  National Insitute for Basic Biology

DIVISION OF CELLULAR REGULATION

Professor:: Norio Murata

Associate Professor:: Hidenori Hayashi

Research Associates:: Takao Kondo; Ikuo Nishida; Dmitry A. Los; Atsushi Sakamoto

Monbusho Foreign Scientist:: Laszlo Mustardy¹⁾

Visiting Scientists:: Yasushi Tasaka; Byoung Yong Moon²⁾; Michael P. Malakhov³⁾; Zoltan Gombos⁴⁾; Carl H. Johnson⁵⁾

Graduate Students:: Katsuzo Noguchi; Patcharaporn Deshnium; Sayamrat Panpoom

Technical staffs:: Sho-Ichi Higashi; Miki Ida

(¹⁾ from Biological Research Center, Szeged, Hungary)
(²⁾ from Inje University, Pusan, Korea)
(³⁾ from Plant Physiology Institute, Moscow, Russia)
(⁴⁾ from Biological Research Center, Szeged, Hungary)
(⁵⁾ from Vanderbilt University, TN, USA)

The research effort of this division is directed toward understanding the tolerance and acclimation of plants to temperature extremes, with particular emphasis on the molecular mechanisms by which plants acclimate to or tolerate temperature conditions. In 1994, several significant achievements were made in the following areas with cyanobacteria and higher plants as experimental materials.

I. Molecular cloning and characterization of cyanobacterial desaturases.

Higher plants and most cyanobacteria have high levels of membrane lipids containing polyunsaturated fatty acids, which are important in their response to ambient temperature. We have isolated the desA, desB, and desC genes of Synechocystis sp. PCC 6803 that encode the Æ12, w3, and Æ9 desaturases, respectively, of the acyl-lipid type. We overexpressed the desA and desC genes in Escherichia coli using the bacteriophage T7 RNA polymerase system. The desaturases, thus overexpressed in E. coli, were active in vitro when reduced ferredoxin was supplemented as an electron donor. The mode of fatty acid desaturation in the transformed E. coli cells demonstrates that the Æ9 and Æ12 desaturases are specific to the sn-1 position of the glycerol moiety and the C18 fatty acids, and are nonspecific with regard to the polar head group of the lipid.

II. Regulation of the expression of the desaturase genes.

Living organisms are exposed to changes in ambient temperatures. However, they can maintain levels of molecular motion, or "fluidity," of membrane lipids by regulating the level of their fatty acid unsaturation. For example, cyanobacterial cells respond to a temperature decrease 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 the fatty acids of the membrane lipids is regulated at the level of the expression of the desaturase genes.

We have shown that the levels of the transcripts of the desA, desB, and desD (for Æ6 desaturase) genes increase about 10-fold during a downward shift of temperature from 34°C to 22°C. However, the level of the transcript of the desC gene remains constant after the temperature shift, suggesting that the expression of the gene is constitutive. Determination of the 5'-termini of the desaturases indicated that the sites of initiation of transcription are significantly similar among all four desaturase genes. Identification of the regulatory elements of the desaturase genes and cloning of their transacting factors are under way.

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

To understand the role of unsaturation of membrane lipids, we disrupted the desA and desD genes in Synechocystis sp. PCC 6803 by inserting antibiotic resistance gene cartridges. This mutation greatly modified the extent of unsaturation of the fatty acids of the membrane lipids (Figure 1). In the wild-type strain, each of the saturated, monounsaturated, diunsaturated, and triunsaturated lipid molecules amounted to 15% of the total membrane lipids; in the desD ^- mutant, the triunsaturated lipid molecule was not present; in the desA ^-/desD ^- mutant, 80% of the total membrane lipids were monounsaturated lipid molecules and only 15% were fully saturated molecules. These decreases in the unsaturation of membrane lipids greatly reduced the tolerance of the cyanobacterium to low temperature in the presence of light.

Figure.1
Changes in composition of lipid molecular species by step-wise depletion of desaturases in Synechocystis sp. PCC 6803. WT, wild-type strain; desD ^-, a mutant of A6 desaturase in which the desD gene was disrupted by insertion of a chloramphenicol resistance gene cartridge; desA ^-/desD ^-, double mutant of Æ6 and Æ12 desaturases in which the desA gene in the desD ^- mutant was mutated by insertion of a kanamycin resistance gene cartridge.

