Introduction

Rice (Oryza sativa) is an essential global staple crop, and ensuring its yield is critical for global food security. The growth and yield of rice are influenced by both intrinsic genetic traits and external environmental factors, with fertilization playing a necessary role. Fertilization contributed to over 40% of the increase in grain production1. While chemical fertilizers provide a clear short-term yield boost, their prolonged use without organic supplementation has diminished their long-term effectiveness. For example, each kilogram of nitrogen fertilizer increased grain production by 9.5 kg in the 1960s and 1970s but only 5.5 kg from the 1970s to the 1980s, similarly, the phosphorus fertilizer efficiency dropped from 35.5 kg to 15.5 kg during the same period2,3. This decline is attributed to the reduced application of organic fertilizers, nutrient losses, and suboptimal usage of chemical fertilizers, which also pose ecological risks and hinder the sustainable development of farmlands.

The study of long-term fertilization’s impact on crop growth and soil fertility is of both theoretical and practical significance. Long-term experiments provide unique insights into the cumulative effects of fertilization, surpassing conventional experiments in scope and relevance4,5,6. Numerous studies have demonstrated that the combined application of chemical and organic fertilizers achieves the highest and most consistent yield increases over time7,8,9,10. Research has revealed that long-term fertilization enhances soil fertility by improving soil organic matter10,11,12,13, nutrient content4,7,14,15, pH balance16, aggregate formation17,18, enzyme activity19, and microbial community diversity and function20,21. Additionally, studies show that integrated fertilization promotes dry matter accumulation, nutrient transport to grains, enzyme activity, root growth, and photosynthesis22,23,24,25,26,27,28.

Despite these advances, most studies on long-term fertilization have focused on soil properties, physical structure, and microbial diversity, with fewer investigations into crop growth dynamics and molecular regulation mechanisms. Addressing this gap, this study examines the effects of long-term fertilization on early-season rice (Oryza sativa) in China, where double-cropping rice systems dominate the Yangtze River region. Early-season rice, characterized by early transplantation and maturation, often suffers from low yield and quality, leading to limited enthusiasm among farmers. Fertilization plays a crucial role in enhancing its production.

Using data from a 42-year long-term field experiment, this research explores the physiological and molecular mechanisms of prolonged fertilization on early-season rice. Specifically, it investigates leaf gene expression at full heading stage, dry matter accumulation, yield components, and SPAD values. This study aims to elucidate the regulatory mechanisms underlying rice growth and provide strategies for high-yield, efficient fertilization in early-season rice production.

Materials and methods

Field description

The long-term field experiment was initiated in 1981 at the Red Soil and Germplasm Resources Research Institute in Zhanggong Town, Jinxian County, Nanchang City, Jiangxi Province, China (116° 20′ 24″ N, 28° 15′ 30″ E). The site is situated at an altitude of 30 m above sea level and experiences a mid-subtropical monsoon climate. Key climatic features include an average annual temperature of 17.6 °C, an effective cumulative temperature is 5528 °C, annual precipitation of 1785 mm, approximately 280 frost-free days per year, and 1950 h of annual sunshine. The cropping system involves double-season rice cultivation.

The soil is classified as paddy soil derived from quaternary red clay. Initial soil properties prior to the experiment were as follows: pH of 6.9, soil organic carbon (SOC) 16.3 g kg−1, total nitrogen (TN) of 1.49 g kg−1, total phosphorus (TP) 0.48 g kg−1, total potassium (TK) of 10.39 g kg−1, alkali-hydrolyzed nitrogen (AN) 150.4 mg kg−1, available phosphorus (AP) 4.15 mg kg−1, and available potassium (AK) 80.52 mg kg−1.

Experimental design

The experiment was conducted using a randomized complete block design with three replications. Each plot measured 50 m². Three treatments were selected from the original nine treatments:

  1. 1.

    NPK: A combination of chemical nitrogen (N), phosphorus (P), and potassium (K).

