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. 1999 Nov;121(3):1037–1045. doi: 10.1104/pp.121.3.1037

Gibberellin Biosynthesis in Maize. Metabolic Studies with GA15, GA24, GA25, GA7, and 2,3-Dehydro-GA91

Gordon Davis 1, Masatomo Kobayashi 1, Bernard O Phinney 1,*, Theo Lange 1, Steve J Croker 1, Paul Gaskin 1, Jake MacMillan 1
PMCID: PMC59468  PMID: 10557253

Abstract

[17-14C]-Labeled GA15, GA24, GA25, GA7, and 2,3-dehydro-GA9 were separately injected into normal, dwarf-1 (d1), and dwarf-5 (d5) seedlings of maize (Zea mays L.). Purified radioactive metabolites from the plant tissues were identified by full-scan gas chromatography-mass spectrometry and Kovats retention index data. The metabolites from GA15 were GA44, GA19, GA20, GA113, and GA15-15,16-ene (artifact?). GA24 was metabolized to GA19, GA20, and GA17. The metabolites from GA25 were GA17, GA25 16α,17-H2-17-OH, and HO-GA25 (hydroxyl position not determined). GA7 was metabolized to GA30, GA3, isoGA3 (artifact?), and trace amounts of GA7-diene-diacid (artifact?). 2,3-Dehydro-GA9 was metabolized to GA5, GA7 (trace amounts), 2,3-dehydro-GA10 (artifact?), GA31, and GA62. Our results provide additional in vivo evidence of a metabolic grid in maize (i.e. pathway convergence). The grid connects members of a putative, non-early 3,13-hydroxylation branch pathway to the corresponding members of the previously documented early 13-hydroxylation branch pathway. The inability to detect the sequence GA12 → GA15 → GA24 → GA9 indicates that the non-early 3,13-hydroxylation pathway probably plays a minor role in the origin of bioactive gibberellins in maize.

The biosynthesis of the gibberellins (GAs) has been recently reviewed (MacMillan, 1997). In all systems studied, the pathway has been shown to proceed from the cyclic diterpene ent-kaurene to GA12 aldehyde then to GA12. Depending on the sequence of hydroxylation at the 3β- and 13-positions, parallel pathways branch from GA12 to the C19-GAs, the number of these branch pathways varying from species to species. For maize (Zea mays L.) we previously demonstrated (see Fig. 1) the presence of the early 13-hydroxylation branch pathway, a pathway originating from GA12 and leading to the hydroxylated C19-GAs, GA1, GA3, and GA5 (Kobayashi et al., 1996 and refs. therein; Spray et al., 1996). As shown in Figure 1, the steps from GA12 to bioactive GA1, GA3, and GA5, the early 13-hydroxylation branch pathway, have been established by feeding studies using labeled substrates; the immediate metabolites were identified by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention index (KRI) data (Fujioka et al., 1990; Kobayashi et al., 1996). All members of this branch pathway are native to maize (Fujioka et al., 1988a, 1988b).

Figure 1.

Figure 1

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Maize branch pathways from GA12: right vertical row, the early 13-hydroxylation branch pathway; left vertical row, the presumptive non-early 3,13-hydroxylation branch pathway. All of the GAs are endogenous to maize except 2,3-dehydroGA9, shown in brackets. ➞, Steps established in this paper; →, steps previously established; - - ➛, steps tested, not observed.

There is indirect evidence for the presence of a second pathway from GA12, the non-early 3,13-hydroxylation branch pathway. The pathway originates from GA12 and leads via GA9 to the 3β-hydroxylated C19-GAs GA4, and GA7 (see Fig. 1). While the pathway has been shown to be present in a number of plant species (for review, see MacMillan, 1997), its presence in maize is based solely on the identification from maize of the five members GA15, GA24, GA9, GA4, and GA7. Moreover, in vivo feeding studies have provided no evidence for the metabolism of GA12 to GA15 (Kobayashi et al., 1996), GA9 to GA4 (Davis et al., 1998), and GA4 to GA7 (Kobayashi et al., 1993).

