Metabolism by Elongating Pea Pericarp'

Received for publication March 11, 1991 Accepted July 18, 1991

Plant Physiol. (1991) 97, 1359-1366 0032-0889/91/97/1 359/08/$01 .00/0

[14C]GA12-Aldehyde, [14C]GA12, and [2H]- and [14C]GA53 Metabolism by Elongating Pea Pericarp' Sonja L. Maki* and Mark L. Brenner Department of Biology, Carleton College, Northfield, Minnesota 55057 (S.L.M.), and Department of Horticultural Science, University of Minnesota, St. Paul, Minnesota 55108 (M.L.B.) ABSTRACT

(10, 1 1) or when the shoot apex is removed above an unfertilized ovary (4). In the latter case, GAI, and possibly precursors of GA1, are transported from the maternal plant to the fruit (22). However, inasmuch as emasculated flowers do not develop into fruits on untopped plants, levels of GAs imported from the maternal plant normally must be low (22). GAs might also be produced within the elongating pericarp tissue. Potts (24) used bioassay techniques to determine GA levels in the pericarp and seeds of fully elongated fruits of near-isogenic tall (Na) and dwarf (na) lines of pea. Although similar levels of bioactivity were detected in seeds from both lines, much less activity was found in the pericarp tissue of a dwarf (na) line. Thus, Potts suggested that the na gene is operative in blocking the production of biologically active GAs in the pericarp and that biologically active GAs in the pericarp of Na lines are produced in situ with little leakage of GAs from the seeds to the pericarp. However, since these fruits were fully elongated, it is not known if the pericarp of younger fruits in the na line contains higher levels of GAs. Not much is known about whether the pericarp produces GAs; however, the pericarp does expand rapidly and rapidly expanding internodes of vegetative peas contain high rates of ent-kaurene (a GA precursor) synthesis activity (7). This observation prompted us to question whether the pericarp has the capacity to produce GAs for growth. Chung and Coolbaugh (7) suggest ent-kaurene synthesis may reflect GA biosynthesis. As young pea fruits (obtained from withered flowers) also synthesize ent-kaurene (14) and seeds younger than 10 days have low ent-kaurene synthesis capacity (6), GAs may be produced in pericarp tissue. This paper describes metabolic studies with [14C]GA12ald, [14C]GA12, and [2H]- and [14C]GA53 in pericarp tissue of 4-dold pea fruits to determine if the pericarp tissue contains the enzymatic pathway to produce biologically active GAs.

Gibberellins (GAs) are either required for, or at least promote, the growth of the pea (Pisum sativum L.) fruit. Whether the pericarp of the pea fruit produces GAs in situ and/or whether GAs are transported into the pericarp from the developing seeds or matemal plant is currently unknown. The objective of this research was to investigate whether the pericarp tissue contains enzymes capable of metabolizing GAs from [14C]GA12-7-aldehyde (I14C]GA12ald) to biologically active GAs. The metabolism of GAs early in the biosynthetic pathway, ['4C]GA12 and [14C]GA12ald, was investigated in pericarp tissue isolated from 4-day-old pea fruits. [14C]GA12ald was metabolized primarily to [14C]GA,2aid-conjugate, [14C]GA12, [14C]GA1,, and polar conjugate-like products by isolated pericarp. In contrast, [14C]GA12 was converted primarily to [14C]GAsI and polar conjugate-like products. Upon further investigations with intact 4-day-old fruits on the plant, [14C]GA12 was found to be converted to a product which copurified with endogenous GA2o. Lastly, [2H]GA2o and [2HJGAI were recovered 48 hours after application of [2H]- and [14C]GA5w to pericarp tissue of intact 3-day-old pea fruits. These results demonstrate that pericarp tissue metabolizes GAs and suggests a function for pericarp GA metabolism during fruit growth.

GAs2 are important in fruit growth of many species (13). The pea (Pisum sativum L.) plant has been used as a model system to study the role of GAs in fruit growth (see reviews by Sponsel [26] and Pharis and King [23]). GAs from the early 1 3-hydroxylation pathway (GA1, GA8, GA20, and GA29) have been identified in separate extracts of pericarp and seeds of fruits obtained 5 DAF (12). Although the source of these GAs, some of which promote pericarp growth in pea, is unknown, they could originate in the developing seeds, the maternal plant, and/or in the pericarp tissue. Developing seeds may provide GAs to the fruit, because pod growth stops when developing seeds are killed, but can be restored by applying certain GAs (9, 25). Parthenocarpic fruit growth can be either induced when ovary explants are treated with GA

MATERIALS AND METHODS Plant Material Seeds of pea, Pisum sativum L. line I3 (Alaska-type), were obtained from the late Dr. Gerald Marx (New York Agricultural Experiment Station, Cornell University, Geneva, New York). Five seeds were planted in 4-L plastic pots containing a 1:1:1 ratio of soil:sand:peat. The plants were grown in a growth chamber (Conviron) at 17°C/15°C (day/night) with cool-white fluorescent and incandescent lights and an 18-h photoperiod. The lights were kept 30 cm above the top of the canopy and the PPFD at this level was 700 ,mol m2 sec'.

