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The human-directed, global selection for glyphosate resistance in weeds has revealed a fascinating diversity of evolved resistance mechanisms, including herbicide sequestration in the vacuole, a rapid cell death response, nucleotide polymorphisms in the herbicide target (5-enolpyruvylshikimate-3-phosphate synthase, EPSPS) and increased gene copy number of EPSPS. For this latter mechanism, two distinct molecular genetic mechanisms have been observed, a tandem duplication mechanism and a large extrachromosomal circular DNA (eccDNA) that is tethered to the chromosomes and passed to gametes at meiosis. These divergent mechanisms have a range of consequences for the spread, fitness, and inheritance of resistance traits, and, particularly in the case of the eccDNA, demonstrate how evolved herbicide resistance can generate new insights into plant adaptation to contemporary environmental stress.

As it represents the target of the successful herbicide glyphosate, great attention has been paid to the shikimate pathway enzyme 5‐enol‐pyruvyl‐shikimate‐3‐phosphate (EPSP) synthase. However, inconsistent results have been reported concerning the sensitivity of the enzyme from cyanobacteria, and consequent inhibitory effects on cyanobacterial growth. The properties of EPSP synthase were investigated in a set of 42 strains representative of the large morphological diversity of these prokaryotes. Publicly available protein sequences were analyzed, and related to enzymatic features. In most cases, the native protein showed an unusual homodimeric composition and a general sensitivity to micromolar doses of glyphosate. By contrast, eight out of 15 Nostocales strains were found to possess a monomeric EPSP synthase, whose activity was inhibited only at concentrations exceeding 1 mM. Sequence analysis showed that these two forms are only distantly related, the latter clustering separately in a clade composed of diverse bacterial phyla. The results are consistent with the occurrence of a horizontal gene transfer event involving an evolutionarily distant organism. Moreover, data suggest that the existence of class I (glyphosate‐sensitive) and class II (glyphosate‐tolerant) EPSP synthases representing two distinct phylogenetic clades is an oversimplification because of the limited number of analyzed samples.

[This corrects the article DOI: 10.3389/fpls.2025.1516963.].

The phenylpropanoid pathway serves as a rich source of metabolites in plants and provides precursors for lignin biosynthesis. Lignin first appeared in tracheophytes and has been hypothesized to have played pivotal roles in land plant colonization. In this review, we summarize recent progress in defining the lignin biosynthetic pathway in lycophytes, monilophytes, gymnosperms, and angiosperms. In particular, we review the key structural genes involved in p -hydroxyphenyl-, guaiacyl-, and syringyl-lignin biosynthesis across plant taxa and consider and integrate new insights on major transcription factors, such as NACs and MYBs. We also review insight regarding a new transcriptional regulator, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, canonically identified as a key enzyme in the shikimate pathway. We use several case studies, including EPSP synthase, to illustrate the evolution processes of gene duplication and neo-functionalization in lignin biosynthesis. This review provides new insights into the genetic engineering of the lignin biosynthetic pathway to overcome biomass recalcitrance in bioenergy crops.

Summary Glyphosate is a popular, systemic, broad‐spectrum herbicide used in modern agriculture. Being a structural analog of phosphoenolpyruvate (PEP), it inhibits 5‐enolpyruvylshikimate 3‐phosphate synthase (EPSPS) which is responsible for the biosynthesis of aromatic amino acids and various aromatic secondary metabolites. Taking a lead from glyphosate‐resistant weeds, two mutant variants of the rice EPSPS gene were developed by amino acid substitution (T173I + P177S; TIPS‐OsEPSPS and G172A + T173I + P177S; GATIPS‐OsEPSPS). These mutated EPSPS genes were overexpressed in rice under the control of either native EPSPS or constitutive promoters (maize ubiquitin [ZmUbi] promoter). The overexpression of TIPS‐OsEPSPS under the control of the ZmUbi promoter resulted in higher tolerance to glyphosate (up to threefold of the recommended dose) without affecting the fitness and related agronomic traits of plants in both controlled and field conditions. Furthermore, such rice lines produced 17%–19% more grains compared to the wild type (WT) in the absence of glyphosate application and the phenylalanine and tryptophan contents in the transgenic seeds were found to be significantly higher in comparison with WT seeds. Our results also revealed that the native promoter guided expression of modified EPSPS genes did not significantly improve the glyphosate tolerance. The present study describing the introduction of a crop‐specific TIPS mutation in class I aroA gene of rice and its overexpression have potential to substantially improve the yield and field level glyphosate tolerance in rice. This is the first report to observe that the EPSPS has role to play in improving grain yield of rice.

