水稻是我国最重要的粮食作物之一，全国约60%的人口以大米为主食。化肥施用是保障水稻高产的关键措施，但过量使用氮肥不仅会造成资源浪费，还会引发一系列环境问题。土壤中残留的氮素通过径流和淋溶作用进入水体，加剧河流湖泊的富营养化；长期高量施用氮肥会导致土壤酸化，破坏微生物群落结构并影响养分循环平衡。当前，我国稻田氮肥施用呈现“总量高、效率低、区域不均”的特点。尽管在政策引导和技术优化的推动下有所改善，但仍需通过进一步技术创新实现减量增效，以平衡粮食安全生产与生态环境可持续性发展的双重目标。

实验室前期通过重离子诱变在武运粳7号(WYJ7)背景下创制了一批突变体材料。我们通过对其根系的硝态氮(NO₃^-)吸收速率进行测定，筛选到了一个NO₃^-吸收速率显著高于野生型的突变体l1。为进一步解析其遗传机制，利用突变体l1与籼稻品种华粳籼74 (HJX74)杂交构建作图群体，结合图位克隆技术，鉴定到一个调控NO₃^-吸收速率的关键基因RNU1。

为验证RNU1的功能，我们在中花11 (ZH11)背景下构建了RNU1的过量表达株系pAct::RNU1-Flag和敲除系rnu1，发现RNU1负调控水稻的NO₃^-吸收速率。与野生型ZH11相比，RNU1过量表达材料的分蘖减少、株高降低，一级枝梗数、二级枝梗数、每穗粒数和单株产量均明显下降，而敲除系rnu1则表现出相反表型。进一步证实了RNU1在调控水稻氮肥利用效率中的关键作用。进一步研究发现，RNU1还负调控水稻硝酸盐还原酶NR与亚硝酸盐还原酶NiR的活性。通过检测不同氮浓度处理下，野生型ZH11中的RNU1转录丰度与蛋白丰度，发现RNU1的转录本丰度和蛋白丰度随着氮浓度的升高而降低，表明RNU1是受低氮诱导的。

RNU1编码一个转录因子。通过酵母体系和水稻原生质体体系的转录激活实验，发现RNU1作为一个转录抑制子行使功能，其抑制功能域为120-173aa。综合运用酵母单杂交、RT-qPCR及水稻原生质体转录激活实验，证实了RNU1通过直接结合在氮代谢相关基因OsNRT2.1、OsNAR2.2、OsNPF7.3、OsNR2、OsNiR1、OsNiR2的启动子区域，显著抑制这些基因的转录活性。这些结果系统揭示了RNU1作为转录抑制因子调控水稻氮代谢的分子机制。

综上所述，本研究鉴定到一个负调控水稻NO₃^-吸收速率的基因RNU1，并对RNU1功能进行初步研究，为解析其调控水稻氮肥利用效率(Nitrogen use Efficiency,NUE)和产量的分子机制奠定基础。

Rice is one of the most important food crops in China, and about 60% of the country's population depends on rice as their staple food. Fertilizer application is a key measure to ensure high rice yield, but excessive use of nitrogen fertilizer will not only cause waste of resources, but also cause a series of environmental problems. The residual nitrogen in the soil enters the water body through runoff and leaching, which aggravates the eutrophication of rivers and lakes. Long-term application of nitrogen fertilizer in high amounts can lead to soil acidification, disrupt the microbial community structure and affect the balance of nutrient cycling. At present, nitrogen fertilizer application in paddy fields in China is characterized by "high total amount, low efficiency and regional unevenness". Although there have been improvements driven by policy guidance and technological optimization, it is still necessary to achieve reduction and efficiency increase through further technological innovation to balance the dual goals of food security production and sustainable development of the ecological environment.

