Nitrogen regulates pollen tube elongation under low-light stress during anthesis to prevent spikelet abortion in rice

Nitrogen regulates pollen tube elongation under low-light stress during anthesis to prevent spikelet abortion in 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 Research Article Nitrogen regulates pollen tube elongation under low-light stress during anthesis to prevent spikelet abortion in rice Yichang Zhong, Hubo Li, Feifei Li, Guanfu Fu, Qiao Deng, Dali Zeng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4737345/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Plant Growth Regulation → Version 1 posted 6 You are reading this latest preprint version Abstract Low-light has emerged as a primary environmental stressor limiting rice production, yet there is currently limited research on relevant regulatory measures. In this study, a low-light-intolerant RGA1 gene mutant ( d1 ), and its wild type (Zhonghua 11, WT), along with an overexpressed line (OE-1), were used to investigate the effects of nitrogen levels on rice yield formation under low-light during anthesis, using field shading treatment. Our results indicated that low-light significantly decreased spikelet fertility, with hindered pollen tube elongation identified as a key factor leading to spikelet abortion. Under low-light conditions, the medium nitrogen treatment (160 kg N·ha − 1 , MN) notably increased the ratio of pollen tube entry into the ovule and spikelet fertility compared to the low nitrogen treatment (60 kg N·ha − 1 , LN), while the high nitrogen treatment (260 kg N·ha − 1 , HN) decreased spikelet fertility and yield in WT and OE-1 plants. For the d1 mutant, except for the LN treatment, other nitrogen treatments had minimal effect on spikelet fertility and yield. Furthermore, compared to the LN treatment, the activities of invertase and sucrose synthase, as well as the content of ATP, and ATPase in the spikelets of WT and OE-1 significantly increased when treated with MN under low-light conditions. In conclusion, moderately increasing nitrogen levels can enhance sucrose metabolism, maintain energy balance, and prevent low-light stress from impeding pollen tube elongation and spikelet fertility. Rice Low-light Nitrogen Pollen Tube Energy Homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Rice is a primary staple for over 50% of the world's population (Muthayya et al., 2014 ). As global population rises, the production of rice becomes increasingly vital for ensuring global food security (Searching et al., 2018). However, during the actual production process, rice yield can be easily restricted by various environmental factors, such as light availability. Being a sun-loving crop, rice requires approximately 1500 hours of bright daylight from transplantation to maturity to thrive (Panigrahy et al., 2020 ). Insufficient light severely inhibits rice growth, resulting in decreased yields. In parts of China, recent environmental pollution has exacerbated climate deterioration, leading to frequent long rainy weather conditions that pose a threat to rice yields (Guo et al., 2021 ). Furthermore, the increasing air pollution is reducing the solar radiation reaching the Earth's surface each year, potentially causing a loss of approximately 0.4–1.8% of global crop production in the future (Müller et al., 2014 ). It is evident that the occurrence of low-light has emerged as one of the primary stresses impeding rice growth. The anthesis stage of rice marks a crucial period for spikelet fertility and yield formation. When encountering abiotic stress (high temperature, drought, salinity, etc.), fertilization failure can lead to reduce grain yields (Jagadish et al., 2020; Xiang et al., 2019 ; Xu et al., 2017 ). Previous research has indicated that low-light exposure during the anthesis of rice could hinder anther dehiscence, diminish pollen grain numbers, and suppress pollen germination (Cecchetti et al., 2008 ; Wilson et al., 2011 ). Recent studies have further revealed that inadequate light during anthesis inhibits pollen tube elongation in rice, resulting in significant yield losses (Li et al., 2023 ). Hence, it is imperative to develop relevant cultivation strategies to mitigate rice yield losses attributed to low-light stress. Nitrogen (N) is an essential nutrient element for the growth and development of crops, regulating their resistance to abiotic stresses by enhancing physiological characteristics and organic structure (Ma et al., 2022 ). Numerous studies have demonstrated that the appropriate application of nitrogen fertilizer can mitigate crop yield losses under various stresses such as drought, flooding, heat, salinity, alkalinity, heavy metals, and others during the anthesis period (Liu et al., 2019 ; Xiong et al., 2020 ; Tian et al., 2022 ; Zhou et al., 2022 ). For instance, heat stress can lead to spikelet abortion, but higher nitrogen levels can reduce the number of degenerated spikelets under such conditions (Liu et al., 2019 ). Nitrogen stored in anthers and pollen grains, along with its metabolic activity, is beneficial for stress resistance (Santiago and Sharkey, 2019 ). Furthermore, when nitrogen was deficient in the medium, the losses of nitrogen transporter TIP1;3 and TIP5;1 in the pollen tube would inhibit the pollen tube elongation in Arabidopsis thaliana (Soto et al., 2008 ; Soto et al., 2010 ), possibly due to nitrogen's regulation of energy status in the pistil. Ma et al. ( 2022 ) demonstrated that nitrogen (N), phosphorus (P), and potassium (K) could all improve plant energy status and affect rice growth and development, with nitrogen having the greatest effect. Pollen tube elongation is a highly energy-consuming physiological activity, and the lack of energy caused by low-light can hinder rice pollen tube elongation (Li et al., 2023 ). And low-light stress can reduce the net photosynthetic rate of leaves to decrease rice's energy supply (Deng et al., 2021 ). Given this, we proposed that regulating nitrogen levels under low-light stress may alleviate the inhibition of pollen tube elongation by adjusting energy levels. RGA1 is responsible for encoding the heterotrimeric G protein α subunit of rice (Ishikawa et al., 1995 ). The Gα subunit plays a role in mediating cellular response to extracellular stimuli, transmitting signals from receptors to downstream effectors, and regulating rice tolerance to abiotic stressors such as high temperature, drought, and salinity (Misra et al., 2007 ; Ferrero-Serrano and Assmann, 2016 ; Jangam et al., 2016 ). Previous research has also established a link between RGA1 and rice's response to light, demonstrating its ability to enhance energy conversion through increased sucrose metabolic rates, maintaining ATP production efficiency, and thereby bolstering rice resilience to low-light stress (Li et al., 2023 ). Nitrogen, as well, influences rice growth and development by improving plant energy status (Ma et al., 2022 ), but its specific effects on pollen tube elongation under low-light conditions during anthesis and the associated regulatory mechanisms require further elucidation. Therefore, this study utilized RGA1 mutants ( d1 ), their corresponding wild types (WT), and over-expressed plants (OE-1) as experimental materials. Different nitrogen fertilizer levels were applied concurrently with shading treatment during anthesis to investigate the mechanism by which nitrogen levels affect pollen tube elongation under low-light conditions during anthesis. Materials and methods Plant materials and growth condition The research was conducted from June to September 2022 on rice experimental station at the China Rice Research Institute (CNRRI) in Hangzhou, Zhejiang Province. The low-light-intolerant RGA1 gene mutant ( d1 ), and its wild type (Zhonghua 11, WT), along with a RGA1 gene overexpressed line (OE-1) were documented in previous study (Li et al., 2023 ). A split-split-plot design was used in the experiment. Main plots were treated with shading or not; spilt-plots were treated with low nitrogen (60 kg N·ha − 1 , LN), medium nitrogen (160 kg N·ha − 1 , MN) and high nitrogen (260 kg N·ha − 1 , HN); and split-split-plots consist of three rice cultivars (WT、 d1 and OE-1). Each treatment was repeated 3 times. The physical and chemical properties of soil are shown in the Online Resource 2. Rice was sown on May 19 and transplanted on June 17, 29 days post sowing. The rice was single planted, with the density of 20 cm × 25 cm and each plot area of 10 m 2 . The application of nitrogen fertilizer (urea) followed the ratio of basal fertilizer: tillering fertilizer: panicle fertilizer = 4:3:3. Phosphate fertilizer (P 2 O 5 ) was applied as a single application for the base fertilizer at a rate of 80 kg P·ha − 1 . Potassium fertilizer (K 2 O) was applied according to the ratio of basal fertilizer: panicle fertilizer = 6:4, with the application amount of 160 kg K·ha − 1 . To ensure the stability of nitrogen level in different plots, all the plots were covered with black plastic film to the bottom of the fields. At the anthesis stage, a double layer black shading net was used to simulate low-light treatment, and the treatment time was 7 days. The height of the shading net was 50 cm from the top of the rice to ensure air exchange under the shading net, so as to maintain the consistency of the treatment and field temperature as far as possible. The climatic conditions during the experiment are shown in Online Resource 