NOTES
419
The non-light-dependentreduction of 2,6-dichlorophenolindophenol by cells of the blue-green alga Anacystis nidulans Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
BAILEYW A R D Department of Biology, Unit,ersity of Mississippi, Universiry, Mississippi 38677 Accepted October 3 I , 1974
WARD,B. 1975. The non-light-dependent reduction of 2,6dichlorophenolindophenol by cells of the blue-green alga Atlacystis t1idr11ot1.s.Can. J . Microbiol. 21: 419-422. Whole cells of the blue-green alga Atfacystis t~idltlans reduced, in the dark, the oxidation-reduction dye, 2,6dichlorophenolindophenolat rates severalfold higher than those of the other algae tested. Under anaerobiosis, the endogenous reductant was depleted after up to 80 nmol of dye were reduced per microliter of cells. Cells held in darkness for several hours exhibited lowered dark reduction rates relative to cells held in light. Treatment with lysozyme and ethylenediaminetetraacetic acid yielded cells that would photoreduce the dye, whereas untreated cells would not. Comparisons of photoreduction and dark reduction revealed that the dark reduction proceeded independently of the photoreduction. It was concluded that the dark reduction represents a pool of endogenous reductant of sufficiently low oxidation-reduction potential to reduce completely 2,6dichlorophenolindophenol.Additionally, untreated cells were shown to be permeable to the dye although they did not photoreduce it; thus lysozyme/ ethylenediaminetetraacetic acid treatment was considered to make the oxidant accessible to the photosynthetic machinery. WARD,B. 1975. The non-light-dependent reduction of 2,6dichlorophenolindophenol by cells of Can. J. Microbiol. 21: 419-422. the blue-green alga At~crcystistiidrrltr~~.~. En ['absence de lumiere, des cellules entieres de Atlcicystis nidlrlatls, une algue bleu-vert, reduisent la teinture oxydante-reductrice 2,6dichlorophCnolindophCnoli d e s taux plusieurs fois plus Clevesque ceux obtenus d'autres algues mises il'essai. Sous des conditions anatrobiques, le reducteur endogene peut reduire jusqu'i 80 nmol de teinture par microlitre de cellules, apres quoi I'activitt devient nulle. Les cellules maintenues a I'obscurite durant plusieurs heures, comparativement a celles qui sont garddes en lumiere, ont des taux de reduction inferieurs. Le traitement par la lysozyme et par I'acide ethylenediaminetetraacetique rend les cellules capables de photoreduction de la teinture, tandis que les cellules non traitees en sont incapables. Les comparaisons entre la photoreduction et la reduction en obscuritk revelent que la reduction en obscurite se fait independamment de la photoreduction. I1 en est conclu que la reduction en obscurite constitue un pool de reducteur endogltne a potentiel oxydo-reducteur suffisamment faible pour riduire completement le 2,6-dichlorophCnolindophCnol.De plus, les cellules non traitkes s'averent permeables i la teinture bien qu'elles ne puissent la rtduire photosynthetiquement et, de ce fait, le traitement lysozyme/~thylenediaminetCtraacCtique est condidere cornme pouvant rendre I'oxydant accessible au mecanisme photosynthktique. [Traduit par le journal]
The blue-green alga Anacystis nidulans has been used extensively for photosynthetic measurements using the light-dependent reduction of various dyes, including the electropositive compound 2,6-dichlorophenolindopheno1 (DPIP) (E,' = 0.224 V). Although intact cells of algae do not generally photoreduce DPIP, membrane preparations (2, 4) and lysozyme-treated whole cells (9) have exhibited photoreductive capabilities. During earlier studies on photosynthetic electron transport in Anacystis (8, 9), a non-light-dependent reduction of DPIP was observed. Other workers 'Received July 19, 1974.
