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Differences in the Requirement for Endogenous Ethylene During Germination of Dormant and Non-Dormant Seeds of Chenopodium album L.
J. PlantPhysiol. Vol. 138. pp. 97-101 {1991}
Differences in the Requirement for Endogenous Ethylene During Germination of Dormant and N on-Dormant Seeds of Chenopodium album L. SYLVIE MACHABEE
and
HARGURDEEP S. SAINI
Institut de recherche en biologie vegetale, Universite de Montreal, 4101, rue Sherbrooke est, Montreal, Quebec, Canada H1X 2B2 Received July 27, 1990 . Accepted November 10, 1990
Summary Requirement for endogenous ethylene in the germination of two lots of Chenopodium album L. seeds, with different inherent degrees of dormancy, was studied. The seeds produced ethylene upon imbibition, and an increase in ethylene emanation either preceded or coincided with the earliest occurrence of radicle emergence. The amounts of ethylene produced were directly proportional to the final germination percentage achieved, regardless of the seed lot or conditions of germination. Breakage of dormancy by a combined treatment with GA4 + 7 , NaN0 3 and white light (stimulated seeds) substantially increased ethylene production and germination. Although an application of 2-aminoethoxyvinyl glycine strongly suppressed ethylene production, it had no effect on the germination of non-dormant or stimulated dormant seeds. Application of 2,5-norbornadiene to antagonize ethylene action did not inhibit germination of non-dormant seeds. However, norbornadiene strongly inhibited the germination of stimulated dormant seeds, and this inhibition was overcome by the application of exogenous ethylene. It is concluded that, in order to germinate, non-dormant seeds do not require the ethylene they produce. However, induction of germination in dormant seeds depends on the action of ethylene produced during this process. The passage from dormancy to germination may, therefore, involve two steps: an ethylene-requiring transition to non-dormant state, followed by the germination itself that does not depend on ethylene.
Key words: Chenopodium album L., dormancy, ethylene action, ethylene synthesis, germination, plant hormones, seed. Abbreviation list: NBD
=
2,5-norbornadiene; AVG
Introduction Seeds of Chenopodium album L. (lamb's-quarters) are generally dormant at maturity, although the percentage of dormant seeds can vary considerably among different seed lots (Bessett and Crompton 1978). Dormancy of this species can be broken by various chemical and physical treatments (Henson 1970, Roberts and Benjamin 1979, Saini et al. 1985 a). Among these, treatments with ethylene or ethephon (2-chloroethylphosphonic acid; an ethylene releasing compound) are perhaps the most effective ways to break dormancy (Karssen 1976, Olatoye and Hall 1972, Saini et al. © 1991 by Gustav Fischer Verlag. Stuttgart
=
2-aminoethoxyvinyl glycine.
1985 a, b, Taylorson, 1979). Sensitivity of the seeds toward exogenous ethylene and other hormones is strongly enhanced by the availability of nitrate, either through exogenous supply or as an endogenous constituent of the seeds, which is in turn dependent on the level of nitrate nutrition received by the parent plant (Saini et al. 1985 a, b, Saini and Spencer 1987). Germinating seeds of several species produce ethylene, and endogenous ethylene has been shown to play an essential role in the germination of Amaranthus caudatus L., Cucumis anguria L., Lactuca sativa L. and Xanthium pennsylvanicum Wallr. (Abeles 1986, Cardoso and Felippe 1987, Kc:pczynski
98
SYLVIE MACHABEE and HARGURDEEP S. SAINI
1986a, b, K"pczynski and Karssen 1985, Ketring 1977, Ketring and Morgan 1972, Khan and Huang 1988, Khan and Prusinski 1989, Saini et al. 1986, 1989, Satoh et a1. 1984). Despite several detailed studies on the dormancy-breaking effects of exogenous ethylene on C album seed germination, the role of endogenous ethylene in the germination of this species has never been examined. It is conceivable that the variations in the degree of dormancy among different seeds of C album may be related to their ability to produce sufficient endogenous ethylene to break dormancy. The purpose of this study was to determine whether endogenously produced ethylene controls dormancy and germination of C album seeds. This was done by correlating ethylene production with the degree of dormancy and time of germination, followed by manipulations of ethylene synthesis and action via appropriate inhibitors.
Materials and Methods Seeds of Chenopodium album L. (Lamb's-quarters) were collected from the vicinity of Vermilion (seed lot-A) and Edmonton (seed lotB), Alberta during the fall of 1985. The seeds were stored dry in a refrigerator until use. Only seeds free from visible abnormalities or damage were used for experiments. Three replicates per treatment, each consisting of 100 seeds per container (Petri dish or Erlenmeyer flask), were used in all experiments. In a preliminary experiment to determine the appropriate treatments for further work (Table 1), seeds were imbibed in 100 x 15-mm disposable Petri dishes, each lined with one Whatman No.3 filter paper soaked in 5 mL of the relevant solution. Petri dishes were sealed with parafilm to prevent moisture loss. In all other experiments, seeds were imbibed on three layers of Whatman No.1 filter paper moistened with 3 mL solution, either in 57-mL (net volume) Erlenmeyer flasks (ethylene measurement experiments) or 60 x 15-mm glass petri dishes (all other experiments). Regardless of the method of imbibition, the seeds were incubated at 22 ± 1 oC, in the dark or in continuous white fluorescent light (17 pmol/m 2/s). For measurement of ethylene evolution from seeds, the Erlenmeyer flasks containing seeds were sealed with rubber sleeve stoppers (Fisher Scientific), which were experimently confirmed to be impervious to ethylene. One mL gas samples were drawn from the flasks with a gas-tight syringe at 24-h intervals for analysis with flame ionization detection on a Hewlett-Packard 5830A gas chromatograph fitted with a Porapak-Q packed column (Supleco Canada). One mL of air was injected back into the flasks after the removal of each gas sample. Because germination in this species is asynchronous and takes several days, this method of sampling for ethylene measurement was chosen despite an alternative technically superior, flow-through system (Saini et al. 1989) to ensure that period(s) of ethylene production would not go undetected. The flow-through method was impractical in this case because of the need for frequent manual sampling around the clock over 6 days. However, to avoid any modification of germination behaviour resulting from the changes in gaseous composition in the closed system (e.g. see Eastwell et al. 1978, Keys et al. 1975), effects of manipulation of ethylene synthesis on seed germination were studied through parallel experiments in a flow-through system as follows: Three uncovered petri dishes were placed inside each of 3.74-L airtight glass bottles fitted with two ports for gas connections. The bottles were supplied with 30mLlmin flow of compressed air from which hydrocarbons (including ethylene) had been removed by platinum-catalyzed thermal oxidation (Eastwell et al. 78). The air was humidified by bubbling through distilled water prior to entry into the bottles. The bottles
Table 1: Effects of GA4 + 7 (OAmM), NaN0 3 (10mM), andlor light on the germination of two seed lots of C. album. Germination (%)1 Seed lot-A Treatment Dark Light H 2O 53±3.2 85±3.3 75±2.9 87±3.6 GA4 + 7 NaN0 3 63±5A 85± 1.8 70± 1.4 94±2.5 GA4 + 7 +NaN0 3 1 Values represent mean±S.E.
