Perinatal exposure to high concentration glyphosate-based herbicides induces intestinal apoptosis by activating endoplasmic reticulum stress in offspring.

Glyphosate-based herbicides (GBHs), the most widely used pesticide worldwide, have been reported to impair organ function in humans and animals. However, research on the effect of maternal GBHs exposure on the intestinal health of offspring has received little attention. Based on the glyphosate limits defined by Codex Alimentarius Commission and European Food Safety Authority, this study established pregnant sow exposure models to investigate the influence of low (L-GBHs, 20 mg/kg) and high concentration GBHs (H-GBHs, 100 mg/kg) on the intestinal health of offspring and proposed the protective mechanism mediated by betaine. The results showed that the intestinal morphology and barrier function of suckling piglets were damaged in the H-GBHs group. H-GBHs increased the activity of glutathione peroxidase (GPX) and levels of methane dicarboxylic aldehyde (MDA), hydrogen peroxide (H2O2) and inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-10 (IL-10)) in suckling piglets and activated Nrf2-mediated antioxidant signaling pathway. Subsequently, we found that exposure to H-GBHs triggered endoplasmic reticulum stress (ERS) and further induced apoptosis by upregulating the expression of Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and Caspase12. Moreover, H-GBHs exposure perturbed mitochondrial membrane fusion and electron transport in mitochondrial respiratory chains by increasing the mRNA expression of mitofusin-2 (MFN2) and succinate dehydrogenase subunit A (SDHA), causing mitochondrial dysfunction. Dietary supplementation with betaine provided modest protection against GBHs-induced intestinal damage in suckling piglets. These findings reveal the mechanism of GBHs-induced intestinal damage in offspring, improving our understanding of the risk of GBHs exposure in pregnant women and suggesting the potential protective effects of betaine against GBHs poisoning.

FIT-based risk-stratification model effectively screens colorectal neoplasia and early-onset colorectal cancer in Chinese population: a nationwide multicenter prospective study.

No fully validated risk-stratification strategies have been established in China where colonoscopies resources are limited. We aimed to develop and validate a fecal immunochemical test (FIT)-based risk-stratification model for colorectal neoplasia (CN); 10,164 individuals were recruited from 175 centers nationwide and were randomly allocated to the derivation (n = 6776) or validation cohort (n = 3388). Multivariate logistic analyses were performed to develop the National Colorectal Polyp Care (NCPC) score, which formed the risk-stratification model along with FIT. The NCPC score was developed from eight independent predicting factors and divided into three levels: low risk (LR 0-14), intermediate risk (IR 15-17), and high risk (HR 18-28). Individuals with IR or HR of NCPC score or FIT+ were classified as increased-risk individuals in the risk-stratification model and were recommended for colonoscopy. The IR/HR of NCPC score showed a higher prevalence of CNs (21.8%/32.8% vs. 11.0%, P < 0.001) and ACNs (4.3%/9.2% vs. 2.0%, P < 0.001) than LR, which was also confirmed in the validation cohort. Similar relative risks and predictive performances were demonstrated between non-specific gastrointestinal symptoms (NSGS) and asymptomatic cohort. The risk-stratification model identified 73.5% CN, 82.6% ACN, and 93.6% CRC when guiding 52.7% individuals to receive colonoscopy and identified 55.8% early-onset ACNs and 72.7% early-onset CRCs with only 25.6% young individuals receiving colonoscopy. The risk-stratification model showed a good risk-stratification ability for CN and early-onset CRCs in Chinese population, including individuals with NSGS and young age.

Perinatal exposure to glyphosate-based herbicides impairs progeny health and placental angiogenesis by disturbing mitochondrial function.

Glyphosate-based herbicides (GBHs) are the most widely used pesticide worldwide and can provoke placental injury. However, whether and how GBHs damage angiogenesis in the placenta is not yet known. This work evaluated the safety of glyphosate on pregnant sows based on the limit level by governments and investigated the effects and mechanism of Low-GBHs (20 mg/kg) and High-GBHs (100 mg/kg) exposure on placental angiogenesis. Results showed that gestational exposure to GBHs decreased placental vessel density and cell multiplication by interfering with the expression of VEGFA, PLGF, VEGFr2 and Hand2 (indicators of angiogenesis), which may be in relation to oxidative stress-induced disorders of mitochondrial fission and fusion as well as the impaired function of the mitochondrial respiratory chain. Additionally, GBHs destroyed barrier function and nutrient transport in the placenta, and was accompanied by jejunum oxidative stress in newborn piglets. However, GBHs exposure had no significant differences on sow reproductive performance. As a natural antioxidant, betaine treatment protected placenta and newborn piglets against GBHs-induced damage. In conclusion, GBHs impaired placental angiogenesis and function and further damaged the health of postnatal progeny, these effects may be linked to mitochondrial dysfunction. Betaine treatment following glyphosate exposure provided modest relief.

