The molecular basis of sulfonylurea herbicide resistance in tobacco

The enzyme acetolactate synthase (ALS) is the target enzyme for the sulfonylurea and imidazolinone herbicides. We describe the isolation and characterization of the ALS genes from two herbicide-resistant mutants, C3 and S4-Hra, of Nicotiana tabacum. There are two distinct ALS genes in tobacco which are 0.7% divergent at the amino acid sequence level. The C3 mutant has a single Pro-Gln replacement at amino acid 196 in one ALS gene. This gene is termed the class I gene and is equivalent to the SuRA locus. The S4-Hra mutant has two amino acid changes in the other ALS gene. This gene is termed the class II gene or the SuRB locus. The S4-Hra mutant includes a Pro-Ala substitution at amino acid 196 and a Trp-Leu substitution at amino acid 573. Gene reintroduction experiments have confirmed that these amino acid substitutions are responsible for the herbicide resistance phenotypes. Transgenic plants carrying these genes are highly resistant to sulfonylurea herbicide applications.     

Molecular Basis of Imidazolinone Herbicide Resistance in Arabidopsis thaliana var Columbia

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, isoleucine, and valine, is inhibited by imidazolinone herbicides. To understand the molecular basis of imidazolinone resistance, we isolated the ALS gene from an imazapyr-resistant mutant GH90 of Arabidopsis thaliana. DNA sequence analysis of the mutant ALS gene demonstrated a single-point mutation from G to A at nucleotide 1958 of the ALS-coding sequence. This would result in Ser to Asn substitution at residue 653 near the carboxyl terminal of the matured ALS. The mutant ALS gene was introduced into tobacco using Agrobacterium-mediated transformation. Imidazolinone-resistant growth of transformed calli and leaves of transgenic plants was 100-fold greater than that of nontransformed control plants. The relative levels of imidazolinone-resistant ALS activity correlated with the amount of herbicide-resistant growth in the leaves of transgenic plants. Southern hybridization analysis confirmed the existence of transferred ALS gene in the transformant showing high imazapyr resistance. The results demonstrate that the mutant ALS gene confers resistance to imidazolinone herbicides. This is the first report, to our knowledge, of the molecular basis of imidazolinone resistance in plants.     

Isolation and characterization of plant genes coding for acetolactate synthase, the target enzyme for two classes of herbicides

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathways to valine, isoleucine, and leucine. It is the target of two structurally unrelated classes of herbicides, the sulfonylureas and the imidazolinones. Genomic clones encoding ALS have been isolated from the higher plants Arabidopsis thaliana and Nicotiana tabacum, using a yeast ALS gene as a heterologous hybridization probe. Clones were positively identified by the homology of their deduced amino acid sequences with those of yeast and bacterial ALS isozymes. The tobacco and Arabidopsis ALS genes have approximately 70% nucleotide homology, and encode mature proteins which are approximately 85% homologous. Little homology is seen between the amino acid sequences of the presumptive N-terminal chloroplast transit peptides. Both plant genes lack introns. The tobacco ALS gene was isolated from a line of tobacco which is resistant to the sulfonylurea herbicides due to an alteration in ALS. The tobacco gene which was isolated codes for an ALS that is sensitive to the herbicides, as assayed by transformation of the gene into sensitive tobacco cells.     

A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.     

Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8).     

TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants

Nuclear matrix attachment regions (MARs) are thought to influence the expression of the flanking genes. TM2, a new DNA fragment isolated from tobacco, can bind with the rice nuclear matrix in vitro. In this study, we investigated the effect of TM2 on transgene expression under the control of three different promoters in stably transformed rice calli and plants. The presence of TM2 flanking the transgene increased the expression of constructs based on the constitutive CaMV 35S and maize ubiquitin gene promoters in both resistant calli and transformed plants. The GUS expression directed by the photosynthetic-tissue-specific PNZIP promoter was also increased in photosynthetic tissues of transformants. However, TM2 did not change the gene expression pattern controlled by the PNZIP promoter. The effect of TM2 in transgenic plants was stronger than that in transgenic calli based on all three promoters. Our results indicate that TM2, as a novel strong MAR, can be used to increase the transgene expression levels in the whole plant or in particular tissues of monocotyledons.     