In the early stage, a batch of mutant materials were created in the background of WYJ7 through heavy ion mutagenesis. By measuring the nitrate nitrogen (NO₃^-) uptake rate in its roots, we screened a mutant l1 with a significantly higher NO₃^- uptake rate than the wild-type one. In order to further elucidate the genetic mechanism, a key gene RNU1 regulating the rate of NO₃^- absorption was identified by crossing the mutant l1 with the indica rice variety HJX74 to construct a mapping population, combined with map cloning technology.

In order to verify the function of RNU1, we constructed the overexpression line pAct::RNU1-Flag and the knockout line rnu1 under the background of ZH11, and found that RNU1 negatively regulated the NO₃^- uptake rate of rice. Compared with wild-type ZH11, the tillering and plant height of RNU1 overexpressed material decreased, and the number of first-order branches, second-class branches, grains per panicle and yield per plant were significantly decreased, while the knockout line rnu1 showed the opposite phenotype. The key role of RNU1 in regulating nitrogen use efficiency in rice was further confirmed. Further studies showed that RNU1 also negatively regulated the activities of rice nitrate reductase NR and nitrite reductase NiR. By measuring the transcriptional abundance and protein abundance of RNU1 in wild-type ZH11 under different nitrogen concentrations, it was found that the transcript abundance and protein abundance of RNU1 decreased with the increase of nitrogen concentration, indicating that RNU1 was induced by low nitrogen.

RNU1 encodes a transcription factor. Through transcriptional activation experiments in yeast system and rice protoplast system, it was found that RNU1 performed its function as a transcriptional repressor, and its inhibitory domain was 120-173aa. Yeast monohybridization, RT-qPCR and rice protoplast transcriptional activation experiments were used to confirm that RNU1 significantly inhibited the transcriptional activity of nitrogen metabolism-related genes OsNRT2.1, OsNAR2.2, OsNPF7.3, OsNR2, OsNiR1 and OsNiR2 by directly binding to the promoter regions of these genes. These results systematically revealed the molecular mechanism of RNU1 as a transcriptional repressor in regulating nitrogen metabolism in rice.

In summary, this study identified a gene RNU1 that negatively regulates the nitrate absorption rate in rice and conducted preliminary research on the function of RNU1, laying the foundation for elucidating the molecular mechanisms underlying its regulation of nitrogen use efficiency (NUE) and yield in rice.

[1]郭韬, 余泓, 邱杰,等. 中国水稻遗传学研究进展与分子设计育种[J]. 中国科学: 生命科学, 2019, 49: 1185-1212.

[2]Alamin Alfatih, Jie Wu, Zi-Sheng Zhang, et al. Rice NIN-LIKE PROTEIN 1 rapidly responds to nitrogen deficiency and improves yield and nitrogen use efficiency[J]. Journal of Experimental Botany, 2020, 71(19): 6032-6042.

[3]Bai S, Yu H, Wang B, Li J. Retrospective and perspective of rice breeding in China[J]. Journal of Genetics and Genomics, 2018, 45(11): 603-612. 

[4]Bao A, Zhao Z, Ding G, et al. Accumulated expression level of cytosolic glutamine synthetase 1 gene (OsGS1;1 or OsGS1;2) alter plant development and the carbon-nitrogen metabolic status in rice[J]. PLoS One, 2014, 9(4): e95581.

[5]Behl R, Tischner R, Raschke K. Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism[J]. Planta, 1988, 176(2): 235-240.

[6]Bouguyon E, et al. Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1[J]. Nature Plants, 2015, 1: 15015.

[7]Braaksma FJ, Feenstra WJ. Isolation and characterization of nitrate reductase-deficient mutants of Arabidopsis thaliana[J]. Theoretical and Applied Genetics, 1982, 64(1): 83-90.

[8]Cai C, Zhao X, Zhu Y, et al. Regulation of the high-affinity nitrate transport system in wheat roots by exogenous abscisic acid and glutamine[J]. Journal of Integrative Plant Biology, 2007, 49(12): 1719-1725.

