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Nitrogen form plays an important role in the growth of moso bamboo (Phyllostachys edulis) seedlings

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Plant Biology

Introduction

Moso bamboo (Phyllostachys edulis) is a species of large monopodial bamboo that is native to China and extensively cultivated throughout China, Japan, Korea, and Vietnam (Rao, Rao & Williams, 1998). Moso bamboo, which covers more than 3.87 hectares, representing 70% of the Chinese bamboo forest areas and 80% of the global distribution of moso bamboo, is the most important bamboo species in China (Song et al., 2016b). Moso bamboo can be planted by transplanting mother bamboo or planting seedling; the former method has generally been used for afforestation throughout China because vegetative propagation with mother bamboo typically takes only 5 years for it to form a grove compared with approximately 10 years by planting seedlings (Banik, 1980; Fu, 2000). However, both removal and replanting of the mother bamboo are labor intensive, high cost with low efficiency. In addition, human consumption of young edible shoots and environmental disturbance restrict the naturally rapid expansion of moso bamboo into a grove (Cai et al., 2008). In contrast, planting seedlings has many advantages, including easy handling, transporting, strong suitability and high survival rate with low cost (Fu, 2000). This provides a new approach for the regeneration and introduction of flowering bamboo forests. In fact, planting seedlings has been successfully applied in Guangxi Province, China to establish new bamboo groves (Qin, 2009; Chen, 2019). However, few studies have been conducted on the nutrient requirements of moso bamboo seedlings during cultivation and breeding. In addition, many bamboo forests are facing abandonment, with the forests potentially reverted to an unmanaged stand associated with a decline in soil organic matter accumulation and soil fertility (Christanty, Kimins & Mailly, 1997; Nakashima, 2001; Chen, Wang & Wang, 2016).

Nitrogen (N) is often an important factor for plant growth and productivity in terrestrial ecosystems. Under natural conditions, ammonium (NH4+) and nitrate (NO3) are the two primary forms of inorganic N available to plants in soil (Xu, Fan & Miller, 2012). In well-aerated agricultural soils and disturbed or early-successional natural ecosystems, NO3 is the major N source, whereas in flooded environments or acidic mature forests, NH4+ is the dominant N source (Glass et al., 2002; Britto & Kronzucker, 2006). Due to differences in environmental conditions, plant species, and the nutritional characteristics of N sources, plants have adapted to different N forms during long-term evolution and may show optimized growth with specific N forms (Britto & Kronzucker, 2013). For example, many conifers, ericaceous species and rice show improved growth with available NH4+, whereas some crops and early-successional pioneer species prefer NO3 (Kronzucker, Siddiqi & Glass, 1997; Britto & Kronzucker, 2013). A previous study conducted by Li et al. (2014b) showed that the growth of P. edulis seedlings are slightly improved with available NO3. However, Song et al. (2013) found that P. edulis tends to absorb NH4+under natural conditions. Gu et al. (2016) indicated that the N form preferred by P. edulis is related to the N concentration. Our study showed that under the low N concentrations (0.1, 0.4 mmol/L), there is no apparent N form preference, but the growth of bamboo seedlings especially the aboveground parts is improved with elevated NH4+ available from 2 to 40 mmol/L (Gu et al., 2016; Zou et al., 2020). Although NH4+can be used as a sole N source and an essential intermediate, it can also result in toxicity symptoms in many plant species, especially when high NH4+concentrations are provided as a sole N source or in combination with low levels of potassium (K) (Ten Hoopen et al., 2010). A previously study showed that shoot and root growth is significantly suppressed in cucumber grown with 10 mmol/L of NH4+ (Roosta & Schjoerring, 2008). Similar results have also been found in Arabidopsis thaliana, barley, tomato, and beans after high NH4+ treatments (Britto & Kronzucker, 2002; Britto & Kronzucker, 2006; Li et al., 2014a). In terms of moso bamboo, Li et al. (2014b) found that when the proportion of NH4+ exceeds 50% of the total N provided (40 mg/L), the growth of P. edulis seedlings is inhibited, and all of the seedlings eventually are died when NH4+ is supplied as a sole N source. However, Zou et al. (2020) showed that higher NH4+ concentrations (16∼40 mmol/L) are beneficial for the growth of aboveground organs, although root growth is suppressed to some extent with NH4+ levels ranging from 24 to 40 mmol/L. The contradictions in these studies indicate that N form preference and NH4+ tolerance of moso bamboo seedlings require further study.

