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2-Plant: New herbicide-tolerant GE rice uses human gene



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TITLE:  A WIDE CROSS-TOLERANCE OF TRANSGENIC RICE CONTAINING HUMAN CYP2B6
        TO VARIOUS CLASSES OF HERBICIDES
SOURCE: ISB News Report, USA, by Sakiko Hirose and Hiroyuki Kawahigashi *
        http://www.isb.vt.edu/news/2005/news05.aug.htm#aug0504
DATE:   Aug 2005

------------------- archive: http://www.genet-info.org/ -------------------


A WIDE CROSS-TOLERANCE OF TRANSGENIC RICE CONTAINING HUMAN CYP2B6 TO
VARIOUS CLASSES OF HERBICIDES

Pesticides have played very important roles against food shortages and
plant diseases, and our modern life would be vastly different without
their use. Weed infestation adversely affects crop production by reducing
yields and decreasing market prices of the crop. For cost-effective land
use, crop yield and price must be maximized, and the cost of weed control
minimized. Herbicides substantially lighten the farmer's heavy physical
workload and improve crop yield and product quality. Herbicides are now
widely used for crop cultivation and for management of lawns, railroad
rights-of-way, highway margins, and other purposes, although there
remains the possibility of non-target effects of pesticides to the
environment caused by agricultural runoff.

One of the major problems with the use of herbicides is that repeated use
of the same herbicide in a field tends to promote the emergence of
herbicide tolerant weeds. Indeed, over 290 biotypes of herbicide tolerant
weeds have been reported in agricultural fields and gardens worldwide
(International survey of herbicide tolerant weeds, http://
www.weedscience.org/in.asp). They are categorized into 18 HRAC (The
Herbicide Resistance Action Committee) groups (Classification of
Herbicides According to Mode of Action 2005, http://
www.plantprotection.org/hrac/Bindex.cfm?doc=moa2002.htm). Fifty percent
of the biotypes are resistant to inhibitors of acetolactate synthase,
such as sulfonylureas, and inhibitors of photosynthesis at photosystem
II, such as triazines.

Several strategies are used to prevent or reduce herbicide tolerance in
weed populations. Using either several herbicides in rotation or in a
mixture including lower doses of herbicides was proposed as the most
practical approach to prevent or delay the appearance of tolerance,
because most cases of herbicide tolerance are due to a single major gene,
and also because the repeated use of a single herbicide for a long time
tends to promote weeds that are tolerant1.

To enable crop plants to grow under such an herbicide regime, it would be
helpful to develop transformed plants containing a gene for a single
mammalian P450 enzyme, which would detoxify several types of herbicides,
and thereby give these plants cross-tolerance to herbicides2.

The cytochrome P450 monooxygenase (P450) system, consisting of cytochrome
P450 and NADPH-cytochrome P450 oxidoreductase, catalyzes monooxygenation
of lipophilic xenobiotic compounds, including herbicides. It has been
reported that several microsomal P450s are involved in xenobiotic
metabolism in mammals. The individual xenobiotic-degrading P450s appear
to show overlapping, broad substrate specificity and thus improve the
animal's ability to catabolize a variety of unknown lipophilic compounds,
including herbicides, in liver3.

We introduced a human gene for CYP2B6, a cytochrome P450 monooxygenase
that inactivates xenobiotic chemicals, into Oryza sativa cv. 'Nipponbare'
by Agrobacterium-mediated transformation. In our study, we demonstrated
that transgenic rice expressing human CYP2B6 under the control of CaMV
35S showed strong herbicide tolerance that resulted from the
detoxification of several types of herbicides by the CYP2B64.

The CYP2B6 rice plants showed normal growth in morphology, including
plant height, leaf color, flowering time, fertility, and seed size,
compared with non-transgenic Nipponbare plants in a greenhouse. The
CYP2B6 rice plants were physiologically the same as non-transgenic
Nipponbare, except for the feature derived from the introduced CYP2B6 gene.

The CYP2B6 rice plants showed high tolerance to 13 out of 17 herbicides,
which belong to different chemical families. These were chloroacetoamides
(acetochlor, alachlor, metolachlor, pretilachlor, and thenylchlor),
oxyacetamides (mefenacet), pyridazinones (norflurazon), 2,6-
dinitroanilines (trifluralin and pendimethalin), phosphoamidates
(amiprofos-methyl), thiocarbamates (pyributicarb), and ureas (chlortoluron).