[9]Cai C, Wang JY, Zhu YG, et al. Gene structure and expression of the high-affinity nitrate transport system in rice roots[J]. Journal of Integrative Plant Biology, 2008, 50(4): 443-451.

[10]Cai H, Xiao J, Zhang Q, et al. Co-suppressed glutamine synthetase2 gene modifies nitrogen metabolism and plant growth in rice[J]. Chinese Science Bulletin-Chinese, 2010, 55: 823-833.

[11]Cai H, Zhou Y, Xiao J, et al. Overexpressed glutamine synthetase gene modifies nitrogen metabolism and abiotic stress responses in rice[J]. Plant Cell Reports, 2009, 28: 527-537.

[12]Cai S, Zhao X, Pittelkow CM, et al. Optimal nitrogen rate strategy for sustainable rice production in China[J]. Nature. 2023, 615(7950): 73-79.

[13]Chardin C, Girin T, Roudier F, et al. The plant RWP-RK transcription factors: key regulators of nitrogen responses and of gametophyte development[J]. Journal of Experimental Botany, 2014, 65(19): 5577-5587.

[14]Chen H, Ning Xu, Qi Wu, et al. OsMADS27 regulates the root development in a NO3-Dependent manner and modulates the salt tolerance in rice (Oryza sativa L.)[J]. Plant Science, 2018, 277: 20-32.

[15]Cheng CL, Dewdney J, Nam HG, et al. A new locus (NIA1) in Arabidopsis thaliana encoding nitrate reductase[J]. The EMBO Journal, 1988, 7: 3309-3314

[16]Chiba Y, Shimizu T, Miyakawa S, et al. Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant hormones[J]. Journal of Plant Research, 2015, 128(4): 679-686.

[17]Chopin F, Orsel M, Dorbe MF, et al. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds[J]. The Plant Cell, 2007, 19(5): 1590-1602.

[18]Choi HK, Kleinhofs A, et al. Nucleotide sequence of rice nitrate reductase genes[J]. Plant Molecular Biology, 1989, 13(6): 731-733.

[19]Coschigano KT, Melo-Oliveira R, Lim J, et al. Arabidopsis gls mutants and distinct Fd-GOGAT genes. Implications for photorespiration and primary nitrogen assimilation[J]. The Plant Cell, 1998, 10: 741-752.

[20]Crawford NM, Glass ADM, et al. Molecular and physiological aspects of nitrate uptake in plants[J]. Trends in Plant Science, 1998, 3: 389-395.

[21]Dechorgnat J, Nguyen C T, Armengaud P, et al. From the soil to the seeds: the long journey of nitrate in plants[J]. Journal of Experimental Botany, 2011, 62: 1349-1359.

[22]Deng P, Jing W, Cao C, et al. Transcriptional repressor RST1 controls salt tolerance and grain yield in rice by regulating gene expression of asparagine synthetase[J]. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(50): e2210338119.

[23]Di D, Sun L, Wang M, et al. WRKY46 promotes ammonium tolerance in Arabidopsis by repressing NUDX9 and indole-3-acetic acid-conjugating genes and by inhibiting ammonium efflux in the root elongation zone[J]. The New Phytologist, 2021, 232(1): 190-207.

[24]Dubos C, Stracke R, Grotewold E, et al. MYB transcription factors in Arabidopsis[J]. Trends in Plant Science, 2010, 15(10): 573-581.

[25]Fan SC, Lin CS, Hsu PK, et al. The Arabidopsis nitrate transporter NRT1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate[J]. The Plant cell, 2009, 21(9): 2750-2761.

[26]Fan X, Tang Z, Tan Y, et al. Overexpression of a pH-sensitive nitrate transporter in rice increases crop yields[J]. Proceedings of the National Academy of Sciences of the United States of America, 2016b, 113(26): 7118-7123.

[27]Fan X, Feng H, Tan Y, et al. A putative 6-transmembrane nitrate transporter OsNRT1.1b plays a key role in rice under low nitrogen[J]. Journal of Integrative Plant Biology, 2016a, 58(6): 590-599.