  2. 2.

    HNPK: A double dose of chemical N, P, and K.

  3. 3.

    NPKM: A combination of chemical N, P, and K fertilizer, along with organic fertilizer.

The fertilizer application rates for each treatment are detailed in Table 1. Urea served as the N source, applied as a base (60%) and tillering fertilizer (40%). Potassium chloride was used for K application, applied entirely as a tillering fertilizer. Calcium magnesium phosphate was used for P application as a base fertilizer. Base fertilizers were applied 1–2 days before transplanting, and tillering fertilizers were applied after rice began re-green.

In the NPKM treatment, chemical fertilizers were applied in the same manner and quantity as the NPK treatment. Organic fertilizers were applied once as a base fertilizer, with winter green manure (vetch) used for early-season rice at a rate of 22,500 kg hm−2. Rice cultivars were rotated every five years, with Lingliangyou 739 used in 2022. The rice was seeded on March 25 and transplanted on April 24. Manual transplantation followed a line-and-row design with 20 cm x 20 cm spacing. Alternate wetting and drying irrigation were employed throughout the growing season. Other agronomic practices, including pest and disease management, followed local high-yield cultivation guidelines.

Table 1 Fertilization application rates for each treatment (kg hm−2).
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Analysis of yield and yield components

Rice was harvested at the mature stage on July 18th 2022. The yield was calculated based on the harvested weight and hill number per plot using the following formula:

$${\text{Yield}} = \frac{{{\text{harvested}}\;{\text{rice}}\;{\text{weight}}}}{{{\text{harvested}}\;{\text{hill}}\;{\text{number}}}}/0.2/0.2 \times 10,000$$
(1)

Three representative plants from each plot were analyzed for yield components, including panicle length, grains per panicle, setting rate, and 1000-grain weight.

Analysis of tiller dynamics

Ten fixed hills were marked in each plot to monitor tiller numbers weekly, starting seven days after transplanting until the numbers stabilized. The tillering rate and productive tiller percentage were calculated as follows:

$${\text{Tillering}}\;{\text{rate}} = \frac{{{\text{maximum}}\;{\text{tiller}}\;{\text{number}} - {\text{basic}}\;{\text{seedling}}\;{\text{number}}}}{{{\text{basic}}\;{\text{seedling}}\;{\text{number}}}} \times 100\%$$
(2)
$${\text{Productive}}\;{\text{tiller}}\;{\text{percentage}} = \frac{{{\text{effective}}\;{\text{panicle}}}}{{{\text{maximum}}\;{\text{tiller}}\;{\text{number}}}} \times 100\%$$
(3)

Measurement of dry matter accumulation and leaf SPAD value

Aboveground segments of rice were collected at the tillering, full heading, filling, and mature stages. Samples were separated into stem, leaf, and spike components, then baked in an oven at 105 °C for 30 min and subsequently dried at 85 °C for 6 h to determine dry weight. A chlorophyll meter (SPAD-502, Minolta Camera Co., Osaka, Japan) was used to measure the SPAD value (chlorophyll content) on 20 fully expanded top leaves per plot at each growth stage.

Leaf sample preparation and RNA-seq

Leaf samples were collected at the full heading and filling stages, flash-frozen in liquid nitrogen, and stored at −80 °C. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). RNA quality was assessed using the Agilent 2100 Bioanalyzer. Sequencing libraries were analyzed via Illumina HiSeq2000 (Illumina Inc., San Diego, CA, USA).

Raw sequencing data were filtered to remove low-quality reads, including reads with adapters and poly-N stretches > 10%. Clean reads were aligned to the rice reference genome database (ftp://ftp.ensemblgenomes.org/pub/release42/plants/fasta/oryza_sativa/dna/Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.gz) using HISAT (Hierarchical Indexing for Spliced Alignment of Transcripts)29. Expression levels were quantified using FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) calculated as:

FPKM = (Number of cDNA fragments uniquely aligned to Gene A / Total number of fragments uniquely aligned to all reference genes) × 106/ (Transcript Length in kilobases of Gene A’s exonic regions)30. Differentially expressed genes (DEGs) were identified using DEseq2 with thresholds set at a fold change ≥ 2 and a P-value < 0.05 for significance.