K is another major plant nutrient that affects plant growth and metabolism (Wang et al., 2013). K plays a vital role in defending against biotic and abiotic stresses, and its role in the alleviation of NH4+ toxicity has been widely reported (Britto & Kronzucker, 2002; Szczerba et al., 2007; Wang et al., 2013). With similar hydration diameter charge and effects on membrane potential, K+ and NH4+ compete each other for the limited ion channel proteins on the cell membrane (Ten Hoopen et al., 2010; Kong et al., 2014; Coskun, Britto & Kronzucker, 2017). K+ can reduce the transport and accumulation of NH4+by direct competition during uptake and can also alleviate the rapid NH4+ cycling at the plasma membrane, thus reducing NH4+ toxicity. On the other hand, K+ can enhance NH4+ utilization by activating the enzymes of NH4+ assimilation and amino acid transport in plant cells (Wang, Siddiqi & Glass, 1996), which promotes NH4+ metabolism to reduce NH4+ toxicity (Ten Hoopen et al., 2010; Zhang et al., 2010).

Orthogonal arrays (often referred to as Taguchi methods) can be used to examine large numbers of factors in a much smaller number of experiments, allowing for the exploration of a unique subset of factor combinations. Therefore, it is a sophisticated time- and cost-efficient testing strategy (Lin, 1987; Qiao et al., 2013). Orthogonal experiments have been used to test the optimization of liquid fertilizer formulation, callus induction, plant regeneration medium in tissue culture and other hydroponic nutrient solution protocols for Sorghum bicolor (Gutiérrez-Miceli et al., 2008), Dendrocalamus latiflorus (Qiao et al., 2013), and Ipomoea spp. (Zhou & Lu, 2013). Using orthogonal arrays, we have studied the effects of different N and K+ concentrations and N form on the growth response and N acquisition of P. edulis seedlings. We have also investigated the extent to which the growth indices of P. edulis seedlings are related to each other. The overall aims of this study were to (1) further clarify the N form preference of P. edulis seedlings and (2) determine the effects of K on the response of moso bamboo to different N forms. This study presents the appropriate fertilization requirements of moso bamboo cultivation based on the experimental data.

Materials and Methods

Plant materials and growth conditions

Seeds of P. edulis were collected from GuanYang City, Guangxi Province, China and stored at 4 °C before sowing. The moso bamboo seeds were soaked overnight in water at 40 °C, shelled, sterilized by soaking in 20% NaClO for 20 min, rinsed in sterile water at least five times, and then germinated in plastic pots (diameter of 150 mm, height of 130 mm) filled with vermiculite. One month later, approximately five cm tall, three foliate seedlings were selected for different treatments.

N and K treatments

L8 (4 × 24) orthogonal arrays were employed to study K+ concentrations (0, 0.5, 1.5, 3 mmol/L), N forms (NH4+, NO3), and N concentrations (8, 32 mmol/L) on seedling growth and N uptake (Table 1). The nutrient solution modified from Norisada & Kojima (2005) contains 2.5 mmol/L Ca2+ as CaCl2 ⋅ 2H2O, 0.25 mmol/L MgSO4 ⋅ 7H2O, 0.6 mmol/L Na2HPO4 ⋅ 10H2O, 0.01 mmol/L Fe-EDTA, 0.02 mmol/L H3BO3, 2 µmol/L MnCl2 ⋅ 4H2O, 2 µmol/L ZnSO4 ⋅ 7H2O, 2 µmol/L CuSO4 ⋅ 5H2O, 0.5 µmol/L Na2MoO4 ⋅ 2H2O, and 0.5 µmol/L CoCl2 ⋅ 6H2O. The K+, NH4+, and NO3 were supplied by using KCl, (NH4)2SO4, and NaNO3, respectively. A nitrification inhibitor C2H4N4 (7 µM) was added to all treatments to prevent nitrification. One replicate consisted of six pots with two seedlings planted per pot, there were three replicates per treatment. Plants were grown in a greenhouse at 25/18 °C ± 3 °C day/night temperature, 65∼70% relative humidity, and 14/10 h day/night photoperiod. Pots were rotated every week to eliminate location effects. After treatment for 2 months, aboveground growth traits, root systems, and N content were analyzed.

