Amaranth's Growth and Physiological Responses to Salt Stress and the Functional Analysis of AtrTCP1 Gene.

Amaranth species are C4 plants that are rich in betalains, and they are tolerant to salinity stress. A small family of plant-specific TCP transcription factors are involved in the response to salt stress. However, it has not been investigated whether amaranth TCP1 is involved in salt stress. We elucidated that the growth and physiology of amaranth were affected by salt concentrations of 50-200 mmol·L-1 NaCl. The data showed that shoot and root growth was inhibited at 200 mmol·L-1, while it was promoted at 50 mmol·L-1. Meanwhile, the plants also showed physiological responses, which indicated salt-induced injuries and adaptation to the salt stress. Moreover, AtrTCP1 promoted Arabidopsis seed germination. The germination rate of wild-type (WT) and 35S::AtrTCP1-GUS Arabidopsis seeds reached around 92% by the seventh day and 94.5% by the second day under normal conditions, respectively. With 150 mmol·L-1 NaCl treatment, the germination rate of the WT and 35S::AtrTCP1-GUS plant seeds was 27.0% by the seventh day and 93.0% by the fourth day, respectively. Under salt stress, the transformed 35S::AtrTCP1 plants bloomed when they grew 21.8 leaves after 16.2 days of treatment, which was earlier than the WT plants. The transformed Arabidopsis plants flowered early to resist salt stress. These results reveal amaranth's growth and physiological responses to salt stress, and provide valuable information on the AtrTCP1 gene.

Isoform-resolved genome annotation enables mapping of tissue-specific betalain regulation in amaranth.

Betalains are coloring pigments produced in some families of the order Caryophyllales, where they replace anthocyanins as coloring pigments. While the betalain pathway itself is well studied, the tissue-specific regulation of the pathway remains mostly unknown. We enhance the high-quality Amaranthus hypochondriacus reference genome and produce a substantially more complete genome annotation, incorporating isoform details. We annotate betalain and anthocyanin pathway genes along with their regulators in amaranth and map the genetic control and tissue-specific regulation of the betalain pathway. Our improved genome annotation allowed us to identify causal mutations that lead to a knock-out of red betacyanins in natural accessions of amaranth. We reveal the tissue-specific regulation of flower color via a previously uncharacterized MYB transcription factor, AhMYB2. Downregulation of AhMYB2 in the flower leads to reduced expression of key betalain enzyme genes and loss of red flower color. Our improved amaranth reference genome represents the most complete genome of amaranth to date and is a valuable resource for betalain and amaranth research. High similarity of the flower betalain regulator AhMYB2 to anthocyanin regulators and a partially conserved interaction motif support the co-option of anthocyanin regulators for the betalain pathway as a possible reason for the mutual exclusiveness of the two pigments.

RNA Isolation Method in Marginal Crops with High Agronomic Potential.

Ribonucleic Acid (RNA) isolation is a basic technique in the field of molecular biology. The purpose of RNA isolation is to acquire pure and complete RNA that can be used to evaluate gene expression. Many methods can be used to perform RNA isolation, all of them based on the chemical properties of nucleic acids. However, some of them do not achieve high RNA yields and purity levels when used in a number of marginally studied crops of agronomic importance, such as grain and vegetable amaranth plants. In the method described here, the use of guanidinium thiocyanate and two additional precipitation steps with different reagents designed to obtain high yields and RNA purity levels from diverse plant species employed for plant functional genomics studies is described.

Development of a rapid detection assay for acetolactate synthase inhibitors resistance in three Amaranthus weed species through loop-mediated isothermal amplification.

