Planta
Planta (Berl.) 132, 1 3 - 17 (1976)
9 by Springer-Verlag 1976
Some Characteristics of Ethylene Production in Peach (Prunus persica L.) Seeds P.H. Jerie and D.J. Chalmers Horticultural Research Station, Victorian Department of Agriculture, Tatura, Vic. 3616, Australia
Summary. The seed of peach fruits develop the capacity to produce ethylene with a lag phase of about 1 h after excision. The site of ethylene synthesis is in the seed coat and rates as high as 6,000 gl kg-1 h-1 were recorded. Ethylene production was reduced to less than 1% of the control by 10 gg/ml cycloheximide. Although the tissue had only a small methionine pool, supplying the seed with exogenous methionine did not influence ethylene production at any stage of seed development. Label from [U-14C]methionine was readily incorporated into ethylene.
Introduction
Many plant tissues produce ethylene depending on their stage of development or in response to injury, stress, or auxin treatments (for a review, see Pratt and Goeschl, 1969). Synthesis of stress ethylene is sensitive to protein inhibitors and commonly has a lag phase of approximately 1 h. In many vegetative tissues and in apple and avocado fruit tissue ethylene is derived from carbons 3 and 4 of methionine while carbon 1 is converted to CO2 and the remainder of the molecule is recycled (Burg and Clagett, 1967; Baur and Yang, 1972). The hormones of the seed have frequently been proposed as agents controlling fruit growth though few convincing relationships have been described (Crane, 1964). For example, in peach fruit the levels of auxins and gibberellins in the seed are related to the ontogeny of seed tissues rather than growth of the whole fruit (Powell and Pratt, 1966; Jackson, 1968). Recently ethylene has been recognised as an important regulator of the growth of peach fruits (Chalmers et al., 1976; Jerie and Chalmers, 1976) and therefore the possibility that the seed is a site for this ethylene synthesis was investigated.
Material and Methods Fruits from the Cling peach cultivar Golden Queen and from an unknown cultivar of almonds were picked in the orchard at the Tatura Horticultural Research Station as required. The rate of ethylene production from peach fruits (Jerie and Chalmers, 1976) and seeds was measured at weekly intervals throughout the growing season. However, the seeds used in the experiments reported in the figures and tables in this paper were taken from fruits harvested during freshweight-stage II (see Chalmers and van den Ende, 1975). During this period the average fresh weight of fruit and seeds increased from 10.7 to 14.6 g and from 0.26 to 0.8 g, respectively. The seed was obtained without visible damage by splitting the endoearp open along the suture. The different tissues of the seed were separated by cutting the seed longitudinally and lifting out the embryo and endosperm pieces. The nucellus was gently scraped from the inside of the seed coat. Before the stone had hardened the seed could be half exposed by cutting across the mesocarp and snapping the fruit in half. Whole seeds or individual tissues were treated with cycloheximide (CHI, 3.6 x 10-SM), methionine (mM), or [U-14C]methionine (12.6 pM, 256 mCi/mmole; New England Nuclear Corp., Boston, Mass., USA), by immersing the tissues in aqueous solutions for 20 min, rinsing under tap water, and gently blotting dry with paper tissues. Uptake of [U-14C]methionine was measured by burning similarly treated seeds in a Packard Instruments (Downers Grove, III., USA) model 305 tissue oxidizer and determining activity with a Packard model 2003 scintillation spectrometer. Seeds oxidized immediately after treatment contained 85 nmole [U-14C]methionine per g fresh weight which was the equivalent of approximately 223 nmole per replication. Ethylene and CO2 was trapped by injecting a volume of gas into a vial containing 1 ml of 0.25 M mercuric perchlorate in 2 M perchloric acid plus a filter paper wick soaked in 2 M NaOH and suspended from the lid. After 1 h at 2 ~ the wick was removed, oven-dried, and the activity counted in a toluene scintillator. The proportion of known standards of 14CO2 recovered by this method was low (64%) but consistent. The values for 14CO2 produced after treatment of seeds with [Ul~C]methionine have been adjusted accordingly. Ten ml of Packard "Instagel" was added to the mercuric perchlorate for counting of [14C] ethylene. Although Thompson and Spencer (1966) have used a similar method for trapping [14C]ethylene Burg and Burg (1964) have warned that ethanol may also be trapped in mercuric perchlorate. This would be unlikely to be a significant problem in these determinations since the seeds
14
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
were excised and therefore in aerobic conditions for a considerable period before [14C]ethylene was produced. The counting efficiencies for [14C]ethylene and 14CO2 were 78 and 75% respectively. Less than 0.01% of 14CO2 was detected in the mercuric perchlorate fraction. Methionine was extracted by macerating the tissue in phosphate buffer (pH 5.5) at 0~ The crude extract was partially purified on an H+-form Dowex-50 column. Amino acids were eluted with 2 M NH4OH. Methionine concentration was determined with a Beckman Instruments (Fullerton, Cal., USA) amino-acid analyser. There was no methionine sulphone and only a trace of methionine sulphoxide present in the extract. The method recovered 95% of known methionine added to the seeds at the start of the extraction procedure. Total N was determined according to Mitchell (1972). To measure ethylene production, disposable plastic syringes fitted with silicone rubber septae instead of needles were used as tissue chambers of variable size. Larger amounts of tissue were enclosed in plastic (Perspex) chambers sealed with cellulose tape. The tissue was sealed in the chambers for periods between 30 and 60 min depending on the rate of ethylene production of the tissue. The chambers were vented for 2 min between successive gas samplings. Ethylene in the gas sample was measured with a Shimadzu (Kyoto, Japan) GC-1C gas chromatograph using an alumina column at 100~ and a hydrogen flame detector.