Table 1:
L8(4×24) orthogonal array design of nutrient solution composition with the concentrations and forms of N and K.
Orthogonal combination K concentration (mmol/L) N form N concentration (mmol/L)
1 0 NH4+ 8
2 0 NO3 32
3 0.5 NH4+ 32
4 0.5 NO3 8
5 1.5 NH4+ 32
6 1.5 NO3 8
7 3 NH4+ 8
8 3 NO3 32
DOI: 10.7717/peerj.9938/table-1

Growth analysis and root morphology

Height was measured with a ruler. The number of unfurled leaves above the cotyledonary node were counted. The chlorophyll content (SPAD value) of the leaves was determined with a chlorophyll meter (SPAD-502, Minolta). Root morphology, including root total length (RL), average diameter (AD), root surface area (RS), root volume (RV) and root tips were determined using an automatic scanning apparatus (EPSON color image scanner LA1600+, Toronto, Canada) equipped with WinRHIZO 2012 software (Regent Instruments, Quebec, Canada).

Measurement of dry weight and N content

When the above experimental treatment was completed, plants were dried at 105 °C for 30 min, and then dried to a constant weight at 70 °C for biomass determination. The samples were digested with H2SO4-H2O2, and the total N content was determined according to the Kjeldahl method (Chen et al., 2013).

Statistical analysis

The data were analyzed to determine the range (R and R’) of orthogonal tests using DPS7.05 statistical software (http://www.statforum.com). Using the same software, an analysis of variance (ANOVA) was conducted and post hoc comparisons were conducted using Duncan’s multiple comparison test with differences considered significant at p < 0.05.

Results

Effects of N and K on the growth of moso bamboo seedlings

The effects of N and K on bamboo seedlings are listed in Table 2. The range analysis (R’ values) of the orthogonal array indicated that N form had a significant impact on the seedling height, biomass, leaf number, and SPAD value of P. edulis. Impacts to a lesser extent were observed for N concentration, while K+ concentration had the least effect (R’N form >  R’N concentration >R’K+concentration, Table 3), suggesting that N form is more important than nitrogen and potassium concentration on growth of moso bamboo seedling. After two months of treatment with the different N forms (Table 1), the seedlings of moso bamboo performed better in the treatments of NH4+ (treatments 1, 3, 5, 7) than the treatments of NO3 (treatments 2, 4, 6, 8) , and seedlings showed greener, heathier leaves and less necrosis in the NH4+ treatments than in the NO3 treatments (Fig. S1). The growth index of seedling height, biomass, leaf number and SPAD value increased 9.98%, 100%, 27.86%, and 257.14%, respectively, by comparing average value of these parameters between the treatments of 1, 3, 5, 7 and the treatments of 2, 4, 6, 8 (Table 2, Fig. 1). For the N concentrations (Table 1), the results showed that seedling height, biomass, leaf number and SPAD value increased by 8.27%, 33.33%, 11.03%, and 22.96%, respectively (Fig. 1), with the nutrition solutions of 8 mmol/L of nitrogen (treatments 1, 4, 6, 7) compared to 32 mmol/L of nitrogen (treatments 2, 3, 5, 8; Table 2).

