Plant Physiol.

(1983) 72, 7-15

0032-0889/83/72/0007/09/$00.50/0

Effect of Cyanide in Dark and Light on the Membrane Potential

and the ATP Level of Young and Mature Green Tissues of
Higher Plants'
Received for publication April 12, 1982 and in revised form December 28, 1982

CORNELIA I. ULLRICH-EBERIUS, ANTON NOVACKY, AND ERIKA BALL

Institutffur Botanik, Technische Hochschule Darmstadt, D-6100 Darmstadt, Federal Republic of Germany (C.
U.-E., E. B.); and Department of Plant Pathology, University of Missouri, Columbia, Missouri 65211 (A. N.)

ABSTRACr than EK. A comparison of this effect with that of DCMU on Em

in the light (26) suggests a small CN-insensitive activity of PSII.
The effect of CN- ad N2 on the dectriCal e pott (E.) In Mnium, Em was reduced to EK+ by CN- in the dark as well as
was with that of CN- on the ATP levels i cotyldos of in the light (6). In higher plants, only experiments on the effect of
Gossypiwn hisawn and In Lemma gibba L In mature cotton tissue, CN- CN- in the nonphotosynthetic tissue of pea epicotyL oat coleop-
E, to the energy-n potential (ED) In the
tiles, carrot, maize roots, and red beet have been reported (1, 3,
dark. In the ight E,, recovered trstl. The same was observed In 12, 15, 17). In autotrophic tissues, energy is provided by respira-
leaves of Nkoedaa, Avena, I iens, c , and In Lecma. In con- tion, or photosynthesis, or both. Therefore, it was important to
trast, In yomg cotton cotyledons and tobacco leaves and, to a large extent,
investigate effects of CN- on Em in photosynthetic tissue under
In +sucrose-grown Lemna, E, was depolarzed to ED aso In the light In a
dark and light conditions in order to know the magnitude and
similar way as In the dark
source of energy for the energy-dependent component of Em.
In Lemn grown without sucrose, the energy-dependent component of Inasmuch as it is known that plant mitochondria can develop
E,. was o* paaf dpled by CN- In dark or igbht Cyanide plus a CN-resistant respiration during wounding, aging, or inhibition
salcyihydroxamic acid completely redced Em to EA ab ed of the Cyt oxidase (5, 28), in the present paper the effect of CN-
and pothe, and severely d the ATP level. Thi suggests on Em is compared with the effect of anaerobiosis on leaf segments
the operatin of a CN-4nsensltive i In ujured Lemcw The
and unwounded Lemna plants.
Idtil CN--duced decay of the ATP level In cotton and Lemma was more Metabolic energy supply and energy requirement change during
rapid than the decay of E,, CN-Induced osciations of the ATP level the development of leaves (8, 27). Therefore, the effect of CN- on
were followed by simiar but slower oscillations of E.. Ti suppworb the Em was investigated with tissues of various plant species at differ-
view of a general -e ce of E.
eped on ATP. Discrepancies between ent physiological age.
Ihb or-Inluced cuanges of E,,. and ATP levels are sugsIt to result
CN- was often supposed to have direct depolarizing effects on
from additionalregulatin of Em by the cyoimtic pH valu
the plasmalemma (10, 12, 25) due to its solubility in lipid phases,
A cmr of ED In youg and mature cotto co EIn the dark most likely as HCN at physiological pH. To discriminate between
and in the liht suggests that In growing young cotyledons the different such a direct membrane effect and the effect on energy supply,
effect of CN- In the light is due to a less effective photosynthesis the CN--dependent decay time of Em was compared with the
together with higb mito l respiratin In Lemma and In mature decay time of the ATP level.
cotton tissue, E. In the light is mataIe by noncyclc photopbosphory-
latd and photosystem II, which is only ptly hbed by CN-, thus
In an mlete rn and recovery of E,,. Complete MATERIALS AND METHODS
n on of photosynthetic 02 evoludon and ane depaization by