' Supported in part by the Minnesota Agricultural Experiment Station, a Doctoral Dissertation Fellowship from the University of Minnesota Graduate School, and the National Science Foundation (NSF/DMB-8607749). Paper No. 18776, Scientific Journal Series. 2Abbreviations: GA, gibberellin; GA12ald, gibberellin A12-7-aldehyde; BHT, butylated hydroxytoluene; EtOAc, ethyl acetate; PVPP, polyvinylpolypyrrolidone; HOAc, acetic acid; GC-SIM, gas chromatography-selected ion monitoring.

1359

1 360

MAKIAND BRENNER

Plants were thinned to three per pot 1 week after emergence and fertilized weekly with 300 ppm 20-20-20 for 3 weeks and with 400 ppm weekly thereafter. Flowering began approximately 30 d after planting and d 0 of fruit development was defined as the time when the petals were fully reflexed.

Plant Physiol. Vol. 97, 1991

Samples were dissolved in 50 ,uL methanol followed by 50 an additional 700 IAL of solvent A. Prior to injection into the respective columns, samples were filtered with disposable 0.45 ,m pore size filters (Acro LC3A, Gelman). Radioactivity was detected by an on-line heterogeneous scintillation system (Packard Trace 7140).

,uL solvent A and then

Chemicals

['4C]GA12ald and ['4CJGA12 were synthesized enzymatically from R,S-[4,5-'4C]mevalonic acid (110 ,uCi/,umol) in a cellfree system of pumpkin endosperm using the method of Birnberg et al. (1) with the following modifications. Prior to HPLC purification, the reaction mix containing the radioactive products was purified by charcoal chromatography as described by Zhu et al. (28). The specific activities of the ['4C] GA12ald and ['4C]GA,2 were determined from their mass spectra (3) to be 196.6 uCi/,umol and 125.1 uCi/,mol, respectively. Incubation of Pericarp Tissue with Radiolabeled Substrates Pea fruits were collected 4 DAF. Pericarp tissue was isolated by splitting the fruit along the sutures. Seeds were removed and the pericarp tissue was placed (with the exterior side down) on moistened filter paper inside a Petri dish. The radioactive substrate (150,000 dpm) was streaked across the fruit tissue in 1 gL of 95% ethanol. To study the metabolism in growing fruits in situ, 150,000 dpm of the radioactive substrate was applied to the exterior of one side of a fruit in 1 uL ethanol. Four hours after applying the radioactive substrate to the fruits in situ, the immature seeds, subtending leaves, and pericarp tissue were harvested for analysis. The tissue was frozen on dry ice and kept at -80°C until extraction. For the initial 4-h application study, each labeled compound was applied to five pericarp halves which were combined and extracted after the treatment period. Tissues from other application studies (time course and intact) were extracted and

analyzed separately. Extraction

Frozen pericarp tissue was extracted twice in 10 mL of cold 80% methanol (aqueous) with BHT (10 mg/L) using a Polytron homogenizer (Brinkman Instruments, NY). The extracts were centrifuged for 30 min at 10,000g and the supernatant was evaporated to dryness in silanized-glass scintillation vials using a SpeedVac concentrator (Savant, Farmingdale, NY). HPLC Systems System 1 employed a 150 x 4.6 mm Nucleosil 5 ,um C18 column with a flow rate of 1 mL/min. Solvent A was 0.1 N HOAc (aqueous) and solvent B was 0.1 N HOAc in CH3CN with the following linear gradient segments: 100% A to 80% A/20% B in 2 min, gradient to 35% B in 15 min, gradient to 75% B in 15 min, gradient to 100% B in 2 min, isocratic 100% B for 7 min. System 2 employed a 10 x 150 mm RoSil 10 IAm C18 column with a flow rate of 4 mL/min and the same solvents and gradient program as in system 1.

