Low Nitrogen Stress Promotes Root Nitrogen Uptake and Assimilation in Strawberry: Contribution of Hormone Networks
低氮胁迫促进草莓根系氮素吸收和同化：激素网络的贡献

Abstract  抽象

Low nitrogen stress severely impedes crop growth and productivity. There has been substantial research on root adaptation to low nitrogen conditions in many plant species. However, the mechanism underlying the morphological response of the strawberry (Fragaria × ananassa Duch.) root to low-NO₃⁻ or low-NH₄⁺ stress remains poorly understood. Strawberry plants were hydroponically cultivated under 1 mM NO₃⁻, 1 mM NH₄⁺, and control (15 mM NO₃⁻) conditions to assess the physiological responses of their roots to low nitrogen stress. As a result, low nitrogen stresses increased the fresh weight of root, lateral root density, and root surface area, as well as enhanced the accumulation of indole-3-acetic acid and jasmonic acid while significantly reducing salicylic acid in the roots. Correspondingly, low nitrogen stresses increased PM H⁺-ATPase activity. Low-NO₃⁻ stress enhanced the activities of nitrate reductase and glutamine synthetase, whereas low-NH₄⁺ treatment led to higher glutamine synthetase and glutamate synthase activities. Collectively, the present results demonstrate that low nitrogen stresses enhance nitrogen uptake of strawberry roots by regulating hormones (indole-3-acetic acid, jasmonic acid, and salicylic acid) and thereby mediating PM H⁺-ATPase activity, while promoting nitrogen metabolism by upregulating the activities of nitrate reductase, glutamine synthetase, and glutamate synthase. In conclusion, low nitrogen conditions may facilitate more efficient acquisition of available N from the soil by strawberry root system.
低氮胁迫严重阻碍了作物的生长和生产力。关于许多植物物种的根系对低氮条件的适应已经进行了大量研究。然而，草莓 （Fragaria × ananassa Duch.） 根对低 NO₃^- 或低 NH₄⁺ 胁迫的形态反应机制仍然知之甚少。草莓植株在 1 mM NO₃⁻、1 mM NH₄⁺ 和对照 （15 mM NO₃⁻） 条件下水培培养，以评估其根系对低氮胁迫的生理反应。因此，低氮胁迫增加了根的鲜重、侧根密度和根表面积，增强了吲哚-3-乙酸和茉莉酸的积累，同时显著降低了根系中的水杨酸。相应地，低氮胁迫增加了 PM ^H+-ATP 酶活性。低 NO₃⁻ 胁迫增强了硝酸盐还原酶和谷氨酰胺合成酶的活性，而低 NH₄⁺ 处理导致更高的谷氨酰胺合成酶和谷氨酸合酶活性。总的来说，目前的结果表明，低氮胁迫通过调节激素（吲哚-3-乙酸、茉莉酸和水杨酸）来增强草莓根的氮吸收，从而介导 PM H⁺-ATP 酶活性，同时通过上调硝酸还原酶、谷氨酰胺合成酶和谷氨酸合酶的活性来促进氮代谢。总之，低氮条件可能有助于草莓根系更有效地从土壤中获取有效氮。

1. Introduction  1. 引言

Nitrogen (N) is one of the most important nutrient elements involved in many key physiological and metabolic reactions in crop growth [1]. Low N supply limits crop growth and therefore reduces its yield [2]. In pursuit of higher crop yields, growers tend to apply excessive N, which far exceeds the actual demand of crops. Globally, about 100 million tons of N is consumed for agricultural production every year [3]. However, there has been significant loss of N due to the low N use efficiency (25–50%) [4], which further causes the significant loss of economic and environmental benefits [5]. Therefore, it is urgent to develop crops with low N demand and high N use efficiency for environment-friendly and sustainable agriculture.
氮 （N） 是参与作物生长中许多关键生理和代谢反应的最重要的营养元素之一 [1]。氮供应不足限制了作物的生长，从而降低了作物的产量 [2]。为了追求更高的作物产量，种植者倾向于施用过量的氮，这远远超过了作物的实际需求。在全球范围内，每年约有 1 亿吨氮用于农业生产 [3]。然而，由于氮肥利用效率低（25-50%）[4]，氮素损失显著，进一步导致了经济和环境效益的显著损失[5]。因此，当务之急是发展低氮需求、高氮利用效率的作物，以实现环境友好型和可持续农业。

Nitrate (NO₃⁻) and ammonium (NH₄⁺) are the two major sources of N in soil, and act as important signaling molecules to modulate various physiological reactions of plants, including gene expression and root architecture modifications [6,7]. The root is a primary organ for nutrient uptake in plants. It has been well documented that root architecture remodeling is a main strategy for plants to adapt to changing environments such as soil nutrient fluctuations [8]. For instance, maize roots respond to low N stress by enhancing root horizontal and vertical extension [9,10], and the formation of lateral roots can be stimulated in response to phosphorus deficiency in rice [11]. Therefore, understanding the mechanisms by which crops respond to nutrient stress through root plasticity can help advance the breeding targets for improving crop nutrient use efficiency via root phenotypes.
硝酸盐 （NO₃⁻） 和铵态氮 （NH₄⁺） 是土壤中氮的两种主要来源，是调节植物各种生理反应的重要信号分子，包括基因表达和根结构修饰 [6,7]。根是植物吸收养分的主要器官。有充分的证据表明，根系结构重塑是植物适应不断变化的环境（如土壤养分波动）的主要策略[8]。例如，玉米根系通过增强根系的水平和垂直伸展来响应低氮胁迫[9,10]，而水稻缺磷可以刺激侧根的形成[11]。因此，了解作物通过根系可塑性响应养分胁迫的机制有助于推进通过根系表型提高作物养分利用效率的育种目标。

Many studies have demonstrated the pivotal role of hormones in regulating root growth under N deficiency. For example, auxin has been widely recognized as a pivotal basis for other hormones to affect root development [12]. Jasmonic acid (JA) is a critical phytohormone regulating crop growth and various defense responses [13,14]. Previous studies have demonstrated that JA can suppress primary root development [15], stimulate lateral root initiation [16], and induce root regeneration [17]. In addition to indole-3-acetic acid (IAA) and JA, salicylic acid (SA) is also an essential component in the complex network of hormonal regulation during plant resistance response. It has been reported that SA can significantly inhibit the development of primary roots, but promote lateral root growth [18] and the formation of root primordia [19]. Several studies have established that roots respond to N fluctuations under the synergistic effect of multiple hormones [20], and the response is largely dependent on the genotypes, environmental conditions, and intensity or duration of low N stress.
许多研究表明，激素在缺氮情况下调节根系生长中起着关键作用。例如，生长素已被广泛认为是其他激素影响根系发育的关键基础 [12]。茉莉酸 （JA） 是一种调节作物生长和各种防御反应的关键植物激素 [13,14]。既往研究表明，JA 可以抑制初级根发育 [15]，刺激侧根启动 [16]，并诱导根再生 [17]。除了吲哚-3-乙酸 （IAA） 和 JA，水杨酸 （SA） 也是植物抗性反应期间激素调节复杂网络的重要组成部分。据报道，SA 可以显着抑制主根的发育，但促进侧根生长 [18] 和根原基的形成 [19]。几项研究已经确定，在多种激素的协同作用下，根对氮波动做出反应[20]，这种反应在很大程度上取决于基因型、环境条件以及低氮胁迫的强度或持续时间。

