Introduction

The root system is the most sensitive plant organ to soil conditions, and changes to the soil first affect the respiration rate and carbon-nitrogen metabolism of the root system (Zhang et al., 2014). Root respiration drives root growth, root maintenance, ion absorption, and ion transport into the xylem (Lambers et al., 2008), and it is also a sensitive indicator of root function that indicates root matter and energy changes (Wang et al., 2002). Root system nonstructural carbohydrates (NSCs) are the main product of carbon-nitrogen metabolism, and NSCs play an important role in plant growth and development (Philipson, 1988), carbon-nitrogen metabolism (Mei et al., 2015), and resistance (Duque and Setter, 2019). Therefore, root respiration rate and carbon-nitrogen metabolism have a significant effect on the growth of plant parts belowground (Rewald et al., 2016), and the primary abiotic factor driving this effect is nitrogen. Recent studies have found that nitrogen increases the storage of NSCs in the larch [Larix gmelinii (Rupr.) Kuzen.] root system (Mei et al., 2015). The application of nitrogen fertilizer helps transform organic carbon in the Pinus tabuliformis Carr. root system, which is achieved by inhibiting root respiration (Zhang et al., 2019) since plant root respiration determines the total amount of carbon allocated to the root (Haynes and Gower, 1995). In woody plants, roots can be differentiated into levels based on root diameter, with corresponding differences in root morphology and physiology. Fine roots have higher respiration rates, but coarse roots have higher NSC content (Yu et al., 2011; Abramoff and Finzi, 2016). Recent studies have shown that in larch and ash (Fraxinus mandshurica Rupr.), root NSC content increases as root diameter increases (Jia et al., 2013). For example, in red oak (Quercus rubra Linn.) and white ash (Fraxinus americana Linn.), coarse roots have higher NSC content than fine roots (Abramoff and Finzi, 2016). However, studies of the effect of nitrogen concentration on root respiration and NSC content have focused on tree species that are economically important for their wood, while the effects on other tree species have rarely been reported.

To further explore the complex relationships between nitrogen and root respiration and nonstructural carbohydrate content of walnut seedlings, we measured the root respiration rate and NSC content of walnut (Juglans regia Linn.) seedlings under favorable growth conditions with the off-body root system and the ketone color ratio methods, respectively. We aimed to evaluate the regulation mechanism of nitrogen on the root respiration rate and nonstructural carbohydrates content of walnut seedlings.

Materials and Methods

Experimental Site and Plant Material

The experiment was conducted at the Key Laboratory of Forestry Ecology and Industry Technology in Arid Region, Education Department of Xinjiang in Xinjiang Province from March to August 2019. One-year-old walnut seedlings (Juglans regia Linn.) were used and were grown for a year under favorable conditions; they experienced consistent growth.

Experimental Design

Seedlings were planted during the walnut leaf budding period (March 27). The seedlings were removed from the field; soil was then washed off the walnut seedlings, and one walnut seedling was planted in each flower pot. Unfertilized pearl stone and zircon were used as a planting matrix and mixed at a 2:1 ratio. The pots had a bottom diameter of 22 cm, upper diameter of 30 cm, and height of 50 cm and contained 6 kg of planting matrix.

After 2 weeks (11 April), we began adding whole nutrient solution, with 200 mL of nutrition solution added to each pot once every 7 days. To ensure adequate moisture for walnut seedlings, 2,000 mL of water was added to each pot every day between 8:00 and 9:00 am. Thirty days after planting (25 April), all walnut seedlings were treated with different nitrogen concentrations, while the whole nutrient and moisture supply schedules did not change, and we applied nitrogen concentration treatment once every 20 days three times. The nutrient solution formation followed Jarkko and Toini (2001) and Ren (2009). Due to the large amount of measured data in this experiment, according to the absorption characteristics of walnut seedlings and the needs of our experiment, we appropriately adjusted and formulated the nutrient solution based on previous research methodology (Table 1). The pH of the nutrient solution was adjusted to between 5.8 and 6.0 with Ca(OH)₂ of 1 mmol·L^-1. We applied nutrient solutions at seven concentrations of N availability (0, 1, 2, 4, 8, 12, and 16 mmol·L^-1) by changing the concentration of NH₄NO₃ in the nutrient solution. The control concentration in the experiment was 0 mmol·L^-1, and the concentration of other nutrients did not change. Each nutrient concentration treatment was replicated and applied to six pots.

We applied seven different nitrogen concentration treatments to six pots of walnut seedlings with the same growth conditions at each concentration. Forty-two pots of walnut seedlings were used in this study. In addition, to increase the reliability and accuracy of the data, we repeated the experiments three times for each walnut seedling treated, further increasing the data volume.