Statistical analysis

One-way analysis of variance (ANOVA) was performed to assess the effects of long-term fertilization of yield, yield components, dry matter accumulation, and SPAD value, with significance set at p < 0.05. Fisher’s least significant difference (LSD) post hoc test was used to determine significant differences among treatments. Graphs were generated using Origin 9.0 software (Microcal Software, Northampton, MA).

Results

Impact of long-term method of fertilization on early rice yield

Long-term method of fertilization significantly affected early-season rice yield (Fig. 1). Both the 42-year average yield (Fig. 1a) and the 42th year’s yield (Fig. 1b) followed the sequence: NPKM > HNPK > NPK. Compared to the NPK treatment, the yields of HNPK and NPKM treatments increased by 56.64% and 90.33%, respectively (p < 0.05) after 42 years of continuous fertilization (Fig. 1b).

Yield components were also significantly impacted by long-term method of fertilization (Figs. 2 and 3a). Compared to the NPK treatment, the number of effective panicles increased by 18.95% and 57.54% under HNPK and NPKM, respectively (Fig. 3a). Spikelet density increased by 9.16% and 22.89% (Fig. 2b), while 1000-grain weight increased 3.74% and 4.28%, respectively (Fig. 2d). The grains per panicle were significantly higher in the NPKM treatment than in the chemical fertilizer treatments (NPK and HNPK) (Fig. 2a), with increases of 20.69–28.55% (Fig. 2c). However, the seed-setting rate showed a slight decline of 3.69–4.51%.

Fig. 1
figure 1

Effect of long-term fertilization on grain yield of early-season rice. Note: Letters upon the same color columns indicated significant differences among treatments at p < 0.05 level, same as below.

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Fig. 2
figure 2

Effect of long-term fertilization on the yield components of early-season rice.

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Tiller dynamics and productive tiller percentage

Fertilization treatments significantly influenced the tiller dynamics of early-season rice (Fig. 3b). The NPK and HNPK treatments exhibited a similar tillering process, where tiller numbers increased between 2 and 7 weeks after transplanting and subsequently decreased. In contrast, the NPKM treatment prolonged the tillering process, with tiller number peaking in the 8th week after transplanting.

Compared to the NPK treatment, the tillering rate increased by 43.84% and 72.73% under HNPK and NPKM, respectively (Fig. 3c). The productive tiller percentage was significantly higher in NPK and NPKM treatments than in HNPK (Fig. 3c), there was no significant difference between the NPK and NPKM treatments in their productive tiller percentage (Fig. 3c), indicating that the NPKM treatment was more effective than both NPK and HNPK.

Fig. 3
figure 3

Effect of long-term fertilization on tillering and panicles formation on early-season rice.

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Dry matter accumulation

Dry matter accumulation in early-season rice was significantly influenced by long-term fertilization (Fig. 4). Across all growth stages—tillering, full heading, filling, and maturity—the total dry matter weight followed the trend: NPKM > HNPK > NPK. Significant differences were observed among the three fertilization treatments at full heading, filling, and maturity stages, except at the tillering stage, where no significant difference was found between NPK and HNPK treatments (Fig. 4d).

Compared to the NPK treatment, the stem dry matter accumulation of HNPK and NPKM increased significantly, ranging from 36.95 to 46.33% across all growth stages (Fig. 4a). Similarly, leaf dry matter accumulation during the tillering stage showed the sequence HNPK > NPKM > NPK, with HNPK and NPKM significantly outperforming NPK (p < 0.05) (Fig. 4b). No significant differences were noted in grain dry matter among treatments at the full heading stage; however, significant increases were observed during the filling and mature stages, following the trend NPKM > HNPK > NPK (p < 0.05, Fig. 4c).