CN- plus saliylbydroxamic acid are suggested to result from pbotooxi- Plants Gossypium hirsutum cv Acala 44 was grown in soil in
dation. growth chambers for 7 to 14 d with light of 90 to 120 w m-2 at
26°C/16 h day to 22°C/8 h night. Tobacco, oat, and garden
balsam plants were grown in vermiculite, irrigated with Hoagland
solution, illuminated with 90 w m 2, at 24°C/14 h day to 21°C/10
h night. Leaves of Nicotiana tabacum, cv KY/6, Burley, were used:
either the first leaf (size 5-8 mm = young) or the third leaf of 8-
to 10-leaf plants (mature). From Avena sativa cv Victory, the 10-
d-old first leaf was used. From Impatiens balsamina, 16-d-old
Cyanide is often used to differentiate between the energy-de- cotyledons were used at the beginning of the 4-leaf stage. Leaves
pendent and energy-independent components of the E.2 in plant of Kalanchoe daigremontiana Hamet et Perrier were used after
cells. In some aquatic plants like Nitella, E. in the dark is similar growth for 6 months in soil in a greenhouse.
to the EK+, and CN- has no effect on Em in the dark (26). In the The leaf sections (10 x 15 mm) were washed (aged) for 2 to 15
light, CN- depolarized the membrane to 11 mv more negative h in aerated experimental standard solution (lx): I mM KCI, I
mM Ca(NO3)2, 1 mm NaH2PO4, 0.25 mm MgSO4, pH 5.7 (11).
'Supported by the Deutsche Forschungsgemeinschaft and a National Lemna gibba L. GI (from the Lemna collection of Professor R.
Science Foundation Grant (PCM 77-25575). Kandeler, Vienna) was grown axenically under SD conditions
2
Abbreviations: Em, electrical transmembrane potential; Ed, diffusion (28°C/8 h day to 23°C/16 h night) at a light intensity of 25 w
potential; E+, Nernst potential of potassium; SHAM, salicylhydroxamic m 2, on a medium containing as macronutrients 3.96 mam KNO3,
acid. 5.47 mM CaCI2, 1.22 mM MgSO4, 1.47 mM KH2PO4, and 29 mM
7
8 ULLRICH-EBERIUS ET AL. Plant Physiol. Vol. 72, 1983

GOSSYPIUM HIRSUTUM into the plant cells with a Leitz micromanipulator. Em was re-
corded with a high impedance electrometer amplifier (WPI model
701, or Keithley 604) and a chart recorder. In light experiments,
-88 light illumination of 150 w m-2 was provided by a quartz halogen bulb
and conducted through glass fiber optics. Further details have
been described earlier (18-20).
A -1677
-9 1d ATP Determination. Cotyledon segments or Lemna plants (150
= -116 dark mg fresh weight) were preincubated for 1 h in light or dark by
+CN- -99
light floating on 10 ml lx solution in 50-ml glass beakers, covered with
B . / ---------- N2 / light Parafim, before CN- (pH 5.7) was injected. The beakers were
/ ~~~~~~~~aiirI gently shaken on a Plexiglas rack in a photo-Warburg apparatus.
The incubation was stopped by rapidly freezing the tissue with
liquid N2. After immediate homogenization with a microdismem-
C-1681 brator (Braun), ATP was extracted with cold 5% HC104 and
determined by the luciferin-luciferase assay as reported earlier
(19).
air/ dark 02 Measurements. These were done polarographically with a
Rank O2 electrode (Clark type) in 3 or 5 ml of lx solution with
/ - 98 dark 100 to 150 mg fresh weight tissue samples. The light source was
the same as during Em measurements. Some experiments were