Before utilization by plants, NO₃⁻ and NH₄⁺ must undergo an assimilation process regulated by a series of enzymes. After absorption by the roots, NH₄⁺ is converted into amino acid by glutamine synthetase (GS) and glutamate synthase (GOGAT). For NO₃⁻, after being absorbed through the root, it will be reduced into NO₂⁻ by nitrate reductase (NR), and then into NH₄⁺ by nitrite reductase (NiR), which is finally assimilated to organic N. Moreover, extensive research has demonstrated the critical role of NO₃⁻ and NH₄⁺ as signaling molecules in inducing the expression of many genes associated with N assimilation [21]. For example, NH₄⁺ can induce GOGAT gene expression in root tips [22].
在被植物利用之前，NO₃^- 和 NH₄⁺ 必须经过由一系列酶调节的同化过程。NH₄⁺ 被根吸收后，被谷氨酰胺合成酶 （GS） 和谷氨酸合酶 （GOGAT） 转化为氨基酸。对于 NO₃⁻，在通过根部吸收后，它会被硝酸盐还原酶 （NR） 还原成 NO₂⁻，然后被亚硝酸盐还原酶 （NiR） 还原成 NH₄⁺，最后被有机氮同化。此外，广泛的研究表明，NO₃⁻ 和 NH₄⁺ 作为信号分子在诱导许多与 N 同化相关的基因表达中起关键作用 [21]。例如，NH₄⁺ 可以在根尖诱导 GOGAT 基因表达 [22]。

As a significant ion pump in cell membranes, plasma membrane (PM) H⁺-ATPase can establish a transmembrane chemical gradient of H⁺ [23], which drives the acidification of the cell wall and thereby promotes root growth [24,25]. Several reports have noted that PM H⁺-ATPase activity is modulated by various environmental signals, including some classical plant hormones and N (reviewed in [23,26]).
质膜 （PM） H⁺-ATP 酶作为细胞膜中的重要离子泵，可以建立 H⁺ 的跨膜化学梯度 [23]，从而驱动细胞壁的酸化，从而促进根系生长 [24,25]。一些报告指出，PM H⁺-ATP 酶活性受各种环境信号的调节，包括一些经典的植物激素和 N（在 [23,26] 中已论述）。

Strawberry is a popular fresh fruit widely grown in most parts of the world. N acts as a major contributor to strawberry growth and fruit quality [27]. Low N severely inhibits strawberry growth, resulting in leaf chlorosis [28] and a decrease in leaf area, dry matter [29], and amino acids content in fruit [30]. Although there have been some reports about the morphological changes of roots in response to N deficiency in strawberry, the regulatory mechanism of hormones on strawberry root remodeling under N deficiency remains poorly understood. Our hypothesis is that low N stimulates root remodeling and enriches root structure for better N acquisition, which is regulated by hormonal interactions. Therefore, this study aims to evaluate the physiological and morphological responses of strawberry roots to low N stress, and preliminarily elucidates the possible regulatory mechanism. The findings are expected to afford a theoretical basis for the genetic improvement and more efficient N use in strawberry production.
草莓是一种流行的新鲜水果，在世界大部分地区广泛种植。氮是草莓生长和果实品质的主要贡献者 [27]。低氮严重抑制草莓生长，导致叶片萎黄[28]，果实中叶面积、干物质[29]和氨基酸含量减少[30]。尽管有一些关于草莓缺氮响应根系形态变化的报道，但激素对草莓缺氮下根系重塑的调控机制仍知之甚少。我们的假设是，低 N 刺激根重塑并丰富根结构以更好地获取 N，这是受荷尔蒙相互作用调节的。因此，本研究旨在评价草莓根系对低氮胁迫的生理和形态响应，并初步阐明可能的调控机制。这些发现有望为草莓生产中的遗传改良和更有效的氮利用提供理论基础。

2. Materials and Methods  2. 材料和方法

2.1. Cultivation Conditions
2.1. 栽培条件

Strawberry stolons (Miaoxiang 3) were selected from the greenhouse, soaked in fungicide, and then transferred into distilled water. When new leaves appeared, uniform seedlings were cultured in plastic basins containing nutrient solution (five seedlings per pot). The seedlings were grown in an artificial climate chamber at 25/20 °C (day/night) with a light cycle of 16/8 h (light/dark), 70% relative humidity, and an average light intensity of 350 μmol m⁻² s⁻¹ PPFD.
从温室中挑选草莓匍匐茎（苗香 3 号），用杀菌剂浸泡，然后转移到蒸馏水中。当新叶出现时，将均匀的幼苗在含有营养液的塑料盆中培养（每盆 5 棵幼苗）。幼苗在 25/20 °C（昼/夜）的人工气候室中生长，光照周期为 16/8 小时（亮/暗），相对湿度为 70%，平均光照强度为 350 μmol ^{m-2 s-1} PPFD。

The nutrient solution contained 2.05 mM KCl, 2.001 mM K₂HPO₄·3H₂O, 1.0 mM MgSO₄·7H₂O, 0.03 mM EDTA-Fe, 0.025 mM H₃BO₄, 2.0 μM MnSO₄·H₂O, 0.78 μM CuSO₄, 3.21 μM ZnSO₄·H₂O, and 0.5 μM Na₂MoO₄. Strawberry seedlings were supplied with a half-strength nutrient solution containing 15.0 mM NO₃⁻ for 2 d [31], and then cultured in a solution containing 1 mM NO₃⁻ (LN treatment), 1 mM NH₄⁺ (LA treatment), or 15 mM NO₃⁻ (Control). NO₃⁻ was added as Ca (NO₃)₂; NH₄⁺ was added as NH₄Cl; and CaCl₂ was used to maintain the same Ca²⁺ concentration for all treatments. The pH of the nutrient solution was held at 6.5 with KOH and HCl. The nutrient solution was renewed every two days and the pots were randomly positioned.
营养液中含有 2.05 mM KCl、2.001 mM K₂HPO₄·3H₂O、1.0 mM MgSO₄·7H₂O、0.03 mM EDTA-Fe、0.025 mM H₃BO₄、2.0 μM MnSO₄·H₂O、0.78 μM CuSO₄、3.21 μM ZnSO₄·H₂O 和 0.5 μM Na₂MoO₄。草莓幼苗用含有15.0 mM NO₃⁻的半强度营养液保存2 d [31]，然后在含有1 mM NO₃⁻（LN处理）、1 mM NH₄⁺（LA处理）或15 mM NO₃⁻（对照）的溶液中培养。NO₃⁻ 被添加为 Ca （NO₃）₂;NH₄⁺ 添加为 NH₄Cl;CaCl₂ 用于在所有处理中维持相同的 Ca²⁺ 浓度。营养液的 pH 值保持在 6.5，KOH 和 HCl 。营养液每两天更新一次，花盆随机放置。

At each sampling time, seedlings were first washed with distilled water and dried with absorbent paper, and then were divided into two parts. The fresh sample was used for biomass, root morphology, and lateral root density determination. The other part was treated with liquid nitrogen and stored at −80 °C for the determination of soluble proteins, enzyme activities, and hormones.
在每个采样时间，先用蒸馏水洗涤幼苗，用吸水纸干燥，然后分成两部分。新鲜样品用于生物量、根形态和侧根密度测定。另一部分用液氮处理并储存在 -80 °C 下，用于测定可溶性蛋白质、酶活性和激素。

2.2. Biomass Determination
2.2. 生物量测定

Strawberry seedlings were washed with distilled water and dried with absorbent paper at 24, 48, and 96 h after treatment. The stems and roots were weighed separately.
草莓幼苗用蒸馏水洗涤，并在处理后 24 、 48 和 96 h 用吸水纸干燥。茎和根分别称重。