[28]Fang Z, Xia K, Yang X, et al. Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency, growth and grain yield in rice[J]. Plant Biotechnology Journal, 2013, 11(4): 446-458.

[29]Fang Z, Bai G, Huang W, et al. The rice peptide transporter OsNPF7.3 is induced by organic nitrogen, and contributes to nitrogen allocation and grain yield[J]. Frontiers in Plant Science, 2017, 8: 1338.

[30]Feng H, Yan M, Fan X, et al. Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status[J]. Journal of Experimental Botany, 2011, 62(7): 2319-2332.

[31]Ferreira L M, de Souza V M, Tavares O C H, et al. OsAMT1.3 expression alters rice ammonium uptake kinetics and root morphology[J]. Plant Biotechnology Reports, 2015, 9(4): 221-229.

[32]Forde B G and Clarkson D T. Nitrate and ammonium nutrition of plants: physiological and molecular perspectives[M]. Advances in botanical research. Academic Press, 1999: 1-90.

[33]Forde B G. Nitrate transporters in plants: structure, function and regulation[J]. Biochimica et Biophysica Acta, 2000, 1465(1-2), 219-235.

[34]Gan Y, Bernreiter A, Filleur S, et al. Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development[J]. The Plant Cell Physiology, 2012, 53: 1003-1016.

[35]Gan Y, Filleur S, Rahman A, et al. Nutritional regulation of ANR1 and other root-expressed MADS-box genes in Arabidopsis thaliana[J]. Planta, 2005, 222(4): 730-742.

[36]Gao Y, Zuopeng Xu, Zhang L, et al. MYB61 is regulated by GRF4 and promotes nitrogen utilization and biomass production in rice.Nature Communications, 2020, 11(1): 5219.

[37]Gao Z, Wang Y, Chen G, et al. The indica nitrate reductase gene OsNR2 allele enhances rice yield potential and nitrogen use efficiency[J]. Nature Communications, 2019, 10(1): 1-10.

[38]Gaudinier A, Rodriguez-Medina J, Zhang L, et al. Transcriptional regulation of nitrogen-associated metabolism and growth[J]. Nature, 2018, 563(7730): 259-264.

[39]Gazzarrini S, Lejay L, Gojon A, et al. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots[J]. The Plant Cell, 1999, 11(5): 937-948.

[40]Gifford M L, Dean A, Gutierrez R A, et al. Cell-specific nitrogen responses mediate developmental plasticity[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(2): 803-808.

[41]Glass A and Siddiqi M 1995. Nitrogen nutrition in higher plants[M]. Associated Publishing Company.

[42]Gruber BD, Giehl RF, Friedel S, et al. Plasticity of the Arabidopsis root system under nutrient deficiencies[J]. Plant Physiology, 2013, 163(1): 161-179.

[43]Guerrero M G, Vega J M, Losada M. The assimilatory nitrate-reducing system and its regulation[J]. Annual Review of Plant Physiology, 1981, 32: 169-204.

[44]Guo F, Wang R, Crawford NM.The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1(CHL1) is regulated by auxinin both shoots and roots[J]. Journal of Experimental Botany, 2002, 53(370): 835-844.

[45]Hamat HB, Kleinhofs A, Warner RL. Nitrate reductase induction and molecular characterization in rice (Oryza sativa L.)[J]. Molecular Genetics and Genomics,1989, 218(1): 93-98.

[46]Han X, Wu K, Fu X, Liu Q. Improving coordination of plant growth and nitrogen metabolism for sustainable agriculture[J]. aBIOTECH. 2020, 1(4): 255-275.

[47]Harberd NP, Belfield E, Yasumura Y. The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: how an "inhibitor of an inhibitor" enables flexible response to fluctuating environments[J]. The Plant Cell, 2009, 21(5): 1328-1339.