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP.     

Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected]

We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.     

Biolistic transfer of large DNA fragments to tobacco cells using YACs retrofitted for plant transformation

To determine whether large DNA molecules could be transferred and integrated intact into the genome of plant cells, we bombarded tobacco suspension cells with yeast DNA containing artificial chromosomes (YACs) having sizes of 80, 150, 210, or 550 kilobases (kb). Plant selectable markers were retrofitted on both YAC arms so that recovery of each arm in transgenic calli could be monitored. Stably transformed calli resistant to kanamycin (300 mg/L) were recovered for each size of YAC tested. Two of 12 kanamycin-resistant transformants for the 80 kb YAC and 8 of 29 kanamycin-resistant transformants for the 150 kb YAC also contained a functional hygromycin gene derived from the opposite YAC arm. Southern analyses using probes that spanned the entire 55 kb insert region of the 80 kb YAC confirmed that one of the two double-resistant lines had integrated a fully intact single copy of the YAC DNA while the other contained a major portion of the insert. Transgenic lines that contained only one selectable marker gene from the 80 kb YAC incorporated relatively small portions of the YAC insert DNA distal to the selectable marker. Our data suggest genomic DNA cloned in artificial chromosomes up to 150 kb in size have a reasonable likelihood of being transferred by biolistic methods and integrated intact into the genome of plant cells. Biolistic transfer of YAC DNA may accelerate the isolation of agronomically useful plant genes using map-based cloning strategies.     

Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis

As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.     

Role of nucleotidyl transferase motif V in strand joining by chlorella virus DNA ligase.

ATP-dependent DNA ligases, NAD(+)-dependent DNA ligases, and GTP-dependent RNA capping enzymes are members of a covalent nucleotidyl transferase superfamily defined by a common fold and a set of conserved peptide motifs. Here we examined the role of nucleotidyl transferase motif V ((184)LLKMKQFKDAEAT(196)) in the nick joining reaction of Chlorella virus DNA ligase, an exemplary ATP-dependent enzyme. We found that alanine substitutions at Lys(186), Lys(188), Asp(192), and Glu(194) reduced ligase specific activity by at least an order of magnitude, whereas substitutions at Lys(191) and Thr(196) were benign. The K186A, D192A, and E194A changes had no effect on the rate of single-turnover nick joining by preformed ligase-adenylate but affected subsequent rounds of nick joining at the ligase adenylation step. Conservative substitutions K186R, D192E, and E194D partially restored activity, whereas K186Q, D192N, and E194Q substitutions did not. Alanine mutation of Lys(188) elicited distinctive catalytic defects, whereby single-turnover nick joining by K188A-adenylate was slowed by an order of magnitude, and high levels of the DNA-adenylate intermediate accumulated. The rate of phosphodiester bond formation at a pre-adenylated nick (step 3 of the ligation pathway) was slowed by the K188A change. Replacement of Lys(188) by arginine reversed the step 3 arrest, whereas glutamine substitution was ineffective. Gel-shift analysis showed that the Lys(188) mutants bound stably to DNA-adenylate. We infer that Lys(188) is involved in the chemical step of phosphodiester bond formation.     

