Session F: Environmental Biotechnology

Chlamydomonas W80, a green alga highly tolerant to cadmium: a basic
study for its application for phytoremediation
Corresponding Applicant: Hitoshi Miyasaka (The Kansai Electric Power Company)
Co-researchers: Kazumasa Hirata and Kazuhisa Miyamoto (Osaka University)
Discover the cadmium-tolerant
mechanisms of Chlamydomonas W80
Extrusion?

Periplasm
accumulation?

Future application tohigher plants(Introduction of algal
genes into higherplants)

Plant cell

Cell wall
accumulation

Vacuole storageIntracellulardetoxification?

Heavy metals

IntracellulardetoxificationGoal & Contribution to well-being for all humanity
Heavy metal contamination of soil and groundwater is a serious
threat to the well-being of all humanity, and phytoremediation is
one of the most promising counter measures for this problem.
Chlamydomonas W80 is a unicellular marine green alga thatshows a high tolerance to the cadmium ion (up to 10 mM CdCl2).
In this study, we will discover the cadmium-tolerant mechanisms,
such as intracellular detoxification and/or extrusion, of this alga.
The mechanism will be applied in the future to generatetransgenic plants, which can effectively extract cadmium (andother toxic heavy metals) from the contaminated soil.

Transgenic plant forphytoextraction of heavy metals

Method / Approach
Cadmium tolerant mechanism

What we know about Chlamydomonas W80 at present:Highly tolerant to cadmium ion; cells grow in the medium with
0.5 to 1mM CdCl2, and tolerate (but no growth) up to 10mM.
Unexpectedly low intracellular concentrations of cysteine,
glutathione, and phytochelatin (PC) in comparison with anothercadmium tolerant green alga Dunaliella tertiolecta, suggestingthat the thiol (-SH) mediated detoxification system is not themajor tolerant mechanism in C. W80.

What we are going to discover:The mechanism of cadmium tolerance of C. W80(i) Are there any intracellular detoxification mechanisms?
If so:
What are the chelating compounds?
Where is the place of cadmium accumulation? (cellularlocalization)(ii) Are there any extruding mechanisms?
Cloning of the genes related to heavy metal tolerance

Examine the changes in the intracellular cadmium concentrationof C. W80 cells cultured in the medium with 10mM CdCl2 to findout if C. W80 cells accumulate and/or extrude the cadmium ion.
The distribution of cadmium ion in the cells will aslo be examinedby energy dispersive spectroscopy (EDS).
Isolate the cadmium binding cellular molecules by metal affinity
chromatography, and analyze them with various methods (e.g.
SDS-PAGE, gel filtration, HPLC, etc.).

Cloning of the genes related to heavy metal tolerance
Approaches for the screening of the cDNA library are as follows:(i) Screening based on the homology with the previously
reported genes (e.g. heavy metal transporters).
(ii) Functional expression screening based on the acquisition ofheavy metal tolerance of the host cells expressing the genes ofC. W80 (host: E. coli and yeast).
(iii) Isolation of heavy metal binding proteins by metal affinity
chromatography, and screening the cDNA library of C. W80
with the antibody raised against the isolated proteins.
(iv) Cloning of the cadmium-inducible genes by subtractive
hybridization and differential display.

How is the response of C. W80 to other heavy metals?
Call for collaboration: We focus mainly on the heavy metal detoxifting proteins and peptides, but there could be some other unknown (novel) cadmiumchelating cellular compounds other than proteins and peptides. If this is the case, we need a collaboration with a chemist whose expertise is chelate chemistry.