Nitric oxide donor sodium nitroprusside enhances iron deficiency tolerance in M7 apple plants through coordinated regulation of Fe acquisition genes and physiological responses

Iron (Fe) deficiency is a widespread constraint on crop productivity, particularly in calcareous soils where elevated bicarbonate levels impair Fe uptake and utilization. Nitric oxide (NO) is a key regulator of Fe homeostasis; however, the physiological effects of exogenous NO donors such as sodium nitroprusside may reflect combined actions of NO and associated by-products. This study evaluated the effects of SNP on M7 apple plants (Malus domestica) under direct low-iron stress (45 and 2 µM Fe) and bicarbonate-induced Fe deficiency in a controlled substrate-based greenhouse system. Low Fe availability significantly reduced growth, chlorophyll content, photosynthetic rate, and leaf active Fe concentration. SNP supplementation, particularly at 200 µM, partially alleviated these constraints and enhanced ferric chelate reductase activity together with the expression of Fe-acquisition genes (FIT, FRO2-like, and IRT1). Notably, sustained activation of these genes despite partial recovery of leaf active Fe indicates that NO reinforces Strategy I Fe-deficiency signaling rather than fully restoring Fe nutritional status. Under bicarbonate stress, these responses persisted, suggesting additional physiological limitations beyond rhizosphere alkalinization. Although soils can emit NO through microbial processes, its bioavailability is variable and may not consistently sustain Fe acquisition under severe Fe limitation. Therefore, exogenous NO application under controlled conditions provides mechanistic insight into NO-mediated regulation of Fe homeostasis. These findings represent an integrated physiological response to SNP treatment and require validation across genotypes and field environments to confirm NO-specific effects and practical relevance.