Somaclonal variation at the nucleotide sequence level in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) as revealed by RAPD and ISSR markers,and by pairwise sequence analysis

The nature of somaclonal variation at the nucleotide sequence level was studied in rice cv. Nipponbare. First, we investigated genomic variations by using 2 molecular marker systems: RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This was followed by sequencing of selected bands that represented genomic variations, and pairwise sequence analysis taking advantage of the whole genome sequence of rice. In addition, transpositional activity of the active MITE, mPing, was analysed by locus-specific PCR amplifications. The 2-year-old calli and their regenerated plants, analysed with 24 RAPD and 20 ISSR primers, showed moderate levels of genomic variation (20.83% and 17.04%, respectively). To test whether DNA methylation plays a role in somaclonal variation, the calli were treated with 5-azacytidine, a chemical agent that reduces cytosine methylation by blocking the activity of DNA methyltransferase. Though dwarfism occurred in regenerants from treated calli (a hallmark of the drug treatment), there was only a slight increase in the frequency of somaclonal variation detected in the treated calli and their regenerated plants relative to untreated controls. The transposon mPing also remained immobile in both treated and untreated calli. Nevertheless, dendrograms constructed according to the Jaccard coefficient calculated by UPGMA of the ISSR bands revealed that the 5-azacytidine-treated and untreated somaclones were grouped into 2 distinct clusters, suggesting a possible indirect effect of the treatment on the genomic changes, depending on the marker used. Sequence analysis indicated a low level of variation (0.31%), with single-base-pair substitutions predominating.     

Selection of regenerable maize callus cultures resistant to 5-methyl-DL-tryptophan,S-2-aminoethyl-L-cysteine and high levels of L-lysine plus L-threonine

Tissues resistant to lethal levels of equimolar L-lysine plus L-threonine (LT), 5-methyl-DL-tryptophan (5MT, a tryptophan analog), or S-2-aminoethyl-L-cysteine (AEC, a lysine analog) were selected from maize callus capable of plant regeneration (H99 and W77-R3019 genotypes).Resistance to LT resulted from resistant calli having a 19 times greater level of free threonine than wild type tissues. The resistance was expressed in roots of whole plants; threonine levels were two to nine times greater in leaves and kernels of resistant plants than in wild type plants. Slightly greater levels of isoleucine, lysine and methionine were also noted, particularly in the kernel. Genetic studies with individual resistant plants did not always produce inheritance ratios typical of simple Mendelian inheritance, but by the third generation after plant regeneration a trend towards homozygosity was apparent and the data suggests that LT resistance is inherited as a single dominant nuclear gene.Resistance to 5MT resulted from resistant calli having a 133 to 161 times greater level of free tryptophan than wild type tissues. Also, phenylalanine was 22 to 30 times as great and histidine, tyrosine and valine were about two times as great as in wild type tissues. Resistance was expressed in roots of whole plants, and tryptophan levels were at least 2000 times greater in resistant than in wild type plants. Phenylalanine was also 32 times greater. All regenerant plants resistant to 5MT were both male and female sterile.Resistance to AFC was caused by decreased AEC uptake by the callus tissue and was not due to increased levels of free lysine. Plants were not regenerated from this callus.     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

O⁶-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O⁶-methylguanine and O⁴-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.     

Requirement of the Pro-Cys-His-Arg sequence for O6-methylguanine-DNA methyltransferase activity revealed by saturation mutagenesis with negative and positive screening

O⁶-Methylguanine-DNA methyltransferase catalyzes transfer of a methyl group from O⁶-methylguanine and O⁴-methylthymine of DNA to a cysteine residue of the enzyme protein, thereby repairing the mutagenic and carcinogenic lesions in a single-step reaction. There are highly conserved amino acid sequences around the methyl-accepting cysteine site in eleven molecular species of methyltransferases. To elucidate the significance of the conserved sequence, amino acid substitutions were introduced by site-directed mutagenesis of the cloned DNA for Escherichia coli Ogt methyltransferase, and the activity and stability of mutant forms of the enzyme were examined. When cysteine-139, to which methyl transfer occurs, was replaced by other amino acids, all of the mutants showed the methyltransferase-negative phenotype. Methyltransferase-positive revertants, isolated from one of the negative mutants, had restored codons for cysteine. Thus the cysteine residue is essential for acceptance of the methyl group and is not replaceable by other amino acids. Using this negative and positive selection procedure, the analysis was extended to other residues near the acceptor site. At the histidine-140 and arginine-141 sites, all the positive revertants isolated carried codons for amino acids identical to those of the wild-type protein. At proline-138, five substitutions (serine, glutamine, threonine, histidine, and alanine) exhibited the positive phenotype but levels of methyltransferase activity in extracts of cells harboring these mutant forms were very low. This suggests that the proline residue at this site is important for maintaining the proper conformation of the protein. With valine-142 substitutions there were seven types of positive revertants, among which mutants carrying isoleucine, cysteine, leucine, and alanine showed relatively high levels of methyltransferase activity. These results indicate that the sequence Pro-Cys-His-Arg is a sine qua non for methyltransferase to exert its function.     