Table 2:
N and K on growth, N content, and root architecture of moso bamboo seedlings.
Analytical results are means ± SE (n = 36). Mean values followed by the same letter (a, b, c, d, e, or f) are not significantly different within the same column according to a Duncan’s multiple comparison test at P < 0.05.
Treatment Mortality rate/(%) Height (cm) Leaf number SPAD Biomass (g) N content (%) Root length (cm) Root surface area (cm2) Root volume (cm3) Average diameter (cm) Root tips
1 8.00a 16.63a 11.44a 27.55a 0.22a 0.92ab 292.86a 30.80a 0.26a 0.34ab 1438.72a
2 0.00a 13.13b 7.75f 6.81d 0.08de 0.84bc 117.98cd 12.34cd 0.10c 0.33ab 612.75cd
3 0.00a 15.70ab 11.0ab 20.52c 0.20a 0.72bc 209.02b 23.81b 0.22ab 0.37ab 915.33b
4 0.00a 14.37ab 9.50de 6.49d 0.12cd 0.39d 186.95b 18.58bc 0.15bc 0.32b 1019.58b
5 0.00a 13.73b 10.17cd 21.51bc 0.14bc 1.14a 99.45d 11.59cd 0.11c 0.37a 393.25d
6 0.00a 14.60ab 9.00e 7.59d 0.11cde 0.60cd 176.63bc 18.13bc 0.15bc 0.32ab 812.17bc
7 0.00a 15.67ab 10.33bc 25.42ab 0.18ab 0.45d 196.28b 21.25b 0.18b 0.34ab 802.50bc
8 8.00a 14.03ab 7.36f 5.70d 0.07e 0.39d 77.79d 8.43d 0.07c 0.34ab 366.22d
DOI: 10.7717/peerj.9938/table-2
Table 3:
Range analysis of the concentrations and forms of N and K on the growth indices of the seedlings.
R is the range of each factor level (R=XmaxXmin); R’ is the adjusted range.
Range and adjusted range of growth indices K concentration N form N concentration
RSeedling height 0.87 1.40 1.17
R’Seedling height 0.55 1.99 1.66
RBiomass 0.04 0.09 0.03
R’Biomass 0.02 0.13 0.05
Rleaf number 1.40 2.33 1
R’leaf number 0.89 3.31 1.42
RSPADvalue 3.68 17.10 3.13
R’SPADvalue 2.34 24.28 4.44
RRoot length 68.39 59.57 87.12
R’Root length 43.52 84.58 123.71
RRoot surface area 6.73 7.50 8.15
R’Root surface area 4.28 10.65 11.57
RRoot volume 0.06 0.07 0.06
R’Root volume 0.04 0.10 0.08
RAverage diameter 0.01 0.03 0.02
R’Average diameter 0.004 0.043 0.028
RRoot tips 441.38 184.77 446.35
R’Root tips 280.89 262.37 633.82
RN content 0.75 0.46 0.06
R’N content 0.26 0.29 0.09
DOI: 10.7717/peerj.9938/table-3
Effects of N and K on aboveground growth of moso bamboo seedlings.

Figure 1: Effects of N and K on aboveground growth of moso bamboo seedlings.

A to D indicates the variation tendency of seedling height (A), biomass (B), leaf number (C) and SPAD value (D) respectively to different K concentrations, N form and N concentrations.

The analysis of variance (Table 4) showed that the N form and N concentration had significant effects on seedling height, biomass, leaf number and SPAD value. The K+ concentration also had significant effects on the leaf number of P. edulis seedlings. Therefore, moso bamboo seedlings treated with a normal nutrient solution supplemented with 8 mmol/L NH4+ and 0.5 mmol/L K+ would show overall better growth indices.

Table 4:
Variance analysis of the concentrations and forms of N and K on the growth indices of the seedlings.
Source of variation The test statistic F
K concentration N form N concentration
Height 0.53 6.94* 4.82*
Leaf number 8.65** 143.16** 26.29**
SPAD value 2.58 307.62** 10.29*
Biomass 2.70 61.25** 8.26*
Root length/(cm) 7.14** 18.34** 39.24**
Root surface area/(cm2) 5.71** 22.50** 26.59**
Root volume/(cm3) 3.94* 22.39** 14.22**
Average diameter/(cm) 0.16 7.72* 3.42
Root tips 16.26** 10.13** 59.13**
Nitrogen content 18.13** 21.62** 11.48**
DOI: 10.7717/peerj.9938/table-4

Notes:

indicates the significance level at p < 0.05.
indicates the significance level at P < 0.01.

