N and Ca are essential nutrients for apple growth and development. Studies have found that Ca content was not low under high N conditions but was poorly available. However, the underlying physiological mechanism through which N regulates Ca availability remains unclear. In this study, apple plants were supplied with N and Ca to analyse the content, in situ distribution, and forms of Ca using noninvasive micro-test technique, electron probe microanalysis, Fourier transform infrared spectroscopy, and transcriptome analysis. A potential interaction was observed between N and Ca in apple leaves. The application of high N and Ca concentration led to a CaOx content of 12.51 g/kg, representing 93.54% of the total Ca in the apple leaves. Electron probe microanalysis revealed that Ca deposited in the phloem primarily existed as CaOx rhombus-shaped crystals. Additionally, high N positively regulated oxalate accumulation in the leaves, increasing it by 40.79 times compared with low N concentration. Specifically, N induced oxalate synthesis in apple leaves by upregulating the MdICL, MdOXAC, and MdMDH genes, while simultaneously inhibiting degradation through downregulation of the MdAAE3 gene. Transcriptome and correlation analyses further confirmed oxaloacetate as the precursor for the synthesis of CaOx crystals in the apple leaves, which were produced via the ‘photosynthesis/glycolysis -oxaloacetate -oxalate -CaOx’ pathway. WGCNA identified potential regulators of the CaOx biosynthesis pathway triggered by N. Overall, the results provide insights into the regulation of Ca availability by N in apple leaves and support the development of Ca efficient cultivation technique.

中文翻译：

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氮通过促进苹果叶片中草酸盐的生物合成来降低钙的可用性


N 和 Ca 是苹果生长发育所必需的营养物质。研究发现，在高氮条件下，Ca 含量并不低，但可用性很差。然而，N 调节 Ca 可用性的潜在生理机制仍不清楚。在这项研究中，为苹果植株提供 N 和 Ca，以使用无创微测试技术、电子探针微分析、傅里叶变换红外光谱和转录组分析来分析 Ca 的含量、原位分布和形式。在苹果叶中观察到 N 和 Ca 之间存在潜在的相互作用。施用高浓度的 N 和 Ca 导致苹果叶片中 CaOx 含量为 12.51 g/kg，占苹果叶片中总 Ca 含量的 93.54%。电子探针微分析表明，沉积在韧皮部中的 Ca 主要以 CaOx 菱形晶体的形式存在。此外，高 N 正向调节叶片中草酸盐的积累，与低 N 浓度相比，草酸盐积累增加了 40.79 倍。具体来说，N 通过上调 MdICL 、 MdOXAC 和 MdMDH 基因来诱导苹果叶片中的草酸盐合成，同时通过下调 MdAAE3 基因来抑制降解。转录组和相关分析进一步证实草酰乙酸是苹果叶中 CaOx 晶体合成的前体，这些晶体是通过“光合作用/糖酵解 -草酰乙酸 -草酸盐 -CaOx”途径产生的。WGCNA 确定了 N 触发的 CaOx 生物合成途径的潜在调节因子。总体而言，结果为苹果叶片中 N 对 Ca 可用性的调节提供了见解，并支持 Ca 高效培养技术的发展。