The Application of Nitrogen Source in Regulating Lignin Biosynthesis, Storage Root Development and Yield of Sweet Potato

Abstract

The understanding of the effects of nitrogen sources on lignin synthesis in sweet potato during storage root formation is limited. In this study, we investigated the effects of different nitrogen source on sweet potato storage root formation and development, as well as lignin synthesis in potential storage roots. The sweet potato cultivars Shangshu 19 and Jixu 23 were used in field experiments in 2019 and 2020. Three treatments were tested: (a) no nitrogen fertilizer application (control); (b) 60 kg hm⁻² ammonium nitrogen; and (c) 60 kg hm⁻² amide nitrogen. The results indicate that during sweet potato storage root formation, ammonium nitrogen significantly enhanced root activity compared to that of the control. The ammonium nitrogen treatment promoted IbEXP1 and inhibited Ibkn1 and Ibkn2 expression during the early stages of storage root formation, then increased gibberellic acid and decreased zeatin riboside content, enhanced phenylalanine ammonia lyase and peroxidase activities, and promoted lignin synthesis in potential storage roots. The opposite effects of ammonium nitrogen treatment on gene expression, hormone contents, and enzyme activity were observed in the late stages of storage root formation. Relative to the control, the ammonium nitrogen treatment significantly increased the number of storage roots during canopy closure. The ammonium nitrogen treatment produced the highest storage root yield and number of storage roots per plant. These results indicated that the ammonium nitrogen can inhibit root lignin synthesis, then promote storage root formation and increase the yield of sweet potato.

1. Introduction

Sweet potato (Ipomoea batatas L.) is an important tuberous crop, ranking after potato and cassava in the world [1]. After seedlings are transplanted, the root primordia in the underground stem procambium differentiate to form adventitious roots [2]. These, in turn, further differentiate into fibrous roots, thick roots, and storage roots (SRs). The adventitious roots first differentiate into white fibrous roots (<2 mm) which will then become pigmented and increase in width to form thick roots (2–5 mm). A part of each thick root will ultimately develop into a SR (>5 mm). Previous studies have extensively investigated the anatomy of sweet potato root systems. The stele cells in fibrous roots are highly lignified and have weak vascular cambium activity. In thick roots, the stele cells have a high degree of lignification and strong vascular cambium activity. There is minimal lignification of the SR stele cells but strong vascular cambial activity and accessory cambium formation. Thick roots have more primary xylem than fibrous roots. This structure is conducive to vascular cambium formation and activity and facilitates SR formation [3,4,5]. Lignin content is the most direct physiological indicator of sweet potato lignification [6]. Lignin is synthesized by phenylpropanoid metabolism. Specifically, phenylalanine ammonia lyase (PAL) deaminates L-phenylalanine to form trans-cinnamic acid. It is the first rate-limiting enzyme in the lignin biosynthesis pathway [7,8,9,10]. Peroxidase (POD) is the last key enzyme in this metabolic pathway. It decomposes H₂O₂ to polymerize monolignols and form lignin [11].

Sweet potato SR formation is closely correlated with endogenous zeatin riboside (ZR) content [12,13], which is mainly distributed in the primary cambium of young sweet potato. Endogenous ZR participates in primary cambium development in young roots. It also activates and promotes SR formation [14]. In studies on tobacco and carrot, respectively, Biemelt et al. [15] and Wang et al. [16] found that gibberellin (GA) promotes lignin accumulation and stele cell lignification. Wang et al. [17] examined sweet potato cultivars and their wild relatives. They found that 5–30 days after planting (DAP), there was no significant difference in root system GA₄ content between cultivars and the wild type. In both cases, there was virtually no root formation. Therefore, they proposed that GA₄ level is not correlated with SR formation. Other researchers found that the application of exogenous GA before and after adventitious root formation delayed SR production and reduced the number of SRs formed. The physiological effects of GA on SR formation remain to be determined [6,13,18].

KNOX1 proteins positively regulate cytokinin biosynthesis [19,20,21] and negatively regulate GA biosynthesis [22,23,24]. Mele et al. [25] found that BREVIPEDICELLUS (BP), the KNOX1 protein in Arabidopsis, controls cell differentiation by negatively regulating lignin biosynthesis. Nevertheless, few studies have examined the relationship between the sweet potato KNOX1 gene and lignification. Ibkn1 and Ibkn2 are KNOX1 genes expressed in sweet potato roots. Their expression levels are twice as high in young SRs than they are in fibrous roots [26,27]. Expansins are proteins participating in cell wall relaxation [28,29,30,31]. Noh et al. [32] discovered an expansin gene (IbEXP1) in sweet potato that inhibits SR formation by positively regulating lignin biosynthesis.

