World Journal
of Microbiology
& Biotechnology
11, 649-653
Effect of gibberellic acid on photosynthesis and glycollate dehydrogenase in Anacystis nidulans A.-K.J.
Sallal
Gibberellic acid at lop4 M was optimal for enhancement of growth, 0, evolution, photosystem activity of glycollate dehydrogenase of Anacystis niduhns. A stimulatory effect was observed on Other concentrations of gibberellic acid were inhibitory to 0, evolution and photosystem phycocyanin, phycoerythrin and p-carotene were significantly enhanced after 48 h incubation with at 10e3 M but the chlorophyll content began to increase 3 h after adding 10m4 M gibberellic acid. Key words: Anacystis nidubns, gibberellic
acid, glycollate
dehydrogenase,
Gibberellic acid (GA), a plant growth regulator, plays a major role in the development of higher plants. It affects the fundamental process of cell expansion (Jones 1973; MacMillan 1980) and also causes quantitative and qualitative changes in certain membrane systems in the cell which precede the stimulation of RNA and protein synthesis (Wareing & Phillips 1978). Recent studies have focused on the effect of GA on gene regulation of many enzymes, including cr-amylase, serine carboxypeptidase and cysteine proteinases (Koehler and Ho, 1990; Dal Degan et al. 1994; Tanida et al. 1994). Although the effect of GA on growth and nitrogenase activity in the cyanobacterium, Anabaena cylindrica, has been studied (Sallal et al. 1994) there have been no reports on its effect on the various photosynthetic reactions involving photosynthetic pigments in this microbe. This is the subject of the present study.
Materials
and Methods
Organism and Growth Conditions Anacystis nidulans (kindly supplied by Professor sity of Dundee, UK) was grown aseptically flasks, each containing 100 ml BG-II medium
G.A. Codd, Univerin 250-ml conical (Stanier el al. 1971).
The author is with the Department of Biological Sciences, Faculty of Science, University of Science and Technology, Irbid, Jordan: fax: 9622 259123. @ 1995 Rapid Science
II and I and the photosystem II. I. Syntheses of gibberellic acid
photosynthesis,
Gibberellic acid was added to flasks in duplicate to the desired concentration and pH was re-adjusted to 7.4. Medium was sterilized by filtration through 0.45~pm pore membrane filters. The culture flasks were incubated at 25°C in an orbital shaker at 100 rev/min. ‘Gro-lux’ fluorescent tubes (Atlas-GEC, Manchester, UK) provided light of 60 fluE m-‘.s at the surface of the flasks. Cell Disruption and Cen+gation About 10 ml culture were harvested by centrifugation at 5000 x g for 20 min. The cells were resuspended in 5 ml 75 mM Tricine buffer, pH 7.5, containing 10 mM NaCI, and disrupted by four, 15-s bursts of ultrasonication, punctuated by 15-s rest periods in an ice-bath. The disrupted cells were centrifuged at 2500 X g for 15 min and the supematant used to study the photosynthetic electron transport system. 0, Evolution 0, evolution was measured at 25°C using a Pt/Ag 0, electrode with an automatic recorder. Cell suspension (3.0 ml) was incubated in the dark for 10 min and then exposed to light at I20 PE mm2.s for another 10 min. The 0, electrode was calibrated according to Lessler (1970). Ferricyanide-Hill Reaction The femicyanide-Hill reaction was used to measure the transfer of electrons from water to ferricyanide, via photosystem II, according to Nishimura et al. (1964), with minor modifications. The 3.0~ml reaction mixture contained 2.3 ml Hi11 buffer (50 rnM Tricine, 0.4 M sucrose and 10 mM NaCI, pH 7.5), I pmol MgCl,, 1 pmol potassium ferricyanide and 0.5 ml cell-free extract. The reaction
mixture was illuminated (120 ,uE m-‘.s) in cuvettes with a tungsten filament lamp. The measured at 420 nm.
net photoreduction
of ferricyanide
was
Publishers WorLd ~oumal of Mrrobmlogy
6 Biotechnology. Vof 1I. 199.5
649
A.-K.].
