Nitrogen supply forms alter adaptive properties of Moso bamboo seedlings in low phosphorus conditions

doi:10.21203/rs.3.rs-4817491/v1

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Research Article

Nitrogen supply forms alter adaptive properties of Moso bamboo seedlings in low phosphorus conditions

https://doi.org/10.21203/rs.3.rs-4817491/v1

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Aims

Soil phosphorus loss often occurs in subtropical areas, resulting in extremely low phosphorus levels in forest land, thus affecting soil fertility and hindering normal plant growth. Further research is needed to understand plant preference for different nitrogen sources and to select appropriate nitrogen sources to improve fertilizer use efficiency. The relationship between soil nutrient dynamics and seedling physiology was studied, and the effects of different nitrogen fertilizers on the growth of Phyllostachys edulis seedlings under low phosphorus conditions were evaluated.

Methods

Under four different forms of nitrogen supply (NO₃⁻-N; NH₄⁺-N; NO₃⁻-N + NH₄⁺-N; Org-N) and two phosphorus treatment conditions (deficient, 5.0 mg·kg^− 1; sufficient, 20 mg·kg^− 1).

Results

Low phosphorus can alter the nutrient environment of some substrates and inhibit the growth of bamboo seedlings, but it improves the efficiency of plant phosphorus utilization; Low phosphorus has a strong inhibitory effect on bamboo, and under low phosphorus conditions, the proportion of amino acid components in bamboo is imbalanced. But it will enhance its adaptability to low phosphorus stress by coordinating the root to shoot ratio, changing the root morphology, improving the efficiency of nitrogen transport and metabolism in roots, stems, and leaves, enhancing the efficiency of leaf electron transfer, and redistributing nutrient resources. In addition, supplying NH₄⁺-N fertilizer under low phosphorus conditions can increase the root to shoot ratio of bamboo, promote the absorption of phosphorus and metal ions by roots, improve nitrogen transport and metabolic efficiency, enhance leaf light utilization capacity, and alleviate the inhibitory effect of bamboo under low phosphorus stress. However, supplying NO₃⁻-N + NH₄⁺-N fertilizer under suitable phosphorus conditions is more conducive to improving the photosynthetic capacity, nitrogen transport and metabolic efficiency of bamboo seedlings, resulting in a significant increase in their total biomass. Therefore, in the actual production and cultivation of bamboo seedlings, attention should be paid to the supply form of nitrogen fertilizer.

Conclusions

Single ammonium nitrogen fertilizer was more suitable for planting bamboo seedlings in natural forest land with seriously low phosphorus, while mixed inorganic nitrogen fertilizer was more suitable for planting bamboo seedlings in soil with reasonable phosphorus fertilizer. It provides theoretical basis for the cultivation and nutrient management practice of bamboo industry.

Low phosphorus stress

Nitrogen form

Phyllostachys edulis seedlings

Physiological characteristics

Bamboo growth

Global climate change represents a paramount environmental challenge in the 21st century, with escalating frequencies of extreme weather events particularly affecting tropical and subtropical regions [53,54]. Amidst these changes, the trophic imbalance between nitrogen (N) and phosphorus (P) in biospheres has gained insufficient attention despite its significance. The global N/P ratio of anthropogenic inputs has surged from 19:1 in the 1980s to 30:1 in 2020 [40]. Approximately 7 billion hectares of land worldwide face P deficiency, posing a critical limitation to plant yield increase [52]. Subtropical regions, characterized by hot temperatures and rainy climates, often experience P loss from soil [45], resulting in critically low P levels in forest land.

A balanced nutrient supply is imperative for achieving high crop yields and quality products, while the current global P shortage significantly affects soil fertility [36,47]. Studies have demonstrated that low P stress impedes the growth of various plants, impacting biomass accumulation, germination rate, chlorophyll content, the Rubisco, root morphological indicators as well the content and distribution of amino acid [20,30,48–50,61]. Concomitantly, the form of N fertilizer significantly influences P nutrient uptake, and superior N fertilizers can improve this process [34]. Nonetheless, extensive utilization of N fertilizers in production is extremely likely to lead to sudden acidification, affecting plant quality and yield [46]. As commonly acknowledged, plants absorb mineral N mainly as nitrate (NO₃⁻) and ammonium (NH₄⁺) [14]. There are differences in the preference for different forms of N fertilizer uptake by different plants, and within the same plant, there are preferences for N fertilizer uptake by different tissues [28]. Thus, proper N source selection is critical for enhancing plant N absorption, stimulating growth, and minimizing fertilizer waste [5].

Nitrogen source preferences are usually influenced by genetic and environmental factors [37]. Some plants exhibit preference or tolerance for specific N forms, influencing their growth efficiency [2,23]. For instance, nitrogen forms affect greenhouse corn plant growth and P uptake, with ammonium N being more effective for its P uptake, NH₄⁺ favored maize growth more than NO₃⁻ [11,15]. Different N sources affect the acquisition and partitioning of photoassimilation products in Scots pine (Pinus sylvestris (L.)) seedlings [32]. In many cases, assigning preferences is not a simple task and requires consideration of a complex set of physiological and environmental features that interact in ways that are not yet well understood [9]. Notably, there is a lack of research on Moso bamboo (Phyllostachys edulis), which holds significant economic value as a versatile resource for construction, paper production, and culinary use, while simultaneously offering ecological benefits such as soil erosion prevention, carbon sequestration, and habitat provision for diverse wildlife, are impacted by N source forms [61]. The growth performance of bamboo seedlings was significantly affected by the form of N used [63]. According to previous studies, indicating variations in response to different forms of N in Moso bamboo [18], particularly showing a preference for NH₄⁺ [64]. Additionally, traditional N fertilization employs inorganic N fertilizers, assuming organic N must be mineralized for plant absorption. Yet, recent advances suggest that plants can directly absorb small organic N molecules, indicating the potential use of amino acid organic N fertilizers [16,19,41]. Thus, we examined how Moso bamboo seedlings respond to low P stress under various N sources. Our goal was to identify the optimal N source combination for Moso bamboo seedling growth. We aimed to determine the most suitable combination of N sources for the growth of Moso bamboo seedlings.

