Molecular Characterization of Resistance to Nicosulfuron in Setaria viridis.

The green foxtail, Setaria viridis (L.) P. Beauv. (Poales: Poaceae), is a troublesome and widespread grass weed in China. The acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron has been intensively used to manage S. viridis, and this has substantially increased the selection pressure. Here we confirmed a 35.8-fold resistance to nicosulfuron in an S. viridis population (R376 population) from China and characterized the resistance mechanism. Molecular analyses revealed an Asp-376-Glu mutation of the ALS gene in the R376 population. The participation of metabolic resistance in the R376 population was proved by cytochrome P450 monooxygenases (P450) inhibitor pre-treatment and metabolism experiments. To further elucidate the mechanism of metabolic resistance, eighteen genes that could be related to the metabolism of nicosulfuron were obtained bythe RNA sequencing. The results of quantitative real-time PCR validation indicated that three ATP-binding cassette (ABC) transporters (ABE2, ABC15, and ABC15-2), four P450 (C76C2, CYOS, C78A5, and C81Q32), and two UDP-glucosyltransferase (UGT) (UGT13248 and UGT73C3), and one glutathione S-transferases (GST) (GST3) were the major candidates that contributed to metabolic nicosulfuron resistance in S. viridis. However, the specific role of these ten genes in metabolic resistance requires more research. Collectively, ALS gene mutations and enhanced metabolism may be responsible for the resistance of R376 to nicosulfuron.

EST-SSR Markers' Development Based on RNA-Sequencing and Their Application in Population Genetic Structure and Diversity Analysis of Eleusine indica in China.

Goosegrass (Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats (3591, 42.79%), followed by trinucleotides (3162, 37.68%). The most dominant mononucleotide and trinucleotide repeat motifs were A/T (3406, 40.59%) and AAT/ATT (103, 1.5%), respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (Na) ranging from two to eleven per locus, and the effective number of alleles (Ne) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.

Nitric oxide alleviates salt stress through protein S-nitrosylation and transcriptional regulation in tomato seedlings.

MAIN CONCLUSION: NO enhances the resistance of tomato seedlings to salt stress through protein S-nitrosylation and transcriptional regulation, which involves the regulation of MAPK signaling and carbohydrate metabolism. Nitric oxide (NO) regulates various physiological and biochemical processes and stress responses in plants. We found that S-nitrosoglutathione (GSNO) treatment significantly promoted the growth of tomato seedling under NaCl stress, indicating that NO plays a positive role in salt stress resistance. Moreover, GSNO pretreatment resulted in an increase of endogenous NO level, S-nitrosothiol (SNO) content, S-nitrosoglutathione reductase (GSNOR) activity and GSNOR expression under salt stress, implicating that S-nitrosylation might be involved in NO-alleviating salt stress. To further explore whether S-nitrosylation is a key molecular mechanism of NO-alleviating salt stress, the biotin-switch technique and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) were conducted. A total of 1054 putative S-nitrosylated proteins have been identified, which were mainly enriched in chloroplast, cytoplasm and mitochondrion. Among them, 15 and 22 S-nitrosylated proteins were involved in mitogen-activated protein kinase (MAPK) signal transduction and carbohydrate metabolism, respectively. In MAPK signaling, various S-nitrosylated proteins, SAM1, SAM3, SAM, PP2C and SnRK, were down-regulated and MAPK, MAPKK and MAPKK5 were up-regulated at the transcriptional level by GSNO treatment under salt stress compared to NaCl treatment alone. The GSNO pretreatment could reduce ethylene production and ABA content under NaCl stress. In addition, the activities of enzyme identified in carbohydrate metabolism, their expression at the transcriptional level and the metabolite content were up-regulated by GSNO supplication under salt stress, resulting in the activation of glycolysis and tricarboxylic acid cycle (TCA) cycles. Thus, these results demonstrated that NO might beneficially regulate MAPK signaling at transcriptional levels and activate carbohydrate metabolism at the post-translational and transcriptional level, protecting seedlings from energy deficiency and salinity, thereby alleviating salt stress-induced damage in tomato seedlings. It provides initial insights into the regulatory mechanisms of NO in response to salt stress.

Multiple resistance to ACCase- and ALS-inhibiting herbicides in black-grass (Alopecurus myosuroides Huds.) in China.

