Effect of long-term fertilization on the growth and yield formation of early rice

Effect of long-term fertilization on the growth and yield formation of early rice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effect of long-term fertilization on the growth and yield formation of early rice Zhihua Hu, Lailou Liu, Xiaolin Xu, Dandan Hu, Huijie Song, Yan Wu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4640724/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Fertilization is crucial for rice growth and yield formation. We conducted a 42-year long-term fixed experiment in southeast China, examining nine treatments. This study focused on three treatments: a combination of chemical N, P, and K (NPK), a double dose of chemical N, P, and K (HNPK), and a combination of chemical fertilizer and organic fertilizers (NPKM). We assessed rice yield, yield components, tiller dynamics, dry matter accumulation, chlorophyll dynamics, and leaf transcriptome at the full heading stage. Results indicated that early rice yield followed the order of NPKM > HNPK > NPK. Compared to NPK, HNPK and NPKM significantly increased spikelet density, effective panicles, and 1000-grain weight, while also promoting tillering. NPKM and HNPK significantly enhanced dry matter accumulation from the full heading stage to the filling stage and facilitated the transport of dry matter from leaves and stems to spikes during the filling to mature stages. NPKM consistently maintained higher chlorophyll content than HNPK and NPK at all stages, significantly reducing chlorophyll decline from the full heading stage to the filling stage. Correlation analysis revealed a significant positive relationship between yield and both chlorophyll content and dry matter accumulation under long-term fertilization. There was also a significant negative correlation between yield and chlorophyll reduction from the full heading stage to the filling stage. Differential gene expression analysis at the full heading stage showed significant enrichment in photosynthesis and plant senescence metabolism pathways among different fertilization treatments. Overall, the combined application of chemical and organic fertilizers significantly increased early rice yield by enhancing tillering, regulating photosynthesis and senescence-related gene expression, boosting dry matter accumulation from the full heading stage to the filling stage, and improving dry matter transport to spikes from the filling to the mature stage. Biological sciences/Plant sciences Earth and environmental sciences/Environmental sciences long-term fertilization early rice dry matter accumulation chlorophyll differently expressed genes (DEGs) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Rice ( Oryza sativa ) is an essential global staple crop. Ensuring rice yield is critical for maintaining worldwide food security. The growth and yield of rice are influenced by its intrinsic genotype and external environmental factors. Research has demonstrated that fertilization contributed to over 40% increase in grain production [ 1 ]. Although applying chemical fertilizers produced a clear short-term yield increase, prolonged administration of less or no organic fertilizer significantly reduced the effectiveness of fertilizers. From the 1960s to the 1970s, each kilogram of nitrogen fertilizer elevated grain production by 9.5 kg, which decreased to just 5.5 kg from the 1970s to the 1980s. For phosphorus fertilizer, it decreased from 35.5 kg to 15.5 kg [ 2 , 3 ]. The low usage of chemical fertilizers and substantial nutrient loss represent not only wasted resources but also pose a substantial threat to the ecological environment, which is detrimental to the green and sustainable development of farmlands. Fertilization is critical to guaranteeing a high and stable yield, indicating there is theoretical and practical value in studying the long-term effects of fertilization on crop yield [ 4 ]. Long-term fixed research was adopted to study the influence of fertilization on soil fertility and crop growth, with advantages like long duration and climatic complexity unmatched by conventional experiments [ 5 , 6 ]. Thus, examining the growth features of early-season rice under prolonged fertilization is essential for rational fertilization and efficient rice field cultivation. Numerous studies [ 7 – 10 ] have demonstrated long-term fertilization significantly impacts crop yield, with the highest yields observed under the combined application of chemical and organic fertilizers, demonstrating consistent yield-increasing effects over time. Xu MG et al. systematically uncovered the mechanisms by which long-term fertilization impacts soil fertility, encompassing soil organic matter [ 10 – 13 ], soil nutrients [ 4 , 7 , 14 , 15 ], pH [ 16 ], soil aggregate formation [ 17 , 18 ], soil enzyme activity [ 19 ], and the diversity of microbial communities and functions [ 20 , 21 ]. Additionally, several studies have investigated the impact of long-term fertilization on crop growth. Wu JF et al. [ 22 – 24 ] indicated that integrated application of organic and inorganic fertilizers promoted dry matter production in rice alongside the accumulation and transportation of nutrients to the grains. Yuan YH et al. [ 25 – 28 ] revealed the regulatory mechanisms of prolonged fertilization on crop enzyme activity, root growth, and photosynthetic features. Prior studies on long-term fertilization primarily focused on soil nutrients, physical structure, and microbial community diversity, with some studies on the physiological traits associated with crop growth. However, fewer reports have investigated the impact of long-term fertilization on the growth of crops and associated molecular regulation mechanisms. Drawing on a 42-year long-term fixed experiment, this study explored the gene expression features of leaves at the full heading stage of early rice exposed to prolonged fertilization, as well as its influence on dry matter accumulation, yield component, and chlorophyll characteristics, aiming to elucidate the physiological and molecular regulatory mechanisms of long-term fertilization on early rice yield. This research refines the response mechanisms of rice growth to various fertilization methods and offers references for high-yield and efficient fertilization strategies for rice. 2. Materials and Methods 2.1 Field description The field experiment began in 1981 located in the Red Soil and Germplasm Resources Research Institute in Zhanggong Town, Jinxian County, Nanchang City, Jiangxi Province (116°20′24″N, 28°15′30″E). The site has a mid-subtropical monsoon climate, 30 m above sea level. The average annual temperature is 17.6°C, the effective cumulative temperature is 5528°C, and the annual precipitation is 1785 mm, with approximately 280 frost-free days per year, and the annual sunshine duration is 1950 hours. The cropping system utilized in the experiment included double-season rice. The soil was paddy soil derived from quaternary red clay. The initial soil properties prior to the experiment were as follows: soil pH of 6.9, soil organic carbon (SOC) 16.3 g·kg − 1 , soil total nitrogen (TN) of 1.49 g·kg − 1 , soil total phosphorus (TP) of 0.48 g·kg − 1 , soil total potassium (TK) of 10.39 g·kg − 1 , soil alkali-hydrolyzed nitrogen (AN) of 150.4 mg·kg-1, soil available phosphorus (AP) of 4.15 mg·kg − 1 , and soil available potassium (AK) of 80.52 mg·kg − 1 . 2.2 Experiment Design The experiment was designed as a randomized complete block with three replications. Each plot was 50 m². Three treatments were chosen from the nine in this study: (1) a combination of chemical N, P, and K(NPK); (2) a double dose of chemical N, P, and K(HNPK); (3) a combination of chemical N, P, and K fertilizer, as well as organic fertilizer (NPKM). The amount of fertilizer applied for each treatment is presented in Table 1 . Urea was employed as the nitrogen fertilizer, applied at a ratio of 6:4 as the base and tillering fertilizers, respectively. Potassium chloride, utilized as the potassium fertilizer, was applied entirely as a tillering fertilizer. Calcium magnesium phosphate, utilized as the phosphorus fertilizer, was applied as the base fertilizer only. The base fertilizers were applied 1 or 2 days prior to transplanting, and tillering fertilizers were administered after the rice started to re-green. In the NPKM treatment, the chemical fertilizers were applied in the same manner and quantity as the NPK treatment, and the organic fertilizer was applied with vetch for early rice and fresh pig manure for late rice at a rate of 22, 500 kg·hm − 2 . The organic fertilizers were applied as base fertilizers once. Other field measures, including seeding, transplanting, irrigation, and pest and disease management, were the same as the local high-yield cultivation practices. Table 1 The fertilization application of each treatment (kg·hm 2 ) Treatment Chemical fertilizer Organic fertilizer N P 2 O 5 K 2 O N P 2 O 5 K 2 O Early rice NPK 90 45 75 0 0 0 HNPK 180 90 150 0 0 0 NPKM 90 45 75 123.1 9 60.75 Late rice NPK 90 45 75 0 0 0 HNPK 180 90 150 0 0 0 NPKM 90 45 75 90 55.1 66.15 2.3 Analysis of yield and yield components Rice was harvested at the mature stages, and the yield was calculated according to the weight and plot area. Three representative plants were obtained to determine yield components, including ear length, grains per panicle, setting rate, and 1000-grain weight. 