Planta
Planta (1984)161:186-192
9 Springer-Verlag 1984
Metabolism of gibberellins by immature barley grain Sarah J. Gilmour*, Paul Gaskin, Valerie M. Sponsel and Jake MacMillan Agricultural Research Council Research Group, School of Chemistry, University of Bristol, Bristol BS8 ITS, UK
Abstract. Gibberellins Ax, A4, A9, A~2-aldehyde, A20 and A51, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented. Key words: Gibberellin (metabolism) - Grain, immature (gibberellin metabolism) - Hordeum (gibberellin).
Introduction
The endogenous gibberellins (GAs) of immature grain of barley (Hordeum vulgare cv. Proctor) have recently been identified by combined capillary gas chromatography-mass spectrometry (GCMS) (Gaskin etal. 1983). Three weeks after anthesis, grains contained GA1 (Fig. 1, structure 12), GA 4 (structure 8), GA8(13), GA12(3), GAa 7 (5), GA2o (9), GA25 (4), GA34 (11), GA4s (19), 18-hydroxy-GA 4 (20), 12fl-hydroxy-GA 9 (16), 18hydroxy-GA34(21 ) (tentative), 12fl-hydroxy-GA51 (14) (tentative) and 18-hydroxy-GA4a(22 ) (tentative). The major GAs appeared to be 18-hydroxyGA 4, 18-hydroxy-GA34 and GA4s, whereas GA1, GA4, GA s and G A 2 o w e r e only present in trace amounts (Gaskin et al. 1983 ; Gilmour 1983). Gibberellin metabolic pathways have been studied in detail in immature endosperm of pumpkin (Cucurbita maxima) (Graebe et al. 1974a, b, *Present address: MSU-DOE Plant Research Laboratory,
Michigan State University, East Lansing, MI 48824, USA Abbreviations: GA~ = gibberellin A~ ; GC-MS =combined gas
chromatography-mass spectrometry; mance liquid chromatography
HPLC = high-perfor-
1980; Hedden et al. 1984) and in developing seeds of pea (Pisum sativum) (Frydman and MacMillan 1975; Sponsel and MacMillan 1977, 1978, 1980; Kamiya and Graebe 1983). However, information on GA metabolism in cereal grains is fragmentary. Isolated aleurone layers of barley (cv. Himalaya) take up [aH]GA1 and [3H]GA5 and metabolize them to unidentified products (Musgrave et al. 1972). Aleurone tissue and embryoless half-seeds of barley (cv. Himalaya) have been reported to convert [3H]GA1 to [3H]GA8 (Nadeau etal. 1972; Stolp et al. 1973). In this paper the metabolism of several GAs, labelled with both radioactive and stable isotopes, in developing grain of Hordeum vulgare cv. Proctor is described. Identifications of metabolites were made using capillary GCMS. Material and methods Labelled GAs. Stable-isotope and radio-labelled GAs used as
substrates were: [l~,2~-3H2]GA~a (specific activity 1.58. 1012 Bq mmol -z) (Nadeau and Rappaport 1974), 80% radiochemically pure as determined by high-performance liquid chromatography (HPLC), but also thought to contain some 3H20; [30~-2H1]GA1 (0.83 atoms 2H/molecule)b, >99% pure by gas chromatography (GC); [17-13C1,3Hz]GA~C'd (0.90 atoms ~3C/molecule, specific activity 8.62.10 l~ Bq mmol-~), pure by GC-MS; [17-3H2]GA9 c (specific activity 1.06109 Bq mmol 1) (Bearder et al. 1976b), 97% pure by GC-radio-counting; [3c~-ZH1]GA9 (0.87 atoms 2H/molecule) (Beale et al. 1980), homogenous by thin-layer chromatography, GC and GC-MS; [6c~-3Hx]GA12-aldehyde (specific activity 2.57a The value for the specific activity of the [~H]GA 1 is based on the molecule being labelled in the 1- and 2-positions to the extent of 1.1.10 lz Bq mmo1-1 and randomly throughout the molecule to the extent of 4.8.101~ Bq mmol-~ (Nadeau and Rappaport 1974) b Prepared by Dr. C. Willis Scrambing of the [3H] label between the 15- and 17-positions (Bearder et al. 1976a) occurred, but the molecule is designated [17-3H2] for convenience a Prepared by Dr. C. Spray
187
S.J. Gitmour et aI. : Gibberellin metabolism in immature barley grain
R~
VR2
R2
R3
R~
(1) CHO " CH2 H (2) [HO ~,-OH,EH20H H (3) EO2H CH2 H
CH3 ZH3 CH3
(~,) C02H (5) IZO2H
CO2H (C,A25) ED2H (fiA17)
CHZ CH2
H OH
RI
(GA12-aldehyde) (16~, 17-dihydroxy-SA12(GA12) a dehyde)
Rt~
R1
I
o
~
17
R 2 ~ / ~ ' - - ~ H C02H 1B
[02H R1
R
(61 H (7) OH (81 H
R2
R3
H H OH
H H H
(I(}) OH
H
(GA9) (5A51} (fiA41 OH (GA20) OH (fiAz9]
(11) OH (12) H (13) OH
OH OH OH
H OH OH
(9)
H
H
(GA3&) (SA1) (F,A8)
',
H
O
~
"'OH
Rz R RI
(lt,| OH (15) H
R2
H H
(16) H
H
(1"7) H
OH
(181 (19) (20) (21) (22]
OH OH OH OH OH
OH OH H OH OH
R3
1231 (GA2)
RL,
CH3 H,~B-OH (121~-hydroxy-(3A511 CH3 H,~(-OH (12e(-hydroxy-OA9) [H 3 H,JB-OH [H 3 H,13-OH EH3 H,c(-OH EH3 H,~I-OH EH2OH H CH2OH H CH20H H,pOH
(12~-hydroxy-GA 91 (12~-hydroxy-GAt~) (GA49) (GA481 (18-hydroxy-fiA4) (18-hydroxy-GA341 (18-hydroxy-GA/,s)
1010 B q m m o l -~) (Bearder et al. 1973); [18-2H1]GA12-alde hyde (0.80 atoms 2H/molecule) (Down et al. 1983), containing approx. 10% [2H]GA12; [17-13C1,3H2]GA2or (0.88 atoms 13C/molecule, specific activity 1.27.109 B q m m o t -1) (Ingram et al. 1984), 97% radiochemicalty pure by HPLC, 99% pure by GC and HPLC with UV detection; [2c~-2H1,17-3H2] GAs l ~ (0.95 atoms ZH/molecule, specific activity 7.7-101~ Bq mmol 1) (Beale and MacMillan 1981), 95% pure by GC-radio-counting Plant material. Seeds of barley (Hordeum vulgare L.) cv. Proctor were obtained from the National Seed Development Organisation, Cambridge, UK. They were planted in 180-mm-diameter pots (five plants per pot) in John Innes No. 1 potting compost. When the seedlings were approx 2 cm high they were sprayed with a suspension of "Milgo E " fungicide (I.C.I., Fernhurst, Surrey, UK) to control powdery mildew. The plants were grown in a controlled-environment chamber, model S/10H (Conviron, Winnipeg, Manitoba, Canada) under a photoperiod of 18 h at 19~ and with 6 h dark at 15 ~ C. Light was provided by 12 "cool-white" fluorescent tubes (48 inches in length) and nine 50-W tungsten bulbs. The photosynthetic photon flux density was 155 lamol s-~ m -2 at the top of the plant canopy (approx. I m from the light source). Humidity was not controlled, and plants were watered as necessary. Each inflorescence was tagged at anthesis. Feed of labelled GAs and extraction procedure. When doubly labelled GAs were not available, a mixture was prepared containing both radioactive and stable-isotope-labelled GA. A solution of the labelled G A in 50% aqueous ethanol (1 gl) was injected in the endosperm of each of the grains of 3-4 ears.
