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Response of a rice field cyanobacterium Anabaena sp. to physiological stressors
Erznimmtiand~iBa~y,
Vol. 36,No. 2,pp. 147-155, 1996 Cootiht 0 1996 F&via Science B.V.
SOO98-8472(96)01007-6
RESPONSE
OF A RICE FIELD CYANOBACTERIUM PHYSIOLOGICAL STRESSORS RlyESHWAR
P. SINI-IA and DONAT-P.
RNABA&l’X SP. TO
JhiDER*
Institut fur Botanik und Pharmazeutische Biologic, Friedrich-Alexander-Universitat, Staudtstr. 5, D-9 1058 Erlangen, Germany (Received 5 Septmber 1995; accepted in revtiedfom
28 December 1995)
Sinha R.P. and Hader D.-P. Respollseof a tiefield yanobacteium Anabaena up.tophysiological stressors. Environmental and Experimental Botany 36,147-155,1996. - The impact ofphysiological factors such as heat, sahnity and L-methionine-DL-sulfoximine (MSO) on growth and total protein profile was studied in a rice field cyanobacterium, Anabaaa sp. There was a gradual decrease in growth rate of the organism with increase in incubation time at 47%; by 168 h of incubation there was essentially no growth. NaCl concentration > 5 mM inhibited growth of the organism, and there was apparently no growth at >200 mM NaCl-supplemented media. Similarly, growth was inhibited with MS0 concentration >5 PM and there was no growth in cultures treated with > 100 PM MSO. SDSPAGE protein profile after heat stress revealed a decline in the synthesis of several proteins but at the same time, synthesis of a new set of proteins of approximately 60-65 kDa was induced after 12 and 24 h of incubation and the same was completely eliminated after 96 h of incubation. Cultures treated with 10 mM of NaCl did not show any change in the protein pattern. In contrast, 100 mM NaCl treated cultures elicited a number of new proteins at around 29, 32, 40 and 70 kDa. Most of the protein bands disappeared in the cultures treated with 500 mM NaCl. Cultures treated with 10 PM MS0 did not show any change in the appearance of the protein bands, whereas the cultures treated with 100 PM MS0 showed a drastic decline in the intensity of all protein bands. The results indicate that different stressors exert specific effects on cyanobacterial protein synthesis. Keyworu!s: Anabaena, cyanobacteria, growth, heat stress, L-methionine-DL-sulfoximine (MSO) treatment, protein profile, sahnity stress.
system for analysing the adaptive mechanisms developed in response to changing stress conditions.(26) The physical environment of an organism is not constant; fluctuations occur in time and space. However, organisms have evolved to
INTRODUCTION
Stressors such as heat, cold, salinity and drought play a key role in determining the growth and development of cyanobacterial populations in their particular habitat.(s,28) Being cosmopolitan in clistribution, cyanobacteria are thought to have been exposed to different levels and types of stressors during their development, thus providing a suitable
survive such conditions and show adaptation to the stressor( Each stress variable usually has a minimum and a maximum level beyond which an organism cannot survive.(‘5)
*Author to whom correspondence should be addressed. 147
148
R. P. SINHA and D.-P. H;iDER
There is also some interaction between the severity of a stressor and its duration. A long exposure at a moderately high temperature may be as injurious as a brief exposure to an extreme temperature. Temperature fluctuations in the environment can occur over a matter of minutes or seasons. Longterm environmental changes may also occur, such as global warming that is suggested to occur because of increased greenhouse gas emissions.(“) It has been postulated that particular proteins whose synthesis is induced by stress conditions are critical to the survival of the organism. Changes in gene activation, transcription, and translation often occur during the acclimation process and thus are thought to be involved in the induction of tolerance. It is a common phenomenon that above a threshold temperature the normal pattern of protein synthesis is repressed and a new set of proteins is synthesized from newly transcribed mRNA, called heat-shock proteins (HSPs). (‘Sag)There are a number of major families of HSPs, classified on the basis of their molecular weight; high molecular weight HSPs (80 kDa), the HSPs 70 family (molecular weight approximately 70 kDa) and low molecular weight HSPs (14-30 kDa). The heat shock response is finely tuned to its thermal adaptation.(“) Like other organisms, cyanobacteria have been reported to possess a heat shock response.(3,4p’g) Salinity has become an ever-increasing problem in irrigated agriculture. Certain bacterial strains such as Rhizobium, which nodulate a number of economically important crops, are highly sensitive to NaC1.(31) Cyanobacterial N,-fixation is supposed to be the most sensitive process in response to enhanced salt concentrations, followed by photosynthesis; respiration has been reported to be increased in salt-adapted cyanobacteria.(2g’33) Decreased photosynthesis and Chl a contents have been reported in Microcys&j?ma and Synechocystis sp. PCC 6803(7) following salinity stress. NJixing cyanobacteria are being used as nitrogen biofertilizers in rice paddy fields in several countries where rice is the major staple dietc3’) A number of strains which thrive in rice fields release small of the major fertilizing product, quantities ammonia, during their active growth, while most of the fixed product is made available to the plants mainly through autolysis and microbial decomposition. Under these circumstances, it is difficult to control the flow of nitrogen compounds needed for