Seed lot-B Dark Light 22±4.3 61±0.7 42±3.3 78±3.0 39±2.3 82±3.3 49±0.8 81±2.8
were placed inside an incubator, and the gaseous effluent from each bottle was vented out of the incubator with a tube. Daily analysis of samples of in-coming air confirmed it to be free from ethylene throughout each experiment. The effects of NBD, with or without ethylene, were measured by placing open Petri dishes in the 3.74-L bottles as above, except that the gas-connection ports were sealed with rubber septa. Liquid NBD was injected into the bottles through one rubber septum onto a piece of cotton wool suspended inside the bottle; NBD completely evaporated within 10 min to give the desired vapor-phase concentration. Ethylene was injected through the septa to obtain the appropriate concentrations, which were confirmed gas-chromatographically. Germination percentage (radicle protrusion from all covering structures) was recorded after 6 days, a period that was confirmed to give maximal germination. Experiments were repeated at least once with similar results.
Results Preliminary experiments In an experiment to determine the percentages of non-dormant and dormant seeds, and appropriate treatments for dormancy breakage, 53 % and 22 % seeds of the lots-A and -B, respectively, germinated without treatment in closed Petri dishes in the dark (Table 1). For lot-B, this figure was somewhat higher than in other experiments, probably owing to the stimulation by accumulated ethylene in the Petri dishes; in the larger containers used for later experiments, the germination was consistently around 12 % (data not shown; comparable lot-A germination was somewhat more variable, 53-59%). Thus, assuming 100% viability, the proportions of non-dormant vs. dormant seeds in the lot-A and lot-B were approximately 55/45 % and 12/88 %, respectively. Applications of GA4 + 7 , NaN0 3 , and/or light promoted germination; best results were obtained with a combination of all three treatments (Table 1). On the basis of these results, seeds incubated in H 20 in the dark are termed «non-stimulated», whereas those treated with a combination of GA4 + 7 , NaN0 3 and light are referred to as «stimulated». For economy, further results are presented primarily using two cases with the highest germination percentages in their respective categories: non-stimulated seeds of lot-A and stimulated seeds of lot-B. These represented the overall typical behaviour of non-dormant and dormant seeds, respectively.
Ethylene synthesis and germination Non-stimulated seeds of the non-dormant lot-A produced considerably more ethylene than the comparable seeds of the
99
Ethylene and seed germination 12
14 ... Seed lot-A
12 WI
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2
2
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1
2
4 3 5 Days from imbibition
6
Fig. 1: Amount of ethylene produced by the seeds of C. album L. imbibed on distilled water (i.e. non-stimulated seeds) and incubated in airtight flasks (57 mL). Arrow indicates the time when radicle emergence was first observed in any seed. Vertical bars on points represent respective S.E.
III
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1:1
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Table 2: Effect of 2-aminoethoxyvinyl glycine (AVG; 1 mM), applied in the continuous-flow system, on the seed germination of C. album. Seed lot-A was germinated in H 2 0 and darkness, and seed lot-B was stimulated to germinate by treatment with a combination of GA4 + 7 (0.4 mM), NaN0 3 (10 mM) and light.
0
1
5
6
0
1 2 4 3 5 Days after imbibition
6
1
-AVG
+AVG
51±3.3
61±4.0 80±2.8
84± 1.5
Values represent mean±S.E.
dormant lot-B (Fig. 1). Ethylene evolution was correlated with germination time; ethylene production increased coincident with or just prior to the start of germination, and ethylene evolution in the faster germinating lot-A started and peaked earlier than in lot-B. Stimulated, dormant (lot-B) seeds produced more ethylene than non-stimulated, non-dormant seeds of lot-A (Fig. 2). This difference was proportional to their respective germination percentages (e.g. Table 1). Again, ethylene evolution started and peaked earlier in the faster germinating seeds of lot-A. Application of AVG, an inhibitor of pyridoxalphosphate-mediated reactions such as the conversion of Sadenosyl-methionine to 1-aminocyclopropane-1-carboxylic acid in ethylene biosynthesis (Yang et al. 1980), inhibited ethylene evolution from both types of seeds (Fig. 2). Indeed the AVG-treated seeds in both situations produced less ethylene than did the dormant, non-stimulated seeds (d. Figs. 1 and 2). Despite this substantial suppression of ethylene synthesis, there was no reduction in germination percentage, even in the continuous flow system, which would prevent any accumulation of residual seed-produced ethylene (Table 2). Ethylene action and germination
Application of NBD, a potent competltlve inhibitor of ethylene action in various systems including seed germina-
3
4
20 18 16 14 12 10 8 6 4 2 0
Germination (%)1 Seed lot-A Seed lot-B
2
Fig. 2: Amount of ethylene produced by the seeds of C. album L., germinating in airtight flasks (57 mL), in the presence or absence of 2-aminoethoxyvinyl glycine (1 mM). Arrows indicate the time when radicle emergence was first noticed in any seed. Vertical bars on points represent respective S.E. (A). Seeds of less dormant lot-A imbibed on distilled water and incubated in the dark (i.e. non-stimulated non-dormant seeds). (B). Seeds of more dormant lot-B imbibed in a solution of GA4 + 7 (O.4mM) and NaN0 3 (lOmM) and incubated in white light (i.e. stimulated dormant seeds).
