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The Cytokinin Content of Aseptically Cultured Pea Fruits
Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa
The Cytokinin Content of Aseptically Cultured Pea Fruits
J. VAN STADEN and J. BUTTON With 1 figure Received November 2, 1977 . Accepted November 23, 1977
Summary The cytokinin levels of young pea fruits (Pisum sativum L.) cultured aseptically, decreased with time. During the entire experimental period the seeds contained higher levels of cytokinins than the pod walls, suggesting that the seeds remained the major sink for nutrients. It is suggested that the complexity of the seed cytokinins is the result of these organs having the ability to metabolize or convert cytokinins derived from the rest of the plant. No evidence could be found in the present investigation that young pea seeds actually synthesize cytokinins. Key words: Cytokinin synthesis, seeds, Pimm sativum.
Introduction Developing fruits and seeds generally contain high levels of endogenous cytokinins (MILLER, 1965; BURROWS and CARR, 1970; BLUMENFELD and GAZIT, 1970; KOSHIMIZU et al., LETHAM, 1973; SANDSTEDT, 1974; SCHULMAN and LAVEE, 1976; DAVEY and VAN STADEN, 1978). These high levels of cytokinins in the fruits and seeds of intact plants could be the result of preferential transport from the roots, which have the capacity to synthesize cytokinins (VAN STADEN and SMITH, 1978); they could have been synthesized within the seeds (LETHAM, 1969; BLUMENFELD and GAZIT, 1970); or they could have accumulated as a result of a combination of the above two possibilities. While it cannot be regarded as unequivocal, and is frequently based on indirect evidence, most of the available data favours the roots as a source of cytokinin supply to the seeds. In Perilla, fruit development is accompanied by an increased supply of cytokinins from the root system (BEEVER and WOOLHOUSE, 1973). The distribution of these cytokinins appears to be controlled by the relative sink strengths of the different organs. Removal of tomato leaves caused an increase in fruit cytokinins (VARGA and BRUINSMA, 1974), while fruit removal in grapes brought about increased cytokinin levels in the leaves (HOAD et aI., 1977). Probably the most direct evidence that cytokinins move into the fruits of intact plants is the observation that the sap passing Z. Pf1anzenphysiol. Bd. 87. S. 129-135. 1978.
130
J.
VAN STADEN and
J.
BUTTON
into developing Lupinus fruits contains high levels of cytokinins (DAVEY and VAN 1978). That fruits and seeds may have the ability to synthesize cytokinins is suggested by the fact that these organs can develop to varying degrees on rootless plants (PETERSON and FLETCHER, 1973). These findings can however, not be used strongly in support of fruits and seeds as being sites of cytokinin synthesis as it has been reported that cytokinins increased in rootless cuttings during storage (SKENE, 1972; HEWETT and WAREING, 1973). The strongest evidence supporting the hypothesis that seeds are a site of synthesis is that the cytokinin levels of seeds in pea pods, cultured independently of a root system, increased as the seeds developed (HAHN et aI., 1974). In Lupinus the pod wall has been shown to contain cytokinins (DAVEY and VAN STADEN, 1977) and it is possible that any increase in the seeds during fruit development may be the result of redirection of cytokinins from the pod tissues rather than to their synthesis within the seeds themselves (DAVEY and VAN STADEN, 1978). In view of this possibility the distribution of cytokinins in aseptically cultured pea fruits was investigated. STADEN,
Materials and Methods Five days after pollination, fruits (pods) were detached from pea plants (Pisum sativum L.) growing under natural conditions and surface sterilized for 10 minutes with a saturated calcium hypochlorite solution. After being rinsed in sterile distilled water the pods were cultured with the cut pedicel inserted in a nutrient medium described by NITSCH (1951) to which was added 5 Ofo sucrose and 1 Ofo agar. Each pod was cultured in a 45 ml culture tube containing 10 ml of medium. The cultures were kept at 23 °e and were subjected to 16 hours of light daily. After 0, 5, 10, and 15 days the seeds were removed from the pods and the seeds and pods extracted separately for cytokinins. The seeds were ground with 80 % ethanol in a glass homogenizer, filtered, concentrated to 1 ml, and fractionated on a Sephadex LH-20 column with 35 Ofo ethanol (ARMSTRONG et aI., 1969). Forty millilitre fractions were collected, dried in 25 ml flasks and each fraction assayed for cell division activity with the soybean callus bioassay (MILLER, 1965). The pod wall material was homogenized with 100 ml of 80 Ofo ethanol and extracted at 5°e for 24 h. After filtration the ethanolic extracts were taken to dryness in vacuo at 35 °e and the residues redissolved in 50 ml 80 Ofo ethanol. The pH of these solutions were adjusted to 2.5 with Hel. The acidified extracts were then passed through Dowex 50 W-X 8 cation exchange resin (H+ form; 20-50 mesh; column 2 X 5 em) at a flow rate of 15 mllh. The columns were washed with 60 ml 80 Ofo ethanol and substances responsible for cell division activity were eluted from the exchange resin with 100 ml 5N NH 40H. The ammonia was removed in an evaporator and the residue dissolved in 1 ml 35 Ofo ethanol. This ethanolic extract was fractionated on Sephadex LH-20 in a similar way to the seeds. For purposes of comparison the fractions of the different seed and pod extracts were assayed simultaneously.