[48]Hasegawa H, Katagiri T, Ida S, Yatou O, Ichii M. Characterization of a rice (Oryza sativa L.) mutant deficient in the heme domain of nitrate reductase[J]. Theoretical and Applied Genetics, 1992, 84(1-2): 6-9.

[49]Hasegawa H, Yatou O, Katagiri T, Ichii M. Screening for nitrate reductase-deficient mutants in rice (Oryza sativa L.)[J].Japanese Journal of Breeding, 1991, 41(1): 95-101.

[50]Ho CH, Lin SH, Hu HC, Tsay YF. CHL1 functions as a nitrate sensor in plants[J]. Cell, 2009, 138(6), 1184-1194.

[51]Hu B, Wang W, Ou S, et al. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies[J]. Nature Genetics, 2015, 47(7): 834-838.

[52]Hu B, Jiang Z, Wang W,et al. Nitrate-NRT1.1B-SPX4 cascade integrates nitrogen and phosphorus signalling networks in plants[J]. Nature Plants, 2019, 5(4): 401-413.

[53]Huang NC, Chiang CS, Crawford NM, Tsay YF. CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots[J]. The Plant Cell, 1996, 8(12): 2183-2191.

[54]Huang NC, Liu KH, Lo HJ, Tsay YF. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake[J]. The Plant Cell, 1999, 11(8): 1381-1392.

[55]Huang W, Nie H, Feng F, Wang J, Lu K, Fang Z. Altered expression of OsNPF7.1 and OsNPF7.4 differentially regulates tillering and grain yield in rice[J]. Plant Science, 2019, 283: 23-31.

[56]Huang X, Qian Q, Liu Z, et al. Natural variation at the DEP1 locus enhances grain yield in rice[J]. Nature Genetics, 2009, 41(4): 494-497.

[57]Huang Y, Ji Z, Tao Y, et al. Improving rice nitrogen-use efficiency by modulating a novel monouniquitination machinery for optimal root plasticity response to nitrogen[J]. Nature Plants, 2023, 9(11): 1902-1914.

[58]Huang S, Liang Z, Chen Si, et al. A transcription factor, OsMADS57, regulates long-distance nitrate transport and root elongation[J]. Plant Physiology, 2019, 180(2): 882-895.

[59]Hu B et al. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies[J]. Nature Genetics, 2015, 47(7): 834-838..

[60]Hu R, Qiu D, Chen Y, et al. Knock-down of a tonoplast localized low-affinity nitrate transporter OsNPF7.2 affects rice growth under high nitrate supply[J]. Frontiers in Plant Science, 2016, 7: 1529.

[61]Hsieh PH, Kan CC, Wu HY, et al. Early molecular events associated with nitrogen deficiency in rice seedling roots[J]. Scientific Reports, 2018, 8(1): 12207. 

[62]James D, Borphukan B, Fartyal D, et al. Concurrent overexpression of OsGS1;1 and OsGS2 genes in transgenic rice (Oryza sativa L.): impact on tolerance to abiotic stresses[J]. Frontiers in Plant Science, 2018, 9: 786.

[63]Jia Z, Giehl R, von Wiren N. Local auxin biosynthesis acts downstream of brassinosteroids to trigger root foraging for nitrogen[J]. Nature Communications, 2021, 12: 5437.

[64]Jia Z, Giehl R, von Wiren N. Nutrient-hormone relations: driving root plasticity in plants[J]. Molecular Plant, 2022, 15: 86-103.

[65]Jia Z, Wiren NV. Signaling pathways underlying nitrogendependent changes in root system architecture: from model to crop species[J]. Journal of Experimental Botany, 2020, 71(15): 4393-4404.

[66]Kaiser BN, Rawat SR, Siddiqi MY, et al. Functional analysis of an Arabidopsis T-DNA knockout of the high-affi nity NH4+ transporter AtAMT1;1[J]. Plant Physiology, 2002, 130(3): 1263-1275.

