Planta
Planta 149, 472-475 (1980)
9 by Springer-Verlag 1980
Zeatin-9-Glucoside, a Major Endogenous Cytokinin of Vinca rosea L. Crown Gall Tissue I.M. Scott, R. H o r g a n , a n d B.A. M c G a w Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U.K.
Abstract. C u l t u r e d c r o w n gall tissue of V i n c a r o s e a L. was f o u n d to c o n t a i n , in a d d i t i o n to the previously reported c y t o k i n i n s zeatin, zeatin riboside, a n d the 0-glucosides of these two c o m p o u n d s , relatively high levels of z e a t i n - 9 - D - g l u c o p y r a n o s i d e . This is the first conclusive identification of a n e n d o g e n o u s c y t o k i n i n 9-glucoside. Key words: C r o w n gall - C y t o k i n i n
Vinca
in b e a n leaves ( W a n g et al. 1977) a n d lupin pods ( S u m m o n s et al. 1979). Miller a n d associates have identified Z 0 G a n d its riboside in c u l t u r e d c r o w n gall t u m o u r tissue of V i n c a r o s e a (Peterson a n d Miller 1977; M o r r i s 1977). This crown gall tissue has been m a i n t a i n e d in o u r l a b o r a t o r y a n d we have f o u n d that it also c o n t a i n s relatively large quantities of Z9G. This is the first u n a m b i g u o u s identification of a n end o g e n o u s c y t o k i n i n 9-glucoside.
Materials and Methods Introduction Studies on the m e t a b o l i s m of radioactively labelled zeatin in a n u m b e r of p l a n t species have revealed that this n a t u r a l l y occurring c y t o k i n i n is c o m m o n l y converted to glucosyl conjugates in which the sugar m a y be linked to the side c h a i n hydroxyl or to nitrogen a t o m s 7 or 9 of the p u r i n e ring. The type of glucoside f o r m e d a p p a r e n t l y d e p e n d s o n the p l a n t tissue. The principal m e t a b o l i t e of zeatin in radish c o t y l e d o n s was the 7-glucoside (ZTG) (Parker et al. 1972), whereas in maize roots the 9-glucoside (Z9G) p r e d o m i n a t e d (Parker et al. 1973). Zeatin-0-glucoside (ZOG) was the m a i n glucoside formed in s o y b e a n callus ( H o r g a n 1975), l u p i n seedlings (Parker et al. 1978) a n d p o p l a r leaves ( D u k e et al. 1979). The occurrence of e n d o g e n o u s c y t o k i n i n glucosides has n o w also been reported. S u m m o n s et al. (1977) identified Z 7 G in radish seeds. 0-glucosides of d i h y d r o z e a t i n or its riboside have been identified Abbreviations: GC=gas chromatography; HPLC=high-perfor-
mance liquid chromatography; I.D.=internal diameter; R F E = rotary film evaporation; TLC=thin layer chromatography; TMS =trimethylsilyl; UV=ultraviolet; Z=zeatin; Z7G=zeatin-7-glucoside; Z9G=zeatin-9-glucoside; ZOG=zeatin-0-glucoside; Z R = zeatin riboside; ZROG=zeatin riboside-0-glucoside
0032-0935/80/0149/0472/$01.00
Plant Material. Cultured crown gall tnmour tissue of Vinca rosea
L. (the generous gift of Professor C.O. MilIer) was maintained on the medium of Miller (1974). Tissue was harvested two months after subculturing and stored at -15 ~ C prior to extraction. Purification Procedure. 1,500 g of frozen tissue was thawed in 6 dm 3
of methanol at 4~ C and then homogenized in the same solvent in a Waring blendor. The homogenate was left to stand at 4~ C for 17 h, and then filtered through cheesecloth and paper. The filtrate was reduced to 500 cm3 of aqueous solution by RFE at 35~ C, adjusted to pH 3.5 with acetic acid, frozen and thawed, and then centrifuged at 18,000 g for 40 rain. The supernatant was filtered through paper and percolated through a column of PVP (300 cm3 bed volume). The column was washed with a further 900 cm 3 of H20 (pH 3.5), and the total effluent adjusted to pH 3 and percolated through a column of cellulose phosphate (NH~ form, pH 3, 1 dms bed volume). The column was washed with 5 dm3 HzO (pH 3) and then basic compounds were eluted with 3 dm3 of 2 M NH4OH. The basic fraction was reduced to 45 cm3 by RFE, adjusted to pH 8 with KOH, and partitioned 5 times against equal volumes of H20-saturated butan-l-oI. The combined butan-l-ol fractions were reduced to dryness by RFE and the residue redissolved in 2.5 cm3 of 35% (v/v) ethanol at 60~ C. The solution was cleared by centrifugation and filtration through glass fibre paper then chromatographed on a column (2.5.70 cm) of Sephadex LH-20 in 35% ethanol at a flow-rate of 30 cm3h 1 in the descending direction. 30 cm3 fractions were collected and the following portions of the eluate (corresponding respectively to the elution volumes of ZROG, ZOG, ZR and Z) were retained for further analysis by reversed phase HPLC: A (241 270 cm3), B (271 330 cm3), C (361-420 cm 3) and D (451-510 cm3).