also have observed endogenous {dark) dye reduction in blue-green algae (3, 6), but the rate and extent of reduction of DPIP in these reports, as well as for other blue-greens tested in our laboratory are severalfold less than that exhibited by Anacystis. This communication compares characteristics of the endogenous dark reduction of DPIP by cells of Anacystis nidlrlans with that observed in other blue-green algae and describes several salient features of the Anacystis dark reduction phenomenon. The blue-green algae tested and the media in which they were grown were Anacystis nidulans, Kratz and Allen strain (modified
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
420
CAN. J. MICROBIlDL. VOL. 21, 1975
medium C (9)); Nostoc muscorwn G (medium B (5)); Anabaena cylindrica (modified medium C); Nostoc sp. (TX-50) (medium B); Phormidium luridium var. olivaceae (medium B); and Agmenellztm quadruplicatum (BG-1) (ASP-2 (7)). The first three cultures were obtained from the laboratory of Dr. Jack Myers, University of Texas, U.S.A. Culture histories of these algae have been reported (5). Agmenellum quadruplicatum was a gift of Dr. L. 0 . Ingram, University of Florida, U.S.A., and Phormidium luridium var. olivaceae was obtained from the Indiana University culture collection, U.S.A. Axenic cultures were maintained at about 160 ft-c of cool white fluorescent light in a water bath thermostated at 30°C. All cultures were aerated with sterile air delivered through glass tubes to about 150 ml of cell suspension in cotton-plugged 38 x 300 mm Pyrex test tubes. Aseptic technique was observed throughout and cultures were checked for contamination before assay to ensure that only blue-green algae were present. For most assays, cells were harvested by centrifugation from midlog phase of growth, washed once in 0.05 M NaH2P04/Na2HP04 buffer, pH 6.5, and resuspended in the same buffer. The reduction of DPIP was followed spectrophotometrically at 600 nm using a Bausch and Lomb Spectronic 505 dual beam recording spectrophotometer. A millimolar extinction coefficient of 18 (pH 6.5) (1) was used for all measurements except those made at different pH values, in which case the absorption at 522 nm (isosbestic point) was followed and a millimolar extinction coefficient of 8.6 was applied (1). "Zero" was set on the spectrophotometer using cells vs. cells after which about 25 nmol of DPIP was added to the measuring-beam cuvette which was quickly inverted to mix and immediately returned to the measuring beam. About 30 s was consumed in this operation. Permeaplasts of Anacystis nidulans were prepared according to the method of Ward and Myers (9). The use of TES (N-tris(hydroxymethy1)methyl-2-amino-ethane sulfonic acid) buffer instead of phosphate was necessary for successful treatment. Photoreduction of DPIP by these treated cells was measured by exposing the cell suspensions plus DPIP to rate-saturating incandescent light provided by a Kodak Carousel 850 projector focused through a 12-cm water
cell. A special cuvette holder mounted in the measuring beam of the spectrophotometer provided a means by which the actinic beam could be applied without removal of the cell suspension. For measurements, a dark reduction rate was determined as described above after which the photocell was inactivated, the door opened, and the actinic beam applied for 5 to 10 min. Changes in absorption at 600 nm subsequent to illumination were taken to represent photosynthetic reduction of DPIP. These photoreductive rates were abolished by M DCMU (3-(3,6dichlorophenyl)- I, I-dimethylurea). Anaerobiosis was achieved by evacuating Thunberg tubes, with constant tapping to dislodge gas bubbles, and replacing the atmosphere with nitrogen. Addenda were tipped in from a side reservoir after evacuation. The rates of dye reduction reported in this paper were taken from the first 1 to 2 min of reaction. Cell quantities were determined by packed cell volume using calibrated blood sedimentation tubes in a horizontal-head centrifuge. Suspensions were centrifuged until no further packing occurred. Cell concentrations for assay were between 1 and 2 p1 cells/ml. A broad pH optimum near 6.5 was determined after making measurements from pH 5.5 to 8.5 in 0.5-pH unit increments; this value was used for all measurements reported herein. Representative rates of DPIP dark reduction in the blue-green algae tested are presented in Table 1. Anacystis nidulans reduced the dye at rates severalfold faster than any other alga; thus this alga was used to study characteristics of the dark reduction.
