Plant Physiol. (I1987) 83, 1048-10530032-0889/87/83/ 1048/06/$0 1.00/0

Internode Length in Pisum1GENE na MAY BLOCK GIBBERELLIN SYNTHESIS BETWEEN ent-7a-HYDROXYKAURENOIC ACIDAND GIBBERELLIN Al2-ALDEHYDE

Received for publication June 18, 1986 and in revised form December 8, 1986

TIMOTHY J. INGRAM AND JAMES B. REID*School ofChemistry, University ofBristol, Bristol, BS8 ITS, United Kingdom, (T.J.I.); and Department ofBotany, University of Tasmania, Hobart, Tasmania, 7001, Australia (J.B.R.)

ABSTRACT

The elongation response of the gibberellin (GA) deficient genotypesna, Is, and lh of peas (Pisum sativum L.) to a range of GA-precursorswas examined. Plants possessing gene na did not respond to precursorsin the GA biosynthetic pathway prior to GAj2-aldehyde. In contrast,plants possessing Ih and Is responded as well as wild-type plants (dwarfedwith AMO-1618) to these compounds. The results suggest that GAbiosynthesis is blocked prior to ent-kaurene in the Ih and Is mutants andbetween ent-7a-hydroxykaurenoic acid and GAi2-aldehyde in the namutant. Feeds of ent-VIHlkaurenoic acid and I2HJGAi2-aldehyde to arange of genotypes supported the above conclusions. The na line WL1766was shown by gas chromatography-mass spectrometry (GC-MS) tometabolize [2HjGA12-aldehyde to a number of12H1C19-GAs including GA,.However, there was no indication in na genotypes for the metabolism ofent-VIH-kaurenoic acid to these GAs. In contrast, the expanding shoottissue of all Na genotypes examined metabolised ent-VHjkaurenoic acidto radioactive compounds that co-chromatographed with GA,, GA,, GA2,and GA29. However, insufficient material was present for unequivocalidentification of the metabolites. The radioactive profiles from HPLC ofextracts of the node treated with ent-[H]kaurenoic acid were similar forboth Na and na plants and contained ent-16a,17-dihydroxykaurenoic acidand ent-6a,7a,16,,17-tetrahydroxykaurenoic acid (both characterized byGC-MS), suggesting that the metabolites arose from side branches ofthe main GA-biosynthetic pathway. Thus, both Na and na plants appearcapable of ent-7a-hydroxylation.

Dwarfing genes have been identified in many species of higherplants. A large number of these result in reduced levels ofbiologically active GAs in the shoot (13, 21, 22, 25). However,their action has only been determined conclusively for the dwarfmutants d5 and di in maize (9, 31) and le in peas (13, 14). Genesle and di both block a late step in the GA2 biosynthetic pathway,the 343-hydroxylation of GA20 to GA1, while d5 blocks an earlystep, the cyclization of copalyl pyrophosphate to ent-kaurene.The site of action of other GA-synthesis genes has been indicatedin some instances from the growth responses ofthe mutant typesto intermediates in the GA-biosynthetic pathway (21, 24, 33).

In peas (Pisum sativum L.) eight well established genes con-trolling internode length have been described (4, 19, 20, 26, 32)

'Financial support was provided by the Royal Society (T. J. I.) andthe Australian Research Grants Scheme (J. B. R.).

2Abbreviations: GAn, gibberellin An; AMO-1618, 2-isopropyl-4-di-methyl-amino-5-methylphenyl- I -piperidine-carboxylate methyl chlo-ride.

and four of these reduce GA-synthesis. Three of these genes, na,lh, and Is, result in substantially reduced levels of all biologicallyactive GAs within the shoot (13, 25-27), suggesting that theyoperate relatively early in the GA-biosynthetic pathway.

Gibberellin biosynthesis in developing seeds of peas has beenwell documented. The pathway to GA12-aldehyde, the first pre-cursor bearing the ent-gibberellane carbon skeleton, is in com-mon with all other higher plants investigated (3, 8). Thereafter,GA12-aldehyde is metabolized via the early C- 13 hydroxylationpathway (15, 29). In the shoot this leads to the production ofGA,, which is regarded as the active GA controlling stem elon-gation (12-14, 23).The present study examines the growth response of plants

possessing genes na, lh, and Is to a series of early intermediatesin the GA-biosynthetic pathway. The metabolic site of action ofthe na mutation was probed further by feeds ofent-[3H]kaurenoicacid and [2H]GA12-aldehyde to intact Na and na plants.

