Planta
Planta 146,649-656 (1979)
9 by Springer-Verlag 1979
Auxin-induced Ethylene Biosynthesis in Subapical Stem Sections of Etiolated Seedlings of Pisum sativum L. Jennifer F. Jones and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. 1-Aminocyclopropane-l-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections ofetiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 m M ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-14C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections i n c u bated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.
One control point for IAA-induced ethylene formation is the level of free IAA in the tissue, and this is closely related to the rates of conjugation of IAA (Kang et al., 1971). Another possible control point lies in the rates of formation and degradation of the ethylene-forming systems (Kang et al., 1971 ; Lieberman and Kunishi, 1975; Sakai and Imaseki, 1971). The pathway of ethylene biosynthesis has recently been identified in apples (Adams and Yang, 1979) and in tomato fruit (Boller et al., 1979) as the sequence methionine~S-adenosylmethionine (SAM) ~l-aminocyclopropane- 1-carboxylic acid (ACC) --,ethylene. In stem sections of peas and in flower tissue of Ipornoea tricolor (Konze et al., 1978), selenomethionine has been found to be a better precursor in this pathway than methionine. It has been proposed that the conversion of SAM to ACC is the step at which ethylene biosynthesis is regulated (Adams and Yang, 1979 ; Boller et al., 1979). Yu et al. (1979) have further proposed that IAA induces the synthesis of the ACC-forming enzyme in mung bean hypocotyls. In this paper, we report on experiments which were designed to determine a) whether or not ACC is an intermediate in the pathway leading from methionine to ethylene in pea stem sections treated with IAA, and b) the point of action of IAA in the ethylene-forming system in this tissue.
Key words: 1-Aminocyclopropane-l-carboxylic acid Auxin - Ethylene - P i s u m Rhizobitoxine analog. Material and Methods Introduction
Plant Material and Conditions of Incubation
IAA induces the formation of ethylene in stem sections of etiolated pea seedlings (Burg and Burg, 1966).
Pea seeds, Pisum sativum L. cv. Alaska (Vaughan-Jacklin Corp., Ovid, Mich., USA), were imbibed overnight in aerated tap water, sown in Vermiculite, and grown in the dark at 25~ for 5-7 days. Subapical stem sections (0.6 cm) were excised directly below the apical hook, and were placed in 25-ml Erlenmeyer flasks (12 sections per flask) on a disc of Whatman No. 1 filter paper wetted with 1 ml water or test solution. All manipulations were
ACC= 1-aminocyclopropane-l-carboxylic acid; AVG=aminoethoxyvinylglycine,the aminoethoxy analog of rhizobitoxine; IAA=indole-3-acetic acid; SAM-S-adenosylmethionine ; SMM = S-methylmethionine Abbreviations:
0032-0935/79/0146/0649/$01.60
650 performed under a green (530-590 nm) safelight at an energy fluence of less than 0.001 W m- 2 and the sections remained in the dark until they were extracted.
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum were scraped off and counted by scintillation spectrometry using ACS scintillation solvent (Amersham Corp., Arlington Heights, I11., USA). The major peaks were identified by co-chromatography with the authentic compounds; S-methylmethionine (SMM) was further identified by electrophoresis (Hanson and Kende, 1976).
Determination of Ethylene Production and ACC Content Ethylene production was measured by gas chromatography as described by Kende and Hanson (1976). In order to determine the content of ACC, 12 pea stem sections were ground in a glass homogenizer with 2 ml 80% (v/v) ethanol containing 5 mM mercaptoethanol. The initial extract and a further 1 ml wash were combined and left for 1 h at 0~ to precipitate the protein. The extract was then centrifuged at 12,000 x g for 15 min in a Sorvall Model RC-2B centrifuge using an SS-34 rotor (DuPont Instruments-Sorvall, Wilmington, Del., USA). The pellet was extracted twice more with 1 ml extraction medium, and the combined supernatants were dried under nitrogen at 45 ~ C. The residue was taken up in 75 gi 50% (v/v) ethanol of which 10 gl were spotted in a line on 5-cm wide strips of precoated 0.1-mm cellulose MN 300 plates (Brinkman Instruments, Westbury, N.Y., USA). The chromatograms were run in chloroform-methanol-58% (w/v) ammonia (2:2: 1, v/v) or in 1-butanol-glacial acetic acid-water (60:15:25, v/v) until the solvent front had migrated 15 cm. The position of an ACC standard run on a separate plate was determined by spraying the plate with ninhydrin. The zones on the chromatograms which corresponded to the position of ACC were scraped off and eluted with 1.8 ml 100 mM sodium-phosphate buffer, pH 11.5. The fractions were centrifuged for 2-3 min to sediment the cellulose. Two aliquots of 0.7 ml were removed from each fraction, and 10 nmol authentic ACC was added to one of these aliquots prior to assaying both portions for ACC as described previously (Boller et al., 1979). The addition of a known quantity of ACC to one aliquot from each extract allowed corrections to be made for inhibition of the assay by the extract. This inhibition was never greater than 50%. Losses of ACC during extraction and chromatography were determined by the addition of a known quantity of ACC to a duplicate sample of tissue at the beginning of the extraction. Recovery ranged from 66-78% and was independent of the concentration of ACC applied. The assay for ACC worked well on chromatographed extracts of pea stem sections, provided that only a small quantity of material was used. An amount of extract equivalent to one half of a section, 0.6 cm in length and ca. 12 mg in weight, was routinely assayed. Larger quantities of material inhibited the assay, and neither passing the extracts through a cation exchange resin (Dowex-50W) or charcoal, or partitioning with butanol, ethyl acetate or ether, nor chromatography with different TLC solvents alleviated this inhibition.
ACC-forming Enzyme: Preparation and Assay Sections from etiolated pea seedlings were treated with IAA as described above and extracted with 2 ml extraction buffer per g fresh weight of tissue, consisting of 100 mM N-2-hydroxyethylpiperazinc-N'-2-ethanesulfonicacid (HEPES) buffer, pH 8.0, containing 4 mM D,L-dithiothreitol (DTT) and 0.4 gM pyridoxal phosphate. For some experiments, bovine serum albumin (BSA) was added to the extraction buffer at 1 mg per ml. The homogehates were filtered through a 50-gm-mesh nylon net and centrifuged at 12,000 x g for 20 min. The supernatant was layered onto columns (10 or 40 cm in length, 10 or 200 ml bed volume, respectively) of Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, N.J., USA). The columns were equilibrated and eluted with 2 mM KHEPES buffer at pH 8.0 containing 0.1 mM DTT and 0.2 pM pyridoxal phosphate. Fractions of 1.6 ml were collected from the large column and of 0.8 ml from the small column. Alternatively, acetone powders were prepared from the pea sections, dessicated, and stored at.4 ~ C overnight prior to use. The powder was then dissolved in column buffer (4 ml per g original fresh weight of tissue) at 4~ C, left to stand for 1 h, centrifuged at 12,000• for 20 min, and the supernatant layered on a column of Sephadex G-50 and eluted as described above. The fractions containing protein were detected by testing an aliquot from each fraction by the method of Bradford (1976). The fractions containing protein were combined and used as the enzyme preparation. All manipulations were performed at 0-4 ~ C. The enzyme preparation was assayed as described by Boller et al. (1979), except that ATP was added to the incubation mixture to give a final concentration of 5 mM. The concentration of protein in the enzyme assay was determined according to Bradford (1976).