TABLE 1 Rates of DPIP dark reduction by several blue-green algae. Data represent ranges of initial rates taken from several trials for each alga. Conditions of assay were the same for each measurement: DPIP at 25 nmol/ml; 25"C, 0.05 M PO,, pH 6.5 Organism Anacystis nidulans Nostoc mriscorum Phormidium luridium Anabaena cylindrica Agmenell~imq~iadruplicaturn N O S ~ OSP. C (TX-50)
nmol DPIP/uI cells/min 9.1 to 10.2 1 . 5 to 2 . 0 l.0t01.7 0 . 5 to 1 . 0 0 . 6 to 1 .O 0.2 t00.3
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
NOTES
In Anacystis, the dark reduction of DPIP began immediately upon addition of the dye and usually gave a reasonably close fit to first-order kinetics. Under anaerobiosis, up to 80 nmol of DPIP was reduced per microliter of cells in a period of about 30 min, after which only a slow (5-10% of initial), constant, background rate was evident. With oxygen present, a portion of the reduced DPIP was constantly reoxidized preventing measurement of actual amounts of DPIP reduced. When cells from a dark reductant-depleted preparation were centrifuged from the medium, aeration of the supernatant resulted in reoxidation of the DPIP to about 90% of initial. Neither the growth medium nor phosphate buffer in which cells had been suspended exhibited detectable dark reduction activity. Cell suspensions held in darkness for several hours before assay yielded lower initial dark reduction relative to cells held in the light; upon a return to light, the dark reduction activity tended to be restored. The results of one such experiment are presented in Table 2. These data indicated a possibility that the dark reductant (or reductants) was being generated by the photosynthetic process. Permeaplasts of Anacystis were used to study the relation of the dark reduction to photosynthesis. A harvested and washed cell suspension was divided into two fractions, one of which was incubated for 30 min at 35°C in 1.0 mM ethylenediaminetetraacetic acid (EDTA) plus 1.5 mg lysozyme (9000 unitslmg) per microliter of cells in 0.05 M TES buffer, pH 7.5 (optimum for lysozyme). Control cells were incubated in TES buffer only. After incubation, the cells were washed twice and resuspended in TABLE 2 Effects of incubation in light or dark on dark reduction of DPIP in Anacystis. Cultures were held under otherwise normal culture conditions and assayed for dark reduction immediately after time period. Assay as described for Table 1 nmol DPIP/pI cellslmin
Treatment, 8h
Rate after incubation
Fraction returned to dark for 120 min
Light Dark
8.10 2.50
2.85 2.52
Fraction returned to light for 120 min 8.01 4.25
TABLE 3 Dark- and light-dependent DPIP reduction rates in untreated cells and in permeaplasts of Anacysfis. Permeaplasts prepared by treatment with EDTA/lysozyme. Data represent four to six trials for each parameter. Assay conditions as for Table 1 except that cells were suspended in 0.05 M TES buffer nmol DPIP/pI cellslmin
Initial dark rate Photoreductive rates after depletion of dark reductant Rates in light with full "pool" of dark reductant
Untreated cells
Permeaplasts
8-10
3-5*
0
9-1 1
8-10
11-15
'Some dark-reductive activity could be recovered in lysozyme/
EDTA treatment supernatant.
TES buffer, pH 6.5. Anaerobic suspensions of untreated control cells and permeaplasts were exposed to just enough DPIP to deplete completely the dark reductant pool, after which 25 nmol/ml of DPIP was added. The suspensions were then illuminated and the extent of photoreduction was determined. Table 3 contains data from such an experiment. From the table, it can be seen that apparent light rates in cells with full dark reductant pools were greater than light rates in pool-depleted cells. The differences almost equalled the dark rates alone, that is, the light and dark reductions proceeded independently of one another. Photoreductive rates in the absence of concomitant dark reduction exhibited zero-order kinetics. Measurements of photoreduction in cells still having a full pool of dark reductant did not yield a fit to either zero- or first-order curves, further indicating independence of light and dark reduction reactions. It should be noted that permeaplasts gave lower rates of dark reduction than did untreated cells. However, some darkreductant activity was recovered in the treatment supernatant. Since permeaplasts are "leaky" and lose not only phycocyanin but also other water-soluble constituents, it is not surprising that an endogenous reducing pool would leach out. It is concluded that the dark reduction of DPIP, by Anacystis at least, represents a pool or pools of reductant of sufficiently low redox potential to reduce completely DPIP, in the absence of light. The dark re-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
422
CAN. J. MICROBIOL. VOL. 21. 1975
duction proceeds independently of photoreduction of DPIP and should be taken into account when making such measurements. Likewise, even the slower rates of the other blue-green algae tested should be corrected for in photoreductive studies. The dark reductant(s) seems to be generated in the light and consumed by cellular processes in the dark. The dark reduction of DPIP must take place in the cell; thus oxidized DPIP must readily move into the cell and reduced DPIP must readily move out of the cell. The inability of untreated Anacystis cells to photoreduce DPIP must be due to a lack of accessibility to the photosynthetic machinery rather than to impermeability to DPIP as has generally been thought. Treatment with lysozyme/EDTA must expose or otherwise make added oxidants accessible to the photosynthetic light reaction.