2.3. Root Morphology Examination
2.3. 牙根形态检查

At 0, 48, and 96 h after treatment, the roots were washed with distilled water and floated in a PVC tray containing 2–3 mm of water, and then scanned with an EPSON V850 PRO scanner (EPSON, Beijing, China). Total root length, root surface area, average diameter, and the number of root tips per plant in the images were analyzed using WinRHIZO 2017a (Regent Instruments Inc., Quebec, QC, Canada).
在处理后 0、48 和 96 小时，用蒸馏水洗涤根部并漂浮在装有 2-3 毫米水的 PVC 托盘中，然后用 EPSON V850 PRO 扫描仪（EPSON，中国北京）扫描。使用 WinRHIZO 2017a（Regent Instruments Inc.，魁北克，QC，加拿大）分析图像中总根长、根表面积、平均直径和每株植物的根尖数。

2.4. Lateral Root Density Determination
2.4. 侧根密度测定

After washing the adventitious roots with distilled water at 48 h after treatment, the number of emerged lateral roots and the length of the adventitious root were counted under a microscope. Lateral root density (LRD) was recorded as the ratio of the number of emerged lateral roots to the distance, which was expressed as cm⁻¹ root length [32].
处理后 48 h 用蒸馏水清洗不定根后，在显微镜下计数出出的侧根数量和不定根的长度。侧根密度 （LRD） 记录为出现的侧根数量与距离的比值，以 ^cm-1 根长表示 [32]。

2.5. Enzyme Activity Assay
2.5. 酶活性测定

Strawberry roots were washed with distilled water and dried with absorbent paper at 12 and 48 h after treatment. After being ground into powder, these roots were used for the determination of enzyme activity.
草莓根用蒸馏水洗涤，并在处理后 12 和 48 h 用吸水纸干燥。研磨成粉末后，这些根用于测定酶活性。

NR activity was determined by reference to the method of Majláth et al. [33]. Strawberry roots (1 g) were well ground in 1 mL 0.2 M phosphate buffer (pH 7.5) and centrifuged at 12,000× g for 5 min at 4 °C. About 0.5 mL 0.1 M KNO₃ and 0.3 mL 2.0 mg mL⁻¹ NADH were added into 0.2 mL supernatant and the mixture reaction was conducted in a water bath at 25 °C for 30 min. Then, the reaction was terminated by the addition of 1 mL 30% trichloroacetic acid. The reaction mixture was added with 2 mL 1% sulfonamide and 2 mL 0.2% α-naphthylamine and mixed well. After standing for 15 min, colorimetry was performed at a 520 nm wavelength by using a Multiskan GO 1510 spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland). One unit of NR activity (U g⁻¹) was defined as the amount of 1 μmol NADH consumed per gram of sample per hour.
NR 活性是通过参考 Majláth 等人 [33] 的方法确定的。将草莓根 （1 g） 在 1 mL 0.2 M 磷酸盐缓冲液 （pH 7.5） 中充分研磨，并在 4 °C 下以 12,000× g 离心 5 分钟。 将约 0.5 mL、0.1 M KNO₃ 和 0.3 mL、2.0 mg ^mL-1 NADH 加入 0.2 mL 上清液中，并在 25 °C 的水浴中反应 30 分钟。然后，通过加入 1 mL 30% 三氯乙酸终止反应。将反应混合物加入 2 mL 1% 磺酰胺和 2 mL 0.2% α-萘胺中，并充分混合。静置 15 分钟后，使用 Multiskan GO 1510 分光光度计（Thermo Fisher Scientific，Vantaa，Finland）在 520 nm 波长下进行比色法测定。一个单位的 NR 活性 （U ^g-1） 定义为每小时每克样品消耗的 1 μmol NADH 量。

NiR activity was assayed under the conditions adapted from the study of Caroline et al. [34]. Briefly, 0.1 g strawberry root and 1 mL buffer (pH 8.0) were ground in a frozen mortar and centrifuged at 12,000× g for 2 min at 4 °C. The buffer was a mixture of 50 mM Tris-HCl and 3 mM EDTA. About 0.1 mL of supernatant was added to a 1.2 mL reaction solution (including 100 mM potassium phosphate buffer (pH 7.5), 10 mM KNO₂, 15 mg mL⁻¹ methyl viologen, H₂O, and 5% sodium dithionite). The mixture reaction was conducted in a water bath at 25 °C for 30 min. Then, 1 mL 1% sulfonamide and 1 mL 0.2% α-naphthylamine were added and shaken well, and the absorbance was measured at 540 nm after standing for 30 min. One unit of NiR activity (U g⁻¹) was defined as the amount of reduction of 1 μmol NO₂⁻ g⁻¹ h⁻¹.
在适应 Caroline 等人 [34] 研究的条件下测定 NiR 活性。简而言之，将 0.1 g 草莓根和 1 mL 缓冲液 （pH 8.0） 在冷冻研钵中研磨，并在 4 °C 下以 12,000 × g 离心 2 分钟。 缓冲液是 50 mM Tris-HCl 和 3 mM EDTA 的混合物。向 1.2 mL 反应溶液中加入约 0.1 mL 上清液（包括 100 mM 磷酸钾缓冲液 （pH 7.5）、10 mM KNO₂、15 mg mL⁻¹ 甲基紫精、H₂O 和 5% 连二亚硫酸钠）。混合物反应在 25 °C 水浴中反应 30 min。然后，加入 1 mL 1% 磺酰胺和 1 mL 0.2% α-萘胺并充分摇匀，静置 30 min 后在 540 nm 处测定吸光度。一个单位的 NiR 活性 （U ^g-1） 定义为 1 μmol NO ^{2-g-1 h-1} 的还原量。

GOGAT activity was measured according to Singh et al. [35], with minor modifications. Strawberry roots (0.1 g) were well ground in 1 mL 25 mM Tris-HCl buffer and centrifuged at 8000× g for 10 min at 4 °C. The supernatant was mixed thoroughly with 20 mM glutamine, 100 mM α-ketoglutarate, and 3 mM NADH. Then, the activity of GOGAT was measured at 340 nm, and expressed as the amount of 1 nmol NADH consumed g⁻¹ min⁻¹.
根据Singh等[35]测量GOGAT活性，并稍作修改。将草莓根 （0.1 g） 在 1 mL 25 mM Tris-HCl 缓冲液中充分研磨，并在 4 °C 下以 8000 × g 离心 10 分钟。 将上清液与 20 mM 谷氨酰胺、100 mM α-酮戊二酸和 3 mM NADH 充分混合。然后，在 340 nm 处测量 GOGAT 的活性，并表示为消耗的 1 nmol NADH 的量 ^{g-1 min-1}。

GS activity was determined according to the method described by Wang et al. [36]. Strawberry roots (1 g) and 4 mL 0.05 mM phosphate buffer (containing 0.4 M sucrose and 4 mM L-cysteine) were thoroughly ground in a frozen mortar. After centrifugation at 12,000× g and 4 °C for 15 min, 1 mL of supernatant was added to 3 mL enzyme reaction solution, which was composed of 50 mM L-glutamate sodium, 4 mM ATP-2Na, 40 mM hydroxylamine, 20 mM magnesium sulfate, 10 mM L-cysteine, and 40 mM phosphate buffer. The reaction was carried out at 30 °C for 15 min. Then, 30% trichloroacetic acid, 5.5 M HCl, and 8% FeCl₃ were added to the mixture to terminate the reaction. After standing for 10 min, the absorbance was measured at 540 nm. GS activity was expressed as the amount of 1 nmol of γ-glutamylhydroxamate generated g⁻¹ min⁻¹.
根据Wang等[36]描述的方法测定GS活性。将草莓根 （1 g） 和 4 mL 0.05 mM 磷酸盐缓冲液（含有 0.4 M 蔗糖和 4 mM L-半胱氨酸）在冷冻研钵中彻底研磨。在 12,00×0 g 和 4 °C 下离心 15 分钟后，将 1 mL 上清液加入 3 mL 酶反应溶液中，该溶液由 50 mM L-谷氨酸钠、4 mM ATP-2Na、40 mM 羟胺、20 mM 硫酸镁、10 mM L-半胱氨酸和 40 mM 磷酸盐缓冲液组成。反应在 30 °C 下进行 15 分钟。然后，向混合物中加入 30% 三氯乙酸、5.5 M HCl 和 8% FeCl₃ 以终止反应。静置 10 分钟后，在 540 nm 处测量吸光度。GS 活性表示为 1 nmol γ-谷氨酰异羟肟酸盐产生的量 ^{g-1 min-1}。

