氮（N）是植物生长发育必需的大量元素，在光合碳同化、蛋白质生物合成及能量代谢等关键生理过程中发挥着不可替代的作用。植物根系主要通过对铵态氮（NH₄⁺-N）和硝态氮（NO₃⁻-N）的跨膜转运完成氮的吸收，这两种氮的形态在代谢途径、转运机制及信号调控等方面均存在显著差异。锌（Zn）是植物必需的微量元素，其吸收和转运受F-bZIP转录因子调控。F-bZIP转录因子OsbZIP48是水稻缺锌反应的核心调节因子，直接正向调控ZIP（Zinc-regulated transporter, Iron-regulated transporter-like (ZRT-IRT) Protein）蛋白的表达。

锌和氮之间存在相互作用，铵态氮是水稻吸收氮素的主要形式，，铵态氮是否以及如何参与锌吸收与转运的调控尚未明确。本研究以水稻为研究材料，通过分子生物学、生物化学及细胞生物学等分析手段，系统解析了OsbZIP48参与铵态氮调控锌吸收与转运的分子机制。具体研究结果如下：

1. 在不同形态（NH₄NO₃/(NH₄)₂SO₄）及浓度梯度的氮源处理下，发现硝态氮（NH₄NO₃）存在时，正常锌（0.4 μM）有利于水稻日本晴生长；氮浓度的升高促进水稻地上部生长，但高NH₄NO₃（1.25 mM）导致根部生物量显著减小；而低锌（0.004 μM）条件下植株矮小，难以利用氮素，高NH₄NO₃显著抑制水稻对锌的吸收（根部抑制达38.6 %，地上部抑制达57.7 %）；当仅铵态氮（(NH₄)₂SO₄）存在时，高铵（2.5 mM、5 mM）显著抑制水稻生长，且同样显著抑制锌的吸收（根部抑制58.9 %，地上部抑制66.7 %）。同时，相较于正常锌处理，低锌导致水稻对铵态氮的吸收与积累量增加（根部增加35.4 %，地上部增加21.9 %），铵态氮积累的增加可能进一步增强了水稻对铵毒的敏感性。因此，我们推断铵态氮在抑制过程中发挥重要作用。

2. 为了明确OsbZIP48是否参与铵态氮抑制锌吸收，本研究以（NH₄NO₃、(NH₄)₂SO₄、KNO₃）三种氮形式培养野生型水稻。结果显示，铵态氮显著抑制OsbZIP48蛋白的表达（23.5 %），缺锌（0 μM）时，铵态氮显著诱导OsbZIP48蛋白表达（20.0 %）；高浓度的NH₄NO₃同样显著诱导OsbZIP48蛋白的表达（正常锌诱导19.0 %，缺锌诱导47.0 %）；高硝态氮也显著诱导OsbZIP48蛋白表达，并且诱导倍数更高（正常锌诱导149.0 %，缺锌诱导66.2 %）。为进一步确认铵态氮对OsbZIP48蛋白的作用，使用不同浓度锌与铵营养液对ProOsbZIP48::OsbZIP48-GFP回补水稻进行处理，在激光共聚焦显微镜下观察根尖细胞中GFP荧光信号强度变化，结果发现，正常锌处理的水稻中，高铵时的细胞GFP信号显著弱于低铵，进一步说明正常锌供应时高铵抑制OsbZIP48蛋白的表达。

3.为了探究高铵抑制水稻对锌的吸收是否与ZIP家族锌转运蛋白基因的表达相关，本研究在正常锌和低锌条件下，对负责锌吸收与转运的主要基因OsZIP4、OsZIP8与OsZIP9的表达进行了检测，结果发现，正常锌时，高铵显著抑制根部OsZIP9基因表达，上调OsZIP8表达，且铵浓度越高，对OsZIP9的抑制作用越强，表明OsZIP9调控水稻根际对锌的吸收能力受高铵抑制。同时发现，低锌诱导OsZIP4与OsZIP8的表达，高铵进一步诱导地上部OsZIP4的表达。由此可知在高铵胁迫下，水稻根部OsZIP9的表达量显著下调，致使降低了锌离子的吸收；与此同时，铵态氮在地上部大量积累，进而加剧了水稻对铵毒的敏感性。