2.4 Analysis of tiller dynamics Ten fixed hills were identified in each plot to count the tillers starting at seven days after transplanting at a 7-day interval until the number diminished. The tillering rate and the ear-bearing tiller percentage were computed as below: tillering rate \(=\frac{\text{m}\text{a}\text{x}\text{i}\text{m}\text{u}\text{m} \text{t}\text{i}\text{l}\text{l}\text{e}\text{r} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r}-\text{b}\text{a}\text{s}\text{i}\text{c} \text{s}\text{e}\text{e}\text{d}\text{l}\text{i}\text{n}\text{g} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r}}{\text{b}\text{a}\text{s}\text{i}\text{c} \text{s}\text{e}\text{e}\text{d}\text{l}\text{i}\text{n}\text{g} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r}}\times 100\%\) (1) ear-bering tiller percentage \(\frac{ \text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\text{i}\text{v}\text{e} \text{p}\text{a}\text{n}\text{i}\text{c}\text{l}\text{e}}{ \text{m}\text{a}\text{x}\text{i}\text{m}\text{u}\text{m} \text{t}\text{i}\text{l}\text{l}\text{e}\text{r} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} }\times 100\%\) (2) 2.5 Measurement of dry matter accumulation and leaf chlorophyll The aboveground segments of rice were collected at the tillering stage, full heading stage, filling stage, and mature stage. The plant samples were separated into stem, leaf, and spike, and then baked in an oven at 105°C for 30 minutes, then dried at 85°C for 6 hours before determining dry weight. A chlorophyll meter [SPAD-502, Soil and plant analysis development (SPAD), Minolta Camera Co. Osaka, Japan] was utilized for chlorophyll measurement on the 20 top fully expanded leaves per plot at the tillering stage, full heading stage, filling stage, and mature stage. 2.6 Leaf sample preparation and RNA-Seq Leaf samples were harvested at the full heading and filling stages, flash-frozen using liquid nitrogen, and stored at -80°C before being sent to Qinke-Tech (Beijing, China) for RNA-seq by Illumina HiSeq2000 (Illumina Inc., San Diego, CA, USA). Raw data were filtered by removing reads, including adapters, poly-N > 10%, and low-quality reads, to obtain clean reads for further analysis. 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 ) by Hisat (Hierarchical Indexing for Spliced Alignment of Transcripts) [ 29 ]. FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) was employed to characterize the gene expression level and calculated as: FPKM = (Number of cDNA fragments uniquely aligned to Gene A / Total number of fragments uniquely aligned to all reference genes) × 10 6 / (Transcript Length in kilobases of Gene A's exonic regions)[ 30 ]. DEseq2 was employed for differential expression analysis; the criteria set for significant differences were a fold change ≥ 2 and a P -value < 0.05. 2.7 Statistical analysis We used one-way analysis of variance (ANOVA) to characterize differences in the responses of yield, yield component, dry matter accumulation, and SPAD value of long-term fertilization and applied a significance level of P < 0.05. Graphs were generated using Origin 9.0 software (Microcal Software, Northhampton, MA). 2.7 Data availability Data available on request from the corresponding author. 3. Results 3.1 Impact of Long-Term Fertilization on Early Rice Yield Long-term fertilization has significantly impacted early rice yield (Fig. 1a). The yield sequence progressed from NPKM > HNPK > NPK. Compared to NPK, both HNPK and NPKM treatments significantly elevated the yield of double-season early rice ( P < 0.05). Following 42 years of continuous fertilization, the yields increased by 56.64% and 90.33%, respectively. The yield components were significantly impacted by long-term fertilization treatments. Compared to NPK, HNPK and NPKM significantly increased the number of effective panicles (Fig. 1b), spikelet density (Fig. 1d), and 1000-grain weight (Fig. 1f). The grains per panicle of NPKM were significantly higher than chemical fertilizer treatments (NPK, HNPK) (Fig. 1c), demonstrating the opposite trend in seed setting (Fig. 1e). 3.2 The tiller dynamic and ear-bearing Long-term fertilization significantly impacted the tiller dynamic of early rice (Fig. 2a). The chemical fertilizer treatments (NPK, HNPK) maintained the same tillering process, and the tiller number increased from 2 to 7 weeks after transplanting and then decreased. The NPKM treatment prolonged the tillering process. The HNPK and NPKM treatments significantly increased the tiller number of early rice compared to NPK, with the tillering rate increasing by 43.84% and 72.73%, respectively (Fig. 3b). The ear-bearing tillering percentage of HNPK was significantly lower than NPK and NPKM (Fig. 2b). 3.3 Dry matter accumulation As depicted in Fig. 3, the dry matter accumulation of early rice was significantly affected by long-term fertilization. The total dry matter weight of the tillering stage, full heading stage, filling stage, and mature stage followed a trend of NPKM > HNPK > NPK, and significantly differed between the three fertilization treatments except between NPK and HNPK at the tillering stage (Fig. 3d). Compared to NPK, HNPK and NPKM significantly increased the stem dry matter of each growth stage, with an increase of 36.95%-46.33%, 19.43%-34.41%, 43.59%-61.51%, and 39.23%-46.58%, respectively (Fig. 3a). The dry matter of leaves in the tillering stage followed the trend of HNPK > NPKM > NPK, with HNPK and NPKM significantly higher than NPK ( P < 0.05) (Fig. 3b). There was no significant difference in grain dry material across the three fertilization treatments at the full heading stage, and significant differences were identified at the filling and mature stages (Fig. 3c), and followed a trend of NPKM > HNPK > NPK, with significant differences between treatments ( P < 0.05). Compared to NPK and HNPK, NPKM significantly elevated the dry matter accumulation from the tillering to full heading stage, with an increase of 90.78%-107.56%. Compared to NPK, NPKM and HNPK increased dry matter accumulation from the full heading to the filling stage by 216.09% and 141.46%. The dry matter accumulation from the filling to the mature stage showed no significant difference among the three fertilization treatments. The dry matter translocation from the stem and leaves to spike was 5.51 g/plant (NPKM), 2.2 g/plant (HNPK), and 1.26 g/plant (NPK). Our findings indicated that the combination of fertilizers with organic fertilizers could significantly promote dry matter accumulation in early rice and increase the transfer of dry matter from the stem and leaf to the spike during the filling stage to maturity. 3.4 Chlorophyll dynamic Long-term fertilization significantly affected the early rice chlorophyll content of leaves (Fig. 4). Compared to NPK, HNPK significantly increased leaf chlorophyll content during the filling and mature stages with an increase of 14.39% and 29.68%, respectively ( P < 0.05). Under the NPKM treatment, leaf chlorophyll content was significantly higher than in NPK and HNPK treatments during the tillering, full heading, grain-filling, and mature stages, with increases of 22.05%-25.58%, 38.80%-58.78%, and 57.47%-104.21%, respectively. The chlorophyll levels of sword leaves from tillering to the full heading stage increased by 5.24 (NPK), 5.09 (HNPK), and 6.11 (NPKM). It decreased by 6.94% (NPKM), 18.23% (HNPK), and 26.29% (NPK) from the full heading to the filling stage. The decreases in chlorophyll content from filling to mature stage of NPK, HNPK, and NPKM were 5.56, 13.74, and 13.96, respectively. These findings indicated that integrating chemical fertilizers with organic fertilizers can significantly enhance the chlorophyll content of sword leaves in early rice and significantly mitigate the reduction of chlorophyll from the full heading to the filling stage. 3.5 Relationship between yield, chlorophyll, and dry matter accumulation Correlation analysis demonstrated that chlorophyll and dry matter accumulation significantly influenced early rice yield under long-term fertilization (Fig. 5). The yield was significantly positively correlated with the change in chlorophyll from full heading to filling stage, and was significantly positively correlated with dry matter accumulation from tillering to full heading stage (△DM1) and full heading to filling stage (△DM2). 3.6 DEGs in rice leaves at full heading stage induced by long-term fertilization Long-term fertilization significantly influenced leaf gene expression in early rice (Fig. 6). There were 3585 differentially expressed genes (DEGs) between NPKM and NPK treatments, of which 1478 genes were up-regulated and 2107 genes were down-regulated. There were 2051 DEGs between NPKM and HNPK, of which 1251 were up-regulated and 800 were down-regulated. There were 3518 DEGs between HNPK and NPK, including 1213 up-regulated and 2305 down-regulated genes. There were 320 DEGs in NPK vs. NPKM, NPK vs. HNPK, and HNPK vs. NPKM, with 903, 882, and 413 unique DEGs, respectively. 3.7 Senescence and photosynthesis-related DEGs Senescence and photosynthesis were considerably influenced by long-term fertilization. Gene Ontology (GO) enrichment (Table 2 ) demonstrated that there were 20 DEGs enriched in rice senescence, mainly involved in encoding proteins containing DUF584 domain, aging-related cysteine protease and its precursor proteins, and aging-related coenzymes. KEEG analysis indicated that DEGs enriched in the photosynthesis pathway were mainly involved in photosystem I, photosystem II, cytochrome, ferredoxin, and other related proteins (Fig. 7). Table 2 The DEGS enriched in the senescence process Gene ID FPKM DEGS Gene function NPK HNPK NPKM Ⅰ Ⅱ Ⅲ Os06g50330 20.324 30.823 38.798 U N N protein EARLY-RESPONSIVE TO DEHYDRATION 7, chloroplastic Os01g52740 3.463 1.328 1.025 D D N response to endogenous stimulus Os03g02280 2.192 0.186 0.681 D N N DUF584 domain containing protein, putative, expressed Os05g45450 0.409 1.277 0.597 N U N DUF584 domain containing protein, putative, expressed Os04g43990 18.011 34.5230 30.241 N U N DUF584 domain containing protein, putative, expressed Os01g52730 5.196 2.715 5.436 N N U DUF584 domain containing protein, putative, expressed Os02g41840 2.031 2.818 10.769 U N U DUF584 domain containing protein, putative, expressed Os10g27350 3.529 0.793 1.405 D D D DUF584 domain containing protein, putative, expressed Os10g33990 7.975 7.638 24.841 U N U DUF584 domain containing protein, putative, expressed Os04g33760 3.445 4.248 6.476 U N N DUF584 domain containing protein, putative, expressed Os03g54130 10.688 2.385 1.382 D D N senescence-specific cysteine protease SAG39 Os04g13140 8.189 0.274 1.442 D D U senescence-specific cysteine protease SAG39 Os01g42780 79.563 56.836 1.776 D N D cysteine proteinase EP-B 2 precursor Os05g27580 0.427 1.725 2.070 U U N wound-induced protein WI12, Os07g49114 10.991 5.614 2.984 D N N wound-induced protein WI12 Os09g01000 221.300 817.118 314.060 N U D putative senescence-associated protein, partial Os10g07010 1.051 0.251 0.115 D D N 3-ketoacyl-CoA synthase Os01g21250 302.410 25.303 34.678 D D N late embryogenesis abundant protein Os11g25160 13.029 33.960 36.780 U U N tropinone reductase homolog At5g06060 isoform X1 Os01g59570 0.318 0.307 0.101 D N D probable LRR receptor-like serine/threonine-protein kinase At1g51810 Note : The DEGs I, Ⅱ, and Ⅲ represented NPK vs. NPKM, NPK vs. HNPK, and HNPK vs. NPKM, respectively. The letters D, U, and N indicated the gene expression of each group exhibiting down-regulated, up-regulated, and non-regulated. 