Plant CellPhysiol. 37(4): 545-549 (1996)
JSPP © 1996
Involvement of Cyanogens in the Promotion of Germination of Cocklebur
Seeds in Response to Various Nitrogenous Compounds, Inhibitors of
Respiratory and Ethylene
Yohji Esashi, Akiko Maruyama, Satoshi Sasaki, Akinobu Tani and Makoto Yoshiyama
Laboratory of Environmental Biology, Botanical Garden, Faculty of Science, Tohoku University, North Campus, Kawauchi, Aobaku,
Sendai, 980-77 Japan
Nitrogenous inhibitors of respiration, namely, KCN,
NaN3 and NH2OH, which promote the germination of
cocklebur {Xanthium pennsylvaniicum Wallr.) seeds, enhanced the accumulation of cyanogenic compounds. Urea
and cyanamide, which were ineffective, did not. Part of the
exogenously applied KCN was converted to cyanogenic glycosides and lipids, but these compounds were only produced at low levels in NaN3-treated seeds. Exposure to NH2OH
caused a slight accumulation of both types of cyanogenic
compounds. Thiourea, effective in stimulating germination, did not increase the cyanogenic compounds, which
suggests that the mechanism of its effect on germination is
different from that of other nitrogenous compounds. Thiocyanate had a slightly promotive effect and caused minor increases in the levels of cyanogenic compounds.
Ethylene increased the metabolic utilization of the accumulated cyanogens in response to KCN or NaN3. The response to ethylene of seeds in secondary dormancy was
restored only after pre-treatment with KCN or NaN3. The
response occurred once the level of cyanogens had increased. By contrast, enhancement of the effects of KCN
and NaN3 on germination by propyl gallate or benzohydroxamate, inhibitors of CN-resistant respiration, was accompanied by the increased accumulation of cyanogens within
the seeds that had been exposed to KCN or NaN3 in combination with the other types of respiratory inhibitors.
Thus, it is suggested that endogenous cyanogens might be
involved in the germination of cocklebur seeds.
Key words: Cocklebur — Cyanogen — Ethylene — Respiration inhibitor — Seed germination.
Various nitrogenous compounds, such as KNO3 (see
Bewley and Black 1982), thiourea (see Mayer and Poljakoff-Mayber 1975) and cyanamide (Spiegel-Roy et al.
1987), and various inhibitors of respiration, such as KCN,
NaN3 and NH2OH (see Bewley and Black 1982), stimulate
the germination of seeds of many plant species. Cocklebur
Abbreviations: BHAM, benzohydroxamic acid; CAS, ficyanoalanine synthase; PG, propyl gallate; SHAM, salicylhydroxamic acid.
seeds can germinate in the presence of thiourea at 30 raM
or more, as well as in response to the inhibitors of respiration listed above (at 0.5 mM to 30 mM), but they do not
respond to 30 mM KNO3 (Esashi et al. 1979) or 30 mM
cyanamide (unpublished). More recently, we found that
thiocyanates stimulate the germination of cocklebur seeds
(unpublished), and that both KNO3 and NH4C1, applied at
200 mM as priming reagents, were effective in stimulating
their germination (Yoshiyama et al. 1996a). At 200 mM,
KNO3 increases the levels of cyanogenic glycosides and
lipids in the cotyledonary tissues. Moreover, the effect of
KNO3 is further enhanced by exposure to C2H4. According
to Yoshiyama et al. (1996b), this enhancement is accompanied by an increase in the size of the pool size of amino
acids. These findings suggest the involvement of accumulated cyanogenic compounds in the germination process of
cocklebur seeds.
Esashi et al. (1991a) reported that not only the seeds of
cyanogenic plants but also those of non-cyanogenic plants,
including cocklebur, contain cyanogenic compounds as
storage substances and release HCN during the pre-germination period. Hasegawa et al. (1994) found that seeds of
many non-cyanogenic plants, as well as those of cyanogenic plants, display /?-cyanoalanine synthase (CAS: EC
4.4.1.9) activity even before imbibition, and the activity of CAS increases during the pre-germination period.
The activity at this stage is enhanced by exposure to C2H4.