4.由于正常锌条件下，高铵抑制OsbZIP48蛋白的表达，且抑制OsZIP9的表达。为了验证OsbZIP48在铵态氮调控锌吸收与转运中的具体作用，本研究在硝酸铵和硫酸铵处理下，观察OsbZIP48的突变体（osbzip48-1）与野生型日本晴的生理表型，发现硝态氮存在的情况下，osbzip48-1突变体的锌含量较野生型显著减少；而在铵态氮作为唯一氮源时，OsbZIP48的突变的锌含量较野生型无显著差异，则表明高铵对锌吸收的抑制作用依赖于OsbZIP48功能。

综上所述，本研究初步揭示了铵态氮调控水稻锌吸收与转运的分子机制：详细剖析不同形态氮源在水稻锌吸收过程中所产生的影响并初步阐明OsbZIP48在该过程中发挥的作用，为深入理解植物氮锌协同利用的分子机制提供新的理论依据。本研究对培育氮锌利用效率双高的水稻新品种、制定更为科学合理的锌肥施用策略，以及夯实粮食安全根基，具有重要的理论价值与实践指导意义。

Nitrogen (N), an essential macronutrient for plant growth and development, plays an irreplaceable role in critical physiological processes such as photosynthetic carbon assimilation, protein biosynthesis, and energy metabolism. Plant roots primarily accomplish nitrogen uptake through transmembrane transport of ammonium nitrogen (NH₄⁺-N) and nitrate nitrogen (NO₃⁻-N), with these two nitrogen forms exhibiting significant differences in metabolic pathways, transport mechanisms, and signaling regulation. Zinc (Zn), a vital micronutrient for plants, has its uptake and transport regulated by F-bZIP transcription factors. The F-bZIP transcription factor OsbZIP48 serves as a core regulator of rice's zinc-deficiency response, directly and positively controlling the expression of ZIP (Zinc-regulated transporter, Iron-regulated transporter-like (ZRT-IRT) Protein) proteins.

The interaction between zinc and nitrogen has been noted, with ammonium nitrogen being the main form of nitrogen absorbed by rice. However, whether and how ammonium nitrogen is involved in the regulation of zinc absorption and transport remains unclear. In this study, using rice as the research material, we systematically analyzed the molecular mechanism by which OsbZIP48 is involved in ammonium nitrogen-regulated zinc absorption and transport through molecular biology, biochemistry, and cell biology analysis methods. The specific research results are as follows:

Under treatments with different forms (NH₄NO₃/(NH₄)₂SO₄) and concentration gradients of nitrogen sources, it was found that when nitrate nitrogen (NH₄NO₃) was present, normal zinc (0.4 μM) was beneficial to the growth of Nipponbare rice; an increase in nitrogen concentration promoted the aboveground growth of rice, but high NH₄NO₃ (1.25 mM) led to a significant reduction in root biomass. Under low zinc (0.004 μM) conditions, plants were dwarfed and difficult to utilize nitrogen, and high NH₄NO₃ significantly inhibited zinc absorption in rice (root inhibition reached 38.6 %, aboveground inhibition reached 57.7 %). When only ammonium nitrogen ((NH₄)₂SO₄) was present, high ammonium (2.5 mM, 5 mM) significantly inhibited rice growth and also significantly inhibited zinc absorption (root inhibition 58.9%, aboveground inhibition 66.7 %). Meanwhile, compared with normal zinc treatment, low zinc led to an increase in the absorption and accumulation of ammonium nitrogen in rice (35.4 % increase in roots, 21.9 % increase in aboveground parts), and the increased accumulation of ammonium nitrogen may further enhance the sensitivity of rice to ammonium toxicity. Therefore, we infer that ammonium nitrogen plays an important role in the inhibition process.