Dietary Glucose Ameliorates Impaired Intestinal Development and Immune Homeostasis Disorders Induced by Chronic Cold Stress in Pig Model.

Endotherms are easily challenged by chronic cold stress. In this study, the development and injury of the small intestine in the Min pig model and Yorkshire pig model under chronic cold stress, and the molecular mechanisms by which glucose supplementation reduces small intestinal mucosal damage were investigated. The results showed that morphological structure lesions of the jejunal mucosa and ileal mucosa were visible in Yorkshire pigs under chronic cold stress. Meanwhile, the Occludin mRNA and protein expression in jejunal mucosa of Yorkshire pigs was decreased. Chronic cold stress enhanced the expression of Toll-like receptor 4 (TLR4), the myeloid differentiation main response 88 (MyD88), nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3), cleaved caspase-1, mature-IL-1ß, and high-mobility group box 1 (HMGB 1) mRNA and protein expression in jejunal mucosa of Yorkshire pigs, whereas the mRNA and protein of Bax was triggered in ileal mucosa. In Min pigs, no such deleterious consequences were observed. Dietary glucose supplementation ameliorates small intestinal mucosal injury, declined TLR4 and MyD88 expression in jejunal mucosa. In conclusion, chronic cold stress induced the small intestinal mucosa damage in Yorkshire pigs, whereas glucose supplementation mitigated the deleterious effects of chronic cold stress on the small intestine.

Correction: Yield performance of early-season rice cultivars grown in the late season of double-season crop production under machine-transplanted conditions.

[This corrects the article DOI: 10.1371/journal.pone.0213075.].

Glyphosate-based herbicides induces autophagy in IPEC-J2 cells and the intervention of N-acetylcysteine.

Glyphosate-based herbicides (GBHs) are the most widely used pesticide in the world, and its extensive use has increased pressures on environmental safety and potential human and livestock health risks. This study investigated the effects of GBHs on antioxidant capacity, inflammatory cytokines, and autophagy of porcine intestinal epithelial cells (IPEC-J2) and its molecular mechanism. Also, the protective effects of N-acetylcysteine (NAC) against the toxicity of GBHs were evaluated. Our results showed that the activities of antioxidant enzymes (SOD, GSH-Px) were decreased by GBHs. GBHs increased inflammatory factors (IL-1ß, IL-6, TNF-α) and the mRNA expression of iNOS and COX-2. GBHs induced the up-regulation of Nrf2/HO-1 pathway and the phosphorylation of IκB-α and NFκB p65, up-regulation of LC3-II/LC3-I, and down-regulation of P62, and NFκB inhibitor decreased the mRNA expression of inflammatory cytokines (IL-1ß, IL-6, IL-8). Moreover, NAC reduced the cytotoxicity by suppressing ROS levels, and changed the autophagy-related proteins such as the suppression of LC3-II conversion and up-regulation of P62. Our findings unveil a novel mechanism of GBHs effects on IPEC-J2 cells and NAC can reverse cytotoxicity to some extent.

Establishment method affects rice root plasticity in response to drought and its relationship with grain yield stability.

By responding to the variable soil environments in which they are grown, the roots of rice crops are likely to contribute to yield stability across a range of soil moistures, nutrient levels, and establishment methods. In this study, we explored different approaches to quantification of root plasticity and characterization of its relationship with yield stability. Using four different statistical approaches (plasticity index, slope, AMMI, and factor analytic) on a set of 17 genotypes including several recently-developed breeding lines targeted to dry direct-seeding, we identified only very few direct relationships between root plasticity and yield stability. However, genotypes identified as having combined yield stability and root plasticity showed higher grain yields across trials. Furthermore, root plasticity was expressed to a greater degree in puddled transplanted trials rather than under dry direct-seeding. Significant interactions between nitrogen and water resulted in contrasting relationships between nitrogen-use efficiency and biomass stability between puddled-transplanted and direct-seeded conditions. These results reflect the complex interaction between nitrogen, drought, and even different types of drought (as a result of the establishment method) on rice root growth, and suggest that although rice root plasticity may confer stable yield across a range of environments, it might be necessary to more narrowly define the targeted environments to which it will be most beneficial.