This study aims to evaluate the impact of various N fertilizers on Moso bamboo seedling growth under low P conditions, considering soil nutrient dynamics and sapling physiology. By employing a seeding nursery and sand cultivation technique, we investigated the influence of different N supply forms on substrate nutrient environments and the growth of Moso bamboo saplings, shedding light on optimal N sources for Moso bamboo growth under varying P treatments.

Plant materials

Moso bamboo seeds were sourced from Lingchuan County, Guilin, Guangxi Province (110°32′E,25°40′N; average elevation 176 m.a.s.l.) in September 2020. After air-drying for 24 h at 22–25℃, the seeds were stored in a kraft envelope at 4℃ and 60% humidity for nine months. Prior to sowing in June 2021, the seeds underwent a 0.3% potassium permanganate disinfection followed by a rinse with sterile water. A 24-hour water soak was then performed to accelerate germination. On June 14, 2021, the preprocessed seeds were sown in plastic pots (14.0 cm height, 11.8 cm bottom diameter, and 14.0 cm caliber diameter, eight seeds per pot) filled with a perlite-soil mixture (v:v = 1:3). The soil, collected from a Moso bamboo forest in Qingshan, Lin'an District, Hangzhou, Zhejiang Province, contained 5.02 g·kg^− 1 of soil organic matter, 460 mg·kg^− 1 of total N, 240 mg·kg^− 1 of total P, 1.301 g·kg^− 1 of total potassium, 93.63 mg·kg^− 1 of alkaline N, 2.58 mg·kg^− 1 of available P, and 54.99 mg·kg^− 1 of available potassium. The bulk density of the soil was 0.98 g·cm^− 3 and the pH value was 4.83. The pots were placed in a greenhouse at Zhejiang Agriculture and Forestry University (119°72'E,30°23'N), maintaining average day/night temperatures of approximately 28℃ and 16℃, daily light intensity of 860 µmol·m^− 2·s^− 1, and 45%-70% humidity. The seedlings were watered as needed and the weeds were eliminated by hand, and the emerged seedlings were thinned when four true leaves had completely expanded, leaving two healthy and uniformly growing seedlings in each pot.

Experimental design and measurements

Immediately after seedling thinning, a randomized design applied two soil P levels: low-P (P1, with deficient available P of 5.0 mg·kg^− 1), and high-P (P2, with sufficient available P of 20 mg·kg^− 1). These rates fall in the natural distribution range of available soil P content in subtropic forest ecosystems [17]. The low-P level was achieved by watering the soil with 100 ml of 0.307 g·L^− 1 KH₂PO₄ solution per pot, and the high-P level with 100 ml of 1.228 g·L^− 1 KH₂PO₄ solution. To balance the potassium (K) added between treatments, 0.092 g of K₂SO₄ was supplemented to the KH₂PO₄ solution that was added to the low-P seedlings [1]. No additional N fertilizer was applied. As a result, 600 pots of seedlings were set up for each soil P level. The seedlings were cultivated using conventional cultivation management until the next June in 2022.

On 13 June 2022, a total of 30 seedlings from each soil P level were sampled to access their morphological status and N, P content, and another 240 seedlings from each P level were randomly selected and lifted, and then transplanted into new pots (consistent with that used in the previous year) contained 2 kg of a perlite-sand mix (v:v = 1:3), one seedling per pot. The sand was acid-washed to prevent any interference from the originally contained nutrients [51]. To avoid the influence of the residual N in the rhizosphere soil, seedling roots were gently rinsed off with running water. The status of the seedling at transplanting is shown in Table 1. After establishing (new leaves started occurrence), seedlings from each soil P level were randomly assigned to four groups, receiving different forms of N fertilization. Fertilizers included nitrate, ammonium, ammonium nitrate, and amino acids mixture. Each treatment was fertilized for 10 weeks (once a week) at 15 mg N seedling^− 1 per single application, for a total of 150 mg N seedling^− 1, with a nutrient solution differing only in N form: (i) nitrate [NO₃⁻, applied as Ca(NO₃)₂]; (ii) ammonium [NH₄⁺, applied as (NH₄)₂SO₄]; (iii) ammonium nitrate [NO₃⁻−NH₄⁺, applied as NH₄NO₃]; and (iv) amino acids [Org − N, applied as mixture of leucine (10.19%), aspartic acid (9.51%), lysine (8.29%), alanine (7.20%), valine (6.72%), histidine (6.65%), glutamic acid (6.62%), phenylalanine (5.29%) and others such as glycine, arginine and serine (less than 5% each); Bioactive peptide® 12-0-0, Xiangyang Winn Biotechnology Co., LTD, Hubei, China]. Fertilization ended on 1 September 2022, when the seedlings had ceased their height flush. All nutrient solutions were prepared with distilled water and equal amounts of the required micronutrients as elaborated in Table 2. After each fertilization, seedlings were watered to imminent saturation to allow rapid dispersion of nutrients to avoid toxicity from excessive salt accumulation, after which regular water management was carried out.

