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This article was submitted to Functional Plant Ecology, a section of the journal Frontiers in Plant Science

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

^{-2} year^{-1}) for

^{-2} year^{-1}) but decreased at high N addition levels (22.4 g N m^{-2} year^{-1}). (4) The RGRs of the whole seedlings and leaves were not significantly correlated with their N:P ratios at low and high N addition levels. By contrast, the RGRs of the stems and roots showed a significantly positive correlation with their own N:P ratio only at low N addition level.

Increasing global atmospheric N deposition could influence the stoichiometry of plant aboveground organs and roots and thus alter the physiological activity and growth rates (

Previous studies on the relationship between plant stoichiometry and environmental changes focused only on plant aboveground organs or leaf stoichiometry (

An increasing number of studies showed that N addition induced varied effects on the stoichiometry of roots with different orders and diameters. For example, ^{-2} year^{-1}, which is insufficient to change soil N content from limitation to saturation for plant growth in most ecosystems (

The growth rate hypothesis (GRH) indicates that fast-growing plants have low N:P and C:P ratios because of the differential allocation to P-rich ribosomal RNA, which results in a negative relation between growth rate and N:P ratio (

In this study, we tested the two hypotheses by determining the growth rates and stoichiometry of leaves, stems, and roots with four diameters (<0.5, 0.5–1, 1–2 and >2 mm) of ^{-2} year^{-1} to 22.4 g N m^{-2} year^{-1} to determine the possible critical values that promote or limit plant growth and alter the relationship between growth rate and N:P ratio from coupling to decoupling.

The experiment was conducted at the Institute of Soil and Water Conservation in Yangling, Shaanxi Province, China (107°38′E, 33°40′N). The region has a classic temperate continental climate with a mean annual precipitation of 674.3 mm, a mean annual temperature of 13.2°C, a sunshine period of 1993.7 h, and a frost-free period of 225 days.

Gray forest soil (Gray Luvisols, FAO soil classification) was collected from Yichuan in Shaanxi Province, China. The soil was sieved through a 2 mm mesh and then transferred to 175 pots. Each pot weighted 18 kg (depth of 35 cm, diameter of 40 cm). One-year-old ^{-2} year^{-1}) in the form of urea. Urea (Fumin Agriculture Product Company, Xi’an, China) was dissolved in 10 mL of distilled water and evenly added to the pot during a rain fall event in March of each year from 2011 to 2014. The levels of N addition for the five fertilization treatments were 0 (control, CK), 2.8, 5.6, 11.2, and 22.4 g N m^{-2} year^{-1} (0, 0.57, 1.15, 2.3, and 4.6 g urea pot^{-1} year^{-1}). The five N addition treatments were observed, and 35 pots were used for each N treatment; this study had a total of 175 pots.

Samples were harvested at two growth stages. In stage I, 1-year-old ^{-1}) was determined through H_{2}SO_{4}–K_{2}Cr_{2}O_{7} oxidation (^{-1}) was determined by persulfate oxidation followed by colorimetric analysis (^{-1}) was determined colorimetrically by Kjeldahl acid digestion with an Alpkem auto-analyzer (Kjektec System 1026 distilling unit, Sweden) after extraction with sulfuric acid (

_{t1}: Initial organ (leaves, stems, and roots) biomass at stage I (g). _{t2}: Final organ [leaves, stems, and whole roots (with four root diameters)] biomass at stage II (mg). Δ

Differences in element content and stoichiometric ratio between any two N addition treatments were tested using one-way ANOVA on SPSS 20.0 statistical software package (SPSS, Inc., United States). Linear regression analyses were used to test the stoichiometric relationships and element contents between leaves and four-diameter root classes, as well as the relationships between RGR and N:P ratio.

Across all seedling organs, the leaves and stems had higher C content than the whole roots. The C content of the roots increased with increasing root diameter. The leaves had higher N and P contents than the other organs. The N and P contents of the roots decreased with increasing root diameter (

Mean (mg g^{-1}) and relative standard deviation (RSD, %) of element contents across N addition gradient.

