Selectable Tolerance to Herbicides By Mutated Acetolactate Synthase ...

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To examine the amount of transplastomic chloroplast genome in eachline, the population representing the native chloroplast genome wasdetermined by PCR with rbcL-Fd and accD-Rv. Two different linestransformed with the same construct were subjected to PCR analysis.The wild-type lines markedly amplified the 0.6-kb product (Fig. 2D,top; lanes 9 and 10), whereas the 0.6-kb products were barelyamplified in lanes 1 to 8 (Fig. 2D, top), indicating that thenative regions amplified by rbcL-Fd and accD-Rv were split by thetransgenes. The internal insert sequence was detected to a greaterextent using primers aadA-Fd2 and ALS-270-Rv in all transgenicplants (Fig. 2D, bottom). However, the 0.8-kb products were notdetected in wild-type plants. Therefore, it is concluded that themajority of the chloroplast genome in these transgenic plants wastransplastomic. Subsequent studies used the transplastomic plantsof lane 2 for A122V, lane 4 for G121A, lane 6 for P197S, and lane 8for W574L/S653I.
ALS Activity in Leaves of Transplastomic Plants
The activity of native ALS from wild-type tobacco in the absence ofALS-inhibiting herbicides was determined by a colorimetric assayand appeared as a red color in samples (Fig. 3A). The red colorchanged to a transparent or pale yellow color following theaddition of SU herbicide (0.1 [mu]M bensulfuron-methyl [BM]), PCherbicide (0.1 [mu]M pyrithiobac-sodium [PS]), and IM herbicide (5[mu]M imazapyr [IP]; Fig. 3A), indicating that these herbicidesinhibited ALS activity. This assay was employed for the evaluationof mALS activity in transplastomic plants (G121A, A122V, P197S, andW574L/ S653I), as determined by the PCR methods described above.The ALS activity of G121A plants was strongly resistant to PS,weakly resistant to BM and sensitive to IP (Fig. 3B), whereas A122Vplants were specifically resistant to IP (Fig. 3B), and P197Splants were strongly resistant to BM, middle resistant to PS, andsensitive to IP (Fig. 3B). The ALS activity of W574L/S653I plantswas strongly resistant to PS, BM, and IP (Fig. 3B). These resultsshow a mALS- mediated herbicide-specific resistance resulting froma transgene introduced by chloroplast transformation.
Effects of Herbicides on Plant Growth
Herbicide resistance was examined using a combination of threedifferent herbicides and a variety of mALSs. In an effort toexamine the in vivo effect of each herbicide on transplastomicplants, the leaves of plants were transferred to regenerationmedium containing 0.5 g L^sup -1^ spectinomycin (SP), 0.1 [mu]M BM,0.1 [mu]M PS, or 1 [mu]M IP, and cultured for 3 weeks. Wildtypetobacco was unable to grow on medium containing any of theherbicides. On the other hand, G121A plants regenerated on 0.1[mu]M PS medium (Fig. 4), and A122V plants regenerated on 1 [mu]MIP medium (Fig. 4). P197S plants were tolerant to 0.1 [mu]M BM and0.1 [mu]M PS (Fig. 4). W574L/S653I plants regenerated on mediumcontaining 0.1 [mu]M BM, 0.1 [mu]M PS, or 1 [mu]M IP (Fig. 4).
T1 seeds, the self-pollinated progeny of A122V plants, and wild-type seeds were planted on medium supplemented with 1 [mu]M IP or0.5 g L^sup -1^ SP. Both seed types were able to grow on Murashigeand Skoog (Fig. 5A). Although wild-type plants were sensitive to IPand SP, all A122V seeds were uniformly resistant to SP and IP (Fig.5, B and C). Green tissues of A122V plants on 1 [mu]M IP mediumgrew in a manner similar to that of wild-type tissue grown onherbicide- free medium (Fig. 5C), although the root length of theformer was shorter. The accumulation of mALS protein did not appearto be sufficient to impart resistance to the herbicide in rootplastids because mALS expression was driven by the psbA promoter,which is known to be a strong promoter in green tissues.
