Introduction
In this paper, we present the results of our study of the resistance mechanism of the tomato (Solanum lycopersicum L.) mutant line HRT1, previously obtained by ethyl methanesulfonate (EMS) mutagenesis of seeds of the commercial tomato line ‘M82’. The mutant was found to be highly resistant to the imidazolinone herbicides imazamox, imazapic, and imazapyr; pyrithiobac-sodium (pyrimidinylthiobenzoic acid group); and propoxycarbazone sodium (sulfonylaminocarbonyl triazolinone group) (Han et al., Reference Han, Dong, Sun, Li and Zheng2012). HRT1 did not differ from M82 in its response to the sulfonylurea herbicides trifloxysulfuron, sulfosulfuron, and chlorsulfuron (Dor et al. Reference Dor, Smirnov, Galili, Guy and Hershenhorn2016).
Wide use of acetohydroxyacid synthase (AHAS)-inhibiting herbicides has resulted in the appearance of AHAS herbicide–resistant weed populations (Gaines et al. Reference Gaines, Duke, Morran, Rigon, Tranel, Küpper and Dayan2020; Owen et al. Reference Owen, Goggin and Powles2012). Nevertheless, AHAS-inhibiting herbicides remain important in the continuously decreasing repertoire of herbicides, due to their broad spectrum of weed control, low toxicity to mammals, high selectivity, and high activity. which allows for low application rates (Owen et al. Reference Owen, Goggin and Powles2012), and in particular, their effectiveness against broomrapes (Orobanche and Phelipanche species) (Dor et al. Reference Dor, Smirnov, Galili, Guy and Hershenhorn2016).
In addition, resistance to AHAS-inhibiting herbicides has been obtained by mutagenesis in many crop lines, such as corn (Zea mays L.) (Newhouse et al. Reference Newhouse, Singh, Shaner and Stidham1991), Arabidopsis thaliana L. (Haughn and Somerville Reference Haughn and Somerville1986), sugar beet (Beta vulgaris L.) (Hart et al. Reference Hart, Saunders and Penner1992; Wright and Penner Reference Wright and Penner1998), canola (Brassica napus L.) (Guo et al., Reference Guo, Liu, Long, Gao, Zhang, Chen, Pu and Hu2022; Swanson et al. Reference Swanson, Herrgesell, Arnoldo, Sippell and Wong1989), soybean [Glycine max (L.) Merr.] (Sebastian et al. Reference Sebastian, Fader, Ulrich, Forney and Chalef1989; Ustun and Uzun Reference Ustun and Uzun2023), tobacco (Nicotiana tabacum L.) (Chaleff and Ray Reference Chaleff and Ray1984), cotton (Gossypium hirsutum L.) (Chen et al. Reference Chen, Ling, Yu and Zhang2023; Rajasekaran et al. Reference Rajasekaran, Grula and Anderson1996), rice (Oryza sativa L.) (Croughan Reference Croughan1998; Piao et al. Reference Piao, Wang, Wei, Zonta, Wan, Bai, Wu, Wang and Fang2018), wheat (Triticum aestivum L.) (Chen et al. Reference Chen, Wang, Heng, Li, Pei, Cao, Deng and Ma2021; Pozniak and Hucl Reference Pozniak and Hucl2004), barley (Hordeum vulgare L.) (Lee et al. Reference Lee, Rustgi, Kumar, Burke, Yenish, Gill, von Wettstein and Ullrich2011), and chickpea (Cicer arietinum L.) (Galili et al. Reference Galili, Hershenhorn, Edelman, Sobolev, Smirnov, Amir-Segev, Bellalou and Dor2021). Imidazolinone herbicides act by inhibiting AHAS (Duggleby and Pang Reference Duggleby and Pang2000; Iwakami et al. Reference Iwakami, Uchito, Watanabe, Yamasue and Inamura2012; Owen et al. Reference Owen, Goggin and Powles2012; Schloss Reference Schloss and Setter1995), which is a key enzyme in the biosynthetic pathway of the branched-chain amino acids leucine, isoleucine, and valine. The consequent deficiency in these amino acids results in plant death (Han et al., Reference Han, Dong, Sun, Li and Zheng2012; Iwakami et al. Reference Iwakami, Uchito, Watanabe, Yamasue and Inamura2012; McCourt et al. 2005). In most cases, resistance is associated with mutations in the catalytic large-subunit AHAS gene family resulting in the substitution of a single highly conserved amino acid residue in the channel leading to the herbicide-binding site of the AHAS protein (Duggleby et al. Reference Duggleby, McCourt and Guddat2008; Lonhienne et al. Reference Lonhienne, Garcia, Low and Guddat2022b; Tranel and Wright Reference Tranel and Wright2002; Walsh et al. Reference Walsh, Babiker, Burke and Hulbert2012). At least 18 amino acid residues have been identified in bacteria, fungi, or plants in which mutation provides resistance to AHAS-inhibiting herbicides. Among them, mutations to amino acid residues Ala-122, Met-124, Pro-197, Arg-199, Thr-203, Ala-205, Lys-256, Met-351, His-352, Asp-375, Met-570, Trp-574, Phe-578, and Ser-653 (numbering according to A. thaliana) have been reported to be involved in plants’ resistance to imidazolinones (Duggleby et al. Reference Duggleby, McCourt and Guddat2008; Galili et al. Reference Galili, Hershenhorn, Edelman, Sobolev, Smirnov, Amir-Segev, Bellalou and Dor2021). Mutations can lead to AHAS inhibitor cross-tolerance, and some lead to broad cross-resistance to all classes of AHAS inhibitors (Duggleby et al. Reference Duggleby, McCourt and Guddat2008).
