NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Cellular Regulation

Professor:
Norio Murata
Associate Professor:
Hajime Wada1) (adjunct)
Research Associates:
Atsushi Sakamoto
Yoshitaka Nishiyama
Iwane Suzuki
Monbusho Foreign Scientists:
Malay Kumar Ray2)
Zoltan Gombos3)
NIBB Fellow:
Akira Katoh
JSPS Visiting Scientists:
Ding-Ji Shi4)
Éva Hideg3)
P. Pardha Saradhi5)
Diego de Mendoza6)
Richard A. Dilley7)
Peddisetty Sharmila5)
JSPS Postdoctoral Fellows:
Alia5)
Michael P. Malakhov8)
EC Fellow:
Ana Maria Otero Casal9)
NIBB Visiting Scientists:
Yasushi Tasaka
Oxana Malakhov10)
Monika Debreczeny3)
Prasanna Mohanty11)
Byong Yong Moon12)
Leonid Kislov8)
Kostas Stamatakis13)
Silvia Franceschelli14)
Laszlo Mustardy3)
Nelly Tsvetkova15)
Tony H. H. Chen16)
Otto Zsiros3)
Dmitry A. Los8)
Julian Eaton-Rye17)
Suleyman I. Allakhverdiev18)
Graduate Students:
Sayamrat Panpoom
Masami Inaba
Hiroshi Yamamoto
Yuji Tanaka
Fumiyasu Yamaguchi
Technical Staffs:
Sho-Ichi Higashi
Hideko Nonaka
1) from Kyushu University, Fukuoka, Japan
2) from Centre for Cellular and Molecular Biology, Hyderabad, India
3) from Biological Research Center, Szeged, Hungary
4) from Institute of Botany, Academia Sinica, Beijing, China
5) from Jamia Millia Islamia University, New Delhi, India
6) from Universidad Nacional de Rosario, Rosario, Argentina
7) from Purdue University, Lafayette, IN, USA
8) from Plant Physiology Institute, Moscow, Russia
9) from Universidad de Santiago, Santiago de Compostela, Spain
10) from Institute of General Genetics, Moscow, Russia
11) from Jawaharlal Nehru University, New Delhi, India
12) from Inje University, Kimhae, Korea
13) from National Centre for Scientific Research ³Demokritos², Athens, Greece
14) from International Institute for Genetics and Biophysics, Naples, Italy
15) from Bulgarian Academy of Sciences, Sofia, Bulgaria
16) from Oregon State University, Corvallis, OR, USA
17) from Otago University, Dunedin, New Zealand
18) from Institute of Soil Science and Photosynthesis, Moscow, Russia


The research effort of this division is aimed at establishing a full understanding of the molecular mechanisms by which plants can acclimate to and tolerate stresses that arise from changes in environment conditions, with particular emphasis on temperature stress and salt stress. In 1996, significant progress was made in research on the following topics in studies with cyanobacteria and higher plants as experimental materials.


I. The temperature-regulated expression of genes for acyl-lipid desaturases in a cyanobacterium

Most living organisms are exposed to changes in the temperature of their environment. In response to a decrease in temperature, an increase occurs in the extent of unsaturation of membrane lipids, which enhances the fluidity of the membranes. This mechanism provides compensation for the decrease in the molecular motion of membrane lipids, which is caused by the decrease in ambient temperature. Individual acyl-lipid desaturases introduce a double bond into fatty acids that are esterified to the glycerol moiety of membrane lipids. The cyanobacterium Synechocystis sp. PCC 6803 has four genes, desA, desB, desC and desD, that encode the D12, w3, D9 and D6 acyl-lipid desaturases, respectively. We investigated the regulation of the expression of the genes for these desaturases in response to changes in temperature. We found that low temperatures enhance the steady-state levels of mRNAs for all the desaturases with the exception of the D9 desaturase. Functional analysis of promoters, using a bacterial gene for luciferase as a reporter, revealed that, to some extent, it is the activation of the promoters of the genes for the D12 and w3 desaturases that is responsible for the increases in the levels of their mRNAs after a downward shift in temperature. In addition, the lifetimes of the mRNAs for the D6, D12, and w3 desaturases were extended at lower temperatures, indicating that the stability of mRNAs might also be involved in the control of the levels of the desaturases. Western blotting analysis demonstrated that the levels of the D6, D12 and w3 desaturases increase at low temperatures, while the level of the D9 desaturase remains constant. These results indicate that the expression of the gene for the D9 desaturase is basically independent of temperature, while the expression of the genes for the D6, D12, and w3 desaturases is regulated by temperature.


II. Localization of acyl-lipid desaturases in cyanobacterial cells

An immunocytochemical study was performed in an attempt to localize the four acyl-lipid desaturases in Synechocystis sp. PCC 6803 using antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-termini of the individual desaturases. The individual preparations of antibodies were specific to the respective desaturases. Immunogold labeling and electron microscopy revealed the specific distribution of the desaturases in regions that corresponded to both the cytoplasmic and the thylakoid membranes, suggesting that all four desaturases are located in both types of membrane. Localization of the desaturases in thylakoid membranes was further confirmed by Western blotting of proteins from isolated membranes. These findings indicate that the desaturation of the fatty acids of membrane lipids occurs within the thylakoid membranes as well as within the cytoplasmic membranes in the cyanobacterial cells.


