Alleviating effect of exogenous humic acid on drought stress in maize at the jointing stage

Drought stress during the jointing stage critically constrains maize productivity, particularly under the context of increasing frequency of prolonged drought events. Humic acid water-soluble fertilizer (HA-WSF) has emerged as a promising biostimulant for enhancing crop stress tolerance, yet its dynamic regulatory mechanisms remain inadequately understood, especially regarding stage-specific responses and recovery processes. To investigate the physiological regulation and stage-specific response characteristics of exogenous humic acid in alleviating drought stress in maize, a pot experiment was conducted using the maize variety Zhengdan 958. HA-WSF at concentrations of 0, 0.50, 0.75, 1.00, 1.25, and 1.50 g/L was foliar-applied at the six-leaf stage. Plants were subsequently subjected to a 12-day moderate drought treatment at the jointing stage, followed by an 8-day rewatering period to simulate post-rainfall recovery. Physiological parameters including leaf relative water content (RWC), water loss rate (WLR), SPAD value, antioxidant enzyme activities (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD), oxidative damage markers (malondialdehyde, MDA; hydrogen peroxide, H₂O₂), osmotic regulators (proline, Pro; soluble sugar, SS), and soluble protein (SP) were systematically measured at four time points (0, 6, 12, and 20 days after drought initiation).Results revealed that HA-WSF application alleviated drought-induced damage through integrated physiological pathways, with effects exhibiting pronounced stage- and concentration-dependent patterns. At 6 days of drought, a concentration of 0.75-1.00 g/L primarily maintained leaf hydration by reducing water loss and activating SOD and CAT, constituting an early physical–antioxidant defense. At 12 days of drought, the optimal concentration shifted to 1.25 g/L, with regulatory focus transitioning to osmotic adjustment driven by coordinated accumulation of Pro and SS, alongside sustained SOD and CAT activities. Partial least squares path modeling further demonstrated that the antioxidant system served as the core mediator in reducing oxidative damage, exhibiting a direct negative effect on oxidative damage (path coefficient –1.05, P < 0.001) and explaining 72.2% of its variance, whereas osmotic adjustment showed no direct effect. The total effect of HA-WSF concentration on oxidative damage was –0.30, with the indirect effect via the antioxidant system accounting for 192% of the total effect, underscoring the predominant role of antioxidant defense. Notably, POD exhibited distinct functional divergence from SOD and CAT, showing significant positive correlations with MDA and H₂O₂, suggesting its involvement in structural defense rather than direct ROS scavenging under prolonged stress. Following rewatering, optimal recovery of SPAD value and SP content was achieved at 1.00 g/L HA-WSF, indicating accelerated photosynthetic restoration and highlighting the carryover benefits of pre-stress biostimulant application. Collectively, the optimal foliar application concentration range for enhancing drought tolerance was identified as 0.75–1.25 g/L.This study systematically reveals a multi-stage physiological integration mode whereby HA-WSF sequentially improves maize drought tolerance and post-rewatering recovery capacity via synergistic pathways coordinating water balance, antioxidant defense, and osmotic adjustment, which provides a reference perspective for exploring the dynamic regulatory mechanisms of humic acid under crop abiotic stress.