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Effects of High Boron Concentrations on Nitrate Utilization and Photosynthesis in Blue-green Algae Anabaena PCC 7119 and Anacystis nidulans
Effects of High Boron Concentrations on Nitrate Utilization and Photosynthesis in Blue-green Algae Anabaena PCC 7119 and Anacystis nidulans P. MATEO, F. MARTINEZ, 1. BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAESO Departamento de Biologia General, Facultad de Ciencias, Universidad Aut6noma de Madrid, 28049, Madrid, Espana Received July 30, 1986 . Accepted October 20, 1986
Summary The effect of excess boron on growth, cell composition, photosynthesis, and nitrogen metabolism, in two species of blue green algae (cyanobacteria), Anabaena PCC 7119 and Anacystis nidulans, was examined. High concentrations of boron in culture media inhibited growth and decreased the contents of phycobiliproteins and chlorophyll. In addition, an accumulation of carbohydrates and a decrease in lipids were observed in both species. Parallel to the fall in photosynthetic pigments, an inhibition of photosynthetic oxygen evolution was observed, after 72 h of culture, in Anabaena PCC 7119. However, initial exposure of the cultures to high concentrations of boron did not affect photosynthesis. In contrast, nitrate uptake decreased in both species with initial exposure to excess concentrations of boron. This inhibition was followed by a decrease in the nitrate reductase activity.
Key words: Anabaena; Anacystis; Boron toxicity; Nitrate uptake; Photosynthesis.
Introduction In higher plants, the range in boron concentration optimal for growth is very narrow, approximately 0.01 to 4mgBl-' (Gupta, 1979). Therefore, the ratio of toxic to adequate levels of boron is smaller than the ratio in any other micronutrient (Reisenauer et aI. 1973) with the toxic effects appearing at concentrations close to the normal requirement of boron (Mengel and Kirkby, 1982). There are many reports stating the symptoms of boron toxicity on plants. These symptoms are reported to be similar in most plants, and consist of a marginal and tip chlorosis which is quickly followed by a necrosis (Shorrocks, 1974). However, there is very little information regarding the actual manner in which boron is toxic to plants. In previous studies with sugar beet we have shown that boron toxicity resulted in an accumulation of nitrates in the sap and in a decrease of the nitrate reductase activity (Carpena et al., 1978; Bonilla et aI., 1980). Similar results have also been obtained in studies with tomato (Bonilla et aI., 1984). Other researches have linked boron toxicity with photosynthesis, Lovatt and Bates (1984) concluded in studies with Cucurbita pepo that an excess of boron lowered the chlorophyll contents and the photosynthetic activity in leaves.
J Plant.
Physiol. Vol. 128. pp. 161-168 {1987}
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P. MATEO, F. MARTINEZ, L BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAEso
In Chlarella pyrenaidasa, however, boron toxicity did not appear to affect the chlorophyll contents although there was an inhibition of the photosynthesis which was explained by an alteration in the organization of the chloroplasts (Sanchez Maeso et aI., 1985). However, the preceding data do not allow any conclusions about the actual manner in which boron is toxic to plants. In this report we report our studies on the effect of high concentrations of boron on the growth, cell composition, photosynthesis, and nitrogen metabolism of two blue-green algae (cyanobacteria): Anabaena pee 7119 and Anarystis nidulans. Data are presented indicating a primary effect of boron toxicity on nitrate uptake and assimilation. Materials and Methods Organisms and culture conditions Anabaena PCC 7119, previously classified as Nostoc muscorum, was a gift of Dr. Rivera, University of Sevilla. Anacystis nidulans was obtained from Prof. Dr. Rodriguez-Lopez, C.S.LC., Madrid. Batch cultures were grown routinely at 26°C in a Kratz and Myers C medium (1955), gassed with air containing 2 % CO2 and illuminated with cool-white fluorescent lamps (lOW m -2). Where appropriate, boron was added as boric acid at concentrations of 50,75, and 100mgBI- I . Culture density was determined at 600 nm using a Beckman DB-GT spectrophotometer. Cells were filtered (using a 0.45 JLm filter paper) or centrifuged and dried at 70°C for 24 h to determine dry weight. Analytical methods
Protein was determined by the method of Lowry et aL (1951) in extracts obtained by treatment of cells with 1 N NaOH at 80°C for 1 h using serum albumin as standard. Carbohydrates were estimated according to Dubois et al. (1956) after extraction with 2N HCI at 100°C for 1 h. Lipids were determined gravimetrically after extraction by the method of Folch et al. (1957). Chlorophyll was estimated according to Marker (1972). Phycobiliproteins were determined in aqueous extracts of cells treated with toluene at 260 nm (1 O.D. = 135 phycobiliproteins) (Blumwald ant Tel-Or, 1982). Photosynthesis
Oxygen evolution was measured with a Clarktype oxygen electrode (Hach Chemical Company). Aliquots of 3 ml with a cell density of 0.1 mgml- I were placed in a temperature-controlled cuvette and illuminated with a quantum flux density of 300 JLE m - 2S - I. Electron Microscopy
Cells were fixed with glutaraldehyde 2 %, and post-fixed with KMn04, 2 %. Dehydration was carried out with water-acetone solutions. Samples, embedded in Vestopal, were sectioned and stained with uranyl acetate and observed in a Phillips EM 300. Nitrate uptake
Nitrate uptake was assayed by measuring the disappearance of nitrate from the medium of culture or in short-term experiments.