2.6. Soluble Protein Content Determination
2.6. 可溶性蛋白含量测定

Soluble protein content was determined according to the method described by Bradford et al. [37], with minor modifications. At 12 h and 48 h after treatment, strawberry roots (0.2 g) were fully ground and centrifuged at 5000× g for 10 min at 4 °C. About 0.1 mL supernatant was mixed with 5 mL of Komas Brilliant Blue G-250 solution (consisting of 100 mg L⁻¹ Komas Brilliant Blue G-250, 4.7% ethanol (v/v) and 8.5% (w/v) phosphoric acid), and the absorbance was measured at 595 nm after standing for 2 min. The soluble protein content was calculated using a standard curve based on bovine serum protein.
根据Bradford等[37]描述的方法测定可溶性蛋白含量，并稍作修改。处理后 12 h 和 48 h，将草莓根 （0.2 g） 充分研磨，并在 4 °C 下以 5000× g 离心 10 分钟。 将约 0.1 mL 上清液与 5 mL Komas Brilliant Blue G-250 溶液（由 100 mg L ^-1 Komas Brilliant Blue G-250、4.7% 乙醇 （v/v） 和 8.5% （w/v） 磷酸组成）混合，静置 2 分钟后在 595 nm 处测量吸光度。使用基于牛血清蛋白的标准曲线计算可溶性蛋白含量。

2.7. Total N Content Measurement
2.7. 总氮含量测量

The total N content in strawberry roots was determined using the Kjeldahl method [38]. At 48 h after treatment, dried strawberry roots (0.1 g) were digested in a Kjeldahl flask with sulfuric acid (5 mL) and hydrogen peroxide. After the mixture was clear, the distilled solution was obtained using a Kjeldahl apparatus (FOSS Kjeltec™ 8400, Hilleroed, Denmark) and titrated with 0.01 mol L⁻¹ 1/2 H₂SO₄.
使用凯氏定氮法测定草莓根中的总氮含量 [38]。处理后 48 小时，将干草莓根 （0.1 g） 放入装有硫酸 （5 mL） 和过氧化氢的凯氏定氮瓶中消化。混合物澄清后，使用凯氏定氮仪（FOSS Kjeltec™ 8400，Hilleroed，丹麦）获得蒸馏液，并用 0.01 mol ^L-1 1/2 H₂SO₄ 滴定。

2.8. PM H⁺-ATPase Activity Assay
2.8. PM H⁺-ATPase 活性测定

PM H⁺-ATPase activity was determined according to Zhang et al. [39]. At 12 h and 48 h after treatment, strawberry roots were homogenized in buffer (containing 250 mM sucrose, 4 mM DTT, 7.2 μg mL⁻¹ PMSF, 50 mM Tris, 8 mM EDTA, and 1.5% PVP). After centrifugation at 10,000× g for 15 min at 4 °C, the supernatant was centrifuged again at 10,000× g for 30 min at 4 °C. The precipitate was re-solubilized in a buffer containing 250 mM sucrose, 2 mM DTT, and 5 mM Pipes. After treatment with sucrose gradient solution and KCl, the obtained precipitate was added into 0.5 mL of reaction solution consisting of 250 mM HEPES-Tris, 25 mM ATP-Na₂, 3 mM Na₂MoO₄, 1 mM NaN₃, 1 mM EDTA, and 0.02% Triton X-100. After the reaction was terminated, 50 μL 10% ascorbic acid was added, and the absorbance was measured at 660 nm after standing for 40 min.
根据Zhang等[39]测定PM H ⁺ -ATP酶活性。在处理后 12 小时和 48 小时，将草莓根在缓冲液（含有 250 mM 蔗糖、4 mM DTT、7.2 μg ^mL-1 PMSF、50 mM Tris、8 mM EDTA 和 1.5% PVP）中匀浆。在 4 °C 下以 10,00×0 g 离心 15 分钟后，将上清液在 4 °C 下再次以 10,000 × g 离心 30 分钟。 将沉淀物重新溶解在含有 250 mM 蔗糖、2 mM DTT 和 5 mM 管道的缓冲液中。用蔗糖梯度溶液和 KCl 处理后，将获得的沉淀加入 0.5 mL 反应溶液中，该溶液由 250 mM HEPES-Tris、25 mM ATP-Na₂、3 mM Na₂MoO₄、1 mM NaN₃、1 mM EDTA 和 0.02% Triton X-100 组成。反应终止后，加入 50 μL 10% 抗坏血酸，静置 40 分钟后在 660 nm 处测定吸光度。

2.9. Determination of Plant Hormones
2.9. 植物激素的测定

Hormones in strawberry roots were determined according to Yang et al. [40], with slight modifications. At 12 h and 48 h after treatment, strawberry roots (0.1 g) were ground in liquid nitrogen and added to 1 mL of extraction solution (consisting of 0.4 mL methanol, 0.4 mL acetonitrile, and 0.2 mL water), and then extracted for 12 h at 4 °C with protection from light before centrifugation at 14,000× g for 10 min. About 0.8 mL supernatant was dried using a nitrogen evaporator, then re-dissolved in 0.2 mL 50% methanol and centrifuged at 14,000× g for 10 min to obtain the supernatant.
根据Yang等[40]测定草莓根中的激素，并稍作修改。处理后 12 h 和 48 h，将草莓根 （0.1 g） 用液氮研磨并加入 1 mL 提取溶液（由 0.4 mL 甲醇、0.4 mL 乙腈和 0.2 mL 水组成）中，然后在 4 °C 下避光提取 12 h，然后以 14,00×0 g 离心 10 g。使用氮气蒸发器干燥约 0.8 mL 上清液， 然后重新溶解在 0.2 mL 50% 甲醇中，并以 14,000× g 离心 10 分钟，得到上清液。

The supernatant was separated by an ExionLC™ AD series high-performance liquid chromatography system (AB SCIEX, Framingham, MA, USA) and a Kinetex^® C18 column (1.7 μm, 150 × 2.1 mm; Phenomenex, Torrance, CA, USA). Distilled water containing 0.04% (v/v) formic acid was used for mobile phase A, and methanol was used for mobile phase B. The elution gradients were as follows: 0–5.5 min, mobile phase B varied linearly from 10% to 95%; 5.6–7 min, mobile phase B remained at 95%; 7.1–7.5 min, mobile phase B varied linearly from 95% to 10%; 7.6–10 min, mobile phase B remained at 10%. Mass spectrometry in the positive/negative ion mode was completed using an AB Sciex Triple Quad 3500 (AB SCIEX, Framingham, MA, USA). Multi Quant software was used to extract the peak areas and retention time of the chromatograms. Hormone contents in strawberry roots were calculated from the standard curve.
通过 ExionLC™ AD 系列高效液相色谱系统 （AB SCIEX， Framingham， MA， USA） 和 Kinetex^® C18 色谱柱 （1.7 μm， 150 × 2.1 mm;Phenomenex， Torrance， CA， USA）。流动相A采用含0.04% （v/v）甲酸的蒸馏水，流动相B采用甲醇。洗脱梯度如下：0–5.5 min，流动相B在10%至95%范围内线性变化;5.6–7 min，流动相B保持在95%;7.1–7.5 min，流动相B在95%至10%范围内线性变化;7.6–10 min，流动相 B 保持在 10%。使用 AB Sciex Triple Quad 3500（AB SCIEX，Framingham，MA，USA）完成正/负离子模式下的质谱分析。使用 Multi Quant 软件提取色谱图的峰面积和保留时间。根据标准曲线计算草莓根中的激素含量。