Fig. 1. Chemical structures of compounds referred to in the text
The stage at which each G A was fed is shown in Table 1. After 7 d the grains were extracted with 80% methanol. After evaporation, the resulting aqueous extracts were partitioned against petroleum ether (boiling point 60-80 ~ C) and ethyl acetate at pH 8, and against ethyl acetate at pH 2.5 as described previously (Gaskin et al. 1983). In some cases the awns and stems were also extracted with methanol. Aliquots of all fractions were radio-counted. The acidic ethyl-acetate fraction was further purified using reverse-phase HPLC according to the procedure described by Gaskin etal. (1983). For the feeds of [13C, 3H]GA4 and [2H][3H]GA9 the acetic-acid concentration was 1%, but in all other feeds the acetic acid was reduced to 0.01% in both the water and methanol. The solvent was applied as a linear gradient of 30 100% methanol in 1% (or 0.01%) acetic acid over 25 min except for the [2H][3H]GA1 feed, when a parabolic gradient was used (No. 10, model 660 solvent programmer; Waters Associates, Milford, Mass., USA). Aliquots of each fraction were removed and radio-counted.
Derivatization and capillary GC-MS. Radioactive HPLC fractions were evaporated to dryness under reduced pressure, converted to their methyl timethylsilyl derivatives, and analyzed by capillary GC-MS as described previously (Gaskin et al. 1983).
Results Doubly labelled GAs were fed to developing barley g r a i n (cv. P r o c t o r ) as d e s c r i b e d in t h e M a t e r i a l
188
S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
Table 1. Labelled GAs fed to immature barley grain for 7 d GAs
Stage fed (days after anthesis)
Level of GA fed (I.tg/grain)
Total amount of GA fed ([xg)
Radioactivity fed (Bq)
Content of stable isotope in substrate (atoms/molecule)
[ZH][3H]GA1 [13C, 3H] GA4 [2HI [3H] GA 9 [ZH][3H]GA9
7 10 10 17 4 6 4
1 1 1 2 1 1 1
87 77 97 170 97 98 89
1.4"105 2.1.105 1.2.105 2.2.105 8.1.103 4,0" 105 2.3" 105
Approx.
[2H][3H]GA1z-aldehyde [13C, 3H]GAzo [2H, 3H] GAs1
Approx.
0.83 0.90 0.50 0.50 0.66 0.88 0.95
Table 2. Percentage radioactivity recovered from GA feeds to developing barley. Results are expressed as percentages of radioactivity fed Fraction
GA fed
Radioactivity fed Awns and rachis (methanolic extract) Grain (methanolic extract) Grain (neutral basic ethyl acetate fraction) Grain (acidic ethyl acetate fraction) Grain (butanol fraction) Grain (aqueous fraction) Fed at 1 gg/grain
[2H][3H] [13C,3H]
[2H][3H]
[2H][3H]
[2HI[3H]
[13C, 3H]
[2H,3H]
GAs
GA9 a
GA9 b
GA12-aldehyde
GA2o
GAs1
100 19 52 3
100 12 62 3
100 20 70 15
100 24 61 2
100 20 64 3
28 17 4
34 23 10
31 27 4
32 26 2
35 22 8
GA 4
100 100 . . . . 50 60 10 1 26 16 8
b Fed at 2 !.tg/grain
38 18 4 ~ Not determined
and methods (see Table 1). Gibberellin A 9 ( 6 ) , GA~2-aldehyde (1) and GAs1 (7), although used as substrates, are not known to be endogenous to immature barley grain. Gibberellin A1(12), GA4(8 ) and GA2o(9 ) are present at levels below 5 ng/grain (Gaskin et al. 1983 ; Gilmour 1983), but were fed at levels which greatly exceeded this in order to obtain GC-MS identifications of metabolites. The percentage radioactivity recovered in the different fractions during purification of the extracts is shown in Table 2. Extracts of awns and rachis, when counted, contained approx. 20% of the radioactivity fed (see Table 2), probably as a result of contamination during application of the labelled substrates. The acidic ethyl-acetate fractions from grain extracts were fractionated by reverse-phase HPLC. Radioactive fractions were pooled as shown in Table 3, and metabolites were identified by capillary GC-MS (Table 3) using reference spectra and Kovats retention indices (Kovats 1958) for comparison. In some cases metabolites could not be readily identified. Some of the metabolites shown in Table 3 had a low incorporation of stable isotope, probably as a consequence
of dilution of the label by endogenous GA, or alternatively as a result of low metabolism of an unnatural substrate. Since most of the pooled radioactive HPLC fractions contained more than one metabolite, it was not possible to calculate percentage yields of individual metabolites. The radioactivity associated with pooled HPLC fractions (Table 3), when expressed as a percentage of radioactivity fed, gives an indication of the overall yield of metabolites. A proposed scheme of GA metabolism derived from the data given in Table 3 is shown in Fig. 2. The solid lines represent conversions which have been demonstrated by feeds, and the dashed lines are speculative steps. For clarity of presentation the conversions involving more than one step, shown in Table 3, are not shown in Fig. 2.
Feed of [2H][3H]GA12-aldehyde3(1).
Stable label was incorporated into [2H][ H]GAl(12), [2H] [3H] GA 8 (13) and [2H] [3H] GA48 (19), clearly demonstrating the conversion of [2H][3H]GA12aldehyde (1) t o C 1 9 G A s (Table 3). No unmetabolized substrate was detected; however, [2H][3H]GA~2(3) was identified. The oxidation of
Table 3. Metabolites identified by GC-MS from in-vivo feeds to immature barley grain. Metabolites marked * are endogenous G A fed
HPLC fractions
[3H] in HPLC fractions As percentage in HPLC run
[2H] [3H]G A 1
38.9
8.9
GA 8*
13
0.98
8-10
21.5
4.9
( GAs* t oxo-GAa
13 -
0.98 0.84
11-14
24.9
5.7
~ GA I * monoOH-GA 1
12
0.83
6-8
9.7
2.0
9-11
32.4
6.6
16-J8
12.8
2.6
19-20
28.2
5.9
GAs* GA49 18-OH-GA48 *a GA48 * 12fl-OH-GA 4 * diOH-GA~ GA34* 18-OH-GA4* 18-OH-GA34 *a GA z GA 4. 18-OH-GA4*
13 18 22 19 17 11 20 21 23 8 20
0.20 0.83 0.19 0.63 0.90 0.48 0.20 0.13 0.22 0.87 0.85 0.13
8-10
25.5
5.6
GA48* 12fl-OH-GA51 *"
19 14
0.17 0.45
12 14 16 21-22
12.5 2.8 37.0
2.7 0.7 7.3
12fl-OH-GA9 * 18-OH-GA34 *a GA 9
16 21 6
0.52 0.10 0.54
GA48 * GA,~9 12fl-OH-GA 9 * triOH-GA 9 12fl-OH-GA51 *a 12fl-OH-GA 9 * monoOH-GA 18-OH-GA34 *~
19 18 16
0.17 0.31 0.46 0.35 0.41 0.46 0.45 0.13 0.09 0.19 approx. 0.50
9-11
22.1
5.1
13-14
9.4
2.6
16
4.2
1.5
18
3.8
1.4
21-22
44.6
12.3
6-12
19.8
4.6
13 19
50.8
11.9
20 25
28.9
6.8
4-7
33.6
8.6
8 10
13.9
3.6
12-14
28.9
7.4
4-7
10.1
2.7
8-10
31.9
8.6
11-14
21.8
5.9
15 17
28.4
7.7
{ GA3~* 18-OH-GA4 * GA 9
[2H] [3H] G A 12-aldehyde
[2H, 3H]-GA51
A m o u n t of stable isotope (atoms/molecule)
4-7
[2H] [3H] G A 9 (2 lag/grain)
[13C, 3H]-GAzo
Structure (Fig. 1)
As percentage fed
[13C, 3H] GA4
[2H] [all] G A 9 (i lag/grain)
Metabolites identified
a Tentative identification since authentic compound is u n k n o w n b Tentative identification due to weak spectrum
14 16 21 11 20 6
GA * GA 8* GA,~8* 16~, 17-diOHG A 12-aldehyde GAI2 *
12 13 19 2
0.58 0.62 0.15
3
0.66
f GA 8* / GA29 GA * monoOH-GA2o diOH-GA2o GA2o*
13 10 12
0.90 0.91 0.89 0.96 _ 0.89
{
GAs *b GA48* 12•_OH.GA51 ,a monoOH-GAs 1 GA34* GAs1
9 13 19 14 11 7
0.52 0.79 0.95 0.19 0.93
190
S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
OH "
",,
~,
0
:
",,,
....
