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PHYSIOLOGIA PLANTAR13M, VOL. 1, 1948
The Effect of Sodium Azide on the Exudationand Oxygen Consumption of Excised Plant Roots
By
GORAN STENLID
Institute of Plant Physiology, Royal Agricultural College, Uppsala 7(Received May 14th, 1948)
I. Introduction
During recent years sodium azide has become one of the respiratorypoisons most frequently used. It has an effect similar to that of the cyanides,but it has some -advantages over these, sodium azide being less volatile andless hydrolysable than \he cyanides.
By combining with atoms of heavy metals azide inhibits about the samerespiratory enzymes as the cyanides. Out of the enzymes that Keilin (1936)reports as possible to inhibit with azide, especially cytochrome oxidase,peroxidase, and catechol oxidase may be mentioned. If an inbibition ofoxygen consumption is obtained with azide, this suggests that some enzymecontaining atoms of a heavy metal is affected. In higher plants especiallythe enzymes mentioned above may be involved. One may not, however, asis often done, without any further evidence interpret an inhibition withazide as an inhibition of cytochrome oxidase. Nor does the fact that therespiration of an organ is unaffected by azide always prove that the threeenzymes mentioned above are absent. This paper will give some notes aboutone of the .causes of this fact, namely the pronounced pH sensitivity ofazide toxication.
Keilin showed (1936) that the oxygen consumption of baker's yeast isinhibited to 98 "/o at pH 5.6, while there is no inhibition at pH 7.5. Experi-ments with embryonic heart muscle of fish (Armstrong & Fisher 1940) andwith the luminescense of the ostracode Cypridina (Chase 1942) also, pointin the same direction. In the investigations on the azide inhibition of the
[185]
186 GORAN STENLID
respiration of higher plants (Henderson & Stauffer 1944, Machlis 1944,Rosene 1947) only slight regard has been paid to this pH sensitivity. Mostof the experiments have been performed only at a single pH value. It istherefore difficult to compare the different data, as one does not knowwhether the inhibitions in the materials in question are pH-sensitive. Marshand Goddard (1939), who made experiments with carrot tissue, report thatthe effect of sodium azide does not markedly increase from pH 5.7 to pH 4.5.
II. Exudation experiments
In a previous communication (Stenlid 1947) it was shown that exudationfrom excised pea roots is increased after addition of cyanide. No parallelrespiration experiments were made, but the concentrations applied (0.001—0.01 M) are known to stop a large fraction of the oxygen consumptionin plant roots. To investigate whether an inhibition of respiration is alwaysaccompanied by an increased exudation the effect of some other poisonswas also tried. Here some of the experiments with sodium azide will bepresented.
The exudation was measured by determining light absorption in ultra-violet light(see Stenlid 1947). The values given in this paper were measured with a Becknianquartz .photometer instead of the apparatus used before. In most of the experiments3—4 days' otd excised roots of wheat (variety »Dianiant II»), barley (variety »Maja-korn»), rye (variety »Stalrag»), oats (variety »Segerhavre») and pea (variety sTors-dagsart II») were used. For further methods see Stentid 1947. For the extinctiondeterminations the samples were acidified witli HCt to pH about 1, as the extinctionof a solution varies with the acidity. Between pH 5 and pH 1 the variation is, how-ever, slight for the solutions mentioned in this paper. The extinction due to buffersolutions and sodium azide may be neglected at wave lengths above 2500 A.
From Tables 1—4 it is obvious that the effect of azide on exudation varieswith the pH of the surrounding solution. Tables 2—4 show that withbarley roots at pH 4.5 a marked effect is obtained in the whole concentrationregion 0.03—0.7 mmol/l, whereas at pH 6.9 only the strongest solution hadany effect. At a concentration of 0.03 mmol/l exudation is even slightlyinhibited, which may, however, be accidental. It should be mentioned,however, that Rosene (1947) found that azide at low concentrations sti-mulated respiration and water transfer in onion roots, while both processes.were inhibited at higher concentrations.
