure to ethylene resulted in a decrease in the endogenous cytokinin levels in treated stolons that did not tuberize. "Hybrid" swellings contained lower levels of cytokinin than little tubers (Fig. 3). The activity on the paper chromatograms co~hromatographed with zeatin and zeatin riboside. No activity was detected on the chromatogram where zeatin glucoside normally occurs. This, however, does not exclude the possibility of glucosylated cytokinins being present, as impurities may inhibit the growth of soybean zG
2.0
zR
z
!.0
34
zn
:n
..K.
_L
z...Gs
~1.~
!'0
0
L.rI
n
0
0-S
I
I
t.0 0
Rf
~
I_
0,5
1,0
i 0
4
i I00
~ i 3~
I 48O
L.e -a
LJ
l $40
l 8OO
t.J i NO
l 1120
~LUT/ON VOLUMEml
Fig. 3. Soybean bioMsays of 30 grof little potato tubers (A) and "hybrid" swellings (B) purified by Dowex 50 and ehromatographed in isopropanol--ammonia---water (10 : 1 : 1 v/v). Z, zeatin; ZR, zeatin riboside; ZG, zeatin glueoside. Fig. 4. Soybean biomays of 30 g little potato tubers (A) and "hybrid" swellinp (B) after fractionation on Sephadex LH-20. The broken line indicates the callus yield obtained with 10/~g/l kinetin. Z, zeatin; ZR, zeatin riboside; ZG, zeatin glucoside.
22
callus in the Rf-strips concerned (Rf 0.20--0.50). Fractionation on Sephadex LH. 20 has shown that cytokinin glucosides are indeed present in potato extracts [8]. The highest levels being recorded in old tubers and young elongating sprouts. In the stolon tips and young tubers only low levels of cytokinin glucoside could be detected [8]. Sephadex LH-20 fractionation [9] of extracts of little tubers and "hybrid" swellings which had been chromatographed on paper (Rf 0.20-0.90) revealed the presence of cell division compounds that coelute with zeatin, zeatin riboside and zeatin glucoside (Fig. 4). In the little tubers the peak co-eluting with zeatin riboside was responsible for the highest callus yield while in the "hybrid" swellings most activity was found where zeatin glucoside elutes. It is difficult to compare the activity of the different free bases with the cytokinin glucosides as their relative activity in the soybean bioassay is not yet known. Van Staden (unpublished) has found that high con-
Fig. 5. Electron micrograph showing the presence of starch (S) in "hybrid" swellings.
23
centrations of zeatin glucoside are less toxic to soybean callus than similar concentrations of zeatin and zeatin riboside. When zeatin glucoside is applied to soybean callus, free zeatin can be extracted from the dividing callus suggesting that the callus has the ability to hydrolyse zeatin glucoside as required. These findings are consistent with the view that cytokinin glucosides are storage or inactive forms of hormone [10,11]. In potatoes, it would appear as if low ethylene concentrations can bring about the conversion of free cytokinins to their glucosides. Alternatively, the higher level of cytokinin glucoside in "hybrid" swellings could have been the result of an inability of the tissue to hydrolyse the cytokinin glucoside. The resulting accumulation of inactive or storage cytokinin could explain why tuberization of ethylene treated stolons is inhibited. This may occur by eliminating the formation of a metabolic sink [12], by inhibiting cell division in the sub-apicai meristem [1], or by preventing starch synthesis [3]. Catchpole and Hillman [4] reported that swellings on potato sprouts induced by ethrel did not contain starch. An ultrastructurai investigation revealed that "hybrid" swellings formed in the presence of a low ethrel concentration did contain limited amounts of starch (Fig. 5). Lateral expansion of the procambial tissue was, however, inhibited. This may account for the development of "hybrid" swellings. If the endogenous cytokinins are indeed involved in tuberization the effect of ethylene may well be to prevent them from functioning, particularly in the cell division process. ACKNOWLEDGEMENTS The financial assistance of the C.S.I.R., Pretoria, to the senior author is grate fully acknowledged. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12
G.G. Dimalla and J. van Staden, Plant Physiol., in press. A.M. Mingo-Castel, F.B. Negrn and O.E. Smith, Plant Physiol., 58 (1974) 798. A.M. Mingo-Cmztel, O.E. Smith and J. Kumamoto, Plant Physiol., 57 (1976) 480. A.H. Catchpole and J. Hillman, Nature, 223 (1969) 1387. J. van Staden and G.G. Dimalla, Ann. Bot., 40 (1976) 1117. J. van Staden, Physiol. Plant., 38 (1976) 240. C.O. Miller, Proc. Natl. Acad. Sci. (Wash.), 54 (1965) 1052. J. van Staden and G.G. Dimalla, Ann. Bot.~ in press. J. van Staden, Physiol. Plant., 36 (1976) 123. C.W. Parker and D.S. Letham, Planta, 114 (1973) 199. J. van Staden, Physiol. Plant., 38 (1976) 1. A.R. Langflle and P.L. Fomline, Plant Sci. Left., 2 (1974) 189.