THE COUNTERACTING EFFECT OF POTASSIUM CYANIDE IN SODIUM AZIDE-INHIBITED GERMINATION OF PAULOWNIA TOMENTOSA STEUD . SEEDS

The effect of some respiratory inhibitors on light-induced Paulownia tomentosa Steud. seed germination was studied. Millimolar solution of sodium azide was sufficient to completely prevent germination induced by a 5-min red light pulse. The inhibitory effect of azide was absent if seeds were rinsed before phytochrome activation by light. Sodium azide was effective only if present in the period of Pfr activity. The escape time from azide inhibition, compared to the escape from far-red light action, was delayed for about 24 hours. When azide was applied after phytochrome activation, its effect depended on how long it was present in the incubation medium. The removal of azide allowed full restoration of germination by another red light pulse and the far-red escape time did not differ from the escape of untreated, i.e., water-imbibed seeds. Potassium cyanide alone did not produce any effect in light-stimulated germination of these seeds. However, it counteracted the inhibitory effect of azide in light-stimulated germination, if applied simultaneously at a concentration three times higher. UDC 582.916.21 : 581.142.044


INTRODUCTION
The germination of empress tree (Paulownia tomentosa Steud.)seeds is phytochrome-controlled.Depending on seed maturation conditions, the light requirement for maximum germination varies from a very brief exposure to several hours of irradiation (in some cases up to 18 h) (Borthwick et al. 1964;Grubišić et. al. 1985).Prolonged imbibition in darkness or in heavy water leads to an increase in the light requirement for maximum germination (Grubišić andKonjević, 1986, 1990).In seeds with natural and induced long-term light requirements, two short (5-min) red light pulses, separated by a certain period of darkness, could substitute for this requirement.Moreover, the long light requirement can be reduced to a single 5-min red light pulse by the application of inorganic nitrates and nitrites (Grubišić and Konjević, 1990), substances with electron-accepting properties (Giba et al. 1994), or nitric oxide-releasing compounds (Giba et al. 1998).
Azide is a well known dormancy-breaking agent for a variety of seeds (Bewleyand Black, 1982).Seeds of pigweed (Taylorsonand Hendricks, 1973), apple (Dziewanowska et al. 1979), wild oat (Adkins et al. 1984), dormant rice (Cohnand Hughes, 1986) and oat (Côme et al. 1988) can be stimulated to germinate by NaN 3 .The same is true of some other respiratory inhibitors (cyanide, carbon monoxide, sulfide).On the other hand, azide and cyanide do not substitute for the light requirement for breaking dormancy in empress tree seeds (Grubišić, 1980).The opposing effect of these inhibitors in germination is not uncommon.Hendricks and Taylorson(1972) showed that azide inhibited, but cyanide stimulated germination of seeds of Lactuca sativa and Amaranthus albus.
In the study presented here, we investigated the effects of sodium azide on light-induced seed germination, focusing on the period of P fr activity.It is demonstrated that azide reversibly inhibits germination of P. tomentosa seeds.In addition, cyanide was found to counteract the inhibitory effect of azide in this species.

Plant material and seed manipulation
Seeds of empress tree (Paulownia tomentosa Steud.) were collected in the Botanical Garden of the University of Belgrade and stored at room temperature until use.Lots of 100 seeds were placed in 6-cm diameter Petri dishes, with 2 ml of distilled water or aqueous solution of the substance to be tested.Seeds were rinsed three times with 3 ml of distilled water before replacing the test solutions.The pH values of the solutions were recorded at the start of imbibition and before removal of the seeds.Diluted HCl and NaOH were used to adjust the pH of the test solutions to the appropriate pH value.Germination was performed at 25±1 o C, in darkness.Specific experimental protocols and irradiation regimes are described in the Figure Legends and Tables.Only for one subset of experiments (Table 1) was a batch of freshly harvested seeds used.These seeds could not be induced to germinate with a 5-min red light pulse, but required several hours of long red light irradiation.
Germination was scored seven days after the inductive red-light treatments or ten days after the start of imbibition.All experiments were repeated three times, with 3-5 replicates each.The data points represent means of pooled results; standard errors are not shown since they never exceeded 3%. .The pH value of the medium was recorded using a laboratory pH-meter (InoLab, pH Level 1, WTW, Weilheim, Germany).