Effects of different N and K on root system architecture of moso bamboo seedlings

According to the resultant R’ values of root architecture parameters (Table 3), N form and N concentration (Table 1) played a significant role on the root growth of P. edulis, while K+ concentration showed the least effect. Root architecture parameters were improved with NH4 + compared to NO3, and root traits were improved under the 8 mmol/L nitrogen (treatments 1, 4, 6 and 7; Tables 1 and 2) compared with the 32 mmol/L nitrogen (treatments 2, 3, 5 and 8; Tables 1 and 2), although the higher N concentration resulted in a greater increase in the root diameter of P. edulis (Table 2, Fig. 2). K had inconsistent effects on the root architecture of P. edulis. The total root length, root surface area and the root tips smaller with the increasing K+ concentrations. In contrast, the root volume and root diameter showed improved growth at a K+ concentration of 0.5 and 1.5 mmol/L, respectively (Table 2; Fig. 2).

Effects of N and K on root system architecture of moso bamboo seedlings.

Figure 2: Effects of N and K on root system architecture of moso bamboo seedlings.

A to E indicates root system architecture of root total length (A), root surface area (B), root volume (C), average diameter (D) and root tips (E) to different K concentrations, N form and N concentrations.

The analysis of variance (Table 4) showed that K+ concentration, N form and N concentration had significant effects on most of the root system parameters. However, there was no evident influence of K+ and N concentrations on average root diameter. In general, P. edulis seedlings treated with 8 mmol/L NH4+ (treatment 1) showed significantly better root architecture parameters than seedlings cultured in the other combinations/treatments (Table 2).

Effects of different N and K+ concentrations on the N content of moso bamboo seedlings

According to the resultant R’ values, the N form and K+ concentration (Table 1) played a more significant role than N concentration on the N content of moso bamboo seedlings (R’N form >  R’K+concentration >  R’N concentration, Table 3). NH4+ was a better N form than the NO3 for the N content of the seedlings, increasing 44.64% by comparing the average value of treatments 1, 3, 5, and 7 and treatments 2, 4, 6, and 8 (Tables 1 and 2, Fig. 3). The N content decreased when the K+ concentration increased from 0 to 0.5 mmol/L, whereas the N content increased when the K+ concentration further increased from 0.5 to 1.5 mmol/L. The optimal K+ concentration for the N content of moso bamboo seedlings was 0.5 to 1.5 mmol/L. The N content of the seedlings increased by 32.2% when the N concentration (Table 1) increased from 8 mmol/L (treatments 1, 4, 6 and 7) to 32 mmol/L (treatments 2, 3, 5 and 8; Tables 1 and 2, Fig. 3).

Effects of N and K on N content of moso bamboo seedlings.

Figure 3: Effects of N and K on N content of moso bamboo seedlings.

The analysis of variance showed that the effects of N form, N concentration, and K+ concentration (Table 1) on the N content of the seedlings were very significant (Table 4). Furthermore, the results indicate that moso bamboo seedlings had higher N content in treatment 5 (32 mmol/L NH4+ + 1.5 mmol/L K+) compared with the other treatments, except for treatment 1 (32 mmol/L NH4+ + 0 mmol/L K+) (Table 2).

Correlation analysis of the growth indices of moso bamboo seedlings

The growth indices of seedling height, leaf number, SPAD value and biomass were positively correlated with each other, and the correlations were also positive for root system architecture parameters such as root length, root surface area, root volume and root tips (Table 5). Growth and biomass accumulation were positively correlated with the main root system architecture parameters, but negatively or not correlated with N content. There were no correlations between root system architecture and the N content of the seedlings (Table 5).