DISCUSSION
ALS Activity in Transplastomic Plants
Herbicide-specific resistance conferred by mALSs and directed bytransplastomic genes in the chloroplast genome has beendemonstrated by this investigation, even though mALS genes hadpreviously only been delivered into the nuclear genomes of certainspecies (Kawai et al., 2007; Okuzaki et al., 2007). ALS activity isthought to be controlled by the regulatory subunit, which alsoplays a role in feedback regulation by Val, Ile, and Leu (Lee andDuggleby, 2001). Although the tobacco mosaic virus 35S promoter hasfrequently been used in nuclear transformation studies, our studyused the psbA promoter because it is the strongest known promoterin chloroplasts (Hayashi et al., 2003). Moreover, given that 100 to1,000 copies of the chloroplast genome exist in a cell, it would beinteresting to determine whether highly expressed mALSs inchloroplasts can influence plant growth. This study has revealedthat transplastomic plants with mALSs grow normally on Murashigeand Skoog medium without significant differences when compared towild-type plants, showing that hyperexpression of mALSs does notinfluence plant growth.
We investigated the involvement of the regulatory subunit in ALSactivity. Regulatory subunits play roles in feedback regulation andfull enzyme activity (Lee and Duggleby, 2001). The determination ofALS activity in leaves where regulatory subunits exist was done inthe presence of 1,1-cyclopropanedicarboxylic acid, which blockedthe acetolactate metabolism, resulting in no feedback regulation.The selectable tolerance of plants transplastomic with G121A,A122V, and W574L/ S653I (Fig. 3) were similar to those obtainedwhen using the same recombinant mALSs that only expressed thecatalytic subunit in E. coli, with which endogenous E. coliregulatory subunits was not concluded to be associated (Kawai etal., 2008). Therefore, the regulatory subunits do not affect thesensitivity of these mALSs to herbicides in transplastomic plants.On the other hand, P197S mALS exhibited different behavior from theabove mutations. The novel tolerance of P197S plants to PC and SUherbicides was demonstrated with regard to mALS activity inresponse to herbicides in leaves (Fig. 3), whereas P197S mALSexpressed in E. coli was resistant to SU herbicides, but not to PCherbicides (Kawai et al., 2008). This result suggested that theregulatory subunit contributed to the acquisition of resistance ofP197S activity to the PC herbicides.
In an effort to investigate the influence of feedback regulation,transplastomic plants were grown on medium containing an herbicide.We analyzed herbicide resistance in transplastomic plants harboringfour different mALSs. W574L/S653I plants showed synergistictolerance, similar to that observed when the corresponding mALSgene was introduced into the nuclear genome of rice (Kawai et al.,2007). The tolerance of P197S plants to PC and SU herbicides wasdemonstrated also in plant growth (Fig. 4). In addition, two othertransplastomic plants (G121A and A122V) showed sensitivities toherbicides both in the activity in leaves (Fig. 3) and in plantgrowth (Fig. 4). Our results provide evidence suggesting that thesensitivity of mALSs to herbicides in plants is not affected byfeedback regulation. The highly expressed mALS molecules may not befully active due to the resultant stoichiometrically insufficientnumber of regulatory subunits (Lee and Duggleby, 2001). Therefore,the ALS activity of transplastomic plants was almost equivalent tothat of wild-type plants in the absence of herbicides. It isconcluded that transplastomic mALSs are useful as sustainablemarkers that can be employed to distinguish GM and non-GM plantswhen using appropriate herbicides in the field.
Benefit of mALSs as Sustainable Markers Herbicide-resistant weedshave been reported in many countries (Tranel and Wright, 2002;Tranel et al., 2007) and include weeds resistant to ALS-inhibitingherbicides. New technology is required to assist in the managementof weeds resistant to these herbicides. We propose a strategyinvolving herbicide rotation to overcome the aforementionedproblem. To this end, we have developed transplastomic plants thatpossess tolerance to PC, IM, and SU/PC. Although it is known thatsome ALS mutations are associated with plant resistance to a singleherbicide, there have been no reports detailing the relationshipbetween a single ALS mutation and resistance to the three classesof herbicide PC, SU, and IM. In this article, transplastomic plantswere generated and their resistance to herbicides wascharacterized.We determined three kinds of ALS mutations thatconferred specific resistance to the three classes of herbicidesused with the transplastomic plants and showed that G121A, A122V,and P197S plants were resistant to PC, IM, and SU/PC herbicides,respectively (Fig. 4). Use of these transplastomic markers in cropplants would enable a new strategy based on the rotation of threeor more combinations of herbicides. The advanced technologydescribed in this article allows for the efficient and strictmanagement of weeds resistant to ALS-inhibiting herbicides.
