Exogenous N Supply on N Transportation and Reuse during the Rice Grain-Filling Stage and Its Relationship with Leaf Color-Changing Parameters

Abstract

During the later reproductive period of rice growth, the chlorophyll in the leaves degraded, accompanied by the nitrogen (N) transportation from leaves to panicle, resulting in a change in leaf color from green to yellow. This study aimed to investigate the effects of exogenous N supply on leaf color-changing, N accumulation, N transportation, and N loss of indica-japonica hybrid rice during the grain-filling stage. Two indica-japonica hybrid rice cultivars, Chunyou 167 (CY167) and Chunyou 927 (CY927), which exhibited significant differences in leaf color-changing during the grain-filling stage, were selected as materials for field experiment and hydroponic experiment with low, medium, and high N treatments (LN, MN, and HN). The dynamic changes in SPAD value from heading to maturity were measured and fitted with quadratic function to extract leaf color-changing parameters; labeled ¹⁵N was used as N source after heading to trace the source of N in the panicle and the remobilization of vegetative organ N. The results showed that 67.37–72.38% of the panicle N was transported from vegetative organs, the N transport efficiency was the upper three leaves > lower leaves > stem, and about 3.1–35.0% of the transported N was lost via volatilization. The effects of exogenous N concentration on N harvest index, N dry matter/grain production efficiency, N reuse efficiency, and N loss were closely related to leaf color-changing parameters. In MN and HN treatment, the N loss was negatively correlated with the onset time of leaf color-changing (T₀) and the final leaf color index (CI_f), but positively correlated with the leaf color-changing rate (R_mean). Increasing the supply of exogenous N increased T₀ and CI_f, but decreased R_mean, N transport/reuse efficiency, N harvest index, and N dry matter/grain production efficiency. Compared to the cultivar CY167 with normal leaf color-changing, the “stay-green” cultivar CY927 had higher T₀, CI_f, and lower R_mean, resulting in less N volatilization loss, lower N harvest index and N transport efficiency, while higher N reuse efficiency. In conclusion, the exogenous N supply affects leaf color by influencing the transportation and reuse of leaf N during the grain-filling stage.

1. Introduction

Nitrogen (N) is a crucial nutrient element that affects the growth and development of crops, and its application rate directly influences the rice dry matter and N accumulation [1]. However, the unreasonable application of N has the adverse effect of causing environmental pollution [2,3]. Therefore, it is essential to develop high-yielding and highly efficient rice production combined with green ecological sustainability. During the later reproductive period of rice, chloroplasts in the leaves degrade, causing the leaf color to gradually turn from green to yellow. The leaves’ color-changing is a typical characteristic of plant aging, which leads to a decrease in the photosynthetic capacity of leaves [4]. Over the years, rice production aimed at achieving high yields and delayed rice leaf color-changing in the later reproductive period through postponing N fertilizer application. This was implemented to extend the duration of the photosynthetic function of the plants, thereby increasing the production and accumulation of photosynthetic substances during the rice grain-filling stage. However, the rice panicle N mainly originates from the translation and reuse of N in vegetative organs, particularly the leaves. Excessively delayed leaf color-changing significantly reduced the N supply to the grains and affected N use efficiency [5,6]. It is found that in rice production, rice cultivars with yellow leaves and green stems at maturity tend to have relatively full grains and a high N harvest index (NHI). In rice breeding, the ability to undergo a favorable leaf color-change during the grain-filling stage has become an advantage of new cultivars. Nevertheless, due to the lack of quantitative indicators to measure and evaluate leaf color-changing characteristics, leaf color-changing is still a relatively vague concept, and there is currently no report on the relationship between leaves’ color-changing and N transportation from vegetative organs to grains during the process of grain-filling.

During rice growth, some N is released from the plant into the atmosphere as a gas, including ammonia (NH₃), N dioxide (NO₂), and nitrous oxide (N₂O). This process is collectively referred to as N volatilization. It was demonstrated that the rice canopy is capable of absorbing and releasing NH₃ [7]. It is generally accepted that well-developed plants are NH₃ absorbers, however, they are transformed into NH₃ releasers when there is an excess of NH_x-N in the plant; especially in the later reproductive period, a considerable amount of N is lost from the aboveground parts of the plant in the form of NH₃ [8]. We hypothesized that aboveground N volatilization in rice plants during the later reproductive stage is related to leaves’ color-changing and extensive degradation of the light-harvesting chlorophyll-protein complex (LHC). However, the specific relationship between leaves’ color-changing, vegetative organs N, and aboveground N loss is unknown, and the effect of aboveground N loss on N use efficiency (NUE) is not understood.

The effects of N fertilizer application rate on dry matter and N accumulation and transportation in rice have been investigated in many studies [9,10,11,12,13], but the results are inconclusive, which may be related to the difference in experimental design, including soil fertility, cultivars, fertilizer and water management. The previous studies on N mobilization of vegetative organs mainly adopted the “apparent remobilization method” (calculating the difference in N accumulation between anthesis and harvest in different plant parts) [14], but this method has a significant margin of error. In comparison, ¹⁵N isotope tracing represents a viable alternative, facilitating the estimation of vegetative organs N remobilization and reuse during the grain-filling stage with reduced bias and enhanced precision [15]. In this experiment, two indica-japonica hybrid rice cultivars exhibiting different leaf color at maturity were selected as materials for a field experiment and ¹⁵N isotope tracing hydroponic experiment, the objective was to investigate the effects of exogenous N supply on leaves’ color-changing and on the N accumulation and N transportation during the rice grain-filling stage.

2. Materials and Methods

2.1. Experimental Design

The experiment was conducted in 2023 at the China National Rice Research Institute (CNRRI) (Hangzhou, Zhejiang: 120.2° E, 30.3° N, 11 m above sea level), and was divided into 2 parts: a field experiment and a hydroponic experiment. Two representative indica-japonica hybrid rice cultivars (Chunyou 167 and Chunyou 927) with similar characteristics of plant type, plant height, leaves position and leaves size, but with obvious differences in leaves’ color-changing characteristics, were selected as experimental materials. Chunyou 167 (CY167) showed obvious color-changing of functional leaves during the grain-filling stage, its flag leaves and inverted 2 leaves (2nd leaves) were yellowish-green at maturity. Chunyou 927 (CY927) showed a slower color-changing of functional leaves during the grain-filling stage, its flag leaves and 2nd leaves and other functional leaves were “stay-green” at maturity.