MATERIALS AND METHODS

Plant Material. The pure lines used in this study come fromthe collection at Hobart (Department of Botany, University ofTasmania). All lines used carry dominant genes at the internodelength loci Lk, Lm, and La and/or Cry. Lines WL1766 (inter-node length genotype Le na Lh Ls), 81 (le na Lh Ls), K202 (LeNa Lh Is), 181 (Le Na Lh Is), M26 (le Na Lh Is), and K511 (LeNa Ih Ls) all possess short internodes compared with the wild-type tall lines WL1771 and Torsdag (both Le Na Lh Ls). LinesK511, 181, K202, and Torsdag are closely related, as are linesWL1 771 and WL1766. Further details about the origin of theselines and their genotypes can be found (26, 28), with the excep-tion of line 181 which was developed at Hobart from cross K202x Torsdag.

Plants were grown in controlled environment cabinets (modelSIOL, Conviron, Winnipeg, Canada) under the following con-ditions: day temperature 20°C; night temperature 15°C; day-length 18 h; light intensity 210 sE m-2 s-' (PAR). All plants weregrown in John Innes No. 2 soil based compost (John InnesInstitute, Norwich, England).Chemical Treatments. All GA precursors used in bioassay

experiments were chemically characterized crystalline com-pounds obtained from natural sources or by partial chemicalsynthesis (5, 7). Their purity was checked prior to use by capillaryGC and/or HPLC. Substrates, dissolved in 10 ul of ethanol, wereapplied to the youngest fully expanded leaf, at doses of 20 ,ug(GA12-aldehyde, GA53-aldehyde, and ent-kaurenoic acid, Fig. 1)or 50 ,ug (ent-kaurene, ent-kaurenoic acid and ent-7a-hydroxy-kaurenoic acid, Fig. 2) per plant. Control plants were treatedwith ethanol alone. Plants were treated at 10 to 12 d aftergermination when 3 to 4 leaves had expanded. A minimum of

1048

INTERNODE LENGTH IN PISUM

5-6 7-8

WL1766

3-4 5-6 7-8Internode

FIG. 1. Internode length plotted against internode number for plantsof lines 181 (Lh Is Na), K511 (lh Ls Na), WL1766 (Lh Ls na), andTorsdag (Lk Ls Na) treated with either 20 gg of GA53-aldehyde (A),GAr2-aldehyde (U), or ent-kaurenoic acid (V) or left untreated (0).Torsdag plants were dwarfed by the application of 100 Ug ofAMO-I6 18.SE were generally too small to indicate on the figure but varied between0.01 and 0.5 cm for individual points with an average of 0. I1 cm. n =

6.

6, but generally between 8 and 12, plants were used for eachtreatment. Plants ofthe tall line Torsdag were routinely shortenedby the application of 100 Mg of AMO-1618 (Serva, Heidelberg)in 10 ,l of ethanol to the testa of the dry seed before planting.In Figure 2 plants of lines KS 11 (Ih) and 181 (Is) were similarlytreated in order to reduce their internode length to that observedin the na line WL1766. Internode lengths were scored after 9 to10 internodes had fully expanded.

1.2

0.8

0.4

'I-,

E O0

2.4

c

0)

4)2.00

C

1.2

0.8

0.4

0 3-4 5-6 7-8 3-4Internode

5-6 7-8

FIG. 2. Internode length plotted against internode number for plantsof lines 181, (Lh Is Na), Torsdag (Lh Ls Na), K5 11 (Ih Ls Na), andWL1766 (Lh Ls na) treated with either 50 gg of ent-kaurene (A), ent-kaurenoic acid (V), or ent-7a-hydroxykaurenoic acid (U) or left untreated(@). The Torsdag, K5 1 1 and L18 1 plants were dwarfed by the applicationof 100 jig of AMO-1618. SE were generally too small to indicate on thefigure but varied between 0.001 and 0.19 cm for individual points withan average of 0.05 cm. n > 7.

