Thus, Radley (19) couldnot show incor- poration of ['4C]mevalonic acid into GAs in whole barley grains. Murphy and Briggs (12), using both whole grains and.
Received for publication January 11, 1991 Accepted April 3, 1991
Plant Physiol. (1991) 96, 1099-1104 0032-0889/91/96/1 099/06/$01 .00/0
ent-Kaurene Biosynthesis in Germinating Barley (Hordeum vulgare L., cv Himalaya) Caryopses and Its Relation to a-Amylase Production' Elke GroBelindemann, Jan E. Graebe*, Dietmar Stockl2, and Peter Hedden Pflanzenphysiologisches Institut der Universitat, Untere KarspOle 2, D-3400 Gottingen, Germany (E.G., J.E.G., D.S.) and Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, United Kingdom (P.H.) added GA has been studied extensively and its reproducibility is well established, but the origin of the endogenous GA and its role in a-amylase production have not been defined conclusively despite several attempts. The hypothesis rests on the apparent production of GAlike substances by isolated embryos as shown by bioassay in several experiments (5, 12, 18-20, 24) and the suppression of this production by ent-kaurene biosynthesis inhibitors such as CCC and phosphon D (18, 20, 24). Results from these early experiments on the sites and timing of GA production were conflicting, as were those on the identities and amounts of the GAs formed. Later, Yamada (22) using GC-MS identified GA, and GA3 in shoot tissue of 2-day old germinating Betzes (two-row) barley and Gaskin et al. (6), using the same method, identified GA,, GA3 (with reservation), GA17, GA19, GA20, GA34, GA48, and 18-hydroxy-GA34 in 3-d-old shoots of a different variety (Maris Otter). These experiments established conclusively the presence of GAs in germinating barley, but the amounts formed, the sites of synthesis, and any possible correlation of GA concentrations with a-amylase formation were not investigated. Attempts to demonstrate GA biosynthesis directly have been unsuccessful. Thus, Radley (19) could not show incorporation of ['4C]mevalonic acid into GAs in whole barley grains. Murphy and Briggs (12), using both whole grains and cell-free extracts of the embryos, failed to show incorporation of ['4C]mevalonic acid and ['4C]isopentenyl pyrophosphate into either ent-kaurene or GAs. Since these same authors identified ent-kaurene in mature dry grains and found that its amount decreased during germination, they proposed that ent-kaurene acted as a stored precursor which became converted to GA during germination (13). Although they failed to show the conversion of ent-['4C]kaurene to GAs, they later demonstrated the stepwise conversion of ent-kaurenol to ent7a-hydroxykaurenoic acid in a microsomal preparation of embryo extracts, thus lending some support to their hypothesis (14). Atzorn and Weiler (1), on the basis of immunoassay measurements of GAs in germinating barley caryopses, concluded that GA, production in the embryo was not involved in aamylase induction, which latter was dependent on the biosynthesis of GA4 in the aleurone layers. However, their results were not reproducible (8) and can, therefore, no longer be considered conclusive.
ABSTRACT ent-Kaurene biosynthesis as a prerequisite for gibberellin (GA) biosynthesis was studied in germinating Hordeum vulgare L., cv Himalaya caryopses and correlated, in time, with the appearance of a-amylase activity. The rate of ent-kaurene biosynthesis was estimated by inhibiting its further metabolism with plant growth retardants (triapenthenol or tetcyclacis) and measuring its accumulation by isotope dilution using combined gas chromatographymass spectrometry. In the inhibitor-treated caryopses, ent-kaurene accumulation began approximately 24 hours after imbibition and proceeded at a rate of about I to 2 picomoles per hour per caryopsis, depending on the batch of seeds. In the absence of inhibitor, ent-kaurene did not accumulate, indicating that it is normally turned over rapidly, presumably to further intermediates of the GA biosynthesis pathway and eventually to GAs. entKaurene accumulation occurred almost exclusively in the shoot, which is, therefore, probably the site of biosynthesis. a-Amylase production began between 30 and 36 hours after imbibition and, thus, correlated well with de novo GA biosynthesis, as estimated from ent-kaurene accumulation. However, inhibition of ent-kaurene oxidation by plant growth retardants did not reduce the aamylase production significantly, although it did reduce shoot elongation. We conclude that ent-kaurene is produced in the shoot and is continuously converted to GA, which is essential for normal shoot elongation, but not for the production of a-amylase in the aleurone layer.