1000
97
/~___--#----g
100
~.
lO
"'
1
In
I
I
2
4
6
I
I
I
I
24
26
28
30
T i m e {h)
5000 10o0 500
E T
100
""
50
c ~o ~., r
10 5.0
/
Results
The fresh weight of fruit, whole seed and seed tissues used in the experiments reported below is shown in Table 1. Peach seeds did not produce measurable amounts of ethylene until 50-90 rain after excision. After this lag phase ethylene production rose exponentially (Fig. 1 a). This pattern was followed by seeds sampled throughout the growing season though, as the seed matured and the seed coat browned, the maximum rate became progressively lower. Ethylene production was generally reduced if the seed was injured by cutting or by scraping the surface (Fig. 1 a). Ethylene
Iff
8
W
1"0 0-5 2
4
6
24
26
Time (h)
Fig. 1 a and b. Ethylene production by peach seeds, a Whole seeds excised at zero time; o undamaged seeds; 9 surface scraped; [] seeds cut into four sections, b Whole seeds and different tissues: •, 9 whole seeds; A, 9 embryo, endosperm and nucellus; o, 9 seed coat. Open and closed symbols are replicates.
Table 1. Fresh weights and their standard deviations of fruit, seeds and seed parts used in experiments described in Figures 1-3 and Table 3 The mean weight of the combined tissues in all treatments and duplicates is given for each experiment, f.w. =fresh weight; S.D. =standard deviation Date
Experiment described
No. of seeds or fruit per sample
Divided or whole fruit
Sample f.w. (g) 7 Nov 17 Dec
Figure l a Figure 1b
13
Nov
Figure 2
21 2
Nov Dec
Figure 3 Table 3
2 2 3 2 2 3 3
23.63
S.D.
Whole seed
Seed coat
Sample f.w. (g)
S.D.
Sample f.w. (g)
S.D.
Sample f.w. (g)
S.D.
0.55 1.65
0.11 0.06 0.90
0.05
1.35
0.09
1.37 0.73 1.70 1.63
0.04 0.17 0.04
Nucellus + endosperm + embryo
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
I
,0
I ....
