Plant Cell Reports
P|ant Cell Reports (1982) 1 : 285-287
© Springer-Verlag 1982
Effect of 2,4,5-Trichlorophenol on Hyphal Membrane Potentials in Rhythmic Mutants of Neurospora crassa D. K n o p p i k 1 and G. Lysek 2 Lehrstuhl ffir Physik, Technische Universit/it Miinchen, D-8050 Freising-Weihenstephan, Federal Republic of Germany 2 Institut ftir Systernatische Botanik und Pflanzengeographie, Freie Universit~it Berlin, D-1000 Berlin 33, Altensteinstr. 6 Received September 14, 1982/November 4, 1982
~stract The u n c o u p l e r 2 , 4 , 5 - t r i c h l o r o p h e n o l (TCP) was used to test for d i f f e r e n c e s in maintaining the h y p h a l m e m b r a n e p o t e n t i a l s in the w i l d type and rhythmic m u t a n t s of N e u r o s p o r a crassa. T r i c h l o r o p h e n o l (O.1 mmol-l-1) resulted in a d e p o l a r i z a t i o n of 93 mV (wild type) and 144 m? in the m u t a n t clock° A total recovery was a c h i e v e d in both strains after w a s h i n g out the uncoupler. The c i r c a d i a n c o n i d i a t i o n m u t a n t band was more s e n s i t i v e than the two other strains and showed two d i f f e r e n t r e a c t i o n patterns: one w i t h d e l a y e d reaction, total b r e a k d o w n and w i t h o u t recovery; the other one with n e a r l y i m m e d i a t e reaction, slow but not e n t i r e l y total decline and p a r t i a l recovery. These d i f f e r e n c e s are d i s c u s s e d in their relation to the c i r c a d i a n r h y t h m of conidiation. Abbreviations PD, p o t e n t i a l d i f f e r e n c e TCP, t r i c ~ r o p h e n o l Introduction R h y t h m i c m y c e l i a l growth and f r u c t i f i c a t i o n r e p r e s e n t s a type of p e r i o d i c a c t i v i t i e s specific to fungi. It is linked to the biological clock of n o n f u n g a l e u c a r y o t i c organisms by p h e n o m e n a like the c i r c a d i a n conidiation of the m u t a n t b a n d of Neurospora crassa (B~nning 1973). Recent i n v e s t i g a t i o n s usin~ P o d o s p o r a anserina s u g g e s t e d that the p r o t o n m o v e m e n t s at the hyphal p l a s m a l e m m a m i g h t be i n v o l v e d in c r e a t i n g the r h y t h m i c growth. These proton m o v e m e n t s at the p l a s m a l e m m a are due to an e l e c t r o g e n i c ATPase, w h i c h extrudes the protons and a c c u m u l a t e s potassium. Both movements are active and d i r e c t e d a g a i n s t the c o n c e n t r a t i o n g r a d i e n t (Slayman and Slayman 1968). In the o p p o s i t e d i r e c t i o n protons flux from the m e d i u m into the c y t o p l a s m due to leakage or p r o t o n - s y m p o r t e d uptake of nutrients. Recent i n v e s t i g a t i o n s have shown that rhythmic growth in fungi m i g h t be due to i n c r e a s e d p r o t o n m o v e m e n t s into the cytop l a s m and a s u b s e q u e n t i n c r e a s e d e x t r u s i o n (Lysek and Schr~fer 1981; Gall und Lysek 1981; Lysek and Jennings unpubl.)
These a l t e r a t i o n s of p r o t o n m o v e m e n t s are a s s u m e d to affect the hyphal p o t e n t i a l s or their s e n s i t i v i t y to e x o g e n o u s factors. After various attempts, the u n c o u p l e r t r i c h l o r o phenol p r o m p t e d results w h i c h are c o m p a t i b l e with this concept and, in addition, showed differences w h i c h p a r a l l e l d i f f e r e n t types of r h y t h m s found in N__eurospora crassa. Materialsand
Methods
Strains: The f o l l o w i n g strains of N e u r o s p o r a crassa Shear et Dodge were used in the experiments: STA4 band
(No 262 FGSC) w i l d type; (b_dd) (No 1858 FGSC) c i r c a d i a n conidiation; clock (cl) (No 1166 FGSC) p e r i o d i c a l t e r a t i o n of b r a n c h i n g pattern.