S&al Mehler Reaction The Mehler reaction, with a 2,6-dichlorophenolindophenol (DCPIP) ascorbate couple as electron donor, was used to measure the transfer of electrons from DCPIP/ ascorbate to methylviologen via photosystem I (PSI) (Schmid et al. 1975). The J.&ml reaction mixture contained 2.3 ml Mehler buffer (75 mM Tricine, 0.2 M KCl), 2.5 pmol DCPIP, 60 pmol ascorbate, 2.5 pmol KCN, 0.1 pm01 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), 0.1 pmol methylviologen and 0.5 ml cell extract. The reaction mixture was equilibrated in the 0, electrode in the dark for 5 min before light-dependent 0, consumption was measured using a light source of 120 PE m-‘.s.
Incubation
Glycollate Dehydrogenase Glyoxylate formation was measured according to the method of Codd & Schmid (1972). The 3.0-ml reaction mixture contained in 2.2 ml 0.1 M KH,PO, buffer, pH 8.2, 10 pmol cysteine. HCl, 10 pmol phenylhydrazine and 0.5 ml cell extract. The reaction was started by the addition of 30 pmol sodium glycollate and the increase in A,,, was then read in a double-beam spectrophotometer.
time (days)
Figure 1. The effect of gibberellic acid, at 10m6 (A), 10e5 (H), 10W4 (0) 10e3 (a) and 10m2 M (A), on the growth of Anacystis nidulans. Each point represents the mean from four experiments, each performed in duplicate. O-Control (without gibberellic acid).
Chlorophyll Chlorophyll
Deterwzinafion a was measured according to Kirk (1967).
a
Biliproteins and p-carotene Determinations The absorption of phycocyanin, phycoerythrin and p-carotene for all cultures of A. nidulans incubated with different concentrations of GA was studied according to Cohen-Bazire & Bryant (1982).
a
Results
E P ,
T
00
c
00
, 1
2
3
Incubation
4
1 5
, 8
7
time (days)
Flgure 2. The effect of gibberellic acid, at 10m6 (A), 10m5 (m). 10e4 (0) 10e3 (0) and 10m2 M (A), on 02 evolution by (a) and photosystem II (b) and I (c) of Anacystic nidulans. Each point represents the mean from two (a, c) or four (b) experiments, each performed in duplicate. O-Control (without gibberellic acid).
650
World ~oumal of Microbiology & Biotechnology. Vol I 1, 1995
Maximum growth stimulation occurred with 10e4 M GA (Figure I). In cultures with 10m2 M GA, complete cell lysis was observed after 24 h (Figure 1). The rates of 0, evolution with various GA concentrations showed a similar trend to growth (Figure 2a), 10m4 M GA increasing evolution by 33% over 7 days. GA also stimulated the photosynthetic electron transport system by a considerable amount, particularly the photosystemreaction (Figure 2b, c). A concentration of 10v4 M was again found to be optimal, increasing the rate of photosystem II by about 95% and that of photosystem I by 42% after 7 days. In contrast, 10m5 and 10m6 M GA only enhanced photosystemactivity by 39% and 25%, respectively, and 10 - ’ M caused 78% inhibition (Figure 2b). The chlorophyll a, phycobiliproteins and p-carotene contents of A. nidulans grown with different concentrations of GA were studied. The only increase in chlorophyll a was with 10m4 M GA, in comparison with the content in control culture (4.1 mg/ml) (see Figure 1 and Table I). All the other GA concentrations tested reduced chlorophyll a contents. The changes in the other photopigment contents in A. nidulans in response to different concentrations of GA are also shown in Table I. The contents of these three pigments increased when GA was present in the medium after 5 days of growth at 25°C. Synthesis of phycocyanin and phycoerythrin was increased 2- to s-fold when A. nidulans
Effect of gibberellic acid on A. nidulans Table 1. Photopfgment nidulans after 5 days’
contents (% of those In controls) growth with gibberellic acid.*
Photopigment
Gibberellic 10-s
Chlorophyll Phycocyanin Phycoerythrin B-Carotene
a
Values
represent
l
means
lo-’
10-e
93 145 190 147
81 97 164 113
122 193 192 167 of four
replicates,
each
Table 2. Changes in the absorption of photopigments or without glbberellic acid for 7 days.’ Incubation (days)
2 3 4 5 6 7
with
In cell-free
of Anecystfs
value
for control
0.86 0.76 0.53 0.9
a standard
Culturet
deviation
extracts
< 5%.