BACKGROUND: The early detection of herbicide resistance in weeds is a key factor to avoid herbicide waste and improve agriculture sustainability. The present study aimed to develop and validate an allele-specific loop-mediated isothermal amplification (AS-LAMP) assay for the quick on-site detection of the resistance-endowing point mutation Trp-574-Leu in the acetolactate synthase (ALS) gene in three widely diffused Amaranthus weed species: Amaranthus retroflexus, Amaranthus hybridus and Amaranthus tuberculatus. RESULTS: The AS-LAMP protocol was developed on wild-type and ALS-mutant plants of the three species and revealed that the amplification approach with only the primer set specific for the mutant allele (574-Leu) was the most promising. The validation and estimation of the AS-LAMP performance evaluated by comparing the results with those of the molecular marker (cleaved amplified polymorphic sequences) indicated that, although the sensitivity and specificity were relatively high in all species (overall 100 and > 65%, respectively), precision was high for A. hybridus L. and A. retroflexus L. (75 and 79%, respectively), but quite low for A. tuberculatus (Moq.) J. D. Sauer (59%). The LAMP assay was also effective on crude genomic DNA extraction, allowing the quick detection of mutant plants in field situation (on site resistance detection). CONCLUSION: The proposed AS-LAMP method has proven to be a promising technique for rapid detection of resistance as a result of Trp-574-Leu on the two monoecious weedy Amaranthus species but resulted less effective in the genetically variable dioecious species A. tuberculatus. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Dual plastid targeting of protoporphyrinogen oxidase 2 in Amaranthaceae promotes herbicide tolerance.

Plant tetrapyrrole biosynthesis (TPB) takes place in plastids and provides the chlorophyll and heme required for photosynthesis and many redox processes throughout plant development. TPB is strictly regulated, since accumulation of several intermediates causes photodynamic damage and cell death. Protoporphyrinogen oxidase (PPO) catalyzes the last common step before TPB diverges into chlorophyll and heme branches. Land plants possess two PPO isoforms. PPO1 is encoded as a precursor protein with a transit peptide, but in most dicotyledonous plants PPO2 does not possess a cleavable N-terminal extension. Arabidopsis (Arabidopsis thaliana) PPO1 and PPO2 localize in chloroplast thylakoids and envelope membranes, respectively. Interestingly, PPO2 proteins in Amaranthaceae contain an N-terminal extension that mediates their import into chloroplasts. Here, we present multiple lines of evidence for dual targeting of PPO2 to thylakoid and envelope membranes in this clade and demonstrate that PPO2 is not found in mitochondria. Transcript analyses revealed that dual targeting in chloroplasts involves the use of two transcription start sites and initiation of translation at different AUG codons. Among eudicots, the parallel accumulation of PPO1 and PPO2 in thylakoid membranes is specific for the Amaranthaceae and underlies PPO2-based herbicide resistance in Amaranthus species.

Quantifying the role of genome size and repeat content in adaptive variation and the architecture of flowering time in Amaranthus tuberculatus.

Genome size variation, largely driven by repeat content, is poorly understood within and among populations, limiting our understanding of its significance for adaptation. Here we characterize intraspecific variation in genome size and repeat content across 186 individuals of Amaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. Sequence-based genome size estimates vary by up to 20% across individuals, consistent with the considerable variability in the abundance of transposable elements, unknown repeats, and rDNAs across individuals. The additive effect of this variation has important phenotypic consequences-individuals with more repeats, and thus larger genomes, show slower flowering times and growth rates. However, compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size is a marginal contributor. Differences in flowering time are reflected by genome size variation across sexes and marginally, habitats, while polygenic variation and a gene copy number variant within the ATP synthesis pathway show consistently stronger environmental clines than genome size. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, demonstrating the numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation.

Transcriptomic analysis of Amaranthus retroflex resistant to PPO-inhibitory herbicides.

