NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

DIVISION OF CELLULAR REGULATION

Professor:
Norio Murata
Associate Professor:
Koji Mikami
Research Associates:
Atsushi Sakamoto
Yoshitaka Nishiyama
Iwane Suzuki
Monbusho Foreign Scientists:
Zolt‡n Gombos 1)
Rajinder S. Dhindsa 2)
Institute Research Fellow:
Akira Katoh
JSPS Visiting Scientists:
Govindjee 3)
Vyacheslav V. Klimov 4)
P. Pardha Saradhi 5)
Peddisetty Sharmila 5)
JSPS Postdoctoral Fellows:
Alia 5)
Michael P. Malakhov 6)
EC Fellow:
Ana Maria Otero Casal 7)
Visiting Scientists:
Prasanna Mohanty 8)
George C. Papageorgiou 9)
Tony H.H. Chen 10)
Sisinthy Shivaji 11)
Tom Wydrzynski 12)
Balazs Szalontai 1)
Dmitry A. Los 6)
Rajni Govindjee 3)
Suleyman I. Allakhverdiev 4)
Julian Eaton-Rye1 3)
Kostas Stamatakis 9)
Liberato Marzullo 14)
Nikolai Moseiko 15)
Otto Zsiros 1)
Oxana Malakhova 16)
Visiting Fellow:
Yasushi Tasaka
Graduate Students:
Sayamrat Panpoom
Masami Inaba
Hiroshi Yamamoto
Yuji Tanaka
Fumiyasu Yamaguchi
Ryoma Suzuki
Technical Staffs:
Sho-Ichi Higashi
Hideko Nonaka
1) from Biological Research Center, Szeged, Hungary
2) from McGill University, Montreal, Canada
3) from University of Illinois, Urbana, USA
4) from Institute of Soil Science and Photosynthesis, Pushchino, Russia
5) from Jamia Millia Islamia University, New Delhi, India
6) from Plant Physiology Institute, Moscow, Russia
7) from Universidad de Santiago, Santiago de Compostela, Spain
8) from Jawaharlal Nehru University, New Delhi, India
9) from National Centre for Scientific Research ŇDemokritosÓ, Athens, Greece
10) from Oregon State University, Corvallis, USA
11) from Centre for Cellular and Molecular Biology, Hyderabad, India
12) from Australian National University, Canberra, Australia
13) from Otago University, Dunedin, New Zealand
14) from International Institute of Genetics and Biophysics, Naples, Italy
15) from Institute of Molecular Genetics, Moscow, Russia
16) from Institute of Common Genetics, Moscow, Russia


The research effort of this division is aimed at developing a full understanding of the molecular mechanisms by which plants are able to acclimate to and tolerate stresses that arise from changes in environmental conditions, with particular emphasis on temperature and salt stresses. In 1997, significant progress was made in research on the following topics in studies with cyanobacteria and higher plants as experimental materials.


I. Improved performance of photosynthesis with polyunsaturated membrane lipids

We previously inactivated genes for fatty-acid desaturases by targeted mutagenesis in the cyanobacterium Synechocystis sp. PCC 6803 and we produced several mutant strains that contained abnormal numbers of unsaturated bonds in the fatty acids of their membrane lipids. By comparing these strains, we demonstrated that polyunsaturated membrane lipids are important in the ability of the photosynthetic machinery to tolerate low temperatures. In order to extend this finding by an alternative approach, we introduced the desA gene for the D12 fatty-acid desaturase of Synechocystis sp. PCC 6803 into cells of Synechococcus sp. PCC 7942 that normally contain monounsaturated fatty acids but no polyunsaturated fatty acids. This transformation resulted in replacement of half of the monounsaturated fatty acids in the membrane lipids by diunsaturated fatty acids. Comparison of the transformed cells with the wild-type cells revealed that the increase in the number of double bonds in the membrane lipids enhanced the ability of the cells to resist photoinhibition at low temperatures by accelerating the recovery of the photosystem II complex from the photoinhibitory damage. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from damage caused by strong light at low temperatures.