of A. nidulans
grown
with
lo-’
M
Photopigment Chlorophyll
1
extracts
Absorption
acid (m)
lo-’
87 358 396 239
in cell-free
C T C T C T C T C T C T C T
a
0.38 0.29 0.42 0.33 0.62 0.51 0.75 0.62 0.80 0.71 1.10 0.96 1.21 0.65
* Values represent the means of four readings, t C-Control (without gibberellic acid); T-test
Phycocyanin
Phycoerythrin
0.34 0.25 0.39 0.27 0.56 0.12 0.70 2.12 0.73 2.65 0.98 3.10 1.10 2.99 each with a standard (with 10m3 M gibberellic
was incubated with 10-j M GA (Tables 1 and 2 and Figure I). This concentration of GA also increased the synthesis of p-carotene by about 140% after 5 days at 25°C. Phycocyanin, phycoerythrin and p-carotene contents increased from 48 h, and peaking on day 5. GA stimulated the synthesis of phycoerythrin more than that of phycocyanin (Tables I and 2). GA at IO-“ M stimulated the activity of the photorespiratory carbon oxidation cycle enzyme, glycollate dehydrogenase, by 26% after 7 days (Figure 3). However, 10e5 and lop6 M GA only enhanced this activity by 16% and 2.9%, respectively, and 10 - ' M GA actually inhibited the activity, by 38% after 7 days (Figure 3). Direct addition of GA (10m4 M) to any of the photosynthetic on glycollate dehydrogenase reaction mixtures did not stimulate the reactions. Since 10m4 M GA was found to be the optimum culture concentration for enhancing the photosynthetic and enzyme reactions after 24 h, the effect of this concentration on the reactions was tested after 0, 3, 6 and 12 h, incubation, using concentrated, log-phase cultures of A. niduluns. It is clear from the results (Table 3) that GA effected the first
0.26 0.19 0.28 0.21 0.45 0.94 0.52 1.70 0.52 2.05 0.76 2.73 0.80 2.10 deviation acid).
/I-Carotene 0.44 0.32 0.48 0.38 0.73 0.84 0.67 1.10 0.91 2.20 1.31 1.82 1.40 1.50
< 4%.
generation of A. nidulans in the cultures, indicating that it may have an intracellular effect on the parts of the cell genome controlling the reactions studied. Electron microscopy of the cyanobacterial cells after culture with various concentrations of GA, using 1% uranyl acetate to negatively stain the cells, indicated that GA had no effect on cell volume.
Discussion The photosynthetic processes in cyanobacteria, eukaryotic plants and algae appear to be similar (Ho & Krogmann 1982). This similarity encouraged the present work on cyanobacteria, since there are no detailed reports on the effect of GA on the photosynthetic reactions of these microorganisms. Gibberellic acid stimulated growth and photosynthetic and glycollate dehydrogenase activities of A. niduluns at a concentration of 10e4 M. Other concentrations of GA exerted an inhibitory effect on certain photosynthetic electron transport reactions. Ahmad (1973) found that lop9 M GA did not promote the growth of A. niduh
World]oumal
of Microbiology 6 Biotechnology. Vol 11, 1995
651
A.-K.]. Sallal Table 3. Effect A. nidulans.*
01 gibberellic
acid
on growth,
Parameter
Photosystem duced h-‘.mg
/ l
2
Incubation
4
C T
0.45 0.45
0.72 0.80
0.80 0.95
1.00 1.20
C T
10 10
15 17
20 24
36 37
re-
C T
12 12
15 22
21 36
33 61
taken
up
C T
13 13
11 12
22 24
30 34
@mol
gly-
C T
0.60 0.60
0.70 0.75
0.90 1.00
1.20 1.35
evolved
0,
5
of four acid);
0
readings, T-test
7
time (days)
Figure3. The effect of gibberellic acid, at 10-e (A), 10m5 (m), 10m4 (0). low3 (0) and lo-* M (A), on the glycollate dehydrogenase activity of Anacystis nidulans. Each point represents the mean from two experiments, each performed in duplicate. O-Control (without gibberellic acid).
