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S-metolachlor is a selective pre-emergence herbicide used in dryland. However, it is challenging to employ in paddy fields due to its phytotoxic effects on rice. As a common phytohormone, Gibberellin-3 (GA3) is inferred to have the ability to alleviate herbicide phytotoxicity. This study first quantitatively verified the phytotoxicity of s-metolachlor to rice and then demonstrated the mitigative effect of GA3 on these adverse reactions. Furthermore, a transcriptome of rice seedlings subjected to different treatments was constructed to assemble the reference genes, followed by comparative metabolomics and proteomics analyses. Metabolomics revealed an enrichment of flavonoid metabolites in the group of adding GA3, and these flavonoids can eliminate ROS in plants. Proteomics analysis indicated that differential proteins were enriched in the phenylpropanoid biosynthesis pathway responsible for the synthesis of flavonoids and that the functions of most differential proteins are associated with peroxidase. The proteome, combined with the transcriptome, revealed that the expressions of proteins and genes was related to the POD activity in the group of adding GA3. It was speculated that the elimination of ROS is key to alleviating the stress of s-metolachlor on rice growth. It was inferred that the mechanism of GA3 in alleviating the phytotoxicity of the substance s-metolachlor is by increasing the activity of the POD and influencing the growth of rice seedlings through the restoration of flavonoid synthesis. In this study, we screened GA3 as a safener to alleviate the phytotoxicity of s-metolachlor on rice. On this basis, the mechanism of alleviating phytotoxicity was studied. The application range of s-metolachlor might be expanded, providing a new supplementary method for weed control and herbicide resistance management.

Plants have evolved circadian clock systems that enable biological processes to occur in tandem with periodic changes in the environment. However, it is largely unknown whether crosstalk occurs between the circadian clock and the response to herbicide in rice. We identified 19 conserved rhythmic metabolites which were response to pesticide application and their metabolic abundance peaked mainly at ZT2 or ZT14-ZT18. We found a series of glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 response genes were expressed following stable circadian rhythms. In order to determine the patterns of their temporal expression, co-expression network analysis was done on 10,467 genes that were periodically expressed throughout a 24-hour period. Next, we identified 4,031 potential direct target genes of OsCCA1 in using DAP-seq data for OsCCA1. Of these, 339, 22, 53, 53 and 63 genes showed a response to glyphosate, s-Metolachlor, fenclorim, metcamifen and GA3 application, respectively. And they were mainly phased from dusk to midnight. Interestingly, we identified significant OsCCA1 binding peaks in the promoter regions of four herbicide resistance genes, including OsCYP81A12, OsCYP81E22, OsCYP76C2, and OsCYP76C4. Finally, we found that herbicide application could affects the expression of some of the central oscillator genes of the rice circadian clock. Here, we used multi-omics data to reveal the crosstalk between the circadian clock and herbicide response processes at the epigenomics, transcriptome, and metabolome levels in rice. This work will serve as a theoretical guide for identifying rhythmic herbicide targets, leading to the creation of new herbicides or the breeding of crops resistant to herbicides.

It is necessary to study the combined toxicity of an herbicide and its safener because the two are often used in combination. S-metolachlor and its safener benoxacor have been detected in aquatic environments and can individually damage the oxidative stress system in zebrafish embryos (Danio rerio). However, only their separate toxicity in zebrafish (Danio rerio) embryo development has been reported. This study assessed the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, including acute toxicity, developmental toxicity, oxidative damage, and cell apoptosis. The 96-h LC50 values were higher in mixtures of benoxacor and S-metolachlor than in benoxacor alone. The treatments included S-metolachlor, Mix-1 (0.1 mg/L benoxacor + 0.1 mg/L S-metolachlor), Mix-2 (0.1 mg/L benoxacor + 0.3 mg/L S-metolachlor) and benoxacor alone. Embryos exposed to Mix-1 and Mix-2 had lower developmental toxicities, superoxide dismutase (SOD) activity, osx and cat expression levels than those exposed to benoxacor alone. Moreover, glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) activities, and the expressions of tbx16, nrf2, bcl2, and caspase9 were higher in the mixtures than in the benoxacor group. High-throughput RNA sequencing revealed that benoxacor had a greater effect on gene regulation than Mix-1 and Mix-2. The malformation rate, different enrichment gene numbers, and gene expression levels of hatched embryos were higher in Mix-1 than in Mix-2. The results indicate that a mixture of S-metolachlor and benoxacor has antagonistic effects in the early stage of embryo development. The mixtures can break the reactive oxygen species balance, causing abnormal cell apoptosis and developmental malformation in embryos. Besides investigating the combined toxicity of benoxacor and S-metolachlor in zebrafish embryo development, this study provides a risk assessment basis for a herbicide combined with its safener.

