NATIONAL INSTITUTE FOR BASIC BIOLOGY

DIVISION OF CELLULAR REGULATION

Professor:
Norio Murata

Associate Professor:
Hidenori Hayashi

Research Associates:
Takao Kondo
Ikuo Nishida
Dmitry A. Los

Monbusho Foreign Scientist:
George C Papageorgiou 1)

JSPS Fellow:
Wei-Ai Su 2)

Visiting Scientists:
Yasushi Tasaka
Michael P. Malakhov 3)
Zoltan Gombos 4)
Marie-Helene Macherel 5)
Ding-Ji Shi 6)
Byoung Yong Moon 7)
Ana Maria Otero Casal 8)
Tomoko Shinomura 9)
Masaya Ishikawa 10)

Graduate Students:
Toshio Sakamoto
Katsuzo Noguchi
Patcharaporn Deshnium
Yoshitaka Nishiyama 11)

Technical staffs:
Sho-Ichi Higashi
Miki Ida

(1) from National Research Center Demokritos, Greece)
(2) from Shanghai Institute of Plant Physiology, P.R. China)
(3) from Plant Physiology Institute, Moscow, Russia)
(4) from Biological Research Center Szeged, Hungary)
(5) ECfellow, France)
(6) from Institute of Botany, Academia Sinica, P.R. China)
(7) KOSEF fellow, from Inje University, Korea)
(8) from University of Santiago, Spain)
(9) from Advanced Research Laboratory, Hitachi Ltd. )
(10) from National Institute of Agrobiological Resources)
(11) from the University of Tokyo)

The research effort of this division is directed toward the understanding of to lerance and adaptation of plants to temperature extremes, with particular emphasis on the molecular mechanisms by which plants acclimate or tolerate these temperature conditions. In 1993, several significant advances were made in the area of temperature response and related areas in the study of cyanobacteria.

I. Cyanobacterial desaturases.

Higher plants, and most cyanobacterial strains, contain high levels of polyunsaturated fatty acids, which are important in their response to ambient temperature.

We isolated the desA genes for (DELTA)12 desaturases of the acyl-lipid type from the cyanobacteria, Synechocystis PCC6803, Synechocystis PCC6714, Synechococcus PCC7002, and Anabaena variabilis and found four conserved sequence domains. We over-expressed the desA gene of Synechocystis PCC6803 in Escherichia coli using the bacteriophage T7 polymerase system. The (DELTA)12 desaturase, thus over-expressed in E. coli, was active in vitro when reduced ferredoxin was added as an electron donor. This result indicates that the cyanobacterial desaturase is similar to the plastidial desaturases in terms of the electron-donating system, but is dissimilar to the cytoplasmic desaturases that use cytochrome b5 as an electron donor.

We isolated the desB and desC genes of Synechocystis PCC6803, which encode the (OMEGA)3 and (DELTA)9 desaturases, respectively, of the acyl-lipid type. We transformed another cyanobacterial strain, Synechococcus PCC7942, with the desA and desB genes. This strain contains only the (DELTA)9 desaturase. The mode of fatty acid desaturation in the transformed cells demonstrates that the (OMEGA)3 desaturase can introduce a double bond at the (OMEGA)3 position of fatty acids that contain an unsaturated bond at the (DELTA)12 position.

II. Importance of membrane-lipid unsaturation in tolerance to low-temperature photoinhibition.

To understand the roles of unsaturation of membrane lipids, we transformed Synechococcus PCC7942 with the desA gene. This transformation greatly modified the extent of unsaturation of the fatty acids of membrane lipids. In the wild-type strain, only monounsaturated lipid molecules existed; in the transformant, each of the monounsaturated and diunsaturated lipid molecules contributes to about 50percent of the total membrane lipids. This change in the unsaturation of membrane lipids greatly reduced susceptibility to low temperature.

These results suggest that diunsaturated fatty acids play an important role in protection against damage to photosynthetic processes by low temperature. By contrast, photosynthetic electron transport, measured at various temperatures, and susceptibility to high temperature were not affected by changes in the extent of unsaturation of the fatty acids.