In this study, we identified a mutation in the AHAS gene in HRT1 tomato plants and characterized the sensitivity of the enzyme to the imidazolinone herbicides imazapic and imazapyr, as well as the sulfonylurea herbicides rimsulfuron and sulfosulfuron. The prevention of HRT1’s death due to branched-chain amino acid starvation following imazapic treatment was also shown. To ascertain the heredity of the resistance trait in the HRT1 line and to determine whether these alleles are recessive or dominant, segregation analysis was conducted. Ligand–protein contact analysis further allowed us to explain the changes in binding forces after modification of the protein–ligand binding region.
Materials and Methods
Plant Material
Tomato (Solanum lycopersicum L.) seeds of ‘M82’ were obtained from Tarsis Agricultural Chemicals (Petah Tikva, Israel). HRT1, a tomato mutant that is highly resistant to imidazolinone herbicides, was obtained by EMS mutagenesis (Dor et al. Reference Dor, Smirnov, Galili, Guy and Hershenhorn2016).
Determination of AHAS Activity
Response of the enzyme AHAS to the herbicides was determined in vivo using crude enzyme extracts isolated and partially purified from young M82 and HRT1 seedlings, as described in Dor et al. (Reference Dor, Galili, Smirnov, Hacham, Amir and Hershenhorn2017). Stock solutions of the tested herbicides were prepared in tetrahydrofuran. Aliquots of these solutions were taken and dried in test tubes. Tetrahydrofuran without herbicides was used in control tubes. AHAS activity was expressed as percentage of the control treatment containing no herbicides. The experiment was conducted in four replicates. Final herbicide concentrations in the reaction mixture were: imazapic (Cadre®, 240 g ai L−1, BASF, Research Triangle Park, NC, www.basf.com) and imazapyr (Arsenal®, 240 g ai L−1, BASF) at 1, 5, 10, 50, 100, and 200 µM; sulfonylurea rimsulfuron (Titus®, 250 g kg−1, Corteva Agriscience UK, Melbourn, Cambridgeshire, UK, www.corteva.co.uk) at 0.05, 0.1, 0.5, 1, 5, and 10 µM; and sulfosulfuron (Monitor®, 750 g ai L−1, Monsanto, St Louis, MO, USA, www.monsanto.com) at 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 µM.
Determination of Free Amino Acids
M82 and homozygote HRT1 plants were grown in 2-L pots in a greenhouse. At the 4-true-leaf stage, five plants of each line were sprayed with imazapic at a rate of 14.4 g ai ha−1. After 3 wk, leaf samples of treated and non-treated plants were analyzed for free amino acids content. Extraction, derivatization, and amino acid analysis were conducted according to Dor et al. (Reference Dor, Galili, Smirnov, Hacham, Amir and Hershenhorn2017). All tests were performed in five replicates.
Sequencing of AHAS Genes
The tomato genome contains three large AHAS genes located on chromosomes 3, 6, and 7 (Tomato Genome Consortium 2012). To determine the DNA sequences of the three AHAS genes, total genomic DNA was extracted from young leaves of M82 and HRT1 plants at the 4-true-leaf stage, as previously described in Fulton et al. (Reference Fulton, Chunwongse and Tanksley1995). All three AHAS genes of M82 and HRT1 plants were PCR amplified, and the amplified fragments were sequenced at Hylabs Laboratory (Rehovot, Israel, https://www.hylabs.co.il/). For mutation determination, the DNA sequences of the amplified fragments of each AHAS gene were compared to their corresponding wild-type (WT) sequences published by the Tomato Genome Consortium utilizing DNAMAN 4.2 (Lynnon Biosoft, San Ramon, CA, USA).