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For determination of nitrate in the medium, cells were removed from culture media samples by centrifugation and aliquots of the media were used for ion determination. Nitrates were measured with an ionanalyzer (Mod. 901 Orion Research) using Orion ionselective electrodes (Mod. 9307 Orion Research). Short-term experiments were carried out by the method of Flores et al. (1980). Cells from 2 or 3-day-old cultures (density about 0.5mgml- l ) were used. After harvesting by filtration, the cells were washed with 25mM Tricine-NaOH buffer pH 8.3, and finally resuspended in the same buffer to a density of 1 mg ml- I . Uptake assays were carried out with continuous shaking in the light (lOW m -2, white light) at 26°C, in conical flasks open to the air, and were started by the addition of KN0 3 (0.4 mM final concentration) to cell suspensions which had been preincubated for 10 min under the above conditions. When the effects of high concentrations of boron were tested, boron was present along the preincubation period previous to the addition of nitrate. Nitrate uptake was determined following the disappearance of nitrate from the assay mixture by estimating the concentration of the ion in aliquots of the cell suspension after rapid removal of the cells by filtration. Nitrate was determined by optical absorption at 210nm in acid solution, according to Cawse (1967). Determination of Nitrate Reductase Activity
The cellular nitrate reductase was assayed in situ in toluene-permeabilized cells according to the method of Herrero et al. (1981).
Results and Discussion Batch cultures of Anabaena pee 7119 exposed to a growth medium containing 50, 75, or 100 mg B .1- 1, reduced their growth and showed a drastic drop in the contents of nitrogenous compounds such as proteins, chlorophyll, and phycobiliproteins when compared with control cultures (Fig. 1). In addition the cells exposed to high boron concentrations showed a decrease in lipid contents and an increase in carbohydrates (Fig. 2). Similar results were obtained with the unicellular strain Anacystis nidulans (Table 1). The photosynthetic activity of the cells of Anabaena exposed to high boron concentrations showed no change during the first hours of culture (Table2). Long exposure to excess boron, however, resulted in a decrease of the photosynthetic oxygen evolution (Table 2) and in an alteration of the normal arrangement of thylakoids (Fig. 3). These data are consistent with the loss of the photosynthetic pigments shown by Anabaena and Anacystis and indicate that the increase of carbohydrates cannot be explained by an enhancement of its synthesis, but by an inhibition of its degradation (Lehmann and Wober, 1976). In addition to the change of the photosynthetic apparatus, the cells of Anabaena exposed to high boron concentrations showed (Fig.3) structured granules which resembled the granules of cyanophycin, a polymer of L-arginine and L-aspartic acid (Simon and Weathers, 1976), characteristic of cyanobacteria, formed from the products of catabolism of cell protein (Allen and Weathers, 1980), and that appeared under a number of environmental conditions and particularly in older cultures. On the other hand, the cells of Anabaena and Anacystis cultured under high boron concentrations showed an alteration in nitrate assimilation: For Anabaena cells, the uptake of nitrates was inhibited during the first hours of culture under excess boron
J Plant.
Physiol. Vol. 128. pp. 161 -168 (1987)
P. MATEO, F. MARTINEZ, 1. BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAESO
164
Fig. 1: Effect of high boron concentrations on the growth, and protein, chlorophyll, and phycoControl, 0 50mgBI-l, 6 biliprotein contents of Anabaena PCC 7119. • 75mgBI- 1 D100mgBI-I.
