Different nitrogen concentrations affect strawberry seedlings nitrogen form preferences through nitrogen assimilation and metabolic pathways

doi:10.1016/j.scienta.2024.113236

Scientia Horticulturae 园艺科学

Volume 332, 1 June 2024, 113236
第 332 卷，2024 年 6 月 1 日，113236

https://doi.org/10.1016/j.scienta.2024.113236 Get rights and content 获取权利和内容

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Highlights 突出

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Strawberry seedlings showed a preference for NH₄⁺-N, NO₃⁻-N, and NH₄⁺-N under low nitrogen, medium nitrogen, and high nitrogen conditions, respectively.
草莓幼苗在低氮、中氮和高氮条件下分别表现出对 NH ₄^+-N、NO₃⁻-N 和 NH₄⁺-N 的偏好。
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The distribution of NH₄⁺-N and NO₃⁻-N in various organs was changed by the change in the concentration of N.
NH₄⁺-N 和 NO₃⁻-N 在各器官中的分布随 N 浓度的变化而变化。
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GS activity not only determines the absorption of NH₄⁺-N, but also significantly correlates with the absorption of NO₃⁻-N.
GS 活性不仅决定 NH₄⁺-N 的吸收，而且与 NO₃⁻-N 的吸收显著相关。
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NRT1.1 is the key factor affecting the preference of NO₃⁻-N in strawberry seedlings.
NRT1.1 是影响草莓幼苗 NO ₃⁻-N 偏好性的关键因子。

Abstract 抽象

The preference for different forms of nitrogen (N) is an essential means to improve plant nitrogen utilization efficiency (NUE). Currently, little information is known about the N uptake preferences of strawberries and their interaction with N concentration. In this study, the preference and distribution of nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N) in strawberry seedlings were investigated in response to different N concentrations. This study used two labeled N sources (¹⁵NH₄NO₃ and NH₄¹⁵NO₃) for tracer labeling. Three levels of NH₄NO₃ were set: low N 5 mM (N1), medium N 15 mM (N2), and high N 25 mM (N3). Each N level included two ¹⁵N labeling treatments, totaling six treatments. The results showed that under low N, medium N, and high N conditions, strawberry seedlings exhibited preferences for NH₄⁺-N, NO₃⁻-N, and NH₄⁺-N, respectively. Compared with N1 and N3 treatments, N2 treatment significantly increased the activity of NR, NiR, GS, and GOGAT in strawberry seedlings roots and leaves, and the relative expression levels of FaNRT1.1, FaNRT2.1, and FaAMT1.1 in roots were significantly upregulated; When the N supply concentration increases, the distribution center of NO₃⁻-N in strawberry seedlings shifts from stem to leaf, and under high N conditions, NH₄⁺-N accumulates significantly in the strawberry roots. According our research, the N assimilation and metabolic capacity of strawberries themselves are important factors affecting N form preferences.
偏爱不同形式的氮（N）是提高植物氮利用效率（NUE）的重要手段。目前，关于草莓的氮吸收偏好及其与氮浓度的相互作用的信息知之甚少。本研究研究了不同氮浓度对草莓幼苗中硝态氮（NO 3^−-N）和铵态氮（NH₄+-N）的偏好和分布。本研究使用两个标记的 N 来源（¹⁵NH₄NO₃ 和 NH₄¹⁵NO₃）进行示踪剂标记。 NH ₄NO₃ 的三个水平被设定为：低 N 5 mM （N1）、中 N 15 mM （N2）和高 N 25 mM （N3）。每个 N 水平包括 2 个 ¹⁵N 标记处理，共 6 个处理。结果表明：在低氮、中氮和高氮条件下，草莓幼苗分别表现出对NH₄^+-N、NO₃⁻-N和NH₄⁺-N的偏好。与 N1 和 N3 处理相比，N2 处理显著提高了草莓幼苗根和叶中 NR、NiR、GS 和 GOGAT 的活性，根中 FaNRT1.1 、 FaNRT2.1 和 FaAMT1.1 的相对表达水平显著上调;当氮素供应浓度增加时，草莓幼苗中NO 3−-N的分布中心由茎向叶移动，在高氮条件下，NH₄⁺-N在草莓根系中显著积累。根据我们的研究，草莓本身的氮同化和代谢能力是影响 N 形态偏好的重要因素。

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Keywords 关键字

Ammonium nitrogen

Nitrate nitrogen

Assimilation

Metabolism

Strawberry

铵态氮

硝酸盐氮

同化

代谢

草莓

Abbreviation 缩写

Nitrogen

Nitrate reductase

NiR

Nitrite reductase

Glutamine synthetase

GOGAT

glutamate synthase

NUE

nitrogen use efficiency

NH₄⁺-N

Ammonium N

NO₃⁻-N

Nitrate N

HATS

High-affinity transport system

LATS

Low-affinity transport system

AMT

Ammonium transporters

NRT

Nitrate transporters

氮

硝酸盐还原酶

NiR

亚硝酸盐还原酶

谷氨酰胺合成酶

GOGAT

谷氨酸合酶

NUE

氮利用效率

NH₄⁺-N

铵 N

NO₃⁻-N

硝酸盐 N

HATS

高亲和力运输系统

LATS

低亲和力运输系统

AMT

铵态氮转运蛋白

NRT

硝酸盐转运蛋白

1. Introduction 1. 引言

Strawberry (Fragaria × ananassa Duch.) is a horticultural crop with high demand for N fertilizer, and its shallow root causes poor ability to obtain nutrients. Therefore, soil fertility and N fertilization application amount are crucial factors for the growth and yield of horticultural strawberry. China has the largest strawberry planting area, reaching 1.4 × 10⁵ hm² (Wang, 2022a), with a yield of 3.7 × 10⁶ t (Wang, 2022b). In China, strawberry has higher economic benefits than other fruits and cash crops. Therefore, farmers' tendency and willingness to obtain high yield through high N fertilizer input are stronger. The investigation showed that the amount of N fertilizer input for strawberry was about 480 kg·hm⁻² (Qian et al., 2020). However, more than 90 % of farmers exceed the recommended amount of N fertilizer application (Lian et al., 2018). High input and low efficiency N fertilizer application has brought many soil and environmental problems, such as soil acidification, soil secondary salinization, water eutrophication, groundwater NO₃⁻exceeding the standard, etc. (Alam et al., 2023). Improving the nitrogen use efficiency (NUE) of strawberry is essential for sustainable development while maintaining yield and quality.
草莓（Fragaria × ananassa Duch.）是一种对氮肥需求量大的园艺作物，其根系较浅，导致获取养分的能力较差。因此，土壤肥力和施氮量是影响园艺草莓生长和产量的关键因素。中国的草莓种植面积最大，达到 1.4 × 10⁵ hm² （Wang， 2022a），产量为 3.7 × 10⁶ t （Wang， 2022b）。在中国，草莓比其他水果和经济作物具有更高的经济效益。因此，农民通过高氮肥投入获得高产的倾向和意愿更强。调查表明，草莓的氮肥投入量约为 480 kg·hm⁻² （Qian et al.， 2020）。然而，超过 90% 的农民超过了推荐的氮肥施用量（Lian 等，2018）。高投入低效氮肥施用带来了许多土壤和环境问题，如土壤酸化、土壤二次盐碱化、水体富营养化、地下水 NO₃ ^超标等（Alam et al.， 2023）。提高草莓的氮利用效率（NUE）对于保持产量和质量的可持续发展至关重要。