Table 2. Changes in the N pool and ethylene production of peach seed coats (~tmole g- 1 fresh wt) 0
9J
9
o - - / j ' ' - - o " . ' = 4 ~8-~K'8
_,:r .//"
r .-e_ 0.05 3 L
//-
L
"
0
/
9" I 0~ ~ll j4 ,,
,~,..',o" ~9
15
Time after excision (h)
C2H4/h
Methionine
Serine
N in Dowex column eluate
0 8
0 0.089
0.038 0.041
7.5 10
59 93
/
iI i
,,~
;l
,,
Table 3. Metabolism of [U14C)nethionine
0.005
~
o.oo,
";' 2'4
2'6
Results are means of two experiments carried out in triplicate
2;
3'o
Time (h)
Time after feeding (rain)
Ethylene gl
CO z nCi mmole- 1 dpm min 1
dpm rain- 1
[U-14C]methionine at zero time
Fig. 2. The effect of partly excising the seed from the fruit on ethylene production, zx, 9 Pericarp only; o, 9 pericarp and seeds separated at zero time and placed the same chamber; o, 9 seeds and pericarp half exposed at zero time subsequently separated completely (arrows indicate time of complete separation). Open and closed symbols are replicates
170 290 340 620
0.0039 0.049 0.133 1.78
0.508 0.163 0.079 0.022
1.16 5.81 6.10 13.99
8.1 4.0 5.2 3.6
18.17 16.28
10.l 4.5
[U-14C]methionine at 330 min 70 300
0.40 3.12
0.032 0.012
~ooo ~ /
0~0
/ 1.0
~.o
01
2
~
" 2'2
2'4
25
2'8
3b
Time (h) Fig. 3. The effect of methionine and cycloheximide on ethylene production of whole peach seeds. 9 control; o ImM methionine at zero time and 4 h; • CHI at zero time; 9 CHI at 1.5 b; 9 CHI at 4 h. Arrows indicate the time of CHI treatment
was synthesised in the seed coat while the other tissues produced barely measureable amounts (Fig. 1b). The volume of ethylene produced by the isolated seed coat was not significantly larger than that produced by the whole seed, the rate being higher because of
the removal of inactive tissue. However on occasions it appeared that isolating the seed coat could stimulate ethylene production early in the experiment and could reduce the lag phase. This point requires further clarification. In seed coats, rates as high as 6,000 l,tl kg- 1 h-1 were observed in some experiments. Similar results were obtained with almond seeds. When the seed was left attached to part of the fruit (i.e. one third of the seed exposed) ethylene production was reduced compared to fully exposed seeds (Fig. 2). After separation ethylene production rose to, or slightly above, the control rate. Treatment with CHI at zero time inhibited ethylene production to less than 1% of the control (Fig. 3). Later CHI treatments resulted in the eventual decline to rates generally above but near those of the zero-time treatment. At no time did methionine treatment stimulate ethylene producton even though at rates of 2,000 gl kg-a h 1 the tissue must have been turning over approximately 13.4 ~tg methionine g-1 fresh weight h- 1. Endogenous levels of methionine were low both at zero time and 8 h later (Table 2) and would require the entire methionine pool to be turned over about every 30 min. During the 8-h incubation the total amino-acid pool increased by about 60%. When seeds were treated at zero time with [U14C]methionine label appeared immediately in CO2
16 and then later, as its synthesis commenced, in ethylene (Table 3). The specific activity of ethylene 170 rain after feeding [U14C]methionine (0.5 nCi/nmole) was similar to that reported by Abeles and Abeles (1972) in unstressed bean leaves. After 620 rain the rate of ethylene production had increased by a factor of 450 while the specific activity of ethylene had declined to 1/20 the 170-min value. When [U14C]methionine was fed after ethylene production had commenced (5.5 h) 3.4% of the label in carbons 3 and 4 of methionine appeared in ethylene in the following 70 min but at a very low specific activity.
Discussion
When first removed from the fruit, whole seeds do not produce measurable amounts of ethylene but it is difficult to relate this to what occurs in the whole fruit. In situ the seed is surrounded by the pericarp which offers a resistance to gas diffusion. Thus, although too low to be quickly measured, the level of ethylene synthesis of the seed may be in equilibrium with a physiologically significant concentration of the gas within the fruit tissue. Chalmers et al. (1976) have shown that in peach fruit, ethylene is an essential hormone for dry-weight growth though it was also shown that the whole fruit produces very little ethylene during stone hardening (Jerie and Chalmers, 1976). After the above consideration it is not possible to discount the seed as a significant source of ethylene for its own development and the development of the fruit during lignification of the endocarp, even though on a whole-fruit basis ethylene may be undetectable at that time. At other stages of development the mesocarp produces large quantities of ethylene and the contribution of the seed, if any, would not be important. After excision from the fruit the peach seed develops the capacity for ethylene synthesis in the seed coat but not in the other tissues. The maximum observed rate of 6,000 gl kg- 1 h- ~ is very high compared to other plant tissues. Even at the more frequently obtained rate of 2,000 pl kg- ~ h- 1 a complete turnover of the methionine pool would be required in less than 30 min. After excision the pool of free amino acids increases as shown by the increase in nitrogen retained in the Dowex-50 column (Table 2). The concentration of serine, the only intermediate of methionine that was measured, also increased by 25%. While these changes could be expected to facilitate methionine synthesis, particularly if the - S - and - C H 3 fragments are recycled as they are in apple tissue (Baur and Yang, 1972), it is nevertheless surprising that