4. Discussion 4.1 Impact of Long-Term Fertilization on the Yield of Early Rice Fertilization is a critical agronomic practice for maintaining paddy productivity and guaranteeing high and stable rice yields. Shen [ 31 ] summarized long-term fertilization experiments and found that organic and chemical fertilizers significantly enhanced crop yields. Additionally, numerous studies [ 4 , 7 , 8 , 14 , 15 , 22 , 24 , 27 , 32 , 33 ] have demonstrated that the maximum yield increases were achieved when combining organic with chemical fertilization, consistent with this study's findings in which early rice yields followed a trend of NPKM > HNPK > NPK. Previous studies investigated the mechanisms of long-term fertilization impacted crop yields. A study by Mao et al. [ 32 ] demonstrated that chemical fertilizers offer rapid nutrients while organic fertilizers improve soil physical and chemical properties. Research by Duan et al. [ 34 , 35 ] indicated that crop yield was positively correlated with soil fertility, whereas long-term chemical fertilization alone showed no significant enhancement in soil fertility [ 31 ], and organic fertilizers application significantly improved soil fertility and enhanced soil enzyme activity and microbial community functional diversity [ 19 , 20 , 21 ], to thereby improve soil productivity. Zhou et al. [ 15 , 36 ] suggested that long-term exclusive use of organic fertilizers cannot attain the critical nutrient requirements of crops. Combining chemical and organic fertilization was critical in providing immediate nutrients and improving soil fertility [ 32 , 37 ], explaining why the highest crop yields were achieved under this fertilization method over prolonged durations. Furthermore, this study found that HNPK treatments significantly elevated early rice yields compared to NPK treatments, suggesting that increasing chemical fertilization could significantly improve crop yields. This contrasts with previous findings that conventional chemical fertilization satisfies normal crop growth but that increased rates do not enhance yield [ 15 , 38 ]. This discrepancy might be associated with the initially low fertilization levels in this experiment, suggesting that as breeding techniques improve and novel rice varieties demand increased nutrients, normal fertilization levels may not suffice, limiting the yield potential. Increased chemical fertilization could achieve these enhanced nutrient demands and increase rice yield to some extent. Furthermore, our findings demonstrated the combination of chemical and organic fertilizers significantly improved yield components. Amanullah, H. [ 39 ] also identified the combined use of mineral fertilizer and organic fertilizer produced improved yield and yield components. This may be due to the easily available nutrient supply from chemical fertilizer at the early growth stage, and organic fertilizer could reduce nutrient loss and maintain the supply of nutrients in later growth stages [ 40 – 42 ]. 4.2 Impact of long-term fertilization on the growth of early rice and associated gene expression Crop growth and development were influenced by genotype and environmental factors, with genotype exerting a decisive role. However, environmental factors like climate and fertilization impact the expression of crop-related genes. This study identified that long-term fertilization significantly influenced gene expression in early rice leaves at the full heading stage. Liu et al. [ 43 , 44 ] also observed characteristic gene expression in crops under long-term fertilization. Through GO and KEEG functional analysis of DEGs, this study identified significant enrichment in genes associated with photosynthesis at the full heading stage of early rice, especially involved in Photosystem I, Photosystem II, cytochromes, and ferredoxin. The accumulation of dry matter and chlorophyll post-heading is consistent with crop yield. This indicated that long-term fertilization impacts the expression of genes related to photosynthesis in early rice and regulates the formation of chlorophyll and dry matter in leaves, thereby impacting yield. Dry matter accumulation is the material foundation of crop yield, and the accumulation, transfer, and distribution of dry matter significantly affect crop yields [ 45 ]. The findings demonstrate that dry matter accumulated from the full heading to the filling stage was significantly positively correlated with yield, while from filling to the maturity stage, there was no significant correlation. The NPKM treatment significantly increased dry matter accumulation from the full heading to the filling stage compared to NPK, and there was no significant difference in dry matter accumulation among the three fertilization methods from the filling to the mature stage. However, the increase in panicle dry matter during the same period was higher with combined NPK and organic fertilizers, further illustrating that this combination can significantly increase dry matter accumulation from full heading to the grain-filling stage and promote the transfer of stem and leaf dry matter to panicles during the filling to mature stages. Xu et al. [ 46 ] showed that organic fertilizers, in combination with chemical fertilizers, decompose to replenish nutrients for rice, increasing the accumulation and distribution of dry matter in various organs and enhancing post-heading assimilation of nutrients. Most dry matter originates from leaf photosynthesis, governed by the rate of accumulation and duration [ 47 – 49 ]. Chloroplasts are the primary sites of photosynthesis, and chlorophyll content was closely tied to the photosynthetic productivity of rice [ 50 ]. Correlation analysis demonstrated that chlorophyll content was significantly positively correlated with dry matter and yield in rice, with nitrogen, phosphorus, and potassium combined with organic fertilizers leading to significantly higher chlorophyll content during the full heading, filling, and maturity stages compared to chemical fertilizers alone, indicating that this combination enhanced photosynthesis in early rice, promoted dry matter accumulation and yield formation. Additionally, combining chemical and organic fertilizers promoted the tillering of early rice, increasing effective panicles. Moreover, the integration of chemical and organic fertilizers delayed the reduction of chlorophyll content in leaves during later stages. Correlation analysis also demonstrated that the decrease in leaf chlorophyll content from the full heading to the filling stage was significantly negatively correlated with rice yield and dry matter accumulation, indicating that the combination of chemical and organic fertilizers, to some extent, delayed rice senescence, extended the duration of dry matter accumulation, and enhanced yield. Zhang et al. [ 51 ] obtained similar results, likely associated with the increased activity of protective enzymes in rice leaves and the lowered content of secondary metabolites like malondialdehyde (MDA) and free proline (Pro) under combined chemical and organic fertilizers [ 25 , 52 ]. This study's transcriptome analysis also demonstrated that genes expressed in leaves at the full heading stage are significantly enriched in senescence, primarily involving proteins containing the DUF584 domain, associated with aging-associated cysteine proteases and precursors and related cofactors. Proteins containing the DUF584 domain played an essential role in rice aging, regulating the synthesis and breakdown of abscisic acid within the plant [ 53 ]. Cysteine aids in promoting GSH formation and scavenging superoxide radicals, revealing the molecular mechanisms by which long-term fertilization with chemical and organic fertilizers regulates the content of MDA, Pro, and other substances, delaying rice aging, enhancing dry matter accumulation, and improving yield formation. 5. Conclusion Long-term fertilization significantly influenced the gene expression of early rice leaves at the full heading stage, with DEGs being enriched in metabolic pathways associated with photosynthesis and aging. These genes were primarily involved in Photosystem I, Photosystem II, cytochromes, ferredoxin, proteins containing the DUF584 domain, cysteine proteases associated with aging, and the encoding and synthesis of their related precursors and cofactors. The application of chemical fertilizer in combination with organic fertilizers significantly increased tillering and enhanced the accumulation of dry matter in early rice, particularly from the full heading to the filling stage, strengthening the transport of dry matter from the stem and leaves to the panicles from the filling to the maturity stage. Additionally, this fertilization strategy significantly increases the chlorophyll content in the sword leaves of rice and effectively alleviates chlorophyll degradation in leaves from the full heading to the filling stage. Declarations Author Contributions: Conceptualization, Zhihua Hu; methodology, Zhihua Hu, Kailou Liu; writing—original draft preparation, Zhihua Hu, Kailou Liu, Xiaolin Xu; writing—review and editing, Dandan Hu, Huijie Song, Yan Wu, Jianfu Wu; funding acquisition, Kailou Liu, Jianfu Wu. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the National Natural Science Foundation of China (42207398; 32260808); the key research and development plan "open ranking" project of Jiangxi Province, China(20223BBF61016). Data Availability Statement: Data reported within the article. Conflicts of Interest: The authors declare no conflicts of interest. 