64 L8 1

4-

>2

0co6

5 lb 1,5 2.0 25 30Fraction no.

FIG. 3. Analysis by HPLC of methylated [3H]GA12-aldehyde metab-olites in acidic ethyl acetate extracts of the tissue expanded above thetreated node of pea lines 81 (le na) and WL1766 (Le na). Standardmethyl-GAs eluted in the fractions indicated.

Torsdag31p

21-

1 F

0

31-

0)%-

cmC

a)0')0C

C

K511 WL1766

I * * I * * * * * * I I

L181 Torsdag A

1XI _ _ _ . _ _ a

21-

1 _

OI- 3-4

I

a a I I i I I I I

- . - - - - - -% - 0%

1049

a

1Plant Physiol. Vol. 83, 1987

100 a)} M* 507

1 207A--73900

%1001d) 207 ? 375M?O41

O-- ,,,.'L - s...- .

100 200 300 400 500 600

m/e

FIG. 4. [2HJGA,2-aldehyde feed to line WL1766. Mass spectra of theMeTMS derivatives of (a) [2H]GA,, (b) [2H]GA8, (c) [2H]GA2o, and (d)[2H]GA29. The Kovat's retention indices were 2670 for GA,, 2819 forGA8, 2486 for GA20, and 2685 for GA29.

Table I. Gibberellin A,2-aldehyde Feed to the na Line WLI 766[2HI-isotope incorporation in metabolites.

Percent IsotopicCompound Composition

[2H] ['H]GA,2 aldehyde 76a 23GA, 75b 25GA8 70b 30GA20 85a 13GA29 74b 26

a Determined from the molecular ion cluster of the mass spectrum(30). bJDetermined by selected ion monitoring of the molecular ions(10).

Details of the labeled compounds used in metabolic studiesare as follows: ent-[17-3H2]kaurenoic acid (1.28 G Bq mmol-')was prepared by Dr. M. H. Beale, University of Bristol (unpub-lished data) by reaction of the norketone with tritiated Wittigreagent (1); [6-3H,]GA,2-aldehyde (0.22 G Bq mmol-') and[ 1 8-2HI]GA,2-aldehyde (76% [2H]isotope incorporation) wereprepared as described by Down et al. (5). The purity of substrateswas checked by HPLC-radiocounting and GC-MS. Plants weretreated with ethanolic solutions of the substrates as above. Thefeeds were conducted as follows: ent-[3H]kaurenoic acid (17 KBq = 4 usg/plant) to 20 plants of each genotype (12 d aftergermination), plants harvested after 2 d; [3HJGA,2-aldehyde (1.8K Bq = 2.5 ,ug/plant) to 8 plants each of lines 81 and WL1766(18 d after germination), plants harvested after 6 d; [2H]GA,2-aldehyde (10 g/plant) to 47 plants of line WL1766 (23 d aftergermination), plants harvested after 5 d.

Extraction and Purification of Metabolites. Plant material wasfrozen in liquid N2 and extracted overnight in methanol:water(4:1). The methanol was removed in vacuo at 40°C and an equalvolume ofpH 8 phosphate buffer (0.25 M) added to the aqueousextract. Samples from ent-[3H]kaurenoic acid feeds were parti-tioned with diethyl ether at pH 8 before adjusting the pH to 2.8and obtaining the acidic ethyl acetate fraction. This procedureremoved excess substrate but prevented the loss of most of therelatively nonpolar metabolites into the neutral organic fraction.All other samples were partitioned with ethyl acetate at pH 8and pH 2.8.

Acidic ethyl acetate fractions were purified by reverse phaseC,8 HPLC. Samples from ent-[3H]kaurenoic acid feeds werechromatographed on an analytical column (250 x 4.6 mm i.d.)packed with Spherisorb ODS (5 tm) (Anachem Ltd., Luton,Bedfordshire, U.K.), eluted with methanol in distilled H20 (con-taining 0.25% H3PO4), delivered by two pumps controlled by asolvent programmer (Laboratory Data Control, Stone, Staffs,U.K.). Solvent conditions were: 30 to 100% methanol, lineargradient over 60 min, 1.5 ml min-' (Figs. 5 and 7), or 30 to 70%methanol, exponential gradient over 45 min, 70% for 7.5 min,100% for 7.5 min, 1.0 ml min-' (Fig. 6). In both cases 1.5 mlfractions were collected. Samples from GA,2-aldehyde feeds werepurified under conditions similar to those described elsewhere(1 1).Throughout the work radioactivity was determined by liquid