Table 1

**Physiological status of** **Phyllostachys edulis** **seedlings at transplantation in June 2022.** P1(5 mg·kg^− 1), P2(20 mg·kg^− 1).
Soil P level	Seedling height (cm)	Seedling mass (g·plant^− 1)	N content (mg·plant^− 1)	P content (mg·plant^− 1)
P1	9.460 ± 0.180	0.464 ± 0.045	4.594 ± 0.493	0.244 ± 0.030
P2	10.490 ± 0.460	4.513 ± 0.472	42.839 ± 4.897	4.827 ± 0.464

Table 2

Formulation of intermediate and micronutrient nutrient solutions providing different forms of nitrogen fertilizers used for fertilization.
Fertilizers (AR)	Application amount(mM)
MgSO₄·7H₂O	2.0000
EDTA-Fe	0.0800
H₃BO₃	0.0600
MnCl₂·4H₂O	0.0200
ZnSO₄·7H₂O	0.0060
CuSO₄·5H₂O	0.0030
NaMoO₄·2H₂O	0.0005
CoCl₂·6H₂O	0.0005

Two weeks after the last fertilization event (T1), four seedlings per treatment were randomly sampled for photosynthetic pigment content and Rubisco enzyme activity analysis in leaves, as well as the free amino acid determination in leaves, stems, and roots. The photosynthetic pigment content of leaves was determined using the ethanol extraction method [6]. The leaf Rubisco enzyme activity was determined using the ELISA kit produced by Shanghai Enzymes Biotechnology Co., Ltd. The total free amino acid content of fresh plant samples was determined using the ninhydrin colorimetric method [10,21], and the full spectrum amino acid composition and content were determined using ultra-high performance liquid chromatography.

At the end of the growing season in the middle of December (T2), the roots of 4 seedlings from each treatment, which can present the average status of the treatment, were gently lifted, cleaned, and scanned using the scanner for root system (Epson V500, USA), followed by analyzing with a plant image analyzer (Wessn LA-S, Hangzhou, China), to assess their root morphological attributes. Synchronously, another 20 seedlings from each treatment were randomly sampled for component mass and mineral nutrient content analysis. The sampled seedlings were washed gently to free the attachments and each seedling was separated into root, stem, and leaf sections, followed by a mixing of each component from 5 seedlings into a merged sample, yielding 4 replicates per treatment. The merged samples were then oven-dried at 70℃ for at least 48 hours until they attained a constant mass. Subsequently, each of the dried samples was ground and passed through a 0.25-mm screen after which, it was wet digested using the H₂SO₄-H₂O₂ method [38] for the determination of total N, P, potassium, magnesium, and iron content. The total N content was determined using the Kjeltec 2300 FOSS automatic N analyzer; the total P content was determined by molybdenum antimony resistance colorimetry on UV2500 ultraviolet spectrophotometer; the total potassium, total magnesium, and total iron contents were determined with the flame spectrophotometry on ICE3300 atomic absorption spectrometer.

At the same time at T2, the rhizosphere soil, which consisted of the soil that remained attached to the roots after gentle shaking, as well as the bulk soil, which is defined as soil that falls from the roots with gentle shaking, was separately collected from 5 pots of each treatment to measure indices related to plant N and P source utilization, such as soil pH, soil organic matter content, total N and P content, following the procedures illustrated in Xing et al. (2023) [55]. The nitrate and ammonium content of the soil samples were also determined using the salicylic acid colorimetric method [44] and indophenol blue colorimetry [12,57], respectively.

Statistical analyses

All statistical analyses were conducted using SPSS 26.0 (IBM, Chicago, IL, USA). Histograms were generated using GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA). Variations of leaf photosynthetic parameters, plant and soil mineral nutrient contents, and amino acids among treatments were assessed using a two-way analysis of variance (ANOVA). The data exhibited a normal distribution according to the Shapiro-Wilk test and homogeneous variance according to Levene's test, thus no transformation of raw data was conducted. Multiple comparisons of means were determined using a Duncan test at α = 0.05. Principal component analysis (PCA) was employed to visualize the relationship between amino acids in roots, mineral nutrients, biomass, as well as photosynthetic parameters. Based on the results of dissimilarity tests and PCA, redundancy analysis (RDA) was performed to examine and verify the relationships between these indicators. All PCA and RDA graphs were created using Origin 2024 for microcomputers (OriginLab, Northampton, Massachusetts, USA).

Soil nutrient environment variations

The measured indices exhibited significant variation between the rhizosphere soil and the bulk soil samples. The interaction between soil P level and N form notably influenced the measured soil environmental indices in both rhizosphere and bulk soil, except for the organic matter content in the bulk soil (Table S1 and S2).

Regarding soil pH, the combined application of NO₃⁻-N and NH₄⁺-N led to a noteworthy decrease in rhizosphere soil under initially low-P condition (P1), but a considerable increase under high-P condition (P2) (Fig. 1, a), compared to other N forms. A similar trend was observed in the bulk soil, where the application of NH₄⁺-N fertilizer also reduced soil pH under low-P condition. These observations indicate diverse physiological and biochemical responses of roots to varying soil P conditions. Conversely, the combined application of NO₃⁻-N and NH₄⁺-N resulted in relatively higher organic matter content in the rhizosphere soil under low-P conditions, significantly surpassing the application of other N forms (Fig. 1, b). Notably, NO₃⁻-N led to the lowest organic matter content in both rhizosphere and bulk soils under either P condition.