Nitrogen source is closely correlated with crop growth and yield. Previous researchers have investigated the absorption and utilisation of various forms of nitrogen by sweet potato, as well as the yield and quality of the plants receiving them [33]. On the other hand, few studies have been conducted on the regulatory effects of various nitrogen sources on SR formation in sweet potato. The effects of nitrogen source on lignin biosynthesis in sweet potato and its relationship with SR formation have also been seldomly reported. In this study, two sweet potato cultivars with significantly different numbers of SRs were selected to determine the effects of different nitrogen source (at optimal levels) on lignin metabolism during adventitious root development, differentiation of young roots into SRs, and the number of SRs produced. The objective was to identify the regulatory effects of various nitrogen source on the number of SRs produced. This information could be used in the cultivation of high-quality, high-yielding sweet potato.

2. Materials and Methods

2.1. Plant Material and Growth Conditions

We used the cultivars of sweet potato Ipomoea batatas (L.) Lam. ‘Shangshu 19’ (S19) and ‘Jixu 23’ (J23) as the test material. The SRs number of S19 is significantly higher than that of J23 [34]. Approximately 25-cm long plant cuttings (slips) were cut from germinated SRs. Three unfolding leaves (the third, fourth, and fifth leaf from the apex) were retained on the cuttings and all other leaves were excised. All axillary and terminal buds were also removed.

Two-year field experiments were conducted at the agricultural base of Hainan University, Haikou, China (20°06′ N, 110°33′ E) from November 2019 to April 2021. Climate data were provided by the Haikou Meteorological Bureau, Haikou, China (

Table 1. Climate during the sweetpotato growing season.

Year	Month	Temperature (°C)			Precipitation (mm)
		Max	Average	Min
2019	11	27.58	23.61	19.92	10.80
	12	25.06	21.16	17.98	6.60
2020	01	24.63	20.41	17.41	11.30
	02	24.66	20.81	18.21	5.60
	03	30.09	25.37	21.98	12.20
	04	28.82	24.59	21.23	36.10
	11	26.11	23.41	20.88	14.80
	12	21.59	19.07	17.04	6.20
2021	01	20.22	16.36	12.97	19.10
	02	24.83	19.82	15.78	26.20
	03	28.69	24.21	21.22	2.00
	04	30.61	26.27	23.35	61.20

).

The soil type is sandy loam with a clay content of 49.32%. The concentration of organic matter was 1.17% in the 0–20 cm soil layer of the field, and the total and available nitrogen was 1.24 g kg⁻¹ and 80.66 mg kg⁻¹, respectively. Available phosphorus was 17.49 mg kg⁻¹, and available potassium concentration was 79.18 mg kg⁻¹.

2.2. Field Experiments

A two-factor split-plot design was employed in this study, with S19 and J23 being assigned to the main plot and the different nitrogen treatments assigned to the subplots. The fertilizers used were ammonium sulfate (N 21%), urea (N 46%), calcium superphosphate (P₂O₅, 16%), and potassium sulfate (K₂O 50%) provided by Sinofert Holdings Limited (Beijing, China). Three different fertilizer treatments were used: 60 kg·hm⁻² ammonium nitrogen (AN), 60 kg·hm⁻² amide nitrogen (XN), and no nitrogen fertilizer (N0). The nitrogen level was calculated as N. Potassium fertilizer at a level of 240 kg·hm⁻² (calculated as K₂O) and phosphate fertilizer at a level of 110 kg·hm⁻² (calculated as P₂O₅) were applied in all treatments. Sublimed Sulphur powder was applied to ensure that all the treatments had the same Sulphur levels. Ammonium nitrogen and amide nitrogen treatments were treated with nitrification inhibitors (3pyr4-dimethylpyrazole phosphate, DMPP) to inhibit nitrification, the dosage was 7% of the amount of nitrogen application. In the amide nitrogen treatment, additional urease inhibitors (N-butylthiophosphoryltriamine, NBPT) were applied to slow down the decomposition of urea, and the dosage was 1% of the amount of nitrogen application. The fertilizers were applied at the bottom of each row to reduce nitrification. Each of the six treatment groups were in quadruplicate and randomly assigned to different subplots. The area of each plot was 16 m^2, with row spacing of 0.8 m. The cuttings spacing was 0.25 m, planted at a depth of approximately 0.10 m in soil beds.

2.3. Sampling

The samples were collected at the early stage of SR formation (14 to 21 DAP) and the late stage of SR formation (28 to 35 DAP) in the field. The thickest 3–4 roots of each plant, namely potential storage roots (PSRs), were selected, cut into approximately 1-cm pieces, and mixed evenly. The segments were rapidly frozen with liquid nitrogen and stored at −80 °C for later enzymatic activity measurements.

In 2019 and 2020, five plants were selected to analyze the number of PSRs (diameter > 0.5 cm) [27,32] and to determine the mean fresh weight at the canopy closure stage (45 DAP). At harvest, roots that were greater than 1.00 cm in diameter were selected as SRs, the fresh weight of SR was weighed, and the total number of SRs in each subplot was also counted.

