This is the accepted version of the journal article: Hei, Jie; Yin, Xiaolei; Wang, Weiqi; [et al.]. «N-enriched biochar increases carbon, nitrogen, and phosphorus accumulation associated with changes in plant ecological stoichiometry in subtropical rice paddy fields». Experimental Agriculture, Vol. 59 (2023), art. e11. DOI 10.1017/S001447972300008X This version is available at https://ddd.uab.cat/record/284317 under the terms of the license 1 N-enriched biochar increases carbon, nitrogen, and phosphorus accumulation 2 associated with changes in plant ecological stoichiometry in subtropical rice paddy 3 fields 4 5 Jie Heia,b#, Xiaolei Yina,b#, Weiqi Wanga,b*, Jordi Sardansc,d*, Chun Wanga,b, Xiaoxuan 6 Chena,b, Akash Tariqe,f, Fanjiang Zenge,f, Abdulwahed Fahad Alrefaeig, Josep 7 Peñuelasc,d 8 a 9 Key Laboratory of Humid Subtropical Eco-geographical Process, Ministry of 10 Education, Fujian Normal University, Fuzhou 350117, China b 11 Institute of Geography, Fujian Normal University, Fuzhou 350117, China c 12 CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08913 Bellaterra, Catalonia, Spain d 13 CREAF. 08913 Cerdanyola del Vallès, Catalonia, Spain e 14 State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and 15 Geography, Chinese Academy of Sciences, Urumqi, 830011, China f 16 Cele National Station of Observation and Research for Desert-Grassland Ecosystems, 17 Cele, 848300, China g 18 Department of Zoology, College of Science, King Saud University, P.O. Box 2455, 19 Riyadh 11451, Saudi Arabia 20 # Equal contribution 21 22 * Corresponding authors 23 Weiqi Wang, Jordi Sardans 24 Key Laboratory of Humid Sub-tropical Eco-geographical Process of Ministry of 25 Education, Fujian Normal University, Fuzhou 350117, China 26 E-mail: wangweiqi15@163.com 27 CREAF. 08913 Cerdanyola del Vallès, Catalonia, Spain 28 E-mail: j.sardans@creaf.uab.cat 29 Summary 30 31 N-enriched biochar can increase the accumulation of carbon (C), nitrogen (N), 32 phosphorus (P) and biomass in rice plants. On the other hand, the biomass and C, N and 33 P contents of plant organs are important indicators to reflect plant C, N and P storages. 34 We established control, 4 t ha-1, and 8 t ha-1 N-enriched biochar treatment plots in a 35 subtropical paddy field in China to investigate the effect of these treatments on C, N, 36 and P storages, ecological stoichiometry in various rice-plant organs, and their 37 relationships with edaphic factors. The application of N-enriched biochar increased the 38 biomass and storages of C, N and P in rice roots, stems, leaves and grains, mainly at 4 39 t ha-1. The application of N-enriched biochar decreased the C/N and C/P ratios of rice 40 organs, but increased their N/P ratio. Changes in C/N were mainly due to the changes 41 in storage, while N/P was positively correlated with N storage of stems, leaves and 42 grains, and negatively correlated with P storage in roots. Pearson correlation analysis 43 revealed that pH was negatively correlated and soil N content was positively correlated 44 with P storage in various organs of rice. In addition, soil P content and chlorophyll were 45 positively correlated with N storage. In conclusion, we found that the application of N- 46 enriched biochar improved plant N- and P-storage and stoichiometrical relations among 47 rice organs. 48 49 Keywords: N-enriched biochar; Rice organs; Soil environmental factors; Carbon, 50 nitrogen and phosphorus; Ecological stoichiometry 51 52 Introduction 53 54 The balance and cycles operating among structural elements (such as C), limiting 55 elements (as N and P), and their ecological stoichiometric ratio in plant organs have 56 always been a focus of interest (Jia et al., 2018). C/N and C/P ratios determine the plant 57 ability to assimilate C and P, to a certain extent, reflecting N and P use efficiency, 58 respectively (Sun et al., 2016). N/P ratio can serve as an index to establish N and P 59 saturations and nutrient supply from soils to plants (von Oheimb et al., 2010), as N and 60 P are essential and critical for improving biomass and productivity, but limiting 61 elements in agricultural ecosystems, forests, and wetlands (Elser et al., 2007). 62 Fertilization and agricultural management are traditional ways of boosting crop 63 productivity and promoting ecosystem health (Sanaullah et al., 2020). Awareness and 64 use of biochar and fertilizer mixtures are fast gaining interest as alternatives amongst 65 modern-day sustainable agricultural practices (Wang et al., 2020). Biochar has a special 66 pore structure and singular physicochemical properties, which can improve physical 67 and chemical properties of soils, and consequently increase nutrient use whilst reducing 68 greenhouse gas emissions (Yin et al., 2021a). Intrinsically, biochar is of immense value 69 to both agriculture and the environment (Xu et al., 2016). However, further research is 70 required in defining the nutrient contents, storage and ecological stoichiometry of plant 71 organs under a combination of nitrogen fertilizers and biochar. Studies of the 72 stoichiometric ratios among C, N, and P contents and storages in plant organs following 73 the application of N-enriched biochar can help develop a robust theoretical basis for 74 understanding the drivers of biogeochemical cycles. 75 The root system is an important and environmentally sensitive organ involved in 76 nutrient absorption and storage (Cao et al., 2020). Stems are vital for nutrient transport 77 and coordination among plant organs (Fortunel et al., 2012), while leaves are primary 78 sites of photosynthesis (He et al., 2020). Then, the contents and distribution of N and P 79 among plant organs affect the photosynthesis of plants (Dordas, 2009). Plant organs 80 have different nutrient use strategies and ecological functions, and cooperate with each 81 other in terms of nutrition and function (Kleyer and Minden, 2015; Zhao et al., 2019). 