Table 5:
Correlation analysis of the growth indices of the seedlings.
Correlations n = 8 Height Leaf number SPAD Biomass N content Root length Root surface Root volume Average diameter Root tips
Height 1
Leaf number .804* 1
SPAD .727* .926** 1
Biomass .890** .975** .903** 1
N content −0.053 0.395 0.459 0.296 1
Root length .889** .787* 0.582 .827* 0.042 1
Root surface .917** .832* 0.653 .884** 0.093 .992** 1
Root volume .923** .880** .709* .928** 0.157 .966** .990** 1
Average diameter 0.155 0.5 0.588 0.448 0.554 −0.094 0.023 0.155 1
Root tips .785* 0.696 0.418 0.701 0.002 .970** .939** .893** −0.239 1
DOI: 10.7717/peerj.9938/table-5

Notes:

Correlation is significant at the 0.05 level (2-tailed).
Correlation is significant at the 0.01 level (2-tailed).

Discussion

Moso bamboo seedlings showed improved growth under the NH4+ nitrogen form

All of the tested growth parameters and total N content were significantly increased with the NH4+ treatments compared to the NO3 treatments, suggesting that the seedlings of moso bamboo prefer NH4+ (Figs. 13). Our results are consistent with those presented by Gu et al. (2016) and Zou et al. (2020) who showed that the growth of bamboo seedlings displayed a strong NH4+ preference. Similarly, Song et al. (2013) showed that N uptake in the field is mainly in the form of NH4 +, accounting for 93.6% of the total inorganic N in bamboo-dominated forests. In addition, Ueda (1960) reported that the application of ammonium sulfate ((NH4)2SO4) strongly promotes the production of new culms in the first year after fertilization of P. edulis and P. reticulata groves. However, such results contradict the findings presented by Li et al. (2014b) that moso bamboo shows limited preference for NO3. Considering the cultivation medium, pH and nutrient supply are different among these experiments (Li et al., 2014b; Gu et al., 2016; Zou et al., 2020), it is hypothesized that the interactions between plant acquisition of N and multiple environmental variables may produce a complex of effects that can greatly influence and shift plant growth responses to variable N sources (Britto & Kronzucker, 2013). Although environmental factors, such as variations in pH, can also affect preferences for N form (Britto & Kronzucker, 2013), it has been reported that the effect of N form on plant performance is independent of growth medium pH (3.8, 5.8) for the P. edulis (Gu et al., 2016). As pH differences among treatments were negligible in our study, we believe, therefore, that performance differences are attributable to the N form rather than to the pH. However, the reasons underlying the preference of NH4+ by P. edulis are poorly understood and may arise from the atrophied nitrate uptake systems in the roots of P. edulis, as there only six nitrate transporters compared to twenty ammonium transporters were identified in the P. edulis genome (Hou, 2020), which was similar to the “ammonium specialists” of many conifers (Kronzucker, Siddiqi & Glass, 1997). Furthermore, moso bamboo is mainly distributed in subtropical acidic soils of southern areas of China, with N mineralization dominated by ammonification and NH4+ is the dominant inorganic N form in the soil (Song et al., 2013; Song et al., 2016a; Li et al., 2017). Therefore, moso bamboo preferentially utilizing NH4+ may be due to an adaptation to the native N nutritional habitat presenting over the course of evolution, similar to most late-successional conifer species growing on acidic soils (Kronzucker, Siddiqi & Glass, 1997; Britto & Kronzucker, 2013).

In aboveground organs, N is a structural element of chlorophyll, which affects the formation of chloroplasts and the accumulation of chlorophyll (Tucker, 2004). A previous study has reported that the chlorophyll content is closely linked to the leaf N content, and N deficiency leads to loss of the green color in the leaves, a reduced leaf area and photosynthetic intensity (Bojović & Marković, 2009). In the present work, the green color in the leaves and leaf area of the seedlings treated with NO3are decreased compared to those treated with NH4+ (Fig. S1), which may be associated with the leaf N content. However, there was no correlation between the total N content and chlorophyll content (SPAD value) or other growth characters (Table 5). One reason for this may be that we measured the N content of the whole plant instead of determining the N content of the root, stem, and leaf separately, so the total N content is inconsistent with the leaf N content. Alternatively, the correlation between the total N content and other growth characters may have not been established during our short-term experiment. Different forms of N have a large effect on leaf growth because N increases the leaf area of plants, chlorophyll content of leaf blades and photosynthetic rate, influencing photosynthesis (Li, Wang & Stewart, 2013). Therefore, the greater growth of moso bamboo seedlings with NH4+than with NO3 might be also associated with the increased photosynthesis of the aboveground organs.