2.10. Statistical Analysis
2.10. 统计分析

Data analysis was performed using SPSS V.26 (SPSS Statistics, Armonk, NY, USA). Duncan’s test was used to detect significant differences at the 95% probability level. If the difference is significant between treatments, a different letter is used.
使用 SPSS V.26 （SPSS Statistics， Armonk， NY， USA） 进行数据分析。Duncan 检验用于检测 95% 概率水平的显著差异。如果处理之间的差异很大，则使用不同的字母。

3. Results  3. 结果

3.1. Biomass of Strawberry Roots
3.1. 草莓根的生物量

Compared with the control, low N stresses showed no significant effect on shoot fresh weight at 24, 48, and 96 h (Figure 1a), but significantly increased the fresh weight of strawberry roots at 48 h (Figure 1b).
与对照相比，低氮胁迫在 24 、 48 和 96 h 对地上部鲜重没有显著影响 （图 1a），但在 48 h 时显著增加了草莓根的鲜重 （图 1b）。

Figure 1. Effect of low N stress on the fresh weight of strawberry shoots and roots at 24 h, 48 h, and 96 h. (a) Shoot fresh weight. (b) Root fresh weight. Values represent mean ± SEM of five biological replicates. Different letters indicate significant differences at p < 0.05.
图 1. 低氮胁迫对 24 h、48 h 和 96 h 草莓芽和根鲜重的影响。（a） 地上部鲜重。（b） 根鲜重。值表示 5 次生物学重复的 SEM ±平均值。不同字母表示在 p < 0.05 处差异显著。

3.2. Morphology of Strawberry Roots
3.2. 草莓根的形态

Low N stresses altered the root structure of strawberry. The total root length under LN treatment increased by 30.34% at 48 h and 27.71% at 96 h in comparison with that under the control but by 23.67% at 48 h and 8.5% at 96 h under LA treatment (Figure 2a).
低 N 胁迫改变了草莓的根系结构。与对照相比，LN 处理下 48 h 和 96 h 的总根长分别增加了 30.34% 和 27.71%，但在 LA 处理下 48 h 和 96 h 分别增加了 23.67% 和 8.5%（图 2a）。

Figure 2. Root morphology of strawberry at 0 h, 48 h, and 96 h under different N treatments. (a) Total root length. (b) Total surface area. (c) Total number of root tips. (d) Average root diameter. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 2. 不同氮处理下草莓 0 h、48 h 和 96 h 的根系形态。（a） 总根长。（b） 总表面积。（c） 根尖总数。（d） 平均根直径。值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

At 48 h, compared with that of the control, the root surface area under LN and LA treatments increased by 21.23% and 22.93%, respectively. However, both LN and LA treatments resulted in no significant difference in the root surface area relative to the control at 96 h (Figure 2b).
48 h时，LN和LA处理下根系表面积较对照分别增加21.23%和22.93%。然而，LN 和 LA 处理在 96 小时时相对于对照的根表面积没有显着差异（图 2b）。

The number of root tips increased significantly under low N stresses (Figure 2c). Compared with that of the control, the number of root tips under LN and LA treatments increased by 15.63% and 8.26% at 48 h, respectively, and a similar trend was observed at 96 h.
在低氮胁迫下，根尖的数量显著增加（图 2c）。与对照相比，LN 和 LA 处理在 48 h 时根尖数量分别增加了 15.63% 和 8.26%，在 96 h 时也观察到类似的趋势。

However, the average root diameter decreased by 9.02% and 7.46% under LN and LA treatments, respectively, compared with that of the control at 48 h (Figure 2d). At 96 h, compared with that of the control, the average root diameter decreased by 11.34% and 6.40%, respectively.
然而，与 48 h 对照相比，LN 和 LA 处理下的平均根直径分别减少了 9.02% 和 7.46%（图 2d）。96 h时，与对照相比，平均根径分别减少了11.34%和6.40%。

3.3. Lateral Root Density
3.3. 侧根密度

LN and LA treatments significantly promoted the formation of lateral roots at 48 h (Figure 3). Compared with that of the control (4.28 ± 0.14 cm⁻¹ root length), LRD increased by 36.69% under LN treatment (5.86 ± 0.32 cm⁻¹ root length) and 24.82% under LA treatment (5.35 ± 0.38 cm⁻¹ root length).
LN 和 LA 处理显着促进了 48 h 侧根的形成（图 3）。与对照 （4.28 ± 0.14 cm⁻¹ 根长） 相比，LN 处理下 LRD 增加了 36.69% （5.86 ± 0.32 ^cm-1 根长），LA 处理下增加了 24.82% （5.35 ± 0.38 ^cm-1 根长）。

Figure 3. Lateral root density under three treatments at 48 h. Values represent mean ± SEM of five biological replicates. Different letters indicate significant differences at p < 0.05.
图 3. 48 小时时 3 次处理下的侧根密度。值表示 5 次生物学重复的 SEM ±平均值。不同字母表示在 p < 0.05 处差异显著。

3.4. Hormone Contents in Strawberry Roots
3.4. 草莓根中的激素含量

Compared with the control, the LN and LA treatment increased the IAA concentration in strawberry roots by 42.37% and 49.51% at 12 h, respectively (Figure 4a). However, no significant difference was detected in root IAA level at 48 h among the three treatments.
与对照相比，LN 和 LA 处理在 12 h 时草莓根中的 IAA 浓度分别提高了 42.37% 和 49.51%（图 4a）。然而，在 3 种处理中，48 h 根系 IAA 水平没有显著差异。

Figure 4. IAA concentration (a), JA concentration (b), and SA concentration (c) in strawberry roots at 12 h and 48 h under different N treatments. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 4. 不同氮处理下草莓根系 IAA 浓度 （a） 、 JA 浓度 （b） 和 SA 浓度 （c） 在 12 h 和 48 h 时。值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

The JA concentration increased significantly under low N stresses (Figure 4b). Compared with that of the control, the JA concentration under LN and LA treatments increased by 64.49% and 36.68% at 12 h, respectively, and a similar trend was observed at 48 h. Moreover, the root JA concentration under LN treatment was 49.13% higher than that under LA treatment.
在低 N 应力下，JA 浓度显著增加（图 4b）。与对照相比，LN 和 LA 处理下 JA 浓度在 12 h 分别提高了 64.49% 和 36.68%，在 48 h 时也观察到类似的趋势。LN 处理下根系 JA 浓度比 LA 处理下高 49.13%。

However, the root SA concentration was significantly lower under low N stresses compared with that under the control (Figure 4c). The root SA concentration decreased by 35.55% and 47.69% under LN and LA treatments, respectively, compared with that of the control at 12 h, and a similar trend was observed at 48 h. However, there was no significant difference between LN and LA treatments at 12 h and 48 h.
然而，与对照相比，低氮胁迫下的根系 SA 浓度显著降低（图 4c）。12 h时，LN和LA处理下根系SA浓度较对照分别下降了35.55%和47.69%，48 h时也呈类似趋势。然而，LN 和 LA 处理在 12 h 和 48 h 时没有显著差异。

