Target-Site and Metabolic Resistance Mechanisms to Penoxsulam in Late Watergrass (Echinochloa phyllopogon) in China.

Echinochloa phyllopogon, a malignant weed in Northeast China's paddy fields, is currently presenting escalating resistance concerns. Our study centered on the HJHL-715 E. phyllopogon population, which showed heightened resistance to penoxsulam, through a whole-plant bioassay. Pretreatment with a P450 inhibitor malathion significantly increased penoxsulam sensitivity in resistant plants. In order to determine the resistance mechanism of the resistant population, we purified the resistant population from individual plants and isolated target-site resistance (TSR) and nontarget-site resistance (NTSR) materials. Pro-197-Thr and Trp-574-Leu mutations in acetolactate synthase (ALS) 1 and ALS2 of the resistant population drove reduced sensitivity of penoxsulam to the target-site ALS, the primary resistance mechanisms. To fully understand the NTSR mechanism, NTSR materials were investigated by using RNA-sequencing (RNA-seq) combined with a reference genome. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis further supported the enhanced penoxsulam metabolism in NTSR materials. Gene expression data and quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation confirmed 29 overexpressed genes under penoxsulam treatment, with 16 genes concurrently upregulated with quinclorac and metamifop treatment. Overall, our study confirmed coexisting TSR and NTSR mechanisms in E. phyllopogon's resistance to ALS inhibitors.

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor.

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Here, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in <i>Nicotiana benthamiana</i> transient assays to target a transgene expressing <i>GFP</i> under the control of the T7 promoter and observed C-to-T conversions. We then targeted the T7 promoter-driven <i>acetolactate synthase</i> sequence that had been stably integrated in the rice genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the <i>acetolactate synthase</i> sequence, resulting in the enrichment of herbicide-responsive residues. We then validated these herbicide-responsive regions in the transgenic rice plants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance.

Toxicity of the herbicides used on herbicide-tolerant crops, and societal consequences of their use in France.

In France, the implementation of mutant herbicide-tolerant crops and the use of the related herbicides - sulfonylureas and imidazolinones - have triggered a strong societal reaction illustrated by the intervening actions of environmentalist groups illegally mowing such crops. Trials are in progress, and therefore should be addressed the questions of the environmental risks and the toxicity of these herbicides for the animals and humans consuming the products derived from these plants. Regulatory authorities have allowed these mutant and herbicide-tolerant plants arguing that the herbicides against which they resist only target an enzyme found in 'weeds' (the acetolactate synthase, ALS), and that therefore all organisms lacking this enzyme would be endowed with immunity to these herbicides. The toxicological literature does not match with this argument: 1) Even in organisms displaying the enzyme ALS, these herbicides impact other molecular targets than ALS; 2) These herbicides are toxic for animals, organisms that do not possess the enzyme ALS, and especially invertebrates, amphibians and fish. In humans, epidemiological studies have shown that the use and handling of these toxins are associated with a significantly increased risk of colon and bladder cancers, and miscarriages. In agricultural soils, these herbicides have a persistence of up to several months, and water samples have concentrations of some of these herbicides above the limit value in drinking water.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

The trichome-specific acetolactate synthase NtALS1 gene, is involved in acylsugar biosynthesis in tobacco (Nicotiana tabacum L.).

MAIN CONCLUSION: NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco. ABTRACT: The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase (ALS) genes in tobacco through homology-based gene prediction using Arabidopsis ALS. Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.

Rational design, synthesis and structure-activity relationship of novel substituted oxazole isoxazole carboxamides as herbicide safener.