Proteomic profiling reveals differentially expressed proteins associated with amylose accumulation during rice grain filling.

BACKGROUND: Amylose accumulation in rice grains is controlled by genetic and environmental factors. Amylose content is a determinant factor of rice quality in terms of cooking and eating. Great variations in amylose content in indica rice cultivars have been observed. The current study was to identify differentially expressed proteins in starch and sucrose metabolism and glycolysis/gluconeogenesis pathways and their relationships to amylose synthesis using two rice cultivars possess contrasting phenotypes in grain amylose content. RESULTS: Synthesis and accumulation of amylose in rice grains significantly affected the variations between rice cultivars in amylose contents. The high amylose content cultivar has three down-regulated differentially expressed proteins, i.e., LOC_Os01g62420.1, LOC_Os02g36600.1, and LOC_Os08g37380.2 in the glycolysis/gluconeogenesis pathway, which limit the glycolytic process and decrease the glucose-1-phosphate consumption. In the starch and sucrose metabolic pathway, an up-regulated protein, i.e., LOC_Os06g04200.1 and two down-regulated proteins, i.e., LOC_Os05g32710.1 and LOC_Os04g43360.1 were identified (Figure 4). Glucose-1-phosphate is one of the first substrates in starch synthesis and glycolysis that are catalyzed to form adenosine diphosphate glucose (ADPG), then the ADPG is catalyzed by granule-bound starch synthase I (GBSS I) to elongate amylose. CONCLUSIONS: The results indicate that decreasing the consumption of glucose-1-phosphate in the glycolytic process is essential for the formation of ADPG and UDPG, which are substrates for amylose synthesis. In theory, amylose content in rice can be regulated by controlling the fate of glucose-1-phosphate.

Genotypic variation in the grain photosynthetic contribution to grain filling in rice.

Grain filling in rice, a staple cereal crop worldwide, is a critical determinant of grain yield and quality. However, there is little available information on the relationship between grain filling and grain photosynthetic capacity in rice. This study evaluated the genetic diversity among six rice cultivars for their grain filling rate (GR0) and the relationships with the grain chlorophyll contents and grain net photosynthetic rate (PN). Significant variations in GR0, PN, and the chlorophyll contents (a, b, and total) in the grains of the cultivars were observed. Approximately 90 % of the variation in GR0 was explained by the grain PN. General linear model regression revealed significant positive correlations between PN/GR0 and the chlorophyll contents (a, b, and total) in the grains. There was also a significant positive correlation between PN and GR0. These positive correlations suggest a direct positive relationship between the grain filling rate and grain chlorophyll contents, which is indicative of the high photosynthetic capacity of the grains during the early grain filling period. These results suggest that the grain chlorophyll contents could be used as a molecular marker in marker-assisted breeding programs for rice cultivars with high grain net photosynthetic capacity during the early period of grain filling to improve grain yield.

Cover crops and chicken grazing in a winter fallow field improve soil carbon and nitrogen contents and decrease methane emissions.

Using symbiotic farming methods [cover crops and chicken grazing (+ C)] in a winter fallow field, we found that the soil organic matter and total nitrogen of the + C treatment were 5.2% and 26.6% higher, respectively, than those of a treatment with cover crops and no chicken grazing (- C). The annual rice grain yield of the + C treatment was 3.8% higher than that of the - C treatment and 12.3% higher than that of the bare fallow field (CK), while the annual CH4 emissions of the + C treatment were 26.9% lower than those of the - C treatment and 10.6% lower than those of the CK treatment. The 100-year global warming potential of the + C treatment was 6.2% lower than that of the - C treatment. Therefore, the use of winter cover crops and chicken grazing in a winter fallow field was effective at reducing CH4 emissions and significantly improving soil nutrients and rice yield.

Primary-tiller panicle number is critical to achieving high grain yields in machine-transplanted hybrid rice.