The influence of N form on both soil total phosphorus (TP) content and available phosphorus (AP) content exhibited similar trends in both rhizosphere and bulk soil under low-P condition (Fig. 1, c, d). Treatments solely involving NO₃⁻-N or NH₄⁺-N resulted in relatively higher TP and AP content compared to treatments involving combined application of NH₄⁺-N and NO₃⁻-N (NO₃⁻-N + NH₄⁺-N) or Org-N. Similarly, under high-P condition, the influence of N form on TP and AP content in rhizosphere soil followed a consistent pattern, whereas in bulk soil, the sole application of NO₃⁻-N resulted in the lowest TP and AP content. Notable, the TP content showed no significant difference among treatments involving NH₄⁺-N, combined application of NH₄⁺-N and NO₃⁻-N, or Org-N under high-P condition, while the AP content exhibited a tendency of Org-N > NO₃⁻-N + NH₄⁺-N > NH₄⁺-N > NO₃⁻-N.

In general, treatments involving Org-N application consistently exhibited the lowest nitrate content in both rhizosphere and bulk soil, regardless of the soil P conditions (Fig. 1, e). The presence of NO₃⁻ in the N application generally resulted in relatively higher nitrate content in the soil compared to treatments without NO₃⁻ application, except for the treatment involving combined application of NH₄⁺-N and NO₃⁻-N under the low-P condition. It is also interesting to note that the nitrate content was significantly higher in treatments involving NO₃⁻-N than in those involving combined application of NH₄⁺-N and NO₃⁻-N in bulk soil under high-P condition. Conversely, although the sole application of NH₄⁺-N resulted in a significantly higher ammonium content in rhizosphere soil compared to other N forms under low-P conditions (Fig. 1, f), the presence of NH₄⁺-N in the N application rarely led to a corresponding higher ammonium N content, especially under high-P condition, where no significant difference was observed among treatments involving different N forms in both rhizosphere and bulk soil.

Plant growth attributes

The root growth of P. edulis seedlings under the two tested soil P levels exhibits significant differences under different N form treatments. The interaction between soil P level and N form significantly affects root surface area, volume, and fine root surface area, but has no significant effect on total root length (Table S3). Under low-P condition, the N supply form had no significant influence on the total root length, surface area, volume, and fine root surface area. Conversely, under high-P condition, except for the total root length difference between the NO₃⁻-N treatment group and the Org-N treatment group being insignificant, all root growth-related indicators of the NO₃⁻-N treatment group are significantly lower than those of other N form treatment groups, while the root total length, total surface area, and fine root surface area of the NH₄⁺-N treatment group are significantly higher than those of the Org-N treatment group, but not significantly different from the combined application of NH₄⁺-N and NO₃⁻-N treatment group (Fig. 2).

The influence of soil P levels and N form on the root growth of the seedlings can further extend to seedling biomass and its partitioning within various tissues. Under low P condition, application of different N forms did not significantly affect the total seedling biomass, but significantly influenced the partitioning of total mass among different tissues. Specifically, the NO₃⁻-N addition group exhibited the lowest proportion of root mass allocation and the highest proportion of stem and leaf mass allocation (Table 3). It is also noteworthy that there was no significant difference in the partitioning of root, stem, and leaf mass of P. edulis seedlings in the NH₄⁺-N addition treatment group compared to other N form addition treatments, indicating that NH₄⁺-N addition treatment may play a balancing regulatory effect on underground nutrient acquisition and aboveground growth of P. edulis seedlings under low P stress condition. However, under high-P condition, both the total seedling mass and its partitioning within different tissues are significantly affected by N addition forms, especially the application of NO₃⁻-N, which resulted in the smallest total seedling mass among the treatments applied with four N forms, but the highest partitioning of root mass; conversely, the application of NH₄⁺-N led to the largest total seedling mass among the four N forms, but the lowest partitioning of root mass. Regarding root to shoot mass ratio (R/S), compared to other N forms, the application of NO₃⁻-N significantly reduced the R/S under low-P condition, while under high-P condition, the R/S was significantly higher than that of the NH₄⁺-N group.

Table 3

Distribution of biomass among tissues in live *Phyllostachys edulis* seedlings at T2 stage. Values are mean mean ± SE (n = 4). Values with different letters in the same column differ significantly (α = 0.05).
P level	N form	Plant mass (g·plant^− 1)	Root mass partitioning (%)	Stem mass partitioning (%)	Leaf mass partitioning (%)	Root/Shoot
P1	N1	0.41 ± 0.05 d	54.28 ± 2.37 c	23.21 ± 1.14 a	22.51 ± 3.12 a	1.21 ± 0.12 d
	N2	0.62 ± 0.11 d	60.65 ± 1.85 ab	22.64 ± 0.77 ab	16.72 ± 1.51 ab	1.56 ± 0.12 abcd
	N3	0.53 ± 0.15 d	62.01 ± 0.98 ab	17.74 ± 2.91 b	20.25 ± 0.87 a	1.64 ± 0.07 abc
	N4	0.30 ± 0.34 d	65.15 ± 2.78 a	22.10 ± 1.04 ab	12.75 ± 1.61 b	1.93 ± 0.25 a
P2	N1	2.71 ± 0.25 c	63.12 ± 1.05 a	19.24 ± 0.91 ab	17.65 ± 0.24 ab	1.72 ± 0.08 ab
	N2	6.38 ± 1.06 a	56.56 ± 0.52 bc	23.02 ± 2.57 a	20.42 ± 2.27 a	1.30 ± 0.03 cd
	N3	5.17 ± 0.73 ab	62.00 ± 1.78 ab	20.44 ± 0.97 ab	17.56 ± 1.83 ab	1.65 ± 0.12 abc
	N4	3.80 ± 0.41 bc	59.85 ± 1.70 ab	19.04 ± 0.41 ab	21.12 ± 0.80 a	1.50 ± 0.10 bcd