In the present study, we describe the metabolism of [17-14C]GA15, [17-14C]GA24, [17-14C]GA25, and [17-14C]GA7 in seedlings of tall, dwarf-1 (d1), and dwarf-5 (d5) maize. Given the previous demonstration of the sequence GA20 → GA5 (2, 3-dehydro-GA20) → GA3 in maize (Fujioka et al., 1990), the possible existence of a parallel sequence of GA9 → 2,3-dehydro-GA9 → GA7 was tested by feeding 2,3-dehydro-[17-14C]GA9, a GA not reported to be present in maize (Fujioka et al., 1988b). The data obtained, together with the previous results from the metabolism of [17-13C,3H]GA9 and [17-13C,3H]GA4, are discussed in terms of the biosynthesis of GAs in maize.

MATERIALS AND METHODS

Plant Material

Normal (tall), dwarf-1 (d1), and dwarf-5 (d5) maize (Zea mays L.) seedlings came from seed stocks of known genotype (Spray et al., 1996). The seeds were pre-soaked in water for 12 h and planted in vermiculite:soil (1:1). The seedlings were then grown in the greenhouse at the University of California, Los Angeles. Three- to four-week-old seedlings (three- to four-leaf stage) were used for feeds.

Labeled Substrates

[17-14C]GA15 (2.07 TBq mol−1), [17-14C]GA24 (2.07 TBq mol−1), and [17-14C]GA7 (2.07 TBq mol−1) were purchased from Prof. L.N. Mander (Australian National University, Canberra). [17-14C]GA25 (2.07 TBq mol−1) was prepared from [17-14C]GA24 (300 kBq; a gift from Prof. L.N. Mander) with cell lysates (3.5 mL) from Escherichia coli NM522 containing clone E5 by methods detailed by Lange (1997) and purified as described by Lange and Graebe (1993). 2,3-Dehydro-[17-14C]GA9 (1.75 TBq mol−1) was prepared as described in MacMillan et al. (1997).

Treatment, Purification, and Analysis

Each of the five labeled GAs, [17-14C]GA15, [17-14C]GA24, [17-14C]GA25, [17-14C]GA7, and 2,3-dehydro-[17-14C]GA9, was dissolved in 90 μL of ethanol:water (1:1). Two microliters of the [17-14C]GA15 solution (1,570 Bq; 250 ng) were individually injected into the coleoptile nodes of three sets of 10 plants (normal, d1, and d5). Similar injections were made with [17-14C]GA24 (1,490 Bq; 250 ng), [17-14C]GA25 (1,420 Bq; 250 ng), and [17-14C]GA7 (1,550 Bq; 250 ng). One set of 10 d5 seedlings was used for the 2,3-dehydro-[17-14C]GA9 injections (485 Bq; 88 ng).

The seedlings were incubated in the greenhouse for 24 h, harvested as sets of 10, frozen with dry ice, and stored at −80°C. Each set of frozen seedlings was homogenized, extracted, and solvent-fractionated to give an acidic ethyl acetate-soluble (AE) fraction. Each fraction was concentrated and purified using Bond Elut (Varian, Harbor City, CA) columns and two steps of HPLC (Davis et al., 1998). All samples were methylated and the GAs in each sample were identifed by full-scan GC-MS and KRI (Gaskin and MacMillan, 1991; Spray et al., 1996).

Isotopic Dilution

To determine whether 2,3-dehydro-GA9 is endogenous to maize, [17-14C]2,3-dehydro-GA9 (1.75 TBq mol−1) was used in an isotopic dilution experiment. Fifteen nanograms (3 Bq) was dissolved in 100 μL of 50% (v/v) aqueous ethanol and added to a homogenate from 50 normal maize seedlings (200 g fresh weight). The homogenate was extracted immediately and solvent fractionated to give an AE fraction. The fraction was processed for the determination of isotopic dilution using the isotope dilution fit program described by Croker et al. (1994).