82 The contents of N and P in plant organs will be different due to organ function, as organs 83 with intense metabolism tend to enrich more in N and P and form higher biomass 84 (Zhang et al., 2018). In addition, agricultural management patterns and global changes 85 will affect the accumulation of C, N and P by plants and change the ecological 86 stoichiometric ratio (Elser et al., 2010; Sardans et al., 2021). For example, C/N changes 87 can reflect changes in N use efficiency by plants (Zhang et al., 2020). In fact, the lack 88 of studies on disentangling nutrient allocation strategies from ecological stoichiometry 89 of plant organs have hindered our understanding. 90 Organ function, climate, latitude, and soil nutrients all affect the allocation of C, N, 91 and P and the stoichiometric ratios in plant organs (He et al., 2015). Generally, climate 92 and latitude determine nutrient allocation patterns in plants. For example, plants in 93 temperate regions at high latitudes are most limited by N, while in tropical regions at 94 low latitudes are limited by P (Reich and Oleksy, 2004). Soil nutrient concentrations 95 also affect root absorption and use efficiencies (Zeng et al., 2016). From a macroscopic 96 perspective, climate and soil environment will jointly affect plant growth (Lin et al., 97 2022). Soil nutrient content and plant nutrient use strategies can determine plant growth 98 more than climate factors in desert areas (Luo et al., 2021). Regardless of the 99 environment, chlorophyll is of great significance for biomass production and crop yield 100 in the ecosystem (Cai et al., 2012; Li et al., 2018), being its content affected by N and 101 P in leaves (Allen and Williams, 1998). 102 China's subtropical region is an important rice production area with a large demand 103 for N and P fertilizers (Li et al., 2010; Wang et al., 2016). As a new soil conditioner, 104 biochar has a positive effect on plant growth in subtropical acidic soil (Li et al., 2012; 105 Hossain et al., 2020). In our previous studies, the application of N-enriched biochar in 106 subtropical rice fields increased the soil concentrations of C, N, P and available P, 107 changing the composition of microbial communities and reducing greenhouse gas 108 emissions (Yin et al., 2021a; Yin et al., 2021b). The C, N, P contents of plant organs 109 constitute good proxies of overall plant nutrient status and can provide information of 110 how crop amendments impact on plant nutritional status (Ogle et al., 2012; Yu et al., 111 2018). However, little is known about how C, N and P storages in rice organs are 112 changed when soil C, N and P contents are increased as well as about the relationships 113 among ecological stoichiometric ratios of C, N and P, soil and plant factors. We 114 collected samples of rice roots, stems, leaves and grains at different growth stages along 115 a year to investigate whether N-enriched biochar would change the concentrations and 116 storage of C, N and P, and C/N/P ratios in different rice organs; and also how soil 117 environmental factors can influence C, N, P storage of rice organs and the regulation of 118 stoichiometric ratio. 119 120 Materials and Methods 121 122 Experimental area 123 The study was performed in the Wufeng Experimental Base of the Rice Research 124 Institute of the Fujian Academy of Agricultural Sciences, China (26.1°N, 119.3°E, 3 to 125 5 m altitude) in the subtropical marine monsoon climate region. The early and late rice 126 (Oryza sativa L.) cultivars were conventional indica rice Hesheng 10 (Jiangxi Academy 127 of Agricultural Sciences, China) and Qinxiangyou 212 (Fujian Academy of Agricultural 128 Sciences, China). Within a year, the farming practice was two successive rice crops 129 (early and late rice), and then vegetables (Ipomoea aquatica Forsk) (Yin et al., 2021a). 130 The growth period of early rice was from April to July, and that of late rice was from 131 July to November. The average air temperature and humidity during the early rice and 132 late rice cycles were 27.1°C and 84.9%, and 26.5°C and 78.9%, respectively (Yang 133 et al., 2022). The rice paddy cultivation soil layer consisted of 28% sand, 60% silt, and 134 12% clay silty loam. The soil pH was 6.5, and soil contents of C, N, and P were 18.2, 135 1.9, and 1.8 g kg-1, respectively. The available P, NH4+-N and NO3−+NO2− in soil were 136 0.3, 4.6 and 29.6 mg kg-1 (Yin et al., 2021b). 137 Rice seedlings were transplanted and sown by a transplanter, with plant spacing 138 and line spacing of 14 and 28 cm, respectively. Compound fertilizers (N:P2O5:K2O, 139 16:16:16) and urea (46% N) were the main fertilizers used to provide 42 kg N ha-1, 40 140 kg P2O5 ha-1, and 40 kg K2O ha-1 before rice planting, and then 35 kg N ha-1, 20 kg P2O5 141 ha-1, and 20 kg K2O ha-1 during tillering. After tillering, 18 kg N ha-1, 10 kg P2O5 ha-1, 142 and 10 kg K2O ha-1 were supplied again to plants. Throughout the tillering stage, water 143 levels were maintained at 5 to 7 cm above the soil surface using an automatic water 144 level controller. After the tillering stage, the dry wet alternate mode of intermittent 145 irrigation was implemented. Water was drained from the paddies two weeks prior to 146 harvest. 147 148 Field experiments 149 The experiment was designed based on the planting habits of Chinese subtropical 150 farmers and previous research (Yin et al., 2021a; Yin et al., 2021b). Spring and summer 151 were the main periods for planting early rice, while summer and autumn were for late 152 rice. In April 2017, plants of the same size and height in the early rice experimental 153 field were selected, and control, 4 t ha-1, and 8 t ha-1 N-enriched biochar treatment 154 groups were established. Three plots per treatment were established in a total of nine 155 plots over an area of 10 m2, 0.5-cm thick and 30-cm high PVC plates were placed 156 between each plot to isolate replicate areas. 