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds the level of methionine did not decrease as ethylene synthesis was activated. The label experiment showed that methionine acts as a substrate for ethylene synthesis but, although the endogenous methionine pool must have been rapidly turned over, exogenously supplied methionine did not stimulate ethylene synthesis. In some other tissues where methionine has been convincingly shown to be an intermediate in ethylene synthesis (Burg and Clagget, 1967; Baur and Yang, 1969), exogeneous methionine does stimulate ethylene production. Exogenous methionine does not stimulate ethylene production in mung-bean hypocotyls but in that tissue there is a relatively large methionine pool (Sakai and Imaseki, 1972). Since the rate of [U14C]methionine utilization increased substantially as ethylene production increased (Table 3) it follows that the methionine pathway of ethylene synthesis was activated when seeds were removed from the fruit. However, the specific activity of ethylene dropped greatly as the rate of ethylene production increased. A similar but smaller effect was observed in stressed bean and tobacco leaves (Abeles and Abeles, 1972) and may indicate the existence of a second pathway for ethylene synthesis which becomes more important as the tissue produces ethylene at higher rates. In our experiments this possibility is supported by the unchanged methionine level (Table 2) and the failure of exogenous methionine to stimulate ethylene production. The decline in specific activity and the failure of exogenous methionine to stimulate ethylene synthesis could be accounted for if the movement of exogenous methionine into the metabolic pool was limiting. However this is clearly not so as [U14C]methionine applied when ethylene production was high (after 330 min) rapidly became incorporated into ethylene in relatively large amounts (Table 3). The rate of [U14C]methionine uptake and incorporation (18.3 dpm min -~) at that time would have been sufficient to maintain the specific activity of ethylene at the value observed at 170 min until 290 min by which time the specific activity had in fact dropped by 2/3. The mechanism that limits ethylene production by the seed in situ is of interest particularly if ethylene from the seed is involved in the control of fruit growth. This effect can be only partly accounted for by the barrier to diffusion of ethylene and other gases caused by the pericarp. Exposure of one third of the seed surface would substantially eliminate the barrier as there are numerous channels between the testa and the endocarp through which gas should freely diffuse. Yet this treatment released the inhibition of ethylene production only partially and an explanation of the residual inhibition is needed. It is possible that
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
inhibitory compounds exported from the tree via the fruit limit ethylene production by the seed. However, inactivation or turnover of the chemical inhibitor would need to be rapid to account for these results. We thank Mr. C. Rayner for amino-acid and N analyses and Miss J. Holdsworth for technical assistance.
References Abeles, A.L., Abeles, F.B. : Biochemical pathway of stress induced ethylene. Plant Physiol. 50, 496498 (1972) Baur, A.H., Yang, J.F.: Precursors of ethylene. Plant Physiol. 44, 1347-1349 (1969) Baur, A.H., Yang, S.F.: Methionine metabolism in apple tissue in relation to ethylene biosynthesis. Phytochemistry 11, 32073214 (1972) Burg, S.P., Burg, E.A.: Biosynthesis of ethylene. Nature (Lond.) 203, 869 870 (1964) Burg, S.P., Clagett, C.O.: Conversion of methionine to ethylene in vegetative tissue and fruit. Biochem. Biophys. Res. Comm. 27, 125-130 (1967) Chalmers, D.J., van den Ende, B.: A reappraisal of the growth
17 and development of peach fruit. Aust. J. Plant Physiol. 2, 623634 (1975) Chalmers, D.J., van den Ende, B., Jerie, P.H. : The effect of (2chloroethyl)phosphonic acid on the sink strength of peach fruit. Planta (Berl.), in press (1976) Crane, J.C.: Growth substances in fruit setting and development. Ann. Rev. Plant Physiol. 15, 303-326 (1964) Jackson, D.I.: Gibberellin and the growth of peach and apricot fruit Aust. J. Biol. Sci. 21, 209-215 (1968) Jerie, P.H., Chalmers, D.J. : Ethylene as a growth hormone in peach fruit. Aust. J. Plant Physiol., (in press) (1976) Mitchell, H. : Micro-determination of nitrogen in plant tissues. J. Assoc. Offic. Agric. Chem. 55, 1 3 (1972) Powell, L.E., Pratt, C. : Growth promoting substances in the developing fruit of peach (Prunus persica L.) J. hort. Sci. 41, 331-348 (1966) Pratt, H.K., Goeschl, J.D.: Physiological roles of ethylene in plants. Ann. Rev. Plant Physiol. 20, 541-584 (1969) Sakai, S., Imaseki, H.L.: Ethylene biosynthesis: Methionine as an in-vivo precursor of ethylene in auxin-treated mung bean hypocotyl segments. Planta (Berl.) 105, 165-173 (1972) Thompson, J.E., Spencer, M.: Preparation and properties of an enzyme system for ethylene production. Nature (Lond.) 210, 595-597 (1966)
Received 16 February , accepted 4 June 1976
Planta (Berl.) 132, 1 3 - 17 (1976)
9 by Springer-Verlag 1976
Some Characteristics of Ethylene Production in Peach (Prunus persica L.) Seeds P.H. Jerie and D.J. Chalmers Horticultural Research Station, Victorian Department of Agriculture, Tatura, Vic. 3616, Australia
Summary. The seed of peach fruits develop the capacity to produce ethylene with a lag phase of about 1 h after excision. The site of ethylene synthesis is in the seed coat and rates as high as 6,000 gl kg-1 h-1 were recorded. Ethylene production was reduced to less than 1% of the control by 10 gg/ml cycloheximide. Although the tissue had only a small methionine pool, supplying the seed with exogenous methionine did not influence ethylene production at any stage of seed development. Label from [U-14C]methionine was readily incorporated into ethylene.