Inhibition by azide
The effects of different concentrations of sodium azide and potassium cyanide on P. tomentosa seed germination are shown in Fig. 1.Azide, up to 10 -4 M, applied at the onset of imbibition, was ineffective in inhibiting red light-induced germination, while addition of 10 -3 M azide completely suppressed it.On the other hand, cyanide concentrations as high as 10 -2 M, applied in the same way, failed to affect germination.For further experiments sodium azide was used in the inhibitory concentration (10 -3 M) and applied at the beginning of each of the three germination phases, i.e. imbibition (3 days), the phase of P fr activity (3 days after the red light pulse), and the phase of radicle elongation (4 days).At the end of each phase, the seeds were washed out and the incubation medium replaced by distilled water.The inhibitory effect of azide Seeds were imbibed either in water or in increasing concentrations (x-axis) of test solutions for 3 days at 25 0 C, irradiated with 5-min red light, and left in darkness.Germination was scored 7 days after the red light treatment.˜-sodium azide; £potassium cyanide.Insert: Effect of sodium azide applied in different phases of P. tomentosa seed germination.Seeds were supplied with distilled water or 10 -3 M solution of sodium azide at different germination phases.Following these phases, the seeds were rinsed.In all treatments, seeds were irradiated 3 days after the onset of imbibition with 5-min red light.Germination was scored 7 days after the light treatment.£-distilled water; ¢-sodium azide; I-imbibition; II-P fr activity; III-radicle elongation; R-5-min red light.

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PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.comwas evident only if it was administered during the period of P fr activity (insert in Fig. 1).
In addition to this, the escape from azide inhibition was followed.A comparison of azide-inhibition escape and escape from the inhibitory effect of far-red light is shown in Fig. 2. In both experiments escape was completed 72 h after the red light pulse.However, while the inhibition to 50% of red light-induced germination by 5min of far-red light was estimated to be at 31 h, the same effect of azide occurred about 20 h later.That was confirmed by probit analyses (insert in Fig. 2).Accordingly, there is an obvious 24-h shift of the escape from azide inhibition.If the seeds were supplied with 10 -3 M sodium azide after the inductive 5-min red light pulse, the percent of germination decreased with the duration of sodium azide treatment.Subsequent stimulation of seed germination by an additional red light pulse, after rinsing, revealed that the azide inhibitory effect in the P fr activity phase is reversible (Fig. 3).
In a similar experimental approach (seeds treated by azide, then rinsed), the far-red light escape time was rechecked and compared to the escape time for the far-red inhibitory effect in seeds imbibed only in water.Pretreatment with 10 -3 M azide did not affect the escape time (Fig. 4), indicating full restoration of the phytochrome pigment system.
A subsequent set of experiments was performed in an attempt to understand the azide inhibitory effect in light-induced germination.A batch of P. tomentosa seeds requiring long light irradiation for maximum germination was used.As was shown earlier (Grubišić and Konjević,1990), two red light pulses of 5-min separated by a 12 hours-long period of darkness can substitute for the continuous light requirement of these seeds.The application of a 10 -3 M concentration of azide inhibited germination if applied after the second red light pulse only (Table 1).Seeds were imbibed in water for 3 days in darkness at 25 0 C, irradiated with 5-min red light, and returned to darkness.After that, they were either irradiated with 5min far-red light at the indicated times (x-axis) and again returned to darkness (£); or transferred to 10 -3 M solution of sodium azide in darkness for 72 h (period of P fr activity), rinsed and irradiated with 5-min red light, and then irradiated with 5-min far-red light at the indicated time intervals (x-axis) and again returned to darkness (˜).Germination was scored 7 days after the last red light treatment.
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The pH dependence of azide inhibition
Empress tree seeds germinate in the incubation medium over a wide pH range (Turner et al. 1988).To evaluate the pH dependence of azide inhibition, seeds were treated with 10 -3 M sodium azide solution adjusted to different pH.The inhibitory effect of azide was assayed at an initial pH range of 2.9 to 10.1.The degree of inhibition due to azide was found to vary as the pH varied (Fig. 5).Complete inhibition was achieved at higher pH values of the incubation medium, i.e. no lightinduced seed germination occurred at pH of 4.6.