Two black-grass (Alopecurus myosuroides Huds.) populations (R2105 and R1027) that were suspected to be resistant to clodinafop-propargyl, an acetyl-CoAcarboxylase (ACCase) inhibitor, were found in winter wheat fields in China. Research was carried out to investigate whether resistance to clodinafop-propargyl was present and the molecular mechanism of herbicide resistance in these two populations. Dose-response assays confirmed high level resistance to clodinafop-propargyl in both R2105 and R1027 populations, with resistance indexes 25.1 and 22.1. ACCase gene sequence comparison revealed three amino acid mutations (Trp-1999-Leu, Ile-2041-Asn, or Asp-2078-Gly) in R2105 population and Ile-2041-Asn mutation in R1027 population. Sensitivity to other herbicides assays indicated that R2105 and R1027 populations were cross resistant to fenoxaprop-P-ethyl and multiple resistant to pyroxsulam and mesosulfuron-methyl. The ALS gene sequence analysis revealed that all resistant individuals in R2105 and R1027 populations had the Trp-574-Leu mutation. Applying malathion, significantly decreased the rate of metabolism of clodinafop-propargyl in both R2105 and R1027 populations. This is the first report of multiple resistance to ACCase- and ALS-inhibiting herbicides conferred by target-site mutations and enhanced metabolism in black-grass in China.

Target gene mutation and enhanced metabolism confer fomesafen resistance in an Amaranthus retroflexus L. population from China.

Amaranthus retroflexus L., a troublesome annual dicotyledonous weed species, is highly competitive with soybean (Glycine max L.). A single-dose herbicide-resistance screening assay identified an A. retroflexus population with suspected resistance to fomesafen. Whole-plant dose-response assays demonstrated that the resistant population (2492) was resistant to protoporphyrinogen oxidase (PPO)-inhibiting herbicides (50.6-fold fomesafen resistance and > 8.1-fold lactofen resistance) compared to a susceptible (S) population. PPX2 gene sequence analysis showed an Arg128Gly amino acid substitution in the 2492 population. Moreover, pretreatment of malathion and the fomesafen metabolic assays through HPLC-MS demonstrated enhanced fomesafen metabolism in the 2492 population. Additionally, the 2492 population was 10.4-fold more resistant to the ALS-inhibiting herbicide imazethapyr and 16.8-fold more resistant to thifensulfuron-methyl than the S population. ALS gene sequence analysis showed an Ala205Val amino acid substitution in the 2492 population. This population of A. retroflexus has coexisting target-site resistance and non-target-site mechanisms for resistance to fomesafen. Multiple herbicide resistance may mean it is necessary to adjust weed management strategies to better control the resistant population.

Multiple resistance to ALS-inhibiting and PPO-inhibiting herbicides in Chenopodium album L. from China.

Common lambsquarters (Chenopodium album L.) is a broadleaf weed that can severely damage soybean fields. Two C. album populations (1744 and 1731) suspected resistant to imazethapyr were investigated for resistance levels to imazethapyr, thifensulfuron-methyl, and fomesafen and their resistance mechanisms were investigated. Whole-plant dose-response assays revealed that, compared to the susceptible (S) population, the 1744 population was 16.5-fold resistant to imazethapyr, slightly resistant to thifensulfuron-methyl (resistance index [R/S], <3). The 1731 population was 18.8-fold resistant to imazethapyr, 2.9-fold resistant to thifensulfuron-methyl, and 5.1-fold resistant to fomesafen. In vitro acetolactate synthase (ALS) assays showed 17.1-fold and 19.3-fold resistance levels of 1744 and 1731 populations to imazethapyr respectively. ALS gene sequence analysis identified Ala122Thr amino acid substitution in the 1744 population and Ser653Thr amino acid substitution in the 1731 population. No mutations of the protoporphyrinogen oxidase (PPO) gene were detected. However, pre-treatment with malathion reversed fomesafen resistance, suggesting nontarget-site resistance mechanisms likely play a role in the 1731 population.

Investigation of resistance mechanisms to bentazone in multiple resistant Amaranthus retroflexus populations.

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.

Nitric Oxide Enhanced Salt Stress Tolerance in Tomato Seedlings, Involving Phytohormone Equilibrium and Photosynthesis.