although IO-* M GA, in combination with lo-* M kinetin, did stimulate growth of Chlorogloea fritschii. Large increases in the absorbances of phycocyanin, phycoerythrin and j?carotene (Tables I and 2) had relatively little effect on the activity of photosystem II. However, the largest increase in chlorophyll a content was found with 10e4 M GA; other concentrations led to decreases (Table 1). Although, phycobiliproteins are accessory pigments for the operation of photosystem II (Glazer 1981), their rapid synthesis in the cells of A. nidufans grown with GA failed to enhance the
652
time (h) 12
represent the means (without gibberellic
3
of
6
0,
Glycollate dehydrogenase oxylate h-‘.mg protein)
Incubation
dehydrogenase
3
II bmol ferricyanide chlorophyll)
Photosystem I @mol h-‘.mg chlorophyll)
glycollate
0 @g ml-‘)
0, evolution &mol h-‘.mg chlorophyll)
Values C-Control
and
Culture
Chlorophyll
l
photosynthesis
World Journal of Microbiology 6 Biotechnology, Vol 11. 19%
each with standard deviation (with low4 M gibberellic acid).
< 5%.
activity of this photosystem (Figure 2b). This emphasises the role of chlorophyll a as a primary light receptor for the operation of photosystem II (Lemasson et al. 1973). Allen & Smith (1969) reported the rapid disappearance of phycocyanin in Synechococctrs 6301 grown in a nitrate-depleted medium, whereas chlorophyll content was little affected. In the present study, the specific activity of glycollate dehydrogenase was little effected by GA concentrations that led to rapid synthesis of phycobiliproteins. Although GA can increase the size of cells of higher plants, it did not effect the cell volume of A. nidttlans (present study) or that of the marine diatom, Cychfella cryptica (Adair & Miller 1982). In conclusion, relative to the controls, 10e4 M GA had a great effect on the growth, photosynthesis and glycollate dehydrogenase activity of A. nidtrlans. This stimulatory effect started after 3 h, although GA had no direct effect when added to the reaction mixtures. The enhancement of the biosynthesis of phycobiliproteins could be helpful in their commercial production.
Acknowledgement N.A. Nimer is thanked for excellent technical assistance.
References Adair,
O.V.
& Miller,
M.W.
1982
Growth
and responses
of the
diatom Cyclotellu crypb’cu (Bacillariophyceae) to gibberellic acid. ]oumal of Phycology 18, 587. Ahmad, M.R. 1973 The effect of gibberellic acid, kinetin and indole-s-acetic acid on the growth of blue-green algae in cukure. Review ofAlgo&y 11, 154-160. Allen, M.M. & Smith, A.J. 1969 Nitrogen chlorosis in blue-green algae. Archives of Microbiology 69, 114-120. Codd, G.A. & Schmid, G.H. 1972 Serological characterization of the glycollate oxidising enzymes from tobacco, Euglena gracihs
Effect and a yellow
mutant
of ChloreNa
vwlguris.
Hunt
Physiology
50,
769-773. Cohen-Bazire, G. & Bryant, D.A. 1982 Phycobilisomes: composition and structure. In The Biology of Cyunobacferia, eds Carr, N.G. & Whitton, B.A. pp. 143-190: London, Blackwell Scientific. Dal Degan, F., Rocher, A., Cameron-Mills, V. & Von Wettstein, D. 1994 The expression of serine carboxypeptidases during maturation and germination of the barley grain. Proceedingsofthe National Academy of Sciences of the United States of America 91, 8209-8213. Glazer, A.N. 1981 Photosynthetic accessory proteins with bilin prosthetic groups. In Biochemistry of Plants, eds Hatch, M.D. & Boardman, N.K. pp. 51-96. New York: Academic Press. Ho, K.K. & Krogmann, D.W. 1982 Photosynthesis In The Biology of Cyunobacferia, eds Can; N.G. & Whitton, B.A. pp. 191-214. London: Blackwell Scientific. Jones, R. 1973 Gibberellins, their physiological role. Ann& Review of Plant Physiology 24, 571-598. Kirk, J.T.O. 1967 Studies on the dependence of chlorophyll synthesis on protein synthesis in Etcglenu gracilis, together with a nomogram for determination of chlorophyll concentration.