Results
In culture the pea seeds increased from an original average fresh weight of 1.0 mg to 10.0 mg over a period of 15 days. Although the seeds increased in size they did Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
Cytokinin synthesis in seeds
131
not all develop normally. Only 8 % of the seeds in the pods cultured for 15 days showed signs of developing to maturity. The pod wall increased in weight only during the first five days of the experiment, whereafter no further growth occurred. The fruits as a whole showed a very similar growth pattern to that of the pod wall. Growth was rapid during the first five days of culture and then levelled off. The increase in fruit fresh weight recorded between five and 15 days after pod excision is attributable to the increased growth of some of the seeds within the pods. At the start of the experiment seeds contributed only 3.4 % to the total fresh weight of the fruits. After 15 days of culture this figure however, had increased to 14.9 % (Table 1). Table 1: The growth of excised pea fruits cultured aseptically. Average fresh weight (mg)
Culture time (days) 10 15 5
0
Whole fruit Pod wall Seed
241 233 1.0
332 315 2.5
346 306 8.3
352 299 10.0
3.4
5.1
11.9
14.9
Contribution of seeds to the fruit weight (010)
04
0·2
0·5
o U
160
320
480
640
800
960
1120
ELUTION VOLUME ml
Fig. 1: Cytokinin activity in the seeds (170 mg) and pod walls (3.2 g) of pea fruits cultured aseptically for five days. The seed and pod wall extracts were fractionated on Sephadex LH-20. Z = zeatin; ZR = zeatin riboside; ZG = zeatin-O-glucoside. Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
132
J.
VAN STADEN and
J.
BUTTON
All the seed and pod wall extracts analyzed contained compounds that stimulated the growth of soybean callus. After fractionation on Sephadex LH-20, four peaks of activity were recorded in the seed extracts, and two in the pod wall extracts (Fig. 1). In the seeds the most polar peak had an elution volume of 200-320 ml while the other three peaks co-eluted with zeatin-O-glucoside, zeatin riboside and zeatin respectively. In the pod wall the two polar peaks were absent and the recorded activity co-eluted with zeatin and zeatin riboside. Both quantitative and qualitative changes in the cytokinins in the seeds were recorded during the course of the experiment. If the results obtained after Sephadex LH-20 fractionation are expressed as .ug zeatin equivalents/g of fresh seed material analyzed, the activity recorded in the most polar peak (elution volume 200-320 ml) decreased with time (Table 2). Immediately after excision of the pea pods (five days after pollination) the seeds apparently did not contain any cytokinins that co-eluted with zeatin-O-glucoside. Ten day old seeds (five days in culture) did however, contain such compounds. The presence of these compounds coincided with the presence of endosperm in the pea seeds. As the endosperm disappeared the activity of this peak also decreased. Most of the cytokinin activity in the young seeds co-eluted with zeatin riboside and zeatin. The activity co-eluting with these two compounds decreased with time. The total cytokinin activity within the seeds decreased after excision of the pods from the mother plants. Table 2: The cytokinin activity of seeds obtained from excised pea pods cultured aseptically. Ethanolic extracts were fractionated on a Sephadex LH-20 column. The peaks of activity with an elution volume of 200-280 ml and those co-eluting with zeatin-O-glucoside zeatin riboside, and zeatin are expressed as ,ug zeatin equivalents/ g of fresh seed material.