scintillation counting following addition of 10 ml of Unisolve E(Koch Light, Haverhill, Suffolk, U.K.) to the samples.Gas Chromatography-Mass Spectrometry. Appropriate frac-

tions from HPLC were combined and reduced to dryness. Thesamples were analyzed as the methyl ester trimethylsilyl ethers.GC-MS was performed as described before (13). Characterizationof metabolites was based on Kovats retention indices (6, 17) andcomparison with authentic spectra.Chemical Structures. The structures ofthe compounds referred

to in the text are shown in Figure 9.

RESULTSActivity of GA-Precursors. Lines carrying the genes na

(WL1766), Is ( 181) and Ih (K5 1 1), show a highly significant stemelongation response (P < 0.00 1) to both GA12-aldehyde andGA53-aldehyde (Fig. 1). Likewise, the tall (wild-type) cv Torsdag,dwarfed with AMO-16 18, responds well to these compounds,although they are substantially less active than GA, (27). The Nalines, K5 11, 181, and Torsdag, also responded significantly (P <0.01) to the GA precursors ent-kaurene and ent-kaurenoic acid.

Table II. ent-[1 7-3H21 Kaurenoic Acid FeedsRadioactivity recovered in the tissue expanded above the treated node. Total ent-[3H] kaurenoic acid fed =

3.33 x I05 Bq to 20 plants of each genotype.

Radioactivity (Bq) inLine Genotype

pH 8 diethyl ether pH 2.8 ethyl acetate

Experiment IWL1766 naLeLhLs 50a 8K202 Na Le Lh Is 150a 53WL1771 NaLeLhLs 20a 72

Experiment 281 naleLhLs b 22WL1766 naLeLhLs 10M26 Nale Lh Is 48Dippes gelbe Viktoria Na le Lh Ls 48181 Na Le Lh Is 42K5 11 Na Lk Ih Ls 64

'Analysis by HPLC indicated one radioactive peak co-chromatographing with ent-[3H] kaurenoicacid. b Not measured.

1050 INGRAM AND REID

INTERNODE LENGTH IN PISUM

co 20 K202

1055;a

0 10 20 30 40 50 60

Fraction no.FIG. 5. Analysis by HPLC of ent-[3H]kaurenoic acid metabolites in

acidic ethyl acetate extracts of the tissue expanded above the treatednode of pea lines WL1766 (Ls na), K202 (Is Na), and WL1771 (Ls Na).Standard compounds eluted in the fractions indicated.

10 20 30 40Fraction no.

10 20 30

FIG. 6. Analysis by HPLC of ent-[3H]kaurenoic acid metabolites inacidic ethyl acetate extracts of the tissue expanded above the treatednode of pea lines 81 (le Lh Ls na), WL1766 (Le Lh Ls na), M26 (le LhIs Na), Dippes gelbe Viktoria (DGV, le Lh Ls Na), 181 (Le Lh Is Na),and K5 1 1 (Le Ih Ls Na). Standard GAs eluted in the fractions indicated.

44

24

CT

_0

:5coir

64

44

4(

2(

10 20 30 40 50 60Fraction no.

FIG. 7. Analysis by HPLC of ent-[3H]kaurenoic acid metabolites inacidic ethyl acetate extracts of the treated tissue of pea lines WL1766 (Lsna), K202 (Is Na), and WL1771 (Ls Na). Standard compounds elutedin the fractions indicated.

