Atrazine Resistance Research Articles

Low frequency paternal transmission of plastid genes in Brassicaceae.

Plastid-encoded genes are maternally inherited in most plant species. Transgenes located on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 × 10(-5). We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants. A frequency of <2.6 × 10(-5) was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology.

Effect of silencing lycB gene on the carotenoid synthesis in Haematococcus pluvialis

Haematococcus pluvialis is a freshwater planktonic single-cell microalgae. It will accumulate high amount of carotenoids under unfavorable environmental conditions. As one of carotenoids, lycopene is an important intermediate in the carotenoid biosynthesis pathway. Lycopene β-cyclase (LycB) is the key enzyme that catalyzes the circularization of lycopene to form β-carotene. In this study, we constructed a p1301-BS-RNAi vector using lycB from Dunaliella salina as the interference sequence with kanamycin and atrazine resistance marker, and then transformed it into H. pluvialis by electroporation. Sixteen independent transgenic lines were obtained after resistance selection, genome PCR, and RT-PCR analyses. Seven well-grown lines were selected to determine the contents of carotenoids by HPLC analysis after inducing by high light. The lycopene content in these lines was increased by 99.4% while the β-carotene content was decreased by 48.4%, indicating that the interference by heterogenous lycB could inhibit the conversion of lycopene into β-carotene. The amount of increase in lycopene was only 5% of the amount of decrease in β-carotene, indicating that 95% of the decreased β-carotene was converted into other metabolites. Therefore, in order to largely increase the lycopene content in H. pluvialis, it is necessary to coordinatively regulate other metabolic pathways.

Multiple Resistance to Herbicides from Four Site-of-Action Groups in Waterhemp (Amaranthus tuberculatus)

In 2006 and 2007, farmers from two counties in Illinois reported failure to control waterhemp with glyphosate. Subsequent onsite field experiments revealed that the populations might be resistant to multiple herbicides. Greenhouse experiments therefore were conducted to confirm glyphosate resistance, and to test for multiple resistance to other herbicides, including atrazine, acifluorfen, lactofen, and imazamox. In glyphosate dose-response experiments, both populations responded similarly to a previously characterized glyphosate-resistant population (MO1). Both Illinois populations also demonstrated high frequencies of resistance to the acetolactate synthase (ALS) inhibitor, imazamox. Additionally, one of the populations demonstrated high frequencies of resistance to both atrazine and the protoporphyrinogen oxidase (PPO) inhibitor, lactofen. Furthermore, using combinations of sequential and tank-mix herbicide applications, individual plants resistant to herbicides spanning all four site-of-action groups were identified from one population. Molecular experiments were performed to provide an initial characterization of the resistance mechanisms and to provide confirmation of the presence of multiple resistance traits within the two populations. Both populations contained the W574L ALS mutation and the ΔG210 PPO mutation, previously shown to confer resistance to ALS and PPO inhibitors, respectively. Atrazine resistance in both populations is suspected to be metabolism-based, because a triazine target-site mutation was not identified. A P106S EPSPS mutation, previously reported to confer glyphosate resistance, was identified in one population. This mutation was identified in both resistant and sensitive plants from the population; however, and so more research is needed to determine the glyphosate-resistance mechanism(s). This is the first known case of a weed population in the United States possessing multiple resistance to herbicides from four site-of-action groups.

Distinct Detoxification Mechanisms Confer Resistance to Mesotrione and Atrazine in a Population of Waterhemp