1. After the treatment, the shading net was removed to allow the rice to grow to maturity under natural conditions. Diseases, pests and grasses were strictly controlled during the whole growth period. Spikelet fertility, grain weight and yield When rice was harvested, samples were taken from each plot according to 5-point method, dried in oven to constant weight, and then threshed to investigate spikelet fertility and 1000-grain weight. At the time of harvest, samples were also taken separately by the 5-point method for yield measurement. After drying and threshing, the grain was weighed, and the yield per hectare was converted. Pollen grain number on stigma, pollen germination rate and pistil tube elongation On the day 4 of low-light treatment, flowering spikelets were taken from the field, removed the pistils, and immediately fixed in FAA fixation solution. Subsequently, according to the method of Zhang et al. ( 2018a ), the fixed pistil tissues were placed in 10 mol/L NaOH solution, incubated at 55℃ for 10 min, washed with distilled water, and stained with 0.1% aniline blue solution for more than 2h. Then, the growth of pollen tube was observed at 350nm using fluorescence microscopy (DM4000B, Leica, Wetzlar, Germany). Randomly divide 50 pistils into 5 groups, and calculate the elongation ratio of pollen tubes in each group based on the standard of pollen tube reaching the ovule. And the number of stigma pollen grains and germination pollen were counted. Pollen germination rate = germination pollen number/ (germination pollen number + non-germination pollen number) ×100%. Nitrogen content On the day 4 of low-light treatment, the flowering spikelets were taken from the field and dried to constant weight after sampling. The dried samples were first digested with H 2 SO 4 -H 2 O 2 , and then the total nitrogen content was determined by the Kjeldahl method. Non-structural carbohydrate content On the day 4 of low-light treatment, the flowering spikelets were taken from the field and frozen with liquid nitrogen to be measured. Referring to the method of Dubois et al. ( 1956 ), anthrone sulfate colorimetry was used to determine the contents of total soluble sugars and starch. Sucrose, glucose, and fructose are extracted in the same way as total soluble sugars. The method of Zhang (2018b) was used to determine the content of sucrose, glucose and fructose. The total non-structural carbohydrate (NSC) content is the sum of soluble sugar and starch content. Sucrose synthase (SUS) and invertase (INV) activities 0.1g spikelet sample frozen with liquid nitrogen was added with 3 ml HEPS-NaOH (pH7.5, containing 50 mmol/L HEPES, 10 mmol/L MgCl 2 , 2 mmol/L EDTA, 1% PVP40, 12.5% glycerol, 5 mmol/L DTT) extract and was grounded into a homogenization. Then centrifuged at 10, 000 rpm/min for 10 min at 4℃. Then 0.1 ml supernatant was added into 0.9 ml working solution (containing 50 mmol/L HEPES, 5 mmol/L UDP, 5 mmol/L magnesium acetate, 50 mmol/L sucrose, 5 mmol/L DTT, the reaction solution needs to be adjusted to pH7.5 by NaOH), and the reaction solution was bathed in water at 30℃ for 20 min. The reaction was terminated in boiling water bath for 5 min. After cooling, added 1 ml DNS and bathed in boiling water for 5 min After cooling, the color was compared at 540 nm with ultraviolet spectrophotometer, and the boiled dead enzyme solution was used as the control. The SUS activity was calculated by using glucose standard solution as the standard curve. The extraction method of INV is the same as SUS. 0.05 ml of supernatant was added to 0.95 ml of acetate-K 3 PO 4 buffer (pH4.7, containing 50 mmol/L sucrose). After 20 min of water bath at 30℃ and 5 min of boiling water bath, the reaction was terminated. After cooling, added 1 ml of DNS with boiling water bath for 5 min. The color was compared at 540 nm by ultraviolet spectrophotometer after cooling, and the boiled dead enzyme solution was used as the control. INV activity was calculated using glucose standard solution as standard curve. ATP and ATPase content 0.1 g frozen sample were ground in liquid nitrogen and ground into homogenate in PBS with pH7.0. After centrifugation, the precipitation was discarded and the supernatant was taken to be measured. The contents of ATP and ATPase were determined by ELISA method, and the assay kit was produced by Shanghai Enzyme-Linked Biotechnology Co., Ltd., China. Statistical analysis Data were analyzed by SPSS 11.5 software (IBM Corp., Armonk, NY, USA) with at least 3 independent replicates for each data. Results Effect of different nitrogen levels on spikelet fertility, grain weight and yield of rice under low-light At the LN level, low-light treatment significantly decreased spikelet fertility of WT, d1 , and OE-1 by 14.7%, 31.7%, and 12.5%, respectively. At the MN level, low-light treatment significantly reduced spikelet fertility of WT and d1 by 10.1% and 28.8%, respectively. Under HN treatment, only d1 showed a significant reduction in spikelet fertility under low-light treatment. (Fig. 1 a, b and c). Low-light treatment had minimal impact on the 1000-grain weight of rice across different nitrogen levels (Fig. 1 d, e and f). The changes in rice yield mirrored those of spikelet fertility. At the LN level, the yields of WT, d1 , and OE-1 were significantly reduced by 17.2%, 35.7%, and 13.7%, respectively. At the MN level, low-light treatment notably suppressed the yield of WT and d1 by 13.7% and 28.8%, respectively. At the HN level, d1 yields were the only ones significantly reduced under low-light conditions, with both WT and OE-1 yields showing a slight increase compared to the controls. (Fig. 1 g, h and i). Effect of different nitrogen levels on pollen grain number, pollen germination rate and pollen tube elongation in pistil under low-light Under control conditions, pollen tube elongation towards the ovule was observed in all rice pistils. However, under low-light treatment, pollen tube rupture occurred in rice pistils (Fig. 2A). It was observed that low-light conditions led to a slight decrease in stigma pollen grain number, albeit within a small range of variation. Similarly, the impact of different nitrogen levels on stigma pollen grain number was also minimal (Fig. 2B a, b and c). In comparison to the control group, low-light treatment inhibited the pollen germination rate of WT and d1 . The extent of inhibition diminished with higher nitrogen levels; however, these differences were not deemed statistically significant (Fig. 2B d, e and f). At the LN level, low-light treatment decreased the proportion of pollen tube elongation to the ovule in WT, d1 , and OE-1 pistils by 21.5%, 34.5%, and 12.8%, respectively. At the MN level, low-light treatment significantly reduced the proportion of pollen tube elongation to the ovule in WT and d1 pistils by 17.7% and 35.4%, respectively. Under HN conditions, only the proportion of pollen tube elongation to the ovule in d1 pistils was significantly reduced under low-light conditions (Fig. 2B g, h and i). Effect of different nitrogen levels on the content of nitrogen in spikelets under low-light As illustrated in Fig. 3, the impact of nitrogen levels on nitrogen content in spikelets varied among different rice lines under low-light. The response pattern of nitrogen content in WT and OE-1 was consistent. Across all nitrogen levels, low-light led to an increase in nitrogen content in spikelets, with the most significant increase observed at the MN level (Fig. 3a and c). However, the nitrogen content in spikelets of d1 remained unaffected by low-light treatment across all nitrogen levels (Fig. 3b). Effect of different nitrogen levels on the starch, soluble sugar, and non-structural carbohydrate (NSC) contents in spikelets under low-light At the LN level, low-light treatment led to a significant decrease in starch content in WT and d1 spikelets by 6.5% and 11.8%, respectively. At MN and HN levels, starch content in d1 spikelets decreased significantly under low-light conditions (Fig. 4a, b and c). Soluble sugar content in spikelets of WT, d1 and OE-1 was inhibited by low-light treatment across all nitrogen levels, with significance observed at the LN level but not at the MN and HN levels (Fig. 4d, e and f). Furthermore, low-light treatment significantly suppressed NSC content in d1 spikelets at all nitrogen levels, while it did not notably affect WT at the HN level, and OE-1 spikelets at the HN and MN levels (Fig. 4g, h and i). Effect of different nitrogen levels on sucrose metabolism in spikelets under low-light Low-light treatment inhibited sucrose content in WT, d1 , and OE-1 spikelets across all nitrogen levels. Compared to WT and OE-1, d1 spikelets exhibited a lesser decrease in sucrose content. But notably, sucrose content in OE-1 spikelets decreased significantly only at the MN and HN levels under low-light treatment (Fig. 5 a, b and c). At the LN level, glucose content decreased in WT, d1 , and OE-1 spikelets under low-light treatment compared to control conditions, with significant decreases observed only in WT and d1 spikelets. In contrast, at the MN and HN levels, low-light treatment led to a decrease in glucose content in WT and d1 spikelets, although these changes were not significant. Glucose content in OE-1 spikelets slightly increased at the MN level under low-light treatment but decreased at the HN level (Fig. 5 d, e and f). The variation trend of fructose content in WT, d1 , and OE-1 spikelets after low-light