In contrast, the na line WL1766 showed no significant responseto these compounds (Figs. 1 and 2). The lack of response inplants possessing gene na has occurred in five experiments usinga range of na lines, application techniques and dose rates (up to100 lAg/plant). Furthermore, na plants showed no response toent-kaurenol, ent-kaurenal (results not shown), and ent-7a-hy-droxykaurenoic acid (Fig. 2). The latter compound causes asmaller, but significant, promotion (P < 0.01 for internodes 5-8) of elongation in lines K5 11 (1h) and 181 (Is) than ent-kaureneand ent-kaurenoic acid, and on cv Torsdag (wild-type) resultedin either no significant effect (Fig. 2) or, in some experiments, asmall promotion of elongation (data not shown). These resultssuggest that the na allele blocks GA synthesis between ent-7a-hydroxykaurenoic acid and GAI2-aldehyde, whereas genes lh andls block the pathway prior to ent-kaurene.Feeds with jHJ- and [2H]-GA12-Aldehyde and ent-VHjKauren-

oic Acid. Biochemical support for the conclusions drawn fromthe bioassay data was sought by examining the metabolism ofGA12-aldehyde and ent-kaurenoic acid in a range of internodelength genotypes.

[6-3H1]GA12-aldehyde was metabolized to radioactive com-pounds that co-chromatograph on HPLC with GA,, GA8, andGA29 in the nana lines 81 (na le) and WL1766 (na Le) (Fig. 3).As expected, the levels of putative 3fl-hydroxylated metabolites(GA, and GA8) were considerably lower in the le line 81 than inline WL1766 (12, 13). The identity of these metabolites wasconfirmed by large scale feeds of [188-2H1]GA12-aldehyde to lineWL1766. Five days after feeding the 2H substrate, the tissue thathad expanded above the treated node contained [2HJGA20, [2H]GA,, [2H]GA8, and [2H]GA29 (Fig. 4). Calculation of isotopeincorporation in these compounds showed no apparent dilutionby endogenous ['HJGAs (Table I). As anticipated from thebioassay responses in the preceding section, these results show

GA2gGA1 G420 GA53ald 70oHKA (1Al2aId KA

00 WL1766

K202

00

WL1771

A2 3

16

12

8

4

0

8

acr

CID

%-

4._co0

co

GA8 GA2g GA1 GA20 GA8 GA2g GA1 GA20H H H F NMH m F

L81 DGV

WL1766 LI81

K51 1

- -ir.- .- - . 1-

1051

2(

6(

WL1766 L181

M26mr-W

-rLA I 'Lll.

a) 1477M, 670 1

96 269 6557%I I'.. II

b1 ) 117 391 M+ 494

O .. , ,207,, ..I,,,l..

100 200 300 400 500 600m/e

FIG. 8. ent-[3H]Kaurenoic acid feed to line WL177 1. Mass spectra of the MeTMS derivatives of(a) ent-6a,7a, 16#, 17-tetrahydroxykaurenoic acid(Kovat's retention index of 2984) and (b) ent- 16a, 17-dihydroxykaurenoic acid (Kovat's retention index of 2787).

/his f 0

ent - kaurene

CO2H

ent-16a. 17(OH)h kaurenoic acid

OH

CO2H

ent - 6a,7a,O16H217(OH)4 kaurenoic acid

OH

0H

HO HH2H

GA,

However, only negligible levels of radioactivity in these regionsof the HPLC profile were observed in either of the na lines 81and WL1766 (Figs. 5, 6). As expected, the presence ofthe le gene

CH2OH in lines M26 and Dippes gelbe Viktoria reduced the levels ofent - kaurenol metabolites co-chromatographing with the 3,B-hydroxylated GAs,

GA,, and GA8. However, in accord with their presumed actionearlier in the GA biosynthetic pathway, neither Is nor lh influ-enced the metabolism of ent-[3H]kaurenoic acid. Although con-firmation of the identity of the metabolites of ent-[3H]kaurenoicacid was not possible (since they ranged between only 50 to 500pg/plant), the results strongly suggest that the na gene preventsthe metabolism of ent-kaurenoic acid to GAs.

CHO Little evidence was found for the metabolism of ent-[3H]ent - kaurenal kaurenoic acid to GAs at the point of application in either Na

or na plants. Rather, analysis by HPLC of acidic ethyl acetateextracts from the treated leaf disclosed a complex (and similar)radioactive profile in all three lines WL1766 (na), K202 (Na),and WL1771 (Na) (Fig. 7). Examination of prominent HPLCpeaks from WL1771 by GC-MS revealed the presence of ent-16a,17-dihydroxykaurenoic acid (fractions 29-30) and ent-

COHN 6a,7a,16(3,7-tetrahydroxykaurenoic acid (fractions 26-27) (Fig.it - kaurenoic acid 8), suggesting that the metabolites of ent-[3H]kaurenoic acid

present in the treated tissue arose from side branches ofthe mainGA-biosynthetic pathway (8).