Leaves | 461.22 (3.58)^{a} |
10.96 (12.77)^{a} |
0.638 (20.85)^{a} |

Stems | 461.94 (4.53)^{a} |
3.37 (16.61)^{e} |
0.365 (24.93)^{d} |

Whole roots | 390.82 (6.94)^{cd} |
6.10 (19.18)^{d} |
0.491 (15.07)^{c} |

Finest roots | 331.27 (9.55)^{e} |
8.40 (22.02)^{b} |
0.687 (14.41)^{a} |

Finer roots | 379.17 (8.60)^{d} |
7.23 (24.48)^{c} |
0.555 (17.66)^{b} |

Middle roots | 402.45 (8.68)^{c} |
5.93 (24.96)^{d} |
0.459 (17.86)^{c} |

Coarse roots | 420.93 (6.07)^{b} |
4.24 (30.66)^{e} |
0.366 (26.50)^{d} |

Effects of N addition on the element content of seedling organs, ^{-1}) among different N addition gradients (

Across all seedling organs, the leaves had the highest N:P ratio, and the stems had the highest C:N and C:P ratios among all organs tested. The C:N and C:P ratios of the roots increased with increasing root diameter (

Mean and relative standard deviation (RSD, %) of stoichiometric ratios across N addition gradient.

Leaves | 42.66 (11.88)^{e} |
754.12 (21.48)^{c} |
17.84 (22.53)^{a} |

Stems | 140.59 (15.95)^{a} |
1334.24 (23.61)^{a} |
9.56 (18.31)^{c} |

Whole roots | 66.09 (18.69)^{cd} |
815.91 (19.05)^{bc} |
12.52 (18.21)^{b} |

Finest roots | 40.91 (20.53)^{e} |
491.82 (17.43)^{d} |
12.25 (15.59)^{b} |

Finer roots | 55.10 (23.52)^{d} |
714.31 (26.68)^{c} |
13.19 (21.61)^{b} |

Middle roots | 71.73 (26.78)^{c} |
913.16 (24.74)^{b} |
13.03 (21.57)^{b} |

Coarse roots | 108.02 (28.96)^{b} |
1234.77 (29.72)^{a} |
11.98 (38.73)^{b} |

Effects of N addition on stoichiometric ratio of seedling organs,

In most cases the element contents and the C:N:P ratios of the leaves correlated significantly with those of the root system, except for the five relationships that were non-significant (

Correlation coefficient and slope of the linear regression (in the parentheses) for the linear regression analysis of element content (upper) and stoichiometry ratio (lower) between the leaves and roots with four diameters.

Leaves | Finest roots | NS | (0.533^{∗}, 0.404) |
(0.479^{∗}, 0.640) |

Leaves | Finer roots | (0.620^{∗}, 0.314) |
(0.595^{∗}, 0.470) |
(0.546^{∗}, 0.736) |

Leaves | Middle roots | (0.781^{∗}, 0.370) |
(0.645^{∗}, 0.609) |
NS |

Leaves | Coarse roots | (0.635^{∗}, 0.411) |
(0.469^{∗}, 0.503) |
(0.717^{∗}, 0.982) |

Leaves | Whole roots | (0.774^{∗}, 0.471) |
(0.744^{∗}, 0.888) |
(0.716^{∗}, 1.289) |

Leaves | Finest roots | NS | (0.807^{∗}, 1.694) |
(0.418^{∗}, 0.252) |

Leaves | Finer roots | (0.550^{∗}, 0.468) |
(0.715^{∗},0.007) |
(0.628^{∗}, 0.246) |

Leaves | Middle roots | (0.462^{∗}, 0.331) |
(0.498^{∗}, 0.712) |
(0.633^{∗}, 0.167) |

Leaves | Coarse roots | (0.705^{∗}, 0.311) |
NS | NS |

Leaves | Whole roots | (0.717^{∗}, 0.747) |
(0.635^{∗}, 1.120) |
(0.712^{∗}, 0.292) |

^{∗}indicates a significant relationship at

Relative growth rate varied among different seedling organs. Compared with the other organs, the stems had higher RGR whereas the roots had lower RGR (^{-2} year^{-1} treatments and then significantly decreased at 22.4 g N m^{-2} year^{-1} treatments compared with the control.

Effects of N addition on relative growth rate of whole seedling and organs.