3.5. PM H⁺-ATPase Activity
3.5. PM H⁺-ATPase 活性

At 12 h, compared with that of the control, the PM H⁺-ATPase activity was significantly higher under the LN treatment, but only slightly increased under LA treatment with no significant difference (Figure 5). The PM H⁺-ATPase activity was significantly increased at 48 h under both LN and LA treatments compared with that of the control.
在 12 小时时，与对照相比，LN 处理下 PM H⁺-ATP 酶活性显着升高，但在 LA 处理下仅略有增加，无显著差异（图 5）。与对照相比，LN 和 LA 处理下 48 h PM H⁺-ATP 酶活性显著增加。

Figure 5. Effect of different N treatments on H⁺-ATPase enzyme activity in strawberry roots at 12 h and 48 h. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 5. 不同氮处理对草莓根系 12 h 和 48 h H⁺-ATPase 酶活性的影响值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

3.6. Enzyme Activity Related to Nitrogen Assimilation
3.6. 与氮同化相关的酶活性

There was no significant difference in the root NR activity of strawberry between low N treatments and the control at 12 h (Figure 6a). At 48 h, the LN and LA treatments increased the NR activity by 19.83% and 10.48%, respectively. However, both LN and LA treatments resulted in no significant difference in NiR activity relative to the control at 12 h and 48 h (Figure 6b).
在 12 h 时，低氮处理与对照之间草莓的根 NR 活性没有显著差异（图 6a）。48 h 时，LN 和 LA 处理使 NR 活性分别提高了 19.83% 和 10.48%。然而，LN 和 LA 处理在 12 小时和 48 小时时相对于对照的 NiR 活性没有显着差异（图 6b）。

Figure 6. Effects of low N stresses on NR (a), NiR (b), GS (c), and GOGAT (d) activity in strawberry roots at 12 h and 48 h. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 6. 低氮胁迫对草莓根系 12 h 和 48 h NR （a） 、 NiR （b） 、 GS （c） 和 GOGAT （d） 活性的影响。值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

Compared with the control, the LN treatment significantly increased the GS activity at 12 h and 48 h, while the LA treatment only significantly increased the GS activity at 48 h (Figure 6c).
与对照相比，LN 处理在 12 h 和 48 h 时显着增加 GS 活性，而 LA 处理仅在 48 h 时显着增加 GS 活性（图 6c）。

The LN treatment resulted in similar GOGAT activity to the control at 12 h and 48 h (Figure 6d). However, the LA treatment increased the GOGAT activity by 15% and 14.85% at 12 h and 48 h, respectively, compared with the control.
LN 处理在 12 小时和 48 小时时产生与对照相似的 GOGAT 活性（图 6d）。然而，与对照相比，LA 处理在 12 h 和 48 h 时分别使 GOGAT 活性提高了 15% 和 14.85%。

3.7. Soluble Protein Content in Strawberry Roots
3.7. 草莓根中的可溶性蛋白含量

Low N stresses showed no significant effect on the soluble protein content in strawberry roots at 12 h (Figure 7). In contrast, at 48 h, the LN and LA treatment increased the soluble protein content by 96.97% and 84.25%, respectively, compared with the control. At 12 h and 48 h, the LN treatment led to a slightly higher soluble protein content than the LA treatment without significant difference.
低氮胁迫对 12 h 时草莓根中的可溶性蛋白含量没有显着影响（图 7）。相比之下，在 48 h 时，LN 和 LA 处理的可溶性蛋白含量分别比对照提高了 96.97% 和 84.25%。在 12 h 和 48 h 时，LN 处理导致可溶性蛋白含量略高于 LA 处理，无显著差异。

Figure 7. Soluble protein content at 12 h and 48 h under different N treatments. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 7. 不同 N 处理下 12 h 和 48 h 的可溶性蛋白含量。值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

3.8. Total Nitrogen Content in Strawberry Roots
3.8. 草莓根中的总氮含量

At 48 h, both LN and LA treatments significantly reduced the total N content in strawberry roots (Figure 8). Compared with the control, the LN and LA treatment reduced the total N content by 17.86% and 24.4%, respectively.
48 h时，LN 和 LA 处理均显著降低草莓根系的总 N 含量（图 8）。与对照相比，LN 和 LA 处理分别降低了 17.86% 和 24.4% 的总 N 含量。

Figure 8. Total N content at 48 h under different N treatments. Values represent mean ± SEM of three biological replicates. Different letters indicate significant differences at p < 0.05.
图 8. 不同 N 处理下 48 h 全 N 含量。值表示三个生物学重复的平均 ± SEM。不同字母表示在 p < 0.05 处差异显著。

4. Discussion  4. 讨论

4.1. Root Architecture Changes for Better N Uptake
4.1. 根结构变化以获得更好的 N 吸收

Root structural plasticity is critical to plant adaptive response to complex and varying living environments. To date, numerous studies have demonstrated that changes in root architecture can dramatically improve crop nutrient efficiency [41]. Correspondingly, root structure can be influenced by nutrient conditions such as N supply [42]. The results of this experiment showed a significant increase in the root fresh weight at 48 h under low N conditions. Therefore, we focused more on the analysis of the roots. Further examination of root architecture revealed that low N treatments significantly increased the total root length, root surface area, and total root tip number, but obviously reduced the average root diameter at 48 h compared with the control. These results are consistent with previous studies, which reported that crops would develop a deeper root system and larger root surface area for more efficient acquisition of N to better adapt to N deficiency [43,44,45].
根系结构可塑性对于植物对复杂多变的生活环境的适应性反应至关重要。迄今为止，大量研究表明，根系结构的变化可以显著提高作物的养分效率[41]。相应地，根系结构会受到营养条件的影响，如氮供应[42]。本试验结果表明，在低氮条件下，48 h时根系鲜重显著增加。因此，我们更侧重于根的分析。进一步检查根结构表明，与对照相比，低氮处理显著增加了总根长、根表面积和总根尖数，但明显降低了 48 h 时的平均根直径。这些结果与以前的研究一致，这些研究报告称，作物会发育出更深的根系和更大的根表面积，从而更有效地获取氮，从而更好地适应氮缺乏[43,44,45]。

Plant hormones play important roles in controlling root development, including lateral root growth and root hair formation [46], and have significant interactions with N [47]. Jia et al. [48] revealed that low N stress could upregulate the transcription of YUC8 and its homologs and the TAA1 gene to enhance local IAA biosynthesis in Arabidopsis roots. Some recent studies have demonstrated that NO₃⁻ and NH₄⁺ signaling can mediate the shoot-to-root transport of auxin [49], and regulate its accumulation in the epithelial cells of lateral root primordia, which in turn stimulates the emergence and growth of lateral roots [50]. Sun et al. [51] reported that low N stress could increase auxin accumulation in roots, enhancing root development through several auxin-mediated pathways. We observed a significant increase in IAA concentration at 12 h under low N treatments in this study. Based on the above results, it could be speculated that low-NO₃⁻ and low-NH₄⁺ signaling could induce local IAA biosynthesis in strawberry plants and enhance its intercellular transport, thereby increasing root IAA concentration and promoting root growth.
植物激素在控制根系发育方面起重要作用，包括侧根生长和根毛形成[46]，并与N有显著的相互作用[47]。Jia等[48]揭示了低氮胁迫可以上调YUC8及其同源物和TAA1基因的转录，从而增强拟南芥根中局部IAA的生物合成。最近的一些研究表明，NO₃⁻ 和 NH₄⁺ 信号转导可以介导生长素从芽到根的转运 [49]，并调节其在侧根原基上皮细胞中的积累，进而刺激侧根的出现和生长 [50]。Sun等[51]报道，低氮胁迫可以增加根系中生长素的积累，通过几种生长素介导的途径促进根系发育。在本研究中，我们观察到在低 N 处理下 12 h IAA 浓度显着增加。基于上述结果，可以推测低 NO₃^- 和低 NH₄⁺ 信号可以诱导草莓植株局部 IAA 生物合成并增强其细胞间转运，从而提高根 IAA 浓度并促进根系生长。