These results together with other observations suggest that unsaturated fatty acids play an important role in protection of the photosynthetic machinery meant to protect against low-temperature photoinhibition. Because the extent of photoinhibition in intact cells reflects a balance between the light-induced inactivation of the photosystem II protein complex and the recovery of the complex from the inactivation, we separated the two processes using protein synthesis inhibitors. The results demonstrated that the unsaturation of the membrane lipids has no effect on the light-induced inactivation process, but accelerates the recovery of the photosystem II complex from the photoinactivated state. Essentially, the same results were obtained from studies of a higher plant, namely, tobacco, in which the level of unsaturation of the thylakoid membrane lipids was genetically modulated by overexpression of glycerol-3-phosphate acyltransferase from squash.

IV. Heat stability of photosynthesis.

We also focused on the response of plants to high-temperature stress. Because photosynthesis is one of the physiological processes that is most susceptible to heat stress in plants, stabilization of the photosynthetic machinery against heat helps plants to tolerate higher temperature.

We have studied an effect for the heat stability of photosynthesis in the cyanobacterium, Synechococcus sp. PCC 7002. When the 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 obtained with Triton X-100, we purified a protein that re-established the heat stability of oxygen evolution. The protein was identified as cytochrome c-550 which has a low redox potential and a molecular mass of 16 kDa. 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. We isolated the gene encoding this cytochrome of Synechococcus sp. PCC 7002. Disruption of this gene in Synechococcus sp. PCC 7002 is in progress in this laboratory. We also studied the heat tolerance of photosynthesis in cultured soybean cells. The heat stability of oxygen evolution in these cells was enhanced by 3°C upon an increase of growth temperature from 25°C to 35°C.

V. Two genes homologous to groEL in cyanobacteria.

GroEL is a highly conserved heat shock protein in prokaryotes and a homologue of the eukaryotic heat shock protein with a molecular mass of about 60 kDa (HSP60). These heat shock proteins act as molecular chaperones that assist proper folding and assembly of other proteins. We discovered two kinds of groEL homologue in Synechococcus sp. PCC 7002 and designated them as groEL-a and groEL-ß. The groEL-a gene forms an operon together with the upstream component of the groES gene, as is the case with the groEL of E. coli. The groEL-ß gene is not accompanied by the groES gene, but groEL-ß retains the carboxyl terminal repeat of Gly-Gly-Met as in the E. coli GroEL.

We mutated the groEL-ß gene in Synechococcus sp. PCC 7002 by inserting an antibiotic resistance gene cartridge and examined the heat shock response of the mutant cells. The heat shock response was greatly reduced in the groEL-ß mutant cells. This was evaluated from examining the viability at high temperatures after heat shock treatment, for example, viability at 48°C after exposure to 45°C. These results demonstrate that not only the groEL-a but also the groEL-ß gene products contribute to the heat shock response in the cyanobacterium. However, the thermal tolerance of photosynthetic oxygen evolution was not modified by the groEL-ß mutation, indicating that GroEL-ß does not contribute to the thermal tolerance of photosynthesis.

VI. Genetic modification of salinity tolerance of a cyanobacterium.

Under saline conditions, some plants produce compatible solutes to avoid deleterious effects caused by salt intolerance. Glycine betaine, a quaternary ammonium compound, is one of such solute found in halotolerant plants and bacteria. We discovered that this compound protects the photosystem II protein complex from inactivation of oxygen evolution in saline conditions. To examine the effect of glycine betaine on photosynthesis in vivo, we transformed the salinity-sensitive cyanobacterium, Synechococcus sp. PCC 7942, with the codeA gene for choline oxidase of Arthrobacter globiformis, which can oxidize choline into glycine betaine. The transformed cells accumulated glycine betaine to a concentration of about 85 mM and grew in the medium containing 0.4 M NaCl (lethal conditions for the wild-type cells) (Figure 2). The salinity tolerance of the oxygen-evolving activity was enhanced by the transformation. These observations demonstrate the in vivo action of glycine betaine in protecting the cyanobacterial cells from salinity-induced stresses.