Introduction
Many plant tissues produce ethylene depending on their stage of development or in response to injury, stress, or auxin treatments (for a review, see Pratt and Goeschl, 1969). Synthesis of stress ethylene is sensitive to protein inhibitors and commonly has a lag phase of approximately 1 h. In many vegetative tissues and in apple and avocado fruit tissue ethylene is derived from carbons 3 and 4 of methionine while carbon 1 is converted to CO2 and the remainder of the molecule is recycled (Burg and Clagett, 1967; Baur and Yang, 1972). The hormones of the seed have frequently been proposed as agents controlling fruit growth though few convincing relationships have been described (Crane, 1964). For example, in peach fruit the levels of auxins and gibberellins in the seed are related to the ontogeny of seed tissues rather than growth of the whole fruit (Powell and Pratt, 1966; Jackson, 1968). Recently ethylene has been recognised as an important regulator of the growth of peach fruits (Chalmers et al., 1976; Jerie and Chalmers, 1976) and therefore the possibility that the seed is a site for this ethylene synthesis was investigated.
Material and Methods Fruits from the Cling peach cultivar Golden Queen and from an unknown cultivar of almonds were picked in the orchard at the Tatura Horticultural Research Station as required. The rate of ethylene production from peach fruits (Jerie and Chalmers, 1976) and seeds was measured at weekly intervals throughout the growing season. However, the seeds used in the experiments reported in the figures and tables in this paper were taken from fruits harvested during freshweight-stage II (see Chalmers and van den Ende, 1975). During this period the average fresh weight of fruit and seeds increased from 10.7 to 14.6 g and from 0.26 to 0.8 g, respectively. The seed was obtained without visible damage by splitting the endoearp open along the suture. The different tissues of the seed were separated by cutting the seed longitudinally and lifting out the embryo and endosperm pieces. The nucellus was gently scraped from the inside of the seed coat. Before the stone had hardened the seed could be half exposed by cutting across the mesocarp and snapping the fruit in half. Whole seeds or individual tissues were treated with cycloheximide (CHI, 3.6 x 10-SM), methionine (mM), or [U-14C]methionine (12.6 pM, 256 mCi/mmole; New England Nuclear Corp., Boston, Mass., USA), by immersing the tissues in aqueous solutions for 20 min, rinsing under tap water, and gently blotting dry with paper tissues. Uptake of [U-14C]methionine was measured by burning similarly treated seeds in a Packard Instruments (Downers Grove, III., USA) model 305 tissue oxidizer and determining activity with a Packard model 2003 scintillation spectrometer. Seeds oxidized immediately after treatment contained 85 nmole [U-14C]methionine per g fresh weight which was the equivalent of approximately 223 nmole per replication. Ethylene and CO2 was trapped by injecting a volume of gas into a vial containing 1 ml of 0.25 M mercuric perchlorate in 2 M perchloric acid plus a filter paper wick soaked in 2 M NaOH and suspended from the lid. After 1 h at 2 ~ the wick was removed, oven-dried, and the activity counted in a toluene scintillator. The proportion of known standards of 14CO2 recovered by this method was low (64%) but consistent. The values for 14CO2 produced after treatment of seeds with [Ul~C]methionine have been adjusted accordingly. Ten ml of Packard "Instagel" was added to the mercuric perchlorate for counting of [14C] ethylene. Although Thompson and Spencer (1966) have used a similar method for trapping [14C]ethylene Burg and Burg (1964) have warned that ethanol may also be trapped in mercuric perchlorate. This would be unlikely to be a significant problem in these determinations since the seeds
14
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
were excised and therefore in aerobic conditions for a considerable period before [14C]ethylene was produced. The counting efficiencies for [14C]ethylene and 14CO2 were 78 and 75% respectively. Less than 0.01% of 14CO2 was detected in the mercuric perchlorate fraction. Methionine was extracted by macerating the tissue in phosphate buffer (pH 5.5) at 0~ The crude extract was partially purified on an H+-form Dowex-50 column. Amino acids were eluted with 2 M NH4OH. Methionine concentration was determined with a Beckman Instruments (Fullerton, Cal., USA) amino-acid analyser. There was no methionine sulphone and only a trace of methionine sulphoxide present in the extract. The method recovered 95% of known methionine added to the seeds at the start of the extraction procedure. Total N was determined according to Mitchell (1972). To measure ethylene production, disposable plastic syringes fitted with silicone rubber septae instead of needles were used as tissue chambers of variable size. Larger amounts of tissue were enclosed in plastic (Perspex) chambers sealed with cellulose tape. The tissue was sealed in the chambers for periods between 30 and 60 min depending on the rate of ethylene production of the tissue. The chambers were vented for 2 min between successive gas samplings. Ethylene in the gas sample was measured with a Shimadzu (Kyoto, Japan) GC-1C gas chromatograph using an alumina column at 100~ and a hydrogen flame detector.