70 60 dP
1:1 0
.
I1S
50 40
.
s::
30
GI
20
e
t!l
10 0
o
2
4
8
2, 5-Norbornadiene (mL/L)
Fig. 3: Effect of 2,5-norbornadiene on the germination of lot-A of C. album seeds, on distilled water in the dark (i.e. non-stimulated nondormant seeds). Vertical lines over the bars indicate respective S.E.
tion (Abeles 1986, Ktrpczynski and Karssen 1985, Saini et al. 1986, 1989, Sisler and Yang 1983), failed to block the germination of non-stimulated, non-dormant seeds of lot-A, even at the extremely high concentration of 8 mLiL (Fig. 3).
100
SYLVIE MACHABEE and HARGURDEEP S. SAINI 100~-------------------------'
80 60 40
20
o
o
2
4
8
2, 5-Norbornadiene (mL/L)
Fig.4: Effects of various concentrations of 2,5-norbornadiene and exogenous ethylene on the germination of lot-B and C. album, treated with a combination of GA4 + 7 (OAmM), NaN0 3 (10mM) and light (i.e. stimulated dormant seeds). Vertical lines over the bars indicate respective S.E.
In contrast, application of NBD to stimulated, dormant seeds (lot-B) strongly inhibited germination in proportion to the concentration. Indeed, germination at 8 mL/L NBD was reduced to the level of non-stimulated seeds (Fig. 4). Application of exogenous ethylene in combination with NBD overcame the inhibition caused by NBD (Fig. 4). The interaction between different concentrations of ethylene and NBD was indicative of competitive antagonism. Discussion The results show that endogenous ethylene is essential for germination of dormant seeds, but not for the germination of non-dormant seeds of C. album. All seeds produced ethylene during germination, and there was a direct relationship between the amount of ethylene produced and the final germination percentage, whether the seeds germinated with or without an external stimulus (Figs. 1 and 2). Differences in ethylene production under various situations probably reflect the number of seeds that were in the process of germination, or past this stage, at a given time. It is quite likely that the seeds that did not germinate produced little or no ethylene. In all cases, an increase in ethylene production started before or coincident with the first visible sign of germination (radicle protrusion). The results suggest a possible role of ethylene in germination. However, the experiments with the inhibitors of ethylene synthesis and action indicate a rather complex picture: Ethylene produced by non-dormant seeds appears to play no mediatory role in germination because an inhibition of ethylene synthesis or action in these seeds failed to block their germination (Figs. 2A and 3, Table2). In contrast, breakage of dormancy through external stimulants appears to be mediated by endogenous ethylene. Inhibition of ethylene action completely blocked the germination of the dormant fraction of these seeds, and exogenous ethylene overcame this blockage (Fig. 4). An apparent inconsistency here was the failure of AVG to inhibit germination, despite a
depression of ethylene production to levels even below those produced by non-stimulated dormant seeds (Figs. 1 and 2 B). Similar results were also previously reported for peanut and Amaranthus caudatus seeds (Hoffman et al. 1983, K~pczynski and Karssen 1985). Germination of Lactuca sativa seeds is only partially blocked by A VG (Saini et al. 1986, 1989). One possible explanation is that ethylene is required for dormancy breakage in such minute quantities that the residual amounts produced even upon AVG treatment are sufficient for germination. It is curious that germination of dormant seeds requires ethylene action whereas the naturally non-dormant seeds do not need ethylene they produce during germination. The non-dormant seeds of A. caudatus, L. sativa and X. pennsylvanicum do require endogenous ethylene for germination (Abeles 1986, K~pczynski and Karssen, 1985, Satoh et al. 1984). In L. sativa, thermoinhibition (a prelude to the induction of thermodormancy) is apparently not caused by a reduction in the ability of seeds to produce ethylene (Abeles 1986, Burdett 1972, Dunlap and Morgan 1977). High temperature may, however, raise the threshold concentration of ethylene required for germination (Dunlap and Morgan 1977). Our findings with C. album suggest that the passage from the dormant to the germination stage may involve two steps: a transition from a dormant to a non-dormant state that requires ethylene, and the next step of germination itself that proceeds without a need for ethylene-action. This would fit with an earlier observation by Karssen (1976) that germination of dormant C. album seeds involves two sites of hormone action: the initiation of growth that is induced by ethylene or gibberellins, and the later stages of growth that lead to radicle protrusion. The exact nature of ethylene-action in seed germination remains obscure, although Abeles (1989) presented evidence suggesting that one action of ethylene could be the promotion of radicle cell expansion in embryonic hypocotyl in L. sativa. Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada, grant No. 37284 to H. S. Saini. The authors wish to thank Ms. Sylvie Lalonde for assistance.