Cytokinin activity Activity at elution volume 200-280 ml Co-eluting with zeatin-O-glucoside (360-440 ml) Co-eluting with zeatin riboside (480-560 ml)
0
Culture time (days) 10 15 5
0.93
0.67
0.53
0.44
0.00
1.75
0.32
0.26
5.60
0.76
0.28
0.04
(600-720 ml)
3.10
2.74
1.09
0.60
Total activity/g seed
9.63
5.92
2.22
1.34
Co-eluting with zeatin
In the pod wall material only two peaks of activity that co-eluted with zeatin and zeatin riboside were recorded (Table 3). During the first five days in culture the total activity in the pod wall increased but then decreased markedly. Expressed on Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
Cytokinin synthesis in seeds
133
Table 3: The cytokinin activity of pod wall tissue obtained from excised pea pods cultured aseptically. Dowex purified extracts were concentrated and fractionated on a Sephadex LH-20 column. The peaks of activity co-eluting with zeatin riboside and zeatin are expressed as ,ug zeatin equivalents/g of fresh pod wall tissue analyzed. Culture time (days) 5 10 15
Cytokinin activity
0
Co-eluting with zeatin riboside (480-560 ml)
0.01
0.29
0.14
0.07
Co-eluting with zeatin (600-720 ml)
1.62
2.26
0.30
0.11
Total activity/g pod wall
1.63
2.55
0.44
0.18
a fresh weight basis, the activity within the seeds was always higher than in the pod walls. Discussion Starting with ten-day old pods HAHN et aI., (1974) concluded that pea seeds have the capacity to produce the cytokinins required for their development. Two important factors were however, not considered in the above study. Firstly the pod walls of the fruits may contain cytokinins, and as is the case in Lupinus (DAVEY and VAN STAOEN, 1978) this tissue may play an important role in supplying the seeds with cytokinins. Secondly, the ten-day old seeds may have contained sufficient cytokinins at the time of culture (10 days old) to ensure their normal development within the excised pods. Using five-day old pea pods in which the average fresh weight of the seeds was only 1 mg compared to the 8 mg or more used by HAHN et aI. (1974) no support could be found for the hypothesis that seeds synthesize cytokinins. The seeds within the pods did not grow normally and only 8 % of them showed signs of development. The highest cytokinin levels were recorded in the young five-day old seeds. This level decreased during culture, indicating that the cytokinins were utilized but not replenished by synthesis. The cytokinins in the pod walls also decreased and reached their lowest level after 15 days in culture. Despite the fact that their cytokinin content decreased, the seeds however at all times contained higher levels of cytokinin than the pod wall tissue. This probably ensured that, as is the case in pods of intact plants (DAVEY and VAN STAOEN, 1978), the seeds in the cultured pods remained the major sink for nutrients. From the present evidence; the fact that cytokinins are present in the sap passing into Lupinus fruits (DAVEY and VAN STAOEN, 1978); and that removal of grape berries resulted in increased cytokinin levels in the leaves (HOAO et aI., 1977), it would appear that the cytokinins in fruits are mainly derived from the roots which have been shown by VAN STAOEN and SMITH (1978) to synthesize cytokinins when grown independently of the shoot.
z.
Pf1anzenphysiol. Bd. 87. S. 129·-135. 1978.