[67]Kiba T, Feria-Bourrellier AB, Lafouge F, et al. The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants[J]. The Plant Cell, 2012, 24(1), 245-258.

[68]Kim JH, Choi D, Kende H. The AtGRF family of putative transcription factor is involved in leaf and cotyledon growth in Arabidopsis[J]. The Plant Journal : for cell and molecular biology, 2003, 36(1): 94-104.

[69]Kotur Z, Mackenzie N, Ramesh S, et al. Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1[J]. The New Phytologist, 2012, 194(3): 724-731.

[70]Krouk G, Lacombe B, Bielach A, et al. Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants[J]. Developmental Cell, 2010, 18(6): 927-937.

[71]Kuijt SJ, Greco R, Agalou A, et al. Interaction between the GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX family of transcription factor[J].Plant Physiology, 164(4), 1952-1966.

[72]Kusano M, Tabuchi M, Fukushima A, et al. Metabolomics data reveal a crucial role of cytosolic glutamine synthetase 1;1 in coordinating metabolic balance in rice[J].The Plant Journal: for cell and molecular biology, 2011, 66(3): 456-466.

[73]Lea PJ, Miflin BJ. Alternative route for nitrogen assimilation in higher plants[J]. Nature, 1974, 251(5476): 614-616.

[74]Lea PJ, Miflin BJ. Glutamate synthase and the synthesis of glutamate in plants[J]. Plant Physiol Biochem, 2003, 41(6-7): 555-564.

[75]Lee BH, Ko JH, Lee S, Lee Y, Pak JH, Kim JH. The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties[J]. Plant Physiology, 2009, 151(2): 655-68.

[76]Léran S, Edel K H, Pervent M, et al. Nitrate sensing and uptake in Arabidopsis are enhanced by ABI2, a phosphatase inactivated by the stress hormone abscisic acid[J]. Science Signaling, 2015, 8(375): ra43.

[77]Lezhneva L, Kiba T, Feria-Bourrellier AB, et al. The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants[J]. The Plant Journal: for cell and molecular biology, 2014, 80(2): 230-241.

[78]Li B, Merrick M, Li S-m, Li H, Zhu S, Shi W, Su Y. Molecular basis and regulation of ammonium transporter in rice[J]. Rice Science, 2009a, 16: 314-322.

[79]Li G, Li B, Dong G, Feng X, Kronzucker H, Shi W. Ammonium-induced shoot ethylene production is associated with the inhibition of lateral root formation in Arabidopsis[J]. Journal of Experimental Botany, 2013, 64(5): 1413-1425.

[80]Li Q, Li BH, Kronzucker HJ, Shi WM. Root growth inhibition by NH4+ in Arabidopsis is mediated by the root tip and is linked to NH4+ efflux and GMPase activity[J]. Plant, Cell & Environment, 2010, 33(9): 1529-1542.

[81]Li JY, Fu YL, Pike SM, et al. The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance[J]. The Plant Cell, 2010, 22(5): 1633-1646.

[82]Lin CM, Koh S, Stacey G, Yu SM, Lin TY, Tsay YF. Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice[J]. Plant Physiology, 2000, 122(2): 379-388.

[83]Lin SH, Kuo HF, Canivenc G, et al. Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport[J]. The Plant Cell, 2008, 20(9): 2514-2528.

[84]Liu H, Guo S, Xu Y, et al. OsmiR396-regulating OsGRFs function in floral organogenesis in rice through binding to their targets OsJNJ706 and OsCR4[J]. Plant Physiology, 2014, 165(1): 160-174.

[85]Liu H, Fu X. Phytohormonal networks facilitate plant root developmental adaptations to environmental changes[J]. Science Bulletin, 2024, 69(6): 709-713.

[86]Liu KH, Tsay YF. Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphory-lation[J]. The EMBO Journal, 2003, 22(5): 1005-1013.

[87]Liu KH, Huang CY, Tsay YF. CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake[J]. The Plant Cell, 1999, 11(5): 865-874.