Nitrogen is the most demanded mineral element in the process of plant growth and development. Ammonium N (NH₄⁺-N) and nitrate N (NO₃⁻-N) are the two main forms of inorganic N absorbed by plants from soil (Li et al., 2013). Plants have evolved two absorption systems for NH₄⁺-N and NO₃⁻-N respectively to efficiently utilize inorganic N in soil, including low affinity transport systems (LATS) and high affinity transport systems (HATS) (Wang et al., 2012). Under natural conditions, soil nutrients are spatially heterogeneous and change with climate. The root system shows preference and plasticity in adapting to the changes of N concentration and form (Luo et al., 2023; Zhu et al., 2021). The absorption and distribution strategies of NH₄⁺-N and NO₃⁻-N in plants are dynamic, influenced by different biological and abiotic factors (Bai et al., 2017; Heng et al., 2022; Huang et al., 2019). Nitrate uptake by Suaeda salsa was significantly positively correlated with soil water content and N application rate, but drought and salt stress had no significant effect on ammonium ion absorption (Bai et al., 2017). Populus cathayana absorbed more NH₄⁺ than NO₃⁻ under intensive drought stress, which was related to the decrease of root vitality and NR activity (Luo et al., 2023). The change of N strategy is an important means for plants to improve NUE and deal with environmental stress.
氮是植物生长发育过程中最需要的矿物元素。铵态氮（NH₄⁺-N）和硝酸盐氮（NO₃⁻-N）是植物从土壤中吸收的两种主要无机氮形式（Li et al.， 2013）。植物已经进化出两种分别用于 NH₄⁺-N 和 NO₃⁻-N 的吸收系统，以有效利用土壤中的无机氮，包括低亲和力运输系统（LATS）和高亲和力运输系统（HATS）（Wang et al.， 2012）。在自然条件下，土壤养分在空间上是异质的，并随气候而变化。根系在适应 N 浓度和形态的变化方面表现出偏好和可塑性（Luo et al.， 2023;Zhu et al.， 2021）。NH₄⁺-N 和 NO₃^--N 在植物中的吸收和分配策略是动态的，受不同生物和非生物因素的影响（Bai等人，2017 年;Heng et al.， 2022;Huang et al.， 2019）。盐地碱地对硝酸盐的吸收与土壤含水量和氮肥施用量呈显著正相关，但干旱和盐胁迫对铵离子吸收没有显著影响（Bai et al.， 2017）。在强烈干旱胁迫下，山杨吸收的 NH ₄⁺ 比 NO₃⁻ 多，这与根系活力和 NR 活性的下降有关（Luo et al.， 2023）。氮策略的改变是植物提高氮利用率和应对环境胁迫的重要手段。

The external N concentration directly affects the N absorption and transport system of plants. More than 75 % of high-affinity NO₃⁻ absorption in plants is contributed by NRT1.1 (Ye et al., 2022). AtNRT1.1 and OsNRT1.1B in Arabidopsis thaliana and rice have dual affinity characteristics (Lin, 2000; Ye et al., 2019). AtNRT2.1 has high affinity activity, and low N conditions can induce its expression in Arabidopsis thaliana (Krouk et al., 2010). AMT1.1, AMT1.2, AMT1.3 are highly expressed in root apices and epidermal cells, collectively contributing to 90 % of the high-affinity transport system's ability to absorb NH₄⁺ (Loqué et al., 2010; Yuan et al., 2007). OsAMT1.1 is highly expressed in root epidermal and stele cells, transporting 25 % of the NH₄⁺ roots absorbed and regulating the transport of NH₄⁺ from underground to aboveground (Li et al., 2016). When the external NH₄⁺ concentration is high, the low affinity AtAMT1.2 can mediate the NH₄⁺ transmembrane transport in the apoplastic and enter the symplastic pathway (Duan et al., 2018; Yuan et al., 2007).
外部氮浓度直接影响植物的氮吸收和运输系统。植物中超过 75% 的高亲和力 NO₃⁻ 吸收是由 NRT1.1 贡献的（Ye等人，2022 年）。拟南芥和水稻中的 AtNRT1.1 和 OsNRT1.1B 具有双重亲和特性（Lin，2000 年;Ye et al.， 2019）。AtNRT2.1 具有高亲和力活性，低 N 条件可诱导其在拟南芥中的表达（Krouk et al.， 2010）。AMT1.1、AMT1.2、AMT1.3 在根尖和表皮细胞中高度表达，共同贡献了 90% 的高亲和力运输系统吸收 NH₄⁺ 的能力（Loqué等人，2010 年;Yuan et al.， 2007）。OsAMT1.1 在根表皮和柱状细胞中高度表达，运输 25% 的 NH₄⁺ 根被吸收并调节 NH₄⁺ 从地下到地上的运输（Li et al.， 2016）。当外部 NH₄ ⁺ 浓度高时，低亲和力 AtAMT1.2 可以介导质外质体中的 NH ₄ ⁺ 跨膜转运并进入共生途径（Duan等人，2018 年;Yuan et al.， 2007）。

The response of strawberries to different N concentrations varies depending on the specific periods, varieties, and cultivation conditions. Under matrix cultivation, different strawberry varieties require different optimal N levels, and appeared to be 10 mM N for ʻAltaking’, ʻKuemsil’ and ʻMaehyang’, and 20 mM N for ʻVitaberry’ (Farjana et al., 2023). When the N application level was 210 mg·kg⁻¹, the activities of glutamate dehydrogenase (GDH),glutamine synthetase (GS) and glutamate synthase (GOGAT) of ʻAkihime’ strawberry were most significantly affected (Fu et al., 2022). When the N supply concentration changed from normal to low N stress, low concentrations of NO₃⁻ and NH₄⁺ induced increased the activities of NR, GS and GOGAT, and enhanced N metabolism (Zhang et al., 2023). Under high NO₃⁻stress, the net photosynthetic rate of strawberry seedling leaves decreased, and the electron transfer of PS Ⅱ was blocked (Han et al., 2015). Chen et al. (Chen et al., 2012) found that the chlorophyll content and net photosynthetic rate of ʻSachinoka’ strawberry were highest at 15 mM NO₃⁻ concentration, and began to decrease when the NO₃⁻ concentration reached 20 mM .
草莓对不同 N 浓度的响应因特定时期、品种和栽培条件而异。在基质培养下，不同的草莓品种需要不同的最佳氮水平，“Altaking”、“Kuemsil”和“Maehyang”的氮含量为 10 mM，“Vitaberry”的氮含量为 20 mM（Farjana等人，2023 年）。当氮肥施用量为 210 mg·kg⁻¹ 时，秋姬草莓谷氨酸脱氢酶（GDH）、谷氨酰胺合成酶（GS）和谷氨酸合酶（GOGAT）活性受到最显著影响（Fu et al.， 2022）。当氮供应浓度从正常氮胁迫变为低氮胁迫时，低浓度的 NO₃⁻ 和 NH₄⁺ 诱导的 NR 、 GS 和 GOGAT 活性增加，并增强氮代谢（Zhang et al.， 2023）。在高NO₃^-胁迫下，草莓幼苗叶片的净光合速率降低，PS II.的电子转移受阻（Han et al.， 2015）。Chen 等人（Chen et al.， 2012）发现，Sachinoka' 草莓的叶绿素含量和净光合速率在 15 mM NO₃⁻ 浓度时最高，当 NO₃⁻ 浓度达到 20 mM 时开始下降。

For most crops, mixed N sources are more beneficial to plant growth and N absorption than single N sources (Dong et al., 2012; Sun et al., 2017). When Pang et al. (Pang et al., 2019) used one of NH₄⁺-N and NO₃⁻-N as the sole N source in the nutrient solution, it has a certain inhibitory effect on plant height, stem diameter, root number, and root length. A large number of studies have shown that when the ratio of NH₄⁺-N and NO₃⁻-N is 1:1, strawberry growth is better than other ratios (Roosta, 2014; Shi et al., 2021; Tabatabaei et al., 2008). In addition, our previous research observed that under the equal supply of NH₄⁺ and NO₃⁻ with a total N concentration of 15 mM in the nutrient solution, the strawberry plant growth was better than that of 5 mM and 25 mM (Jia et al., 2022). When NH₄⁺-N and NO₃⁻-N coexist, there is an interaction between them. Compared with the supply of NO₃⁻ alone, mixed N supply improves the activity of nitrate reductase (NR) and glutamine synthetase (GS) and the content of amino acids in maize roots, which is also considered to be the main driving force for maize growth (Wang et al., 2023). Zhu et al. (Zhu et al., 2019) reported that NO₃⁻ has a positive effect on NH₄⁺ absorption, while NH₄⁺ has an inhibitory effect on NO₃⁻ absorption. Similarly, similar effects were observed in wheat (Zhong et al., 2014); however, in tea trees, the opposite effect was observed (Ruan et al., 2016).
对于大多数作物，混合氮源比单一氮源更有利于植物生长和氮吸收（Dong et al.， 2012;Sun等人，2017 年）。当 Pang et al. （Pang et al.， 2019）使用 NH₄⁺-N 和 NO₃⁻-N 中的一种作为营养液中的唯一 N 源时，对株高、茎粗、根数和根长具有一定的抑制作用。大量研究表明，当 NH₄⁺-N 和 NO₃⁻-N 的比例为 1：1 时，草莓的生长优于其他比例（Roosta，2014 年;Shi et al.， 2021;Tabatabaei et al.， 2008）。此外，我们之前的研究观察到，在营养液中总 N 浓度为 15 mM 的 NH ₄⁺ 和 NO₃⁻ 等量供应下，草莓植株的生长优于 5 mM 和 25 mM（Jia et al.， 2022）。当 NH₄⁺-N 和 NO₃⁻-N 共存时，它们之间存在相互作用。与单独供应 NO₃⁻ 相比，混合氮供应提高了硝酸还原酶（NR）和谷氨酰胺合成酶（GS）的活性以及玉米根系氨基酸的含量，这也被认为是玉米生长的主要驱动力（Wang et al.， 2023）。Zhu 等人。（Zhu et al.， 2019）报道 NO₃⁻ 对 NH ₄⁺ 吸收有积极影响，而 NH₄⁺ 对 NO ₃⁻ 吸收有抑制作用。同样，在小麦中也观察到了类似的效果（Zhong et al.， 2014）;然而，在茶树中，观察到了相反的效果（Ruan et al.， 2016）。