Significantly high tolerance was observed to the five chloroacetoamides,
which inhibit the synthesis of very long chain fatty acids (VLCFA). For
example, the CYP2B6 rice seeds were able to germinate and grow in the
medium containing 80 _M metolachlor (about 15 times the dose of practical
use in cornfields), while non-transgenic Nipponbare did not germinate in
the presence of 2 _M metolachlor (about one-third the dose of practical
use in cornfields). Another VLCFA synthesis-inhibiting herbicide,
mefenacet, inhibited the germination of Nipponbare, but had little effect
on the growth of CYP2B6 rice plants.

 Microtubule assembly-inhibiting herbicides, pendimethalin and
trifluralin, and unknown function herbicide, pyributicarb, inhibited the
root growth of Nipponbare, but CYP2B6 rice plants produced roots and grew
better than Nipponbare. A photosynthesis-inhibiting herbicide,
chlortoluron, inhibited the growth of Nipponbare plants, but the CYP2B6
plants grew vigorously.

CYP2B6 rice was slightly tolerant to norflurazon, which caused bleaching
of shoots of Nipponbare by the inhibition of carotenoid synthesis. The
CYP2B6 rice metabolized the herbicides during and after germination,
keeping the concentration of the herbicide in plant tissues under the
lethal threshold. Therefore, the CYP2B6 rice plants could metabolize a
broad spectrum of herbicides and showed cross-tolerance to several
herbicides having different chemical structures and different modes of action.

The results of thin layer chromatography analysis revealed that the
amounts of metolachlor decreased in CYP2B6 rice plants and in the medium
of CYP2B6 rice faster than those of non-transgenic Nipponbare. In this
study, the CYP2B6 rice plants rapidly metabolized metolachlor to its
demethylated metabolite. The metabolism of metolachlor seemed to be
enhanced by the introduced CYP2B6 in the transgenic plants, although
metolachlor was metabolized not only by CYP2B6 rice but also by control
plants.

In the greenhouse, CYP2B6 rice plants grew vigorously in an enamel pot
with soil and water that also contained metolachlor at the same dose of
practical use in cornfields. On the other hand, non-transgenic Nipponbare
plants were almost killed by metolachlor. This result indicated that
CYP2B6 rice plants were practically useful as an herbicide tolerant crop
under the conditions of a paddy field.

The wider cross-tolerance to herbicides having different modes of action
and different chemical structures seems to be a special feature of
transgenic plants expressing mammalian P450 genes. This cross-tolerance
would prove useful to prevent the development of herbicide resistance of
weeds, because the use of several herbicides in rotation would not harm
the crop.

The herbicide cross-tolerance during germination should be important for
weed control in rice fields, especially with the direct-seeding system.
In the transplanting-cultivation system of rice seedling, standing water
in paddies prevents the germination of many weeds, and as a result, few
kinds of plants are major weeds. However, in the direct-seeding system
without water cover, the germinating rice must compete with many kinds of
weeds. The transgenic rice with cross-tolerance to various types of
herbicide should be an ideal plant for weed control with herbicide
mixtures, especially in direct seeding.

We expect that CYP2B6 rice will also prove useful in degrading and thus
decreasing the environmental loads of herbicides, insecticides,
industrial chemicals, and endocrine-disrupting pollutants in paddy fields
and the connected water streams. In the future, transgenic plants
expressing P450 species should be good not only for developing herbicide-
tolerant rice but also for reducing the environmental impact of agrochemicals.


References

1. Putwain PD. (1990) The resistance of plants to herbicides. In Weed
control handbook: Principles, R Hance and K Holly, Eds., Blackwell:
Oxford, 217-242

2. Ohkawa H, Tsujii H, & Ohkawa Y. (1999) The use of cytochrome P450
genes to introduce herbicide tolerance in crops: a review. Pestic Sci,
55, 867-874

3. Inui H et al. (2001) Metabolism of herbicides and other chemicals in
human cytochrome P450 species and in transgenic potato plants co-
expressing human CYP1A1, CYP2B6 and CYP2C19. J Pestic Sci 26, 28-40

4. Hirose S et al. (2005) Transgenic Rice Containing Human CYP2B6
Detoxifies Various Classes of Herbicides. J Agric Food Chem 53, 3461-3467

Sakiko Hirose and Hiroyuki Kawahigashi*
Plant Biotechnology Department National Institute of Agrobiological
Sciences Ibaraki, Japan
*shiwak@affrc.go.jp


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