BACKGROUND: The commercial utilization of genetically modified soybeans has yielded substantial economic advantages. Nevertheless, the genetic drift towards wild soybeans is one of the main ecological risks that needs to be addressed. Previous experiments demonstrated the absence of fitness cost or florescence overlap in hybrid offspring resulting from the crossbreeding of transgenic soybean GTS40-3-2 and Zhengzhou wild soybeans. In this study, hybrid progeny was systematically crossed with wild soybeans to establish a backcross progeny system. This system was employed to evaluate the ecological risk associated with the backcross progeny of transgenic and wild soybeans.
RESULTS: The findings indicated that the offspring from the backcross exhibited glyphosate tolerance. Furthermore, the expression of foreign proteins in the backcross offspring was notably lower than in the transgenic soybean, and there was no significant difference when compared to the hybrid progeny. Parameters such as germination rate, aboveground biomass, pods per plant, full seeds per plant, and 100-grain weight exhibited no significant differences between the negative and positive lines of the backcross progenies, and no fitness cost was identified in comparison to wild soybeans. These results underscore the potential for foreign genes to propagate within other wild soybeans, which requires continuous attention.
CONCLUSIONS: The widespread adoption of genetically modified soybeans has undeniably led to substantial economic gains. However, the research findings emphasize the critical importance of addressing the ecological risks posed by genetic drift towards wild soybeans. The backcross progeny system established in this study indicates that the potential for foreign gene dissemination to wild soybean populations warrants continued attention and mitigation strategies.

Histidine biosynthesis is essential for the growth and development of plants, where it occurs within chloroplasts. The eleven reactions are catalyzed by eight enzymes, known as HISN1-8, each acting sequentially. Here, we present the crystal structures of a 5\'-ProFAR isomerase (HISN3) from the model legume Medicago truncatula bound to its enzymatically synthesized substrate (ProFAR) and product (PrFAR). The active site of MtHISN3 contains a sodium cation that participates in ligand recognition, a feature not observed in bacterial and fungal structures of homologous enzymes. The steady-state kinetics of wild-type MtHISN3 revealed a slightly higher turnover rate compared to its bacterial homologs. Plant HISN3 sequences contain an unusually elongated Lys60-Ser91 fragment, while deletion of the 74-80 region resulted in a 30-fold loss in catalytic efficiency compared to the wild-type. Molecular dynamics simulations suggested that the fragment facilitates product release, thereby contributing to a higher kcat. Moreover, conservation analyses suggested a non-cyanobacterial origin for plant HISN3 enzymes, which is another instance of a non-cyanobacterial enzyme in the plant histidine biosynthetic pathway. Finally, a virtual screening campaign yielded five molecules, with the energy gains ranging between -13.6 and -13.1 kcal/mol, which provide new scaffolds for the future development of herbicides.

Herbicide-resistant Conyza spp. are a threat to many crops. These widespread weeds are closely related species and often cooccur. To characterize the origins of their resistance and the mechanisms underlying their spread, we assessed the genomic variation in glyphosate-resistant Conyza spp. in Brazil. Twenty populations were sampled from soybean fields across four macroregions (MRSs). A genotyping-by-sequencing study resulted in 2,998 single-nucleotide polymorphisms (SNPs) obtained for C. bonariensis (L.) and the closely related C. sumatrensis (Retz) E. Walker. Higher genomic diversity (π) and heterozygosity (HO/HE) and lower inbreeding coefficient (FIS) values were detected in populations of Conyza spp. from MRS 1 (southern) than in those from other MRSs. Strong genomic structure clustered individuals into three groups (FST = 0.22; p value = 0.000) associated with the MRSs. Thus, resistance to glyphosate originated from independent selection in different MRSs across Brazil. Our dataset supports the occurrence of intraspecific gene flow in Brazil and identified individuals of C. bonariensis that did not group within species. These findings suggest that allelic introgressions within and among species have impacted the evolution and spread of resistance to glyphosate in Conyza spp. We discuss how to mitigate new resistance cases, particularly for the released stacked traits herbicide tolerance in soybeans.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (Ki of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.

This study aimed to compare the impact of iodosulfuron-methyl-sodium and an iodosulfuron-based herbicidal ionic liquid (HIL) on the microbiomes constituting the epiphytes and endophytes of cornflower (Centaurea cyanus L.). The experiment involved biotypes of cornflower susceptible and resistant to acetolactate synthase inhibition, examining potential bacterial involvement in sulfonylurea herbicide detoxification. We focused on microbial communities present on the surface and in the plant tissues of roots and shoots. The research included the synthesis and physicochemical analysis of a novel HIL, evaluation of shifts in bacterial community composition, analysis of the presence of catabolic genes associated with sulfonylurea herbicide degradation and determination of their abundance in all experimental variants. Overall, for the susceptible biotype, the biodiversity of the root microbiome was higher compared to shoot microbiome; however, both decreased notably after herbicide or HIL applications. The herbicide-resistant biotype showed lower degree of biodiversity changes, but shifts in community composition occurred, particularly in case of HIL treatment.

Discovering and engineering herbicide-resistant genes is a crucial challenge in crop breeding. This study focuses on the 4-hydroxyphenylpyruvate dioxygenase Inhibitor Sensitive 1-Like (HSL) protein, prevalent in higher plants and exhibiting weak catalytic activity against many β-triketone herbicides (β-THs). The crystal structures of maize HSL1A complexed with β-THs were elucidated, identifying four essential herbicide-binding residues and explaining the weak activity of HSL1A against the herbicides. Utilizing an artificial evolution approach, we developed a series of rice HSL1 mutants targeting the four residues. Then, these mutants were systematically evaluated, identifying the M10 variant as the most effective in modifying β-THs. The initial active conformation of substrate binding in HSL1 was also revealed from these mutants. Furthermore, overexpression of M10 in rice significantly enhanced resistance to β-THs, resulting in a notable 32-fold increase in resistance to methyl-benquitrione. In conclusion, the artificially evolved M10 gene shows great potential for the development of herbicide-resistant crops.