Chemica~ L-[U-~4C]Methionine (specific activity 250-260 mCi/mmol) was purchased from New England Nuclear, Boston, Mass., USA; D, L-methylmethinone sulfonium chloride from U.S. Biochemical Corp., Cleveland, O., USA; and all other biochemicals either from Calbiochem-Behring Corp., La Jolla, Cal., USA or Sigma Chemical Co., St. Louis, Mo., USA. Aminoethoxyvinylglycine (AVG) was a gift from Dr. M. Lieberman (U.S. Department of Agriculture, Beltsville, Md., USA).
Radiotracer Experiments with L-[ U-14C] Methionine Twelve pea stem sections were incubated in 25-ml Erlenmeyer flasks containing 2 g Ci L[U-~*C]methionine (258 mCi/mmol) dissolved in 0.9 ml water and a CO z trap consisting of a suspended vial with 751al of 1 N NaOH saturated with Ba(OH)2 and a fluted filter paper wick. After 1 h, 100 ~1 of either 1 mM IAA or water were injected through the serum-vial caps into the flasks. The specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC were determined as described by Hanson and Kende (1976) and Boller et al. (1979). The pea stem sections were extracted as described above for the determination of ACC and chromatographed in the acidic TLC solvent. The radioactivity on the plates was located with a Radiochromatogram Scanner, Model 7201 (Packard Instruments, Downers Grove, I11., USA). The zones on the plates which corresponded to the major radioactive peaks
Results Effect o f Application o f A C C When freshly excised pea stem sections were placed in 0.1 m M I A A , t h e r e w a s a lag p e r i o d o f 2 - 3 h b e f o r e an increase in e t h y l e n e p r o d u c t i o n b e c a m e e v i d e n t (Fig. 1). W h e n t h e s e c t i o n s w e r e p l a c e d o n a s o l u t i o n of 1 mM ACC or 1 mM ACC+IAA, t h e rise in e t h ylene production was immediate, and the pea sections p r o d u c e d m u c h g r e a t e r (ca. 3 - f o l d ) q u a n t i t i e s o f e t h ylene than those incubated on IAA alone. IAA + ACC
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651 Table 1. The effect of different concentrations of 1-aminocyclopropane-l-carboxylic acid (ACC) on ethylene production in subapical stem sections of etiolated pea seedlings. Pea sections, 0.6 cm in length, were incubated on water or ACC (0.1, 1, 2, 3, 5, and 10 mM) overnight (5:00 p.m.-8:30 a.m.) and transferred to 25-ml Erlenmeyer flasks containing water or 0.1 mM IAA in the morning. Ethylene production was measured 7 and 19 h after addition of IAA. The average of two replicates is given
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did not stimulate the production of ethylene to any greater extent than ACC alone. On the contrary, from 6 h after the addition of ACC or ACC+ IAA, more ethylene was evolved from sections incubated with ACC alone than from those incubated with A C C + IAA. When sections were excised and incubated on ACC overnight prior to the addition of IAA, the pattern of stimulation of ethylene production was similar to that seen with freshly cut sections, even though ethylene production was reduced. However, in this case there was little difference between the quantities of ethylene produced by sections incubated with A C C + I A A compared to those incubated with ACC alone at any time after the addition of IAA (Table 1). Less ethylene was produced by sections incubated with 0.1 mM ACC than by those incubated in water alone. The reason for this is not clear. The increase in ethylene production in response to ACC was saturated at a concentration of 1 mM ACC, both in the presence and absence of IAA (Table 1). In contrast, Konze and Kende (1979b) found that conversion of ACC to ethylene in homogenates of pea stems could only be saturated at very high ACC concentrations (above 400 mM) or could not be saturated at all. This discrepancy either reflects differences in the mechanisms by which ACC is converted to ethylene in intact tissue versus the cell-free system or it indi-
H20 IAA 0.1 m M 0.1 m M 1.0 mM 1.0 m M 2.0 mM 2.0 mM 3.0 mM 3.0 m M 5.0 mM 5.0 mM 10.0 mM 10.0 m M
ACC ACC + IAA ACC ACC + IAA ACC ACC+IAA ACC ACC § IAA ACC ACC+IAA ACC ACC+IAA
Ethylene production (nmol/12 sections -+ S.D.9 7h
19h
1.08_+0.06 3.74 + 0.0I 0.01 +_0 2.07 -+ 0.44 5.81-+0.79 6.68 + 0.56 5.73 -+ 0.57 6.62_+0.33 5.98 + 0.45 7.25 +_0 5.26-+0.32 6.34+_0.64 5.30-+0.57 6.47_+0.08
1.55_+0.21 6.01 + 0.45 0.05-+0.02 4.19 -+ 0.70 15.05_+2.05 14.85 -+ 1.20 16.60 _+2.26 t5.50+_0.85 17.30 + 0.92 15.45 + 0.50 16.80+_ 1.13 15.35_+ 1.91 14.30_+0.71 14.55+_0.35
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cates the presence of a saturable uptake system for ACC in pea stem sections.
Endogenous Content of ACC When freshly excised pea stem sections were treated with IAA, the level of ACC increased in a manner similar to the increase in ethylene production (Fig. 2B). The increases in both ACC level and ethylene formation were evident after a lag of 2 h. The rate of ACC formation induced by IAA was much higher than the rate of ethylene production, however. AVG, an inhibitor of ethylene formation (see review by Lieberman, 1979) which had been shown to block the synthesis of ACC in apple tissue (Adams and Yang, 1979) and the ACC-forming enzyme from tomato (Boller et al., 1979), inhibited both IAA-induced ACC and ethylene production (Fig. 2A). Little ACC or ethylene was present in sections incubated with water alone (Fig. 2A). A comparison was made between ethylene production and ACC levels in sections incubated in different concentrations of IAA, (Fig. 3). The increases in the production of ethylene (Fig. 3 A) closely paralleled the increases in the levels of ACC (Fig. 3B), although again the increases in ACC were larger than the increases in ethylene production. The
652
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Fig. 2A and B. The effect of IAA and aminoethoxyvinylglycine (AVG) on ethylene production by and 1-aminocyclopropane-1carboxylic acid (ACC) content of subapical stem sections of etiolated pea seedlings. Twelve sections each were incubated on water alone (o), on 0.1 m M IAA (e) and 0.1 m M I A A + 0 . 1 m M AVG ( ' ) . At 0, 2, 4, 6 and 8 h after the sections were placed in their respective incubation media, they were extracted with 80% (v/v) ethanol+5 m M mercaptoethanol and chromatographed on cellulose thin-layer plates with chloroform-methanol-58 % (w/v) ammonia (2:2: 1, v/v). The zone which co-chromatographed with authentic ACC was assayed for ACC in a reaction which yielded ethylene. The experiment was performed in duplicate. - - -Ethylene production ; - ACC level. Note differences in the scale of the ordinates in A and B. The bar indicates the standard deviation
Labelling Studies
optimal concentration of IAA for both was between 0.1 and 1.0 mM. Since selenoethionine and selenomethionine stimulated IAA-induced ethylene production in subapical stem sections of etiolated pea seedlings (Konze et al., 1978), it was of interest to determine the effect of these selenoamino acids on the levels of ACC and [AA-induced ethylene production. Ethylene production and the levels of ACC in pea stem sections treated with IAA were greatly increased in the presence of the selenoamino acids (Fig. 4). The increase in ACC levels in sections treated with selenomethionine and selenoethionine became apparent prior to the increase in ethylene production. The selenoamino acids had little effect on ACC levels in the absence of IAA.