Segregation Analysis
To ascertain the heredity of the resistance trait in the HRT1 line and to determine whether these alleles are recessive or dominant, we grew 10 plants that were homozygous for the resistance mutation and 10 homozygous M82 plants. F2 plants derived from a cross between these two lines followed by self-pollination were screened for resistance to imazapic in a greenhouse and under field conditions as follows: 75 plants (5 groups of 15 plants) were planted in 2-L pots in Newe Ya’ar soil (medium-heavy clay–loam soil containing, on a dry weight basis, 55% clay, 23% silt, 20% sand, 2% organic matter, pH 7.1), 1 plant per pot, in a greenhouse; an additional 74 plants were planted in an open field at Newe Ya’ar research center (32.70917°N, 35.17989°E). M82 and HRT1 plants (10 of each) were planted in the greenhouse and in the field (for a total of 40 plants) as positive a negative control. At the 6-true-leaf stage, five M82, five HRT1, and all F2 plants were sprayed with imazapic at a rate of 24 g ai ha−1. The other five M82 and five HRT1 plants were sprayed with water (control). The number of resistant and sensitive plants was evaluated visually 3 wk after treatment. Plant injury was assessed on a scale of 5 (healthy, no damage) to 1 (death). For the segregation pattern, Pearson’s chi-square analysis with JMP5 software (SAS Institute, Cary, NC, USA) was used to evaluate the suitability of the single-gene model.
Ligand–Protein Contact Analysis
Ligand–protein contact analysis, which predicts the binding forces obtained after chemical modification of the protein–ligand binding region, was conducted using LPC software (Sobolev et al. Reference Sobolev, Sorokine, Prilusky, Abola and Edelman1999). The model for protein-structure prediction was built as described in Sobolev et al. (Reference Sobolev, Eyal, Gerzon, Potapov, Babor, Prilusky and Edelman2005).
Statistical Analysis
Data on herbicide influence on AHAS activity were computed by nonlinear regressions using Sigma-Plot v. 11.01 (SPSS, Chicago, IL) as Y = y0
+
${{a}\over{{1 + {{\left( {{{x}\over{{x0}}}} \right)}^b}}}}\;$
for imazapic and sulfosulfuron, and Y =
${{a}\over{{1 + {{\left( {{{x}\over{{x0}}}} \right)}^b}}}}\;$
for imazapyr and rimsulfuron. The amino acid content results were subjected to ANOVA using JMP Software v. 5.0 (SAS Institute). The data were separated by standard error of the mean (SEM) and compared by Student’s t-test (P < 0.05).
Results and Discussion
Determination of the Mutation
Mechanisms accounting for herbicide resistance in plants include increased metabolism, sequestration, reduced uptake and/or translocation, and modification of the herbicide target site (Lonhienne et al. Reference Lonhienne, Garcia, Low and Guddat2022b; Sala et al. Reference Sala, Bulos and Echarte2008). In most cases describing the resistance mechanism to AHAS-inhibiting herbicides, resistance is due to a point mutation(s) in the gene(s) encoding the AHAS catalytic subunit, reducing the enzyme’s sensitivity to herbicides (Han et al. Reference Han, Dong, Sun, Li and Zheng2012; Lonhienne et al. Reference Lonhienne, Cheng, Garcia, Hu, Low, Schenk, Williams and Guddat2022a, Reference Lonhienne, Garcia, Low and Guddat2022b).
Sequence analysis of the three AHAS genes revealed a single point transition mutation of C to T in AHAS1 at position 581 (Figure 1A) located on chromosome 3 (Tomato Genome Consortium 2012). This resulted in a substitution of alanine by valine at position 194, corresponding to Ala-205 in Arabidopsis (Figure 1B). This is a common mutation providing resistance to imidazolinones (Jain and Tar’an Reference Jain and Tar’an2014), as reported for chickpea (Thompson and Tar’an Reference Thompson and Tar’an2014), sunflower (Helianthus annuus L.) (White et al. Reference White, Graham and Owen2003), and the weeds redroot pigweed (Amaranthus retroflexus L.) and eastern black nightshade (Solanum ptycanthum Dunal) (Ashigh and Tardif Reference Ashigh and Tardif2007; Beckie and Tardif Reference Beckie and Tardif2012; McNaughton et al. Reference McNaughton, Letarte, Lee and Tardif2005). To the best of our knowledge, this is the first report of an alanine to Val-194 (according to Arabidopsis 205) mutation in imidazolinone-resistant tomatoes. No additional mutations were found in this gene, or in AHAS2 or AHAS3 located on chromosomes 7 and 6, respectively (data not shown).