The dry matter accumulation from the tillering to full heading stage was significantly greater under NPKM, with increases of 90.78–107.56% (Table 2). From the full heading to filling stage, dry matter accumulation under NPKM and HNPK increased by 216.09% and 141.46%, respectively, compared to NPK (Table 2). No significant differences were observed in dry matter accumulation from the following to maturity stage among treatments (Table 2).

Dry matter translocation from stem and leaves to spike during the filling to maturity stage was the highest under NPKM (5.51 g/plant), followed by HNPK (2.2 g/plant) and NPK (1.26 g/plant). These results indicate that integrating chemical and organic fertilizers (NPKM) significantly enhanced dry matter accumulation and improved dry matter translocation to spikes during grain filling and maturity.

Fig. 4
figure 4

Dry matter accumulation characteristics of early-season rice under long-term fertilization. Note: TS, FHS, FS, and MS represent the tillering stage, full heading stage, filling stage, and maturity stages, respectively.

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SPAD value dynamic of leaves

Long-term fertilization significantly influenced the chlorophyll content (SPAD value) of early-season rice leaves (Fig. 5). Compared to NPK, the HNPK treatment significantly increased SPAD values during the filling and maturity stages by 14.39% and 29.68%, respectively (p < 0.05). The NPKM treatment consistently produced significantly higher SPAD values across all growth stages, with increases of 22.05–25.58% at the tillering stage, 38.80–58.78% at the full heading stage, and 57.47–104.21% at the filling and maturity stages (p < 0.05).

From the tillering to full heading stage, SPAD values increased 5.24 (NPK), 5.09 (HNPK), and 6.11 (NPKM) (Table 2). From the full heading to filling stage, SPAD values declined by 6.94% (NPKM), 18.23% (HNPK), and 26.29% (NPK). Between the filling and maturity stages, SPAD values decreased by 13.96 (NPK), 13.74 (HNPK), and 15.56 (NPKM).

These findings suggest that the NPKM treatment significantly enhanced SPAD value and mitigated its decline from the full heading to the filling stage.

Fig. 5
figure 5

Chlorophyll SPAD dynamics of early-season rice leaves under long-term fertilization.

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Table 2 Changes in SPAD and dry matter accumulation between adjacent growth stages.
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Relationship between yield, SPAD, and dry matter accumulation

Correlation analysis revealed significant relationships between yield, SPAD values, and dry matter accumulation (Fig. 6). Yield was positively correlated with dry matter accumulation from tillering to full heading stage (ΔDM1). Conversely, yield was negatively correlated with the decline in SPAD values from full heading to filling stage (ΔS2).

Fig. 6
figure 6

Correlation analysis of yield, SPAD, and dry matter accumulation in early-season rice under long-term fertilization. Note: Letters S1, S2, S3, S4, DM1, DM2, DM3 and DM4 represent the SPAD and total dry matter of early season rice at tillering stage, fullheading stage, filling stage and mature sage, respectively. Symbols *and ** in the circle indicate significant correlations at the p < 0.05 and p < 0.01 levels.

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DEGs in rice leaves at full heading stage induced by long-term fertilization

Long-term fertilization significantly influenced leaf gene expression in early-season rice leaves at the full heading stage (Fig. 7). A total of 3,585 DEGs were identified between NPKM and NPK treatments, with 1,478 genes upregulated and 2,107 genes downregulated. Comparatively, 2,051 DEGs were identified between NPKM and HNPK treatments, of which 1,251 were upregulated and 800 were down-regulated, while 3,518 DEGs were detected between HNPK and NPK treatments, including 1,213 upregulated and 2,305 downregulated genes. Additionally, 320 DEGs overlapped across the three treatment comparisons, with 903, 882, and 413 unique DEGs identified in NPK versus NPKM, NPK versus HNPK, and HNPK versus NPKM, respectively.