In order to establish that ACC is an intermediate in the pathway leading from methionine to ethylene in pea stem sections, freshly excised sections were incubated on L-[U-14C]methionine with or without IAA. The radioactivity incorporated into ACC, SMM and homoserine+methionine sulfoxide was determined for sections treated with and without IAA, and the specific radioactivity of the ethylene produced by the sections was compared with that of carbon atoms 2 and 3 of ACC. The total amount of radioactivity in the ethanol-soluble extract of the sections was found to increase with time of incubation on L-[U-14C]methionine; sections treated with IAA incorporated more radioactivity than sections incubated on water (Fig. 5). Radioactivity in trapped CO2 was also higher in flasks containing pea stem sections treated with IAA (data not shown). Radiochromatogram scans of the ethanol extracts showed three major peaks of radioactivity; these peaks had the same Rf
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values as authentic SMM, methionine and ACC, respectively. SMM was also identified by electrophoresis and ACC by the A C C assay. The peak of radioactivity with the Rf of ACC was only present in extracts from sections which had been treated with I A A for more than 2 h. The zones of the TLC plates which corresponded to these three peaks, and a fourth smaller and broader peak with the Rf value of homoserine and methionine sulfoxide, were scraped off, and the radioactivity in each zone was determined by liquid scintillation spectrometry. Most of the radioactivity was found to be associated with SMM (Fig. 5). A small amount of radioactivity had also been incorporated into compound(s) migrating with the Rf of homoserine and methionine sulfoxide. Radioactivity was also found in ACC but, in contrast to the other compounds studied, only in extracts from sections that had been treated with I A A (Fig. 5). The time course of incorporation of radioactivity from L-[U-14C]methionine into ethylene and the carbon atoms 2 and 3 of ACC, released as ethylene during the assay for ACC, were studied (Fig. 6). Radioac-
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Fig. 5. Radioactivity in the total ethanol extract and incorporated into S-methylmethionine(SMM), homoserine+ methionine sulfoxide, and 1-aminocyclopropane-l-carboxylicacid (ACC) of subapical stem sections from etiolated pea seedlings after treatment with L-[U-14C]methionine (2 gCi, 258 mCi/mmol) for 1 h prior to the addition of water or IAA (0.1 mM) for a further 0, 2, 4, 6 a n d 8 h. The ethanol extracts were chromatographed on cellulose thinlayer plates in 1-butanol-glacial acetic acid-water (60 : 15:25, v/v). The plates were scanned for radioactivity. The zones which cochromatographed with authentic SMM, homoserine+methionine sulfoxide, and ACC were scraped from the plates and the radioactivity was determined by liquid scintillation spectrometry. 9 plus IAA; o minus IAA
tivity was incorporated into ethylene produced by IAA-treated pea stem sections, and this incorporation closely paralleled the rise in ethylene production (Fig. 6A). In a similar manner, the radioactivity in carbon atoms 2 and 3 of A C C increased in parallel with the increase in A C C levels (Fig. 6 B). A comparison was made between the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC. The experiment was repeated twice, and the results of both experiments are given in Table 2. After treatment of pea stem sections with I A A for
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Fig. 6A. Total ethylene and [~4C]ethylene from 12 subapical stem sections of etiolated pea seedlings treated wit L-[U-14C]methionine (2 pCi, 258 mCi/mmol) for 1 h prior to the addition of water (o) or IAA (e) for2, 4, 6, and 8h. For the determination of radioactivity ethylene was removed from one flask at each time point and absorbed in mercuric perchlorate. The radioactivity in mercuric perchlorate was counted by liquid scintillation spectrometry. --[14C]ethylene; . . . . total ethylene. B 1-Aminocyclopropane-l-carboxylic acid (ACC) content and [14C]ethylene released in the ACC assay. Subapical pea stem sections were treated as described in Fig. 6A. Batches of 12 pea stem sections were extracted with 80% (v/v) ethanol, ehromatographed on cellulose thin-layer plates with chloroform-methanoI-58% (w/v) ammonia (2: 2:1, v/v), and the zone which co-chromatographed with authentic ACC was assayed for ACC in a reaction which yielded ethylene. The radioactivity in the ethylene released from the ACC using the ACC assay was determined as described for Fig. 6A. [14C]ethylene released from ACC; . . . . total ACC
more than 2 h, the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC were similar, indicating that all of the ethylene produced was derived from ACC. The specific radioactivities of ethylene and carbon atoms 2 and 3 of ACC, obtained from sections treated with IAA for 2 h, were dissimilar, that of ethylene being about double that of ACC. The radioactive ethylene produced after 2 h, which
would also include wound ethylene (Saltveit and Dilley, 1978), might have been derived from labelled ACC which had not yet fully equilibrated with the pool of unlabelled ACC in the tissue. An alternative explanation for the difference in the specific radioactivities of ethylene and C-2 and C-3 of ACC after 2 h is the large margin of error in this measurement resulting from the low quantities of ethylene, ACC and radioactivity produced.
ACC-Forming Enzyme Since IAA appears to regulate the formation of ACC, the effect of IAA on the level of the enzyme which catalyzes the conversion of SAM to ACC was investigated. It proved to be very difficult to recover measurable activities of this enzyme from pea homogenates. Since the activity of the corresponding enzyme preparation from tomato was inhibited when pea stem sections were ground with tomato pericarp, it appeared that one or more inhibitors were present in the pea homogenates. A variety of methods were employed in attempts to improve the recovery of the ACCforming enzyme from peas, ammonium sulfate precipitation, preparation of an acetone powder, addition of bovine serum albumin, ethylene diaminetetraacetic acid (EDTA) or sucrose to the extraction medium, and addition of different concentrations of pyridoxal phosphate, ATP and E D T A to the assay mixture. None of these procedures increased the activity of the enzyme, however. Ascorbate, iodoacetamide and diethyldithiocarbamate (DIECA) were not tried
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum Table 3. Activity of the 1-aminocyclopropane-l-carboxylic acid (ACC)-forming enzyme in subapical stem sections of etiolated pea seedlings after treatment with IAA. Pea stem sections were incubated with 0.1 m M I A A or water, homogenized and the homogenates were chromatographed on columns of Sephadex G-50 (10 cm in length). Fractions of 0.8 ml were collected. The protein-containing fractions were combined a n d used as the enzyme preparation. Bovine serum albumin (BSA) (1 mg/ml) was added to the extraction buffer in one experiment, a n d this added protein was accounted for in the determination of the specific activity of the enzyme. An acetone powder was prepared for another experiment, dissolved in column buffer and desalted on a Sephadex G-50 column. Aliquots of 400 gl were taken from the enzyme preparations and incubated with HEPES buffer, p H 8.0, 5 m M A T P (homogenate experiment, only) and 45 g M S-adenosylmethionine for 3 h at 30 ~ C. Four replicates were assayed for each treatment
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as components of the extraction buffer since they either inhibited (ascorbate and iodoacetamide) the tomato enzyme preparation or increased the blank value of the assay (DIECA). Although the activity of the ACC-forming enzyme from pea stem sections was low, it could be shown that it was higher in homogenates of IAA-treated than in control stem sections (Table 3).