Nitric oxide (NO), as a ubiquitous gas signaling molecule, modulates various physiological and biochemical processes and stress responses in plants. In our study, the NO donor nitrosoglutathione (GSNO) significantly promoted tomato seedling growth under NaCl stress, whereas NO scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide potassium (cPTIO) treatment reversed the positive effect of NO, indicating that NO plays an essential role in enhancing salt stress resistance. To explore the mechanism of NO-alleviated salt stress, the transcriptome of tomato leaves was analyzed. A total of 739 differentially expressed genes (DEGs) were identified and classified into different metabolic pathways, especially photosynthesis, plant hormone signal transduction, and carbon metabolism. Of these, approximately 16 and 9 DEGs involved in plant signal transduction and photosynthesis, respectively, were further studied. We found that GSNO increased the endogenous indoleacetic acid (IAA) and salicylic acid (SA) levels but decreased abscisic acid (ABA) and ethylene (ETH) levels under salt stress conditions. Additionally, GSNO induced increases in photosynthesis pigment content and chlorophyll fluorescence parameters under NaCl stress, thereby enhancing the photosynthetic capacity of tomato seedlings. Moreover, the effects of NO mentioned above were reversed by cPTIO. Together, the results of this study revealed that NO regulates the expression of genes related to phytohormone signal transduction and photosynthesis antenna proteins and, therefore, regulates endogenous hormonal equilibrium and enhances photosynthetic capacity, alleviating salt toxicity in tomato seedlings.

Optimization of Headspace Solid-Phase Micro-Extraction Conditions (HS-SPME) and Identification of Major Volatile Aroma-Active Compounds in Chinese Chive (Allium tuberosum Rottler).

In order to rapidly and precisely identify the volatile compounds in Chinese chive (Allium tuberosum Rottler), seven key parameters of headspace solid-phase micro-extraction conditions (HS-SPME) from Chinese chive were optimized. A total of 59 volatile compounds were identified by using the optimized method, including 28 ethers, 15 aldehydes, 6 alcohols, 5 ketones, 2 hydrocarbons, 1 ester, and 2 phenols. Ethers are the most abundant, especially dimethyl trisulfide (10,623.30 µg/kg). By calculating the odor activity values (OAVs), 11 volatile compounds were identified as the major aroma-active compounds of Chinese chive. From the analysis of the composition of Chinese chive aroma, the "garlic and onion" odor (OAV = 2361.09) showed an absolute predominance over the other 5 categories of aroma. The results of this study elucidated the main sources of Chinese chive aroma from a chemical point of view and provided the theoretical basis for improving the flavor quality of Chinese chive.

Characterization of glyphosate and quizalofop-p-ethyl multiple resistance in Eleusine indica.

Glyphosate and Acetyl-coenzyme A Carboxylase (ACCase) inhibitors are popular herbicides that control goosegrass. However, some populations are difficult to control due to resistance resulting from the increasing selection pressure. The objectives of this research were to detect the multiple resistance levels, resistance mechanisms, and fitness costs of two goosegrass populations collected in China. The resistance indices of two resistant populations (denominated as R1 and R2) to glyphosate were 3.8 and 2.3, respectively; and it was 18.0 and 14.2 to quizalofop-p-ethyl, respectively. Shikimate accumulation in R1 and R2 populations was only 8% of that of the susceptible population after glyphosate treatment. A Pro-106-Ala mutation in EPSPS and an Asp-2078-Gly mutation in ACCase were present in both resistant populations. Both the expression level of EPSPS and ACCase in resistant populations were similar to that of susceptible populations. The leaf area of the individuals in wild-type populations was more than three times of the leaf area in the resistant populations. Similarly, resistant plants were 45-49% shorter, had 70-76% less fresh shoot weight, and 67-69% fewer seeds than wild-type plants. Goosegrass populations have evolved multiple resistance to glyphosate and the ACCase inhibitor quizalofop-p-ethyl in China. The Pro-106-Ala mutation in the EPSPS and the Asp-2078-Gly mutation in the ACCase were responsible for this resistance. In addition, a fitness cost exists in the resistant populations, and more work should conduct to clear which mutation is responsible for the fitness penalty.

Target site mutations and cytochrome P450s-involved metabolism confer resistance to nicosulfuron in green foxtail (Setaria viridis).

Green foxtail [Setaria viridis (L.) P.Beauv.] is a troublesome grass weed that is widely distributed in maize (Zea mays L.) fields across China. Many populations of S. viridis have evolved resistance to the acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron. The objectives of this research were to confirm nicosulfuron resistance in these populations and to investigate the basis of nicosulfuron resistance. Whole-plant dose-response experiments showed 6 out of 13 S. viridis populations were highly resistance (20-30 times) to nicosulfuron. Sequencing of the ALS gene revealed two amino acid mutations, Asp-376-Glu and Pro-197-Ala, in the nicosulfuron-resistant populations. A malathion pretreatment study revealed that the R376 and R197 subpopulations might have cytochrome P450s-mediated herbicide metabolic resistance. The resistant populations were cross-resistant to imazethapyr but sensitive to topramezone and quizalofop-p-ethyl. This is the first report of resistance to ALS inhibitors conferred by target site mutations (Asp-376-Glu or Pro-197-Ser) and possible cytochrome P450s-involved metabolism in S. viridis.