Amaranthus retroflexus L. is one of the malignant weeds which can cause a reduction in the soybean yield. We found a population of A. retroflexus (R-Q) resistant to fomesafen through the initial screening of whole-plant dose response bioassay in the research. The resistance index of the population (R-Q) was 183 times of the sensitive population (S-N). The resistant and sensitive populations were used as experimental materials in the paper. Strand-specific RNA-Seq analyses of R‒Q and S‒N populations obtained from herbicide-treated and mock-treated leaf samples after treatment were conducted to generate a full-length transcriptome database. We analyzed differentially expressed genes (DEGs) among the R-Q and S‒N A. retroflexus populations treated with recommended dose and mock-treated on the 1st (24 h) and 3rd (72 h) days to identify genes involved in fomesafen resistance. All 82,287 unigenes were annotated by Blastx search with E-value < 0.00001 from 7 databases. A total of 94,815 DEGs among the three group comparisons were identified. Two nuclear genes encoding PPO (PPX1 and PPX2) and five unigenes belonging to the AP2-EREBP, GRAS, NAC, bHLH and bZIP families exhibited different expression patterns between individuals of S‒N and R-Q populations. The A. retroflexus transcriptome and specific transcription factor families which can respond to fomesafen in resistant and susceptible genotypes were reported in this paper. The PPX1 and PPX2 genes of the target enzyme were identified. The study establishes the foundation for future research and provides opportunities to manage resistant weeds better.

Comparative Transcriptomic Analysis of the Metabolism of Betalains and Flavonoids in Red Amaranth Hypocotyl under Blue Light and Dark Conditions.

Amaranth plants contain abundant betalains and flavonoids. Anthocyanins are important flavonoids; however, they cannot coexist in the same plant with betalains. Blue light influences metabolite synthesis and hypocotyl elongation; accordingly, analyses of its effects on betalain and flavonoid biosynthesis in Amaranthus tricolor may provide insight into the distribution of these plant pigments. We analyzed the betalain and flavonoid content and transcriptome profiles in amaranth hypocotyls under blue light and dark conditions. Furthermore, we analyzed the expression patterns of key genes related to betalains and flavonoids. Amaranth hypocotyls were shorter and redder and showed higher betalain and flavonoid content under blue light than in dark conditions. Key genes involved in the synthesis of betalains and flavonoids were upregulated under blue light. The gene encoding DELLA was also upregulated. These results suggest that blue light favors the synthesis of both betalains and flavonoids via the suppression of bioactive gibberellin and the promotion of DELLA protein accumulation, which also suppresses hypocotyl elongation. The metabolite profiles differed between plants under blue light and dark conditions. These findings improve our understanding of the environmental cues and molecular mechanisms underlying pigment variation in Amaranthus.

Genetic basis and origin of resistance to acetolactate synthase inhibitors in Amaranthus palmeri from Spain and Italy.

BACKGROUND: Amaranthus palmeri is an aggressive annual weed native to the United States, which has become invasive in some European countries. Populations resistant to acetolactate synthase (ALS) inhibitors have been recorded in Spain and Italy, but the evolutionary origin of the resistance traits remains unknown. Bioassays were conducted to identify cross-resistance to ALS inhibitors and a haplotype-based genetic approach was used to elucidate the origin and distribution of resistance in both countries. RESULTS: Amaranthus palmeri populations were resistant to thifensulfuron-methyl and imazamox, and the 574-Leu mutant ALS allele was found to be the main cause of resistance among them. In two Spanish populations, 376-Glu and 197-Thr mutant ALS alleles were also found. The haplotype analyses revealed the presence of two and four distinct 574-Leu mutant haplotypes in the Italian and Spanish populations, respectively. None was common to both countries, but some mutant haplotypes were shared between geographically close populations or between populations more than 100 km apart. Wide genetic diversity was found in two very close Spanish populations. CONCLUSION: ALS-resistant A. palmeri populations were introduced to Italy and Spain from outside Europe. Populations from both countries have different evolutionary histories and originate from independent introduction events. ALS resistance then spread over short and long distances by seed dispersal. The higher number and genetic diversity among mutant haplotypes from the Spanish populations indicated recurrent invasions. The implementation of control tactics to limit seed dispersal and the establishment of A. palmeri is recommended in both countries. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Comparative transcriptomic analysis of male and females in the dioecious weeds Amaranthus palmeri and Amaranthus tuberculatus.