the development of the rice plants. To overcome this problem, MSO, which inhibits the primary ammonia assimilating enzyme, glutamine synthetase (GS), has been reported to be extensively used for the sustained release of ammonia. High concentrations of MS0 might be toxic for the growth of cyanobacteria, and hence changes in total protein profile are expected.(30) In the present investigation, an attempt was made to study the effects of heat, salinity and MS0 on growth and total protein profile of Anabaena sp. isolated from an Indian rice field. During the hot summer season, cyanobacterial populations in Indian rice paddy fields are exposed to high temperatures (47-48%) for several weeks. The above mentioned stressors either alone or in combination may have a detrimental effect on cyanobacterial populations in the tropical rice paddy fields. Any adverse impact on N,-fixing cyanobacteria will affect the productivity of higher plants because N2fixing capacities and photosynthetic abilities in one organism are unique to cyanobacteria only. This prompted us to search for the possible effects on the growth pattern as well as on total protein profile of Anabaena sp. This strain was selected for study largely due to its widespread occurrence in the Indian rice paddy fields, having a fast growing rate as well as an exceedingly high amount of pigment proteins. MATERIALS
AND METHODS
Isolation, punjfication and growth conditions of the organism The heterocystous and filamentous N,-fixing cyanobacterium Anabaena sp. was isolated from a rice paddy field near Varanasi, India. The specimen collected from the fields was thoroughly washed by double distilled water and, after mild homogenisation, spread on solid agar (Difco-Bacto; 1.5%) in Petri dishes (75 mm) containing different synthetic media such as Allen and Amon’s, BG-1 l(‘l) or modified Chu-lO(**) and incubated in fluorescent white light (14.4 W m-2) at 27°C. After 10-12 days of incubation, minute colonies started appearing on the agar plates. Well spread individual colonies were isolated and restreaked on fresh agar plates. In parallel, individual colonies were transferred into 10 ml of the above mentioned liquid media in a test tube and incubated in continuous light. After 7-l 0 days of incubation, cultures growing in liquid media were harvested by centrifugation ( 1500s for 10 min)
STRESS RESPONSES and the resulting pellet was washed, suspended in a minimum volume of liquid media and the filaments were broken by gentle shaking with sterilized glass beads. The homogeneous suspension containing mostly individual cells or short filaments was spread on solid agar Petri plates of the above mentioned synthetic media. Individual cells were marked and after 7-10 days of growth, colonies appearing on agar plates were examined microscopically and retransferred onto solid agar Petri plates. Restreaking and subculturing were repeated several times to obtain a single colony apparently free from contamination. These colonies were manually isolated and routinely grown in liquid medium. Out of all the media tested, Anabaena sp. showed its best growth in the modified Chu-10 medium.@‘) Cultures were routinely grown in an autoclaved modified Chu- 1O(“) liquid medium (devoid of any nitrogen sources) in Erlenmeyer flasks filled to 40% of their nominal volume and placed in a culture room at 27°C and illuminated with white fluorescent light of 14.4 W m-‘. Heat, salin@ and MS0 (L-methionine-DL-suyoximine) treatment Exponentially growing (7-l 0 day old) suspension cultures of Anabozna sp. were harvested by centrifugation (J2-21M/E) using a JA 20 rotor (Beckman Instruments, Palo Alto, CA) at 15008 for 10 min at room temperature, and transferred into fresh Chu-10’22’ liquid medium. The culture was then placed in an incubator at a temperature of 47°C and an irradiance of 14.4 W m-‘. At predetermined intervals, aliquots (2 ml) were withdrawn and growth as well as total protein profile was analysed. A separate control was maintained at a temperature of 27°C and an irradiance of 14.4 W m-‘. Cultures were also inoculated with various concentration of NaCl and/or MS0 and placed in a culture room at a temperature of 27°C and an irradiance of 14.4 W m-*. At predetermined intervals, aliquots (2 ml) were withdrawn and subjected to growth measurement as well as SDS-PAGE analyses. Even though being thermotolerant, the cells grew equally well at 27°C. Gowth estimation Growth of the organism was ascertained either by measuring Chl a content or by estimating protein content. For extracting Chl a, a known volume of
IN ANABAENA SP.
149
culture was centrifuged and the pellet was resuspended in a known volume of 80% acetone, shaken for 5 min and kept overnight in a refrigerator at 4-V. The resulting suspension was centrifuged and the supernatant was used for the estimation of Chl a content. The concentration of protein was determined by the method of Bradford.@) Bovine serum albumin was used as a standard. Absorption spectroscopy Acetone soluble Chl a was transferred into a quartz cuvette having an optical path length of 10 mm (2 mm thickness, Hellma, Mullheim, Germany): absorption spectra were measured in a single beam DU 70 spectrophotometer (Beckman, Palo Alto, CA). The raw spectra were transferred to a microcomputer and treated mathematically and statistically using the software provided by the manufacturer (Beckman, Palo Alto, CA). Gradient gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was undertaken in a vertical system (200 1, Pharmacia, LKB, Uppsala, Sweden) with gels of 155 x 130 mm, 1.5 mm thick, using the method of Laemmli(‘3) with a gradient (55 15% T) in the resolving gel. Gels were stained with Coomassie brilliant blue R 250 and dried in a gel dryer (Bio-Rad, Richmond, CA). Experiments were performed at least 3 times. RESULTS Eflects of heat, salin@ and MS0 on the growth of Anabaena sp. Fig. 1 shows the spectra of acetone soluble pigments after the growth of Anabaena sp. at normal (27%) and elevated (47°C) temperatures. It is evident from the spectra that when cultures were grown at normal growth temperature (27“C), there was no change in the absorption up to 24 h; thereafter, there was a linear increase in absorption with increasing incubation time, the maximum being at 168 h. The cultures growing at an elevated temperature (47°C) showed a gradual loss in absorption with increasing incubation time, and by 168 h all peaks disappeared, denoting that there was no growth in the culture at 47%. The growth response of the test organism to various concentrations of NaCl is illustrated in Fig. 2.