Previous research reported the first case of resistance to mesotrione and other 4-hydroxyphenylpyruvate dioxygenase (HPPD) herbicides in a waterhemp (Amaranthus tuberculatus) population designated MCR (for McLean County mesotrione- and atrazine-resistant). Herein, experiments were conducted to determine if target site or nontarget site mechanisms confer mesotrione resistance in MCR. Additionally, the basis for atrazine resistance was investigated in MCR and an atrazine-resistant but mesotrione-sensitive population (ACR for Adams County mesotrione-sensitive but atrazine-resistant). A standard sensitive population (WCS for Wayne County herbicide-sensitive) was also used for comparison. Mesotrione resistance was not due to an alteration in HPPD sequence, HPPD expression, or reduced herbicide absorption. Metabolism studies using whole plants and excised leaves revealed that the time for 50% of absorbed mesotrione to degrade in MCR was significantly shorter than in ACR and WCS, which correlated with previous phenotypic responses to mesotrione and the quantity of the metabolite 4-hydroxy-mesotrione in excised leaves. The cytochrome P450 monooxygenase inhibitors malathion and tetcyclacis significantly reduced mesotrione metabolism in MCR and corn (Zea mays) excised leaves but not in ACR. Furthermore, malathion increased mesotrione activity in MCR seedlings in greenhouse studies. These results indicate that enhanced oxidative metabolism contributes significantly to mesotrione resistance in MCR. Sequence analysis of atrazine-resistant (MCR and ACR) and atrazine-sensitive (WCS) waterhemp populations detected no differences in the psbA gene. The times for 50% of absorbed atrazine to degrade in corn, MCR, and ACR leaves were shorter than in WCS, and a polar metabolite of atrazine was detected in corn, MCR, and ACR that cochromatographed with a synthetic atrazine-glutathione conjugate. Thus, elevated rates of metabolism via distinct detoxification mechanisms contribute to mesotrione and atrazine resistance within the MCR population.

Synergistic Effects of Atrazine and Mesotrione on Susceptible and Resistant Wild Radish (Raphanus raphanistrum) Populations and the Potential for Overcoming Resistance to Triazine Herbicides

The synergistic interaction between mesotrione, a hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide, and atrazine, a photosystem II (PS II)-inhibiting herbicide, has been identified in the control of several weed species. A series of dose–response studies examined the synergistic effect of these herbicides on a susceptible (S) wild radish population. The potential for this interaction to overcome target-sitepsbA gene-based atrazine resistance in a resistant (R) wild radish population was also investigated. Control of S wild radish with atrazine was enhanced by up to 40% when low rates (1.0 to 1.5 g ha−1) of mesotrione were applied in combination. This synergistic response was demonstrated across a range of atrazine–mesotrione rate combinations on this S wild radish population. Further, the efficacy of 1.5 g ha−1mesotrione increased control of the R population by a further 60% when applied in combination with 400 g ha−1of atrazine. This result clearly demonstrated the synergistic interaction of these herbicides in overcoming the target-site resistance mechanism. The mechanism responsible for the observed synergistic interaction between mesotrione and atrazine remains unknown. However, it is speculated that an alternate atrazine binding site may be responsible. Regardless of the biochemical nature of this interaction, evidence from whole-plant bioassays clearly demonstrated that synergistic herbicide combinations improve herbicide efficiency, with lower application rates required to control weed populations. This, combined with the potential to overcomepsbA gene-based triazine resistance, and, thereby, regain the use of these herbicides, will result in more sustainable herbicide use.

Conserved structure of the chloroplast-DNA encoded D1 protein is essential for effective photoprotection via non-photochemical thermal dissipation in higher plants

Naturally selected atrazine-resistant (AR) weeds possessing a Ser(264) --> Gly D1 protein encoded by a mutant psbA allele in the chloroplast-DNA have increased photosensitivity and lower fitness. The D1 mutant lines of S. nigrum revealed impaired regulation of photosystem II (PSII) activity as compared with the wild-type plants resulting in a less effective photochemical light utilization and in addition, a lower capacity of non-photochemical thermal dissipation (NPQ), one of the main photoprotective mechanisms in oxygenic photosynthetic organisms. In this work, comparative chlorophyll fluorescence analysis in attached leaves of wild-type and AR Solanum nigrum L. and in their reciprocal crosses has been used to establish how the lower NPQ is inherited. Both a 50% reduction in steady-state NPQ and a 60-70% reduction in the rapidly reversible, energy-dependent (qE) component of NPQ were common phenomena in the parent and hybrid lines of D1 mutant S. nigrum. The nuclear hybrid status of the F2 plant material was confirmed by morphological observations on fully developed leaves. No alteration was found in the nucleotide sequence and the deduced amino acid sequences of the nuclear psbS gene isolated from different biotypes of S. nigrum, and there were no differences in the expressions of both the PsbS and the D1 proteins. All things considered, co-inheritance of the lower photoprotective NPQ capacity and the Ser(264) --> Gly D1 protein mutation was clearly observed, suggesting that the evolutionarily conserved D1 structure must be indispensable for the efficient NPQ process in higher plants.