Acknowledgment This work was supported in part by a grant from the Committee on Faculty Research of the University of Mississippi.
1. ARMSTRONG, J. McD. 1964. The molar extinction coefficient of 2,6-dichlorophenol indophenol. Biochim. Biophys. Acta, 86: 194-197. 2. BIGGINS,J . 1967. Photosynthetic reactions by-lysed protoplasts and particle preparations from the bluegreen alga, Phormidiltm luridiurn. Plant Physiol. 42: 1447-1456. 3. CARR,N. G., and M. HALLAWAY. 1965. Reduction in dark and light by of phenolindo-2,6-dichlorophenol J. Gen. Mithe blue-green alga, Arl~baenri~~tiritrbilis. crobiol. 39: 335-344. 4. FREDRICKS, W. W., and A. T. JAGENDORF. 1964. A soluble component of the Hill reaction in Anacystis t~idrrlons.Arch. Biochem. Biophys. 104: 39119. 5. STEVENS, S . E., JR., C. 0 . PATTERSON, and J. MYERS. 1973. The production of hydrogen peroxide by bluegreen algae: A survey. J. Phycol. 9: 4271130. 6. VAN BAALEN, C. 1955. Dissertation, University of Texas, Austin. C. 1962. Studies on marine blue-green 7. VANBAALEN, algae. Bot. Mar. 4: 130-139. 8. WARD,B. 1971. Dissertation, University of Texas, Austin. 9. WARD,B., and J . MYERS.1972. Photosynthetic properties of perrneaplasts of Anticystis. Plant Physiol. 50: 547-550.
419
The non-light-dependentreduction of 2,6-dichlorophenolindophenol by cells of the blue-green alga Anacystis nidulans Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
BAILEYW A R D Department of Biology, Unit,ersity of Mississippi, Universiry, Mississippi 38677 Accepted October 3 I , 1974
WARD,B. 1975. The non-light-dependent reduction of 2,6dichlorophenolindophenol by cells of the blue-green alga Atlacystis t1idr11ot1.s.Can. J . Microbiol. 21: 419-422. Whole cells of the blue-green alga Atfacystis t~idltlans reduced, in the dark, the oxidation-reduction dye, 2,6dichlorophenolindophenolat rates severalfold higher than those of the other algae tested. Under anaerobiosis, the endogenous reductant was depleted after up to 80 nmol of dye were reduced per microliter of cells. Cells held in darkness for several hours exhibited lowered dark reduction rates relative to cells held in light. Treatment with lysozyme and ethylenediaminetetraacetic acid yielded cells that would photoreduce the dye, whereas untreated cells would not. Comparisons of photoreduction and dark reduction revealed that the dark reduction proceeded independently of the photoreduction. It was concluded that the dark reduction represents a pool of endogenous reductant of sufficiently low oxidation-reduction potential to reduce completely 2,6dichlorophenolindophenol.Additionally, untreated cells were shown to be permeable to the dye although they did not photoreduce it; thus lysozyme/ ethylenediaminetetraacetic acid treatment was considered to make the oxidant accessible to the photosynthetic machinery. WARD,B. 1975. The non-light-dependent reduction of 2,6dichlorophenolindophenol by cells of Can. J. Microbiol. 21: 419-422. the blue-green alga At~crcystistiidrrltr~~.~. En ['absence de lumiere, des cellules entieres de Atlcicystis nidlrlatls, une algue bleu-vert, reduisent la teinture oxydante-reductrice 2,6dichlorophCnolindophCnoli d e s taux plusieurs fois plus Clevesque ceux obtenus d'autres algues mises il'essai. Sous des conditions anatrobiques, le reducteur endogene peut reduire jusqu'i 80 nmol de teinture par microlitre de cellules, apres quoi I'activitt devient nulle. Les cellules maintenues a I'obscurite durant plusieurs heures, comparativement a celles qui sont garddes en lumiere, ont des taux de reduction inferieurs. Le traitement par la lysozyme et par I'acide ethylenediaminetetraacetique rend les cellules capables de photoreduction de la teinture, tandis que les cellules non traitees en sont incapables. Les comparaisons entre la photoreduction et la reduction en obscuritk revelent que la reduction en obscurite se fait independamment de la photoreduction. I1 en est conclu que la reduction en obscurite constitue un pool de reducteur endogltne a potentiel oxydo-reducteur suffisamment faible pour riduire completement le 2,6-dichlorophCnolindophCnol.De plus, les cellules non traitkes s'averent permeables i la teinture bien qu'elles ne puissent la rtduire photosynthetiquement et, de ce fait, le traitement lysozyme/~thylenediaminetCtraacCtique est condidere cornme pouvant rendre I'oxydant accessible au mecanisme photosynthktique. [Traduit par le journal]