Size and architecture of the root system are important factors of nutrient acquisition efficiency as they ensure the total volume of soil explored by the plant, and the total surface of exchange between roots and the soil solution (Nacry, Bouguyon & Gojon, 2013). Root system architecture is highly plastic, strongly modulated by N availability. A change in the root system can greatly impact nutrient acquisition from soil. In the present study, N form has a dramatic impact on the root system architecture of moso bamboo seedlings. Accordingly, the overall plant height, leaf number and biomass could be improved with the greater root length, root surface area and root volume in the bamboo seedlings treated with NH4+ (Figs. 1 and 2), due to positive correlations between most parameters of root morphology and aboveground indices (Table 5).

Moso bamboo suffers from the toxicity due to excessive NH4+

Although NH4+ is a preferred N source, excessive NH4+ such as 32 mmol/L (treatments 3 and 5), suppress aboveground growth, biomass accumulation and root system, while the total N content is increased by 35.77% compared to treatments with 8 mmol/L NH4+(treatments 1 and 7) (Table 2), suggesting that the P. edulis seedlings suffer from the toxicity due to excessive NH4+.

The uptake of NH4+ and NO3 by the root system is determined by different affinities. In the low concentration range, the uptake is mediated by high-affinity transport systems (HATS), while under high concentrations (typically >0.5∼1.0 mmol/L), the activity of low affinity transport systems (LATS) takes over from HATS (Nacry, Bouguyon & Gojon, 2013). Unlike HATS, the NH4+ uptake mediated by the LATS is not saturated and poorly regulated. It generally shows a linear increase with the increase of the external concentration, when NH4+ absorbed by roots far exceeds the amount of assimilation, it will cause excessive levels of NH4+ and thus plant NH4+ toxicity (Givan, 1979; Glass et al., 2002; Nacry, Bouguyon & Gojon, 2013). On the other hand, with the high similarity in the charge, size and hydration energy between NH4+ and K+, the K+ion transporters and channels do not discriminate between NH4+ and K+. The NH4+ can be transported through K+ transporters and channels as well as nonselective cation channels (NSCC), which may also contribute to NH4+ toxicity, especially at low K+ levels (Ten Hoopen et al., 2010). Although moso bamboo showed growth suppression with the high NH4+ treatments, there was no difference in the survive rate compared to the 8 mmol/L NH4+ treatments (Table 2), indicating moso bamboo seedlings were tolerant to high concentrations of ammonium.

Balkos, Britto & Kronzucker (2010) and Li et al. (2012) have underscored the central importance of NH4+/K+ ratios to determine NH4+ toxicity and tolerance in plants. However, the alleviation of NH4+ toxicity by K+ addition is not obvious for P. edulis seedlings. Under high NH4+ conditions, elevated exogenous K+ from 0.5 mmol/L to 1.5 mmol/L (treatments 3 and 5) caused a further inhibition of plant growth and increased NH4+ accumulation in moso bamboo seedlings. The external K+ may not be high enough to alleviate NH4+ toxicity, as described previously for Arabidopsis treated with 5∼20 mmol/L KNO3 (Zou et al., 2012). Alternately, moso bamboo may have evolved mechanisms of vacuolar nitrogen storage and downward transport of nitrogen from aerial parts to roots, allowing the plant to maintain nitrogen homeostasis, by which they may survive exposure to potentially toxic NH4+ concentrations, similar to the findings from other studies (Britto et al., 2001; Kronzucker et al., 2001; Britto & Kronzucker, 2002). Further studies need to be conducted to determine whether there are other mechanisms and the exact mode of actions of K+ and NH4+ relevant to the toxicity and tolerance.