D
+CN-//E
/ ,ight
done manometrically in a photo-Warburg apparatus in which
light was supplied by Krypton lamps. In dark experiments, 250
-170 - CN- ;12 p1 I M KCN solution in the center vessel of the Warburg flasks
+CN- 4 maintained a constant CN- concentration and simultaneously
-162 absorbed evolving CO2. In the light experiments, different CO2
E1 concentrations in the gas phase were maintained by 0.1 mg
carbonic anhydrase (Serva) in a mixture of 3 M KHCO3/K2CO3
light in the center vessel of the Warburg flask (30). Because of their
large aerenchyma, duckweed plants assemble at the top of the 02
electrode chamber. Therefore the recorded rates, especially the
higher ones, are considerably lower than those obtained by man-
ometry. Manometric control rates are 20.3 ± 2.1 (10) .mol 02 g1
N2/1 ight
fresh weight h-1 at 0.03% CO2 and 178.1 ± 4.1 (14) beween I and
j8 2 2.5% CO2. In the presence of CN-, it was not possible to maintain
air/dark -183
I I 1 i a constant CO2 partial pressure in the Warburg vessels at pH 5.7,
0 5 10 15 20 25 since CN- inactivates the zinc-containing carbonic anhydrase
time (min) (Solomonson in 28).
FIG. 1. Effect of I mm CN- on Em in segments of cotton cotyledons, in K' Determination. After extraction of the leaf tissue with boiling
7-d (A-C) and 14-d-old (D-F) plants, in the light (A, E) and in the dark water for 1 h, the K+ content was determined by flame photometry.
(B, D). C and F, effect of N2 and Em recovery in air/dark or N2/light. All experiments (Em, ATP, and gas exchange) were performed
Numbers at the traces denote recorded mv. at 25°C and with a final CN- concentration of 1 mm.
sucrose (22). RESULTS
Duckweed may exist in two different energy states. Both plant
types were used in the present experiments. Plants of the standard Electrical Membrane Potential In 7-d cotton cotyledons (G.
culture are in a high-energy state with an ATP level of about 100 hirsutum), Em was reduced to a similar level by CN- in both light
nmol ATP g-' fresh weight in the dark, a high respiration rate of and dark (Fig. 1, A and B). To determine if CN- abolished the
8,umol 02 g-' fresh weight h-1, and a high Em of -220 mv in the total energy-dependent component of Em, cotyledon segments
dark (19). Upon transferring plants to a sucrose-free medium and were flushed with N2 in the dark. Anaerobiosis resulted in a
by additional acceleration of the metabolism by growth under LD similar effect on Em as CN- (Fig. IC). Thus, CN- completely
conditions for about 7 d, the plants get into a highly energy- inhibited the energy-dependent component of Em in dark and
deficient state after 6 h up to 3 d under dark conditions. The ATP light in 7-d-old cotyledons.
level decreases to about 22 nmol g-1 fresh weight, respiration to 2 In 14-d-old cotton cotyledons, in the dark CN- had a similar
,umol 02 g1 fresh weight h-1, and Em to -90 mv in the dark (19). effect as in 7-d cotyledons (Fig. ID). In the light, CN- had a
After keeping these plants in darkness for more than 4 d, they smaller effect on Em which transiently recovered even in the
recover with an ATP level of 65 nmol g-1 fresh weight, a respira- presence of CN- (Fig. IE). These Em values were reduced to ED
tion rate of 4 ,umol 02 g- fresh weight h-1, and again a high Em by additional application of SHAM (Table I). Inasmuch as in
of -220 mv. skunk cabbage the half-maximal inhibition of mitochondrial res-
Electrophyslological Measurements. For Em measurements, the piration was exerted by 0.26 mm SHAM (see ref. in Palmer in 28),
leaf sections of plants were mounted in a verticaL 4-ml Plexiglas we used 1 mm. Under anaerobiosis in the dark, the energy-
cuvette, perfused with lx solution at a flow rate of 10 ml mm dependent component decreased to the level measured under CN-
For CN- experiments in lx solution, the 1 mm KCI was substi- in the dark (Fig. IF).
tuted by 1 mm KCN (13), and the solution was adjusted to pH 5.7 In 14-d-old cotyledons, after depolarization by N2 in the dark,
with HCI. Glass micropipettes were pulled with a vertical puller Em recovered with similar velocity upon switching from N2 to air
(D. Kopf) from fiber-filled borosilicate capillaries (WPI and Hil- as from N2/dark to N2/light. In contrast, in 7-d-old cotyledons,
genberg). Micropipettes with a tip diameter below 1 jum and filled Em recovered much faster than in 14.d cotyledons in air/dark; but,
with 3 M KCI (Eo = -5 to -15 mv; R,p = 10 to 20 MO2) were under N2/light, it repolarized very slowly (Fig. 1, C and F). This
used as microsaltbridges to Ag/AgCl electrodes and were inserted suggests differences in energy production by young and older
 CN- EFFECT ON MEMBRANE POTENTIAL AND ATP 9
Table I. Membrane Potential (E.) and Respiration in L. gibba and in Cotyledons of G. hirsutum
Effect of 1 mM CN-, CN- + 1 mM SHAM in 0.3% ethanol N2, or 10 Am DCMU in 1% ethanol. Duckweed grown in ± sucrose nutrient solution. All
measurements in lx solution at pH 5.7, containing 1 mim KCI. Em values from maximum depolarization between 5 and 15 min. For long-time treatment,
the plants were kept either in dark or in light for 15 h. D, dark; L, light; n.d., experiments not done. Values are means ± SE and numbers of experiments
(n).
Plants Control CN- 15 h CN- SHAM CN-
+ SHAM
15 h CN
+ SHAM
N2 DCMU
Membrane
potential
(mv) High-energy
L.gibba D-sucrose -216±8 -157±9 -146±6 -219±6 -91 ±3 -99±7 -116±2 -233±3
(10) (7) (3) (4) (8) (5) (4) (3)
D+sucrose -209±5 -131 ±7 -135±5 -212+3 -93±4 -93±7 -92±7 -233± 1
(9) (14) (6) (3) (9) (3) (2) (3)
L-sucrose -248±7 -214±9 -159± 10 -233+5 -121 ±4 -60±9 -219±4 -230±3
(8) (8) (5) (2) (8) (5) (3) (3)
L+sucrose -232±7 -131 ±6 -152±3 -239+6 -94±2 -48±2 -233±6 -241 +3
(4) (8) (3) (8) (3) (5) (7) (6)
Low-energy
L gibba D-sucrose -99 + 6 -96 ± 5 n.d. n.d. -96 + 5 n.d. -118 ± 2 -118 ± 5
(10) (6) (3) (4) (3)
L -sucrose -218 ± 5 -137 ± 4 n.d. n.d. -95 ± 4 n.d. -264 ± 5 -115 ± 7
(8) (3) (3) (8) (3)
Respiration
(pmol 02
g fresh
wt.h) L. gibba D +sucrose -7.38 ± 0.3 -3.63 + 0.1 -2.5 ± 0.2 -7.6 ± 1.1 -0.17 ± 0.06 -0.1 + 0.1 0 (7) n.d.
(30) (43) (8) (8) (12) (6)
Membrane
potential
(mv) G. hirsutum D 7-d -180 ± 3 -96 ± 3 n.d. n.d. -89 ± 3 n.d. -96 ± 2 -171 ± 4
(21) (5) (4) (6) (9)
D 14-d -174±3 -95± 1 n.d. -174±2 -93+5 n.d. -97±2 -175+2
(17) (6) (2) (3) (25) (3)
L 7-d -172 3 -92 4 n.d. n.d. -98 + 5 n.d. -160 ± 8 -167 + 6
(11) (5) (6) (3) (5)
L14-d -169±1 -126±4 n.d. -162+1 -95+4 n.d. -183±5 -165±3
(25) (21) (2) (3) (5) (6)

