Plant Cell Reports (1985) 4:140-143
PlantCell Reports
© Springer-Verlag1985
Changes of cytokinin nucleotides in an anise cell culture (Pimpinella anisum L.) during growth and embryogenesis Dietrich Ernst and Dieter Oesterhelt Max-Planck-Institut flit Biochemie, D-8033 Martinsried, FRG Received November 13, 1984 / Revised version received March 29, 1985 - Communicated by M. H. Zenk
Abstract
Endogenous levels of cytokinin nucleotides in an anise cell culture were determined during proembryonal, as well as embryonal development. In both cultures the maximum level of isopentenyladenine nucleotides was found during the f i r s t four days of incubation which correlated with the be~nnir~g of iogaiitli,~ic gro~vLh (embrvonai: 8 ng g ' fresh weight; proembryonal: 17.4 ng g-1 fresh weight). The concentration of zeatin nucleotides remained constant at a very low l e v e l . The present data and those of Ernst et aI.(1984) and Ernst and Oesterhelt (1984) are concerned in ascribing a major role to cytokinins in c e l l d i v i s i o n , but not in embryo differentiation. Abbreviations: 2,4-D:2,4-dichloroDhenoxy acid; FW=fresh weight; RIA=radioimmunoassay
acetic
Introduction Although much is known about cytokinin nucleotides during cytokinin biosynthesis and metabolism using radiolabeled precursors (Bopp and Erichsen 19~I; Burrows and Fuell 1981; Chen 1981; Horgan et ai.1981; Laloue et al.19B1; Maa5 and Kl~mbt 1981), few reports e x i s t on the nature of cytokinin nucleotides during growth and development of plants. C h i l l i n g seeds of Leucadendron daohnoldes resulted in a progressive decrease in the water soluble cytokinins, accompanied by an increase in butanol soluble cytokinins (Brown and Van Staden 1973). Smith and Van Staden (1978) showed the presence of cytokinin nucleotides in excised embryos and endosperm of Zea mays. In maize kernels the concentration of cytokinin ribonucleotides decreased s l i g h t l y a f t e r imbibition and then maintained constant (Julin-Tegelman 1979). Embryos of Acer pseudoDlatanus L. f r u i t s showed an increase in free, as well as bound cytokinins during the f i r s t 24 h of incubation and then a decline after longer incubation periods (Julin-Tegelman and Pinfield 1982). Koda (1982) reported changes of the water-soluble cytokinins during the l i f e - c y c l e of Dotato tubers. All these reports suggest a breakdown of the cytokinin nucleotides, leading to the corresponding cytokinin ribosides and bases. In a previous paper we reDorted a c o r r e l a t i o n
Offprint requests to:
D. Ernst
in
the cytokinin concentration to the logarithmic growth of both a Droembrvonic and an embryonic anise c e l l culture (Ernst et ai.1984). To c l a r i f y the c o r r e l a t i o n of cytokinins to c e l l growth and embryogenesis we analyzed the same cultures for the presence of cytokinin nucleotides during growth and embryogenesis. Materials and Methods
Chemicals Cellulose phosphate P11 was from Whatman (Springfield M i l l , U K ) , p o l y v i n y l p o l y p y r r o l i d o n e , 2,4-D, AMP and a l k a l i n e phosphatas@~(calf i n t e s t i n e type I) from Sigma (Munich~ FRG), --C-6-benzylaminopurine (1.9-2.2 GBq mmol-') from Amersham-Buchler (Braunschweig, FRG), reference compounds for the RIA were as those described before (Ernst et ai.1983,1984). All other chemicals were obtained from Merck (Darmstadt, FRG). Plant Material and Extraction of the Cells Proembryonic (+2,4-D) and embryonic (-2,4-D) c e l l cultures were grown in 1.8 i Fernbach flasks under conditions as described by Ernst et ai.