Moreover, not only KCN but also cysteine, as a co-substrate of CAS, promotes the germination of cocklebur
seeds (Hasegawa et al. 1995). Hydrogen sulfide, a product
of the CAS-catalyzed reaction, was also effective in promoting germination (unpublished). From these observations, we suggested that CAS might be involved in seed
germination by supplying a sulfhydryl group and by augmenting the pool of amino acids, and that C2H4 might act
to promote germination by activating CAS. Furthermore,
the application to seeds of C2H4 in combination with KCN
was effective in promoting germination (Hasegawa et al.
1995). The decreased responsiveness of secondarily dormant
cocklebur seeds to C2H4 was restored only when they were
pre-treated with KCN or NaN3, but not when they were
post-treated (Esashi et al. 1982). Nitrogenous inhibitors of
respiration might directly or indirectly stimulate seed germination via the accumulation of cyanogens in seed tissues,
545
546
Y. Esashi et al.
and C2H4 might accelerate the germination by promoting
the utilization of the accumulated cyanogens through the
activation of CAS. The first objective of the present study
was to identify nitrogenous compounds that could stimulate the germination of cocklebur seeds by enhancing the
cyanogen content of seed tissues. The second objective
was to determine whether or not the enhancement of germination of cocklebur seeds by the application of KCN or
NaN3 together with an inhibitor of cyanide-resistant respiration, PG or BHAM (Esashi et al. 1981), might be due to
the accumulation of cyanogens.
Materials and Methods
Seeds and seed tissues—The upper and lower seeds of
cocklebur (Xanthium pennsylvanicum Wallr.), which had been
stored at 8°C, were used. In one experiment, the cotyledonary segments isolated from the lower seeds were pre-soaked for 6 h and
used after de-coating. Upper seeds were used in the state of secondary dormancy. To induce this state, they were pre-soaked in
water at 23 °C for either 55 (Table 3) or 300 (Table 5) d. Longterm soaking for 300 d was used to obtain a population of
seeds in deep secondary dormancy that had lost their responsiveness to KCN and NaN3. All treatments were carried out in 125-ml
flasks that contained two disks of filter paper wetted with 4 ml of a
solution of various chemicals, with 3 or 4 replicates. Each flask
also contained 1 g FW of cotyledonary segments, or 23 upper or
20 lower seeds. Treated segments of seeds or seeds in each flask
were rinsed with running tap water, gathered together in groups,
and again washed with deionized water. Some of the seeds were
used for extraction of cyanogens. The remaining seeds were distributed into three flasks that contained two disks of filter paper
wetted with 3.5 ml of distilled water for prescribed periods with or
without 30 fi\ liter"1 C2H4 for germination tests or extraction of
cyanogens.
Assays of cyanogens and cyanogenic glycosides and lipids—
The bound HCN (cyanogens), cyanogenic glycosides and cyanogenic lipids in seeds or cotyledonary tissues were quantitated as
described by Esashi et al. (1991a). Cyanogens, cyanogenic glycosides and lipids were hydrolyzed by 8 M H2SO4, ^-glucosidase and
lipase, respectively, in flasks that each contained a small glass
tube. The liberated HCN was absorbed by 2.5 M NaOH in the
tube, and the amount of trapped HCN was assayed quantitatively
with a gas chromatograph (Shimadzu GC-14A; Kyoto) with a
flame thermionic detector after liberation by acidification with
HC1 of the solution of NaOH. Standard errors of the mean (n=4)
were determined from the data, which represented the differences
between the amounts obtained by acidification before and after
various hydrolytic treatments.
Results and Discussion
Production of cyanogens in response to various nitrogenous compounds—Treatment with KCN of cyanogenic
apple seeds (Bogatek et al. 1991) and non-cyanogenic
cocklebur seeds (Esashi et al. 1991b) stimulates germination and augments the cyanogen contents. When inhibitors
of respiration, namely, KCN, NaN3 and NH 2 OH, were applied at concentrations that were effective in inducing germi-
nation of cocklebur seeds (Esashi et al. 1979), levels of
cyanogens increased significantly during an incubation period of only 18 h. Thiourea, which is very effective in causing germination of cocklebur seeds at 100 mM, did not
promote the accumulation of cyanogens. Urea, as well
as KNO3 and NH4C1, which were ineffective (Esashi et al.
1979), failed to promote the accumulation of cyanogens
(Table 1).
