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Effect of the natural algicide, cyanobacterin, on a herbicide-resistant mutant of anacystis nidulans R2
Plant Science, 46 (1986) 5--10
5
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF THE NATURAL ALGICIDE, CYANOBACTERIN, ON A HERBICIDERESISTANT MUTANT OF ANACYS'TIS NIDULANS R2
FLORENCE
K. G L E A S O N ,
DEBORAH
E. CASE, K E V I N
D. S I P P R E L L and T I M O T H Y
S. M A G N U S O N
Gray Freshwater Biological Institute, University of Minnesota, P.O. Box 100, Navarre, M N 55392 (U.S.A.) (Received March 19th, 1986) (Revision received M a y 5th, 1986) (Accepted M a y 12th, 1986) Cyanobacterin, a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni, inhibits the growth of algae and plants. This compound is a potent inhibitor of photosynthetic electron transport and acts at a site in photosystem II (PS II). To further define the site of action of cyanobacterin, the effects of this natural product were investigated in a herbicide-resistant mutant of the cyanobacterium, Anacystis nidulans R2D2-X1. A. nidulans R 2 D 2 - X 1 was reported to grow and maintain photosynthetic electron transport in the presence of 20 u M 3-(3,4-dichlorophenyl)-l,l-dimethylurea ( D C M U ) and 6.0 ~ M atrazine. Resistance was attributed to an altered 32 kDa (quinone-binding, QB) protein [6]. In the presence of Hill electron acceptors, K3Fe(CN)6 and dichlorophenol-indophenol (DCPIP), spheroplasts of A. nidulans R2D2-X1 were inhibited by cyanobacterin at the same concentration as wild type spheroblasts. Under these same conditions, spheroplasts of the mutant maintained their resistance to D C M U . Similar results were obtained with isolated thylakoid m e m branes. In contrast, silicomolybdate reduction, which is resistant to D C M U inhibition, was very sensitive to cyanobacterin. W e conclude that cyanobacterin inhibits electron transport in PS II at a unique site which is different from that of D C M U .
Key words: photosynthetic electron transport; cyanobacteria; 3-(3,4-dichlorophenyl)-l,l-dimethylurea; photo-
system II
Introduction
The
cyanobacterium
(blue-green
alga),
S. hofmanni (University of Texas Culture Collection, Austin, TX (UTEX) 2 3 4 9 ) w a s found to inhibit the growth of other algae in two species cultures. Inhibition was due to the production and excretion of a secondary metabolite by S. hofrnanni [1]. The active metabolite has been isolated and chemically characterized. This metabolite, called cyanobacterin, is a diaryl-substituted Abbreviations: ATCC, American Type Culture Collection, Rockville, MD; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; atrazine, 2~chloro-4~thylamino-6-isopropyl-S-triazine; DCPIP, dichlorophenolindophenol; HQNO, 2-n-heptyl-4-hydroxyquinolineN-oxide; PS II, photosystem II; QB, quinone-binding protein; UTEX, University of Texas Culture Collection, Austin, TX.
gamma lactone with a chlorine substituent on one of the aromatic rings [2]. Investigation of the mechanism of action of cyano~acterin showed that the compound inhibited photosynthetic electron transport in the unicellular cyanobacterium, Synechococcus sp. (American Type Culture Collection, Rockville, MD (ATCC) 27146). In photosynthetically active spheroplasts of Synechococcus, cyanobacterin inhibited electron transfer to the Hill acceptors, DCPIP and K3Fe(CN)6, indicating that the probable site of action is in PS II [3]. Cyanobacterin was also shown to inhibit the Hill reaction in pea chloroplasts thus confirming its general herbicidal activity [4]. The concentrations of cyanobacterin needed to inhibit photosynthetic electron transport in both the cyanobacterial and pea chloroplast systems were comparable to that of the known
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
PS II inhibitor, DCMU. DCMU and chemically related herbicides, such as atrazine, have been reported to bind to a 32 kDa or QB protein on the reducing side of PS II and prevent electron transfer from a primary quinone acceptor [ 5]. Herbicide-resistant mutants of both plants and algae often have an altered QB protein. A DCMU-resistant m u t a n t of the cyanobacterium A. nidulans was reported to have such an altered herbicide-binding protein [6]. Cross-resistance of this m u t a n t to cyanobacterin would indicate that the natural product acts at the same site as these classical PS II inhibitors. We report here the effects of cyanobacterin on photosynthetic electron transport in a DCMU-resistant m u t a n t of the cyanobacterium, A. nidulans.
Materials and methods
Growth o f the cyanobacteria A. nidulans, strain R2 and a m u t a n t strain derived from it, R2D2-X1, were obtained from L.A. Sherman (University of Missouri, Columbia, MO). Both strains were maintained in liquid Bg 11 medium [ 7 ], at room temperature under constant illumination. Experimental cultures were grown in the same medium at 30°C and 66 pE m -2 s -1 photosynthetically active radiation. Cell numbers were determined by direct counting in a Petroff-Hausser counting chamber.