Figure 1. Sequence analysis of AHAS1 located on chromosome 3. (A) AHAS1 nucleotide sequences (541–597) of wild-type (WT) and HRT1 tomato. The C to T transition at position 581 is highlighted in green. (B) WT and HRT1 tomato AHAS1 amino acids 181–199 (192–210 according to Arabidopsis thaliana [ARA]). The alanine to valine transition at position 194 (205 according to Arabidopsis).
Response of AHAS to Herbicides
The activity of AHAS extracted from HRT1 and M82 tomato lines in the presence of imidazolinone or sulfonylurea herbicides was determined in vitro using crude enzyme extracts. AHAS enzyme extracted from the parental tomato line M82 was considerably more sensitive to imazapic than that extracted from HRT1 (Figure 2A). The activity of the M82 enzyme was already significantly decreased at 1 µM imazapic, a concentration that did not affect the AHAS extracted from HRT1; complete inhibition of the M82 enzyme was obtained at 10 µM, whereas the HRT1 enzyme was still active at 100 µM; LD50 for M82 was 0.47 µM compared with 3.31 µM for HRT1. M82 AHAS was also more sensitive to imazapyr than the HRT1 enzyme (Figure 2B); LD50 for M82 was 2.52 µM compared with 6.65 µM for HRT1. On the other hand, AHAS enzymes of both lines were extremely sensitive to the sulfonylurea herbicides rimsulfuron and sulfosulfuron (Figure 2C and 2D). LD50 values were 0.02 and 0.04 µM for rimsulfuron and 0.0008 and 0.0018 µM for sulfosulfuron, for M82 and HRT1, respectively. The HRT1 resistance to the imidazolinone group herbicides was thought to be due to a change in the herbicide’s target site on the AHAS protein. Interestingly, at all rates of imidazolinones, AHAS of HRT1 retained its activity at 25% to 45% of the control, indicating that only one of the three AHAS enzymes had become resistant. Resistance caused by point mutations in the AHAS gene may be specific to imidazolinone herbicides, to sulfonylurea herbicides, or to a broad spectrum of AHAS inhibitors (McCourt et al. Reference McCourt, Pang, King-Scott, Guddat and Duggleby2006). For example, substitutions of Pro-197 usually provide resistance to sulfonylurea but not imidazolinones, whereas substitutions of Ala-122 result in imidazolinone but not sulfonylurea resistance. In many cases, alterations of Ala-205 to Val-205 have been reported to provide resistance to both groups of herbicides (Saari et al. Reference Saari, Cotterman, Thill, Powles and Holtum2018; Tranel and Wright Reference Tranel and Wright2002). Both the sulfonylureas and imidazolinones inhibit the enzyme by binding within and obstructing the channel leading to the active site. However, only 10 amino acid residues are involved in the binding of both sulfonylureas and imidazolinones. The other residues interact only with sulfonylureas or only with imidazolinones. Thus, the binding sites of the two classes of herbicides only partially overlap (McCourt et al. Reference McCourt, Pang, King-Scott, Guddat and Duggleby2006). Unfortunately, we did not find any resistance of the HRT1 mutant to sulfonylurea, aside from partial resistance to foramsulfuron (Equip®, 22.5 g ai L−1, Bayer AG, Leverkusen, Germany, www.bayer.com/) (Dor et al. Reference Dor, Smirnov, Galili, Guy and Hershenhorn2016).
