Gene mutation is a basic evolutionary mechanism in plants under selection pressure of herbicides. Such mutation has pleiotropic effects on plant growth. We systemically investigated the effects of Pro106Leu (P106L), Pro106Ser (P106S), and Thr102Ile + Pro106Ser (TIPS) mutations on EPSPS functionality and fitness traits in Eleusine indica at the biochemical and physiological levels. The affinity of natural EPSPS for glyphosate was 53.8 times higher than that for phosphoenolpyruvate (PEP), as revealed by the dissociation constant; the constant decreased in both the P106L (39.9-fold) and P106S (46.9-fold) mutants but increased in the TIPS (87.5-fold) mutant. The Km (PEP) values of the P106L, P106S, and TIPS mutants were 2.4-, 0.7-, and 4.1-fold higher than that of natural EPSPS, corresponding to resistance levels of 2.5, 1.9, and 11.4, respectively. The catalytic efficiency values (maximum reaction rates) were 0.89-, 0.94-, and 0.26-fold higher than that of natural EPSPS. The levels of metabolites related to amino acids and nucleotides were significantly reduced in the mutated plants. The fitness costs were substantial for the biomass, total leaf area, seed number, and seedling emergence throughout the growth period in the plants with P106L and TIPS mutations. These results provide insights into EPSPS kinetics and their effect on plant growth.

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Aegilops tauschii Coss. (2n = 2x = 14, DD) is a problematic weed and a rich source of genetic material for wheat crop improvement programs. We used physiological traits (plant height, dry weight biomass, Na⁺ and K⁺ concentration) and 14 microsatellite markers to evaluate the genetic diversity and salinity tolerance in 40 Ae. tauschii populations. The molecular marker allied with salinity stress showed polymorphisms, and a cluster analysis divided the populations into different groups, which indicated diversity among populations. Results showed that the expression level of AeHKT1;4 and AeNHX1 were significantly induced during salinity stress treatments (50 and 200 mM), while AeHKT1;4 showed relative expression in roots, and AeNHX1 was expressed in leaves under the control conditions. Compared with the control conditions, the expression level of AeHKT1;4 significantly increased 1.7-fold under 50 mM salinity stress and 4.7-fold under 200 mM salinity stress in the roots of Ae. tauschii. AeNHX1 showed a relative expression level of 1.6-fold under 50 mM salinity stress and 4.6-fold under 200 mM salinity stress compared with the control conditions. The results provide strong evidence that, under salinity stress conditions, AeHKT1;4 and AeNHX1 synergistically regulate the Na⁺ homeostasis through regulating Na⁺ transport in Ae. tauschii. AeNHX1 sequestrated the Na⁺ into vacuoles, which control the regulation of Na⁺ transport from roots to leaves under salinity stress conditions in Ae. tauschii.