Treatment (g N m^{-2} year^{-1}) |
Whole plant relative growth rate | Leaves relative growth rate | Stems relative growth rate | Whole roots relative growth rate | Whole plant biomass (g seedling^{-1}) |
---|---|---|---|---|---|

(mg g^{-1}d^{-1}) |
(mg g^{-1}d^{-1}) |
(mg g^{-1}d^{-1}) |
(mg g^{-1}d^{-1}) |
||

CK (0) | 1.596 ± 0.056^{c} |
1.803 ± 0.086^{ab} |
1.639 ± 0.048^{d} |
1.090 ± 0.046^{b} |
365.41 ± 29.04^{c} |

N1 (2.8) | 1.746 ± 0.058^{b} |
1.809 ± 0.080^{ab} |
2.077 ± 0.049^{b} |
1.107 ± 0.085^{b} |
504.38 ± 40.77^{b} |

N2 (5.6) | 1.745 ± 0.081^{b} |
1.889 ± 0.099^{a} |
1.988 ± 0.084^{c} |
1.073 ± 0.051^{b} |
558.64 ± 63.23^{b} |

N3 (11.2) | 2.049 ± 0.048^{a} |
1.784 ± 0.068^{ab} |
2.398 ± 0.040^{a} |
2.069 ± 0.039^{a} |
837.44 ± 57.06^{a} |

N4 (22.4) | 1.603 ± 0.033^{c} |
1.731 ± 0.032^{b} |
1.986 ± 0.048^{c} |
0.949 ± 0.010^{c} |
316.26 ± 14.50^{c} |

^{-2}year

^{-1}, respectively.

The relationships between RGR and N:P ratio varied under different N addition ranges. The RGR of the whole seedlings and leaves showed no significant correlation with their N:P ratios at 0–11.2 and 11.2–22.4 g N m^{-2} year^{-1} treatments. The RGRs of the stems and whole roots had significantly positive correlation with their own N:P ratios at 0–11.2 g N m^{-2} year^{-1} but not at 11.2–22.4 g N m^{-2} year^{-1} treatments (

Relationships between N:P ratio and relative growth rate (mg g^{-1} d^{-1}) for

In this study, the stoichiometric ratios varied among the different plant organs. N addition significantly changed the stoichiometric ratios and element contents (except P content) of the plant organs. In addition, stoichiometry of root was more sensitive to the changes of soil available N content compared with those of the other organs. These results partly support our first hypothesis.

Many studies found that the stoichiometry of different organs varies (

Stoichiometry mainly reflects the ability of plants to utilize C, N, and P, which are susceptible to environmental changes (

The effects of N addition on plant stoichiometry are different not only in different organs but also in the same organ. In this study, N addition significantly changed the C:N ratio of the finest roots and the C:P ratios of the finest and finer roots. Moreover, N addition significantly increased the N:P ratios of the finest, finer, and middle roots. In general, the changes in the stoichiometric ratio were greater in finer roots than in coarse roots. The reasons for these changes may be as follows: N addition would change the morphological and physiological characteristics of the root system; this change increases the number, length, production, turnover, and biomass of finer roots but does not affect coarse roots (

Homeostasis is the ability to maintain stable nutrient content of plant organs despite fluctuation in environmental resources (

The RGR differed among seedling organs. N addition significantly increased the RGRs at 0–11.2 g N m^{-2} year^{-1} treatments but significantly decreased the RGRs at 22.4 g N m^{-2} year^{-1} treatments. Previous studies found that the research region is an N-limited region (^{-2} year^{-1}. However, N addition at 22.4 g N m^{-2} year^{-1} decreased plant growth rates, possibly indicating that the critical value for N saturation is between 11.2 and 22.4 g N m^{-2} year^{-1}. In addition, the relationships between RGR and N:P ratio varied among the different seedling organs, and these relationships were determined by N addition level. These results partly supported our second hypothesis.

The N:P ratio of whole seedlings showed no significant correlation with its own RGR, which does not support the RGH at the whole seedling level. This result can be attributed to the phenomenon that N addition exerted no significant influence on N:P ratios but increased seedling growth at low N addition and restrained growth at high N addition. Our results are consistent with the findings of

The responses of stoichiometry to N addition varied among ^{-2} year^{-1}) but was restrained at high N addition levels (11.2–22.4 g N m^{-2} year^{-1}). A positive correlation between RGR of the stems and roots with their own N:P ratio was observed at low N addition levels. These findings are inconsistent with GRH. At high N addition level, however, the correlations were decoupling. Our conclusion are based on the seedling plant, and the studies on mature tree are needed in the future.

GW, GL, and SX conceived and designed the study. HZ and MD performed the experiments. HJ wrote the paper. GW, HJ, and GL reviewed and edited the manuscript. All authors read and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.