40
20
~ 30
5
I<{
o~ If)
a:
10~ n... .....
o >:r o ~ 10
-.J
5
<{
u
a
Fig. 2: Carbohydrate and lipid content (in % of dry weight) at 96 h of culture in Anabaena cells grown under different B concentrations. • Carbohydrates, 0 Lipids.
50 75 100 mg B 1-1
Table 1: Biochemical composition of Anacystis nidulans cells after 72 h of culture. Values, expressed in % of cell dry weight, are the means of five experiments ± SD.
J Plant.
Constituents
Control
100mg B 1-1
Proteins Chlorophyll Carbohydrates Lipids Nucleic acids
57. 1± 5.2 2.1±O.1 15.5± 1.3 10.7±O.9 12.0± 1.2
26.7±5.3 O.9±O.1 46.6±2.5 7.0±O.8 12.9±2.4
Physiol. Vol. 128.pp. 161-168(1987)
Boron toxicity in blue-green algae
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Table 2: Effect of high boron concentrations on photosynthetic oxygen evolution of cells of Anabaena PCC 7119. The values are the means of four experiments ± SD. /Lmol O 2 evolved mg Chl- I h 2
Control 100 mg B I-I
h
72
157.3±12.3 151.7± 9.1
I
h
152.5± 11.8 5.6± 3.1
B Fig.3: Electron micrographs of Anabaena PCC 7119. A) Cell, in control medium. B) Cell, grown in 100 mg B 1- I. Bar represents 1 /Lm.
as the results of short-term experiments presented in Fig. 4 show. Similar results were obtained in Anacystis by measuring the disappearance of nitrates from the medium in long-term experiments (Fig. 5). In addition, the nitrate reductase activity was affected by boron toxicity. As shown in Table3, the nitrate reductase activity of both Anaj. Plant. Physiol. Vol. 128. pp. 161-168 (1987)
166
P. MATEO, F. MARTINEZ, 1. BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAESO
L
2
o
---~
400
w L
........
.."....
.....
~ ~200 w ::t
I- -
«
0:::
I-
Z
a
90
150
Fig. 4: Effect of high boron concentrations on nitrate uptake of Anabaena PCC 7119 .• Control, o 100mgBI- I.
210
TIME[min.)
o
2
3
Fig. 5: Time-course of nitrate utilization by Anacystis nidulans grown under high boron concentrations .• Control, 0 100mgBI- I.
4
DAYS
Table 3: Effect of high boron concentrations on cellular nitrate reductase activity in Anabaena and Anacystis. Activity units correspond to /Lmol of nitrite produced per min. The values are the means of three experiments ± SD. Nitrate reductase activity (mU mg protein -I) Control 100 mg B I-I
Anabaena 7119
Anacystis nidulans
7.1±0.8 3.7±0.4
6.1±0.6 3.2±0.5
baena and Anacystis was inhibited by 47 % after 72 h of culture under high boron concentrations. These data clearly show that, in blue-green algae, the effect of boron toxicity on nitrogen metabolism preceded the alteration of the photosynthesis. Nitrate uptake was found to be inhibited whithin the first hour of culture. This inhibition of nitrate uptake was followed by a decrease of nitrate reductase activity and by symptoms of nitrogen starvation such as loss of phycobiliproteins and chlorophyll and increase of carbohydrates (Allen and Smith, 1969). The impairment of photosynthesis appeared at a later stage of boron toxicity and probably was a secondary effect of the nitrogen starvation caused by the primary effect of nitrate uptake.