150
R. P. SINHA and D.-P. I-L%DER
0.2
0.1
0
0.0 350
400
450
500
550
so0
aia
Wavelength [nm]
There was no significant difference in the growth of the organism treated with 2 mM NaCl in com-
parison with the control culture (without NaCl). Growth was inhibited at NaCl concentration >5 mM. There was apparently no growth at 200 and 500 mM NaCl-supplemented media in any of the replicate experiments. Growth of Anabama sp. at varying concentration of MS0 is illustrated in Fig. 3. Growth was severely inhibited in cultures treated with MS0 up to 3 days; thereafter, growth resumed in cultures containing up to 5 PM MSO. There was no significant difference in final growth yield in the control and 250 N8CI 2mM OmM
5mM
10 mm 100mM 2WmM 5WmM 0
( 0
I 1
2
5
4
5
5
7
Time [d]
Fig. 2. Growth of Anabaem sp. at various concentration of NaCl at a normal growth temperature of 27°C and an irradiance of 14.4 W m-‘. Data are for three separate but identical experiments + S.D.
2
3
4
5
6
7
Time [d]
700
Fig. 1. Absorption spectra showing the growth ofAnabaena sp. under control (27%) and elevated (47%) temperature conditions after increasing exposure times. Data are for three separate but identical experiments.
I 1
Fig. 3. Growth of Anabaem sp. at various concentration of MS0 at a normal growth temperature of 27“C and an irradiance of 14.4 W m-*. Data are for three separate but identical experiments + S.D.
in cultures treated with 2 PM MSO. Growth was inhibited with MS0 concentration 5 PM. There was no growth in cultures treated with 100 PM MSO.
ofhat, salinip and MS0 on the protein pro& of Anabaena 4. The SDS-PAGE protein profile of Anabama sp. after heat stress is illustrated in Fig. 4. It is evident from the electrophoretic pattern that exposure to heat stress resulted in alterations in cyanobacterial protein synthesis. Synthesis of several proteins declined with increasing exposure duration to 47°C. At the same time, synthesis of a new set of proteins was induced. Exposure at 47% for 12 h resulted in a decline in protein bands between 16 and 22 kDa which represent the monomers of phycocyanin (lane 4). There was further loss in these protein bands at 48 h of exposure (lane 6); these bands were completely lost after 96 h of incubation (lane 8). Synthesis of a new set of proteins of approximately 6Ck65 kDa was observed after 12 and 24 h of incubation at 47°C (lanes 4 and 10, E8ect.s
respectively). However, these protein bands started to disappear after 48 h and were completely eliminated after 96 h of incubation (lanes 6 and 8). One very prominent band of approximately 55 kDa was less affected than the rest of the protein bands even after 96 h of incubation at 47% (lane 8). Cultures were supplemented with various concentrations (2-500 mM) of NaCl, and after 72 h of growth were subjected to SDS-PAGE analyses (Fig.
STRESS RESPONSES
IN A&m
151
SP.
12345678910
MW (kDa)
Fig. 4. Vertical SDS-PAGE (gradient 5-15% T) protein profile ofAnabaena sp. with increasing time period of heat treatment. Lane 1: marker proteins (Sigma, MWS-877 L), lane 2: control (27°C; 0 h), lane 3: control (12 h), lane 4: 47°C (12 h), lane 5: control (48 h), lane 6: 47% (48 h), lane 7: control (96 h), lane 8: 47% (96 h), lane 9: control (24 \ h) andlane 10: 47’C (24 h). Equal amounts ofproteins were loaded into each well. I
1234
Fig. 5. Vertical SDS-PAGE (gradient 5-15% T) protein profile of An&mu sp. with increasing concentrations of NaCl after 72 h of growth. Lane 1: control, lane 2: 2 n% lane 3 5 mM, lane 4: 10 mM, lane 5: 100 mM> lane 6: 200 mM, lane 7: 500 mM and lane 8: marker proteins (Sigma, MWS-877 L). Equal amounts ofproteins were loaded into each well.
56
7
8
9
Fig. 6. Vertical SDS-PAGE (gradient 5-15% T) protein proiile of Anabama sp. with increasing concentrations of MS0 after 72 h of growth. Lane 1: marker proteins (Sigma, Mb%877 L), lane 2: control, lane 3: 1 PM, lane 4: 2 PM, lane 5: 5 PM, lane 6: 10 PM, lane 7: 100 PM, lane 8: 200 m, and lane 9: 500 PM. Equal amounts of proteins were loaded into each well.
STRESS RESPONSES IN AJviUMNA SF’. 5). Cultures treated with 2 mM NaCl showed more intense protein bands in comparison with control cultures (without NaCl). Cultures treated with 5 and 10 mM NaCl had a protein pattern identical to that of the 2 mM NaCl-treated cultures. In contrast, 100 r&I NaCl-treated cultures showed a number of new proteins at around 29, 32, 40 and 70 kDa. There was a drastic decline in the intensity of all the protein bands in the cultures treated with 200 mM NaCl. The majority of protein bands disappeared in the cultures treated with 500 mM NaCI. The SDS-PAGE protein profile of Anabma sp. grown at various concentration of MS0 is presented in Fig. 6. It is evident from the results that there was no difference in the appearance of the protein bands in cultures treated with up to 10 PM MS0 in comparison with control cultures. However, cultures treated with 100 PM MS0 showed a drastic decline in the intensity of all the protein bands, especially between the molecular weights of 22-55 kDa and 55 to above 66 kDa. Loss in the protein bands was very obvious in the cultures treated with 200 and 500 PM MSO. DISCUSSION The data obtained in the present study support the hypothesis stated in the introduction and indicate that protein synthesis is significantly suppressed in the test organism when the cells are subjected to elevated levels of heat, salinity and/or MSO. The degree of suppression depends on the severity of the stressor. Suppression of normal protein synthesis during stress is commonly observed among other experimental systems including the extensively studied heat-shock response in Drosophild2) and soybean.(“) It has been documented that most organisms investigated to date respond to shock treatment by synthesizing a new set of proteins;(3,5,24,30)our results indicate that the cyanobacterium Anabaena sp. also responds in the same manner. Despite the widespread existence of shock proteins, very little is known about their precise physiological function(s). Several lines of evidence suggest that one of the major functions of the stress proteins is to enable the organisms to cope with the stressor. Induction and accumulation of heat shock proteins (HSPs) are closely correlated with the development of ther-
153
motolerance, i.e. the ability of an organism to withstand an elevated, normally lethal temperature.@) During heat stress, there is not only a transcriptional activation of heat shock genes but also transcriptional repression of most previously active genes, possibly due to temperature-induced alterations in the transcriptional machinery.(‘8’23) An alternative/additional mechanism was suggested by who found that heat shock factor Westwood et ~1.~~~) binds not only to puff sites on Drosophila chromosomes associated with heat shock but also to a large number of other sites, suggesting that heat shock factor binding may lead to the repression of normal gene activity. Repression of normal protein synthesis is supposed to be also under translational control, exerted over the pre-existing, normal cellular mRNAs. These are normally not degraded during heat stress but persist in a translatable form.(“) On recovery from heat stress, these mRNAs are translated.(“) Recently, Herman et CZ~.(‘~) reported that regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli.