BACKGROUND: Waterhemp (Amaranthus tuberculatus (Moq.) Sauer) and Palmer amaranth (Amaranthus palmeri S. Wats.) are two dioecious and important weed species in the world that can rapidly evolve herbicide-resistance traits. Understanding these two species' dioecious and sex-determination mechanisms could open opportunities for new tools to control them. This study aims to identify the differential expression patterns between males and females in A. tuberculatus and A. palmeri. Multiple analyses, including differential expression, co-expression, and promoter analyses, used RNA-seq data from multiple tissue types to identify putative essential genes for sex determination in both dioecious species. RESULTS: Genes were identified as potential key players for sex determination in A. palmeri. Genes PPR247, WEX, and ACD6 were differentially expressed between the sexes and located at scaffold 20 within or near the male-specific Y (MSY) region. Multiple genes involved with flower development were co-expressed with these three genes. For A. tuberculatus, no differentially expressed gene was identified within the MSY region; however, multiple autosomal class B and C genes were identified as differentially expressed and possible candidate genes. CONCLUSIONS: This is the first study comparing the global expression profile between males and females in dioecious weedy Amaranthus species. Results narrow down putative essential genes for sex-determination in A. palmeri and A. tuberculatus and also strengthen the hypothesis of two different evolutionary events for dioecy within the genus.

Extrachromosomal circular DNA-mediated spread of herbicide resistance in interspecific hybrids of pigweed.

Extrachromosomal circular DNAs (eccDNAs) are found in many eukaryotic organisms. EccDNA-powered copy number variation plays diverse roles, from oncogenesis in humans to herbicide resistance in crop weeds. Here, we report interspecific eccDNA flow and its dynamic behavior in soma cells of natural populations and F1 hybrids of Amaranthus sp. The glyphosate-resistance (GR) trait is controlled by eccDNA-based amplification harboring the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (eccDNA replicon), the molecular target of glyphosate. We documented pollen-mediated transfer of eccDNA in experimental hybrids between glyphosate-susceptible Amaranthus tuberculatus and GR Amaranthus palmeri. Experimental hybridization and fluorescence in situ hybridization (FISH) analysis revealed that the eccDNA replicon in Amaranthus spinosus derived from GR A. palmeri by natural hybridization. FISH analysis also revealed random chromosome anchoring and massive eccDNA replicon copy number variation in soma cells of weedy hybrids. The results suggest that eccDNAs are inheritable across compatible species, contributing to genome plasticity and rapid adaptive evolution.

Comparative analysis of dioecious Amaranthus plastomes and phylogenomic implications within Amaranthaceae s.s.

BACKGROUND: The genus Amaranthus L. consists of 70-80 species distributed across temperate and tropical regions of the world. Nine species are dioecious and native to North America; two of which are agronomically important weeds of row crops. The genus has been described as taxonomically challenging and relationships among species including the dioecious ones are poorly understood. In this study, we investigated the phylogenetic relationships among the dioecious amaranths and sought to gain insights into plastid tree incongruence. A total of 19 Amaranthus species' complete plastomes were analyzed. Among these, seven dioecious Amaranthus plastomes were newly sequenced and assembled, an additional two were assembled from previously published short reads sequences and 10 other plastomes were obtained from a public repository (GenBank). RESULTS: Comparative analysis of the dioecious Amaranthus species' plastomes revealed sizes ranged from 150,011 to 150,735 bp and consisted of 112 unique genes (78 protein-coding genes, 30 transfer RNAs and 4 ribosomal RNAs). Maximum likelihood trees, Bayesian inference trees and splits graphs support the monophyly of subgenera Acnida (7 dioecious species) and Amaranthus; however, the relationship of A. australis and A. cannabinus to the other dioecious species in Acnida could not be established, as it appears a chloroplast capture occurred from the lineage leading to the Acnida + Amaranthus clades. Our results also revealed intraplastome conflict at some tree branches that were in some cases alleviated with the use of whole chloroplast genome alignment, indicating non-coding regions contribute valuable phylogenetic signals toward shallow relationship resolution. Furthermore, we report a very low evolutionary distance between A. palmeri and A. watsonii, indicating that these two species are more genetically related than previously reported. CONCLUSIONS: Our study provides valuable plastome resources as well as a framework for further evolutionary analyses of the entire Amaranthus genus as more species are sequenced.