Cultures: Cultures were i n o c u l a t e d from stock cultures to solid m e d i a c o n t a i n i n g 2 % agar and 3 % malt extract. The media were c o v e r e d w i t h s c r a t c h e d and s t e r i l i z e d c e l l o p h a n e (type 600 PIO, Kalle, Darmstadt, W.Germany) and i n c u b a t e d at 25°C for 15 h (STA4), 20 h (band) or 24 h (clock). M e a s u r e m e n t s : For m e a s u r e m e n t s , pieces of 7 x 20 mm 2 were cut out from the c e l l o p h a n e and t r a n s f e r r e d into a c h a m b e r a c c o r d i n g to G r a d m a n n and Slayman (1975), where the mycelium a t t a c h e d to the c e l l o p h a n e was b a t h e d in a standard solution (see below). M e a s u r e ments were made using c o n v e n t i o n a l c ~ p i l l a r y m i c r o e l e c t r o d e s filled w i t h 3 mol'l- KCl. The p o t e n t i a l d i f f e r e n c e was m e a s u r e d by a h i g h - i m p e d a n c e e l e c t r o m e t e r a m p l i f i e r model 610 C K e i t h l e y instruments, c o n n e c t e d to a M e t r a w a t t recorder. Solutions: The standard solution for recording the p o t e n t i a l d i f f e r e n c e ~ o n t a i n e d 10 mmol'l -I CaCl 2 and 133 m m o l . l sucrose (Slayman and Slayman 1968). A b o u t 10 min after i n s e r t i o n of the electrode, i.e., after h a v i n g r e a c h e d a c o n s t a n t reading of the p o t e n t i a l difference, the s t a n d a r d solution was s u b s t i t u t e d by the same solution containing in addition O.1 mmol.l -I 2 , 4 , 5 - t r i c h l o r o phenol (TCP); EGA-Chemie, Steinheim, Germany. Results In Fig.
la the typical
reaction
of the w i l d
0721-7714/82/0001/0285/$
01.00
286 -I type after addition of O.1 mmol-1 trichlorophenol to the b a t h i n g m e d i u m is seen. The w i l d strain shows an i m m e d i a t e l y starting d e p o l a r i zation of about 93 mV (Table I). Fig.2 gives evidence that the effect depends on the applied c o n c e n t r a t i o n of trichlorophenol. A f t e r r e m o v i n g the inhibitor, a total recovery of the p o t e n t i a l is achieved. The m u t a n t b a n d w h i c h differs from the w i l d type by its c i r c a d i a n conidiation, contrasts to this reaction pattern by a lag phase of about 8 min b e t w e e n the arrival of the uncoupler in the cuvette and the start of a visible reaction (Fig. Ib and Table I). The reaction itself is very fast, in most cases it lasts less than I min. The final level differs by 172 mJ] (mean) from the original resting potential. Since the residual value results from a mere d i f f u s i o n potential, this reaction leads to a total b r e a k - d o w n of the m e m b r a n e potential.
PD/ mV -200 -150
~ - 1 0 8 m V
- 50
'
I
I
5
10
15
PD/mV
-200
I
210
t/min
25
, TCP
-150 -100 -
50
---11mY 0
Fig.
0
{
1.0
I%
20
2'5
t/min
I: R e s t i n g p o t e n t i a l d i f f e r e n c e (PD) and the typical reaction after application of O.1 m m o l . 1 - 1 T C P in the w i l d s t r ~ n STA4 (a) and in the m u t a n t b a n d (b) of N e u r o s p o r a crassa. The arrow marks t~e arrival of the T C P - s o l u t i o n in the chamber used for the measurements.