157 The experimental N-enriched biochar had total carbon concentration (TC) 6%, 158 total nitrogen concentration (TN) 8%, and total phosphorus concentration (TP) 0.4%, 159 with other components as detailed in Suppl. Material Tab. S1, and the electron 160 microscopy image of N-enriched biochar in Suppl. Material Fig. S1. The process of 161 preparation of biochar includes slowly pyrolysis of biomass at 600 ℃ for 90 min, and 162 thereafter mixing with nitrogen fertilizer at a ratio of about 1:3 (Yin et al., 2021b). The 163 main reason for considering application of N-enriched biochar is based on the 164 characteristics of subtropical rice fields, where soil N is an important limiting factor 165 (Inamura et al., 2009; Yin et al., 2021a). In particular, the role of eutrophic biochar (N, 166 P, etc.) is increasingly valued because it is designed for specific areas and may have 167 great potential for sustainable agricultural development (Karim et al., 2022). 168 169 Plant analyses 170 Three samples of plant roots, stems, leaves, and grains from the control, 4 t ha-1, and 8 171 t ha-1 treatment groups were randomly collected during the tillering and maturity stages 172 from both early and late rice crops. Fresh leaves were also collected and used to measure 173 chlorophyll concentration, while the remainder of the plant was oven-dried to constant 174 weight for biomass measurement. The leaf surface area was calculated through the 175 equation 1 by measuring the leaf length and maximum leaf width (Umashankar et al., 176 2005). 177 178 𝐿𝑒𝑎𝑓 𝑠𝑢𝑟𝑓𝑎𝑐𝑒 𝑎𝑟𝑒𝑎 = 𝑚𝑎𝑥𝑖𝑚𝑢𝑚 𝑙𝑒𝑎𝑓 𝑤𝑖𝑑𝑡ℎ ∗ 𝑙𝑒𝑎𝑓 𝑙𝑒𝑛𝑔𝑡ℎ ∗ 0.75 (1) 179 180 Biomass of rice roots, stems, and leaves was determined by drying at 60 °C. The 181 dried roots, stems, leaves and grains were ground with a ball mill (KZ-ll, China). The 182 plant C and N contents were measured by dry combustion using CN element analyzer 183 (Vario EL, Germany) following Leuschner et al. (2006), while plant P content was 184 determined after sulfuric acid and perchloric acid digestion, using a continuous flow 185 analyzer (Skalar analytical SAN++, Netherlands), as reported by Van Staalduinen et al. 186 (2010). 187 For chlorophyll determination, leaf samples were ground to homogenate, and 188 chlorophyll was extracted with anhydrous ethanol to a fixed volume of 25 mL. The 189 chlorophyll concentrations in rice leaves were measured using an ultraviolet visible 190 spectrophotometer (Shimadzu UV-2450, Kyoto, Japan) and estimated as follows (Zou, 191 2000): 192 193 𝐶ℎ𝑙𝑎 (𝑚𝑔 𝐿−1 ) = 13.95 ∗ 𝐷665 − 6.88 ∗ 𝐷649 （2） 194 𝐶ℎ𝑙𝑏 (𝑚𝑔 𝐿−1 ) = 24.96 ∗ 𝐷665 − 7.32 ∗ 𝐷649 （3） 𝑉 195 𝐶ℎ𝑙 (𝑚𝑔 𝑔−1 ) = (𝐶ℎ𝑙𝑎 + 𝐶ℎ𝑙𝑏) ∗ 𝑀 (4) 196 197 where Chla and Chlb are the concentrations of chlorophyll a and b in the extract, Chl 198 is the concentration on mass basis, D649 and D665 represent absorbance at a given 199 wavelength (665 nm and 649 nm), M is the sample weight, and V is the volume (mL). 200 201 Soil analyses 202 Soil C and N contents were determined by dry combustion using CN element analyzer 203 (Vario MAX CN, Elementar, Germany), following Amishev and Fox (2006). Soil P 204 content was digested by sulfuric acid and perchloric acid, and determined by continuous 205 flow analyzer (Skalar analytical SAN++, Netherlands) (Yu et al., 2010). Soil salinity, 206 pH, and temperature were measured at a depth of 15 cm with a conductivity meter 207 (2265FS EC Meter, Spectrum Technologies Inc., USA) and pH/temperature meter 208 (Starter 300, USA), respectively. 209 210 Allometric growth 211 The proportional allometric model 𝑦 = 𝑎𝑥 𝑘 was used to explore the proportional 212 relationship among C, N, and P in rice plant organs. Firstly, the logarithm of both sides 213 was transformed as follows: 214 𝑙𝑜𝑔(𝑦)−𝑙𝑜𝑔(𝑎) 215 𝑘= (5) 𝑙𝑜𝑔(𝑥) 216 217 where k is the slope (allometric growth index), and x and y the C, N, and P contents and 218 storage. During the analysis, the original data was converted into a base-10 logarithmic 219 form. When k = 1, the ratio of any two elements in the roots, stems, and leaves of the 220 rice fields was isokinetic, whereas when k > 1 or k < 1, the ratio of the two elements 221 was allokinetic. Regression analysis was used to analyze the model parameters (Niklas 222 and Cobb, 2005). 223 224 Calculation of storage of C, N, and P in rice organs 225 The C, N, and P storages per unit area (𝑌𝑠𝑡𝑜𝑟𝑎𝑔𝑒 ) in both early and late rice were 226 estimated according to Ogle et al. (2012): 227 228 𝑌𝑠𝑡𝑜𝑟𝑎𝑔𝑒 = 𝑋𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ∗ 𝐵𝑏𝑖𝑜𝑚𝑎𝑠𝑠 (6) 229 230 where 𝑋𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 is the content of C, N, or P in a given organ and 𝐵𝑏𝑖𝑜𝑚𝑎𝑠𝑠 is the 231 biomass of a given organ. 232 233 Statistical analysis 234 All the experimental data were tested for normality and homogeneity of variance before 235 analysis and inconsistent data were standardized. Data were plotted using Excel 2019 236 (Microsoft, USA), Origin 2020 (OriginLab, USA) and SPSS 20.0 (SPSS Inc., USA) 237 statistical analysis software. A one-way analysis of variance (ANOVA) and repeated- 238 measures ANOVA in SPSS 22.0 were used to analyze differences in C, N, and P 239 contents, storages and ecological stoichiometric ratio of rice roots, stems, leaves, and 240 grains after application of the N-enriched biochar. While one-way ANOVA was used to 241 analyze the treatment effects in each sampling time, repeated-measures ANOVA was 242 used to analyze the overall treatment effect along time and the interactions of time with 243 treatment. The minimum significant difference (LSD, at p<0.05) was used for 244 comparisons. The storages, ecological stoichiometric ratios, contents of C, N, and P in 245 roots, stems, leaves, and grains were mapped using a linear fitting of the allometric 246 growth in Origin 2020. The redundancy analysis (RDA), the Pearson correlation 247 analysis, and the mapping of the environmental factors, plant C, N, and P contents, 248 storages and their stoichiometric ratios were carried out with Canoco 5 and R language 249 corrplot package (McKenna et al., 2016). 