Molecular Characterization of Acetolactate ... - ACS Publications

Chapter 22

Downloaded by OHIO STATE UNIV LIBRARIES on September 7, 2012 | http://pubs.acs.org Publication Date: March 8, 2005 | doi: 10.1021/bk-2005-0899.ch022

Molecular Characterization of Acetolactate Synthase in Resistant Weeds and Crops 1

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Tsutomu Shimizu , Koichiro Kaku , Kiyoshi Kawai , Takeshige Miyazawa , and Yoshiyuki Tanaka 1

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Kumiai Chemical Industry Company, Ltd., Kakegawa, Shizuoka 436-0011, Japan National Institute of Agrobiological Sciences, Tsukuba, Ibaragi 305-0856, Japan

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The goal of this paper is to outline the studies on the molecular characteristics of acetolactate synthase (ALS) in herbicide resistant weeds and crops. For this purpose, papers and patents concerning this field were reviewed, and our studies involving novel mutated ALS genes from rice cells, synthesized rice ALS genes, performance of the gene as a selectable marker for the genetic transformation, generation of transgenic plants with the gene, and the use of the recombinant ALS's for the herbicide resistance management were briefly summarized.

Introduction Acetolactate synthase, ALS, also referred to as acetohydroxy acid synthase, AHAS, is the first common enzyme in the biosynthetic pathway to the branched-chain amino acids; valine, leucine and isoleucine (Fig. 1). This pathway exists in plants and microorganisms such as bacteria, fungi and algae. ALS is the primary target site of action for at least four structurally distinct classes of herbicides including the sulfonylureas (SU), the imidazolinones (IM), the triazolopyrimidine sulfonamides (TP) and our pyrimidinyl carboxy herbicides (PC)(Fig. 2).

© 2005 American Chemical Society In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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256

Threonine TPP 2-Aminobutyrate

active aldehyde FAD, Mg++

EC Norvaline


Norleucine

Pyruvate

2-Ketobutyrate ^

Acetolactate synthase

χ

2-Acetolactate

2-Aceto-2-hydroxybutyrate

t 2,3-Dihydroxy-3-methyl valerate

2,3-Dihydroxyiso valerate

\

I

2-Ketoisovalerate

2-Keto-3-methyl valerate

î Valine

isoleucine

Leucine

Ο

Fig. 1. Biosynthetic pathway of branched-chain amino acids Pyrimidinyl carboxy (PC) herbicides Bispyribac-sodium (BS) for use in rice and vegitation management

Pyriminobac-methyl for use in rice

Pyrfthiobac-sodium for use in cotton

a

pCH3

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Chlorsulftiron as a representative of the SU

H Cv^ 3

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Imazaquin as a representative of the IM

0
CMB,SCM, and SDM mean the conventional eel setecfon, the somatic cell mutation, and the site-dree ted mit agenesis, respectively.

A number of plants and cultured plant cells resistant to ALS-inhibiting herbicides have been generated using the conventional mutation breeding, the somatic cell selection, and the site-directed mutagenesis. ALS genes encoding for the catalytic subunits have been cloned from some of these plants, and their sequences are shown to differ from their wild-types. For example, the mutations of proline at position 196 to glutamine, alanine and serine in the tobacco ALS have been found through somatic cells selection. The most commonly encountered mutations involve the residues of alanine and proline in the upstream region, and tryptophan and serine in the down stream region. These mutation patterns are very similar to those of the herbicide-resistant weeds described above (3). It was expected that novel mutated ALS genes that had different mutations from those reported in papers and patents were obtained through the selection of

In Environmental Fate and Safety Management of Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

263


plant cells under the pressure of BS. Fig. 8 shows the method for the generation of PC-resistant rice cells and isolation of the ALS cDNAs from phage libraries.

Fig. 8. Isolation of ALS genes from BS-resistant rice cells First, the callusfromrice seeds was induced. The calli were then cultured with 1 micromolar of BS for about 2 months so that the BS-resistant cells were generated. The cells were next cultured with higher concentrations of BS. And finally, several kinds of spontaneous BS-resistant cells that could grow under the pressure of 100 micromolar of BS were obtained. A wild-type ALS gene and a mutated ALS gene have been clonedfromthe BS-resistant cells using the partial cDNA that is an expressed sequence tag obtained from the MAFF DNA bank of Japan as a homologous hybridization probe. Fig. 9 shows a comparison of the deduced amino acid sequences between the wild-type ALS and the mutated ALS. The first amino acid shows the sequences between position 361 and the C-terminal position 644 in the mutated ALS, and second amino acid sequence does that in the wild-type ALS. The mutations involved the residues of tryptophan 548 to leucine and serine 627 to isoleucine. This double mutation on rice is a new combination of spontaneous mutations with the novel substitution at the serine position (7). One-point mutated ALS genes were then prepared to compare the sensitivities of their recombinant ALS's to the ALS-inhibiting herbicides with that of the two-point mutant (Fig. 10).