Plant physiological attributes

Generally, low-P condition expectedly reduced the accumulation of P nutrients in the roots, stems, and leaves of bamboo seedlings, as well as N in the stems and leaves but in the roots. Under low-P condition, P and N content peaked in the leaves, while under high-P condition, P content peaked in the stems and N content peaked in the leaves. Notably, under high-P condition, either application of NO₃⁻-N and NH₄⁺-N led to higher N content in various tissues, though the differences were not always significant compared to other N forms. However, under low-P condition, the application of NO₃⁻-N led to the lowest N content in the roots, stems and leaves (Fig. 3a, b, & c). For P content in the seedling components, application of NH₄⁺-N under high-P condition consistently resulted to the lowest P content in roots, stems and leaves, while under low-P condition, application of NH₄⁺-N resulted in a relatively higher P content in stems, with the application of NO₃⁻-N resulting in higher P content in roots and leaves, although the differences were not always significant (Fig. 3, d, e, f).

Low-P condition also led to significantly decreased content of the total chlorophyll, chlorophyll a, chlorophyll b, and carotenoids, as well as Rubisco enzyme activity in the leaves, comparing to the high-P condition (Fig. 4). Notably, application of NH₄⁺-N tended to increase the chlorophyll and carotenoids contents under low-P condition, although the differences were not always significant (Fig. 4, a, b, c). Conversely, Org-N tended to decrease the chlorophyll and carotenoids content in the leaves under low-P condition, but led to higher carotenoids content in leaves compared to NO₃⁻-N application under high-P condition. Regarding the Rubisco enzyme activity in the leaves, the combined application of NH₄⁺-N and NO₃⁻-N resulted in the highest value under high-P condition, but a relatively lower value compared to NH₄⁺-N and Org-N under low-P condition (Fig. 4, e).

The content of potassium, iron, and magnesium in the seedlings also varied significantly with application of different forms of N fertilizer under both low-P and high-P condition, with differing trends in different tissues (Table 4). Under low-P condition, the combined application of NH₄⁺-N and NO₃⁻-N significantly increased magnesium and iron content in roots compared to other N application treatments, and the potassium content was significantly higher in the Org-N treatment compared to combined application of NH₄⁺-N and NO₃⁻-N; for potassium, iron, and magnesium content in stems, the effects of NO₃⁻-N were generally adverse compared to other N forms, with combined application of NH₄⁺-N and NO₃⁻-N resulting in relatively higher potassium content, Org-N in higher iron and magnesium content, although no significant differences were observed between Org-N and the combined application of NH₄⁺-N and NO₃⁻-N.

Table 4

Elemental contents of various tissues in live *Phyllostachys edulis* seedlings at the T2 period. Values are mean ± SE (n = 4). Values with different letters in the same column differ significantly (α = 0.05).
Component	P level	N form	Total potassium content (g·kg^− 1)	Total iron content (g·kg^− 1)	Total magnesium content (g·kg^− 1)
Root	P1	N1	20.76 ± 0.25 cd	0.86 ± 0.03 c	0.79 ± 0.00 c
		N2	23.14 ± 1.55 bc	0.89 ± 0.08 b	0.81 ± 0.01 c
		N3	26.21 ± 1.27 ab	1.69 ± 0.15 a	0.83 ± 0.00 a
		N4	27.89 ± 1.13 a	0.88 ± 0.11 c	0.78 ± 0.00 c
	P2	N1	21.98 ± 0.74 bcd	1.57 ± 0.16 a	0.84 ± 0.00 ab
		N2	14.30 ± 1.13 c	1.19 ± 0.09 ab	0.82 ± 0.00 bc
		N3	19.39 ± 3.04 cd	1.36 ± 0.13 a	0.83 ± 0.01 ab
		N4	17.84 ± 1.25 de	1.69 ± 0.26 a	0.83 ± 0.00 a
Stem	P1	N1	16.95 ± 0.45 bc	0.46 ± 0.03 d	0.75 ± 0.00 bc
		N2	20.25 ± 0.78 abc	0.66 ± 0.02 c	0.78 ± 0.01 ab
		N3	21.87 ± 1.99 ab	0.73 ± 0.10 bc	0.79 ± 0.01 a
		N4	15.49 ± 0.66 c	0.72 ± 0.09 ab	0.82 ± 0.02 a
	P2	N1	24.60 ± 2.02 a	0.46 ± 0.03 a	0.82 ± 0.00 bc
		N2	19.16 ± 0.28 bc	0.39 ± 0.05 abc	0.81 ± 0.01 c
		N3	24.86 ± 1.76 a	0.48 ± 0.09 abc	0.81 ± 0.01 bc
		N4	21.65 ± 2.56 ab	0.38 ± 0.02 a	0.83 ± 0.01 c
Leaf	P1	N1	18.99 ± 0.30 c	0.78 ± 0.01 c	0.67 ± 0.01 b
		N2	24.76 ± 1.43 b	0.99 ± 0.06 b	0.75 ± 0.01 a
		N3	22.77 ± 1.84 b	1.06 ± 0.07 b	0.75 ± 0.05 a
		N4	29.76 ± 1.64 a	0.44 ± 0.09 a	0.82 ± 0.01 cd
	P2	N1	21.34 ± 0.61 bc	0.71 ± 0.09 ab	0.80 ± 0.00 b
		N2	21.24 ± 0.72 bc	0.33 ± 0.02 ab	0.78 ± 0.01 d
		N3	22.07 ± 0.39 bc	0.58 ± 0.10 ab	0.78 ± 0.00 bc
		N4	22.17 ± 0.76 bc	0.41 ± 0.07 ab	0.80 ± 0.01 cd

