ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 6, pp. 751–755. © MAIK “Nauka /Interperiodica” (Russia), 2006. Original Russian Text © S.Yu. Selivankina, N.K. Zubkova, E.V. Kupriyanova, T.V. Lyukevich, V.V. Kusnetsov, D.A. Los, O.N. Kulaeva, 2006, published in Fiziologiya Rastenii, 2006, Vol. 53, No. 6, pp. 851–856.
Cyanobacteria Respond to Cytokinin S. Yu. Selivankina, N. K. Zubkova, E. V. Kupriyanova, T. V. Lyukevich, V. V. Kusnetsov, D. A. Los, and O. N. Kulaeva Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia; fax: 7 (495) 977-8018; e-mail:
[email protected] Received January 10, 2006
Abstract—We studied the effects of trans-zeatin (10–9–10–5 M) on the RNA synthesis in the in vitro transcriptional system containing DNA and RNA polymerase of a cyanobacterium Synechocystis sp. PCC 6803. The cytokinin enhanced transcription in this system, and its action depended on the concentration applied: trans-zeatin exerted its highest effects at the concentrations of 10–8–10–6 M. Adenine, which derivatives are trans-zeatin and other purine cytokinins, did exert such an effect. Its wide range of concentrations (10–7–10–4 M) did not affect 3H-UMP incorporation into RNA. This indicates a specificity of trans-zeatin action. Culturing of cyanobacteria for 3 days in the presence of synthetic cytokinin benzyladenine (10–6 M) also enhanced 3H-UMP incorporation into RNA. A cytokinin-binding protein isolated earlier from plant chloroplasts, which is involved in the cytokinin-dependent control of chloroplastic transcription, enhanced substantially the response of the cyanobacterial transcriptional system to cytokinin. The results obtained show that cyanobacteria, evolutionary ancestors of chloroplasts, own the system of cytokinin signal recognition, which might be transferred to the plant cell. This inspires further investigation of the systems of cytokinin signal perception and transduction in cyanobacteria and elucidation of common and different traits of these systems in plants and cyanobacteria. DOI: 10.1134/S1021443706060045 Key words: cyanobacteria - cytokinin - transcription - chloroplastic cytokinin-binding protein
INTRODUCTION According to prevailing hypothesis, chloroplast originated as a result of endocytosis of ancient photosynthesizing cyanobacteria into the eukaryotic cell with their subsequent evolutionary modification under the effects of the nucleus and cytoplasm [1]. A comparative analysis of chloroplasts and cyanobacteria can help considerably in the understanding of this evolutionary route, in particular in the elucidation of the origin of chloroplast regulatory systems. A substantial cytokinin role in the control of chloroplast biogenesis is now established [2–5]. Cytokinins stimulate the synthesis of proteins involved in the chloroplast electron transfer chain [6]; they also regulate expression of the gene encoding protochlorophyllide oxidoreductase, a most important enzyme on chlorophyll biosynthesis [7]. Chloroplasts are of importance for a general cell response to cytokinins [8]. Chloroplasts contain a large set of natural cytokinins [9]. However, the place of chloroplast cytokinin synthesis is not established, and it is unknown which genome comprises genes encoding the enzymes of this synthesis. Chloroplast receptors of cytokinins involved in their signal transduction is not detected as well. In this connection, the chloroplastic cytokininbinding protein (chlCBP), found by the authors, Abbreviations: BA—benzyladenine; CBP—cytokinin-binding protein; chlCBP—chloroplastic cytokinin-binding protein.