Results similar to those in Tables 1—4 were also obtained with 3—4 days'old excised roots of oats, rye and pea, and in experiments with excised roottips from 14 days' old wheat plants cultivated in nutrient solution.
THE EFFECT OF SODIUM AZIDE 187
Table 1. Exudation from excised wheat roots (30—W mm tong) in about 0.01 M citrate-phosphate buffers with and without azide. The values give the extinction in a 1 cm layer.
Duration of the experiment 225 minutes.
^ " ~ " - ^ - ^ ^ ^ Final pH
2900 A2800 A2700 A2600 A2500 A
K(bufrer + NaN3>
a = 260^0 A)
6.20.1
— mmol/1NaN,
0.096 0.0910.124 0.1200.149 0.1490.161 0.1610.153 0.153
1.0
5.80.1
mmol/1NaN,
0.105 0.129. 0.134 0.163
0.164 0.2030.179 0.2250.169 0.209
1.3
5.00.1
— nimol/1NaNj
0.129 0.2350.154 0.2830.188 0.3510.204 0.3860.190 0.340
1.9
4.50.1
— mmol/1NaN,
0.192 0.4140.225 0.4820.275 0.5980.295 0.6500.265 0.560
2.2
Table 2. Exudation from excised barley roots (30—iO mm tong) in about 0.01 M citrate-phosphate buffers with and without azide. The values give the extinction in a 1 cm layerat 2600 A. The pH values are the mean of the solutions with and without NaN3. The
solution was changed after 70 minutes.
pH
1 initial
4.5
5.2
5.9
6.4
6.9
final
4.6
5.3
5.9
6.4
6.9
0—70
mmol/1NaNg
0.7
0.7
0.7
0.7
0.7
minutes
p
0.2020.0830.1600.0830.1470.1150.1600.1150.1550.135
'"-(bufTer -\- NaNj)
''-'(buffer)
2.4
1.9
1.3
1.4
1.1
pH
initial
4.5
5.2
5.9
6.4
6.9
final
4.7
5.3
5.9
6.4
6.9
70—28C
mmol/1
0.7
0.7
0.7
0.7
0.7
minutes
E '
0.4560.1360.4410.1230.3360.1190.2150.1000.1150.106
'"•'(buffer + NaNa)
'^(buffer)
3.4
3.6
2.8
'1.2
1.1
Tahle 3. The same as the experiment in Tabte 2, but lower concentration of azide.Duration of the experiment 215 minutes.
pH
initial
4.5
5.9
6.9
final
4.6
5.9
6.9
mmol/1 NaNj
0.2
0.2
0.2
li
0.3130.1150.1810.1270.1380.138
''^buffer + NiiN's)
''^(buffer)
2.7
1.4
1.0
188 GORAN STENLID
Table 4. The same as the experiment in Table 2, hut lower concentration of azide.Duration of the experiment 215 minutes.
initial pH
4.5
5.2
5.9
6.4
6.9
nimol/I NaNj
0.03
0.03
0.03
0.03
0.03
K
0.5200.3200.3580.2850.2740.3000.2480.2950.2660.299
'"-'(l)uirer + NaNa)
'"'(bufrer)
1.6
1.3
0.9
0.8
0.9
III. Respiration experimentsTo be able to compare the influence of azide on respiration and exudation it
was necessary to make some respiration experiments under the same conditionsand witb tbe same plant material as in tbe exudation experiments. Oxygenconsumption was determined by means of a Warburg-Barcroft apparatus. Tbeexperiments were made at 25° C witb about 15 root tips (10—15 mm long) ineacb flask. Altbough the plant material was cultivated under almost constantconditions tbe plants did not react in exactly tbe same manner on different days.Only tbe parallels in the same Table are therefore directly comparable. Tbe variationfrom one day to anotber was, however, not very pronounced. After starting anexperiment, some time had to pass before tbe oxygen consumption became constant.Tbe time 0 is chosen after the oxygen consumption had grown approximatelyconstant and does not mean tbat tbe experiments were started at tbat tinie.