As mentioned above, JA and SA are also closely associated with signaling pathways regulating root structure. Wang et al. [52] and Sun et al. [53] suggested that jasmonate application could increase the lateral root number in rice and Arabidopsis. Gutierrez et al. [54] reported that JA negatively affected adventitious root formation. Moreover, extensive research has revealed a close correlation between JA and auxin. Several studies have suggested that the JA signaling pathway is positively associated with auxin homeostasis by regulating the expression of the auxin-related gene [16,55]. Xu et al. [12] revealed that JA promotes auxin transport by regulating PIN genes, while auxin regulates JA homeostasis by modulating GH3.3/5/6 genes. Our study revealed significant increases in LRD, IAA, and JA concentration under low N stresses in strawberry. Therefore, it can be inferred that there is a synergistic association between JA and IAA to facilitate lateral root formation under N deficiency.
如上所述，JA 和 SA 也与调节根结构的信号通路密切相关。Wang等[52]和Sun等[53]认为，茉莉酸酯的应用可以增加水稻和拟南芥的侧根数。Gutierrez等[54]报道，JA对不定根形成产生负面影响。此外，广泛的研究揭示了 JA 和生长素之间的密切相关性。几项研究表明，JA信号通路通过调节生长素相关基因的表达与生长素稳态呈正相关[16,55]。Xu等[12]揭示了JA通过调节PIN基因促进生长素转运，而生长素通过调节GH3.3/5/6基因来调节JA稳态。我们的研究揭示了在低氮胁迫下草莓 LRD 、 IAA 和 JA 浓度的显著增加。因此，可以推断 JA 和 IAA 之间存在协同关联，以促进缺氮下侧根的形成。

Previous research has highlighted the role of SA in reducing the number of lateral roots in Arabidopsis seedlings [18,19,56]. Moreover, several studies have indicated that SA promotes or inhibits lateral root growth depending on its own concentration [57] and the IAA concentration [58]. Research in this area has documented the interaction between SA and IAA. Kitakura et al. [59] observed that a high level of SA would interfere with auxin distribution. Llorente et al. [60] found that SA could improve the stability of auxin repressor proteins in the process of auxin signal transduction by binding to them, thereby short circuiting the auxin signal network. Similarly, the antagonistic relationship between SA and JA in regulating root morphology has been widely investigated [61,62]. SA-induced expression of ANAC032 and GRX480 can inhibit JA signaling in plant immunity [63,64]. Interestingly, JA signaling also blocks SA biosynthesis by modulating the activities of multiple NAC transcription factors (NAM, ATAF, and CUC transcription factors) [65]. In this study, we observed a significant decrease in SA concentration in strawberry roots under low N stresses, which was contrary to the trend of IAA and JA. Hence, it is reasonable to speculate that SA may negatively regulate lateral roots by interfering with the signal transduction of IAA and JA.
以前的研究强调了 SA 在减少拟南芥幼苗侧根数量方面的作用 [18,19,56]。此外，几项研究表明，SA 促进或抑制侧根生长取决于其自身的浓度 [57] 和 IAA 浓度 [58]。该领域的研究记录了 SA 和 IAA 之间的相互作用。Kitakura等[59]观察到，高水平的SA会干扰生长素的分布。Llorente等[60]发现，SA可以通过与生长素抑制蛋白结合来提高生长素抑制蛋白在生长素信号转导过程中的稳定性，从而使生长素信号网络短路。同样，SA 和 JA 在调节根形态中的拮抗关系也得到了广泛的研究 [61,62]。SA 诱导的 ANAC032 和 GRX480 表达可以抑制植物免疫中的 JA 信号传导 [63,64]。有趣的是，JA 信号转导还通过调节多种 NAC 转录因子（NAM、ATAF 和 CUC 转录因子）的活性来阻断 SA 生物合成 [65]。本研究观察到低氮胁迫下草莓根系 SA 浓度显著降低，这与 IAA 和 JA 的趋势相反。因此，可以合理地推测 SA 可能通过干扰 IAA 和 JA 的信号转导来负向调节侧根。

Cell wall loosening, which is facilitated by H⁺ efflux provided by PM H⁺-ATPase [66], is the direct reason for cell expansion and plant growth [67]. Sperandio et al. [68] assessed the importance of PM H⁺-ATPase activity in the adaptation to N deficiency in rice. Further research revealed that the starvation and resupply of N could promote PM H⁺-ATPase activity and root growth [69]. It is widely acknowledged that IAA can induce PM H⁺-ATPase activity according to the acid growth theory [24]. Furthermore, recent research has confirmed that PM-ATPase activity is related to auxin-binding protein 1 (ABP1), which can be activated by auxin accumulation [70]. In this study, we observed the same increasing trend of LRD, IAA concentration, and H⁺-ATPase activity in strawberry under low N stresses. In general, it seemed that the accumulation of IAA increased PM H⁺-ATPase activity, which in turn promoted lateral root initiation and primordium development. This speculation is consistent with findings in the studies of maize and wheat by Sun et al. [51] and Lv et al. [71], who reported that an increase in IAA in maize and wheat roots under low N stress led to an increase in H⁺ efflux and acidification of apoplastic space, and ultimately boosted lateral root growth. Moreover, the addition of sodium orthovanadate (Na₃VO₄, an inhibitor of PM H⁺-ATPase activity) or 2,3,5-triiodobenzoic acid (TIBA, an inhibitor of the polar transport of auxin) eliminated the enhancing effect of N deficiency on root elongation.
PM H⁺-ATP酶提供的H⁺外排促进了细胞壁松动[66]，这是细胞扩增和植物生长的直接原因[67]。Sperandio 等 [68] 评估了 PM H⁺-ATP 酶活性在水稻适应缺氮中的重要性。进一步的研究表明，氮的饥饿和再供应可以促进 PM H⁺-ATP 酶活性和根系生长 [69]。根据酸生长理论，IAA 可以诱导 PM H⁺-ATP 酶活性 [24]。此外，最近的研究证实，PM-ATP酶活性与生长素结合蛋白1（ABP1）有关，生长素积累可以激活[70]。在这项研究中，我们观察到在低氮胁迫下草莓中 LRD 、 IAA 浓度和 H⁺-ATP 酶活性也有相同的增加趋势。一般来说，IAA 的积累似乎增加了 PM H⁺-ATP 酶活性，进而促进了侧根起始和原基发育。这一推测与Sun等[51]和Lv等[71]对玉米和小麦的研究结果一致，他们报道，在低氮胁迫下，玉米和小麦根系IAA的增加导致H⁺外排增加和质外体空间酸化，并最终促进侧根生长。此外，添加原钒酸钠 （Na₃VO_4，PM H⁺-ATP 酶活性抑制剂） 或 2,3,5-三碘苯并酸 （TIBA，生长素极性转运抑制剂） 消除了缺氮对根伸长的增强作用。