which controls cytokinin-dependent transcription in the chloroplastic in vitro system is of a great importance [10, 11]. It is not so far established whether this protein is encoded in the nucleus or chloroplasts; the origin of the systems for the cytokinin regulation of chloroplast biogenesis is not known as well. Therefore, it is of crucial interest to learn whether chloroplasts could obtain these systems from cyanobacteria. It is accepted that hormones appeared in evolution to coordinate functions of specialized cells in the system of the multicellular organism. However, the molecular basis of their appearance remains completely unstudied: we do not know to what extent the regulatory systems of unicellular organisms were used in evolution of the higher plant hormonal system. It is established that membrane histidine kinases, the components of the two-component receptor system, are involved in the perception of cytokinin and ethylene signals by plant cells. This system is widely presented in the cells of prokaryotes [12], in cyanobacteria in particular [13–16]. The genes displaying a high affinity for the cytokinin membrane receptor CRE1 and ethylene receptors were found in the cyanobacterial genome (http://www.kazusa.or.jp/cyano/Synechocystis/cgi-bin/ orfinfo.cgi?title=Chr&name=slr1759&iden=1). This permits a supposition that cytokinins and ethylene are the components of the cyanobacterial regulatory system, which could be transferred to chloroplasts. Later, the genes controlling cytokinin action might be
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The molecular mechanisms controlling cyanobacterial responses to stresses were elucidated [13–16].
incorporation into RNA, cpm/µg DNA × 10 –3
120
3H-UMP
100 80 60 40 20 0
3
5
6
7
8
9
pH Fig. 1. Transcriptional activity of the Synechocystis supernatant as dependent on pH.
partially or completely transferred from the chloroplast to nuclear genome, thus determining semi-autonomy or complete dependence of chloroplasts on the nucleus and cytoplasm in their response to cytokinins. As far as we know, nothing is known currently about cytokinin regulatory function in cyanobacteria. Some data published 36 years ago [17] are not reliable and require a critical review. At the same time, a great progress was achieved in our knowledge concerning the cyanobacterial genome and the mechanism of transcription [18, 19]. A close similarity was found between RNA polymerases of blue-green algae, eubacteria, and plastids. As distinct from chloroplasts comprising two RNA polymerases of nuclear and chloroplastic coding, blue-green algae possess only a single RNA polymerase composed of five major subunits. Some difference was detected in the operon organization of the genes encoding subunits of RNA polymerase in blue-green algae and eubacteria. In Synechocystis the genes encoding all eight sigma factors involved in the control of transcription were identified, RNA polymerase subunits were isolated and its core enzyme active in in vitro transcription was reconstructed [20]. Table 1. Transcriptional activity of the Synechosystis supernatant as dependent on the amount of DNA in the reaction medium DNA amount, µg
3H-UMP incorporation into RNA, cpm/sample
1.4
77565 ± 1021
2.8
93187 ± 1122
7.0
122851 ± 4205
14.0
87825 ± 1053
In the light of the outlined facts, the objective of this work was to elucidate the possibility of cytokinin action on cyanobacterial transcription. Cyanobacterial lysate was used as a transcription system; this lysate contained DNA and RNA polymerase providing for the RNA synthesis in vitro. We believed that a detection of cytokinin effect in this system would be a start point for subsequent detail analysis of the cyanobacterial response to cytokinin and its comparison with that of chloroplasts. It is important for the elucidation of the evolutionary origin of chloroplast biogenesis regulation by cytokinins. We could admit that cytokinins would not affect the bacterial system. This would indicate that chloroplast responses to cytokinins appeared in higher plants without the contribution of cyanobacteria. MATERIALS AND METHODS We used cyanobacterium Synechocystis sp. PCC 6803 strain Du Pont (Du Pont, United States), obtained from the Institute of General Biology (Okazaki, Japan). Cyanobacteria were cultured photoautrophically under sterile conditions in BG11 medium containing 20 mM Hepes–NaOH, pH 7.5 [21] at 34°C, continuous illumination (70 µmol/(m2 s)), and bubbling with air–gas mixture containing 2% CO2 [22]. After 3 days of culturing, the cells were sedimented by centrifugation at 15500 g (K-23 centrifuge) for 15 min. The pellet was washed with the medium containing 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM KCl, and 4 mM 2-mercaptoethanol. Thereafter, the cells were destroyed with glass beads (G4649, Sigma, United States), and the homogenate was centrifuged at 15 000 g for 15 min at 4°ë. Aliquots were taken from both the supernatant and sedimented membrane fraction to evaluate a distribution of transcriptional activity between these two fraction. It turned out that cyanobacterial DNA and RNA polymerase were present in both fractions. In both cases, RNA polymerase activity was estimated in the reaction medium 100 µl in volume containing about 10 µg DNA, 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 10 mM 2-mercaptoethanol, and ATP, CTP, and GTP 0.2 mM each. 3H-UTP (1.28 TBq/mM, 10 µM) was used as a radioactive precursor. The reaction was run at 25°ë for 25 min and stopped by the addition of 100 µl of cold 10% TCA containing 0.9% sodium pyrophosphate. Radioactivity was measured with a scintillation counter (LKB, Sweden). Optimum pH values (Fig. 1), Mg2+ concentration (Fig. 2) and DNA content in the reaction medium (Table 1) were found in preliminary experiments. All experiments were performed in at least three replicates with three or four recordings each. Tables present mean values and their standard errors.