Effects of azide in the presence of cyanide
Potassium cyanide alone did not affect the germination of P. tomentosa seeds.In contrast, when seeds were treated with a mixture of azide and cyanide solution in a two-factorial experiment, germination was significantly promoted.Although cyanide did not influence light-stimulated germination of these seeds, it did revert the inhibitory effect of azide if applied simultaneously at three-fold higher concentration (Fig. 6).
Since it is known that cyanide raises the pH of the incubation medium, both the initial and final pH values at the highest concentrations of inhibitors were monitored.It turned out that the final pH values never fell below 6.4 (data not shown).Azide completely inhibited lightinduced germination of P. tomentosa seeds at any pH value above 4.6 (Fig. 5).Therefore, the counteracting effect of cyanide in azide-inhibited germination of seeds Table 1.Effect of sodium azide in germination of P. tomentosa seeds with a natural long-term light requirement.Seeds were imbibed for 3 days in darkness at 25 0 C in distilled water (£) or in 10 -3 M solution of sodium azide (¢), irradiated with 5-min red light, and returned to darkness.They then were rinsed and the solutions were replaced.After a corresponding interval of 12 h, seeds were irradiated with another 5-min red light pulse, rinsed again, and transferred to distilled water or sodium azide solution.Germination was scored 7 days after the first red light treatment.I-imbibition (3 days); II-phytochrome activity (3 days); III-radicle elongation (4 days); R-5-min red light.PDF created with FinePrint pdfFactory Pro trial version www.pdffactory.com of this species cannot be ascribed to cyanide-changed pH values of the incubation medium.

DISCUSSION
In light-induced seeds of P. tomentosa, the presence of 10 -3 M sodium azide in the incubation medium inhibited germination.Potassium cyanide did not affect this process.In the presence of azide, an inductive 5-min red light pulse was completely ineffective.After rinsing and replacement of azide solution by distilled water, seeds still did not germinate and required an additional red light pulse for germination instead (Fig. 3).A direct azide interaction with the phytochrome molecule is questionable.Azide seemed to affect one of the (early) steps in the phytochrome transduction chain.This assumption is supported by the fact that escape from azide inhibition existed, the time needed for 50% escape being about 20 h longer than that for escape from far-red light inhibition (Fig. 2).
It was anticipated earlier that pH control of the incubation medium, when utilizing dormancy-breaking chemicals that are weak acids or bases, might improve their effectiveness and reproducibility.It has been shown in dehulled red rice seeds that the dormancy-breaking activity of azide, cyanide, and hydroxylamine are pHdependent.In each case, activity was observed at pH values that favor formation of the uncharged form of azide (pK a =4.7), cyanide, (pK a =9.3), and some other compounds (Cohnand Hughes, 1986).
Azide did not substitute for light in breaking dormancy of P. tomentosa seeds.However, the inhibitory effect of azide in light-induced germination of seeds of this species was pH-dependent and occurred when the initial pH value of the incubation medium was 4.6 (Fig. 5).In addition, the final pH value of the azide solution differed from the initial one, varying from 4.1 to 7.3 (data not shown).Otherwise, under such experimental conditions the molecules of the inhibitor would have been overwhelmingly ionized (deprotonated).Taking into account these findings, we speculate that N 3 represents the inhibitory form of the azide molecule in light-induced germination of empress tree seeds.
In seeds with a long-light requirement induced to germinate by two pulses of red light separated by a period of darkness, azide prevented the effect of the second pulse only (Table 1).It was previously shown that the need for two red light pulses in these seeds can be modified by addition of nitrates (Grubišić and Konjević, 1990) or different NO-releasing compounds (Giba et al. 1998), or by upward and downward temperature shifts (Grubišić and Konjević, 1992).All of these treatments make germination possible under suboptimal light conditions, i.e. after induction by one red light pulse.
It is surprising that cyanide, ineffective by itself in P. tomentosa seed germination, can overcome the inhibition of azide if applied simultaneously.The counteracting cyanide effect was noticed only when both inhibitors were present in the incubation medium in higher concentrations, i.e., above the millimolar range.The finding that the effect of one factor (cyanide) was evident only in the presence of inhibitory concentrations of another one (azide) implies that there is an interaction between the two factors studied.
It has been suggested that the apparent resistance of germination to cyanide is an experimental artefact due to extreme cyanide volatility at the usual pH used in germination experiments (Yu et al. 1981).In light-induced germination of P. tomentosa seeds, the relief of azide inhibition by cyanide was not a result of altered pH in the incubation medium.Thus, the possibility that the "cyanide effect" might not be cyanide-specific was ruled out.
Data of the kind presented here on the effects of coincidental application of azide and cyanide have not been reported so far in studies of seed germination.However, a similar approach has been applied in experiments with isolated animal cells and tissues.For instance, the vasorelaxant effect of azide was partially reversed or prevented by an excess of free cyanide (Smithand Wilcox, 1994).In addition, the cyanide effect appeared to be competitive and reversible, although the same concentrations of cyanide alone remained without effect (Kruszyna et al. 1982(Kruszyna et al. , 1985)).Thus, azide-inhibited germination of P. tomentosa seeds may turn out to be an appropriate tool for further studies of phytochrome-controlled germination.

