Objectives Investigation of the growth, nitrogen uptake and assimilation of apple callus under nitrogen deficiency can help us to understand the response mechanism of apple growth and development, and provide theoretical basis for further study of molecular mechanism of nitrogen deficiency affecting callus.

Methods Leaf callus of ‘Gala 3’ apple induced from tissue culture seedlings were used as tested materials. Differentiation medium (MS) with deficient and normal levels of NO₃^–-N (0 mol/L and 0.039 mol/L) were prepared, and the NH₄NO₃ and KNO₃ in the deficient MS were replaced by NH₄Cl and KCl. The same age of functional leaves were selected, and cut perpendicular to the vein and removed the petioles and tips using the sterilized surgical blade. The back of leaves were laid flat on MS normal differentiation medium, dark cultured for 3 days and then 7 days under light. The leaves were transferred to prepared MS medium immediately and cultured for 3 weeks. The callus was collected from the wound of leaves after cultured for 0, 1, 3, 7, 14, 21 days. The cell morphology, NO₃^–-N content, NO₃^– uptake flux, N assimilation enzyme activity and relative expression of N assimilation enzyme genes were observed and tested.

Results One day after NO₃^–-N deficiency treatment, the cells of callus became smaller in volume, and arranged in loose with larger intercellular space. After 7 days, the cells were deformed and arranged irregularly. After 7 days, the NO₃^– content in callus reached the peak at 1.54 mg/g, which was significantly higher than that of the control. The maximum decrease rate was 13.64% after 7 days. Before NO₃^–-N deficiency treatment, the NO₃^– absorption rate was the highest, which was 22.38 pmol/(cm²·s). With the prolongation of treatment time, the absorption rate decreased sharply and the decrease in the first treatment day was as high as 84.1%. After treatment for 7 days, NO₃^– was changed from uptake to efflux and the deficit was 24.45 pmol/(cm²·s). There was no significant change in NR activity within 7 days treatment, while increased rapidly in NR activity with increase rate of 19.26% after 7 days treatment. There was no significant difference in NiR activity within 14 days treatment, but the increase rate of NiR activity reached to 21.83% after 14 days treatment. After 1 day of nitrate deficiency treatment, GS activity was the lowest as 0.22 U/g. After 7 days, with a slight increase of 22.9%. The GOGAT activity was the lowest after treatment for 3 days, which was 0.088 U/g and then the enzyme activity increased and remained stable, but still lower than the control. When treated for 3 days, the relative expression of MdNR2 gene began to be higher than that of the control. After 21 days treatment, the relative expression of MdNR2, MdNIR, MdGS2, and MdGOGAT genes reached the peak, which were 3.36, 2.52, 11.37 and 2.29 times higher than those in the control, respectively.

Conclusions Apple callus cells are extremely sensitive to N deficiency. The cell response occurs after one day of nitrate deficiency treatment, the content of nitrate nitrogen increased gradually, but the absorption rate of NO₃^– decreased sharply. The activity of assimilating enzyme of nitrogen become decreasing and the expression of nitrogen assimilation enzyme gene is up-regulated. After 7 days of nitrogen deficiency treatment, the absorption of NO₃^– is transformed into efflux; the nitrogen assimilation enzyme activity increases generally, and the gene expression of nitrogen assimilation enzyme is further increased. To sum up, nitrogen deficiency leads to unbalanced nitrogen metabolism, the morphological structure of cells is seriously affected, resulting in abnormal growth and development of callus.