2.1.1. N Fertilizer Treatment in the Field

The experimental field was a blue purple soil with oilseed rape as a previous crop. The 0–20 cm tillage layer of soil contained 3.46% organic matter, 0.21% total N, 1.58% total K, 0.06% total P, available N of 156.96 mg kg⁻¹, available P of 38.05 mg kg⁻¹, available K of 77.13 mg kg⁻¹, and pH of 6.82. The field experiment was conducted in a split-plot design, with N application as the main plot and cultivar as the split-plot. Three N fertilizer dosage treatments, 0 kg ha⁻¹ (LN), 150 kg ha⁻¹ (MN), and 300 kg ha⁻¹ (HN), were set up and the N fertilizer was applied in the form of granular urea 3 times in a ratio of basal-N: tiller-N: panicle-N as 4:3:3. Each treatment was replicated 3 times, with a plot of 60 m² and a split-plot area of 30 m². The plots were separated by ridges and the ridges were covered with plastic film to ensure separate drainage and irrigation between treatments. Rice was transplanted on 13 June 2023, at a planting density of 20.0 cm × 25.0 cm, with 2 plants per hill. All treatments (including the no N fertilizer treatment) were applied with the same amount of superphosphate (12% P₂O₅) and potash (K₂O) at 97.5 kg ha⁻¹ and 189 kg ha⁻¹, respectively. Phosphorous was applied as a one-time basal application before transplanting, and potassium was used in two application doses: 50% as a basal dressing and the remaining 50% top dressed at panicle initiation stage. The field was flooded after transplanting, and a depth of 1 to 4 cm of flooding water was maintained until 7 days before maturity except for drainage at the maximum tillering stage to reduce unproductive tillers. Pests and diseases were intensively controlled with chemicals to avoid biomass and yield losses.

2.1.2. ¹⁵N Fertilizer Treatment in Hydroponics

Three distinct N concentration levels were set in hydroponic experiments: 0.17 mmol L⁻¹ (LN), 0.54 mmol L⁻¹ (MN), and 1.62 mmol L⁻¹ (HN), with 2 cultivars, for a total of 6 treatments with 3 replications per treatment; each replication was a hydroponic tank 2.0 m in length, 1.0 m in width, and 0.5 m in height. The hydroponic tank was placed in a plastic greenhouse to prevent rain interference, filled with nutrient solution to a height of 40 cm. Perforated foam boards were then placed on top of the solution, with holes of 3 cm diameter and 10 cm spacing between holes. Rice seedlings were transplanted on 8 June 2023 and fixed in the holes of foam board with sponge with 2 seedlings per hole. The formulation of the nutrient solution was referred to as Kimura b [16], and unlabeled (NH₄)₂SO₄ was used as N source before heading, while ¹⁵N-labeled (NH₄)₂SO₄ (10.09 atomic % ¹⁵N, Shanghai Research Institute of Chemical Industry, Shanghai, China) was used as N source after heading. The pH was adjusted to 5.5–6.0 with HCl/NaOH every 2 days. Pest and weed control were managed according to local high-yield rice production.

2.2. Measurement and Analysis

2.2.1. SPAD Value in the Leaves

SPAD values of 10 representative flag leaves were measured at 5-day intervals from flowering to maturity using a chlorophyll meter (Konica Minolta Co., Tokyo, Japan) in three replications, with the measurement site being the upper 1/3 of the leaf blade, avoiding the leaf vein.

2.2.2. Chlorophyll Content in the Leaves

We measured the chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of flag leaves via the 95% ethanol extraction method [17].

2.2.3. Leaf Color-Changing Parameters

To describe the dynamics of changes in rice leaves color with days (d) during heading to maturity, and to eliminate the differences in leaves color among cultivars, the degree of leaves color-changing was normalized to a color index (CI) using the following formula:

CI = SPAD value at n days after heading (DAF n)/SPAD value at heading

(1)

The larger the value of CI, the smaller the degree of leaf color-changing. With CI as the dependent variable and days after heading as the independent variable, according to the methods of Chang et al. [18], the quadratic function CI = at² + bt + c, was used to simulate leaf color-changing dynamics, and four parameters related to color change were extracted, including leaf color-changing onset time (T₀), final leaf color index (CI_f), leaf color-changing duration (T₁₀₀), and the mean rate of the leaf color-changing (R_mean, Figure 1).

2.2.4. Plant N Content and N Accumulation

Five hills of plants were sampled randomly from each subplot at heading and maturity, and divided into 4 parts: stem plus sheath (stem), upper 3 leaf blades (upper 3 leaves), lower leaf blades (lower leaves), and panicle. The dry weight of each part was determined after oven-drying at 105 °C for 30 min and then at 80 °C for 48 h to achieve a constant weight. Plant dry weight at heading and maturity was calculated as the sum of the weights of all four parts. The N content in the leaves, stem and panicle was determined using the Kjeldahl digestion and steam distillation methods, and the N accumulation of stems, leaves, and panicles was the product of their dry matter accumulation and N content, respectively.

2.2.5. Plant N Transportation and N Use Efficiency (NUE) Calculations

In the hydroponics experiment, ¹⁴N and ¹⁵N were used as the N source before and after heading. N content was determined via the Kjeldahl digestion and steam distillation methods [19]. And a suitable quantity of the absorbent from the aforementioned reaction was reacted with sodium hypobromite (NaBrO) solution under high vacuum. The N₂ produced was then directly introduced into the MAT-271 gas isotope mass spectrometer (Thermo Finnigan, San Jose, CA, USA) for the detection of ¹⁵N isotope abundance. The ionization source was an electron bombardment type (EI) with a Faraday cup detector. The instrument’s parameters were set as follows: ion source emission current of 0.04 mA, electron energy of 98.60 eV, trap voltage of 133.15 V, ion source temperature of 160 °C, and high voltage of 9 kV. The absolute ratios of gas isotopes were determined using mass spectral peak height ratios. And plant percentage of ¹⁵N derived from fertilizer (Ndf) was calculated as follows [19]:

Ndf (%) = (a − b)/(c − d)

(2)

where ‘a’ is the ¹⁵N atom% in the plant sample; ‘b’ is the ¹⁵N atom% in the control sample from unlabeled nutrient culture; ‘c’ is the atom% in (NH₄)₂SO₄; and ‘d’ is the natural ¹⁵N atom % in air (0.3664 ¹⁵N atom %).

