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Vol. 13 No. 2 - April 2007
‘Golden Jubilee Number’

Should we choose, to eat Cd contaminated rice
or to use GM plant-microbes?

By: Yoshikatsu Murooka*

The advancements in science and technology in the 20th century provided us a comfortable and convenient life. At the same time, however, the advancements provided many unexpected problems as well. Mankind has suffered from unknown diseases for the past several decades due to exposure to industrial wastes.

In Asia, a majority of chemical pollutants responsible for several diseases are heavy metals. Similar conditions must exist worldwide.  Widespread pollution by heavy metals that are generated by various industries has serious adverse effects on human health and the environment.  Decontamination of the soil and water around industrial plants presents major challenges for a long time. Japanese people, especially farmers and fishermen, were confronted with unidentifiable symptoms for years. 

Extensive research later identified these symptoms as ‘Itai-itai’ disease and ‘Minamata’ disease. Japanese scientists determined that these diseases resulted from rice grains containing large amounts of cadmium and fishes containing methyl-mercury, respectively. ‘Itai-itai’ and ‘Minamata’ diseases have yet to be completely eradicated in Japan and their incidence is increasing worldwide.

Anthropogenic sources of heavy metal deposition are on the increase as a result of the current industrial revolution, especially in developing countries.  Agriculture, mining, smelting, electroplating, and other industrial activities are to blame for undesirable concentrations of metals, such as As, Cd, Cr, Cu, Ni, Pb and Zn, in the soil. Although trace metals are an important part of the soil ecosystem, accumulation of these metals is harmful to people, animals, plants and other organisms coming in contact with the soil and groundwater

In my laboratory, we analyzed the cadmium content in soil samples from various rice fields in Japan. One soil sample taken from a rice field, located downstream from an old mine which was closed more than 30 years ago, had an extremely high concentration of cadmium. This data is highly confidential and unpublished to date since if I were to release this information to the press, the prefecture governments involved would panic. Consumers won’t buy any rice produced from this prefecture, even though most of rice had no contamination of cadmium. It would be better to tell the farmer to stop the cultivation of rice in this kind of field and clean up the soil as soon as possible.

Unlike many other pollutants, heavy metals are difficult to remove from the environment.  These metals cannot be chemically or biologically degraded, and are ultimately indestructible.

In the rhizosphere, plant roots secrete various nutrients and attractants stimulating microbes to swarm plant roots. Certain microbes have the ability to gather and carry metal ions to plant roots. The metal ions released by these microorganisms are taken in by plants. These phenomena gave me an idea for bioremediation using plants and their microbes.

Let us recall plant symbiosis. A typical plant symbiosis is a leguminous plant, the soybean, and its nitrogen-fixing rhizobia, Bradirhizobium japonicum. 1/4th to 1/3rd of the world’s soils are acidic and/or nutrient poor. Since these acidic soils contain far fewer microorganisms and limited species, we need to inoculate soils with appropriate rhizobia.

Dr. Hiep of Cantho University, Vietnam and Dr. Nantakorn  Boonkerd, Suranalee Institute of Science and Technology, Thailand have shown the significant formation of nitrogen-fixing nodules on the soybean roots by inoculation of the soils with Bradirhizobium species in Mekong Delta in Vietnam and Thailand, respectively. The yields of soybeans were increased.

The soybean plant produces sugars by photosynthesis. The sugars are supplied to their rhizobia as an energy source. Rhizobia present in the nodule fix atmospheric nitrogen converting it into ammonia with energy supplied by the plant, and the ammonia is supplied to the plant as nitrogen source. Most plant roots form mycorrhizal association, which enhances the uptake of  water and phosphate ions from water and phasphorus deficient soils. Therefore, you do not supply chemical fertilizer or at least you decrease the addition of chemical fertilizer. These symbioses are beneficial to agriculture and interesting with regard to their molecular mechanisms.

Another idea that we had for heavy metal accumulation in symbiosis was to introduce useful gene, such as metal-binding protein, into a Rhizobium. Genes for metal-binding proteins introduced into a Rhizobium will be expressed in bacteroids in nodules.