The blue-green alga Anacystis nidulans has been used extensively for photosynthetic measurements using the light-dependent reduction of various dyes, including the electropositive compound 2,6-dichlorophenolindopheno1 (DPIP) (E,' = 0.224 V). Although intact cells of algae do not generally photoreduce DPIP, membrane preparations (2, 4) and lysozyme-treated whole cells (9) have exhibited photoreductive capabilities. During earlier studies on photosynthetic electron transport in Anacystis (8, 9), a non-light-dependent reduction of DPIP was observed. Other workers 'Received July 19, 1974.
also have observed endogenous {dark) dye reduction in blue-green algae (3, 6), but the rate and extent of reduction of DPIP in these reports, as well as for other blue-greens tested in our laboratory are severalfold less than that exhibited by Anacystis. This communication compares characteristics of the endogenous dark reduction of DPIP by cells of Anacystis nidlrlans with that observed in other blue-green algae and describes several salient features of the Anacystis dark reduction phenomenon. The blue-green algae tested and the media in which they were grown were Anacystis nidulans, Kratz and Allen strain (modified
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
420
CAN. J. MICROBIlDL. VOL. 21, 1975
medium C (9)); Nostoc muscorwn G (medium B (5)); Anabaena cylindrica (modified medium C); Nostoc sp. (TX-50) (medium B); Phormidium luridium var. olivaceae (medium B); and Agmenellztm quadruplicatum (BG-1) (ASP-2 (7)). The first three cultures were obtained from the laboratory of Dr. Jack Myers, University of Texas, U.S.A. Culture histories of these algae have been reported (5). Agmenellum quadruplicatum was a gift of Dr. L. 0 . Ingram, University of Florida, U.S.A., and Phormidium luridium var. olivaceae was obtained from the Indiana University culture collection, U.S.A. Axenic cultures were maintained at about 160 ft-c of cool white fluorescent light in a water bath thermostated at 30°C. All cultures were aerated with sterile air delivered through glass tubes to about 150 ml of cell suspension in cotton-plugged 38 x 300 mm Pyrex test tubes. Aseptic technique was observed throughout and cultures were checked for contamination before assay to ensure that only blue-green algae were present. For most assays, cells were harvested by centrifugation from midlog phase of growth, washed once in 0.05 M NaH2P04/Na2HP04 buffer, pH 6.5, and resuspended in the same buffer. The reduction of DPIP was followed spectrophotometrically at 600 nm using a Bausch and Lomb Spectronic 505 dual beam recording spectrophotometer. A millimolar extinction coefficient of 18 (pH 6.5) (1) was used for all measurements except those made at different pH values, in which case the absorption at 522 nm (isosbestic point) was followed and a millimolar extinction coefficient of 8.6 was applied (1). "Zero" was set on the spectrophotometer using cells vs. cells after which about 25 nmol of DPIP was added to the measuring-beam cuvette which was quickly inverted to mix and immediately returned to the measuring beam. About 30 s was consumed in this operation. Permeaplasts of Anacystis nidulans were prepared according to the method of Ward and Myers (9). The use of TES (N-tris(hydroxymethy1)methyl-2-amino-ethane sulfonic acid) buffer instead of phosphate was necessary for successful treatment. Photoreduction of DPIP by these treated cells was measured by exposing the cell suspensions plus DPIP to rate-saturating incandescent light provided by a Kodak Carousel 850 projector focused through a 12-cm water