The plant total ¹⁵N accumulation after heading, N transportation, and N reuse related indicators were calculated as follows:

¹⁵N accumulation after heading (mg hill⁻¹) = N accumulation at maturity × Ndf/100

(3)

where ¹⁵N accumulation of upper 3 leaves, lower leaves, stems, and panicle was calculated respectively, and ¹⁵N accumulation of vegetative organs was the sum of the upper 3 leaves, lower leaves, stems, and panicle.

Transported N (mg hill⁻¹) = N accumulation at heading − (N accumulation at maturity − ¹⁵N accumulation after heading)

(4)

where the transported N of the upper 3 leaves, lower leaves, and stems was calculated respectively, and the total transported N was the sum of the upper 3 leaves, lower leaves, and stems.

Total effective transported N (mg hill⁻¹) = panicle N accumulation at maturity − panicle N accumulation at heading − panicle ¹⁵N accumulation after heading

(5)

N effective transportation percentage (%) = Total effective transported N/Total transported N × 100

(6)

N loss (mg hill⁻¹) = Total transported N − Total effective transported N

(7)

N loss rate (%) = N loss/N accumulation at heading_{(leaves+stems)} × 100

(8)

N transport efficiency (%) = transported N/N accumulation at heading × 100

(9)

where N transport efficiency of upper 3 leaves, lower leaves, and stems were calculated respectively, and the total N transport efficiency was the N accumulation_{(leaves+stems)}/N accumulation_{(leaves+stems)} at heading.

N reuse efficiency (%) = Total effective transported N/N accumulation_{(leaves+stems)} at heading

(10)

N harvest index (%) = Panicle N accumulation at maturity/N accumulation_{(leaves+stems+panicle)} at maturity × 100

(11)

N grain production efficiency (g grain g⁻¹) = Yield/N accumulation_{(leaves+stems+panicle)} at maturity

(12)

N dry matter production efficiency (g grain g⁻¹) = Dry matter accumulation at maturity/N accumulation at maturity

(13)

2.2.6. Yield and Its Components

At maturity, the effective panicles of 20 rice hills were counted, and 5 hills that had average effective panicle numbers were sampled from each subplot. The grains of the sampled plants were threshed and classified into filled and unfilled grains by submerging them into tap water, and unfilled grains were further classified into empty and half-filled grains via winnowing. The number of filled, half-filled, and empty grains was determined using a yield trait scorer (YTS-4, Green Pheno Technology Co., Ltd., Wuhan, Hubei, China). The grain yield was determined from harvesting 50 hills away from the side rows in each plot and was converted to 14.5% moisture content.

2.3. Statistical Analysis

Statistical analyses were performed using Microsoft Excel 2021 and SAS version 9.2. A two-factor split-plot analysis of variance was conducted for all of the above-mentioned parameters from 3 replicates. The Duncan’s tests were used in the analysis of variance to detect significant differences among the mean values for different treatments at p < 0.05. Correlation analysis and graph plotting were performed using Origin 2021 and GraphPad 9.5.

3. Results

3.1. Effect of Exogenous N Supply on Leaf Color-Changing and Its Varietal Differences

3.1.1. Leaf Chlorophyll Content

As shown in Figure 2 and

Table 1. Chlorophyll content of flag leaf of two indica-japonica hybrid rice cultivars at the heading stage and maturity stage under different N levels.

Cultivar	Treatment	Flag Leaf Chlorophyll Content (mg g−1)
		Field Experiment		Hydroponic Experiment
		At Heading	At Maturity	At Heading	At Maturity
CY167	LN	2.94 ± 0.02 d	0.82 ± 0.03 e	3.06 ± 0.09 c	0.20 ± 0.01 e
	MN	3.07 ± 0.10 cd	1.49 ± 0.05 d	3.42 ± 0.09 b	0.41 ± 0.02 d
	HN	3.18 ± 0.15 bcd	1.53 ± 0.02 cd	3.45 ± 0.26 b	1.41 ± 0.04 b
CY927	LN	3.31 ± 0.26 bc	1.57 ± 0.07 c	3.06 ± 0.09 c	0.27 ± 0.01 e
	MN	3.45 ± 0.18 ab	2.04 ± 0.05 b	3.42 ± 0.09 b	0.66 ± 0.07 c
	HN	3.71 ± 0.24 a	2.34 ± 0.07 a	3.89 ± 0.08 a	2.84 ± 0.13 a

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

, a gradual decline in SPAD value was observed from heading to maturity, exogenous N supply increased both the chlorophyll content and SPAD value of flag leaves at heading and maturity, slowed down the rate of SPAD value decline, and prolonged the duration of the grain-filling stage. The grain-filling stage of both cultivars lasted 40 d in LN treatment, 43–45 d in MN treatment, and up to 50 d in HN treatment. The SPAD value of flag leaves was HN > MN > LN, with an average SPAD of 16.58 to 18.39% higher in MN than in LN, and an increase of 25.75 to 29.69% in HN compared to LN. Compared with cultivar CY167, CY927 is a cultivar with dark green leaf color, and had significantly higher leaf SPAD values during the grain-filling stage.

3.1.2. Leaf Color-Changing Parameters

The leaf color-changing characteristic parameters of the two cultivars were extracted as shown in

Table 2. Differences in the characteristic parameters of leaf color-changing between the two cultivars under different N treatments.

Cultivar	Treatment	T0 (d)	T100 (d)	Rmean (1 × 10−3 d−1)	CIf
CY167	LN	3.7 ± 0.3 e	36.3 ± 0.3 c	15.5 ± 0.1 a	0.35 ± 0.01 e
	MN	5.5 ± 0.4 d	37.5 ± 0.4 b	11.8 ± 0.1 b	0.45 ± 0.01 d
	HN	11.2 ± 1.2 b	38.8 ± 1.2 a	9.9 ± 0.2 c	0.59 ± 0.04 b
CY927	LN	6.1 ± 0.8 d	34.0 ± 0.8 d	12.5 ± 1.1 b	0.52 ± 0.04 c
	MN	8.4 ± 0.3 c	36.6 ± 0.3 c	9.4 ± 0.1 c	0.62 ± 0.01 b
	HN	13.3 ± 0.4 a	36.8 ± 0.4 bc	6.3 ± 0.1 d	0.77 ± 0.01 a

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

.

