Exogenous Proline Modulates Physiological Responses and Induces Stress Memory in Wheat Under Repeated and Delayed Drought Stress

Drought stress negatively affects plant metabolism and growth, triggering complex defence mechanisms to limit damage. This study evaluated the effectiveness of a single foliar application of 1 mM L-proline (Pro) in winter wheat (Triticum aestivum L., cv. Bohemie) in two separate experiments differing in the time interval between application and drought&mdash:7 days (experiment 1) and 35 days (experiment 2). Net photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), leaf water potential (&Psi:w), intrinsic water use efficiency (WUEi), endogenous proline content (Pro), malondialdehyde content (MDA), and maximum quantum yield of photosystem II (Fv/Fm) were measured. In experiment 1, drought markedly reduced net photosynthetic rate, transpiration rate, stomatal conductance, and leaf water potential in both drought-stressed treatments, namely, without priming plants (S) and with Pro priming plants (SPro). Pro and MDA content increased under stress. Higher E and gs in the SPro treatment indicated more effective stomatal regulation and a distinct water use strategy. Pro content was significantly lower in SPro compared to S, whereas differences in MDA levels between these treatments were not statistically significant. The second drought period (D2) led to more pronounced limitations in gas exchange in both S and SPro. Enhanced osmoregulation was reflected by lower &Psi:w (S &lt: SPro) and higher Pro accumulation in S (S &gt: SPro). The effect of exogenous Pro persisted in the form of reduced endogenous Pro synthesis and improved photosystem II protection. Rehydration of stressed plants restored all monitored physiological parameters, and Pro-treated plants exhibited a more efficient recovery of gas exchange. Experiment 2 demonstrated a long-lasting priming effect that improved the preparedness of plants for future drought events. In the SPro treatment, this stress memory supported more efficient osmoregulation, reduced lipid peroxidation, improved protection of photosystem II integrity, and a more effective restart of gas exchange following rehydration. Our findings highlight the potential of exogenous proline as a practical tool for enhancing crop resilience to climate-induced drought stress.