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Cyanobacteria were grown in nutrient medium for 3 days in the presence or absence of synthetic cytokinin benzyladenine (BA). Then, algal cells were homogenized using glass beads, and the homogenate was centrifuged at 15000 g for 15 min. Transcriptional activity was detected in both the supernatant and the pellet (Table 2). Thus, both fractions contained DNA, RNA polymerase, and required transcription factors. DNA of these two fractions could differ in its connection with membranes, the extent of fragmentation, binding to proteins, etc. However, cytokinin addition to the medium of cyanobacteria culturing enhanced considerably the transcriptional activities in both the supernatant and the pellet (Table 2). BA effect was manifested at its concentration in the medium of 10–7 M. This value is within the range of cytokinin concentrations enhancing transcription in plants. Such activation of bacterial transcription by cytokinin added to culturing medium might occur due to enhanced synthesis of RNA polymerase in bacterial cells or due to its activation. In order to elucidate whether cytokinin could directly affect cyanobacterial transcriptional activity, in the next series of experiments, we added a natural highly active higher plant cytokinin trans-zeatin immediately to the reaction medium for measuring cyanobacterial RNA polymerase activity. We found that, in this case, trans-zeatin also enhanced in vitro transcription in a concentration-dependent mode (Table 3). At the concentration of 10–9 M, trans-zeatin weakly affected incorporation of the labeled precursor into RNA. At the concentration of 10–8 M, cytokinin enhanced in vitro transcription substantially, and its action was maintained in the concentration range from 10–8 to 10–6 M. Further concentration increase to 10–5 M reduced the cytokinin effect. In order to check a specificity of trans-zeatin action, we added adenine to the reaction medium. Natural plant cytokinins are known to be adenine derivatives, which contain a definite radical in the 6th position of the purine ring. Adenine does not display cytokinin activity [23]. The addition of a wide range of adenine concentrations (10–7 to 10–4 M) to the reaction medium
3H-UMP
RESULTS AND DISCUSSION
incorporation into RNA, cpm/µg DNA × 10–3
CYANOBACTERIA RESPOND TO CYTOKININ 90 80 70 60 50 40 30 20 10 0
0
2
753
4 6 8 10 12 16 Mg2+ concentration, mM
20
Fig. 2. Transcriptional activity of the Synechocystis supernatant as dependent on Mg2+ concentration.
