氮素亏缺对苹果愈伤组织硝态氮吸收及同化的影响

【目的】研究硝态氮亏缺对苹果叶片愈伤组织生长及硝态氮吸收同化的影响，了解苹果愈伤组织对硝态氮亏缺的响应机制，为进一步研究缺氮处理影响愈伤组织生长发育的分子机理提供理论依据。【方法】以‘嘎拉3’组培苗叶片愈伤组织为试材进行组培试验，设置培养基中NO₃–-N亏缺和适宜两个水平 (NO₃–-浓度分别为0 mol/L和0.039 mol/L)。选取叶龄一致的功能性叶片，用灭菌手术刀片沿垂直叶脉方向划伤叶片并切除叶柄和叶尖，叶背向上平铺于MS分化培养基，暗培养3天然后转至光下7天，将长出愈伤组织的叶片分别转移至MS正常分化培养基 (CK) 和MS NO₃–-N亏缺分化培养基 (T，用NH₄Cl、KCl分别代替MS中的NH₄NO₃、KNO₃)，培养3周。在转板第0、1、3、7、14、21天分别取叶片伤口处的愈伤组织，观察其细胞形态，测定硝态氮含量、NO₃–流速、氮素同化酶活性和氮素同化酶基因相对表达量。【结果】苹果愈伤组织经NO₃–-N亏缺处理1天后，细胞体积变小，间隙变大，排列疏松，7天后细胞变形，排列无规则。愈伤组织中硝态氮含量在处理7天时达到峰值，为1.54 mg/g，显著高于对照，最大降幅出现在7天后，为13.64%。NO₃–-N亏缺处理前，NO₃–吸收速率最大，为22.38 pmol/(cm2·s)，处理1天后降幅为84.1%，处理至7天时，NO₃–已经由吸收变为外排，逆差为24.45 pmol/(cm2·s)。NR活性在处理至7天时无显著变化，7天后快速增加，增幅为19.26%。NiR活性在处理至14天时，无显著性差异，14天后上升幅度为21.83%，缺氮处理1天后，GS活性最低，为0.22 U/g，7天后稍有增加，增幅为22.9%。处理组GOGAT活性在第3天时最低，为0.088 U/g，随后酶活性增加并保持稳定，但是仍低于对照组。处理组氮代谢关键酶基因MdNR2、MdNIR、MdGS2、MdGOGAT的表达量在处理至21天时达到峰值，分别为对照组表达量的3.36、2.52、11.37和2.29倍。【结论】苹果愈伤组织对缺氮非常敏感，从第一天起就可以观测到细胞间隙变大且体积变小，对NO₃–的吸收速率逐渐降低，氮素同化酶活性基本呈逐渐降低的趋势，氮素同化酶基因表达量逐渐升高。缺氮7天后，苹果愈伤组织硝态氮含量趋于稳定，并开始外排NO₃–；氮素同化酶活性基本呈逐渐升高的趋势，氮素同化酶基因表达量进一步升高。总之，氮素亏缺处理前期提高了苹果愈伤组织对NO₃–的吸收，随着处理时间的延长，氮素代谢失衡，严重影响了细胞的形态结构，导致愈伤组织生长发育异常。

Effects of nitrogen deficiency on nitrate uptake and assimilation of apple callus

【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.