Separation of Conjugates from "Free" Metabolites Silica partition chromatography was used to test for the presence of conjugates. The procedure of Koshioka et al. (18) was followed with the following modifications. Column dimensions were 25 x 7 mm and free GAs were eluted with EtOAc:HOAc (99:1, v/v). Under these conditions an apolar conjugate, etiocholane-3-a-ol-17-one glucuronide methyl ester, eluted in the methanol fraction and GA8 eluted in the EtOAc/HOAc fractions as reported elsewhere (2). Hydrolysis of Conjugates

For enzymatic hydrolysis of conjugates, radioactive metabolites were dried in 1.5 mL Eppendorf tubes and incubated with 200 ML each of the following enzymes dissolved in a potassium-phosphate buffer (0.1 M, pH 5.0.): Aspergillus niger cellulase, Trichoderma viridae cellulase, almond ,B-glucosidase (3 mg/mL) and pectinase (Macerase, 1 mg/mL). During hydrolysis the tubes were shaken at room temperature for 24 h. One relatively apolar conjugate, which was resistant to enzymatic hydrolysis, was chemically hydrolyzed with 0.1 N KOH at 50°C for 4 h. Purification of Endogenous GAs from a Bulk Extract Pea fruits (4-d-old) were split open, the seeds were separated from pericarp, and both pericarp and seeds were frozen on dry ice and stored at -80°C. Pericarp tissue from 240 fruits (92 g fresh weight) was extracted in 5 volumes (w/v) 80% methanol containing 10 mg/L BHT using a commercial blender. The extract was stirred overnight at 4°C. Following vacuum filtration through Whatman No. 1 filter paper, the residue was extracted a second time, stirred at 4°C for 4 h, and filtered as before. The metabolites obtained from [14C] GA12 applied to isolated pericarp tissue and attached pods that eluted at 22.2 min and 18.2 min, respectively, were added to the filtrate (80,000 dpm each). The methanol was evaporated in vacuo and the aqueous fraction was adjusted to pH 8.0 with 0.1 N NH40H and partitioned against an equal volume of hexane. After vigorous shaking, the hexane was removed by evaporation causing some of the Chl to form globules. The aqueous fraction was then passed through a 6 x 2 cm pad of PVPP with much of the chlorophyll being retained on the PVPP. The PVPP was then rinsed with three column volumes of water (pH 8.0), and the filtrate was applied to a charcoal:Celite column 6 cm in diameter (3 g of a 1:2 mix ofcharcoal:Celite per 10 g fresh weight). The column was rinsed with 3 volumes of 0.1 N HOAc prior to elution of the GAs with 80% methanol followed by 100% 0.1 N HOAc in acetone. The column was washed with 0.1 N HOAc in ethyl acetate. The solvents were evaporated to approximately 2 mL of water prior to filtration and purification on HPLC system

GIBBERELLIN METABOLISM IN PEA FRUITS

2. Individual peaks were collected, methylated (5) and rechromatographed on HPLC system 1 prior to GC-MS analysis.

[2H]- and [14C]GA53 Metabolism in 3-d-Old Pea Fruits in Situ

['4C]GA53 was obtained from a 4-h incubation of ['4C] GA12ald with five 20-d-old pea cotyledons excised from developing seeds (19). The ["4C]GA53 (325,000 dpm, approximately 125 uCi/,Mmol) was separated from other products by C18 HPLC and combined with [17-2H2]GA53 in 95% ethanol. Two micrograms of [2H2]GA53 were injected into the pericarp tissue of 20 3-d-old pea fruits (1 ML per fruit). Thus, the pericarp tissue of each fruit was injected with approximately 100 ng of [2H2]GA53 and 16,250 dpm of ['4C]GA53. After 48 h the fruits were excised from the plants and the immature seeds and pericarp tissue were separated and frozen on dry ice. The plant material was stored at -80°C until extraction. Pericarp tissue was divided into four equal parts and each part was homogenized (Polytron, Brinkman Instruments) in 20 mL of 80% aqueous methanol containing 10 mg/L BHT and centrifuged for 30 min at 5,000g. The extracts were pooled yielding an 80 mL final extract. The pooled extract was passed through disposable C18 columns for purification according to Koshioka et al. (18) as follows: the extract was filtered (Whatman No. 1 filter paper) and then passed through a 10 g C18 column (Mega Bond Elut, Analytichem International) which was rinsed with an additional 20 mL of 80% methanol. The concentration of methanol in the eluant was adjusted to 50% (v/v) by the addition of 52 mL H20 (pH 6.5) before passing through another 10 g column followed by a final rinse with 40 mL of 50% methanol to complete elution of the free and conjugated GAs. The eluent was then taken to dryness with excess methanol using a rotary evaporator. The immature seeds were homogenized as above using 25% of the volumes used for the pericarp. Smaller (1 g) C18 columns (Sep-Pak, Waters Associates) were used for these smaller samples. SiO2 partition chromatography was performed on each extract to separate free GAs from GA glucosyl conjugates ( 18). The residue resulting from the methanol extraction was dissolved sequentially in 1 mL of methanol and 1 mL of water, absorbed onto 0.6 g of Celite, and dried overnight under a gentle stream of N2. The Celite was placed at the top of a SiO2 column (15 mm i.d.) which had been slurried and packed with formic acid-saturated hexane:ethyl acetate (5:95, v/v). Columns were packed with 5 g and 2.5 g of SiO2 for the pericarp and immature seed extracts, respectively. Free GAs in the pericarp extract or the immature seed extract were eluted under vacuum first from the column with 70 mL or 35 mL, respectively, of formic acid-saturated hexane:ethyl acetate (5:95, v/v). GA glucosyl conjugates were eluted under vacuum with two methanol washes of either 70 mL each for the pericarp extract or 35 mL for the immature seed extract. The fractions were dried under reduced pressure in a rotary evaporator with excess methanol. HPLC analysis of the free and conjugate fractions of the pericarp and the immature seed extracts was performed as follows. Samples were dissolved in 20 uL of methanol followed

