Plant Cell Physiol. 37(8): 1054-1058(1996)
JSPP © 1996
Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic
Compounds
Akiko Maruyama', Makoto Yoshiyama1, Yasuhiro Adachi', Akinobu Tani 1 , Ryo Hasegawa2 and
Yohji Esashi3
1
2
Laboratory of Environmental Science, Botanical Garden, Faculty of Science, Tohoku University, North Campus, Kawauchi,
Aobaku, Sendai, 980-77 Japan
Agricultural Science Research Laboratory, Takarazuka Research Center, Sumitomo Chemical Co., Ltd., Takarazuka, 655 Japan
The effects of allyl, sulfur and cyanogenic compounds
on the germination of upper cocklebur (Xanthium pennsylvanicum Wallr.) seeds were examined. Mercaptoethanol
and methylmercaptan as well as KCN, substrates for pcyanoalanine synthase (CAS), and H2S and thiocyanate,
the products of the CAS catalyzing reaction, were effective
in promoting germination, suggesting the involvement of
CAS in germination.
Most of allyl compounds, especially allylthiourea, as
well as ethylene which activated CAS [Hasegawa et al.
(1994) Physiol. Plant. 91: 141], promoted the germination
in an abnormal type which occurred by the predominant
growth of cotyledons as did C2H4 [Katoh and Esashi (1975)
Plant Cell Physiol. 16: 687]. However, they failed to activate CAS unlike ethylene, and to liberate free ethylene
during an incubation period. It was thus possible that an
C2H4-like double bond within allyl compounds can act to
promote seed germination.
Key words: Allyl compound — Cocklebur — yS-Cyanoalanine synthase (EC 4.4.1.9) — Cyanogenic compound —
Seed germination — Sulfur compound.
Both ethylene (Katoh and Esashi 1975) and KCN
(Esashi et al. 1979, Esashi 1991) promote the germination
of cocklebur seeds. Esashi et al. (1991) found that the seeds
of cyanogenic plants and non-cyanogenic plants, including
cocklebur, contain cyanogenic compounds as storage substances and release HCN during the pre-germination period. Hasegawa et al. (1994) reported that /?-cyanoalanine
synthase (CAS; EC 4.4.1.9), which catalyzes HCN and cysteine to yS-cyanoalanine and H2S (Miller and Conn 1980) is
present in many dry seeds, including cocklebur, and its activity increases during pre-germination periods. In cocklebur, rice (Hasegawa et al. 1994), cucumbur, radish and
barley (Hasegawa et al. 1995b), the activities of CAS
was further increased by ethylene application during preAbbreviation: CAS, yS-cyanoalanine synthase.
Correspondence author: Yohji Esashi Laboratory of Environmental Science, Botanical Garden, Faculty of Science, North Campus of Tohoku University, Kawauchi, Aobaku, Sendai, 980-77,
Japan. Fax: 81-22-263-9279.
3
germination periods. Moreover, not only KCN but also cysteine, substrates of CAS, promotes the germination of
cocklebur seeds (Hasegawa et al. 1995a). From these facts,
Hasegawa et al. (1995a) discussed that CAS may participate in the germination process by making an increased
supply of the SH-group derived from cysteine, in addition
to enlarging the amino acid pool.
Chemicals containing CN-moiety such as cyanamide
(Henzell and Briscoe 1975, Shulman et al. 1986, SpiegelRoy et al. 1987) and thiocyanates (Thompson and Kosar
1939), besides KCN, have been known to break bud dormancy and to promote seed germination, respectively.
Similarly, the sulfur containing chemicals such as cysteine
(Roberts and Smith 1977, Hasegawa et al. 1995a) as a cosubstrate of CAS, and thiourea (Mayer and PoljakoffMayber 1975, Stokes 1965) stimulate seed germination.
Moreover, some allyl compounds having carbon double
bond like C2H4 have a dormancy-breaking actions (Hosoki
et al. 1985, Corbineau et al. 1991). This work was carried
out to survey more precisely the effects of various compounds containing CN moiety, S element and allyl base on
cocklebur seed germination and to confirm the possibility
of CAS involvement.