Comprehensive Evaluation of Amino Acids and Polyphenols in 69 Varieties of Green Cabbage (Brassica oleracea L. var. capitata L.) Based on Multivariate Statistical Analysis.

The biological activities of the primary metabolites and secondary metabolites of 69 green cabbage varieties were tested. The LC-MS detection method was used to determine the content of 19 free amino acids (lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, valine, arginine, asparagine, glycine, proline, tyrosine, glutamine, alanine, aspartic acid, serine, and glutamate). The content of 10 polyphenols (chlorogenic acid, gallic acid, 4-coumaric acid, ferulic acid, gentisic acid, cymarin, erucic acid, benzoic acid, rutin, and kaempferol) was determined by the HPLC detection method. Considering the complexity of the data obtained, variance analysis, diversity analysis, correlation analysis, hierarchical cluster analysis (HCA), and principal component analysis (PCA) were used to process and correlate amino acid or polyphenol data, respectively. The results showed that there were significant differences between the different amino acids and polyphenols of the 69 cabbage varieties. The most abundant amino acids and polyphenols were Glu and rutin, respectively. Both amino acids and polyphenols had a high genetic diversity, and multiple groups of significant or extremely significant correlations. The 69 cabbage varieties were divided into two groups, according to 19 amino acid indexes, by PCA. Among them, seven varieties with high amino acid content all fell into the fourth quadrant. The HCA of amino acids also supports this view. Based on 10 polyphenols, the 69 cabbage varieties were divided into two groups by HCA. Based on 29 indexes of amino acids and polyphenols, 69 cabbage varieties were evaluated and ranked by PCA. Therefore, in this study, cabbage varieties were classified in accordance with the level of amino acids and polyphenols, which provided a theoretical basis for the genetic improvement of nutritional quality in cabbage.

Glyphosate resistance in Eleusine indica: EPSPS overexpression and P106A mutation evolved in the same individuals.

Goosegrass is one of the most widespread weeds in orchards and tea plantations in China, and glyphosate is a popular herbicide used to control it. However, high glyphosate selection pressure has led to some populations becoming resistant. The objectives of this research were to determine resistance levels and possible resistance mechanisms of goosegrass populations from several tea plantations in Zhejiang Province in China. The resistance indexes in four goosegrass populations (SH, SY, CA and CX) ranged from 4.9 to 13.4, and lower shikimate accumulation in these populations compared with a glyphosate-susceptible (GS) population confirmed their resistance to glyphosate. No mutations in the target gene EPSPS were found in populations SH and SY, however, the expression of EPSPS in these two populations was 9.3 and 29.7 times higher than that in the GS population, respectively. In the CX population, a P106S mutation in EPSPS was found in 6.7% of the individuals and another 80.0% of individuals had EPSPS amplification. In population CA, all the individuals had a P106A mutation and 86.7% of them had amplification in EPSPS. The EPSPS copy numbers ranged from 5.2 to 62.3 in these four resistant populations. There was a positive correlation between signal intensities of primary anti-EPSPS antibody and the copy number of the EPSPS protein, as indicated by immunoblot analysis. In population CA, with high-level resistance to glyphosate, both P106A mutation and amplification in EPSPS evolved in the same individuals in this population.

Investigation of resistance mechanism to fomesafen in Amaranthus retroflexus L.

Amaranthus retroflexus L. is one of the most troublesome weeds in autumn-crop fields in Northeast China. In recent years, field applications of fomesafen have failed to control an A. retroflexus population in Heilongjiang Province, China. Therefore, in this study, experiments were conducted to determine the resistance of A. retroflexus to fomesafen and investigate the molecular basis of herbicide resistance. Whole-plant dose-response experiments showed that the resistant (R) population exhibited 41.8-fold resistance to fomesafen compared with the susceptible (S) population. Target-gene sequence analysis revealed an Arg-128-Gly substitution in the protoporphyrinogen oxidase (PPO) in the R population. The response of PPO2 transgenic Arabidopsis thaliana to fomesafen demonstrated that the Arg-128-Gly substitution conferred high resistance to fomesafen. Cross- and multiple-resistance analyses indicated that the R population was cross-resistant to lactofen and carfentrazone-ethyl but was sensitive to imazethapyr, thifensulfuron-methyl, atrazine, and glyphosate. This study indicated that the Arg-128-Gly substitution is the main reason for A. retroflexus resistance to fomesafen. To our knowledge, this is the first report of a target-site based mechanism for the resistance to a PPO-inhibiting herbicide in A. retroflexus.