Molecular characterization of atrazine resistance in common ragweed (Ambrosia artemisiifolia L.)

Common ragweed (Ambrosia artemisiifolia L.) is the most frequent weed in the Carpathian Basin and is spreading fast in other parts of Europe. In recent years, besides the wild type, a mutant genotype resistant to atrazine herbicides has evolved and is now widespread in many areas. The present study demonstrates that the atrazine resistance of ragweed is maternally inherited, and is caused by a point mutation in the psbA chloroplast gene. The promoter 5'-untranslated region and the open reading frame regions of the gene were analysed, and a homology search was performed. Both the atrazine-resistant and susceptible types of cpDNA were present in atrazine-resistant plants, while the mixed presence of both genotypes in the same plant, known as heteroplasmy, was not unequivocally detectable in susceptible plants.

Interactions of Mesotrione and Atrazine in Two Weed Species with Different Mechanisms for Atrazine Resistance

The joint activity of mesotrione and atrazine can display synergistic effects on the control of both triazine-sensitive and site-of-action-based triazine-resistant (TR) broadleaf weeds. The first objective of this study was to evaluate a PRE application of atrazine followed by a POST application of mesotrione for potential interactions in both site-of-action-based TR redroot pigweed and metabolism-based atrazine-resistant (AR) velvetleaf. Results from these sequential experiments demonstrated that synergism was detected in reducing biomass of the TR redroot pigweed but not in the AR velvetleaf with metabolism-based resistance. The second objective was to evaluate the joint activity of mesotrione and atrazine in a tank-mix application in the AR velvetleaf biotype. Greenhouse studies with the AR biotype indicated that synergism resulted from a tank mix with a constant mesotrione rate of 3.2 g ai ha−1in mixture with atrazine ranging from 126 to 13,440 g ai ha−1. Chlorophyll fluorescence imaging also revealed a synergistic interaction on the AR biotype when 3.2 g ha−1of mesotrione was applied with 126 g ha−1of atrazine beginning 36 h after treatment and persisting through 72 h.

Physiological and Molecular Characterization of Atrazine Resistance in a Wild Radish (Raphanus raphanistrum) Population

This study documents the physiology and genetics of evolved atrazine resistance in a wild radish population from Western Australia. Plant response to atrazine treatment confirmed a high level of resistance in population WARR5. At 0.25 kg atrazine/ha, all plants from a susceptible population were killed, whereas resistant WARR5 was unaffected at the highest dose tested (4 kg atrazine/ha). Leaf photosynthesis in susceptible plants was inhibited after 1 kg atrazine/ha treatment, whereas leaf photosynthesis in WARR5 plants was unaffected. Furthermore, atrazine resistance was maternally inherited. Sequencing of apsbAgene fragment in resistant WARR5 and susceptible plants revealed a single point mutation resulting in a coding change from Ser264to Gly of the D1 protein in resistant plants. We are confident that this mutation is the basis of resistance to the photosystem II inhibitors in this wild radish population.

Morphophysiological traits and atrazine sensitivity in Chenopodium album L.