134
]. VAN STADEN and]. BUTTON
The cytokinin composition of the seeds and pod walls of pea fruits not only differed quantitatively but also qualitatively. Only two compounds that co-eluted with zeatin and zeatin riboside were detected in the pod walls. In the seeds themselves, four peaks of activity were recorded. One of these peaks co-eluted with zeatin-O-glucoside and was present in the highest concentration when the endosperm volume of the seeds was at its maximum (ten-day old seeds). The presence of cytokinin glucosides in the endosperm of seeds is well established (VAN STADEN, 1976; DAVEY and VAN STADEN, 1977; SMITH and VAN STADEN, 1977). These glucoside compounds probably represent inactive storage forms of the cytokinins and can be hydrolyzed when required (VAN STADEN and PAPAPHILIPPOU, 1977). It would thus appear that while the seeds are dependent for their cytokinins on the rest of the plant they do have the capacity to metabolize or convert imported cytokinins. Acknowledgements The financial assistance of the Council for Scientific and Industrial Research, Pretoria is gratefully acknowledged. References ARMSTRONG, D. J., W. ]. BURROWS, P. F. EVANS, and F. SKOOG: Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451-456 (1969). BEEVER, ]. E. and H. W. WOOLHOUSE: Increased cytokinin from roots of Perilla /rutescens during flower and fruit development. Nature 246,31-32 (1973). BLUMENFELD, A. and S. GAZIT: Cytokinin activity in avocado seeds during fruit development. Plant Physiol. 46, 331-333 (1970). BURROWS, W. ]. and D. ]. CARR: Cytokinin content of pea seeds during their growth and development. Physiol. Plant. 23, 1064-1070 (1970). DAVEY, J. E. and]. VAN STADEN: A cytokinin complex in the developing fruits of Lupinus albus. Physiol. Plant. 39, 221-224 (1977). - - Cytokinins in lupin seeds. Plant Physiol. 69 (S), 75 (1977). - Endogenous cytokinins in Lupinus albus: III. Distribution III fruits. Physiol. Plant. In Press. HAHN, H., R. DE ZACKS, and H. KENDE: Cytokinin formation in pea seeds. Naturwissenschaften 61,170 (1974). HEWETT, E. W. and P. F. WAREING: Cytokinins in Populus x robusta: Changes during chilling and bud burst. Physiol. Plant. 28, 393-399 (1973). HOAD, G. V., B. R. LovEYs, and K. G. M. SKENE: The effect of fruit-removal on cytokinins and gibberellin-like substances in grape leaves. Planta 136, 25-30 (1977). KOSHIMIZU, K., S. MATSUBARA, T. KUSAKI, and T. MITSUI: Isolation of a new cytokinin from immature yellow lupin seeds. Agr. Biol. Chern. 31, 795-801 (1967). LETHAM, D. S.: Regulators of cell division in plant tissues. VIII. The cytokinins of the apple fruit. Physiol. Plant. 22, 925-936 (1969). - Cytokinins from Zea mays. Phytochemistry 12, 2445-2455 (1973). MILLER, C. 0.: Evidence for the natural occurrence of zeatin and derivations: Compounds from maize which promote cell division. Proc. nat!. Acad. Sci. USA. 54, 1052-1058 (1965). NITSCH, J. P.: Growth and development in vitro of excised ovaries. Amer. J. Bot. 38,566-577 (1951).
Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
Cytokinin synthesis in seeds
135
J. Bot. 51,1899-1905 (1973). SANDSTEDT, R.: Relative activities of some cytokinin fractions of developing cotton fruit. Physiol. Plant. 30, 168-171 (1974). SCHULMAN, Y. and S. LAVEE: Endogenous cytokinins in maturing manzanillo olive fruits. Plant Physiol. 57, 490-492 (1975). SKENE, K. G. M.: Cytokinins in the xylem sap of grape vine canes: changes in activity during cold-storage. Planta 104, 89-92 (1972). SMITH, A. R. and]. VAN STADEN: Distribution of cytokinins in seeds of Zea mays L. prior to and during germination. Plant Physiol. 59 (5), 75 (1977). VARGA, A. and]. BRUINSMA: The growth and ripening of tomato fruits at different levels of endogenous cytokinins. J. hort. Sci. 49,135-142 (1974). VAN STADEN, J.: The identification of zeatin glucoside from coconut milk. Physiol. Plant. 36,123-126 (1976). VAN STADEN, J. and A. P. PAPAPHILIPPOU: Biological activity of O-~-D glucopyranosylzeatin. Plant Physiol. 60, 649-650 (1977). VAN STADEN, J. and A. R. SMITH: The synthesis of cytokinins in excised roots of maize and tomato under aseptic conditions. Ann. Bot. In Press. PETERSON, C. A. and R. A. FLETCHER: Formation of fruits on rootless plants. Can.
Prof. J. VAN STADEN, Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa.
