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Commelina communis L. is a troublesome weed in agronomic fields and increasingly threatens the yield security of corn in north-eastern China. Previously, we found that a C. communis population (JL-1) has evolved resistance to atrazine. Although the potential genetic and enzymic differences contributing to atrazine resistance in this population have been investigated, the specific molecular mechanisms underlying C. communis resistance are still poorly understood. Here, the expression level of the target gene PsbA and the non-target-site resistance (NTSR) mechanism for this population were studied. The results showed that the decline in chlorophyll content in JL-1 leaves was less than in the susceptible JS-10 population following atrazine treatment. JL-1 exhibited an enhanced expression of the PsbA gene compared with JS-10 of 7.28- and 14.28-fold higher at 0 and 24 h after treatment with atrazine, respectively. The cytochrome P450 monooxygenase (P450) inhibitor piperonyl butoxide (PBO) increased the phytotoxicity of atrazine in both populations of C. communis. Seven candidate genes associated with NTSR of Jl-1 were identified through RNA-seq and validated by quantitative real-time PCR, including 5 upregulated genes involved in herbicide metabolism. In addition, the activities of glutathione S-transferases and P450s in JL-1 were increased compared with JS-10. Collectively, PsbA gene overexpression and enhanced metabolism are likely to be responsible for JL-1 resistance to atrazine.

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Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.

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Quercus (oak) is an important economic and ecological tree species in the world, and it is the necessary feed for oak silkworm feeding. Chloroplasts play an important role in green plants but the codon usage of oak chloroplast genomes is not fully studied. We examined the codon usage of the oak chloroplast genomes in detail to facilitate the understanding of their biology and evolution. We downloaded all the protein coding genes of 26 non-redundant chloroplast reference genomes, removed short ones and those containing internal stop codons, and finally retained 50 genes shared by all genomes for comparative analyses. The base composition, codon bias, and codon preference are not significantly different between genomes but are significantly different among genes within these genomes. Oak chloroplast genomes prefer T/A-ending codons and avoid C/G-ending codons, and the psbA gene has the same preference except for the codons encoding amino acid Phe. Complex factors such as context-dependent mutations are the major factors affecting codon usage in these genomes, while selection plays an important role on the psbA gene. Our study provided an important understanding of codon usage in the oak chloroplast genomes and found that the psbA gene has nearly the same codon usage preference as other genes in the oak chloroplasts.

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Redroot amaranth (Amaranthus retroflexus L.) is a noxious weed that affects soybean production in China. Experiments were conducted to determine the molecular basis of resistance to bentazone. Whole-plant dose-response experiments showed that two populations (R1 and R2) exhibited resistance to bentazone with resistance indices of 9.01 and 6.85, respectively. Sequencing of the psbA gene revealed no amino acid substitution in the two populations. qRT-PCR analysis verified that psbA gene expression in R1 and R2 populations was increased significantly after treatment with bentazone, which was 3-fold and 5-fold higher than that in S1 and S2 populations, respectively. The P450 inhibitor malathion significantly reduced the level of resistance in the R1 and R2 populations when used prior to bentazone treatment. The R1 population exhibited multiple resistance to thifensulfuron-methyl and lactofen, caused by target site mutations (Asp-376-Glu in ALS, Arg-128-Gly in PPO2). In conclusion, increased gene expression of the psbA gene and enhanced herbicide metabolism seem to be the basis of resistance to bentazone in these A. retroflexus populations.

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Asiatic dayflower (Commelina communis L.) is a detrimental weed that mainly infests corn and soybean fields in China. Recently, some C. communis populations have exhibited resistance to atrazine, intensifying the difficulties in controlling the weed. However, little is known on the mechanism underlying C. communis resistance to atrazine. Therefore, two populations collected from Jilin (JL-1) and Jiangsu (JS-10) provinces of China were used to evaluate their growth responses to atrazine. The results showed that the JL-1 population displayed a low level of resistance to atrazine compared with JS-10 population, with the resistant index (RI) value of 2.9. To determine if a mutation in the psbA gene was the basis for varied resistance to this herbicide, the full-length gene encoding 353 amino acids with no intron was sequenced by using genome-walking techniques. No mutation known to confer resistance to atrazine was observed in either JL-1 or JS-10 populations. The malondialdehyde (MDA) contents relative to the control group were significantly higher in JS-10 population than in JL-1 population at 7 days after treatment with atrazine, suggesting that atrazine induced severer oxidant damage on JS-10 population. Additionally, significantly enhanced activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX), were detected in the JL-1 population, which was most likely to confer resistance to atrazine. To the best of our knowledge, this is the first investigation into the potential genetic and enzymatic differences contributing to atrazine resistance in this population.