250 251 Results 252 253 N-enriched biochar, phenotypic parameters and biomass of rice organs 254 During the tillering and maturity stages, 4 t ha-1 N-enriched biochar increased leaf 255 surface area in both early and late rice (Fig. 1a), and the chlorophyll concentrations in 256 early rice (Fig. 1b). Applying 4 t ha-1 N-enriched biochar during the tillering stage 257 increased root biomass in early and late rice, and at maturity stage, 4 t ha-1 N-enriched 258 biochar increased root biomass of only early rice (Fig. 1c). The 4 t ha-1 N-enriched 259 biochar increased stem biomass at maturity for both early and late rice (Fig. 1d). The 4 260 t ha-1 N-enriched biochar during the tillering and maturity stages in both early and late 261 rice increased leaf biomass (Fig. 1e) and increased grain biomass in both early rice and 262 late rice (Fig. 1f). In comparison, application of 8 t ha-1 N-enriched biochar (cf. control) 263 generally had no effect on the measured phenotypic parameters, except chlorophyll 264 concentrations of early rice at tillering. 265 266 N-enriched biochar and C, N, and P storages in rice organs 267 In early rice, 4 t ha-1 N-enriched biochar dose increased C storage in roots and leaves 268 at the tillering stage (Fig. 2a,c), and in stems, leaves, and grains at the maturity stage 269 (Fig. 2b-d). In late rice, 4 t ha-1 biochar increased C storage in stems and leaves at the 270 tillering and maturity stages (Fig. 2b,c). Yet, 8 t ha-1 biochar dose generally had no effect 271 on C storage in roots and stems, but it did increase C storage in leaves both in early and 272 late rice. 273 In early rice, 4 and 8 t ha-1 doses of N-enriched biochar increased N storage in 274 roots, stems, and leaves at the tillering stage (Fig. 2e-g), and in stems and leaves, at the 275 maturity stage (Fig. 2f,g). In late rice, 8 t ha-1 of N-enriched biochar increased N storage 276 in roots and stems at the tillering stage (Fig. 2e,f), and 4 t ha-1 and 8 t ha-1 doses 277 increased N storage in leaves at the tillering and maturity stage (Fig. 2g). In both early 278 and late rice, 4 t ha-1 N-enriched biochar dose increased N storage in grains (Fig. 2h). 279 In both early and late rice, 4 and 8 t ha-1 doses of N-enriched biochar increased P 280 storage in leaves during the tillering and maturity stages (Fig. 2k). In early rice, 4 and 281 8 t ha-1 doses increased P storage in stems at the maturity stage (Fig. 2g). In both early 282 and late rice, 4 t ha-1 biochar dose increased P storage in grains (Fig. 2l). In addition, 283 repeated-measures ANOVA showed N-enriched biochar treatment had a great impact 284 on C, N and P storages in rice organs as a whole, while time and biochar × time 285 interaction had a great impact on N storage of rice roots, stems and leaves (Suppl. 286 Material Tab. S2). 287 288 N-enriched biochar and the ecological stoichiometric ratios 289 In early rice, 4 and 8 t ha-1 doses of N-enriched biochar reduced the C/N in roots at 290 tillering and in stems and leaves at both tillering and maturity stages (Fig. 3a-c). At the 291 maturity stage, 4 and 8 t ha-1 doses of N-enriched biochar decreased the C/N in grains 292 of early rice (Fig. 3d). Four and 8 t ha-1 doses of N-enriched biochar increased root C/P 293 in early rice at tillering (Fig. 3e), but decreased stem and leaf C/P at maturity (Fig. 3f,g). 294 The 4 and 8 t ha-1 doses of N-enriched biochar reduced leaf C/P of late rice at the 295 maturity stage (Fig. 3g). At the tillering stage, 4 and 8 t ha-1 doses of N-enriched biochar 296 significantly increased N/P in roots, stems, and leaves in early rice (Fig. 3i-k). In early 297 rice, 8 t ha-1 N-enriched biochar dose increased N/P in all rice organs at the maturity 298 stage (Fig. 3i-l). On the other hand, 8 t ha-1 N-enriched biochar dose increased N/P only 299 in stems and leaves of late rice at the tillering stage (Fig. 3j,k). Overall, N-enriched 300 biochar treatment had a great impact on the C/N and C/P of rice roots and stems, while 301 time and biochar × time interaction impacted N/P of rice roots and stems (Suppl. 302 Material Tab. S2). 303 Based on the principal component analysis, we found significant differences 304 between C, N and P storages of roots, stems and leaves and the ecological 305 stoichiometric ratio at the tillering stage. Among them, the difference between stems 306 and roots was the most obvious (Fig. 4a,b). C, N and P storages of stems and grains 307 were higher than those of roots and leaves in both early and late rice (Suppl. Material 308 Fig. S2). 309 310 Allometric growth, C, N and P storages and ecological stoichiometric ratio 311 The allometric growth model of ecological stoichiometric ratio and C, N and P storages 312 revealed that root N storage was negatively correlated with C/N (Fig. 5a) and root P 313 storage was negatively correlated with C/N and positively correlated with N/P (Fig. 314 5a,c). Root P storage was negatively correlated with C/N and positively correlated with 315 N/P (Fig. 5a,c). Root C storage was negatively correlated with C/P and N/P (Fig. 5b,c). 316 The N storage of stems, leaves and grains were negatively correlated with C/N and 317 positively correlated with N/P (Fig. 5d,f,g,i,j,l), while grain P storage was negatively 318 correlated with C/P (Fig. 5k). 