Amino acid content and distribution

The interaction between soil P level and N form had a significant impact on the total free amino acid content in the leaves and stems of the seedlings, but not the total free amino acid content in roots, which was mainly affected by the N form (Table S4). Generally, the amino acid content in root was relatively lower in treatments applied with NH₄⁺-N or Org-N, irrespective the soil P levels. However, the amino acid content in stems and leaves of the seedlings varied differently with the N form under contrasting soil P levels (Fig. 5, a). Under low-P condition, application of NO₃⁻-N resulted in the lowest amino acid content in stems and NH₄⁺-N resulted in the lowest amino acid content in leaves. However, under high-P condition, application of NH₄⁺-N consistently results in the lowest amino acid content in leaves, with Org-N interestingly increasing amino acid content in leaves, compared to other N forms.

Twenty-nine free amino acids were detected in the seedlings, with the roots comprising more than 5% of the total amino acids including ASN, ASP, gamma-GABA, GLU, and SER (Fig. 5, b). While the predominant amino acids in the stems were GLN, ASN, SER, gamma-GABA, and ASN (Fig. 5, c), and the leaf exhibits high levels of SER, GLN, ASN, gamma-GABA, ALA, and GLU (Fig. 5, d). The percentages of ASN, ASP, gamma-GABA, and GLU in roots ranged from 14.5–18.7%, 15.2–21.2%, 12.5–16.7%, and 8.9–11.3% respectively, which were higher compared to the percentages found in stems and leaves (Table S5). SER yielded higher content in leaves than in roots; ALA content levels ranked in order of leaf > root > stem, while ETH was more consistent among tissues under high-P condition, ranging from about 1–2%. However, the percentage of ARG was greater in stems, ranging from about 7.2–12.7%, with only a 0.4–2% occurrence in leaves and roots. GLN accounted for 3.0–7.2% in roots, 21.4–29.8% in stems, and 9.8–16.1% in leaves. Meanwhile, LYS accounted for 4.8–16.1% in leaves and was higher in leaves than in stems or roots. SER was higher than root, and ALA accounted for the highest concentration in leaves, followed by roots and then stems. ETH accounted for 4.4–4% in leaves, while SER accounted for the highest concentration in leaves, followed by roots and stems. In roots, the concentration was only 1.3–1.9%. The concentration in leaves ranged from 4.6-7.0%.

Notably, there was significant variation in the proportion of amino acid fractions among treatments with different N forms under low-P condition (Fig. 5). Despite the type of N fertilizer used, the content of ETH in the root system was only 0.7%-1.2% of the total amino acids, while in the leaves it increased to 16.2%-22.0%, representing the highest proportion of total amino acids in the leaves, and differing from the performance noted at the low-P level. In conjunction with the type of N fertilizer initially added to the substrate, our study revealed that although the substrate displayed the highest LEU when organic N fertilizer was first added, it only constituted a small proportion in the plant, specifically 1.39% in the root system under low-P condition and merely 0.7% in the root system under high-P condition.

In the roots of the seedlings, LYS and ALA were positively correlated with root N content, while ALA and GLY were negatively correlated with root P content; GLY also showed a strong positive correlation with root K content, whereas LYS and ALA were negatively correlated with K content (Fig. 6, a). LYS, ALA, and GLY in seedling roots were consistently positively correlated with root biomass, with ALA and LYS showing negative correlations with total root length, root volume, fine root surface area and total root surface area (Fig. 6, b). In seedling stems, GLY exhibited a positive correlation with stem mass and iron content, but a negative correlation with N, P, K, and Mg content; whereas LYS and ALA were positively correlated with N, P, K, and Mg content but negatively correlated with iron content (Fig. 6, c ,d). In the leaves, LYS and ALA were positively correlated with P and Mg content, as well as with photosynthetic pigments, Rubisco enzyme activity, so as to leaf biomass (Fig. 6, e, f).