for in vitro RNA synthesis did not affect transcriptional activity (Table 3). This indicates a specificity of transzeatin action. The results obtained permit a conclusion that the reaction medium contained a target for cytokinin action, and the interaction between cytokinin and this target provided for transcription activation. A comparison of this system with cytokinin action on nuclear or chloroplastic transcription systems shows some differences. Trans-zeatin enhanced in vitro transcription in the system comprising RNA polymerase bound with chromatin, but only in the presence of cytokinin-binding protein (CBP) isolated from nuclei or cytosol [24, 25]. Each of the components (cytokinin or CBP) separately could not activate transcription. CBP mediated cytokinin-induced activation of transcription catalyzed by RNA polymerases. In chloroplast lysates, the presence of chlCBP was also required for trans-zeatin action [10, 11]. As distinct from purified chromatin and chloroplast lysate, the supernatant of the cyanobacterial homogenate evidently comprised some factors providing for cytokinin-induced activation of transcription. We also tested the effect of barley chlCBP on in vitro RNA synthesis in the cyanobacterial transcription synthesis. This protein mediated trans-zeatin-induced
Table 2. The effects of BA, added to the medium for cyanobacteria culturing, on in vitro transcription in the supernatant and pellet obtained by centrifugation of the cyanobacterial homogentate at 15500 g for 15 min Supernatant BA concentration, M
3H-UMP
Pellet 3H-UMP
incorporation into RNA
incorporation into RNA
cpm/10 µg of DNA
%
cpm/10 µg of DNA
%
0
48988 ± 3429
100
27750 ± 2570
100
10–7
219445 ± 1536
408
76202 ± 1891
275
10–6
50428 ± 3012
103
44403 ± 3215
160
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Table 3. The effect of trans-zeatin and adenine on in vitro RNA synthesis in the cyanobacterial transcription system 3H-UMP
Concentration, M
incorporation into RNA
cpm/10 µg of DNA
%
0
16226 ± 325
100
10–9
20009 ± 800
123
10–8
28361 ± 934
175
10–7
29200 ± 876
180
10–6
30096 ± 1504
185
10–5
22384 ± 895
138
0
21011 ± 630
100
10–7
19740 ± 789
94
10–6
16469 ± 210
103
10–5
18333 ± 733
88
10–4
21713 ± 868
103
Trans-zeatin
Adenine
Note: The supernatant obtained by centrifugation of the cyanobacterial homogentate at 15500 g for 15 min was used as a source of DNA and RNA polymerase.
Table 4. The effect of chlCBP from barley leaves on in vitro transcription in the system from cyanobacteria 3H-UMP
Treatment
incorporation into RNA
cpm/10 µg of DNA
%
Control
2913 ± 182
100
Trans-zeatin, 10–8 M
8228 ± 164
283
chlCBP, 10 µg
9690 ± 960
333
21315 ± 852
732
Trans-zeatin, 10–8 M + chlCBP, 10 µg
Note: The pellet obtained by centrifugation of the cyanobacterial homogentate at 15500 g for 15 min was used as a source of DNA and RNA polymerase. chlCBP was isolated from barley leaves as described earlier [10, 11].
activation of transcription in the chloroplast lysates from barley leaves, which served a source for chlCBP isolation [10, 11]. Table 4 shows that chlCBP enhanced label incorporation into RNA in the transcription system containing DNA and RNA polymerase from cyanobacteria. Trans-zeatin addition to this system also enhanced transcription, whereas simultaneous addition of both chlCBP and trans-zeatin induced most strong transcription activation.