The development of machine-transplanted hybrid rice is a feasible approach to meet the needs of both high grain yield and high labor efficiency in China, but limited information is available on the critical plant traits associated with high grain yields in machine-transplanted hybrid rice. This study was carried out to identify which type of culms (i.e., main stems and primary and secondary tillers) and which yield components of this culm are critical to achieving high grain yields in machine-transplanted hybrid rice. Field experiments were conducted with two hybrid rice cultivars grown under two densities of machine transplanting in two years. Results showed that total grain yield of main stems and primary and secondary tillers was not significantly affected by cultivar but was significantly affected by density and year. Averaged across cultivars, densities, and years, main stems and primary and secondary tillers contributed about 15%, 50%, and 35% to total grain yield, respectively. Total grain yield was not significantly related to grain yields of main stems and secondary tillers but was positively and significantly related to grain yield of primary tillers. Approximately 85% of the variation in total grain yield was explained by grain yield of primary tillers, which was positively and significantly related to primary-tiller panicles per m2 but not to spikelets per panicle, spikelet filling percentage, or grain weight of primary tillers. Based on these results, it is concluded that primary-tiller panicle number is essential for achieving high grain yields in machine-transplanted hybrid rice.

Decreasing hill density combined with increasing nitrogen rate led to yield decline in hybrid rice under low-light conditions.

Low light is a common environmental factor that adversely affects rice yields. This study was conducted to evaluate the combined effect of hill density and nitrogen (N) fertilizer rate on yield attributes in hybrid rice under low-light conditions. Field experiments were conducted in 2014 and 2015. Two hybrid rice cultivars (Y-liangyou 1 and Luoyou 9348) were grown under combinations of three hill density levels (high, 40 × 104 hills ha-1; moderate, 27 × 104 hills ha-1; low, 14 × 104 hills ha-1) and two N rate levels (high, 240 kg ha-1; moderate, 143-148 kg ha-1), and shaded from heading to maturity. Grain yield was highest in the combination of high hill density and moderate N rate and significantly declined with decreasing hill density combined with increasing N rate for both cultivars in both years. Averaged across two cultivars and two years, grain yield declined by about 4% for each 10% decrease in hill density combined with each 10% increase in N rate. A significant reduction in spikelet filling percentage was observed with decreasing hill density combined with increasing N rate in Y-liangyou 1 in 2015 and Luoyou 9348 in 2014. The same trend was observed for grain weight in Y-liangyou 1 in 2014 and Luoyou 9348 in 2015. These results indicate that adopting the practice of decreasing hill density combined with increasing N rate can result in poor grain filling and consequently yield decline in hybrid rice under low-light conditions.

Grain filling of early-season rice cultivars grown under mechanical transplanting.

High yields of mechanized intensive rice-based cropping systems, e.g. double-season cropping using early- and late-season rice, are important to ensure national food security in China. However, few studies addressing the relationship between grain weight and grain yield of early-season rice under machine-transplanted conditions. A field experiment was conducted to determine the critical grain-filling characteristics and related physiological aspects that contribute to high grain weight in machine-transplanted early-season rice. The results showed that grain yield was significantly positively correlated with grain weight but not with panicles per m2, spikelets per panicle, and spikelet-filling percentage. Furthermore, this study demonstrated that there was a significant positive correlation between grain weight and mean grain-filling rate, which was significantly positively correlated with harvest index and grain cytokinin content. These results indicate that high grain-filling rate driven by good transport of assimilates to grains and strong grain sink strength is responsible for high grain weight in machine-transplanted early-season rice.

Contrasting change in biomass translocation with environment in two rice hybrids.

Translocation of biomass produced during pre-heading to grains is a determinant of grain yield, but also plays an important role in adaptation to unfavorable environments during post-heading in rice. In this study, field experiments were conducted to determine the critical factors that regulate biomass translocation in rice. Biomass translocation and production characteristics of two rice hybrids (Guiliangyou 2 and Y-liangyou 1) were compared between two site-year environments (Naning-2014 and Yongan-2018). Results showed that biomass translocation parameters (biomass translocation amount and rate and contribution of biomass translocation to filled grain weight) and ratio of biomass production during pre-heading to post-heading (BPpre/BPpost ratio) decreased in Guiliangyou 2 but increased in Y-liangyou 1 with the environment change from Nanning-2014 to Yongan-2018. The decreased BPpre/BPpost in Guiliangyou 2 was attributable to increased biomass production during post-heading (BPpost), while the increased BPpre/BPpost ratio in Y-liangyou 1 was due to increased biomass production during pre-heading (BPpre). Higher cumulative incident solar radiation and larger diurnal temperature variation were responsible for the increased BPpost in Guiliangyou 2 and the increased BPpre in Y-liangyou 1 grown in Yongan in 2018 compared to in Nanning in 2014. The results of this study indicate that changes in biomass translocation and production with environment (climate) in rice are dependent on genotype and that the BPpre/BPpost ratio is an important factor regulating biomass translocation in rice.