Z. Pjlanzenphysiol. Bd. 87. S. 129-135. 1978.
The Cytokinin Content of Aseptically Cultured Pea Fruits
J. VAN STADEN and J. BUTTON With 1 figure Received November 2, 1977 . Accepted November 23, 1977
Summary The cytokinin levels of young pea fruits (Pisum sativum L.) cultured aseptically, decreased with time. During the entire experimental period the seeds contained higher levels of cytokinins than the pod walls, suggesting that the seeds remained the major sink for nutrients. It is suggested that the complexity of the seed cytokinins is the result of these organs having the ability to metabolize or convert cytokinins derived from the rest of the plant. No evidence could be found in the present investigation that young pea seeds actually synthesize cytokinins. Key words: Cytokinin synthesis, seeds, Pimm sativum.
Introduction Developing fruits and seeds generally contain high levels of endogenous cytokinins (MILLER, 1965; BURROWS and CARR, 1970; BLUMENFELD and GAZIT, 1970; KOSHIMIZU et al., LETHAM, 1973; SANDSTEDT, 1974; SCHULMAN and LAVEE, 1976; DAVEY and VAN STADEN, 1978). These high levels of cytokinins in the fruits and seeds of intact plants could be the result of preferential transport from the roots, which have the capacity to synthesize cytokinins (VAN STADEN and SMITH, 1978); they could have been synthesized within the seeds (LETHAM, 1969; BLUMENFELD and GAZIT, 1970); or they could have accumulated as a result of a combination of the above two possibilities. While it cannot be regarded as unequivocal, and is frequently based on indirect evidence, most of the available data favours the roots as a source of cytokinin supply to the seeds. In Perilla, fruit development is accompanied by an increased supply of cytokinins from the root system (BEEVER and WOOLHOUSE, 1973). The distribution of these cytokinins appears to be controlled by the relative sink strengths of the different organs. Removal of tomato leaves caused an increase in fruit cytokinins (VARGA and BRUINSMA, 1974), while fruit removal in grapes brought about increased cytokinin levels in the leaves (HOAD et aI., 1977). Probably the most direct evidence that cytokinins move into the fruits of intact plants is the observation that the sap passing Z. Pf1anzenphysiol. Bd. 87. S. 129-135. 1978.
130
J.
VAN STADEN and
J.
BUTTON
into developing Lupinus fruits contains high levels of cytokinins (DAVEY and VAN 1978). That fruits and seeds may have the ability to synthesize cytokinins is suggested by the fact that these organs can develop to varying degrees on rootless plants (PETERSON and FLETCHER, 1973). These findings can however, not be used strongly in support of fruits and seeds as being sites of cytokinin synthesis as it has been reported that cytokinins increased in rootless cuttings during storage (SKENE, 1972; HEWETT and WAREING, 1973). The strongest evidence supporting the hypothesis that seeds are a site of synthesis is that the cytokinin levels of seeds in pea pods, cultured independently of a root system, increased as the seeds developed (HAHN et aI., 1974). In Lupinus the pod wall has been shown to contain cytokinins (DAVEY and VAN STADEN, 1977) and it is possible that any increase in the seeds during fruit development may be the result of redirection of cytokinins from the pod tissues rather than to their synthesis within the seeds themselves (DAVEY and VAN STADEN, 1978). In view of this possibility the distribution of cytokinins in aseptically cultured pea fruits was investigated. STADEN,
Materials and Methods Five days after pollination, fruits (pods) were detached from pea plants (Pisum sativum L.) growing under natural conditions and surface sterilized for 10 minutes with a saturated calcium hypochlorite solution. After being rinsed in sterile distilled water the pods were cultured with the cut pedicel inserted in a nutrient medium described by NITSCH (1951) to which was added 5 Ofo sucrose and 1 Ofo agar. Each pod was cultured in a 45 ml culture tube containing 10 ml of medium. The cultures were kept at 23 °e and were subjected to 16 hours of light daily. After 0, 5, 10, and 15 days the seeds were removed from the pods and the seeds and pods extracted separately for cytokinins. The seeds were ground with 80 % ethanol in a glass homogenizer, filtered, concentrated to 1 ml, and fractionated on a Sephadex LH-20 column with 35 Ofo ethanol (ARMSTRONG et aI., 1969). Forty millilitre fractions were collected, dried in 25 ml flasks and each fraction assayed for cell division activity with the soybean callus bioassay (MILLER, 1965). The pod wall material was homogenized with 100 ml of 80 Ofo ethanol and extracted at 5°e for 24 h. After filtration the ethanolic extracts were taken to dryness in vacuo at 35 °e and the residues redissolved in 50 ml 80 Ofo ethanol. The pH of these solutions were adjusted to 2.5 with Hel. The acidified extracts were then passed through Dowex 50 W-X 8 cation exchange resin (H+ form; 20-50 mesh; column 2 X 5 em) at a flow rate of 15 mllh. The columns were washed with 60 ml 80 Ofo ethanol and substances responsible for cell division activity were eluted from the exchange resin with 100 ml 5N NH 40H. The ammonia was removed in an evaporator and the residue dissolved in 1 ml 35 Ofo ethanol. This ethanolic extract was fractionated on Sephadex LH-20 in a similar way to the seeds. For purposes of comparison the fractions of the different seed and pod extracts were assayed simultaneously.
