Abstract
Humic substances (HS) are widely recognized as plant biostimulants, yet the molecular mechanisms underlying their mode of action remain incompletely defined. Here, we used RNA sequencing to investigate the early transcriptional responses of rice roots exposed for only 4 h to vermicompost-derived humic acid (HA). Our results reveal a rapid and pronounced transcriptional reprogramming consistent with the establishment of a eustress-like physiological state. HA treatment induced 231 genes, whereas only seven were repressed, indicating a predominantly stimulatory effect on gene expression. The induced genes were significantly enriched in functional categories related to redox homeostasis, glutathione metabolism, oxidoreductase and peroxidase activities, and cellular detoxification, suggesting an immediate adjustment of intracellular redox balance and antioxidant capacity. Concomitantly, pathways associated with phenylpropanoid metabolism, oxylipin biosynthesis, and jasmonate-mediated signaling were activated, together with the induction of transcription factors from the WRKY and C2H2 zinc finger families, supporting early regulatory control of defense-related networks. The coordinated activation of redox- and hormone-associated pathways indicates that HA rapidly modulates ROS-dependent signaling and integrates it with jasmonate-centered responses. Importantly, this transcriptional signature is consistent with a eustress-like state in which defense and adaptive mechanisms are mobilized without evidence of acute stress injury. Collectively, our findings establish a mechanistic framework for HS action in which HA functions as a chemical eustressor that rapidly reconfigures redox-hormone crosstalk, positioning reactive oxygen species signaling as a central integrative hub underlying the biostimulant and adaptive effects of humic substances in plants.