treatment under different nitrogen levels mirrored that of glucose, with no significant differences observed (Fig. 5 g, h and i). At the LN level, the ratio of sucrose to total sugar (sucrose + glucose + fructose) in WT, d1 , and OE-1 rice spikelets increased after low-light treatment compared to the control condition, albeit insignificantly. In contrast, at the MN and HN levels, the proportion of sucrose in WT and OE-1 spikelets decreased under low-light treatment, with a significant decrease observed in OE-1 at the MN level. Moreover, d1 showed less sensitivity to changes in different nitrogen levels (Fig. 5 j, k and l). The effects of different nitrogen levels on INV and SUS activities in WT, d1 , and OE-1 spikelets varied under low-light treatment. At the LN level, INV and SUS activities decreased in WT spikelets under low-light compared to the control, with only INV activities showing a significant decrease. At the MN level, INV and SUS activities in WT spikelets increased, but not significantly, under low-light treatment. At the HN level, low-light treatment led to an increase in INV activity and a decrease in SUS activity in WT spikelets, albeit not significantly in both (Fig. 5 m and p). Low-light treatment significantly reduced INV and SUS activity in d1 spikelets across all nitrogen levels; INV activity decreased by 15.4%, 14.7%, and 14.5% at LN, MN, and HN levels, while SUS activity decreased by 12.0%, 11.8%, and 11.9%, respectively (Fig. 5 n and q). Low-light treatment significantly enhanced the INV and SUS activities of OE-1 at the MN level, but had little effect on INV and SUS activities at the LN and HN levels (Fig. 5 o and r). Effect of different nitrogen levels on the content of ATP and ATPase in spikelets under low-light Low-light treatment significantly increased ATP content in WT spikelets at the MN level, and significantly decreased ATP and ATPase content in WT spikelets at the LN level (Fig. 6a and d). Across all nitrogen levels, low-light treatment significantly inhibited ATP and ATPase content in d1 spikelets (Fig. 6b and e). At the MN level, low-light treatment significantly increased the contents of ATP and ATPase in OE-1 spikelets (Fig. 6c and f). Discussion In this experiment, the shading efficiency of the double-layer black shading net was approximately 73% (Online Resource 1), which is similar to the light intensity on overcast and rainy days in summer as estimated by Lv et al. (2021), indicating that this shading method to some extent simulates the severe overcast and rainy weather encountered by rice during anthesis. Consistent with previous results (Li et al., 2023 ), low-light had different inhibitory effects on spikelet fertility and yield in WT, d1 , and OE-1, with the reduction magnitude being d1 > WT > OE-1, mainly due to differences in pollen tube elongation (Figs. 1 and 2). Furthermore, appropriate nitrogen fertilization can regulate pollen tube elongation in rice pistils under low-light to decrease yield losses, but different rice lines respond differently to nitrogen. Under low-light conditions, as nitrogen increased to the MN level, the inhibition of pollen tube elongation in pistils of WT and OE-1 was alleviated; and the difference with the control conditions also narrowed at the HN level. As for d1 , the influence of different nitrogen levels on its pollen tube elongation under low-light treatment was minimal, suggesting that this may be related to its insensitivity to nitrogen. Sun et al. ( 2014 ) demonstrated that RGA1 deficiency causes nitrogen insensitivity in rice plants. In this experiment, low-light increased the nitrogen content in in the spikelets of both WT and OE-1, and appropriate nitrogen application also increased the nitrogen content (Fig. 3). These results indicate that rice tends to increase the nitrogen content in spikelets in response to low-light. In contrast, the nitrogen content in spikelets of d1 , as a dull-sensitive genotype, was not affected by low-light, which is similar to the results of Marmagne et al. ( 2022 ). Therefore, the difference in nitrogen sensitivity between WT, OE-1, and d1 leads to different regulatory effects of nitrogen on their pollen tube elongation. Nitrogen can affect the sugar metabolism in spikelets of rice under low-light. In this study, nitrogen application under low-light treatment decreased the proportion of sucrose to total sugars (sucrose + glucose + fructose) in the spikelets of both WT and OE-1 and increased the activities of INV and SUS (Fig. 5 ). These findings suggest that elevated nitrogen levels promote sucrose metabolism into monosaccharides, which may be related to the regulatory effect of plants on carbon and nitrogen balance. Studies have indicated that the accumulation of nitrogen metabolites under low-light conditions can suppress nitrate reductase expression, and exogenous sugar addition can promote carbon-nitrogen balance to alleviate this inhibition (Vincentz et al., 1993 ). Liu et al. ( 2022 ) also demonstrated that the application of exogenous sucrose enhanced SUS activity in plants with excessively high nitrogen levels, thereby facilitating carbohydrate metabolism to uphold the equilibrium of carbon and nitrogen metabolism. Consequently, the elevation of nitrogen content in WT and OE-1 spikelets under low-light conditions may trigger sucrose metabolism activation to sustain the equilibrium of carbon and nitrogen. On the other hand, as described by Paul and Foyer ( 2001 ), the utilization of sucrose depends on the plant's ability to produce amino acids through nitrogen metabolism, and the production of amino acids requires the conversion of sucrose into ATP, both of which require the maintenance of energy homeostasis for coordination. In this experiment, improving nitrogen levels under low-light treatment increased ATP and ATPase contents in the spikelets of WT and OE-1 (Fig. 6), indicating that nitrogen played a regulatory role in energy homeostasis within spikelets under low-light conditions. Recent studies have reported similar findings, showing that nitrogen can modulate rice respiration by enhancing ATP content and the activities of NADH dehydrogenase, cytochrome oxidase, and ATPase in plants (Ma et al., 2022 ). Moreover, improving INV activity to stimulate sucrose metabolism and maintain energy homeostasis under stress conditions can ensure pollen tube elongation in the pistil of rice (Jiang et al., 2020 ; Li et al., 2023 ). Thus, the regulatory impact of nitrogen on energy homeostasis proves beneficial for the pollen tube elongation process, which requires substantial ATP consumption (Rounds et al., 2011 ). In summary, moderately increasing nitrogen levels can promote sucrose metabolism, maintain energy balance, and prevent low-light stress induced inhibition of pollen tube elongation and spikelet fertility. Conclusion Low-light treatment led to a decrease in spikelet fertility and rice yield, primarily attributed to the impediment of pollen tube elongation. In WT and OE-1, compared to the LN level, the MN level was more favorable for pollen tube elongation in the female stamens during anthesis under low-light conditions, thereby reducing the loss of spikelet fertility and yield, while HN levels inhibited spikelet fertility and yield. The nitrogen content in WT and OE-1 spikelets increased under low-light conditions. Conversely, d1 exhibited no significant response in terms of spikelet fertility, yield, pollen tube elongation, and nitrogen content to changes in nitrogen levels due to its nitrogen-insensitive nature. Under low-light treatment, the MN level enhanced INV and SUS activities in spikelets compared to the LN level, leading to decreased starch metabolism and increased sucrose conversion efficiency. Additionally, ATP and ATPase contents in WT and OE-1 spikelets remained at elevated levels. These findings suggest that moderately increasing nitrogen levels could stimulate sucrose metabolism, uphold energy equilibrium, and prevent obstacles to pollen tube elongation and spikelet abortion induced by low-light stress. Declarations Acknowledgements This work was supported by the Zhejiang A&F University Scientific Research Development Fund Projects (2023LFR038 to Linzhou Huang and 2024LFR026 to Hubo Li). Funding This work was supported by the Zhejiang A&F University Scientific Research Development Fund Projects (2023LFR038 to Linzhou Huang and 2024LFR026 to Hubo Li). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author contributions Linzhou Huang and Dali Zeng designed the research. Yichang Zhong, Hubo Li, Feifei Li and Qiao Deng performed the experiments. Yichang Zhong, Hubo Li and Guanfu Fuanalyzed the data. Yichang Zhong, Hubo Li and Linzhou Huang wrote the paper with contributions from all the authors. 