Results
In culture the pea seeds increased from an original average fresh weight of 1.0 mg to 10.0 mg over a period of 15 days. Although the seeds increased in size they did Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
Cytokinin synthesis in seeds
131
not all develop normally. Only 8 % of the seeds in the pods cultured for 15 days showed signs of developing to maturity. The pod wall increased in weight only during the first five days of the experiment, whereafter no further growth occurred. The fruits as a whole showed a very similar growth pattern to that of the pod wall. Growth was rapid during the first five days of culture and then levelled off. The increase in fruit fresh weight recorded between five and 15 days after pod excision is attributable to the increased growth of some of the seeds within the pods. At the start of the experiment seeds contributed only 3.4 % to the total fresh weight of the fruits. After 15 days of culture this figure however, had increased to 14.9 % (Table 1). Table 1: The growth of excised pea fruits cultured aseptically. Average fresh weight (mg)
Culture time (days) 10 15 5
0
Whole fruit Pod wall Seed
241 233 1.0
332 315 2.5
346 306 8.3
352 299 10.0
3.4
5.1
11.9
14.9
Contribution of seeds to the fruit weight (010)
04
0·2
0·5
o U
160
320
480
640
800
960
1120
ELUTION VOLUME ml
Fig. 1: Cytokinin activity in the seeds (170 mg) and pod walls (3.2 g) of pea fruits cultured aseptically for five days. The seed and pod wall extracts were fractionated on Sephadex LH-20. Z = zeatin; ZR = zeatin riboside; ZG = zeatin-O-glucoside. Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
132
J.
VAN STADEN and
J.
BUTTON
All the seed and pod wall extracts analyzed contained compounds that stimulated the growth of soybean callus. After fractionation on Sephadex LH-20, four peaks of activity were recorded in the seed extracts, and two in the pod wall extracts (Fig. 1). In the seeds the most polar peak had an elution volume of 200-320 ml while the other three peaks co-eluted with zeatin-O-glucoside, zeatin riboside and zeatin respectively. In the pod wall the two polar peaks were absent and the recorded activity co-eluted with zeatin and zeatin riboside. Both quantitative and qualitative changes in the cytokinins in the seeds were recorded during the course of the experiment. If the results obtained after Sephadex LH-20 fractionation are expressed as .ug zeatin equivalents/g of fresh seed material analyzed, the activity recorded in the most polar peak (elution volume 200-320 ml) decreased with time (Table 2). Immediately after excision of the pea pods (five days after pollination) the seeds apparently did not contain any cytokinins that co-eluted with zeatin-O-glucoside. Ten day old seeds (five days in culture) did however, contain such compounds. The presence of these compounds coincided with the presence of endosperm in the pea seeds. As the endosperm disappeared the activity of this peak also decreased. Most of the cytokinin activity in the young seeds co-eluted with zeatin riboside and zeatin. The activity co-eluting with these two compounds decreased with time. The total cytokinin activity within the seeds decreased after excision of the pods from the mother plants. Table 2: The cytokinin activity of seeds obtained from excised pea pods cultured aseptically. Ethanolic extracts were fractionated on a Sephadex LH-20 column. The peaks of activity with an elution volume of 200-280 ml and those co-eluting with zeatin-O-glucoside zeatin riboside, and zeatin are expressed as ,ug zeatin equivalents/ g of fresh seed material.