Growth traits of moso bamboo are not improved by elevated K+

A previous study indicated that K+ is the major osmotically active cation contributing to the maintenance of root cell turgor and expansion, therefore plant roots are usually poorly developed in the absence of K+ (Dolan & Davies, 2004; Tsay et al., 2011). However, in the present study, K+ did not seem to play an important role in the root development, as most of the measured root growth traits are not improved by the increased K+ or are even better without K+(Fig. 2). Gao (2010) has also reported that there was a negative linear correlation between the available potassium storage in the soil layer and the average height of P. edulis, yet there was no obvious correlation between the storage amount of available potassium and the diameter of P. edulis. When K+ concentration was elevated from 0 to 3 mmol/L, the N content of P. edulis reduced by 104.44% from treatments 1 to 7 when treated with 8 mmol/L NH4+ (Table 2). Similar findings have also been observed in barley and rice, where NH4+ absorption of barley roots decreases by 60% when external K+ increases from 0.1 mmol/L to 1.5 mmol/L (Szczerba et al., 2007; Balkos, Britto & Kronzucker, 2010). Therefore, we speculate that the inhibition of root growth due to the elevated exogenous K+ may be related to the reduced NH4+ uptake in moso bamboo.

In contrast to the competitive uptake between NH4+ and K+, the acquisition of K+ and NO3 is usually cooperative (Tsay et al., 2011; Coskun, Britto & Kronzucker, 2017). The Dijkshoorn-Benzioni hypothesis has provided an intriguing example of the cooperative action of K + and NO3 in plants, namely, that NO3 is transported from roots to shoots in the xylem, using K+ as a counter ion (Benzioni, Vaadia & Lips, 1971; Coskun, Britto & Kronzucker, 2014; Dijkshoorn, 1958). It may be due to the charge balance of K+ and NO3, which could be substituted by other cations, in particular Mg 2+ (Hagin, Olsen & Shaviv, 1990; Coskun, Britto & Kronzucker, 2014), low supply of K+ can activate NO3 assimilation, reinforcing the NO3 reduction in roots (Balkos, Britto & Kronzucker, 2010; Zhang et al., 2010). In this study, increasing K+ from 0.5 mmol/L to 1.5 mmol/L (treatments 4 and 6) with 8 mmol/L NO3 treatment enhances the N content of moso bamboo seedlings by 53.85% (Table 2) in spite of a slightly growth inhibition of NH4+-preferring moso bamboo (Table 2), which might be related to the inhibited nitrate reductase activity due to the high endogenous NO3 concentrations (Reddy & Menary, 1990). However, further studies are needed to determine the role of K in the response of moso bamboo to different N forms and the molecular physiological mechanisms of the low potassium requirements.

Conclusions

N form and concentration play a significant role in the growth and N content of moso bamboo seedlings, while K+ concentration has a limited effect. The results from our experiments demonstrate improved growth of moso bamboo seedlings when treated with 8 mmol/L NH4+. Under elevated N concentrations, overall growth is inhibited though N content of the seedlings increases. In addition, root growth is inhibited with increasing K+ concentrations. Although moso bamboo seedlings display some level of NH4+-tolerance, high concentrations of NH4+can inhibit their growth, and increasing K+ concentration in the nutrient solution does not relieve the inhibitory effect of high NH4+ (32 mmol/L). Therefore, it is recommended that moso bamboo seedlings should be fed 8 mmol/L NH4+ fertilizer with a moderate K+ concentration.

Supplemental Information

Phenotypes of P. edulis (moso bamboo) seedlings under different treatments

The seedlings showed greener, heathier leaves and less necrosis in the NH 4 + treatments 1, 3, 5 and 7 than the NO 3 treatments 2, 4, 6 and 8, respectively. The image was taken after two months of different treatments. Bars = 30 cm.

DOI: 10.7717/peerj.9938/supp-1

Original data of growth index under each treatment

The raw data were used for range, variance and correlation analysis to compare the importance of N form, N and K concentrations on the growth of bamboo seedlings.

DOI: 10.7717/peerj.9938/supp-2