Our data also show that protein synthesis including HSPs synthesis and salinity-induced (NaCl > 100 mM) protein synthesis completely ceases if the test organism is exposed to a longer duration of heat shock or to a higher concentration of NaCl (> 100 mM). Comparable observations were recorded for cells treated with higher concentrations of MS0 (> 10 PM). MSO-induced inhibition of protein synthesis in the organisms might be due to the strong inhibition of glutamine synthetase (GS) activity resulting in the release of excess nitrogenous materials and thus causing nitrogen starvation. It is hard to demonstrate unambiguously that a particular observed change in protein expression contributes to the survival process. Changes in the expression of large numbers of proteins occur due to stress, yet it is probable that only some of these proteins are directly involved in stress tolerance. It is possible that in some cases the synthesis of a protein indicates sensitivity to a stressor rather than being part of a tolerance mechanism. In general, the results indicate that protein synthesis in the test organism eventually succumbed to high temperature (as has also been reported by Lai et Al.) as well as higher concentrations of NaCl and MSO.“” Our earlier observations of UV-B stress also revealed a gradual decline in the growth rate as well
R. P. SINI-IA and D.-P. HADER as protein content in several rice field cyanobacteria including Anabama SP.(‘~) In conclusion, our results indicate that any substantial increase in the above mentioned stressors in nature, might be detrimental to the ecologically and economically important cyanobacterial cornmunities, which, in turn, may affect the productivity of higher plants. With increasing presence of stressot-s, there might be a setback in the agricultural economy of all countries where cyanobacteria are being considered as an alternate natural source of nitrogenous fertilizers for rice paddies and other crops. work was financially supported by the Deutsche Akademische Austauschdienst and the Ministry of H.R.D., Government of India to R.P.S. and the European Community (EG-EV5V-CT91-0026) and the BayFORKLIM (DIII 1) to D.-P.H.
Acknowkz&mmts--This
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2. Ballinger D.G. and Pardue M.I. (1982) The subcellular compartmentalization of mRNA in heat shocked Drosophila cells. Pages 183-190 in MJ. Schlesinger, M. Ashburner and A. Tissieres, eds, Heat shockjom bacteria to man. Cold Spring Harbor, New York. 3. Bhagwat A. A. and Apte S. K. (1989) Comparative analysis of proteins induced by heat shock, salinity and osmotic stress in the nitrogen-fixing cyanobacterium Anabaena sp. strain L-31.3. Bactiol. 171, 5187-5189. 4. Borbely G., Suranyi G. and Korcz A., Pahi Z. (1985) Effect of heat shock on protein synthesisin the cyanobacterium Synechacoccus sp. strain PCC 6301. 3. Bacterial. 161, 1125-l 130. 5. Borbtly G. and Suranyi G. (1988) Cyanobacterial heat-shock proteins and stress responses. Method. Enzy. 167,622+27.
6. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
7. Erdmann N., Fulda S. and Hagemann M. (1992) Glucosylglycerol accumulation during salt acclimation of two unicellular cyanobacteria. 3. Gen.Microbiol. 138,363-368.
8. Fay P. (1992) Oxygen relations of nitrogen fixation in cyanobacteria. Miuobiul. Rev. 56, 34&373. g. Gurley W. B. and Key J. L. (1991) Transcriptional regulation of the heat-shock response: a plant perspective. Biochaistp 30, 1-12. 10 Herman C., Lecat S., D’Ari R. and Bouloc P. (1995) ’ Regulation of the heat-shock response depends on divalent metal ions in an hJIB mutant of Escherichia coli. Molecul. Microbial. 18, 247-255.
11. Howarth C. J. and Ougham H. J. (1993) Tansely Review No. 5 1, Gene expression under temperature stress. Nm Phytal. 125, l-26. 12. Kimpel J. A. and Key J. L. (1985) Heat shock in plants. fiends Biochem. Sci. 10, 353-357. 13. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Na&ure 227,680-685. 14 Lai Y-K., Li C-W. and HU C-H., Lee M-L. (1988) ’ Quantitative and qualitative analyses of protein synthesis during heat shock in the marine diatom Jvittschia alba (Baeilluriophyceae).3. Phycol. 24, 50+5
14.
15. LevittJ. (1980) Responses of plants to environmental stress. Vol. 1, Chilli&?ee,tiq and high temperaturestress. Academic Press, New York. 16. Lin C-Y., Roberts J. K. and Key J. L. (1984) Aquisition of thermotolerance in soybean seedlings. Synthesis and accumulation of heat shock protein and their cellular localization. Plant Physiol. 74, 152-160. 17. Lindquist S. (1981) Regulation of protein synthesis during heat shock. Nature 293,3 1 l-3 14. la. Miller M. W. and Ziskin M. C. (1989) Biological consequences of hyperthermia. Ultrasound Med. Biol. 15, 707-722.