Globally, CRISPR-Cas9–based genome editing has ushered in a novel era of crop advancements. Weeds pose serious a threat to rice crop productivity. Among the numerous herbicides, glyphosate [N-(phosphonomethyl)-glycine] has been employed as a post-emergent, broad-spectrum herbicide that represses the shikimate pathway via inhibition of EPSPS (5′-enolpyruvylshikimate-3-phosphate synthase) enzyme in chloroplasts. Here, we describe the development of glyphosate-resistant rice lines by site-specific amino acid substitutions (G172A, T173I, and P177S: GATIPS-m OsEPSPS ) and modification of phosphoenolpyruvate-binding site in the native OsEPSPS gene employing fragment knockout and knock-in of homology donor repair (HDR) template harboring desired mutations through CRISPR-Cas9–based genome editing. The indigenously designed two-sgRNA OsEPSPS -NICTK-1_pCRISPR-Cas9 construct harboring rice codon-optimized Sp Cas9 along with OsEPSPS -HDR template was transformed into rice. Stable homozygous T 2 edited rice lines revealed significantly high degree of glyphosate-resistance both in vitro (4 mM/L) and field conditions (6 ml/L; Roundup Ready) in contrast to wild type (WT). Edited T 2 rice lines (ER 1–6 ) with enhanced glyphosate resistance revealed lower levels of endogenous shikimate (14.5-fold) in contrast to treated WT but quite similar to WT. ER 1–6 lines exhibited increased aromatic amino acid contents (Phe, two-fold; Trp, 2.5-fold; and Tyr, two-fold) than WT. Interestingly, glyphosate-resistant Cas9-free EL 1–6 rice lines displayed a significant increment in grain yield (20%–22%) in comparison to WT. Together, results highlighted that the efficacy of GATIPS mutations in OsEPSPS has tremendously contributed in glyphosate resistance (foliar spray of 6 ml/L), enhanced aromatic amino acids, and improved grain yields in rice. These results ensure a novel strategy for weed management without yield penalties, with a higher probability of commercial release.

Glyphosate is a widely used non-selective, broad-spectrum, systemic herbicide by interfering with the biosynthesis of aromatic amino acids. However, the emergence of glyphosate-resistant weeds has driven the need for enhanced herbicide resistance in crops. In this study, we engineered two mutant variants of the tobacco EPSPS gene through amino acid substitution (TIPS-NtEPSPS and P180S-NtEPSPS). These mutated EPSPS genes were overexpressed in tobacco under the control of CaMV35S promoters. Our results demonstrate that overexpression of TIPS-NtEPSPS significantly enhances glyphosate tolerance, allowing plants to withstand up to four times the recommended dose without compromising their fitness. This research highlights the potential of the TIPS-NtEPSPS mutant to improve herbicide resistance in tobacco, offering a viable approach for effective weed management.