Preparation o f spheroblasts and thylakoids Spheroblasts were prepared as previously described for Synechococcus sp. ATCC 27146 except that incubation with lysozyme was done at 30°C for 4 h [3]. Spheroblasts were suspended in a buffer containing 330 mM sorbitol; 2 mM EDTA; 1 mM MgC12; 0.5 mM K2HPO4; 5 mM Na4P20~ and 50 mM HEPES--NaOH buffer (pH 7.6). Spheroplasts were used immediately after preparation. Thylakoid membranes were prepared from wild type and m u t a n t cultures of A. nidulans. Cells were harvested in mid,exponential growth phase by centrifugation at 2000 × g
for 10 min. The cells were resuspended in 4 vols. (w/v) of buffer containing: 50 mM HEPES (pH 7.6); 330 mM sorbitol; 25 mM MgC12; 10 mM CaCl2 and 0.5 mM K2HPO 4. The cells were disrupted in a French Pressure cell at 18--20 000 psi. Unbroken cells and debris were removed by centrifugation at 2000 × g for 5 min. The pellets were discarded and the supernatant fraction was centrifuged at 20 000 × g for 20 min. The thylakoid-containing pellets were resuspended in the above buffer plus Ficoll (7.5 mg/ml). The membrane suspensions were used immediately or stored at --30°C. Stored thylakoid preparations retained activity for approx. I month.
Hill reaction Rates of oxygen evolution were measured with a Clark-type oxygen electrode set into a 1.5-ml reaction chamber. The chamber contained approx. 1 ml of the above buffers and spheroplast or thylakoid preparations were added to produce measurable rates of oxygen evolution. The temperature w,o maintained at 30°C. Full scale deflection of the recorder was set by bubbling air through the suspe~asion medium for 20 min. The concentration of oxygen in the chamber was estimated to be 237/~M at 30°C [8]. The zero point was set by adding sodium dithionite to the chamber and depleting oxygen below detectable levels. Spheroplast and thylakoid preparations were kept in the dark under nitrogen for at least 20 min prior to experimentation. Illumination of the reaction chamber was from a projector lamp set to deliver 150 to 1000 /~E m -2 s -1. Oxygen evolution from spheroplast~ was measured in the presence of 2.5 mM K3Fe(CN)~. Reduction of DCPIP by spheroplasts was monitored spectrophotometrically at 600 nm in a Hewlett-Packard 8450A instrument. Spheroplasts were illuminated by a mirrored projection lamp set to deliver 430 /JE m -2 s -1. The concentration of DCPIP in the spectrophotometric assay was 50 uM. The Hill reaction using thylakoi.ts
was monitored polarographically in the presence of 0.25 mM DCPIP or 0.2 mM silicomolybdic acid. The chlorophyll concentrations were estimated from the absorbance at 664 nm following extraction with 90% (v/v) acetone [9]. DCMU (Sigma Chemical Co., St. Louis, MO) was dissolved in anhydrous diethyl ether. Cyanobacterin was purified as previously described [2] and dissolved in anhydrous diethyl ether. Silicomolybdic acid (Pfaltz and Bauer, Inc., Stanford, CT) was dissolved in dimethyl sulfoxide: water, 1 : 1.
Table I. Effect of DCMU and cyanobacterin on oxygen evolution of spheroplasts of A. nidulans in the presence of 2.5 mM K3Fe(CN)~. Spheroplast samples were adjusted to 20--25 ug of chlorophyll a per reaction in a total volume of 1.5 ml. Samples were suspended in a buffer containing: 50 mM HEPES (pH 7.6); 330 mM sorbitol; 1 mM MgCl2; 2 mM EDTA; 0.5 mM K~HPO, and 5 mM Na,P~O~. Illumination was at 150 # E m -2 s-'. Data for A. nidulans R2 are from 2 experiments; and those for R2D2-X1 are from 3 separate trials. Rates of oxygen evolution were those established in the first 5 min following additions. Zero rates were sustained for a minimum of 20 rain. Strain
Addition
moles 02 evolved h- 1 (rag chl a )-'
Wild type
None a DCMU (40 nM) DCMU (85 nM} Cyanobacterin (3 nM) Cyanobacterin (15.5 nM) None a DCMU (8.5 × 103 nM) DCMU (25.5 × 103 nM) Cyanobaeterin (3nM) Cyanobacterin (15.5 aM)
20.4 12 0 9.2
DCMU-resistant mutant
aControl experiments received equivalent to the volume used (5--20 ul/reaction).