319 320 Contents, storages, and ecological stoichiometric ratio as affected by environmental 321 factors 322 The cumulative interpretation degrees of soil environmental factors to the contents, 323 storages and ecological stoichiometric ratio of C, N and P in rice roots, stems, leaves 324 and grains were 27.9%, 59.9%, 59.9% and 91.5%, respectively (Suppl. Material Fig. 325 S3a-d). Among them, soil pH was the main factor affecting roots, and soil pH, bulk 326 density and water content were the main factors affecting stems. The soil P content, 327 bulk density and leaf surface area were the main factors affecting leaves, and soil C 328 content and leaf surface area were the main factors affecting grains (Suppl. Material 329 Fig. S3a-d). From the relationship between soil environmental factors and C, N, P 330 storages and ecological stoichiometric ratio, soil pH was negatively correlated with P 331 storage in various rice organs (Fig. 6a-d), negatively correlated with C/P in roots, stems 332 and leaves (Fig. 6a-c), and negatively correlated with grain N/P in various organs (Fig. 333 6d). Soil salinity was positively correlated with N storage in roots, stems and leaves, 334 and negatively correlated with C/N (Fig. 6a-c). Soil N content was positively correlated 335 with P storage and C/P in roots, stems and leaves of rice (Fig. 6a-c). Soil P content was 336 positively correlated with N storage and negatively correlated with C/N in roots and 337 stems (Fig. 6a,b). Leaf surface area was positively correlated with C storage and 338 chlorophyll was positively correlated with N storage and negatively correlated with 339 C/N in various organs (Fig. 6a-d). 340 341 Discussion 342 343 C, N, and P storages and ecological stoichiometry in rice organs as affected by N- 344 enriched biochar 345 Biochar can affect plant growth by changing the composition and then physical 346 and chemical properties of soil microbial community (Liao et al., 2016), which is an 347 important additive affecting the cycling of nutrients such as N and P (Hossain et al., 348 2020). During both early and late rice cultivation, 4 t ha-1 N-enriched biochar increased 349 biomass, and storage of C, N, and P in grains, and 4 and 8 t ha-1 N-enriched biochar 350 increased storages of C, N and P in leaves (Figs. 1c-f and 2c,d,g,h,k,l). During early 351 rice cultivation, 4 and 8 t ha-1 N-enriched biochar increased the content of N in roots, 352 stems and grains, increased the contents of C, N and P in leaves, and increased C, N 353 storages of roots and N storage of stems (Fig. 2a,e,f and Suppl. Material Fig. 354 S4c,e,f,g,h,k). These changes supported the hypothesis that N-enriched biochar would 355 increase the contents and storages of C and N in some rice tissues. Our results are also 356 in agreement with previous reports on the application of P-enriched biochar and other 357 types of biochar (Biederman and Harpole, 2013; Wang et al., 2014). The most direct 358 influencing factor is that N-enriched biochar improves the plant accumulation of N, P 359 and biomass (Yuan et al., 2018). Because the application of N-enriched biochar 360 increased the contents of soil C, N and P through the increase of fungal and bacterial 361 abundance and the improvement of soil physics and chemistry, P sequestration in 362 subtropical paddy soil was increased and N limitation alleviated (Yin et al., 2021b). In 363 addition, the N-enriched biochar applied in this experiment contained elements such as 364 iron, aluminium, manganese, and silicon (Suppl. Material Tab. S1), which can meet 365 other nutrient needs of plants, combine with cations in soil minerals and reduce nutrient 366 loss (Yin et al., 2021a). 367 N-enriched biochar has unique pore structure and high specific surface area, which 368 improve soil physical and chemical conditions to increase the nutrient retention 369 capacity of soils, and then improve the nutrient use efficiency of plants (Jin et al., 2019; 370 Hossain et al., 2020). For example, N-enriched biochar can improve soil cation and 371 anion exchange capacity to reduce nutrient gaseous volatilization and leaching loss 372 (Tomczyk et al., 2020), and reduce soil bulk density to improve soil water holding 373 capacity and aggregate stability (Shaheen and Turaib Ali Bukhari, 2018). Therefore, N- 374 enriched biochar can play a role in nutrient fixation and provide potential nutrient 375 sources for plants. However, due to the influence of environmental factors such as soil 376 physics and chemistry and temperature, the relationship between the application 377 amount of biochar and the absorption of nutrients by plants is not linear (Yuan et al., 378 2018). Our study revealed that the application of 4 t ha-1 N-enriched biochar had better 379 effects on C, N and P storage and phenotypic parameters of rice than 8 t ha -1 (Figs. 1 380 and 2). In fact, excessive fertilization can cause nutrient overload or even disrupt the 381 ecological stoichiometric balance between C, N, and P in plant organs, negatively 382 affecting plant growth (Hoegberg et al., 2006; Penuelas et al., 2013). 383 The ecological stoichiometric ratio of C, N, and P elements in plants can reveal 384 nutritional limitations, as well as changes in use of nutrients and growth rate (Luo et al., 385 2020). Herein, we found that after the application of N-enriched biochar, the C/N of 386 rice organs decreased in early rice (Fig. 3a-d), which also supported the hypothesis that 387 N-enriched biochar would reduce the C/N in some rice tissues. This finding indicates 388 that the limitation of N in rice organs decreased, and the N use-efficiency increased, 389 which was conducive to the provision of biomass and yield (Hurtado et al., 2020; He 390 et al., 2015). In addition, N and C/N, P and C/P in rice organs were negatively correlated, 391 suggesting that the nutritional limitation imposed by N and P decreased after the 392 application of N-enriched biochar (Fig. 6). Plant N/P can indicate the degree of 393 limitation of N and P. When N/P<14, plant growth tends to be affected by N restrictions, 394 whereas N/P>16 suggests plant growth tends to be restricted by P. When N/P is between 395 14 and 16, plant growth may be affected by N and P, or not limited by them (Koerselman 396 and Meuleman, 1996). Our study showed that N/P of rice-plant organs was less than 14 397 (Fig. 3). During early rice cultivation at tillering stage, 4 and 8 t ha-1 N-enriched biochar 398 increased N/P of rice roots (1.7 and 2.8 times, respectively), stems (97% and 1.4 times, 399 respectively) and leaves (32% and 33%, respectively), as shown in Fig. 3i-k. During 400 early rice cultivation at maturity stage, 8 t ha-1 N-enriched biochar increased the N/P of 401 grains in 62% (Fig. 3l). Taken together, our data suggest that rice was mainly affected 402 by N, but the application of N-enriched biochar increased the N/P ratio in rice and so 403 compensated for the N limitation. 