Sheath blight disease of rice caused by the fungal pathogen R. solani AG1‐IA remains a big threat to rice production worldwide. A limited genetic variation in rice for tolerance to this pathogen and little success in understanding how it defeats host defence are major reasons behind it. In this study, we attempted to decode the virulence spectrum of R. solani AG1‐IA in rice using time‐course transcriptome analysis and functional genomics tools. Several stage‐specific and commonly expressed genes were identified. Notably, the shikimate pathway emerged as an important pathway and was implicated in the virulence of R. solani AG1‐IA. Inhibition of the shikimate pathway by glyphosate, a known inhibitor of 5‐enolpyruvylshikimate‐3‐phosphate (EPSP) synthase, reduced the vegetative growth of R. solani AG1‐IA and several other phytopathogens. Our results were complemented by in vitro inhibition studies of RsEPSP synthase using a recombinantly expressed protein. Comparative sequence analysis of RsEPSP synthase with plants and known major phytopathogens revealed a distinct region in RsEPSP synthase. Using Nicothaiana benthamiana as a model system and stable rice transgenic lines, we demonstrated that targeting this distinct region through host‐induced gene silencing (HIGS) compromises the growth and virulence of R. solani AG1‐IA. The study lays a foundation for a deeper understanding of the identified virulence genes and establishes the shikimate pathway as a central target to control phytopathogens.

Herbicides are commonly deployed as the front-line treatment to control infestations of weeds in native ecosystems and among crop plants in agriculture. However, the prevalence of herbicide resistance in many species is a major global challenge. The specificity and effectiveness of herbicides acting on diverse weed species are tightly linked to targeted proteins. The conservation and variance at these sites among different weed species remain largely unexplored. Using novel genome data in a genome-guided approach, 12 common herbicide-target genes and their coded proteins were identified from seven species of Weeds of National Significance in Australia: Alternanthera philoxeroides (alligator weed), Lycium ferocissimum (African boxthorn), Senecio madagascariensis (fireweed), Lantana camara (lantana), Parthenium hysterophorus (parthenium), Cryptostegia grandiflora (rubber vine), and Eichhornia crassipes (water hyacinth). Gene and protein sequences targeted by the acetolactate synthase (ALS) inhibitors and glyphosate were recovered. Compared to structurally resolved homologous proteins as reference, high sequence conservation was observed at the herbicide-target sites in the ALS (target for ALS inhibitors), and in 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (target for glyphosate). Although the sequences are largely conserved in the seven phylogenetically diverse species, mutations observed in the ALS proteins of fireweed and parthenium suggest resistance of these weeds to ALS-inhibiting and other herbicides. These protein sites remain as attractive targets for the development of novel inhibitors and herbicides. This notion is reinforced by the results from the phylogenetic analysis of the 12 proteins, which reveal a largely consistent vertical inheritance in their evolutionary histories. These results demonstrate the utility of high-throughput genome sequencing to rapidly identify and characterize gene targets by computational methods, bypassing the experimental characterization of individual genes. Data generated from this study provide a useful reference for future investigations in herbicide discovery and development.

Weeds and their devastating effects have been a great threat since the start of agriculture. They compete with crop plants in the field and negatively influence the crop yield quality and quantity along with survival of the plants. Glyphosate is an important broad-spectrum systemic herbicide which has been widely used to combat various weed problems since last two decades. It is very effective even at low concentrations, and possesses low environmental toxicity and soil residual activity. However, the residual concentration of glyphosate inside the plant has been of major concern as it severely affects the important metabolic pathways, and results in poor plant growth and grain yield. In this study, we compared the glyphosate tolerance efficiency of two different transgenic groups over expressing proline/173/serine (P173S) rice glyphosate tolerant mutant gene ( ) alone and in combination with the glyphosate detoxifying encoding gene, recently characterized from . The molecular analysis of all transgenic plant lines showed a stable integration of transgenes and their active expression in foliar tissues. The physiological analysis of glyphosate treated transgenic lines at seed germination and vegetative stages showed a significant difference in glyphosate tolerance between the two transgenic groups. The transgenic plants with and genes, representing dual glyphosate tolerance mechanisms, showed an improved root-shoot growth, physiology, overall phenotype and higher level of glyphosate tolerance compared to the transgenic plants. This study highlights the advantage of led detoxification mechanism as a crucial component of glyphosate tolerance strategy in combination with glyphosate tolerant gene, which offered a better option to tackle glyphosate accumulation and imparted more robust glyphosate tolerance in rice transgenic plants.