19. Nicholson P., Varley J. P. A. and Howe C. J. (1991) A comparison of stress responses in the cyanobacterium Phonnidium luminosum.FEMSMicrobiol. Lett. 78, 109-l 14. 20. Parry M. L., Carter T. R. and Porter J. H. (1989) The greenhouse effect and the future of UK agriculture.3. Royal&. Sot. Erg. 150, 12&181. 21. Rippka R., Deruelles J., Waterbury J. B., Herdman M. and Starrier R. Y. (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. 3. Gen.Microbial. 111, l-6 1. 22. Safferman R. S. and Morris M-E. (1964) Growth characteristics of the blue-green algal virus LPP- 1.3. Ba&iol. 88, 771-775. 23. Schoffl F., Ross01 I. and Angermtiller S. (1987) Regu-
lation of the transcription of heat shock genes in nuclei from soybean (GbC;nemax)seedlings. Plant Cell Environ.10, 113-120. 24. Schubert H., Fulda S. and Hagemann M. (1993) Effects of adaptation to different salt concentrations on photosynthesis and pigmentation of the cyano-
STRESS bacterium
SyruxhocysEis sp. PCC 6803.3; 142,291-295.
RESPONSES
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25. Sinha R.P., Kumar H.D., Kumar A. and Hader D.P. (1995) Effects of W-B irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in cyanobacteria. Actu Bototool. 34, 187192. 26. Stanier R. Y. and Cohen-Bazire G. (1977) Phototrophic prokaryotes: the cyanobacteria. Annu. Rev.
Microbd. 31,225-274. 27. Storti R. V., Scott M. P., Rich A. and Pardue M. L. (1980) Translational control of protein synthesis in response to heat shock in D. rndanoga.& cells. Cell 22,
825-834. 28. Tandeau
de Marsac N. and Houmard J. (1993) Adaptation ofcynobacteria to environmental stimuli: new steps towards molecular mechanisms. FEMS Microbial. Reu. 104, 119-190. 29. Tel-Or E. (1980) Response of N,-fixing cyanobacteria to salt. Ap~l. Environ. Microbial. 40, 689-693.
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30. Thomas
S. P., Zaritsky A. and Boussiba S. (1990) Ammonia excretion by an L-methionine-DGsulfoximine-resistant mutant of the rice field cyanobacterium Anabatna &men.&. A#d. Environ. Mizrobial.
56,349s3504. 3 1. Upchurch R. G. and Elkan G. H. (1977) Comparision of colony morphology, salt tolerance,and effectiveness in Rhizobium japonicum. Can. j? Microbial. 23, 1118-l 122. 32. Venkataraman G. S. (198 1) Blue-green algae: a possible remedy to nitrogen scarcity. Cum Sti. 50, 253256. 33. Vonshak A. and Guy R., Guy M. (1988) The response of the filamentous cyanobacterium Spirulina platensir to salt stress. Arch. Mkrobiol. 150, 417-
420. 34. Westwood J. T. and Clos J., Wu C. (1991) Stressinduced oligomerization calization of heat-shock 827.
and chromosomal factor. Natire 353,
relo822-
Vol. 36,No. 2,pp. 147-155, 1996 Cootiht 0 1996 F&via Science B.V.
SOO98-8472(96)01007-6
RESPONSE
OF A RICE FIELD CYANOBACTERIUM PHYSIOLOGICAL STRESSORS RlyESHWAR
P. SINI-IA and DONAT-P.
RNABA&l’X SP. TO
JhiDER*
Institut fur Botanik und Pharmazeutische Biologic, Friedrich-Alexander-Universitat, Staudtstr. 5, D-9 1058 Erlangen, Germany (Received 5 Septmber 1995; accepted in revtiedfom
28 December 1995)
Sinha R.P. and Hader D.-P. Respollseof a tiefield yanobacteium Anabaena up.tophysiological stressors. Environmental and Experimental Botany 36,147-155,1996. - The impact ofphysiological factors such as heat, sahnity and L-methionine-DL-sulfoximine (MSO) on growth and total protein profile was studied in a rice field cyanobacterium, Anabaaa sp. There was a gradual decrease in growth rate of the organism with increase in incubation time at 47%; by 168 h of incubation there was essentially no growth. NaCl concentration > 5 mM inhibited growth of the organism, and there was apparently no growth at >200 mM NaCl-supplemented media. Similarly, growth was inhibited with MS0 concentration >5 PM and there was no growth in cultures treated with > 100 PM MSO. SDSPAGE protein profile after heat stress revealed a decline in the synthesis of several proteins but at the same time, synthesis of a new set of proteins of approximately 60-65 kDa was induced after 12 and 24 h of incubation and the same was completely eliminated after 96 h of incubation. Cultures treated with 10 mM of NaCl did not show any change in the protein pattern. In contrast, 100 mM NaCl treated cultures elicited a number of new proteins at around 29, 32, 40 and 70 kDa. Most of the protein bands disappeared in the cultures treated with 500 mM NaCl. Cultures treated with 10 PM MS0 did not show any change in the appearance of the protein bands, whereas the cultures treated with 100 PM MS0 showed a drastic decline in the intensity of all protein bands. The results indicate that different stressors exert specific effects on cyanobacterial protein synthesis. Keyworu!s: Anabaena, cyanobacteria, growth, heat stress, L-methionine-DL-sulfoximine (MSO) treatment, protein profile, sahnity stress.