0 25.2 9.6 0 14.4 0
Table II. Effect of DCMU and cyanobacterin on the Hill reaction of spheroplasts of A. nidulans in the presence of 50 #M DCPIP. Spheroplast samples were suspended in the buffer described in Table I and the concentration adjusted to 1.6--2.0 ~g chlorophyll a per reaction in a total volume of 1.0 ml. Light intensity was 430 uE m -2 s -1. Rates were monitored for 5 rain at 600 nM and an additional 5 rain following additions. Each value is an average of 3 separate trials. Strain
Addition
umoles DCPIP reduced h -I (mg chl a) - '
Wild type
None a DCMU (10 nM) DCMU (20 nM) Cyanobacterin (0.23 nM) Cyanobacterin (2.3 nM) None a DCMU (100 uM) DCMU (250 uM) Cyanobaeterin (0.23 nM) Cyanobacterin (2.3 nM)
60 18 0 9
DCMU-resistant mutant
0 84 60 0 16 0
aControl experiments received solvent equivalent to the volume used with inhibitors.
Results Both 1 # M D C M U [6] and 2.3 # M cyanobacterin [3] are potent inhibitors of the growth of A. nidulans R2. The mutant strain of A. nidulans, R2D2, was selected for its ability to grow in the presence of 10 # M D C M U . D N A was isolated from this m u t a n t and used to transform A. nidulans R2 to the resistant strain designated R2D2X1. DCMU (10 ~M) has no effect on the growth of the transformed cyanobacterium
[6]. diethyl ether with inhibitors
As in our previous report on Synechoccus sp. ATCC 27146 [3], we found that intact cells of A. nidulans R2 or R2D2-X1 failed
to reduce added Hill electron acceptors. Presumably the cell wall prevents the efficient diffusion of most Hill acceptors to the thylakoid membranes. However, after removal of the cell walls with lysozyme, both A. nidulans strains would effectively reduce both K3Fe(CN)6 and DCPIP in the light. The reduction of ferricyanide was followed polarographically. The results are shown in Table I. Oxygen evolution in the presence of ferricyanide was inhibited completely by 85 nM DCMU and 15.5 nM cyanobacterin in A. nidulans, strain R2. In contrast, spheroplasts of the resistant mutant, R2D2-X1, were unaffected by nanomolar concentrations of DCMU and required the addition of 8.5 pM DCMU to observe the Is0 (concentration at which oxygen evolution was inhibited by 50%). Complete inhibition was observed only at 25.5 pM DCMU. In this system, spheroplasts of R2D2-X1 were as sensitive to cyanobacterin as wild t y p e cells. The Is0 for both preparations was 3.0 nM cyanobacterin.
Similar results were obtained when DCPIP was used as an electron acceptor for photosynthetic electron transport. As shown in Table II, the mutant strain R2D2-X1 was remarkably resistant to DCMU under these assay conditions. A concentration of 250 ~M was needed to observe complete inhibition of dye reduction. However, under the same conditions, spheroplasts of this organism were no more resistant to cyanobacterin than those prepared from the parent strain. Reduction of DCPIP was totally inhibited by a 2.3 nM concentration of the natural product in both R2 and R2D2-X1 spheroplasts. Further confirmation of these results were obtained with thylakoid preparations from wild t y p e and mutant strains. Rates of oxygen evolution were monitored in the presence of DCPIP and silicomolybdic acid as electron acceptors. Data are shown in Table III. The DCMU-resistant mutant showed no altered sensitivity to cyanobacterin in the presence of either acceptor.
Table III. E f f e c t o f c y a n o b a c t e r i n a n d D C M U o n p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t in A. nidulans t h y l a k o i d m e m b r a n e p r e p a r a t i o n s . T h y l a k o i d p r e p a r a t i o n s were s u s p e n d e d in b u f f e r c o n t a i n i n g : 50 m M H E P E S (pH 7 . 6 ) ; 330 mM s o r b i t o l ; 25 m M MgCI2; 10 m M CaCI2 a n d 0.5 m M K2HPO 4. F o r t h e s i l i c o m o l y b d a t e r e d u c t i o n , 50 m g of b o v i n e s e r u m a l b u m i n was a d d e d t o t h e r e a c t i o n c h a m b e r . C o n c e n t r a t i o n s were a d j u s t e d t o 1 4 0 - - 1 8 0 ug chl a per r e a c t i o n in a t o t a l v o l u m e o f 1.5 ml. Light i n t e n s i t y was 1 0 0 0 ~ E m -2 s-1 . Strain
Wild t y p e
DCMU-resistant mutant
Addition
#moles 0 2 evolved h -~ ( m g chl a) -~
DCPIP (0.25 m M )
Silicomolybdate (0.20 m M )
None DCMU ( 1 5 0 nM) DCMU ( 2 8 0 nM) DCMU (420 nM) C y a n o b a c t e r i n (30 n M ) C y a n o b a c t e r i n (61 nM) C y a n o b a c t e r i n ( 1 0 7 nM)
4.6 1.5 0 -2.2 0 --
7.2 -3.3 0 -3.8 0