References ABELES, F. B.: Role of ethylene in Lactuca sativa cv. «Grand Rapids» seed germination. Plant Physiol. 81, 780-787 (1986). BASSETT, I. J. and C. W. CROMPTON: The biology of Canadian weeds. 32. Chenopodium album L. Can. J. Bot. 58, 1061-1072 (1978). BURDETT, A. N.: Antagonistic effects of high and low temperature pretreatments on the germination and pre germination ethylene synthesis of lettuce seeds. Plant Physiol. 50, 201-204 (1972). CARDOSO, V. J. M. and G. M. FEUPPE: Endogenous ethylene and the germination of Cucumis anguria L. seeds. Rev. Bras. Bot. 10, 29-32 (1987). DUNLAP,J. R. and P. W. MORGAN: Characterization of ethylene/gibberellic acid control of germination in Lactuca sativa L. Plant Cell Physiol. 18, 561-568 (1977). EASlWELL, K. c., P. K. BASSI, and M. S. SPENCER: Comparison and evaluation of methods for the removal of ethylene and other hy-
Ethylene and seed germination drocarbons from air for biological studies. Plant Physio!' 62, 723 -726 (1978). HANSON, I. E.: The effect of light, potassium nitrate and temperature on the germination of Chenopodium album L. Weed Res. 10, 27 -39 (1970). HOFFMAN, N. E., loR. Fu, and S. F. YANG: Identification and metabolism of 1-(malonylamino)cyclopropane-l-carboxylic acid in germinating peanut seeds. Plant Physio!. 71, 197 -199 (1983). KHAN, A. A. and l PRUSINSKI: Kinetin enhanced 1-aminocyclopropane-I-carboxylic acid utilization during alleviation of high temperature stress in lettuce seeds. Plant Physio!' 91, 733 -737 (1989). KHAN, A. A. and X.-L. HUANG: Synergistic promotion of ethylene production and germination with kinetin and 1-aminocyclopropane-I-carboxylic acid in lettuce seeds exposed to salinity stress. Plant Physio!. 87, 847 - 852 (1988). KARSSEN, C. M.: Two sites of hormonal action during germination of Chenopodium album seeds. Physio!. Plant. 36, 264-270 (1976). I4PCZYNSKI, J.: Ethylene-dependent action of gibberellin in the seed germination of Amaranthus caudatus. Physio!. Plant. 67, 584 - 587 (1986 a). - Inhibition of Amaranthus caudatus seed germination by polyethylene glycol-6000 and abscisic acid and its reversal by ethephon or 1-aminocyclopropane-l-carboxylic acid. Physio!. Plant. 67, 588-591 (1985 b). K~PCZYNSKI, land M. KARSSEN: Requirement for endogenous ethylene during germination of non-dormant seeds of Amaranthuscaudatus. Physio!. Plant. 63, 49-52 (1985). KETRING, D. L.: Ethylene and seed germination. In: KHAN, A. A. (ed.): The Physiology and Biochemistry of Seed Dormancy and Germination, 157 -178. North-Holland Publishing Co., Amsterdam (1977). KETRING, D. L. and P. W. MORGAN: Physiology of oil seeds. IV. Role of endogenous ethylene and inhibitory regulators during natural and induced afterripening of dormant virginia-type peanut seeds. Plant Physio!. 50, 382-387 (1972). KEYs, R. D ., O. E. SMITH, J. KUMAMOTO, and J. L. LYON: Effects of gibberellic acid, kinetin and ethylene plus carbon dioxide on the thermodormancy of lettuce seed (Lactuca sativa L. cv. Mesa 659). Plant Physio!. 56, 825 - 829 (1975).
c.
101
OLATOYE, S. T. and M. A. HALL: Interaction of ethylene and light on dormant weed seeds. In: HEYDECKER, W. (ed.): Seed Ecology, 233 -249. Butterworths, London (1972). ROBERTS, E. H. and S. K. BENJAMIN: The interaction of light, nitrate and alternating temperature on the germination of Chenopodium album, Capsella bursapastoris and Poa annua before and after chilling. Seed Sci. Techno!. 7, 379-392 (1979). SAINI, H. S., P. K. BASSI, and M. S. SPENCER: Seed germination in Chenopodium album L.: Relationship between nitrate and the effects of plant hormones. Plant Physio!. 77, 940-943 (1985 a). - - - Seed germination in Chenopodium album L.: Further evidence for the dependence of the effects of growth regulators on nitrate availability. Plant Cell Environ. 8, 707 -711 ( 1985 b). SAINI, H. S. and M. S. SPENCER: Manipulation of seed nitrate content modulates the dormancy-breaking effect of ethylene on Chenopo· dium album seed. Can. l Bot. 65, 876- 878 (1987). SAINI, H. S., E. D. CONSOLACION, P. K. BASSI, and M. S. SPENCER: Requirement for ethylene synthesis and action during relief of thermoinhibition of lettuce seed germination by combinations of gibberellic acid, kinetin and carbon dioxide. Plant Physio!. 81, 950-953 (1986). - - - - Control processes in the induction and relief of thermoinhibition of lettuce seed germination - actions of phytochrome and endogenous ethylene. Plant Physio!. 90, 311- 315 (1989). SATOH, S., Y. TAKEDA, and Y. ESASHI: Dormancy and impotency of cocklebur seeds. IX. Changes in ACC-ethylene conversion activity and ACC content of dormant and non-dormant seeds during soaking. l Exp. Bot. 35, 1515-1524 (1984). SISLER, E. D. and S. F. YANG: Effect of butenes and cyclic olefins on etiolated pea plants in relation to the ethylene response (abstract 225). Plant Physio!. 72-S, 40 (1983). TAYLORSON, R. B. : Response of weed seeds to ethylene and related hydrocarbons. Weed Sci. 27, 7-10 (1979). YANG, S. F., D .O. ADAMS, C. LIZADA, Y. Yu, K. l BRADFORD, A. C. CAMERON, and N. E. HOFFMAN: Mechanism and regulation of ethylene biosynthesis. In: SKOOG, F. (ed.): Plant Growth Substances 1979, 219-229. Springer-Verlag, Berlin (1980).