',,
9"
Ha
12~B'OH'GA51
'.,
'
12p'OH'SA9 /
,'
'
0
>
GA9
OH
~o~
.o- "i'~ ~-Ao 2 H /~ ,zp-OH-u~
.o,-'-i,-U-~co.H', _ | H ..--f OA+BLU2rl 9",
#l
....................
~ . ~ HO""'i/IH
C02H5A12- ", aldehyde"..
HO,~'-.i~~ ~02H" , 9 n fiA3~. " "'',
~C02H 5A~.
" " " -,
.'7
CH20H 18-OH-~A/*8
- - - -~' HO "-, ,
..
~ H
"~
[flzOH IB-OH-GA4 "~] ./O,,~ ' ~
',
YA
18-OH-fiA3g
~ H O0~
OH
aA2}O2"
Fig. 2. Proposed scheme of GA metabolism in developing barley grain. Dashed lines indicate speculative steps. All GAs shown except for GAg, GA29 and GAs~ have been shown to be endogenous to developing barley grain. For clarity, realized conversions involving more than one step are not shown
[2H][3HlGA12-aldehyde to [2H][3H]GA12 may not have been enzymatic, since aerial oxidation of GA12-aldehyde to G A I / c a n occur (Bearder 1973).
Feeds of [2H][3H]GA9 (6) and [2H][3H] GA5I(7). Although there is no evidence for GA 9(6) or GA51(7) being endogenous to developing barley grain (Gaskin etal. 1983). [2H][3H]GA9 and [ZH,3H]GA51 were metabolized to native GAs. Thus [/H][3H]GA9 was 12flhydroxylated to [2H] [3H] 12]?-hydroxy-GA 9 (16), 2]?- and 12fl-hydroxylated to [2H][3H]12]?-hydroxy-GA51(14), 2]?- and 3]?-hydroxylated to [2H] [3H] GA34 (11), 2]?-, 3]?- and 12]?-hydroxylated to [2H][3H]GA4s (19) and 2]?-, 3]?- and 18-hydroxylated to [2H][3H]18-hydroxy-GA34(21) (Table 3). There was no evidence for [2H] [3H] GA 9 being 2]?-hydroxylated to [2H][3H]GAsl(7), or 3]?-hydroxylated to [2H][3H]GA4(8). However, it is possible that [2H] [3H] GA 9 is a precursor of both, but that neither accumulate (see Fig. 2). The very low endogenous level of GA 4 and the apparent
absence of GAs1 (Gaskin et al. 1983) could indicate that these GAs have a high rate of turnover. When [2H][3H]GA9 was fed at 2 gg/grain, [2H][3H]lS-hydroxy-GA 4 (20), and [2HI[all] GA49 (18) were obtained in addition to the metabolites detailed above. Interestingly, unlabelled 12]?-hydroxy-GA4(17 ) was also observed, which would indicate that this compound is endogenous to developing barley grain, although it was n o t detected by Gaskin et al. (1983). The mass spectrum of 12]?-hydroxy-GA4 has been published previously (Gaskin et al. 1984). Gibberellin A49(18 ) may not be a native GA. [2H] [3H] Gibberellin A 51 (7) was 12]?-hydroxylated to [2Hl[3H] 12]?-hydroxy-GA 51 (14), 3]?-hydroxylated to [2H][3H]GA34(ll) and 3]?- and 12]?hydroxylated to [2H] [all] GA4s (19).
Feed of [13C, 3H]GA4 (8). From Table 3 it is apparent that [13C, 3 H ] G A 4 ( 8 ) w a s 2fl-hydroxylated to [13C,3H]GA34(11), 12fl-hydroxylated to [13C, 3HI 12]?-hydroxy-GA4 (17), 18-hydroxylated
s.J. Gilmour et al. : Gibberellinmetabolism in immature barley grain to [13C,3H]18-hydroxy-GA4(20) and hydroxylated at combinations of these positions to [13C, all] GA4s(19 ), [13C, 3H]lS-hydroxy-GA34(21 ) and [13C, 3H]18-hydroxy-GA4s(22). Speculative routes to these GAs are shown in Fig. 2. Two products which have not been shown to be endogenous to barley (Gaskin et al. 1983), i.e. GA/(23) and GA49(18), were also identified from the feed of [13C,3H]GA4 to developing barley grain (Table 3). [13C, 3H]Gibberellin A a may be an artefact as it is known to be produced from GA 4 by dilute mineral acid (Grove 1961). [13C, 3H]Gibberellin A49 may have arisen as a result of substrate overloading as [2H][3H]GA49 was also produced from the feed of 2 gg/grain of [2HI [3H] G A 9 (Table 3).
Feeds of [2H][3H]GA1 (12) and [13C,3H] GA2o(9 ). From the feeds of [2H][3H]GAI(12) and [13C, 3H]GA2o(9) it is apparent that GA2o is converted to GAs(13 ) via GA 1 (Fig. 2). [13C, 3H]Gibberellin A20 is also converted to [13C, 3H]GA29(10) (see Fig. 2), but this may not reflect the normal in-vivo situation, as GA29 has not been shown to be a native barley G A (Gaskin et al. 1983). Discussion
It should be stressed that the results shown in Table 3 were obtained from feeds of doubly labelled GAs, fed at experimentally convenient stages of development of the barley grain, at levels which greatly exceeded the endogenous. Nevertheless, the substrates were predominantly metabolized to native GAs (Table 3). Of the substrates fed, [2H][3H]GA12-aldehyde, [2H][3H]GA9 and [ZH][3H]GAsl have not been identified as native to barley, although they were metabolized to native GAs (Table 3). In these circumstances the schematic summary of the results shown in Fig. 2 can only be regarded as a working hypothesis for future metabolic work. [2HJ [3H] Gibberellin A 12-aldehyde was metabolized to di- and tri-hydroxylated C19GAs. The only C2oGAs to accumulate were GA12 , which may arise non-enzymatically, and 16,17-dihydroxy-GAlz-aldehyde, which may be an artefact of substrate overloading. Hydroxylation at the 13position possibly occurs early in the pathway as [2H][3H]GA1 was formed from [2H][3H]GAlzaldehyde, but not from the C 19 GAs, [2H][3H]GA9 or [13C,3H]GA4. In addition, 13hydroxylated C20GAs are present in developing (GA17) and germinating (GA17 and GA19 ) barley
191
grain (Gaskin et al. 1983). An early 13-hydroxylation pathway appears to be of widespread occurrence in plants (see Sponsel 1983 for examples). The non-hydroxylated C19GA , GA9, was extensively hydroxylated at the 2/?-, 3/?-, 12/?- and 18-positions. Likewise GA 4 was hydroxylated at the 2/?-, 12/?- and 18-positions. Whether GA 9 is a precursor of GA 4 in barley has not been established. [2H] [3H] Gibberellin A 4 did not accumulate in feeds of [2H][3H]GA9, although other 3-hydroxylated metabolites were observed. As the level of native GA 4 is very low, the failure to detect [2H][3H]GA4 in feeds of [2H][3H]GA9 may be caused by its very rapid turnover. The order and importance of the multiple hydroxylation of C19 GAs in barley is difficult to establish. Some key intermediates were not available for feeding, and several metabolites remain unidentified. However some patterns of GA metabolism in barley are apparent. For instance, in the present feeds no 13-hydroxylated GAs were shown to be 12/?- or 18-hydroxylated. Furthermore, 18-hydroxylation only occurred on those GAs which were already 3-hydroxylated. Whether these results are caused by our inability to recognise and identify new metabolites, or whether they reflect a measure of substrate selectivity by the enzymes involved remains to be established. On the other hand, 2/?-hydroxylated GAs, which are biologically inactive and which are not usually further hydroxylated in other plants, do appear to be extensively metabolized in barley. Whether this reflects normal metabolism or merely the low substrate selectivity of barley hydroxylases remains to be investigated. The presence of metabolites which are not native to barley is probably a result of feeding excessive levels of labelled substrates. Labelled GA49 , which was observed in feeds of [2H] [3H] GA 9 and [13C, 3H]GA4 is 120~-hydroxylated whereas the 12/?-epimer, GA4s , is native. Similarly, Railton et al. (1974), Frydman and MacMillan (1975) and Sponsel and MacMillan (1977) observed the nonnative 12~-hydroxy-GA 9 (H2-GA 31) (15) (Gaskin et al. 1984) in high-dose feeds of [3H]GA9 to Pisum sativum seedlings and seeds. Substrate overloading probably also results in conjugation of excess substrate and extraneous metabolites. However, the substantial radioactivity observed in acidic butanol fractions of grain extracts (Table 2) was not further analysed. An attempt to identify metabolites after a feed of 5 ng/grain [13C, all] GA4, which more nearly reflects the endogenous level (Gilmour 1983), was unsuccessful because of excessive dilution of polar
192
S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
products by endogenous GAs. Several approaches are envisaged in order to overcome this problem. The use of inhibitors of GA biosynthesis or of a GA-less barley mutant would reduce background GA levels, thereby allowing the identification of metabolites in feeds conducted at physiological concentrations. Alternatively, the use of cell-free enzyme preparations, from which the endogenous GAs can be removed, would allow the sequence of hydroxylation of C 1 9 G A s to be studied more precisely. We would like to thank the Agricultural Research Council for financial support for this work. We are also grateful to the following for synthesis of labelled gibberellins: Dr. M. Beale, Dr. J. Bearder, Mr. M. Lee, Dr. C. Spray and Dr. C. Willis. In addition we would like to thank Dr. L. Rappaport, University of California, Davis, USA for the gift of [3H]GA1.