In terms of leaf color-changing onset time (T₀), the cultivar CY927 with dark green leaves exhibited a delayed leaf color-changing onset time T₀ than CY167, and was delayed by 2.4, 2.9, and 2.1 d in LN, MN, and HN treatments, respectively. However, the leaf color-changing duration (T₁₀₀) of CY167 was 1.7–2.3 d longer than CY927. Exogenous N supply delayed T₀ and prolonged T₁₀₀, but the delaying effect on T₀ was significantly greater than the prolonging effect on T₁₀₀. Compared with LN, HN delayed T₀ by 7.2–7.5 d, but only prolonged T₁₀₀ by 2.5–2.8 d. No significant difference was observed in the effect of exogenous N supply on the leaf color-changing time T₀ and T₁₀₀ between cultivars.

In terms of leaf color-changing degree, the leaf color index at maturity (CI_f) of the rive cultivar CY927 with dark green leaves was significantly higher than that of CY167 by 48.57%, 37.78%, and 30.51%, in LN, MN, and HN treatments, respectively. Exogenous N supply significantly increased the CI_f. Compared to LN, the CI_f of CY167 increased by 28.57% and 68.57% in MN and HN, respectively, while for CY927, the increases were 19.23% and 48.08%, respectively; the increase in CI_f by exogenous N supply was greater in CY167 than in CY927.

In terms of leaf color-changing rate, cultivar CY167 had faster leaf color-changing rate than CY927, and the mean leaf color-changing rate (R_mean) in LN, MN, and HN were 24.00%, 25.53%, and 57.14% faster, respectively, with significant differences between cultivars. Exogenous N supply significantly reduced the R_mean of both cultivars, the R_mean of CY167 was decreased by 23.87% and 36.13% in MN and HN treatments, respectively, compared to LN, CY927 was reduced by 25.53% and 57.14%, respectively. Notably, exogenous N supply had a greater effect on the color-changing rate of CY927 than CY167, especially under HN treatment.

3.2. Effects of Exogenous N Supply on N Accumulation and N Transportation during the Grain-Filling Period

As the concentration of exogenous N increased, there was a significant increase in the N content and dry matter accumulation in various organs (leaves, stem, panicle) of both cultivars at both the heading stage and maturity stage, showing HN > MN > LN (Tables S1 and S2). There were no significant differences in N accumulation between cultivars at heading, while at maturity, only the LN level showed no significant differences between cultivars, both the MN and HN treatments were significantly higher in CY927 than in CY167 (

Table 3. N accumulation and N loss in 2 rice cultivars at full heading stage and maturity.

Cultivar	Treatment	At Heading	During Grain-filling	At Maturity
		N Accumulation (mg hill−1)	15N Uptake (mg hill−1)	N Accumulation (mg hill−1)
CY167	LN	175.78 ± 3.22 c	12.76 ± 0.17 f	185.70 ± 3.01 e
	MN	278.66 ± 12.83 b	20.25 ± 1.41 d	254.71 ± 16.82 d
	HN	498.60 ± 21.14 a	44.35 ± 0.66 b	440.72 ± 8.76 b
CY927	LN	191.11 ± 14.10 c	14.47 ± 0.18 e	189.22 ± 4.57 e
	MN	285.94 ± 6.73 b	23.53 ± 0.91 c	284.89 ± 9.70 c
	HN	501.79 ± 13.46 a	47.50 ± 1.35 a	492.40 ± 13.10 a

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

). Further ¹⁵N tracing experiment found the ¹⁵N accumulation after heading accounted for 6.87–10.06% of the plant N accumulation at maturity. The ¹⁵N accumulation increased significantly with the increase in the exogenous N supply, with MN increasing by 58.7–62.6% compared to LN and HN increasing by 101.87–119.01% compared to MN. There were significant differences in ¹⁵N accumulation between cultivars, and CY927 was significantly higher than CY167 in all N treatment.

Further divided rice plants into four parts: the upper three leaves, lower leaves, stem, and panicle, to analyze the changes in N accumulation in different parts during the grain-filling stage, as shown in

Table 4. N accumulation and transportation in leaves and stems during grain-filling period.

Position	Cultivar	Treatment	At Heading	During Grain-Filling		At Maturity	N Transport Efficiency (%)
			N Accumulation (mg hill−1)	N Transportation (mg hill−1)	15N Uptake (mg hill−1)	N Accumulation (mg hill−1)
Upper leaves	CY167	LN	60.04 ± 1.99 c	46.25 ± 1.74 e	0.49 ± 0.09 e	14.28 ± 0.68 e	77.02 ± 0.85 b
		MN	85.58 ± 6.20 b	67.23 ± 5.43 c	0.66 ± 0.02 d	19.01 ± 0.77 d	78.52 ± 0.65 a
		HN	142.30 ± 14.22 a	102.28 ± 11.00 a	2.45 ± 0.17 b	42.46 ± 3.44 b	71.84 ± 1.03 c
	CY927	LN	62.68 ± 4.79 c	44.54 ± 3.75 e	0.69 ± 0.03 d	18.83 ± 1.03 d	71.09 ± 0.58 c
		MN	86.09 ± 3.73 b	59.60 ± 3.06 d	1.11 ± 0.03 c	27.60 ± 0.70c	69.21 ± 0.64 d
		HN	140.96 ± 4.98 a	95.14 ± 3.80 b	2.66 ± 0.04a	48.49 ± 1.19 a	67.48 ± 0.34 e
Lower leaves	CY167	LN	29.64 ± 0.63 c	17.88 ± 0.68 d	0.25 ± 0.02 e	12.01 ± 1.16 d	60.37 ± 3.19 d
		MN	62.60 ± 2.28 b	45.55 ± 1.60 c	0.41 ± 0.03 d	17.46 ± 1.16 c	72.77 ± 1.27 a
		HN	128.35 ± 8.39 a	87.83 ± 6.28 a	1.44 ± 0.06 a	41.97 ± 2.16 a	68.41 ± 0.59 b
	CY927	LN	30.92 ± 2.86 c	19.20 ± 2.17 d	0.27 ± 0.02 e	11.99 ± 0.73 d	62.00 ± 1.49 d
		MN	62.20 ± 4.13 b	40.94 ± 3.45 c	0.55 ± 0.01 c	21.80 ± 0.68 b	65.77 ± 1.23 bc
		HN	123.94 ± 6.37 a	80.89 ± 4.83 b	1.33 ± 0.04 b	44.38 ± 1.85 a	65.25 ± 0.82 c
Stem	CY167	LN	55.43 ± 2.02 d	27.22 ± 1.37 d	0.98 ± 0.01 e	29.19 ± 0.71 d	49.09 ± 0.80 d
		MN	91.08 ± 5.73 c	60.74 ± 4.59 b	1.34 ± 0.05 cd	31.68 ± 1.93 cd	66.66 ± 1.69 a
		HN	171.20 ± 4.70 b	101.57 ± 2.73 a	5.41 ± 0.34 b	75.03 ± 6.82 b	59.38 ± 2.95 b
	CY927	LN	67.31 ± 6.23 d	40.73 ± 4.37 c	1.16 ± 0.03 de	27.74 ± 2.12 d	60.45 ± 1.52 b
		MN	102.86 ± 2.24 c	67.11 ± 2.63 b	1.65 ± 0.04 c	37.39 ± 1.58 c	65.26 ± 2.82 a
		HN	186.09 ± 13.22 a	100.04 ± 8.72 a	5.96 ± 0.38 a	92.00 ± 6.22 a	53.74 ± 2.03 c