2.4. Determination of Root Activity

The triphenyl tetrazolium chloride (TTC) test was modified from the method of Chen et al. [35] for the determination of root activity. Prior to TTC incubation, the root tip around 2 cm of sweet potato roots were washed with sterile water for 10 min and placed in 15 mL of TTC solution (0.08% TTC in 0.05 M sodium phosphate buffer, pH 7.4) and placed in dark at 30 °C for 24 h. The TTC solution was drained and washed with sterile water. Roots for the measurement of TTC were excised and cut into segments of 1 cm. The reduced TTC, formazan, was extracted twice with 10 mL 95% ethanol in a water bath with 80 °C for 20 min, and absorbance was measured at the 485 nm wavelength. In each independent experiment, the test was repeated three times.

2.5. Determination of Lignin Content

The lignin test was modified from the method of Bhaskara et al. [36]. The PSR of sweet potato was dried at 80 °C to constant weight. The dry roots were smashed and sieved with a 40-mesh sieve, before 2 mg were sampled and placed in 5 mL of methanol in a glass test tube. The tissue was extracted with four changes of methanol during 48 h. After methanolic extraction, the tissue was ground and placed in a test tube containing 0.5 mL thioglycolic acid (Sigma Chemical Co., Ltd., City of Saint Louis, MO, USA) and 5 mL 2 mol L⁻¹ HCl. The tubes were capped with glass marbles and heated for 4 h at 95 °C. The solids were collected by centrifugation and the supernatant was discarded after cooling. The solids were washed by resuspension in 5 mL water followed by centrifugation. The supernatant was discarded, and the solids were incubated in 5 mL for 18 h 0.5 mol L⁻¹ NaOH to solubilize the ligninthioglycolic acid (LTGA). After this extraction, the solids were removed by centrifugation (two washes, with 2 mL 0.5 N NaOH each time). The NaOH extracts and water washes were combined and poured into a 15-mL conical centrifuge tube, and 1 mL of concentrated HCl was added. The acidified solution was held for 4 h at 4 °C to aid in LTGA precipitation. The precipitated LTGA was collected by centrifugation (500× g, 15 min). The pellet was washed twice by resuspension and centrifugation in 0.1 mol L⁻¹ Hl (2 mL per wash). The final pellet was dissolved in 0.5 mol L⁻¹ NaOH to a final volume of 2.5 mL. The final solution was centrifuged (10,000× g, 3 min) to remove any insoluble material prior to measurement of the absorbance of the solution at 280 nm wavelength.

2.6. Determination of Zeatin Riboside (ZR) and Gibberellin (GA₃) Content

ZR and GA₃were determined by enzyme-linked immunosorbent assays (ELISA) with monoclonal antibodies produced by the Phytohormones Research Institute (China Agricultural University), following the modifications described by Degenhardt et al. [37]. Hence, 0.5 g fresh PSRs were extracted and purified by passing through C18-Sep-Pak cartridges. Each well in 96-well microtitration plates was coated using a 50 μL sample and 50 μL antigens (0.25 μg mL⁻¹) against the hormones. The coated plates in a wet box were incubated at 37 °C for 30 min. After washing four times using phosphate-buffered saline (PBS) + Tween 20 (0.1% (v/v)) buffer (pH 7.4), 100 μL antibodies (20 μg mL⁻¹) was added to each well, and the plates were incubated for a further 30 min at 37 °C in a wet box. The plates were rinsed four times, using PBS + Tween 20 buffer and 100 μL color-development solution containing 2 mg mL⁻¹ O-phenylenediamine (OPD), and 0.008% (v/v) H₂O₂ was added to each well. The reaction progress was stopped by adding 50 μL of 2 mol L⁻¹ H₂SO₄ per well when the 2000 ng mL⁻¹ standard showed a pale color. Color development in each well was detected using an ELISA Reader at optical density A490.

2.7. Determination of PAL and POD Activity

Total soluble protein was extracted from the fresh PSRs by an extraction buffer, and the concentration was determined using the Bradford [38] protein assay. The determination of POD and PAL activities was performed with extracted protein. According to the method described by Kwak et al. [39], pyrogallol was used as a substrate to determine POD activity. The amount of enzyme required to form 1 mg of purpurogallin from pyrogallol in 20 s was defined as a unit of POD activity, as measured by absorbance at 420 nm. Phenylalanine ammonia lyase (PAL) was assayed according to Kamran1 et al. [7]. PAL activity was measured by incubating 0.5 mL of supernatant with 2 mL 0.1 M borate buffer (pH 8.0) containing 3 mM L-phenylalanine at 30 °C for 1 h. PAL activity was assayed spectrophotometrically following the formation of trans-cinnamic acid from L-phenylalanine at 290 nm. The amount of enzyme that causes the increase in absorbance of 0.05 at 290 nm h⁻¹ mg⁻¹ protein was defined as a unit of PAL activity.