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P/rysiol. Vol. 128. pp. 161-168 (1987)
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Recently, much attention has been paid to the influence of boron on membrane integrity and function_ Tanada (1974, 1978, 1983), Pollard et al. (1977), Smyth and Dugger (1980, 1981), Parr and Loughman (1983) and Goldbach (1985) have shown that boron deficiency affects the permeability of the plasmalemma and the translocation of nutrients within the plant- Our results suggest that boron toxicity could also lead to an impairment of membrane permeability which would result in an inhibition of nitrate uptake. References ALLEN, M. M. and A. J. SMITH: Nitrogen chlorosis in blue-green algae. Arch. Microbiol. 69, 114-120 (1969). ALLEN, M. M. and P. WEATHERS: Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308. J. Bacteriol. 141, 959-962 (1980). BLUMWALD, E. and E. TEL-OR: Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum. Arch. Microbiol. 132, 168-172 (1982). BONILLA, I., C. CADAHIA, O. CARPENA, and V. HERNANDO: Effects of boron on nitrogen metabolism and sugar levels of sugar beet. Plant Soil 57, 3 - 9 (1980). BONILLA, I., P. MATEO, A. GARATE, E. FERNANDEZ, and E. SANCHEZ: Effect of boron on nitrate reductase activity and sugar levels in Lycopersicon esculentum. VI. Int. ColI. Optim. Plant Nutrition 1, 77 - 83 (1984). CARPENA, 0., V. HERNANDO, C. CADAHIA, and I. BONILLA: Effects of boron on the activity on the nitrate reductase in sugar beet. VIII Int. Coll. Plant Anal. Fert. Probl. New Zealand, pp. 83-90 (1978). CAWSE, P. A.: The determination of nitrate in soil solution by ultraviolet spectrophotometry. Analyst 92, 311 (1967). DUBOIS, M., R. A. GILLES, J. K. HAMILTON, P. A. ROBERS, and F. SMITH: Colorimetric method for determination of sugar and related substances. Anal. Chern. 28, 350-356 (1956). FLORES, E., M. G. GUERRERO, and M. LOSADA: Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch. Microbiol. 128, 137-144 (1980). FOLCH, J., M. LEES, and G. H. SLOANE STANLEY: A simple method from the isolation and purification of total lipids from animal tissues. J. BioI. Chern. 226, 497 -509 (1957). GOLDBACH, H.: Influence of boron nutrition on net uptake and efflux of 32p and 14C-glucose in Heliantus annuus roots and cell cultures of Daucus carota. J. Plant Physiol. 118,431-438 (1985). GUPTA, V. c.: Boron nutrition of crops. Adv. Agron. 31, 273-315 (1979). HERRERO, A., E. FLORES, and M. G. GUERRERO: Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119 and Nostoc sp. strain 6719. J. Bacteriol. 145, 267-271 (1981). KRATZ, W. A. and J. MYERS: Nutrition and growth of several blue-green algae. Amer. J. Bot. 42, 282-287 (1955). LEHMANN, M. and G. WOBER: Accumulation, mobilization and turnover of glycogen in the blue-green bacterium Anacystis nidulans. Arch. Microbiol. 111,93-97 (1976). LOVATT, C. J. and L. M. BATES: Early effects of excess boron on photosynthesis and growth of Cucurbita pepo. J. Exp. Bot. 35,297 -305 (1984). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARE, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193,265-275 (1951). MARKER, A. F. M.: The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin. Freshwater BioI. 2, 361-385 (1972). MENGEL, K. and E. A. KIRKBY: Principles of plant nutrition pp. 483 - 494. Int. Potash Inst., Bern, Switzerland (1982).
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PARR, A. ]. and B. C. LOUGHMAN: Boron and membrane function in plants. In: Metals and micronutrients uptake and utilization by Plants. pp. 87 -107. ROBB, D. A. and D. A. PIERPOINTS (Eds.) W. S. Academic Press (1983). POLLARD, A. S., A. D. PARR, and B. C. LOUGHMAN: Boron in relation to membrane function in higher plants. J. Exp. Bot. 28, 831-841 (1977). REISENAUER, H. M., L. M. WALSH, and R. G. HOEFT: Testing soils for sulphur, boron, molybdenum, and chlorine. In Soil Testing and Plant Analysis. pp. 173-200. WALSH, L. M. and]. D. BEATON (Eds.). Soil Science Society Annual, Madison, Wisconsin (1973). SANCHEZ, MAEso, E., E. FERNANDEZ VALIENTE, I. BONILLA, and P. MATEO: Accumulation of proteins in giant cells induced by high boron concentrations in Chlorella pyrenoidosa. ]. Plant Physiol.121, 301-311 (1985). SHORROCKS, V. M.: Boron deficiency - Its prevention and cure p. 56. Borax Consolidated Limited, London (1974). SIMON, R. D. and P. WEATHERS: Determination of the structure of the novel which is found in cyanobacteria. Biochem. Biophys. Acta 420, 165-176 (1976). SMYTH, D. A. and W. M. DUGGER: Effect of boron deficiency on 86Rubidium uptake and photosynthesis in the diatom Cylindroteca /usiformis. Plant Physiol. 66, 692 - 695 (1980). - - Cellular changes during boron-deficient culture of the diatom Cylindroteca /usiformis. Physiol. Plant. 51,111-117 (1981). TANADA, T.: Boron-induced bioelectric field change in mung bean hipocotyl. Plant Physiol. 53, 775-776 (1974). - Boron-key element in the actions of phytochrome and gravity? Planta 143, 109-111 (1978). - Localization of boron in membranes.]. Plant Nutr. 6, 743-749 (1983).