Figure 2. Influence of imazapic (A), imazapyr (B), rimsulfuron (C), and sulfosulfuron (D) on AHAS activity of M82 and HRT1 tomato plants. Data were computed by nonlinear regression using Sigma-Plot v. 11.01. (A) Y = y0
+
${{a}\over{{1 + {{\left( {{{x}\over{{x0}}} \right)}^b}}}}\;$
; for M82: y
o = 2.3, a = 100, x
0 = 0.47, b = 1.24, R
2 = 0.98, P < 0.0001; for HRT1: y
o = −2.05, a = 100, x
0 = 3.31, b = 0.74, R
2 = 0.99, P < 0.0001. (B) Y =
${{a}\over{{1 + {{\left( {{x}\over{{x0}}} \right)}^b}}}}$
; for M82: a = 100, x
0 = 2.52, b = 0.97, R
2 = 0.99, P < 0.0001; for HRT1: a = 100, x
0 = 6.55, b = 0.48, R
2 = 0.96, P < 0.0001. (C) Y =
${{a}\over{{1 + {{\left( {{{x}\over{{x0}}}} \right)}^b}}}}$
; for M82: a = 100, x
0 = 0.02, b = 1.3, R
2 = 0.99, P < 0.0001; for HRT1: a = 100, x
0 = 0.04, b = 0.62, R
2 = 0.95, P < 0.0001. (D) Y = y
o +
${{{100}}\over{{1 + {{\left( {{{x}\over{{x0}}}} \right)}^b}}}}$
; for M82: y
o = 1.72, a = 100, x
0 = 0.0008, b = 0.99, R
2 = 0.98, P < 0.0001; for HRT1: y
o = 0.1, a = 100, x
0 = 0.018, b = 1.14, R
2 = 0.99, P < 0.0001.
Influence of Imazapic on Contents of Total and Branched-Chain Amino Acids in Plant Tissues
Three weeks after being sprayed with imazapic, total amino acid content was significantly reduced in leaves of M82, from 2,984 nM g−1 to 1,964 nM g−1, but not in leaves of HRT1 (Figure 3A). In a previous study, imazapic significantly reduced total free amino acids in Phelipanche aegyptiaca (Pers.) Pomel. plants attached to HRT1 plants, but not in the roots of the HRT1 plants (Dor et al. Reference Dor, Galili, Smirnov, Hacham, Amir and Hershenhorn2017). A reduction in total free amino acids was also obtained in the leaves and roots of imazethapyr-treated pea (Pisum sativum L.) (Zabalza et al. Reference Zabalza, Zulet, Gil-Monreal, Igal and Royuela2013), and in canola treated with ZJo273 (a novel AHAS inhibitor) (Tian et al. Reference Tian, Jin, Ali, Guo, Liu, Zhang, Zhang, He and Zhou2014). Similar observations were made for the total content of branched-chain amino acids (Figure 3B) and for isoleucine (Figure 3C) and valine (Figure 3D) content: following imazapic treatment, total branched-chain amino acids in leaves of M82 plants were significantly reduced from 344 to 258 nM g−1; isoleucine content was significantly reduced from 102 to 68 nM g−1, and valine content was significantly reduced from 106 to 78 nM g−1 (Figure 3B–D). A small, nonsignificant reduction (from 136 to 111 nm g−1) was also obtained for leucine content in the leaves of M82 plants after imazapic treatment (data not shown). In HRT1 leaves, there were no significant differences in the total content of branched-chain amino acids (Figure 3B), or in the leucine (data not shown), isoleucine, or valine content (Figure 3C and 3D). A nonsignificant loss in total content of amino acids (2,792 nM g−1 compared with 3,698 nM g−1 in the control) (Figure 3A) was observed. Branched-chain amino acids also were decreased in AHAS inhibitor–treated pea (Ray Reference Ray1984), maize (Anderson and Hibberd Reference Anderson and Hibberd1985), and other plants (Zhou et al. Reference Zhou, Liu, Zhang and Liu2007). In addition, the levels of free leucine, isoleucine, and valine were significantly decreased in imazethapyr-treated chickpea lines sensitive to this herbicide, but not in resistant lines (Prakash et al. Reference Prakash, Singh, Chauhan, Sharma, Bharadwaj, Hegde, Jain, Gaur and Tripathi2017).
Figure 3. Influence of imazapic treatment on the amino acid content in M82 and HRT1 tomato plant leaves. M82 and HRT1 plants were sprayed with imazapic at a rate of 14.4 g ai ha−1. After 3 wk, leaf samples of treated and nontreated plants were taken for analysis of total amino acids (A), total branched-chain amino acids (B), isoleucine (C), and valine (D). Vertical lines present standard error of the mean (SEM); different letters indicate significant differences between control and imazapic-treated plants of the same line according to Student’s t-test.