tissue, ie. higher rates in oxidative phosphorylation than photo- depolarization remained about the same as found after short
phosphorylation in young cotyledons. exposure. The only exception was CN- + SHAM in 15 h contin-
In mature leaves of the terrestrial dicotyledons K. daigremon- uous light, where the values dropped far below ED (Table 1). This
tiana, N. tabacum, and I. balsamina or the monocotyledon A. potential difference, however, can no longer be regarded as Em.
sativa, after an initial inhibition of Em by CN-, qualitatively It is necessary to determine which energy sources contribute to
similar repolarization of Em in the presence of CN- was observed the maintenance of Em in Lemna in the light (ie. mitochondrial
in light in contrast to dark (Fig. 2, A-C, and Fig. 3, B-D). But phosphorylation, or noncyclic or cyclic photophosphorylation, or
again in young leaves of tobacco seedlings, Em did not repolarize all three reactions together) and to the partial CN- insensitivity of
after CN- application in the light (Fig. 3A). Em in the light. To discriminate between energy sources, low-
In contrast to the effect of N2 (Fig. 4D), the Em of unwounded energy and high-energy plants were used as described in "Mate-
L gibba in the dark was only partly depolarized by CN-, even rials and Methods." In high-energy plants, N2 depolarized Em in
after extended incubation (more than 9 h) (Fig. 4, A and C). The the dark to -96 mv (Fig. 4D). Upon switching from N2 to air in
remaining electrogenic portion was still depolarizable by H+/ the dark, Em recovered completely within 10 min. Low-energy
glucose cotransport (Fig. 4C, +glucose). Only the simultaneous plants sustained the high Em value only in the light (Fig. 4, E and
application of CN- with 1 mm SHAM, known to inhibit the CN-- F). In the dark, Em was similar as found in high-energy plants
insensitive alternative oxidase in plant mitochondria, depolarized under anaerobiosis, thus confirming the lack of energy in these
the membrane to a similar value as obtained under anaerobiosis plants. Hence, in low-energy plants in light, Em must be main-
(Table I). tained solely by photosynthesis. To determine which photophos-
As in all other plants tested, the Em in Lemna was much less phorylation, cyclic or noncyclic, provides the energy in the light,
affected by CN- in the light. Membranes repolarized in the light 10 AM DCMU was added to such energy-deficient plants. After
with CN- still present (Fig. 4, B and C). In sucrose-grown plants, switching light on in the presence of DCMU, Em hyperpolarized
the effect of CN- in the light was more pronounced than in only transiently (Fig. 4E). Removal of DCMU immediately re-
sucrose-starved plants (Table I). The simultaneous application of sulted in the usual light-dependent hyperpolarization. This indi-
CN- + SHAM reduced Em to a similar level in light and dark. cates energy supply from noncycic photophosphorylation. Addi-
After prolonged CN- and CN- + SHAM treatment (15 h), tion of CN- and CN- + SHAM in the light in this kind of plant
 v~ (
10 ULLRICH-EBERIUS ET AL. Plant PhysioL Vol. 72, 1983
0 10 20 30 40 1min) 50
KALANCHOE DAIGREMONTIANA I I I

*CN-16 -206 LEMNA GIBBA

A ligh
-210A
- -125

A *CN
darkr light -232 Ar

/-178 -232
A23 ii2tlg
-222
-CN -244
B *CN-
C -11.7
B light-176 9h in CN- -132
-186 dark f
-11.5
c ~~ ~~
-195
_159%
~ .glucose
light
-119
li~ ~ ~ 1ght -131. -CN- -267
-2l0
/-102 -104 -113
+CN- ,light air

0
dar
C 201

20
10 30 40 50
a]D
>
J
D

dark
N2-9-

190
dark

-227
-0

1%
time (min) in air
light1 ethanol .DCMU
FIG. 2. Membrane potential changes in leaf segments of K daigremon- dark -127
twna, affected by 1 mm CN-; A and B, in the light; C, in the dark. Mean E
values ± SE and numbers of experiments (n): A, -CN-, -207 ± 9 mv (17); -197 E1
B, +CN-, -145 ± 15 mv (11). C, -CN-, -210 ± 7 mv (12); +CN-, -98
± 8 mv (6).
E] E
light2 -DCMU
-CN- LIGHT -120 20 min
-151
-86
*CN\ -220
A 181 Nicotiana tabacum
-181 -CN (young) light dark
*CN- I4 dark
--I Nicotiana tabacumE -94
-189
B CN- -194 (mtreE
mature) o ]t
air