cell. A special cuvette holder mounted in the measuring beam of the spectrophotometer provided a means by which the actinic beam could be applied without removal of the cell suspension. For measurements, a dark reduction rate was determined as described above after which the photocell was inactivated, the door opened, and the actinic beam applied for 5 to 10 min. Changes in absorption at 600 nm subsequent to illumination were taken to represent photosynthetic reduction of DPIP. These photoreductive rates were abolished by M DCMU (3-(3,6dichlorophenyl)- I, I-dimethylurea). Anaerobiosis was achieved by evacuating Thunberg tubes, with constant tapping to dislodge gas bubbles, and replacing the atmosphere with nitrogen. Addenda were tipped in from a side reservoir after evacuation. The rates of dye reduction reported in this paper were taken from the first 1 to 2 min of reaction. Cell quantities were determined by packed cell volume using calibrated blood sedimentation tubes in a horizontal-head centrifuge. Suspensions were centrifuged until no further packing occurred. Cell concentrations for assay were between 1 and 2 p1 cells/ml. A broad pH optimum near 6.5 was determined after making measurements from pH 5.5 to 8.5 in 0.5-pH unit increments; this value was used for all measurements reported herein. Representative rates of DPIP dark reduction in the blue-green algae tested are presented in Table 1. Anacystis nidulans reduced the dye at rates severalfold faster than any other alga; thus this alga was used to study characteristics of the dark reduction.
TABLE 1 Rates of DPIP dark reduction by several blue-green algae. Data represent ranges of initial rates taken from several trials for each alga. Conditions of assay were the same for each measurement: DPIP at 25 nmol/ml; 25"C, 0.05 M PO,, pH 6.5 Organism Anacystis nidulans Nostoc mriscorum Phormidium luridium Anabaena cylindrica Agmenell~imq~iadruplicaturn N O S ~ OSP. C (TX-50)
nmol DPIP/uI cells/min 9.1 to 10.2 1 . 5 to 2 . 0 l.0t01.7 0 . 5 to 1 . 0 0 . 6 to 1 .O 0.2 t00.3
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
NOTES
In Anacystis, the dark reduction of DPIP began immediately upon addition of the dye and usually gave a reasonably close fit to first-order kinetics. Under anaerobiosis, up to 80 nmol of DPIP was reduced per microliter of cells in a period of about 30 min, after which only a slow (5-10% of initial), constant, background rate was evident. With oxygen present, a portion of the reduced DPIP was constantly reoxidized preventing measurement of actual amounts of DPIP reduced. When cells from a dark reductant-depleted preparation were centrifuged from the medium, aeration of the supernatant resulted in reoxidation of the DPIP to about 90% of initial. Neither the growth medium nor phosphate buffer in which cells had been suspended exhibited detectable dark reduction activity. Cell suspensions held in darkness for several hours before assay yielded lower initial dark reduction relative to cells held in the light; upon a return to light, the dark reduction activity tended to be restored. The results of one such experiment are presented in Table 2. These data indicated a possibility that the dark reductant (or reductants) was being generated by the photosynthetic process. Permeaplasts of Anacystis were used to study the relation of the dark reduction to photosynthesis. A harvested and washed cell suspension was divided into two fractions, one of which was incubated for 30 min at 35°C in 1.0 mM ethylenediaminetetraacetic acid (EDTA) plus 1.5 mg lysozyme (9000 unitslmg) per microliter of cells in 0.05 M TES buffer, pH 7.5 (optimum for lysozyme). Control cells were incubated in TES buffer only. After incubation, the cells were washed twice and resuspended in TABLE 2 Effects of incubation in light or dark on dark reduction of DPIP in Anacystis. Cultures were held under otherwise normal culture conditions and assayed for dark reduction immediately after time period. Assay as described for Table 1 nmol DPIP/pI cellslmin
Treatment, 8h
Rate after incubation
Fraction returned to dark for 120 min
Light Dark
8.10 2.50
2.85 2.52
Fraction returned to light for 120 min 8.01 4.25
TABLE 3 Dark- and light-dependent DPIP reduction rates in untreated cells and in permeaplasts of Anacysfis. Permeaplasts prepared by treatment with EDTA/lysozyme. Data represent four to six trials for each parameter. Assay conditions as for Table 1 except that cells were suspended in 0.05 M TES buffer nmol DPIP/pI cellslmin
Initial dark rate Photoreductive rates after depletion of dark reductant Rates in light with full "pool" of dark reductant
Untreated cells
Permeaplasts
8-10
3-5*
0
9-1 1
8-10
11-15
'Some dark-reductive activity could be recovered in lysozyme/
EDTA treatment supernatant.