2.8. qRT-PCR Analysis

According to the manufacturer’s protocol, total RNA was isolated from the fresh PSRs using an RNAprep Pure Plant Kit (TianGen, Beijing, China). The qRT-PCR was carried out in a 25-µL reaction volume containing 2X TransStart Top Green q-PCR SuperMix (TransGen BioteCh, Beijing, China). Quantitative analysis was conducted with the Bio-Rad CFX Manager system (Hercules, CA, USA). The expression of a specific gene against a control takes the formula 2^−ΔΔC_T. The mRNA level of actin (a stably expressed gene) was used as control gene for qRT-PCR analysis. The primers of related genes are shown in Supplementary Table S1 [13].

2.9. Statistical Analysis

The analysis was performed as a two-factor split-plot design. Data analysis was performed using IBM SPSS Statistics v. 24.0 (IBM, Armonk, NY, USA). The figures were prepared using SigmaPlot software (SigmaPlot v. 12.5, Systat Software, San Jose, CA, USA).

3. Results

3.1. Effects of Nitrogen Source on Storage Root Formation and Yield

The effects of various nitrogen sources on sweet potato SR traits are shown in

Table 2. Characteristics of the formation of the sweet potato storage root in field trials during canopy closure and harvest period (2019–2020).

Year	Cultivars	Treatment	At the Canopy Closure Period		At Harvest Time
			Potential Storage Root Number Per Plant	Average Fresh Weight of Storage Root (g)	Storage Root Number Per Plant	Average Fresh Weight of Storage Root (g)	Yield (kg hm−2)	Yield Increment (%)
2019	S19	N0	5.00 ± 1.00 de	71.97 ± 0.5 d	3.67 ± 0.58 c	155.00 ± 3.21 g	28,417.55 ± 1136.7 g	-
		AN	5.67 ± 0.58 c	78.69 ± 0.53 cd	4.67 ± 0.58 a	146.89 ± 2.65 h	34,274.1 ± 1370.96 d	20.61
		XN	5.33 ± 0.58 cd	71.12 ± 0.59 e	4.00 ± 0 b	147.65 ± 2.65 h	29,530.2 ± 1181.21 f	3.91
	J23	N0	4.67 ± 0.58 ef	81.86 ± 1 c	2.67 ± 0.58 f	162.27 ± 0.58 f	21,635.82 ± 865.43 j	-
		AN	5.00 ± 1.00 de	95.86 ± 0.86 a	3.33 ± 0.58 d	147.95 ± 2.57 h	24,659.22 ± 986.37 h	13.97
		XN	4.33 ± 0.58 f	82.94 ± 0.79 b	3.00 ± 0 e	158.07 ± 0.58 fg	23,711.45 ± 948.46 i	9.59
2020	S19	N0	6.67 ± 0.58 b	86.86 ± 0.99 i	4.00 ± 0 b	184.06 ± 4.00 d	36,811.44 ± 1472.46 b	-
		AN	8.33 ± 0.58 a	93.66 ± 1 g	4.67 ± 0.58 a	175.25 ± 3.51 e	40,891.22 ± 1635.65 a	11.08
		XN	6.67 ± 0.58 b	85.11 ± 0.81 i	4.00 ± 0 b	184.67 ± 1.53 d	36,935.43 ± 1477.42 b	0.34
	J23	N0	4.33 ± 0.58 f	91.96 ± 1 h	3.00 ± 1.00 e	215.62 ± 1.53 a	32,343.46 ± 1293.74 e	-
		AN	5.67 ± 0.58 c	98.56 ± 0.9 f	3.67 ± 0.58 c	192.83 ± 2.00 c	35,352.49 ± 1414.1 c	9.30
		XN	4.33 ± 0.58 f	92.22 ± 0.6 g	3.33 ± 0.58 d	209.01 ± 1.73 b	34,834.65 ± 1393.39 d	7.70
ANOVA analysis
Year			108.00 **	1614.49 **	2.29	9170.64 **	1875.01 **	-
Cultivars			784.00 **	765.13 **	16.20 *	327.98 **	3450.78 **	-
Treatment			28.80 **	574.84 **	7.87 **	81.14 **	6113.86 **	-
Year × Cultivars			256.00 **	115.55 **	0.20	115.50 **	299.28 **	-
Year × Treatment			7.20 **	20.85 **	0.12	5.91 *	78.30 **	-
Cultivars × Treatment			0.80	17.97 **	0.37	12.16 **	1226.82 **	-
Year × Cultivars × Treatment			0.80	26.82 **	0.13	3.12	81.20 **	-

Note: N0, no nitrogen fertilizer; AN, ammonium nitrogen; XN, amide nitrogen. Split-split-Plot ANOVA, LSD. Values followed by lowercase letters within a column are significantly different among phosphorus treatments (p < 0.05). *—p < 0.05; **—p < 0.01. Yield increment compared to N0 treatment.

. At canopy closure, the ammonium nitrogen treatment (ANT) significantly (p < 0.05) increased the number and weight of PSRs relative to the control. In contrast, the amide nitrogen resulted in a number and weight of PSRs similar to those for the control.