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M., Mohammad, W., Shafi, M., Nawaz, H., Shehzadi, S., Amir, M. Effect of integrated use of organic and inorganic N sources on wheat yield. Sarhad J Agric, 26: 559–563 (2010). Liu, J. T., Zhu, K. L., Zhao, H. C., Li, Y. B., Liu, S. T., Song, X. Y., Li, J. Transcriptome Analysis of Maize Ear Leaves under Long-Term Applications of Nitrogen Fertilizer and its Combinations with Phosphorus and Potassium Fertilizers. Journal of Soil Science and Plant Nutrition, 22(1): 112-120 (2021). Kangi, E., Brzostek, E. R., Bills, R. J., Callister, S. J., Zink, E. M., Kim, Y. M., Larsen, P. E., Cumming, J. R. A multi-omic survey of black cottonwood tissues highlights coordinated transcriptomic and metabolomic mechanisms for plant adaptation to phosphorus deficiency. Frontiers in Plant Science, 15, 1324608 ((2024). Wang, H. C., Yang, B. J., Mahmood, A., Maqbool, R., Hassan, M. U., Khan, T. A., Iqbal, M. M., Huang, G. Q. Winter cropping improves yield, dry matter accumulation and translocation and nitrogen uptake of double-cropping rice. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 51(3), 13299-13299 (2003). Xu, Y. L., Tang, H. M., Cheng, A. W., Xiao, X. P., Guo, L. J., Sun, J. M., Li, W. Y. Effects of different long-term fertilizer management methods on dry matter accumulation and yield of rice in the double cropping rice field. Journal of Anhui Agricultural University, 42 (5): 674-680 (2005). (in Chinese) Ye, Y. L., Wang, G. L., Huang, Y. F., Zhu, Y. J., Meng, Q. F., Chen, X. P., Zhang, F. S., Cui, Z. L. Understanding physiological processes associated with yield-trait relationships in modern wheat varieties. Field Crops Research, 124(3): 316-322 (2012). Mackown, C. T., Van, S. D. A., Zhang, N. Y. Wheat vegetative nitrogen compositional changes in response to reduced reproductive sink strength. Plant Physiology, 99(4): 1469-1474 (1992). Zhou, B. Y., Yue, Y., Sun, X. F., Wang, X. B., Wang, Z. M., Ma, W., Zhao, M. Maize grain yield and dry matter production responses to variations in weather conditions. Agronomy Journal, 108(1): 1-9 (2016). Shiratsuchi, H., Yamagishi, T., Ishii, R. Leaf nitrogen distribution to maximize the canopy photosynthesis in rice. Field Crops Research, 95(2-3): 291-304 (2006). Zhang, Y. P., Liu, Q., Rong, X. M., Xie, G. X., Li, X., Peng, J. W., Song, H. X., Zhang, Z. H. Effects of organic manure and inorganic fertilizer combination on photosynthesis characteristics and enzyme activities of NR and SPS in rice functional leaves. Journal of Hunan Agricultural University (Natural Sciences), 37(5): 540-545 (2011). (in Chinese) Li, S., Zhao, X. H., Ye, X. S., Zhang, L. M., Shi, L., Xu, F. S., Ding, G. D. The effects of condensed molasses soluble on the growth and development of rapeseed through seed germination, hydroponics and field trials. Agriculture, 10(7), 260 (2020).. Wang, Y. T., Li, Y. Y., Tian, H. N., Wang, W., Hussain, S., Yuan, Y., Li, R., Hussain, H., Wang, T. Y., Wang, S. C. AtS40-1, a group I DUF584 protein positively regulates ABA response and salt tolerance in Arabidopsis. Gene, 846 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 12 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 01 Dec, 2024 Reviews received at journal 06 Nov, 2024 Reviewers agreed at journal 14 Oct, 2024 Reviews received at journal 15 Sep, 2024 Reviewers agreed at journal 23 Aug, 2024 Reviews received at journal 23 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers invited by journal 17 Jul, 2024 Editor assigned by journal 17 Jul, 2024 Editor invited by journal 02 Jul, 2024 Submission checks completed at journal 28 Jun, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4640724","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":328654479,"identity":"b623b7b4-293a-4e3e-861f-5afa1dd1f27d","order_by":0,"name":"Zhihua Hu","email":"","orcid":"","institution":"College of Land Resources and Encironment","correspondingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Hu","suffix":""},{"id":328654480,"identity":"ec9c3fa6-98df-4df2-9936-5998eb15de24","order_by":1,"name":"Lailou Liu","email":"","orcid":"","institution":"Jiangxi Institute of Red Soil and Germplasm Resources","correspondingAuthor":false,"prefix":"","firstName":"Lailou","middleName":"","lastName":"Liu","suffix":""},{"id":328654481,"identity":"63302b65-88de-42e4-97bb-77eb40d6f724","order_by":2,"name":"Xiaolin Xu","email":"","orcid":"","institution":"Jiangxi Institute of Red Soil and Germplasm Resources","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Xu","suffix":""},{"id":328654482,"identity":"c5281ed8-9f21-4040-a221-830a17e28234","order_by":3,"name":"Dandan Hu","email":"","orcid":"","institution":"Jiangxi Institute of Red Soil and Germplasm Resources","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Hu","suffix":""},{"id":328654483,"identity":"fa9e09a0-8bd3-4c46-9e9f-1479fd593d94","order_by":4,"name":"Huijie Song","email":"","orcid":"","institution":"Jiangxi Institute of Red Soil and Germplasm Resources","correspondingAuthor":false,"prefix":"","firstName":"Huijie","middleName":"","lastName":"Song","suffix":""},{"id":328654484,"identity":"4f0f3a28-433f-4dd9-a590-b6b3feb96b52","order_by":5,"name":"Yan Wu","email":"","orcid":"","institution":"Jiangxi Institute of Red Soil and Germplasm Resources","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Wu","suffix":""},{"id":328654485,"identity":"f9fee888-b5d9-4c5d-b404-cc3c5ffaa8a3","order_by":6,"name":"Jianfu Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFCCgw8f/zGQkGNjbz5ArJbDxgY8BRbGfDzHEojVwmwmwPOhInGeRI4CcRoMDh5mY5AwkEhvY8hhYPhRsY0ILQcOsz0wMJDIbWM4e4Cx58xtwlrMDpw/bpAA0sLYl8DM2EaUlsNsEgeADmNj5jEgXotkg4FEAhsbsVrsDxxmNmYwkDBs42FLOEiUXyRnHGZ8zPCnTl5+/uODD35UEKGFQeIAgn0AlyJUwN9AnLpRMApGwSgYwQAAj307jfIBjaAAAAAASUVORK5CYII=","orcid":"","institution":"College of Land Resources and Encironment","correspondingAuthor":true,"prefix":"","firstName":"Jianfu","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-06-26 07:24:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4640724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4640724/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-93474-8","type":"published","date":"2025-03-12T15:58:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60700078,"identity":"2ba88108-15db-42c6-abe2-e4ce8ddb72c1","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":62794,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of long-term fertilization on early rice yield and yield components.\u003c/p\u003e\n\u003cp\u003eNote: Letters upon the same color columns indicated significant differences among treatments at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 level, as below.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/750e02197d55a56cf39b1335.png"},{"id":60700076,"identity":"af5aa49d-f190-4772-ac64-d13042c800ec","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":39692,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of long-term fertilization on tillering and ear formation of early rice.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/7e93f72376d2ce7234f7bd4c.png"},{"id":60700077,"identity":"641791c4-dc88-421b-8d73-d66285680ef4","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":60440,"visible":true,"origin":"","legend":"\u003cp\u003eThe characteristics of dry matter accumulation of early rice under long-term fertilization\u003c/p\u003e\n\u003cp\u003eNote: The TS, FHS, FS, and MS represent the tillering stage, full heading stage, filling stage, and mature stages of rice, respectively.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/345d40f0fa43415fe4098b39.png"},{"id":60700475,"identity":"a9700f7b-8925-4b5c-a4cc-d6bea8ec9fba","added_by":"auto","created_at":"2024-07-19 17:51:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34068,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of long-term fertilization on the chlorophyll content of the early rice leaf\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/a8b48e36521747e36450bc8c.png"},{"id":60700082,"identity":"43ed0b23-2302-41c7-ac49-252462efa365","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":130200,"visible":true,"origin":"","legend":"\u003cp\u003eThe correlation of yield, chlorophyll, and dry matter of early rice under long-term fertilization\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e △Chl1, △Chl2, △Chl3, △DM1, △DM2, and △DM3 represent the variation of chlorophyll from the tillering stage to full heading stage, the variation of chlorophyll from full heading stage to filling stage, the variation of chlorophyll from filling stage to mature stage, the accumulation of dry matter from tillering stage to full heading stage, the accumulation of dry matter from full heading stage to filling stage and the accumulation of dry matter from filling stage to mature stage respectively, while *and ** in the circle indicate significant correlations at the \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 and \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01\u003cem\u003e \u003c/em\u003elevels.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/960fc1a5b5204f707cc199a8.png"},{"id":60700080,"identity":"97983ca5-5138-4506-ad56-926c165d8f03","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":107660,"visible":true,"origin":"","legend":"\u003cp\u003eThe DEGS of early rice leaves at the full heading stage under long-term fertilization.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/60cf114ed5bb4511a5acdf39.png"},{"id":60700081,"identity":"e7427534-37b0-4e40-bc07-60e5c95a54e2","added_by":"auto","created_at":"2024-07-19 17:43:26","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":394158,"visible":true,"origin":"","legend":"\u003cp\u003ePhotosynthesis-related KEGG analysis of DEGs in the leaves of rice at full heading stage.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/6c9872e4dc15bcce70191d19.png"},{"id":78689143,"identity":"b4e4053e-26c0-4194-9470-0406a3bcbdbc","added_by":"auto","created_at":"2025-03-17 16:11:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1716273,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4640724/v1/e048d04f-ecf7-4d7a-8e47-ea44911b4692.