I.M. Scott et al. : Zeatin-9-Riboside in Crown Gall
473
BI B2
HPLC. Equipment and chromatographic systems used have been described in detail by H o r g a n and Kramers (1979). Reversed phase H P L C was carried out on a 150 x 10 m m I.D. column of Hypersil ODS eluted at a flow-rate of 5 cm 3 m i n with HaO (adjusted to pH 7 with triethylammonium bicarbonate) containing acetonitrile. T h e following proportions of acetonitrile were used for analysis of the fractions obtained from Sephadex LH-20 chromatography: A, 9 % ; B, 8%; C, 10%; D, 8%. Sample volume was I cm 3, injected into a 1.5 cm 3 loop. N o r m a l phase H P L C was carried out on a 250-4.5 m m I.D. column of Partisil 10 PAC eluted at a flow-rate of 2 cm 3 min -1 with acetonitrile: H 2 0 (9:1). Sample volume was 30 gl, injected into a 50 gl loop.
E
I.U
L) z n,
o m
Planta 149, 472-475 (1980)
9 by Springer-Verlag 1980
Zeatin-9-Glucoside, a Major Endogenous Cytokinin of Vinca rosea L. Crown Gall Tissue I.M. Scott, R. H o r g a n , a n d B.A. M c G a w Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, U.K.
Abstract. C u l t u r e d c r o w n gall tissue of V i n c a r o s e a L. was f o u n d to c o n t a i n , in a d d i t i o n to the previously reported c y t o k i n i n s zeatin, zeatin riboside, a n d the 0-glucosides of these two c o m p o u n d s , relatively high levels of z e a t i n - 9 - D - g l u c o p y r a n o s i d e . This is the first conclusive identification of a n e n d o g e n o u s c y t o k i n i n 9-glucoside. Key words: C r o w n gall - C y t o k i n i n
Vinca
in b e a n leaves ( W a n g et al. 1977) a n d lupin pods ( S u m m o n s et al. 1979). Miller a n d associates have identified Z 0 G a n d its riboside in c u l t u r e d c r o w n gall t u m o u r tissue of V i n c a r o s e a (Peterson a n d Miller 1977; M o r r i s 1977). This crown gall tissue has been m a i n t a i n e d in o u r l a b o r a t o r y a n d we have f o u n d that it also c o n t a i n s relatively large quantities of Z9G. This is the first u n a m b i g u o u s identification of a n end o g e n o u s c y t o k i n i n 9-glucoside.