Cyanogenic glycosides and lipids were included in the
cyanogens that accumulated in the cotyledonary segments
that had been treated with NaN3, NH2OH or KCN (Table 2).
The growth of the segments was completely inhibited by
the inhibitors of respiration but not by 30 mM KNO3. KCN
at 20 mM induced the accumulation of cyanogenic glycosides and lipids to about three and two times the levels
achieved with KNO3, respectively (Table 2). NH 2 OH inhibited the cotyledonary growth as did KCN and induced
the accumulation of both cyanogenic compounds. However, NaN3 did not increase cyanogenic glicosides and
lipids (Table 2), indicating that most cyanogens produced
in the presence of NaN3 and NH 2 OH are different from the
cyanogenic glycosides and lipids.
On the other hand, both cyanamid and urea, which
did not promote germination of cocklebur seeds, hardly increased the contents of the cyanogenic glycosides and lipids
(Table 3). As in Table 1, thiourea did not cause the accumulation of both cyanogenic compounds, though being
very effective in stimulating the seed germination (Esashi et
al. 1979). However, two thiocyanates, slightly effective in
inducing the seed germination, caused a slight accumulation of both cyanogenic compounds (Table 3), which might
be due to the partial incorporation of their CN moieties by
thiosulfate sulfurtransferase (EC 2.8.1.1), which catalyzed
between CN~ and SCN~ and was ubiquitously distributed
in higher plants (Chew 1973). A similar level of cyanogen
accumulation was also recognized with NaN3, the results of
which were different from those in Table 2 where the cotTable 1 Effects of nitrogenous compounds on the levels
of cyanogens in non-dormant lower seeds of cocklebur
Compound
(mM)
H2O (Control)
CCKNH^ (100)
CS(NH2)2 (100)
NH4C1 (30)
KN0 3 (30)
NaN3 (5)
NH2OH (20)
KCN (20)
Bound HCN
(pmol(gFW)-')
81± 8
73± 9
62± 5
79± 10
86± 8
2,290 ±170
l,250±230
15,390±7O0
Seeds were treated with each compound for 18 h at 23°C. The initial level of bound HCN was 68±10 pmol (g FW)" 1 .
Cyanogens in germination of cocklebur seeds
547
Table 2 Effects of various nitrogenous compounds on the levels of cyanogenic glycosides and lipids in the cotyledonary
tissues of the lower seeds of cocklebur
Compound
(mM)
Increase in fresh weight
{%)
Content (HCN pmol initial (g FW)~')
Cyanogenic lipids
Cyanogenic glycosides
47.9±2.5
49±
6
13±
3
NaN3 (5)
0.2±0.1
54±
2
26+
8
NH2OH (20)
1.4±0.4
87 ± 21
52± 10
54.9±2.8
610± 40
910± 40
2,050±150
2,130+230
H2O
KNO3 (30)
0
KCN (20)
De-coated cotyledonary segments were treated with various chemicals for 3 d at 23°C. Then they were weighed and used for assays of
cyanogenic compounds.
yledonary segments were used, suggesting that part of the
cyanogens produced in response to NaN3 can more or less
be converted to cyanogenic glycosides and lipids. NH2OH
brought about the accumulation of both cyanogenic compounds at the same significant levels as those in Table 2,
and KCN caused a tremendous accumulation as shown in
Table 2. Thus, the germination-stimulating actions of
many nitrogenous compounds are more or less associated
with the increased accumulation of cyanogens in seeds, except for thiourea. The accumulated cyanogens would be
used as an amino acid source in imbibed seeds, because 14CKCN was incorporated into various amino acids via /?cyanoalanine (Taylorson and Hendricks 1973, Dziewanowska and Lewak 1982).
The increased contents of cyanogenic compounds by
NH2OH may be due to the production of HCN from
NH2OH in the presence of glyoxylate in green leaves
Table 3 Effects of various nitrogenous compounds on
the levels of cyanogenic glycosides and lipids in upper seeds
in secondary dormancy of cocklebur that had been presoaked for 55 d
(mM)
H2O (Control)
CO(NH2)2 (20)
CS(NH2)2 (20)
NaSCN (20)
NH4SCN (20)
NCNH2 (20)
NH2OH (20)
NaN3 (0.5)
KCN (20)
Content (pmol seed ')
Cyanogenic
Cyanogenic
glycosides
lipids
9± 2
10± 4
14± 5
22± 8
27± 7
12± 7
52±16
30± 9
210±40
5± 1
5± 2
6± 4
30± 9
15± 4
9± 6
44±11
15± 5
210±50
Pre-soaked seeds were treated with chemicals for 3 d at 23°C and
then assayed for cyanogenic compounds.