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A partial rbcL sequence from the type material of Spongites discoideus from southern Argentina showed that it was distinct from rbcL sequences of South African specimens to which that name had been applied based on morpho-anatomy. A partial rbcL sequence from an original syntype specimen, herein designated the lectotype, of Lithophyllum marlothii, type locality Camps Bay, Western Cape Province, South Africa, was identical to rbcL sequences of South African field-collected specimens assigned to S. discoideus. Based on phylogenetic analyses of rbcL and/or psbA sequences, both of these species belong in Pneophyllum and are transferred there as P. discoideum comb. nov. and P. marlothii comb. nov. The two species exhibit a distinct type of development where thick, secondary, monomerous disks are produced from thin, primary, dimerous crusts. Whether this type of development represents an example of convergent evolution or is characteristic of a clade of species within Pneophyllum remains to be resolved.

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There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to metribuzin in a wild radish population. Dose-response studies revealed a higher level of resistance to metribuzin in the resistant (R) compared to the susceptible (S) population. Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In addition, a higher level of [14C]-metribuzin metabolism and, consequently, a lower level of [14C] translocation were also detected in the R plants. These results demonstrated that both psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this wild radish population. Furthermore, this resistant population showed resistance to ALS-inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.

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BACKGROUND: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides. RESULTS: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides. CONCLUSION: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides. © 2018 Society of Chemical Industry.

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Pearl millet has been recommended beneficial for several therapeutic purposes. However, little is known of the physiological responses to abiotic stressors, especially of atrazine. In order to elucidate the physiological and molecular responses of pearl millet to atrazine stress, we studied the response of various biomarkers under increasing herbicide concentrations (0, 5, 10, and 50 mg/kg). We also quantified the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) (H2O2 and O2•-) produced in the leaves to evaluate the extent of oxidative damage. Increasing atrazine concentrations significantly increased ROS and MDA production in the plant leaves. Ascorbate peroxidase (APX) and peroxidase (POD) activities increased, while catalase (CAT) and superoxide dismutase activities reduced with increasing atrazine concentrations. Generally, atrazine applied at 50 mg/kg suppressed chlorophyll contents, whereas, chlorophyll (a/b) ratio was increased. Atrazine applied at 50 mg/kg significantly suppressed antioxidant gene expressions to the lowest. The APX gene showed overall low response to the atrazine treatments. The chloroplastic psbA gene showed highest expression with 10 mg/kg atrazine, whereas atrazine at 50 mg/kg significantly suppressed the gene expression to its lowest. Pearl millet was able to suppress oxidative stress under low atrazine levels, but high atrazine concentration could induce more oxidative damage.

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The psbA gene, which encodes a major photosystem II protein (protein II or D1), is a marker for the presence of phototrophic organisms in water communities. We have pioneered the use of this marker for studying the diversity of phototrophic microflora of freshwater invertebrates. The object of the study is the microbial associations accompanying the endemic Baikal sponge Baikalospongia intermedia and the surrounding aquatic microbial community. Analysis of the psbA gene sequences in the examined microbiomes demonstrates the presence of various phototrophic groups, such as Cyanobacteria, Chlorophyta, Heterokonta, Haptophyta, and Ochrophyta algae, as well as cyanophages. A total of 35 unique psbA gene sequences have been distinguished in the microbial communities of the endemic sponge B. intermedia and 32 unique sequences in the water community surrounding the sponge. These data demonstrate the involvement of sponge symbiotic communities in the accumulation of primary production and carbon cycle in the Lake Baikal ecosystem.