Figure 2.
Growth under saline conditions in control cells (PAM) and transformed cells (PAMCOD) in which the codA gene (for choline oxidase) was introduced in the chromosome of Synechococcus sp. PCC 7942 and was overexpressed under control of the ConII promoter. Cells were grown at 30°C for 6 days in a medium containing O.4 M NaCl.

Publication List:

(1) Original papers: Gombos, Z., Wada, H. and Murata, N. ( 1994) The recovery of photosynthesis from low-temperature photoinhibition is accelerated by the unsaturation of membrane lipids: a mechanism of chilling tolerance. Proc. Natt Acad Sci. U.S.A. 91, 8787-8791.; Gombos, Z., Wada, H., Hideg, E. and Murata, N. (1994) The unsaturation of membrane lipids stabilizes photosynthesis against heat stress. Plant Physiol. 104, 563-567.; Kondo, T. and Ishiura, M. (1994) Circadian rhythms of cyanobacteria: Monitoring the biological clock of individual colonies by bioluminescence. J Bacteriol. 176, 1881-1885.; Kondo, T., Tsinoremas, N.F., Golden, S.S., Johnson, C.H., Kutsuna, S. and Ishiura, M. (1994) Circadian clock mutants of cyanobacteria. Science 266, 1233-1236.; Los, D.A. and Murata, N. (1994) Lowtemperature induced accumulation of the desaturase gene transcript in Synechocystis PCC 6803 results from both acceleration of transcription and increase in mRNA stability. Russian J. Plant Physiol. 41, 147-151.; Malakhov, M.P., Wada, H., Los, D.A., Semenenko, V.E. and Murata, N. (1994) A new type cytochrome c from Synechocystis PCC 6803. J. Plant Physiol. 144, 259-264.; Nishiyama, Y., Hayashi, H., Watanabe, T. and Murata, N. (1994) Photosynthetic oxygen evolution is stabilized by cytochrome c-550 against heat inactivation in Synechococcus sp. PCC 7002. Plant Physiol. 105, 1313-1319.; Sakamoto, T., Los, D.A., Higashi, S.) Wada, H., Nishida, I., Ohmori, M. and Murata, N. (1994) Cloning of w3 desaturase from cyanobacteria and its use in altering the degree of membrane-lipid unsaturation. Plant Mol. Biol. 26, 249-264.; Sakamoto, T., Wada, H., Nishida, I., Ohmori, M. and Murata, N. (1994) ldentification of conserved domains in the Æ 12 desaturase of cyanobacteria. Plant Mol. Biol. 24, 643-650.; Sakamoto, T., Wada, H., Nishida, I., Ohmori, M. and Murata, N. (1994) Æ9 acyl-lipid desaturase of cyanobacteria: Molecular cloning and substrate specificities in terms of fatty acids, snpositions, and polar head groups. J. Biel. Chem. 269, 25576-25580.; Wada, H., Gombos, Z. and Murata, N. (1994) Contribution of membrane lipids to the ability of the photosynthetic machinery to tolerate temperature stress. Proc. Natl. Acad. Sci. U.S.A. 91, 4273-4277.

(2) Reviews: Murata, N. (1994) Genetic and temperature-induced modulation of cyanobacterial membrane lipids. In Temperature Adaptation of Biological Membranes (A.R. Cossins, ed.), Portland Press, London, pp. 155-162.; Murata, N. (1994) Genetic engineering of phosphatidylglycerol and chilling sensitivity in higher plants. In Temperature Adaptation of Biological Membranes (A.R. Cossins, ed.), Portland Press, London, pp. 163-167.

(3) Proceeding: Hayashi, H., Nishida, I., Ishizaki-Nishizawa, O., Nishiyama, Y. and Murata, N. (1994) Genetically engineered modification of plant chilling sensitivity and characterization of cyanobacterial heat shock proteins. In Biochemical and Cellular Mechanisms of Stress Tolerance in Plants (J.H.Cherry, ed.), Springer-Verlag, Berlin, pp. 543-555