None DCMU ( 2 8 0 nM) DCMU (5.7 × 103 nM) DCMU (28.6 x 103 nM) C y a n o b a c t e r i n (30 n M ) C y a n o b a c t e r i n (78 n M )
7.5 -4.0 1.6 2.7 0
9.2 3.7 --3.7 --
Discussion In a previous report, we demonstrated that cyanobacterin inhibits photosynthetic electron transport in spheroplasts of t~i~e unicellular cyanobacterium, Synechococcus sp. at approx. 25 nM [3]. The algicide was subsequently found to inhibit the Hill reaction in pea chloroplasts when ferricyanide, DCPIP, p-benzoquinone and silicomolybdate were used as electron acceptors. Given the number of herbicides which act on the quinone-binding protein in PS II [10], it seems reasonable to propose and test the hypothesis that cyanobacterin also acts at this same site. The A. nidulans strain R2D2, was selected after mutagenesis for its ability to grow in the presence of 10 pM DCMU. The transformant, R 2 D 2 - X l , was produced from this strain. This procedure reduces the possibility of the transformant having multiple genetic lesions. Golden and Sherman demonstrated that the transformant was as resistant to DCMU as the original mutant. Photosynthetic oxygen evolution in membrane preparations of the transformant was also resistant to DCMU. In addition, the cells exhibited cross-resistance to other PS II inhibitors such as atrazine and 2-n-heptyl-4hydroxyquinoline-N-oxide. The mutant was shown to have an altered 32 kDa (QB) protein [6]. However, both spheroplasts and thylakoid membrane preparations of R2D2X l show no resistance to cyanobacterin although they maintain a high resistance to DCMU under our assay conditions. From these data, we conclude that the natural algicide, cyanobacterin, does not act at the same site as DCMU. Herbicide-resistant mutants of other photosynthetic organisms often are found to have some resistance to additional herbicides which are believed to bind to the same QB protein. For example, DCMU-resistant mutants of both the cyanobacterium, Aphanocapsa 6714 and the eukaryotic alga, Chlamydornonas reinhardii, were reported to
grow in the presence of atrazine. However, in Aphanocapsa, cross-resistance was not demonstrated in spheroplasts or thylakoid membrane preparations [ 11]. In the Chlamydomonas mutant, a low b u t definite resistance to atrazine was found in the Hill reaction with thylakoid membrane preparations [12]. These data indicate that the two herbicides share a similar b u t not identical binding site. Similar results have been reported for a variety of higher plant systems (see for example, Ref. 13). The fact that the DCMU-resistant mutant of A. nidulans is not resistant to cyanobacterin at the membrane level indicates that the natural product does not act on the QB protein. It might still be argued that cyanobacterin does interact with the QB protein b u t at a site which is not affected by the mutation. However, our data on inhibition of silicomolybdate reduction support our conclusion that this natural product acts at a completely different site in PS II. Silicomolybdic acid can accept electrons on the oxidizing side of the primary quinone receptor in PS II. This reaction is relatively insensitive to DCMU which interrupts electron flow on the reducing side of the quinone [4]. Our data (Table III) are in agreement. The reduction of silicomolybdate is quite sensitive to cyanobacterin in both cyanobacterial strains and in pea chloroplasts [4]. We conclude that the natural product does not bind to the same site as conventional herbicides but has a unique mode of action. Experiments are in progress to further characterize this site. Acknowledgments Research Contribution No. 192. This work is the result of research sponsored by the Minnesota Sea Grant Program, supported by the NOAA Office of Sea Grant, Department of Commerce, under Grant No. DOC/ NA83AA-D-00056 R/NP-1 to F.K.G. The U.S. Government is authorized to reproduce and distribute reprints for government pur-
10
poses, not withstanding any notation that may appear hereon.
copyright
References 1 C.P. Mason, K.R. Edwards, R.E. Carlson, J. PignateIIo, F.K. Gleason and J.M. Wood, Science, 215 (1982) 400. 2 J.J. Pignatello, J. Porwoll, R.E. Carlson, A. Xavier, F.K. Gleason and J.M. Wood, J. Org. Chem., 48 (1983) 4035. 3 F.K. Gleason and J,L. Paulson, Arch. Microbiol., 138 (1984) 273. 4 F.K. Gleason and D.E. Case, Plant Physiol., 80 (1986) in press. 5 W.F.J. Vermass, G. Renger and C.J. Arntzen, Z. Naturforsch., 39c (1984) 368. 6 S.A. Golden and L.A. Sherman, Biochim. Biophys. Acta, 764 (1984) 239.