(1983,1984). The frozen c e i l s were extracted with ice-cold methanol (80%,v/w) overnight at 4 °C, f i l t e r e d and the residue again extracted with methanol (80%,v/v) and f i l t e r e d . The organic solvent was evaporated from the combined f i l t r a t e s at 40 °C and the pH of the aqueous phase adjusted to oH 3.S w i t h acetic acid. To this splution Dolyvinyloolypyrrolidone was added (0.1 g g-" FW), the suspension was s t i r r e d for 30 min at 4 °C, then f i l t e r e d and the residue washed with d i l u t e acetic acid of pH 3.5. The combined f i l t r a t e s were adjusted to DH 3.1 with acetic acid and applied to a l c e l l u l o s e phosphate column (NH4+; 2 ml bed volume g- FW). The column was eluted with f i v e bed volumes of acetic acid pH 3.1 and the eluate evaporated. The residue was dissolved in 0.1 M T r i s - b u f f e r pH 9.5, containing 0.01M MgCI2. After addition of phosphatase (0.5 mg g-1 FW) the solution was incubated at 37 °C for 24 h and the reaction stopped by the addition of 0.5 volumes ethanol. After f i l t r a t i o n of the suspension, the ethanol was evaporated and the aqueous phase adjusted to pH 8 with HCI before extraction with water-saturated butanol (3x). The combined butanol phases were evaporated to dryness, the residue dissolved in methanol, f i l t e r e d through glass wool and then spotted on Whatman 3MM paper. The chromatogram was developed with lso-propanol / 25% NH40H / water
141 (10:1:1, by vol). After drying, the paper was cut into ten equal Dieces, which were tested in the Amaranthus bioassay according to Biddington and Thomas (1973). For q u a n t i t a t i v e measurements the Rf-region from 0.7 to 1.0 (corresponding to authentic isopentenyladenosine and zeatin riboside) was eluted with methanol/water (80%,v/v). After evaporation of the organic solvent, aliquots of the aqueous phase were tested in the RIA. Determination of Phosphatase A c t i v i t y PhosDhatase a c t i v i t y was assayed with AMP as substrate. The frozen anise c e i l s (0.5-2.0 g) were extracted in the absence and presence of AMP (2 mM), respectively. The homogenate was centrifuged at 2,500xg for 5 min and the supernatant was used for a phosphate determination according to Ames (1966). This inorganic phosphate determination is based on a reduction of a phosphomolybdate complex by ascorbic acid. Under the reaction conditions AMP is stable and therefore i t can be used as a substrate for demonstrating a phosphatase a c t i v i t y . The phosphate content of samples without AMP was substracted from
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PlantCell Reports
© Springer-Verlag1985
Changes of cytokinin nucleotides in an anise cell culture (Pimpinella anisum L.) during growth and embryogenesis Dietrich Ernst and Dieter Oesterhelt Max-Planck-Institut flit Biochemie, D-8033 Martinsried, FRG Received November 13, 1984 / Revised version received March 29, 1985 - Communicated by M. H. Zenk
Abstract
Endogenous levels of cytokinin nucleotides in an anise cell culture were determined during proembryonal, as well as embryonal development. In both cultures the maximum level of isopentenyladenine nucleotides was found during the f i r s t four days of incubation which correlated with the be~nnir~g of iogaiitli,~ic gro~vLh (embrvonai: 8 ng g ' fresh weight; proembryonal: 17.4 ng g-1 fresh weight). The concentration of zeatin nucleotides remained constant at a very low l e v e l . The present data and those of Ernst et aI.(1984) and Ernst and Oesterhelt (1984) are concerned in ascribing a major role to cytokinins in c e l l d i v i s i o n , but not in embryo differentiation. Abbreviations: 2,4-D:2,4-dichloroDhenoxy acid; FW=fresh weight; RIA=radioimmunoassay
acetic
Introduction Although much is known about cytokinin nucleotides during cytokinin biosynthesis and metabolism using radiolabeled precursors (Bopp and Erichsen 19~I; Burrows and Fuell 1981; Chen 1981; Horgan et ai.1981; Laloue et al.19B1; Maa5 and Kl~mbt 1981), few reports e x i s t on the nature of cytokinin nucleotides during growth and development of plants. C h i l l i n g seeds of Leucadendron daohnoldes resulted in a progressive decrease in the water soluble cytokinins, accompanied by an increase in butanol soluble cytokinins (Brown and Van Staden 1973). Smith and Van Staden (1978) showed the presence of cytokinin nucleotides in excised embryos and endosperm of Zea mays. In maize kernels the concentration of cytokinin ribonucleotides decreased s l i g h t l y a f t e r imbibition and then maintained constant (Julin-Tegelman 1979). Embryos of Acer pseudoDlatanus L. f r u i t s showed an increase in free, as well as bound cytokinins during the f i r s t 24 h of incubation and then a decline after longer incubation periods (Julin-Tegelman and Pinfield 1982). Koda (1982) reported changes of the water-soluble cytokinins during the l i f e - c y c l e of Dotato tubers. All these reports suggest a breakdown of the cytokinin nucleotides, leading to the corresponding cytokinin ribosides and bases. In a previous paper we reDorted a c o r r e l a t i o n
