Up-regulation of bZIP88 transcription factor is involved in resistance to three different herbicides in both Echinochloa crus-galli and E. glabrescens.

The resistance of weeds to herbicides poses a major threat to agricultural production, and non-target-site resistance (NTSR) is often a serious problem as its mechanisms can in some cases confer resistance to herbicides with different modes of action. In this study, we hypothesized that bZIP transcription factors (TFs), which regulate abiotic stress responses in many plants, play a regulatory role in NTSR. Whole-plant assays indicated that the wild grasses Echinochloa crus-galli and E. glabrescens are resistant to the herbicides penoxsulam, cyhalofop-butyl, and quintrione. Transcriptome sequencing then identified 101 and 49 bZIP TFs with differential expression following penoxsulam treatment in E. crus-galli and E. glabrescens, respectively. Twelve of these genes had >60% homology with rice genes. The expression of bZIP88 was considerably up-regulated 6 h after treatment with the three different herbicides, and it was similar between resistant and susceptible populations; however, the relative expression levels before herbicide treatment and 24 h after were the same. We used rice (Oryza sativa ssp. japonica cv Nipponbare) as a model system for functional validation and found that CRISPR-Cas9-knockout of the rice bZIP88 ortholog increased the sensitivity to herbicide, whereas overexpression reduced it. The OsbZIP88 protein was localized to the nucleus. Using ChIP coupled with high-throughput sequencing, OsbZIP88 was found to form a network regulatory center with other TFs such as bZIP20/52/59 to regulate OsKS1, OsCOE1, and OsIM1, which are related to auxin, abscisic acid, brassinosteroids, and gibberellic acid. Based on these results, we have established a database of bZIP TFs corresponding to herbicide stress, and resolved the mechanisms of the positive regulation of herbicide resistance by bZIP88, thereby providing new insights for NTSR.