264 1st Amino Acid Sequence; mutant 2nd Amino Acid Sequence; wild-type 361 '

SBAKIVHIDIDPJtëlGKNKQPirVSIC^Via^^ ************************************************************ 361" SRAKIVHIDIDPAEIGKNKQPHVSICADVK^ 421' QKREFPLGYKTFGEEIPPQYAIQVLDELTKGEAIIATGVGQHC^HAAQYYTYKRPRQWLS ************************************************************ 421" QKREFPIX3YKTFGEEIPPQYAIQVLDELTKGEAIIATGVGQHQÊ4WAAQYYTÏKRPRQWLS


481 · SAGLG»MGFGLPJU*AGASVM^^ ************************************************************ 481" SAGLGAMGFGLPAAAGASVANPG^ 541 'HI/34VVQI^DRFYKANRAH1TLGNPECESEIYPDFVTIAK ******* **************************************************** 541" HLGMVVQWEDRFYKANRAHTYLQiPECESEIYPDFVTIAKGFNIPAVRVTKKSEVRAAIK 601' KMLETPGPYLLDIIVPHQEITVLPMIPIGfffl ************************** ***************** 601" K M L E T P G P Y I J L D I I V P H Q E H V L P M I P S G G A F K D M I I I 3 ( S G R T W

548; tryptophan (W)—>leucine (L) 627; serine (S)—Msoleuclne (I)

Fig. 9. Comparison of amino acid sequences between ALS's from the mutant and the wild-type

Fig. 10. Preparation of one-point mutated ALSs by PCR and self-polymerase reaction Each one-point mutant was prepared from the two-point mutant by PCR and the self-polymerase reaction. Recombinant ALS's from these ALS genes were expressed in Eschericia coli as GST-fused proteins (Fig. 11) and the proteins were examined for their sensitivities to herbicides.



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Fig. 11. Expressionof recombinant ALS's as GST-fused proteins The ALS expressed from the wild-type gene showed a similar sensitivity to BS and chlorsulfuron compared with that prepared from the natural source (Fig. 12).

_ 4 _ w i l d type -+-S627I mutant

—m_ W547L mutant - · « . . . S627I/W547L mutant

Fig. 12. Sensitivities of GST-fused ALS's to BS and chlorsulfuron On the contrary, the ALS expressed from the two-point mutated ALS gene showed quite different sensitivities to the herbicides. This ALS showed a


266 stronger resistance to BS than to chlorsulfuron. BS had no effect on the enzyme even at 100 micromolar, which is an approximately 10,000-fold higher concentration than the I value for the wild-type enzyme. It was notable that the two-point mutated gene imparted synergistic resistance to ALS against BS that is stronger than the additive effect predicted from the degree of each resistance of the one-point mutated ALS. 50


Use of the mutated ALS genes for the genetic transformation and the herbicide resistance management As shown above, the novel mutated ALS gene from rice exhibited a high resistance to the PC herbicide. Thus we studied the use of this gene as a selectable marker for the genetic transformation of plants. Promoters and terminators derived from rice were used and a new binary vector was constructed. The two-point mutated ALS gene was driven with a rice callus specific promoter, and the GFP gene was driven with a constitutive promoter. Rice seeds were transformed with this vector by the Agrobacterium method and the transformed cells were selected by the pressure of BS. As result, fluorescence from GFP was detected only in selected cells (Fig. 13), ^eating that the two-point mutated ALS gene was an effective selection marker for rice transformation.

Fig. 13. Use of the mutated ALS gene as a selectable marker for rice transformation


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Transgenic rice plants were then generated to examine whether this gene works normally in the plant or not. The two-point mutated ALS gene was driven with a constitutive 35S promoter cassette with enhanced expression activity. Rice seeds were transformed with this vector and a transgenic rice plant was generated. This transgenic rice plant exhibited resistance to BS and grew normally (Fig. 14) so that it was fertile.

!15§_

NPJH P35S

5'

PMLH7133-ALS

A, a transgenic rice plant (To) transformed with the mutant ALS gene B, a transgenic rice plant transformed with the wild ALS gene

Transgenic rice plants at 5- to 6-leaf stage were treated with bispyribac-sodium (1 kg a.i /ha). The photograph was taken two months after bispyribac-sodium treatment. Plant length of the transgenic rice plant marked by A was 88 cm.

Fig. 14. Sensitivity of the transgenic rice (T ) to BS 0

Ti seeds were collected and the BS-resistant phenotype of T! plants were examined. The result showed that the phenotype was segregated by approximately 3 : 1 according to Mender's law. The plants that exhibited resistance to BS were cultivated on a large scale and several kinds of T seeds were collected. Consequently, homozygotes for the resistant trait were found in these T seeds through examination of their sensitivities to BS. Fig. 15 shows the sensitivities of the homozygote to BS. The right figure is an enlargement of the left figure. 2

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Fig. IS. Sensitivity of the transgenic rice (T ) to BS 2

This homozygote grew normally without BS and exhibited resistance to BS. These results suggested that the two-point mutated rice ALS gene functionally worked in rice and had no bad effects on rice. The marker system is needed not only for the general recombinant technology but also for the gene targeting such as the homologous recombination and the mismatch repair. The mutated rice ALS gene can be used for such gene technologies. (Fig. 16).

Selectable marker Recombinant DNA technology ^

^

m

^ T r a n s g e n i c plants

Promotor from plants

Marker Gene targeting

- | - t > Homologous recombination (including chloroplast gene targeting) L|> Mismatch repair using chimeric RNA /DNA oligonucleotides

^ N o n - t r a n s g e n i c plants?

Marker

Fig. 16. Use of rice mutated ALS genes as markers for gene technologies Using the accumulated knowledge concerning rice ALS genes, rice mutated ALS genes were artificially prepared. (Fig. 17).


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Molecular Characterization of Acetolactate ... - ACS Publications

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