III. Regulation of the expression of the desA gene by changes in the fluidity of the plasma membrane.

Living organisms can maintain the molecular motion, or "fluidity", of membrane lipids by regulating the level of unsaturation in fatty acids. For example, cyanobacterial cells respond to a decrease in temperature by introducing double bonds into the fatty acids of membrane lipids, thus compensating for the temperature-induced decrease in the molecular motion of membrane lipids. Desaturases are responsible for the introduction of these specific double bonds. We have demonstrated that the low temperature-induced desaturation of fatty acids of membrane lipids is regulated at the level of the expression of the desaturase genes.

To study the mechanism of the low temperature-induced regulation of the expression of the desA gene in greater detail, we examined the effect of membrane fluidity on the gene expression by catalytic hydrogenation of the unsaturation bonds of fatty acids of membrane lipids. A 4-min hydrogenation of the Synechocystis PCC6803 cell increased the level of fully saturated lipids at the expenses of diunsaturated lipids in plasma membrane, but had no such effect in thylakoid membranes. After this hydrogenation, the level of mRNA of the desA gene increased 10-fold within 30 min. This increase in the mRNA level after hydrogenation resembles that following a decrease in ambient temperature. These results suggest that the decease in fluidity of plasma membrane is the first response to temperature change.

IV. Heat stability of photosynthesis.

We also focused on the response of plants to high-temperature stress. Since photosynthesis is the physiological process most susceptible to heat stress in plants, its heat stability is an important factor in the tolerance of plants to high temperature.

We studied a component responsible for the heat stability of photosynthesis in the cyanobacterium, Synechococcus PCC7002. When thylakoid membranes isolated from the cyanobacterial cells were treated with a low concentration of Triton X-100, the heat stability of oxygen evolution was decreased by 4'C. From the extracts with Triton X-100, we purified a protein that increased the heat stability of oxygen evolution by 4'C. The protein was identified as a low redox potential cytochrome c-550 having a molecular mass of 16 kDa. We isolated the gene encoding this cytochrome from Synechococcus PCC7002, and determined its nucleotide sequence. The deduced amino-acid sequence revealed that the gene product consists of a transit peptide of 34 residues and a mature protein of 136 residues. These results indicate that cytochrome c-550 is involved in the mechanism of heat stability of oxygen evolution and, therefore, in the heat stability of photosynthesis.

V. Two groEL-homologous genes in cyanobacterial cells.

There are a wide variety of heat shock proteins, most of which are highly conserved in both prokaryotes and eukaryotes. Cellular levels of these proteins dramatically increase upon heat shock or other environmental stresses. Some heat shock proteins act as molecular chaperones that assist in the folding and assembly of other proteins.

To examine the role of heat shock proteins in acclimation to high-temperature stress, we attempted to isolate the genes for the heat shock protein groEL from Synechococcus PCC7002. We discovered that there are two groEL-homologous genes. One of the groEL-homologous genes is accompanied by the groES gene, and the groES and groEL genes constitute the groESL operon as the groESL operon in E. coli. The other groEL-homologous gene is not accompanied by the groES homologous gene. However, this groEL-homologous gene retains the carboxyl-terminal repeat of Gly-Gly-Met, which is typical in the groEL gene of E. coli. Heat shock increased the levels of mRNAs of both groEL-homologous genes. To understand the function of the product of the groEL-homologous genes, we have knocked out one of the groEL homologous genes by insertional disruption with a kanamycin resistance gene cartridge. We are currently investigating the responses of these mutants to high-temperature stress.

Publication List:

(1) Original papers

Allakhverdiev, S.1., Hayashi, H., Fujimura, Y., Klimov, V.V. and Murata, N. (1993) Inactivation of photosynthetic oxygen evolution by (o-phenanthroline and LiCl04 in photosystem 2 of pea. Photosynth. Res. 35, 345-349.

Hayashi, H., Fujimura, Y., Mohanty, P.S. and Murata, N. (1993) The role of CP 47 in the evolution of oxygen and the binding of the extrinsic 33kDa protein to the core complex of Photosystem ll as determined by limited proteolysis. Photosynth. Res. 36, 35-42.

Higashi, S. and Murata, N. (1993) An in vivo study of substrate specificities of acyl-lipid desaturases and acyltransferases in lipid synthesis in Synechocystis PCC6803. Plant Physiol. 102, 1275-1278.

Kondo, T., Strayer, C.A., Kulkarni, R.D., Taylor, W., Ishiura, M. Golden, S.S. and Johnson, C.H. (1993) Circadian rhythms in prokaryotes: Iuciferase as a reporter of circadian gene expression in cyanobacteria. Proc. Natl. Acad. Sci. USA 90, 5672-5676.