Nicosulfuron-resistant biotype (R) and -sensitive biotype (S) Amaranthus retroflexus L. seeds were subjected to different temperature, light, salt, osmotic potential, pH value and burial depth treatments. The difference in germination response of two populations to the above abiotic environmental factors was used to study the fitness cost of nicosulfuron-resistance evolution in A. retroflexus. The aim is to find a powerful tool for weed control in the presence of evolutionary resistance selection. The results of this experiment showed that the germination rate and germination index in S population were generally higher than that in R population. When the salt stress was 80 mM, the water potential was -0.1 Mpa ~ -0.4 Mpa, and under strong acid and alkali conditions, the germination index in S population was prominently higher than that in R population (p<0.05). The delayed seed germination in R population indicated that its nicosulfuron resistance may be linked to seed biochemical compositions that altered seed germination dynamics. The resistant and sensitive biotype of A. retroflexus had differently favourable adaptability in diverse environments. Salt, osmotic potential and pH value are not the major constraints for A. retroflexus germination, however, A. retroflexus are strongly responsive to temperature, light and burial depth. Considering that seeds of A. retroflexus are unable to reach the soil surface beyond the depth of 6 cm, deep inversion tillage before sowing may be an effective and economical weed management tool for the control of nicosulfuron resistant A. retroflexus.

BACKGROUND: Base editing is a powerful tool for artificial evolution to create allelic diversity and improve agronomic traits. However, the great evolutionary potential for every sgRNA target has been overlooked. And there is currently no high-throughput method for generating and characterizing as many changes in a single target as possible based on large mutant pools to permit rapid gene directed evolution in plants.
RESULTS: In this study, we establish an efficient germline-specific evolution system to screen beneficial alleles in Arabidopsis which could be applied for crop improvement. This system is based on a strong egg cell-specific cytosine base editor and the large seed production of Arabidopsis, which enables each T1 plant with unedited wild type alleles to produce thousands of independent T2 mutant lines. It has the ability of creating a wide range of mutant lines, including those containing atypical base substitutions, and as well providing a space- and labor-saving way to store and screen the resulting mutant libraries. Using this system, we efficiently generate herbicide-resistant EPSPS, ALS, and HPPD variants that could be used in crop breeding.
CONCLUSIONS: Here, we demonstrate the significant potential of base editing-mediated artificial evolution for each sgRNA target and devised an efficient system for conducting deep evolution to harness this potential.

Avena fatua L. is one of the most damaging and malignant weeds in wheat fields in China. Fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon, which belong to Acetyl-CoA carboxylase- (ACCase), acetolactate synthase- (ALS), and photosystem II- (PS II) inhibitors, respectively, are commonly used in wheat fields and have a long history of use on A. fatua. An A. fatua population (R) resistant to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon was collected from a wheat field in 2020. This study explored the mechanisms of target site resistance (TSR) and non-target site resistance (NTSR) in the multi-resistant A. fatua. Whole-plant bioassays showed that the R population had evolved high resistance to fenoxaprop-P-ethyl and moderate resistance to mesosulfuron-methyl and isoproturon. However, no mutations were detected in the ACCase, ALS, or psbA genes in the R population. In addition, the ACCase and ALS gene expression levels in the R group were significantly higher than those in the susceptible population (S) after treatment with fenoxaprop-P-ethyl or mesosulfuron-methyl. In vitro ACCase and ALS activity assays showed that ACCase and ALS from the R population were insensitive to fenoxaprop and mesosulfuron-methyl, respectively, with resistance indices 6.12-fold and 17.46-fold higher than those of the S population. Furthermore, pretreatment with P450 inhibitors significantly (P < 0.05) reversed the multi-resistant A. fatua\'s resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon. Sethoxydim, flucarbazone‑sodium, chlortoluron, and cypyrafluone were effective in controlling multi-resistance A. fatua. Therefore, the overexpression of ACCase and ALS to synthesize sufficient herbicide-targeting proteins, along with P450-mediated metabolism, conferred resistance to fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon in the R population.

This study focuses on dilution effect of target-site resistance (TSR) to acetolactate synthase (ALS) inhibitors in Schoenoplectiella juncoides, which harbors two ALS genes, ALS1 and ALS2. We assessed gene expression, enzyme activity, and whole-plant resistance profiles across four S. juncoides lines: the susceptible line, the parental resistant lines with a homozygous mutation in either ALS1 or ALS2, and the bred progeny line with homozygous mutations in both ALS1 and ALS2. Gene expression and enzyme function showed a proportional relationship that the expression ratios of ALS1 to ALS2, approximately 70:30, were consistent with the functional ratio predicted by the double-sigmoidal plateau positions observed in enzyme assays. However, at the whole-plant level, resistance did not correlate to the putative abundance of susceptible enzyme, but the parental lines showed similar resistance to each other despite different enzyme-level resistances. This suggests a non-proportional mechanism in the reflection of physiological enzymatic profiles to whole-plant resistance profiles. These findings highlight the complexity of herbicide resistance and the need for further research to understand the mechanisms that influence resistance outcomes. Understanding these relationships is essential for developing strategies to manage herbicide resistance effectively.