Table 2 shows that, for both years, the ANT resulted in the highest SR yield, which was 9.30–20.61% greater than that of the control. The SR yield under the amide nitrogen treatment (XNT) was similar with that of the control. The ANT increased the number of SRs formed per plant by 16.67% to 29.07% over that of the control but reduced the average SR weight.

3.2. Effect of Nitrogen Source on Root Activity of Sweet potato

Figure 1 shows that the root activity in both cultivars at 15 DAP was significantly higher than that at 30 DAP. At 15 DAP, ammonium and amide nitrogen applications significantly (p < 0.01) enhanced root activity. Nevertheless, the ANT resulted in a greater increase in root activity than did the XNT. At 30 DAP, the root activity in the ANT was significantly (p < 0.05) higher than that of the controls. In contrast, the root activity under the XNT was similar to that of the control.

3.3. Effects of Nitrogen Source on Lignin Content in Potential Storage Root

Figure 2 shows that the lignin content in the PSRs of both cultivars first increased, then decreased, reaching a maximum at 28 DAP. The lignin content of Shangshu 19 was significantly (p < 0.05) lower than that of Jixu 23. At 14–21 DAP, the lignin content of the PSRs under the ANT was significantly (p < 0.05) higher than that of the control. The lignin content of the PSRs receiving amide nitrogen was similar to that of the control. By 28–35 DAP, the lignin content of the PSRs being given ammonium nitrogen was significantly (p < 0.05) lower than that of the control and the roots receiving amide nitrogen. The lignin content of the PSRs under the XNT was lower than that of controls, but the difference was not significant (p > 0.05).

3.4. Effects of Nitrogen Source on the Activity of PAL and POD in Potential Storage Root

Table 3. PAL and POD activity in potential storage roots of sweet potato during storage root formation.

Days after Planting	Cultivars	Treatment	PAL Activity (Units g−1)	POD Activity (Units mg−1 Protein)
14	S19	N0	41.21 ± 0.73 d	14.88 ± 0.31 de
		AN	45.3 ± 0.98 c	16.4 ± 0.4 bc
		XN	45.06 ± 0.61 c	23.06 ± 0.45 a
	J23	N0	39.79 ± 0.85 e	17.52 ± 0.67 b
		AN	50.33 ± 0.67 a	14.23 ± 0.22 e
		XN	49.4 ± 0.56 b	15.51 ± 0.22 cd
21	S19	N0	48.58 ± 1.15 a	21.91 ± 1.97 a
		AN	50.41 ± 1.15 a	13.55 ± 1.78 b
		XN	46.56 ± 1.1 b	16.28 ± 0.89 b
	J23	N0	40.84 ± 1.1 c	15.42 ± 0.62 b
		AN	44.69 ± 0.67 b	20.37 ± 0.22 a
		XN	40.43 ± 0.78 c	20.34 ± 0.81 a
28	S19	N0	56.13 ± 0.51 b	20.31 ± 0.4 b
		AN	60.75 ± 0.43 a	22.91 ± 0.36 a
		XN	49.92 ± 0.69 e	20.9 ± 0.22 e
	J23	N0	53.73 ± 1.1 c	24.01 ± 0.62 c
		AN	60.87 ± 0.43 a	17.14 ± 0.49 a
		XN	51.63 ± 0.73 d	24.48 ± 0.91 d
ANOVA analysis
14	Cultivars		2406.83 **	79.22 *
	Treatment		2841.52 **	192.36 **
	Cultivars × Treatment		538.38 **	288.01 **
21	Cultivars		115.04 **	72.05 *
	Treatment		22.57 **	2.19
	Cultivars × Treatment		1.5	33.36 **
28	Cultivars		27.93 *	96.33 *
	Treatment		655.78 **	41.05 **
	Cultivars × Treatment		27.5 **	152.54 **

Note: N0, no nitrogen fertilizer; AN, ammonium nitrogen; XN, amide nitrogen. Split-Plot ANOVA, LSD. Values followed by lowercase letters within a column are significantly different among phosphorus treatments (p < 0.05). *—p < 0.05; **—p < 0.01.

shows that, at 14–28 DAP, the PAL activity in the PSRs of both cultivars decreased initially, then increased. The POD activity showed the opposite trend to the PAL activity. At 14 and 21 DAP, the PAL and POD activities of the PSRs under the ANT were significantly (p < 0.05) higher than those of the control. On the other hand, the PAL and POD activities of both cultivars receiving amide nitrogen were similar to those of the control. At 28 DAP, the PAL and POD activities in the PSRs being given ammonium nitrogen were significantly (p < 0.05) lower than the control and those getting amide nitrogen. Under the XNT, the PAL activity in the PSRs of both cultivars was significantly (p < 0.05) lower than that in the controls, but their POD activity was similar with that of the control.