[88]Liu KH et al. Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks[J]. Nature, 2017, 545: 311-316.

[89]Liu Q, Chen X, Wu K, Fu X. Nitrogen signaling and use efficiency in plants: what’s new[J]? Current Opinion in Plant Biology, 2015, 27: 192-198.

[90]Liu Q, Han R, Wu K, et al. G-protein βγ subunits determine grain size through interaction with MADS-domain transcription factors in rice[J]. Nature Communications, 2018, 9(1): 852.

[91]Li S, Tian Y, Wu K, et al. Modulating plant growth-metabolism coordination for sustainable agriculture[J]. Nature, 2018, 560(7720): 595-600.

[92]Liu X, Hu B, Chu C. Nitrogen assimilation in plants: current status and future prospects[J]. Journal of Genetics Genomics, 2022, 49(5): 394-404.

[93]Li Y, Ouyang J, Wang YY, et al. Disruption of the rice nitrate transporter OsNPF2.2 hinders root-to-shoot nitrate transport and vascular development[J]. Scientific Reports, 2015, 5: 9635.

[94]Loqué D, Yuan L, Kojima S, et al. Additive contribution of AMT1;1 and AMT1;3 to high-affinity ammonium uptake across the plasma membrane of nitrogen-deficient Arabidopsis roots[J]. The Plant Journal: for cell and molecular biology, 2006, 48(4): 522-534.

[95]Ludewig U, Neuhauser B, Dynowski M. Molecular mechanisms of ammonium transport and accumulation in plants[J]. FEBS letters, 2007, 581(12): 2301-2308.

[96]Ma W, Li J, Qu B, He X, Zhao X, Li B, Fu X, Tong Y. Auxin biosynthetic gene TAR2 is involved in low nitrogen-mediated reprogramming of root architecture in Arabidopsis[J]. The Plant Journal: for cell and molecular biology, 2014, 78(1): 70-79.

[97]Maghiaoui A, Bouguyon E, Cuesta C, et al. The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate[J]. Journal of Experimental Botany, 2020, 71(15): 4480-4494.

[98]Marchive C, Roudier F, Castaings L, et al. Nuclear retention of the transcription factor NLP7 orchestrates the early response to nitrate in plants[J]. Nature Communications, 2013, 4: 1713.

[99]Meier, Markus, et al. Auxin-mediated root branching is determined by the form of available nitrogen[J]. Nature Plants, 2020, 6(9): 1136-1145.

[100]Mészáros A, Pauk J. Chlorate resistance as a tool to study the effect of nitrate reductase antisense gene in wheat[J]. Cereal Research Communication, 2002, 30: 245-252.

[101]More`re-Le, Paven MC, et al. Characterization of a dual-affinity nitrate transporter MtNRT1. 3 in the model legume Medicago truncatula[J]. Journal of Experimental Botany, 2011, 62(15): 5595-5605.

[102]Murase K, Hirano Y, Sun TP, Hakoshima T. Gibberellin-induced DELLA recognition by the gibberellin receptor GID1[J]. Nature, 2008, 456(7221): 459-463.

[103]Neuhäuser B, Dynowski M, Mayer M, Ludewig U. Regulation of NH4+ transport by essential cross talk between AMT monomers through the carboxyl tails[J]. Plant Physiology, 2007, 143(4): 1651-1659.

[104]Nishimura A, Ashikari M, Lin S, Takashi T, Angeles ER, Yamamoto T, Matsuoka M. Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems[J]. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(33): 11940-11944.

[105]Nour-Eldin HH, et al. NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds[J]. Nature, 2012, 488(7412):531-534.

[106]Ogawa T, Fukuoka H, Yano H, Ohkawa Y. Relationships between nitrite reductase activity and genotype-dependent callus growth in rice cell cultures[J]. Plant Cell Reports, 1999, 18(7-8): 576-581.

