Appropriate calcium supply improves photosynthetic nitrogen use efficiency by improving nitrogen distribution and leaf ultrastructure in apple rootstock leaves

The photosynthetic nitrogen (N) use efficiency (PNUE) of apple leaves varies under different calcium (Ca) levels, but the underlying physiological mechanism remains unclear. This study systematically elucidated the synergistic regulatory mechanisms of Ca levels on N uptake, allocation, and photosynthetic performance in apple rootstock by integrating a leaf photosynthetic N allocation model, photosynthetic limiting factor analysis, and microstructural observations. Ca stress (low Ca: 0.1 mM Ca²⁺; high Ca: 15 mM Ca²⁺) significantly suppressed nitrate transporter gene (NRT) expression and reduced the net NO₃⁻ influx rate of root surfaces, thereby hindering N uptake and translocation to shoots. Low Ca treatment increased stomatal and biochemical limitations by inducing stomatal closure and reducing the maximum carboxylation rate (Vₘₐₓ). By contrast, high Ca treatment increased mesophyll conductance limitation by increasing cell wall thickness, leaf thickness, and leaf Ca oxalate content, and by decreasing Ca and N availability. Both low and high Ca treatments disrupted leaf N allocation, characterized by increased proportions of non-protein and storage N and decreased allocation to photosynthetic N components, such as carboxylate system N and electron transport N. Together, these changes reduced the leaf PNUE. By contrast, optimal Ca treatment (5 mM Ca²⁺) significantly improved the leaf net photosynthetic rate and PNUE by optimising photosynthetic N allocation, improving leaf structural integrity and reducing starch grain accumulation. This study provides a theoretical basis for precise Ca management in apple orchards.