Physiological and biophysical evidence associates FeRbohB with rutin-mediated responses to PEG-simulated water deficit during common buckwheat germination

Drought stress limits seed germination in common buckwheat, yet the links between flavonoid treatment, physiological stress responses, and reactive oxygen species (ROS) homeostasis remain incompletely defined. Rutin, a major buckwheat flavonoid, has been associated with stress protection, but its physiological response modules and candidate rutin-associated proteins under PEG-simulated water deficit remain unclear. Here, we combined assays using PEG-simulated water deficit, integrated physiological analysis, RNA-seq-based candidate prioritization, structural modelling, construct-level interaction assessment, quantitative real-time PCR ( qRT-PCR), and heterologous transient expression to investigate rutin-associated responses under PEG-simulated water deficit. Rutin improved seed germination, was associated with lower superoxide-related, hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA) endpoints, and enhanced antioxidant enzyme activities and osmotic-adjustment compounds. Integrated correlation analysis, principal component analysis (PCA), grey relational analysis, and exploratory partial least squares structural equation modelling (PLS-SEM) described covariance among physiological indicators. The PLS-SEM showed a positive association between antioxidant-system and osmotic-adjustment scores (β = 0.968), whereas the partial path from the antioxidant system to oxidative-damage markers (β = −0.880) had a bootstrap interval spanning zero and did not establish a protective causal pathway. Intersecting 88 homology-mapped rutin-associated candidate proteins with genes differentially expressed under PEG-simulated water deficit identified seven candidates. The original protein–protein interaction (PPI) and single-fit Random Forest (RF) analyses prioritized FeRbohB; both were interpreted as exploratory computational evidence rather than independent biological validation. FeRbohB was retained for downstream characterization because it was an RBOH-family candidate with direct relevance to ROS biology. Molecular docking and a 100 ns molecular dynamics (MD) trajectory provided an exploratory structural hypothesis, and microscale thermophoresis (MST) supported an apparent micromolar-affinity interaction between rutin and the engineered soluble His-FeRbohB ΔTM -mCherry construct under the assay conditions; this construct-level result does not establish interaction with native full-length FeRbohB in planta. qRT-PCR in common buckwheat provided evidence at the expression level, whereas transient expression in Nicotiana benthamiana provided phenotypic support in a heterologous system. Collectively, these findings support FeRbohB as a candidate rutin-associated protein linked to ROS homeostasis during PEG-simulated water deficit, but they do not establish that FeRbohB is required for rutin-associated protection or that rutin regulates the NADPH oxidase activity of native full-length FeRbohB.