Discussion
Since application of exogenous ACC to pea stem sections stimulated the production of ethylene (Fig. 1), it seemed probable that ACC was an intermediate in the production of ethylene in this tissue. This hypothesis was supported by our finding that endogenous levels of ACC increased at the same time as ethylene production in pea stem sections treated with IAA (Fig. 2). Furthermore, AVG inhibited both IAAinduced ethylene production and ACC formation (Fig. 2). Selenoethionine and selenomethionine stimulated ethylene synthesis in subapical sections of etiolated pea seedlings (Konze et al., 1978). Experiments with senescing flower tissue of Ipornoea tricolor showed that selenomethionine was a better substrate of me-
655
thionine adenosyltransferase, the enzyme that converts methionine to SAM, than was methionine (Konze and Kende, 1979a). Since selenomethionine apparently stimulated the production of ethylene by enhancing the conversion of methionine to SAM, SAM was proposed to be an intermediate in all systems where these selenamino acids enhanced the production of ethylene. Application of selenoethionine and selenomethionine resulted in increased levels of ACC and in enhanced ethylene production in pea stem sections (Fig. 4); therefore, the occurrence of the pathway methionine ~ SAM --, ACC ---,ethylene was substantiated for this tissue. This conclusion was further strengthened by results of labelling experiments which showed conclusively that methionine was converted to ethylene via ACC. Determinations of the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC after feeding L-[U-14C]methio nine to sections treated with IAA demonstrated that all of the ethylene formed in response to IAA could be accounted for as originating from ACC (Table 2). The labelling experiments also showed that the rate of ACC formation in pea stem sections treated with IAA and I A A + selenoamino acids was higher than the rate of ethylene formation under the same conditions (Figs. 2 4 ) . Apart from ACC, SMM was a major metabolite of methionine. This compound did not appear to be a close precursor of ethylene, however (Schilling and Kende, 1979). Application of exogenous ACC resulted in the immediate production of ethylene in pea stem sections whereas application of IAA was followed by a lag period of 2-3 h before a rise in ethylene production was apparent (Fig. 1). This, together with the fact that ACC stimulated the production of ethylene even in the absence of IAA (Fig. 1), indicated that the formation of ACC was the rate-limiting step in ethylene formation by etiolated pea stem sections. This hypothesis was supported by our finding that IAA induced the formation of ACC at the same time as it induced the synthesis of ethylene (Fig. 2). Furthermore, the amount of ACC formed was directly related to the concentration of IAA applied (Fig. 3). The ACC-forming enzyme isolated from tomatoes is inhibited by AVG, an antagonist of pyridoxalphosphate-dependent enzymes (Boller et al., 1979). The formation of ACC in pea stem sections is probably also mediated by a pyridoxal-phosphate-dependent enzyme since AVG inhibits both IAA-induced formation of ACC and ethylene (Fig. 2). Therefore, it is very likely that the same or a similar enzyme is present in both pea stem sections and tomato fruit, despite the low activity of the enzyme found in peas. IAA enhances the activity of this enzyme, but it is not known whether this increase in activity is based
656
on de-novo synthesis or activation of the enzyme. Studies with the protein synthesis inhibitor cycloheximide, have shown that IAA-induced ethylene production requires continuous protein synthesis (Kang et al. 1971 ; Lieberman and Kunishi, 1975). The ACCforming enzyme may, therefore, be synthesized denovo in response to IAA treatment. It can be concluded that IAA induces the synthesis of ethylene in etiolated pea stem sections through a stimulation of the activity of the enzyme which converts SAM to ACC. A similar hypothesis was proposed by Yu et al. (1979) who showed that the conversion of ACC to ethylene was not dependent on IAA in mung-bean hypocotyls. This research was supported by the U.S. Department of Energy under Contract No. EY-76-C-02-1338, and by the National Science Foundation through Grant No. PCM 77-08522 to H.K.
References Adams, D.O., Yang, S.F.: Ethylene biosynthesis: Identification of 1-aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Nat. Acad. Sci. USA 76, 170-174 (1979) Boller, T., Herner, R.C., Kende, H.: Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1carboxylic acid. Planta 145, 293 303 (1979) Bradford, M.M. : A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976) Burg, S.P., Burg, E.A. : The interaction between auxin and ethylene and its role in plant growth. Proc. Nat. Acad. Sci. USA 55, 262-269 (1966)
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum Hanson, A.D., Kende, H.: Methionine metabolism and ethylene biosynthesis in senescent flower tissue of morning-glory. Plant Physiol. 57, 528-537 (1976) Kang, B.G., Newcomb, W., Burg, S.P.: Mechanism of auxininduced ethylene production. Plant Physiol. 47, 504-509 (1971) Kende, H., Hanson, A.D.: Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiol. 57, 523-527 (1976) Konze, J.R., Schilling, N., Kende, H.: Enhancement of ethylene formation by selenoamino acids. Plant Physiol. 62, 397 401 (1978) Konze, J.R., Kende, H. : Interactions of methionine and selenomethionine with methionine adenosyltransferase and ethylene generating systems. Plant Physiol. 63, 507 510 (1979a) Konze, J.R., Kende, H.: Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings. Planta 146, 293-301 (1979b) Lieberman, M., Kunishi, A.T.: Ethylene forming systems in etiolated pea seedling and apple tissue. Plant Physiol 55, 1074-1078 (1975) Lieberman, M.: Biosynthesis and action of ethylene. Ann. Rev. Plant Physiol. 30, 533-591 (1979) Sakai, S., Imaseki, H.: Auxin-induced ethylene production by mung bean hypocotyl segments. Plant Cell Physiol. 12, 349-359 (1971) Saltveit, M.E., Dilley, D.R. : Rapidly induced wound ethylene from excised segments of etiolated Pisum sativum L., cv. Alaska. Plant Physiol. 61,447-450 (1978) Schilling, N., Kende, H. : Methionine metabolism and ethylene formation in etiolated pea stem sections. Plant Physiol. 63, 639-642 (1979) Yu, Y., Adams, D.O., Yang, S.F.: Regulation of auxin-induced ethylene production in mung bean hypocotyls. Role of 1-aminocyclopropane-l-carboxylic acid. Plant Physiol. 63, 589 590 (1979)
Received 7 June; accepted 18 July 1979
Planta 146,649-656 (1979)
9 by Springer-Verlag 1979
Auxin-induced Ethylene Biosynthesis in Subapical Stem Sections of Etiolated Seedlings of Pisum sativum L. Jennifer F. Jones and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. 1-Aminocyclopropane-l-carboxylic acid (ACC) stimulated the production of ethylene in subapical stem sections ofetiolated pea (cv. Alaska) seedlings in the presence and absence of indole-3-acetic acid (IAA). No lag period was evident following application of ACC, and the response was saturated at a concentration of 1 m M ACC. Levels of endogenous ACC paralleled the increase in ethylene production in sections treated with different concentrations of IAA and with selenoethionine or selenomethionine plus IAA. The IAA-induced formation of both ACC and ethylene was blocked by the rhizobitoxine analog aminoethoxyvinylglycine (AVG). Labelling studies with L-[U-14C]methionine showed an increase in the labelling of ethylene and ACC after treatment with IAA. IAA had no specific effect on the incorporation of label into S-methylmethionine or homoserine. The specific radioactivity of ethylene was similar to the specific radioactivity of carbon atoms 2 and 3 of ACC after treatment with IAA, indicating that all of the ethylene was derived from ACC. The activity of the ACC-forming enzyme was higher in sections i n c u bated with IAA than in sections incubated with water alone. These results support the hypothesis that ACC is the in-vivo precursor of ethylene in etiolated pea tissue and that IAA stimulates ethylene production by increasing the activity of the ACC-forming enzyme.