Table
/{f J
100
50
' 0,025
0
Fig.
PD
' 0,05
To test w h e t h e r the d e s c r i b e d a l t e r a t i o n s of the t r i c h l o r o p h e n o l effect are typical for the m u t a n t band (and thus for its c i r c a d i a n c o n i d i a t i o n pattern), another rhythmic mutant, the strain clock was included. It shows a p e r i o d i c a l hyphal b r a n c h i n g (zonating) w h i c h is neither c i r c a d i a n nor s y n c h r o n i z e d to e n v i r o n m e n t a l cycles. T~e PD a l t e r a t i o n after a d d i t i o n of O.1 m m o l . l t r i c h l o r o p h e n o l is quite similar to that o b s e r v e d in the w i l d type, the d i f f e r e n c e is a longer decline (36 min instead of 24 min) and a r e s u l t i n g larger d e p o l a r i z a t i o n (144 mV as c o m p a r e d to 93 mV in the w i l d type; see Table I). After r e p l a c i n g the t r i c h l o r o p h e n o l solution by the original b a t h i n g medium, a total recovery is observed. Thus, the overall pattern of clock resembles that of the w i l d type.
of O.1 mmol'l -I TCP
in hyphae
Depolarization in TCP (mV)
Start of r e a c t i o n after TCP addition (min)
Time b e t w e e n start and maximal reaction (min)
Recovery after TCP t r e a t m e n t
93+ 9
0.4+0.2
24.0+4.0
total
-213+10
144+13
0.8+0.3
36.0+7.0
band
-186+
8
172+ 8
7.7+1.4
0.6+0.2
-211+10
134+25
1.7+O.5
18.2+2.8
6
T C P / m m o [ . I-1
M a r k e d d i f f e r e n c e s b e t w e e n the w i l d strain and band are also found in the r e c o v e r y after r e m o v i n g the t r i c h l o r o p h e n o l solution. No recovery was found after the typical "fast reaction" w i t h its total breakdown. After the " l o w - s p e e d reaction" a p a r t i a l but not complete r e c o v e r y was o b s e r v e d in but two cases.
STA4 (wild type) clock
-198+
' 0,10
2: C o r r e l a t i o n b e t w e e n applied concentrations of TCP and d e p o l a r i z a t i o n in the w i l d type STA4 of N e u r o s p o r a crassa.
I: Mean resting p o t e n t i a l s and reactions after a p p l i c a t i o n of the listed rhythmic strains of N e u r o s p o r a crassa. Resting (mV)
/
In some cases a slower r e a c t i o n is found, w h i c h also results in a very low residual potential, but not in a total b r e a k d o w n (Table I). The d i f f e r e n c e s b e t w e e n these two reaction patterns of b a n d are not yet comp l e t e l y understood, p r e s u h a b l y they are due to d i f f e r e n t c i r c a d i a n phases.