F
'CN
C -201 15 patiens balsamina
-CN I

ICN .101 0 20 time (min) 40 60

D -134 -121 Avena sativa FIG. 4. Membrane potential changes in L gibba. A-C, induced by I
.I__ mM CN-, A and C in dark, B in light. C, E. recorded after 9 h in dark in
0 10 20 30 40 50 CN, recovery of E., upon removal of CN, after switching light on in the
time (min) presence of CN-, and depolarizatio of E. by 50 mm glucose. D, Depo-
FIG. 3. Membrane depolarization by I mm CN-. A, in N. tabacum larization of high-energy duckweed by N2 in dark, hypeolrization in
yroung leaves (10); B, in N. tabacum segents of mature leaves (8); C, iA light or dark/air. E, Effect of 10 DCMU in 1% ethanol on E. of low-
oaf segments of L balsa (3); D, in leaf smts ofA. sativa (7); in the ener duckweed: initial, partial, and transient hyperpolarization and
lillight. Number of experiments (n). stationary depolarization in iht. F, Effect of CN- and CN- plus SHAM
on E. in the light in low-energy plants.
revealed also that the light reaction is partly inhibited by CN-
and completely by CN- + SHAM (Fig. 4F; Table I). is in the same range as the ED values measured in the presence of
pH. The low-energy L gibba (see "Materials and Methods") CN- or under anaerobiosis. Thus, ED must be predominantly
was exposed to CN- at various extracellular pH values in order to determined by EK in both young and mature tissues.
test an influence of ApH on the energy requirement for the Table III shows the intracellular K+ content ofduckweed grown
maintenance of the membrane potential. As shown in Figure 5, at under various ionic conditions. EK+ of these plants was in the
pH 5.7 these plants maintained only a diffusion potential and same range as ED measured in the dark in the presece of CN-
CN- had no effect on Em. After removal of CN-, the pH of the + SHAM. Only in plants grown on nutriet solution without
perfusion solution was increased to 8, and within 2 h the mem- sucrose for 7 d (5.43 mm K+) and me in lx soltion (1 mm
brane hyperpolarized to -210 mv in the dark. The subsequent K+) was ED different from ER+, due to a K+ distribution not in
strong depolarization by CN- proved that this membrane poten- steady-state.
tial, generated by loweing the extracllular H+ concentration, was ATP LeveL In both 7- and 14-d-old cotton cotyledons, the
still energy dependent. At pH 8 in the absence of any ApH, average ATP level was higher in the dark than in the light (Fig.
protons are still pumped out aainst the electrical grdint of 6). In 7-d cotyledons, the dark ATP level was about twice that of
-210 mv (AGO 20.3 kJ mol'). Switching light on additionally 14-d cotyledons. In all cases except for 74 cotlons in the light,
proved that the HW-extrusion pump could be activated. ATP decay after CN- appication was more rapid than the CN--
EK4. Table II shows the intracellular K4 content of 7- and 14-d- induced deneof E. (able IV). In the dark in CN-, E,. was
old cotton cotyledons or segments washed for 15 h in lx solution. equal to ED, but ATP emained at a level of only 50% inhibition
The value of -96 mv for EK, calculated by the Nernst equation, (Fig. 6). In the light, after the initial inhibition, the ATP level
 CN- EFFECT ON MEMBRANE POTENTIAL AND ATP 11

dark air CN IX/pH8

X/pH8 ~~~~~LEMNA GIBBA
pH5.7 -96 v JVX/pH8

2h
CN- __,
' Jo | | l a~~~~~~~~~-
0 10 20 0 10 20 30 40
time (min)
FIG. 5. Effect of extracellular pH on the membrane potential (E.) in low-energy L gibba and response of Em to I mm CN- (five experiments). Trace
on the right: Em in the same experiment after 2 h continuous measurement at pH 8.