1361

by 750 ML of 0.1 N HOAc (aqueous), centrifuged at 5,000g and the supernatant was filtered through a 0.45 MAm pore size filter. Filtered samples were then injected onto a 5 MAm Spherisorb C18 column (150 mm x 4.6 mm) with a flow rate of 1 mL/min and eluted using the conditions already described as system 1. One-half minute fractions were collected and pooled according to the elution position of GA standards. GC-MS GC-MS was performed with a Hewlett-Packard GC model 5890 which was connected to a Hewlett-Packard MSD model 5970 with data processing by a Hewlett-Packard Chem Station with a Hewlett-Packard 9000 Series 300 computer. Following methylation (5), samples were taken to dryness and transferred in redistilled methanol to 200 MuL silanized glass GC vial inserts. The methanol was evaporated and the samples were kept dry over P205 and under vacuum in a desiccator. Samples were converted to their MeTMSi derivatives by incubation in 20 ML pyridine/bis-trimethylsilyltrifluoroacetamide + 1% trimethylchlorosilane (1:1, v/v) at 60°C for 60 min. The sample containing putative GA53 was analyzed by GC-SIM as its methyl ester. The samples were taken to dryness and redissolved in 5 to 10 ML CH2Cl2 prior to on-column injection of 1 ML onto a DB-5 fused silica column (30 m x 0.249 mm; 1.0 Mm film thickness). Helium was used as the carrier gas at a flow rate of 1 mL per min. The column temperature was initially 38°C, then was ramped to 200°C in 2 min, held for 2 min, then ramped to 280°C at 4°C/min and held for 25 min. The mass range scanned was 200-550 atomic mass units for full scan mass spectrometry of the 18.2 min [14C]GA12 metabolite, whereas GC-SIM was used for the analysis of the [2H]- and [14C]GA53 metabolites monitoring at least four prominent ions for each GA of interest. RESULTS [14C]GA12aid and [14C]GA12 Metabolism in Isolated Pericarp Tissue Incubating pericarp tissue with ['4C]GA12ald and ['4C]GA12 for 4 h resulted in the formation of many metabolites (Fig. 1), with the spectrum of metabolites dependent on the substrate. The metabolite which eluted at 22.0 to 22.2 min (the elution position of GA53) was observed with both substrates. Most of the ['4C]GA12ald metabolites eluted as conjugates on the silica partition columns (Table I). However, a portion of the combined 16.8 and 17.8 min peak, the majority of the 22.0 min peak, and the 24.0 min peak eluted as free GAs. A greater proportion of the metabolites of [14C]GA12 eluted as free GAs (Table I); approximately half of the radioactivity associated with the 16.4 and 18.2 min peak and over 90% of the 22.2 min peak were eluted with EtOAc. The metabolites which eluted between 10.0 and 14.0 min behaved as

conjugates. The nature of several of the conjugate-like metabolites of

['4C]GA12 and ['4C]GA12ald formed in pericarp tissue was investigated further by enzymatic and chemical hydrolysis (Table I). The retention times of the 10.4 min and 11.4 min metabolites of ['4C]GA12 shifted to 13.6 min and 14.4 min, respectively, following enzymatic hydrolysis. Similar results