Cytokinin activity Activity at elution volume 200-280 ml Co-eluting with zeatin-O-glucoside (360-440 ml) Co-eluting with zeatin riboside (480-560 ml)
0
Culture time (days) 10 15 5
0.93
0.67
0.53
0.44
0.00
1.75
0.32
0.26
5.60
0.76
0.28
0.04
(600-720 ml)
3.10
2.74
1.09
0.60
Total activity/g seed
9.63
5.92
2.22
1.34
Co-eluting with zeatin
In the pod wall material only two peaks of activity that co-eluted with zeatin and zeatin riboside were recorded (Table 3). During the first five days in culture the total activity in the pod wall increased but then decreased markedly. Expressed on Z. P/lanzenphysiol. Bd. 87. S. 129-135. 1978.
Cytokinin synthesis in seeds
133
Table 3: The cytokinin activity of pod wall tissue obtained from excised pea pods cultured aseptically. Dowex purified extracts were concentrated and fractionated on a Sephadex LH-20 column. The peaks of activity co-eluting with zeatin riboside and zeatin are expressed as ,ug zeatin equivalents/g of fresh pod wall tissue analyzed. Culture time (days) 5 10 15
Cytokinin activity
0
Co-eluting with zeatin riboside (480-560 ml)
0.01
0.29
0.14
0.07
Co-eluting with zeatin (600-720 ml)
1.62
2.26
0.30
0.11
Total activity/g pod wall
1.63
2.55
0.44
0.18
a fresh weight basis, the activity within the seeds was always higher than in the pod walls. Discussion Starting with ten-day old pods HAHN et aI., (1974) concluded that pea seeds have the capacity to produce the cytokinins required for their development. Two important factors were however, not considered in the above study. Firstly the pod walls of the fruits may contain cytokinins, and as is the case in Lupinus (DAVEY and VAN STAOEN, 1978) this tissue may play an important role in supplying the seeds with cytokinins. Secondly, the ten-day old seeds may have contained sufficient cytokinins at the time of culture (10 days old) to ensure their normal development within the excised pods. Using five-day old pea pods in which the average fresh weight of the seeds was only 1 mg compared to the 8 mg or more used by HAHN et aI. (1974) no support could be found for the hypothesis that seeds synthesize cytokinins. The seeds within the pods did not grow normally and only 8 % of them showed signs of development. The highest cytokinin levels were recorded in the young five-day old seeds. This level decreased during culture, indicating that the cytokinins were utilized but not replenished by synthesis. The cytokinins in the pod walls also decreased and reached their lowest level after 15 days in culture. Despite the fact that their cytokinin content decreased, the seeds however at all times contained higher levels of cytokinin than the pod wall tissue. This probably ensured that, as is the case in pods of intact plants (DAVEY and VAN STAOEN, 1978), the seeds in the cultured pods remained the major sink for nutrients. From the present evidence; the fact that cytokinins are present in the sap passing into Lupinus fruits (DAVEY and VAN STAOEN, 1978); and that removal of grape berries resulted in increased cytokinin levels in the leaves (HOAO et aI., 1977), it would appear that the cytokinins in fruits are mainly derived from the roots which have been shown by VAN STAOEN and SMITH (1978) to synthesize cytokinins when grown independently of the shoot.
z.
Pf1anzenphysiol. Bd. 87. S. 129·-135. 1978.