RESULTS AND DISCUSSION

Metabolism [17-14C]GA15

The recovered [14C]labeled metabolites GA44, GA19, GA20, GA113, and GA15-15,16-ene (artifact?) are shown in Table I, and are based on identification by the full-scan GC-MS and KRI data presented in Table II. The step from GA15 to GA44 (Fig. 1) is a direct 13-hydroxylation that is new for maize. The observed 13-hydroxylation of GA15 to GA44 in maize (Fig. 1) has also been reported in a cell-free preparation from germinating barley (Grosslindemann et al., 1992). In addition, the opened lactone of GA15 is metabolized to GA44 from in vitro studies using seeds of pea (Kamiya and Graebe, 1983) and bean (Takahashi et al., 1986). The steps GA44 → GA19 and GA19 → GA20 have been previously demonstrated in maize seedlings (Kobayashi et al., 1996). The step from GA15 to GA113 (Fig. 2) is a direct 12α-hydroxylation, which is new for maize and for higher plants. GA113 has not been found to occur naturally in maize but has been recently isolated from the seeds and shoots of the Japanese radish (Nakayama et al., 1998). The relatively high levels of endogenous GA44 and GA19 present in the normal and d1 seedlings compared with the d5 seedlings (Fujioka et al., 1988a) may create feedback inhibition and thus account for the absence of the labeled metabolite GA19, in the normal and d1 seedlings, in contrast to the recovery of [14C]GA19 from d5 seedlings.

Table I.

Analysis of metabolites from feeds of [17-14C]GA15 (250 ng, 1.57 × 103 Bq each) to normal, d1, and d5 seedlings of maize

Plant Material ODS-HPLC Fraction N(CH3)2-HPLC Fraction Radioactivity [14C]Producta
Bq
Normal (12.0 g) 18–21 31–34 128 GA113
22–25 31–34 49 GA44
30–34 26–29 34 GA15-15,16-ene
30–34 30–33 628 GA15 (feed)
d1 (7.2 g) 22–25 31–32 109 GA44
30–34 28–31 452 GA15 (feed)
d5 (7.0 g) 18–21 31–34 228 GA20, GA113
22–25 28–30 102 GA44
22–25 45–47 94 GA19
30–34 28–31 726 GA15 (feed)
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a

Identified by data shown in Table II. 

Table II.

Representative GC-MS and KRI data used for the identification of GA metabolites (listed in Table I) from the feeds of [17-14C]GA15 to maize

[14C]GA Metabolite/Ref. Compound KRIa Diagnostic Ions
[14C]GA15 2,587 m/z 346 314 300 286 241 213 195
intensity 19 19 13 54 100 9 30
GA15 ref. 2,605 m/z 344 312 298 284 239 211 193
intensity 25 27 18 70 100 13 33
[14C]GA15-15,16-ene 2,542 m/z 346 314 288 286 243 229 217 199 185 159
intensity 30 59 53 62 100 42 66 24 28 54
GA15-15,16-ene ref. 2,551 m/z 344 312 286 284 243 227 217 197 183 159
intensity 18 60 39 69 100 30 36 19 23 41
[14C]GA19 2,584 m/z 464 436 404 376 347 317 287 258 241 210
intensity 17 100 24 52 18 18 23 28 46 40
GA19 ref. 2,596 m/z 462 434 402 374 345 315 285 258 239 208
intensity 4 100 7 4 24 5 21 30 33 32
[14C]GA20 2,473 m/z 420 405 377 303 237 209 207
intensity 100 3 47 19 15 52 58
GA20 ref. 2,482 m/z 418 403 375 301 235 207
intensity 100 16 46 12 8 30
[14C]GA44 2,768 m/z 434 419 375 240 209 182
intensity 60 7 11 32 100 12
GA44 ref. 2,786 m/z 432 417 373 238 207 180
intensity 46 6 14 33 100 11
[14C]GA113 2,801 m/z 434 402 374 312 298 284 239 227
intensity 75 26 18 49 35 86 100 30
GA113 ref. 2,801 m/z 432 400 372 310 296 282 237 225
intensity 100 31 27 56 36 83 82 38
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a

The discrepancies between the KRI values for the metabolites and for the standards (ref.) are due to batch-to-batch variations in the GC columns used. 

Figure 2.

Figure 2

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Structures of metabolites not shown in Figure 1.