3.5. Effects of Nitrogen Source on the Content of GA₃ and ZR in Potential Storage Root

The GA₃ content in the PSRs of both cultivars decreased with root growth (

Table 4. ZR and GA₃ contents in potential storage roots of sweet potato during storage root formation.

Days after Planting	Cultivars	Treatment	ZR (ng g−1 FW)	GA3 (ng g−1 FW)
14	S19	N0	8.39 ± 0.24 cd	6.92 ± 0.2 c
		AN	7.41 ± 0.46 e	8.36 ± 0.16 b
		XN	8.11 ± 0.11 d	7.31 ± 0.22 c
	J23	N0	9.21 ± 0.48 a	8.14 ± 0.23 b
		AN	8.53 ± 0.15 bc	9.28 ± 0.2 a
		XN	8.87 ± 0.11 ab	8.28 ± 0.28 b
21	S19	N0	10.55 ± 0.14 c	6.37 ± 0.31 c
		AN	10.01 ± 0.37 d	7.72 ± 0.28 b
		XN	10.27 ± 0.22 cd	6.8 ± 0.27 c
	J23	N0	12.69 ± 0.47 a	7.48 ± 0.18 b
		AN	11.92 ± 0.15 b	8.54 ± 0.2 a
		XN	12.47 ± 0.07 a	7.92 ± 0.13 b
28	S19	N0	9.06 ± 0.46 e	5.99 ± 0.24 ab
		AN	10.29 ± 0.13 bc	5.06 ± 0.04 c
		XN	9.34 ± 0.24 de	5.71 ± 0.04 b
	J23	N0	9.89 ± 0.47 cd	6.22 ± 0.18 a
		AN	11.4 ± 0.71 a	5.17 ± 0.27 c
		XN	10.62 ± 0.3 b	6.29 ± 0.26 a
35	S19	N0	7.43 ± 0.2 e	5.08 ± 0.36 a
		AN	9.09 ± 0.29 b	4.15 ± 0.13 b
		XN	7.95 ± 0.14 d	5.06 ± 0.14 a
	J23	N0	7.67 ± 0.3 e	5.11 ± 0.37 a
		AN	9.5 ± 0.05 a	4.41 ± 0.3 b
		XN	8.38 ± 0.42 c	5.18 ± 0.02 a
ANOVA analysis
14	Cultivars		571.07 **	281.73 **
	Treatment		24.67 **	62.85 **
	Cultivars × Treatment		1.29	0.86
21	Cultivars		108669 **	78.68 *
	Treatment		19.77 **	38.76 **
	Cultivars × Treatment		1.07	0.78
28	Cultivars		72.01 *	19.07 *
	Treatment		42.82 **	37.37 **
	Cultivars × Treatment		1.15	1.83
35	Cultivars		172.24 **	1.5
	Treatment		219.57 **	20.55 **
	Cultivars × Treatment		0.75	0.29

Note: N0, no nitrogen fertilizer; AN, ammonium nitrogen; XN, amide nitrogen. Split-Plot ANOVA, LSD. Values followed by lowercase letters within a column are significantly different among phosphorus treatments (p < 0.05). *—p < 0.05; **—p < 0.01.

). At 14–21 DAP, the GA₃ content in the PSRs receiving ANT was significantly (p < 0.05) higher than those of the control and amide-nitrogen treatments. At 28–35 DAP, the GA₃ content in the PSRs getting ammonium nitrogen was significantly (p < 0.05) lower than those of the control and amide-nitrogen treatments.

Table 4 also shows that the ZR content of the PSRs of both cultivars initially increased, then decreased, reaching a maximum at 21 DAP. At 14–21 DAP, the ZR content in the PSRs receiving ammonium nitrogen was significantly (p < 0.05) lower than those of the control and the XNTs. At 28–35 DAP, the ZR content in the PSRs getting ammonium nitrogen was significantly (p < 0.05) higher than those of the control and the XNTs.

3.6. Effects of Nitrogen Source on the Regulation of Lignin Biosynthesis Gene Expression in Potential Storage Roots

Figure 3 shows that at 14–28 DAP, the expression levels of Ibkn1 and Ibkn2 in the PSRs of both cultivars changed unimodally during their development. The expression levels peaked at 21 DAP. The expression level of IbEXP1 decreased with root development in both cultivars.

Nitrogen source influenced the expression levels of Ibkn1 and Ibkn2 in the PSRs. At 14–21 DAP, the expression levels of Ibkn1 and Ibkn2 in the PSRs receiving ANT were significantly (p < 0.05) lower than those of the control. At 28 DAP, the expression levels of Ibkn1 and Ibkn2 in the PSRs getting ammonium nitrogen were significantly (p < 0.05) higher than those of the controls. At 14–28 DAP, the expression levels of Ibkn1 and Ibkn2 in the PSRs under the XNT was similar with those of the control.