F
t
I
TI F I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Table II. K+ Content in 7- and 14-Day-Old Cotton Cotyledons
EK+ = calculated Nernst potential. lx solution contains 1 mm KV.
Values are means ± SE and numbers of experiments (n).
GOSSYPIUM HIRSUTUM
K+ Content
7-d cotyledons 14-d cotyledons
o-- -o light >7d
* *e dark
mol K+ g-l fresh wt
Whole cotyledons 43.83 ± 0.82 (11) 51.85 ± 1.3 (12)
--- l{ight > 14d
200[1 * * dark
Sections, 15 h in Ix 43.83 ± 0.80 (5) 44.70 ± 0.59 (11)

EK+ -96 mv -96 mv

Table III. K+ Content, ED, and EK+ of L gibba

ED of plants from permanent, sucrose-containing culture was measured
in the culture solution (K: = 5.43 mm) and that of plants from the 7-d
sucrose-free medium (K: = 5.43 mm) was measured in lx solution (K:
= I mm), both in the presence of I mM CN- plus I mM SHAM, in the <150 -+'
dark. Plants kept in lx solution for 7 d were measured in lx solution.
Values are means ± SE and numbers of experiments (n).
EKx o 100- _ ' F%

Preculture K+ Content ED Measured K: Calcu-

lated
4A
mol g-'fresh wt mv mM mv
+ Sucrose 136.67 ± 0.67 (15) -84 ± 2.07 (22) 5.43 -83
7 d - sucrose 121.49 ± 0.49 (10) -91 ± 4.00 (16) 1.00 -123
7 din lx 116.79 ± 0.45 (32) -117 ± 1.44 (43) 1.00 -122

recovered similarly to Em, but faster. It then decreased to a low

level of only 10 to 15% of the control in tissues of both ages.
The ATP level of L gibba was markedly higher in the light 0
than in the dark (Fig. 7) in contrast to cotton tissue. But the 0 5 10 time(min)20 30
oscillations of the ATP levels upon addition of CN- were similar FIG. 6. Time course of the effect of I mm CN- on the ATP level of 7-
to those in cotton. In the dark, ATP changes were similar to Em and 14-d-old cotton cotyledons in dark and light. Mean values of each
levelling off at about 50% inhibition. In the light in the presence seven experiments ± SE.
of CN-, the ATP level recovered considerably more slowly than
Em (Fig. 4, B and C) within 18 h to 75% of the controL As in 50% in 14-d cotyledons, whereas 02 uptake was not affected, the
cotton, the initial decay of the ATP level was more rapid than the respiratory ATP production must be partially uncoupled and
CN-induced decay of Em (Table IV). Application of CN- + partially CN- insensitive.
SHAM in dark or light reduced the ATP level of Lemna to the The CN- inhibition of 02 uptake also in Lemna was dependent
very low value of about 15 nmol g-1 fresh weight (Fig. 7). This on the magnitude of respiration. In plants continuously grown on
suggests that in Lemna the CN-insensitive portion of the active a plus sucrose medium, the respiration was 7 to 9 ,mol 02 g 1
component of Em must indeed be due to photosynthesis or the fresh weight h-' (Fig. 9). In these plants, CN- had a strong effect
operation of a CN-resistant alternative oxidase in their mito- of more than 60% inhibition. With decreasing respiration rate due
chondria, though Lemna tissue was neither cut nor 'aged.' to prolonged growth without sucrose, the CN- inhibition became
Respiration. The dark 02 uptake rate was 5-fold higher in 7-d smaller and finally, when the rate of 02 uptake was less than 2
than in 14-d cotton cotyledons (Fig. 8). It was very sensitive to pmol g-' fresh weight h-', it was even stimulated by CN-. SHAM
CN- and was inhibited to the CN-unaffected 02 uptake rate of together with CN- completely abolished 02 uptake (Table I).
14-d cotyledons (Fig. 8, A and B). As the ATP level decreased by SHAM alone had no effect on respiration, Em, or the ATP level.
12 ULLRICH-EBERIUS ET AL. -PI-a-nt Physiol. Vol. 72, 1983

150
LEMNA GIBBA
125

100
3.
U- l ight, CN-
7 75
dark CN
< 50
E
' 25
.'- ---- light,
CN-
- ' liht.SHAM
~---w4
() t

0 10 30 50 70 90 18 h
time (min)
FIG. 7. Time course of the effect of 1 mM CN- on the ATP level of L
gibba, in dark and light, and effect of I mm CN- plus 1 mm SHAM, in
0.3% ethanol. The arrow indicates the time (zero) of addition of the
inhibitors. Mean values of five experiments ± SE each.
Table IV. CNI-Induced Decay ofthe A TP Level and of Em in Cotton
Cotyledons and in Lemna
-/2 half-time of decay in seconds required to reach the first minimum.
For ATP: total level - 100% (at zero time). For Em: AEm = Em - ED =
100% (at zero time).
Age of
Plants
Experimental
Conditions t1/2(ATP) tii2(Em)
d s 0 10 20 30
7 Dark 63.8 71.0
time (min)
FIG. 8. Respiration in G. hirsutum cotyledons. Effect of I mm CN- on
7 Light 176.5 72.5 7- and 14.d-old cotyledons in the dark. Polarographic measurements.
Cotton Trace A, 112 mg fresh weight; trace B, 200 fresh weight. Numbers at the
14 Dark 88.2 135.0 traces denote rates of 02 uptake in pmol 02 g-1 fresh weight h-' (20
14 Light 30.0 176.5 experiments).
Lemna Dark 139.5 253.5
Light 333.0 525.0 c 0
0 601
.0
Photosynthetic 02 Evolution. COrdependent photosynthetic 02 c ._
40-
evolution rates were similar in 7- and 14-d-old cotton cotyledons, 0
._

