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:
Suleyman I. Allakhverdiev1)
Institute Research Fellow:
Akira Katoh
Mikio Kinoshita
Sayamrat Panpoom
JSPS Visiting Scientists:
Vyacheslav V. Klimov1)
JSPS Postdoctoral Fellows:
Eira Kanervo2)
EU Fellow:
Silvia Franceschelli3)
Visiting Scientists:
Adam Gilmore4)
Cai-Xia Hou5)
Larissa Kisseleva6)
Dmitry A. Los6)
Zolt*n Magyar7)
Bruno Maresca3)
Liberato Marzullo3)
Bal*zs Szalontai7)
Roumiana Todorova8)
Tom Wydrzynski4)
Graduate Students:
Masami Inaba
Hiroshi Yamamoto
Yuji Tanaka
Ryoma Suzuki
Yu Kanesaki
Technical Staff:
Sho-ichi Higashi
Hideko Nonaka
1) from the Institute of Basic Biological Problems, Pushchino, Russia
2) from the University of Turku, Turku, Finland
3) from the International Institute of Genetics and Biophysics, Naples, Italy
4) from the Australian National University, Canberra, Australia
5) from Xinjiang University, Urumqi, China
6) from the Institute of Plant Physiology, Moscow, Russia
7) from the Biological Research Center, Szeged, Hungary
8) from the Institute of Biophysics, Sofia, Bulgaria


The research efforts of this division are 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 stress and salt stress. In 1998, we made significant progress in the following areas, using higher plants and cyanobacteria as our experimental materials.


I. Enhancement of the stress tolerance of plants by genetic engineering of the synthesis 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 high concentrations of salt and other types of environmental stress. To examine the effects of betaine in vivo on the protection against high-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. Transformed Arabidopsis plants were able to synthesize betaine and to tolerate high-salt stress. Moreover, transformation with the codA gene enhanced the protection of the photosynthetic machinery against low-temperature photoinhibition and accelerated the recovery of the machinery from photo-induced damage. Betaine also accumulated in the seeds of the transformed plants. Such seeds were more tolerant than seeds of wild-type plants to low temperatures during imbibition and germination. The growth of young seedlings of transformed plants was also accelerated at low temperatures. Furthermore, transformation with the codA gene significantly enhanced the tolerance of seeds to high temperatures during both imbibition and germination (Fig. 1), and young seedlings also exhibited enhanced tolerance to high temperatures. Thus, accumulation of betaine increased the tolerance of Arabidopsis to various stresses, namely, high-salt stress, light stress, low-temperature stress and high-temperature stress.

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, as a consequence, exhibited tolerance to high-salt stress. During treatment with 0.15 M NaCl (high-salt stress), 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, transformed plants began to grow again at the normal rate after a significantly shorter time than the wild-type plants. The photosynthetic machinery of the transformed plants was more tolerant to salt stress and low-temperature stress than was that of the wild-type plants. Our results in Arabidopsis and rice demonstrate the potential usefulness of the codA gene in the genetic engineering of stress tolerance in a wide variety of agronomically important crops.

Fig. 1.
Effects of high temperature during imbibition on the subsequent germination of seeds from wild-type and transformed plants of Arabidopsis thaliana. Dry seeds were allowed to imbibe water at 22, 40, 50 and 55ƒC for 60 min. Then seeds were inoculated on plates of MS medium. After incubation at 4ƒC for 2 days, the seeds were incubated at 22ƒC. Three days later, after germination had occurred, plates were photographed. Each plate was 9 cm in diameter.


II. Important roles of polyunsaturated membrane lipids in stress tolerance

In studies conducted prior to 1998, we succeeded in inactivating genes for fatty-acid desaturases by targeted mutagenesis in the cyanobacterium Synechocystis sp. PCC 6803 and we produced several mutant strains in which the fatty acids of the membrane lipids contained abnormal numbers of unsaturated bonds. By comparing various properties of 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 the wild-type strain Synechococcus sp. PCC 7942, which normally contains monounsaturated fatty acids and no polyunsaturated fatty acids. We designated the transformed strain desA+. The transformation resulted in replacement of half of the monounsaturated fatty acids in the membrane lipids by diunsaturated fatty acids. Comparison of desA+ cells with wild-type cells revealed that the increased number of double bonds in the membrane lipids enhanced the ability of cells to resist photoinhibition at low temperatures by accelerating the recovery of the photosystem II complex from photoinhibitory damage. Our findings indicate that polyunsaturated membrane lipids are important in protecting the photosynthetic machinery from damage caused by strong light at low temperatures.

The D1 protein at the catalytic center of the photosystem II complex of Synechococcus sp. PCC 7942 exists as two isoforms, designated D1:1 and D1:2, and transfer of cells to low temperatures results in the replacement of D1:1, the prevailing form, by D1:2. The extent of such replacement in desA+ cells at low temperatures was greater than that in wild-type cells. It seems likely that polyunsaturated membrane lipids facilitate the exchange of isoforms of the D1 protein and, in this way, they maintain the photochemical activity of the photosystem II complex at low temperatures.