404 405 C, N and P storages and ecological stoichiometric allocation strategies in rice organs 406 as affected by N-enriched biochar 407 The ecological stoichiometric ratio of C, N and P among plant organs can reflect 408 nutrient acquisition strategies, functional differentiation and nutrient cycle patterns 409 (Luo et al., 2021). The results of PCoA showed that the distance between roots and 410 leaves and grains were greater than that of stems. Differences in C, N and P storages 411 among roots, leaves and grains due to N-enriched biochar application (Fig. 4) are 412 consistent with a general change in functioning of rice organs and with the ecological 413 stoichiometry paradigms (Sardans et al., 2017). The allometric growth theory indicates 414 that C, N, and P contents in leaves have positive allometric correlation (Suppl. Material 415 Fig. S5). As important metabolic organs, leaves capture CO2 through photosynthesis 416 and increase the accumulation of plant organic matter (Gao et al., 2020). In structural 417 organs such as roots and stems, P is stored to meet the needs of rice growth (Schreeg et 418 al., 2014). During the maturation, grain growth needs more nutrients to promote rice 419 heading and high yields (Deng et al., 2015). Therefore, physiological functions and 420 nutrient use strategy enable plants to selectively adjust their nutrients to support growth 421 (Yan et al., 2016; Schreeg et al., 2014). Our results also show that plants can coordinate 422 the distribution and accumulation of nutrients in organs through the ecological 423 chemometrics of C, N and P (Fig. 5), thus supporting our hypothesis that N-enriched 424 biochar changes the stoichiometry of C, N and P accumulation among rice organs. 425 There was a negative allometric relationship between N storage and C/N in all organs 426 of rice (Fig. 5a,d,g,j). Herein, low C/N corresponded to higher N storage (Fig. 5d,e,g,j), 427 which was caused by N limitation in subtropical rice fields where rice with fast growth 428 rate but short cycle should absorb more nutrients (Wang et al., 2015). In addition, N/P 429 of rice stems, leaves and grains showed positive allometric correlation with N storage, 430 and N/P of roots showed negative allometric correlation with P storage (Fig. 5c,f,i,l). 431 Enrichment of N in stems, leaves and grains after the application of N-enriched biochar 432 would alleviate N limitation and increase N stock. The enrichment of P in roots would 433 increase P stock as well. This may be related to the differential distribution of N and P 434 among rice organs (Suppl. Material Fig. S2b,c). The aboveground parts (stems, leaves 435 and grains) usually have richer chlorophyll concentration, and then N should be 436 concentrated to meet the photosynthetic requirement in a shorter life cycle (Ullah et al., 437 2019). Because of P limitation, roots should absorb more P in subtropical soil to 438 promote rice growth (Elser et al., 2007; Hurtado et al., 2020). Therefore, the 439 accumulation of C, N and P is affected by the nutrient allocation strategy and the C, N 440 and P ecological stoichiometry of rice organs. 441 442 C, N, and P storages in rice organs as affected by environmental factors 443 Our results showed that soil environmental factors such as soil pH, salinity, soil N 444 and P contents affected the C, N and P storages and ecological stoichiometric ratio of 445 rice organs (Fig. 6). In order to adapt to a changing environment, the nutrient dynamics 446 and nutrient absorption strategy of plants should change and improve allocation to 447 match growth (Leishman et al., 2007). Correlation analysis revealed that low pH was 448 associated to reduced P storage in roots, stems and leaves of rice (Fig. 6a-c). Soil P is 449 easily fixed by iron and aluminium in acid soil, making it difficult for plants to use this 450 nutrient (Elser et al., 2007). However, the N-enriched biochar contains alkaline metal 451 ions, which improve soil pH, improve the acidic soil environment, and help rice to 452 obtain more available P from the soil (Hossain et al., 2020). For example, P content in 453 rice roots, stems and leaves was positively correlated with soil pH (Fig. 6a-c). In 454 addition, soil salinity had a positive relationship with N storage of rice roots, stems and 455 leaves (Fig. 6a-c). Higher investment in P mineralization and uptake is common when 456 N availability increases in terrestrial plant communities due to higher capacity of 457 phosphatases and root growth (Sardans and Peñuelas 2012). Based on the special 458 specific surface area and pore structure, N-enriched biochar increases the content of 459 soluble NO3−, K+ and Ca2+ by affecting soil water holding capacity and pH (Igalavithana 460 et al., 2017). However, excessive salinity would reduce the absorption of nutrients such 461 as N and P and this effect should be considered when applying N-enriched biochar. 