IC0H

ent - 7a hydroxykaurenoic acid

na

HOCO2H

GA12 - aldehyde

FIG. 9. The possible action of the genes Ih, Is, and na on GA biosyn-thesis in the shoots of P. sativum L.

that the na mutation does not prevent the further metabolism ofGA12-aldehyde.The metabolism of ent-[1 7-3H2]kaurenoic acid was investi-

gated in genotypes varying at the Na, Le, Lh, and Ls loci.Extremely low levels of radioactivity were exported to the tissuethat had expanded above the treated node (Table II), probablydue to poor uptake of this nonpolar substrate. Analysis of theacidic ethyl acetate fractions from this tissue by HPLC revealedthe presence of 3H metabolites co-eluting with GA8, GA29, GA,,and GA20 in all of the Na genotypes examined (Figs. 5, 6).

DISCUSSIONBoth the results from application experiments with GA-pre-

cursors and metabolic studies with ent-[3H]kaurenoic acid sug-gest that the gene na prevents the conversion of ent-7a-hydroxy-kaurenoic acid to GA12-aldehyde (Fig. 9). The GA-precursorsent-kaurene, ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, andent-7a-hydroxykaurenoic acid were all inactive when applied tona plants, even at a dose rate of 100 jig per plant. By comparison,all Na lines tested showed a significant growth response to 50 Agof ent-kaurene and ent-kaurenoic acid. ent-7a-Hydroxykauren-oic acid was also effective in these plants but its activity appearedsubstantially less than ent-kaurene and ent-kaurenoic acid. Asimilar result has been obtained in experiments with the GA-deficient tomato mutants ga-I and ga-3 (JAD Zeevaart, personalcommunication) and is consistent with work on the fungusGibberellafujikuroi, in which ent-7a-hydroxykaurenoic acid wasshown to be less efficiently converted to GAs than ent-kaurenoicacid (2, 18). The cause of this reduced conversion of the ent-7a-hydroxykaurenoic acid is not clear. As observed in other suchstudies (21, 24; JAD Zeevaart, personal communication), theactivity of precursors early in the GA pathway is considerablylower than the biologically active compound, GA,, possiblybecause oftheir nonpolar nature. It should be noted that althoughthe present biochemical evidence suggests that gene na com-pletely blocks GA biosynthesis, genetic evidence suggests thatgene na (and also Ih and Is) is 'leaky' to a minor extent (26).

Although the metabolites of ent-[3H]kaurenoic acid could notbe positively identified in extracts from the expanded shoottissue, the differences in the HPLC profiles from Na and na linesstrongly suggest that the latter cannot metabolize ent-kaurenoic

1052 INGRAM AND REID Plant Physiol. Vol. 83, 1987

ell

l~

INTERNODE LENGTH IN PISUM

acid to C1g-GAs. In comparison, the na line WL1766 readilymetabolized [2H]GAI2-aldehyde to the C19-GAs, GA,, GA8,GA20, and GA29. In the treated tissue the major metabolites ofent-[3H]kaurenoic acid appear similar in both Na and na lines.Since ent-6a,7a, 16f,17-tetrahydroxykaurenoic acid was identi-fied from this region of the HPLC profile, it is likely that bothNa and na plants can carry out the ent-7a-hydroxylation of ent-kaurenoic acid. No evidence was obtained for the build up ofent-7a-hydroxykaurenoic acid in na plants. Evidence for theconversion of ent-[3H]kaurenoic acid to GAs within the treatedtissue of Na lines was obscured by the high levels of nonpolarmetabolites. The hydroxylated ent-kaurenoic acids observed maybe artifacts arising from substrate overloading.The proposed site of action of gene na in the GA biosynthetic

pathway is different from that proven for other GA synthesismutants (23). However, application experiments to tomato sug-gest that the dwarf mutant ga-2 (-Ve-270) may also block theGA-biosynthetic pathway between ent-7a-hydroxykaurenoicacid and GA12-aldehyde (33; JAD Zeevaart, personal commu-nication).