Quantifying accumulation characteristics of glutelin and prolamin in rice grains.

Glutelin and prolamin are the two major proteins in rice grains. Grain content of glutelin is considerably higher than that of prolamin in rice, but there is limited information on the factors determining the different grain contents of glutelin and prolamin. To address this knowledge gap, the present study compared final weight per grain and accumulation characteristics of glutelin and prolamin in four rice cultivars. Results showed that final glutelin weight per grain was 3.24-3.95 times higher than final prolamin weight per grain. Glutelin and prolamin accumulation processes were well fitted by the logistic equation. The initial, maximum, and mean accumulation rates of glutelin were 1.69-4.67 times higher than those of prolamin. The active accumulation duration of glutelin was 2.9-5.1 d longer than that of prolamin. These results indicate that both higher accumulation rate and longer active accumulation duration are responsible for the higher final weight per grain of glutelin compared to prolamin in rice.

Yield performance of machine-transplanted double-season rice grown following oilseed rape.

Growing oilseed rape in the fallow season may be a feasible alternative to growing green manure (e.g. Chinese milk vetch) for improving rice productivity. The objective of this study was to determine the yield performance of machine-transplanted double-season rice (i.e. early- and late-season rice) grown following oilseed rape. Field experiments were conducted to compare machine-transplanted double-season rice grown following oilseed rape, Chinese milk vetch and fallow (i.e. no crop) at Hengyang and Yueyang, Hunan Province, China in three cropping cycles from 2014 to 2017. Results showed that machine-transplanted double-season rice grown following oilseed rape and Chinese milk vetch produced similar grain yield, which was higher than that grown following fallow across two sites and three cropping cycles. The higher grain yield of machine-transplanted double-season rice grown following oilseed rape and Chinese milk vetch was attributable to improvement in both sink size (spikelet number per m2) and source capacity (total biomass). However, the reasons for the improved sink size of machine-transplanted double-season rice grown following oilseed rape and Chinese milk vetch were not entirely the same. Growing oilseed rape increased panicle size (spikelet number per panicle) and panicle number in early- and late-season rice, respectively, while growing Chinese milk vetch increased panicle number in both the early- and late-season rice. Our study suggests that growing oilseed rape in the fallow season is a useful alternative strategy for improving productivity of machine-transplanted double-season rice.

Yield performance of early-season rice cultivars grown in the late season of double-season crop production under machine-transplanted conditions.

In order to solve the problem of labor shortage in double-season rice production areas, machine transplanting, as opposed to manual transplanting, has become the more popular alternative method in rice cultivation. However, the most existing late rice cultivars are not suitable for machine double-season rice cultivation due to their long duration of growth. Therefore, based on the previous studies we chose early season rice cultivars to meet the needs of machine double-season rice cultivation. In this study, field experiments were conducted during the late season in 2015 and 2016 in Liuyang County, Hunan Province, China. Grain yield and yield-related traits were compared among eight early-season cultivars (Liangyou 6, Lingliangyou 211, Lingliangyou 268, Zhuliangyou 819, Xiangzaoxian 32, Xiangzaoxian 42, Zhongjiazao 17, and Zhongzao 39) in 2015 and four cultivars (Lingliangyou 268, Zhuliangyou 819, Zhongjiazao 17, and Zhongzao 39) in 2016, selected from the highest yielding cultivars grown in 2015. Lingliangyou 268 produced 8-44% higher grain yield than did the other cultivars except Zhongjiazao17 in 2015. This higher grain yield was driven by grain weight and aboveground biomass. The greater aboveground biomass in Lingliangyou 268 was mainly attributed to higher apparent radiation use efficiency (aboveground biomass/incident solar radiation). Our study suggests that improvement in grain weight and apparent radiation use efficiency were critical to the high grain yield of early-season rice cultivars grown in late season under machine transplanting conditions.