One control point for IAA-induced ethylene formation is the level of free IAA in the tissue, and this is closely related to the rates of conjugation of IAA (Kang et al., 1971). Another possible control point lies in the rates of formation and degradation of the ethylene-forming systems (Kang et al., 1971 ; Lieberman and Kunishi, 1975; Sakai and Imaseki, 1971). The pathway of ethylene biosynthesis has recently been identified in apples (Adams and Yang, 1979) and in tomato fruit (Boller et al., 1979) as the sequence methionine~S-adenosylmethionine (SAM) ~l-aminocyclopropane- 1-carboxylic acid (ACC) --,ethylene. In stem sections of peas and in flower tissue of Ipornoea tricolor (Konze et al., 1978), selenomethionine has been found to be a better precursor in this pathway than methionine. It has been proposed that the conversion of SAM to ACC is the step at which ethylene biosynthesis is regulated (Adams and Yang, 1979 ; Boller et al., 1979). Yu et al. (1979) have further proposed that IAA induces the synthesis of the ACC-forming enzyme in mung bean hypocotyls. In this paper, we report on experiments which were designed to determine a) whether or not ACC is an intermediate in the pathway leading from methionine to ethylene in pea stem sections treated with IAA, and b) the point of action of IAA in the ethylene-forming system in this tissue.
Key words: 1-Aminocyclopropane-l-carboxylic acid Auxin - Ethylene - P i s u m Rhizobitoxine analog. Material and Methods Introduction
Plant Material and Conditions of Incubation
IAA induces the formation of ethylene in stem sections of etiolated pea seedlings (Burg and Burg, 1966).
Pea seeds, Pisum sativum L. cv. Alaska (Vaughan-Jacklin Corp., Ovid, Mich., USA), were imbibed overnight in aerated tap water, sown in Vermiculite, and grown in the dark at 25~ for 5-7 days. Subapical stem sections (0.6 cm) were excised directly below the apical hook, and were placed in 25-ml Erlenmeyer flasks (12 sections per flask) on a disc of Whatman No. 1 filter paper wetted with 1 ml water or test solution. All manipulations were
ACC= 1-aminocyclopropane-l-carboxylic acid; AVG=aminoethoxyvinylglycine,the aminoethoxy analog of rhizobitoxine; IAA=indole-3-acetic acid; SAM-S-adenosylmethionine ; SMM = S-methylmethionine Abbreviations:
0032-0935/79/0146/0649/$01.60
650 performed under a green (530-590 nm) safelight at an energy fluence of less than 0.001 W m- 2 and the sections remained in the dark until they were extracted.
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum were scraped off and counted by scintillation spectrometry using ACS scintillation solvent (Amersham Corp., Arlington Heights, I11., USA). The major peaks were identified by co-chromatography with the authentic compounds; S-methylmethionine (SMM) was further identified by electrophoresis (Hanson and Kende, 1976).
Determination of Ethylene Production and ACC Content Ethylene production was measured by gas chromatography as described by Kende and Hanson (1976). In order to determine the content of ACC, 12 pea stem sections were ground in a glass homogenizer with 2 ml 80% (v/v) ethanol containing 5 mM mercaptoethanol. The initial extract and a further 1 ml wash were combined and left for 1 h at 0~ to precipitate the protein. The extract was then centrifuged at 12,000 x g for 15 min in a Sorvall Model RC-2B centrifuge using an SS-34 rotor (DuPont Instruments-Sorvall, Wilmington, Del., USA). The pellet was extracted twice more with 1 ml extraction medium, and the combined supernatants were dried under nitrogen at 45 ~ C. The residue was taken up in 75 gi 50% (v/v) ethanol of which 10 gl were spotted in a line on 5-cm wide strips of precoated 0.1-mm cellulose MN 300 plates (Brinkman Instruments, Westbury, N.Y., USA). The chromatograms were run in chloroform-methanol-58% (w/v) ammonia (2:2: 1, v/v) or in 1-butanol-glacial acetic acid-water (60:15:25, v/v) until the solvent front had migrated 15 cm. The position of an ACC standard run on a separate plate was determined by spraying the plate with ninhydrin. The zones on the chromatograms which corresponded to the position of ACC were scraped off and eluted with 1.8 ml 100 mM sodium-phosphate buffer, pH 11.5. The fractions were centrifuged for 2-3 min to sediment the cellulose. Two aliquots of 0.7 ml were removed from each fraction, and 10 nmol authentic ACC was added to one of these aliquots prior to assaying both portions for ACC as described previously (Boller et al., 1979). The addition of a known quantity of ACC to one aliquot from each extract allowed corrections to be made for inhibition of the assay by the extract. This inhibition was never greater than 50%. Losses of ACC during extraction and chromatography were determined by the addition of a known quantity of ACC to a duplicate sample of tissue at the beginning of the extraction. Recovery ranged from 66-78% and was independent of the concentration of ACC applied. The assay for ACC worked well on chromatographed extracts of pea stem sections, provided that only a small quantity of material was used. An amount of extract equivalent to one half of a section, 0.6 cm in length and ca. 12 mg in weight, was routinely assayed. Larger quantities of material inhibited the assay, and neither passing the extracts through a cation exchange resin (Dowex-50W) or charcoal, or partitioning with butanol, ethyl acetate or ether, nor chromatography with different TLC solvents alleviated this inhibition.
ACC-forming Enzyme: Preparation and Assay Sections from etiolated pea seedlings were treated with IAA as described above and extracted with 2 ml extraction buffer per g fresh weight of tissue, consisting of 100 mM N-2-hydroxyethylpiperazinc-N'-2-ethanesulfonicacid (HEPES) buffer, pH 8.0, containing 4 mM D,L-dithiothreitol (DTT) and 0.4 gM pyridoxal phosphate. For some experiments, bovine serum albumin (BSA) was added to the extraction buffer at 1 mg per ml. The homogehates were filtered through a 50-gm-mesh nylon net and centrifuged at 12,000 x g for 20 min. The supernatant was layered onto columns (10 or 40 cm in length, 10 or 200 ml bed volume, respectively) of Sephadex G-50 (Pharmacia Fine Chemicals, Piscataway, N.J., USA). The columns were equilibrated and eluted with 2 mM KHEPES buffer at pH 8.0 containing 0.1 mM DTT and 0.2 pM pyridoxal phosphate. Fractions of 1.6 ml were collected from the large column and of 0.8 ml from the small column. Alternatively, acetone powders were prepared from the pea sections, dessicated, and stored at.4 ~ C overnight prior to use. The powder was then dissolved in column buffer (4 ml per g original fresh weight of tissue) at 4~ C, left to stand for 1 h, centrifuged at 12,000• for 20 min, and the supernatant layered on a column of Sephadex G-50 and eluted as described above. The fractions containing protein were detected by testing an aliquot from each fraction by the method of Bradford (1976). The fractions containing protein were combined and used as the enzyme preparation. All manipulations were performed at 0-4 ~ C. The enzyme preparation was assayed as described by Boller et al. (1979), except that ATP was added to the incubation mixture to give a final concentration of 5 mM. The concentration of protein in the enzyme assay was determined according to Bradford (1976).