Activation of in vitro transcription by chlCBP is of a great interest. We can suppose two possibilities for explanation of this phenomenon. Firstly, our preliminary data indicate the presence of endogenous cytokinins in Synechocystis, and they could enhance transcription in combination with chlCBP. Second possibility is that trans-zeatin is not the only factor affecting chlCBP activity. It might be that other so far unknown factors are involved in transcription activation together with chlCBP. Certainly, these suppositions require experimental confirmation. Thus, the transcription system of cyanobacteria could recognize chlCBP, respond to its presence, and provide its enhancement in the presence of chlCBP and trans-zeatin. Therefore, we might suppose that chlCBP mediates cyanobacterial transcription activation induced by cytokinin. The results obtained permit the conclusion that cyanobacteria possess molecular targets for cytokinin action providing for cytokinin-induced activation of in vitro transcription. As far as we know, this is the first report about cyanobacterial response to cytokinin. Such results are of principal importance because they indicate the occurrence of cyanobacterial regulatory systems involving cytokinins. This makes sense to further study the effects of cytokinin on cyanobacteria and to compare cyanobacterial and chloroplast responses to cytokinin. As was mentioned in the Introduction section, the investigations in this direction can highlight the evolutionary origin of the cytokinin regulatory role, primarily in the control of chloroplast biogenesis. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, project no. 05-04-48289, and by the Grant of the President of Russian Federation Supporting the leading scientific schools, project no. NSh3692.2006.4. REFERENCES 1. Danilenko, N.G. and Davydenko, O.G., Miry genomov organell (Organelle Genomes), Minsk: Tekhnologiya, 2003. 2. Parthier, B., The Role of Phytohormones (Cytokinins) in Chloroplast Development, Biochem. Physiol. Pflanz., 1979, vol. 174, pp. 173–214. 3. Chory, J., Reinnecke, D., Sim, S., Washburn, T., and Brenner, M., A Role of Cytokinins in De-Etiolation in Arabidopsis, Plant Physiol., 1994, vol. 104, pp. 339– 347. 4. Chen, C.-M., Jin, G., Andersen, B.R., and Ertl, J.R., Modulation of Plant Gene Expression by Cytokinin, Aust. J. Plant Physiol., 1993, vol. 20, pp. 609–619. 5. Kulaeva, O.N. and Kusnetsov, V.V., Recent Advances and Horizons of the Cytokinin Studying, Fiziol. Rast. (Moscow), 2002, vol. 49, pp. 626–640 (Russ. J. Plant Physiol., Engl. Transl., pp. 561–575).
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CYANOBACTERIA RESPOND TO CYTOKININ 6. Kusnetsov, V.V., Oelmuller, R., Sarwat, M.I., Porfirova, S.A., Cherepneva, G.N., Herrmann, R.G., and Kulaeva, O.N., Cytokinins, Abscisic Acid and Light Affect Accumulation of Chloroplast Proteins in Lupinus luteus Cotyledons without Notable Effect on Steady State mRNA Levels, Planta, 1994, vol. 194, pp. 318– 327. 7. Kusnetsov, V.V., Herrmann, R.G., Kulaeva, O.N., and Oelmuller, R., Cytokinin Stimulates and Abscisic Acid Inhibits Greening of Etiolated Lupinus luteus Cotyledons by Affecting the Expression of the Light-Sensitive Photochlorophyllide Oxidoreductase, Mol. Gen. Genet., 1998, vol. 259, pp. 21–28. 8. Kulaeva, O.N., Burkhanova, E.A., Karavaiko, N.N., Selivankina, S.Yu., Porfirova, S.A., Maslova, G.G., Zemlyachenko, Ya.V., and Börner, Th., Chloroplasts Affect the Leaf Response to Cytokinin, J. Plant Physiol., 2002, vol. 159, pp. 1309–1316. 9. Benková, E., Witters, E., van Dongen, W., Kolárˇ , J., Motyka, V., Brzobohaty, B., van Onckelen, H.A., and Machá c ková , I., Cytokinins in Tobacco and Wheat Chloroplasts. Occurrence and Changes due to Light/Dark Treatment, Plant Physiol., 1999, vol. 121, pp. 245–251. 