Paleg (15) and Yomo (23) discovered independently that GA3 could substitute for the embryo in initiating a-amylase formation in germinating barley grains. This finding and the demonstration by Chrispeels and Varner (4) that isolated aleurone layers produced a-amylase in response to added GA3 led to the well-known hypothesis that GA3 produced in the embryo diffuses to the aleurone tissue where it induces the formation of a-amylase and other hydrolytic enzymes. The induction of a-amylase in the aleurone layer in response to XSupported by grants from the Deutsche Forschungsgemeinschaft. 2Present address: Instand e.V., Johannes-Weyer-Str. 1, D-4000 Dusseldorf 1, FRG. 3Abbreviation: GA, gibberellin.
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Gilmour et al. (7) showed the conversion of GA12-aldehyde to several GAs, including GA,, in immature, developing
barley grains. Theoretically, GAs biosynthesized during grain development could be stored in the mature grain, either free or conjugated, causing a-amylase induction during germination. However, dry, mature barley embryos generally are reported to have no GA-like activity and no evidence for the release of GAs from conjugates has been found in barley caryopses (1, 24). We have initiated a new investigation of GA biosynthesis in germinating barley (Hordeum vulgare L., cv Himalaya) grains. In this paper we describe studies on the biosynthesis of ent-kaurene, an important GA precursor, in vivo by using a previously unreported method. The oxidation of ent-kaurene is prevented by imbibition with solutions of plant growth retardants which inhibit the steps between ent-kaurene and ent-kaurenoic acid (17; JE Graebe, K Lurssen unpublished results). As a result, ent-kaurene accumulates, presumably at the rate of its biosynthesis, and it can be quantified by isotope dilution as determined using GC-selected ion monitoring. In this way we demonstrate that the onset of ent-kaurene biosynthesis precedes a-amylase production in the germinating caryopses. However, inhibition of ent-kaurene metabolism does not affect a-amylase production which, therefore, appears to be independent of de novo GA biosynthesis. MATERIALS AND METHODS Labeled ent-Kaurene ent-['4C]Kaurene (7.6 TBq. mol-') was prepared from R-2['4C]mevalonic acid (1.96 TBq.mol-'; Amersham-Buchler, Braunschweig, FRG) using a cell-free system from Cucurbita maxima L. endosperm as described by Graebe et al. (9) with the modifications specified by Turnbull et al. (21) and with the addition of 10 nM (S)-triapenthenol to prevent ent-kaurene oxidation by the enzyme system. The final product was purified by HPLC and identified by GC-MS. Plant Growth Retardants
(S)-Triapenthenol [(S)-l-cyclohexyl-4,4-dimethyl-2-(1,2,4 -triazol-1-yl)-1-penten-3-ol] was a gift from Dr. K. Lurssen, Bayer AG, Leverkusen, FRG. Tetcyclacis[5-(4-chlorophenyl)3,4,5,9,10-pentaazatetracyclo-5,4, 102,6,08,1"-dodeca-3,9diene] was a gift from Dr. W. Rademacher, BASF, Limbur-
gerhof, FRG. Concentrated solutions of the plant growth retardants in methanol were added under vigorous stirring to 99 volumes of water to give the appropriate retardant concentrations in 1% (v/v) methanol. Plant Material
Barley grains (Hordeum vulgare L., cv Himalaya) were a gift from Prof. R. L. Jones, Berkeley, CA (1982 harvest) or were purchased from the Crops and Soils Club, Department of Agronomy and Soils, Washington State University, Pullman, WA (1985 harvest). The grains were soaked in 1% (v/ v) sodium hypochlorite solution for 5 min, rinsed thoroughly with distilled water, and imbibed in covered Petri dishes (9 cm diameter) on filter paper soaked with 1% aqueous meth-
Plant Physiol. Vol. 96, 1991
anol or plant growth retardant solution. After 24 h, batches of 50 germinating caryopses were transferred to 400 mL beakers containing 20 g of vermiculite moistened with 1% methanol or growth retardant solution. Water and growth retardants were not replenished for the duration of the experiments. The beakers were covered with transparent plastic wrap film ("Julia") in which holes were punched for air exchange. Both imbibition and germination occurred under a 14 h photoperiod at 22°C during the day and 18°C during the night. Light was supplied by "Osram" L65 W/25 whiteuniversal tubes, located 80 cm above the plants, giving a photon fluence rate of 162 Mmol* m2 s-'. At the time of sampling, the plants were rinsed, weighed, measured, immediately frozen in liquid N2, and stored at -30°C until analyzed for ent-kaurene contents. Aleurone layers were prepared by the method of Chrispeels and Varner (4).