Lehel, C., Los, D., Wada, H., Gyorgyei, J., Horvath, I., Kovacs, E., Murata, N . and Vigh, L. (1993) A second groEL-Iike gene, organized in a groESL operon is present in the genome of Synechocystis sp. PCC6803. J. Biol. Chem. 268, 1799-1804.

Los, D., Horvath, I., Vigh, L. and Murata, N. (1993) The temperature-dependent expression of the desaturase gene desA in Synechocystis PCC6803. FEBS Lett. 318, 57-60.

Malakhov, M.P., Wada, H., Los, D.A., Sakamoto, T. and Murata, N. (1993) Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9. Plant Mol. Biol. 21, 913-918.

Mamedov, M., Hayashi, H. and Murata, N. (1993) Effects of glycinebetaine and unsaturation of membrane lipids on heat stability of photosynthetic electron-transport and phosphorylation reactions in Synechocystis PCC6803. Biochim. Biophys. Acta 1142, 1-5.

Mohanty, P., Hayashi, H., Papageorgiou, G.C. and Murata, N. (1993) Stabilization of the Mn-cluster of the oxygenevolving complex by glycinebetaine. Biochim. Biophys. Acta 1144, 92-96.

Nishida, I., Tasaka, Y., Shiraishi, H. and Murata, N. (1993) The gene and the RNA for the precursor to the plastidlocated glycerol-3-phosphate acyltransferase of Arabidopsis thaliana. Plant Mol. Biol. 21, 267-277.

Nishiyama, Y., Kovacs, E., Lee, C.B., Hayashi, H., Watanabe, T. and Murata, N. (1993) Photosynthetic adaptation to high temperature associated with thylakoid membranes of Synechococcus PCC7002. Plant Cell Physiol. 34, 337-343.

Vigh, L., Los, D.A., Horvath, I. and Murata, N. (1993) The primary signal in the biological perception of temperature: Pd-catalyzed hydrogenation of membrane lipids stimulated the expression of the desA gene in Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 90, 9090-9094.

Wada, H., Schmidt, H., Heinz, E. and Murata, N. (1993) In vitro ferredoxindependent desaturation of fatty acids in cyanobacterial thylakoid membranes. J. Bacteriol. 175, 544-547.

Wada, H., Macherel, M.-H. and Murata, N. (1993) The desA gene of the cyanobacterium Synechocystis sp. strain PCC6803 is the structural gene for A 12 desaturase. J Bacteriol. 175, 6056-6058.

(2) Reviews
Murata, N., Wada, H., Gombos, Z. and Nishida, I . (1993) The molecular mechanism of the low-temperature tolerance of plants studied by gene technology of membrane lipids. In Interacting Stresses on Plants in a Changing Climate (M.B. Jackson and C.R. Black, eds.). Springer-Verlag, Berlin, pp. 7 15-723.

Nishida, I., Imai, H., Ishizaki-Nishizawa, O., Tasaka, Y., Shiraishi, H., Higashi, S., Hayashi, H., Beppu, T., Matsuo, T. and Murata, N. (1993) Molecular and physiological studies of glycerol-3-phosphate acyltransferase, acyl-ACP hydrolase and stearoyl-ACP desaturase. In Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants (N. Murata and C.R. Somerville, eds.). The American Society of Plant Physiologists, Rockville, Maryland, pp. 79-88.

Wada, H., Gombos, Z., Sakamoto, T., Higashi, S., Los, D.A., Heinz, E., Schmidt, H. and Murata, N. (1993) Fatty acid desaturation in cyanobacteria. In Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants (N. Murata and C.R. Somerville, eds.). The American Society of Plant Physiologists, Rockville, Maryland, pp. 67-78.

Wada, H., Gombos, Z., Sakamoto, T. and Murata, N. (1993) Role of lipids in low-temperature adaptation. In Photosynthetic Responses to the Environment (H.Y. Yamamoto and C.M. Smith, eds.). The American Society for Plant Physiologists, Rockville, Maryland, pp. 78-87.

(3) Book
Murata, N. and Somerville, C., Eds. (1993) Biochemistry and Molecular Biology of Membrane and Storage Lipids of Plants, the American Society of Plant Physiologists, Rockville, Maryland.