Table 5. Determination of 0« consumption in a Warburg-Barcroft apparatus. Every flaskcontained 0.8 ml citrate-phosphate buffer and 15 root tips of wheat. 0.15 ml 20"/o NaOHin centre well. At 4- sodium azide was added to flasks 1—3 to give a concentration of1 mmol/1; to flasks 4—5 an equivalent quantity of water was added. Initial pH 4.4, finalpH 4.8 (mean of all lhe flasks). The O2 consumption is given in arbitrary units (change
in mm of the manometer readings, corrected for the thermobarometer readings).
FlaskNo.
1
4
0/0
Time in~̂̂ minutes
^ ^ ^ - - ^
12345
-3(mean)
— ̂ (mean)
inhibition
0-15
1311111112
t.Ol
15-30
1512121214
1.02
0
30-45
1514131315
0.98
98
45-00•1
1815141618
0.<j3
70-85\
311
1213
O.!3
87
S5-100
110
1516
0.0-i
08
100-130
000
2527
0.00 -
100
130-160
00
— 12629
- O . O i
100
160-190
100
2629
O.Oi
99
190-220
100
2732
O.Oi
99
THE EFFECT OF f̂ ODIUM AZIDE 189
Table 6. The same as the experiment in fable 5, but initial pH 5.4, final pfl 5.6.
^^•^^^^^^ Time in^^^-^-^^ minutes
Flask ^"^"^--^No. ^~^~- - - , ^
12345
< *j
^ (mean)4 ^ 5 / N^ (mean)
O/o inhibition
0—15
15
n1515](>
1.00
1
15-35i
20•y,\
202022
1.00
.00
45-60
554
1214
0.36
64
60—75
3S3
1213
0.24
76
75—90
3t3
1214
0.21
79
90 — 150
5
64854
0.15
85
Table 7. The same as the experiment in Table 5, but initiat pH 1.0, finat pH 73.
^^^-^^ Time in,̂ ^-^—^ minutesHask ^^..^
No. \ ^
12345
^ •'(mean)
4 —5(mean)
"/o inhibition
0—i51o—S0 30—45
1012131212
0.98
1118121213
0.96
0.97
911111011
0.98
55-70I-
1113111011
1.11
— 14
70-85
1112111011
1.08
— 11
85-100 100-115
811109 ,
IQ
1.02
— 5
1012119
1ft
1.16
— 20
115—130 130-160
811101011
0.92
+ :->
19221918
1.05
— 8
100-205
25323026
1.03
— 6
Table 8. The same as the experiment in Tabte 5, but concentration of azide 0.1 mmotlt.Initial pH 4.4, final pH 4.7.
FlaskNo.
1-
4
0/0
Time in^̂ ^̂ ^ minutes
12345
-3(.neun>
~-\mean)
inhibition
0-15
1513121316
0.92
15-30
1514141416
0.95
0.92
30-45-1
1312131217
0.88
55—70
5339
11
0.37
60
70—85
655
1318
0.34
63
85—100
432
1114
0.24
74
100—125
422
2128
0.11
88
190 GORAN STENLID
Table 9. The same as the experiment in Table 5, but concentration of azide O.I mmol/lInitial pH 5.4, final pH 5.6.
FlaskNo.
1 -
3 -
Time in^minutes
1234
-2(n,ean)
-'^(mean)
o/n inhibition
0—15
17151318
1.03
15—30
17161216
1.18
1.11
30 — 45
19171418
1.12
55 — 70
14151316
1.00
10
70 — 85
11111316
0.76
32
85 — 100
10101115
0.77
31
100—115
109
1113
0.79
29
115—145
25232631
0.84
24
145—175
18182228
0.72
25
175—235
39394658
0.75
32
Table 10. The same as the experiment in Table 5, bat concentration of azide O.I mmotlt.Initial pH 7.0, final pH 7.1.
FlaskNo.