A series of novel substituted oxazole isoxazole carboxamides derivatives were designed on the basis of active subunit combination. Forty-four novel compounds were synthesized by an efficient one-pot procedure under microwave irradiation. The bioactivity was evaluated as herbicide safener against the injury of chlorsulfuron. It was found that most of the synthesized compounds displayed remarkable protection against chlorsulfuron via enhanced glutathione content and glutathione S transferase activity. Especially compound I-11 exhibited better bioactivity than the safeners isoxadifen-ethyl and R-28725. Molecular docking simulations suggested that the target compounds could compete with chlorsulfuron in the active site of acetolactate synthase, which could explain the protective effects of safeners. The present work demonstrates that the target compounds containing oxazole isoxazole groups could be considered as potential candidates for developing novel safeners in the future.

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of Mycobacterium tuberculosis.

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus Mucor irregularis, which was obtained from the Cameroonian medicinal plant Moringa stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis (MIC90 1.56 µM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 µM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated and was superior to streptomycin treatment.

Cloning and characterization of GST fusion tag stabilized large subunit of Escherichia coli acetohydroxyacid synthase I.

There are three acetohydroxyacid synthase (AHAS, EC 4.1.3.18) isozymes (I, II, and III) in the enterobacteria Escherichia coli among which AHAS I is the most active. Its large subunit (LSU) possesses full catalytic machinery, but is unstable in the absence of the small subunit (SSU). To get applicable LSU of AHAS I, we prepared and characterized in this study the polypeptide as a His-tagged (His-LSU) and a glutathione S-transferase (GST)-tagged (GST-LSU) fusion protein, respectively. The results showed that the His-LSU is unstable, whereas the GST-LSU displays excellent stability. This phenomenon suggests that the GST polypeptide fusion tag could stabilize the target protein when compared with histidine tag. It is the first time that the stabilizing effect of the GST tag was observed. Further characterization of the GST-LSU protein indicated that it possesses the basic functions of AHAS I with a specific activity of 20.8 µmol min(-1) mg(-1) and a Km value for pyruvate of 0.95 mM. These observations imply that introduction of the GST fusion tag to LSU of AHAS I does not affect the function of the protein. The possible reasons that the GST fusion tag could make the LSU stable are initially discussed.

Characterization of the acetohydroxyacid synthase multigene family in the tetraploide plant Chenopodium quinoa

Background Currently, the technology called Clearfield® is used in the development of crops resistant to herbicides that inhibit the enzyme acetohydroxy acid synthase (AHAS, EC 2.2.1.6). AHAS is the first enzyme of the biosynthetic pathway that produces the branched-chain of the essential amino acids valine, leucine, and isoleucine. Therefore, multiple copies of the AHAS gene might be of interest for breeding programs targeting herbicide resistance. In this work, the characterization of the AHAS gene was accomplished for the Chenopodium quinoa Regalona-Baer cultivar. Cloning, sequencing, and Southern blotting were conducted to determine the number of gene copies. Results The presence of multiple copies of the AHAS gene as has been shown previously in several other species is described. Six copies of the AHAS gene were confirmed with Southern blot analyses. CqHAS1 and CqAHAS2 variants showed the highest homology with AHAS mRNA sequences found in the NR Database. A third copy, CqAHAS3, shared similar fragments with both CqAHAS1 and CqAHAS2, suggesting duplication through homeologous chromosomes pairing. Conclusions The presence of multiple copies of the gene AHAS shows that gene duplication is a common feature in polyploid species during evolution. In addition, to our knowledge, this is the first report of the interaction of sub-genomes in quinoa.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Utilizing next-generation sequencing to study homeologous polymorphisms and herbicide-resistance-endowing mutations in Poa annua acetolactate synthase genes.