a
-I00
~U/mV
total no partial
287 Discussion The results d e m o n s t r a t e that the m u t a n t band contrasts to the w i l d type and to a n o t h e r rhythmic strain by its p e c u l i a r pattern of d e p o l a r i z a t i o n after a p p l i c a t i o n of TCP. In the w i l d strain STA4 the u n c o u p l e r affects the p o t e n t i a l d i f f e r e n c e as expected. At the hyphal p l a s m a l e m m a it is m a i n t a i n e d by an A T P - d e p e n d e n t "pump" w h i c h extrudes protons (and Na + ions) and a c c u m u l a t e s K + ions (Slayman and Slayman 1968; Galpin et al. 1978). The u n c o u p l i n g by t r i c h l o r o p h e n o l reduces the ATP c o n t e n t of the h y p h a e and thus reduces the active t r a n s p o r t across the p l a s m a l e m m a . The A T P a s e s can no longer keep up w i t h the p a s s i v e fluxes of the ions, e s p e c i a l l y w i t h the r a p i d l y m o v i n g protons and the hyphal p o t e n t i a l declines until it reaches a new e q u i l i b r i u m level. The removal of the inhibitor c o n s e q u e n t l y restores the original steady state. This r e a c t i o n p a t t e r n m i g h t be r e g a r d e d as "normal" since it is also found in clock, the only d i f f e r e n c e b e i n g the larger d e p o l a r i z a tion. This, however, is in a c c o r d a n c e with the c o n c l u s i o n of Lysek and Jennings (unpubl.) that the clock c h a r a c t e r in P o d o s p o r a anserina is due to the h y p h a l r e a c t i o n against an i n c r e a s e d p r o t o n influx into the c y t o p l a s m (Gradmann and Slayman 1975). This r e a c t i o n consumes part of the m e t a b o l i c e n e r g y and this enhances the s e n s i t i v i t y of the p o t e n t i a l d i f f e r e n c e a g a i n s t the uncoupler. A c c o r d i n g l y , the e q u i l i b r i u m level r e a c h e d after application of t r i c h l o r o p h e n o l is more d i f f e r e n t from the o r i g i n a l value than in the w i l d type. The o b s e r v e d r e c o v e r y is in a c c o r d a n c e with this view. The i n c r e a s e d s e n s i t i v i t y of b a n d may be due to a similar stress of the p r o t o n e x t r u s i o n system. For the delay of the r e a c t i o n and the f o l l o w i n g very fast breakdown, no explanation can be given actually. The lack of r e c o v e r y after w a s h i n g out the TCP, however, points to a p e r m a n e n t lesion in b a n d w h i c h may be due to the a l t e r a t i o n s in the m i t o c h o n drial A T P a s e of this strain found by D i e c k m a n and Brody (1980).
The c o r r e l a t i o n of these a l t e r a t i o n s in band with its c i r c a d i a n c o n i d i a t i o n is a s c e r t a i n e d by the occuzmence of the second r e a c t i o n pattern, w h i c h is more similar to that of the other two strains. The c h a n g i n g sensitivity indicates a linkage to the c i r c a d i a n phase. W o o d w a r d and Sargent (]973) found an a l t e r n a t i n g r e s p i r a t o r y a c t i v i t y in band. Their view of a "circadian u n c o u p l i n g " agrees with the findings r e p o r t e d here. Attempts are now made to test the r e a c t i o n p a t t e r n of this strain over the entire c i r c a d i a n period. This proves d i f f i c u l t since the light n e c e s s a r y for h a n d l i n g the samples and for m i c r o s c o p i c o b s e r v a t i o n is a c t i n g as "Zeitgeber". The authors thank Mrs. W. S c h w e r d t n e r for t e c h n i c a l a s s i s t a n c e and the D e u t s c h e Fors c h u n g s g e m e i n s c h a f t , B o n n - B a d Godesberg, for financial support.
References B O n n i n g , E (1973) The p h y s i o l o g i c a l clock; c i r c a d i a n rhythms and b i o l o g i c a l chronometry. 3rd revised ed. H e i d e l b e r g Sci. Libr., vol I; The E n g l i s h Univ. Press Ltd., London, and Springer-Verlag, New York - H e i d e l b e r g Berlin. Dieckman, 896-898.