Differences in the Requirement for Endogenous Ethylene During Germination of Dormant and N on-Dormant Seeds of Chenopodium album L. SYLVIE MACHABEE
and
HARGURDEEP S. SAINI
Institut de recherche en biologie vegetale, Universite de Montreal, 4101, rue Sherbrooke est, Montreal, Quebec, Canada H1X 2B2 Received July 27, 1990 . Accepted November 10, 1990
Summary Requirement for endogenous ethylene in the germination of two lots of Chenopodium album L. seeds, with different inherent degrees of dormancy, was studied. The seeds produced ethylene upon imbibition, and an increase in ethylene emanation either preceded or coincided with the earliest occurrence of radicle emergence. The amounts of ethylene produced were directly proportional to the final germination percentage achieved, regardless of the seed lot or conditions of germination. Breakage of dormancy by a combined treatment with GA4 + 7 , NaN0 3 and white light (stimulated seeds) substantially increased ethylene production and germination. Although an application of 2-aminoethoxyvinyl glycine strongly suppressed ethylene production, it had no effect on the germination of non-dormant or stimulated dormant seeds. Application of 2,5-norbornadiene to antagonize ethylene action did not inhibit germination of non-dormant seeds. However, norbornadiene strongly inhibited the germination of stimulated dormant seeds, and this inhibition was overcome by the application of exogenous ethylene. It is concluded that, in order to germinate, non-dormant seeds do not require the ethylene they produce. However, induction of germination in dormant seeds depends on the action of ethylene produced during this process. The passage from dormancy to germination may, therefore, involve two steps: an ethylene-requiring transition to non-dormant state, followed by the germination itself that does not depend on ethylene.
Key words: Chenopodium album L., dormancy, ethylene action, ethylene synthesis, germination, plant hormones, seed. Abbreviation list: NBD
=
2,5-norbornadiene; AVG
Introduction Seeds of Chenopodium album L. (lamb's-quarters) are generally dormant at maturity, although the percentage of dormant seeds can vary considerably among different seed lots (Bessett and Crompton 1978). Dormancy of this species can be broken by various chemical and physical treatments (Henson 1970, Roberts and Benjamin 1979, Saini et al. 1985 a). Among these, treatments with ethylene or ethephon (2-chloroethylphosphonic acid; an ethylene releasing compound) are perhaps the most effective ways to break dormancy (Karssen 1976, Olatoye and Hall 1972, Saini et al. © 1991 by Gustav Fischer Verlag. Stuttgart
=
2-aminoethoxyvinyl glycine.
1985 a, b, Taylorson, 1979). Sensitivity of the seeds toward exogenous ethylene and other hormones is strongly enhanced by the availability of nitrate, either through exogenous supply or as an endogenous constituent of the seeds, which is in turn dependent on the level of nitrate nutrition received by the parent plant (Saini et al. 1985 a, b, Saini and Spencer 1987). Germinating seeds of several species produce ethylene, and endogenous ethylene has been shown to play an essential role in the germination of Amaranthus caudatus L., Cucumis anguria L., Lactuca sativa L. and Xanthium pennsylvanicum Wallr. (Abeles 1986, Cardoso and Felippe 1987, Kc:pczynski
98
SYLVIE MACHABEE and HARGURDEEP S. SAINI
1986a, b, K"pczynski and Karssen 1985, Ketring 1977, Ketring and Morgan 1972, Khan and Huang 1988, Khan and Prusinski 1989, Saini et al. 1986, 1989, Satoh et a1. 1984). Despite several detailed studies on the dormancy-breaking effects of exogenous ethylene on C album seed germination, the role of endogenous ethylene in the germination of this species has never been examined. It is conceivable that the variations in the degree of dormancy among different seeds of C album may be related to their ability to produce sufficient endogenous ethylene to break dormancy. The purpose of this study was to determine whether endogenously produced ethylene controls dormancy and germination of C album seeds. This was done by correlating ethylene production with the degree of dormancy and time of germination, followed by manipulations of ethylene synthesis and action via appropriate inhibitors.
Materials and Methods Seeds of Chenopodium album L. (Lamb's-quarters) were collected from the vicinity of Vermilion (seed lot-A) and Edmonton (seed lotB), Alberta during the fall of 1985. The seeds were stored dry in a refrigerator until use. Only seeds free from visible abnormalities or damage were used for experiments. Three replicates per treatment, each consisting of 100 seeds per container (Petri dish or Erlenmeyer flask), were used in all experiments. In a preliminary experiment to determine the appropriate treatments for further work (Table 1), seeds were imbibed in 100 x 15-mm disposable Petri dishes, each lined with one Whatman No.3 filter paper soaked in 5 mL of the relevant solution. Petri dishes were sealed with parafilm to prevent moisture loss. In all other experiments, seeds were imbibed on three layers of Whatman No.1 filter paper moistened with 3 mL solution, either in 57-mL (net volume) Erlenmeyer flasks (ethylene measurement experiments) or 60 x 15-mm glass petri dishes (all other experiments). Regardless of the method of imbibition, the seeds were incubated at 22 ± 1 oC, in the dark or in continuous white fluorescent light (17 pmol/m 2/s). For measurement of ethylene evolution from seeds, the Erlenmeyer flasks containing seeds were sealed with rubber sleeve stoppers (Fisher Scientific), which were experimently confirmed to be impervious to ethylene. One mL gas samples were drawn from the flasks with a gas-tight syringe at 24-h intervals for analysis with flame ionization detection on a Hewlett-Packard 5830A gas chromatograph fitted with a Porapak-Q packed column (Supleco Canada). One mL of air was injected back into the flasks after the removal of each gas sample. Because germination in this species is asynchronous and takes several days, this method of sampling for ethylene measurement was chosen despite an alternative technically superior, flow-through system (Saini et al. 1989) to ensure that period(s) of ethylene production would not go undetected. The flow-through method was impractical in this case because of the need for frequent manual sampling around the clock over 6 days. However, to avoid any modification of germination behaviour resulting from the changes in gaseous composition in the closed system (e.g. see Eastwell et al. 1978, Keys et al. 1975), effects of manipulation of ethylene synthesis on seed germination were studied through parallel experiments in a flow-through system as follows: Three uncovered petri dishes were placed inside each of 3.74-L airtight glass bottles fitted with two ports for gas connections. The bottles were supplied with 30mLlmin flow of compressed air from which hydrocarbons (including ethylene) had been removed by platinum-catalyzed thermal oxidation (Eastwell et al. 78). The air was humidified by bubbling through distilled water prior to entry into the bottles. The bottles
Table 1: Effects of GA4 + 7 (OAmM), NaN0 3 (10mM), andlor light on the germination of two seed lots of C. album. Germination (%)1 Seed lot-A Treatment Dark Light H 2O 53±3.2 85±3.3 75±2.9 87±3.6 GA4 + 7 NaN0 3 63±5A 85± 1.8 70± 1.4 94±2.5 GA4 + 7 +NaN0 3 1 Values represent mean±S.E.
