Abstract: The nitric oxide (NO) and Ca²⁺ transduction pathways have been implicated in mediating stress response and tolerance in plants. However, little is known about the interaction between NO and Ca²⁺ signals, in regulation of photosynthesis and antioxidant capacity of alfalfa seedlings under drought stress. This research aimed to explore the mechanism by which Ca²⁺ signaling mediated by nitric oxide (NO) in Medicago sativa seedlings regulates photosynthesis and antioxidant enzyme activity under osmotic stress. Solutions with:(i) differing osmotic potentials obtained using poly ethylene-glycol-6000 (PEG); (ii) a nitric oxide donor sodium nitroprusside (SNP); (iii) an exogenous Ca²⁺ signaling donor, calcium chloride (CaCl₂); (iv) an NO blocker (methylene blue, MB); and (v) a Ca²⁺ channel blocker (LaCl₃), were pre-sprayed onto the leaves of alfalfa seedlings in different combinations. The effect of the NO-mediated Ca²⁺ signaling on the photosynthetic pigment content, gas exchange parameters, antioxidant enzyme activity, on the levels of malondialdehyde (MDA) and proline, and on the peroxidase (POD) isoenzyme electrophoresis atlas were studied in leaves of alfalfa under osmotic stress simulated by 15% PEG. Results show that the photo-physiological characteristics of alfalfa seedling were significantly affected by osmotic stress. NO and Ca²⁺ application alleviated the decrease of chlorophyll a, carotenoid contents and the total chlorophyll contents, increased net photosynthetic rate (P_n), stomatal conductance (G_s) and stomatal limited value (L_s), but reduced intercellular CO₂ concentration (C_i) in alfalfa seedling leaves. Furthermore, compared to SNP or CaCl₂ treatment alone, SNP and CaCl₂ treatments applied together significantly increased the activities of SOD, POD, CAT and the content of proline by 39.29%, 30.41%, 56.24% and 45.59%, respectively, and decreased the content of MDA by 45.59%. The POD is ozymeprofile exhibited the greatest band number and strongest enzyme activity on the fourth day, and new band appeared after combined NO and Ca²⁺ treatment. Interestingly, when exogenous Ca²⁺was added together with MB, the promotional activity of Ca²⁺was inhibited.In addition, La³⁺ inhibited the promotional effect of NO. This indicated that in leaves of alfalfa seedlings under osmotic stress, Ca²⁺ mediated signaling transduction pathways, when induced by NO, helped to maintain antioxidant activity and prevent damage to photosynthesis systems.

Key words: nitric oxide, Ca²⁺, Medicago sativa, osmotic stress, photosynthesis, antioxidant enzymes