1000
97
/~___--#----g
100
~.
lO
"'
1
In
I
I
2
4
6
I
I
I
I
24
26
28
30
T i m e {h)
5000 10o0 500
E T
100
""
50
c ~o ~., r
10 5.0
/
Results
The fresh weight of fruit, whole seed and seed tissues used in the experiments reported below is shown in Table 1. Peach seeds did not produce measurable amounts of ethylene until 50-90 rain after excision. After this lag phase ethylene production rose exponentially (Fig. 1 a). This pattern was followed by seeds sampled throughout the growing season though, as the seed matured and the seed coat browned, the maximum rate became progressively lower. Ethylene production was generally reduced if the seed was injured by cutting or by scraping the surface (Fig. 1 a). Ethylene
Iff
8
W
1"0 0-5 2
4
6
24
26
Time (h)
Fig. 1 a and b. Ethylene production by peach seeds, a Whole seeds excised at zero time; o undamaged seeds; 9 surface scraped; [] seeds cut into four sections, b Whole seeds and different tissues: •, 9 whole seeds; A, 9 embryo, endosperm and nucellus; o, 9 seed coat. Open and closed symbols are replicates.
Table 1. Fresh weights and their standard deviations of fruit, seeds and seed parts used in experiments described in Figures 1-3 and Table 3 The mean weight of the combined tissues in all treatments and duplicates is given for each experiment, f.w. =fresh weight; S.D. =standard deviation Date
Experiment described
No. of seeds or fruit per sample
Divided or whole fruit
Sample f.w. (g) 7 Nov 17 Dec
Figure l a Figure 1b
13
Nov
Figure 2
21 2
Nov Dec
Figure 3 Table 3
2 2 3 2 2 3 3
23.63
S.D.
Whole seed
Seed coat
Sample f.w. (g)
S.D.
Sample f.w. (g)
S.D.
Sample f.w. (g)
S.D.
0.55 1.65
0.11 0.06 0.90
0.05
1.35
0.09
1.37 0.73 1.70 1.63
0.04 0.17 0.04
Nucellus + endosperm + embryo
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
I
,0
I ....
Table 2. Changes in the N pool and ethylene production of peach seed coats (~tmole g- 1 fresh wt) 0
9J
9
o - - / j ' ' - - o " . ' = 4 ~8-~K'8
_,:r .//"
r .-e_ 0.05 3 L
//-
L
"
0
/
9" I 0~ ~ll j4 ,,
,~,..',o" ~9
15
Time after excision (h)
C2H4/h
Methionine
Serine
N in Dowex column eluate
0 8
0 0.089
0.038 0.041
7.5 10
59 93
/
iI i
,,~
;l
,,
Table 3. Metabolism of [U14C)nethionine
0.005
~
o.oo,
";' 2'4
2'6
Results are means of two experiments carried out in triplicate
2;
3'o
Time (h)
Time after feeding (rain)
Ethylene gl
CO z nCi mmole- 1 dpm min 1
dpm rain- 1
[U-14C]methionine at zero time
Fig. 2. The effect of partly excising the seed from the fruit on ethylene production, zx, 9 Pericarp only; o, 9 pericarp and seeds separated at zero time and placed the same chamber; o, 9 seeds and pericarp half exposed at zero time subsequently separated completely (arrows indicate time of complete separation). Open and closed symbols are replicates
170 290 340 620
0.0039 0.049 0.133 1.78
0.508 0.163 0.079 0.022
1.16 5.81 6.10 13.99
8.1 4.0 5.2 3.6
18.17 16.28
10.l 4.5
[U-14C]methionine at 330 min 70 300
0.40 3.12
0.032 0.012
~ooo ~ /
0~0
/ 1.0
~.o
01
2
~
" 2'2
2'4
25
2'8
3b
Time (h) Fig. 3. The effect of methionine and cycloheximide on ethylene production of whole peach seeds. 9 control; o ImM methionine at zero time and 4 h; • CHI at zero time; 9 CHI at 1.5 b; 9 CHI at 4 h. Arrows indicate the time of CHI treatment
was synthesised in the seed coat while the other tissues produced barely measureable amounts (Fig. 1b). The volume of ethylene produced by the isolated seed coat was not significantly larger than that produced by the whole seed, the rate being higher because of
the removal of inactive tissue. However on occasions it appeared that isolating the seed coat could stimulate ethylene production early in the experiment and could reduce the lag phase. This point requires further clarification. In seed coats, rates as high as 6,000 l,tl kg- 1 h-1 were observed in some experiments. Similar results were obtained with almond seeds. When the seed was left attached to part of the fruit (i.e. one third of the seed exposed) ethylene production was reduced compared to fully exposed seeds (Fig. 2). After separation ethylene production rose to, or slightly above, the control rate. Treatment with CHI at zero time inhibited ethylene production to less than 1% of the