Seed lot-B Dark Light 22±4.3 61±0.7 42±3.3 78±3.0 39±2.3 82±3.3 49±0.8 81±2.8
were placed inside an incubator, and the gaseous effluent from each bottle was vented out of the incubator with a tube. Daily analysis of samples of in-coming air confirmed it to be free from ethylene throughout each experiment. The effects of NBD, with or without ethylene, were measured by placing open Petri dishes in the 3.74-L bottles as above, except that the gas-connection ports were sealed with rubber septa. Liquid NBD was injected into the bottles through one rubber septum onto a piece of cotton wool suspended inside the bottle; NBD completely evaporated within 10 min to give the desired vapor-phase concentration. Ethylene was injected through the septa to obtain the appropriate concentrations, which were confirmed gas-chromatographically. Germination percentage (radicle protrusion from all covering structures) was recorded after 6 days, a period that was confirmed to give maximal germination. Experiments were repeated at least once with similar results.
Results Preliminary experiments In an experiment to determine the percentages of non-dormant and dormant seeds, and appropriate treatments for dormancy breakage, 53 % and 22 % seeds of the lots-A and -B, respectively, germinated without treatment in closed Petri dishes in the dark (Table 1). For lot-B, this figure was somewhat higher than in other experiments, probably owing to the stimulation by accumulated ethylene in the Petri dishes; in the larger containers used for later experiments, the germination was consistently around 12 % (data not shown; comparable lot-A germination was somewhat more variable, 53-59%). Thus, assuming 100% viability, the proportions of non-dormant vs. dormant seeds in the lot-A and lot-B were approximately 55/45 % and 12/88 %, respectively. Applications of GA4 + 7 , NaN0 3 , and/or light promoted germination; best results were obtained with a combination of all three treatments (Table 1). On the basis of these results, seeds incubated in H 20 in the dark are termed «non-stimulated», whereas those treated with a combination of GA4 + 7 , NaN0 3 and light are referred to as «stimulated». For economy, further results are presented primarily using two cases with the highest germination percentages in their respective categories: non-stimulated seeds of lot-A and stimulated seeds of lot-B. These represented the overall typical behaviour of non-dormant and dormant seeds, respectively.
Ethylene synthesis and germination Non-stimulated seeds of the non-dormant lot-A produced considerably more ethylene than the comparable seeds of the
99
Ethylene and seed germination 12
14 ... Seed lot-A
12 WI
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10
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iii
4
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2
2
0
0 0
1
2
4 3 5 Days from imbibition
6
Fig. 1: Amount of ethylene produced by the seeds of C. album L. imbibed on distilled water (i.e. non-stimulated seeds) and incubated in airtight flasks (57 mL). Arrow indicates the time when radicle emergence was first observed in any seed. Vertical bars on points represent respective S.E.
III
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Table 2: Effect of 2-aminoethoxyvinyl glycine (AVG; 1 mM), applied in the continuous-flow system, on the seed germination of C. album. Seed lot-A was germinated in H 2 0 and darkness, and seed lot-B was stimulated to germinate by treatment with a combination of GA4 + 7 (0.4 mM), NaN0 3 (10 mM) and light.
0
1
5
6
0
1 2 4 3 5 Days after imbibition
6
1
-AVG
+AVG
51±3.3
61±4.0 80±2.8
84± 1.5
Values represent mean±S.E.
dormant lot-B (Fig. 1). Ethylene evolution was correlated with germination time; ethylene production increased coincident with or just prior to the start of germination, and ethylene evolution in the faster germinating lot-A started and peaked earlier than in lot-B. Stimulated, dormant (lot-B) seeds produced more ethylene than non-stimulated, non-dormant seeds of lot-A (Fig. 2). This difference was proportional to their respective germination percentages (e.g. Table 1). Again, ethylene evolution started and peaked earlier in the faster germinating seeds of lot-A. Application of AVG, an inhibitor of pyridoxalphosphate-mediated reactions such as the conversion of Sadenosyl-methionine to 1-aminocyclopropane-1-carboxylic acid in ethylene biosynthesis (Yang et al. 1980), inhibited ethylene evolution from both types of seeds (Fig. 2). Indeed the AVG-treated seeds in both situations produced less ethylene than did the dormant, non-stimulated seeds (d. Figs. 1 and 2). Despite this substantial suppression of ethylene synthesis, there was no reduction in germination percentage, even in the continuous flow system, which would prevent any accumulation of residual seed-produced ethylene (Table 2). Ethylene action and germination
Application of NBD, a potent competltlve inhibitor of ethylene action in various systems including seed germina-
3
4
20 18 16 14 12 10 8 6 4 2 0
Germination (%)1 Seed lot-A Seed lot-B
2
Fig. 2: Amount of ethylene produced by the seeds of C. album L., germinating in airtight flasks (57 mL), in the presence or absence of 2-aminoethoxyvinyl glycine (1 mM). Arrows indicate the time when radicle emergence was first noticed in any seed. Vertical bars on points represent respective S.E. (A). Seeds of less dormant lot-A imbibed on distilled water and incubated in the dark (i.e. non-stimulated non-dormant seeds). (B). Seeds of more dormant lot-B imbibed in a solution of GA4 + 7 (O.4mM) and NaN0 3 (lOmM) and incubated in white light (i.e. stimulated dormant seeds).