being saturated at 0.5% CO2 (Fig. IOA). At 0.03% CO2 in 7-d -

-

201
._L
cotyledons, the rate was lower than the respiration rate, thus In
U)
°1 /
resulting in a shift of the compensation point to a higher CO2
concentraton (from 0.01-0.04% C02; Fig. 10, A-C in comparson c
0
0
1 5 7 9
with trace E). In cotyledons of both ages, 02 evolution was rapidly 02 uptake rate
affected by CN-, at low (0.03%) and at high (1%) CO2 concentra- z 1-
C
0
20[ */ gF'
O29~FW-h- -11
tions (Fig. 10, C-E). After switching the light off, 02 uptake %-
0

increased, suggesting that the C02-dependent photosynthesis was 4U 401

not completely inhibited by CN-. E
Also, in Lemna CN- did not completely inhibit ligHt-dependent 0) In 60'
02 evolution (Fig. 1 IA). Only additional application of SHAM
gradually suppressed dark 02 uptake and light 02 evolution (Fig.
0

11, A and B). Upon switching light on after application of SHAM FIG. 9. Respiration in L gibba Effect of 1 mm CN- on plants with
(trace A), the 02 evolution was not increased by the amount of high or low respiration rates in dark. Manometric measurements. Mean
dark 02 consumption prior to addition of SHAM. This means that values of 30 experiments.
either CN-in2sensitive2 uptake by the mitochondria is sup-
prssed by light, or SHAM has an additional inhibitory effect also the primary site of action of CN- must be the ATP production
on photosynthesis. and not directly the plasmalemma. The same conclusion was
drawn earlier for Neurospora (25).
DISCUSSION ATP Level and E,m. A comparison of the effect of CN on Em
and on the ATP level shows the general dependence of Em on the
The present results clearly show that in dark-incubated cotton ATP level, mainly from the similarity of their oscillations in light
cotyledons CN- is a useful tool to disciminate between the and dark. ATP changes were followed in time by very similar E.
energy-dependeat and energy-independent component of Em. Val- changes. However, the correlation between Em and ATP was not
ues obtained after application of anaerobiosis and those for the as strict as it was reported for Chara and red beet (14, 15, 17, 24).
K+ distribution are in ageement with the CN- experi*nts. It is This may become obvious from three amples. (a) While in the
also shown by comparison of the CN-induced slower decay of light, the ATP level was reduced by CN- to 10% in 14-d cotton
Em with the faster initial decline of the ATP level (Table IV) that cotyledons and to 30% of the control in Lemna, the energy-
 IL
a,

E
'v
CN- EFFECT ON MEMBRANE POTENTIAL AND ATP

B
SHg4A
L
+CN-

E,
74

.8
LCm-
D

- -
L D +SAMj

loonni ltr-
D

FIG. 1 1. Effect of I mM CN- and of I ml CN- plus1 mM SHAM on

L

photosynthesis and respiration in L gibba A, CN- applied prior to

SHAM. B, SHAM addition followed by successive additions of 1 mm CN-
each. 500 mg fresh weight in 3 ml Ix solution, 1% C02, polarographic
13