Phnfu 78, 200-207. Koehler, S.M. & Ho, T.H. 1990 Hormonal regulation, processing and secretion of cysteine proteinases in barley aleurone layers. Plant Cell 2, 769-783. Lemasson, C., Tandeau De Marsac, N. & Cohen-Bazire, G. 1973 Role of allophycocyanin as a light-harvesting pigment in cyanobacteria. Proceedings of the National Academy of Sciences of the United Sfufes of America 70, 3130-3133.
of
gibberellic acid on A. nidulans
Lessler, M.A. 1970 Oxygen electrode measurements in biochemical analysis. M&o& of Biochemicu\ Analysis 17, l-29. MacMillan, J. 1980 Hormonal regulation of development I. Molecular aspects of plant hormones. In Encyclopedia of Plant Physiology, Vol. 9, eds Pirson, A. & Zimmermann, M.H. New York: Springer. Nishimura, M., Sakurai, H. & Takamiya, A. 1964 Wavelength dependency on the inhibition of the Hill reaction and the analysis of the process by flashing light. Biochimica et Biophysicu Acta 79, 241-248. Sallal, A.K., Nimer, N.A. & El-Durini, N.M. 1994 Effect of gibberellit acid on photosynthetic electron transport reactions and nitrogenase activity in Anabaena cylindrica. Microbios 78, IT25. Schmid, G.H., Radunz, A. & Menke, A.W. 1975 The effect of an antiserum to plastocyanin on various chloroplast preparations. Zeifschriff fiir Nufurforschung 3Oc, 201-212. Stanier, R.V., Kunisawa, R., Mandel, M. & Cohen-Bazire, A. 1971 Purification and properties of unicellular blue-green algae (order Chroococcales). Bac~eriologicul Reviews 35, 171-205. Tanida, I., Kim, J.K. & Wu, R. 1994 Functional dissection of a rice high-PI alpha-amylase gene promoter. Molecular and General Genetics 244, 127-134. Wareing, P.F. & Phillips, LDJ. 1978 The Control of Growth and Differentiafion in Plants, 2nd edn. New York: Pergamon Press.
(Received in revised form 22 June 1995; accepted 24 ]une
World louml
of
Minobiologg
6 Biotechdogy.Vol I?,
1995
1995}
653
of Microbiology
& Biotechnology
11, 649-653
Effect of gibberellic acid on photosynthesis and glycollate dehydrogenase in Anacystis nidulans A.-K.J.
Sallal
Gibberellic acid at lop4 M was optimal for enhancement of growth, 0, evolution, photosystem activity of glycollate dehydrogenase of Anacystis niduhns. A stimulatory effect was observed on Other concentrations of gibberellic acid were inhibitory to 0, evolution and photosystem phycocyanin, phycoerythrin and p-carotene were significantly enhanced after 48 h incubation with at 10e3 M but the chlorophyll content began to increase 3 h after adding 10m4 M gibberellic acid. Key words: Anacystis nidubns, gibberellic
acid, glycollate
dehydrogenase,
Gibberellic acid (GA), a plant growth regulator, plays a major role in the development of higher plants. It affects the fundamental process of cell expansion (Jones 1973; MacMillan 1980) and also causes quantitative and qualitative changes in certain membrane systems in the cell which precede the stimulation of RNA and protein synthesis (Wareing & Phillips 1978). Recent studies have focused on the effect of GA on gene regulation of many enzymes, including cr-amylase, serine carboxypeptidase and cysteine proteinases (Koehler and Ho, 1990; Dal Degan et al. 1994; Tanida et al. 1994). Although the effect of GA on growth and nitrogenase activity in the cyanobacterium, Anabaena cylindrica, has been studied (Sallal et al. 1994) there have been no reports on its effect on the various photosynthetic reactions involving photosynthetic pigments in this microbe. This is the subject of the present study.
Materials
and Methods
Organism and Growth Conditions Anacystis nidulans (kindly supplied by Professor sity of Dundee, UK) was grown aseptically flasks, each containing 100 ml BG-II medium
G.A. Codd, Univerin 250-ml conical (Stanier el al. 1971).