70 60 dP
1:1 0
.
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50 40
.
s::
30
GI
20
e
t!l
10 0
o
2
4
8
2, 5-Norbornadiene (mL/L)
Fig. 3: Effect of 2,5-norbornadiene on the germination of lot-A of C. album seeds, on distilled water in the dark (i.e. non-stimulated nondormant seeds). Vertical lines over the bars indicate respective S.E.
tion (Abeles 1986, Ktrpczynski and Karssen 1985, Saini et al. 1986, 1989, Sisler and Yang 1983), failed to block the germination of non-stimulated, non-dormant seeds of lot-A, even at the extremely high concentration of 8 mLiL (Fig. 3).
100
SYLVIE MACHABEE and HARGURDEEP S. SAINI 100~-------------------------'
80 60 40
20
o
o
2
4
8
2, 5-Norbornadiene (mL/L)
Fig.4: Effects of various concentrations of 2,5-norbornadiene and exogenous ethylene on the germination of lot-B and C. album, treated with a combination of GA4 + 7 (OAmM), NaN0 3 (10mM) and light (i.e. stimulated dormant seeds). Vertical lines over the bars indicate respective S.E.
In contrast, application of NBD to stimulated, dormant seeds (lot-B) strongly inhibited germination in proportion to the concentration. Indeed, germination at 8 mL/L NBD was reduced to the level of non-stimulated seeds (Fig. 4). Application of exogenous ethylene in combination with NBD overcame the inhibition caused by NBD (Fig. 4). The interaction between different concentrations of ethylene and NBD was indicative of competitive antagonism. Discussion The results show that endogenous ethylene is essential for germination of dormant seeds, but not for the germination of non-dormant seeds of C. album. All seeds produced ethylene during germination, and there was a direct relationship between the amount of ethylene produced and the final germination percentage, whether the seeds germinated with or without an external stimulus (Figs. 1 and 2). Differences in ethylene production under various situations probably reflect the number of seeds that were in the process of germination, or past this stage, at a given time. It is quite likely that the seeds that did not germinate produced little or no ethylene. In all cases, an increase in ethylene production started before or coincident with the first visible sign of germination (radicle protrusion). The results suggest a possible role of ethylene in germination. However, the experiments with the inhibitors of ethylene synthesis and action indicate a rather complex picture: Ethylene produced by non-dormant seeds appears to play no mediatory role in germination because an inhibition of ethylene synthesis or action in these seeds failed to block their germination (Figs. 2A and 3, Table2). In contrast, breakage of dormancy through external stimulants appears to be mediated by endogenous ethylene. Inhibition of ethylene action completely blocked the germination of the dormant fraction of these seeds, and exogenous ethylene overcame this blockage (Fig. 4). An apparent inconsistency here was the failure of AVG to inhibit germination, despite a
depression of ethylene production to levels even below those produced by non-stimulated dormant seeds (Figs. 1 and 2 B). Similar results were also previously reported for peanut and Amaranthus caudatus seeds (Hoffman et al. 1983, K~pczynski and Karssen 1985). Germination of Lactuca sativa seeds is only partially blocked by A VG (Saini et al. 1986, 1989). One possible explanation is that ethylene is required for dormancy breakage in such minute quantities that the residual amounts produced even upon AVG treatment are sufficient for germination. It is curious that germination of dormant seeds requires ethylene action whereas the naturally non-dormant seeds do not need ethylene they produce during germination. The non-dormant seeds of A. caudatus, L. sativa and X. pennsylvanicum do require endogenous ethylene for germination (Abeles 1986, K~pczynski and Karssen, 1985, Satoh et al. 1984). In L. sativa, thermoinhibition (a prelude to the induction of thermodormancy) is apparently not caused by a reduction in the ability of seeds to produce ethylene (Abeles 1986, Burdett 1972, Dunlap and Morgan 1977). High temperature may, however, raise the threshold concentration of ethylene required for germination (Dunlap and Morgan 1977). Our findings with C. album suggest that the passage from the dormant to the germination stage may involve two steps: a transition from a dormant to a non-dormant state that requires ethylene, and the next step of germination itself that proceeds without a need for ethylene-action. This would fit with an earlier observation by Karssen (1976) that germination of dormant C. album seeds involves two sites of hormone action: the initiation of growth that is induced by ethylene or gibberellins, and the later stages of growth that lead to radicle protrusion. The exact nature of ethylene-action in seed germination remains obscure, although Abeles (1989) presented evidence suggesting that one action of ethylene could be the promotion of radicle cell expansion in embryonic hypocotyl in L. sativa. Acknowledgements This research was supported by the Natural Sciences and Engineering Research Council of Canada, grant No. 37284 to H. S. Saini. The authors wish to thank Ms. Sylvie Lalonde for assistance.