[17-14C]GA24

The recovered [14C]labeled metabolites, GA19, GA20, and GA17 are shown in Table III, and are based on identification by the full-scan GC-MS and KRI data presented in Table IV. The step from GA24 to GA19 (Fig. 1) is a direct 13-hydroxylation and is new for maize seedlings. The step from GA19 to GA20 has been previously established for maize (Kobayashi et al., 1996) with no evidence for the conversion of GA19 to GA17. However, the conversion of GA19 to GA17 has been demonstrated using GA 20-oxidases from spinach (Wu et al., 1996) and pumpkin (Lange et al., 1994), which have been cloned and expressed in E. coli.

Table III.

Analysis of metabolites from feeds of [17-14C]GA24 (250 ng, 1.49 × 103 Bq each) to normal, d1, and d5 seedlings of maize

Plant Material ODS-HPLC Fraction N(CH3)2-HPLC Fraction Radioactivity [14C]Productsa
Bq
Normal (20.8 g) 19–21 33–36 117 GA20
22–23 42–45 4,290 GA19
29–30 40–43 663 GA24 (feed)
d1 (9.5 g) 19–21 33–36 452 GA20
29–30 40–43 852 GA24 (feed)
d5 (10.8 g) 19–21 33–36 710 GA20
24–25 33–35 218 GA17
29–30 40–43 347 GA24 (feed)
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a

Identified by data shown in Table IV. 

Table IV.

Representative GC-MS and KRI data used for the identification of GA metabolites (listed in Table III) from the feeds of [17-14C]GA24 to maize

[14C]GA Metabolite/Ref. Compound KRIa Diagnostic Ions
[14C]GA17 2,563 m/z 494 462 435 434 403 375 374 253 210 195
intensity 64 37 28 37 19 26 23 27 100 21
GA17 ref. 2,575 m/z 492 460 433 432 401 373 372 251 208 193
intensity 43 23 26 15 11 23 14 24 100 22
[14C]GA19 2,584 m/z 464 436 404 376 347 317 287 258 241 210
intensity 17 100 24 52 18 18 23 28 46 40
GA19 ref. 2,596 m/z 462 434 402 374 345 315 285 258 239 208
intensity 4 100 7 4 24 5 21 30 33 32
[14C]GA20 2,473 m/z 420 405 377 361 303 237 209 194 182 169
intensity 100 12 50 14 14 6 32 8 8 8
GA20 ref. 2,482 m/z 418 403 375 359 301 235 207 192 180 167
intensity 100 16 6 2 2 8 30 8 6 7
[14C]GA24 2,426 m/z 376 348 344 316 312 288 287 284 256 229 228 227
intensity 3 8 33 91 42 72 50 34 58 58 83 100
GA24 ref. 2,442 m/z 374 346 342 314 310 286 285 282 254 227 226 225
intensity 4 8 30 80 26 79 72 42 29 70 100 78
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a

The discrepancies between the KRI values for the metabolites and for the standards (ref.) are due to batch-to-batch variations in the GC columns used. 

[17-14C]GA25

The recovered [14C]labeled metabolites GA17, GA25 16α,17-H2-17-OH, and HO-GA25 (hydroxyl position not determined) are shown in Table V, based on identification by the full-scan GC-MS and KRI data presented in Table VI. The metabolism of GA25 to GA17 (Fig. 2) is a result of direct 13-hydroxylation. This step is new for plants.

Table V.

Analysis of metabolites from feeds of [17-14C]GA25 (250 ng, 1.42 × 103 Bq each) to normal, d1, and d5 seedlings of maize

Plant Material ODS-HPLC Fraction N(CH3)2-HPLC Fraction Radioactivity [14C]Producta
Bq
Normal (17.1 g) 19–21 23–25 88 GA25 16α, 17-H2 17-OH
19–21 26–28 101 HO-GA25, unknown position of hydroxyl
23–25 27–29 412 GA17
29–31 26–28 498 GA25 (feed)
d1 (6.0 g) 19–21 26–28 35 HO-GA25, unknown position of hydroxyl
23–25 27–29 560 GA17
29–31 26–28 365 GA25 (feed)
d5 (5.2 g) 19–21 23–25 41 GA25 16α, 17-H2 17-OH
19–21 26–28 78 HO-GA25, unknown position of hydroxyl
23–25 27–29 563 GA17
29–31 26–28 595 GA25 (feed)
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a

Identified by data shown in Table VI. 