C
, Brody, S
Gall, A, Lysek, G 13
(1981)
(1980)
Science
Neurosp
Newsl
Galpin, M F J, Jennings, D H, Oates, Hobot, A (1978) Exp Mycol 2: 258-269 Gradmann, D, Slayman, Biol 23: 181-212
C L
Lysek, G, Schr~fer, Ges 94: 105-112
(1981)
Slayman, Physiol
K
C L, Slayman, 52: 424-443
C W
(1975)
207: 28: K,
J Xembrane
Ber Dtsch (1968)
Bot
J Gen
Woodward, D O, Sargent, M L (1973) In: A P~rez-Miravete (ed) "Behaviour of m i c r o - o r g a nisms", P r o c . 1 0 th Intern. Congress for Microbiol., M e x i c o City. pp.282-296
P|ant Cell Reports (1982) 1 : 285-287
© Springer-Verlag 1982
Effect of 2,4,5-Trichlorophenol on Hyphal Membrane Potentials in Rhythmic Mutants of Neurospora crassa D. K n o p p i k 1 and G. Lysek 2 Lehrstuhl ffir Physik, Technische Universit/it Miinchen, D-8050 Freising-Weihenstephan, Federal Republic of Germany 2 Institut ftir Systernatische Botanik und Pflanzengeographie, Freie Universit~it Berlin, D-1000 Berlin 33, Altensteinstr. 6 Received September 14, 1982/November 4, 1982
~stract The u n c o u p l e r 2 , 4 , 5 - t r i c h l o r o p h e n o l (TCP) was used to test for d i f f e r e n c e s in maintaining the h y p h a l m e m b r a n e p o t e n t i a l s in the w i l d type and rhythmic m u t a n t s of N e u r o s p o r a crassa. T r i c h l o r o p h e n o l (O.1 mmol-l-1) resulted in a d e p o l a r i z a t i o n of 93 mV (wild type) and 144 m? in the m u t a n t clock° A total recovery was a c h i e v e d in both strains after w a s h i n g out the uncoupler. The c i r c a d i a n c o n i d i a t i o n m u t a n t band was more s e n s i t i v e than the two other strains and showed two d i f f e r e n t r e a c t i o n patterns: one w i t h d e l a y e d reaction, total b r e a k d o w n and w i t h o u t recovery; the other one with n e a r l y i m m e d i a t e reaction, slow but not e n t i r e l y total decline and p a r t i a l recovery. These d i f f e r e n c e s are d i s c u s s e d in their relation to the c i r c a d i a n r h y t h m of conidiation. Abbreviations PD, p o t e n t i a l d i f f e r e n c e TCP, t r i c ~ r o p h e n o l Introduction R h y t h m i c m y c e l i a l growth and f r u c t i f i c a t i o n r e p r e s e n t s a type of p e r i o d i c a c t i v i t i e s specific to fungi. It is linked to the biological clock of n o n f u n g a l e u c a r y o t i c organisms by p h e n o m e n a like the c i r c a d i a n conidiation of the m u t a n t b a n d of Neurospora crassa (B~nning 1973). Recent i n v e s t i g a t i o n s usin~ P o d o s p o r a anserina s u g g e s t e d that the p r o t o n m o v e m e n t s at the hyphal p l a s m a l e m m a m i g h t be i n v o l v e d in c r e a t i n g the r h y t h m i c growth. These proton m o v e m e n t s at the p l a s m a l e m m a are due to an e l e c t r o g e n i c ATPase, w h i c h extrudes the protons and a c c u m u l a t e s potassium. Both movements are active and d i r e c t e d a g a i n s t the c o n c e n t r a t i o n g r a d i e n t (Slayman and Slayman 1968). In the o p p o s i t e d i r e c t i o n protons flux from the m e d i u m into the c y t o p l a s m due to leakage or p r o t o n - s y m p o r t e d uptake of nutrients. Recent i n v e s t i g a t i o n s have shown that rhythmic growth in fungi m i g h t be due to i n c r e a s e d p r o t o n m o v e m e n t s into the cytop l a s m and a s u b s e q u e n t i n c r e a s e d e x t r u s i o n (Lysek and Schr~fer 1981; Gall und Lysek 1981; Lysek and Jennings unpubl.)
These a l t e r a t i o n s of p r o t o n m o v e m e n t s are a s s u m e d to affect the hyphal p o t e n t i a l s or their s e n s i t i v i t y to e x o g e n o u s factors. After various attempts, the u n c o u p l e r t r i c h l o r o phenol p r o m p t e d results w h i c h are c o m p a t i b l e with this concept and, in addition, showed differences w h i c h p a r a l l e l d i f f e r e n t types of r h y t h m s found in N__eurospora crassa. Materialsand
Methods
Strains: The f o l l o w i n g strains of N e u r o s p o r a crassa Shear et Dodge were used in the experiments: STA4 band
(No 262 FGSC) w i l d type; (b_dd) (No 1858 FGSC) c i r c a d i a n conidiation; clock (cl) (No 1166 FGSC) p e r i o d i c a l t e r a t i o n of b r a n c h i n g pattern.