7 R.Y. Stanier, R. Kunisawa, M. Mandel and G. Cohen-Bazire, Bacteriol. Rev., 35 (1971) 171. 8 T.G. Cooper, The Tools of Biochemistry, John Wiley and Sons, N.Y., 1977, p. 32. 9 D.I. Arnon, B.D. McSwain, H.Y. Tsujimoto and K. Wada, Biochim. Biophys. Acta, 357 (1974) 321. 10 D.J. Kyle, Photochem. Photobiol., 41 (1985) 107. 11 C. Astier, C. Vernotte, J. Der-Vartanian and F. Joset-Espardellier, Plant Cell Physiol., 70 (1979) 1501. 12 R.E. Galloway and L. Mets, Plant Physiol., 70 (1982) 1673. 13 J. Hirschberg, A. Bleecker, D.J. Kyle, L. McIntosh and C.J. Arntzen, Z. Naturforsch., 39c (1984) 412. 14 R. Barr, F.L. Crane and R.T. Giaquinta, Plant Physiol., 55 (1975) 460.
5
Elsevier Scientific Publishers Ireland Ltd.
EFFECT OF THE NATURAL ALGICIDE, CYANOBACTERIN, ON A HERBICIDERESISTANT MUTANT OF ANACYS'TIS NIDULANS R2
FLORENCE
K. G L E A S O N ,
DEBORAH
E. CASE, K E V I N
D. S I P P R E L L and T I M O T H Y
S. M A G N U S O N
Gray Freshwater Biological Institute, University of Minnesota, P.O. Box 100, Navarre, M N 55392 (U.S.A.) (Received March 19th, 1986) (Revision received M a y 5th, 1986) (Accepted M a y 12th, 1986) Cyanobacterin, a secondary metabolite produced by the cyanobacterium, Scytonema hofmanni, inhibits the growth of algae and plants. This compound is a potent inhibitor of photosynthetic electron transport and acts at a site in photosystem II (PS II). To further define the site of action of cyanobacterin, the effects of this natural product were investigated in a herbicide-resistant mutant of the cyanobacterium, Anacystis nidulans R2D2-X1. A. nidulans R 2 D 2 - X 1 was reported to grow and maintain photosynthetic electron transport in the presence of 20 u M 3-(3,4-dichlorophenyl)-l,l-dimethylurea ( D C M U ) and 6.0 ~ M atrazine. Resistance was attributed to an altered 32 kDa (quinone-binding, QB) protein [6]. In the presence of Hill electron acceptors, K3Fe(CN)6 and dichlorophenol-indophenol (DCPIP), spheroplasts of A. nidulans R2D2-X1 were inhibited by cyanobacterin at the same concentration as wild type spheroblasts. Under these same conditions, spheroplasts of the mutant maintained their resistance to D C M U . Similar results were obtained with isolated thylakoid m e m branes. In contrast, silicomolybdate reduction, which is resistant to D C M U inhibition, was very sensitive to cyanobacterin. W e conclude that cyanobacterin inhibits electron transport in PS II at a unique site which is different from that of D C M U .
Key words: photosynthetic electron transport; cyanobacteria; 3-(3,4-dichlorophenyl)-l,l-dimethylurea; photo-
system II
Introduction
The
cyanobacterium
(blue-green
alga),
S. hofmanni (University of Texas Culture Collection, Austin, TX (UTEX) 2 3 4 9 ) w a s found to inhibit the growth of other algae in two species cultures. Inhibition was due to the production and excretion of a secondary metabolite by S. hofrnanni [1]. The active metabolite has been isolated and chemically characterized. This metabolite, called cyanobacterin, is a diaryl-substituted Abbreviations: ATCC, American Type Culture Collection, Rockville, MD; DCMU, 3-(3,4-dichlorophenyl)-l,l-dimethylurea; atrazine, 2~chloro-4~thylamino-6-isopropyl-S-triazine; DCPIP, dichlorophenolindophenol; HQNO, 2-n-heptyl-4-hydroxyquinolineN-oxide; PS II, photosystem II; QB, quinone-binding protein; UTEX, University of Texas Culture Collection, Austin, TX.
gamma lactone with a chlorine substituent on one of the aromatic rings [2]. Investigation of the mechanism of action of cyano~acterin showed that the compound inhibited photosynthetic electron transport in the unicellular cyanobacterium, Synechococcus sp. (American Type Culture Collection, Rockville, MD (ATCC) 27146). In photosynthetically active spheroplasts of Synechococcus, cyanobacterin inhibited electron transfer to the Hill acceptors, DCPIP and K3Fe(CN)6, indicating that the probable site of action is in PS II [3]. Cyanobacterin was also shown to inhibit the Hill reaction in pea chloroplasts thus confirming its general herbicidal activity [4]. The concentrations of cyanobacterin needed to inhibit photosynthetic electron transport in both the cyanobacterial and pea chloroplast systems were comparable to that of the known
0168-9452/86/$03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
PS II inhibitor, DCMU. DCMU and chemically related herbicides, such as atrazine, have been reported to bind to a 32 kDa or QB protein on the reducing side of PS II and prevent electron transfer from a primary quinone acceptor [ 5]. Herbicide-resistant mutants of both plants and algae often have an altered QB protein. A DCMU-resistant m u t a n t of the cyanobacterium A. nidulans was reported to have such an altered herbicide-binding protein [6]. Cross-resistance of this m u t a n t to cyanobacterin would indicate that the natural product acts at the same site as these classical PS II inhibitors. We report here the effects of cyanobacterin on photosynthetic electron transport in a DCMU-resistant m u t a n t of the cyanobacterium, A. nidulans.