system for analysing the adaptive mechanisms developed in response to changing stress conditions.(26) The physical environment of an organism is not constant; fluctuations occur in time and space. However, organisms have evolved to
INTRODUCTION
Stressors such as heat, cold, salinity and drought play a key role in determining the growth and development of cyanobacterial populations in their particular habitat.(s,28) Being cosmopolitan in clistribution, cyanobacteria are thought to have been exposed to different levels and types of stressors during their development, thus providing a suitable
survive such conditions and show adaptation to the stressor( Each stress variable usually has a minimum and a maximum level beyond which an organism cannot survive.(‘5)
*Author to whom correspondence should be addressed. 147
148
R. P. SINHA and D.-P. H;iDER
There is also some interaction between the severity of a stressor and its duration. A long exposure at a moderately high temperature may be as injurious as a brief exposure to an extreme temperature. Temperature fluctuations in the environment can occur over a matter of minutes or seasons. Longterm environmental changes may also occur, such as global warming that is suggested to occur because of increased greenhouse gas emissions.(“) It has been postulated that particular proteins whose synthesis is induced by stress conditions are critical to the survival of the organism. Changes in gene activation, transcription, and translation often occur during the acclimation process and thus are thought to be involved in the induction of tolerance. It is a common phenomenon that above a threshold temperature the normal pattern of protein synthesis is repressed and a new set of proteins is synthesized from newly transcribed mRNA, called heat-shock proteins (HSPs). (‘Sag)There are a number of major families of HSPs, classified on the basis of their molecular weight; high molecular weight HSPs (80 kDa), the HSPs 70 family (molecular weight approximately 70 kDa) and low molecular weight HSPs (14-30 kDa). The heat shock response is finely tuned to its thermal adaptation.(“) Like other organisms, cyanobacteria have been reported to possess a heat shock response.(3,4p’g) Salinity has become an ever-increasing problem in irrigated agriculture. Certain bacterial strains such as Rhizobium, which nodulate a number of economically important crops, are highly sensitive to NaC1.(31) Cyanobacterial N,-fixation is supposed to be the most sensitive process in response to enhanced salt concentrations, followed by photosynthesis; respiration has been reported to be increased in salt-adapted cyanobacteria.(2g’33) Decreased photosynthesis and Chl a contents have been reported in Microcys&j?ma and Synechocystis sp. PCC 6803(7) following salinity stress. NJixing cyanobacteria are being used as nitrogen biofertilizers in rice paddy fields in several countries where rice is the major staple dietc3’) A number of strains which thrive in rice fields release small of the major fertilizing product, quantities ammonia, during their active growth, while most of the fixed product is made available to the plants mainly through autolysis and microbial decomposition. Under these circumstances, it is difficult to control the flow of nitrogen compounds needed for
the development of the rice plants. To overcome this problem, MSO, which inhibits the primary ammonia assimilating enzyme, glutamine synthetase (GS), has been reported to be extensively used for the sustained release of ammonia. High concentrations of MS0 might be toxic for the growth of cyanobacteria, and hence changes in total protein profile are expected.(30) In the present investigation, an attempt was made to study the effects of heat, salinity and MS0 on growth and total protein profile of Anabaena sp. isolated from an Indian rice field. During the hot summer season, cyanobacterial populations in Indian rice paddy fields are exposed to high temperatures (47-48%) for several weeks. The above mentioned stressors either alone or in combination may have a detrimental effect on cyanobacterial populations in the tropical rice paddy fields. Any adverse impact on N,-fixing cyanobacteria will affect the productivity of higher plants because N2fixing capacities and photosynthetic abilities in one organism are unique to cyanobacteria only. This prompted us to search for the possible effects on the growth pattern as well as on total protein profile of Anabaena sp. This strain was selected for study largely due to its widespread occurrence in the Indian rice paddy fields, having a fast growing rate as well as an exceedingly high amount of pigment proteins. MATERIALS
AND METHODS
Isolation, punjfication and growth conditions of the organism The heterocystous and filamentous N,-fixing cyanobacterium Anabaena sp. was isolated from a rice paddy field near Varanasi, India. The specimen collected from the fields was thoroughly washed by double distilled water and, after mild homogenisation, spread on solid agar (Difco-Bacto; 1.5%) in Petri dishes (75 mm) containing different synthetic media such as Allen and Amon’s, BG-1 l(‘l) or modified Chu-lO(**) and incubated in fluorescent white light (14.4 W m-2) at 27°C. After 10-12 days of incubation, minute colonies started appearing on the agar plates. Well spread individual colonies were isolated and restreaked on fresh agar plates. In parallel, individual colonies were transferred into 10 ml of the above mentioned liquid media in a test tube and incubated in continuous light. After 7-l 0 days of incubation, cultures growing in liquid media were harvested by centrifugation ( 1500s for 10 min)