1 -
4 -
Time in^^^minutes
^ \
12345
-\,ne.-,n)
-5(n,BaiO
O/o inhibition
0-20
2017181522
0.99
20—40
1817161519
1.00
1.00
40—60
2119171721
1.00
70—90
1916171621
0.94
6
90-110
1715151519
0.92
8
110-150
3330302937
0.92
8
150—190
3332342840
0.97
3
Table 11. Inhibition of O2 consumption at different pH values and concentrations ofCombination of Tables 5—10.
^ ~ ^ - \ , ^ ^ pHmmol/l ^~^--,..,̂
1.00.1
4.6
87—10060— 88
5.5
64—8510—32
7.1
(-20)-53 —8
THE EFFECT OF SODIUM AZIDE 191
Results similar to those given in Tables 5—11 were also obtained withroot tips from 3—4 days' old barley plants and wilh root tips from wheatplants cultivated for 14 days in nutrient solutions.
Summing up the experiments on Oj consumption it can be said that theresults agree with those from the exudation experiments. The exudation isincreased when the respiration is inhibited. In both cases the effect is in-creased with increasing acidity, concentration, and duration of the experi-ments.
IV. Discussion
Several weak acids (as hydrofluoric and malonic acids) seem to behavein a similar manner as hydrazoic acid. Bonner & Wildman (1946) showedthat the Og consumption of spinach leaves at pH 4.5 was reduced 80 "/o bymalonate, whereas it had no inhibitory effect at pH 7.0. Turner & Hanly(1947) showed the same for carrot tissue. They emphasize the fact thatthe contradictory data concerning the malonate effect on respiration areexplicable if the acidity is considered. For malonic acid as well as forhydrofluoric acid (for literature see Borei 1945) decreased dissociation andincreased permeability are suggested to cause the greater effect in acidsolutions.
Hydrazoic acid is a weak acid with its pK = 4.6—4.8. This means that itis dissociated to 50 »/o at pH 4.7, to 90 "/o at pH 5.7, and to 99 "/o at pH 6.7.If the undissociated hydrazoic acid penetrates more easily than the anionthis must result in a stronger effect in more acid solutions. The concentrationof undissociated hydrazoic acid in the svirrounding medium and not thetotal concentration of azide should thus determine the toxic effect (cf. Keilin1936). The results in Tables 1—10 support this view. Further it may bementioned that the same effect was obtained in experiments with wheatroots at pH 4.5 and 0.2 mmol/l azide as at pH 6.7 and 15 mmol/1 azide. Inboth cases the exudation was increased ca. 100 "/o by the azide and in bothcases the concentration of undissociated acid was ca. 0.15 mmol/1.
It must be stressed, however, that also several other factors than thedissociation of the acid may contribute to the stronger effect in acidsolutions. The pH of the plasm and sap and the dissociation of theirconstituents are also altered (data for barley and pea roots are given byTheron 1924 and for wheat roots by Keyssner 1931) and this may give thesame result as altered dissociation of the poison. Recently it was shownthat in ui'fro hydrazoic acid and not the azide anion combines with cyto-chrome oxidase (Stannard «& Horecker 1948). In acid solutions the changedionic balance causes an increased absorption of anions (see Lundegardh
192 GORAN STENLID
Table 12. The same as ttie experiment in Tabte 1, but with acetic acid added in.-itead ofazide. The values give the extinction at 2600 A. Duration of the experiment 180 minutes.
Total concentration of acetate (HAc4-NaAc) 0.008 M.
Experiment A
Experiment B
pH
initial
4.754.755.85.86.756.75
4.94.95.45.45.95.9
final
4.84.855.855.86.756.75
Ac
+ 1 + 1 + 1
i + 1
+ 1
-f
E
0.4940.1740.1580.1340.1420.140
0.3770.1880.2160.1440.1500.149
''-'(bulTer -1- Ac)
^"(buffer)
2.8
1.2
1.0
2.0
1.5
1.0
1945) which may increase the ahsorption of azide ions although theirconcentration is diminished.
Summing up one may say that the probable cause of the greater inhibitoryeffect in acid solutions is that the azide combines more easily with therespiratory enzymes. Several factors may contribute to this effect but thechief one seems to be that the undissociated acid permeates more easily.