Chemica~ L-[U-~4C]Methionine (specific activity 250-260 mCi/mmol) was purchased from New England Nuclear, Boston, Mass., USA; D, L-methylmethinone sulfonium chloride from U.S. Biochemical Corp., Cleveland, O., USA; and all other biochemicals either from Calbiochem-Behring Corp., La Jolla, Cal., USA or Sigma Chemical Co., St. Louis, Mo., USA. Aminoethoxyvinylglycine (AVG) was a gift from Dr. M. Lieberman (U.S. Department of Agriculture, Beltsville, Md., USA).
Radiotracer Experiments with L-[ U-14C] Methionine Twelve pea stem sections were incubated in 25-ml Erlenmeyer flasks containing 2 g Ci L[U-~*C]methionine (258 mCi/mmol) dissolved in 0.9 ml water and a CO z trap consisting of a suspended vial with 751al of 1 N NaOH saturated with Ba(OH)2 and a fluted filter paper wick. After 1 h, 100 ~1 of either 1 mM IAA or water were injected through the serum-vial caps into the flasks. The specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC were determined as described by Hanson and Kende (1976) and Boller et al. (1979). The pea stem sections were extracted as described above for the determination of ACC and chromatographed in the acidic TLC solvent. The radioactivity on the plates was located with a Radiochromatogram Scanner, Model 7201 (Packard Instruments, Downers Grove, I11., USA). The zones on the plates which corresponded to the major radioactive peaks
Results Effect o f Application o f A C C When freshly excised pea stem sections were placed in 0.1 m M I A A , t h e r e w a s a lag p e r i o d o f 2 - 3 h b e f o r e an increase in e t h y l e n e p r o d u c t i o n b e c a m e e v i d e n t (Fig. 1). W h e n t h e s e c t i o n s w e r e p l a c e d o n a s o l u t i o n of 1 mM ACC or 1 mM ACC+IAA, t h e rise in e t h ylene production was immediate, and the pea sections p r o d u c e d m u c h g r e a t e r (ca. 3 - f o l d ) q u a n t i t i e s o f e t h ylene than those incubated on IAA alone. IAA + ACC
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651 Table 1. The effect of different concentrations of 1-aminocyclopropane-l-carboxylic acid (ACC) on ethylene production in subapical stem sections of etiolated pea seedlings. Pea sections, 0.6 cm in length, were incubated on water or ACC (0.1, 1, 2, 3, 5, and 10 mM) overnight (5:00 p.m.-8:30 a.m.) and transferred to 25-ml Erlenmeyer flasks containing water or 0.1 mM IAA in the morning. Ethylene production was measured 7 and 19 h after addition of IAA. The average of two replicates is given
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did not stimulate the production of ethylene to any greater extent than ACC alone. On the contrary, from 6 h after the addition of ACC or ACC+ IAA, more ethylene was evolved from sections incubated with ACC alone than from those incubated with A C C + IAA. When sections were excised and incubated on ACC overnight prior to the addition of IAA, the pattern of stimulation of ethylene production was similar to that seen with freshly cut sections, even though ethylene production was reduced. However, in this case there was little difference between the quantities of ethylene produced by sections incubated with A C C + I A A compared to those incubated with ACC alone at any time after the addition of IAA (Table 1). Less ethylene was produced by sections incubated with 0.1 mM ACC than by those incubated in water alone. The reason for this is not clear. The increase in ethylene production in response to ACC was saturated at a concentration of 1 mM ACC, both in the presence and absence of IAA (Table 1). In contrast, Konze and Kende (1979b) found that conversion of ACC to ethylene in homogenates of pea stems could only be saturated at very high ACC concentrations (above 400 mM) or could not be saturated at all. This discrepancy either reflects differences in the mechanisms by which ACC is converted to ethylene in intact tissue versus the cell-free system or it indi-
H20 IAA 0.1 m M 0.1 m M 1.0 mM 1.0 m M 2.0 mM 2.0 mM 3.0 mM 3.0 m M 5.0 mM 5.0 mM 10.0 mM 10.0 m M
ACC ACC + IAA ACC ACC + IAA ACC ACC+IAA ACC ACC § IAA ACC ACC+IAA ACC ACC+IAA
Ethylene production (nmol/12 sections -+ S.D.9 7h
19h
1.08_+0.06 3.74 + 0.0I 0.01 +_0 2.07 -+ 0.44 5.81-+0.79 6.68 + 0.56 5.73 -+ 0.57 6.62_+0.33 5.98 + 0.45 7.25 +_0 5.26-+0.32 6.34+_0.64 5.30-+0.57 6.47_+0.08
1.55_+0.21 6.01 + 0.45 0.05-+0.02 4.19 -+ 0.70 15.05_+2.05 14.85 -+ 1.20 16.60 _+2.26 t5.50+_0.85 17.30 + 0.92 15.45 + 0.50 16.80+_ 1.13 15.35_+ 1.91 14.30_+0.71 14.55+_0.35
Standard deviation
cates the presence of a saturable uptake system for ACC in pea stem sections.
Endogenous Content of ACC When freshly excised pea stem sections were treated with IAA, the level of ACC increased in a manner similar to the increase in ethylene production (Fig. 2B). The increases in both ACC level and ethylene formation were evident after a lag of 2 h. The rate of ACC formation induced by IAA was much higher than the rate of ethylene production, however. AVG, an inhibitor of ethylene formation (see review by Lieberman, 1979) which had been shown to block the synthesis of ACC in apple tissue (Adams and Yang, 1979) and the ACC-forming enzyme from tomato (Boller et al., 1979), inhibited both IAA-induced ACC and ethylene production (Fig. 2A). Little ACC or ethylene was present in sections incubated with water alone (Fig. 2A). A comparison was made between ethylene production and ACC levels in sections incubated in different concentrations of IAA, (Fig. 3). The increases in the production of ethylene (Fig. 3 A) closely paralleled the increases in the levels of ACC (Fig. 3B), although again the increases in ACC were larger than the increases in ethylene production. The
652
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Fig. 2A and B. The effect of IAA and aminoethoxyvinylglycine (AVG) on ethylene production by and 1-aminocyclopropane-1carboxylic acid (ACC) content of subapical stem sections of etiolated pea seedlings. Twelve sections each were incubated on water alone (o), on 0.1 m M IAA (e) and 0.1 m M I A A + 0 . 1 m M AVG ( ' ) . At 0, 2, 4, 6 and 8 h after the sections were placed in their respective incubation media, they were extracted with 80% (v/v) ethanol+5 m M mercaptoethanol and chromatographed on cellulose thin-layer plates with chloroform-methanol-58 % (w/v) ammonia (2:2: 1, v/v). The zone which co-chromatographed with authentic ACC was assayed for ACC in a reaction which yielded ethylene. The experiment was performed in duplicate. - - -Ethylene production ; - ACC level. Note differences in the scale of the ordinates in A and B. The bar indicates the standard deviation
Labelling Studies
optimal concentration of IAA for both was between 0.1 and 1.0 mM. Since selenoethionine and selenomethionine stimulated IAA-induced ethylene production in subapical stem sections of etiolated pea seedlings (Konze et al., 1978), it was of interest to determine the effect of these selenoamino acids on the levels of ACC and [AA-induced ethylene production. Ethylene production and the levels of ACC in pea stem sections treated with IAA were greatly increased in the presence of the selenoamino acids (Fig. 4). The increase in ACC levels in sections treated with selenomethionine and selenoethionine became apparent prior to the increase in ethylene production. The selenoamino acids had little effect on ACC levels in the absence of IAA.