Extraction and Purification of ent-Kaurene Extracts were prepared from mature seeds, germinating seeds, isolated aleurone layers, or parts of seedlings (100 in each case) by homogenizing the frozen material in four volumes (w/v) cold 80% aqueous methanol in a cold (-20°C) Waring Blendor. Immediately after homogenization, 740 to 3700 Bq ent-['4C]kaurene was added and the extract was then stirred at 4°C. After 2 h the mixture was centrifuged at 3000 rpm for 10 min, and the pellet was resuspended in the initial volume of cold 80% methanol. Centrifugation and resuspension were repeated after a further 2 and 18 h, after which the pellet was discarded. NaCl (5 g) was added to the combined methanol supernatants and ent-kaurene was extracted with three volumes petroleum ether (boiling range 60-80°C). The methanolic phase was decanted from undissolved NaCl and discarded. The remaining NaCl was dissolved in as little water as possible and this solution was extracted with an equal volume of petroleum ether. The pooled petroleum ether extracts were concentrated to 5 mL at 4°C (the cold preventing evaporation of ent-kaurene) and applied to a Sep-Pak silica cartridge (Waters, Milford, MA), which was then washed with 5 mL petroleum ether. The combined eluate and washing were taken to dryness, dissolved in 5 mL petroleum ether and chromatographed again in the same way. Chromatography over two cartridges was necessary to remove fatty acids, which otherwise plugged the HPLC pre-column. The combined eluate and washing were evaporated to dryness in a gentle stream of N2 at 4°C.
HPLC The chromatographic apparatus, consisting of a pre-column packed with Polygosil 60-10 C18, a Radial-Pak main column (15 cm long, 8 mm diameter) packed with Novapak C18 and a gradient elution system with an on-line radioactivity detector has been described in detail (10). The dried samples were dissolved in 100 ,uL methanol, and the solutions were injected via a Rheodyne sample injector (100 ,uL loop) onto the column system, which had been equilibrated with 75% (v/v) methanol in 10 mm aqueous acetic acid. Samples were eluted by a 5-min linear gradient from 75 to 100% (v/v) methanol in 10 mm acetic acid followed by 23 min isocratic elution
ent-KAURENE BIOSYNTHESIS IN GERMINATING BARLEY
with methanol at 1 mL-min-'. The retention time of entkaurene was 24 min, the overall recovery was about 70%.