J Plant. Physiol. Vol. 128.pp. 161-168 (1987)
Summary The effect of excess boron on growth, cell composition, photosynthesis, and nitrogen metabolism, in two species of blue green algae (cyanobacteria), Anabaena PCC 7119 and Anacystis nidulans, was examined. High concentrations of boron in culture media inhibited growth and decreased the contents of phycobiliproteins and chlorophyll. In addition, an accumulation of carbohydrates and a decrease in lipids were observed in both species. Parallel to the fall in photosynthetic pigments, an inhibition of photosynthetic oxygen evolution was observed, after 72 h of culture, in Anabaena PCC 7119. However, initial exposure of the cultures to high concentrations of boron did not affect photosynthesis. In contrast, nitrate uptake decreased in both species with initial exposure to excess concentrations of boron. This inhibition was followed by a decrease in the nitrate reductase activity.
Key words: Anabaena; Anacystis; Boron toxicity; Nitrate uptake; Photosynthesis.
Introduction In higher plants, the range in boron concentration optimal for growth is very narrow, approximately 0.01 to 4mgBl-' (Gupta, 1979). Therefore, the ratio of toxic to adequate levels of boron is smaller than the ratio in any other micronutrient (Reisenauer et aI. 1973) with the toxic effects appearing at concentrations close to the normal requirement of boron (Mengel and Kirkby, 1982). There are many reports stating the symptoms of boron toxicity on plants. These symptoms are reported to be similar in most plants, and consist of a marginal and tip chlorosis which is quickly followed by a necrosis (Shorrocks, 1974). However, there is very little information regarding the actual manner in which boron is toxic to plants. In previous studies with sugar beet we have shown that boron toxicity resulted in an accumulation of nitrates in the sap and in a decrease of the nitrate reductase activity (Carpena et al., 1978; Bonilla et aI., 1980). Similar results have also been obtained in studies with tomato (Bonilla et aI., 1984). Other researches have linked boron toxicity with photosynthesis, Lovatt and Bates (1984) concluded in studies with Cucurbita pepo that an excess of boron lowered the chlorophyll contents and the photosynthetic activity in leaves.
J Plant.
Physiol. Vol. 128. pp. 161-168 {1987}
162
P. MATEO, F. MARTINEZ, L BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAEso
In Chlarella pyrenaidasa, however, boron toxicity did not appear to affect the chlorophyll contents although there was an inhibition of the photosynthesis which was explained by an alteration in the organization of the chloroplasts (Sanchez Maeso et aI., 1985). However, the preceding data do not allow any conclusions about the actual manner in which boron is toxic to plants. In this report we report our studies on the effect of high concentrations of boron on the growth, cell composition, photosynthesis, and nitrogen metabolism of two blue-green algae (cyanobacteria): Anabaena pee 7119 and Anarystis nidulans. Data are presented indicating a primary effect of boron toxicity on nitrate uptake and assimilation. Materials and Methods Organisms and culture conditions Anabaena PCC 7119, previously classified as Nostoc muscorum, was a gift of Dr. Rivera, University of Sevilla. Anacystis nidulans was obtained from Prof. Dr. Rodriguez-Lopez, C.S.LC., Madrid. Batch cultures were grown routinely at 26°C in a Kratz and Myers C medium (1955), gassed with air containing 2 % CO2 and illuminated with cool-white fluorescent lamps (lOW m -2). Where appropriate, boron was added as boric acid at concentrations of 50,75, and 100mgBI- I . Culture density was determined at 600 nm using a Beckman DB-GT spectrophotometer. Cells were filtered (using a 0.45 JLm filter paper) or centrifuged and dried at 70°C for 24 h to determine dry weight. Analytical methods
Protein was determined by the method of Lowry et aL (1951) in extracts obtained by treatment of cells with 1 N NaOH at 80°C for 1 h using serum albumin as standard. Carbohydrates were estimated according to Dubois et al. (1956) after extraction with 2N HCI at 100°C for 1 h. Lipids were determined gravimetrically after extraction by the method of Folch et al. (1957). Chlorophyll was estimated according to Marker (1972). Phycobiliproteins were determined in aqueous extracts of cells treated with toluene at 260 nm (1 O.D. = 135 phycobiliproteins) (Blumwald ant Tel-Or, 1982). Photosynthesis
Oxygen evolution was measured with a Clarktype oxygen electrode (Hach Chemical Company). Aliquots of 3 ml with a cell density of 0.1 mgml- I were placed in a temperature-controlled cuvette and illuminated with a quantum flux density of 300 JLE m - 2S - I. Electron Microscopy
Cells were fixed with glutaraldehyde 2 %, and post-fixed with KMn04, 2 %. Dehydration was carried out with water-acetone solutions. Samples, embedded in Vestopal, were sectioned and stained with uranyl acetate and observed in a Phillips EM 300. Nitrate uptake
Nitrate uptake was assayed by measuring the disappearance of nitrate from the medium of culture or in short-term experiments.