The author is with the Department of Biological Sciences, Faculty of Science, University of Science and Technology, Irbid, Jordan: fax: 9622 259123. @ 1995 Rapid Science
II and I and the photosystem II. I. Syntheses of gibberellic acid
photosynthesis,
Gibberellic acid was added to flasks in duplicate to the desired concentration and pH was re-adjusted to 7.4. Medium was sterilized by filtration through 0.45~pm pore membrane filters. The culture flasks were incubated at 25°C in an orbital shaker at 100 rev/min. ‘Gro-lux’ fluorescent tubes (Atlas-GEC, Manchester, UK) provided light of 60 fluE m-‘.s at the surface of the flasks. Cell Disruption and Cen+gation About 10 ml culture were harvested by centrifugation at 5000 x g for 20 min. The cells were resuspended in 5 ml 75 mM Tricine buffer, pH 7.5, containing 10 mM NaCI, and disrupted by four, 15-s bursts of ultrasonication, punctuated by 15-s rest periods in an ice-bath. The disrupted cells were centrifuged at 2500 X g for 15 min and the supematant used to study the photosynthetic electron transport system. 0, Evolution 0, evolution was measured at 25°C using a Pt/Ag 0, electrode with an automatic recorder. Cell suspension (3.0 ml) was incubated in the dark for 10 min and then exposed to light at I20 PE mm2.s for another 10 min. The 0, electrode was calibrated according to Lessler (1970). Ferricyanide-Hill Reaction The femicyanide-Hill reaction was used to measure the transfer of electrons from water to ferricyanide, via photosystem II, according to Nishimura et al. (1964), with minor modifications. The 3.0~ml reaction mixture contained 2.3 ml Hi11 buffer (50 rnM Tricine, 0.4 M sucrose and 10 mM NaCI, pH 7.5), I pmol MgCl,, 1 pmol potassium ferricyanide and 0.5 ml cell-free extract. The reaction
mixture was illuminated (120 ,uE m-‘.s) in cuvettes with a tungsten filament lamp. The measured at 420 nm.
net photoreduction
of ferricyanide
was
Publishers WorLd ~oumal of Mrrobmlogy
6 Biotechnology. Vof 1I. 199.5
649
A.-K.].
S&al Mehler Reaction The Mehler reaction, with a 2,6-dichlorophenolindophenol (DCPIP) ascorbate couple as electron donor, was used to measure the transfer of electrons from DCPIP/ ascorbate to methylviologen via photosystem I (PSI) (Schmid et al. 1975). The J.&ml reaction mixture contained 2.3 ml Mehler buffer (75 mM Tricine, 0.2 M KCl), 2.5 pmol DCPIP, 60 pmol ascorbate, 2.5 pmol KCN, 0.1 pm01 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), 0.1 pmol methylviologen and 0.5 ml cell extract. The reaction mixture was equilibrated in the 0, electrode in the dark for 5 min before light-dependent 0, consumption was measured using a light source of 120 PE m-‘.s.
Incubation
Glycollate Dehydrogenase Glyoxylate formation was measured according to the method of Codd & Schmid (1972). The 3.0-ml reaction mixture contained in 2.2 ml 0.1 M KH,PO, buffer, pH 8.2, 10 pmol cysteine. HCl, 10 pmol phenylhydrazine and 0.5 ml cell extract. The reaction was started by the addition of 30 pmol sodium glycollate and the increase in A,,, was then read in a double-beam spectrophotometer.
time (days)
Figure 1. The effect of gibberellic acid, at 10m6 (A), 10e5 (H), 10W4 (0) 10e3 (a) and 10m2 M (A), on the growth of Anacystis nidulans. Each point represents the mean from four experiments, each performed in duplicate. O-Control (without gibberellic acid).
Chlorophyll Chlorophyll
Deterwzinafion a was measured according to Kirk (1967).
a
Biliproteins and p-carotene Determinations The absorption of phycocyanin, phycoerythrin and p-carotene for all cultures of A. nidulans incubated with different concentrations of GA was studied according to Cohen-Bazire & Bryant (1982).
a
Results
E P ,
T
00
c
00
, 1
2
3
Incubation
4
1 5
, 8
7
time (days)
Flgure 2. The effect of gibberellic acid, at 10m6 (A), 10m5 (m). 10e4 (0) 10e3 (0) and 10m2 M (A), on 02 evolution by (a) and photosystem II (b) and I (c) of Anacystic nidulans. Each point represents the mean from two (a, c) or four (b) experiments, each performed in duplicate. O-Control (without gibberellic acid).