Root Respiration Measurements

Root respiration rate was determined with the off-body root method (Clark et al., 2010). We selected three walnut seedlings from each nitrogen concentration treatment, removed each walnut seedling root system, washed it quickly with clean water, and then used the cursor caliper to divide the root system into three stages according to root diameter (d): d ≤ 1 mm, 1 mm ≤ d ≤ 2 mm, and d > 2 mm. We quickly weighed the root of each diameter stage in segments (d ≤ 1 mm cut into 8 cm segments, 1 mm ≤ d ≤ 2 mm cut into 5-cm segments, d > 2 mm cut into 2-cm segments). We immersed the selected root samples in a constant temperature circulating water bath at 18°C and allowed them to equilibrate for 30 min. Root respiration was determined at 18°C by measuring O₂ consumption using gas-phase O₂ electrodes (Model Chlorolab-2, Hansatech Instruments Ltd, King’s Lynn, UK) connected to the circulating water baths. Oxygen consumption measurements started when the respiratory response reached a stable state; the oxygen consumption of the living root at each diameter level in each cell was measured three times, and the respiration measurements were completed within a 2-h period. Following respiration measurements, the same samples from each root order were scanned using an Expression v700 photo scanner (Epson Telford Ltd, Telford, UK). Root images were analyzed by WinRHIZO (Pro2009a) software (Regent Instruments Company, Canada) for the diameter and length of each individual root. The same root samples were then oven-dried at 80°C for 24 h and weighed. The specific root respiration rate (SRR) was calculated using the following formulae (Eq. 1):

where, V_S indicates total respiration rate of unit length root (nmol·min^-1), V_T is the volume of unit length root (cm³), and m is the weight of the unit length root system (g).

Nonstructural Carbohydrate Content Measurements

Nonstructural carbohydrates (NSCs)—soluble total sugar (C) and starch content (X)—were measured using the ketone color ratio. The content of soluble total sugar and starch was calculated using the following formulae (Eq. 2 and Eq. 3):

where, C₁ indicates the amount of grapes found from the standard curve (µg), V_T indicates the total volume of the sample extract (mL), V₁ represents the volume of the sample extracted when color rendering (mL), W indicates the determination of sample fresh weight (g), and 0.9 is the coefficient of conversion from glucose to starch.

Statistical Analysis

The data were analyzed using Microsoft Excel 2010, and correlation analysis and significant statistical analysis of variables were conducted using SPSS 20. Differences in the measured properties across treatments were tested at the level of α = 0.05 using one-way analysis of variance (ANOVA) with tests for normal distribution and homogeneity of variance. A linear regression analysis was conducted to measure the relationships between study parameters.

Results

Correlation between Respiration Rate and Nonstructural Carbohydrates

Nitrogen addition can increase the respiration rate of walnut roots and have little effect on NSC content in the root system. Both the root diameter and the specific root respiration rate were significantly correlated with the root content of soluble total sugar and starch (p < 0.01) (Table 2). The specific root respiration rate was significantly correlated with the root diameter level (p < 0.01), and there was a significant correlation between soluble total sugar content and starch content (p < 0.01). However, the nitrogen concentration was not significantly related to total sugar content and soluble starch (p > 0.05). Root diameter was the major factor affecting the specific root respiration rate of walnut and NSC content.

Linear Relationship between Respiration Rate and Nonstructural Carbohydrates

The specific root respiration rate and the NSC content had a negative linear correlation at different nitrogen concentrations (Table 3). The R² values between the specific root respiration rate and the soluble total sugar content and the starch content were between 0.672 ‑ 0.997 and 0.661 ‑ 0.947, respectively, and all the correlations were significant. The correlations between specific root respiration rate and the content of soluble total sugar and starch were the highest when the nitrogen concentration was N2. This shows that NSC content has the greatest effect on specific root respiration rate at N2 levels and explains the specific root respiration rate of 94.7% to 99.7% at different nitrogen concentrations.

Effects of Nitrogen on Respiration Rate and Nonstructural Carbohydrates

As illustrated in Table 4, in roots with diameters of d ≤ 1 mm, 1 mm < d ≤ 2 mm, and d > 2 mm, soluble total sugar and starch contents and the specific root respiration rate were significantly lower in N0 than other nitrogen concentrations (p < 0.05), while they were significantly higher than other nitrogen concentrations, such as N16 (p < 0.05). Total soluble sugar and starch contents in roots increased with increased root diameter under the same nitrogen concentration, but specific root respiration rate decreased with increased root diameter, and the specific root respiration rate decreased with the increase of soluble total sugar and starch content.

Discussion

Previous studies of the effects of nitrogen on plant growth have shown that nitrogen fertilizer has a growth-promoting effect and that increasing nitrogen fertilizer increases the number of plant cytokines (Bloom et al., 2005), separates tissue cells, and promotes cell growth (Lawlor, 2002). Therefore, respiration provides energy for growing trees to form new tree structures (Ramos, 1985). The effect of nitrogen addition on the respiration rate of forest roots has been reported for Acer negundo Linn.(Burton et al., 2012), Pinus tabuliformis Carr. (Zhang et al., 2019), Populus tremula Linn.(Ceccon et al., 2016), and Cunninghamia lanceolata (Lamb.) Hook.(Fan et al., 2017). These prior studies found that higher nitrogen concentrations in the root soil significantly contributed to the respiration rate of forest fine roots while also significantly increasing the biomass of the coarse roots. Jia et al. (2011) reported that nitrogen concentration of the root system has a highly significant linear relationship with root respiration rate. We observed a significant positive correlation between the specific root respiration rate and nitrogen application levels of walnut seedlings, and the specific root respiration rate of walnut seedlings increased as root diameter decreased at the same nitrogen level, which is consistent with previous studies (Chen et al., 2010; Bravo et al., 2017). Fine roots play a vital role in root systems because they have high physiological activity during nutrient and water uptake from soil. This characteristic of fine roots enables them to be used as an indicator for plant physiological status during environmental changes (Razaq et al., 2017).