Fig. 1 .
Fig. 1.Effect of sodium azide and potassium cyanide on light-induced germination of P. tomentosa seeds.Seeds were imbibed either in water or in increasing concentrations (x-axis) of test solutions for 3 days at 25 0 C, irradiated with 5-min red light, and left in darkness.Germination was scored 7 days after the red light treatment.˜-sodium azide; £potassium cyanide.Insert: Effect of sodium azide applied in different phases of P. tomentosa seed germination.Seeds were supplied with distilled water or 10 -3 M solution of sodium azide at different germination phases.Following these phases, the seeds were rinsed.In all treatments, seeds were irradiated 3 days after the onset of imbibition with 5-min red light.Germination was scored 7 days after the light treatment.£-distilled water; ¢-sodium azide; I-imbibition; II-P fr activity; III-radicle elongation; R-5-min red light. 31

Fig. 2 .
Fig. 2. Escape time for far-red light and inhibitory effect of sodium azide in lightinduced germination of P. tomentosa seeds.After imbibition in water for 3 days in darkness at 25 0 C, seeds were irradiated with 5-min red light.After that, they were either irradiated with 5-min far red light at the indicated time intervals and returned to darkness (£) or transferred after rinsing to 10 -3 M sodium azide solution and returned to darkness (˜).Insert: Probit analysis of escape time from azide inhibition and far-red light inhibitory action.

Fig. 4 .
Fig. 4. Escape time from far-red light inhibitory action in water-imbibed and sodium azide-treated seeds of empress tree.Seeds were imbibed in water for 3 days in darkness at 25 0 C, irradiated with 5-min red light, and returned to darkness.After that, they were either irradiated with 5min far-red light at the indicated times (x-axis) and again returned to darkness (£); or transferred to 10 -3 M solution of sodium azide in darkness for 72 h (period of P fr activity), rinsed and irradiated with 5-min red light, and then irradiated with 5-min far-red light at the indicated time intervals (x-axis) and again returned to darkness (˜).Germination was scored 7 days after the last red light treatment.

Fig. 5 .
Fig.5.The pH dependance of azide inhibition.Seeds were imbibed for 3 days in darkness at 25 0 C in distilled water (£) or in 10 -3 M sodium azide solution (˜) with initial pH values in the range of 2.76-10.28.After that, they were irradiated with a 5-min red light pulse and returned to darkness.Germination was scored 7 days after the light treatment.
Fig.6.Effect of sodium azide and potassium cyanide on light-induced germination of P. tomentosa seeds.Seeds were incubated in a mixture of different concentrations of sodium azide and potassium cyanide (10 -2 M-10 -6 M) for 3 days in darkness at 25 0 C, irradiated with 5-min red light, and returned to darkness.Germination was scored 7 days after the light treatment.