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FEBS Lett 582(29):4077-82. https://doi.org/10.1016/j.febslet.2008.11.002 Soto G, Fox R, Ayub N, Alleva K, Guaimas F, Erijman EJ, Mazzella A, et al (2010) TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is probably involved in nitrogen remobilization in Arabidopsis thaliana. Plant J 64(6):1038-47. https://doi.org/10.1111/j.1365-313X.2010.04395.x Sun H, Qian Q, Wu K, Luo J, Wang S, Zhang C, Ma Y, et al (2014) Heterotrimeric G proteins regulate nitrogen-use efficiency in rice. Nat Genet 46(6): 652-6. https://doi.org/10.1038/ng.2958 Tian T, Wang J, Wang H, Cui J, Shi X, Song J, Li W, et al (2022) Nitrogen application alleviates salt stress by enhancing osmotic balance, ROS scavenging, and photosynthesis of rapeseed seedlings (Brassica napus). Plant Signal Behav 17(1):2081419. https://doi.org/10.1080/15592324.2022.2081419 Vincentz M, Moureaux T, Leydecker MT, Vaucheret H, Caboche M (1993) Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant J 3(2): 315-24. https://doi.org/10.1111/j.1365-313x.1993.tb00183.x Wilson ZA, Song J, Taylor B, Yang C (2011) The final split: the regulation of anther dehiscence. J Exp Bot 62(5):1633-49. https://doi.org/10.1093/jxb/err014 Xiang X, Zhang P, Yu P, Zhang Y, Yang Z, Sun L, Wu W, et al (2019) LSSR1 facilitates seed setting rate by promoting fertilization in rice. Rice 12(1): 31. https://doi.org/10.1186/s12284-019-0280-3 Xiong Q, Zhong L, Du J, Zhu C, Peng X, He X, Fu J, et al (2020) Ribosome profiling reveals the effects of nitrogen application translational regulation of yield recovery after abrupt drought-flood alternation in rice. Plant Physiol Biochem 155:42-58. https://doi.org/10.1016/j.plaphy.2020.07.021 Xu Y, Yang J, Wang Y, Wang J, Yu Y, Long Y, Wang Y, et al (2017) OsCNGC13 promotes sxianged-setting rate by facilitating pollen tube growth in stylar tissues. PLoS Genet 13(7): e1006906. https://doi.org/10.1371/journal.pgen.1006906 Zhang C, Li G, Chen T, Feng B, Fu W, Yan J, Islam MR, et al (2018a) Heat stress induces spikelet sterility in rice at anthesis through inhibition of pollen tube elongation interfering with auxin homeostasis in pollinated pistils. Rice , 11(1): 14. https://doi.org/10.1186/s12284-018-0206-5 Zhang C, Feng B, Chen T, Fu W, Li H, Li G, Jin Q, et al (2018b) Heat stress-reduced kernel weight in rice at anthesis is associated with impaired source-sink relationship and sugars allocation. Environ Exp Bot 155: 718-733. https://doi.org/10.1016/j.envexpbot.2018.08.021 Zhou Q, Wang H, Xu C, Zheng S, Wu M, Zhang Q, Liao Y, et al (2022) Nitrogen application practices to reduce cadmium concentration in rice ( Oryza sativa L.) grains. Environ Sci Pollut Res Int 29(33):50530-50539. https://doi.org/10.1007/s11356-022-19381-x Supplementary Files ESM1.pdf ESM2.pdf Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2024 Read the published version in Plant Growth Regulation → Version 1 posted Editorial decision: Major revisions 14 Aug, 2024 Reviewers agreed at journal 22 Jul, 2024 Reviewers invited by journal 22 Jul, 2024 Editor invited by journal 22 Jul, 2024 Editor assigned by journal 16 Jul, 2024 First submitted to journal 13 Jul, 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. 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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-4737345","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330008183,"identity":"f695f382-60db-42e3-bddd-05bcbcb349e6","order_by":0,"name":"Yichang Zhong","email":"","orcid":"","institution":"Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Yichang","middleName":"","lastName":"Zhong","suffix":""},{"id":330008184,"identity":"f93aa03a-d1c8-44c8-9864-9ba0c8fddcee","order_by":1,"name":"Hubo Li","email":"","orcid":"","institution":"Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Hubo","middleName":"","lastName":"Li","suffix":""},{"id":330008185,"identity":"ed5f5915-d130-4cdd-b40d-aabe79381abd","order_by":2,"name":"Feifei Li","email":"","orcid":"","institution":"Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Feifei","middleName":"","lastName":"Li","suffix":""},{"id":330008186,"identity":"93436ba0-62c1-4b2b-8f32-53fd14f5c77d","order_by":3,"name":"Guanfu Fu","email":"","orcid":"","institution":"China National Rice Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Guanfu","middleName":"","lastName":"Fu","suffix":""},{"id":330008187,"identity":"aa72affd-b3cd-4f01-8139-c527300e4258","order_by":4,"name":"Qiao Deng","email":"","orcid":"","institution":"Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Qiao","middleName":"","lastName":"Deng","suffix":""},{"id":330008188,"identity":"a17b51a1-ad35-499a-bf32-17ffebfc9e96","order_by":5,"name":"Dali Zeng","email":"","orcid":"","institution":"Zhejiang A and F University","correspondingAuthor":false,"prefix":"","firstName":"Dali","middleName":"","lastName":"Zeng","suffix":""},{"id":330008189,"identity":"c40b1c6d-0ad7-4404-b3b4-8dd2eab6a293","order_by":6,"name":"Linzhou Huang","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3990-765X","institution":"Zhejiang A and F University","correspondingAuthor":true,"prefix":"","firstName":"Linzhou","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-07-14 06:56:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4737345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4737345/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10725-024-01269-0","type":"published","date":"2024-12-31T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62482685,"identity":"1d16dfd9-b38f-48f4-a0f2-13165174b174","added_by":"auto","created_at":"2024-08-14 17:23:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on spikelet fertility, grain weight and yield of rice under low-light treatment.\u003c/strong\u003e a-c, Spikelet fertility of WT, \u003cem\u003ed1\u003c/em\u003e and the OE-1 rice plants under different levels of nitrogen (LN, low nitrogen; MN, medium nitrogen; HN, high nitrogen); d-f, 1000-grain weight of WT, \u003cem\u003ed1\u003c/em\u003e and the OE-1 rice plants under different nitrogen levels; g-i, Rice yield of WT, \u003cem\u003ed1\u003c/em\u003e and the OE-1 rice plants under different nitrogen levels. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and * indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/f68abe0eb1c4ad230d012eb8.png"},{"id":62482686,"identity":"37f9e895-6aa2-427b-a4e1-6b7111f7fccf","added_by":"auto","created_at":"2024-08-14 17:23:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":75654,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on the number of pollen grains on stigma, pollen germination rate, and pollen tube elongation in pistils of rice under low-light treatment.\u003c/strong\u003e A, Morphology of pistils (scale bar = 500 μm); B, a-c, Number of pollen grains on stigma of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under low-nitrogen (LN), medium-nitrogen (MN) and high-nitrogen (HN) conditions; d-f, Pollen germination rate the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different nitrogen levels; h-j, Ratio of pollen tubes in ovules of the the WT, \u003cem\u003ed1\u003c/em\u003eand OE-1 rice plants under different nitrogen levels. The white triangles indicate the end point of pollen tube elongation. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and * indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/e69370d4fbdcb40a029e8e9c.png"},{"id":62482687,"identity":"031cc953-3025-43e9-8e29-ca9cc2083da0","added_by":"auto","created_at":"2024-08-14 17:23:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":21268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on the N content in spikelets of rice under low-light treatment.\u003c/strong\u003e a-c, N content in spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and * indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/583c937b4fd7bfc8a8ad51c5.png"},{"id":62482363,"identity":"7697e23b-d3f7-4efa-8bd4-757a5e538c57","added_by":"auto","created_at":"2024-08-14 17:15:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":51632,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on the non-structural carbohydrate (NSC) content in spikelets of rice under low-light treatment.\u003c/strong\u003e a-c, Starch content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; d-f, Soluble sugar content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; g-i, NSC content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and *indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/2596f31424b6cb356972b0f2.png"},{"id":62482688,"identity":"c9b3a596-03e9-43d3-a78e-a13a0ae3eaae","added_by":"auto","created_at":"2024-08-14 17:23:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on the conversion of sucrose in spikelets of rice under low-light treatment.\u003c/strong\u003e a-c, sucrose content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; d-f, Glucose content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; g-i, Fructose content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; j-l, Sucrose / (sucrose+ glucose+fructose) in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; m-o, the activity of INV in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; p-r, the activity of SUS in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and * indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/686bef79dbe002ef305cb3d4.png"},{"id":62482917,"identity":"8dfbfc7c-ddf6-4d24-a570-02cba443fcab","added_by":"auto","created_at":"2024-08-14 17:31:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":35567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of different nitrogen levels on the content of ATP and ATPase in spikelets of rice under low-light treatment.