Early humic acid-induced eustress triggers biostimulant responses in Arabidopsis thaliana: RBOHC-derived H2O2 drives growth promotion while RBOHD enables nutritional reprogramming.
Abstract
Root-applied humic acids (HAs) promote plant development, yet the early signaling mechanisms underlying their biostimulant action remain incompletely understood. Here, we show that treatment of Arabidopsis thaliana roots with a sedimentary humic acid (SHA) triggers a transient eustress-like response, characterized by early inhibition of shoot and root growth and increased H2O2 accumulation in root hairs and apical roots. This initial oxidative phase was followed by enhanced shoot and root growth, supporting the view that SHA acts as an adaptive plant modulator through a controlled stress-related signal. Pharmacological H2O2 scavenging and genetic analysis of NADPH oxidase mutants revealed that SHA-induced growth promotion depends on RBOHC/RHD2-mediated H2O2 production in roots. In rhd2 plants, SHA failed to induce the characteristic H2O2 increase and did not promote shoot or root growth. By contrast, rbohd plants retained SHA-induced growth stimulation but showed impaired activation of nutrient transporter and plasma membrane H+-ATPase genes, limited improvement in shoot nutrient use efficiency, and partial root architectural remodeling. These results indicate that RBOHC-derived H2O2 is required for the growth-promoting response, whereas RBOHD cooperates with RBOHC to engage broader nutritional and developmental programs. SHA also induced extensive hormonal reprogramming in roots and shoots; however, disruption of individual ABA, auxin, jasmonate, or cytokinin signaling components did not abolish growth promotion, suggesting partially redundant hormonal regulation downstream of ROS. Overall, our findings identify an RBOHC-H2O2 module as a primary entry point for SHA-mediated biostimulant action and support a model in which root-localized oxidative eustress coordinates hormonal, developmental, and nutritional reprogramming in Arabidopsis.