References Beale, M.H., MacMillan, J. (1981) Partial syntheses of [2c~-ZH]and [2a-3H]-gibberellin Az9 and [2e-2H, 15,17-3H]gibberellin A51 from gibberellin A 3. J. Chem. Soc. Perkin Trans. 1,394-400 Beale, M.H., Gaskin, P., Kirkwood, P.S., MacMillan, J. (1980) Partial synthesis of gibberellin A 9 and [3~- and 3fl-ZH1]gibberellin A9; gibberellin A 5 and [lfl, 3-all2 and -3Hz]gibberellin As; and gibberellin A2o and [lfl, 3c~-2H2 and -3H2] gibberellin Azo. J. Chem. Soc. 885-890 Bearder, J.R. (1973) Diterpenoid biosynthesis in mutants of Fusarium moniliforme. Ph.D. thesis, University of Bristol Bearder, J.R., Frydman, V.M., Gaskin, P., MacMillan, J., Wels, C.M., Phinney, B.O. (1976a) Fungal products. Part XVI. Conversion of isosteviol and steviol acetate into gibberellin analogues by mutant B1-41 a of Gibberellafujikuroi and preparation of [3H]-gibberellin Azo. J. Chem. Soc. Perkin Trans. 1, 173-178 Bearder, J.R., Frydman, V.M., Gaskin, P., Hatton, I.K., Harvey, W.E., MacMillan, J., Phinney, B.O. (1976b) Fungal products. Part XVII. Microbiological hydroxylation of gibberellin A 9 and its methyl ester. J. Chem. Soc. 178-183 Bearder, J.R., MacMillan, J., Phinney, B.O. (1973) 3-Hydroxylation of gibberellin A12-aldehyde in Gibberella fujikuroi strain REC-193A. Phytochemistry 12, 2173-2179 Down, G.J., Lee, M., MacMillan, J., Staples, K.S. (1983) Preparation of 13-hydroxygibberellin Axz-7-aldehyde. J. Chem. Soc. 1103 1108 Frydman, V.M., MacMillan, J. (1975) The metabolism of gibberellins Ag, A2o and A29 in immature seeds of Pisum satirum cv. Progress No. 9. Planta 125, 181-195 Gaskin, P., Gilmour, S.J., Lenton, J.R., MacMillan, J., Sponsel, V.M. (1983) Endogenous gibberellins and kauranoids identified from developing and germinating barley grain. J. Plant Growth Regul. 2, 229-242 Gaskin, P., Hutchison, M., Lewis, N., MacMillan, J., Phinney, B.O. (1984) Microbial conversion of 12-oxygenated and other derivatives ofent-kaur-16-en-19-oic acid by Gibberella fujikuroi, mutant B1-41a. Phytochemistry 559-564 Gilmour, S.J. (1983) Some studies on the endogenous gibberel-
lins of barley (Hordeum vulgare). Ph.D. thesis, University of Bristol Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974a) Biosynthesis of gibberellins A12, A15, A24, A36 and A37 by a cell-free system from Cucurbita maxima. Phytochemistry 13, 1433-1440 Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974b) The biosynthesis of a C19-gibberellin from mevalonic acid in a cell-free system from a higher plant. Planta 120, 307-309 Graebe, J.E., Hedden, P., Rademacher, W. (1980) Gibberellin biosynthesis. In: Gibberellins chemistry, physiology and use. British Plant Growth Regulator Group, Monograph 5, pp. 31-47, Lenton, J.R., ed. BPGRG, Wantage Grove, J.F. (1961) Gibberellin A z, J. Chem. Soc. 3545-3547 Hedden, P., Graebe, J.E., Beale, M.H., Gaskin, P., MacMillan, J. (1984) The biosynthesis of 12cr gibberellins in a cell-free system from Cucurbita maxima endosperm. Phytochemistry 569-574 Ingrain, T.J., Reid, J.B., Muffet, I.C., Gaskin, P., Willis, C.L., MacMillan, J. (1984) Internode length in Pisum. The Le gene controls the 3fl-hydroxylation of gibberellin A2o to gibberellin A 1. Planta 160, 455-463 Kamiya, Y., Graebe, J.E. (1983) The biosynthesis of all major pea gibberellins in a cell-free system from Pisum sativum. Phytochemistry 22, 681-689 Kovats, E. (1958) Gas-chromatographische characterisierung organischer verbindungen, Tell 1: Retentions Indices aliphatischer Halogenide, Alkohole, Aldehyde und Ketone. Helv. Chim. Acta 41, 1915-1932 Musgrave, A., Kays, S.E., Kende, H. (1972) Uptake and metabolism of radioactive gibberellins by barley aleurone layers. Planta 102, 1 10 Nadeau, R., Rappaport, L. (1974) The synthesis of [3H]gibberellin A 3 and [3H] gibberellin A 1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A 3. Phytochemistry 13, 1537-1545 Nadeau, R., Rappaport, L., Stolp, C.L. (1972) Uptake and metabolism of 3H-gibberellin A 1 by barley aleurone layers : response to abscisic acid. Planta 107, 315-324 Railton, I.D., Durley, R.C., Pharis, R.P. (1974) Metabolism of tritiated gibberellin A 9 by shoots of dark-grown dwarf pea, cv. Meteor. Plant Physiol. 54, 6-12 Sponsel, V.M. (1983) In vivo gibberellin metabolism in higher plants. In: Biochemistry and physiology of gibberellins, vol. 1, pp 151-250, Crozier, A., ed. Praeger, New York Sponsel, V.M., MacMillan, J. (1977) Further studies on the metabolism of gibberellins (GAs) A 9, A/o and A29 in immature seeds of Pisum sativum cv. Progress No. 9. Planta 135, 129-136 Sponsel, V.M., MacMillan, J. (1978) Metabolism of gibberellin Az9 in seeds of Pisum sativum cv. Progress No. 9; use of [2H] and [3H] GAs, and the identification of a new GA catabolite. Planta 144, 69-78 Sponsel, V.M., MacMillan, J. (1980) Metabolism of [13C~]gibberellin A29 to [~3C~]gibberellin-catabolite in maturing seeds of Pisum sativum cv. Progress No. 9. Planta 150, 46-52 Stolp, C.V. Nadeau, R., Rappaport, L. (1973) Effect of abscisic acid on uptake and metabolism of 3H-gibberellin A 1 and 3H-pseudo-gibberellin A 1 by barley half-seeds. Plant Physiol. 52, 546-548 Received 10 January; accepted 2 February 1984
Planta (1984)161:186-192
9 Springer-Verlag 1984
Metabolism of gibberellins by immature barley grain Sarah J. Gilmour*, Paul Gaskin, Valerie M. Sponsel and Jake MacMillan Agricultural Research Council Research Group, School of Chemistry, University of Bristol, Bristol BS8 ITS, UK
Abstract. Gibberellins Ax, A4, A9, A~2-aldehyde, A20 and A51, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented. Key words: Gibberellin (metabolism) - Grain, immature (gibberellin metabolism) - Hordeum (gibberellin).