Patterns of ISSR and REMAP DNA markers after cryogenic preservation of spring wheat calli by dehydration method

Inter-Simple Sequence Repeat (ISSR) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were applied to study the influence of successive steps of dehydration cryopreservation on DNA in recovered calli and regenerated plants of spring wheat (Triticum aestivum L.). The precultivation step had no influence on the genetic stability of plant material. After the dehydration step, a new fragment appeared in the REMAP profiles for one DNA sample of calli of Nv16 line. A fragment of similar length was observed in one DNA sample for calli regenerated after complete procedure of cryopreservation in liquid nitrogen (−196°C). However, in samples of calli cultured in vitro for two and four weeks after any type of treatments, the amplicon spectra exhibited no difference from those of starting materials. The amplicon profiles of plants regenerated from calli after successive steps of cryopreservation were also identical to the profiles of the mother plants.     

Herbicide Resistance in Datura innoxia: Kinetic Characterization of Acetolactate Synthase from Wild-Type and Sulfonylurea-Resistant Cell Variants

Acetolactate synthase (ALS, EC 4. 1.3. 18), the first enzyme in the biosynthesis of branched-chain amino acids, was isolated from wild-type and sulfonylurea-resistant Datura innoxia cell variants and characterized. Apparent K_m values of the ALS for pyruvate from three sulfonylurea-resistant variants (CSR2, CSR6, and CSR10) were manyfold greater than that of the wild type. The inhibition of wild-type and herbicide-resistant ALS activity by chlorsulfuron (CS), a sulfonylurea herbicide, and l-leucine (l-Leu), one of the feedback inhibitors of the enzyme, was examined. ALS from two CS-resistant variants exhibited severalfold greater resistance to CS than did the wild-type enzyme. Inhibition of ALS by l-Leu fitted a partially competitive pattern most closely. It is proposed that the herbicide resistance mutation accentuated the partial inhibition characteristics of ALS by l-Leu. ALS from one of the two CS-resistant variants (CSR6) had a K_i for l-Leu an order of magnitude greater than that of the wild-type enzyme. The alterations in kinetic properties observed in the ALS from sulfonylurea-resistant variants are discussed in relation to the possible evolutionary significance of the herbicide binding site of this enzyme, the physiological effects of such biochemical alterations, and their practical utility in genetic studies.     

Chimeric vector construction for higher-plant transformation

A chimeric vector pKR612B1 was developed containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV), and was used to transform higher plant protoplasts. Plasmid pDOB612, the parental vector of pKR612B1, has two unique restriction sites, SmaI and BamHI, positioned just downstream of the CaMV gene VI promoter sequence. These unique cloning sites can be used for any kind of gene insertion into this vector. Using the polyethylene glycol transformation procedure, a large number of turnip and tobacco protoplasts were transformed and proved to be resistant to kanamycin (Km). From tobacco protoplasts whole Km-resistant plants were regenerated and shown to contain the integrated foreign gene. APH activity was detected in both transformed calli and in regenerated plants. DNA from transformed clones was analysed by Southern blot hybridization, showing the presence of the Tn5-derived gene.