Nitrogen source affected the expression level of IbEXP1 in the PSRs. At 14–21 DAP, the expression level of IbEXP1 in the PSRs under the ANT was significantly (p < 0.05) higher than that of the control. At 28 DAP, the expression level of IbEXP1 in the PSRs treated with ammonium nitrogen was significantly (p < 0.05) lower than that of the control. At 14 DAP, the expression level of IbEXP1 in the PSRs of S19 receiving amide nitrogen was significantly (p < 0.05) higher than that of the control, whereas that of J23 getting XNT was similar to that of the control. At 21–28 DAP, the expression level of IbEXP1 in the PSRs under the XNT was similar with that of the control.

3.7. Correlation Analysis

At 14–35 DAP, in the correlation analysis of lignin content with GA₃ and ZR contents, the PAL and POD activities and the Ibkn1, Ibkn2, and IbEXP1 expression levels (

Table 5. Coefficients of correlation of GA₃ and ZR content; PAL and POD activity; Ibkn1, Ibkn2, and IbEXP1 expression with lignin content.

Cultivars	Items	Days after Planting
		14	21	28	35
S19	GA3	0.766 *	0.928 **	0.785 *	0.810 **
	ZR	−0.881 **	−0.905 **	−0.882 **	−0.760 *
	PAL	0.844 **	0.935 **	−0.839 **	—
	POD	0.719 *	0.877 **	−0.792 *	—
	Ibkn1	−0.800 **	−0.893 **	−0.844 **	—
	Ibkn2	−0.835 **	−0.910 **	−0.914 **	—
	IbEXP1	0.781 *	0.828 **	0.896 **	—
J23	GA3	0.982 **	0.942 **	0.926 **	0.865 **
	ZR	−0.786 *	−0.945 **	−0.876 **	−0.864 **
	PAL	0.983 **	0.946 **	0.912 **	—
	POD	0.975 **	0.954 **	0.929 **	—
	Ibkn1	−0.867 **	−0.960 **	−0.803 **	—
	Ibkn2	−0.960 **	−0.870 **	−0.799 **	—
	IbEXP1	0.950 **	0.938 **	0.898 **	—

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

) showed that the GA₃ content, PAL and POD activities, and IbEXP1 expression levels were significantly (p < 0.05) positively correlated with the lignin content, whereas the ZR content and Ibkn1 and Ibkn2 expression levels were significantly (p < 0.05) negatively correlated with the lignin content.

4. Discussion

4.1. Effects of Nitrogen Source on the Root Activity and the Number of Storage Roots

Previous studies have shown that, at the same nitrogen levels, ammonium nitrogen fertilizer resulted in a higher SR yield and nitrogen utilisation efficiency than XNT [12,33,40]. The present study demonstrated that nitrogen application during SR formation (15–30 DAP) significantly (p < 0.01) enhanced root activity and that treatment with ammonium nitrogen increased root activity more than the XNT. These findings are consistent with those reported by Shi et al. (2010) after canopy closure in sweet potato (40–120 DAP). At that time, the number and weight of PSRs under ANT were significantly higher than those of the control and the XNT. During sweet potato harvest, ammonium nitrogen significantly (p < 0.05) increased the number and yield of SRs. Our analysis indicated that ANT increases the number of SRs at harvest by promoting the growth of young roots and by increasing the number of PSRs. These effects ultimately increase yield as well.

4.2. Effect of Nitrogen Source on Lignin Content in Potential Storage Roots and Its Correlation with the Number of Storage Roots

Primary xylem maturation in young sweet potato roots generally occurs at 20–25 DAP. At that time, the vascular cambium is highly active. It grows inwards to form secondary xylem and outwards to produce secondary phloem. At 25–30 DAP, accessory cambium appears in the thin-walled stele tissue surrounding the primary xylem vessels. The accessory cambium forms mainly around the primary xylem. The high number of primary xylem vessels induces accessory cambium activity which, in turn, inhibits stele cell lignification and favors SR formation [3,4,5]. Lignin content is the most direct physiological indicator of sweet potato lignification [6]. A higher root lignin content facilitates water and nutrient transport [16]. The present study found that at 14–21 DAP, the lignin content of the PSRs under the ANT was significantly higher than those of the control and the amide-nitrogen treatment. At 28–35 DAP, the lignin content of the PSRs receiving ammonium nitrogen was significantly lower than those of the control and the amide-nitrogen treatment. Our analysis indicates that ammonium nitrogen promotes lignin biosynthesis in the PSRs during primary xylem development (early stage of SR formation). This effect enhances root activity, facilitates water and nutrient transport, and promotes the formation of accessory cambium. Inhibiting lignin biosynthesis in the PSRs after accessory cambium formation (late stage of SR formation) will promote accessory cambium activity which, in turn, facilitates SR formation and increases the number of SRs.