Peruvian Amaranth (kiwicha) Accumulates Higher Levels of the Unsaturated Linoleic Acid.

Grain amaranth (Amaranthus spp.) is an emerging crop rich in proteins and other valuable nutrients. It was domesticated twice, in Mexico and Peru. Although global trade is dominated by Mexican species of amaranth, Peruvian amaranth (A. caudatus, kiwicha) has remained neglected, although it harbours valuable traits. In the current study, we investigate the accumulation of polyunsaturated fatty acids, comparing four genotypes of A. caudatus with K432, a commercial variety deriving from the Mexican species A. hypochondriacus under the temperate environment of Southwest Germany. We show that the A. caudatus genotypes flowered later (only in late autumn), such that they were taller as compared to the Mexican hybrid but yielded fewer grains. The oil of kiwicha showed a significantly higher content of unsaturated fatty acids, especially of linoleic acid and α-linolenic acid compared to early flowering genotype K432. To gain insight into the molecular mechanisms behind these differences, we sequenced the genomes of the A. hypochondriacus × hybridus variety K432 and the Peruvian kiwicha genotype 8300 and identified the homologues for genes involved in the ω3 fatty-acid pathway and concurrent oxylipin metabolism, as well as of key factors for jasmonate signalling and cold acclimation. We followed the expression of these transcripts over three stages of seed development in all five genotypes. We find that transcripts for Δ6 desaturases are elevated in kiwicha, whereas in the Mexican hybrid, the concurrent lipoxygenase is more active, which is followed by the activation of jasmonate biosynthesis and signalling. The early accumulation of transcripts involved in cold-stress signalling reports that the Mexican hybrid experiences cold stress already early in autumn, whereas the kiwicha genotypes do not display indications for cold stress, except for the very final phase, when there were already freezing temperatures. We interpret the higher content of unsaturated fatty acids in the context of the different climatic conditions shaping domestication (tropical conditions in the case of Mexican amaranth, sharp cold snaps in the case of kiwicha) and suggest that kiwicha oil has high potential as functional food which can be developed further by tailoring genetic backgrounds, agricultural practice, and processing.

Genomic profiling of dioecious Amaranthus species provides novel insights into species relatedness and sex genes.

BACKGROUND: Amaranthus L. is a diverse genus consisting of domesticated, weedy, and non-invasive species distributed around the world. Nine species are dioecious, of which Amaranthus palmeri S. Watson and Amaranthus tuberculatus (Moq.) J.D. Sauer are troublesome weeds of agronomic crops in the USA and elsewhere. Shallow relationships among the dioecious Amaranthus species and the conservation of candidate genes within previously identified A. palmeri and A. tuberculatus male-specific regions of the Y (MSYs) in other dioecious species are poorly understood. In this study, seven genomes of dioecious amaranths were obtained by paired-end short-read sequencing and combined with short reads of seventeen species in the family Amaranthaceae from NCBI database. The species were phylogenomically analyzed to understand their relatedness. Genome characteristics for the dioecious species were evaluated and coverage analysis was used to investigate the conservation of sequences within the MSY regions. RESULTS: We provide genome size, heterozygosity, and ploidy level inference for seven newly sequenced dioecious Amaranthus species and two additional dioecious species from the NCBI database. We report a pattern of transposable element proliferation in the species, in which seven species had more Ty3 elements than copia elements while A. palmeri and A. watsonii had more copia elements than Ty3 elements, similar to the TE pattern in some monoecious amaranths. Using a Mash-based phylogenomic analysis, we accurately recovered taxonomic relationships among the dioecious Amaranthus species that were previously identified based on comparative morphology. Coverage analysis revealed eleven candidate gene models within the A. palmeri MSY region with male-enriched coverages, as well as regions on scaffold 19 with female-enriched coverage, based on A. watsonii read alignments. A previously reported FLOWERING LOCUS T (FT) within A. tuberculatus MSY contig was also found to exhibit male-enriched coverages for three species closely related to A. tuberculatus but not for A. watsonii reads. Additional characterization of the A. palmeri MSY region revealed that 78% of the region is made of repetitive elements, typical of a sex determination region with reduced recombination. CONCLUSIONS: The results of this study further increase our understanding of the relationships among the dioecious species of the Amaranthus genus as well as revealed genes with potential roles in sex function in the species.

