Nitrate reductase rather than nitric oxide synthase activity is involved in 24-epibrassinolide-induced nitric oxide synthesis to improve tolerance to iron deficiency in strawberry (Fragaria x annassa) by up-regulating the ascorbate-glutathione cycle

Involvement of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme in 24-epibrassinolide (EB)-triggered nitric oxide (NO) synthesis to improve iron deficiency (ID) tolerance in strawberry plants was studied. EB was sprayed to strawberry plants every two days for two weeks. Then, the EB-treated plants were pre-treated with inhibitors of NR, tungstate, or NOS, L-NAME for 3 h. During the first three weeks, Fe was supplied as 100 mu M EDTA-Fe or FeSO4 to Fe-sufficient or Fe-deficient plants, respectively. Thereafter, plants were subjected for further three weeks to control (100 mu M EDTA-Fe) and Fe deficiency (ID; without Fe). ID reduced biomass, chlorophyll, and chlorophyll fluorescence, while increased oxidative stress parameters, ascorbate (AsA), glutathione (GSH), endogenous NO, and the activities of NR, NOS, and antioxidant enzymes. Pre-treatments with EB and EB + SNP improved ID tolerance of strawberry by improving leaf Fe2+, plant growth, and antioxidant enzyme activities, and causing a further elevation in AsA, GSH, NO, NR and NOS. L-NAME application reversed NOS activity, but it did not eliminate NO, however, tungstate application reversed both NR activity and NO synthesis in plants exposed to ID + EB, suggesting that NR is the main contributor of EB-induced NO synthesis to improve ID tolerance in strawberry plants.