Materials and methods
Growth o f the cyanobacteria A. nidulans, strain R2 and a m u t a n t strain derived from it, R2D2-X1, were obtained from L.A. Sherman (University of Missouri, Columbia, MO). Both strains were maintained in liquid Bg 11 medium [ 7 ], at room temperature under constant illumination. Experimental cultures were grown in the same medium at 30°C and 66 pE m -2 s -1 photosynthetically active radiation. Cell numbers were determined by direct counting in a Petroff-Hausser counting chamber.
Preparation o f spheroblasts and thylakoids Spheroblasts were prepared as previously described for Synechococcus sp. ATCC 27146 except that incubation with lysozyme was done at 30°C for 4 h [3]. Spheroblasts were suspended in a buffer containing 330 mM sorbitol; 2 mM EDTA; 1 mM MgC12; 0.5 mM K2HPO4; 5 mM Na4P20~ and 50 mM HEPES--NaOH buffer (pH 7.6). Spheroplasts were used immediately after preparation. Thylakoid membranes were prepared from wild type and m u t a n t cultures of A. nidulans. Cells were harvested in mid,exponential growth phase by centrifugation at 2000 × g
for 10 min. The cells were resuspended in 4 vols. (w/v) of buffer containing: 50 mM HEPES (pH 7.6); 330 mM sorbitol; 25 mM MgC12; 10 mM CaCl2 and 0.5 mM K2HPO 4. The cells were disrupted in a French Pressure cell at 18--20 000 psi. Unbroken cells and debris were removed by centrifugation at 2000 × g for 5 min. The pellets were discarded and the supernatant fraction was centrifuged at 20 000 × g for 20 min. The thylakoid-containing pellets were resuspended in the above buffer plus Ficoll (7.5 mg/ml). The membrane suspensions were used immediately or stored at --30°C. Stored thylakoid preparations retained activity for approx. I month.
Hill reaction Rates of oxygen evolution were measured with a Clark-type oxygen electrode set into a 1.5-ml reaction chamber. The chamber contained approx. 1 ml of the above buffers and spheroplast or thylakoid preparations were added to produce measurable rates of oxygen evolution. The temperature w,o maintained at 30°C. Full scale deflection of the recorder was set by bubbling air through the suspe~asion medium for 20 min. The concentration of oxygen in the chamber was estimated to be 237/~M at 30°C [8]. The zero point was set by adding sodium dithionite to the chamber and depleting oxygen below detectable levels. Spheroplast and thylakoid preparations were kept in the dark under nitrogen for at least 20 min prior to experimentation. Illumination of the reaction chamber was from a projector lamp set to deliver 150 to 1000 /~E m -2 s -1. Oxygen evolution from spheroplast~ was measured in the presence of 2.5 mM K3Fe(CN)~. Reduction of DCPIP by spheroplasts was monitored spectrophotometrically at 600 nm in a Hewlett-Packard 8450A instrument. Spheroplasts were illuminated by a mirrored projection lamp set to deliver 430 /JE m -2 s -1. The concentration of DCPIP in the spectrophotometric assay was 50 uM. The Hill reaction using thylakoi.ts
was monitored polarographically in the presence of 0.25 mM DCPIP or 0.2 mM silicomolybdic acid. The chlorophyll concentrations were estimated from the absorbance at 664 nm following extraction with 90% (v/v) acetone [9]. DCMU (Sigma Chemical Co., St. Louis, MO) was dissolved in anhydrous diethyl ether. Cyanobacterin was purified as previously described [2] and dissolved in anhydrous diethyl ether. Silicomolybdic acid (Pfaltz and Bauer, Inc., Stanford, CT) was dissolved in dimethyl sulfoxide: water, 1 : 1.