1362

MAKI AND BRENNER


obtained for the conjugate-like metabolites of [14C] GA12ald, with one exception. The 28.4 min metabolite was resistant to enzymatic hydrolysis, although a trace of radioactivity occurred in the elution position of ['4C]GA12ald (34.8 min). Base hydrolysis of the 28.4 min peak indeed yielded a radioactive peak at the elution position of GA12ald (Table I), suggesting that this metabolite of [14C]GA12ald feeds was a conjugate of ['4C]GA12ald. In the time course experiment, metabolism of ['4CJGA12ald in isolated, 4-d-old pericarp tissue was rapid. After 2 h less than 5% of the total radioactivity remained as ['4C]GA12ald (Fig. 2A). The peak at 28.4 min (the putative ['4C]GA12ald conjugate) rose as the concentration of ['4C]GA12ald fell. The rate of ['4C]GA12 metabolism (Fig. 2B) was similar to that of ['4C]GA12ald, with less than 10% of the total radioactivity remaining as ['4C]GA12 after 2 h. Putative ['4C]GA53 (Ri; 22.2 min) and the 13.6 min peak, a putative conjugate, were formed coincident with the metabolism of ['4C]GA12. The peaks at 10.4 and 11.4 min, which also behaved as conjugates, accumulated slowly over the course of 4 h.

were

0

x

02 'C 1-

X

0

1-

Time (min)

[14C]GA12 Metabolism in Pericarp Tissue in Situ Because [14C]GA12ald

Figure 1. Total radiolabeled products obtained after 4 h applications of (A) [14C]GA12ald and (B) [14C]GA12 to 4-d-old isolated pericarp tissue and separated on HPLC system 1. Retention times (min) for some GA standards are as follows: GA53,22.2; GA20, 18.2, GA1, 11.6; GA8, 9.0.

was

converted primarily to a conju-

[14C]GA12 was metabolized along the 13-hydroxy pathway, the metabolism of [14C]GA12 was further investi-

gate while

gated in elongating fruits in situ. A representative chromatogram of the radioactive metabolites obtained from 4 and 24

Table I. Conjugate Nature of the 14C Metabolites Percentage of radioactivity for the 14C metabolites and their identification as a free GA or GA conjugate as determined by their elution from a silicic acid partition column with ethyl acetate:acetic acid (99:1) or subsequently with methanol. If more than 75% of the radioactivity eluted in the ethyl acetate fraction, the nature of the peak was designated as free. If more than 75% of the radioactivity eluted in the methanol fraction, the nature of the peak was designated as conjugate. Retention times on HPLC system 1 for products formed following enzymatic or chemical hydrolysis are also given. Retention Time Percentage Natureof Percentage of 1 HPLC System Typeof of Hydrolysis with System Elutedl with Eluted Peak Hydrolysis Time Retention Products Methanol Ethyl Acetate

Neatur

HPLCtio

Hyperofyi

min

min

[14C]GA12ald metabolites: 14 11 7 8 28 10 79 48 12

86 89 93 92 72 90

Conjugate Enzymatic 12.2, 16.8, 17.8 Conjugate Enzymatic 12.2, 16.8,17.8 Conjugate Enzymatic 16.8, 17.8 a Conjugate

21 52 88

Free Mixed

10.4

21

11.4 12.6 and 13.6 15.4 16.4

11 7 34

79 89 93 66

Conjugate Enzymatic 13.6 Conjugate Enzymatic 14.4 Conjugate

53 50 91 23

47 50 9 77

9.4 10.8 12.2 14.0 16.8 and 17.8 20.0 22.0 24.0 28.4

Mixed

Conjugate Enzymatic 25.8 Conjugate Enzymatic 28.4, 34.8 (trace) Chemical 34.8

[14C]GA12 metabolites:

18.2 22.2 25.0 a Not done.

Mixed Mixed Mixed Free

Conjugate


pericarp tissue. GA20 was identified in the sample which copurified with the radiolabeled 18.2 min peak recovered from feeds to intact fruits (Table II); however, there was no evidence of any ["1C] ions in the spectra of GA20.

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1363

[2H]- and [14CJGA53 Metabolism by Pericarp Tissue in Situ Nearly all (90%) of the radioactivity was recovered in the free and conjugate fractions of the pericarp tissue with only

|

trace levels in the immature seed extract (Fig. 4). A major radioactive peak eluted at the position of GA53 in the conjuA-A 28.4' - -GA gate fraction of the pericarp extract (data not shown). Since can occur when samples are absorbed onto Celite 12trapping 1 2 IG A121D (18), the conjugate fractions obtained after HPLC analysis | were combined, adsorbed onto a glass-fiber filter (instead of Celite) and subjected to a second SiO2 chromatographic step. The free fraction from this second SiO2 chromatographic step was injected onto the HPLC column, and 0.5 min fractions were collected into the respective original vials from the first SiO2 partition column and subsequent HPLC column. Substantially more GA53 as well as the 14.4 min and the 15.4 min metabolites were recovered and they eluted in the free fraction " 10.4 *411.4' (data not shown); this confirmed that some trapping had occurred during the initial SiO2 chromatographic step. k 6

>13

2020-

0-10~~4 CA12 I I I I 0 30 60 90 120 150 180 210 240 T

3000I

I

.