Table VI.

Representative GC-MS and KRI data used for the identification of GA metabolites (listed in Table V) from the feeds of [17-14C]GA25 to maize

[14C]GA Metabolite/Ref. Compound KRIa Diagnostic Ions
[14C]GA17 2,539 m/z 494 462 435 434 403 375 374 253 210 195
intensity 77 94 67 94 33 41 41 11 100 11
GA17 ref. 2,575 m/z 492 460 433 432 401 373 372 251 208 193
intensity 43 23 26 15 11 23 14 24 100 22
[14C]GA25 2,455 m/z 406 374 314 255 286 227 199
intensity 0 19 63 9 100 37 6
GA25 ref. 2,440 m/z 404 372 312 253 284 225 197
intensity 0 13 82 8 100 41 4
[14C]HO-GA25, unknown position of hydroxylb 2,667 m/z 494 462 460 434 432 402 400 374 372
intensity 3 77 19 36 14 41 17 100 31
[14C]GA25 16α,17-H2 17-OH 2,738 m/z 496 464 436 404 376 342 314 286 227
intensity 0 69 22 100 66 28 36 57 37
[14C]GA25 16α,17-H2 17-OH ref. 2,760 m/z 494 462 434 402 374 340 312 284 225
intensity 0 86 26 100 63 30 78 97 93
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a

The discrepancies between the KRI and ion abundance values for the metabolites and for the standards (ref.) are due to the change in the GC-MS instrument from a DANI-3800 GC-VG Analytical 70–250 (Micromass, Beverly, MA) mass spectrometer to a Thermoquest GCQ (Thermoquest, San Jose, CA) gas chromatograph with a WCOT BPX5 capillary column (25-m × 0.22-mm × 0.25-μm film thickness; Scientific Glass Engineering). 

b

No reference data available; identification by analogy with known HO-GA25 examples. 

[17-14C]GA7

The [14C]labeled metabolites GA30, GA3, isoGA3, and GA7-diene-diacid (trace amounts) are shown in Table VII, and are based on identification by the full-scan GC-MS and KRI data shown in Table VIII. However, in each case, most of the radioactivity was recovered in fractions (Table VII) that contained products not analyzable by GC-MS. The products are presumed to be conjugates.

Table VII.

Analysis of metabolites from feeds of [17-14C]GA7 (250 ng, 1.55 × 103 Bq each) to seedlings of normal, d1, and d5 seedlings of maize

Plant Material ODS-HPLC Fraction N(CH3)2-HPLC Fraction Radioactivity [14C]Productb
Bq
Normal (14.2 g) 8–9 34–35 17a GA30
10–11 33–34 23a GA3, isoGA3
19–22 33–35 18a GA7-diene-diacid
24–26 31–34 216a c
24–26 44–47 265a c
d1 (8.3 g) 8–9 34–35 33 GA30
10–11 33–34 54 GA3, isoGA3
19–22 33–35 24 GA7-diene-diacid
24–26 31–34 432 c
24–26 44–47 723 c
d5 (8.1 g) 8–9 34–35 53 GA30
10–11 33–34 98 GA3, isoGA3
19–22 33–35 15 GA7-diene-diacid
24–26 31–34 470 c
24–26 44–47 393 c
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a

One-half of the original feed. 

b

Identified by data shown in Table VIII. 

c

No 14C-labeled compounds were identified by GC-MS. 

Table VIII.