462 Previously, the application of N-enriched biochar increased the contents of soil C, N 463 and P (Yin et al., 2021b). Our results showed that N-enriched biochar increased and 464 strengthened the relationships between soil N and P contents and the N and P storages 465 in rice organs (Fig. 6). Soil N and P contents were interrelated with plant N and P 466 storages, and leaf surface area was positively correlated with C storage in rice organs 467 while chlorophyll was positively correlated with N storage in rice organs (Fig. 6). In 468 fact, rice with short growth cycle needs more chlorophyll – and then N – to accumulate 469 organic matter (Wang et al., 2015). In conclusion, we found that organs responded 470 differently to environmental factors, most likely due to different nutrient use strategies. 471 472 Overall impact of N-enriched biochar on economic and environmental benefits in 473 rice paddy fields 474 The application of N-enriched biochar can change the physical and chemical 475 properties of soil, leaf surface area and chlorophyll and increase the storages of C, N 476 and P, which are regulated by the ecological stoichiometric ratio of C, N and P in rice 477 organs (Fig. 7). A negative linear relationship between rice N storage and C/N was 478 found, while N/P has a negative linear relationship with P storage of rice roots, and a 479 positive linear relationship with N storage of rice stems, leaves and grains (Fig. 7). 480 These findings can be explained by nutrient utilization strategy and the optimal ratio 481 among plant organs (Zhang et al., 2020). Our study showed that the application of N- 482 enriched biochar will be conducive to the accumulation of rice biomass and N and P 483 storage and stimulate plants to use and remobilize nutrients from rice organs, especially 484 under 4 t ha-1 of N-enriched biochar. At the same time, N-enriched biochar help to 485 reduce costs and accumulation of pollutants in farmland, thereby providing both 486 economic and environmental benefits (Wang et al., 2020). For example, Yin et al. 487 (2021a) found that the application of N-enriched biochar in subtropical rice fields 488 reduced greenhouse gas emissions while increasing rice yield. Previous studies have 489 also shown that the application of N-enriched biochar in subtropical rice fields alleviate 490 the limitation of N in soil and increase total and available soil P by improving soil 491 physicochemical properties and microbial community richness, thus promoting rice 492 growth (Yin et al., 2021b). 493 The preparation of N-enriched biochar in this study used waste straw and wood, 494 with a cost about US$ 222.40 per ton, while the price of nitrogen fertilizer was about 495 US$ 355.85 per ton. By using agricultural residues such as waste straw and wood, we 496 can reduce transportation costs and environmental pollution caused by incineration 497 (Galinato et al., 2011). It is worth noting that the carbon trading system may also bring 498 some economic income to farmers in the future (Pandit et al., 2018). From the 499 perspective of soil environment improvement, plant nutrient absorption and reduction 500 of greenhouse gas emission, the application of 4 t ha-1 N-enriched biochar would 501 promote the sustainable development of subtropical rice production and offer farmers 502 a new way of increasing rice yields. However, we must recognize that the rice response 503 to 8 t ha-1 N-enriched biochar in terms of biomass and yield was low, which indicates 504 that excessive dosage of biochar may produce low economic and ecological benefits. 505 This may be due to the negative effect of high salinity, as commented previously 506 (Biederman et al., 2013; Hossain et al., 2020). 507 508 Conclusion 509 510 In general, this study comprehensively analyzed the changes of C, N and P storages in 511 rice organs and their responses to soil environmental factors and ecological 512 stoichiometry. The application of 4 t ha-1 N-enriched biochar played a positive role in 513 the nutrient accumulation in rice organs. Our study also revealed that in the subtropical 514 paddy field dominated by N limitation, the storage of C, N and P among organs of rice 515 varied, and the accumulation of C, N and P and their stoichiometric relations were 516 affected mainly by soil pH, salinity, and N and P concentrations. Finally, the application 517 of N-enriched biochar in subtropical areas can promote the absorption and 518 accumulation of plant nutrients such as C, N and P, and provide good economic and 519 ecological benefits. 520 521 Acknowledgements 522 523 This study was supported by the National Natural Science Foundation of China 524 (41571287). JP and JS were funded by the Spanish Government projects PID2019- 525 110521GB-I00 and PID2020115770RB-I, Fundación Ramón Areces project 526 ELEMENTAL-CLIMATE, and Catalan government project SGR2017-1005. We 527 extend our appreciation to the Researchers Supporting Project no.504 RSP-2021/218, 528 King Saud University, Riyadh, Saudi Arabia. 529 530 Declaration of interests 531 532 The authors declare that they have no known competing financial interests or personal 533 relationships that could have appeared to influence the work reported in this paper. 534 535 536 537 538 539 540 541 References 542 543 Allen J. P. and Williams J. C. (1998). Photosynthetic reaction centers. FEBS letters 544 438(1-2), 5-9. 545 Amishev D. Y. and Fox T. R. (2006). The effect of weed control and fertilization on 546 survival and growth of four pine species in the Virginia Piedmont. Forest Ecology 547 and Management 236(1), 93-101. 548 Biederman L. A. and Harpole W. S. (2013). Biochar and its effects on plant 549 productivity and nutrient cycling: a meta‐analysis. GCB bioenergy 5(2), 202-214. 550 Cai H., Chu Q., Yuan L., Liu J., Chen X., Chen F., Mi G. and Zhang F. 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China Agricultural Press, 761 Beijing. 