Based on the previous studies mentioned above, our scientific speculation is that there is an interaction between strawberry's preference for different N forms and N concentration. Therefore, this study used a sand-cultivated pot experiment, selecting ammonium nitrate (NH₄NO₃) as the N source for strawberries.The NH₄⁺ and NO₃⁻ in NH₄NO₃ were labeled with ¹⁵N isotopes, respectively. Three N levels were set. The objectives of this study are: (1) to clarify the preference and accumulation differences of strawberry for two N forms (NH₄⁺-N and NO₃⁻-N) under different N concentrations; (2) tried to uncover the possible explanations for the different N forms preference by assessing changes of genes and enzyme activity involved in N utilization, assimilation.
基于上述前述研究，我们的科学推测是，草莓对不同 N 形式的偏好与 N 浓度之间存在交互作用。因此，本研究采用沙培盆栽实验，选择硝酸铵（NH₄NO₃）作为草莓的氮源。 NH₄NO₃ 中的 NH ₄⁺ 和 NO₃⁻ 分别用 ¹⁵N 同位素标记。设置了三个 N 水平。本研究的目的是：（1）阐明不同 N 浓度下草莓对两种 N 形式（NH₄^+-N 和 NO₃⁻-N）的偏好和积累差异;（2）试图通过评估参与 N 利用、同化的基因和酶活性的变化来揭示不同 N 形式偏好的可能解释。

2. Materials and methods 2. 材料和方法

2.1. Experimental sites and materials
2.1. 实验地点和材料

The experiment was conducted in the greenhouse of Xuzhou Agricultural Science Research Institute. The test strawberry variety was Ssanta. In March 10, 2022, strawberry seedlings with consistent growth and health were selected (plant height of about 5 cm, two leaves) and planted in plastic pots filled with vermiculite and perlite (1:1). The pot body had a diameter of 12 cm at the top and 10.5 cm at the bottom, and a height of 12 cm. Within 7 days after planting, 200 ml of deionized water was poured every day. After 7 days, nutrient solution was poured, with 300 ml of nutrient solution poured every 3 days. The nutrient solution was a modified Japanese Yamazaki formula nutrient solution, including 40 mg·L ⁻ ¹ NH₄NO₃ (Contains 10 % labeled NH₄NO₃), 217.5 mg·L ⁻ ¹ K₂SO₄, 68 mg·L ⁻ ¹ KH₂PO₄, 111 mg·L ⁻ ¹ CaCl₂, 123 mg·L ⁻ ¹ MgSO₄·7H₂O, 2.68 mg·L ⁻ ¹ H₃BO₃, 2.13 mg·L ⁻ ¹ MnSO₄·4H₂O, 0.22 mg·L ⁻ ¹ ZnSO₄·7H₂O, 0.08 mg·L ⁻ ¹ CuSO₄·5H₂O, 0.02 mg·L ⁻ ¹ (NH₄)₆Mo₇O₂₄·4H₂O, and 30.00 mg·L ⁻ ¹ EDTA-NaFe. All nutrient solutions were supplemented with nitrification inhibitor dicyandiamide (C₂H₄N₄, 7 μM) to inhibit nitrification.
该实验在徐州市农业科学研究院的温室中进行。测试的草莓品种是 Ssanta。2022 年 3 月 10 日，选择生长健康一致的草莓幼苗（株高约 5 厘米，两片叶子），种植在装满蛭石和珍珠岩的塑料盆中（1：1）。壶身顶部直径为 12 厘米，底部直径为 10.5 厘米，高度为 12 厘米。种植后 7 天内，每天倒入 200 ml 去离子水。7 天后，倒入营养液，每 3 天倒入 300 ml 营养液。营养液为改良日本山崎配方营养液，含40 mg·L⁻¹ NH₄NO₃（含10 %标记NH₄NO₃），217.5 mg·L⁻¹ K₂SO₄， 68 mg·L⁻¹ KH₂PO₄， 111 mg·L⁻¹ CaCl₂， 123 mg·L⁻¹ 硫酸镁₄·7H₂O， 2.68 mg·L⁻¹ H₃BO₃， 2.13 mg·L⁻¹ MnSO₄·4H₂O， 0.22 mg·L⁻¹ ZnSO₄·7H₂O， 0.08 mg·L⁻¹ 硫酸铜₄·5H₂O， 0.02 mg·L⁻¹ （NH₄）₆Mo₇O₂₄·4H₂O，30.00 mg·L-1 EDTA-NaFe。所有营养液均补充硝化抑制剂双氰胺（C₂H₄N₄， 7 μM）以抑制硝化作用。

2.2. Experimental design and sampling
2.2. 实验设计和采样

The experiment prepared nutrient solutions with different concentrations of NH₄NO₃ by changing the amount of NH₄NO₃ in the aforementioned nutrient solutions. Based on previous research (Jia et al., 2022), set low N 5 mM (N1), medium N 15 mM (N2), and high N 25 mM (N3) NH₄NO₃ concentrations. Each N level includes two ¹⁵N labeling treatments. Low N treatment was tracer labeled with ¹⁵NH₄NO₃ (10.12 atom% ¹⁵N-NH₄⁺) and NH₄¹⁵NO₃ (10.12 atom% ¹⁵N-NO₃⁻), denoted as AN1, NN1. The same applies to medium N and high N treatments, denoted as AN2, NN2, AN3, and NN3, respectively There are a total of 6 treatments 10 pots per treatment, a total of 60 pots.
本实验通过改变上述营养液中 NH 4 NO ₃ 的含量，制备了不同浓度 NH 4 NO ₃ 的营养液。根据以前的研究（Jia et al.， 2022），设置低 N 5 mM （N1）、中 N 15 mM （N2）和高 N 25 mM （N3） NH₄NO₃ 浓度。每个 N 水平包括两个 ¹⁵N 标记处理。低氮处理用 ¹⁵NH₄NO₃ （10.12 原子% ¹⁵N-NH₄⁺）和 NH₄¹⁵NO₃ （10.12 原子% ¹⁵N-NO₃⁻）标记，记为AN1、NN1。这同样适用于中氮和高氮处理，分别表示为 AN2、NN2、AN3 和 NN3 共 6 次处理每个处理 10 罐，共 60 罐。

2.3. Analytical methods 2.3. 分析方法

2.3.1. Root morphology assays
2.3.1. 根形态测定

After 45 days of nutrient solution cultivation, 3 strawberry seedlings were taken from each treatment. The vermiculite and perlite on the roots were gently washed away with water, followed by root scanning. The root was scanned using Epson Perfection V850 Pro scanner (EPSON, Beijing, China) and analyzed using WinRHIZO 2017a (Regent Instruments Inc., Quebec, QC, Canada).
营养液培养 45 d 后，每个处理取 3 株草莓幼苗。用水轻轻洗去根部的蛭石和珍珠岩，然后进行根部扫描。使用 Epson Perfection V850 Pro 扫描仪（EPSON，中国北京）扫描根部，并使用 WinRHIZO 2017a（Regent Instruments Inc.，魁北克，QC，加拿大）进行分析。