control (Fig. 3). Later CHI treatments resulted in the eventual decline to rates generally above but near those of the zero-time treatment. At no time did methionine treatment stimulate ethylene producton even though at rates of 2,000 gl kg-a h 1 the tissue must have been turning over approximately 13.4 ~tg methionine g-1 fresh weight h- 1. Endogenous levels of methionine were low both at zero time and 8 h later (Table 2) and would require the entire methionine pool to be turned over about every 30 min. During the 8-h incubation the total amino-acid pool increased by about 60%. When seeds were treated at zero time with [U14C]methionine label appeared immediately in CO2
16 and then later, as its synthesis commenced, in ethylene (Table 3). The specific activity of ethylene 170 rain after feeding [U14C]methionine (0.5 nCi/nmole) was similar to that reported by Abeles and Abeles (1972) in unstressed bean leaves. After 620 rain the rate of ethylene production had increased by a factor of 450 while the specific activity of ethylene had declined to 1/20 the 170-min value. When [U14C]methionine was fed after ethylene production had commenced (5.5 h) 3.4% of the label in carbons 3 and 4 of methionine appeared in ethylene in the following 70 min but at a very low specific activity.
Discussion
When first removed from the fruit, whole seeds do not produce measurable amounts of ethylene but it is difficult to relate this to what occurs in the whole fruit. In situ the seed is surrounded by the pericarp which offers a resistance to gas diffusion. Thus, although too low to be quickly measured, the level of ethylene synthesis of the seed may be in equilibrium with a physiologically significant concentration of the gas within the fruit tissue. Chalmers et al. (1976) have shown that in peach fruit, ethylene is an essential hormone for dry-weight growth though it was also shown that the whole fruit produces very little ethylene during stone hardening (Jerie and Chalmers, 1976). After the above consideration it is not possible to discount the seed as a significant source of ethylene for its own development and the development of the fruit during lignification of the endocarp, even though on a whole-fruit basis ethylene may be undetectable at that time. At other stages of development the mesocarp produces large quantities of ethylene and the contribution of the seed, if any, would not be important. After excision from the fruit the peach seed develops the capacity for ethylene synthesis in the seed coat but not in the other tissues. The maximum observed rate of 6,000 gl kg- 1 h- ~ is very high compared to other plant tissues. Even at the more frequently obtained rate of 2,000 pl kg- ~ h- 1 a complete turnover of the methionine pool would be required in less than 30 min. After excision the pool of free amino acids increases as shown by the increase in nitrogen retained in the Dowex-50 column (Table 2). The concentration of serine, the only intermediate of methionine that was measured, also increased by 25%. While these changes could be expected to facilitate methionine synthesis, particularly if the - S - and - C H 3 fragments are recycled as they are in apple tissue (Baur and Yang, 1972), it is nevertheless surprising that
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds the level of methionine did not decrease as ethylene synthesis was activated. The label experiment showed that methionine acts as a substrate for ethylene synthesis but, although the endogenous methionine pool must have been rapidly turned over, exogenously supplied methionine did not stimulate ethylene synthesis. In some other tissues where methionine has been convincingly shown to be an intermediate in ethylene synthesis (Burg and Clagget, 1967; Baur and Yang, 1969), exogeneous methionine does stimulate ethylene production. Exogenous methionine does not stimulate ethylene production in mung-bean hypocotyls but in that tissue there is a relatively large methionine pool (Sakai and Imaseki, 1972). Since the rate of [U14C]methionine utilization increased substantially as ethylene production increased (Table 3) it follows that the methionine pathway of ethylene