Soaking with methyl jasmonate could significantly increase the H⁺-ATPase activity and H⁺ transmembrane transport in ginger rhizomes [72]. Chen et al. [73] reported the enhancement of H⁺-ATPase activity mediated by JA signaling under multiple stresses (herbivore stress and salt stress). In addition, SA is involved in the regulation of PM H⁺-ATPase. Recent research has demonstrated that H⁺-ATPase activity can be upregulated in SA-pretreated seedlings, resulting in an enhanced tolerance to salt stress [74]. Interestingly, previous studies of temperature stress have shown that SA pretreatments can also stimulate PM H⁺-ATPase activity in grapes [75] and peas [76]. In this study, significant increases in JA concentration and H⁺-ATPase activity while a significant decrease in SA concentration were observed under low N stress in strawberry. Taking into account the synergistic and/or antagonistic effects among IAA, JA, and SA, it can be speculated that low N stress stimulates an asymmetric activation of the crosstalk network between hormones and contributes to a final balance of interaction, which may lead to increases in H⁺-ATPase activity and H⁺ efflux, thereby promoting lateral root formation and growth.
用茉莉酸甲酯浸泡可以显著提高姜根茎中 H⁺-ATP 酶活性和 H⁺ 跨膜转运 [72]。Chen等[73]报道了在多种胁迫（食草动物胁迫和盐胁迫）下JA信号传导介导的H⁺-ATP酶活性增强。此外，SA 还参与 PM H⁺-ATP 酶的调节。最近的研究表明，H⁺-ATP酶活性在SA预处理的幼苗中可以上调，从而增强对盐胁迫的耐受性[74]。有趣的是，先前对温度胁迫的研究表明，SA 预处理还可以刺激葡萄 [75] 和豌豆 [76] 中的 PM H⁺-ATP 酶活性。在本研究中，在低氮胁迫下，草莓的 JA 浓度和 H⁺-ATP 酶活性显著增加，而 SA 浓度显著降低。考虑到 IAA、JA 和 SA 之间的协同和/或拮抗作用，可以推测低 N 应激刺激激素之间串扰网络的不对称激活，并有助于相互作用的最终平衡，这可能导致 H⁺-ATP 酶活性和 H⁺ 外排的增加，从而促进侧根的形成和生长。

Lv et al. [71] observed significant decreases in root NO₃⁻ and NH₄⁺ influx under low N conditions, which was consistent with the decrease in root total N content. This finding is generally in agreement with our study. Moreover, root weight under low N conditions was significantly higher than that of the control, which may be related to the redistribution of photosynthate [77,78].
Lv等[71]观察到，在低氮条件下，根系NO₃⁻和NH₄⁺流入量显著减少，这与根系总氮含量的降低一致。这一发现与我们的研究基本一致。此外，在低氮条件下，根重显著高于对照，这可能与光合产物的再分配有关[77,78]。

4.2. Improving N Utilization via Enzyme Activities Changes
4.2. 通过改变酶活性提高氮利用率

After NO₃⁻ is absorbed by roots, NR and NiR initiate the first stage of N assimilation, followed by the GS/GOGAT cycle responsible for the conversion of inorganic N into organic N [79]. NR is the first and also a rate-limiting enzyme in N assimilation [80]. GS is a multifunctional enzyme, and its level can reflect the strength of N assimilation [81]. Li et al. [82] observed that NR activity in the growth medium of Pseudochlorococcum sp. gradually increased with N depletion. In contrast to NR, NiR does not appear to be affected by N level and form [83]. Xiong et al. [83] demonstrated that a low N level stimulates but a high N level inhibits GS activity in citrus. Zhou et al. [84] showed that lower N treatments induced higher GOGAT activity in lettuce. Here, we found that low NO₃⁻ resulted in higher NR and GS activities, whereas low NH₄⁺ led to higher GS and GOGAT activities. Considering the significant increase in soluble protein content in strawberry roots at 48 h under low N stresses, we speculate that low N promotes N assimilation mainly by inducing NR, GS, and GOGAT activities, thereby promoting protein biosynthesis.
NO₃⁻ 被根吸收后，NR 和 NiR 开始第一阶段的氮同化，然后是负责将无机氮转化为有机氮的 GS/GOGAT 循环 [79]。NR 是 N 同化中的第一种，也是一种限速酶 [80]。GS是一种多功能酶，其水平可以反映氮同化的强度[81]。Li等[82]观察到，随着氮的消耗，假绿球菌生长培养基中的NR活性逐渐增加。与 NR 相比，NiR 似乎不受 N 水平和形态的影响 [83]。Xiong等[83]证明，低氮水平刺激柑橘，但高氮水平抑制柑橘的GS活性。周等[84]表明，低氮处理诱导生菜中较高的GOGAT活性。在这里，我们发现低 NO₃⁻ 导致较高的 NR 和 GS 活性，而低 NH₄⁺ 导致较高的 GS 和 GOGAT 活性。考虑到低氮胁迫下草莓根系可溶性蛋白含量在 48 h 时的显著增加，我们推测低氮主要通过诱导 NR、GS 和 GOGAT 活性来促进氮同化，从而促进蛋白质生物合成。

It has been well documented that the activity of enzymes related to N assimilation is also regulated by phytohormones. In a previous study, with exogenous IAA application under copper (Cu) stress, spinach seedlings exhibited higher NR, GS, GOGAT activities, and soluble protein content [85]. Similarly, Parihar et al. [86] observed that the application of methyl jasmonate could increase the NR, GS, and GOGAT activities in luffa. Several studies have suggested that low concentrations of SA could increase the NR activity in wheat [87] and maize [88]; on the other hand, an inhibitory effect was observed at a high SA concentration [89]. In this study, compared with the control, low N stress significantly changed the NR, GS, and GOGAT activities as well as IAA, JA, and SA concentrations. Therefore, it is very possible that the crosstalk between hormones may increase the activities of N assimilation-related enzymes, which help the strawberry roots to maintain higher levels of N assimilation and protein biosynthesis under low N conditions.
有充分的证据表明，与 N 同化相关的酶的活性也受植物激素的调节。在以前的研究中，在铜 （Cu） 胁迫下施用外源 IAA，菠菜幼苗表现出更高的 NR、GS、GOGAT 活性和可溶性蛋白含量 [85]。同样，Parihar等[86]观察到，茉莉酸甲酯的应用可以增加丝瓜中的NR、GS和GOGAT活性。几项研究表明，低浓度的 SA 可以增加小麦 [87] 和玉米 [88] 中的 NR 活性;另一方面，在高 SA 浓度下观察到抑制作用 [89]。在本研究中，与对照相比，低氮胁迫显著改变了 NR 、 GS 和 GOGAT 活性以及 IAA 、 JA 和 SA 浓度。因此，激素之间的串扰很可能会增加 N 同化相关酶的活性，这有助于草莓根在低 N 条件下保持更高水平的 N 同化和蛋白质生物合成。

5. Conclusions  5. 结论

This study elucidates the central role of hormones in the complex regulatory network under N deficiency in strawberry. There are two possible mechanisms for the changes in root architecture of strawberry plants for better N acquisition. First, a larger root surface area is formed to enhance the ability of root to explore the soil for more N. This process may be mediated by the final balance of hormonal interactions (including IAA, JA, and SA), which can increase PM H⁺-ATPase activity and ultimately accelerate cell wall acidification, thereby enhancing lateral root formation and growth. Second, NR, GS, and GOGAT activities are enhanced to improve root N assimilation and protein biosynthesis, thus promoting lateral root growth. This process may also be associated with the regulation of hormonal networks. In conclusion, strawberry roots can better adapt to the N-deficient environment by increasing N absorption area and N assimilation.
本研究阐明了激素在草莓缺氮下复杂调控网络中的核心作用。草莓植株根结构的变化有两种可能的机制，可以更好地获取氮。首先，形成更大的根表面积以增强根在土壤中探索更多氮的能力。这个过程可能由激素相互作用（包括 IAA、JA 和 SA）的最终平衡介导，这可以增加 PM H⁺-ATP 酶活性并最终加速细胞壁酸化，从而增强侧根的形成和生长。其次，NR、GS 和 GOGAT 活性增强，以改善根 N 同化和蛋白质生物合成，从而促进侧根生长。这个过程也可能与荷尔蒙网络的调节有关。综上所述，草莓根系可以通过增加氮吸收面积和氮同化来更好地适应缺氮环境。