2.3.2. Biomass 2.3.2. 生物质

Plants were harvested 45 days after treatment and were divided into leaves, stems and roots. A portion of strawberry seedlings were quickly frozen with liquid N and then transferred to an ultra low temperature refrigerator (−80 °C) for further analysis. Another part of strawberry seedlings was heated at 105 °C for 30 min and dried at 80 °C for 5 days. Dry weight and ¹⁵N abundance were measured after drying.
植物在处理后 45 天收获，并分为叶、茎和根。用液态氮快速冷冻一部分草莓幼苗，然后转移到超低温冰箱（-80 °C）中进行进一步分析。另一部分草莓幼苗在105 °C下加热30 min，并在80 °C下干燥5 d。干燥后测定干重和 ¹⁵N 丰度。

2.3.3. Determining the activities of the key enzymes participating in n metabolism
2.3.3. 确定参与 N 代谢的关键酶的活性

The dry weight was recorded as the biomass. The activities of NR, NiR, GS and Glutamate Synthase (GOGAT) were investigated using specific assay kits (Boxbio, Beijing, China).
干重记录为生物量。使用特异性检测试剂盒（Boxbio， Beijing， China）研究 NR、NiR、GS 和谷氨酸合酶（GOGAT）的活性。

2.3.4. Contents of ¹⁵N
2.3.4. ¹⁵N 的含量

The content of N was determined using the Kjeldahl method, and the abundance of ¹⁵N was determined using a ZHT-03 mass spectrometer produced by Beijing Analytical Instrument Factory, China (Chinese Academy of Agricultural Sciences).

Total N in each organ (g) = \frac{each organ N concentration (%)}{100 \times organ dry weight (g)}

% Ndff = \frac{abundance of^{15} N in plant - natural abundance of^{15} N}{abundance of^{15} N in fertilizer - natural abundance of^{15} N} \times 100 %

^{15} N distribution rate (%) = \frac{^{15} N absorbed by each organ from fertilizer (mg)}{total^{15} N absorbed by plant from fertilizer (mg)} \times 100 %

^{15} N absorbed by each organ from fertilizer (mg) = total N in each organ (g) \times % Ndff

采用凯氏定氮法测定 N 含量，使用中国北京分析仪器厂（中国农业科学院）生产的 ZHT-03 质谱仪测定 ¹⁵ N 的丰度。

Total N in each organ (g) = \frac{each organ N concentration (%)}{100 \times organ dry weight (g)}

% Ndff = \frac{abundance of^{15} N in plant - natural abundance of^{15} N}{abundance of^{15} N in fertilizer - natural abundance of^{15} N} \times 100 %

^{15} N distribution rate (%) = \frac{^{15} N absorbed by each organ from fertilizer (mg)}{total^{15} N absorbed by plant from fertilizer (mg)} \times 100 %

^{15} N absorbed by each organ from fertilizer (mg) = total N in each organ (g) \times % Ndff

2.3.5. RNA extraction, cDNA preparation, and qRT-PCR
2.3.5. RNA 提取、cDNA 制备和 qRT-PCR

The frozen samples were quickly ground into powder in liquid N. 100 mg of tissue samples were taken. The total RNA of the samples was extracted using the polysaccharide and polyphenol plant total RNA extraction kit produced by Proteinssci. The synthesis of the first strand of cDNA was performed according to the instructions of the kit Primer Script™ RT ReagentKit With gDNA Eraser (Takara). In qRT-PCR analysis, SYBR Premix Ex Taq (TaKaRa Biotechnology Co., Ltd., Dalian, China) kit was used for real-time fluorescence quantitative analysis. Three replicates were performed for each gene and straw-berry actin was used as the internal reference gene. The qRT-PCR primer sequence is shown in Supplementary Table S1. The experimental results were analyzed quantitatively using the 2^−ΔΔCT method.
将冷冻样品在液体 N 中迅速研磨成粉末，取 100 mg 组织样品。使用 Proteinssci 生产的多糖和多酚植物总 RNA 提取试剂盒提取样品的总 RNA。根据试剂盒 Primer Script™ RT ReagentKit With gDNA Eraser （Takara）的说明进行第一链 cDNA 的合成。在 qRT-PCR 分析中，使用 SYBR Premix Ex Taq （TaKaRa Biotechnology Co.， Ltd.， Dalian， China）试剂盒进行实时荧光定量分析。每个基因进行 3 次重复，草莓肌动蛋白作为内部参考基因。qRT-PCR 引物序列如补充表 S1 所示。采用 2^−ΔΔCT 方法对实验结果进行定量分析。

2.4. Statistical analysis
2.4. 统计分析

Origin 8.0 (OriginLab Corporation, Northhampton, MA, USA) was used for figure drawing. Data were analyzed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) by using one-way factorial analysis of variance (ANOVA). In all cases, differences were considered significant at a probability level of P<0.05. Furthermore, correlation analyses using Spearman's correlation were performed.
Origin 8.0 （OriginLab Corporation， Northhampton， MA， USA）用于人物绘图。使用 SPSS 17.0 （SPSS， Inc.， Chicago， IL， USA）使用单因素方差分析（ANOVA）分析数据。在所有情况下，在概率水平为 P<0.05 时，差异被认为是显著的。此外，使用 Spearman 相关性进行了相关性分析。

3. Results 3. 结果

3.1. Biomass and root morphology
3.1. 生物量和根系形态

As shown in Table 1, different N concentrations had significant effects on the dry weight of rhizomes and leaves and root morphology of strawberry seedlings. With the increase of N supply level, the dry weight of root, stem and leaf of strawberry increased first and then decreased, and the dry weight of root, stem, leaf and whole plant was the highest under NN2 and AN2 N levels. With the increase of N supply level, root length, root surface area and root volume all increased first and then decreased. Under NN3 and AN3 treatments, root length, root surface area and root volume were the smallest.
如表 1 所示，不同 N 浓度对草莓幼苗根茎和叶片干重以及根系形态有显著影响。随着氮素供应水平的增加，草莓根、茎、叶干重呈先增加后降低的趋势，其中NN2和AN2 N水平下草莓根、茎、叶和全株干重最高。随着氮素供应水平的增加，根长、根表面积和根体积均呈先增加后减少的趋势。在 NN3 和 AN3 处理下，根长、根表面积和根体积最小。

Table 1. Effects of different N concentrations on biomass and root morphology of strawberry seedlings.
表 1.不同氮浓度对草莓幼苗生物量和根系形态的影响

Treatments 治疗	Root dry weight 根干重	Stem dry weight 茎干重	Leaf dry weight 叶片干重	Total dry weight 总干重	Total root length 根总长度	Root surface area 根表面积	Total root volume 总根卷
Empty Cell	(g) （七）	(g) （七）	(g) （七）	(g) （七）	(cm) （厘米）	(cm²) （厘米²）	(cm³) （厘米³）
NN1	1.3 ± 0.07c	1.3 ± 0.07b	1.7 ± 0.07b	4.4 ± 0.27c	2552±174ab 2552±174起	229±47ab 229±47起价	2.64±0.86ab 2.64±0.86起价
NN2	2.3 ± 0.07a 2.3 ± 0.07 安培	2.2 ± 0.27a 2.2 ± 0.27 安培	3.1 ± 0.09a 3.1 ± 0.09 安培	7.6 ± 0.22a 7.6 ± 0.22 安培	2709±254a 编号 2709±254A	258±19a 258±19 个	2.81±0.13ab 2.81±0.13来自
NN3	1.9 ± 0.11b	1.4 ± 0.08b	3.3 ± 0.10a 3.3 ± 0.10 安培	7.0 ± 0.27b	2307±134b	203±06b	2.16±0.08ab 2.16±0.08从
AN1	1.4 ± 0.17c	1.3 ± 0.08b	1.7 ± 0.12b	4.3 ± 0.06c	2538±247ab 2538±247起	223±34ab 223±34起价	2.46±0.49ab 2.46±0.49从
AN2	2.3 ± 0.04a 2.3 ± 0.04 安培	2.3 ± 0.27a 2.3 ± 0.27 安培	3.1 ± 0.09a 3.1 ± 0.09 安培	7.7 ± 0.26a 7.7 ± 0.26 安培	2722±31a 2722±31 个	242±21ab 242±21起价	3.12±0.69a 3.12±0.69 个
AN3	1.9 ± 0.09b	1.3 ± 0.11b	3.2 ± 0.19a 3.2 ± 0.19 安培	6.7 ± 0.26b	2368±32b	194±30b 194±30 字节	1.91±0.03b