In order to establish that ACC is an intermediate in the pathway leading from methionine to ethylene in pea stem sections, freshly excised sections were incubated on L-[U-14C]methionine with or without IAA. The radioactivity incorporated into ACC, SMM and homoserine+methionine sulfoxide was determined for sections treated with and without IAA, and the specific radioactivity of the ethylene produced by the sections was compared with that of carbon atoms 2 and 3 of ACC. The total amount of radioactivity in the ethanol-soluble extract of the sections was found to increase with time of incubation on L-[U-14C]methionine; sections treated with IAA incorporated more radioactivity than sections incubated on water (Fig. 5). Radioactivity in trapped CO2 was also higher in flasks containing pea stem sections treated with IAA (data not shown). Radiochromatogram scans of the ethanol extracts showed three major peaks of radioactivity; these peaks had the same Rf
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J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum 1.5
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tivity was incorporated into ethylene produced by IAA-treated pea stem sections, and this incorporation closely paralleled the rise in ethylene production (Fig. 6A). In a similar manner, the radioactivity in carbon atoms 2 and 3 of A C C increased in parallel with the increase in A C C levels (Fig. 6 B). A comparison was made between the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC. The experiment was repeated twice, and the results of both experiments are given in Table 2. After treatment of pea stem sections with I A A for
654
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E
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Table 2. A comparison of the specific radioactivity of ethylene produced by pea stem sections with that of carbon atoms 2 and 3 of endogenous 1-aminocyclopropane-l-carboxylic acid (ACC). Batches of 12 pea stem sections were incubated in 25-ml Erlenmeyer flasks in L-[U-14C]methionine (2 ~tCi, 258 mCi/mmol) for 1 h before the addition of IAA (0.1 mM). After 2, 4, 6 and 8 h, the level and radioactivity of ethylene in one flask was determined and the pea stem sections from that flask were extracted. The extracts were chromatographed on cellulose thin-layer plates. The amount of ACC and radioactivity of carbon atoms 2 and 3 of ACC were determined with the ACC assay using the appropriate chromatographic zone
O
Length of treatment with IAA (h)
k5 c
15
B
2
4
8
6
,/""
30 !
Specific radioactivity (nCi/nmol) Ethylene
C-2 and C-3 of ACC
2 4 6
1.69 (1.28) a 1.09 (0.96) 1.01 (n.a.) b
0.53 (0.43) 0.97 (0.85) 0.77 (n.a.)
8
0.99 (0.91)
O.78 (O.93)
W I
u
Results of a 2nd experiment are given in paranthesis n.a. = n o t assayed
2 0 ea
c
5
.,4,"
-llo
J 0
~ ~ - - - 9 ...... 0- . . . . . . . 2 4 6 Hours from IAA oddition
uu
/" 0 j
8
Fig. 6A. Total ethylene and [~4C]ethylene from 12 subapical stem sections of etiolated pea seedlings treated wit L-[U-14C]methionine (2 pCi, 258 mCi/mmol) for 1 h prior to the addition of water (o) or IAA (e) for2, 4, 6, and 8h. For the determination of radioactivity ethylene was removed from one flask at each time point and absorbed in mercuric perchlorate. The radioactivity in mercuric perchlorate was counted by liquid scintillation spectrometry. --[14C]ethylene; . . . . total ethylene. B 1-Aminocyclopropane-l-carboxylic acid (ACC) content and [14C]ethylene released in the ACC assay. Subapical pea stem sections were treated as described in Fig. 6A. Batches of 12 pea stem sections were extracted with 80% (v/v) ethanol, ehromatographed on cellulose thin-layer plates with chloroform-methanoI-58% (w/v) ammonia (2: 2:1, v/v), and the zone which co-chromatographed with authentic ACC was assayed for ACC in a reaction which yielded ethylene. The radioactivity in the ethylene released from the ACC using the ACC assay was determined as described for Fig. 6A. [14C]ethylene released from ACC; . . . . total ACC
more than 2 h, the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC were similar, indicating that all of the ethylene produced was derived from ACC. The specific radioactivities of ethylene and carbon atoms 2 and 3 of ACC, obtained from sections treated with IAA for 2 h, were dissimilar, that of ethylene being about double that of ACC. The radioactive ethylene produced after 2 h, which
would also include wound ethylene (Saltveit and Dilley, 1978), might have been derived from labelled ACC which had not yet fully equilibrated with the pool of unlabelled ACC in the tissue. An alternative explanation for the difference in the specific radioactivities of ethylene and C-2 and C-3 of ACC after 2 h is the large margin of error in this measurement resulting from the low quantities of ethylene, ACC and radioactivity produced.
ACC-Forming Enzyme Since IAA appears to regulate the formation of ACC, the effect of IAA on the level of the enzyme which catalyzes the conversion of SAM to ACC was investigated. It proved to be very difficult to recover measurable activities of this enzyme from pea homogenates. Since the activity of the corresponding enzyme preparation from tomato was inhibited when pea stem sections were ground with tomato pericarp, it appeared that one or more inhibitors were present in the pea homogenates. A variety of methods were employed in attempts to improve the recovery of the ACCforming enzyme from peas, ammonium sulfate precipitation, preparation of an acetone powder, addition of bovine serum albumin, ethylene diaminetetraacetic acid (EDTA) or sucrose to the extraction medium, and addition of different concentrations of pyridoxal phosphate, ATP and E D T A to the assay mixture. None of these procedures increased the activity of the enzyme, however. Ascorbate, iodoacetamide and diethyldithiocarbamate (DIECA) were not tried
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum Table 3. Activity of the 1-aminocyclopropane-l-carboxylic acid (ACC)-forming enzyme in subapical stem sections of etiolated pea seedlings after treatment with IAA. Pea stem sections were incubated with 0.1 m M I A A or water, homogenized and the homogenates were chromatographed on columns of Sephadex G-50 (10 cm in length). Fractions of 0.8 ml were collected. The protein-containing fractions were combined a n d used as the enzyme preparation. Bovine serum albumin (BSA) (1 mg/ml) was added to the extraction buffer in one experiment, a n d this added protein was accounted for in the determination of the specific activity of the enzyme. An acetone powder was prepared for another experiment, dissolved in column buffer and desalted on a Sephadex G-50 column. Aliquots of 400 gl were taken from the enzyme preparations and incubated with HEPES buffer, p H 8.0, 5 m M A T P (homogenate experiment, only) and 45 g M S-adenosylmethionine for 3 h at 30 ~ C. Four replicates were assayed for each treatment
Treatment
Length of treatment
Specific activity_+ S.D. (nmol A C C m g - 1 protein 3 h - 1)
(h)
IAA H20 IAA H20 a
b
3 3 6 6
Homogenate
Acetone powder
Homogenate + BSA
0.080_+0.002 0 b 0.180+0.007 0b
n.a. a n.a. 0.210+_0.034 0.008_+0.008
n.a. n.a. 0.056+0.013 0.013+0.006
n . a . = n o t assayed N o detectable enzyme levels
as components of the extraction buffer since they either inhibited (ascorbate and iodoacetamide) the tomato enzyme preparation or increased the blank value of the assay (DIECA). Although the activity of the ACC-forming enzyme from pea stem sections was low, it could be shown that it was higher in homogenates of IAA-treated than in control stem sections (Table 3).