To clarify whether OsbZIP48 is involved in the inhibition of zinc absorption by ammonium nitrogen, this study cultured wild-type rice with three nitrogen forms (NH₄NO₃, (NH₄)₂SO₄, KNO3). The results showed that ammonium nitrogen significantly inhibited the expression of OsbZIP48 protein (23.5 %), and when zinc was deficient (0 μM), ammonium nitrogen significantly induced the expression of OsbZIP48 protein (20.0 %); high concentration of NH₄NO₃ also significantly induced the expression of OsbZIP48 protein (19.0 % induction under normal zinc, 47.0 % induction under zinc deficiency); high nitrate nitrogen also significantly induced the expression of OsbZIP48 protein, and the induction fold was higher (149.0 % induction under normal zinc, 66.2 % induction under zinc deficiency). To further confirm the effect of ammonium nitrogen on OsbZIP48 protein, ProOsbZIP48::OsbZIP48-GFP complemented rice was treated with different concentrations of zinc and ammonium nutrient solutions, and the change in GFP fluorescence signal intensity in root tip cells was observed under a laser confocal microscope. It was found that in rice with normal zinc treatment, the cellular GFP signal under high ammonium was significantly weaker than that under low ammonium, further indicating that high ammonium inhibits the expression of OsbZIP48 protein under normal zinc supply.

To explore whether high ammonium inhibition of zinc absorption in rice is related to the expression of ZIP family zinc transporter genes, this study detected the expression of the main genes responsible for zinc absorption and transport, OsZIP4, OsZIP8, and OsZIP9, under normal zinc and low zinc conditions. The results showed that under normal zinc, high ammonium significantly inhibited the expression of OsZIP9 gene in roots and up-regulated the expression of OsZIP8, and the higher the ammonium concentration, the stronger the inhibitory effect on OsZIP9, indicating that OsZIP9-regulated zinc absorption capacity in the rhizosphere of rice is inhibited by high ammonium. It was also found that low zinc induced the expression of OsZIP4 and OsZIP8, and high ammonium further induced the expression of OsZIP4 in the aboveground parts. It can be seen that under high ammonium stress, the expression level of OsZIP9 in rice roots is significantly down-regulated, resulting in reduced zinc ion absorption; at the same time, ammonium nitrogen accumulates in large quantities in the aboveground parts, thereby exacerbating the sensitivity of rice to ammonium toxicity.

Since under normal zinc conditions, high ammonium inhibits the expression of OsbZIP48 protein and the expression of OsZIP9. To verify the specific role of OsbZIP48 in ammonium nitrogen-regulated zinc absorption and transport, this study observed the physiological phenotypes of the OsbZIP48 mutant (osbzip48-1) and wild-type Nipponbare under ammonium nitrate and ammonium sulfate treatments. It was found that in the presence of nitrate nitrogen, the zinc content of the osbzip48-1 mutant was significantly lower than that of the wild type; while when ammonium nitrogen was the only nitrogen source, the zinc content of the OsbZIP48 mutant had no significant difference from that of the wild type, indicating that the inhibitory effect of high ammonium on zinc absorption is dependent on OsbZIP48 function.

In conclusion, this study preliminarily reveals the molecular mechanism by which ammonium nitrogen regulates zinc absorption and transport in rice: it elaborates on the effects of different forms of nitrogen sources on zinc absorption in rice and preliminarily clarifies the role of OsbZIP48 in this process, providing a new theoretical basis for in-depth understanding of the molecular mechanism of synergistic nitrogen and zinc utilization in plants. This study has important theoretical value and practical guiding significance for breeding new rice varieties with high nitrogen and zinc utilization efficiency, formulating more scientific and reasonable zinc fertilizer application strategies, and consolidating the foundation of food security.

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