Data presented are means±SD (n = 3). In each column, different lowercase letters show significant differences of means (P < 0.05). A means NH₄⁺ was labeled;N means NO₃⁻ was labeled. N1, N2, and N3 represent N supply concentrations of 5 mM, 15 mM, and 25 mM, respectively.
提供的数据是均值 ±SD （n = 3）。在每列中，不同的小写字母表示均值差异显著（P < 0.05）。A 表示标记了 NH ₄⁺;N 表示标记了 NO₃⁻。N1、N2 和 N3 分别代表 5 mM、15 mM 和 25 mM 的 N 供应浓度。

3.2. N metabolism-related enzyme activities in different organs
3.2. 不同器官中与 N 代谢相关的酶活性

As shown in Fig. 1, N levels significantly affected the activities of N metabolizing enzymes in rhizomes and leaves. In different organs, the highest activity of NR, NiR and GOGAT enzymes was found in leaves, while the highest activity of GS enzymes was found in roots. In rhizomes and leaves, NR, NiR, GS and GOGAT under N2 treatment were significantly higher than those under N1 and N3 treatment. These results indicated that N2 treatment had the fastest rate of N assimilation.
如图 1 所示，N 水平显著影响根茎和叶片中 N 代谢酶的活性。在不同器官中，NR 、 NiR 和 GOGAT 酶在叶片中的活性最高，而 GS 酶在根中的活性最高。在根茎和叶片中，N2 处理下的 NR 、 NiR 、 GS 和 GOGAT 显著高于 N1 和 N3 处理下。这些结果表明，N2 处理的 N 同化率最快。

3.3. N accumulation and nitrogen use efficiency
3.3. 氮素积累和氮素利用效率

Different N concentrations significantly affected the utilization and accumulation of different forms of N in strawberry seedlings (Table 2). The total N content, ¹⁵N content, Ndff% and NUE of NN1 treatment were significantly lower than NN2 treatment. Increasing N concentration improved the absorption of NO₃⁻-N by strawberry. However, continued increase of N concentration did not improve the NO₃⁻-N absorption capacity of strawberries, and NN3 treatment NUE decreased by 2.99 % compared with NN2. With the increase of N concentration, the ¹⁵N accumulation of NH₄⁺-N in strawberry gradually increased, and the differences of AN1, AN2 and AN3 reached a significant level. The utilization rate of NH₄⁺-N in strawberry was the highest in AN1 treatment, and Ndff% and N utilization rate showed a trend of increasing after decreasing.
不同 N 浓度显著影响草莓幼苗中不同形式 N 的利用和积累（表 2）。 NN1 处理的总 N 含量、¹⁵N 含量、Ndff% 和 NUE 显著低于 NN2 处理。增加 N 浓度提高了草莓对 NO₃⁻--N 的吸收。然而，氮浓度的持续增加并未提高草莓的 NO ₃⁻-N 吸收能力，NN3 处理的 NUE 与 NN2 相比降低了 2.99 %。随着 N 浓度的增加，草莓中 NH ₄⁺-N 的 ¹⁵N 积累量逐渐增加，AN1、AN2 和 AN3 的差异达到显著水平。AN1处理草莓中NH 4+-N的利用率最高，Ndff%和N利用率呈先降低后上升的趋势。

Table 2. Effects of different N levels on absorption of NH₄⁺-N and NO₃⁻-N in Strawberry Seedlings.
表 2.不同氮水平对草莓幼苗 NH ₄⁺--N 和 NO₃⁻-N 吸收的影响

Treament 处理	Total nitrogen content (g) 总氮含量（g）	Ndff%	¹⁵N accumulation (mg) ¹⁵N 积累（mg）	NUE of labeled nitrogen forms(%) 标记氮形式的氮肥利用率（%）
NN1	0.05±0.0032b 0.05±0.0032 倍	2.07±0.14e 2.07±0.14	0.32±0.03e 0.32±0.03 元	3.51±0.35c 3,51±0.35 摄氏度
NN2	0.13±0.0071a 0.13±0.0071 个	4.56±0.16b	2.01±0.05b 2.01±0.05 字节	7.45±0.19a 7.45±0.19 安
NN3	0.14±0.0093a 0.14±0.0093 个	4.6 ± 0.41b	2.01±0.17b	4.46±0.38b
AN1	0.05±0.0002b 0.05±0.0002 磅	3.46±0.28c 3.46±0.28摄氏度	0.64±0.06d 0.64±0.06 天	7.16±0.63a 7.16±0.63 节
AN2	0.14±0.0108a 0.14±0.0108 安培	2.75±0.14d 2.75±0.14 天	1.22±0.15c 1.22±0.15摄氏度	4.52±0.56b
AN3	0.13±0.0075a 0.13±0.0075 个电流	5.36±0.14a 5.36±0.14 安	2.34±0.13a 2.34±0.13 安	5.19±0.29b

Data presented are means±SD (n = 3). In each column, different lowercase letters indicate significant differences in average values (P < 0.05). Nue represents nitrogen utilization efficiency. A means NH₄⁺ was labeled;N means NO₃⁻ was labeled. N1, N2, and N3 represent N supply concentrations of 5 mM, 15 mM, and 25 mM, respectively.
提供的数据是均值 ±SD （n = 3）。在每列中，不同的小写字母表示平均值存在显著差异（P < 0.05）。Nue 代表氮利用效率。A 表示标记了 NH ₄⁺;N 表示标记了 NO₃⁻。N1、N2 和 N3 分别代表 5 mM、15 mM 和 25 mM 的 N 供应浓度。

Under low N concentration, Ndff%, ¹⁵N content and NUE under AN1 treatment were significantly higher than those under NN1 treatment, indicating that strawberry plants had stronger competition and absorption capacity for NH₄⁺-N. Under these conditions, strawberry plants prefer to absorb NH₄⁺-N. At medium N concentration, Ndff% and NUE of NN2 treatment were significantly higher than that of AN2, showing a preference for NO₃⁻-N. At high N concentration, Ndff% of AN3 treatment was the highest, reaching 5.36 % compared with other treatments. There was no significant difference in NUE between NN3 and AN3 treatments.
在低氮浓度下， AN1 处理下的 Ndff%、¹⁵N 含量和 NUE 显著高于 NN1 处理，表明草莓植株对 NH 4+-N 具有更强的竞争和吸收能力。在这些条件下，草莓植物更喜欢吸收 NH₄⁺-N。在中等氮浓度下，NN2 处理的 Ndff% 和 NUE 显著高于 AN2，表现出对 NO₃⁻--N 的偏好。在高氮浓度下，AN3 处理的 Ndff% 最高，与其他处理相比达到 5.36 %。NN3 和 AN3 治疗之间的 NUE 无显著差异。

3.4. Accumulation of ¹⁵N in each organ
3.4. 每个器官中 ¹⁵ N 的积累

As shown in Fig. 2, the accumulation of NH₄⁺-N and NO₃⁻-N in roots, stems and leaves of strawberry seedlings was affected by the concentration of N supply. During each treatment, the NH₄⁺-N accumulation in roots, stems and leaves increased with the increase of N supply concentration. At the concentration of N3, the NH₄⁺-N accumulation in roots increased by 1.54 times compared with N2 treatment, and the NO₃⁻-N accumulation in roots at the same concentration was 2.24 times, reaching 1.15 mg. The accumulation of NO₃⁻-N in strawberry root was the highest in N2 treatment, followed by N3 treatment (0.68 mg and 0.51 mg, respectively). At different concentrations of N, NO₃⁻-N accumulation in stems was significantly higher than NH₄⁺-N. The cumulative amount of NO₃⁻-N in stems and leaves treated with N2 and N3 was not significantly different, but the cumulative amount of NO₃⁻-N in roots treated with N3 was significantly decreased compared with N2.
如图 2 所示，草莓幼苗根、茎和叶中 NH 4+-N 和 NO₃−-N 的积累受氮供应浓度的影响。在每次处理过程中，NH₄⁺-N 在根、茎和叶中的积累量随着 N 供应浓度的增加而增加。在 N3 浓度下，NH₄⁺-N 在根中的积累量比 N2 处理增加了 1.54 倍，相同浓度下 NO ₃⁻-N 在根中的积累量为 2.24 倍，达到 1.15 mg。 N2处理中草莓根中NO ₃⁻-N的积累量最高，其次是N3处理（分别为0.68 mg和0.51 mg）。在不同浓度的 N 下，茎中 NO₃⁻--N 积累量显著高于 NH₄⁺-N。N2 和 N3 处理的茎叶中 NO 3−-N 的累积量无显著差异，但与 N2 处理的根中 NO 3−--N 的累积量相比，NO ₃⁻--N 的累积量显著降低。