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 Figure captions 794 795 Fig. 1 Effects of N-enriched biochar (4 and 8 t ha-1) on leaf surface area (a), chlorophyll 796 concentration (b), and root (c), stem (d), leaf (e) and grain (f) biomass of early and late 797 rice crops. Mean values and standard errors. Uppercase letters indicate differences 798 among treatments in a given growth stage (p<0.05). In legend, T stands for tillering 799 stage and M stands for maturity stage. 800 801 Fig. 2 Effects of N-enriched biochar (4 and 8 t ha-1) on C (a-d), N (e-h) and P (i-l) 802 storages in root (a,e,i), stem (b,f,j), leaf (c,g,k) and grain (d,h,l) of early and late rice 803 crops. Mean values and standard errors. Uppercase letters indicate differences among 804 treatments in a given growth stage (p<0.05). In legend, T stands for tillering stage and 805 M stands for maturity stage. 806 807 Fig. 3 Effects of N-enriched biochar (4 and 8 t ha-1) on C/N (a-d), C/P (e-h) and N/P (i- 808 l) ratios in root (a,e,i), stem (b,f,j), leaf (c,g,k) and grain (d,h,l) of early and late rice 809 crops. Mean values and standard errors. Uppercase letters indicate differences among 810 treatments in a given growth stage (p<0.05). In legend, T stands for tillering stage and 811 M stands for maturity stage. 812 813 Fig. 4 Principal component analysis of C, N and P storage and ecological stoichiometric 814 ratio in rice organs at the tillering (a) and maturation stages (b). The data includes early 815 and late rice. The horizontal and vertical axes represent the first and second principal 816 coordinates, respectively. The percentage marked on the horizontal and vertical axis is 817 the contribution of the principal coordinate to the difference of the sample matrix data 818 variance. 819 820 Fig. 5 Correlations between C, N and P storage (gray square, red circle, and blue 821 triangle, respectively) and ecological stoichiometric ratios C/N (a,d,g,j), C/P (b,e,h,k) 822 and N/P (c,f,i,l) in root (a-c), stem (d-f), leaf (g-i) and grain (j-l) of rice plants. r is the 823 correlation coefficient, * and ** stand for p<0.05 and p<0.01, respectively. 824 825 Fig. 6 Correlation analysis of C, N and P concentrations, storage, ecological 826 stoichiometric ratios and soil environmental factors, considering root (a), stem (b), leaf 827 (c) and grain (d) of rice plants. WC: Soil water content; CL: Chlorophyll; BD: Soil bulk 828 density; T: Soil temperature; SL: Soil salinity; LA: Leaf surface area; SC: Soil C; SN: 829 Soil N; SP: Soil P, CS: C storage, NS: N storage, PS: P storage. 830 831 Fig. 7 A conceptual model of the effects of N-enriched biochar on the concentrations, 832 storage and ecological stoichiometric ratios of C, N and P in rice organs. 833 834 Early rice 早稻 Late rice 晚稻 Early rice Late rice Early rice Late rice (a) (b) (c) B B B B B A M-8t ha-1 T-8t ha-1 A A A A A A AB AB A A B B B B B A B A A A A A A A M-4t ha-1 T-4t ha-1 A AB AB A AB A 60 40 20 0 20 40 60 10 5 0 5 10 60 40 20 0 20 40 60 Leaf surface area (cm2) Chlorophyll (mg g-1) Root biomass (g m-2) (d) (e) (f) M-Control A B B B T-Control A A A A C B A B B B A A B B B B A A A A B C B C B B 240 160 80 0 80 160 240 90 60 30 0 30 60 90 900 600 300 0 300 600 900 Stem biomass (g m-2) Leaf biomass (g m-2) Grain biomass (g m-2) 835 836 Fig. 1 Root Stem Leaf Grain Early rice Late rice Early rice Late rice Early rice Late rice Early rice Late rice (a) (b) A (c) (d) C storage (kg hm-2) B A B B C A A A A A A B B B A A B B B M-8t ha-1 A A T-8t ha-1 A A B B C C A A A A A A B B A a A B B B B 180 120 60 0 60 120 180 1000 500 0 500 1000 400 200 0 200 400 4000 2000 0 2000 4000 M-4t ha-1 T-4t ha-1 (e) (f) (g) (h) N storage (kg hm-2) B B B B C C A B A B A B C B A A A A B A A A M-Control T-Control A A B A B B A A A A A A B B A A A A A A 8 6 4 2 0 2 4 6 8 40 20 0 20 40 40 20 0 20 40180 120 60 0 60 120 180 (i) (j) (k) (l) P storage (kg hm-2) A B A B C C A AB A B A A B B B A A A B B A A A A B B B B A A A AB A A B AB A A A A A A 4 3 2 1 0 1 2 3 4 12 9 6 3 0 3 6 9 12 4 3 2 1 0 1 2 3 4 60 40 20 0 20 40 60 837 838 839 Fig. 2 Root Stem Leaf Grain Early rice Late rice Early rice Late rice Early rice Late rice Early rice Late rice (a) (b) (c) (d) A B A B A A B A B A B A A C C/N C C C C B A B B A A A M-8t ha-1 A A A T-8t ha-1 A C A A A B B A A A B A C A 80 60 40 20 0 20 40 60 80 60 40 20 0 20 40 60 40 20 0 20 40 40 30 20 10 0 10 20 30 40 (e) (f) (g) (h) M-4t ha-1 A B A T-4t ha-1 C A A B AB A A A A A A A B A A A A C/P A A A A A A A A A A A A B A B B M-Control T-Control A A B A B C 140 70 0 70 140 240 160 80 0 80 160 240 300 150 0 150 300 120 80 40 0 40 80 120 (i) (j) (k) (l) C A B B B B B A A B A B B A A A A A A A N/P AB A B A B A B A AB A A A A A B A A A A A A A 9 6 3 0 3 6 99 6 3 0 3 6 9 15 10 5 0 5 10 15 6 4 2 0 2 4 6 840 841 Fig. 3 842 843 844 845 846 847 848 849 Fig. 4 850 851 C storage N storage P storage 3 3 3 (a) Root (b) Root (c) Root r= 0.081 r= 0.415* r= 0.331* log C, N, P storage 2 2 2 r= 0.553** r= 0.180 r= 0.251 1 1 1 0 0 0 r= 0.333* r= 0.193 r= 0.352* 1.2 1.4 1.6 1.8 1.6 1.8 2.0 2.2 0.0 0.2 0.4 0.6 0.8 1.0 log C/N log C/P log N/P (d) Stem r= 0.179 (e) Stem r= 0.047 (f) Stem 3 3 3 r= 0.324 log C, N, P storage 2 2 2 r= 0.615** r= 0.259 r= 0.652** 1 1 1 r= 0.173 r= 0.04 r= 0.220 1.2 1.4 1.6 1.8 1.8 2.0 2.2 2.4 0.4 0.6 0.8 log C/N log C/P log N/P 3 3 3 (g) Leaf (h) Leaf (i) Leaf log C, N, P storage 2 r= 0.305 2 r= 0.218 2 r= 0.254 1 1 1 r= 0.530** r= 0.312* r= 0.043 0 0 0 r= 0.129 r= 0.007 r= 0.212 1.0 1.2 1.4 1.6 2.0 2.2 2.4 0.6 0.8 1.0 1.2 log C/N log C/P log N/P 4 4 4 (j) Grain (k) Grain (l) Grain log C, N, P storage 3 r= 0.185 3 r= 0.059 3 r= 0.154 r= 0.330 r= 0.622** r= 0.809** 2 2 2 r= 0.396 r= 0.648** r= 0.045 1 1 1 1.3 1.4 1.5 1.8 1.9 2.0 2.1 0.3 0.4 0.5 0.6 0.7 0.8 log C/N log C/P log N/P 852 853 854 Fig. 5 855 856 857 858 859 860 Fig. 6 861 862 863 864 865 866 Fig. 7