Materials and Methods
Upper cocklebur (Xanthium pennsylvanicum Wallr.) seeds
which were harvested in 1991 or 1992 and stored at 8°C were used
throughout this experiment. For the prepaparation of secondarily
dormant seeds, the seeds were pre-soaked at 23°C for 2 to 5
weeks. For non-volatile chemical treatments, a lot of 22 to 24
seeds were incubated on 2 layers of filter paper wetted with 4 ml of
H2O or chemical solution in each petri dish 9 cm in diameter at
23°C for 3 days. For volatile chemical treatments, including allylcyanide, allylethylether, allylmethylsulfide, allylisothiocyanate,
allylamine, allylmercaptan, allylchloride, allylalcohol, allylether,
methylmercaptan, mercaptoethanol, ethanol, acetone, /i-propanol, propylether, propylmercaptan, Petri dishes containing the
seeds were placed in a 2,000 ml desiccator. Just before the desiccator sealed, a small vial filled with the chilled test solutions at
different volumes calculated as they completely volatiled was placed in the container as quickly as possible. In the case of H 2 S, its
necessary volume was directly injected using a hypodermic syringe
into the desiccator through a rubber stopper.
Adequate concentrations for chemical treatments were preliminarily investigated to induce germination in the extent ranging from 0.01 to 100 mmol liter"1 head space. All experiments
1054
Allyl, sulfur, seed germination
were at least repeated twice and the data obtained at the most effective concentration were shown as a representative.
After incubation, the numbers of germinated seeds were
counted, and the non-germinated seeds were thoroughly washed
with 30°C tap water and then transferred to other petri dishes with
2 disks of filter paper moistened with 3.5 ml of H2O for counting
the subsequent germination. The petri dishes were placed at 23°C
throughout the experimental periods. Evolved C2H4 was assayed
as described (Katoh and Esashi 1975).
Results and Discussion
All allyl compounds tested, except for allylthiourea,
had little germination promoting effects during an initial
test period of 3 days (Table 1), even if they were applied at
varying concentrations over 5 orders starting from 0.001
mM (data not shown). Seeds were dead at higher concetrations of volatile allyl compounds. Germination of
cocklebur seeds was stimulated by allylthiourea alone during the first 3 days. However, the seeds contacted with
allylurea, allylcyanide and allylmercaptan began to germinate only after removal of the chemicals by rinsing. At that
time, allylthiourea at 30 mM caused full germination, and
allylmercaptan was very effective at a very low concentration of 0.3 mM. Propanol and propylmercaptan did not
show any stimulation (data not shown) which was inconsis-
1055
tent with the findings by Cohn et al. (1988). Expectedly,
H2S exhibited some germination-promoting effect even during a test period for the first 3 days. Mercaptoethanol and
methyl mercaptan, which can react as a substrate of CAS instead of KCN (Akopyan et al. 1975), did not show any promotive effect in unimbibed seeds. Interestingly, cyanamide
is effective in breaking bud dormancy had little promotive
effect. Both ammonium and sodium thiocyanates were ineffective only during the first 3 days, but they exhibited
some promotive actions on the subsequent germination,
unlike cyanamide. Both cyanamide and thiocyanates did
not act as the cosubstrate of CAS instead of KCN (data not
shown). The facts that both H2S and thiocyanates as CAS
products (Akopyan et al. 1975) stimulated the germination
of cocklebur seeds further suggest the possible involvement
of CAS in the germination process of cocklebur seeds, although thiocyanates can also be produced from cyanide by
means of thiosulfate sulfurtransferase (EC 2.8.1.1) (Sorbo
1953).