J Plant.
Physiol. Vol. 128. pp. 161-168 {1987}
Boron toxicity in blue-green algae
163
For determination of nitrate in the medium, cells were removed from culture media samples by centrifugation and aliquots of the media were used for ion determination. Nitrates were measured with an ionanalyzer (Mod. 901 Orion Research) using Orion ionselective electrodes (Mod. 9307 Orion Research). Short-term experiments were carried out by the method of Flores et al. (1980). Cells from 2 or 3-day-old cultures (density about 0.5mgml- l ) were used. After harvesting by filtration, the cells were washed with 25mM Tricine-NaOH buffer pH 8.3, and finally resuspended in the same buffer to a density of 1 mg ml- I . Uptake assays were carried out with continuous shaking in the light (lOW m -2, white light) at 26°C, in conical flasks open to the air, and were started by the addition of KN0 3 (0.4 mM final concentration) to cell suspensions which had been preincubated for 10 min under the above conditions. When the effects of high concentrations of boron were tested, boron was present along the preincubation period previous to the addition of nitrate. Nitrate uptake was determined following the disappearance of nitrate from the assay mixture by estimating the concentration of the ion in aliquots of the cell suspension after rapid removal of the cells by filtration. Nitrate was determined by optical absorption at 210nm in acid solution, according to Cawse (1967). Determination of Nitrate Reductase Activity
The cellular nitrate reductase was assayed in situ in toluene-permeabilized cells according to the method of Herrero et al. (1981).
Results and Discussion Batch cultures of Anabaena pee 7119 exposed to a growth medium containing 50, 75, or 100 mg B .1- 1, reduced their growth and showed a drastic drop in the contents of nitrogenous compounds such as proteins, chlorophyll, and phycobiliproteins when compared with control cultures (Fig. 1). In addition the cells exposed to high boron concentrations showed a decrease in lipid contents and an increase in carbohydrates (Fig. 2). Similar results were obtained with the unicellular strain Anacystis nidulans (Table 1). The photosynthetic activity of the cells of Anabaena exposed to high boron concentrations showed no change during the first hours of culture (Table2). Long exposure to excess boron, however, resulted in a decrease of the photosynthetic oxygen evolution (Table 2) and in an alteration of the normal arrangement of thylakoids (Fig. 3). These data are consistent with the loss of the photosynthetic pigments shown by Anabaena and Anacystis and indicate that the increase of carbohydrates cannot be explained by an enhancement of its synthesis, but by an inhibition of its degradation (Lehmann and Wober, 1976). In addition to the change of the photosynthetic apparatus, the cells of Anabaena exposed to high boron concentrations showed (Fig.3) structured granules which resembled the granules of cyanophycin, a polymer of L-arginine and L-aspartic acid (Simon and Weathers, 1976), characteristic of cyanobacteria, formed from the products of catabolism of cell protein (Allen and Weathers, 1980), and that appeared under a number of environmental conditions and particularly in older cultures. On the other hand, the cells of Anabaena and Anacystis cultured under high boron concentrations showed an alteration in nitrate assimilation: For Anabaena cells, the uptake of nitrates was inhibited during the first hours of culture under excess boron
J Plant.
Physiol. Vol. 128. pp. 161 -168 (1987)
P. MATEO, F. MARTINEZ, 1. BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAESO
164
Fig. 1: Effect of high boron concentrations on the growth, and protein, chlorophyll, and phycoControl, 0 50mgBI-l, 6 biliprotein contents of Anabaena PCC 7119. • 75mgBI- 1 D100mgBI-I.
40
20
~ 30
5
I<{
o~ If)
a:
10~ n... .....
o >:r o ~ 10
-.J
5
<{
u
a
Fig. 2: Carbohydrate and lipid content (in % of dry weight) at 96 h of culture in Anabaena cells grown under different B concentrations. • Carbohydrates, 0 Lipids.
50 75 100 mg B 1-1
Table 1: Biochemical composition of Anacystis nidulans cells after 72 h of culture. Values, expressed in % of cell dry weight, are the means of five experiments ± SD.