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Herbicide-resistance mutations may impose a fitness penalty in herbicide-free environments. Moreover, the fitness penalty associated with herbicide resistance is not a stable parameter and can be influenced by ecological factors. Here, we used two Brachypodium hybridum accessions collected from the same planted forest, sensitive (S) and target-site resistance (TSR) to photosystem II (PSII) inhibitors, to study the effect of agro-ecological parameters on fitness penalty. Both accessions were collected in the same habitat, thus, we can assume that the genetic variance between them is relatively low. This allow us to focus on the effect of PSII TSR on plant fitness. S plants grains were significantly larger than those of the TSR plants and this was associated with a higher rate of germination. Under low radiation, the TSR plants showed a significant fitness penalty relative to S plants. S plants exhibiting dominance when both types of plants were grown together in a low-light environment. In contrast to previous documented studies, under high-light environment our TSR accession didn't show any significant difference in fitness compared to the S accession. Nitrogen deficiency had significant effect on the R compared to the S accession and was demonstrated in significant yield reduction. TSR plants also expressed a high fitness penalty, relative to the S plants, when grown in competition with wheat plants. Two evolutionary scenarios can be suggested to explain the coexistence of both TSR and S plants in the same habitat. The application of PSII inhibitors may have created selective pressure toward TSR dominancy; termination of herbicide application gave an ecological advantage to S plants, creating changes in the composition of the seed bank. Alternatively, the high radiation intensities found in the Mediterranean-like climate may reduce the fitness penalty associated with TSR. Our results may suggest that by integrating non-herbicidal approaches into weed-management programs, we can reduce the agricultural costs associated with herbicide resistance.

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Calcium (Ca) has been reported to lessen oxidative damages in plants by upregulating the activities of antioxidant enzymes. However, atrazine mediated reactive oxygen species (ROS) reduction by Ca is limited. This study therefore investigated the effect of exogenously applied Ca on ROS, antioxidants activity and gene transcripts, the D1 protein (psbA gene), and chlorophyll contents in Pennisetum seedlings pre-treated with atrazine. Atrazine toxicity increased ROS production and enzyme activities (ascorbate peroxidase APX, peroxidase POD, Superoxide dismutase SOD, glutathione-S-transferase GST); but decreased antioxidants (APX, POD, and Cu/Zn SOD) and psbA gene transcripts. Atrazine also decreased the chlorophyll contents, but increased chlorophyll (a/b) ratio. Contrarily, Ca application to atrazine pre-treated seedlings lowered the harmful effects of atrazine by reducing ROS levels, but enhancing the accumulation of total chlorophyll contents. Ca-protected seedlings in the presence of atrazine manifested reduced APX and POD activity, whereas SOD and GST activity was further increased with Ca application. Antioxidant gene transcripts that were down-regulated by atrazine toxicity were up-regulated with the application of Ca. Calcium application also resulted in up-regulation of the D1 protein. In conclusion, ability of calcium to reverse atrazine-induced oxidative damage and calcium regulatory role on GST in Pennisetum was presented.

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BACKGROUND: Propanil-resistant (R) Cyperus difformis populations were recently confirmed in California rice fields. To date, propanil resistance in other weed species has been associated with enhanced aryl acylamidase (AAA)-mediated propanil conversion into 3,4-dichloroaniline. Our objectives were to determine the level of propanil resistance and cross-resistance to other PSII inhibitors in C. difformis lines, and to elucidate the mechanism of propanil resistance. RESULTS: The propanil-R line had a 14-fold propanil resistance and increased resistance to bromoxynil, diuron and metribuzin, but not to atrazine. The R line, however, displayed a fourfold increased susceptibility to bentazon. Interestingly, susceptible (S) plants accumulated more 3,4-dichloroaniline and were more injured by propanil and carbaryl (AAA-inhibitor) applications than R plants, suggesting that propanil metabolism is not the resistance mechanism. psbA gene sequence analysis indicated a valine-219-isoleucine (Val219 Ile) amino acid exchange in the propanil-R chloroplast D1 protein. CONCLUSION: The D1 Val219 Ile modification in C. difformis causes resistance to propanil, diuron, metribuzin and bromoxynil but increased susceptibility to bentazon, suggesting that the Val219 residue participates in binding of these herbicides. This is the first report of a higher plant exhibiting target-site propanil resistance. Tank mixing of bentazon and propanil, where permitted, can control both propanil-R and propanil-S C. difformis and prevent the spread of the resistant phenotype. © 2016 Society of Chemical Industry.