Fig. 7
figure 7

The DEGS in early-season rice leaves at the full heading stage under long-term fertilization.

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Senescence- and photosynthesis-related DEGs

Gene Ontology (GO) enrichment analysis revealed that 20 DEGs were associated with rice senescence (Table 3). Several DEGs (e.g., Os03g02280, Os05g45450, Os04g43990, Os01g52730, Os02g41840, Os10g27350, Os10g33990, and Os04g33760) were primarily involved in encoding proteins containing DUF584 domains, with others (e.g., Os03g54130, Os04g13140, and Os01g42780) encoded senescence-specific cysteine protease and precursors. Additional DEGs were involved in coenzymes and stress response processes.

KEGG pathway enrichment analysis indicated that photosynthesis-related DEGs were primarily involved in Photosystem I, Photosystem II, cytochrome, ferredoxin, and other related proteins (Fig. 8).

Table 3 DEGs associated with senescence in rice leaves at the full heading stage.
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Fig. 8
figure 8

KEGG pathway enrichment of photosynthesis-related DEGs in rice leaves at the full heading stage. Note: (a) NPK versus HNPK, (b) NPK versus NPKM. The red, green, blue and none indicate upregulated, downregulated, upregulated and downregulated, non-regulated DEGs, respectively.

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Discussion

Impact of long-term fertilization on early rice yield

Fertilization is a critical agronomic practice for maintaining productivity and ensuring stable rice yields. Shen et al.31 and other studies4,7,8,14,15,22,24,27,32,33 have demonstrated that combining organic and chemical fertilizers significantly enhances crop yields, consistent with this study’s findings where early-season rice yields followed the trend of NPKM > HNPK > NPK.

The mechanisms underpinning these effects include the rapid nutrient provision from chemical fertilizers and the enhancement of soil physical and chemical properties by organic fertilizers32,34,35. Long-term chemical fertilization alone has limited impact on soil fertility improvement31, whereas organic fertilizers enhance soil enzyme activity, microbial diversity, and productivity19,20,21. However, exclusive use of organic fertilizers is insufficient for meeting crops’ nutrient needs15,36. Thus, combining chemical and organic fertilizers ensures immediate nutrient supply while improving soil fertility over time, leading to sustained high yields32,37.

This study also found that HNPK significantly increased yields compared to NPK, suggesting that higher chemical fertilizer rates can enhance nutrient availability and yield potential. This contrasts with previous studies indicating that standard chemical fertilization suffices for normal growth15,38. This discrepancy may arise from initially low fertilization levels in this experiment, suggesting that modern rice varieties with higher nutrient demands require adjusted fertilization strategies.

Moreover, combining chemical and organic fertilizers significantly improved yield components, such as effective panicles, spikelet density, and grain weight, Amanullah et al.39 observed similar findings, emphasizing that long-term integration of chemical and organic fertilization enhances the storage capacity of rice to support increased dry matter production and improved yield outcomes.

Dry matter accumulation forms the material foundation for crop yield, with its accumulation, transfer, and distribution significantly influencing crop yields40. Most dry matter originates from photosynthesis, primarily occurring in chloroplasts, and is determined by both the accumulation rate and duration41,42,43. Chlorophyll content, closely associated with photosynthetic productivity, is positively correlated with the leaf photosynthetic rate within a certain range44,45,46. Previous research has established a strong positive correlation between SPAD values and chlorophyll content, making SPAD an effective tool for estimating relative chlorophyll content47,48.

This study found that the combined use of mineral and organic fertilizers significantly increased SPAD values during the rice growth period. This indicates that long-term application of chemical fertilizer with organic fertilizers enhanced photosynthetic intensity, contributing to a stronger photosynthetic “source” in rice. Correlation analysis demonstrated a significant positive relationship between chlorophyll content, dry matter accumulation, and yield in rice. Compared with chemical fertilizers alone, combining chemical and organic fertilizers (NPKM treatment) significantly increased chlorophyll content during the full heading, filling, and maturity stages, thereby enhancing photosynthesis, promoting dry matter accumulation, and improving yield formation.