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

:

The N accumulation in the upper three leaves, lower leaves, and stem at both full heading and maturity stages, as well as transported N during grain-filling stage, all significantly increased with higher exogenous N supply. The N transport efficiency in the upper and lower leaves was 67.49–77.03% and 60.32–72.76%, respectively, both significantly higher than that of the stem (49.11–66.69%). In cultivar CY927, except for the N transport efficiency of LN > MN > HN in the upper three leaves, MN had the highest N transport efficiency in the lower leaves and stems. In cultivar CY167, the N transport efficiency of the upper leaves, lower leaves, and stem was highest in MN. The newly added ¹⁵N accumulation in the leaves and stems after heading only accounted for 0.90–2.80% of the leaf N accumulation and 2.21–5.96% of the stem N accumulation at maturity, indicating that this fraction of N had a minor impact on changes in plant N accumulation during grain-filling stage.

In terms of leaf N accumulation, there was no significant difference between cultivars in both the upper leaves and lower leaves at heading. In the LN treatment, the two cultivars had similar leaf-transported N amounts, but with the exogenous N supply increased, the increase in leaf-transported N in CY167 was significantly greater than that in CY927, especially in the upper leaves, where the N transport efficiency of the upper leaves of CY927 was significantly lower than that of CY167. In addition, CY927 accumulated more ¹⁵N in the upper leaves during the grain-filling stage compared to CY167. Therefore, at maturity, the N accumulation in the upper leaves was significantly lower in CY167 compared to CY927 in all N treatments. However, the differences in N accumulation between the two cultivars were less pronounced in the lower leaves, with only CY927 having a higher N accumulation than CY167 in MN treatment at maturity.

In terms of stem N accumulation, there was no significant difference in the stem N accumulation between cultivars in LN and MN treatments at both the heading and maturity stages, but CY927 was significantly higher than CY167 in the HN treatment. The stem-transported N of CY927 was significantly higher than that of CY167 in LN treatment, but as exogenous N supply increased, the difference between cultivars decreased, while the trend for stem ¹⁵N accumulation was the opposite. There was no significant difference in stem ¹⁵N accumulation between the cultivars in the LN treatment, but the differences between the cultivars increased as exogenous N supply increased, the stem ¹⁵N accumulation of CY927 was significantly higher than that of CY167 in the HN treatment.

Panicle N accumulation increased with the exogenous N supply, and no significant differences were observed between cultivars in both LN and MN treatments at both the heading and maturity stages. However, in the HN level, the panicle N accumulation at heading was opposite to that at maturity, and the order of panicle N accumulation changed from CY167 > CY927 at heading to CY167 < CY927 at maturity (

Table 5. N accumulation in panicle during grain-filling stage.

Cultivar	Treatment	At Heading	During Grain-Filling		At Maturity
		N Accumulation (mg hill−1)	Effective Transported N (mg hill−1)	15N Accumulation (mg hill−1)	N Accumulation (mg hill−1)
CY167	LN	30.67 ± 0.37 d	88.51 ± 3.97 d	11.03 ± 0.23 f	130.21 ± 3.81 d
	MN	39.40 ± 3.35 c	129.32 ± 16.91 c	17.84 ± 1.33 d	186.56 ± 14.49 c
	HN	56.75 ± 2.57 a	189.46 ± 3.11 b	35.04 ± 0.17 b	281.26 ± 2.49 b
CY927	LN	30.19 ± 0.48 d	88.1 ± 2.71 d	12.35 ± 0.12 e	130.65 ± 2.21 d
	MN	34.79 ± 2.90 cd	143.06 ± 8.97 c	20.24 ± 0.84 c	198.09 ± 9.92 c
	HN	50.80 ± 4.68 b	219.17 ± 11.55 a	37.55 ± 1.08 a	307.53 ± 10.28 a

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

). Further analyses revealed that this was related to the higher ¹⁵N uptake in CY927 compared to CY167 under HN, but more importantly, it was due to the significantly higher effective transported N in the panicle of CY927 compared to CY167 in the HN treatment.

3.3. Effect of Exogenous N Supply on N Transport Efficiency and N Use Efficiency

As shown in Table 5 and Figure 3, during the grain-filling stage, the total transported N was much greater than the total effective transported N in the panicle, with approximately 2.84 mg hill⁻¹ to 102.22 mg hill⁻¹ of N lost during transport, accounting for approximately 1.62–20.59% of the plant N accumulation at heading. With increasing exogenous N supply, the total transported N in both cultivars progressively increased, but the N effective transportation percentage and N reuse efficiency kept decreasing, while the N loss rate kept increasing. Comparing the N transport, N loss and reuse between the two cultivars, it was found although CY167 had higher transported N than CY927 in all N treatments, and the differences between cultivars were significant at LN and HN treatments. However, the N loss of CY927 in MN and HN treatments was significantly lower than CY167 by 44.39% and 43.78%, CY927 had a significantly higher N reuse efficiency.

There was no significant difference in yield between cultivars CY167 and CY927 (

Table 6. Effect of exogenous N supply on rice grain yield, dry matter accumulation, and N use efficiency (field experiment).