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BACKGROUND: Evolution of multiple herbicide resistance in weeds is a serious threat to weed management in crop production. Kochia is an economically important broadleaf weed in the U.S. Great Plains. This study aimed to confirm resistance to four sites of action of herbicides in a single kochia (Kochia scoparia L. Schrad.) population from a crop field near Garden City (GC), Kansas, and further determine the underlying mechanisms of resistance. RESULTS: One-fourth of the GC plants survived the labeled rate or higher of atrazine [photosystem II (PSII) inhibitor], and the surviving plants had the Ser-264 to Gly mutation in the psbA gene, the target site of atrazine. Results showed that 90% of GC plants survived the labeled rate of dicamba, a synthetic auxin. At least 87% of the plants survived up to 72 g a.i. ha(-1) of chlorsulfuron [acetolactate synthase (ALS) inhibitor], and analysis of the ALS gene revealed the presence of Pro-197 to Thr and/or Trp-574 to Lue mutation(s). Most GC plants also survived the labeled rate of glyphosate [5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor), and the resistant plants had 5-9 EPSPS gene copies (relative to the ALS gene). CONCLUSION: We confirm the first case of evolution of resistance to four herbicide sites of action (PSII, ALS and EPSPS inhibitors and synthetic auxins) in a single kochia population, and target-site-based mechanisms confer resistance to atrazine, glyphosate and chlorsulfuron.

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In this study, the effects of short-term aluminium toxicity and the application of spermidine on the lichen Xanthoria parietina were investigated at the physiological and transcriptional levels. Our results suggest that aluminium stress leads to physiological processes in a dose-dependent manner through differences in lipid peroxidation rate, chlorophyll content and glutathione reductase (EC 1.6.4.2) activity in aluminium and spermidine treated samples. The expression of the photosystem II D1 protein (psbA) gene was quantified using semi-quantitative RT-PCR. Increased glutathione reductase activity and psbA mRNA transcript levels were observed in the X. parietina thalli that were treated with spermidine before aluminium-stress. The results showed that the application of spermidine could mitigate aluminium-induced lipid peroxidation and chlorophyll degradation on lichen X. parietina thalli through an increase in psbA transcript levels and activity of glutathione reductase (GR) enzymes.

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BACKGROUND: Resistance of Chenopodium album to triazinones and triazines can be caused by two amino acid exchanges, serine-264-glycine (Ser(264) Gly) and alanine-251-valine (Ala(251) Val), in the chloroplast D1 protein. This paper describes the identification of a biotype with a leucine-218-valine (Leu(218) Val) switch found in German sugar beet fields with unsatisfactory weed control. A greenhouse experiment has been performed to compare the resistance profile of the newly identified biotype with biotypes that carry the Ser(264) Gly and Ala(251) Val mutations. RESULTS: Application rate-response curves obtained from the greenhouse experiment showed that the Leu(218) Val exchange induced significant resistance against the triazinones but not against terbuthylazine. The level of resistance against the triazinones was higher in the Ser(264) Gly and Ala(251) Val biotypes compared with the Leu(218) Val biotype. All biotypes tested were more resistant to metribuzin than to metamitron. Following terbuthylazine treatment, Ser264 Gly displayed a high level of resistance, Ala(251) Val showed moderate resistance. A PCR-RFLP assay for Ser(264) Gly has been extended to include detection of Ala251 Val and Leu(218) Val mutations. CONCLUSION: The D1 Leu(218) Val substitution in C. album confers significant resistance to triazinones. This suggests that Leu(218) Val is involved in the binding of triazinones. First establishment of the resistance profiles of the three psbA mutations suggests that these mutations have been independently selected.

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The Del1 mutant of the green alga Chlamydomonas reinhardtii with a defined deletion in the chloroplast encoded psbA gene is unable to grow photoautotrophically. We show here that this mutant can be transformed with PCR-generated psbA fragments of varying length to yield photosynthetically growing colonies. PCR fragments need not be purified but can be directly precipitated from the amplification reaction onto tungsten particles, allowing fast and efficient mutagenesis experiments. Flanking regions bordering the deletion breakpoints have been systematically shortened from both sides. The shortest fragment giving rise to significant numbers of transformants contains about 50 bp upstream and 120 bp downstream of the deletion breakpoint. This technique greatly simplifies comprehensive structure-function analyses of the D1 protein in Chlamydomonas, but could perhaps be adapted to other chloroplast genes in this or other organisms as well.