3.5. Distribution of ¹⁵N in each organ
3.5. 每个器官中 ¹⁵ N 的分布

Different N levels changed the distribution of NH₄⁺-N and NO₃⁻-N in strawberry seedlings (Fig. 3). Under low N condition, strawberry stem was the main distribution organ of NO₃⁻-N, and NO₃⁻-N distribution center shifts to leaf with increasing N supply concentration. NH₄⁺-N and NO₃⁻-N showed the opposite distribution characteristics. Under the condition of low N, the distribution rate of ¹⁵N in the root system was the highest.
不同 N 水平改变了草莓幼苗中 NH ₄⁺-N 和 NO₃⁻-N 的分布（图 3）。在低氮条件下，草莓茎是 NO₃⁻-N 的主要分布器官，随着氮素供应浓度的增加，NO 3−-N 分布中心向叶片转移。NH₄⁺-N 和 NO₃⁻--N 表现出相反的分布特征。在低 N 条件下，¹⁵N 在根系中的分配速率最高。

3.6. Relative expression of genes related to N uptake and transport in roots
3.6. 根中与 N 吸收和运输相关的基因的相对表达

FaNRTs and FaAMTs participate in the uptake and transport of NO₃⁻ and NH₄⁺-N in roots, respectively, and this study found that different N concentrations affected their expressions (Fig. 4). In N2 treatment, the relative gene expression levels of FaNRT1.1, FaNRT2.1 and FaAMT1.1 (Fig. 4a,b,c) showed the same trend, increasing first and then decreasing, indicating that appropriate N concentration could induce the up-regulation of some genes of NRT and AMT family. Among them, the expression of FaAMT1.1 in N3 treatment was significantly lower than that in N1 and N2 treatment with high N concentration, and high N inhibited its expression. FaAMT1.2 was not sensitive to changes in N concentration, and its expression was significantly down-regulated only when treated with N3 (Fig. 4d).
FaNRTs 和 FaAMTs 分别参与根中 NO ₃⁻ 和 NH₄⁺-N 的摄取和转运，本研究发现不同的 N 浓度会影响它们的表达（图 4）。在 N2 处理中，FaNRT1.1、FaNRT2.1 和 FaAMT1.1 的相对基因表达水平（图 4a、b、c）呈现相同的趋势，先升高后下降，表明适当的 N 浓度可诱导 NRT 和 AMT 家族部分基因的上调。其中，FaAMT1.1 在 N3 处理中的表达显著低于高 N 浓度的 N1 和 N2 处理，且高 N 抑制其表达。FaAMT1.2 对 N 浓度的变化不敏感，只有在 N3 处理时其表达才显著下调（图 4d）。

3.7. Correlation analysis between ¹⁵N accumulation and related influencing factors
3.7. ¹⁵N 积累与相关影响因素的相关性分析

As shown in Fig. 5, the overall absorption of NO₃⁻-N by strawberries was significantly correlated with GS activity and NRT1.1 expression in stems and leaves. The overall absorption of NH₄⁺-N by strawberry seedlings was significantly correlated with GS activity and NR activity in stems. In addition, the accumulation of NO₃⁻-N in the root system was significantly correlated with NR activity, NRT1.1, and NRT2.1. The accumulation of NH₄⁺-N in the root system was correlated with GS activity in various organs, but not significantly correlated with AMT1.1 and AMT1.2 in the root system. GS activity was not only significantly correlated with NH₄⁺-N in various organs, but also significantly correlated with the accumulation of NO₃⁻-N in strawberry roots, stems, and leaves.
如图 5 所示，草莓对 NO₃⁻-N 的总吸收与 GS 活性和 NRT1.1 在茎和叶中的表达呈显著相关。草莓幼苗对 NH ₄⁺-N 的总体吸收与 GS 活性和 NR 活性在茎中呈显著相关。此外，根系中 NO ₃⁻-N 的积累与 NR 活性、NRT1.1 和 NRT2.1 显著相关。NH₄⁺-N 在根系中的积累与各器官的 GS 活性相关，但与根系中的 AMT1.1 和 AMT1.2 无显著相关性。GS 活性不仅与各器官中的 NH ₄⁺-N 显著相关，而且与草莓根、茎和叶中 NO ₃⁻-N 的积累也显著相关。

4. Discussion 4. 讨论

In general, NH₄⁺-N should be the preferred N source for plants as it requires less energy to absorb (Socci and Templer, 2015). When NH₄⁺-N and NO₃⁻-N are supplied at the same concentration, some plants prefer to absorb NH₄⁺-N rather than NO₃⁻-N (Ruan et al., 2016; Zhong et al., 2014). Liu et al. (Liu et al., 2007) found that when the total N concentration was 7 mM and equal amounts of NH₄⁺-N and NO₃⁻-N were supplied, strawberries absorbed 4.3 % more NH₄⁺-N than NO₃⁻-N. In this study with a N concentration of 5 mM, AN1 treatment of strawberry seedlings resulted in a higher ndff%, ¹⁵N accumulation, and utilization rate than NN1, with increases of 1.39 %, 0.9 mg, and 1.01 %, respectively (Table 2). Strawberries showed a stronger absorption capacity for NH₄⁺-N. This is consistent with the results of Zhang et al., who used lower N concentrations for cultivation (Zhang et al., 2005).
一般来说，NH₄⁺-N 应该是植物首选的氮来源，因为它需要较少的能量来吸收（Socci 和 Templer，2015）。当NH₄ ⁺ -N和NO₃ ^- -N以相同浓度供应时，一些植物更喜欢吸收NH₄ ⁺ -N而不是NO₃ ^- -N（Ruan等人，2016;Zhong et al.， 2014）。Liu 等人（Liu et al.， 2007）发现，当总氮浓度为 7 mM 并且提供等量的 NH₄⁺-N 和 NO₃⁻-N 时，草莓吸收的 NH₄⁺-N 比 NO₃⁻-N 多 4.3%。在这项氮浓度为 5 mM 的研究中，草莓幼苗的 AN1 处理导致 ndff%、¹⁵N 积累和利用率高于 NN1，分别增加了 1.39 %、0.9 mg 和 1.01 %（表 2）。草莓对 NH₄⁺-N 的吸收能力较强。这与 Zhang 等人的结果一致，他们使用较低的 N 浓度进行培养（Zhang et al.， 2005）。

The GS/GOGAT pathway is the main way of assimilating NH₄⁺, which is generated from GS and GOGAT (Coleto et al., 2023). In this study, the activity of GS and GOGAT enzymes was inhibited by N3 treatment (Fig 1). The root system is the main site of NH₄⁺-N assimilation, with most of the NH₄⁺-N being assimilated before transport to the shoot. In addition, AMT1.1 not only regulates NH₄⁺ absorption but also regulates the transport from the underground to the aboveground (Li et al., 2016). The relative expression level of AMT1.1 in the root system under N3 treatment was only 30.6 % of that under N2 treatment (Fig 4). The efficiency of NH₄⁺-N assimilation in Arabidopsis thaliana decreased under high NH₄⁺ stress (Yi et al., 2020), similar to our results. It can be seen that due to the insufficient assimilating capacity of strawberry roots for NH₄⁺+, a large amount of NH₄⁺ accumulates in the roots.
GS/GOGAT 通路是同化 NH₄⁺ 的主要方式，NH 4+ 由 GS 和 GOGAT 产生（Coleto等人，2023 年）。在这项研究中，N3 处理抑制了 GS 和 GOGAT 酶的活性（图 1）。根系是 NH₄⁺-N 同化的主要部位，大部分 NH₄⁺-N 在运输到芽之前被同化。此外，AMT1.1 不仅调节 NH₄⁺ 吸收，还调节从地下到地上的运输（Li et al.， 2016）。N3 处理下 AMT1.1 在根系中的相对表达水平仅为 N2 处理下的 30.6 %（图 4）。在高 NH ₄⁺ 胁迫下，拟南芥中 NH ₄⁺-N 同化的效率降低（Yi等人，2020 年），与我们的结果相似。由此可见，由于草莓根系对NH ₄⁺+的同化能力不足，大量的NH₄⁺在根系中积累。