Segregation Analysis
Mutation inheritance is important for breeding programs in which the resistance is to be introduced into elite cultivars. Segregation analysis indicated that 16 (21%) out of 75 and 21 (28%) out of 74 F2 (M82 × HRT1) plants were resistant to imazapic at a rate of 24 g ai ha−1 under greenhouse and field conditions, respectively. The resistance segregated 1:3 in the progeny (χ2 = 0.46, P = 0.5), indicating that resistance to imidazolinones in line HRT1 is due to a single recessive gene. Although AHAS resistance segregates as a single semi-dominant allele in many plant species, such as chickpea (Thompson and Tar’an Reference Thompson and Tar’an2014), canola (Swanson et al. Reference Swanson, Coumans, Brown, Patel and Beversdorf1988), soybean (Sebastian et al. Reference Sebastian, Fader, Ulrich, Forney and Chalef1989), sunflower (Sala et al. Reference Sala, Bulos and Echarte2008), wheat (Pozniak and Hucl Reference Pozniak and Hucl2004), sorghum [Sorghum bicolor (L.) Moench] (Tesso et al. Reference Tesso, Kershner, Ochanda, Al-Khatib and Tuinstra2011), and maize (Harms et al. Reference Harms, Montoya, Privalle and Briggs1990; Newhouse et al. Reference Newhouse, Singh, Shaner and Stidham1991), in other soybean mutants, it segregates, as in our case, as as a single recessive gene (Sebastian and Chaleff Reference Sebastian and Chaleff1987). Recessive inheritance is advantageous when transferring the resistance trait to target plants, because it is very easy to screen for this trait, and all resistant plants are homozygous. In contrast, dominant inheritance has the advantage of producing inbred seeds, because the trait only needs to be passed on to one of the hybrid parents.
Mutation Changes AHAS1 Structure
The ligand–protein contact server (LPC software; Sobolev et al. Reference Sobolev, Eyal, Gerzon, Potapov, Babor, Prilusky and Edelman2005) analyzes and visualizes atomic interactions within a protein or protein complex, providing characteristics for every atom–atom contact (atom properties, distance, and contact area). We used this software to examine the microenvironment of the mutated residues. The model of Arabidopsis AHAS1 in complex with the imidazolinone imazaquin (IQ) revealed that IQ blocks the active channel of the enzyme formed by the interface of two catalytic monomers (Figure 4A). This corresponds well with work done by McCourt et al. (Reference McCourt, Pang, King-Scott, Guddat and Duggleby2006), who presented the first 3D structure of Arabidopsis thaliana AHAS in complex with the imidazolinone IQ. Ligand–protein contact analysis showed that the closest distance from Ala-205 to IQ is between the hydrophobic CB atom of Ala-205 and the hydrophilic OC atom of IQ (Figure 4B). This distance is large, and the repulsion is very small. Replacement of Ala-205 by the larger hydrophobic residue Val-205 results in a closer distance to the hydrophilic OC atom of IQ, thereby increasing repulsion and making herbicide binding more difficult (Figure 4). This orientation of Val-205 is due to repulsion from the hydrophilic atom of Thr-203. In this orientation, the distance between the hydrophobic C atom of Val-205 and the hydrophilic O atom of IQ is 3.6 Å. This is in agreement with Jain and Tar’an (Reference Jain and Tar’an2014), who proposed that the presence of Ala-205 in the active site allows for imazamox binding, whereas the presence of valine at the same position disrupts this binding. Moreover, in that study, partially hydrophobic cluster analysis showed that the presence of the more hydrophobic residue (valine) instead of alanine results in a conformational change at the protein interface, modifying the herbicide-binding site (Jain and Tar’an Reference Jain and Tar’an2014). Thus, the enzyme becomes inaccessible to imidazolinone herbicides, and branched-chain amino acid starvation is prevented (Figure 2 A and B).
Figure 4. Protein–ligand complex of AHAS1 from Arabidopsis with imazaquin (IQ; PDB entry1Z8N). (A) Two molecules of IQ (purple) block the active channels in the AHAS protein dimer (yellow and green). (B) IQ molecule interaction with amino acid residues of the enzyme. Purple, IQ; blue, valine in position 205; red – Thr-203. The orientation of valine is due to repulsion from the hydrophilic O atom of Thr-203. In this orientation, the distance between the hydrophobic C atom of the valine and hydrophilic atom O of IQ is 3.6 Å.
Funding statement
This research was funded by the chief scientist of the Ministry of Agriculture (“Integrated Broomrape Management Approach in Agricultural Crops” project, 2010).
Competing interests
The authors declare no conflicts of interest.
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