Chen et al. (2017) found that the diameter of the forest root system was related to root growth and carbohydrate distribution and that the NSC concentration of poplar fine roots was lower than that in coarse roots. Poplar may have a higher turnover rate for fine roots than other tree species and therefore does not store large amounts of reserves in fine roots (Regier et al., 2010). Additionally, some studies have reported that NSC content increases with increasing root diameter, but root respiration rate also monotonically decreases with increasing root diameter in the roots of Larix gmelinii Rupr. and Fraxinus mandshurica Rupr.(Jia et al., 2013). This has also been reported in Pinus palustris Mill.(Pregitzer et al., 2002; Aubrey and Teskey, 2018), where root respiration rates increased with increasing nitrogen concentration and the fine roots contained high concentrations of nitrogen when N was fully effective in the soil. Overall, numerous studies have reported that fine roots have higher rates of carbon-nitrogen metabolism than other root sizes (Hishi, 2007). This is because fine roots have a fast metabolism to provide energy for the growth, and development of the root system requires the consumption of large amounts of NSCs, while coarse roots need to store sufficient nutrients for complex organic compounds such as synthetic proteins when forests enter hibernation (Bazot et al., 2013; Martinez- Vilalta et al., 2016). The same results were obtained in this study, where the NSC content of the roots of walnut seedlings increased with the increasing root diameter, which is inversely proportional to the specific root respiration rate, which decreases with increasing root diameter.

Studies have found that low nitrogen levels can significantly increase the NSC content in the roots of seven northern hardwood tree species in the United States (Kobe et al., 2010), and studies have shown that increasing nitrogen application levels has a tendency to reduce the respiration rate of Pinus taeda Linn.fine roots (Drake et al., 2008). Here, we found that the respiration rate and NSC content of the roots of walnut seedlings were higher at N16 levels, and the effect is not significant at lower nitrogen levels. This is inconsistent with previous studies, which may be due in part to the fact that our experimental materials were one-year-old walnut seedlings. At this stage, the walnut seedlings are in a stage of prolonged vertical root growth, with the majority of total root weight of 87.82% in the coarse root and less in the side roots (Yang et al., 1980). In this early stage, the root system is strongly influenced by the external environment and nutrient demand is high. Under high nitrogen concentrations, the physiological activity of roots was stimulated to absorb and store a large amount of nitrogen nutrients, which are then converted into organic substances to meet the needs of their own root growth (Fan et al., 2013). Some studies have shown that an appropriate amount of nitrogen application helps increase the underground biomass of walnut trees in adult stages and promotes the growth of the root system (Liu et al., 2007). However, when the nitrogen concentration increases, the walnut trees will suffer from excessive nutrition, which inhibits the growth of underground biomass (Liu et al., 2007). The response of walnut seedling roots to higher nitrogen concentration is different from that of adult walnut roots to nitrogen nutrition supply, which may be due to the different absorption effects of walnut on nitrogen supply during seedling growth and adult fruiting. Walnut root metabolism is fast in the seedling stage and can promote root growth under the condition of higher nitrogen. In addition, transforming nitrogen absorbed by the root from soil into carbohydrates is highly efficient, and the nonstructural carbohydrate content in the roots increases accordingly (Liu, 2005; Li et al., 2006). However, in adult walnut trees, a higher concentration of nitrogen not only provides nutrients for root growth, but also provides energy for nutrients accumulated in fruits, which adds more processes to the distribution of nitrogen in adult walnut trees, and the metabolism speed is different in each stage. Therefore, a higher concentration of nitrogen in adult trees cannot be distributed to various organs in time, resulting in nitrogen accumulation (Xi et al., 1992). In addition, fertilization depth affects the growth of the walnut root system. Previous research shows that growth of the walnut root system at a depth of 30 cm is higher than that at a depth of 50 cm (Li, 2015). This is because the root tip region of walnut seedlings is the main part for absorbing nitrogen. As the organ with more root tips, the fibrous root system is mainly distributed in a shallow soil layer, while the straight root system plays a supporting role in forest growth and grows in a deeper soil layer (Yang et al., 1980). This is the main reason why walnut seedlings with different fertilization depths have different nitrogen absorption effects. In this study, walnut seedlings were planted in 50-cm-high flowerpots, and the depth of fertilization was relatively shallow, thus making the root system more sensitive to nitrogen concentration. Therefore, a higher nitrogen concentration had obvious effects on the respiration rate of the seedling root system and the content of nonstructural carbohydrates.

In conclusion, a nitrogen concentration was 16 mmol·L^-1 promotes root respiration and increases the content of NSCs in walnut seedlings.