(A) 4 hours

Time (mi) Figure 2. Four hour time course showing products formed by isolated pencarp tissue from (A) [14C]GA12ald and (B) [14C]GA12. Each point represents the mean of three replicates ± the standard error of the mean.

2000l

.. 1000-

C6

h applications to 4-d-old fruits (Fig. 3) shows a large amount of ['4C]GA,2 still remaining. A major peak, which behaved almost entirely as a free GA (data not shown) eluted at 18.2 min. There was very little radioactivity in the elution position of GA53 (22.2 min). The amount of conjugate formation was also less than had occurred in the 4 h feeds to isolated pod tissue. Most of the radioactivity remained in the pericarp, although after 24 h a small amount (approximately 0.5% of applied radioactivity) was observed in the subtending leaves following combustion and subsequent liquid scintillation spectroscopy and less than 0.1 % of applied radioactivity was detected in a 10% aliquot of an 80% methanol extract of the developing seeds (data not shown).

Analysis of the Tissue Extract

0

0

10

20

30

20

30

3000

(B) 24 hours E

2000

ol 1000

[14C]GA12 Metabolites in a Bulk Pericarp

The ["'C]GA,2 metabolite which eluted at 22.2 min was identified as ["'C]GA53 based on GC-SIM (Table II). There was no dilution from endogenous GA53 because the percent of [14C] in the [14C]GA53 recovered following purification was the same as that of the ['4C]GA,2 substrate applied to the

0

0- 0

~~10

Time

(mi)

Figure 3. 14C products recovered in the pericarp after (A) a 4 h and (B) a 24 h application of [14C]GA12 to the pericarp of intact, 4-d-old fruits.

1 364


MAKI AND BRENNER

[2H]GA20 was identified by GC-SIM (Table III) in the HPLC fraction corresponding to the elution position of a standard of GA20 (15.4 min; Fig. 4). One of the 14C metabolites coeluted with the HPLC fraction containing the [2H]GA20. [2H]GA1 was also detected by GC-SIM in the HPLC fraction corresponding to the elution position of a standard of GA, (Table III). There was considerable dilution by endogenous GA20 and GA1 as shown by the increase in the ratio of nondeuterated to deuterated ions (Table III). Though radioactive standards were available for GA8 and GA44, they were not detected (in either natural or deuterated form) in the respective fractions. Traces of the major ions for deuterated and nondeuterated GA29, 508 and 506 respectively, were detected in the fraction corresponding to the elution position of a [3H,'3C]GA29 standard.

DISCUSSION Results from the studies of [14C]GA12 metabolism in isolated pericarp tissue of elongating pea fruits indicate that the early 13-hydroxylation operates since [14C]GA53 is a major product. The rapidity of this conversion suggests a high turnover rate of GA12 in isolated pericarp tissue. However, once [14C]GA53 is formed its metabolism is much slower and [14C] GA53 accumulates over 4 h. Metabolism of [14C]GA12 by pericarp tissue in situ is different from that in isolated pericarp halves. When ['4C]GA12 was applied to the exterior of elongating fruits, only a trace of [14C]GA53 and fewer conjugates were observed. The major metabolite after 4 and 24 h application (the 18.2 min peak) copurified with endogenous GA20. Less conjugation may have occurred because the [14C]GA12 was applied to the exterior of the fruit; the [14C]GA12 was applied to the interior in the isolated pericarp feeds. Application to the exterior may have resulted in slower penetration and less perturbation of endogenous levels of GA12. Another explanation for these differences may be a differential localization of GA metabolic enzymes between inner and outer cell layers of the pericarp, however we did not observe qualitative differences between products formed following a 24 h application of [14C]GA12 to the exterior and interior of isolated pericarp tissue with the

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Pericarp Metabolites C-

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Free Fraction

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GA1

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GA20

GA8~

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o

GA53

40C

200

.9GA44 C E

0-

0~~~~~~~~0

D

20

10

30

Time (min)

Figure 4. Radiolabeled products recovered in the free-GA fraction of the pericarp tissue following a 48 h application (by injection) of [2H]and [14C]GA53 into the pericarp of intact pea fruits. There were no detectable peaks of radioactivity present in either the free-GA or conjugated-GA fractions of the immature seed extract. GA standards eluted in the positions indicated.