\u003c/strong\u003e a-c, ATP content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen; d-f, ATPase content in the spikelets of the WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 rice plants under different levels of nitrogen. The mean values and standard errors in the figures correspond to data from 3 replicates. A student’s \u003cem\u003et\u003c/em\u003e-test was used to compare and analyze the differences between treatments, and * indicates the significant difference (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/8029a84061fd86386fbdff31.png"},{"id":73094746,"identity":"7979befd-28a4-43d1-b326-98c64911efec","added_by":"auto","created_at":"2025-01-06 16:24:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1054858,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/ddb5ea0b-6185-495c-875c-43817a695970.pdf"},{"id":62482365,"identity":"36ea69a4-9f34-4fd2-b421-5e6b3a666c17","added_by":"auto","created_at":"2024-08-14 17:15:07","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":172373,"visible":true,"origin":"","legend":"","description":"","filename":"ESM1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/009681cdec3b94c7c4698c54.pdf"},{"id":62482369,"identity":"6e6e3df1-f88c-4075-adc2-c87a9b27bab4","added_by":"auto","created_at":"2024-08-14 17:15:07","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":118410,"visible":true,"origin":"","legend":"","description":"","filename":"ESM2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4737345/v1/df472147bd96f4677722dbfb.pdf"}],"financialInterests":"","formattedTitle":"Nitrogen regulates pollen tube elongation under low-light stress during anthesis to prevent spikelet abortion in rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice is a primary staple for over 50% of the world's population (Muthayya et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As global population rises, the production of rice becomes increasingly vital for ensuring global food security (Searching et al., 2018). However, during the actual production process, rice yield can be easily restricted by various environmental factors, such as light availability. Being a sun-loving crop, rice requires approximately 1500 hours of bright daylight from transplantation to maturity to thrive (Panigrahy et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Insufficient light severely inhibits rice growth, resulting in decreased yields. In parts of China, recent environmental pollution has exacerbated climate deterioration, leading to frequent long rainy weather conditions that pose a threat to rice yields (Guo et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, the increasing air pollution is reducing the solar radiation reaching the Earth's surface each year, potentially causing a loss of approximately 0.4\u0026ndash;1.8% of global crop production in the future (M\u0026uuml;ller et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). It is evident that the occurrence of low-light has emerged as one of the primary stresses impeding rice growth. The anthesis stage of rice marks a crucial period for spikelet fertility and yield formation. When encountering abiotic stress (high temperature, drought, salinity, etc.), fertilization failure can lead to reduce grain yields (Jagadish et al., 2020; Xiang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previous research has indicated that low-light exposure during the anthesis of rice could hinder anther dehiscence, diminish pollen grain numbers, and suppress pollen germination (Cecchetti et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Wilson et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Recent studies have further revealed that inadequate light during anthesis inhibits pollen tube elongation in rice, resulting in significant yield losses (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Hence, it is imperative to develop relevant cultivation strategies to mitigate rice yield losses attributed to low-light stress.\u003c/p\u003e \u003cp\u003eNitrogen (N) is an essential nutrient element for the growth and development of crops, regulating their resistance to abiotic stresses by enhancing physiological characteristics and organic structure (Ma et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Numerous studies have demonstrated that the appropriate application of nitrogen fertilizer can mitigate crop yield losses under various stresses such as drought, flooding, heat, salinity, alkalinity, heavy metals, and others during the anthesis period (Liu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xiong et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, heat stress can lead to spikelet abortion, but higher nitrogen levels can reduce the number of degenerated spikelets under such conditions (Liu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Nitrogen stored in anthers and pollen grains, along with its metabolic activity, is beneficial for stress resistance (Santiago and Sharkey, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, when nitrogen was deficient in the medium, the losses of nitrogen transporter TIP1;3 and TIP5;1 in the pollen tube would inhibit the pollen tube elongation in Arabidopsis thaliana (Soto et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Soto et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), possibly due to nitrogen's regulation of energy status in the pistil. Ma et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) demonstrated that nitrogen (N), phosphorus (P), and potassium (K) could all improve plant energy status and affect rice growth and development, with nitrogen having the greatest effect. Pollen tube elongation is a highly energy-consuming physiological activity, and the lack of energy caused by low-light can hinder rice pollen tube elongation (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). And low-light stress can reduce the net photosynthetic rate of leaves to decrease rice's energy supply (Deng et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Given this, we proposed that regulating nitrogen levels under low-light stress may alleviate the inhibition of pollen tube elongation by adjusting energy levels.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRGA1\u003c/em\u003e is responsible for encoding the heterotrimeric G protein α subunit of rice (Ishikawa et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). The Gα subunit plays a role in mediating cellular response to extracellular stimuli, transmitting signals from receptors to downstream effectors, and regulating rice tolerance to abiotic stressors such as high temperature, drought, and salinity (Misra et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ferrero-Serrano and Assmann, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jangam et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Previous research has also established a link between \u003cem\u003eRGA1\u003c/em\u003e and rice's response to light, demonstrating its ability to enhance energy conversion through increased sucrose metabolic rates, maintaining ATP production efficiency, and thereby bolstering rice resilience to low-light stress (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nitrogen, as well, influences rice growth and development by improving plant energy status (Ma et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), but its specific effects on pollen tube elongation under low-light conditions during anthesis and the associated regulatory mechanisms require further elucidation. Therefore, this study utilized \u003cem\u003eRGA1\u003c/em\u003e mutants (\u003cem\u003ed1\u003c/em\u003e), their corresponding wild types (WT), and over-expressed plants (OE-1) as experimental materials. Different nitrogen fertilizer levels were applied concurrently with shading treatment during anthesis to investigate the mechanism by which nitrogen levels affect pollen tube elongation under low-light conditions during anthesis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth condition\u003c/h2\u003e \u003cp\u003eThe research was conducted from June to September 2022 on rice experimental station at the China Rice Research Institute (CNRRI) in Hangzhou, Zhejiang Province. The low-light-intolerant \u003cem\u003eRGA1\u003c/em\u003e gene mutant (\u003cem\u003ed1\u003c/em\u003e), and its wild type (Zhonghua 11, WT), along with a \u003cem\u003eRGA1\u003c/em\u003e gene overexpressed line (OE-1) were documented in previous study (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). A split-split-plot design was used in the experiment. Main plots were treated with shading or not; spilt-plots were treated with low nitrogen (60 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, LN), medium nitrogen (160 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MN) and high nitrogen (260 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, HN); and split-split-plots consist of three rice cultivars (WT、\u003cem\u003ed1\u003c/em\u003e and OE-1). Each treatment was repeated 3 times. The physical and chemical properties of soil are shown in the Online Resource 2. Rice was sown on May 19 and transplanted on June 17, 29 days post sowing. The rice was single planted, with the density of 20 cm \u0026times; 25 cm and each plot area of 10 m\u003csup\u003e2\u003c/sup\u003e. The application of nitrogen fertilizer (urea) followed the ratio of basal fertilizer: tillering fertilizer: panicle fertilizer\u0026thinsp;=\u0026thinsp;4:3:3. Phosphate fertilizer (P\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) was applied as a single application for the base fertilizer at a rate of 80 kg P\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Potassium fertilizer (K\u003csub\u003e2\u003c/sub\u003eO) was applied according to the ratio of basal fertilizer: panicle fertilizer\u0026thinsp;=\u0026thinsp;6:4, with the application amount of 160 kg K\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. To ensure the