Soil Environmental Basis of Nitrogen Supply Affecting Bamboo Seedling Growth

The rhizosphere soil acidity in cultivated substrates can vary depending on the type of N plants absorb. The study reveals that the application of NO₃⁻-N fertilizer significantly increased rhizosphere soil pH at all low P levels, stimulating Moso bamboo transplant root systems to absorb more NO₃⁻-N [42]. This process releases more OH⁻ ions to maintain anion-cation balance, resulting in higher rhizosphere soil pH [43]. Conversely, combined application of NH₄⁺-N and NO₃⁻-N decreased rhizosphere soil pH under low P stress, while significantly increasing the organic matter content of the rhizosphere soil. Under suitable P conditions, the organic matter content of the rhizosphere soil in NH₄⁺-N and Org-N groups was significantly higher than that of the NO₃⁻-N group. This difference was attributed to organic N fertilizers containing large amounts of organic carbon. Moreover, Moso bamboo transplants growing under low P stress exhibit poor soil N absorption abilities, leading to increased soil organic matter retention. Mixed N fertilizers may effectively balance the distribution of nitrate and ammonium N within Moso bamboo seedlings, facilitating N absorption and conversion into organic matter for storage [4,24].

The substrate mainly contained nitrate N, ammonium N, and soluble organic N. Under low P stress, inorganic N, particularly nitrate N, dominated the substrate, regardless of the N fertilizer applied. However, increasing P application levels decreased the nitrate N content of the substrate under NO₃⁻-N and NH₄⁺-N forms, with no significant difference observed in combined application of NH₄⁺-N and NO₃⁻-N and Org-N. This phenomenon could be attributed to the free movement of nitrate N in the substrate [35]. Rhizosphere soil containing NO₃⁻-N and combined application of NH₄⁺-N and NO₃⁻-N was primarily stored as inorganic N, whereas NH₄⁺-N and Org-N were predominantly stored as soluble organic N.

Influence of Soil Phosphorus Levels and Nitrogen Forms on P. edulis Seedling Morphological Attributes and Mineral Nutrient Content

Nitrogen and phosphorus, as important components of carbohydrates, proteins, nucleic acids, and other organic compounds in plants, participate in the coordination of many physiological activities. Previous research by Marschner et al. (2012) [35] has shown that soil P levels and N forms interact to influence root morphology. In our study, under low-P condition, the form of N supplied did not significantly impact root growth attributes (Fig. 2, b, c, d). This finding is consistent with Marschner et al. (2012) [35], who noted that low P availability often diminishes the impact of N forms on root growth. Conversely, under high-P condition, seedlings treated with NO₃⁻-N exhibited significantly reduced root growth metrics compared to those receiving other N forms (Fig. 2). This suggests that high P availability combined with nitrate N can inhibit root development, coincide with the results by Liu et al. (2020) [31], which highlighted that an excess of P can alter the root system architecture by inhibiting root growth and development, particularly affecting the lateral roots and root hairs. This may be attributed to that nitrate serves as both a nutrient and a signaling molecule. When combined with high P levels, nitrate can further exacerbate the inhibition of root development by affecting hormonal balances and signaling pathways within the plant [3,26]. Conversely, the NH₄⁺-N treatment under high-P conditions resulted in enhanced root growth parameters, aligning with Ma et al. (2013) [33], suggesting that ammonium N promotes better root expansion and nutrient uptake in P-rich environments [5]. There is also research on Carya illinoinensis demonstrating that the combined application of NH₄⁺-N and P can promote plant growth, nutrient uptake, and N assimilating enzyme activities, which in turn improves root area, root activity, and overall root growth parameters [7].

As well documented by previous studies [22,62], the interaction between soil P levels and N forms significantly affects total seedling biomass and its allocation among different tissues. Our results indicate that under low-P conditions, total biomass remained unaffected by N form, but biomass partitioning varied. Seedlings treated with NO₃⁻-N showed the lowest root biomass allocation, and the highest allocation to stems and leaves. However, our findings that NH₄⁺-N promotes balanced biomass allocation between roots and shoots under low-P condition align with these studies. Additionally, under high-P conditions, the increased total biomass and reduced root mass allocation in the NH₄⁺-N treatment observed in our study are consistent with Zhang et al. (2019) [59], who noted that ammonium N supports overall growth better than nitrate N in high P environments. This suggests that NH₄⁺-N treatment under high-P conditions can lead to enhanced shoot growth and overall biomass production at the expense of root development.

Our study further reveals that soil P levels and N forms significantly affect the physiological attributes of P. edulis seedlings. Low-P condition reduced the accumulation of P and N in stems and leaves, while increased N content was observed in various tissues with NO₃⁻-N and NH₄⁺-N treatments under high-P condition, consistent with Lambers and Oliveira (2019) [27]. These results underline the critical role of nutrient interactions in determining plant growth and development, emphasizing the importance of optimizing nutrient management strategies for better plant performance [58]. Additionally, the enhancement of chlorophyll and carotenoid contents under NH₄⁺-N treatment in low-P condition suggests a mitigating effect of ammonium N on P stress, which can also be supported by Ye et al. (2022) [56]. This may be partially attributed to that NH₄⁺-N can stimulate root exudation, which can help mobilize P in the soil [13], as well as that NH₄⁺-N affects hormone levels like auxins and cytokinins, leading to better growth and nutrient assimilation [8]. Concomitantly, our results show that the combined application of NO₃⁻-N and NH₄⁺-N significantly increased magnesium and iron content in roots under low-P condition, while Org-N resulted in higher potassium content, suggesting that organic or mixed N sources may better support mineral nutrition under P-deficient conditions [25].