exception that more ['4C]GA12 was present when applied to the exterior (data not shown). Metabolism of [14C]GA12 in the pericarp of intact fruits might also have been influenced by the presence of the developing seeds or the maternal plant. Sponsel (25) suggested that the developing seeds may indirectly maintain GA production in the pericarp tissue. ['4C]GA12 is metabolized further down the early 13-hydroxylation pathway to a product which coeluted with GA20 in situ, supporting the idea that the developing seeds are important for GA metabolism in the pericarp. Recently, Ozga and Brenner (21) demonstrated that seeds are important for the metabolism of [(4C]GA19 to ['4C] GA20 in the pericarp. The endogenous level of GA53 is low in the pericarp of 4d-old fruits because there was no dilution of 14C from the applied substrate [14C]GA12. In vegetative shoots of the G2 genotype, the endogenous level of GA53 was calculated to be 0.3 ng/g fresh weight (15). GA53 has been shown to reach levels as high as 7 ng/g fresh weight in shoot-forming tobacco crown gall tumors and levels of GA53 increased as ent-kaurene

Table II. GC-MS Identification of Methyl Ester (Me) or Methyl Ester Trimethylsilyl Ether (MeTMSi) Derivatives of the [14C]GA12 Metabolites of 4-d-old Pericarp Tissue

[14C]GA12-Me (applied to pericarp)

Retention

m/z Characteristic Ions

% 14Ca

on GC min

(Relative Abundance)

(Specific Activity)

24.9 300(100), 240(22), 328(21), (125.1 ACi/,Amole) [14C] ions: 316(33), 256(9), 344(6) 27.7 NAb 318(100), 257(4), 259(24), 284(1), 286(16) [2H]GA53-Me (standard) 27.7 25.4 376(M+,1), 344(21), 316(100), 301(14), GA53-Me (detected in pericarp) (125.6 ,gCi/gmole) 241(14), [14C] ions: 332(34), 257(24) 27.3 NA 418(M+,100), 403(14), 375(58), 359(14), GA2o-MeTMSi (standard) 301(10) NA 27.2 GA20-MeTMSi (detected in pencarp) 418(M+,100), 403(22), 375(81), 359(25), 301(19) (no [14C] ions detected) a The %14C is calculated from the ratio of the major ion for 12C and 14C (27) and the specific activity was calculated according to Bowen et b NA = not applicable. al. (3). 21.8

1 365


Table IlIl. Capillary GC-SIM of Me-TMSi Derivatives of GA Standards and [2H2]GA Metabolites following Application of [14C]- and C'H2]-GA53 to the Pericarp of 3-d-old Pea Fruits An estimate of the 2H2 composition is expressed as the ratio of the nondeuterated to deuterated ions calculated from the abundances of each ion in the ion pair specified for each GA. Abundance values were obtained from integrated peak areas and corrected for natural contributions of isotopes in the protio and deuterated GAs. 1H:2H (ion Pair) GA Constituent Ions m/z (%) GC Rt min

[2H2]GAs, (substrate) Putative [2H2]GA2o Authentic GA20 Putative [2H2]GAj Authentic GA,

26.932 27.099 27.269 31.109 31.301

450(100), 448(1), 391(64), 389(3), 251(5), 253(78) 420(77), 418(100), 405(12), 403(14), 377(55), 375(64) 418(100), 403(14), 375(58), 359(14), 301(10) 508(49), 506(100), 493(5), 491(12), 449(3), 447(10), 315(9) 506(100), 491(9), 447(10), 416(1), 390(3)