Offprint requests to:
D. Ernst
in
the cytokinin concentration to the logarithmic growth of both a Droembrvonic and an embryonic anise c e l l culture (Ernst et ai.1984). To c l a r i f y the c o r r e l a t i o n of cytokinins to c e l l growth and embryogenesis we analyzed the same cultures for the presence of cytokinin nucleotides during growth and embryogenesis. Materials and Methods
Chemicals Cellulose phosphate P11 was from Whatman (Springfield M i l l , U K ) , p o l y v i n y l p o l y p y r r o l i d o n e , 2,4-D, AMP and a l k a l i n e phosphatas@~(calf i n t e s t i n e type I) from Sigma (Munich~ FRG), --C-6-benzylaminopurine (1.9-2.2 GBq mmol-') from Amersham-Buchler (Braunschweig, FRG), reference compounds for the RIA were as those described before (Ernst et ai.1983,1984). All other chemicals were obtained from Merck (Darmstadt, FRG). Plant Material and Extraction of the Cells Proembryonic (+2,4-D) and embryonic (-2,4-D) c e l l cultures were grown in 1.8 i Fernbach flasks under conditions as described by Ernst et ai.(1983,1984). The frozen c e i l s were extracted with ice-cold methanol (80%,v/w) overnight at 4 °C, f i l t e r e d and the residue again extracted with methanol (80%,v/v) and f i l t e r e d . The organic solvent was evaporated from the combined f i l t r a t e s at 40 °C and the pH of the aqueous phase adjusted to oH 3.S w i t h acetic acid. To this splution Dolyvinyloolypyrrolidone was added (0.1 g g-" FW), the suspension was s t i r r e d for 30 min at 4 °C, then f i l t e r e d and the residue washed with d i l u t e acetic acid of pH 3.5. The combined f i l t r a t e s were adjusted to DH 3.1 with acetic acid and applied to a l c e l l u l o s e phosphate column (NH4+; 2 ml bed volume g- FW). The column was eluted with f i v e bed volumes of acetic acid pH 3.1 and the eluate evaporated. The residue was dissolved in 0.1 M T r i s - b u f f e r pH 9.5, containing 0.01M MgCI2. After addition of phosphatase (0.5 mg g-1 FW) the solution was incubated at 37 °C for 24 h and the reaction stopped by the addition of 0.5 volumes ethanol. After f i l t r a t i o n of the suspension, the ethanol was evaporated and the aqueous phase adjusted to pH 8 with HCI before extraction with water-saturated butanol (3x). The combined butanol phases were evaporated to dryness, the residue dissolved in methanol, f i l t e r e d through glass wool and then spotted on Whatman 3MM paper. The chromatogram was developed with lso-propanol / 25% NH40H / water
141 (10:1:1, by vol). After drying, the paper was cut into ten equal Dieces, which were tested in the Amaranthus bioassay according to Biddington and Thomas (1973). For q u a n t i t a t i v e measurements the Rf-region from 0.7 to 1.0 (corresponding to authentic isopentenyladenosine and zeatin riboside) was eluted with methanol/water (80%,v/v). After evaporation of the organic solvent, aliquots of the aqueous phase were tested in the RIA. Determination of Phosphatase A c t i v i t y PhosDhatase a c t i v i t y was assayed with AMP as substrate. The frozen anise c e i l s (0.5-2.0 g) were extracted in the absence and presence of AMP (2 mM), respectively. The homogenate was centrifuged at 2,500xg for 5 min and the supernatant was used for a phosphate determination according to Ames (1966). This inorganic phosphate determination is based on a reduction of a phosphomolybdate complex by ascorbic acid. Under the reaction conditions AMP is stable and therefore i t can be used as a substrate for demonstrating a phosphatase a c t i v i t y . The phosphate content of samples without AMP was substracted from
A
E¢ I
,o
0.10
E
0.0~
Lx.I