134
]. VAN STADEN and]. BUTTON
The cytokinin composition of the seeds and pod walls of pea fruits not only differed quantitatively but also qualitatively. Only two compounds that co-eluted with zeatin and zeatin riboside were detected in the pod walls. In the seeds themselves, four peaks of activity were recorded. One of these peaks co-eluted with zeatin-O-glucoside and was present in the highest concentration when the endosperm volume of the seeds was at its maximum (ten-day old seeds). The presence of cytokinin glucosides in the endosperm of seeds is well established (VAN STADEN, 1976; DAVEY and VAN STADEN, 1977; SMITH and VAN STADEN, 1977). These glucoside compounds probably represent inactive storage forms of the cytokinins and can be hydrolyzed when required (VAN STADEN and PAPAPHILIPPOU, 1977). It would thus appear that while the seeds are dependent for their cytokinins on the rest of the plant they do have the capacity to metabolize or convert imported cytokinins. Acknowledgements The financial assistance of the Council for Scientific and Industrial Research, Pretoria is gratefully acknowledged. References ARMSTRONG, D. J., W. ]. BURROWS, P. F. EVANS, and F. SKOOG: Isolation of cytokinins from tRNA. Biochem. Biophys. Res. Commun. 37, 451-456 (1969). BEEVER, ]. E. and H. W. WOOLHOUSE: Increased cytokinin from roots of Perilla /rutescens during flower and fruit development. Nature 246,31-32 (1973). BLUMENFELD, A. and S. GAZIT: Cytokinin activity in avocado seeds during fruit development. Plant Physiol. 46, 331-333 (1970). BURROWS, W. ]. and D. ]. CARR: Cytokinin content of pea seeds during their growth and development. Physiol. Plant. 23, 1064-1070 (1970). DAVEY, J. E. and]. VAN STADEN: A cytokinin complex in the developing fruits of Lupinus albus. Physiol. Plant. 39, 221-224 (1977). - - Cytokinins in lupin seeds. Plant Physiol. 69 (S), 75 (1977). - Endogenous cytokinins in Lupinus albus: III. Distribution III fruits. Physiol. Plant. In Press. HAHN, H., R. DE ZACKS, and H. KENDE: Cytokinin formation in pea seeds. Naturwissenschaften 61,170 (1974). HEWETT, E. W. and P. F. WAREING: Cytokinins in Populus x robusta: Changes during chilling and bud burst. Physiol. Plant. 28, 393-399 (1973). HOAD, G. V., B. R. LovEYs, and K. G. M. SKENE: The effect of fruit-removal on cytokinins and gibberellin-like substances in grape leaves. Planta 136, 25-30 (1977). KOSHIMIZU, K., S. MATSUBARA, T. KUSAKI, and T. MITSUI: Isolation of a new cytokinin from immature yellow lupin seeds. Agr. Biol. Chern. 31, 795-801 (1967). LETHAM, D. S.: Regulators of cell division in plant tissues. VIII. The cytokinins of the apple fruit. Physiol. Plant. 22, 925-936 (1969). - Cytokinins from Zea mays. Phytochemistry 12, 2445-2455 (1973). MILLER, C. 0.: Evidence for the natural occurrence of zeatin and derivations: Compounds from maize which promote cell division. Proc. nat!. Acad. Sci. USA. 54, 1052-1058 (1965). NITSCH, J. P.: Growth and development in vitro of excised ovaries. Amer. J. Bot. 38,566-577 (1951).
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J. Bot. 51,1899-1905 (1973). SANDSTEDT, R.: Relative activities of some cytokinin fractions of developing cotton fruit. Physiol. Plant. 30, 168-171 (1974). SCHULMAN, Y. and S. LAVEE: Endogenous cytokinins in maturing manzanillo olive fruits. Plant Physiol. 57, 490-492 (1975). SKENE, K. G. M.: Cytokinins in the xylem sap of grape vine canes: changes in activity during cold-storage. Planta 104, 89-92 (1972). SMITH, A. R. and]. VAN STADEN: Distribution of cytokinins in seeds of Zea mays L. prior to and during germination. Plant Physiol. 59 (5), 75 (1977). VARGA, A. and]. BRUINSMA: The growth and ripening of tomato fruits at different levels of endogenous cytokinins. J. hort. Sci. 49,135-142 (1974). VAN STADEN, J.: The identification of zeatin glucoside from coconut milk. Physiol. Plant. 36,123-126 (1976). VAN STADEN, J. and A. P. PAPAPHILIPPOU: Biological activity of O-~-D glucopyranosylzeatin. Plant Physiol. 60, 649-650 (1977). VAN STADEN, J. and A. R. SMITH: The synthesis of cytokinins in excised roots of maize and tomato under aseptic conditions. Ann. Bot. In Press. PETERSON, C. A. and R. A. FLETCHER: Formation of fruits on rootless plants. Can.
Prof. J. VAN STADEN, Department of Botany, University of Natal, Pietermaritzburg 3200, Republic of South Africa.
Z. Pjlanzenphysiol. Bd. 87. S. 129-135. 1978.