J Plant.
Constituents
Control
100mg B 1-1
Proteins Chlorophyll Carbohydrates Lipids Nucleic acids
57. 1± 5.2 2.1±O.1 15.5± 1.3 10.7±O.9 12.0± 1.2
26.7±5.3 O.9±O.1 46.6±2.5 7.0±O.8 12.9±2.4
Physiol. Vol. 128.pp. 161-168(1987)
Boron toxicity in blue-green algae
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Table 2: Effect of high boron concentrations on photosynthetic oxygen evolution of cells of Anabaena PCC 7119. The values are the means of four experiments ± SD. /Lmol O 2 evolved mg Chl- I h 2
Control 100 mg B I-I
h
72
157.3±12.3 151.7± 9.1
I
h
152.5± 11.8 5.6± 3.1
B Fig.3: Electron micrographs of Anabaena PCC 7119. A) Cell, in control medium. B) Cell, grown in 100 mg B 1- I. Bar represents 1 /Lm.
as the results of short-term experiments presented in Fig. 4 show. Similar results were obtained in Anacystis by measuring the disappearance of nitrates from the medium in long-term experiments (Fig. 5). In addition, the nitrate reductase activity was affected by boron toxicity. As shown in Table3, the nitrate reductase activity of both Anaj. Plant. Physiol. Vol. 128. pp. 161-168 (1987)
166
P. MATEO, F. MARTINEZ, 1. BONILLA, E. FERNANDEZ VALIENTE, and E. SANCHEZ MAESO
L
2
o
---~
400
w L
........
.."....
.....
~ ~200 w ::t
I- -
«
0:::
I-
Z
a
90
150
Fig. 4: Effect of high boron concentrations on nitrate uptake of Anabaena PCC 7119 .• Control, o 100mgBI- I.
210
TIME[min.)
o
2
3
Fig. 5: Time-course of nitrate utilization by Anacystis nidulans grown under high boron concentrations .• Control, 0 100mgBI- I.
4
DAYS
Table 3: Effect of high boron concentrations on cellular nitrate reductase activity in Anabaena and Anacystis. Activity units correspond to /Lmol of nitrite produced per min. The values are the means of three experiments ± SD. Nitrate reductase activity (mU mg protein -I) Control 100 mg B I-I
Anabaena 7119
Anacystis nidulans
7.1±0.8 3.7±0.4
6.1±0.6 3.2±0.5
baena and Anacystis was inhibited by 47 % after 72 h of culture under high boron concentrations. These data clearly show that, in blue-green algae, the effect of boron toxicity on nitrogen metabolism preceded the alteration of the photosynthesis. Nitrate uptake was found to be inhibited whithin the first hour of culture. This inhibition of nitrate uptake was followed by a decrease of nitrate reductase activity and by symptoms of nitrogen starvation such as loss of phycobiliproteins and chlorophyll and increase of carbohydrates (Allen and Smith, 1969). The impairment of photosynthesis appeared at a later stage of boron toxicity and probably was a secondary effect of the nitrogen starvation caused by the primary effect of nitrate uptake.
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Recently, much attention has been paid to the influence of boron on membrane integrity and function_ Tanada (1974, 1978, 1983), Pollard et al. (1977), Smyth and Dugger (1980, 1981), Parr and Loughman (1983) and Goldbach (1985) have shown that boron deficiency affects the permeability of the plasmalemma and the translocation of nutrients within the plant- Our results suggest that boron toxicity could also lead to an impairment of membrane permeability which would result in an inhibition of nitrate uptake. References ALLEN, M. M. and A. J. SMITH: Nitrogen chlorosis in blue-green algae. Arch. Microbiol. 69, 114-120 (1969). ALLEN, M. M. and P. WEATHERS: Structure and composition of cyanophycin granules in the cyanobacterium Aphanocapsa 6308. J. Bacteriol. 141, 959-962 (1980). BLUMWALD, E. and E. TEL-OR: Osmoregulation and cell composition in salt-adaptation of Nostoc muscorum. Arch. Microbiol. 