Soil detoxification using genetically modified poplars – impacts on soil fungi in the root area?

Topic

With the help of genetically modified poplars it may in future be possible to detoxify soils with high levels of heavy metal contamination.

Cleaning up areas contaminated by heavy metals represents a major economic and ecological problem in mining areas. One important approach is soil detoxification using trees, particularly poplars. Poplars are characterised by fast growth and, unlike other trees, are relatively resistant to heavy metals. This means that they can store large quantities of heavy metals in their shoots. This ability can be improved still further through genetic modification.

One genetic approach aims to increase the expression of glutathione in poplars. Glutathione plays an important role in binding heavy metals in the plant cells.

But what impacts does the glutathione poplar have on fungus communities in the root area? Heavy metal uptake relies on effective substance exchange in the root are. In forest ecosystems in temperate zones nearly 100 per cent of short roots are mycorrhized . This symbiosis between trees and soil fungi (mycorrhiza) secures the tree’s supply of water and nutrient salt. The ability of tree species to detoxify the soil is therefore heavily dependent on whether they are still able to form these symbioses after genetic modification.

Since the genetic modification (increased expression of the g-glutamylcystein synthetase) in the transgenic poplars leads to an increased sulphur requirement as well as increased storage of heavy metals in the leaves, the aim of this part-project was to investigate whether:

• the plants’ increased sulphur requirement has an effect on the associated mycorrhizal fungi in the root area under outdoor conditions,

• the increased capacity of the transgenic poplars to store heavy metals and to detoxify also applies to mycorrhized outdoor poplars,

• horizontal gene transfer occurs from the poplars to the associated fungi in the root area under outdoor conditions in connection with the symbiosis between mycorrhizal fungi and tree root.

Summary

A total of 100 000 ectomycorrhizas from an outdoor trial were investigated for possible horizontal gene transfer from poplars to their associated ectomycorrhizal fungi. No transfer of the marker gene from the poplars to their fungal partners was found.

The mycorrhization extent of transgenic poplars planted in soils contaminated with different levels of heavy metals was much higher than for non-transgenic plants. However, the transgenic plants in soils contaminated with heavy metals displayed lower growth and higher stress symptoms, which would explain the increased mycorrhization.

Experiment description

Vigour tests on mycorrhizal fungi

Transgenic poplars which develop high glutathione levels have an increased sulphur requirement. The impacts of the increased sulphur content of the transgenic poplars on the fungi-tree root symbiosis was investigated under controlled laboratory conditions. Transgenic poplars were exposed to different levels of sulphur and the growth of the fungi was observed.

Similar experiments were designed to check the impacts of increased heavy metal content (Cd, Pb, Zn etc.) on the vigour of mycorrhizal fungi.

Investigation of a possible horizontal gene transfer from poplars to mycorrhizal fungi in the root area

For this study, transgenic poplars were produced that contained a special marker gene construct that gave them a herbicide resistance. The first release took place in 2000. In the following years samples of mycorrhizal fungi were taken from the root area and tested for herbicide resistance.

Results

Investigations into a possible horizontal gene transfer from poplars to mycorrhizal fungi in the root area

The released transgenic poplars imparted herbicide resistance under the control of a fungal promoter . Should horizontal gene transfer take place, this would enable the expression of the transgene in the fungal partner.

The root systems of 70 transgenic poplars were used for the isolation of mycorrhizas. 2000-4000 mycorrhizas were isolated per plant. The number of associated fungus species per plant varied widely between the sample batches: in the spring of 2002 a much higher number of mycorrhiza species per plant was observed than in the autumn. The samples taken in 2003 produced the opposite result (probably caused by drought).

A total of 100 000 ectomycorrhizas were analysed. Ectomycorrhizal fungi that demonstrated growth on herbicide selection plates were tested for the presence of the marker gene using PCR analysis. Of approx. 2000 herbicide-resistant fungus colonies, however, none contained the marker gene. These findings meant that, as with the previous trials under laboratory conditions, it was not possible to demonstrate any cases of horizontal gene transfer from poplars to the ectomycorrhizal fungi associated with them.

The ectomycorrhizas associated with the poplars were identified using DNA sequence comparison. Overall, five different ectomycorrhizal fungi were identified which account for approx. 90 per cent of the mycorrhizal flora of the poplars.

Mycorrhization of transgenic and non-transgenic poplars under different levels of heavy metal soil contamination

The project assessed the effect of the transgenic trait (increased glutathione biosynthesis) and the effect of different heavy metal concentrations in the soil on the composition of the fungal partners that form a mycorrhizal symbiosis with the plants.

The mycorrhization rate of the transgenic plants was much higher than for the non-transgenic plants on all trial areas. On the control areas/areas with average contamination, 32 per cent on average of the non-transgenic plants’ fine roots were mycorrhized, compared with 50 per cent of the transgenic rootlets. This trend was observed even on the heavily contaminated areas, although the difference between the non-transgenic (20 per cent mycorrhization rate) and transgenic plants (25 per cent mycorrhization rate) was much smaller here.

The species composition is currently being examined in more detail with the help of molecular markers. No difference in the composition of the symbiotic partners was found between the transgenic and non-transgenic poplars using morphological criteria.

Mechanism of heavy metal uptake

Ectomycorrhizal fungi showed an increased expression of a fungal gene –a possible metal transporter gene – under zinc deficiency and when incubated with cadmium. In order to discover the mechanism for heavy metal uptake by the ectomycorrhizal fungi, the expression of a possible metal transporter gene was investigated in yeast cells as a test system. However, these cells proved incapable of surviving. In future the transport characteristics need to be investigated in a different system, e.g. in egg cells of the clawed frog.