We investigated the role of polyunsaturated membrane lipids in the tolerance of the photosynthetic machinery to high-salt stress by comparing the desA-/desD- mutant of Synechocystis sp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. The oxygen-evolving activity of desA-/desD- cells was more sensitive to high-salt stress than was that of wild-type cells. Moreover, the activity of the Na+/H+ antiport in desA-/desD- cells was suppressed to a greater extent than that of the antiport in wild-type cells under high-salt stress. These observations suggest that polyunsaturated membrane lipids might stimulate the activity and/or the synthesis of the Na+/H+ antiport system and protect the photosynthetic machinery against salt-induced inactivation.


III. Molecular mechanisms of the protection of the photosynthetic machinery against high-temperature stress

The oxygen-evolving machinery of the photosystem II complex is extremely susceptible to inhibition at high temperatures. We have been studying the molecular mechanisms that underlie the stabilization of the photosynthetic machinery against the heat-induced inactivation in the cyanobacterium Synechococcus sp. PCC 7002. Biochemical investigations of thylakoid membranes allowed us to identify two proteins, cytochrome c550 and PsbU, as factors that stabilize the oxygen-evolving machinery at high temperatures. To elucidate the role of PsbU in vivo, we inactivated the psbU gene in Synechococcus sp. PCC 7002 by targeted mutagenesis. Mutated cells were not only unable to increase the thermal stability of their oxygen-evolving machinery but they were also unable to develop cellular thermotolerance upon acclimation to high temperatures. These results suggest that PsbU might play an important role in enhancing the thermal stability of the oxygen-evolving machinery at high temperatures and, moreover, that the stabilization of the machinery might be crucial for the acquisition of cellular thermotolerance.


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

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


Publication List:
(1) Original articles
Alia, Hayashi, H., Chen, T.H.H. and Murata, N. (1998) Transformation with a gene for choline oxidase enhances the cold tolerance of Arabidopsis during germination and early growth. Plant Cell Environ., 21, 232-239.
Alia, Hayashi, H., Sakamoto, A. and Murata, N. (1998) Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine. Plant J., 16, 155-161.
Fukuchi-Mizutani, M., Tasaka, Y., Tanaka, T., Ashikari, T., Kusumi, T. and Murata, N. (1998) Characterization of D9 acyl-lipid desaturase homologues from Arabidopsis thaliana. Plant Cell Physiol., 39, 247-253.
Hayashi, H., Alia, Sakamoto, A., Nonaka, H., Chen, T.H.H. and Murata, N. (1998) Enhanced germination under high-salt conditions of seeds of transgenic Arabidopsis with a bacterial gene (codA) for choline oxidase. J. Plant Res., 111, 357-362.
Kanervo, E, Murata, N. and Aro, E.-M. (1998) Massive breakdown of the photosystem II polypeptides in a mutant of the cyanobacterium Synechocystis sp. PCC 6803. Photosynth. Res., 57, 81-91.
Panpoom, S., Los, D.A. and Murata, N. (1998) Biochemical characterization of a D12 acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli. Biochim. Biophys. Acta, 1390, 323-332.
Papageorgiou, G.C., Alygizaki-Zorba, A., Ladas, N. and Murata, N. (1998) A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC 7942. Physiol. Plant., 103, 215-224.
Sakamoto, A., Alia and Murata, N. (1998) Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Mol. Biol., 38, 1011-1019.
Sakamoto, T., Shen, G., Higashi, S., Murata, N. and Bryant, D.A. (1998) Alteration of low-temperature susceptibility of the cyanobacterium Synechococcus sp. PCC 7002 by genetic manipulation of membrane lipid unsaturation. Arch. Microbiol., 169, 20-28.
Sippola, K., Kanervo, E., Murata, N. and Aro, E.-M. (1998) A genetically engineered increase in fatty acid unsaturation in Synechococcus sp. PCC 7942 allows exchange of D1 protein forms and sustenance of photosystem II activity at low temperature. Eur. J. Biochem., 251, 641-648.
Yokoi, S., Higashi, S., Kishitani, S., Murata, N. and Toriyama, K. (1998) Introduction of the cDNA for Arabidopsis glycerol-3-phosphate acyltransferase (GPAT) confers unsaturation of fatty acids and chilling tolerance of photosynthesis on rice. Mol. Breeding, 4, 269-275.
(2) Review articles
Gombos, Z. and Murata, N. (1998) Genetic engineering of the unsaturation of membrane glycerolipid: effects on the ability of the photosynthetic machinery to tolerate temperature stress. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A. Siegenthaler and N. Murata, eds.) pp. 249-262. Kluwer Academic Publishers, Dordrecht.
Los, D.A. and Murata, N. (1998) Structure and expression of fatty acid desaturases. Biochim. Biophys. Acta, 1394, 3-15.
Murata, N. and Siegenthaler, P.-A. (1998) Lipids in photosynthesis: an overview. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A. Siegenthaler and N. Murata, eds.) pp. 1-20. Kluwer Academic Publishers, Dordrecht.
Wada, H. and Murata, N. (1998) Membrane lipids in cyanobacteria. In Lipids in Photosynthesis: Structure, Function and Genetics (P.-A. Siegenthaler and N. Murata, eds.) pp. 65-81. Kluwer Academic Publishers, Dordrecht.


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Last Modified: 12:00, May 28, 1999