synthesis was activated when seeds were removed from the fruit. However, the specific activity of ethylene dropped greatly as the rate of ethylene production increased. A similar but smaller effect was observed in stressed bean and tobacco leaves (Abeles and Abeles, 1972) and may indicate the existence of a second pathway for ethylene synthesis which becomes more important as the tissue produces ethylene at higher rates. In our experiments this possibility is supported by the unchanged methionine level (Table 2) and the failure of exogenous methionine to stimulate ethylene production. The decline in specific activity and the failure of exogenous methionine to stimulate ethylene synthesis could be accounted for if the movement of exogenous methionine into the metabolic pool was limiting. However this is clearly not so as [U14C]methionine applied when ethylene production was high (after 330 min) rapidly became incorporated into ethylene in relatively large amounts (Table 3). The rate of [U14C]methionine uptake and incorporation (18.3 dpm min -~) at that time would have been sufficient to maintain the specific activity of ethylene at the value observed at 170 min until 290 min by which time the specific activity had in fact dropped by 2/3. The mechanism that limits ethylene production by the seed in situ is of interest particularly if ethylene from the seed is involved in the control of fruit growth. This effect can be only partly accounted for by the barrier to diffusion of ethylene and other gases caused by the pericarp. Exposure of one third of the seed surface would substantially eliminate the barrier as there are numerous channels between the testa and the endocarp through which gas should freely diffuse. Yet this treatment released the inhibition of ethylene production only partially and an explanation of the residual inhibition is needed. It is possible that
P.H. Jerie and D.J. Chalmers: Ethylene Production in Peach Seeds
inhibitory compounds exported from the tree via the fruit limit ethylene production by the seed. However, inactivation or turnover of the chemical inhibitor would need to be rapid to account for these results. We thank Mr. C. Rayner for amino-acid and N analyses and Miss J. Holdsworth for technical assistance.
References Abeles, A.L., Abeles, F.B. : Biochemical pathway of stress induced ethylene. Plant Physiol. 50, 496498 (1972) Baur, A.H., Yang, J.F.: Precursors of ethylene. Plant Physiol. 44, 1347-1349 (1969) Baur, A.H., Yang, S.F.: Methionine metabolism in apple tissue in relation to ethylene biosynthesis. Phytochemistry 11, 32073214 (1972) Burg, S.P., Burg, E.A.: Biosynthesis of ethylene. Nature (Lond.) 203, 869 870 (1964) Burg, S.P., Clagett, C.O.: Conversion of methionine to ethylene in vegetative tissue and fruit. Biochem. Biophys. Res. Comm. 27, 125-130 (1967) Chalmers, D.J., van den Ende, B.: A reappraisal of the growth
17 and development of peach fruit. Aust. J. Plant Physiol. 2, 623634 (1975) Chalmers, D.J., van den Ende, B., Jerie, P.H. : The effect of (2chloroethyl)phosphonic acid on the sink strength of peach fruit. Planta (Berl.), in press (1976) Crane, J.C.: Growth substances in fruit setting and development. Ann. Rev. Plant Physiol. 15, 303-326 (1964) Jackson, D.I.: Gibberellin and the growth of peach and apricot fruit Aust. J. Biol. Sci. 21, 209-215 (1968) Jerie, P.H., Chalmers, D.J. : Ethylene as a growth hormone in peach fruit. Aust. J. Plant Physiol., (in press) (1976) Mitchell, H. : Micro-determination of nitrogen in plant tissues. J. Assoc. Offic. Agric. Chem. 55, 1 3 (1972) Powell, L.E., Pratt, C. : Growth promoting substances in the developing fruit of peach (Prunus persica L.) J. hort. Sci. 41, 331-348 (1966) Pratt, H.K., Goeschl, J.D.: Physiological roles of ethylene in plants. Ann. Rev. Plant Physiol. 20, 541-584 (1969) Sakai, S., Imaseki, H.L.: Ethylene biosynthesis: Methionine as an in-vivo precursor of ethylene in auxin-treated mung bean hypocotyl segments. Planta (Berl.) 105, 165-173 (1972) Thompson, J.E., Spencer, M.: Preparation and properties of an enzyme system for ethylene production. Nature (Lond.) 210, 595-597 (1966)
Received 16 February , accepted 4 June 1976