Introduction
The endogenous gibberellins (GAs) of immature grain of barley (Hordeum vulgare cv. Proctor) have recently been identified by combined capillary gas chromatography-mass spectrometry (GCMS) (Gaskin etal. 1983). Three weeks after anthesis, grains contained GA1 (Fig. 1, structure 12), GA 4 (structure 8), GA8(13), GA12(3), GAa 7 (5), GA2o (9), GA25 (4), GA34 (11), GA4s (19), 18-hydroxy-GA 4 (20), 12fl-hydroxy-GA 9 (16), 18hydroxy-GA34(21 ) (tentative), 12fl-hydroxy-GA51 (14) (tentative) and 18-hydroxy-GA4a(22 ) (tentative). The major GAs appeared to be 18-hydroxyGA 4, 18-hydroxy-GA34 and GA4s, whereas GA1, GA4, GA s and G A 2 o w e r e only present in trace amounts (Gaskin et al. 1983 ; Gilmour 1983). Gibberellin metabolic pathways have been studied in detail in immature endosperm of pumpkin (Cucurbita maxima) (Graebe et al. 1974a, b, *Present address: MSU-DOE Plant Research Laboratory,
Michigan State University, East Lansing, MI 48824, USA Abbreviations: GA~ = gibberellin A~ ; GC-MS =combined gas
chromatography-mass spectrometry; mance liquid chromatography
HPLC = high-perfor-
1980; Hedden et al. 1984) and in developing seeds of pea (Pisum sativum) (Frydman and MacMillan 1975; Sponsel and MacMillan 1977, 1978, 1980; Kamiya and Graebe 1983). However, information on GA metabolism in cereal grains is fragmentary. Isolated aleurone layers of barley (cv. Himalaya) take up [aH]GA1 and [3H]GA5 and metabolize them to unidentified products (Musgrave et al. 1972). Aleurone tissue and embryoless half-seeds of barley (cv. Himalaya) have been reported to convert [3H]GA1 to [3H]GA8 (Nadeau etal. 1972; Stolp et al. 1973). In this paper the metabolism of several GAs, labelled with both radioactive and stable isotopes, in developing grain of Hordeum vulgare cv. Proctor is described. Identifications of metabolites were made using capillary GCMS. Material and methods Labelled GAs. Stable-isotope and radio-labelled GAs used as
substrates were: [l~,2~-3H2]GA~a (specific activity 1.58. 1012 Bq mmol -z) (Nadeau and Rappaport 1974), 80% radiochemically pure as determined by high-performance liquid chromatography (HPLC), but also thought to contain some 3H20; [30~-2H1]GA1 (0.83 atoms 2H/molecule)b, >99% pure by gas chromatography (GC); [17-13C1,3Hz]GA~C'd (0.90 atoms ~3C/molecule, specific activity 8.62.10 l~ Bq mmol-~), pure by GC-MS; [17-3H2]GA9 c (specific activity 1.06109 Bq mmol 1) (Bearder et al. 1976b), 97% pure by GC-radio-counting; [3c~-ZH1]GA9 (0.87 atoms 2H/molecule) (Beale et al. 1980), homogenous by thin-layer chromatography, GC and GC-MS; [6c~-3Hx]GA12-aldehyde (specific activity 2.57a The value for the specific activity of the [~H]GA 1 is based on the molecule being labelled in the 1- and 2-positions to the extent of 1.1.10 lz Bq mmo1-1 and randomly throughout the molecule to the extent of 4.8.101~ Bq mmol-~ (Nadeau and Rappaport 1974) b Prepared by Dr. C. Willis Scrambing of the [3H] label between the 15- and 17-positions (Bearder et al. 1976a) occurred, but the molecule is designated [17-3H2] for convenience a Prepared by Dr. C. Spray
187
S.J. Gitmour et aI. : Gibberellin metabolism in immature barley grain
R~
VR2
R2
R3
R~
(1) CHO " CH2 H (2) [HO ~,-OH,EH20H H (3) EO2H CH2 H
CH3 ZH3 CH3
(~,) C02H (5) IZO2H
CO2H (C,A25) ED2H (fiA17)
CHZ CH2
H OH
RI
(GA12-aldehyde) (16~, 17-dihydroxy-SA12(GA12) a dehyde)
Rt~
R1
I
o
~
17
R 2 ~ / ~ ' - - ~ H C02H 1B
[02H R1
R
(61 H (7) OH (81 H
R2
R3
H H OH
H H H
(I(}) OH
H
(GA9) (5A51} (fiA41 OH (GA20) OH (fiAz9]
(11) OH (12) H (13) OH
OH OH OH
H OH OH
(9)
H
H
(GA3&) (SA1) (F,A8)
',
H
O
~
"'OH
Rz R RI
(lt,| OH (15) H
R2
H H
(16) H
H
(1"7) H
OH
(181 (19) (20) (21) (22]
OH OH OH OH OH
OH OH H OH OH
R3
1231 (GA2)
RL,
CH3 H,~B-OH (121~-hydroxy-(3A511 CH3 H,~(-OH (12e(-hydroxy-OA9) [H 3 H,JB-OH [H 3 H,13-OH EH3 H,c(-OH EH3 H,~I-OH EH2OH H CH2OH H CH20H H,pOH
(12~-hydroxy-GA 91 (12~-hydroxy-GAt~) (GA49) (GA481 (18-hydroxy-fiA4) (18-hydroxy-GA341 (18-hydroxy-GA/,s)
1010 B q m m o l -~) (Bearder et al. 1973); [18-2H1]GA12-alde hyde (0.80 atoms 2H/molecule) (Down et al. 1983), containing approx. 10% [2H]GA12; [17-13C1,3H2]GA2or (0.88 atoms 13C/molecule, specific activity 1.27.109 B q m m o t -1) (Ingram et al. 1984), 97% radiochemicalty pure by HPLC, 99% pure by GC and HPLC with UV detection; [2c~-2H1,17-3H2] GAs l ~ (0.95 atoms ZH/molecule, specific activity 7.7-101~ Bq mmol 1) (Beale and MacMillan 1981), 95% pure by GC-radio-counting Plant material. Seeds of barley (Hordeum vulgare L.) cv. Proctor were obtained from the National Seed Development Organisation, Cambridge, UK. They were planted in 180-mm-diameter pots (five plants per pot) in John Innes No. 1 potting compost. When the seedlings were approx 2 cm high they were sprayed with a suspension of "Milgo E " fungicide (I.C.I., Fernhurst, Surrey, UK) to control powdery mildew. The plants were grown in a controlled-environment chamber, model S/10H (Conviron, Winnipeg, Manitoba, Canada) under a photoperiod of 18 h at 19~ and with 6 h dark at 15 ~ C. Light was provided by 12 "cool-white" fluorescent tubes (48 inches in length) and nine 50-W tungsten bulbs. The photosynthetic photon flux density was 155 lamol s-~ m -2 at the top of the plant canopy (approx. I m from the light source). Humidity was not controlled, and plants were watered as necessary. Each inflorescence was tagged at anthesis. Feed of labelled GAs and extraction procedure. When doubly labelled GAs were not available, a mixture was prepared containing both radioactive and stable-isotope-labelled GA. A solution of the labelled G A in 50% aqueous ethanol (1 gl) was injected in the endosperm of each of the grains of 3-4 ears.