Table I. Effect of DCMU and cyanobacterin on oxygen evolution of spheroplasts of A. nidulans in the presence of 2.5 mM K3Fe(CN)~. Spheroplast samples were adjusted to 20--25 ug of chlorophyll a per reaction in a total volume of 1.5 ml. Samples were suspended in a buffer containing: 50 mM HEPES (pH 7.6); 330 mM sorbitol; 1 mM MgCl2; 2 mM EDTA; 0.5 mM K~HPO, and 5 mM Na,P~O~. Illumination was at 150 # E m -2 s-'. Data for A. nidulans R2 are from 2 experiments; and those for R2D2-X1 are from 3 separate trials. Rates of oxygen evolution were those established in the first 5 min following additions. Zero rates were sustained for a minimum of 20 rain. Strain
Addition
moles 02 evolved h- 1 (rag chl a )-'
Wild type
None a DCMU (40 nM) DCMU (85 nM} Cyanobacterin (3 nM) Cyanobacterin (15.5 nM) None a DCMU (8.5 × 103 nM) DCMU (25.5 × 103 nM) Cyanobaeterin (3nM) Cyanobacterin (15.5 aM)
20.4 12 0 9.2
DCMU-resistant mutant
aControl experiments received equivalent to the volume used (5--20 ul/reaction).
0 25.2 9.6 0 14.4 0
Table II. Effect of DCMU and cyanobacterin on the Hill reaction of spheroplasts of A. nidulans in the presence of 50 #M DCPIP. Spheroplast samples were suspended in the buffer described in Table I and the concentration adjusted to 1.6--2.0 ~g chlorophyll a per reaction in a total volume of 1.0 ml. Light intensity was 430 uE m -2 s -1. Rates were monitored for 5 rain at 600 nM and an additional 5 rain following additions. Each value is an average of 3 separate trials. Strain
Addition
umoles DCPIP reduced h -I (mg chl a) - '
Wild type
None a DCMU (10 nM) DCMU (20 nM) Cyanobacterin (0.23 nM) Cyanobacterin (2.3 nM) None a DCMU (100 uM) DCMU (250 uM) Cyanobaeterin (0.23 nM) Cyanobacterin (2.3 nM)
60 18 0 9
DCMU-resistant mutant
0 84 60 0 16 0
aControl experiments received solvent equivalent to the volume used with inhibitors.
Results Both 1 # M D C M U [6] and 2.3 # M cyanobacterin [3] are potent inhibitors of the growth of A. nidulans R2. The mutant strain of A. nidulans, R2D2, was selected for its ability to grow in the presence of 10 # M D C M U . D N A was isolated from this m u t a n t and used to transform A. nidulans R2 to the resistant strain designated R2D2X1. DCMU (10 ~M) has no effect on the growth of the transformed cyanobacterium
[6]. diethyl ether with inhibitors
As in our previous report on Synechoccus sp. ATCC 27146 [3], we found that intact cells of A. nidulans R2 or R2D2-X1 failed
to reduce added Hill electron acceptors. Presumably the cell wall prevents the efficient diffusion of most Hill acceptors to the thylakoid membranes. However, after removal of the cell walls with lysozyme, both A. nidulans strains would effectively reduce both K3Fe(CN)6 and DCPIP in the light. The reduction of ferricyanide was followed polarographically. The results are shown in Table I. Oxygen evolution in the presence of ferricyanide was inhibited completely by 85 nM DCMU and 15.5 nM cyanobacterin in A. nidulans, strain R2. In contrast, spheroplasts of the resistant mutant, R2D2-X1, were unaffected by nanomolar concentrations of DCMU and required the addition of 8.5 pM DCMU to observe the Is0 (concentration at which oxygen evolution was inhibited by 50%). Complete inhibition was observed only at 25.5 pM DCMU. In this system, spheroplasts of R2D2-X1 were as sensitive to cyanobacterin as wild t y p e cells. The Is0 for both preparations was 3.0 nM cyanobacterin.
Similar results were obtained when DCPIP was used as an electron acceptor for photosynthetic electron transport. As shown in Table II, the mutant strain R2D2-X1 was remarkably resistant to DCMU under these assay conditions. A concentration of 250 ~M was needed to observe complete inhibition of dye reduction. However, under the same conditions, spheroplasts of this organism were no more resistant to cyanobacterin than those prepared from the parent strain. Reduction of DCPIP was totally inhibited by a 2.3 nM concentration of the natural product in both R2 and R2D2-X1 spheroplasts. Further confirmation of these results were obtained with thylakoid preparations from wild t y p e and mutant strains. Rates of oxygen evolution were monitored in the presence of DCPIP and silicomolybdic acid as electron acceptors. Data are shown in Table III. The DCMU-resistant mutant showed no altered sensitivity to cyanobacterin in the presence of either acceptor.