A Chenopodium album L. biotype surviving in atrazine-treated Serbian corn fields (VC) was compared against atrazine-susceptible (S) and atrazine-resistant (R) standards. Atrazine (2 kg ha(-1)) killed S and VC shoot biomass 15 days after treatment (DAT), but R was only suppressed by 42% and survived 8 kg ha(-1). Atrazine at 2 kg ha(-1) only inhibited VC height by 60% as against 100 and 0% for S and R respectively. Chlorophyll fluorescence (Fv/Fm) and transpiration were insensitive to atrazine in R, but were inhibited by 90 and 100% in S and by 50 and 60% in VC respectively. Decline of Fv/Fm after 2 kg ha(-1) atrazine was stabilized at 3 DAT for the VC biotype. A toxicity mitigation mechanism could have facilitated VC survival in an atrazine-treated field. Further knowledge on this mechanism is needed to establish if surviving VC plants are indicators of atrazine resistance evolution in these Serbian corn fields. Variables related to foliar function provided better detection of weed mechanisms to survive herbicide action than the usual shoot biomass measurements.

A Ser–Gly substitution in plastid-encoded photosystem II D1 protein is responsible for atrazine resistance in foxtail millet (Setaria italica)

A foxtail millet (Setaria italica L. Beauv.) line resistant to atrazine was obtained through interspecific hybridization between wild S. viridis L. Beauv. and cultivated S. italica. The resistance was proved to be controlled by a chloroplast-inherited gene and it has further been utilized in foxtail millet production. However, the sequence information of the putative atrazine resistance gene, psbA in foxtail millet’s chloroplast genome encoding photosystem II D1 protein (32 kDa thylakoid membrane protein) (photosystem QB protein) and the mutation site responsible for the resistance are not known. In this paper the psbA sequences of six atrazine susceptible/resistant foxtail millet varieties were obtained and compared. The results indicated that there was only one amino acid difference between susceptible and resistance gene, resulting from a single base substitution. It was concluded that a mutant allele of photosystem II protein D1 encoding a Gly residue instead of a Ser residue at position 264 is a major gene of resistance to atrazine. Moreover, the phylogenetic tree based on the psbA coding region of thirty-five plant species was carried out. The phylogenetic relationship between S. italica and other plants and the related evolutionary issues were discussed and it was suggested that psbA sequences could be used in phylogenetic studies in plants.

Altered photosynthetic performance of a natural Arabidopsis accession is associated with atrazine resistance

Natural variation for photosynthetic traits was studied by determining chlorophyll fluorescence parameters in a collection of Arabidopsis accessions. This screen revealed only one single accession (Ely), exhibiting photosynthetic characteristics markedly different from all others, while a few lines showed small but significant variation. Detailed genetic and physiological analyses showed reduced fitness for Ely compared with the standard laboratory strain Ler for various growth parameters. At low temperature (15 degrees C), Ely had a higher electron transport rate than Ler, indicating increased photosystem II efficiency under this condition, while at high temperature (30 degrees C) the opposite was observed. Ely had a high sensitivity to UV-B radiation compared with Ler and was atrazine resistant. This atrazine-resistance and related chlorophyll fluorescence traits were maternally inherited, pointing towards chloroplast-located gene(s). Definite proof that Ely is atrazine-resistant was obtained by sequencing the psbA gene, encoding the D1 protein of photosystem II, revealing a point mutation causing the same amino acid change as found in other atrazine-resistant species. Additional nuclear encoded genetic variation was also present, as was concluded from the small but significant differences in phenotype between Ely and its reciprocal crosses with Ler. It was concluded that the photosynthetic yield is highly conserved and that only severe selection pressure results in marked variations in photosynthetic performance.

Cross-resistance and herbicide metabolism in grass weeds in Europe: biochemical and physiological aspects

In Europe, 18 weedy grass species had been confirmed to have biotypes with resistance to herbicides. The most frequent is that of atrazine resistance, with nine resistant biotypes found. These biotypes are mainly resistant because of changes in the D1 protein of photosystem II. All atrazine-resistant biotypes, except that of bristly foxtail, show cross-resistance to s-triazine and as-triazines. From an agriculture point of view, the most important cases of resistance are those found in blackgrass, wild oat, Italian ryegrass, rigid ryegrass, and barnyardgrass. In these species, cross- and multiple resistances were observed due to metabolism or changes in the target protein by genetic mutations or both. These biotypes are extremely difficult to control with alternative herbicides.