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effect of long-term fertilization on the growth and yield formation of early rice","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e) is an essential global staple crop. Ensuring rice yield is critical for maintaining worldwide food security. The growth and yield of rice are influenced by its intrinsic genotype and external environmental factors. Research has demonstrated that fertilization contributed to over 40% increase in grain production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Although applying chemical fertilizers produced a clear short-term yield increase, prolonged administration of less or no organic fertilizer significantly reduced the effectiveness of fertilizers. From the 1960s to the 1970s, each kilogram of nitrogen fertilizer elevated grain production by 9.5 kg, which decreased to just 5.5 kg from the 1970s to the 1980s. For phosphorus fertilizer, it decreased from 35.5 kg to 15.5 kg [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The low usage of chemical fertilizers and substantial nutrient loss represent not only wasted resources but also pose a substantial threat to the ecological environment, which is detrimental to the green and sustainable development of farmlands. Fertilization is critical to guaranteeing a high and stable yield, indicating there is theoretical and practical value in studying the long-term effects of fertilization on crop yield [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Long-term fixed research was adopted to study the influence of fertilization on soil fertility and crop growth, with advantages like long duration and climatic complexity unmatched by conventional experiments [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Thus, examining the growth features of early-season rice under prolonged fertilization is essential for rational fertilization and efficient rice field cultivation. Numerous studies [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] have demonstrated long-term fertilization significantly impacts crop yield, with the highest yields observed under the combined application of chemical and organic fertilizers, demonstrating consistent yield-increasing effects over time. Xu MG et al. systematically uncovered the mechanisms by which long-term fertilization impacts soil fertility, encompassing soil organic matter [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], soil nutrients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], pH [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], soil aggregate formation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], soil enzyme activity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and the diversity of microbial communities and functions [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, several studies have investigated the impact of long-term fertilization on crop growth. Wu JF et al. [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] indicated that integrated application of organic and inorganic fertilizers promoted dry matter production in rice alongside the accumulation and transportation of nutrients to the grains. Yuan YH et al. [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] revealed the regulatory mechanisms of prolonged fertilization on crop enzyme activity, root growth, and photosynthetic features. Prior studies on long-term fertilization primarily focused on soil nutrients, physical structure, and microbial community diversity, with some studies on the physiological traits associated with crop growth. However, fewer reports have investigated the impact of long-term fertilization on the growth of crops and associated molecular regulation mechanisms.\u003c/p\u003e \u003cp\u003eDrawing on a 42-year long-term fixed experiment, this study explored the gene expression features of leaves at the full heading stage of early rice exposed to prolonged fertilization, as well as its influence on dry matter accumulation, yield component, and chlorophyll characteristics, aiming to elucidate the physiological and molecular regulatory mechanisms of long-term fertilization on early rice yield. This research refines the response mechanisms of rice growth to various fertilization methods and offers references for high-yield and efficient fertilization strategies for rice.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Field description\u003c/h2\u003e \u003cp\u003eThe field experiment began in 1981 located in the Red Soil and Germplasm Resources Research Institute in Zhanggong Town, Jinxian County, Nanchang City, Jiangxi Province (116\u0026deg;20\u0026prime;24\u0026Prime;N, 28\u0026deg;15\u0026prime;30\u0026Prime;E). The site has a mid-subtropical monsoon climate, 30 m above sea level. The average annual temperature is 17.6\u0026deg;C, the effective cumulative temperature is 5528\u0026deg;C, and the annual precipitation is 1785 mm, with approximately 280 frost-free days per year, and the annual sunshine duration is 1950 hours. The cropping system utilized in the experiment included double-season rice. The soil was paddy soil derived from quaternary red clay. The initial soil properties prior to the experiment were as follows: soil pH of 6.9, soil organic carbon (SOC) 16.3 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, soil total nitrogen (TN) of 1.49 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, soil total phosphorus (TP) of 0.48 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, soil total potassium (TK) of 10.39 g\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, soil alkali-hydrolyzed nitrogen (AN) of 150.4 mg\u0026middot;kg-1, soil available phosphorus (AP) of 4.15 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and soil available potassium (AK) of 80.52 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Experiment Design\u003c/h2\u003e \u003cp\u003eThe experiment was designed as a randomized complete block with three replications. Each plot was 50 m\u0026sup2;. Three treatments were chosen from the nine in this study: (1) a combination of chemical N, P, and K(NPK); (2) a double dose of chemical N, P, and K(HNPK); (3) a combination of chemical N, P, and K fertilizer, as well as organic fertilizer (NPKM). The amount of fertilizer applied for each treatment is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Urea was employed as the nitrogen fertilizer, applied at a ratio of 6:4 as the base and tillering fertilizers, respectively. Potassium chloride, utilized as the potassium fertilizer, was applied entirely as a tillering fertilizer. Calcium magnesium phosphate, utilized as the phosphorus fertilizer, was applied as the base fertilizer only. The base fertilizers were applied 1 or 2 days prior to transplanting, and tillering fertilizers were administered after the rice started to re-green. In the NPKM treatment, the chemical fertilizers were applied in the same manner and quantity as the NPK treatment, and the organic fertilizer was applied with vetch for early rice and fresh pig manure for late rice at a rate of 22, 500 kg\u0026middot;hm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e. The organic fertilizers were applied as base fertilizers once. Other field measures, including seeding, transplanting, irrigation, and pest and disease management, were the same as the local high-yield cultivation practices.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe fertilization application of each treatment (kg\u0026middot;hm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eChemical fertilizer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eOrganic fertilizer\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eK\u003csub\u003e2\u003c/sub\u003eO\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eEarly rice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHNPK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPKM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e123.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e60.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eLate rice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHNPK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNPKM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e55.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e66.15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Analysis of yield and yield components\u003c/h2\u003e \u003cp\u003eRice was harvested at the mature stages, and the yield was calculated according to the weight and plot area. Three representative plants were obtained to determine yield components, including ear length, grains per panicle, setting rate, and 1000-grain weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of tiller dynamics\u003c/h2\u003e \u003cp\u003eTen fixed hills were identified in each plot to count the tillers starting at seven days after transplanting at a 7-day interval until the number diminished. The tillering rate and the ear-bearing tiller percentage were computed as below:\u003c/p\u003e \u003cp\u003etillering rate\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(=\\frac{\\text{m}\\text{a}\\text{x}\\text{i}\\text{m}\\text{u}\\text{m} \\text{t}\\text{i}\\text{l}\\text{l}\\text{e}\\text{r} \\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}-\\text{b}\\text{a}\\text{s}\\text{i}\\text{c} \\text{s}\\text{e}\\text{e}\\text{d}\\text{l}\\text{i}\\text{n}\\text{g} \\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}}{\\text{b}\\text{a}\\text{s}\\text{i}\\text{c} \\text{s}\\text{e}\\text{e}\\text{d}\\text{l}\\text{i}\\text{n}\\text{g} \\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r}}\\times 100\\%\\)\u003c/span\u003e\u003c/span\u003e (1)\u003c/p\u003e \u003cp\u003eear-bering tiller percentage \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\frac{ \\text{e}\\text{f}\\text{f}\\text{e}\\text{c}\\text{t}\\text{i}\\text{v}\\text{e} \\text{p}\\text{a}\\text{n}\\text{i}\\text{c}\\text{l}\\text{e}}{ \\text{m}\\text{a}\\text{x}\\text{i}\\text{m}\\text{u}\\text{m} \\text{t}\\text{i}\\text{l}\\text{l}\\text{e}\\text{r} \\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} }\\times 100\\%\\)\u003c/span\u003e\u003c/span\u003e(2)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurement of dry matter accumulation and leaf chlorophyll\u003c/h2\u003e \u003cp\u003eThe aboveground segments of rice were collected at the tillering stage, full heading stage, filling stage, and mature stage. The plant samples were separated into stem, leaf, and spike, and then baked in an oven at 105\u0026deg;C for 30 minutes, then dried at 85\u0026deg;C for 6 hours before determining dry weight. A chlorophyll meter [SPAD-502, Soil