Plants possessing the GA synthesis genes lh and Is (27) re-sponded in a similar way to the application of ent-kaurene aswild-type plants dwarfed with AMO-1618. This suggests thatboth of these genes may block GA-biosynthesis prior to ent-kaurene. The genes d5 and anI in maize (9, 22), dx in rice (21),and ga-I and ga-3 in tomatoes (16, 33; JAD Zeevaart, personalcommunication) also appear to block GA synthesis prior tokaurene. Neither Is nor lh plants appear to possess impairedcarotenoid production suggesting that the blocks are probablybetween geranylgeranylpyrophosphate and ent-kaurene. How-ever, further detailed studies are required to determine the actionof these genes.

Finally, the positive identification of [2H]GAI in feeds of [2H]GA12-aldehyde to WL1766 plants is consistent with the growthresponse of plants treated with GA-precursors resulting fromtheir metabolism to GA. Gibberellin A1 is probably the onlyGA active per se in controlling internode elongation in peas (12,13) and several other species (23).

Acknowledgments-We are grateful to Professor J. MacMillan for help andpractical support in the completion of the present work, Dr. M. H. Beale and Mr.M. J. Lee for preparation of labeled precursors, and Mr. P. Gaskin for capillaryGC-MS and interpretation of spectra.

LITERATURE CITED

1. BEARDER JR, VM FRYDMAN, P GASKIN, IK HATTON, WE HARVEY, J MACMIL-LAN, BO PHINNEY 1976 Fungal products. Part XVII. Microbiological hy-droxylation of gibberellin A9 and its methyl ester. J Chem Soc Perkin Trans1: 178-183

2. BREADER JR, J MACMILLAN, BO PHINNEY 1975 Fungal products. Part XIV.Metabolic pathways from ent-kaurenoic acid to the fungal gibberellins inmutant B1-41a of Gibberellafujikuroi. J Chem Soc Perkin Trans I: 721-726

3. COOLBAUGH RC 1983 Early stages of gibberellin biosynthesis. In A Crozier,ed, The Biochemistry and Physiology of Gibberellins, Vol I. Praeger, NewYork, pp 53-98

4. DE HAAN H 1927 Length factors in Pisum. Genetica 9:481-4975. DOWN GJ, M LEE, J MACMILLAN, KS STAPLES 1983 Preparation of 13-

hydroxy-gibberellin A12-7-aldehyde. J Chem Soc Perkin Trans 1: 1103-11086. GASKIN P, J MACMILLAN, RD FINN, RJ PRYCE 1971 'Parafilm': a convenient

source of n-alkane standards for the determination of gas chromatographicretention indices. Phytochemistry 10: 1155-1157

7. GRAEBE JE, HJ ROPERS 1978 Gibberellins. In DS Letham, PB Goodwin, TJVHiggins, eds, Phytohormones and Related Compounds-A Comprehensive

Treatise, Vol I. Elsevier/North-Holland Biomedical Press, Amsterdam, pp107-204

8. HEDDEN P 1983 In vitro metabolism of gibberellins. In A Crozier, ed, TheBiochemistry and Physiology of Gibberellins, Vol I. Praeger, New York, pp99- 149

9. HEDDEN P. BO PHINNEY 1979 Comparison of ent-kaurene and ent-isokaurenesynthesis in cell-free systems from etiolated shoots of normal and dwarf-5maize seedlings. Phytochemistry 18: 1475-1479

10. INGRAM TJ, JB REID 1987 Internode length in Pisum. Biochemical expressionof the le and na mutations in the slender phenotype. J Plant Growth Regul.In press

11. INGRAM TJ, JB REID, J MACMILLAN 1985 Internode length in Pisum sativumL. The kinetics of growth and [3H] gibberellin A20 metabolism in genotypena Le. Planta 164: 429-438

12. INGRAM TJ, JB REID, J MACMILLAN 1986 The quantitative relationship be-tween gibberellin Al and internode elongation in Pisum sativum L. Planta168: 414-420

13. INGRAM TJ, JB REID, IC MURFET, P GASKIN, CL WILLIS, J MACMILLAN 1984Internode length in Pisum. The le gene controls the 3,B-hydroxylation ofgibberellin A20 to gibberellin Al. Planta 160: 455-463