To characterize biochemically the D12 desaturase of Synechocystis sp. PCC 6803, we overexpressed the enzyme in Escherichia coli. The overexpressed enzyme was active; it was associated with cell membranes and represented about 10% of the total cellular protein. The activity of desaturase was very stable in the presence of 2 M sorbitol between pH 7 and pH 8. Purification of the desaturase is in progress.


II. Enhancement of stress tolerance in plants by genetic engineering: transformation with a gene that confers the biosynthesis of glycinebetaine

Glycinebetaine (hereafter abbreviated as betaine) is a compatible solute that is found in a number of halotolerant species of plants and bacteria. It has been implicated in the protection of cellular functions against salt and other types of environmental stress. To examine the effect of betaine in vivo on the protection of the photosynthetic machinery against salt stress, we transformed Arabidopsis thaliana, which does not normally accumulate betaine, with the codA gene for choline oxidase (which catalyzes the conversion of choline to betaine) from Arthrobacter globiformis. Transgenic Arabidopsis plants had the ability to synthesize betaine and to tolerate salt stress. Furthermore, transformation with the codA gene enhanced the protection against low-temperature photoinhibition and accelerated the recovery from photo-induced damage. Thus, accumulation of betaine both enhanced salt tolerance and contributed to resistance to photoinhibition at low temperature. Betaine was also accumulated in the seeds of transformed plants and, as a result, the seeds were more tolerant than seeds of wild-type plants to low temperatures during imbibition and germination. Furthermore, transformation with the codA gene accelerated the growth of young seedlings at low temperatures.

We also introduced the codA gene into rice plants by Agrobacterium-mediated transformation, aiming to enhance the tolerance of this important crop to salt stress. Transformed rice plants accumulated betaine and, consequently, exhibited enhanced ability to tolerate salt stress. Figure 1 shows wild-type rice plants and transformed plants after growth under salt-stress conditions. During the stress treatment with 0.15 M NaCl, growth of both wild-type and transformed plants was inhibited and obvious damage, such as wilting, bleaching of chlorophyll and necrosis, was visible. After removal of the salt stress, the growth rate of transformed plants returned to normal but that of wild-type plants did not recover. The oxygen-evolving machinery of the transformed plants was more tolerant to salt stress than that of the wild-type plants. Our results in Arabidopsis and rice demonstrate the potential usefulness of the codA gene in the engineering of stress tolerance in a wide variety of agronomically important crops.

Fig. 1.
Wild-type rice plants (WT) and rice plants (ChlCOD and CytCOD) transformed with the codA gene from Arthrobacter globiformis after growth under salt stress. Three-week-old plants that had been grown under normal conditions were subjected to salt stress by exposure to 0.15 M NaCl for one week. They were then allowed to grow under normal conditions for three weeks.


III. A new factor involved in the heat stability of the photosynthetic machinery

The evolution of oxygen is one of the reactions of photosynthesis that is extremely susceptible to high temperature. The molecular mechanism underlying the stabilization of the photosynthetic machinery against heat-induced inactivation has been studied in the cyanobacterium Synechococcus sp. PCC 7002. We demonstrated previously that cytochrome c550, located in the lumenal side of thylakoid membranes, is involved in the stability of the oxygen-evolving machinery at high temperatures. We then initiated a search for other factors that might enhance heat stability and we identified a protein of 13 kDa as an important factor. The gene for the 13-kDa protein was cloned from Synechococcus, and the deduced amino-acid sequence revealed that this protein was homologous to PsbU, an extrinsic protein of the photosystem II complex. Targeted mutagenesis of the psbU gene in Synechococcus sp. PCC 7002 resulted in cells in which oxygen-evolving activity was particularly susceptible to high temperature. These results indicate that PsbU plays an important role in stabilizing the oxygen-evolving machinery at high temperatures.