We used Chinese milk vetch as our symbiotic host plant. Known as renge-soh in Japanese and Astragalus sinicus in Latin, Chinese milk vetch is a green manure legume. This plant has been used as soil fertilizer for rice fields in China for 1000 years and in Japan for two hundred years. In the rainy season, farmers immerse renge-soh plants with their nodules into the paddy water and plow in the fields. As a result of this, no nitrogen fertilizer is required. Mesorhizobium huakuii subsp. rengei strain B3 is a bacterium that establishes a symbiotic relationship with Astragalus sinicus. It would be of considerable interest if we could use this leguminous plant to increase the soil nitrogen content and, at the same time, to remove heavy metals from the soil.

Inoculation of strain B3 resulted in increased accumulation of cadmium not only in nodules but in renge-soh too. We found about 2% cadmium accumulation in this plant per year. Given this accumulation rate for cadmium by renge-soh, it would take about 50 years to eliminate cadmium from polluted soil using phytoremediation. We cannot wait for 50 years. So, we began to genetically bread the renge-soh plant to increase its ability to accumulate heavy metals.

Genetic engineering suggests the possible use of specially designed microbial biosorbents with suitable selectivity and affinity for heavy metals. Normally we have a mechanism in our bodies which produces metallothioneins, metal-binding proteins, to protect us against toxic heavy metals. For the remediation of heavy metals, we selected the metallothionein protein as a metal binding protein. Overexpression of metal-binding proteins by bacterial cells results in enhanced accumulation of cadmium and offers a promising strategy for the development of microbe-based biosorbents for remediation of metal-contaminated soil.

To increase the binding of heavy metal ions, we designed oligometric metallothioneins and succeeded in expression of the tetrameric metallothionein in bacteroid in root nodules of renge-soh.  The tetrameric metallothionein bound 4 times more cadmium and other heavy metals than the naturally occurring monomer metallothionein. The presence of  106 to 108 bacterial progeny of the rhizobia in each nodule on the roots of renge-soh is advantageous for the expression of foreign genes that help to sequester heavy metals in contaminated soil. Once symbiosis is established, the heavy metals should accumulate in such nodules.

Phytochelatins are an attractive alternative to metallothioneins since they offer the potential for enhanced affinity and selectivity for heavy metals. The structure of phytochelatins can be represented by (g-Glu-Cys)n-Gly, where n ranges from 2 to 11. Phytochelatins have higher metal-binding capacity than metallothioneins. Thus, phytochelatins are attractive as metal-binding peptides for the development of a microbe-based biosorbents for remediation of metal-polluted soils. We demonstrated the introduction of the Arabidopsis gene for phytochelatin synthase (PCS); (AtPCS) into strain B3. The gene of MTL4 or AtPCS was expressed under the control of bacteroid-specific promoters, namely, the promoters of the nifH gene and nolB gene. The gene for MTL4 or PCS was expressed in free-living cells under microaerobic conditions when the promoter was activated by nifA. The expression of the MTL4 and AtPCS genes in strain B3 increased the ability of cells to bind cadmium approximately 2-fold and 9- to 19-fold, respectively.

When recombinant strain B3 established the symbiotic relationship with renge-soh, the symbionts increased cadmium accumulation in root nodules by 1.5-1.8 fold. The expression of the both MTL4 and AtPCS genes showed additive effect on cadmium accumulation in nodules. In paddy soil, addition of recombinant strain B3 carrying a plasmid with the both MTL4 and AsPCS genes significantly increased the accumulation of cadmium in roots and nodules of A. sinicus. Our results showed about 9% cadmium

accumulation for this engineered plant. Since we cannot decompose heavy metals, cadmium containing plants are harvested, incinerated, and the ashes are then stocked to recycle the heavy metals.

Thus, this system uses the advantages of both plants and rhizobia, in particular, engineered genes can be transformed to plants through infection with recombinant bacteria.

By our calculations, it would take 3 years to clean up cadmium contaminated soils with the tri-annual cultivation of the engineered plants. However, our government does not permit to use genetically engineered plants and microbes in open fields. GM plants and microbes have great potential for humanity. However, we must carefully test and monitor their safety for humans and the environment. It is not so easy to prove the safety of GM organisms. My contention is that using GM plant-microbes is much safer than taking heavy metal-contaminated grains.

*Professor of Osaka University San Francisco Center for Education & Research, 120 Montgomery St., Suite 1270, San Francisco, CA 94104, USA - E-mail: murooka@bio.eng.osaka-u.ac.jp

This article has been reproduced from the archives of EnviroNews - Newsletter of ISEB India.

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