Chromosome-scale Amaranthus tricolor genome provides insights into the evolution of the genus Amaranthus and the mechanism of betalain biosynthesis.

Amaranthus tricolor is a vegetable and ornamental amaranth, with high lysine, dietary fibre and squalene content. The red cultivar of A. tricolor possesses a high concentration of betalains, which has been used as natural food colorants. Here, we constructed the genome of A. tricolor, the first reference genome for the subgenus Albersia, combining PacBio HiFi, Nanopore ultra-long and Hi-C data. The contig N50 size was 906 kb, and 99.58% of contig sequence was anchored to the 17 chromosomes, totalling 520 Mb. We annotated 27,813 protein-coding genes with an average 1.3 kb coding sequence and 5.3 exons. We inferred that A. tricolor underwent a whole-genome duplication (WGD) and that the WGD shared by amaranths occurred in the last common ancestor of subfamily Amaranthoideae. Moreover, we comprehensively identified candidate genes in betalain biosynthesis pathway. Among them, DODAα1 and CYP76ADα1, located in one topologically associated domain (TAD) of an active (A) compartment on chromosome 16, were more highly expressed in red leaves than in green leaves, and DODAα1 might be the rate-limiting enzyme gene in betalains biosynthesis. This study presents new genome resources and enriches our understanding of amaranth evolution, betalains production, facilitating molecular breeding improvements and the understanding of C4 plants evolution.

Rapid weed adaptation and range expansion in response to agriculture over the past two centuries.

North America has experienced a massive increase in cropland use since 1800, accompanied more recently by the intensification of agricultural practices. Through genome analysis of present-day and historical samples spanning environments over the past two centuries, we studied the effect of these changes in farming on the extent and tempo of evolution across the native range of the common waterhemp (Amaranthus tuberculatus), a now pervasive agricultural weed. Modern agriculture has imposed strengths of selection rarely observed in the wild, with notable shifts in allele frequency trajectories since agricultural intensification in the 1960s. An evolutionary response to this extreme selection was facilitated by a concurrent human-mediated range shift. By reshaping genome-wide diversity across the landscape, agriculture has driven the success of this weed in the 21st century.

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.

Sequence characterization of eccDNA content in glyphosate sensitive and resistant Palmer amaranth from geographically distant populations.

The discovery of non-chromosomal circular DNA offers new directions in linking genome structure with function in plant biology. Glyphosate resistance through EPSPS gene copy amplification in Palmer amaranth was due to an autonomously replicating extra-chromosomal circular DNA mechanism (eccDNA). CIDER-Seq analysis of geographically distant glyphosate sensitive (GS) and resistant (GR) Palmer Amaranth (Amaranthus palmeri) revealed the presence of numerous small extra-chromosomal circular DNAs varying in size and with degrees of repetitive content, coding sequence, and motifs associated with autonomous replication. In GS biotypes, only a small portion of these aligned to the 399 kb eccDNA replicon, the vehicle underlying gene amplification and genetic resistance to the herbicide glyphosate. The aligned eccDNAs from GS were separated from one another by large gaps in sequence. In GR biotypes, the eccDNAs were present in both abundance and diversity to assemble into a nearly complete eccDNA replicon. Mean sizes of eccDNAs were similar in both biotypes and were around 5kb with larger eccDNAs near 25kb. Gene content for eccDNAs ranged from 0 to 3 with functions that include ribosomal proteins, transport, metabolism, and general stress response genetic elements. Repeat content among smaller eccDNAs indicate a potential for recombination into larger structures. Genomic hotspots were also identified in the Palmer amaranth genome with a disposition for gene focal amplifications as eccDNA. The presence of eccDNA may serve as a reservoir of genetic heterogeneity in this species and may be functionally important for survival.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate.

MAIN CONCLUSION: Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha-1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = - 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

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.