650
World ~oumal of Microbiology & Biotechnology. Vol I 1, 1995
Maximum growth stimulation occurred with 10e4 M GA (Figure I). In cultures with 10m2 M GA, complete cell lysis was observed after 24 h (Figure 1). The rates of 0, evolution with various GA concentrations showed a similar trend to growth (Figure 2a), 10m4 M GA increasing evolution by 33% over 7 days. GA also stimulated the photosynthetic electron transport system by a considerable amount, particularly the photosystemreaction (Figure 2b, c). A concentration of 10v4 M was again found to be optimal, increasing the rate of photosystem II by about 95% and that of photosystem I by 42% after 7 days. In contrast, 10m5 and 10m6 M GA only enhanced photosystemactivity by 39% and 25%, respectively, and 10 - ’ M caused 78% inhibition (Figure 2b). The chlorophyll a, phycobiliproteins and p-carotene contents of A. nidulans grown with different concentrations of GA were studied. The only increase in chlorophyll a was with 10m4 M GA, in comparison with the content in control culture (4.1 mg/ml) (see Figure 1 and Table I). All the other GA concentrations tested reduced chlorophyll a contents. The changes in the other photopigment contents in A. nidulans in response to different concentrations of GA are also shown in Table I. The contents of these three pigments increased when GA was present in the medium after 5 days of growth at 25°C. Synthesis of phycocyanin and phycoerythrin was increased 2- to s-fold when A. nidulans
Effect of gibberellic acid on A. nidulans Table 1. Photopfgment nidulans after 5 days’
contents (% of those In controls) growth with gibberellic acid.*
Photopigment
Gibberellic 10-s
Chlorophyll Phycocyanin Phycoerythrin B-Carotene
a
Values
represent
l
means
lo-’
10-e
93 145 190 147
81 97 164 113
122 193 192 167 of four
replicates,
each
Table 2. Changes in the absorption of photopigments or without glbberellic acid for 7 days.’ Incubation (days)
2 3 4 5 6 7
with
In cell-free
of Anecystfs
value
for control
0.86 0.76 0.53 0.9
a standard
Culturet
deviation
extracts
< 5%.
of A. nidulans
grown
with
lo-’
M
Photopigment Chlorophyll
1
extracts
Absorption
acid (m)
lo-’
87 358 396 239
in cell-free
C T C T C T C T C T C T C T
a
0.38 0.29 0.42 0.33 0.62 0.51 0.75 0.62 0.80 0.71 1.10 0.96 1.21 0.65
* Values represent the means of four readings, t C-Control (without gibberellic acid); T-test
Phycocyanin
Phycoerythrin
0.34 0.25 0.39 0.27 0.56 0.12 0.70 2.12 0.73 2.65 0.98 3.10 1.10 2.99 each with a standard (with 10m3 M gibberellic
was incubated with 10-j M GA (Tables 1 and 2 and Figure I). This concentration of GA also increased the synthesis of p-carotene by about 140% after 5 days at 25°C. Phycocyanin, phycoerythrin and p-carotene contents increased from 48 h, and peaking on day 5. GA stimulated the synthesis of phycoerythrin more than that of phycocyanin (Tables I and 2). GA at IO-“ M stimulated the activity of the photorespiratory carbon oxidation cycle enzyme, glycollate dehydrogenase, by 26% after 7 days (Figure 3). However, 10e5 and lop6 M GA only enhanced this activity by 16% and 2.9%, respectively, and 10 - ' M GA actually inhibited the activity, by 38% after 7 days (Figure 3). Direct addition of GA (10m4 M) to any of the photosynthetic on glycollate dehydrogenase reaction mixtures did not stimulate the reactions. Since 10m4 M GA was found to be the optimum culture concentration for enhancing the photosynthetic and enzyme reactions after 24 h, the effect of this concentration on the reactions was tested after 0, 3, 6 and 12 h, incubation, using concentrated, log-phase cultures of A. niduluns. It is clear from the results (Table 3) that GA effected the first
0.26 0.19 0.28 0.21 0.45 0.94 0.52 1.70 0.52 2.05 0.76 2.73 0.80 2.10 deviation acid).