and plant analysis development (SPAD), Minolta Camera Co. Osaka, Japan] was utilized for chlorophyll measurement on the 20 top fully expanded leaves per plot at the tillering stage, full heading stage, filling stage, and mature stage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Leaf sample preparation and RNA-Seq\u003c/h2\u003e \u003cp\u003eLeaf samples were harvested at the full heading and filling stages, flash-frozen using liquid nitrogen, and stored at -80\u0026deg;C before being sent to Qinke-Tech (Beijing, China) for RNA-seq by Illumina HiSeq2000 (Illumina Inc., San Diego, CA, USA). Raw data were filtered by removing reads, including adapters, poly-N\u0026thinsp;\u0026gt;\u0026thinsp;10%, and low-quality reads, to obtain clean reads for further analysis. Clean reads were aligned to the rice reference genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eftp://ftp.ensemblgenomes.org/pub/release42/plants/fasta/oryza_sativa/dna/Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.gz\u003c/span\u003e\u003cspan address=\"http://ftp://ftp.ensemblgenomes.org/pub/release42/plants/fasta/oryza_sativa/dna/Oryza_sativa.IRGSP-1.0.dna.toplevel.fa.gz\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by Hisat (Hierarchical Indexing for Spliced Alignment of Transcripts) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) was employed to characterize the gene expression level and calculated as: FPKM = (Number of cDNA fragments uniquely aligned to Gene A / Total number of fragments uniquely aligned to all reference genes) \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/ (Transcript Length in kilobases of Gene A's exonic regions)[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. DEseq2 was employed for differential expression analysis; the criteria set for significant differences were a fold change\u0026thinsp;\u0026ge;\u0026thinsp;2 and a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eWe used one-way analysis of variance (ANOVA) to characterize differences in the responses of yield, yield component, dry matter accumulation, and SPAD value of long-term fertilization and applied a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Graphs were generated using Origin 9.0 software (Microcal Software, Northhampton, MA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Data availability\u003c/h2\u003e \u003cp\u003eData available on request from the corresponding author.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Impact of Long-Term Fertilization on Early Rice Yield\u003c/h2\u003e\n \u003cp\u003eLong-term fertilization has significantly impacted early rice yield (Fig.\u0026nbsp;1a). The yield sequence progressed from NPKM\u0026thinsp;\u0026gt;\u0026thinsp;HNPK\u0026thinsp;\u0026gt;\u0026thinsp;NPK. Compared to NPK, both HNPK and NPKM treatments significantly elevated the yield of double-season early rice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Following 42 years of continuous fertilization, the yields increased by 56.64% and 90.33%, respectively. The yield components were significantly impacted by long-term fertilization treatments. Compared to NPK, HNPK and NPKM significantly increased the number of effective panicles (Fig. 1b), spikelet density (Fig. 1d), and 1000-grain weight (Fig. 1f). The grains per panicle of NPKM were significantly higher than chemical fertilizer treatments (NPK, HNPK) (Fig. 1c), demonstrating the opposite trend in seed setting (Fig. 1e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 The tiller dynamic and ear-bearing\u003c/h2\u003e\n \u003cp\u003eLong-term fertilization significantly impacted the tiller dynamic of early rice (Fig. 2a). The chemical fertilizer treatments (NPK, HNPK) maintained the same tillering process, and the tiller number increased from 2 to 7 weeks after transplanting and then decreased. The NPKM treatment prolonged the tillering process. The HNPK and NPKM treatments significantly increased the tiller number of early rice compared to NPK, with the tillering rate increasing by 43.84% and 72.73%, respectively (Fig. 3b). The ear-bearing tillering percentage of HNPK was significantly lower than NPK and NPKM (Fig. 2b).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Dry matter accumulation\u003c/h2\u003e\n \u003cp\u003eAs depicted in Fig.\u0026nbsp;3, the dry matter accumulation of early rice was significantly affected by long-term fertilization. The total dry matter weight of the tillering stage, full heading stage, filling stage, and mature stage followed a trend of NPKM\u0026thinsp;\u0026gt;\u0026thinsp;HNPK\u0026thinsp;\u0026gt;\u0026thinsp;NPK, and significantly differed between the three fertilization treatments except between NPK and HNPK at the tillering stage (Fig.\u0026nbsp;3d). Compared to NPK, HNPK and NPKM significantly increased the stem dry matter of each growth stage, with an increase of 36.95%-46.33%, 19.43%-34.41%, 43.59%-61.51%, and 39.23%-46.58%, respectively (Fig.\u0026nbsp;3a). The dry matter of leaves in the tillering stage followed the trend of HNPK\u0026thinsp;\u0026gt;\u0026thinsp;NPKM\u0026thinsp;\u0026gt;\u0026thinsp;NPK, with HNPK and NPKM significantly higher than NPK (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;3b). There was no significant difference in grain dry material across the three fertilization treatments at the full heading stage, and significant differences were identified at the filling and mature stages (Fig.\u0026nbsp;3c), and followed a trend of NPKM\u0026thinsp;\u0026gt;\u0026thinsp;HNPK\u0026thinsp;\u0026gt;\u0026thinsp;NPK, with significant differences between treatments (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Compared to NPK and HNPK, NPKM significantly elevated the dry matter accumulation from the tillering to full heading stage, with an increase of 90.78%-107.56%. Compared to NPK, NPKM and HNPK increased dry matter accumulation from the full heading to the filling stage by 216.09% and 141.46%. The dry matter accumulation from the filling to the mature stage showed no significant difference among the three fertilization treatments. The dry matter translocation from the stem and leaves to spike was 5.51 g/plant (NPKM), 2.2 g/plant (HNPK), and 1.26 g/plant (NPK). Our findings indicated that the combination of fertilizers with organic fertilizers could significantly promote dry matter accumulation in early rice and increase the transfer of dry matter from the stem and leaf to the spike during the filling stage to maturity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Chlorophyll dynamic\u003c/h2\u003e\n \u003cp\u003eLong-term fertilization significantly affected the early rice chlorophyll content of leaves (Fig.\u0026nbsp;4). Compared to NPK, HNPK significantly increased leaf chlorophyll content during the filling and mature stages with an increase of 14.39% and 29.68%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under the NPKM treatment, leaf chlorophyll content was significantly higher than in NPK and HNPK treatments during the tillering, full heading, grain-filling, and mature stages, with increases of 22.05%-25.58%, 38.80%-58.78%, and 57.47%-104.21%, respectively. The chlorophyll levels of sword leaves from tillering to the full heading stage increased by 5.24 (NPK), 5.09 (HNPK), and 6.11 (NPKM). It decreased by 6.94% (NPKM), 18.23% (HNPK), and 26.29% (NPK) from the full heading to the filling stage. The decreases in chlorophyll content from filling to mature stage of NPK, HNPK, and NPKM were 5.56, 13.74, and 13.96, respectively. These findings indicated that integrating chemical fertilizers with organic fertilizers can significantly enhance the chlorophyll content of sword leaves in early rice and significantly mitigate the reduction of chlorophyll from the full heading to the filling stage.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Relationship between yield, chlorophyll, and dry matter accumulation\u003c/h2\u003e\n \u003cp\u003eCorrelation analysis demonstrated that chlorophyll and dry matter accumulation significantly influenced early rice yield under long-term fertilization (Fig. 5). The yield was significantly positively correlated with the change in chlorophyll from full heading to filling stage, and was significantly positively correlated with dry matter accumulation from tillering to full heading stage (△DM1) and full heading to filling stage (△DM2).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 DEGs in rice leaves at full heading stage induced by long-term fertilization\u003c/h2\u003e\n \u003cp\u003eLong-term fertilization significantly influenced leaf gene expression in early rice (Fig. 6). There were 3585 differentially expressed genes (DEGs) between NPKM and NPK treatments, of which 1478 genes were up-regulated and 2107 genes were down-regulated. There were 2051 DEGs between NPKM and HNPK, of which 1251 were up-regulated and 800 were down-regulated. There were 3518 DEGs between HNPK and NPK, including 1213 up-regulated and 2305 down-regulated genes. There were 320 DEGs in NPK vs. NPKM, NPK vs. HNPK, and HNPK vs. NPKM, with 903, 882, and 413 unique DEGs, respectively.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Senescence and photosynthesis-related DEGs\u003c/h2\u003e\n \u003cp\u003eSenescence and photosynthesis were considerably influenced by long-term fertilization. Gene Ontology (GO) enrichment (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) demonstrated that there were 20 DEGs enriched in rice senescence, mainly involved in encoding proteins containing DUF584 domain, aging-related cysteine protease and its precursor proteins, and aging-related coenzymes. KEEG analysis indicated that DEGs enriched in the photosynthesis pathway were mainly involved in photosystem I, photosystem II, cytochrome, ferredoxin, and other related proteins (Fig. 7).