References ABELES, F. B.: Role of ethylene in Lactuca sativa cv. «Grand Rapids» seed germination. Plant Physiol. 81, 780-787 (1986). BASSETT, I. J. and C. W. CROMPTON: The biology of Canadian weeds. 32. Chenopodium album L. Can. J. Bot. 58, 1061-1072 (1978). BURDETT, A. N.: Antagonistic effects of high and low temperature pretreatments on the germination and pre germination ethylene synthesis of lettuce seeds. Plant Physiol. 50, 201-204 (1972). CARDOSO, V. J. M. and G. M. FEUPPE: Endogenous ethylene and the germination of Cucumis anguria L. seeds. Rev. Bras. Bot. 10, 29-32 (1987). DUNLAP,J. R. and P. W. MORGAN: Characterization of ethylene/gibberellic acid control of germination in Lactuca sativa L. Plant Cell Physiol. 18, 561-568 (1977). EASlWELL, K. c., P. K. BASSI, and M. S. SPENCER: Comparison and evaluation of methods for the removal of ethylene and other hy-
Ethylene and seed germination drocarbons from air for biological studies. Plant Physio!' 62, 723 -726 (1978). HANSON, I. E.: The effect of light, potassium nitrate and temperature on the germination of Chenopodium album L. Weed Res. 10, 27 -39 (1970). HOFFMAN, N. E., loR. Fu, and S. F. YANG: Identification and metabolism of 1-(malonylamino)cyclopropane-l-carboxylic acid in germinating peanut seeds. Plant Physio!. 71, 197 -199 (1983). KHAN, A. A. and l PRUSINSKI: Kinetin enhanced 1-aminocyclopropane-I-carboxylic acid utilization during alleviation of high temperature stress in lettuce seeds. Plant Physio!' 91, 733 -737 (1989). KHAN, A. A. and X.-L. HUANG: Synergistic promotion of ethylene production and germination with kinetin and 1-aminocyclopropane-I-carboxylic acid in lettuce seeds exposed to salinity stress. Plant Physio!. 87, 847 - 852 (1988). KARSSEN, C. M.: Two sites of hormonal action during germination of Chenopodium album seeds. Physio!. Plant. 36, 264-270 (1976). I4PCZYNSKI, J.: Ethylene-dependent action of gibberellin in the seed germination of Amaranthus caudatus. Physio!. Plant. 67, 584 - 587 (1986 a). - Inhibition of Amaranthus caudatus seed germination by polyethylene glycol-6000 and abscisic acid and its reversal by ethephon or 1-aminocyclopropane-l-carboxylic acid. Physio!. Plant. 67, 588-591 (1985 b). K~PCZYNSKI, land M. KARSSEN: Requirement for endogenous ethylene during germination of non-dormant seeds of Amaranthuscaudatus. Physio!. Plant. 63, 49-52 (1985). KETRING, D. L.: Ethylene and seed germination. In: KHAN, A. A. (ed.): The Physiology and Biochemistry of Seed Dormancy and Germination, 157 -178. North-Holland Publishing Co., Amsterdam (1977). KETRING, D. L. and P. W. MORGAN: Physiology of oil seeds. IV. Role of endogenous ethylene and inhibitory regulators during natural and induced afterripening of dormant virginia-type peanut seeds. Plant Physio!. 50, 382-387 (1972). KEYs, R. D ., O. E. SMITH, J. KUMAMOTO, and J. L. LYON: Effects of gibberellic acid, kinetin and ethylene plus carbon dioxide on the thermodormancy of lettuce seed (Lactuca sativa L. cv. Mesa 659). Plant Physio!. 56, 825 - 829 (1975).
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OLATOYE, S. T. and M. A. HALL: Interaction of ethylene and light on dormant weed seeds. In: HEYDECKER, W. (ed.): Seed Ecology, 233 -249. Butterworths, London (1972). ROBERTS, E. H. and S. K. BENJAMIN: The interaction of light, nitrate and alternating temperature on the germination of Chenopodium album, Capsella bursapastoris and Poa annua before and after chilling. Seed Sci. Techno!. 7, 379-392 (1979). SAINI, H. S., P. K. BASSI, and M. S. SPENCER: Seed germination in Chenopodium album L.: Relationship between nitrate and the effects of plant hormones. Plant Physio!. 77, 940-943 (1985 a). - - - Seed germination in Chenopodium album L.: Further evidence for the dependence of the effects of growth regulators on nitrate availability. Plant Cell Environ. 8, 707 -711 ( 1985 b). SAINI, H. S. and M. S. SPENCER: Manipulation of seed nitrate content modulates the dormancy-breaking effect of ethylene on Chenopo· dium album seed. Can. l Bot. 65, 876- 878 (1987). SAINI, H. S., E. D. CONSOLACION, P. K. BASSI, and M. S. SPENCER: Requirement for ethylene synthesis and action during relief of thermoinhibition of lettuce seed germination by combinations of gibberellic acid, kinetin and carbon dioxide. Plant Physio!. 81, 950-953 (1986). - - - - Control processes in the induction and relief of thermoinhibition of lettuce seed germination - actions of phytochrome and endogenous ethylene. Plant Physio!. 90, 311- 315 (1989). SATOH, S., Y. TAKEDA, and Y. ESASHI: Dormancy and impotency of cocklebur seeds. IX. Changes in ACC-ethylene conversion activity and ACC content of dormant and non-dormant seeds during soaking. l Exp. Bot. 35, 1515-1524 (1984). SISLER, E. D. and S. F. YANG: Effect of butenes and cyclic olefins on etiolated pea plants in relation to the ethylene response (abstract 225). Plant Physio!. 72-S, 40 (1983). TAYLORSON, R. B. : Response of weed seeds to ethylene and related hydrocarbons. Weed Sci. 27, 7-10 (1979). YANG, S. F., D .O. ADAMS, C. LIZADA, Y. Yu, K. l BRADFORD, A. C. CAMERON, and N. E. HOFFMAN: Mechanism and regulation of ethylene biosynthesis. In: SKOOG, F. (ed.): Plant Growth Substances 1979, 219-229. Springer-Verlag, Berlin (1980).