stability of nitrogen level in different plots, all the plots were covered with black plastic film to the bottom of the fields. At the anthesis stage, a double layer black shading net was used to simulate low-light treatment, and the treatment time was 7 days. The height of the shading net was 50 cm from the top of the rice to ensure air exchange under the shading net, so as to maintain the consistency of the treatment and field temperature as far as possible. The climatic conditions during the experiment are shown in Online Resource 1. After the treatment, the shading net was removed to allow the rice to grow to maturity under natural conditions. Diseases, pests and grasses were strictly controlled during the whole growth period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSpikelet fertility, grain weight and yield\u003c/h2\u003e \u003cp\u003eWhen rice was harvested, samples were taken from each plot according to 5-point method, dried in oven to constant weight, and then threshed to investigate spikelet fertility and 1000-grain weight. At the time of harvest, samples were also taken separately by the 5-point method for yield measurement. After drying and threshing, the grain was weighed, and the yield per hectare was converted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePollen grain number on stigma, pollen germination rate and pistil tube elongation\u003c/h2\u003e \u003cp\u003eOn the day 4 of low-light treatment, flowering spikelets were taken from the field, removed the pistils, and immediately fixed in FAA fixation solution. Subsequently, according to the method of Zhang et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018a\u003c/span\u003e), the fixed pistil tissues were placed in 10 mol/L NaOH solution, incubated at 55℃ for 10 min, washed with distilled water, and stained with 0.1% aniline blue solution for more than 2h. Then, the growth of pollen tube was observed at 350nm using fluorescence microscopy (DM4000B, Leica, Wetzlar, Germany). Randomly divide 50 pistils into 5 groups, and calculate the elongation ratio of pollen tubes in each group based on the standard of pollen tube reaching the ovule. And the number of stigma pollen grains and germination pollen were counted. Pollen germination rate\u0026thinsp;=\u0026thinsp;germination pollen number/ (germination pollen number\u0026thinsp;+\u0026thinsp;non-germination pollen number) \u0026times;100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen content\u003c/h2\u003e \u003cp\u003eOn the day 4 of low-light treatment, the flowering spikelets were taken from the field and dried to constant weight after sampling. The dried samples were first digested with H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e-H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and then the total nitrogen content was determined by the Kjeldahl method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNon-structural carbohydrate content\u003c/h2\u003e \u003cp\u003eOn the day 4 of low-light treatment, the flowering spikelets were taken from the field and frozen with liquid nitrogen to be measured. Referring to the method of Dubois et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1956\u003c/span\u003e), anthrone sulfate colorimetry was used to determine the contents of total soluble sugars and starch. Sucrose, glucose, and fructose are extracted in the same way as total soluble sugars. The method of Zhang (2018b) was used to determine the content of sucrose, glucose and fructose. The total non-structural carbohydrate (NSC) content is the sum of soluble sugar and starch content.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSucrose synthase (SUS) and invertase (INV) activities\u003c/h2\u003e \u003cp\u003e0.1g spikelet sample frozen with liquid nitrogen was added with 3 ml HEPS-NaOH (pH7.5, containing 50 mmol/L HEPES, 10 mmol/L MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mmol/L EDTA, 1% PVP40, 12.5% glycerol, 5 mmol/L DTT) extract and was grounded into a homogenization. Then centrifuged at 10, 000 rpm/min for 10 min at 4℃. Then 0.1 ml supernatant was added into 0.9 ml working solution (containing 50 mmol/L HEPES, 5 mmol/L UDP, 5 mmol/L magnesium acetate, 50 mmol/L sucrose, 5 mmol/L DTT, the reaction solution needs to be adjusted to pH7.5 by NaOH), and the reaction solution was bathed in water at 30℃ for 20 min. The reaction was terminated in boiling water bath for 5 min. After cooling, added 1 ml DNS and bathed in boiling water for 5 min After cooling, the color was compared at 540 nm with ultraviolet spectrophotometer, and the boiled dead enzyme solution was used as the control. The SUS activity was calculated by using glucose standard solution as the standard curve. The extraction method of INV is the same as SUS. 0.05 ml of supernatant was added to 0.95 ml of acetate-K\u003csub\u003e3\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e buffer (pH4.7, containing 50 mmol/L sucrose). After 20 min of water bath at 30℃ and 5 min of boiling water bath, the reaction was terminated. After cooling, added 1 ml of DNS with boiling water bath for 5 min. The color was compared at 540 nm by ultraviolet spectrophotometer after cooling, and the boiled dead enzyme solution was used as the control. INV activity was calculated using glucose standard solution as standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eATP and ATPase content\u003c/h2\u003e \u003cp\u003e0.1 g frozen sample were ground in liquid nitrogen and ground into homogenate in PBS with pH7.0. After centrifugation, the precipitation was discarded and the supernatant was taken to be measured. The contents of ATP and ATPase were determined by ELISA method, and the assay kit was produced by Shanghai Enzyme-Linked Biotechnology Co., Ltd., China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed by SPSS 11.5 software (IBM Corp., Armonk, NY, USA) with at least 3 independent replicates for each data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of different nitrogen levels on spikelet fertility, grain weight and yield of rice under low-light\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the LN level, low-light treatment significantly decreased spikelet fertility of WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 by 14.7%, 31.7%, and 12.5%, respectively. At the MN level, low-light treatment significantly reduced spikelet fertility of WT and \u003cem\u003ed1\u003c/em\u003e by 10.1% and 28.8%, respectively. Under HN treatment, only \u003cem\u003ed1\u003c/em\u003e showed a significant reduction in spikelet fertility under low-light treatment. (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b and c). Low-light treatment had minimal impact on the 1000-grain weight of rice across different nitrogen levels (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed, e and f). The changes in rice yield mirrored those of spikelet fertility. At the LN level, the yields of WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 were significantly reduced by 17.2%, 35.7%, and 13.7%, respectively. At the MN level, low-light treatment notably suppressed the yield of WT and \u003cem\u003ed1\u003c/em\u003e by 13.7% and 28.8%, respectively. At the HN level, \u003cem\u003ed1\u003c/em\u003e yields were the only ones significantly reduced under low-light conditions, with both WT and OE-1 yields showing a slight increase compared to the controls. (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg, h and i).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of different nitrogen levels on pollen grain number, pollen germination rate and pollen tube elongation in pistil under low-light\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder control conditions, pollen tube elongation towards the ovule was observed in all rice pistils. However, under low-light treatment, pollen tube rupture occurred in rice pistils (Fig. 2A). It was observed that low-light conditions led to a slight decrease in stigma pollen grain number, albeit within a small range of variation. Similarly, the impact of different nitrogen levels on stigma pollen grain number was also minimal (Fig. 2B a, b and c). In comparison to the control group, low-light treatment inhibited the pollen germination rate of WT and \u003cem\u003ed1\u003c/em\u003e. The extent of inhibition diminished with higher nitrogen levels; however, these differences were not deemed statistically significant (Fig. 2B d, e and f). At the LN level, low-light treatment decreased the proportion of pollen tube elongation to the ovule in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 pistils by 21.5%, 34.5%, and 12.8%, respectively. At the MN level, low-light treatment significantly reduced the proportion of pollen tube elongation to the ovule in WT and \u003cem\u003ed1\u003c/em\u003e pistils by 17.7% and 35.4%, respectively. Under HN conditions, only the proportion of pollen tube elongation to the ovule in \u003cem\u003ed1\u003c/em\u003e pistils was significantly reduced under low-light conditions (Fig. 2B g, h and i).\u003c/p\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of different nitrogen levels on the content of nitrogen in spikelets under low-light\u003c/h2\u003e\n \u003cp\u003eAs illustrated in Fig. 3, the impact of nitrogen levels on nitrogen content in spikelets varied among different rice lines under low-light. The response pattern of nitrogen content in WT and OE-1 was consistent. Across all nitrogen levels, low-light led to an increase in nitrogen content in spikelets, with the most significant increase observed at the MN level (Fig. 3a and c). However, the nitrogen content in spikelets of \u003cem\u003ed1\u003c/em\u003e remained unaffected by low-light treatment across all nitrogen levels (Fig. 3b).