Additionally, the NPKM treatment significantly increased the transfer of dry matter from stems and leaves to panicles during the filling to maturity stages. This finding suggests that combined fertilization strategies enhance the flow of assimilates, leading to improved yield performance in early-season rice.

Impact of long-term fertilization on growth and gene expression in early rice

Compared with chemical fertilization treatments (NPK and HNPK), the NPKM treatment prolonged the tillering process and increased the number of effective panicles. This can be attributed to the immediate availability of nutrients from chemical fertilizers during the early growth stages and the sustained nutrient supply from organic fertilizers in later stages, reducing nutrient loss and supporting prolonged growth49,50,51.

Crop growth and development are influenced by genotype and environmental factors, with genotype exerting a decisive role. However, environmental factors, such as fertilization, significantly affect gene expression, resulting in phenotypic variations. This study identified that long-term fertilization significantly influenced gene expression in early rice leaves at the full heading stage. Consistent with findings from Liu et al.52,53, GO and KEEG functional analyses of DEGs revealed significant enrichment of photosynthesis-related genes, particularly those involved in Photosystem I, Photosystem II, cytochromes, and ferredoxin. These findings align with the observed differences in SPAD values and dry matter accumulation among treatments, confirming that long-term fertilization regulates photosynthetic gene expression, influencing chlorophyll synthesis, dry matter accumulation, and, ultimately, yield.

The combined application of chemical and organic fertilizers also delayed chlorophyll degradation (as indicated by SPAD values) during the later growth stages. This aligns with Xu et al.54, who demonstrated that organic fertilizers decompose gradually, replenishing nutrients and enhancing post-heading nutrient assimilation. Correlation analysis further revealed a significant negative relationship between the decrease in chlorophyll content from the heading to filling stages and yield, indicating that combined fertilization delayed leaf senescence, extended photosynthetic activity, and promoted dry matter accumulation and yield improvement. Zhang et al.55 reported similar results, attributing them to increased protective enzyme activity and reduced secondary metabolites such as malondialdehyde (MDA) and free proline (Pro) under combined fertilization25,56. Transcriptome analysis in this study showed that genes expressed in leaves at the full heading stage were significantly enriched in senescence-related pathways, involving proteins containing the DUF584 ___domain, aging-related cysteine proteases, and associated cofactors. Proteins with the DUF584 ___domain play essential roles in regulating rice aging by modulating the synthesis and degradation of abscisic acid57. Cysteine further contributes to glutathione (GSH) formation and scavenging superoxide radicals, revealing molecular mechanisms by which combined fertilization reduces MDA and Pro levels, delays aging, enhances dry matter accumulation, and supports yield formation.

Future directions

Late-season rice typically outperforms early-season rice in yield, with distinct growth environments and responses to fertilization. To develop comprehensive fertilization strategies, future studies should investigate the effects of long-term fertilization on late-season rice growth, yield, and regulatory mechanisms.

Conclusion

Long-term method of fertilization significantly influenced the gene expression of early rice leaves at the full heading stage, with DEGs enriched in metabolic pathways associated with photosynthesis and senescence. These genes were primarily involved in Photosystem I, Photosystem II, cytochromes, ferredoxin, DUF584 ___domain-containing proteins, cysteine proteases linked to aging, and the encoding and synthesis of their related precursors and cofactors.

The application of chemical fertilizer in combination with organic fertilizers significantly enhanced tillering and promoted dry matter accumulation in early-season rice, particularly from the full heading to the filling stage. This strategy also strengthened the transport of dry matter from stems and leaves to panicles during the filling to maturity stages. Additionally, combined fertilization significantly increased chlorophyll content in sword leaves and effectively mitigated chlorophyll degradation from the full heading to the filling stage, thereby prolonging photosynthetic activity and contributing to higher yields.