Many allyl compounds, excepting allylcyanide,were
promotive also to pre-soaked secondarily dormant seeds
(Table 2), although generally required higher concentrations to induce some significant effects compared with the
un-imbibed ones. Why allylcyanide became ineffective in
the pre-soaked seeds is an unsolved problem. Neverthe-
Table 1 Effects of allyl, sulfuric and cyanogenic compounds on the germination of unimbibed cocklebur seeds
Chemical
Allyl thiourea (30 mM)
Allyl urea (100 mM)
Allyl cyanide (30mmol liter"1 head space)
Allyl ethyl ether (0.01 mmol liter"1 head space)
Allyl methyl sulfide (3 mmol liter"1 head space)
Allyl isothiocyanate (0.1 mmol liter"' head space)
Allyl amine (0.1 mmol liter"1 head space)
Allyl mercaptan (0.3 mmol liter"1 head space)
Allyl chloride (0.03 mmol liter"1 head space)
Allyl alcohol (0.003 mmol liter"1 head space)
Allyl ether (0.03 mmol liter"1 head space)
H2S (0.03 mmol liter"1 head space)
Methyl mercaptan (0.3 mmol liter"1 head space)
Mercaptoethanol (1 mmol liter"1 head space)
Cyanamide (10 mM)
Dithiothreitol (3 mM)
Ammonium thiocyanate (30 mM)
Sodium thiocyanate (30 mM)
Germination (%)
Before washing
7 days after washing
56.0*
7.7
—
7.4
85.4*
24.0*
36.6*
2.3
—
—
7.0
11.2
47.5*
1.10
15.3*
11.7
1.3
0.8
2.9
—
—
2.6
11.2
44.5*
56.6*
2.6
Germination data were taken at the 3rd day before washing, and the remaining un-germinated seeds were washed and placed for further
1 week on water substratum in air. Data were shown as differences in % germination between the water controls and treated seeds, in
which seeds were contacted with the concentration givingfinallya maximal germination. Control values are 9.5 ±2.5% before washing,
14.6±4.67% after washing. Minus shows % germination below the control, but not significant inhibition. Astral marks show the significant promotion at p=0.05.
1056
Allyl, sulfur, seed germination
Table 2 Effects of allyl, sulfuric and cyanogenic compounds on the germination of secondarily dormant cocklebur seeds
which were pre-soaked for 2 weeks
Chemical
Allyl thiourea (30 mM)
Allyl urea (100 mM)
Allyl cyanide (30 mmol liter" 1 head space)
Allyl methyl sulfide (3 mmol liter" 1 head space)
Allyl isothiocyanate (0.1 mmol liter" 1 head space)
Allyl mercaptan (0.3 mmol liter" 1 head space)
Allyl alcohol (0.003 mmol liter" 1 head space)
Allyl ether (0.03 mmol liter" 1 head space)
H2S (0.5 mmol liter" 1 head space)
Methyl mercaptan (0.3 mmol liter" 1 head space)
Mercaptoethanol (1 mmol liter" 1 head space)
Ammonium thiocyanate (30 mM)
Sodium thiocyanate (30 mM)
Germination
Before washing
7 days after washing
81.0*
—
—
—
62.8*
—
9.2
—
40.6*
28.2*
48.2*
—
—
95.4*
10.1*
—
68.3*
63.2*
24.0*
25.0*
51.5*
39.3*
33.8*
53.0*
21.9*
20.4*
Incubation were performed as in Table 1. Data are shown as in Table 1. Control values are 3.3 ± 10% before washing, 4.6± 1.27% after
washing. All symbols are as in Table. 1.
less, many chemicals, which were ineffective in unimbibed
seeds, exhibited some effects on the pre-soaked seeds. Espedaily, mercaptoethanol and methylmercaptan caused a
marked germination which are available as substrates of
CAS instead of KCN the degrees of which corresponded
roughly to the efficiency as substrates of the CAS catalyzing reaction (Akopyan et al. 1975). In cocklebur seeds,
KCN was capable of causing full germination (Esashi et al.
1979), but mercaptoethanol and methylmercaptan were not
even at their optimal concentrations (Table 2). Moreover,
allyl-methylsulfide and -isothiocyanate which contain both
element S and CN moiety, respectively, were more significantly effective in pre-soaked seeds (Table 2). Furthermore,
even allyl-ether and -alcohol including neither S nor CN
showed slight but significant effects. However, the abundant production of C 2 H 4 from the seeds exposed to these
allyl compounds could not be observed in both unimbibed
and imbibed seeds (data not shown), which imply that an
allyl base of a carbon double bond, within allyl compounds
can act by itself as does C2H4. Other relationships between
structure or reactivity of these chemicals were not found.