(Hucklesby et al. 1982). However, also another possibility
that the conversion of NH2OH to nitrite via the action of
hydroxylamine dismutase in the presense of H2O2 which
was found in cyano-bacteria (Bagchi and Kleiner 1991)
could not be neglected, because nitrate as its nitrification
product was capable of accumulating cyanogens (Table 2).
The increases of cyanogens in the seeds treated with NaN3
do not seem to be due to the incorporation of N in NaN3,
because the degree of cyanogen accumulation was higher at
0.5 mM (Table 3) rather than at 5 mM (Table 2). Since
HCN can also be formed from histidine, tyrosine and
histamine (Gewitz et al. 1976) and since glycine and basic
amino acids are sources of cyanogens in microorganisms
(Knowles 1988), NaN3 might cause the accumulation of
cyanogens indirectly by influencing the metabolism of
amino acids via the strong inhibition of aerobic respiration. Rates of KCN-dependent and NH2OH-dependent
cyanogenesis, unlike NaN3-dependent cyanogenesis, increased as the concentration of the agent was increased
(data not shown).
Recently, Yoshiyama et al. (1996a) found that KNO3
applied to cocklebur seeds at 200 mM, as a priming reagent, caused the significant increases in the levels of
cyanogenic glycosides and lipids and in the size of the pool
of amino acids. As shown in Table 2, similar results were
obtained even at 30 mM KNO3 with de-coated cotyledons
and after long-term exposure for 3 days. Therefore, KNO3mediated cyanogenesis, unlike the NaN3-mediated process,
seems to arise indirectly from the increased size of the pool
of amino acids in the presence of NO3~. Cohn and Castle
(1984) found that the dormancy-breaking effect of NO2 gas
in red rice seeds was independent of the pH of the incubation medium. Moreover, red rice seeds also respond to
KCN, NaN3 and NH2OH (Cohn and Hughes 1986). The
present results suggest that the dormancy-breaking effect of
NO2 gas in red rice seeds might somehow be related to the
production of cyanogens.
Not only KCN and NaN3 but also CO, an inhibitor of
548
Y. Esashi et al.
Table 4 Production of cyanogens in the lower seeds of cocklebur treated with KCN or NaN3
Compound
Cyanogen content
after 18 h
(pmol(gFW)-')
C2H4
HCN evolution during
the following 23 h
(pmol(gFW)- 1 )
Cyanogen content
after 23 h
(pmol(gFW)- 1 )
Cyanogen utilized
during 23 h
(pmol(gFW)-')
KCN
39,800 ±400
—
20,200 ±900
17,500±700
2,120
+
21,000 ±900
12,400±700
5,980
—
+
1,000 ±120
820± 90
120
l,100± 80
450 ± 40
370
NaN3
l,950±290
The evolution of HCN and consumption of cyanogens in response to C2H4 were monitored. Seeds were pre-treated with 30 mM KCN or
5 mM NaN3 for 18 h at 23°C, and, after washing, they were incubated with or without 30/ul liter"1 C2H4 for 23 h. The amounts of
cyanogens consumed are shown as the differences between levels before and after the treatment with C2H4.
Cyt-c oxidase, promoted seed germination (Masuda and
Asahira 1981, Esashi et al. 1991b), but the extent of the promotion by CO was very low. Unlike KCN or NaN,, CO
did not induce the accumulation of cyanogens (Esashi et
al. 1991b). Therefore, the strong germination-stimulating
effects of KCN and NaN3 might be a result of their actions
as inhibitors of respiration, in addition to their stimulation
of the accumulation of cyanogens. Conversely, the limited
effects of thiocyanates on germination could be due to their
failure to function as inhibitors of respiration.