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe DEGS enriched in the senescence process\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGene ID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eFPKM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eDEGS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eGene function\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNPK\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHNPK\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNPKM\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eⅠ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eⅡ\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eⅢ\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs06g50330\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.823\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprotein EARLY-RESPONSIVE TO DEHYDRATION 7, chloroplastic\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs01g52740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.463\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.025\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eresponse to endogenous stimulus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs03g02280\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.192\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.186\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.681\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs05g45450\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.409\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.597\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs04g43990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.5230\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.241\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs01g52730\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.196\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.715\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.436\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs02g41840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.769\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs10g27350\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.529\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.793\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.405\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs10g33990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.975\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.638\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e24.841\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs04g33760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.445\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e4.248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.476\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDUF584 domain containing protein, putative, expressed\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs03g54130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.382\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esenescence-specific cysteine protease SAG39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs04g13140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.189\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.274\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003esenescence-specific cysteine protease SAG39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs01g42780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.563\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.836\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.776\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecysteine proteinase EP-B 2 precursor\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs05g27580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.725\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.070\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ewound-induced protein WI12,\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs07g49114\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.991\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.614\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.984\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ewound-induced protein WI12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs09g01000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e221.300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e817.118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e314.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eputative senescence-associated protein, partial\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs10g07010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.051\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.251\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-ketoacyl-CoA synthase\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs01g21250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e302.410\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.678\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003elate embryogenesis abundant protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs11g25160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.780\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003etropinone reductase homolog At5g06060 isoform X1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOs01g59570\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.318\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eprobable LRR receptor-like serine/threonine-protein kinase At1g51810\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\"\u003e\u003cstrong\u003eNote\u003c/strong\u003e: The DEGs I, Ⅱ, and Ⅲ represented NPK vs. NPKM, NPK vs. HNPK, and HNPK vs. NPKM, respectively. The letters D, U, and N indicated the gene expression of each group exhibiting down-regulated, up-regulated, and non-regulated.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Impact of Long-Term Fertilization on the Yield of Early Rice\u003c/h2\u003e \u003cp\u003eFertilization is a critical agronomic practice for maintaining paddy productivity and guaranteeing high and stable rice yields. Shen [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] summarized long-term fertilization experiments and found that organic and chemical fertilizers significantly enhanced crop yields. Additionally, numerous studies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] have demonstrated that the maximum yield increases were achieved when combining organic with chemical fertilization, consistent with this study's findings in which early rice yields followed a trend of NPKM\u0026thinsp;\u0026gt;\u0026thinsp;HNPK\u0026thinsp;\u0026gt;\u0026thinsp;NPK. Previous studies investigated the mechanisms of long-term fertilization impacted crop yields. A study by Mao et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] demonstrated that chemical fertilizers offer rapid nutrients while organic fertilizers improve soil physical and chemical properties. Research by Duan et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] indicated that crop yield was positively correlated with soil fertility, whereas long-term chemical fertilization alone showed no significant enhancement in soil fertility [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and organic fertilizers application significantly improved soil fertility and enhanced soil enzyme activity and microbial community functional diversity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], to thereby improve soil productivity. Zhou et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] suggested that long-term exclusive use of organic fertilizers cannot attain the critical nutrient requirements of crops. Combining chemical and organic fertilization was critical in providing immediate nutrients and improving soil fertility [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], explaining why the highest crop yields were achieved under this fertilization method over prolonged durations. Furthermore, this study found that HNPK treatments significantly elevated early rice yields compared to NPK treatments, suggesting that increasing chemical fertilization could significantly improve crop yields. This contrasts with previous findings that conventional chemical fertilization satisfies normal crop growth but that increased rates do not enhance yield [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This discrepancy might be associated with the initially low fertilization levels in this experiment, suggesting that as breeding techniques improve and novel rice varieties demand increased nutrients, normal fertilization levels may not suffice, limiting the yield potential. Increased chemical fertilization could achieve these enhanced nutrient demands and increase rice yield to some extent. Furthermore, our findings demonstrated the combination of chemical and organic fertilizers significantly improved yield components. Amanullah, H. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] also identified the combined use of mineral fertilizer and organic fertilizer produced improved yield and yield components. This may be due to the easily available nutrient supply from chemical fertilizer at the early growth stage, and organic fertilizer could reduce nutrient loss and maintain the supply of nutrients in later growth stages [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Impact of long-term fertilization on the growth of early rice and associated gene expression\u003c/h2\u003e \u003cp\u003eCrop growth and development were influenced by genotype and environmental factors, with genotype exerting a decisive role. However, environmental factors like climate and fertilization impact the expression of crop-related genes. This study identified that long-term fertilization significantly influenced gene expression in early rice leaves at the full heading stage. Liu et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] also observed characteristic gene expression in crops under long-term fertilization. Through GO and KEEG functional analysis of DEGs, this study identified significant enrichment in genes associated with photosynthesis at the full heading stage of early rice, especially involved in Photosystem I, Photosystem II, cytochromes, and ferredoxin. The accumulation of dry matter and chlorophyll post-heading is consistent with crop yield. This indicated that long-term fertilization impacts the expression of genes related to photosynthesis in early rice and regulates the formation of chlorophyll and dry matter in leaves, thereby impacting yield. Dry matter accumulation is the material foundation of crop