STRESS RESPONSES and the resulting pellet was washed, suspended in a minimum volume of liquid media and the filaments were broken by gentle shaking with sterilized glass beads. The homogeneous suspension containing mostly individual cells or short filaments was spread on solid agar Petri plates of the above mentioned synthetic media. Individual cells were marked and after 7-10 days of growth, colonies appearing on agar plates were examined microscopically and retransferred onto solid agar Petri plates. Restreaking and subculturing were repeated several times to obtain a single colony apparently free from contamination. These colonies were manually isolated and routinely grown in liquid medium. Out of all the media tested, Anabaena sp. showed its best growth in the modified Chu-10 medium.@‘) Cultures were routinely grown in an autoclaved modified Chu- 1O(“) liquid medium (devoid of any nitrogen sources) in Erlenmeyer flasks filled to 40% of their nominal volume and placed in a culture room at 27°C and illuminated with white fluorescent light of 14.4 W m-‘. Heat, salin@ and MS0 (L-methionine-DL-suyoximine) treatment Exponentially growing (7-l 0 day old) suspension cultures of Anabozna sp. were harvested by centrifugation (J2-21M/E) using a JA 20 rotor (Beckman Instruments, Palo Alto, CA) at 15008 for 10 min at room temperature, and transferred into fresh Chu-10’22’ liquid medium. The culture was then placed in an incubator at a temperature of 47°C and an irradiance of 14.4 W m-‘. At predetermined intervals, aliquots (2 ml) were withdrawn and growth as well as total protein profile was analysed. A separate control was maintained at a temperature of 27°C and an irradiance of 14.4 W m-‘. Cultures were also inoculated with various concentration of NaCl and/or MS0 and placed in a culture room at a temperature of 27°C and an irradiance of 14.4 W m-*. At predetermined intervals, aliquots (2 ml) were withdrawn and subjected to growth measurement as well as SDS-PAGE analyses. Even though being thermotolerant, the cells grew equally well at 27°C. Gowth estimation Growth of the organism was ascertained either by measuring Chl a content or by estimating protein content. For extracting Chl a, a known volume of
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culture was centrifuged and the pellet was resuspended in a known volume of 80% acetone, shaken for 5 min and kept overnight in a refrigerator at 4-V. The resulting suspension was centrifuged and the supernatant was used for the estimation of Chl a content. The concentration of protein was determined by the method of Bradford.@) Bovine serum albumin was used as a standard. Absorption spectroscopy Acetone soluble Chl a was transferred into a quartz cuvette having an optical path length of 10 mm (2 mm thickness, Hellma, Mullheim, Germany): absorption spectra were measured in a single beam DU 70 spectrophotometer (Beckman, Palo Alto, CA). The raw spectra were transferred to a microcomputer and treated mathematically and statistically using the software provided by the manufacturer (Beckman, Palo Alto, CA). Gradient gel electrophoresis Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was undertaken in a vertical system (200 1, Pharmacia, LKB, Uppsala, Sweden) with gels of 155 x 130 mm, 1.5 mm thick, using the method of Laemmli(‘3) with a gradient (55 15% T) in the resolving gel. Gels were stained with Coomassie brilliant blue R 250 and dried in a gel dryer (Bio-Rad, Richmond, CA). Experiments were performed at least 3 times. RESULTS Eflects of heat, salin@ and MS0 on the growth of Anabaena sp. Fig. 1 shows the spectra of acetone soluble pigments after the growth of Anabaena sp. at normal (27%) and elevated (47°C) temperatures. It is evident from the spectra that when cultures were grown at normal growth temperature (27“C), there was no change in the absorption up to 24 h; thereafter, there was a linear increase in absorption with increasing incubation time, the maximum being at 168 h. The cultures growing at an elevated temperature (47°C) showed a gradual loss in absorption with increasing incubation time, and by 168 h all peaks disappeared, denoting that there was no growth in the culture at 47%. The growth response of the test organism to various concentrations of NaCl is illustrated in Fig. 2.
150
R. P. SINHA and D.-P. I-L%DER
0.2
0.1
0
0.0 350
400
450
500
550
so0
aia
Wavelength [nm]
There was no significant difference in the growth of the organism treated with 2 mM NaCl in com-
parison with the control culture (without NaCl). Growth was inhibited at NaCl concentration >5 mM. There was apparently no growth at 200 and 500 mM NaCl-supplemented media in any of the replicate experiments. Growth of Anabama sp. at varying concentration of MS0 is illustrated in Fig. 3. Growth was severely inhibited in cultures treated with MS0 up to 3 days; thereafter, growth resumed in cultures containing up to 5 PM MSO. There was no significant difference in final growth yield in the control and 250 N8CI 2mM OmM
5mM
10 mm 100mM 2WmM 5WmM 0
( 0
I 1
2
5
4
5
5
7
Time [d]
Fig. 2. Growth of Anabaem sp. at various concentration of NaCl at a normal growth temperature of 27°C and an irradiance of 14.4 W m-‘. Data are for three separate but identical experiments + S.D.
2
3
4
5
6
7
Time [d]
700
Fig. 1. Absorption spectra showing the growth ofAnabaena sp. under control (27%) and elevated (47%) temperature conditions after increasing exposure times. Data are for three separate but identical experiments.
I 1
Fig. 3. Growth of Anabaem sp. at various concentration of MS0 at a normal growth temperature of 27“C and an irradiance of 14.4 W m-*. Data are for three separate but identical experiments + S.D.
in cultures treated with 2 PM MSO. Growth was inhibited with MS0 concentration 5 PM. There was no growth in cultures treated with 100 PM MSO.
ofhat, salinip and MS0 on the protein pro& of Anabaena 4. The SDS-PAGE protein profile of Anabama sp. after heat stress is illustrated in Fig. 4. It is evident from the electrophoretic pattern that exposure to heat stress resulted in alterations in cyanobacterial protein synthesis. Synthesis of several proteins declined with increasing exposure duration to 47°C. At the same time, synthesis of a new set of proteins was induced. Exposure at 47% for 12 h resulted in a decline in protein bands between 16 and 22 kDa which represent the monomers of phycocyanin (lane 4). There was further loss in these protein bands at 48 h of exposure (lane 6); these bands were completely lost after 96 h of incubation (lane 8). Synthesis of a new set of proteins of approximately 6Ck65 kDa was observed after 12 and 24 h of incubation at 47°C (lanes 4 and 10, E8ect.s
respectively). However, these protein bands started to disappear after 48 h and were completely eliminated after 96 h of incubation (lanes 6 and 8). One very prominent band of approximately 55 kDa was less affected than the rest of the protein bands even after 96 h of incubation at 47% (lane 8). Cultures were supplemented with various concentrations (2-500 mM) of NaCl, and after 72 h of growth were subjected to SDS-PAGE analyses (Fig.
STRESS RESPONSES
IN A&m
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SP.