Table III. E f f e c t o f c y a n o b a c t e r i n a n d D C M U o n p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t in A. nidulans t h y l a k o i d m e m b r a n e p r e p a r a t i o n s . T h y l a k o i d p r e p a r a t i o n s were s u s p e n d e d in b u f f e r c o n t a i n i n g : 50 m M H E P E S (pH 7 . 6 ) ; 330 mM s o r b i t o l ; 25 m M MgCI2; 10 m M CaCI2 a n d 0.5 m M K2HPO 4. F o r t h e s i l i c o m o l y b d a t e r e d u c t i o n , 50 m g of b o v i n e s e r u m a l b u m i n was a d d e d t o t h e r e a c t i o n c h a m b e r . C o n c e n t r a t i o n s were a d j u s t e d t o 1 4 0 - - 1 8 0 ug chl a per r e a c t i o n in a t o t a l v o l u m e o f 1.5 ml. Light i n t e n s i t y was 1 0 0 0 ~ E m -2 s-1 . Strain
Wild t y p e
DCMU-resistant mutant
Addition
#moles 0 2 evolved h -~ ( m g chl a) -~
DCPIP (0.25 m M )
Silicomolybdate (0.20 m M )
None DCMU ( 1 5 0 nM) DCMU ( 2 8 0 nM) DCMU (420 nM) C y a n o b a c t e r i n (30 n M ) C y a n o b a c t e r i n (61 nM) C y a n o b a c t e r i n ( 1 0 7 nM)
4.6 1.5 0 -2.2 0 --
7.2 -3.3 0 -3.8 0
None DCMU ( 2 8 0 nM) DCMU (5.7 × 103 nM) DCMU (28.6 x 103 nM) C y a n o b a c t e r i n (30 n M ) C y a n o b a c t e r i n (78 n M )
7.5 -4.0 1.6 2.7 0
9.2 3.7 --3.7 --
Discussion In a previous report, we demonstrated that cyanobacterin inhibits photosynthetic electron transport in spheroplasts of t~i~e unicellular cyanobacterium, Synechococcus sp. at approx. 25 nM [3]. The algicide was subsequently found to inhibit the Hill reaction in pea chloroplasts when ferricyanide, DCPIP, p-benzoquinone and silicomolybdate were used as electron acceptors. Given the number of herbicides which act on the quinone-binding protein in PS II [10], it seems reasonable to propose and test the hypothesis that cyanobacterin also acts at this same site. The A. nidulans strain R2D2, was selected after mutagenesis for its ability to grow in the presence of 10 pM DCMU. The transformant, R 2 D 2 - X l , was produced from this strain. This procedure reduces the possibility of the transformant having multiple genetic lesions. Golden and Sherman demonstrated that the transformant was as resistant to DCMU as the original mutant. Photosynthetic oxygen evolution in membrane preparations of the transformant was also resistant to DCMU. In addition, the cells exhibited cross-resistance to other PS II inhibitors such as atrazine and 2-n-heptyl-4hydroxyquinoline-N-oxide. The mutant was shown to have an altered 32 kDa (QB) protein [6]. However, both spheroplasts and thylakoid membrane preparations of R2D2X l show no resistance to cyanobacterin although they maintain a high resistance to DCMU under our assay conditions. From these data, we conclude that the natural algicide, cyanobacterin, does not act at the same site as DCMU. Herbicide-resistant mutants of other photosynthetic organisms often are found to have some resistance to additional herbicides which are believed to bind to the same QB protein. For example, DCMU-resistant mutants of both the cyanobacterium, Aphanocapsa 6714 and the eukaryotic alga, Chlamydornonas reinhardii, were reported to
grow in the presence of atrazine. However, in Aphanocapsa, cross-resistance was not demonstrated in spheroplasts or thylakoid membrane preparations [ 11]. In the Chlamydomonas mutant, a low b u t definite resistance to atrazine was found in the Hill reaction with thylakoid membrane preparations [12]. These data indicate that the two herbicides share a similar b u t not identical binding site. Similar results have been reported for a variety of higher plant systems (see for example, Ref. 13). The fact that the DCMU-resistant mutant of A. nidulans is not resistant to cyanobacterin at the membrane level indicates that the natural product does not act on the QB protein. It might still be argued that cyanobacterin does interact with the QB protein b u t at a site which is not affected by the mutation. However, our data on inhibition of silicomolybdate reduction support our conclusion that this natural product acts at a completely different site in PS II. Silicomolybdic acid can accept electrons on the oxidizing side of the primary quinone receptor in PS II. This reaction is relatively insensitive to DCMU which interrupts electron flow on the reducing side of the quinone [4]. Our data (Table III) are in agreement. The reduction of silicomolybdate is quite sensitive to cyanobacterin in both cyanobacterial strains and in pea chloroplasts [4]. We conclude that the natural product does not bind to the same site as conventional herbicides but has a unique mode of action. Experiments are in progress to further characterize this site. Acknowledgments Research Contribution No. 192. This work is the result of research sponsored by the Minnesota Sea Grant Program, supported by the NOAA Office of Sea Grant, Department of Commerce, under Grant No. DOC/ NA83AA-D-00056 R/NP-1 to F.K.G. The U.S. Government is authorized to reproduce and distribute reprints for government pur-
10
poses, not withstanding any notation that may appear hereon.
copyright
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