BACKGROUND: Detection of single nucleotide polymorphisms (SNPs) related to herbicide resistance in non-model polyploid weed species is fraught with difficulty owing to the gene duplication and lack of reference sequences. Our research seeks to overcome these obstacles by Illumina HiSeq read mapping, SNP calling and allele frequency determinations. Our focus is on the acetolactate synthase (ALS) gene, the target site of ALS-inhibiting herbicides, in Poa annua, an allotetraploid weed species originating from two diploid parents, P. supina and P. infirma. RESULTS: ALS contigs with complete coding regions of P. supina, P. infirma and P. annua were assembled and compared with ALS genes from other plant species. The ALS infirma-homeolog of P. annua showed higher levels of nucleotide sequence variability than the supina-homeolog. Comparisons of read mappings of P. annua and a simulated P. supina × P. infirma hybrid showed high resemblance. Two homeolog-specific primer pairs were designed and used to amplify a 1860 bp region covering all resistance-conferring codons in the ALS gene. Four P. annua populations, GN, RB, GW and LG, showed high resistance to two ALS inhibitors, bispyribac-sodium and foramsulfuron, and two populations, HD and RS, showed lower resistance in the rate-response trial. Mutations conferring Trp-574-Leu substitution were observed in the infirma-homeolog of GN and RB and in the supina-homeolog of GW and LG, but no resistance-conferring mutation was observed in the two populations of lower resistance, HD and RS. CONCLUSION: In this study we have demonstrated the use of NGS data to study homeologous polymorphisms, parentage and herbicide resistance in an allotetraploid weed species, P. annua. Complete coding sequences of the ALS gene were assembled for P. infirma, P. supina, infirma-homeolog and supina-homeolog in P. annua. A pipeline consisting of read mapping, SNP calling and allele frequency calculation was developed to study the parentage of P. annua, which provided a new perspective on this topic besides the views of morphology, karyotype and phylogeny. Our two homeolog-specific primer pairs can be utilized in future research to separate the homeologs of the ALS gene in P. annua and cover all the codons that have been reported to confer herbicide resistance.

Using next-generation sequencing to detect mutations endowing resistance to pesticides: application to acetolactate-synthase (ALS)-based resistance in barnyard grass, a polyploid grass weed.

BACKGROUND: Next-generation sequencing (NGS) technologies offer tremendous possibilities for accurate detection of mutations endowing pesticide resistance, yet their use for this purpose has not emerged in crop protection. This study aims at promoting NGS use for pesticide resistance diagnosis. It describes a simple procedure accessible to virtually any scientist and implementing freely accessible programs for the analysis of NGS data. RESULTS: Three PCR amplicons encompassing seven codons of the acetolactate-synthase gene crucial for herbicide resistance were sequenced using non-quantified pools of crude DNA extracts from 40 plants in each of 28 field populations of barnyard grass, a polyploid weed. A total of 63,959 quality NGS sequence runs were obtained using the 454 technology. Three herbicide-resistance-endowing mutations (Pro-197-Ser, Pro-197-Leu and/or Trp-574-Leu) were identified in seven populations. The NGS results were confirmed by individual plant Sanger sequencing. CONCLUSION: This work demonstrated the feasibility of NGS-based detection of pesticide resistance, and the advantages of NGS compared with other molecular biology techniques for analysing large numbers of individuals. NGS-based resistance diagnosis has the potential to play a substantial role in monitoring resistance, maintaining pesticide efficacy and optimising pesticide applications.

The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species.

Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 µM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

KEY MESSAGE: Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 µg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 µg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 µg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

Transcription activator-like effector nucleases enable efficient plant genome engineering.

The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production.

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19 % in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.

Isolation and expression of genes for acetolactate synthase and acetyl-CoA carboxylase in Echinochloa phyllopogon, a polyploid weed species.

BACKGROUND: Target-site resistance is the major cause of herbicide resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in arable weeds, whereas non-target-site resistance is rarely reported. In the Echinochloa phyllopogon biotypes resistant to these herbicides, target-site resistance has not been reported, and non-target-site resistance is assumed to be the basis for resistance. To explore why target-site resistance had not occurred, the target-site genes for these herbicides were isolated from E. phyllopogon, and their expression levels in a resistant biotype were determined. RESULTS: Two complete ALS genes and the carboxyltransferase domain of four ACCase genes were isolated. The expression levels of ALS and ACCase genes were higher in organs containing metabolically active meristems, except for ACC4, which was not expressed in any organ. The differential expression among examined organs was more prominent for ALS2 and ACC2 and less evident for ALS1, ACC1 and ACC3. CONCLUSION: E. phyllopogon has multiple copies of the ALS and ACCase genes, and different expression patterns were observed among the copies. The existence of three active ACCase genes and the difference in their relative expression levels could influence the occurrence of target-site resistance to ACCase inhibitors in E. phyllopogon.