12345678910
MW (kDa)
Fig. 4. Vertical SDS-PAGE (gradient 5-15% T) protein profile ofAnabaena sp. with increasing time period of heat treatment. Lane 1: marker proteins (Sigma, MWS-877 L), lane 2: control (27°C; 0 h), lane 3: control (12 h), lane 4: 47°C (12 h), lane 5: control (48 h), lane 6: 47% (48 h), lane 7: control (96 h), lane 8: 47% (96 h), lane 9: control (24 \ h) andlane 10: 47’C (24 h). Equal amounts ofproteins were loaded into each well. I
1234
Fig. 5. Vertical SDS-PAGE (gradient 5-15% T) protein profile of An&mu sp. with increasing concentrations of NaCl after 72 h of growth. Lane 1: control, lane 2: 2 n% lane 3 5 mM, lane 4: 10 mM, lane 5: 100 mM> lane 6: 200 mM, lane 7: 500 mM and lane 8: marker proteins (Sigma, MWS-877 L). Equal amounts ofproteins were loaded into each well.
56
7
8
9
Fig. 6. Vertical SDS-PAGE (gradient 5-15% T) protein proiile of Anabama sp. with increasing concentrations of MS0 after 72 h of growth. Lane 1: marker proteins (Sigma, Mb%877 L), lane 2: control, lane 3: 1 PM, lane 4: 2 PM, lane 5: 5 PM, lane 6: 10 PM, lane 7: 100 PM, lane 8: 200 m, and lane 9: 500 PM. Equal amounts of proteins were loaded into each well.
STRESS RESPONSES IN AJviUMNA SF’. 5). Cultures treated with 2 mM NaCl showed more intense protein bands in comparison with control cultures (without NaCl). Cultures treated with 5 and 10 mM NaCl had a protein pattern identical to that of the 2 mM NaCl-treated cultures. In contrast, 100 r&I NaCl-treated cultures showed a number of new proteins at around 29, 32, 40 and 70 kDa. There was a drastic decline in the intensity of all the protein bands in the cultures treated with 200 mM NaCl. The majority of protein bands disappeared in the cultures treated with 500 mM NaCI. The SDS-PAGE protein profile of Anabma sp. grown at various concentration of MS0 is presented in Fig. 6. It is evident from the results that there was no difference in the appearance of the protein bands in cultures treated with up to 10 PM MS0 in comparison with control cultures. However, cultures treated with 100 PM MS0 showed a drastic decline in the intensity of all the protein bands, especially between the molecular weights of 22-55 kDa and 55 to above 66 kDa. Loss in the protein bands was very obvious in the cultures treated with 200 and 500 PM MSO. DISCUSSION The data obtained in the present study support the hypothesis stated in the introduction and indicate that protein synthesis is significantly suppressed in the test organism when the cells are subjected to elevated levels of heat, salinity and/or MSO. The degree of suppression depends on the severity of the stressor. Suppression of normal protein synthesis during stress is commonly observed among other experimental systems including the extensively studied heat-shock response in Drosophild2) and soybean.(“) It has been documented that most organisms investigated to date respond to shock treatment by synthesizing a new set of proteins;(3,5,24,30)our results indicate that the cyanobacterium Anabaena sp. also responds in the same manner. Despite the widespread existence of shock proteins, very little is known about their precise physiological function(s). Several lines of evidence suggest that one of the major functions of the stress proteins is to enable the organisms to cope with the stressor. Induction and accumulation of heat shock proteins (HSPs) are closely correlated with the development of ther-
153
motolerance, i.e. the ability of an organism to withstand an elevated, normally lethal temperature.@) During heat stress, there is not only a transcriptional activation of heat shock genes but also transcriptional repression of most previously active genes, possibly due to temperature-induced alterations in the transcriptional machinery.(‘8’23) An alternative/additional mechanism was suggested by who found that heat shock factor Westwood et ~1.~~~) binds not only to puff sites on Drosophila chromosomes associated with heat shock but also to a large number of other sites, suggesting that heat shock factor binding may lead to the repression of normal gene activity. Repression of normal protein synthesis is supposed to be also under translational control, exerted over the pre-existing, normal cellular mRNAs. These are normally not degraded during heat stress but persist in a translatable form.(“) On recovery from heat stress, these mRNAs are translated.(“) Recently, Herman et CZ~.(‘~) reported that regulation of the heat-shock response depends on divalent metal ions in an hflB mutant of Escherichia coli.
Our data also show that protein synthesis including HSPs synthesis and salinity-induced (NaCl > 100 mM) protein synthesis completely ceases if the test organism is exposed to a longer duration of heat shock or to a higher concentration of NaCl (> 100 mM). Comparable observations were recorded for cells treated with higher concentrations of MS0 (> 10 PM). MSO-induced inhibition of protein synthesis in the organisms might be due to the strong inhibition of glutamine synthetase (GS) activity resulting in the release of excess nitrogenous materials and thus causing nitrogen starvation. It is hard to demonstrate unambiguously that a particular observed change in protein expression contributes to the survival process. Changes in the expression of large numbers of proteins occur due to stress, yet it is probable that only some of these proteins are directly involved in stress tolerance. It is possible that in some cases the synthesis of a protein indicates sensitivity to a stressor rather than being part of a tolerance mechanism. In general, the results indicate that protein synthesis in the test organism eventually succumbed to high temperature (as has also been reported by Lai et Al.) as well as higher concentrations of NaCl and MSO.“” Our earlier observations of UV-B stress also revealed a gradual decline in the growth rate as well
R. P. SINI-IA and D.-P. HADER as protein content in several rice field cyanobacteria including Anabama SP.(‘~) In conclusion, our results indicate that any substantial increase in the above mentioned stressors in nature, might be detrimental to the ecologically and economically important cyanobacterial cornmunities, which, in turn, may affect the productivity of higher plants. With increasing presence of stressot-s, there might be a setback in the agricultural economy of all countries where cyanobacteria are being considered as an alternate natural source of nitrogenous fertilizers for rice paddies and other crops. work was financially supported by the Deutsche Akademische Austauschdienst and the Ministry of H.R.D., Government of India to R.P.S. and the European Community (EG-EV5V-CT91-0026) and the BayFORKLIM (DIII 1) to D.-P.H.
Acknowkz&mmts--This
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