[Herbal textural research, morphologic characteristics and DNA barcoding of botanical origins of Alismatis Rhizoma].

Alismatis Rhizoma(Zexie) is a commonly used traditional Chinese medicine, and it is separated into "Chuan Zexie"(Sichuan and Hubei provinces), "Jian Zexie"(Fujian and Jiangxi provinces) and "Guang Zexie"(Guangxi province) according to different producing areas. Alisma plantago-aquatica and A. orientale were listed as the original plants of Alismatis Rhizoma in different editions of Chinese Pharmacopoeia(Ch.P), respectively. The botanical origins of Alismatis Rhizoma caused much controversy during a period of time. This study aimed to define the botanical origins of Alismatis Rhizoma from different producing areas, and supply scientific evidence for Ch. P 2020 edition. In this paper, we summarized the descriptions of original plants and producing areas of Alismatis Rhizoma in ancient literatures. Flowers and fruits of original plants of Alismatis Rhizoma were collected from different typical areas, and compared with the morphological description of two species from Alisma genus in the Flora of China. Thirty-nine batches of leaves from 8 different areas were identified using DNA barcoding technology. The results showed that original plants of Alismatis Rhizoma from different areas could be distinguished from each other based on morphological characteristics and molecular characteristics. Then, "Jian Zexie" was identified as A. orientale, while "Chuan Zexie" and "Guang Zexie" were identified as A. plantago-aquatica. In conclusion, combining with herbal textural research, morphologic characteristics, DNA barcoding technology and market situation, this paper recommended that the botanical sources of Alismatis Rhizoma could be revised as Alisma orientale(Sam.) Juzep. and Alisma plantago-aquatica Linn. in the Ch. P 2020 edition.

DELAY OF GERMINATION1 (DOG1) regulates both seed dormancy and flowering time through microRNA pathways.

Seed germination and flowering, two critical developmental transitions in plant life cycles, are coordinately regulated by genetic and environmental factors to match plant establishment and reproduction to seasonal cues. The DELAY OF GERMINATION1 (DOG1) gene is involved in regulating seed dormancy in response to temperature and has also been associated genetically with pleiotropic flowering phenotypes across diverse Arabidopsis thaliana accessions and locations. Here we show that DOG1 can regulate seed dormancy and flowering times in lettuce (Lactuca sativa, Ls) and Arabidopsis through an influence on levels of microRNAs (miRNAs) miR156 and miR172. In lettuce, suppression of LsDOG1 expression enabled seed germination at high temperature and promoted early flowering in association with reduced miR156 and increased miR172 levels. In Arabidopsis, higher miR156 levels resulting from overexpression of the MIR156 gene enhanced seed dormancy and delayed flowering. These phenotypic effects, as well as conversion of MIR156 transcripts to miR156, were compromised in DOG1 loss-of-function mutant plants, especially in seeds. Overexpression of MIR172 reduced seed dormancy and promoted early flowering in Arabidopsis, and the effect on flowering required functional DOG1 Transcript levels of several genes associated with miRNA processing were consistently lower in dry seeds of Arabidopsis and lettuce when DOG1 was mutated or its expression was reduced; in contrast, transcript levels of these genes were elevated in a DOG1 gain-of-function mutant. Our results reveal a previously unknown linkage between two critical developmental phase transitions in the plant life cycle through a DOG1-miR156-miR172 interaction.

The basis of resistance mechanism to mesosulfuron-methyl in Tausch's goatgrass (Aegilops tauschii Coss.).