/I-Carotene 0.44 0.32 0.48 0.38 0.73 0.84 0.67 1.10 0.91 2.20 1.31 1.82 1.40 1.50
< 4%.
generation of A. nidulans in the cultures, indicating that it may have an intracellular effect on the parts of the cell genome controlling the reactions studied. Electron microscopy of the cyanobacterial cells after culture with various concentrations of GA, using 1% uranyl acetate to negatively stain the cells, indicated that GA had no effect on cell volume.
Discussion The photosynthetic processes in cyanobacteria, eukaryotic plants and algae appear to be similar (Ho & Krogmann 1982). This similarity encouraged the present work on cyanobacteria, since there are no detailed reports on the effect of GA on the photosynthetic reactions of these microorganisms. Gibberellic acid stimulated growth and photosynthetic and glycollate dehydrogenase activities of A. niduluns at a concentration of 10e4 M. Other concentrations of GA exerted an inhibitory effect on certain photosynthetic electron transport reactions. Ahmad (1973) found that lop9 M GA did not promote the growth of A. niduh
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651
A.-K.]. Sallal Table 3. Effect A. nidulans.*
01 gibberellic
acid
on growth,
Parameter
Photosystem duced h-‘.mg
/ l
2
Incubation
4
C T
0.45 0.45
0.72 0.80
0.80 0.95
1.00 1.20
C T
10 10
15 17
20 24
36 37
re-
C T
12 12
15 22
21 36
33 61
taken
up
C T
13 13
11 12
22 24
30 34
@mol
gly-
C T
0.60 0.60
0.70 0.75
0.90 1.00
1.20 1.35
evolved
0,
5
of four acid);
0
readings, T-test
7
time (days)
Figure3. The effect of gibberellic acid, at 10-e (A), 10m5 (m), 10m4 (0). low3 (0) and lo-* M (A), on the glycollate dehydrogenase activity of Anacystis nidulans. Each point represents the mean from two experiments, each performed in duplicate. O-Control (without gibberellic acid).
although IO-* M GA, in combination with lo-* M kinetin, did stimulate growth of Chlorogloea fritschii. Large increases in the absorbances of phycocyanin, phycoerythrin and j?carotene (Tables I and 2) had relatively little effect on the activity of photosystem II. However, the largest increase in chlorophyll a content was found with 10e4 M GA; other concentrations led to decreases (Table 1). Although, phycobiliproteins are accessory pigments for the operation of photosystem II (Glazer 1981), their rapid synthesis in the cells of A. nidufans grown with GA failed to enhance the
652
time (h) 12
represent the means (without gibberellic
3
of
6
0,
Glycollate dehydrogenase oxylate h-‘.mg protein)
Incubation
dehydrogenase
3
II bmol ferricyanide chlorophyll)
Photosystem I @mol h-‘.mg chlorophyll)
glycollate
0 @g ml-‘)
0, evolution &mol h-‘.mg chlorophyll)
Values C-Control
and
Culture
Chlorophyll
l
photosynthesis
World Journal of Microbiology 6 Biotechnology, Vol 11. 19%
each with standard deviation (with low4 M gibberellic acid).
< 5%.
activity of this photosystem (Figure 2b). This emphasises the role of chlorophyll a as a primary light receptor for the operation of photosystem II (Lemasson et al. 1973). Allen & Smith (1969) reported the rapid disappearance of phycocyanin in Synechococctrs 6301 grown in a nitrate-depleted medium, whereas chlorophyll content was little affected. In the present study, the specific activity of glycollate dehydrogenase was little effected by GA concentrations that led to rapid synthesis of phycobiliproteins. Although GA can increase the size of cells of higher plants, it did not effect the cell volume of A. nidttlans (present study) or that of the marine diatom, Cychfella cryptica (Adair & Miller 1982). In conclusion, relative to the controls, 10e4 M GA had a great effect on the growth, photosynthesis and glycollate dehydrogenase activity of A. nidtrlans. This stimulatory effect started after 3 h, although GA had no direct effect when added to the reaction mixtures. The enhancement of the biosynthesis of phycobiliproteins could be helpful in their commercial production.
Acknowledgement N.A. Nimer is thanked for excellent technical assistance.
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O.V.
& Miller,
M.W.
1982
Growth
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(Received in revised form 22 June 1995; accepted 24 ]une
World louml
of
Minobiologg
6 Biotechdogy.Vol I?,
1995
1995}
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