Cultures: Cultures were i n o c u l a t e d from stock cultures to solid m e d i a c o n t a i n i n g 2 % agar and 3 % malt extract. The media were c o v e r e d w i t h s c r a t c h e d and s t e r i l i z e d c e l l o p h a n e (type 600 PIO, Kalle, Darmstadt, W.Germany) and i n c u b a t e d at 25°C for 15 h (STA4), 20 h (band) or 24 h (clock). M e a s u r e m e n t s : For m e a s u r e m e n t s , pieces of 7 x 20 mm 2 were cut out from the c e l l o p h a n e and t r a n s f e r r e d into a c h a m b e r a c c o r d i n g to G r a d m a n n and Slayman (1975), where the mycelium a t t a c h e d to the c e l l o p h a n e was b a t h e d in a standard solution (see below). M e a s u r e ments were made using c o n v e n t i o n a l c ~ p i l l a r y m i c r o e l e c t r o d e s filled w i t h 3 mol'l- KCl. The p o t e n t i a l d i f f e r e n c e was m e a s u r e d by a h i g h - i m p e d a n c e e l e c t r o m e t e r a m p l i f i e r model 610 C K e i t h l e y instruments, c o n n e c t e d to a M e t r a w a t t recorder. Solutions: The standard solution for recording the p o t e n t i a l d i f f e r e n c e ~ o n t a i n e d 10 mmol'l -I CaCl 2 and 133 m m o l . l sucrose (Slayman and Slayman 1968). A b o u t 10 min after i n s e r t i o n of the electrode, i.e., after h a v i n g r e a c h e d a c o n s t a n t reading of the p o t e n t i a l difference, the s t a n d a r d solution was s u b s t i t u t e d by the same solution containing in addition O.1 mmol.l -I 2 , 4 , 5 - t r i c h l o r o phenol (TCP); EGA-Chemie, Steinheim, Germany. Results In Fig.
la the typical
reaction
of the w i l d
0721-7714/82/0001/0285/$
01.00
286 -I type after addition of O.1 mmol-1 trichlorophenol to the b a t h i n g m e d i u m is seen. The w i l d strain shows an i m m e d i a t e l y starting d e p o l a r i zation of about 93 mV (Table I). Fig.2 gives evidence that the effect depends on the applied c o n c e n t r a t i o n of trichlorophenol. A f t e r r e m o v i n g the inhibitor, a total recovery of the p o t e n t i a l is achieved. The m u t a n t b a n d w h i c h differs from the w i l d type by its c i r c a d i a n conidiation, contrasts to this reaction pattern by a lag phase of about 8 min b e t w e e n the arrival of the uncoupler in the cuvette and the start of a visible reaction (Fig. Ib and Table I). The reaction itself is very fast, in most cases it lasts less than I min. The final level differs by 172 mJ] (mean) from the original resting potential. Since the residual value results from a mere d i f f u s i o n potential, this reaction leads to a total b r e a k - d o w n of the m e m b r a n e potential.
PD/ mV -200 -150
~ - 1 0 8 m V
- 50
'
I
I
5
10
15
PD/mV
-200
I
210
t/min
25
, TCP
-150 -100 -
50
---11mY 0
Fig.
0
{
1.0
I%
20
2'5
t/min
I: R e s t i n g p o t e n t i a l d i f f e r e n c e (PD) and the typical reaction after application of O.1 m m o l . 1 - 1 T C P in the w i l d s t r ~ n STA4 (a) and in the m u t a n t b a n d (b) of N e u r o s p o r a crassa. The arrow marks t~e arrival of the T C P - s o l u t i o n in the chamber used for the measurements.