Barnyardgrass (Echinochloa crus-galli L.) is the most serious weed threatening rice production, and its effects are aggravated by resistance to the quinclorac herbicide in the Chinese rice fields. This study conducted a comparative proteomic characterization of the quinclorac-treated and non-treated resistant and susceptible E. crus-galli using isobaric tags for relative and absolute quantification (iTRAQ). The results indicated that the quinclorac-resistant E. crus-galli had weaker photosynthesis and a weaker capacity to mitigate abiotic stress, which suggested its lower environmental adaptability. Quinclorac treatment significantly increased the number and expression of the photosynthesis-related proteins in the resistant E. crus-galli and elevated its photosynthetic parameters, indicating a higher photosynthetic rate compared to those of the susceptible E. crus-galli. The improved adaptability of the resistant E. crus-galli to quinclorac stress could be attributed to the observed up-regulated expression of eight herbicide resistance-related proteins and the down-regulation of two proteins associated with abscisic acid biosynthesis. In addition, high photosynthetic parameters and low glutathione thiotransferase (GST) activity were observed in the quinclorac-resistant E. crus-galli compared with the susceptible biotype, which was consistent with the proteomic sequencing results. Overall, this study demonstrated that the resistant E. crus-galli enhanced its adaptability to quinclorac by improving the photosynthetic efficiency and GST activity.

Botanical compounds with herbicidal activity exhibit safety, low toxicity, and low chances of herbicide resistance development in plants. They have widespread applications in green agricultural production and the development of organic agriculture. In the present study, dihydrocoumarin showed potential as a botanical herbicide, and its phenotypic characteristics and mechanism of action were studied in barnyardgrass [Echinochloa crus-galli (L.) P.Beauv.] seedlings. The results indicated that dihydrocoumarin inhibited the growth of barnyardgrass without causing significant inhibition of rice seedling growth at concentrations ranging between 0.5 and 1.0 g/L. Additionally, dihydrocoumarin treatment could cause oxidative stress in barnyardgrass, disrupt the cell membrane, and reduce the root cell activity, resulting in root cell death. Transcriptomic analyses revealed that dihydrocoumarin could inhibit barnyardgrass normal growth by affecting the signal transduction of plant hormones. The results showed significant differential expression of plant hormone signal transduction genes in barnyardgrass. Additionally, dihydrocoumarin interfered with the expression of numerous phenylpropanoid biosynthesis genes in barnyardgrass that affect the production of various vital metabolites. We speculate that the barnyardgrass growth was suppressed by the interaction among hormones and phenylpropanoid biosynthesis genes, indicating that dihydrocoumarin can be applied as a bioherbicide to control barnyardgrass growth in rice transplanting fields.

With the expansion of saline-alkaline tolerant rice in China, the effects of salinity stress (NaCl) on quinclorac-resistant Echinochloa crus-galli (L.) Beauv (E. crus-galli) is unclear. In this study, the growth chamber experiment was conducted to test the germination and growth physiology of seven populations of E. crus-galli with quinclorac-different resistance levels which were collected from Hunan province. The results showed that a significant decrease of the germination rate and fresh weight of the plants, as well as the length of the roots and young shoots appeared, along with the increased resistance while treated with NaCl. However, no significant differences were detected between quinclorac-resistant and -susceptible E. crus-galli populations while without NaCl treatment. A further study with spectrophotometer showed that the salinity treatment resulted in the increase of the GST activity in all E. crus-galli populations, which are more obvious in those resistant biotypes, and transcriptomics revealed that salt stress reduces the adaptability of quinclorac-resistant E. crus-galli by reducing the biosynthesis, activities of antioxidant enzymes and metabolic enzyme. This study demonstrated that salinity stress (NaCl) may reduce the adaptability of quinclorac-resistant E. crus-galli.