measurements. Numbers at the traces denote rates of 02 uptake (-) or 02

evolution (+) in pmol 02 g-' fresh weight h-' (eight experiments).
cotton cotyledons, the ATP level increased to 110%, while Em in
the dark was completely depolarized to ED (21). (c) In low-energy
Lemna, fusicoccin hyperpolarized the membrane from -97 to
-250 mv, while the ATP level remained at only 32% of the
standard control value (2).
A discrepancy between the ATP level and Em was also reported
for L. paucicostata (16). In L. gibba, dicyclohexyl carbodiimide
(DCCD) slowly depolarized Em only to 80% of the control in the
light. Only after depolarization by the H+/glycine cotransport did
Em remain depolarized similarly to L. paucicostata (E. Fischer, A.
Novacky, unpublished results). In L. gibba, different from L.
paucicostata (16), 5 mM arsenate had only a transient effect on the
ATP level and a more complex one on Em. In the light, arsenate
did not affect Em in plants grown on phosphate medium, in the
dark it slowly decreased Em to 44% of the control. In phosphate-
starved plants, arsenate induced Em, depolarization, probably via
the H+/phosphate cotransport carrier, and after a transient recov-
ery Em decreased to 27% of the control (C. I. Ullrich-Eberius, A.
Novacky, unpublished results).
The discrepancy between the ATP level and Em may have
several reasons. (a) The ATP concentration in a plant cell is
probably not homogeneous due to compartmentation and trans-
port barriers. (b) Although the ATP levels in dark and light are
similar (14), the turnover of ATP is considerably faster in the light
than in the dark (7), so the ATP level does not necessarily reflect
the energy supply rates. (c) The energy-dependent component of
Em depends on the activity ofthe ATP-dependent H+-efflux pump
and consists of an electrical and a chemical component, ie. AA
and ApH across the plasmalemma. The cytoplasmic pH (pHi) is
regulated by the ATP/H+-extrusion pump and by cell metabolism.
0 10 20 30 time (min) 50 Cell metabolism, ATP content, intracellular pH, and Em are in
mutual interaction. Hence, not only ATP but equally pHi deter-
FIG. 10. Photosynthesis in G. hirsutum cotyledons. A, CO2 concentra- mines Em. In the light, Em is much less ATP dependent than in
tion dependence of 02 evolution in 7- and 14-d-old cotyledons in light. the dark. H+ consumption by reductive processes in the light, ie.
Manometric measurements. Chl content in 7-d cotyledons 1.63 0.15
-
reduction of CO2, N03-, and So42- may lead to a lower demand
mg g-' fresh weight and in 14-d cotyledons 1.29 + 0.07 mg g-1 fresh for ATP by the H+-extrusion pump. Decreasing the ApH across
weight (18). Mean values of six experiments sE. B, Dark-light changes the plasmalemma had a similar effect on Em as exposing the plants
of 02 uptake or evolution in 7-d-old cotyledons at 0.03% C02. C, Imme- to light. Hence, the activity of the pump cannot solely depend on
diate effect of CN- on 02 evolution at 0.03% C02 in 7-d cotyledons. D, ATP. The different redox state of the cell during respiration and/
Immediate effect of CN- on 14-d cotyledons at 0.03% C02; and E, at 1% or photosynthesis (9) may also contribute to the activity of the
CO2 (addition of 10 IL 1 M KHCO3 to 5 ml lx solution, resulting in a pH pump at the plasmalemma.
of 6.95 -2 mM KHCO3). B-E, Polarographic measurements. Numbers at Action of CN-. The effect of CN- on E. and ATP was shown
the traces denote rates of 02 uptake (-) or 02 evolution (+) in umol 02 g' to be different in various plant species. In Lemna, in contrast to
fresh weight h-1. cotton, the inhibition by CN- of the energy-dependent portion of
Em and of the ATP level in the dark was incomplete (Figs. 4C and
dependent portion of Em was depolarized only to 40% in cotton 6; Table I). Only addition of SHAM together with CN- completely
and to 56 to 86% of the control in Lemna. In the dark in both inhibited respiration (Table I), thus reducing the ATP level to 15%
tissues, ATP is reduced to 50% of the control and the energy- of the control (Fig. 7) and Em to ED (Tables I and III). As in
dependent Em is completely depolarized in cotton, to 34 to 53% of cotton, in K daigremontiana CN- reduced Em to ED in the dark
the control in Lemna. (b) In experiments with bacterial-infected (Fig. 2, A and C). Though cotton and Kalanchoe tissues were
14 ULLRICH-EBERIUS ET AL. Plant Physiol. Vol. 72, 1983
sliced and aged for several hours, they did not show a significant pressed by SHAM, but also in the light, simultaneously with 02
CN-resistant Em component as did the unwounded duckweed. evolution (Table I; Fig. 11). The additional inhibition was slower
Nevertheless, in cotton cotyledons respiration and the ATP level than that exerted by CN-; SHAM alone had no immediate effect
proved to be partly CN- resistant in the dark (Figs. 6 and 8). on Em, ATP leveL or photosynthetic 02 evolution. Hydroxamic
In the light, the action of CN- was revealed to be very complex. acids have been found to inhibit peroxidases and lipoxygenases
In all mature tissues tested, whether sections or unwounded tissue, (see Palmer in 28). Therefore, SHAM may cause severe photoox-
Em was initially less reduced. In contrast to dark, Em recovered idation in the chloroplasts, perhaps by poisoning the superoxide
almost completely in the presence of CN- (Figs. 1E; 2, A and B; dismutase. The deleterious effect of CN- plus SHAM treatment
3, B-D; 4B). The same was already reported earlier for mature became obvious in Lemna plants which, after prolonged incuba-
leaf tissue of Saccharnm off cinarum and Zea mays (18). Em finally tion in continuous light, lost their capability to maintain even the
levelled off at intermediate values after 2 to h (cotton at -145 mv; diffusion potential (Table I).
Kalanchoe at -134 mv; and Lemna at -160 mv).
It seems to be a peculiarity of young tissue that Em is as sensitive Acknowledgments-We thank Drs. Ulrich Luttge and Takeshi Kagawa for criti-
cally reading the manuscript and Sylvia Lenz and Waltraud Linker for valuable
to CN- in the light as in the dark. In mature tissue, mitochondrial technical assistance.
respiration is thought to be controlled by photosynthesis via the
adenylate system and the oxidation/reduction state, and its activ- LITERATURE CITED
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 CN- EFFECT ON MEMBRANE POTENTIAL AND ATP 15
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