Amino acid content in rice grains is affected by high temperature during the early grain-filling period.

Amino acid content in grains is an important nutritional quality trait in rice. High temperature can affect rice quality by accelerating grain filling. However, there is limited information available on the influence of high temperature on amino acid content in rice grains, especially under natural conditions. In this study, grain-filling traits and amino acid content in the grain of two rice cultivars (Luliangyou 996 and Lingliangyou 268) were compared between two years (2016 and 2017) with contrasting temperatures during the early grain-filling period under field conditions. Average daily mean temperature during the period of 0-5 days after full heading in 2016 (30.1 °C) was 5.4 °C higher than that in 2017. Initial, maximum, and mean grain-filling rates were 42-307% higher in 2016 than in 2017 for Luliangyou 996 and Lingliangyou 268. The time taken to reach the maximum grain-filling rate and active grain-filling duration were 6.3-10.7 d shorter in 2016 compared to 2017 for Luliangyou 996 and Lingliangyou 268. Grain weight was equal to or significantly higher in 2016 than in 2017 for Luliangyou 996 and Lingliangyou 268. N accumulation and N content in the grain were significantly lower in 2016 than in 2017 for both cultivars. The grain contents of all detected amino acids, except for methionine in Luliangyou 996, were significantly lower in 2016 than in 2017. Our study suggests that high temperature during the early grain-filling period can result in an accelerated grain-filling process, reduced N accumulation and content in rice grains, and consequently reduced amino acid content in the grain.

Contrasting responses of grain yield to reducing nitrogen application rate in double- and single-season rice.

Reducing N rate without sacrificing grain yield is crucial for sustainable rice production in China. In this study, field experiments were conducted in 2016 and 2017 to determine whether the response of grain yield to reducing N rate varied between double-season rice (DSR) and single-season rice (SSR). Three N rates were applied for each rice crop, i.e. 150 kg ha-1 (N150, the locally recommended N rate), 90 kg ha-1 (N90), and 0 kg ha-1 (N0). Grain yield was lower under N90 than under N150 in DSR by 11-18%, whereas the difference was not significant in SSR. Grain yield under N0 was 36-63% lower in DSR than in SSR. There was a significant relationship between percentage change in grain yield comparing N90 to N150 with grain yield under N0. Furthermore, it was observed that grain yield under N0 was not significantly associated with growth duration but was closely associated with daily grain yield under N0, and the relationships of daily grain yield under N0 with seasonal average daily mean temperature and solar radiation were not significant. These results indicate that response of grain yield to reducing N rate was more sensitive in DSR compared to SSR due to lower soil N productivity. Growth duration and climatic conditions did not explain the difference in soil N productivity between DSR and SSR. The findings of this study should encourage more research into comparing the inherent traits of plants, especially the morphology and physiology of the root system, between DSR and SSR.

Continuous applications of biochar to rice: Effects on nitrogen uptake and utilization.

Improving soil quality is critical for increasing rice yield, and biochar could be a beneficial soil amendment for high yield. This study was conducted to determine the effects of continuous (repeated seasonal) applications of biochar on nitrogen (N) uptake and utilization in rice. A fixed field experiment was done in Yongan Town, Hunan Province, China, in six continuous seasons (the early and late rice-growing seasons from 2015 to 2017). Results showed that biochar application did not significantly affect soil N uptake in the first four seasons. The effect of biochar application on fertilizer N uptake was not significant in three of the first four seasons. In the fifth and sixth seasons, biochar application resulted in 14-26% increases in soil N uptake but 19-26% decreases in fertilizer N uptake. Soil N availability did not explain the increased soil N uptake with biochar application. The decreased fertilizer N uptake with biochar application was attributed to both decreased fertilizer N availability and increased N loss through ammonia volatilization. As a consequence of a compensation between the increased soil N uptake and the decreased fertilizer N uptake, the effect of biochar application on total N uptake was not significant in the fifth and sixth seasons. However, biochar application led to 7-11% increases in internal N use efficiency in the fifth and sixth seasons and 6% increase in grain yield in the sixth season. Our study suggests that the effects of repeated seasonal applications of biochar on N uptake and utilization in rice depend on the duration of biochar application. Longer continuous applications of biochar can increase internal N use efficiency and grain yield in rice with insignificant change in total N uptake.