Split-transgene expression in wheat.

The establishment of traits that result from the concerted expression of complementing transgene fragments is a feasible tool for trait control or gene flow control in plants. This chapter describes the methodology for producing herbicide-resistant and pollen-sterile wheat plants by the intein-mediated assembly of inactive precursor protein fragments (protein trans-splicing). We suggest the design of intein-containing vectors for split-transgene expression. We describe transient plant assays that can be used to analyse the functionality of the system and describe the transformation of wheat plants using a split selection marker.We hope that this chapter will be a helpful guideline for researchers who are interested in applying similar split-gene approaches in wheat or other monocotyledonous crops.

Cytosolic localization of acetohydroxyacid synthase Ilv2 and its impact on diacetyl formation during beer fermentation.

Diacetyl (2,3-butanedione) imparts an unpleasant "butterscotch-like" flavor to alcoholic beverages such as beer, and therefore its concentration needs to be reduced below the sensory threshold before packaging. We examined the mechanisms that lead to highly elevated diacetyl formation in petite mutants of Saccharomyces cerevisiae during beer fermentations. We present evidence that elevated diacetyl formation is tightly connected to the mitochondrial import of acetohydroxyacid synthase (Ilv2), the key enzyme in the production of diacetyl. Our data suggest that accumulation of the matrix-targeted Ilv2 preprotein in the cytosol is responsible for the observed high diacetyl levels. We could show that the Ilv2 preprotein accumulates in the cytosol of petite yeasts. Furthermore, expression of an Ilv2 variant that lacks the N-terminal mitochondrial targeting sequence and thus cannot be imported into mitochondria led to highly elevated diacetyl levels comparable to a petite strain. We further show that expression of a mutant allele of the γ-subunit of the F(1)-ATPase (ATP3-5) could be an attractive way to reduce diacetyl formation by petite strains.

Structural and functional evaluation of three well-conserved serine residues in tobacco acetohydroxyacid synthase.

The first step in the common pathway for the biosynthesis of branched-chain amino acids (BCAAs) is catalyzed by acetohydroxyacid synthase (AHAS). The roles of three well-conserved serine residues (S167, S506, and S539) in tobacco AHAS were determined using site-directed mutagenesis. The mutations S167F and S506F were found to be inactive and abolished the binding affinity for cofactor FAD. The Far-UV CD spectrum of the inactive mutants was similar to that of wild-type enzyme, indicating no major conformational changes in the secondary structure. However, the active mutants, S167R, S506A, S506R, S539A, S539F and S539R, showed lower specific activities. Further, a homology model of tobacco AHAS was generated based on the crystal structure of yeast AHAS. In the model, the S167 and S506 residues were identified near the FAD binding site, while the S539 residue was found to near the ThDP binding site. The S539 mutants, S539A and S539R, showed strong resistance to three classes of herbicides, NC-311 (a sulfonylurea), Cadre (an imidazolinone), and TP (a triazolopyrimidine). In contrast, the active S167 and S506 mutants did not show any significant resistance to the herbicides, with the exception of S506R, which showed strong resistance to all herbicides. Thus, our results suggest that the S167 and S506 residues are essential for catalytic activity by playing a role in the FAD binding site. The S539 residue was found to be near the ThDP with an essential role in the catalytic activity and specific mutants of this residue (S539A and S539R) showed strong herbicide resistance as well.