Increased utilization of KCN-induced or NaN3-induced cyanogens in response to C^i4—The decreased responsiveness of cocklebur seeds in secondary dormancy to
C2H4 was restored by pre-treatment with KCN or NaN3
(Esashi et al. 1982). Ethylene did not affect the evolution of
free HCN from KCN- or NaN3-treated seeds, but the levels
of cyanogens 23 h after rinsing were far lower in the presence of C2H4 than in its absence (Table 4). This result sug-
gests that the amounts of cyanogens, which were utilized
during the subsequent period, were about 2- to 3-fold
higher in seeds exposed to C2H4 than in un-exposed seeds,
regardless of whether the cyanogens were derived from
KCN or NaN3. These results imply, moreover, that the
restoration of responsiveness to C2H4 of seeds in secondary
dormancy by pre-treatment with KCN or NaN3 might be
due to the supply of the substrate for CAS, which is activated by C2H4.
Enhanced accumulation of cyanogens in the presence
of KCN or NaN3 plus PG or BHAM—The germination of
non-dormant lower cocklebur seeds at 23°C, in which the
ratio of the flux via alternative respiration to the flux via
cytochrome-mediated respiration was low, was stimulated
by low concentrations of PG or BHAM, which was inhibitors of the alternative respiration. A similar rinding was
made in lettuce seeds treated with SHAM, a similar type of
respiratory inhibitor (Brooks et al. 1985). As shown in
Table 5 Effects of KCN and NaN3 in combination with PG or BHAM on the germination and cyanogen content of upper seeds of cocklebur in secondary dormancy
Treatment
Germination
1
Bound HCN content (pmol(gFW)- )
After 21.5 h
Before rinsing
H 2 0 (Control)
PG
BHAM
0
KCN
KCN+PG
KCN+BHAM
3.8
50.4
63.8
93,700 ±7,400
127,400±9,500
NaN3
NaN3 + PG
NaN3 + BHAM
5.4
42.6
51.2
l,250± 780
2,530± 1,100
l,980± 930
0
0
28 ±
23±
19±
10
8
9
117,300±9,300
25±
17±
Utilized amount
(pmol(gFW)-1)
7
8
2
13± 6
19,100±980
21,500±890
22,300 ±990
110± 10
140± 20
100± 20
6
6
75,000
106,000
95,000
1,100
2,400
1,900
Seeds that had been pre-soaked for 300 d were treated with 30 mM or 5 mM NaN3 with or without 25 mM PG or 20 mM BHAM at
23°C for 15.5 h. Then some treated seeds were assayed for cyanogen content immediately after rinsing. The remaining seeds were placed
on a wet substratum for assays of both their germination ability after 4d and the amount of cyanogens utilized during the next 21.5
h.
Cyanogens in germination of cocklebur seeds
Table 5, however, both PG and BHAM applied singly were
quite ineffective in inducing germination or the accumulation of cyanogens in cocklebur seeds in secondary dormancy, in which the flux of the alternative respiration was extremely low (Esashi et al. 1982). Nevertheless, the deeply
dormant seeds, which had lost even the ability to respond
to KCN or NaN3 by germination, germinated when KCN or
NaN3 was applied together with PG or BHAM (Table 5), as
described in a previous paper (Esashi et al. 1981). In lettuce
also, maximal germination occurred with the combination
of KCN and SHAM (Brooks et al. 1985). In cocklebur,
both inhibitors significantly augmented the accumulation
of cyanogens in response to KCN or NaN3 when applied in
combination with either of them (Table 5). As a result, the
amounts of KCN- or NaN3-induced cyanogens that could
be utilized during the subsequent period were much
hihgher when KCN or NaN3 was supplied in combination
with PG or BHAM than when PG or BHAM was absent.
These results suggest that there is a factor that is more important in the regulation of germination in seeds in secondary dormancy than the balance between the fluxes of
the two respiratory systems.
The increased accumulation of cyanogens in the response to KCN or NaN3 combined with PG or BHAM
could supply the abundant amounts of cyanogens for
utilization by the treated seeds during the subsequent period, probably through the action of CAS (Table 5). The
high levels of cyanogens in seeds that had been pre-treated
with KCN or NaN3 could thus lead to the breaking of secondary dormancy via increases in the size of the amino acid
pool and a supply of numerous sulfhydryl groups.
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(Received December 20, 1995; Accepted April 9, 1996)