Discussion
Since application of exogenous ACC to pea stem sections stimulated the production of ethylene (Fig. 1), it seemed probable that ACC was an intermediate in the production of ethylene in this tissue. This hypothesis was supported by our finding that endogenous levels of ACC increased at the same time as ethylene production in pea stem sections treated with IAA (Fig. 2). Furthermore, AVG inhibited both IAAinduced ethylene production and ACC formation (Fig. 2). Selenoethionine and selenomethionine stimulated ethylene synthesis in subapical sections of etiolated pea seedlings (Konze et al., 1978). Experiments with senescing flower tissue of Ipornoea tricolor showed that selenomethionine was a better substrate of me-
655
thionine adenosyltransferase, the enzyme that converts methionine to SAM, than was methionine (Konze and Kende, 1979a). Since selenomethionine apparently stimulated the production of ethylene by enhancing the conversion of methionine to SAM, SAM was proposed to be an intermediate in all systems where these selenamino acids enhanced the production of ethylene. Application of selenoethionine and selenomethionine resulted in increased levels of ACC and in enhanced ethylene production in pea stem sections (Fig. 4); therefore, the occurrence of the pathway methionine ~ SAM --, ACC ---,ethylene was substantiated for this tissue. This conclusion was further strengthened by results of labelling experiments which showed conclusively that methionine was converted to ethylene via ACC. Determinations of the specific radioactivities of ethylene and of carbon atoms 2 and 3 of ACC after feeding L-[U-14C]methio nine to sections treated with IAA demonstrated that all of the ethylene formed in response to IAA could be accounted for as originating from ACC (Table 2). The labelling experiments also showed that the rate of ACC formation in pea stem sections treated with IAA and I A A + selenoamino acids was higher than the rate of ethylene formation under the same conditions (Figs. 2 4 ) . Apart from ACC, SMM was a major metabolite of methionine. This compound did not appear to be a close precursor of ethylene, however (Schilling and Kende, 1979). Application of exogenous ACC resulted in the immediate production of ethylene in pea stem sections whereas application of IAA was followed by a lag period of 2-3 h before a rise in ethylene production was apparent (Fig. 1). This, together with the fact that ACC stimulated the production of ethylene even in the absence of IAA (Fig. 1), indicated that the formation of ACC was the rate-limiting step in ethylene formation by etiolated pea stem sections. This hypothesis was supported by our finding that IAA induced the formation of ACC at the same time as it induced the synthesis of ethylene (Fig. 2). Furthermore, the amount of ACC formed was directly related to the concentration of IAA applied (Fig. 3). The ACC-forming enzyme isolated from tomatoes is inhibited by AVG, an antagonist of pyridoxalphosphate-dependent enzymes (Boller et al., 1979). The formation of ACC in pea stem sections is probably also mediated by a pyridoxal-phosphate-dependent enzyme since AVG inhibits both IAA-induced formation of ACC and ethylene (Fig. 2). Therefore, it is very likely that the same or a similar enzyme is present in both pea stem sections and tomato fruit, despite the low activity of the enzyme found in peas. IAA enhances the activity of this enzyme, but it is not known whether this increase in activity is based
656
on de-novo synthesis or activation of the enzyme. Studies with the protein synthesis inhibitor cycloheximide, have shown that IAA-induced ethylene production requires continuous protein synthesis (Kang et al. 1971 ; Lieberman and Kunishi, 1975). The ACCforming enzyme may, therefore, be synthesized denovo in response to IAA treatment. It can be concluded that IAA induces the synthesis of ethylene in etiolated pea stem sections through a stimulation of the activity of the enzyme which converts SAM to ACC. A similar hypothesis was proposed by Yu et al. (1979) who showed that the conversion of ACC to ethylene was not dependent on IAA in mung-bean hypocotyls. This research was supported by the U.S. Department of Energy under Contract No. EY-76-C-02-1338, and by the National Science Foundation through Grant No. PCM 77-08522 to H.K.
References Adams, D.O., Yang, S.F.: Ethylene biosynthesis: Identification of 1-aminocyclopropane-l-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. Nat. Acad. Sci. USA 76, 170-174 (1979) Boller, T., Herner, R.C., Kende, H.: Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1carboxylic acid. Planta 145, 293 303 (1979) Bradford, M.M. : A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 (1976) Burg, S.P., Burg, E.A. : The interaction between auxin and ethylene and its role in plant growth. Proc. Nat. Acad. Sci. USA 55, 262-269 (1966)
J.F. Jones and H. Kende: IAA-induced Ethylene Biosynthesis in Pisum Hanson, A.D., Kende, H.: Methionine metabolism and ethylene biosynthesis in senescent flower tissue of morning-glory. Plant Physiol. 57, 528-537 (1976) Kang, B.G., Newcomb, W., Burg, S.P.: Mechanism of auxininduced ethylene production. Plant Physiol. 47, 504-509 (1971) Kende, H., Hanson, A.D.: Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiol. 57, 523-527 (1976) Konze, J.R., Schilling, N., Kende, H.: Enhancement of ethylene formation by selenoamino acids. Plant Physiol. 62, 397 401 (1978) Konze, J.R., Kende, H. : Interactions of methionine and selenomethionine with methionine adenosyltransferase and ethylene generating systems. Plant Physiol. 63, 507 510 (1979a) Konze, J.R., Kende, H.: Ethylene formation from 1-aminocyclopropane-1-carboxylic acid in homogenates of etiolated pea seedlings. Planta 146, 293-301 (1979b) Lieberman, M., Kunishi, A.T.: Ethylene forming systems in etiolated pea seedling and apple tissue. Plant Physiol 55, 1074-1078 (1975) Lieberman, M.: Biosynthesis and action of ethylene. Ann. Rev. Plant Physiol. 30, 533-591 (1979) Sakai, S., Imaseki, H.: Auxin-induced ethylene production by mung bean hypocotyl segments. Plant Cell Physiol. 12, 349-359 (1971) Saltveit, M.E., Dilley, D.R. : Rapidly induced wound ethylene from excised segments of etiolated Pisum sativum L., cv. Alaska. Plant Physiol. 61,447-450 (1978) Schilling, N., Kende, H. : Methionine metabolism and ethylene formation in etiolated pea stem sections. Plant Physiol. 63, 639-642 (1979) Yu, Y., Adams, D.O., Yang, S.F.: Regulation of auxin-induced ethylene production in mung bean hypocotyls. Role of 1-aminocyclopropane-l-carboxylic acid. Plant Physiol. 63, 589 590 (1979)
Received 7 June; accepted 18 July 1979