synthesis capacity increased (20). There was no dilution of the "'C in the recovered ["'C]GA53. We calculated that the concentration of endogenous GA53 required to reduce the specific activity 50% would be about 2.7 ng/g fresh weight. Thus GA53 levels are likely below this in pericarp tissue obtained from 4-d-old fruits. Failure to detect endogenous GA53 is not surprising since it has been reported to be rapidly metabolized in vegetative pea plants (8). GA53 was also not detected in a 25 g fresh weight extract of 22-d-old pea cotyledons which actively synthesize GAs (19). Both [2H]GA2o and [2H]GAI were found in the pericarp tissue from the [2H]- and [14C]GA53 feed to 3-d-old pea fruits while only traces of 14C were detected in the extract of immature seed, strongly suggesting that the enzymes necessary to convert GA53 to GA, exist in the pericarp. The [2H]GA20 and [2H]GAI recovered were also markedly diluted with endogenous GA20 and GA,. The presence of GA20 (12, 16) and GA, (12) has been previously reported in extracts of isolated pericarp tissue. Pericarp tissue has the capacity to metabolize [14C]GA12ald, [14C]GA12, and [2H]- and [14C]GA53 to other GAs. Sponsel (25) found that GA53-aldehyde partially restored pericarp elongation in seed-killed pea fruits. Our results suggest that the restoration of elongation growth observed by Sponsel was a result of metabolism to an active GA. Whether the GA metabolism observed in our studies contributes to the GAs in the pericarp has yet to be determined. Further studies are required to determine turnover rates and endogenous pool sizes of specific GAs to determine what proportion of GAs present are synthesized in the pericarp. Different patterns of metabolism were observed for [14C] GA12ald and [14C]GA12 in isolated pericarp tissue. [14C] GA12ald was metabolized primarily to its conjugate and other conjugates, though a small amount of [14C]GA53 was observed after 4 h and [14C]GA12 was formed in the first hour of the time course study. Enzymatic hydrolysis of several of the early eluting [14C]GA12ald conjugates resulted in more than one product and hydrolysis of the different early eluting conjugates resulted in the same spectrum of metabolites. These results may indicate the presence of a polyhydroxylated GA conjugated at one to several positions. [14C]GA12, on the other hand, was metabolized to more products behaving as free GAs. Kamiya and Graebe (17) also observed [14C]GA12ald conjugate formation with cell-free extracts of pea embryos. They concluded that GA12 was a more suitable substrate for

0.050 (389/391) 1.470 (418/420) Not applicable 3.56 (506/508) Not applicable

studying GA metabolism (17). These differences in metabolism may be due to differential compartmentation of the applied substrates or preferential conjugation of [14C]GA12ald. In summary, these results show that the pericarp can metabolize GAs on the 13-hydroxylation pathway (the major pathway in pea). [14C]GA12 was more suitable as a substrate for studying GA metabolism in pericarp tissue. Pericarp tissue of intact fruits in situ metabolized ['4C]GA12 to GAs farther down the 13-hydroxylation pathway. Lastly, the metabolism of [2H]- and [14C]GA53 to [2H]GA20 and [2H]GAI demonstrates synthesis of biologically active GAs in the pericarp during elongation of pea fruits. ACKNOWLEDGMENTS We thank Dr. Jocelyn Ozga for helpful comments on the manuscript. We also thank Jeny Pierson for technical assistance. [17,172H]GA53 was a gift from Dr. Richard Pharis of the University of Calgary, Alberta, Canada and [17-3H,'3C]GA29 was a gift from Dr. Bernard Phinney of the University of California, Los Angeles.

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3. 4. 5.

6. 7.

8. 9.

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19. Maki SL, Brenner ML, Birnberg PR, Davies PJ, Krick TP (1986) Identification of pea gibberellins by studying ['4C]GA,2aldehyde metabolism. Plant Physiol 81: 984-990 20. Mettrie R, De Greef J, Nakagawa S, Sakurai A (1988) entKaurene synthesis and endogenous levels of gibberellins in a shoot forming tobacco crown gall tissue. Plant Cell Physiol 29: 777-784 21. Ozga J, Brenner ML (1990) The effect of seeds on GA metabolism in pea pericarp (abstract No. 18). Plant Physiol 93: S-5 22. Pereto JG, Beltran JP, Garcia-Martinez JL (1988) The source of gibberellins in the parthenocarpic development of ovaries on topped pea plants. Planta 175: 493-499 23. Pharis RP, King RW (1985) Gibberellins and reproductive development in seed plants. Annu Rev Plant Physiol 36: 517-568 24. Potts WC (1986) Gibberellins in light-grown shoots of Pisum sativum L. and the influence of reproductive development. Plant Cell Physiol 27: 997-1003 25. Sponsel VM (1982) Effects of applied gibberellin and naphthylacetic acid on pod development in fruits of Pisum sativum L. cv. Progress No. 9. J Plant Growth Regul 1: 147-152 26. Sponsel VM (1985) Gibberellins in Pisum sativum-their nature, distribution and involvement in growth and development of the plant. Physiol Plant 65: 533-538 27. Takahashi N, Yamaguchi I, Yamane H (1986) Gibberellins. In N Takahashi, ed, Chemistry of Plant Hormones. CRC Press, Boca Raton, FL, pp 57-151 28. Zhu Y-X, Davies PJ, Halinska A (1988) Gibberellin A12 and gibberellin A12-7-aldehyde as endogenous compounds in developing seeds of Pisum sativum. Phytochemistry 27: 2549-2552