Investigations concerning herbicide resistance have been made usingchloroplast transformation. For example, the petunia EPSPS gene wasintroduced into the tobacco chloroplast genome and resulted intransplastomic plants resistant to glyphosate (Daniell et al.,1998). Similarly, the bar gene for phosphinothricin resistance wasused to investigate the resulting plant phenotype (Lutz et al.,2001). Because this gene is derived from microorganisms and notplants, it is less suitable for use in C-CGTT-based approaches.However, EPSPS is worthy of consideration in strategies involvingherbicide rotation schemes as described above because EPSPS ispresent in higher plants. The glyphosate and ALS-inhibitingherbicides are thought to be nontoxic to living organisms, exceptplants and microorganisms (Peterson and Shama, 2005). PlantderivedEPSPS might be useful as an additional tool for use in an herbiciderotation system for the management of herbicide-resistant weeds.The technology developed in this study may be employed inC-CGTTbased methodologies in association with aadA eliminationfollowing transformation.
MATERIALS AND METHODS
Construction of Plastid Transformation Vectors
Genes for the transit peptide-truncated ALS mutants A122V, P197S,and W574L/S653 placed in pBluescript (pBS; Stratagene; Kawai etal., 2008) were used as templates for PCR. ALS(G121A) was generatedby replacement of the EcoRV-AvrII fragment of pBS-ALS(A122V) with aG121A-containing product amplified using G121A Fd and G121A Rv,followed by digestion with EcoRV and AvrII. pBS-ALS(G121A) was alsoused as a template for PCR. Fragments of ALS mutants were amplifiedusing KOD-plus DNA polymerase for higher fidelity (Toyobo). Primerscorresponding to the ALS coding region were ALS Fd and ALS Rv. ThePCR reaction was performed by employing 30 cycles of denaturationfor 30 s at 94[degrees]C, annealing for 20 s at 55[degrees]C, andextension for 2 min at 68[degrees]C. PCR-generated fragments wereligated to the SphI site of pLD6 (GenBank accession no. CS165374;Adachi et al., 2007) harboring the 16S rRNA gene promoter-aadA-psbAgene terminator, psbA gene promoter-SphI-TpsbA, andPrrn-aadA-TpsbA- PpsbA-SphI-TpsbA to drive mALS gene expressionusing the psbA promoter. Sequencing analysis was performed using aBigDye terminator cycle sequence kit (Applied Biosystems). Vectorsgenerated were named pLD6- mALS, pLD6-A122V, and so on. The SalI-NotI region of pLD6-mALS was introduced into the SalI and NotIsites of pLD200 (GenBank accession no. BD174938; Adachi et al.,2007), which possesses a sequence between rbcL and accD derivedfrom tobacco for homologous recombination. The absence ofunexpected mutations was confirmed by sequence analysis. Finally,each chloroplast transformation vector comprised two transgeneconstructs, PrrnaadA- TpsbA and PpsbA-mALS-TpsbA, referred to aspLD200-mALS. The nucleotide sequences of primers used in this studyare listed in Table I.
Transformation and Transgene Confirmation
Chloroplast transformation and the preparation of genomic DNA toconfirm the presence of transgenes were performed using the tobacco(Nicotiana tabacum) cultivar Xanthi according to previouslydescribed methods (Adachi et al., 2007). SP-resistant shoots wereselected on RMOP medium containing 0.5 g L^sup -1^ SP rooted onMurashige and Skoog agar and selfpollinated to obtaintransplastomic seeds. PCR analysis was performed using thefollowing five primer sets: the rbcL-aadA region of pLD200-mALSswith rbcL-Fd and aadA- Rv, the ALS-accD region with N4-Fd andaccD-Rv, the insert integrated into the chloroplast genome withALS-1597-Fd (annealed with ALS in the vector) and accD-N-Rv (foraccD in the endogenous chloroplast genome, but not included in thevector), the aadA-ALS region with aadAFd2 and ALS-270-Rv, and therbcL and accD regions of the endogenous chloroplast genome withrbcL-Fd and accD-Rv. The nucleotide sequences of primers used inthis study are listed in Table I.