Fig. 1. Chemical structures of compounds referred to in the text
The stage at which each G A was fed is shown in Table 1. After 7 d the grains were extracted with 80% methanol. After evaporation, the resulting aqueous extracts were partitioned against petroleum ether (boiling point 60-80 ~ C) and ethyl acetate at pH 8, and against ethyl acetate at pH 2.5 as described previously (Gaskin et al. 1983). In some cases the awns and stems were also extracted with methanol. Aliquots of all fractions were radio-counted. The acidic ethyl-acetate fraction was further purified using reverse-phase HPLC according to the procedure described by Gaskin etal. (1983). For the feeds of [13C, 3H]GA4 and [2H][3H]GA9 the acetic-acid concentration was 1%, but in all other feeds the acetic acid was reduced to 0.01% in both the water and methanol. The solvent was applied as a linear gradient of 30 100% methanol in 1% (or 0.01%) acetic acid over 25 min except for the [2H][3H]GA1 feed, when a parabolic gradient was used (No. 10, model 660 solvent programmer; Waters Associates, Milford, Mass., USA). Aliquots of each fraction were removed and radio-counted.
Derivatization and capillary GC-MS. Radioactive HPLC fractions were evaporated to dryness under reduced pressure, converted to their methyl timethylsilyl derivatives, and analyzed by capillary GC-MS as described previously (Gaskin et al. 1983).
Results Doubly labelled GAs were fed to developing barley g r a i n (cv. P r o c t o r ) as d e s c r i b e d in t h e M a t e r i a l
188
S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
Table 1. Labelled GAs fed to immature barley grain for 7 d GAs
Stage fed (days after anthesis)
Level of GA fed (I.tg/grain)
Total amount of GA fed ([xg)
Radioactivity fed (Bq)
Content of stable isotope in substrate (atoms/molecule)
[ZH][3H]GA1 [13C, 3H] GA4 [2HI [3H] GA 9 [ZH][3H]GA9
7 10 10 17 4 6 4
1 1 1 2 1 1 1
87 77 97 170 97 98 89
1.4"105 2.1.105 1.2.105 2.2.105 8.1.103 4,0" 105 2.3" 105
Approx.
[2H][3H]GA1z-aldehyde [13C, 3H]GAzo [2H, 3H] GAs1
Approx.
0.83 0.90 0.50 0.50 0.66 0.88 0.95
Table 2. Percentage radioactivity recovered from GA feeds to developing barley. Results are expressed as percentages of radioactivity fed Fraction
GA fed
Radioactivity fed Awns and rachis (methanolic extract) Grain (methanolic extract) Grain (neutral basic ethyl acetate fraction) Grain (acidic ethyl acetate fraction) Grain (butanol fraction) Grain (aqueous fraction) Fed at 1 gg/grain
[2H][3H] [13C,3H]
[2H][3H]
[2H][3H]
[2HI[3H]
[13C, 3H]
[2H,3H]
GAs
GA9 a
GA9 b
GA12-aldehyde
GA2o
GAs1
100 19 52 3
100 12 62 3
100 20 70 15
100 24 61 2
100 20 64 3
28 17 4
34 23 10
31 27 4
32 26 2
35 22 8
GA 4
100 100 . . . . 50 60 10 1 26 16 8
b Fed at 2 !.tg/grain
38 18 4 ~ Not determined
and methods (see Table 1). Gibberellin A 9 ( 6 ) , GA~2-aldehyde (1) and GAs1 (7), although used as substrates, are not known to be endogenous to immature barley grain. Gibberellin A1(12), GA4(8 ) and GA2o(9 ) are present at levels below 5 ng/grain (Gaskin et al. 1983 ; Gilmour 1983), but were fed at levels which greatly exceeded this in order to obtain GC-MS identifications of metabolites. The percentage radioactivity recovered in the different fractions during purification of the extracts is shown in Table 2. Extracts of awns and rachis, when counted, contained approx. 20% of the radioactivity fed (see Table 2), probably as a result of contamination during application of the labelled substrates. The acidic ethyl-acetate fractions from grain extracts were fractionated by reverse-phase HPLC. Radioactive fractions were pooled as shown in Table 3, and metabolites were identified by capillary GC-MS (Table 3) using reference spectra and Kovats retention indices (Kovats 1958) for comparison. In some cases metabolites could not be readily identified. Some of the metabolites shown in Table 3 had a low incorporation of stable isotope, probably as a consequence
of dilution of the label by endogenous GA, or alternatively as a result of low metabolism of an unnatural substrate. Since most of the pooled radioactive HPLC fractions contained more than one metabolite, it was not possible to calculate percentage yields of individual metabolites. The radioactivity associated with pooled HPLC fractions (Table 3), when expressed as a percentage of radioactivity fed, gives an indication of the overall yield of metabolites. A proposed scheme of GA metabolism derived from the data given in Table 3 is shown in Fig. 2. The solid lines represent conversions which have been demonstrated by feeds, and the dashed lines are speculative steps. For clarity of presentation the conversions involving more than one step, shown in Table 3, are not shown in Fig. 2.
Feed of [2H][3H]GA12-aldehyde3(1).
Stable label was incorporated into [2H][ H]GAl(12), [2H] [3H] GA 8 (13) and [2H] [3H] GA48 (19), clearly demonstrating the conversion of [2H][3H]GA12aldehyde (1) t o C 1 9 G A s (Table 3). No unmetabolized substrate was detected; however, [2H][3H]GA~2(3) was identified. The oxidation of
Table 3. Metabolites identified by GC-MS from in-vivo feeds to immature barley grain. Metabolites marked * are endogenous G A fed
HPLC fractions
[3H] in HPLC fractions As percentage in HPLC run
[2H] [3H]G A 1
38.9
8.9
GA 8*
13
0.98
8-10
21.5
4.9
( GAs* t oxo-GAa
13 -
0.98 0.84
11-14
24.9
5.7
~ GA I * monoOH-GA 1
12
0.83
6-8
9.7
2.0
9-11
32.4
6.6
16-J8
12.8
2.6
19-20
28.2
5.9
GAs* GA49 18-OH-GA48 *a GA48 * 12fl-OH-GA 4 * diOH-GA~ GA34* 18-OH-GA4* 18-OH-GA34 *a GA z GA 4. 18-OH-GA4*
13 18 22 19 17 11 20 21 23 8 20
0.20 0.83 0.19 0.63 0.90 0.48 0.20 0.13 0.22 0.87 0.85 0.13
8-10
25.5
5.6
GA48* 12fl-OH-GA51 *"
19 14
0.17 0.45
12 14 16 21-22
12.5 2.8 37.0
2.7 0.7 7.3
12fl-OH-GA9 * 18-OH-GA34 *a GA 9
16 21 6
0.52 0.10 0.54
GA48 * GA,~9 12fl-OH-GA 9 * triOH-GA 9 12fl-OH-GA51 *a 12fl-OH-GA 9 * monoOH-GA 18-OH-GA34 *~
19 18 16
0.17 0.31 0.46 0.35 0.41 0.46 0.45 0.13 0.09 0.19 approx. 0.50
9-11
22.1
5.1
13-14
9.4
2.6
16
4.2
1.5
18
3.8
1.4
21-22
44.6
12.3
6-12
19.8
4.6
13 19
50.8
11.9
20 25
28.9
6.8
4-7
33.6
8.6
8 10
13.9
3.6
12-14
28.9
7.4
4-7
10.1
2.7
8-10
31.9
8.6
11-14
21.8
5.9
15 17
28.4
7.7
{ GA3~* 18-OH-GA4 * GA 9
[2H] [3H] G A 12-aldehyde
[2H, 3H]-GA51
A m o u n t of stable isotope (atoms/molecule)
4-7
[2H] [3H] G A 9 (2 lag/grain)
[13C, 3H]-GAzo
Structure (Fig. 1)
As percentage fed
[13C, 3H] GA4
[2H] [all] G A 9 (i lag/grain)
Metabolites identified
a Tentative identification since authentic compound is u n k n o w n b Tentative identification due to weak spectrum
14 16 21 11 20 6
GA * GA 8* GA,~8* 16~, 17-diOHG A 12-aldehyde GAI2 *
12 13 19 2
0.58 0.62 0.15
3
0.66
f GA 8* / GA29 GA * monoOH-GA2o diOH-GA2o GA2o*
13 10 12
0.90 0.91 0.89 0.96 _ 0.89
{
GAs *b GA48* 12•_OH.GA51 ,a monoOH-GAs 1 GA34* GAs1
9 13 19 14 11 7
0.52 0.79 0.95 0.19 0.93
190
S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
OH "
",,
~,
0
:
",,,
....