Tausch's goatgrass (Aegilops tauschii Coss.) is one of the most troublesome weeds in winter wheat-growing regions of China. In recent years, the recommended field rate of mesosulfuron-methyl failed to control the Tausch's goatgrass population in Shanxi province (SX), China. Experiments were conducted to characterize the herbicide resistance level and investigate the basis of mesosulfuron-methyl resistance in Tausch's goatgrass. Whole-plant dose-response tests showed that the SX population exhibited 11.42-fold resistance to mesosulfuron-methyl than the susceptible HN population, and the resistance level in the SX population could be significantly reduced by malathion, a cytochrome P450 inhibitor. The SX population also exhibited cross-resistance to imazethapyr, pyroxsulam and bispyribac­sodium. Acetohydroxyacid synthase (AHAS) sequencing and enzyme activity assays demonstrated that the mesosulfuron-methyl resistance was not conferred by target-site substitution. A sensitive AHAS, together with the malathion revisable resistance, suggested that herbicide metabolism likely plays a main role in the mechanism of mesosulfuron-methyl resistance in the SX population. To our knowledge, this is the first report elucidating the mesosulfuron-methyl resistance in Tausch's goatgrass.

Investigating the mechanisms of glyphosate resistance in goosegrass (Eleusine indica (L.) Gaertn.) by RNA sequencing technology.

Glyphosate is an important non-selective herbicide that is in common use worldwide. However, evolved glyphosate-resistant (GR) weeds significantly affect crop yields. Unfortunately, the mechanisms underlying resistance in GR weeds, such as goosegrass (Eleusine indica (L.) Gaertn.), an annual weed found worldwide, have not been fully elucidated. In this study, transcriptome analysis was conducted to further assess the potential mechanisms of glyphosate resistance in goosegrass. The RNA sequencing libraries generated 24 597 462 clean reads. De novo assembly analysis produced 48 852 UniGenes with an average length of 847 bp. All UniGenes were annotated using seven databases. Sixteen candidate differentially expressed genes selected by digital gene expression analysis were validated by quantitative real-time PCR (qRT-PCR). Among these UniGenes, the EPSPS and PFK genes were constitutively up-regulated in resistant (R) individuals and showed a higher copy number than that in susceptible (S) individuals. The expressions of four UniGenes relevant to photosynthesis were inhibited by glyphosate in S individuals, and this toxic response was confirmed by gas exchange analysis. Two UniGenes annotated as glutathione transferase (GST) were constitutively up-regulated in R individuals, and were induced by glyphosate both in R and S. In addition, the GST activities in R individuals were higher than in S. Our research confirmed that two UniGenes (PFK, EPSPS) were strongly associated with target resistance, and two GST-annotated UniGenes may play a role in metabolic glyphosate resistance in goosegrass.

Characterization of Eleusine indica with gene mutation or amplification in EPSPS to glyphosate.

The evolution of weed-resistant species threatens the sustainable use of glyphosate, which is the most important herbicide widely used in agriculture worldwide. Moreover, the high glyphosate resistance (>180-fold based on LD50) of Eleusine indica found in Malaysia, which carries a double mutation in its 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), made the control of this species more difficult. By contrast, the same species carrying the same double mutation in EPSPS (T102I+P106S) but found in China only shows a resistance level of not more than 14-fold based on GR50. The resistance level of this population is four times higher than that of the population carrying a single mutation (P106L). Although the members of this population survive under a high glyphosate dosage of 10,080gaeha-1, their growth was significantly inhibited by glyphosate under the recommend dose (840gaeha-1), where in the fresh weight was 85.4% of the control. EPSPS expression, relative copy number, and EPSPS activity in this population were similar to those of the susceptible population. In addition, the expression of two glutathione transferase (GST) genes (GST-U8 and GST-23) and the enzyme activity of the GST in this population did not significantly differ from those of the susceptible population. This finding is important in elucidating the resistance of the naturally evolved glyphosate-resistant (GR) weed species carrying a double mutation in EPSPS to glyphosate.

Target-site basis for resistance to imazethapyr in redroot amaranth (Amaranthus retroflexus L.).

Experiments were conducted to confirm imazethapyr resistance in redroot amaranth (Amaranthus retroflexus L.) and study the target-site based mechanism for the resistance. Whole-plant response experiments revealed that the resistant (R) population exhibited 19.16 fold resistance to imazethapyr compared with the susceptible (S) population. In vitro ALS activity assay demonstrated that the imazethapyr I50 value of the R population was 21.33 times greater than that of the S population. However, qRT-PCR analysis revealed that there is no difference in ALS gene expression between the R and S populations. Sequence analysis revealed an Asp-376-Glu substitution in ALS in the R population. In order to verify that the imazethapyr resistance was conferred by Asp-376-Glu mutation, the ALS-R and ALS-S genes were fused to the CaMV 35S promoter and introduced into Arabidopsis respectively. The expression of ALS-R in transgenic Arabidopsis plants exhibited 13.79 fold resistance to imazethapyr compared to ALS-S transgenic Arabidopsis.