GC-MS
GC-MS was performed using a Finnigan (Munich, FRG) model 4015 GC-MS with data system. The samples were dissolved in 1 IAL petroleum ether and injected via a Grob split-injector (250TC, split 50:1, opened after 1 min) into a Macherey and Nagel (Duren, FRG) fused silica SE-30 capillary column (25 m x 0.32 mm x 0.25 gm film thickness). The He flow rate was 2 mL * min-'. The column temperature was maintained at 9O'C for 3 min, then programmed at 10Cmin-' to 250C and maintained at 250C for 10 min. The ion source temperature was 290C, the electron energy was 70 eV and the emission current was 0.25 mA. Spectra were acquired from 200C. The retention time of ent-kaurene was 3 min after reaching 200C. The curve shown in Figure IC was obtained by the use of a Hewlett Packard 5890 gas chromatograph (Hewlett Packard Ltd., Winnersh, Berks., UK) coupled to a 5970 Mass Selective Detector. The samples (1 gL) were injected into a fused silica OV 1701 capillary column (25 m x 0.2 mm x 0.25 ,m film thickness; Thames Chromatography, Maidenhead, UK) at an He inlet pressure of 0.09 MPa and an oven temperature of 60C with the injector split valve closed. After 0.5 min the split (50:1) was opened and after 1 min the oven temperature was increased at 20C min-' to 200C and then at 40C- min-' to 27O'C. The injector and interface temperatures were 220C and 270C, respectively. The change in instrumentation became necessary because the Finnigan GC-MS ceased functioning. However, the internal standardization ensured fully comparable results with the two instruments. The ['4C]-isotope content was calculated from the relative intensities of the signals in the molecular ion cluster, i.e. at m/z 272, 274, 276, 278, and 280 for M+, [M + 2]+, [M + 4]+, [M + 6]+, and [M + 8]+, respectively, using the formula given in ref. 2. The relative abundance of the isotope peaks (m/z 274-280) in the standard ent-[14C] kaurene reflect the proportions of ent-kaurene molecules containing one, two, three, or four '4C-atoms, respectively, originating from [2-'4C]mevalonic acid. Upon diluting with a nonradioactive sample, the intensity of the '2C-molecular ion (m/z 272) increases relative to the isotope peaks. With knowledge of the original specific activity, the new specific activity, and the amount of endogenous ent-['4C]kaurene standard added, the amount of endogenous ent-kaurene is easily calculated. By this method, less than 5 ng of ent-kaurene can be detected, 10 ng can be reasonably quantified, and 15 ng can be quantified with precision. With a sample size of 100 and a recovery rate of 70%, these amounts correspond to 0.3, 0.5, and 0.8 pmol/caryopsis, respectively. a-Amylase Assay Batches of 50 seedlings were removed from the vermiculite, frozen in liquid N2, and ground to a fine powder in a cold (-20'C) Waring Blendor. The powder was stirred with 6 mL 1 mm sodium acetate buffer (pH 4.8) containing 10 mm CaCl2.
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After centrifugation at 3000 rpm for 10 min, the supernatants were heated to 60'C for 0.5 h to inactivate f3-amylase and centrifuged again. These supernatants were used for the determination of a-amylase activity according to Chrispeels and Varner (4). RESULTS ent-Kaurene Accumulation
Isotope dilution analysis by GC-MS showed that dry mature caryopses contained only 0.4 pmol ent-kaurene per caryopsis and that the contents remained low (average 1.2 pmol entkaurene per caryopsis) for at least 6 d after imbibition in water (Fig. 1A, untreated control). In contrast, when the grains were imbibed with an aqueous solution of 100 uM triapenthenol, ent-kaurene began accumulating between 24 and 48
Figure 1. ent-Kaurene accumulation in germinating barley caryopses and the emerging seedlings as the result of treatment with the plant growth regulators (S)triapenthenol or tetcyclacis. A and B, Two different experiments with caryopses of the 1982 harvest; C, caryopses of the 1985 harvest: 100 gM triapenthenol (-); 10 gM tetcyclacis (A); 100 gM tetcyclacis (A); untreated control (0). D, entKaurene levels in different parts of the caryopses and seedlings (1982 harvest) grown in the presence of 100 AM triapenthenol: roots (); endosperm and aleurone (A); shoots (0).