',,
9"
Ha
12~B'OH'GA51
'.,
'
12p'OH'SA9 /
,'
'
0
>
GA9
OH
~o~
.o- "i'~ ~-Ao 2 H /~ ,zp-OH-u~
.o,-'-i,-U-~co.H', _ | H ..--f OA+BLU2rl 9",
#l
....................
~ . ~ HO""'i/IH
C02H5A12- ", aldehyde"..
HO,~'-.i~~ ~02H" , 9 n fiA3~. " "'',
~C02H 5A~.
" " " -,
.'7
CH20H 18-OH-~A/*8
- - - -~' HO "-, ,
..
~ H
"~
[flzOH IB-OH-GA4 "~] ./O,,~ ' ~
',
YA
18-OH-fiA3g
~ H O0~
OH
aA2}O2"
Fig. 2. Proposed scheme of GA metabolism in developing barley grain. Dashed lines indicate speculative steps. All GAs shown except for GAg, GA29 and GAs~ have been shown to be endogenous to developing barley grain. For clarity, realized conversions involving more than one step are not shown
[2H][3HlGA12-aldehyde to [2H][3H]GA12 may not have been enzymatic, since aerial oxidation of GA12-aldehyde to G A I / c a n occur (Bearder 1973).
Feeds of [2H][3H]GA9 (6) and [2H][3H] GA5I(7). Although there is no evidence for GA 9(6) or GA51(7) being endogenous to developing barley grain (Gaskin etal. 1983). [2H][3H]GA9 and [ZH,3H]GA51 were metabolized to native GAs. Thus [/H][3H]GA9 was 12flhydroxylated to [2H] [3H] 12]?-hydroxy-GA 9 (16), 2]?- and 12fl-hydroxylated to [2H][3H]12]?-hydroxy-GA51(14), 2]?- and 3]?-hydroxylated to [2H] [3H] GA34 (11), 2]?-, 3]?- and 12]?-hydroxylated to [2H][3H]GA4s (19) and 2]?-, 3]?- and 18-hydroxylated to [2H][3H]18-hydroxy-GA34(21) (Table 3). There was no evidence for [2H] [3H] GA 9 being 2]?-hydroxylated to [2H][3H]GAsl(7), or 3]?-hydroxylated to [2H][3H]GA4(8). However, it is possible that [2H] [3H] GA 9 is a precursor of both, but that neither accumulate (see Fig. 2). The very low endogenous level of GA 4 and the apparent
absence of GAs1 (Gaskin et al. 1983) could indicate that these GAs have a high rate of turnover. When [2H][3H]GA9 was fed at 2 gg/grain, [2H][3H]lS-hydroxy-GA 4 (20), and [2HI[all] GA49 (18) were obtained in addition to the metabolites detailed above. Interestingly, unlabelled 12]?-hydroxy-GA4(17 ) was also observed, which would indicate that this compound is endogenous to developing barley grain, although it was n o t detected by Gaskin et al. (1983). The mass spectrum of 12]?-hydroxy-GA4 has been published previously (Gaskin et al. 1984). Gibberellin A49(18 ) may not be a native GA. [2H] [3H] Gibberellin A 51 (7) was 12]?-hydroxylated to [2Hl[3H] 12]?-hydroxy-GA 51 (14), 3]?-hydroxylated to [2H][3H]GA34(ll) and 3]?- and 12]?hydroxylated to [2H] [all] GA4s (19).
Feed of [13C, 3H]GA4 (8). From Table 3 it is apparent that [13C, 3 H ] G A 4 ( 8 ) w a s 2fl-hydroxylated to [13C,3H]GA34(11), 12fl-hydroxylated to [13C, 3HI 12]?-hydroxy-GA4 (17), 18-hydroxylated
s.J. Gilmour et al. : Gibberellinmetabolism in immature barley grain to [13C,3H]18-hydroxy-GA4(20) and hydroxylated at combinations of these positions to [13C, all] GA4s(19 ), [13C, 3H]lS-hydroxy-GA34(21 ) and [13C, 3H]18-hydroxy-GA4s(22). Speculative routes to these GAs are shown in Fig. 2. Two products which have not been shown to be endogenous to barley (Gaskin et al. 1983), i.e. GA/(23) and GA49(18), were also identified from the feed of [13C,3H]GA4 to developing barley grain (Table 3). [13C, 3H]Gibberellin A a may be an artefact as it is known to be produced from GA 4 by dilute mineral acid (Grove 1961). [13C, 3H]Gibberellin A49 may have arisen as a result of substrate overloading as [2H][3H]GA49 was also produced from the feed of 2 gg/grain of [2HI [3H] G A 9 (Table 3).
Feeds of [2H][3H]GA1 (12) and [13C,3H] GA2o(9 ). From the feeds of [2H][3H]GAI(12) and [13C, 3H]GA2o(9) it is apparent that GA2o is converted to GAs(13 ) via GA 1 (Fig. 2). [13C, 3H]Gibberellin A20 is also converted to [13C, 3H]GA29(10) (see Fig. 2), but this may not reflect the normal in-vivo situation, as GA29 has not been shown to be a native barley G A (Gaskin et al. 1983). Discussion
It should be stressed that the results shown in Table 3 were obtained from feeds of doubly labelled GAs, fed at experimentally convenient stages of development of the barley grain, at levels which greatly exceeded the endogenous. Nevertheless, the substrates were predominantly metabolized to native GAs (Table 3). Of the substrates fed, [2H][3H]GA12-aldehyde, [2H][3H]GA9 and [ZH][3H]GAsl have not been identified as native to barley, although they were metabolized to native GAs (Table 3). In these circumstances the schematic summary of the results shown in Fig. 2 can only be regarded as a working hypothesis for future metabolic work. [2HJ [3H] Gibberellin A 12-aldehyde was metabolized to di- and tri-hydroxylated C19GAs. The only C2oGAs to accumulate were GA12 , which may arise non-enzymatically, and 16,17-dihydroxy-GAlz-aldehyde, which may be an artefact of substrate overloading. Hydroxylation at the 13position possibly occurs early in the pathway as [2H][3H]GA1 was formed from [2H][3H]GAlzaldehyde, but not from the C 19 GAs, [2H][3H]GA9 or [13C,3H]GA4. In addition, 13hydroxylated C20GAs are present in developing (GA17) and germinating (GA17 and GA19 ) barley
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grain (Gaskin et al. 1983). An early 13-hydroxylation pathway appears to be of widespread occurrence in plants (see Sponsel 1983 for examples). The non-hydroxylated C19GA , GA9, was extensively hydroxylated at the 2/?-, 3/?-, 12/?- and 18-positions. Likewise GA 4 was hydroxylated at the 2/?-, 12/?- and 18-positions. Whether GA 9 is a precursor of GA 4 in barley has not been established. [2H] [3H] Gibberellin A 4 did not accumulate in feeds of [2H][3H]GA9, although other 3-hydroxylated metabolites were observed. As the level of native GA 4 is very low, the failure to detect [2H][3H]GA4 in feeds of [2H][3H]GA9 may be caused by its very rapid turnover. The order and importance of the multiple hydroxylation of C19 GAs in barley is difficult to establish. Some key intermediates were not available for feeding, and several metabolites remain unidentified. However some patterns of GA metabolism in barley are apparent. For instance, in the present feeds no 13-hydroxylated GAs were shown to be 12/?- or 18-hydroxylated. Furthermore, 18-hydroxylation only occurred on those GAs which were already 3-hydroxylated. Whether these results are caused by our inability to recognise and identify new metabolites, or whether they reflect a measure of substrate selectivity by the enzymes involved remains to be established. On the other hand, 2/?-hydroxylated GAs, which are biologically inactive and which are not usually further hydroxylated in other plants, do appear to be extensively metabolized in barley. Whether this reflects normal metabolism or merely the low substrate selectivity of barley hydroxylases remains to be investigated. The presence of metabolites which are not native to barley is probably a result of feeding excessive levels of labelled substrates. Labelled GA49 , which was observed in feeds of [2H] [3H] GA 9 and [13C, 3H]GA4 is 120~-hydroxylated whereas the 12/?-epimer, GA4s , is native. Similarly, Railton et al. (1974), Frydman and MacMillan (1975) and Sponsel and MacMillan (1977) observed the nonnative 12~-hydroxy-GA 9 (H2-GA 31) (15) (Gaskin et al. 1984) in high-dose feeds of [3H]GA9 to Pisum sativum seedlings and seeds. Substrate overloading probably also results in conjugation of excess substrate and extraneous metabolites. However, the substantial radioactivity observed in acidic butanol fractions of grain extracts (Table 2) was not further analysed. An attempt to identify metabolites after a feed of 5 ng/grain [13C, all] GA4, which more nearly reflects the endogenous level (Gilmour 1983), was unsuccessful because of excessive dilution of polar
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S.J. Gilmour et al. : Gibberellin metabolism in immature barley grain
products by endogenous GAs. Several approaches are envisaged in order to overcome this problem. The use of inhibitors of GA biosynthesis or of a GA-less barley mutant would reduce background GA levels, thereby allowing the identification of metabolites in feeds conducted at physiological concentrations. Alternatively, the use of cell-free enzyme preparations, from which the endogenous GAs can be removed, would allow the sequence of hydroxylation of C 1 9 G A s to be studied more precisely. We would like to thank the Agricultural Research Council for financial support for this work. We are also grateful to the following for synthesis of labelled gibberellins: Dr. M. Beale, Dr. J. Bearder, Mr. M. Lee, Dr. C. Spray and Dr. C. Willis. In addition we would like to thank Dr. L. Rappaport, University of California, Davis, USA for the gift of [3H]GA1.