Representative GC-MS and KRI data used for the identification of GA metabolites (listed in Table VII) from the feeds of [17-14C]GA7 to maize

[14C]GA Metabolite/Ref. Compound KRIa Diagnostic Ions
[14C]GA3 2,685 m/z 506 491 447 433 372 349 313 240 210
intensity 100 6 8 5 12 6 8 12 19
GA3 ref. 2,692 m/z 504 489 445 431 370 347 311 238 208
intensity 100 7 12 9 24 9 14 21 37
iso[14C]GA3 2,625 m/z 506 501 477 447 372 240 223
intensity 100 9 20 17 16 24 22
isoGA3 ref. 2,633 m/z 504 499 475 445 370 238 221
intensity 100 10 12 9 12 28 12
[14C]GA7b (feed) 2,520 m/z 418 386 358 300 284 225 224 195 181 155
intensity 11 12 17 21 25 60 100 28 28 31
GA7 ref.b 2,525 m/z 416 384 356 298 282 223 222 193 179 155
intensity 9 22 22 19 35 73 100 43 42 48
[14C]GA7 di-acid 9,10-ene 2,399 m/z 432 372 313 283 223 195
intensity 14 45 59 100 100 60
GA7 di-acid 9,10-ene ref. 2,405 m/z 430 370 311 281 221 193
intensity 17 23 80 100 77 24
[14C]GA30 2,754 m/z 506 446 416 384 371 315 282 223 195
intensity 11 6 13 18 38 44 34 100 42
GA30 ref. 2,759 m/z 504 444 414 382 369 315 280 221 193
intensity 30 10 26 21 50 17 37 100 47
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a

The discrepancies between the KRI values for the metabolites and for the standards (ref.) are due to batch-to-batch variations in the GC columns used. 

b

Data for [14C]GA7 is reported, although not recovered from feed. 

2,3-Dehydro-[17-14C]GA9

The recovered [14C]labeled metabolites, GA5, GA7 (trace amounts), 2,3 dehydro-GA10 (artifact), GA31, and GA62 are shown in Table IX, based on identification by the full-scan GC-MS and KRI data shown in Table X. Four of the metabolites are formed by hydroxylation at C-1β (GA62, Fig. 2), at C-3β (GA7, Fig. 1), at C-12α (GA31, Fig. 2), and at C-13 (GA5, Fig. 1). 2,3-Dehydro-GA10 (Fig. 2) is the product of hydration of the 16,17-double bond and this step may be non-enzymatic. The metabolism of 2,3-dehydro-[17-2H2]GA9 to [2H2]GA7 has been previously reported from cell-free systems from seeds of wild cucumber and apple (Albone et al., 1990). The metabolism of 2,3-dehydro-GA9 to GA62, to GA31, and to GA5 are the first examples of these conversions in plants.

Table IX.

Analysis of metabolites from feeds of 2,3-dehydro-[17-14C]GA9 (88 ng, 485 Bq each) to d5 maize (10.0 g of plant material)

ODS-HPLC Fraction N(CH3)2-HPLC Fraction Radioactivity [14C]Producta Specific Radioactivity
Bq TBq mol−1
14–15 13 77 GA31 1.81
16–18 9–10 136 2,3-Dehydro-GA10 1.74
16–18 14–15 162 GA5 1.76
19–21 10–13 124 GA62 Not determined
22–24 9–10 14 GA7 (trace) Not determined
26–27 12 381 2,3-Dehydro-GA9 (feed) 1.76
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a

Identified by data shown in Table X. 

Table X.

Representative GC-MS and KRI data used for the identification of GA metabolites (listed in Table IX) from the feeds of 2,3-dehydro-[17-14C]GA9 to d5 maize