10. Kulaeva, O.N., Karavaiko, N.N., Selivankina, N.N., Kusnetsov, V.V., Zemlyachenko, Ya.V., Cherepneva, G.N., Maslova, G.G., Lyukevich, T.V., Smith, A.R., and Hall, M.A., Nuclear and Chloroplast Cytokinin-Binding Proteins from Barley Leaves Participating in Transcription Regulation, Plant Growth Regul., 2000, vol. 32, pp. 329–335. 11. Lyukevich, T.V., Kusnetsov, V.V., Karavaiko, N.N., Kulaeva, O.N., and Selivankina, S.Yu., The Involvement of the Chloroplast Zeatin-Binding Protein in HormoneDependent Transcriptional Control of the Chloroplast Genome, Fiziol. Rast. (Moscow), 2002, vol. 49, pp. 105– 112 (Russ. J. Plant Physiol., Engl. Transl., pp. 92–98). 12. Stock, A.M., Robinson, V.L., and Goudreau, P.N., TwoComponent Signal Transduction, Annu. Rev. Biochem., 2000, vol. 69, pp. 183–216. 13. Suzuki, I., Los, D.A., Kanesaki, Y., Mikami, K., and Murata, N., The Pathway for Perception and Transduction of Low-Temperature Signals in Synechocystis, EMBO J., 2000, vol. 19, pp. 1327–1334. 14. Los, D.A., Structure, Expression Regulation, and Functions of Fatty Acid Desaturase, Usp. Biol. Khim., 2001, vol. 41, pp. 163–198. ^
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15. Inaba, M., Suzuki, I., Szalontai, B., Kanesaki, Y., Los, D.A., Hayashi, H., and Murata, N., GeneticallyEngineered Rigidification of Membrane Lipids Enhances the Cold Inducibility of Gene Expression in Synechocystis, J. Biol. Chem., 2003, vol. 278, pp. 12191–12198. 16. Los, D.A., Low-Temperature and Substrate Induction of the Gene for ∆9 Fatty Acid Desaturase in the Thermophilic Cyanobacterium Synechococcus vulcanus, Fiziol. Rast. (Moscow), 2004, vol. 51, pp. 184–189 (Russ. J. Plant Physiol., Engl. Transl., pp. 164–169). 17. Hoffmann, P. and Rathsack, R., Zur Cytokinin-Wirklung bei Blau Algen, Biochem. Physiol. Pflanz., 1970, vol. 161, pp. 95–96. 18. Thiel, T., Genetic Analysis of Cyanobacteria, The Molecular Biology of Cyanobacteria, Bryant, D.A., Ed., Dordrecht: Kluwer, 1994, pp. 581–611. 19. Curtis, S.E., The Transcription Apparatus and the Regulation of Transcription Initiation, The Molecular Biology of Cyanobacteria, Bryant, D.A., Ed., Dordrecht: Kluˇ wer, 1994, pp. 613–639. 20. Imamura, S., Yoshihara, S., Nakano, S., Shiozaki, N., Yamada, A., Tanaka, K., Takahashi, H., Asayama, M., and Shirai, M., Purification, Characterization, and Gene Expression of All Sigma Factors of RNA Polymerase in a Cyanobacterium, J. Mol. Biol., 2003, vol. 325, pp. 857–872. 21. Stanier, R.Y., Kunisawa, R., Mandel, M., and CohenBazine, G., Purification and Properties of Unicellular Blue-Green Algae (order Chroococales), Bact. Rev., 1971, vol. 35, pp. 171–205. 22. Gombos, Z., Wada, H., Varkonvi, Z., Los, D.A., and Murata, N., Characterization of the Fad12 Mutant of Synechocystis That Is Defective in delta 12 Acyl-Lipid Desaturase Activity, Biochim. Biophys. Acta, 1996, vol. 1299, pp. 117–123. 23. Kulaeva, O.N., Tsytokininy, ikh struktura i funktsiya (Cytokinins: Their Structure and Functions), Moscow: Nauka, 1973. 24. Kulaeva, O.N., Karavaiko, N.N., Selivankina, S.Yu., Zemlyachenko, Ya.V., and Shipilova, S.V., Receptor of trans-Zeatin Involved in Transcription Activation by Cytokinin, FEBS Lett., 1995, vol. 366, pp. 26–28. 25. Selivankina, S.Yu., Karavaiko, N.N., Zemlyachenko, Ya.V., Maslova, G.G., and Kulaeva, O.N., Control of In Vitro Transcription Exerted by the Cytokinin Receptor, Fiziol. Rast. (Moscow), 2001, vol. 48, pp. 434–440 (Russ. J. Plant Physiol., Engl. Transl., pp. 370–376).
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