IV. Stress-dependent enhanced expression of cytochrome cM and suppression of expression of cytochrome c6 and plastocyanin

Cytochrome cM is a c-type cytochrome with a molecular mass of 8 kDa. We identified and cloned the cytM gene for cytochrome cM from Synechocystis sp. PCC 6803. In 1997, we cloned homologs of the cytM gene from other cyanophytes and a prochlorophyte, providing evidence that the cytM gene is probably distributed universally in oxygenic photosynthetic prokaryotes. Northern-blotting analysis revealed that the cytM gene of Synechocystis sp. PCC 6803 was barely expressed under normal growth conditions but that expression was induced when cells were exposed to low temperature and/or high-intensity-light. By contrast, expression of the petJ gene for cytochrome c6 and the petE gene for plastocyanin, which are electron carriers that transport electrons from the cytochrome b6/f complex to the photosystem I complex, was suppressed at low temperatures or under high-intensity-light. These observations suggest that regulation of the expression of the cytM gene was the mirror image of regulation of the petJ and petE genes under the stress conditions examined.


Publication List:
(1) Original articles
Deshnium, P., Gombos, Z., Nishiyama, Y. and Murata, N. (1997) The action in vivo of glycine betaine in enhancement of tolerance of Synechococcus sp. strain PCC 7942 to low temperature. J. Bacteriol. 179, 339-344.
Gombos, Z., Kanervo, E., Tsvetkova, N., Sakamoto, T., Aro, E.-M. and Murata, N. (1997) Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane glycerolipids. Plant Physiol., 115, 551-559.
Hayashi, H., Alia, Must‡rdy, L., Deshnium, P., Ida, M. and Murata, N. (1997) Transformation of Arabidopsis thaliana with the codA gene for choline oxidase; accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J., 12, 133-142.
Hideg, E. and Murata, N. (1997) The irreversible photoinhibition of the photosystem II complex in leaves of Vicia faba under strong light. Plant Sci., 130, 151-158.
Kanervo, E., Tasaka, Y., Murata, N. and Aro, E.-M. (1997) Membrane lipid unsaturation modulates processing of photosystem II reaction-center protein D1 at low temperature. Plant Physiol., 114, 841-849.
Los, D.A., Ray, M.K. and Murata, N. (1997) Differences in the control of the temperature-dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803. Mol. Microbiol., 25, 1167-1175.
Nishiyama, Y., Los, D.A., Hayashi, H. and Murata, N. (1997) Thermal protection of the oxygen-evolving machinery by PsbU, an extrinsic protein of photosystem II, in Synechococcus species PCC 7002. Plant Physiol., 115, 1473-1480.
Sakamoto, T., Higashi, S., Wada, H., Murata, N. and Bryant, D.A. (1997) Low temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002. FEMS Microbiol. Lett., 152, 313-320.
Sato, N., Maruyama, K., Nishiyama, Y. and Murata, N. (1997) Identification of a cold-regulated RNA-binding protein from the marine cyanobacterium Synechococcus sp. PCC 7002. J. Plant Res., 110, 405-410.
(2) Review articles
Hayashi, H. and Murata, N. (1997) Genetically engineered enhancement of salt tolerance in higher plants. In Stress Responses of Photosynthetic Organisms (K. Sato and N. Murata, eds.) pp. 133-148. Elsevier Science, Amsterdam.
Murata, N. and Los, D.A. (1997) Membrane fluidity and temperature perception. Plant Physiol., 115, 875-879.
Murata, N. and Nishiyama, Y. (1997) Molecular mechanisms of the low-temperature tolerance of the photosynthetic machinery. In Stress Responses of Photosynthetic Organisms (K. Sato and N. Murata, eds.) pp. 93-112. Elsevier Science, Amsterdam.
Murata, N. and Tasaka, Y. (1997) Glycerol-3-phosphate acyltransferase in plants. Biochim. Biophys. Acta, 1348, 10-16.


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Last Modified: 12:00, May 28, 1998