Materials and Methods Introduction Studies on the m e t a b o l i s m of radioactively labelled zeatin in a n u m b e r of p l a n t species have revealed that this n a t u r a l l y occurring c y t o k i n i n is c o m m o n l y converted to glucosyl conjugates in which the sugar m a y be linked to the side c h a i n hydroxyl or to nitrogen a t o m s 7 or 9 of the p u r i n e ring. The type of glucoside f o r m e d a p p a r e n t l y d e p e n d s o n the p l a n t tissue. The principal m e t a b o l i t e of zeatin in radish c o t y l e d o n s was the 7-glucoside (ZTG) (Parker et al. 1972), whereas in maize roots the 9-glucoside (Z9G) p r e d o m i n a t e d (Parker et al. 1973). Zeatin-0-glucoside (ZOG) was the m a i n glucoside formed in s o y b e a n callus ( H o r g a n 1975), l u p i n seedlings (Parker et al. 1978) a n d p o p l a r leaves ( D u k e et al. 1979). The occurrence of e n d o g e n o u s c y t o k i n i n glucosides has n o w also been reported. S u m m o n s et al. (1977) identified Z 7 G in radish seeds. 0-glucosides of d i h y d r o z e a t i n or its riboside have been identified Abbreviations: GC=gas chromatography; HPLC=high-perfor-
mance liquid chromatography; I.D.=internal diameter; R F E = rotary film evaporation; TLC=thin layer chromatography; TMS =trimethylsilyl; UV=ultraviolet; Z=zeatin; Z7G=zeatin-7-glucoside; Z9G=zeatin-9-glucoside; ZOG=zeatin-0-glucoside; Z R = zeatin riboside; ZROG=zeatin riboside-0-glucoside
0032-0935/80/0149/0472/$01.00
Plant Material. Cultured crown gall tnmour tissue of Vinca rosea
L. (the generous gift of Professor C.O. MilIer) was maintained on the medium of Miller (1974). Tissue was harvested two months after subculturing and stored at -15 ~ C prior to extraction. Purification Procedure. 1,500 g of frozen tissue was thawed in 6 dm 3
of methanol at 4~ C and then homogenized in the same solvent in a Waring blendor. The homogenate was left to stand at 4~ C for 17 h, and then filtered through cheesecloth and paper. The filtrate was reduced to 500 cm3 of aqueous solution by RFE at 35~ C, adjusted to pH 3.5 with acetic acid, frozen and thawed, and then centrifuged at 18,000 g for 40 rain. The supernatant was filtered through paper and percolated through a column of PVP (300 cm3 bed volume). The column was washed with a further 900 cm 3 of H20 (pH 3.5), and the total effluent adjusted to pH 3 and percolated through a column of cellulose phosphate (NH~ form, pH 3, 1 dms bed volume). The column was washed with 5 dm3 HzO (pH 3) and then basic compounds were eluted with 3 dm3 of 2 M NH4OH. The basic fraction was reduced to 45 cm3 by RFE, adjusted to pH 8 with KOH, and partitioned 5 times against equal volumes of H20-saturated butan-l-oI. The combined butan-l-ol fractions were reduced to dryness by RFE and the residue redissolved in 2.5 cm3 of 35% (v/v) ethanol at 60~ C. The solution was cleared by centrifugation and filtration through glass fibre paper then chromatographed on a column (2.5.70 cm) of Sephadex LH-20 in 35% ethanol at a flow-rate of 30 cm3h 1 in the descending direction. 30 cm3 fractions were collected and the following portions of the eluate (corresponding respectively to the elution volumes of ZROG, ZOG, ZR and Z) were retained for further analysis by reversed phase HPLC: A (241 270 cm3), B (271 330 cm3), C (361-420 cm 3) and D (451-510 cm3).
I.M. Scott et al. : Zeatin-9-Riboside in Crown Gall
473
BI B2
HPLC. Equipment and chromatographic systems used have been described in detail by H o r g a n and Kramers (1979). Reversed phase H P L C was carried out on a 150 x 10 m m I.D. column of Hypersil ODS eluted at a flow-rate of 5 cm 3 m i n with HaO (adjusted to pH 7 with triethylammonium bicarbonate) containing acetonitrile. T h e following proportions of acetonitrile were used for analysis of the fractions obtained from Sephadex LH-20 chromatography: A, 9 % ; B, 8%; C, 10%; D, 8%. Sample volume was I cm 3, injected into a 1.5 cm 3 loop. N o r m a l phase H P L C was carried out on a 250-4.5 m m I.D. column of Partisil 10 PAC eluted at a flow-rate of 2 cm 3 min -1 with acetonitrile: H 2 0 (9:1). Sample volume was 30 gl, injected into a 50 gl loop.
E
I.U
L) z n,
o m