TES buffer, pH 6.5. Anaerobic suspensions of untreated control cells and permeaplasts were exposed to just enough DPIP to deplete completely the dark reductant pool, after which 25 nmol/ml of DPIP was added. The suspensions were then illuminated and the extent of photoreduction was determined. Table 3 contains data from such an experiment. From the table, it can be seen that apparent light rates in cells with full dark reductant pools were greater than light rates in pool-depleted cells. The differences almost equalled the dark rates alone, that is, the light and dark reductions proceeded independently of one another. Photoreductive rates in the absence of concomitant dark reduction exhibited zero-order kinetics. Measurements of photoreduction in cells still having a full pool of dark reductant did not yield a fit to either zero- or first-order curves, further indicating independence of light and dark reduction reactions. It should be noted that permeaplasts gave lower rates of dark reduction than did untreated cells. However, some darkreductant activity was recovered in the treatment supernatant. Since permeaplasts are "leaky" and lose not only phycocyanin but also other water-soluble constituents, it is not surprising that an endogenous reducing pool would leach out. It is concluded that the dark reduction of DPIP, by Anacystis at least, represents a pool or pools of reductant of sufficiently low redox potential to reduce completely DPIP, in the absence of light. The dark re-
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/18/14 For personal use only.
422
CAN. J. MICROBIOL. VOL. 21. 1975
duction proceeds independently of photoreduction of DPIP and should be taken into account when making such measurements. Likewise, even the slower rates of the other blue-green algae tested should be corrected for in photoreductive studies. The dark reductant(s) seems to be generated in the light and consumed by cellular processes in the dark. The dark reduction of DPIP must take place in the cell; thus oxidized DPIP must readily move into the cell and reduced DPIP must readily move out of the cell. The inability of untreated Anacystis cells to photoreduce DPIP must be due to a lack of accessibility to the photosynthetic machinery rather than to impermeability to DPIP as has generally been thought. Treatment with lysozyme/EDTA must expose or otherwise make added oxidants accessible to the photosynthetic light reaction.
Acknowledgment This work was supported in part by a grant from the Committee on Faculty Research of the University of Mississippi.
1. ARMSTRONG, J. McD. 1964. The molar extinction coefficient of 2,6-dichlorophenol indophenol. Biochim. Biophys. Acta, 86: 194-197. 2. BIGGINS,J . 1967. Photosynthetic reactions by-lysed protoplasts and particle preparations from the bluegreen alga, Phormidiltm luridiurn. Plant Physiol. 42: 1447-1456. 3. CARR,N. G., and M. HALLAWAY. 1965. Reduction in dark and light by of phenolindo-2,6-dichlorophenol J. Gen. Mithe blue-green alga, Arl~baenri~~tiritrbilis. crobiol. 39: 335-344. 4. FREDRICKS, W. W., and A. T. JAGENDORF. 1964. A soluble component of the Hill reaction in Anacystis t~idrrlons.Arch. Biochem. Biophys. 104: 39119. 5. STEVENS, S . E., JR., C. 0 . PATTERSON, and J. MYERS. 1973. The production of hydrogen peroxide by bluegreen algae: A survey. J. Phycol. 9: 4271130. 6. VAN BAALEN, C. 1955. Dissertation, University of Texas, Austin. C. 1962. Studies on marine blue-green 7. VANBAALEN, algae. Bot. Mar. 4: 130-139. 8. WARD,B. 1971. Dissertation, University of Texas, Austin. 9. WARD,B., and J . MYERS.1972. Photosynthetic properties of perrneaplasts of Anticystis. Plant Physiol. 50: 547-550.