III. The importance of polyunsaturated membrane lipids in photosynthesis

We demonstrated previously that polyunsaturated membrane lipids are important in the ability of the photosynthetic machinery to tolerate low temperatures by targeted disruption of genes for desaturases in Synechocystis sp. PCC 6803, in which all polyunsaturated fatty acids of membrane lipids were replaced, as a result, by monounsaturated fatty acids. To confirm this finding using an alternative approach, we introduced the desA gene for the D12 desaturase of Synechocystis sp. PCC 6803 into cells of Synechococcus sp. PCC 7942 that contained monounsaturated fatty acids but no polyunsaturated fatty acids. As the result of this transformation, half of the monounsaturated fatty acids in the membrane lipids were replaced 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 photoinhibitory damage. These findings indicate that polyunsaturated fatty acids are important in the protection of the photosynthetic machinery from damage by strong light at low temperatures.


IV. Acclimation to heat of the photosynthetic machinery

The evolution of oxygen is one of the reactions of photosynthesis that is most 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 lumen of thylakoids, is involved in the stability of the oxygen-evolving machinery at high temperatures. We searched for another factor that might enhance the heat stability and recently identified a protein of 13 kDa as an important factor. The gene encoding the 13-kDa protein was cloned from Synechococcus, and the deduced amino-acid sequence revealed that this protein is homologous to the PsbU protein, an extrinsic protein of photosystem II, which has previously been found in thermophilic species of cyanobacteria. Inactivation of the gene in Synechococcus sp. PCC 7942, by insertion of an antibiotic-resistance gene cartridge, had no significant effect on the oxygen-evolving activity or on the photoautotrophic growth at temperatures within the physiological range. However, when mutant cells and wild-type cells that had been grown at a physiological temperature were exposed to a higher temperature, the oxygen-evolving activity was lost much more rapidly from the mutant cells than from the wild type. These results indicate that the PsbU protein plays an important role in stabilizing the oxygen-evolving machinery at high temperatures.


V. Genetic enhancement of stress tolerance in Arabidopsis thaliana and in rice plants by genetic engineering that allowed the biosynthesis of glycinebetaine

Glycinebetaine (hereafter abbreviated as betaine) is a compatible solute that is found in a number of halo-tolerant 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 on the protection of the photosynthetic machinery in vivo 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 (H. Hayashi et al. (1997) Plant J., in press). Transgenic Arabidopsis plants acquired the ability to synthesize betaine and to tolerate salt stress throughout their life cycle. Figure 1 shows the enhanced tolerance to salt stress (100 mM NaCl) of seedlings of transgenic Arabidopsis. Furthermore, the transformation 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.

We also introduced the codA gene of A. globiformis into rice, one of the most important crops worldwide, aiming to enhance the salt tolerance of this cereal crop. Transgenic rice plants expressed the codA gene and accumulated betaine. Furthermore, the oxygen-evolving machinery of the transgenic plants was more tolerant to salt stress than that of the wild-type plants. These results demonstrate the potential usefulness of the codA gene in the engineering of stress tolerance in a wide variety of agronomically important crops.

Fig. 1.
Effects of salt stress on the growth of seedlings after germination of seeds of wild-type and transformed Arabidopsis thaliana. Seeds from wild-type and transformed plants were germinated on a plate of Murashige-Skoog medium supplemented with 0.1 M NaCl and solidified with Gellan gum, and incubated at 22ƒC for 20 days.


Publication List:
(1) Original articles
Allakhverdiev, S. I., Feyziev, Ya. M., Ahmed, A., Hayashi, H., Aliev, Ja. A., Klimov, V. V., Murata, N. and Carpentier, R. (1996) Stabilization of oxygen evolution and primary electron transport reactions in photosystem II against heat stress with glycinebetaine and sucrose. J. Photochem. Photobiol. 34, 149-157.
Gombos, Z., Wada, H., Varkonyi, Z., Los, D. A. and Murata, N. (1996) Characterization of the Fad12 mutant of Synechocystis that is defective in D12 acyl-lipid desaturase activity. Biochim. Biophys. Acta 1299, 117-123.
Ishizaki-Nishizawa, O., Fujii, T., Azuma, M., Sekiguchi, K., Murata, N., Ohtani, T. and Toguri, T. (1996) Low-temperature resistance of higher plants is significantly enhanced by a nonspecific cyanobacterial desaturase. Nature Biotech. 14, 1003-1006.
Malakhov, M., Wada, H., Los, D. A. and Murata, N. (1996) The coxD gene for heme O synthase in Synechocystis. Biochim. Biophys. Acta 1273, 84-86.
Mustardy, L., Los, D. A., Gombos, Z. and Murata, N. (1996) Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: Evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation. Proc. Natl. Acad. Sci. U.S.A. 93, 10524-10527.
Nishida, I., Swinhoe, R., Slabas, A. and Murata, N. (1996) Cloning of Brassica napus CTP: phosphocholine cytidylyltransferase cDNAs by complementation in a yeast cct mutant. Plant Mol. Biol. 31, 205-211.
Tasaka, Y., Gombos, Z., Nishiyama, Y., Mohanty, P., Ohba, T., Ohki, K. and Murata, N. (1996) Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: Evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis. EMBO J. 15, 6416-6425.
(2) Review articles
Murata, N., Deshnium, P. and Tasaka, Y. (1996) Biosynthesis of g-linolenic acid in the cyanobacterium Spirulina platensis. In g-Linolenic Acid (Y.-S. Huang and D. E. Mills, eds.) pp. 22-32. AOCS Press, Champaign, Illinois.
Nishida, I. and Murata, N. (1996) Chilling sensitivity in plants and cyanobacteria: The crucial contribution of membrane lipids. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47, 541-568.

nibb-adm@nibb.ac.jp
Last Modified: 12:00, June 27, 1997