Cultivar	Treatment	Yield	Dry Matter Accumulation	N Harvest Index (%)	N Grain Production Efficiency	N Dry Matter Production Efficiency
		(kg ha−1)	DMA (kg ha−1)	NHI (%)	NGPE (kg grain kg−1)	NDMPE (kg grain kg−1)
CY167	LN	6595.0 ± 303.4 b	15,396.43 ± 315.75 e	68.83 ± 1.58 a	53.36 ± 1.82 a	112.61 ± 5.69 a
	MN	10,616.5 ± 307.5 a	18,648.21 ± 1075.35 d	66.70 ± 0.48 ab	49.89 ± 1.93 ab	92.52 ± 1.94 c
	HN	10,935.5 ± 488.2 a	23,952.32 ± 572.96 a	63.91 ± 0.90 c	45.06 ± 0.99 c	88.01 ± 2.13 c
CY927	LN	6932.1 ± 196.5 b	14,745.85 ± 521.40 e	64.77 ± 2.04 bc	49.56 ± 0.98 b	100.34 ± 3.57 b
	MN	10,540.9 ± 577.4 a	20,086.76 ± 373.44 c	64.66 ± 1.88 bc	48.78 ± 3.36 b	92.67 ± 3.77 c
	HN	10,382.8 ± 383.5 a	22,814.41 ± 210.66 b	56.35 ± 1.91 d	39.35 ± 0.58 d	79.75 ± 4.50 d

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

and

Table 7. Effect of exogenous N supply on rice grain yield, dry matter accumulation, and N use efficiency (hydroponic experiment).

Cultivar	Treatment	Yield	Dry Matter Accumulation	N Harvest Index	N Grain Production Efficiency	N Dry Matter Production Efficiency
		(g hill−1)	(g hill−1)	NHI (%)	NGPE (g grain g−1)	NDMPE (g grain g−1)
CY167	LN	13.55 ± 0.35 c	25.47 ± 0.47 c	64.17 ± 1.05 ab	72.96 ± 2.54 a	137.19 ± 3.54 a
	MN	16.11 ± 0.84 b	29.25 ± 1.09 b	66.17 ± 1.53 a	63.28 ± 1.69 b	114.99 ± 3.82 b
	HN	18.02 ± 1.36 a	34.05 ± 1.48 a	55.88 ± 0.86 c	40.90 ± 3.46 d	77.30 ± 4.10 d
CY927	LN	12.48 ± 0.15 c	25.18 ± 0.38 c	62.52 ± 0.44 b	65.96 ± 2.07 b	133.16 ± 4.16 a
	MN	14.72 ± 0.38 b	28.40 ± 0.38 b	62.40 ± 1.49 b	51.73 ± 2.93 c	99.78 ± 4.60 c
	HN	17.67 ± 1.25 a	35.22 ± 0.81 a	54.82 ± 0.97 c	35.87 ± 1.72 e	71.51 ± 1.28 d

Different lowercase letters indicate that the data of different treatments in the same column are significantly different at p < 0.05.

). Both cultivars responded similarly to exogenous N supply, with comparable yields in HN and MN, which were both significantly higher than LN. Dry matter accumulation increased with increasing N levels, with HN > MN > LN. The difference in dry matter accumulation between cultivars was not significant in the hydroponic experiment, however, in the field experiment, only LN treatment showed no significant difference between cultivars, while the dry matter accumulation of CY927 was significantly higher than that of CY167 in MN, and the value of CY167 was significantly higher than that of CY927 in HN. The N harvest index (NHI), N grain production efficiency (NGPE), and N dry matter production efficiency (NDMPE) all decreased with increasing exogenous N supply, and CY167 had significantly higher NHI, NGPE, and NDMPE than CY927 at the same N treatment.

3.4. Correlation Analysis of Leaves’ Color-Changing Parameters with N Transport and Use Efficiency

Correlation analysis was conducted among leaf color-changing parameters onset time (T₀), duration (T₁₀₀), rate (R_mean), final leaf color index (CI_f), N transport efficiency (NTE), N reuse efficiency (NRE), N harvest index (NHI), N grain production efficiency (NGPE), and N dry matter production efficiency (NDMPE) as shown in Figure 4.

(1)

There was a highly significantly negatively correlation between leaf color-changing onset time T₀ and leaf color-changing rate R_mean (R = −0.92, p < 0.01), as well as a highly significantly positive correlation between T₀ and the final leaf color index CI_f (R = 0.92, p < 0.01).

(2)

The stem, leaf and total N transportation, effective N transportation, and N loss were all highly significantly and positively correlated with T₀, T₁₀₀, and CI_f (R = 0.55–0.92, p < 0.05), and significantly negatively correlated with R_mean, (R = −0.87–−0.55, p < 0.05).

(3)

There were no significant correlations between leaf color-changing parameters and stem N transport efficiency, but there were significant correlations between leaf color-changing parameters and leaf N transport efficiency, and the correlation differed between leaf positions. The N transport efficiency of the upper leaves was significantly negatively correlated with T₀ and CI_f (R = −0.87 to −0.53, p < 0.05), and significantly positively correlated with R_mean (R = 0.54–0.81, p < 0.05). The N transportation efficiency of the lower leaves demonstrated a significant positive correlation with T₁₀₀ (R = 0.61, p < 0.01), but was not significantly correlated with T₀, CI_f, and R_mean.

(4)

N harvest index, N grain production efficiency, N dry matter production efficiency, and N reuse efficiency were all significantly negatively correlated with T₀ and CI_f (R = −0.96–−0.48, p < 0.05), and significantly positively correlated with R_mean (R = 0.56–0.91, p < 0.05). T₁₀₀ was significantly negatively correlated with N grain production efficiency, N dry matter production efficiency, and N reuse efficiency (R = −0.59–0.48, p < 0.05), but not significantly correlated with N harvest index. In addition, N effective transportation percentage showed a significant negative correlation with T₀ and T₁₀₀ (R = −0.58–−0.53, p < 0.05).

Figure 4. Correlation analysis of leaves’ color-changing parameters with N transportation, N transport efficiency, and N use efficiency under different N levels. Leaf color-changing parameters included leaf color-changing onset time (T₀), leaf color-changing duration (T₁₀₀), mean leaf color-changing rate (R_mean), and final leaf color index (CI_f). NT_UL, NT_LL, NT_Leaves, NT_Stem, NT and ENT represent upper 3 leaves N transportation, lower leaves N transportation, leaves N transportation, stem N transportation, vegetative organs N transportation, and effective N transportation, respectively; NTE_UL, NTE_LL, NTE_Leaves, NTE_Stem, and NTE represent N transport efficiency of the upper 3 leaves, the lower leaves, the leaves, the stem, and the vegetative organs, respectively. NETE, NHI, NGPE, NDMPE, and NRE represent the N effective transportation percentage, N harvest index, N grain production efficiency, N dry matter production efficiency, and N reuse efficiency. The circle size and color depth represent the correlation coefficients, with “**” and “*” representing significant differences at p < 0.01 and p < 0.05, respectively.