Table
/{f J
100
50
' 0,025
0
Fig.
PD
' 0,05
To test w h e t h e r the d e s c r i b e d a l t e r a t i o n s of the t r i c h l o r o p h e n o l effect are typical for the m u t a n t band (and thus for its c i r c a d i a n c o n i d i a t i o n pattern), another rhythmic mutant, the strain clock was included. It shows a p e r i o d i c a l hyphal b r a n c h i n g (zonating) w h i c h is neither c i r c a d i a n nor s y n c h r o n i z e d to e n v i r o n m e n t a l cycles. T~e PD a l t e r a t i o n after a d d i t i o n of O.1 m m o l . l t r i c h l o r o p h e n o l is quite similar to that o b s e r v e d in the w i l d type, the d i f f e r e n c e is a longer decline (36 min instead of 24 min) and a r e s u l t i n g larger d e p o l a r i z a t i o n (144 mV as c o m p a r e d to 93 mV in the w i l d type; see Table I). After r e p l a c i n g the t r i c h l o r o p h e n o l solution by the original b a t h i n g medium, a total recovery is observed. Thus, the overall pattern of clock resembles that of the w i l d type.
of O.1 mmol'l -I TCP
in hyphae
Depolarization in TCP (mV)
Start of r e a c t i o n after TCP addition (min)
Time b e t w e e n start and maximal reaction (min)
Recovery after TCP t r e a t m e n t
93+ 9
0.4+0.2
24.0+4.0
total
-213+10
144+13
0.8+0.3
36.0+7.0
band
-186+
8
172+ 8
7.7+1.4
0.6+0.2
-211+10
134+25
1.7+O.5
18.2+2.8
6
T C P / m m o [ . I-1
M a r k e d d i f f e r e n c e s b e t w e e n the w i l d strain and band are also found in the r e c o v e r y after r e m o v i n g the t r i c h l o r o p h e n o l solution. No recovery was found after the typical "fast reaction" w i t h its total breakdown. After the " l o w - s p e e d reaction" a p a r t i a l but not complete r e c o v e r y was o b s e r v e d in but two cases.
STA4 (wild type) clock
-198+
' 0,10
2: C o r r e l a t i o n b e t w e e n applied concentrations of TCP and d e p o l a r i z a t i o n in the w i l d type STA4 of N e u r o s p o r a crassa.
I: Mean resting p o t e n t i a l s and reactions after a p p l i c a t i o n of the listed rhythmic strains of N e u r o s p o r a crassa. Resting (mV)
/
In some cases a slower r e a c t i o n is found, w h i c h also results in a very low residual potential, but not in a total b r e a k d o w n (Table I). The d i f f e r e n c e s b e t w e e n these two reaction patterns of b a n d are not yet comp l e t e l y understood, p r e s u h a b l y they are due to d i f f e r e n t c i r c a d i a n phases.