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of different nitrogen levels on the starch, soluble sugar, and non-structural carbohydrate (NSC) contents in spikelets under low-light\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAt the LN level, low-light treatment led to a significant decrease in starch content in WT and \u003cem\u003ed1\u003c/em\u003e spikelets by 6.5% and 11.8%, respectively. At MN and HN levels, starch content in \u003cem\u003ed1\u003c/em\u003e spikelets decreased significantly under low-light conditions (Fig. 4a, b and c). Soluble sugar content in spikelets of WT, \u003cem\u003ed1\u003c/em\u003e and OE-1 was inhibited by low-light treatment across all nitrogen levels, with significance observed at the LN level but not at the MN and HN levels (Fig. 4d, e and f). Furthermore, low-light treatment significantly suppressed NSC content in \u003cem\u003ed1\u003c/em\u003e spikelets at all nitrogen levels, while it did not notably affect WT at the HN level, and OE-1 spikelets at the HN and MN levels (Fig. 4g, h and i).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of different nitrogen levels on sucrose metabolism in spikelets under low-light\u003c/h2\u003e\n \u003cp\u003eLow-light treatment inhibited sucrose content in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 spikelets across all nitrogen levels. Compared to WT and OE-1, \u003cem\u003ed1\u003c/em\u003e spikelets exhibited a lesser decrease in sucrose content. But notably, sucrose content in OE-1 spikelets decreased significantly only at the MN and HN levels under low-light treatment (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b and c). At the LN level, glucose content decreased in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 spikelets under low-light treatment compared to control conditions, with significant decreases observed only in WT and \u003cem\u003ed1\u003c/em\u003e spikelets. In contrast, at the MN and HN levels, low-light treatment led to a decrease in glucose content in WT and \u003cem\u003ed1\u003c/em\u003e spikelets, although these changes were not significant. Glucose content in OE-1 spikelets slightly increased at the MN level under low-light treatment but decreased at the HN level (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed, e and f). The variation trend of fructose content in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 spikelets after low-light treatment under different nitrogen levels mirrored that of glucose, with no significant differences observed (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eg, h and i). At the LN level, the ratio of sucrose to total sugar (sucrose\u0026thinsp;+\u0026thinsp;glucose\u0026thinsp;+\u0026thinsp;fructose) in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 rice spikelets increased after low-light treatment compared to the control condition, albeit insignificantly. In contrast, at the MN and HN levels, the proportion of sucrose in WT and OE-1 spikelets decreased under low-light treatment, with a significant decrease observed in OE-1 at the MN level. Moreover, \u003cem\u003ed1\u003c/em\u003e showed less sensitivity to changes in different nitrogen levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ej, k and l).\u003c/p\u003e\n \u003cp\u003eThe effects of different nitrogen levels on INV and SUS activities in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1 spikelets varied under low-light treatment. At the LN level, INV and SUS activities decreased in WT spikelets under low-light compared to the control, with only INV activities showing a significant decrease. At the MN level, INV and SUS activities in WT spikelets increased, but not significantly, under low-light treatment. At the HN level, low-light treatment led to an increase in INV activity and a decrease in SUS activity in WT spikelets, albeit not significantly in both (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003em and p). Low-light treatment significantly reduced INV and SUS activity in \u003cem\u003ed1\u003c/em\u003e spikelets across all nitrogen levels; INV activity decreased by 15.4%, 14.7%, and 14.5% at LN, MN, and HN levels, while SUS activity decreased by 12.0%, 11.8%, and 11.9%, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003en and q). Low-light treatment significantly enhanced the INV and SUS activities of OE-1 at the MN level, but had little effect on INV and SUS activities at the LN and HN levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eo and r).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEffect of different nitrogen levels on the content of ATP and ATPase in spikelets under low-light\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eLow-light treatment significantly increased ATP content in WT spikelets at the MN level, and significantly decreased ATP and ATPase content in WT spikelets at the LN level (Fig. 6a and d). Across all nitrogen levels, low-light treatment significantly inhibited ATP and ATPase content in \u003cem\u003ed1\u003c/em\u003e spikelets (Fig. 6b and e). At the MN level, low-light treatment significantly increased the contents of ATP and ATPase in OE-1 spikelets (Fig. 6c and f).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this experiment, the shading efficiency of the double-layer black shading net was approximately 73% (Online Resource 1), which is similar to the light intensity on overcast and rainy days in summer as estimated by Lv et al. (2021), indicating that this shading method to some extent simulates the severe overcast and rainy weather encountered by rice during anthesis. Consistent with previous results (Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), low-light had different inhibitory effects on spikelet fertility and yield in WT, \u003cem\u003ed1\u003c/em\u003e, and OE-1, with the reduction magnitude being \u003cem\u003ed1\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;WT\u0026thinsp;\u0026gt;\u0026thinsp;OE-1, mainly due to differences in pollen tube elongation (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and 2). Furthermore, appropriate nitrogen fertilization can regulate pollen tube elongation in rice pistils under low-light to decrease yield losses, but different rice lines respond differently to nitrogen. Under low-light conditions, as nitrogen increased to the MN level, the inhibition of pollen tube elongation in pistils of WT and OE-1 was alleviated; and the difference with the control conditions also narrowed at the HN level. As for \u003cem\u003ed1\u003c/em\u003e, the influence of different nitrogen levels on its pollen tube elongation under low-light treatment was minimal, suggesting that this may be related to its insensitivity to nitrogen. Sun et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) demonstrated that \u003cem\u003eRGA1\u003c/em\u003e deficiency causes nitrogen insensitivity in rice plants. In this experiment, low-light increased the nitrogen content in in the spikelets of both WT and OE-1, and appropriate nitrogen application also increased the nitrogen content (Fig.\u0026nbsp;3). These results indicate that rice tends to increase the nitrogen content in spikelets in response to low-light. In contrast, the nitrogen content in spikelets of \u003cem\u003ed1\u003c/em\u003e, as a dull-sensitive genotype, was not affected by low-light, which is similar to the results of Marmagne et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, the difference in nitrogen sensitivity between WT, OE-1, and \u003cem\u003ed1\u003c/em\u003e leads to different regulatory effects of nitrogen on their pollen tube elongation.\u003c/p\u003e \u003cp\u003eNitrogen can affect the sugar metabolism in spikelets of rice under low-light. In this study, nitrogen application under low-light treatment decreased the proportion of sucrose to total sugars (sucrose\u0026thinsp;+\u0026thinsp;glucose\u0026thinsp;+\u0026thinsp;fructose) in the spikelets of both WT and OE-1 and increased the activities of INV and SUS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These findings suggest that elevated nitrogen levels promote sucrose metabolism into monosaccharides, which may be related to the regulatory effect of plants on carbon and nitrogen balance. Studies have indicated that the accumulation of nitrogen metabolites under low-light conditions can suppress nitrate reductase expression, and exogenous sugar addition can promote carbon-nitrogen balance to alleviate this inhibition (Vincentz et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Liu et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) also demonstrated that the application of exogenous sucrose enhanced SUS activity in plants with excessively high nitrogen levels, thereby facilitating carbohydrate metabolism to uphold the equilibrium of carbon and nitrogen metabolism. Consequently, the elevation of nitrogen content in WT and OE-1 spikelets under low-light conditions may trigger sucrose metabolism activation to sustain the equilibrium of carbon and nitrogen. On the other hand, as described by Paul and Foyer (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), the utilization of sucrose depends on the plant's ability to produce amino acids through nitrogen metabolism, and the production of amino acids requires the conversion of sucrose into ATP, both of which require the maintenance of energy homeostasis for coordination. In this experiment, improving nitrogen levels under low-light treatment increased ATP and ATPase contents in the spikelets of WT and OE-1 (Fig.