Impact of Nitrogen Forms on Photosynthetic Pigments in Transplanted Bamboo Leaves under Low Phosphorus

The influence of N forms on the photosynthetic pigments of Moso bamboo transplant leaves varied based on P application levels. Under low P stress, Org-N fertilizer application notably reduced total chlorophyll, chlorophyll a, and carotenoids compared to other N fertilizers, with no significant variation in chlorophyll b. This indicates that Org-N fertilizer had the greatest negative impact on photosynthesis in Moso bamboo seedling leaves under low P stress. Conversely, NH₄⁺-N application under suitable P conditions significantly increased total chlorophyll, chlorophyll a, b, and carotenoids compared to NO₃⁻-N. This suggests that NH₄⁺-N fertilizer may be more conducive to photosynthesis in Moso bamboo transplants under suitable P levels compared to NO₃⁻-N. Overall, the application of NO₃⁻-N, NH₄⁺-N, and combined application of NH₄⁺-N and NO₃⁻-N fertilizers under low P stress may increase photosynthetic pigment levels. Additionally, using NH₄⁺-N fertilizers at high P levels may yield better results compared to NO₃⁻-N fertilizers.

Photosynthesis is essential for plants to produce organic matter and acquire energy. Leaves, being primary tissues for photosynthesis, harness, transform, and transmit light energy through chlorophyll. Carotenoids provide protection to chloroplast molecules against photo-oxidative damage and optimize the efficiency of light energy utilization. Rubisco, a crucial enzyme in the Calvin cycle, participates in photosynthetic CO₂ fixation and photorespiratory release of CO₂ [29]. Phosphorus influences the production and distribution of photosynthetic assimilates by promoting or inhibiting the activities of key enzymes like Rubisco [39,60]. This study observed greater Rubisco activity with NH₄⁺-N and Org-N fertilizers under low P stress, indicating their potential to alleviate inhibitory effects on photosynthesis. Further research is needed to confirm these findings and investigate the modulation of Rubisco activity in Moso bamboo transplants at different P application levels.

Effect of Nitrogen Supply Pattern on Amino Acid Content and Distribution of Moso Bamboo Transplants under Low Phosphorus Stress

Plants can convert NH₄⁺-N into amino acids in the root system and transport them aboveground via xylem for metabolic use. Free amino acids provide a crucial material foundation for plant stress resistance, impacting material metabolism and protein synthesis. Phosphorus application level, N supply form, and their interaction significantly affected total free amino acids, with no significant effect on roots, primarily influenced by P application level. The total amount of free amino acids in roots increased with decreasing P application level. Compared to higher P levels, stem and leaf tissues exhibited decreased total free amino acids under low P stress. NH₄⁺-N fertilizer significantly increased amino acid content in stem tissues compared to other N forms. Under moderate P conditions, amino acid fractions remained stable, with a higher ratio of gamma-GABA observed compared to low P levels. However, under low P stress, amino acid fractions were dysregulated, and allocation proportions varied significantly among different N supply forms. Amino acids such as PRO, VAL, LEU, and ILE increased in all tissues of Moso bamboo transplants under low-P stress, particularly with NO₃⁻-N fertilizer, indicating their potential as stress tolerance indicators. Additionally, NH₄⁺-N fertilizer increased GLN content and proportion in stem tissues under low P stress, enhancing the resistance of Moso bamboo seedlings. Moreover, low P stress facilitated the conversion of other amino acid fractions to ETH, stored in leaves to better adapt to low P stress.

The presence of ALA positively correlated with photosynthetic indexes and Rubisco in Moso bamboo leaves, suggesting its role in enhancing photosynthesis. Furthermore, LYS and ALA promoted Mg²⁺ content and photosynthetic pigments, enhancing photosynthesis in Moso bamboo. Further research is needed to explore the potential benefits of these amino acids in Moso bamboo cultivation (Fig. 6, a, b, e, f).

Our study demonstrated that the interaction between soil P levels and N application forms significantly impacts the soil nutrient environment and plant growth of P. edulis seedlings. Under low-P condition, combined NH₄⁺-N and NO₃⁻-N application improved soil pH and organic matter content, whereas under high-P condition, NH₄⁺-N alone enhanced root growth parameters, total seedling biomass, and balanced nutrient acquisition. Notably, NH₄⁺-N application under high-P conditions resulted in higher N content across tissues and increased chlorophyll and carotenoid levels under low-P conditions. Furthermore, the study highlighted the significant influence of N forms on amino acid content and distribution in plant tissues, with NH₄⁺-N optimizing growth by positively correlating with root N content and biomass. This research underscores the importance of tailoring N form applications to soil P levels to maximize plant growth and nutrient utilization in P. edulis seedlings.

Acknowledgements:

We thank the greenhouse managers and workers at Zhejiang A&F University for their valuable help and support.

Author contributions:

WS designed the study, KW, WS and JZ performed the experiments, analyzed the data and co-wrote the manuscript. RX participated in data collection and analysis. WS and YY supervised the study.

Funding:

This work was supported by the Natural Science Foundation of Zhejiang Province (LY22C160004) and the National Natural Science Foundation of China (31901369, 32271971).

Data availability:

Data not included in the manuscript but important for a detailed review can be found online in the Supporting Information section.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.

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Nitrogen supply forms alter adaptive properties of Moso bamboo seedlings in low phosphorus conditions

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Soil nutrient environment variations

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Soil Environmental Basis of Nitrogen Supply Affecting Bamboo Seedling Growth

Impact of Nitrogen Forms on Photosynthetic Pigments in Transplanted Bamboo Leaves under Low Phosphorus

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