References Beale, M.H., MacMillan, J. (1981) Partial syntheses of [2c~-ZH]and [2a-3H]-gibberellin Az9 and [2e-2H, 15,17-3H]gibberellin A51 from gibberellin A 3. J. Chem. Soc. Perkin Trans. 1,394-400 Beale, M.H., Gaskin, P., Kirkwood, P.S., MacMillan, J. (1980) Partial synthesis of gibberellin A 9 and [3~- and 3fl-ZH1]gibberellin A9; gibberellin A 5 and [lfl, 3-all2 and -3Hz]gibberellin As; and gibberellin A2o and [lfl, 3c~-2H2 and -3H2] gibberellin Azo. J. Chem. Soc. 885-890 Bearder, J.R. (1973) Diterpenoid biosynthesis in mutants of Fusarium moniliforme. Ph.D. thesis, University of Bristol Bearder, J.R., Frydman, V.M., Gaskin, P., MacMillan, J., Wels, C.M., Phinney, B.O. (1976a) Fungal products. Part XVI. Conversion of isosteviol and steviol acetate into gibberellin analogues by mutant B1-41 a of Gibberellafujikuroi and preparation of [3H]-gibberellin Azo. J. Chem. Soc. Perkin Trans. 1, 173-178 Bearder, J.R., Frydman, V.M., Gaskin, P., Hatton, I.K., Harvey, W.E., MacMillan, J., Phinney, B.O. (1976b) Fungal products. Part XVII. Microbiological hydroxylation of gibberellin A 9 and its methyl ester. J. Chem. Soc. 178-183 Bearder, J.R., MacMillan, J., Phinney, B.O. (1973) 3-Hydroxylation of gibberellin A12-aldehyde in Gibberella fujikuroi strain REC-193A. Phytochemistry 12, 2173-2179 Down, G.J., Lee, M., MacMillan, J., Staples, K.S. (1983) Preparation of 13-hydroxygibberellin Axz-7-aldehyde. J. Chem. Soc. 1103 1108 Frydman, V.M., MacMillan, J. (1975) The metabolism of gibberellins Ag, A2o and A29 in immature seeds of Pisum satirum cv. Progress No. 9. Planta 125, 181-195 Gaskin, P., Gilmour, S.J., Lenton, J.R., MacMillan, J., Sponsel, V.M. (1983) Endogenous gibberellins and kauranoids identified from developing and germinating barley grain. J. Plant Growth Regul. 2, 229-242 Gaskin, P., Hutchison, M., Lewis, N., MacMillan, J., Phinney, B.O. (1984) Microbial conversion of 12-oxygenated and other derivatives ofent-kaur-16-en-19-oic acid by Gibberella fujikuroi, mutant B1-41a. Phytochemistry 559-564 Gilmour, S.J. (1983) Some studies on the endogenous gibberel-
lins of barley (Hordeum vulgare). Ph.D. thesis, University of Bristol Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974a) Biosynthesis of gibberellins A12, A15, A24, A36 and A37 by a cell-free system from Cucurbita maxima. Phytochemistry 13, 1433-1440 Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J. (1974b) The biosynthesis of a C19-gibberellin from mevalonic acid in a cell-free system from a higher plant. Planta 120, 307-309 Graebe, J.E., Hedden, P., Rademacher, W. (1980) Gibberellin biosynthesis. In: Gibberellins chemistry, physiology and use. British Plant Growth Regulator Group, Monograph 5, pp. 31-47, Lenton, J.R., ed. BPGRG, Wantage Grove, J.F. (1961) Gibberellin A z, J. Chem. Soc. 3545-3547 Hedden, P., Graebe, J.E., Beale, M.H., Gaskin, P., MacMillan, J. (1984) The biosynthesis of 12cr gibberellins in a cell-free system from Cucurbita maxima endosperm. Phytochemistry 569-574 Ingrain, T.J., Reid, J.B., Muffet, I.C., Gaskin, P., Willis, C.L., MacMillan, J. (1984) Internode length in Pisum. The Le gene controls the 3fl-hydroxylation of gibberellin A2o to gibberellin A 1. Planta 160, 455-463 Kamiya, Y., Graebe, J.E. (1983) The biosynthesis of all major pea gibberellins in a cell-free system from Pisum sativum. Phytochemistry 22, 681-689 Kovats, E. (1958) Gas-chromatographische characterisierung organischer verbindungen, Tell 1: Retentions Indices aliphatischer Halogenide, Alkohole, Aldehyde und Ketone. Helv. Chim. Acta 41, 1915-1932 Musgrave, A., Kays, S.E., Kende, H. (1972) Uptake and metabolism of radioactive gibberellins by barley aleurone layers. Planta 102, 1 10 Nadeau, R., Rappaport, L. (1974) The synthesis of [3H]gibberellin A 3 and [3H] gibberellin A 1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A 3. Phytochemistry 13, 1537-1545 Nadeau, R., Rappaport, L., Stolp, C.L. (1972) Uptake and metabolism of 3H-gibberellin A 1 by barley aleurone layers : response to abscisic acid. Planta 107, 315-324 Railton, I.D., Durley, R.C., Pharis, R.P. (1974) Metabolism of tritiated gibberellin A 9 by shoots of dark-grown dwarf pea, cv. Meteor. Plant Physiol. 54, 6-12 Sponsel, V.M. (1983) In vivo gibberellin metabolism in higher plants. In: Biochemistry and physiology of gibberellins, vol. 1, pp 151-250, Crozier, A., ed. Praeger, New York Sponsel, V.M., MacMillan, J. (1977) Further studies on the metabolism of gibberellins (GAs) A 9, A/o and A29 in immature seeds of Pisum sativum cv. Progress No. 9. Planta 135, 129-136 Sponsel, V.M., MacMillan, J. (1978) Metabolism of gibberellin Az9 in seeds of Pisum sativum cv. Progress No. 9; use of [2H] and [3H] GAs, and the identification of a new GA catabolite. Planta 144, 69-78 Sponsel, V.M., MacMillan, J. (1980) Metabolism of [13C~]gibberellin A29 to [~3C~]gibberellin-catabolite in maturing seeds of Pisum sativum cv. Progress No. 9. Planta 150, 46-52 Stolp, C.V. Nadeau, R., Rappaport, L. (1973) Effect of abscisic acid on uptake and metabolism of 3H-gibberellin A 1 and 3H-pseudo-gibberellin A 1 by barley half-seeds. Plant Physiol. 52, 546-548 Received 10 January; accepted 2 February 1984