\u0026nbsp;6), indicating that nitrogen played a regulatory role in energy homeostasis within spikelets under low-light conditions. Recent studies have reported similar findings, showing that nitrogen can modulate rice respiration by enhancing ATP content and the activities of NADH dehydrogenase, cytochrome oxidase, and ATPase in plants (Ma et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, improving INV activity to stimulate sucrose metabolism and maintain energy homeostasis under stress conditions can ensure pollen tube elongation in the pistil of rice (Jiang et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Thus, the regulatory impact of nitrogen on energy homeostasis proves beneficial for the pollen tube elongation process, which requires substantial ATP consumption (Rounds et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In summary, moderately increasing nitrogen levels can promote sucrose metabolism, maintain energy balance, and prevent low-light stress induced inhibition of pollen tube elongation and spikelet fertility.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eLow-light treatment led to a decrease in spikelet fertility and rice yield, primarily attributed to the impediment of pollen tube elongation. In WT and OE-1, compared to the LN level, the MN level was more favorable for pollen tube elongation in the female stamens during anthesis under low-light conditions, thereby reducing the loss of spikelet fertility and yield, while HN levels inhibited spikelet fertility and yield. The nitrogen content in WT and OE-1 spikelets increased under low-light conditions. Conversely, \u003cem\u003ed1\u003c/em\u003e exhibited no significant response in terms of spikelet fertility, yield, pollen tube elongation, and nitrogen content to changes in nitrogen levels due to its nitrogen-insensitive nature. Under low-light treatment, the MN level enhanced INV and SUS activities in spikelets compared to the LN level, leading to decreased starch metabolism and increased sucrose conversion efficiency. Additionally, ATP and ATPase contents in WT and OE-1 spikelets remained at elevated levels. These findings suggest that moderately increasing nitrogen levels could stimulate sucrose metabolism, uphold energy equilibrium, and prevent obstacles to pollen tube elongation and spikelet abortion induced by low-light stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Zhejiang A\u0026amp;F University Scientific Research Development Fund Projects (2023LFR038 to Linzhou Huang and 2024LFR026 to Hubo Li).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Zhejiang A\u0026amp;F University Scientific Research Development Fund Projects (2023LFR038 to Linzhou Huang and 2024LFR026 to Hubo Li).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinzhou Huang and Dali Zeng designed the research. Yichang Zhong, Hubo Li, Feifei Li and Qiao Deng performed the experiments. Yichang Zhong, Hubo Li and Guanfu Fuanalyzed the data. Yichang Zhong, Hubo Li and Linzhou Huang wrote the paper with contributions from all the authors. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCecchetti V, Altamura MM, Falasca G, Costantino P, Cardarelli M (2008) Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation. \u003cem\u003ePlant Cell\u003c/em\u003e 20(7): 1760-1774. https://doi.org/10.1105/tpc.107.057570\u003c/li\u003e\n\u003cli\u003eDeng F, Zeng Y, Li Q, He C, Li B, Zhu Y, Zhou X, et al (2021) Decreased anther dehiscence contributes to a lower fertilization rate of rice subjected to shading stress. \u003cem\u003eField Crops Res\u003c/em\u003e 273: 108291. https://doi.org/10.1016/j.fcr.2021.108291\u003c/li\u003e\n\u003cli\u003eDubois M, Gilles K, Hamilton J, Rebers P, Smith F (1956) Colorimetric method for determination of sugars and related substances. \u003cem\u003eAnal Chem\u003c/em\u003e 28: 350-356. https://doi.org/10.1021/ac60111a017\u003c/li\u003e\n\u003cli\u003eFerrero-Serrano \u0026Aacute;, Assmann SM (2016) The \u0026alpha;-subunit of the rice heterotrimeric G protein, \u003cem\u003eRGA1\u003c/em\u003e, regulates drought tolerance during the vegetative phase in the dwarf rice mutant \u003cem\u003ed1\u003c/em\u003e. \u003cem\u003eJ Exp Bot\u003c/em\u003e 67(11):3433-43. https://doi.org/10.1093/jxb/erw183\u003c/li\u003e\n\u003cli\u003eGuo X, Zhao J, Wang RL, Li XY, Wang MT (2021) Continuous-rain hazard of transplanting and direct-sowing rice in Sichuan Basin, China. \u003cem\u003eChinese J Appl Ecol\u003c/em\u003e 32(9): 3213－3222. https://doi.org/10.13287/j.1001-9332.202109.013\u003c/li\u003e\n\u003cli\u003eIshikawa A, Tsubouchi H, Iwasaki Y, Asahi T (1995) Molecular cloning and characterization of a cDNA for the alpha subunit of a G protein from rice. \u003cem\u003ePlant Cell Physiol \u003c/em\u003e36(2):353-9. https://doi.org/10.1093/oxfordjournals.pcp.a078767\u003c/li\u003e\n\u003cli\u003eLyu HH, Niu YY, Zhang M, Li H (2021) Analysis of variation trend of light intensity and air temperature and humidity in solar greenhouse. \u003cem\u003eTrans Chinese Soc Agric Mach\u003c/em\u003e 52(S0): 410-417. http://dx.doi.org/10.6041/j.issn.1000-1298.2021.S0.052\u003c/li\u003e\n\u003cli\u003eJagadish SVK. 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https://doi.org/10.1007/s11356-022-19381-x\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Rice, Low-light, Nitrogen, Pollen Tube, Energy Homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-4737345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4737345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow-light has emerged as a primary environmental stressor limiting rice production, yet there is currently limited research on relevant regulatory measures. In this study, a low-light-intolerant \u003cem\u003eRGA1\u003c/em\u003e gene mutant (\u003cem\u003ed1\u003c/em\u003e), and its wild type (Zhonghua 11, WT), along with an overexpressed line (OE-1), were used to investigate the effects of nitrogen levels on rice yield formation under low-light during anthesis, using field shading treatment. Our results indicated that low-light significantly decreased spikelet fertility, with hindered pollen tube elongation identified as a key factor leading to spikelet abortion. Under low-light conditions, the medium nitrogen treatment (160 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, MN) notably increased the ratio of pollen tube entry into the ovule and spikelet fertility compared to the low nitrogen treatment (60 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, LN), while the high nitrogen treatment (260 kg N\u0026middot;ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, HN) decreased spikelet fertility and yield in WT and OE-1 plants. For the \u003cem\u003ed1\u003c/em\u003e mutant, except for the LN treatment, other nitrogen treatments had minimal effect on spikelet fertility and yield. Furthermore, compared to the LN treatment, the activities of invertase and sucrose synthase, as well as the content of ATP, and ATPase in the spikelets of WT and OE-1 significantly increased when treated with MN under low-light conditions. In conclusion, moderately increasing nitrogen levels can enhance sucrose metabolism, maintain energy balance, and prevent low-light stress from impeding pollen tube elongation and spikelet fertility.\u003c/p\u003e","manuscriptTitle":"Nitrogen regulates pollen tube elongation under low-light stress during anthesis to prevent spikelet abortion in rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-14 17:15:02","doi":"10.21203/rs.3.rs-4737345/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2024-08-15T01:52:54+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-07-22T11:04:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-22T07:38:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Plant Growth Regulation","date":"2024-07-22T04:02:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-16T07:17:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Growth Regulation","date":"2024-07-14T02:56:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-growth-regulation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"grow","sideBox":"Learn more about [Plant Growth Regulation](https://www.springer.com/journal/10725)","snPcode":"10725","submissionUrl":"https://submission.nature.com/new-submission/10725/3","title":"Plant Growth Regulation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d5242ca9-6461-4768-9a72-c7ba7cefb002","owner":[],"postedDate":"August 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:22:07+00:00","versionOfRecord":{"articleIdentity":"rs-4737345","link":"https://doi.org/10.1007/s10725-024-01269-0","journal":{"identity":"plant-growth-regulation","isVorOnly":false,"title":"Plant Growth Regulation"},"publishedOn":"2024-12-31 15:57:20","publishedOnDateReadable":"December 31st, 2024"},"versionCreatedAt":"2024-08-14 17:15:02","video":"","vorDoi":"10.1007/s10725-024-01269-0","vorDoiUrl":"https://doi.org/10.1007/s10725-024-01269-0","workflowStages":[]},"version":"v1","identity":"rs-4737345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4737345","identity":"rs-4737345","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}