Herbicides Inhibiting ALS Activity
BS, PS, and PM were used as representative PC herbicides,chlorsulfuron (CS) and BM were used as representative SUherbicides, and imazaquin (IQ) and IP were used as representativeIM herbicides (Fig. 1). These chemical compounds were provided byKI Chemical Research Institute Co., Ltd.
ALS Activity Assay in Leaves
ALS activity was determined as follows. A leaf section (50 mg) wasfloated and incubated on 25% Murashige and Skoog medium containing0.5 mM 1,1-cyclopropanedicarboxylic acid, an inhibitor ofacetolactate metabolism, with or without ALS-inhibiting herbicidesfor 42 h under the same conditions used for plant growth (Adachi etal., 2007). The sample was then placed at -80[degrees]C for 1 h,transferred into 200 mL of 0.025% Triton X-100, and incubated at60[degrees]C for 5 min followed by incubation at room temperaturefor 60 min to effect extraction of acetolactate synthesized by ALS.The extract (100 [mu]L) was transferred to a 1.5-mL tube, to whichwas added 10 [mu]L of 1 N H^sub 2^SO^sub 4^. The sample was thenincubated at 60[degrees]C for 30 min to convert acetolactate toacetoin. An aliquot (50 [mu]L) of 0.5% (w/v) creatine and 50 [mu]Lof 5% (w/v) 1-naphthol dissolved in 2.5 N NaOH were added and theresultant mixture was subsequently incubated at 37[degrees]C for 30min. The amount of acetoin formed was determined by a colorimetricassay.
Sequence data from this article can be found in the GenBank/EMBLdata libraries under accession numbers CS165374 (pLD6), BD174938(pLD200), and BT020540 (ALS).
ACKNOWLEDGMENT
We are grateful to Kiyoshi Kawai for technical support concerningthe ALS activity assay.
Received April 15, 2008; accepted May 20, 2008; published May 30,2008.
1 This work was supported by the Intellectual Cluster (Keihanna,2002-2006), Center of Excellence (COE) Program in the 21st Century(2002-2006), and Global COE Program (2007), and Grants-in-Aid fromthe Ministry of Education, Culture, Sports, Science and Technologyof Japan (Monbukagakusho), and by the Goto Research Grant fromUniversity of Shizuoka (to H. Kobayashi). M.S. was a postdoctoralfellow supported by the COE Program in the 21st Century.
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Masanori Shimizu, Maki Goto, Moeko Hanai, Tsutomu Shimizu, NorihikoIzawa, Hirosuke Kanamoto, Ken-Ichi Tomizawa, Akiho Yokota, andHirokazu Kobayashi*
Laboratory of Plant Molecular Improvement and Global Center ofExcellence Program, Graduate School of Nutritional andEnvironmental Science, University of Shizuoka, Shizuoka 422-8526,Japan (M.S., M.G., M.H., H. Kobayashi); Life Science ResearchInstitute, Kumiai Chemical Industry Co., Ltd., Kikugawa, Shizuoka439-0031, Japan (T.S., N.I.); Research Institute of InnovationTechnology for the Earth, Kizugawa, Kyoto 619-0292, Japan (H.Kanamoto); Plant High Technology Institute, Takayama Science Plaza,Ikoma, Nara 630-0101, Japan (K.I.-T.); and Graduate School ofBiological Science, Nara Institute of Science and Technology,Ikoma, Nara 634-0813, Japan (A.Y.)
* Corresponding author; e-mail hirokoba@u-shizuoka-ken.ac.jp.
The author responsible for distribution of materials integral tothe findings presented in this article in accordance with thepolicy described in the Instructions for Authors(www.plantphysiol.org) is: Hirokazu Kobayashi(hirokoba@u-shizuoka-ken.ac.jp).
Copyright American Society of Plant Biologists Aug 2008
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