Figure 4. Correlation analysis of leaves’ color-changing parameters with N transportation, N transport efficiency, and N use efficiency under different N levels. Leaf color-changing parameters included leaf color-changing onset time (T₀), leaf color-changing duration (T₁₀₀), mean leaf color-changing rate (R_mean), and final leaf color index (CI_f). NT_UL, NT_LL, NT_Leaves, NT_Stem, NT and ENT represent upper 3 leaves N transportation, lower leaves N transportation, leaves N transportation, stem N transportation, vegetative organs N transportation, and effective N transportation, respectively; NTE_UL, NTE_LL, NTE_Leaves, NTE_Stem, and NTE represent N transport efficiency of the upper 3 leaves, the lower leaves, the leaves, the stem, and the vegetative organs, respectively. NETE, NHI, NGPE, NDMPE, and NRE represent the N effective transportation percentage, N harvest index, N grain production efficiency, N dry matter production efficiency, and N reuse efficiency. The circle size and color depth represent the correlation coefficients, with “**” and “*” representing significant differences at p < 0.01 and p < 0.05, respectively.

Under the LN level, the N loss during N transportation was at a low level. When the N concentration was higher than 0.54 mmol L⁻¹ (MN), the N loss increased significantly and varied significantly among cultivars. The N loss of both cultivars was significantly negatively correlated with T₀ (R = −0.98–−0.88, p < 0.05) and CI_f (R = −0.98–−0.91, p < 0.05), and was significantly positively correlated with the R_mean (R = 0.92–0.97, p < 0.01, Figure 5).

4. Discussion

4.1. Source Analysis of Panicle N

During the grain-filling stage of cereal crops such as rice, wheat, and maize, protein synthesis in the grain depends on the reuse of N in senescent organs, up to 50 to 90% of the protein synthesis in the grain depends on the reuse of N in aging vegetative organs [20,21,22]. The process of N remobilization and reuse from vegetative organs into grains during the late reproductive phase has been extensively studied across various plants, such as maize, oilseed rape, wheat, and Arabidopsis thaliana [15,22,23,24]. The N transport efficiency and N reuse efficiency of vegetative organs were typically calculated by comparing the difference in N accumulation at anthesis and maturity (apparent remobilization). Due to the inability to accurately identify whether the source of N in the panicle comes from post-anthesis root uptake or N transportation from the vegetative organs, and the inability to eliminate the interference of post-anthesis N volatilization on N reuse, this “apparent remobilization” method has relatively large errors. In order to more accurately distinguish whether the panicle N comes from the remobilization and reuse of vegetative organs N, ¹⁴N and ¹⁵N were used as N sources before heading and after heading in the hydroponic experiment. The results showed that the ¹⁵N absorbed during grain-filling stage only accounted for 6.87–10.06% of the total plant N accumulation and 8.47–12.46% of the panicle N accumulation at maturity, while 67.37–72.38% of the panicle N was transported N from vegetative organs. Our results were consistent with those of Kichey et al. [14], who conducted field experiments using ¹⁵N labeled N fertilizer and showed that an average of 71.2% of grain N was derived from the remobilized vegetative organs. Our study further compared the N transportation in different vegetative organs (upper three leaves, lower leaves, and stem) of rice, and found that the N transport efficiency in the upper three leaves ranged from 67.48 to 78.52%, which was slightly higher than that of the lower leaves (60.37–72.77%), and significantly higher than that in the stem (49.09–66.66%), indicating that the leaves were the primary source of N reuse. Our study also revealed that although most of the ¹⁵N absorbed after heading was preferentially supplied to the panicle (78.83–88.08%) for grain filling, there was also a portion allocated to the stem (6.65–12.53%) and leaves (5.27–8.78%). Traditional “apparent remobilization” methods could not distinguish the newly accumulated N in the vegetative organs and often resulted in lower calculated values for N transportation.

4.2. Effect of Exogenous N Supply on Leaves’ Color-Changing and Its Relation to N Reuse in Vegetative Organs

N plays a pivotal role in the synthesis of chlorophyll, which is intimately associated with the leaves’ color. Leaf color is an outward manifestation of leaf N content [25]. Exogenous N supply delays the onset of leaf color-changing and affects leaf color-changing duration [2]. The “stay-green” cultivars exhibit a delayed onset of color-changing under the same N level. When the supply of exogenous N was limited, chlorophyll synthesis rates decreased, decomposition rates increased, and leaves underwent premature yellowing and senescence, and the green holding period of the leaves was shortened [26]. In our study, as the N supply increased, the duration of leaf color-changing was prolonged. However, despite the fact that cultivar CY927 had a longer leaf color-changing duration T₁₀₀ and a higher final leaf color index CI_f compared with CY167 at the same level of N supply, its yield did not significantly increase. Similarly, no difference in yield was found between “stay-green” cultivar and normal leaf color cultivar in maize [27]. Therefore, high leaf N content did not necessarily increase photosynthetic efficiency, and N in plants might be prioritized for nonphotosynthetic processes [28]. In addition, when excess N fertilizer was applied, the upper leaves grew vigorously with an elevated chlorophyll content, often resulting in the majority of light radiation being intercepted by the upper leaves, causing poor light conditions for the lower leaves [29,30].

Maintaining high photosynthetic capacity and efficient reuse of N during the grain-filling stage is considered central to increasing grain yield [31]. Although there was a close correlation between leaf color-changing and vegetative organ N remobilization, a time lag existed between the N transportation from vegetative organs and leaf color-changing indicating that the two processes were not synchronized during the grain-filling stage. Studies have shown that N transportation in vegetative organs occurs before leaf color-changing, and that stored proteins accumulated in stems, sheaths, and leaves before heading are preferentially remobilized in order to minimize the effect of leaf chloroplast protein degradation on leaf photosynthetic capacity [32,33]. In our study, the SPAD value of flag leaf first increased slightly, and a significant decrease in SPAD values was observed 3–13 d after full heading (Figure 2A). This finding indicated an asynchrony between N transportation and leaf color-changing. As a result, no significant correlation between leaf color-changing parameters and stem N transport efficiency could be observed. In this experiment, compared to the normal color-changing cultivar CY167, the cultivar CY927 with dark green leaves had a higher N storage capacity in its stem and a higher N transport capacity in low N environments (Table 4). Previous studies of gene expression related to leaf aging have found that two genotypes—one with yellow leaves at maturity and the other with green leaves—have the same aging initiation signal [34], but the former has an earlier initiation time for aging genes than the latter. And an N-deficient environment can act as an external signal to accelerate leaf senescence. However, the intrinsic reasons for the differences in the onset of senescence remain unknown [34,35]. We propose that differences in the onset of leaf color-changing/senescence in different cultivars may be related to differences in N available for transport in the stem. To maintain high photosynthetic capacity during the grain-filling stage, plants preferentially utilize N stored in the stem and in the lower leaves. Most of the transported N from the stem was directed towards the panicle for grain filling, but a small portion enters the functioning leaves. Therefore, the SPAD value of the flag leaves increased in a short period of time after heading. When the N transported from the stem was inadequate for meeting the needs of grain filling, the chloroplast proteins in the leaves degraded to become an N source and the leaves began to change color.