a
-I00
~U/mV
total no partial
287 Discussion The results d e m o n s t r a t e that the m u t a n t band contrasts to the w i l d type and to a n o t h e r rhythmic strain by its p e c u l i a r pattern of d e p o l a r i z a t i o n after a p p l i c a t i o n of TCP. In the w i l d strain STA4 the u n c o u p l e r affects the p o t e n t i a l d i f f e r e n c e as expected. At the hyphal p l a s m a l e m m a it is m a i n t a i n e d by an A T P - d e p e n d e n t "pump" w h i c h extrudes protons (and Na + ions) and a c c u m u l a t e s K + ions (Slayman and Slayman 1968; Galpin et al. 1978). The u n c o u p l i n g by t r i c h l o r o p h e n o l reduces the ATP c o n t e n t of the h y p h a e and thus reduces the active t r a n s p o r t across the p l a s m a l e m m a . The A T P a s e s can no longer keep up w i t h the p a s s i v e fluxes of the ions, e s p e c i a l l y w i t h the r a p i d l y m o v i n g protons and the hyphal p o t e n t i a l declines until it reaches a new e q u i l i b r i u m level. The removal of the inhibitor c o n s e q u e n t l y restores the original steady state. This r e a c t i o n p a t t e r n m i g h t be r e g a r d e d as "normal" since it is also found in clock, the only d i f f e r e n c e b e i n g the larger d e p o l a r i z a tion. This, however, is in a c c o r d a n c e with the c o n c l u s i o n of Lysek and Jennings (unpubl.) that the clock c h a r a c t e r in P o d o s p o r a anserina is due to the h y p h a l r e a c t i o n against an i n c r e a s e d p r o t o n influx into the c y t o p l a s m (Gradmann and Slayman 1975). This r e a c t i o n consumes part of the m e t a b o l i c e n e r g y and this enhances the s e n s i t i v i t y of the p o t e n t i a l d i f f e r e n c e a g a i n s t the uncoupler. A c c o r d i n g l y , the e q u i l i b r i u m level r e a c h e d after application of t r i c h l o r o p h e n o l is more d i f f e r e n t from the o r i g i n a l value than in the w i l d type. The o b s e r v e d r e c o v e r y is in a c c o r d a n c e with this view. The i n c r e a s e d s e n s i t i v i t y of b a n d may be due to a similar stress of the p r o t o n e x t r u s i o n system. For the delay of the r e a c t i o n and the f o l l o w i n g very fast breakdown, no explanation can be given actually. The lack of r e c o v e r y after w a s h i n g out the TCP, however, points to a p e r m a n e n t lesion in b a n d w h i c h may be due to the a l t e r a t i o n s in the m i t o c h o n drial A T P a s e of this strain found by D i e c k m a n and Brody (1980).
The c o r r e l a t i o n of these a l t e r a t i o n s in band with its c i r c a d i a n c o n i d i a t i o n is a s c e r t a i n e d by the occuzmence of the second r e a c t i o n pattern, w h i c h is more similar to that of the other two strains. The c h a n g i n g sensitivity indicates a linkage to the c i r c a d i a n phase. W o o d w a r d and Sargent (]973) found an a l t e r n a t i n g r e s p i r a t o r y a c t i v i t y in band. Their view of a "circadian u n c o u p l i n g " agrees with the findings r e p o r t e d here. Attempts are now made to test the r e a c t i o n p a t t e r n of this strain over the entire c i r c a d i a n period. This proves d i f f i c u l t since the light n e c e s s a r y for h a n d l i n g the samples and for m i c r o s c o p i c o b s e r v a t i o n is a c t i n g as "Zeitgeber". The authors thank Mrs. W. S c h w e r d t n e r for t e c h n i c a l a s s i s t a n c e and the D e u t s c h e Fors c h u n g s g e m e i n s c h a f t , B o n n - B a d Godesberg, for financial support.
References B O n n i n g , E (1973) The p h y s i o l o g i c a l clock; c i r c a d i a n rhythms and b i o l o g i c a l chronometry. 3rd revised ed. H e i d e l b e r g Sci. Libr., vol I; The E n g l i s h Univ. Press Ltd., London, and Springer-Verlag, New York - H e i d e l b e r g Berlin. Dieckman, 896-898.
C
, Brody, S
Gall, A, Lysek, G 13
(1981)
(1980)
Science
Neurosp
Newsl
Galpin, M F J, Jennings, D H, Oates, Hobot, A (1978) Exp Mycol 2: 258-269 Gradmann, D, Slayman, Biol 23: 181-212
C L
Lysek, G, Schr~fer, Ges 94: 105-112
(1981)
Slayman, Physiol
K
C L, Slayman, 52: 424-443
C W
(1975)
207: 28: K,
J Xembrane
Ber Dtsch (1968)
Bot
J Gen
Woodward, D O, Sargent, M L (1973) In: A P~rez-Miravete (ed) "Behaviour of m i c r o - o r g a nisms", P r o c . 1 0 th Intern. Congress for Microbiol., M e x i c o City. pp.282-296