Here it can be mentioned that the toxic action of acetic acid i.s alsomarkedly increased at lower pH values, as will be seen from Table 12.pK for acetic acid is 4.75, i.e. the same as for hydrazoic acid, and the effectalso in this case is strong first at pH values in the neighbourhood of pK.
From observations at a single pH value it is obviously not possible todecide how azide affects exudation and oxygen consumption/ Even slightalterations of the acidity in the physiologically important pH range 4.5—6.5may cause great changes in the inhibitory effect. The data stating that afixed part of the respiration of plant organs is resistant to azide are probablyoften not valid at other pH values than those used in the experiments. Withthe methods and the plant material used in this investigation all valuesbetween insensitivity and complete inhibition may be obtained withoutchanging the concentration. This may be interpreted so that all therespiration is sensitive to azide but that the permeability for azide to therespiration system is free only at low pH values. It is also possible thatnot all of the respiration is sensitive to azide but that indirect processes atlower pH values totally stop the oxygen consumption. Warburg (1946)
THE EFFECT OF SODIUM AZIDE 193
Table 13. Oxygen consumption in phosphate buffer (O.OI M; pH 7.0). Flasks Nos. 1—3contain NaNg in a concentration of 1.0 mmol/l. At I glucose was added to all the flasks
to give a concentration of 0.01 M. For further explanations see Table 5.
FlaskNo.
Time inminutes
12345
0—30
2222232424
30—60
2223242524
60—90•
2121212424
100—130
2221213129
130—160
2224233231
160—220
4145486867
critically reviews the data on cyanide insensitive respiration, which isprobably the same fraction that is not affected by azide.' He says that thefact that part of the respiration is unaffected in cyanide poisoned systemsdoes not prove that this part is insensitive to cyanide. There are manyfactors complicating this problem. The chief of these are: 1) HCN is volatileand is rapidly absorbed by the alkaVi used for absorbtion of COo. This errorcan be avoided by using alkali-cyanide mixtures with the same concentrationof free HCN as the experimental fluid (Robbie 1946). 2) HCN rapidly reactsalso on other cell constituents than heavy metal enzymes especially sugars,aldehydes, and keto acids. 3) According to the law of mass action a part ofthe enzyme system always remains unaffected.
If there is any azide sensitive system in the root surface it ought to beaffected by azide in the surrounding solution also at higher pH values,irrespective of the low permeability. As no effect is obtained at pH 7, thismeans either that there is no respiration system sensitive to azide in thesurface, or that it is inactive under the experimental conditions. Possiblythere is too little substrate for the enzyme systems. Experiments were there-fore made with addition of glucose to the surrounding solution. In purebuffer solutions (pH 6.5—7.0) addition of glucose to a concentration of0.01 M increased the oxygen consumption by 10—30 Vo. If azide was added,glucose caused no, or only a slight and retarded, increase (see Table 13).
These results suggest that the utilization of glucose is inhibited by azide.This may be due to inhibition either of a respiratory system in the surfaceor of absorption and transport of the glucose. Lundegardh and Burstrom(1944) discuss the absorption of glucose by wheat roots. They say ». . . glucoseis absorbed from the surrounding medium by means of an active process.This process is apparently located in the surface layer of the root but seemsnot to be identical with the anion respiration.* This view is in good agree-ment with the experiment in Table 13. Spiegelman and collaborators (1946)13
194 GORAN STENLID
using radioactive isotopes made some interesting observations on the phos-phorous metabolism of yeast. They found tbat sodium azide in a con-centration tbat only sligbtly affected respiration, totally stopped tbe pbos-pborylation processes. It is possible tbat azide also in plant roots affectspbospborylation and bence tbe absorption of glucose and tbe utilization ofit in respiration.
Summary
1. Exudation (exosmosis) from excised plant roots is increased by sodiumazide. Tbe effect of azide is greater at pH 4.5 tban at pH 7.