[14C]GA Metabolite/Ref. Compound KRIa Diagnostic Ions
[14C]GA5 2,475 m/z 418 403 359 345 315 301 209
intensity 100 17 24 9 31 402 32
GA5 ref. 2,479 m/z 416 401 357 343 313 299 207
intensity 100 21 24 22 9 58 55
[14C]GA7 2,522 m/z 418 386 358 300 284 225 224
intensity 8 46 8 18 20 60 100
GA7 ref. 2,525 m/z 416 384 356 298 282 223 222
intensity 9 14 22 19 35 30 100
2,3-Dehydro-[14C]GA9 2,298 m/z 299 286 284 227 226 156
intensity 7 41 12 51 100 45
2,3-Dehydro-GA9 ref. 2,301 m/z 297 284 282 225 224 156
intensity 10 46 2 62 100 36
2,3-Dehydro-[14C]GA10b 2,563 m/z 420 361 329 286 227 226 143 132
intensity 24 24 46 52 58 79 52 100
[14C]GA31 2,546 m/z 418 386 371 296 284 268 251 241 225 224 223 195
intensity 7 7 10 14 47 33 15 10 58 100 77 44
GA31 ref. 2,550 m/z 416 384 369 294 282 266 269 239 223 222 221 193
intensity 6 8 10 19 49 33 23 7 71 100 72 36
[14C]GA62 2,424 m/z 418 403 374 315 284 225 224 209
intensity 2 7 13 18 36 100 95 26
GA62 ref. 2,424 m/z 416 401 372 313 282 223 222 207
intensity 0 4 8 10 24 100 93 10
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a

The discrepancies between the KRI values for the metabolites and for the standards (ref.) are due to batch-to-batch variations in the GC columns used. 

b

No reference data available; identification by analogy with known GA-15,16-enes. 

Isotopic Dilution of 2,3-Dehydro-GA9

In view of the observed conversion of 2,3-dehydro-GA9 to GA7, we investigated the possible natural occurrence of 2,3-dehydro-GA9 in maize. Thus, we determined the level of isotopic dilution of 2,3-dehydro-[17-14C]GA9 added to a homogenate of normal maize seedlings. No dilution of label was observed based on a full-scan GC-MS analysis of the recovered 2,3-dehydro-[17-14C]GA9 (data not shown), thus indicating that 2,3-dehydro-GA9 is not endogenous to maize.

General

The structures of the substrates and metabolites presented in this report are shown in Figures 1 and 2, with the exception of the HO-GA25 metabolite for which the hydroxylation site was not determined. In maize, the 13-hydroxylation of GA15 to GA44, GA24 to GA19, GA9 to GA20, and GA4 to GA1 results in the formation of a grid connecting members of the (presumptive) non-early 3,13-hydroxylation pathway to members of the early 13-hydroxylation pathway (Fig. 1). The two steps, GA15 → GA44 and GA24 → GA19, represent the first demonstration of in vivo crossovers between C20-GAs. A similar grid connecting the two branch pathways has been demonstrated from in vitro studies from a number of plant species (Kamiya and Graebe, 1983; Takahashi et al., 1986; Grosslindemann et al., 1992). The 13-hydroxylation of GA15, GA24, GA9, and GA4 in maize may reside in a single 13-hydroxylase with low substrate specificity or with the presence of separate substrate-specific enzymes. The failure to detect the sequence GA12 → GA15 → GA24 → GA9 → GA4 → GA7 could be because the Km values for these substrates are much lower for the 13-hydroxylase(s) than for the 20-oxidase(s).

The two labeled metabolites GA15-15,16-ene and GA7-diene-diacid were probably formed by the non-enzymatic rearrangement of a double bond. Additionally, 2,3-dehydro-GA10 was probably formed as a result of a non-enzymatic hydration of the 16,17-double bond in the substrate 2,3-dehydro-GA9.

Based on the previous demonstration that GA5 is an intermediate between GA20 and GA3 in maize shoots (Fujioka et al., 1990; Spray et al., 1996), we examined the possibility that 2,3-dehydro-GA9 is an intermediate between GA9 and GA7. Our results show that 2,3-dehydro-GA9 is predominantly 13-hydroxylated to GA5, 12α-hydroxylated to GA31, and 1β-hydroxylated to GA62, and converted into GA7 in trace amounts. However, isotope dilution studies gave no evidence for the natural occurrence of 2,3-dehydro-GA9 in maize shoots (data not shown). The metabolic origin of GA15, GA24, GA9, GA4, and GA7 in maize remains unresolved.

Footnotes

1

This work was supported by the National Science Foundation (grant nos. MCB–9604460 and MCB–9306597) and by the U.S. Department of Energy (grant no. DE–FG03–90ER20016). The IACR receives grant-aided support from the Biotechnological and Biological Science Research Council of the United Kingdom.

LITERATURE CITED

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