132, 168-172 (1982). BONILLA, I., C. CADAHIA, O. CARPENA, and V. HERNANDO: Effects of boron on nitrogen metabolism and sugar levels of sugar beet. Plant Soil 57, 3 - 9 (1980). BONILLA, I., P. MATEO, A. GARATE, E. FERNANDEZ, and E. SANCHEZ: Effect of boron on nitrate reductase activity and sugar levels in Lycopersicon esculentum. VI. Int. ColI. Optim. Plant Nutrition 1, 77 - 83 (1984). CARPENA, 0., V. HERNANDO, C. CADAHIA, and I. BONILLA: Effects of boron on the activity on the nitrate reductase in sugar beet. VIII Int. Coll. Plant Anal. Fert. Probl. New Zealand, pp. 83-90 (1978). CAWSE, P. A.: The determination of nitrate in soil solution by ultraviolet spectrophotometry. Analyst 92, 311 (1967). DUBOIS, M., R. A. GILLES, J. K. HAMILTON, P. A. ROBERS, and F. SMITH: Colorimetric method for determination of sugar and related substances. Anal. Chern. 28, 350-356 (1956). FLORES, E., M. G. GUERRERO, and M. LOSADA: Short-term ammonium inhibition of nitrate utilization by Anacystis nidulans and other cyanobacteria. Arch. Microbiol. 128, 137-144 (1980). FOLCH, J., M. LEES, and G. H. SLOANE STANLEY: A simple method from the isolation and purification of total lipids from animal tissues. J. BioI. Chern. 226, 497 -509 (1957). GOLDBACH, H.: Influence of boron nutrition on net uptake and efflux of 32p and 14C-glucose in Heliantus annuus roots and cell cultures of Daucus carota. J. Plant Physiol. 118,431-438 (1985). GUPTA, V. c.: Boron nutrition of crops. Adv. Agron. 31, 273-315 (1979). HERRERO, A., E. FLORES, and M. G. GUERRERO: Regulation of nitrate reductase levels in the cyanobacteria Anacystis nidulans, Anabaena sp. strain 7119 and Nostoc sp. strain 6719. J. Bacteriol. 145, 267-271 (1981). KRATZ, W. A. and J. MYERS: Nutrition and growth of several blue-green algae. Amer. J. Bot. 42, 282-287 (1955). LEHMANN, M. and G. WOBER: Accumulation, mobilization and turnover of glycogen in the blue-green bacterium Anacystis nidulans. Arch. Microbiol. 111,93-97 (1976). LOVATT, C. J. and L. M. BATES: Early effects of excess boron on photosynthesis and growth of Cucurbita pepo. J. Exp. Bot. 35,297 -305 (1984). LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARE, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193,265-275 (1951). MARKER, A. F. M.: The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin. Freshwater BioI. 2, 361-385 (1972). MENGEL, K. and E. A. KIRKBY: Principles of plant nutrition pp. 483 - 494. Int. Potash Inst., Bern, Switzerland (1982).
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PARR, A. ]. and B. C. LOUGHMAN: Boron and membrane function in plants. In: Metals and micronutrients uptake and utilization by Plants. pp. 87 -107. ROBB, D. A. and D. A. PIERPOINTS (Eds.) W. S. Academic Press (1983). POLLARD, A. S., A. D. PARR, and B. C. LOUGHMAN: Boron in relation to membrane function in higher plants. J. Exp. Bot. 28, 831-841 (1977). REISENAUER, H. M., L. M. WALSH, and R. G. HOEFT: Testing soils for sulphur, boron, molybdenum, and chlorine. In Soil Testing and Plant Analysis. pp. 173-200. WALSH, L. M. and]. D. BEATON (Eds.). Soil Science Society Annual, Madison, Wisconsin (1973). SANCHEZ, MAEso, E., E. FERNANDEZ VALIENTE, I. BONILLA, and P. MATEO: Accumulation of proteins in giant cells induced by high boron concentrations in Chlorella pyrenoidosa. ]. Plant Physiol.121, 301-311 (1985). SHORROCKS, V. M.: Boron deficiency - Its prevention and cure p. 56. Borax Consolidated Limited, London (1974). SIMON, R. D. and P. WEATHERS: Determination of the structure of the novel which is found in cyanobacteria. Biochem. Biophys. Acta 420, 165-176 (1976). SMYTH, D. A. and W. M. DUGGER: Effect of boron deficiency on 86Rubidium uptake and photosynthesis in the diatom Cylindroteca /usiformis. Plant Physiol. 66, 692 - 695 (1980). - - Cellular changes during boron-deficient culture of the diatom Cylindroteca /usiformis. Physiol. Plant. 51,111-117 (1981). TANADA, T.: Boron-induced bioelectric field change in mung bean hipocotyl. Plant Physiol. 53, 775-776 (1974). - Boron-key element in the actions of phytochrome and gravity? Planta 143, 109-111 (1978). - Localization of boron in membranes.]. Plant Nutr. 6, 743-749 (1983).
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