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Table 1. ent-Kaurene Levels in the Alwrone Layers of Barley Caryopses Germinating in the Presence or Absence of 100 Mm (S)-Triapenthenol Age Control (S)-Tnapenthenol d pmol/caryopsis 1 0.41 0.46 2 0.11 0.23 3 0.07 0.19
h after imbibition, the accumulation reaching a rate of approximately 1 pmol h-' during two 24-h periods separated by one 24-h period (72-96 h) in which no accumulation took place (Fig. IA). Since triapenthenol inhibits ent-kaurene oxidation (JE Graebe, K Lurssen unpublished), we assume that the accumulation is a measure of ent-kaurene biosynthesis. The low amounts of ent-kaurene in the control indicate that this hydrocarbon intermediate is normally turned over rapidly. Figure lB compares the ent-kaurene accumulation obtained with two different growth retardants, triapenthenol, and tetcyclacis. The curves obtained with 100 Mm triapenthenol and 10 ,uM tetcyclacis are both similar to the curve in Figure IA, which was likewise obtained with 100 MM triapenthenol; in all three cases, ent-kaurene accumulation began after 24 h and there was a break between 72 and 96 h. The curve obtained with a 10-fold higher concentration of tetcyclacis (100 MM, Figure IB) had a different shape and showed no break in the accumulation between 72 and 96 h. Nevertheless, over a 6-d period there was as much ent-kaurene produced as with the lower concentration. The reason for the lower total accumulation of ent-kaurene in the experiment in Figure lB as compared to the one in Figure IA is unknown. Figure IC shows the accumulation of ent-kaurene in caryopses of the same cultivar but of a different harvest (1985). As in the previous experiments, ent-kaurene accumulation began 24 h after imbibition in the presence of the plant growth retardant, but there was no break between 72 and 96 h and the accumulation proceeded at an average rate of 2.3 pmolh-', which is more than twice the rate found in the caryopses of the 1982 harvest. As in previous experiments, there was no accumulation of ent-kaurene in the absence of inhibitor (Fig. IC). Also in caryopses of the 1985 harvest, treatment with 100 MM tetcyclacis inhibited growth completely without affecting total ent-kaurene production, the accumulation being the same as with 10 MM tetcyclacis (data not shown). Figure ID shows that the shoot was the major site of entkaurene accumulation (40 pmol per shoot) in the presence of 100 MM triapenthenol. The combined endosperm-aleurone contained very much less ent-kaurene (average 3.3 pmol per caryopsis) and traces only were found in the roots (1 pmol per caryopsis). The break in ent-kaurene accumulation between 72 and 96 h, noted for the whole seedling was also evident in the shoot. Finally, aleurone layers were separated from treated and untreated caryopses at different times and analyzed for entkaurene (Table I). The aleurone layers contained small amounts of ent-kaurene, but they did not accumulate this compound for the duration of the experiment. Furthermore,
when embryoless half-seeds were incubated with and without 100 AM triapenthenol for 3 d, the isolated aleurone layers contained 0.53 pmol ent-kaurene per aleurone in both cases. These results indicate that, within the first 3 d ofgermination, ent-kaurene is neither imported from the embryo into the aleurone tissue nor biosynthesized in this tissue. With respect to germination, there were no visible effects of 100 jAM triapenthenol and 10 MM tetcyclacis during the first 48 h, radicles and plumules emerging at the same time in treated and untreated caryopses. The differences, however, became obvious as soon as the control seedlings started elongating 48 h after imbibition, while growth in the treated seedlings was delayed by approximately 24 h and resulted in 60 to 70% shorter shoots after 144 h (Fig. 2, A and B). It is noteworthy that control seedlings started elongating at a time when ent-kaurene synthesis and turnover were well advanced (as evidenced by the accumulation in the inhibited seedlings), whereas the treated seedlings commenced growing coincident with the break in ent-kaurene production (cf. Fig. 1, A and B). Treatment with 100 AM tetcyclacis inhibited growth completely, although neither the production of ent-kaurene (Fig. lB) nor, as is shown later, the production of a-amylase was impeded by this concentration. In spite of their higher rate of ent-kaurene production, the seedlings of the 1985 harvest had a lower growth rate than those harvested in 1982 (Fig. 2B). Therefore, it appears that the rate of ent-kaurene biosynthesis is not a limiting factor for growth. Since the ent-kaurene accumulation levels off between 72 and 96 h in rapidly growing shoots only and since this occurs just as the treated seedlings start growing, the retardants may become limiting for a short period at this time. a-Amylase Production The results presented above show that ent-kaurene biosynthesis begins between 24 and 48 h after imbibition and that
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