NATIONAL INSTITUTE FOR BASIC BIOLOGY
DIVISION OF CELLULAR REGULATION
- Norio Murata
- Associate Professor:
- Hidenori Hayashi
- Research Associates:
- Takao Kondo
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
Yoshitaka Nishiyama 11)
- Technical staffs:
- Sho-Ichi Higashi
(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
I. Cyanobacterial desaturases.
- Higher plants, and most cyanobacterial strains, contain high levels of
polyunsaturated fatty acids, which are important in their response to
- 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
- 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
- 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
- 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
- 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.
- (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.