Previous studies have shown that, in maize, cultivars with yellow leaves at maturity exhibited higher N transport efficiency and less post-anthesis period N uptake capacity compared to the cultivar with green leaves at maturity [36]. In our study, we found that the cultivar CY927 with dark green leaves had higher ¹⁵N accumulation after heading than the cultivar CY167 with normal leaf color changing at the same N levels. The cultivar CY167 had a higher N transport efficiency compared to CY927 in the MN and HN treatments, whereas they showed no N transport advantage in the LN treatment. It may be due to the low stem transported N accelerating leave senescence and enhancing color-changing rate, and more transported N efficiently allocated to the panicle, narrowing the gap between the two cultivars [37]. Furthermore, we also found that the N harvest index, N grain production efficiency, and the N dry matter production efficiency were all low in CY927 despite its high N accumulation; the increase in exogenous N increased plant N accumulation and transported N of vegetative organs, yet decreased leaf N transport efficiency, which were consistent with the findings of previous studies [38].

In summary, to achieve high dry matter accumulation and leaf N reuse efficiency during the grain-filling stage, chloroplasts in leaves should be degraded at an appropriate time and rate. However, there is still a lack of clear understanding of when leaf color-changing begins and at what rate it occurs to achieve synergy between yield and N transport efficiency.

4.3. Relationship between Leaf Color-Changing and Plant N Loss during the Grain-Filling Stage

In the later reproductive period of rice growth, in addition to being transported to the panicles for grain filling, a portion of the transported N from vegetative organs is lost to the atmosphere in a gaseous form [19]. NH₃ is the main form of N volatilization loss [39]. Huang et al. [40] found that 16.7% and 13.4% of tillering fertilizer N and panicle fertilizer N, respectively, were lost through volatilization in the late stage of rice reproductive growth; the rates of plant NH₃ volatilization were a significant negative correlation with N accumulation, N grain production efficiency, N agronomic efficiency, and N physiological utilization [39,41]. Our study showed that with the increase in exogenous N supply, the amount and rate of N volatilization loss increased, while vegetative organ N reuse efficiency decreased (Table 3, Figure 3); N loss was significantly negatively correlated with N harvest index, N dry matter productivity, N grain production efficiency, and N reuse efficiency (NRE) (Figure 4). This suggested that a high exogenous N supply enhances N accumulation in plants but also leads to increased N transport loss in vegetative organs. Therefore, the decrease in N use efficiency in high N levels may be due to the augmented N losses.

Previous studies have demonstrated that the NH₃ volatilization in winter wheat predominantly occurs during the anthesis and late grain-filling stage [42]; during tobacco aging, plants with rapidly aging leaves had higher NH₃ emission [43]. Ammonium ion (NH₄⁺) was continuously produced in mesophyll cells through various processes, the most important being the N cycle pathway of the photorespiratory and the protein degradation pathway during plant senescence. Because the photorespiration intensity decreased during the later reproductive period, whereas protein degradation was enhanced [44], it suggested that N loss in rice plants after heading was less related to photorespiration, but was more strongly associated with protein degradation and N transportation during leaf senescence. Previous studies have shown that during plant senescence, the degradation of chlorophyll and LHC proteins in senescent leaves generates free amino acids, oligopeptides, nitrate, and various other nitrogenous compounds [33], most of which could be loaded by transporter proteins and transported via the phloem to the panicle, while a small fraction can be hydrolyzed to produce NH₄⁺ to enter the apoplast. Typically, NH₄⁺ generated by protein degradation in aging tissues or organs, will undergo ammonium assimilation via the glutamine synthetase (GS)-glutamate synthetase (GOGAT) pathway. But as leaves senesce, the activity of GS enzyme decreases, resulting in a considerable portion of NH₄⁺ not being assimilated promptly and instead entering the apoplast. Plants release NH₃ when the NH₃ concentration in the apoplast is higher than that of the atmospheric. Therefore, apoplast NH₄⁺ is the direct source of NH₃ volatilization, while the imbalance between the chloroplast protein degradation and N re-assimilation is the main cause of N volatilization losses in plants [41]. In this study, correlation analysis showed that the N loss was significantly positively correlated with the leaf color-changing rate R_mean at MN and HN levels (Figure 5B), and the N loss of the cultivar CY167 with fast leaf color-changing rate was significantly higher than that of CY927 with slow color-changing rate in MN and HN treatment. In this experiment, although cultivar CY927 had lower transported N and N transport efficiency than CY167, it instead had higher effective transported N and N effective transportation percentage under HN (Table 5 & Figure 2). Therefore, from the point of view of reducing N volatilization losses, slow leaf color-changing is more conducive to enhancing the N transportation efficiency of vegetative organs. This may also be related to the cultivar’s stronger N uptake ability after heading, which delayed chlorophyll degradation and enhanced the activity of enzymes linked to N metabolism, such as GS enzyme (Figure 6).

5. Conclusions

An increased N supply significantly delayed the leaves’ color-changing onset time and extended the leaves’ color-changing duration. At the same N level, “stay-green” cultivars exhibited a later color-changing. Increasing the level of N supply could enhance plant N uptake and transportation, while the N transportation efficiency would subsequently decrease, which might be related to the higher N loss during transportation under HN treatment. The “stay-green” cultivar exhibited high N uptake and low N use efficiency (N harvest index, N grain production efficiency, and N dry matter production efficiency), while its N effective transportation percentage was relatively high, which might be attributed to the slow rate of leaf color-changing, resulting in reduced N loss during the grain-filling stage. For such cultivars, N fertilizer rates can be appropriately reduced in high fertility soils to achieve higher N use efficiency.