Life Sciences Open Day 2002

Objectives of Research:
General: Photosynthetic organisms displaying extreme tolerance to envirnmental stresses offer interesting objects for uncovering unique, or widely-conserved, protective and adaptive mechanisms. Dunaliella - a unicellular green alga photosynthetically similar to higher plants - is able to survive and proliferate in hyper-saline water bodies and under highly intense sunlight and thus provides a particularly suitable model for such studies.

Salt tolerance: Dunaliella withstands extreme salinities while maintaing a low intracellular salt concentration. Osmotic adjustment is achieved by intracellular accumulation of glycerol to a level counterbalancing the external osmoticum. Our goal is to uncover cellular components, and hence mechanisms, which act additionally to osmotic adjustment to enable growth in high salinities.

Defenses against intense light: Light fluxes in excess to those utilized in photosynthesis can irrevocably damage the photosynthetic machinery. Some Dunaliella strains overproduce the pigment [[beta]]-carotene that, together with other carotenoids, is thought to provide photo-protection. Initially using [[beta]]-carotene overproduction as an indicator for light stress, we aimed at uncovering additional light stress responses.

Genetic transformation: Development of a transformation system for Dunaliella is highly desirable in view of the outstanding physiological attributes of these algae. This direction is currently pursued.

Recent Findings:
Identification of mechanisms involved in salt tolerance (partly in collaborarion with U. Pick): Postulating that components of such mechanisms are likely to be induced under high salt, we looked for proteins preferrentially accumulating in high-salt grown cells. Two salt-induced proteins, of 60 kDa and p150 kDa, were identified, purified to homogenity, localized to the plasma membrane and their corresponding cDNAs were cloned.

Based on cDNA cloning, p60 consisted of an internal duplication of a sequence similar to the ~30 kDa animal carbonic anhydrases. Increasing salinities, alkaline shift, or bicarbonate depletion (all CO2-limiting conditions) induced parallel changes in p60 and its mRNA. Carbonic anhydrase activity of p60 was retained in high salinities, in contrast to other algal and animal carbonic anhydrases. These results identified p60 as a structurally novel carbonic anhydrase exhibiting halophilic-like characteristics and transcriptionally regulated by CO2 availability. This enzyme is potentially suited to optimize CO2 uptake by cells growing in hyper-saline media.

The other salt-induced plasma membrane protein, of 150 kD (first described by A. Sadka), was identified as a unique transferrin-like molecule which, unlike its soluble internally-duplicated animal homologs, contains three rather than two potentially lobe-forming units. The algal protein contains no putative trans-membrane domains and the mode of its membrane association still needs to be clarified. Transferrins are ubiquitous in the animal kingdom, where they function primarily as iron binding and transport proteins, but have not been identified yet in plants. The induction of p150 upon removal of iron from a low-salt medium is in keeping with the conclusion that high salinity presumably limits iron availability which might be the critical factor in the induction of p150. Labeling of p150 by ⁵⁹Fe provides preliminary evidence for the role of the protein in iron transport into the cells. Thus, transferrin-like proteins evolved far earlier than previously postulated probably to fulfil roles on the surface of unicellular organisms.

Light stress responses: An Elip-like algal protein - A search for genes activated under light stress in Dunaliella (concomitant with [[beta]]-carotene accumulation) led to to the identification of Cbr, an algal analog of Elips, a group of proteins conserved in higher plants. Cbr/Elips are structurally related to chlorophyll a/b binding proteins. The Cbr protein is present in light-harvesting antennae only during light stress and is possibly bound to zeaxanthin, a carotenoid pigment (xanthophyll) also typically formed under these conditions (previous work of A. Lers and H. Levy). Possibly, zeaxanthin-Cbr complexes (which might also contain additional pigments) act in a photo-protective mechanism. To gain insights into such mechanisms, chlorophyll fluorescence quenching measurements were conducted (in collaboration with S. Malkin). Recent studies defined the action spectrum and dose response of Cbr induction and changes in carotenoid composition .

Additional light-stress activated genes and proteins (partly in collaboration with U. Pick): Several other responses to light stress were identified in addition to [[beta]]-carotene accumulation, violaxanthin conversion into zeaxanthin and Cbr induction. The cDNA for a major protein component of [[beta]]-carotene globules was cloned and found to encode a protein showing no obvious similarity to any of the proteins present in carotenoid assemblies described so far. The protein is transcriptionally activated under a variety of light stress conditions. Under similar conditions, a gene shown to encode a chloroplast envelope protein is also induced. Altogether, light stress conditions which normally down-regulate genes such as cab, bring about the induction of genes involved in photo-protective, or yet undefined, functions. Characteriza-tion of mutants showing light stress responses under relatively low light intensities are expected to provide important insights into the acting regulatory mechanisms.

Recent Publications:

Sadka, A., Himmelhosh, S. and Zamir, A. (1991) A 150 kDa cell surface protein is induced by salt in the halotolerant alga Dunaliella salina. Plant Physiol. 95: 822-831.

Levy, H., Tal, T., Shaish, A. and Zamir, A. (1993) Cbr, an algal homolog of plant early light-induced proteins, is a putative zeaxanthin binding protein. J. Biol. Chem. 268: 20892-20896.

Fisher, M., Pick, U. and Zamir, A. (1994) A salt-induced 60 kDa plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella. Plant Physiol. 106: 1359-1365.

Zamir, A. (1995) Plant defences against excessive light studied in the microalga Dunaliella. Endeavour 19: 152-156.

Braun, P., Banet, G., Malkin, S. and Zamir, A. (1995) Evidence for the role of Cbr, an algal Elip homolog in non-photochemical quenching of chlorophyll fluorescence. Photosynthesis: from Light to Biosphere, Vol. IV: 21-26. Kluwer Academic Publishers.

Fisher, M., Gokhman, I, Pick, U. and Zamir, A. (1996) A plasma-membrane carbonic anhydrase from Dunaliella salina is internally duplicated and exhibits halophilic-like properties. J. Biol. Chem. (In press).

Braun, P., Banet, G., Tal, T., Malkin, S. and Zamir, A. (1996) Possible role of Cbr, an algal Early Light Induced Protein, in non-photochemical quenching of chlorophyll fluorescence. Plant Physiol. (In press).

Fisher, M., Gokhman, I., Pick, U. and Zamir, A. (1996) A unique 150 kDa transferrin-like protein located in the plasma membtrane of Dunaliella is induced by salt and iron limitation. (Submitted)

Banet, G., Pick, U. and Zamir,A (2000) "Light Harvesting Complex Iib pigments and proteins in association with Cbr, a homolog of higher-plant early light-inducible proteins in the unicellular green alga Dunaliella". Planta 210, 947-955