Low frequency transmission of a plastid-encoded trait in Setaria italica.

It has been claimed that engineering traits into the chloroplast will prevent transgene transmission by pollen, precluding transgene flow from crops. A Setaria italica (foxtail or birdseed millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant red-leaf base marker) was crossed with five male-sterile yellow- or green-leafed herbicide susceptible lines. Chloroplast-inherited resistance was consistently pollen transmitted at a 3x10(-4 )frequency in >780,000 hybrid offspring. The nuclear marker segregated in the F(2), but resistance did not segregate, as expected. Pollen transmission of plastome traits can only be detected using both large samples and selectable genetic markers. The risk of pollen transmission at this frequency would be several orders of magnitude greater than spontaneous nuclear-genome mutation-rates. Chloroplast transformation may be an unacceptable means of preventing transgene outflow, unless stacked with additional mechanisms such as mitigating genes and/or male sterility.

Atrazine resistance entails a limited xanthophyll cycle activity, a lower PSII efficiency and an altered pattern of excess excitation dissipation.

Atrazine-resistant (AR) weeds have a modified D1 protein structure, with a Ser264-->Gly mutation on the D1 protein, near the plastoquinone binding niche. The photosynthetic performance, the light response of the xanthophyll cycle and chlorophyll fluorescence quenching-related parameters were compared in attached leaves of susceptible (S) and AR biotypes of the C3 dicot Chenopodium album L., Epilobium adenocaulon Hausskn., Erigeron canadensis L., Senecio vulgaris L. and Solanum nigrum L. and the C4 dicot Amaranthus retroflexus L. grown under natural high-light conditions. No significant difference in CO2 assimilation rate per leaf area unit was found between the S and AR biotypes of the investigated C3 plants, whereas the AR biotype of A. retroflexus exhibited a relatively poor photosynthetic performance. The D1 protein mutant plants expressed a reduced activity of light-stimulated zeaxanthin formation. Neither the lower violaxanthin de-epoxidase activity nor the depletion of ascorbate seems to be the cause of the lower in vivo zeaxanthin formation in the AR plants. All the D1 mutant weeds had limited light-induced non-photochemical (NPQ) and photochemical (qP) quenching capacities, and displayed a higher photosensitivity, as characterized by the ratio (1-qP)/NPQ and a higher susceptibility to photoinhibition. Analysis of the chlorophyll fluorescence parameters showed that a lower proportion of excitation energy was allocated to PSII photochemistry, while a higher excess of excitation remained in the AR weeds relative to the S plants.

A review of physiological and biochemical aspects of resistance to atrazine and paraquat in Hungarian weeds.

The most important results in the field of atrazine and paraquat resistance research by Hungarian researchers are reviewed. Pleiotropic effects accompanying atrazine resistance were investigated in atrazine-resistant (AR) and susceptible (S) biotypes of horseweed (Conyza canadensis (L) Cronq). No significant difference in carbon dioxide assimilation rate was found between the AR and S plants. The rates of the Hill reaction of the AR and S chloroplasts exhibited different temperature dependence. The thylakoid membrane lipids contained a lower amount of polar lipid and the fatty acid content exhibited a higher degree of unsaturation in the AR biotype. Photosynthetic apparatus of the AR biotype had better adaptive ability at low temperature and showed enhanced susceptibility to high-temperature stress. AR horseweed plants had reduced activity of xanthophyll cycle, limited capacity of light-induced non-photochemical and photochemical quenching, higher photosensitivity and susceptibility to photo-inhibition. In the case of paraquat resistance, horseweed found in Hungary exhibited a resistance factor of 450; the resistance is not based on an elevated level and activity of the antioxidant enzyme system. The suggested role of polyamines in the resistance mechanisms can be excluded. The higher putrescine and total polyamine content of paraquat-treated R leaves can be regarded as a general stress response rather than as a symptom of paraquat resistance. A paraquat-inducible, nuclear-coded protein, which presumably functions by carrying paraquat to the vacuole, is supposed to play a role in resistance.