From the perspective of ¹⁵N distribution, NH₄⁺-N accumulated significantly in the roots of strawberry seedlings (Fig. 3), and their root growth was also significantly inhibited (Table 1). The plant showed typical high NH₄⁺ stress, which is also known as ammonium toxicity. GS activity is the key to controlling free NH₄⁺ in plant tissues and determines the utilization and tolerance of NH₄⁺ by plants (Cruz et al., 2011). Coskun et al. (Coskun et al., 2013) confirmed that excessive ammonium assimilation can lead to a reduction in carbon sources (sugars) available for plant growth and energy consumption in the transmembrane cycle of NO₃⁻-N/NH₄⁺-N in root cells. Under high external NH₄⁺ concentrations, plants may activate the NH₄⁺ efflux system to cope with high NH₄⁺ influx (Takushi and Hitoshi, 2017). The efflux of ammonium requires energy, and the higher respiratory rate of barley roots provides energy for the higher rate of ammonium efflux process, which further exacerbates the imbalance of energy supply (Babourina et al., 2007). This further exacerbates the lack of energy supply. High NH₄⁺ in the leaves damaged the oxygen-evolving complex and increased heat dissipation, resulting in disruption of the photosynthetic electron transport chain (Chen et al., 2023). The reduction in photosynthesis in the leaves leads to insufficient carbon sources for NH₄⁺ assimilation and an imbalance in carbon and N metabolism, ultimately resulting in inhibited root growth and reduced biomass.
从 ¹⁵N 分布的角度来看，NH₄⁺-N 在草莓幼苗根系中显著积累（图 3），其根系生长也受到显著抑制（表 1）。该植物表现出典型的高 NH₄⁺ 胁迫，也称为铵态氮毒性。GS 活性是控制植物组织中游离 NH₄⁺ 的关键，并决定了植物对 NH 4+ 的利用和耐受性（Cruz et al.， 2011）。Coskun等人（Coskun等人，2013）证实，过量的铵态氮同化会导致根细胞中NO 3---N/NH 4+-N的跨膜循环中可用于植物生长的碳源（糖）和能源消耗减少。在高浓度的外部 NH₄⁺ 下，植物可能会激活 NH₄⁺ 外排系统以应对高 NH₄⁺ 流入（Takushi 和 Hitoshi，2017）。铵态氮的外排需要能量，而大麦根较高的呼吸速率为较高的铵态氮外流过程提供了能量，这进一步加剧了能量供应的不平衡（Babourina et al.， 2007）。这进一步加剧了能源供应的缺乏。叶片中高 NH₄⁺ 破坏了析氧复合物并增加了散热，导致光合电子传递链中断（Chen等人，2023 年）。叶片光合作用的减少导致 NH₄⁺ 同化的碳源不足，碳和氮代谢失衡，最终导致根生长受抑制和生物量减少。

Strawberry seedlings showed a preference for NO₃⁻-N under medium N conditions. NR plays a key role in Nmetabolism (Mu and Chen, 2021). NR activity is regulated by exogenous NO₃⁻-N, and with the increase in the content of substrate NO₃⁻-N, NR activity also affects plant absorption of inorganic N (Liu et al., 2018; Zhu et al., 2016). It is generally believed that NRT1.1 is a dual-affinity nitrate transporter, while NRT2.1 belongs to a high-affinity nitrate transporter (Liu et al., 2018; Wu and Zhao, 2010). This study found that medium N treatment induced significant upregulation of NR activity in leaves and FaNRT1.1 and FaNRT2.1 expression in roots (Fig 4). Through correlation analysis, we found that FaNRT1.1 and FaNRT2.1 in roots were significantly correlated with the overall NO₃⁻-N absorption of strawberries (Fig 5). Therefore, we speculate that the higher NO₃⁻-N assimilation capacity in leaves greatly promotes the absorption capacity of NO₃⁻-N. This may be the reason why strawberry exhibits a preference for NO₃⁻-N under medium N conditions.
草莓幼苗在中等氮条件下表现出对 NO₃⁻-N 的偏好。NR 在 Nmetabolism 中起关键作用（Mu 和 Chen， 2021）。NR 活性受外源 NO₃^--N 的调节，随着底物 NO₃^--N 含量的增加，NR 活性也会影响植物对无机氮的吸收（Liu等人，2018 年;Zhu et al.， 2016）。通常认为 NRT1.1 是一种双亲和力硝酸盐转运蛋白，而 NRT2.1 属于高亲和力硝酸盐转运蛋白（Liu等人，2018 年;Wu 和 Zhao，2010 年）。本研究发现，中等氮处理诱导叶片中 NR 活性和根中 FaNRT1.1 和 FaNRT2.1 表达的显著上调（图 4）。通过相关性分析，我们发现根系中的 FaNRT1.1 和 FaNRT2.1 与草莓总体 NO₃⁻-N 吸收显著相关（图 5）。因此，我们推测叶片中较高的 NO₃⁻-N 同化能力极大地促进了 NO₃⁻-N 的吸收能力。这可能是草莓在中等氮条件下表现出对 NO ₃⁻--N 的偏好的原因。

The absorption of NO₃⁻-N was inhibited under high N conditions (Table 2, Fig 4a,b). Our results indicated that the accumulation of NO₃⁻-N in plants was not only significantly positively correlated with NRT1.1 in roots, but also significantly positively correlated with GS activity in stems and leaves (Fig 5). This may be due to the fact that when the local upper GS activity decreases, high free NH₄⁺ inhibits photosynthesis, which in turn affects NO₃⁻-N absorption. There was evidence that NRT1.1 and NRT2.1 were downregulated by NH₄⁺, which could reduce root absorption of NO₃⁻-N. Other species also exhibit similar phenomena (Mustapha et al., 2012). In addition, the absorption of NO₃⁻-N is an active and energy-consuming process. In the above discussion, we mentioned that the depletion of the supply of C skeleton due to excessive assimilation of NH₄⁺ in the roots is clearly detrimental to the absorption and assimilation of NO₃⁻-N. Therefore, strawberry seedlings exhibit a preference for NH₄⁺ under high N conditions, which may be due to the decrease in NO₃⁻-N absorption regulation by NH₄⁺
在高 N 条件下，NO ₃⁻-N 的吸收受到抑制（表 2，图 4a，b）。我们的结果表明，植物中 NO ₃⁻-N 的积累不仅与根中的 NRT1.1 呈显著正相关，而且与茎叶中的 GS 活性也呈显著正相关（图 5）。这可能是由于当局部上部 GS 活性降低时，高游离 NH₄⁺ 抑制光合作用，进而影响 NO₃⁻-N 的吸收。有证据表明，NRT1.1 和 NRT2.1 被 NH₄⁺ 下调，这可以减少根对 NO₃⁻-N 的吸收。其他物种也表现出类似的现象（Mustapha et al.， 2012）。此外，NO₃⁻-N 的吸收是一个活跃且耗能的过程。在上面的讨论中，我们提到，由于根中 NH 4+ 的过度同化导致 C 骨架供应的消耗显然不利于 NO ₃⁻--N 的吸收和同化。因此，草莓幼苗在高氮条件下表现出对 NH₄⁺ 的偏好，这可能是由于 NH ₄⁺ 对 NO ₃⁻-N 吸收调节的减少.