4.3. Effect of Nitrogen Source on the Activity of Key Enzymes of Lignin Synthesis in Potential Storage Roots and Their Correlation with Lignin Content

Lignin is synthesized by phenylpropanoid metabolism. PAL catalyzes the deamination of L-phenylalanine to trans-cinnamic acid. PAL is the first rate-limiting enzyme in the lignin biosynthesis pathway [7,8,9,10]. POD is the last key enzyme in this metabolic pathway. It catalyzes the decomposition of H₂O₂ to polymerize monolignols and form lignin [11]. The present study found that at 14 and 21 DAP, the PAL and POD activities under the ANT were significantly (p < 0.05) higher than those under the control and the XNT. At 28 DAP, the PAL and POD activities under the ANT were significantly (p < 0.05) lower than those under the control and the XNT. Correlation analysis indicates that the PAL and POD activities in the PSRs were significantly positively correlated with lignin content.

4.4. The Effect of Nitrogen Source on the GA and ZR Content in Potential Storage Roots and Their Correlation with Lignin Content

In their studies on tobacco and carrot, respectively, Biemelt et al. [15] and Wang et al. [16] found that GA promotes lignin accumulation and stele cell lignification. Endogenous ZR in young sweet potato roots is localized mainly in the primary cambium. ZR participates in the development and activation of the primary cambium in young roots as well as the formation and development of the accessory cambium [14]. Our results indicate that at 14–21 DAP, the GA₃ content in the PSRs receiving ammonium nitrogen was significantly higher than those of the control and the XNT. The ZR content showed the opposite trend. Correlation analysis indicates that in PSRs, GA₃ and lignin content is positively correlated, whereas the ZR and lignin content is negatively correlated.

4.5. Effects of Nitrogen Source on Lignin-Related Gene Expression in Potential Storage Roots and Their Correlation with Lignin Synthesis

Noh et al. [32] found that, compared to the wild type, IbEXP1-antisense sweet potato had significantly less lignification in its fibrous roots and significantly more SRs. Moreover, the transcription levels in the fibrous roots were significantly higher than those in the young SRs. The authors speculated that IbEXP1 restricted early SR thickening by promoting stele cell lignification and inhibiting cambium cell proliferation. In this way, IbEXP1 negatively regulated SR formation. KNOX1 proteins can positively regulate cytokinin synthesis [19,20,21] and negatively regulate GA synthesis [22,23,24]. The KNOX1 protein in Arabidopsis, BREVIPEDICELLUS (BP), controls cell differentiation by negatively regulating lignin biosynthesis [25]. Ibkn1, Ibkn2, and Ibkn3 are KNOX1 genes expressed in sweet potato roots. The expression levels of Ibkn1 and Ibkn2 were more than twice as high in young SRs than in fibrous roots. Ibkn3 showed various expression patterns in different sweet potato cultivars. Some authors have speculated that Ibkn3 may in fact be a pseudogene [26,27]. The present study found that at 14 and 21 DAP, the expression levels of Ibkn1 and Ibkn2 in PSRs under the ANT were significantly (p < 0.05) lower than those in the control and the XNT. The expression levels of IbEXP1 showed the opposite pattern. At 28 DAP, the expression levels of Ibkn1 and Ibkn2 in PSRs under the ANT were significantly (p < 0.05) higher than those in the control and the XNT. The expression levels of IbEXP1 showed the opposite pattern. Correlation analysis showed that in PSRs, the IbEXP1 expression level was positively correlated with lignin content, whereas the Ibkn1 and Ibkn2 expression levels were negatively correlated with lignin content. Our analysis indicates that, compared to the control and the XNT, the ANT significantly reduced the expression levels of Ibkn1 and Ibkn2 in the PSRs during the early stage of SR formation. This effect facilitated the increase in GA₃ content and decrease in ZR content, thereby promoting primary xylem development. During the late stage of SR formation, the ANT significantly (p < 0.05) increased the expression levels of Ibkn1 and Ibkn2 in the PSRs, thereby facilitating decreases in the GA₃ content and increases in the ZR content. In this way, stele cell lignification was inhibited in the PSRs, and the formation of more SRs was promoted. Our results corroborate the conclusion of Noh et al. [32] that IbEXP1 positively regulates lignin metabolism and therefore negatively regulates SR formation.

5. Conclusions

In this study, the possible response mechanism of the SR number and yield of sweet potato to nitrogen source was explained from the aspects of endogenous hormone concentration, lignin content, key enzymes of lignin biosynthesis and related regulatory genes in PSRs of sweet potato. It was proven that in the early stage of SR formation, ammonium nitrogen fertilization will promote primary xylem development in PSRs. In the late stage of SR formation, it will inhibit cell lignification in PSRs, facilitate SR formation, and increase the number of SRs per plant. Thus, it can be seen that nitrogen form is an important factor in regulating lignin biosynthesis in PSRs, in turn affecting the number and yield of SR of sweet potato. However, for safety reasons, nitrate treatment was not set up in this paper, which can be studied in the future, in order to enrich the theory and techniques of nitrogen management and cultivation to promote the formation and development of SRs and improve the yield of sweet potato.