14. INGRAM TJ, JB REID, WC PoTrS, IC MURFET 1983 Internode length in Pisum.IV. The effect of the gene Le on gibberellin metabolism. Physiol Plant 59:607-616

15. KAMIYA Y, JE GRAEBE 1983 The biosynthesis of all major pea gibberellins ina cell-free system from Pisum sativum. Phytochemistry 22: 681-689

16. KOORNNEEF M, JH VAN DER VEEN, CJP SPRUIT, CM KARSSEN 1981 Isolationand use of mutants with altered germination behaviour in Arabidopsisthaliana and tomato. In Induced Mutants as a Tool for Crop Plant Improve-ment. IAEA-SM, Vienna, 251: 227-232

17. KOVATS E 1958 Gas chromatographische charakterisierung organischer verbin-dungen. Teil 1: Retention indices aliphatischer halogenide alkohole, aldehydeund ketone. Helv Chim Acta 41: 1915-1932

18. LEW FT, CA WEST 1971 (-)-Kaur-1 6-en-7,B-ol- I 9-oic acid, an intermediate ingibberellin biosynthesis. Phytochemistry 10: 2065-2076

19. LINDQVisT K 1951 The mutant 'micro' in Pisum. Heriditas 37: 389-42020. MENDEL G 1866 Versuche uber Pflanzenhybriden. Verhand Naturf Ver Brunn

4: 3-47. Reprinted 1951 in J Hered 42: 3-4721. MURAKAMI Y 1972 Dwarfing genes in rice and their relation to gibberellin

biosynthesis. In DJ Carr, ed, Plant Growth Substances, 1970. Springer-Verlag, Berlin, pp 166-174

22. PHINNEY BO 1961 Dwarfing genes in Zea mays and their relation to gibber-ellins. In RM Klein, ed, Plant Growth Regulation. Iowa State UniversityPress, Ames, IA, pp 489-511

23. PHINNEY BO 1984 Gibberellin Al, dwarfism and the control ofshoot elongationin higher plants. In A Crozier, JR Hillman, eds, The Biosynthesis andMetabolism of Plant Hormones, Soc for Exp Biol Seminar Series 23. Cam-bridge Univ Press, London, pp 17-41

24. PHINNEY BO, C SPRAY 1982 Chemical genetics and the gibberellin pathway inZea mays L. In PF Wareing, ed, Plant Growth Substances, 1982. AcademicPress, London, pp 101-110

25. Pom WC, JB REID 1983 Internode length in Pisum. III. The effect andinteraction of the Na/na and Le/le gene differences on endogenous gibber-ellin-like substances. Physiol Plant 57: 448-454

26. REID JB 1986 Internode length in Pisum. Three further loci, lh, Is and Ik. AnnBot 57: 577-592

27. REID JB, WC Porrs 1986 Internode length in Pisum. Two further mutants, Ihand ls, with reduced gibberellin synthesis, and a gibberellin insensitivemutant, lk. Physiol Plant 66: 417-426

28. REID JB, IC MURFET, WC POTTS 1983 Internode length in Pisum. II. Additionalinformation on the relationship and action of loci Le, La, Cry, Na and Lm.J Exp Bot 34: 349-364

29. ROPERS HJ, JE GRAEBE, P GASKIN, J MACMILLAN 1978 Gibberellin biosyn-thesis in a cell-free system from immature seeds ofPisum sativum. BiochemBiophys Res Commun 80: 690-697

30. SPONSEL VM, J MACMILLAN 1978 Metabolism of gibberellin A29 in seeds ofPisum sativum cv Progress No. 9; use of [2H]- and [3H]GAs, and theidentification of a new GA catabolite. Planta 144: 69-78

31. SPRAY C, BO PHINNEY, P GASKIN, SJ GILMOUR, J MACMILLAN 1984 Internodelength in Zea mays L. The dwarf- I mutation controls the 3j3-hydroxylationof gibberellin A20 to gibberellin A,. Planta 160: 464-468

32. WELLENSIEK SJ 1969 The physiological effects of flower forming genes in peas.Z Pflanzenphysiol 60: 388-402

33. ZEEVAART JAD 1984 Gibberellins in single gene dwarf mutants of tomato.Plant Physiol 75: S-186

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