Identification of Two Mechanisms of Atrazine Resistance in Setaria faberi and Setaria viridis Biotypes

Abstract Two resistant (R) biotypes of Setaria faberi and S. viridis have been detected in maize fields continuously treated with triazine herbicides. The R biotypes of S. faberi and S. viridis were 10.0 and 6.5 times, respectively, more resistant to atrazine than their susceptible (S) biotypes. Both R biotype plastids had less affinity for atrazine than the S biotypes based on chlorophyll fluorescence and Hill reaction assays. As expected, they exhibited cross-resistance to a wide variety of photosystem II inhibitors. The hierarchy of resistance level of these two biotypes was chloro- s -triazines ⪢ methoxy- s -triazines > methyltio- s -triazines ≥ as-triazines. There was no difference in the absorption and translocation of [ 14 C]atrazine in R and S biotypes. Fast fluorescence induction curves showed that atrazine inhibited whole-leaf photosynthesis in S biotypes and they later recovered slight fluorescence activity in both populations. All biotypes produced the same pattern of metabolites, but the R biotypes detoxified the atrazine to conjugate-atrazine at a higher rate than the S biotypes (R > 75% > S). The five wild-type Setaria spp. found in Spain showed differences in detoxification of atrazine. The hierarchy of detoxifying level of atrazine of these Setaria spp. was S. verticilata ≃ S. adherens ⪢ S. faberi ≃ S. viridis >⪢ S. glauca .

The Mae I assay for scoring atrazine resistance is codon-usage dependent in legumes

The Mae I assay for scoring atrazine resistance is codon-usage dependent in legumes

A simple procedure for polymerase chain reaction of the PSBA gene in algae: application to the screening of mutations conferring atrazine resistance and discrimination of natural populations of Porphyra linearis

A simplified procedure is described for polymerase chain reaction (PCR) of a partial sequence (bp 601–893) of the plastid gene psbA in the rhodophyte Porphyra linearis and the diatoms Haslea ostreria and Skeletonema costatum. This procedure involves the use of all tissues of P. linearis and live cell suspensions of H. ostreria or S. costatum as DNA templates, without any further purification of DNA. As in the case of PCR with DNA extracts, a single major band of the expected size (292 bp) was obtained after PCR for the three species. Sequences of the amplified fragments were aligned, confirming that the amplified products were part of the psbA gene. The method was then used to screen mutations in partial psbA genes of 23 samples of P. linearis collected at four different stations along the mid-Atlantic coast of France. An alignment was obtained indicating the existence of mutations, though not in codons known for herbicide resistance. All mutations found were silent. However, genetic polymorphism discriminated between samples collected from two stations. The method employed allows rapid amplification of the herbicide target gene and simplifies the procedure for screening mutations or populations in algae. Its application to other genes and species is considered.

Short-Term Responses of Photosystem II to Heat Stress in Cold-Acclimated Atrazine-Resistant and Susceptible Biotypes of Erigeron canadensis (L.)

When leaves of atrazine-resistant (AR) and atrazine-sensitive (S) Erigeron canadensis (L.) plants grown at 5 °C were exposed to an elevated temperature (35 °C) for 30 min, the critical (Tc) and peak temperatures (Tp) of the F0 vs. T curves were considerably higher for the leaves of the S biotype, but not for those of the AR biotype. The temperature dependences of Fv/Fm and ΔF/Fm′ were not greatly different for the heat-treated cold-acclimated AR biotype, in contrast with the situation for the S plants. This short-term heat treatment resulted in a more significant shift in the optimal thermal interval of CO2 fixation for the S than for the AR biotypes