yield, and the accumulation, transfer, and distribution of dry matter significantly affect crop yields [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The findings demonstrate that dry matter accumulated from the full heading to the filling stage was significantly positively correlated with yield, while from filling to the maturity stage, there was no significant correlation. The NPKM treatment significantly increased dry matter accumulation from the full heading to the filling stage compared to NPK, and there was no significant difference in dry matter accumulation among the three fertilization methods from the filling to the mature stage. However, the increase in panicle dry matter during the same period was higher with combined NPK and organic fertilizers, further illustrating that this combination can significantly increase dry matter accumulation from full heading to the grain-filling stage and promote the transfer of stem and leaf dry matter to panicles during the filling to mature stages. Xu et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] showed that organic fertilizers, in combination with chemical fertilizers, decompose to replenish nutrients for rice, increasing the accumulation and distribution of dry matter in various organs and enhancing post-heading assimilation of nutrients. Most dry matter originates from leaf photosynthesis, governed by the rate of accumulation and duration [\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Chloroplasts are the primary sites of photosynthesis, and chlorophyll content was closely tied to the photosynthetic productivity of rice [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Correlation analysis demonstrated that chlorophyll content was significantly positively correlated with dry matter and yield in rice, with nitrogen, phosphorus, and potassium combined with organic fertilizers leading to significantly higher chlorophyll content during the full heading, filling, and maturity stages compared to chemical fertilizers alone, indicating that this combination enhanced photosynthesis in early rice, promoted dry matter accumulation and yield formation. Additionally, combining chemical and organic fertilizers promoted the tillering of early rice, increasing effective panicles.\u003c/p\u003e \u003cp\u003eMoreover, the integration of chemical and organic fertilizers delayed the reduction of chlorophyll content in leaves during later stages. Correlation analysis also demonstrated that the decrease in leaf chlorophyll content from the full heading to the filling stage was significantly negatively correlated with rice yield and dry matter accumulation, indicating that the combination of chemical and organic fertilizers, to some extent, delayed rice senescence, extended the duration of dry matter accumulation, and enhanced yield. Zhang et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] obtained similar results, likely associated with the increased activity of protective enzymes in rice leaves and the lowered content of secondary metabolites like malondialdehyde (MDA) and free proline (Pro) under combined chemical and organic fertilizers [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. This study's transcriptome analysis also demonstrated that genes expressed in leaves at the full heading stage are significantly enriched in senescence, primarily involving proteins containing the DUF584 domain, associated with aging-associated cysteine proteases and precursors and related cofactors. Proteins containing the DUF584 domain played an essential role in rice aging, regulating the synthesis and breakdown of abscisic acid within the plant [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Cysteine aids in promoting GSH formation and scavenging superoxide radicals, revealing the molecular mechanisms by which long-term fertilization with chemical and organic fertilizers regulates the content of MDA, Pro, and other substances, delaying rice aging, enhancing dry matter accumulation, and improving yield formation.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eLong-term fertilization significantly influenced the gene expression of early rice leaves at the full heading stage, with DEGs being enriched in metabolic pathways associated with photosynthesis and aging. These genes were primarily involved in Photosystem I, Photosystem II, cytochromes, ferredoxin, proteins containing the DUF584 domain, cysteine proteases associated with aging, and the encoding and synthesis of their related precursors and cofactors. The application of chemical fertilizer in combination with organic fertilizers significantly increased tillering and enhanced the accumulation of dry matter in early rice, particularly from the full heading to the filling stage, strengthening the transport of dry matter from the stem and leaves to the panicles from the filling to the maturity stage. Additionally, this fertilization strategy significantly increases the chlorophyll content in the sword leaves of rice and effectively alleviates chlorophyll degradation in leaves from the full heading to the filling stage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, Zhihua Hu; methodology, Zhihua Hu, Kailou Liu; writing\u0026mdash;original draft preparation, Zhihua Hu, Kailou Liu, Xiaolin Xu; writing\u0026mdash;review and editing, Dandan Hu, Huijie Song, Yan Wu, Jianfu Wu; funding acquisition, Kailou Liu, Jianfu Wu. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by the National Natural Science Foundation of China (42207398; 32260808); the key research and development plan \u0026quot;open ranking\u0026quot; project of Jiangxi Province, China(20223BBF61016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003eData reported within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFood and Agriculture Organization of the United Nations (FAO). 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Gene, 846 (2022). \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"long-term fertilization, early rice, dry matter accumulation, chlorophyll, differently expressed genes (DEGs)","lastPublishedDoi":"10.21203/rs.3.rs-4640724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4640724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFertilization is crucial for rice growth and yield formation. We conducted a 42-year long-term fixed experiment in southeast China, examining nine treatments. This study focused on three treatments: a combination of chemical N, P, and K (NPK), a double dose of chemical N, P, and K (HNPK), and a combination of chemical fertilizer and organic fertilizers (NPKM). We assessed rice yield, yield components, tiller dynamics, dry matter accumulation, chlorophyll dynamics, and leaf transcriptome at the full heading stage. Results indicated that early rice yield followed the order of NPKM \u0026gt; HNPK \u0026gt; NPK. Compared to NPK, HNPK and NPKM significantly increased spikelet density, effective panicles, and 1000-grain weight, while also promoting tillering. NPKM and HNPK significantly enhanced dry matter accumulation from the full heading stage to the filling stage and facilitated the transport of dry matter from leaves and stems to spikes during the filling to mature stages. NPKM consistently maintained higher chlorophyll content than HNPK and NPK at all stages, significantly reducing chlorophyll decline from the full heading stage to the filling stage. Correlation analysis revealed a significant positive relationship between yield and both chlorophyll content and dry matter accumulation under long-term fertilization. There was also a significant negative correlation between yield and chlorophyll reduction from the full heading stage to the filling stage. Differential gene expression analysis at the full heading stage showed significant enrichment in photosynthesis and plant senescence metabolism pathways among different fertilization treatments. Overall, the combined application of chemical and organic fertilizers significantly increased early rice yield by enhancing tillering, regulating photosynthesis and senescence-related gene expression, boosting dry matter accumulation from the full heading stage to the filling stage, and improving dry matter transport to spikes from the filling to the mature stage.\u003c/p\u003e","manuscriptTitle":"Effect of long-term fertilization on the growth and yield formation of early rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 17:43:21","doi":"10.21203/rs.3.rs-4640724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-02T03:52:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-06T18:19:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251679155707654038120002033121398110585","date":"2024-10-14T04:31:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-15T08:42:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326751967181670415946345042881105220999","date":"2024-08-23T13:31:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-24T02:58:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"321873661240704927816387519208947824971","date":"2024-07-18T01:59:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T10:58:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-17T10:50:09+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-02T12:12:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-28T06:36:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-26T07:23:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"207e27dc-1eea-449f-b26a-df1099c3c16d","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34786170,"name":"Biological sciences/Plant sciences"},{"id":34786171,"name":"Earth and environmental sciences/Environmental sciences"}],"tags":[],"updatedAt":"2025-03-17T16:05:40+00:00","versionOfRecord":{"articleIdentity":"rs-4640724","link":"https://doi.org/10.1038/s41598-025-93474-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-12 15:58:51","publishedOnDateReadable":"March 12th, 2025"},"versionCreatedAt":"2024-07-19 17:43:21","video":"","vorDoi":"10.1038/s41598-025-93474-8","vorDoiUrl":"https://doi.org/10.1038/s41598-025-93474-8","workflowStages":[]},"version":"v1","identity":"rs-4640724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4640724","identity":"rs-4640724","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}