2. Tbe oxygen consumption of excised plant roots is inbibited by sodiumazide. In a 1 mmol/1 solution tbere is no inbibition at pH 7, ca. 70 Vo inbi-bition at pH 5.6, and total inbibition at pH 4.5. Inbibited oxygen consump-tion seems to be correlated witb increased exudation.
3. Addition of glucose in tbe surrounding medium increases oxygen con-sumption of excised wbeat and barley roots by 20—30 "/o. In a 1 mmol/1 azidesolution glucose given at pH 7 produces no effect, altbougb azide does notaffect tbe »basic» oxygen consumption under tbese conditions.
This work was aided by a grant from ^Statens Tekniska Forskningsr3d»,Stockholm.
References
Armstrong, C. W. .1. & Fisher, K. C: A comparison of the effects of the respiratoryinhihitors azide and cyanide on the frequency of the emhryonic fish heart. — J. Cell.Comp. Physiol. 16: 103. 1940.
Bonner, J. & Wildman, S. G.: Enzymatic mechanisms in the respiration of spinach leaves.— Arch. Bioch. 10:497. 1946.
Borei, H.: Inhihition of cellular oxidation hy fluoride. — Ark. for Kemi, Mineral, ochGeol. Stockholm. 20 A N:o 8. 1945.
Chase, A. M.: The reaction of Cypridina luciferin with azide. — J. Cell. Comp. Physiol.19: 173. 1942.
Henderson, J. H. M. & Stauffer, J. F.: The influence of some respiratory inhibitors andintermediates on growth and respiration of excised tomato roots. — Amer. J. Bot.31:528. 1944.
Keilin, D.: The action of sodium azide on cellular respiration and on some catalyticoxidation reactions. — Proc. Royal Soc. 121 B: 165. 1936.
Keyssner, E.: Der Einfluss der Wasserstoffionenkonzentration in der Nahrlosung auf dieReaktion in der Pflanze. — Planta 12: 575. 1931.
Lundegirdh, H.: Absorption, transport, and exudation of inorganic ions hy the root. •—Ark. for Bot. Stockholm. 32 A N:o 12. 1945.
•— & Burstrom, H.: On the sugar consumption and respiration of wheat roots at diffe-rent pH values. — Ann. Agric. Coll. Sweden. 12: 51. 1944.
Machlis, L.: The influence of some respiratory inhihitors and intermediates on respirationand salt accumulation of excised harley roots. — Amer. J. Bot. 31:183. 1944.
THE EFFECT OF SODIUM AZIDE 195
Marsh, P. B. & Goddard, P. R.: Respiration and fermentation in the carrot, Daucus carota.1. Respiration. — Amer. J. Bot. 26: 724. 1939.
Robbie, W. A.: The effect of cyanide on the oxygen consumption and cleavage of the seaurchin egg. — J. Cell. Comp. Physiol. 28:305. 1946.
Rosene, H. F.: Reversible azide inhibition of oxygen consumption and water transfer inroot tissue. — J. Cell. Comp. Physiol. 30: 15. 1947.
Spiegelman, S. & Kamen, M. D.: The site of uncoupling of phosphorylation from carbo-hydrate metabolism in the presence of NaNg. — Federation Proc. 5; 99. 1946.
— & Dunn, R.: Mechanism of azide inhibition of synthetic activity and its relation tophosphorylation. — Federation Proc. 5:99. 1946.
Stannard, J. N. & Horecker, R. L.: The in vitro inhibition of cytochrome oxidase by azideand cyanide. — J. Riol. Chem. 172:599. 1948.
Stenlid, G.: Exudation from excised pea roots as influenced by inorganic ions. — Ann.Agric. Coll. Sweden. 14:301. 1947.
Theron, J. J.: Influence of reaction on the interrelation between the plant and its culturemedium. — Univ. Calif. Publ. Agr. Sci. 4:413. 1924.
Turner, J. S. & Hanly, V.: Malonate and plant respiration. — Nature 160:296. 1947.Warburg, 0.: Schwermctalle als Wirkungsgruppen von Fermenten. — Berlin 1946.