5. Conclusion 5. 总结

To sum up, when the concentration of N supply gradually increased, strawberry seedlings exhibited a change in preference from NH₄⁺-N to NO₃⁻-N, and then to NH₄⁺-N. When N supply was low to medium, the NO₃⁻-N absorption and transport system in strawberry seedlings responds more strongly to increased N concentration. Under the condition of high N, the decrease of GS activity led to the accumulation of NH₄⁺ in the root, inhibited the expression of FaNRT1.1, and made strawberries prefer NH₄⁺-N.
综上所述，当氮素供应浓度逐渐增加时，草莓幼苗表现出从 NH₄⁺-N 到 NO₃⁻-N，再到 NH₄⁺-N 的偏好变化。当氮供应量为中低时，草莓幼苗中的 NO 3−--N 吸收和运输系统对氮浓度的增加反应更强烈。在高 N 条件下，GS 活性的降低导致 NH₄⁺ 在根系中积累，抑制了 FaNRT1.1 的表达，使草莓更喜欢 NH₄⁺-N。

CRediT authorship contribution statement
CRediT 作者贡献声明

Zhihang Jia: Writing – review & editing, Writing – original draft, Visualization, Validation, Resources, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jia Zhang: Writing – review & editing, Software, Conceptualization. Wei Jiang: Supervision, Methodology, Investigation, Conceptualization. Meng Wei: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration. Lin Zhao: Investigation. Gangbo Li: Conceptualization.
贾志航：写作 - 审查和编辑，写作 - 原始草稿，可视化，验证，资源，项目管理，方法论，调查，正式分析，数据管理，概念化。张佳：写作 - 审查和编辑，软件，概念化。江伟：监督、方法、调查、概念化。孟伟：写作 - 审查和编辑，写作 - 原始草稿，可视化，验证，监督，项目管理。赵林：调查。李刚博：概念化。

Declaration of competing interest
利益争夺声明

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
作者声明，他们没有已知的竞争性经济利益或个人关系，这些利益或个人关系似乎可能会影响本文报告的工作。

Acknowledgement 确认

This research was supported by Scientific Research Foundation of Xuzhou Academy of Agricultural Sciences (XM2021008 and XM2023006).
本研究得到了徐州市农业科学院科学研究基金（XM2021008 和 XM2023006）的支持。

Appendix. Supplementary materials
附录。补充材料

Download: Download spreadsheet (20KB)

Data availability 数据可用性

Data will be made available on request.
数据将应要求提供。

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Google Scholar
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View in Scopus Google Scholar
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Physiological and biochemical responses of Phragmites australis correlate with water quality in lagoon wastewater treatment system
2025, Environmental Technology and Innovation
Natural wastewater lagooning is an energy-efficient process, and its effectiveness is significantly influenced by the selection of plant species, whose capacity to purify water is affected by various environmental parameters. This study aimed to establish the correlation between the physiological and biochemical responses of a purifying plant, Phragmites australis, and the water quality index (WQI) of water in a lagoon treatment plant across four seasons. A comparison was made with control water and plants from the Wadi (ephemeral riverbed that contains water only when rain occurs). Several physicochemical parameters were measured before and after the wastewater treatment. The analyses of the plant focused on the carbohydrate and proline content in its roots, stems, and leaves. The physicochemical analysis of the water revealed very high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD5), volatile suspended solids (VSS), and total suspended solids (TSS) in the raw wastewater (RWW). These levels subsequently decreased in the treated water (TWW), contrasting with the control water (Wadi), which exhibited very low levels. In this context, RWW had a WQI of 284.3 ± 40.5, which was highly correlated with the tested parameters, indicating a water quality classification ranging from unsuitable to questionable for irrigation use. In contrast, TWW had a WQI of 172 ± 33.2, reflecting a classification from Doubtful to Permissible, while the control water displayed excellent quality with a WQI of 14.7 ± 2.2. The concentrations observed in TWW were as follows: COD (192.5 ± 58.1 mg/L), BOD5 (6 ± 38 mg/L), VSS (45.8 ± 16.8 mg/L), and TSS (101.6 ± 27 mg/L). A high concentration of NH₄⁺ ions was noted compared to NO₂^– and NO₃^–, which had an insignificant effect. The values of electrical conductivity (EC) were notably high but did not show a significant difference between the water types. The effect of WQI for the three types of water (RWW, TWW, and control) on proline accumulation in the two plant organs (leaves and roots) showed no significant effect, unlike in the stems. However, the variation of this osmoticum was significantly affected by seasonal changes. The highest proline concentrations were recorded during the autumn season in all three organs studied, with values ranging from a minimum of 0.5 µg/g dry matter (DM) to 2 µg/g DM. A similar trend was observed for carbohydrates, with the highest concentrations also noted in the autumn season across all water types and plant organs. The observed values for carbohydrates ranged between a minimum of 0.5 µg/g DM and 1.5 µg/g DM. The results indicated a significantly higher accumulation of proline and carbohydrates in the roots, stems, and leaves during the autumn season. The concentrations recorded in these organs were 1.6 ± 0.2 µg/g DM, 1.6 ± 0.1 µg/g DM, and 1.8 ± 0.04 µg/g DM, respectively. These levels were higher in the control plants compared to those at the treatment station, with the exception of carbohydrates in the stems.
Genome-Wide Identification and Expression Analysis of GS and GOGAT Gene Family in Pecan (Carya illinoinensis) under Different Nitrogen Forms
2024, Phyton-International Journal of Experimental Botany
Ammonium nitrogen (NH₄⁺-N) is one of the main forms of nitrogen absorbed and utilized by plants, and mastering the regulatory mechanism of plant ammonium assimilation is a key way to improve the efficiency of plant nitrogen utilization. Glutamine synthetase (GS) and glutamate synthase (GOGAT), two key enzymes for ammonium assimilation, have rarely been studied in pecan. In this study, GS and GOGAT family members of pecan were identified and analyzed using bioinformatics methods. The results indicated that 6 GS and 4 GOGAT genes were identified. The cis-acting elements can be broadly categorized into light-responsive, hormone-responsive, and stress-responsive elements. The findings from the analysis of homologous evolution revealed that neither of the two gene families experienced tandem duplication events. Additionally, different ratios of ammonium to nitrate nitrogen were set to analyze the activities of GS and GOGAT enzymes and expression levels in pecan. The results demonstrate differences in the activities of GS and GOGAT enzymes and the gene expression levels in various tissues of pecan under different nitrogen form ratios. This study established a foundation for further mastering the molecular regulatory mechanism of nitrogen assimilation in pecan, and provided a theoretical basis for enhancing the ability of pecan to absorb and utilize nitrogen.
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View Abstract

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Research Paper 研究论文Different nitrogen concentrations affect strawberry seedlings nitrogen form preferences through nitrogen assimilation and metabolic pathways不同氮浓度通过氮同化和代谢途径影响草莓幼苗氮形态偏好

Highlights 突出

Abstract 抽象

Questions answered in this article本文中回答的问题

Keywords 关键字

Abbreviation 缩写

1. Introduction 1. 引言

2. Materials and methods 2. 材料和方法

2.1. Experimental sites and materials2.1. 实验地点和材料

2.2. Experimental design and sampling2.2. 实验设计和采样

2.3. Analytical methods 2.3. 分析方法

2.3.1. Root morphology assays2.3.1. 根形态测定

2.3.2. Biomass 2.3.2. 生物质

2.3.3. Determining the activities of the key enzymes participating in n metabolism2.3.3. 确定参与 N 代谢的关键酶的活性

2.3.4. Contents of 15N2.3.4. 15N 的含量

2.3.5. RNA extraction, cDNA preparation, and qRT-PCR2.3.5. RNA 提取、cDNA 制备和 qRT-PCR

2.4. Statistical analysis2.4. 统计分析

3. Results 3. 结果

3.1. Biomass and root morphology3.1. 生物量和根系形态

3.2. N metabolism-related enzyme activities in different organs3.2. 不同器官中与 N 代谢相关的酶活性

3.3. N accumulation and nitrogen use efficiency3.3. 氮素积累和氮素利用效率

3.4. Accumulation of 15N in each organ3.4. 每个器官中 15 N 的积累

3.5. Distribution of 15N in each organ3.5. 每个器官中 15 N 的分布

3.6. Relative expression of genes related to N uptake and transport in roots3.6. 根中与 N 吸收和运输相关的基因的相对表达

3.7. Correlation analysis between 15N accumulation and related influencing factors3.7. 15N 积累与相关影响因素的相关性分析