Multi-Stage Transcriptomics Reveals TF- and lncRNA-Mediated Regulatory Networks Governing Glyphosate Resistance in Cyperus rotundus

Abstract Glyphosate resistance in Cyperus rotundus, a globally invasive weed, is increasingly threatening crop production, yet its molecular mechanisms remain unclear. Here, we performed multi-stage RNA sequencing of tubers from glyphosate-resistant and -susceptible populations across three developmental stages to investigate the transcriptional dynamics underlying resistance. Differential expression analysis revealed extensive stage-specific transcriptional reprogramming, with the strongest divergence at the III developmental stage. Integrating constitutive and stage-dependent expression patterns identified 34 high-confidence candidate resistance-associated genes. Co-expression and regulatory network analyses uncovered coordinated transcriptional modules involving WRKY, NAC, ERF, MYB, bHLH, and ARF transcription factors together with multiple long non-coding RNAs. Notably, all allelic copies of ABCC8 were consistently up-regulated in glyphosate-resistant plants, validated by RT-qPCR, and occupied a central position in the resistance regulatory network, suggesting multilayered regulation of detoxification. These findings demonstrate that glyphosate resistance in C. rotundus is a developmentally coordinated, multilayered non-target-site resistance process.