This possibility was supported by the normality of germinants in response to various chemicals (Table 3), in
which the normalities of the germinants exposed to allyl
compounds were generally lower than those of others as
in C 2 H 4 . These findings suggest that allyl compounds stimulate the cocklebur seed germination by preferentially
accelerating the cotyledonary growth rather than the axial
growth (Esashi et al. 1975). In contrast to this, KCN has
been reported to enhance the normality of the germinants
(Esashi etal. 1979). Also in Table 3, thiocyanates including
CN moiety and H2S as a product of the CAS reaction
caused germination at higher normalities, which suggest
that these compounds predominantly stimulate the axial
Table 3 The normality of cocklebur seedlings germinated in response to the typical chemicals used in Table 1, 2 and 4
Chemical
H 2 O (Control)
C2H4 (0.01 mmol liter" 1 head space)
Thiourea (30 mM)
Allyl thiourea (30 mM)
Allyl mercaptan (0.3 mmol liter" 1 head space)
H2S (0.03 mmol liter" 1 head space)
Ammonium thiocyanate (30 mM)
Sodium thiocyanate (30 mM)
Normality (%)
Unimbibed
Imbibed
72.2
4.2
6.8
3.4
70.1
71.9
81.3
83.2
100
15.6
27.2
0
55.0
100
84.2
100
Normality is shown as a rate of seeds normally germinated by radicle growth to the total numbers of germinated seeds.
Ally], sulfur, seed germination
1057
Table 4 Germination response to urea, thiourea, allylurea and allylthiourea with or without C 2 H 4 of unimbibed and imbibed cocklebur seeds
Chemical
0.01 mmol liter ' head space C 2 H 4
30 mM Urea
30 mM Allyl urea
30 mM U r e a + 0 . 0 1 mmol liter" 1 head space C 2 H 4
30 mM Thiourea
30 mM Allyl thiourea
30 mM Thiourea+0.01 mmol liter" 1 head space C 2 H 4
Germination {%)
Unimbibed
Imbibed
26.5'
42.1"
12.6*
26.7*
29.8*
45.4*
36.1*
3.4
19.4*
67.4*
Data are shown as in Tables 1 and 2. Control values of unimbibed and imbibed are 14.6±4.6% and 4.6± 1.2%. All symbols are as in
Table 1.
growth, unlike the allyl compounds and C 2 H 4 .
To further confirm the action analogy of allyl compounds with C 2 H 4 , the effects of allyl-urea and -thiourea on
the cocklebur seed germination were compared with those
of urea and thiourea applied singly or in combination with
C 2 H 4 (Table 4). In the un-imbibed seeds, thiourea and
allylthiourea were similarly effective, whereas in the imbibed ones, allylthiourea was much more effective than
thiourea. In both seeds, ethylene enhanced the effect of
thiourea.
On the other hand, many compounds having lipophilic properties with a small polality and a small molecular size have been known to be active in breaking the dormancy of many seeds (Adkins et al. 1984a, b , Taylorson
1982, Amritphale et al. 1993). Taylorson and Hendricks
(1979, 1981) proposed the hypothesis that acetone, ethanol
and other organic solvents act at the membrane level
through their properties as anaesthetics. In cocklebur
seeds, however, the significant germination-stimulating
effects of acetone, ethanol and n-propanol were not found
regardless of whether they were unimbibed or imbibed
(data not shown). As shown in Table 2, both ethanol and
ether exhibiting some effects on the germination only when
applied to early secondarily dormant seeds in the form of
molecules with an allyl base. These results do not support
the hypothesis that some organic solvents may act to seed
germination as anaesthetics in the case of cocklebur seeds.
The present results suggest that the carbon double
bond of C 2 H 4 may be capable of acting as a seed germination stimulator even when it exists within a molecule such
as allyl compounds, and that both the S element and CN
moiety are implicated in proceeding the germination process of cocklebur seeds, part of which may be controlled
via the action of CAS. Nevertheless, the fact that allyl compounds unlike C 2 H 4 failed to activate CAS (data not
shown) suggest that allyl compounds act to promote seed
germination through a mechanism different from the activation of CAS. However, the action mechanisms of thiourea
and of the most active allylthiourea in germination control
still remain undetermined.
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(Received June 10, 1996; Accepted August 21, 1996)