[107]Ogura T, Goeschl C, Filiault D, et al. Root System Depth in Arabidopsis is shaped by EXOCYST70A3 via the dynamic modulation of auxin transport[J]. Cell, 2019, 178(2): 400-412.e16.

[108]Ohashi M, et al. Lack of cytosolic glutamine synthetase1;2 in vascular tissues of axillary buds causes severe reduction in their outgrowth and disorder of metabolic balance in rice seedlings[J]. The Plant Journal: for cell and molecular biology, 2015, 81(2): 347-356.

[109]Okamoto, Mamoru, et al. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision[J]. Plant & Cell Physiology, 2003, 44(3), 304-317.

[110]Omidbakhshfard MA, Proost S, Fujikura U, Mueller-Roeber B. Growth-regulating factor (GRFs): a small transcription factor family with inportant functions in plant biology[J]. Molecular Plant, 2015, 8(7): 998-1010.

[111]Orsel M, Filleur S, Fraisier V, Daniel-Vedele F. Nitrate transport in plants: which gene and which control[J]. Journal of Experimental Botany, 2002, 53(370): 825-833.

[112]Ozawa K, Kawahigashi H. Positional cloning of the nitrite reductase gene associated with good growth and regeneration ability of calli and establishment of a new selection system for Agrobacterium-mediated transformation in rice (Oryza sativa L.)[J]. Plant Science, 2006, 170(2): 384-393.

[113]Prabhu, L and Pingali. Green revolution: Impacts, limits, and the path ahead[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(31): 12302-12308.

[114]Puig J, Meynard D, Khong GN, et al. Analysis of the expression of the AGL17-like clade of MADS-box transcription factors in rice[J]. Gene Expression Patterns, 2013, 13(5-6), 160-170.

[115]Ranathunge K, El-Kereamy A, Gidda S, et al. AMT1;1 transgenic rice plants with enhanced NH4+ permeability show superior growth and higher yield under optimal and suboptimal NH4+ conditions[J]. Journal of Experimental Botany, 2014, 65(4): 965-979.

[116]Remans T, Nacry P, Pervent M, et al. The Arabidopsis NRT1. 1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(50): 19206-19211.

[117]Sasaki A, Ashikari M, Ueguchi-Tanaka M, et al. A mutant gibberellin-synthesis gene in rice[J]. Nature, 2002a, 416(6882): 701-702.

[118]Sasaki A, Itoh H, Gomi K, et al. Accumulation of phosphorylated repressor for gibberellin signaling in an F-box mutant[J]. Science, 2003, 299(5614): 1896-1898.

[119]Schauser L, Roussis A, Stiller J, Stougaard J. A plant regulator controlling development of symbiotic root nodules[J]. Nature, 1999, 402(6758): 191-195.

[120]Shao A, Ma W, Zhao X, et al. The auxin biosynthetic TRYPTOPHAN AMINOTRANSFERASE RELATED TaTAR2.1-3A increases grain yield of wheat[J]. Plant Physiology, 2017, 174(4): 2274-2288.

[121]Shen TC. The induction of nitrate reductase and the preferential assimilation of ammonium in germinating rice seedlings[J]. Plant Physiology, 1969, 44(11): 1650-1655.

[122]Shen T-C, Funkhouser EA, Guerrero MG. NADH- and NAD(P)H-nitrate reductases in rice seedlings[J]. Plant Physiology, 1976, 58(3): 292-294.

[123]Shimada A, Ueguchi-Tanaka M, Nakatsu T, et al. Structural basis for gibberellin recognition by its receptor GID1[J]. Nature, 2008, 456(7221): 520-523.

[124]Siddiqi M Y, Glass A D, Ruth T J, et al. Studies of the uptake of nitrate in barley: I. Kinetics of 13NO3- influx[J]. Plant Physiology, 1990, 93(4): 1426-1432.

[125]Sierla M, Hõrak H, Overmyer K, et al. The receptor-like pseudokinase GHR1 is required for stomatal closure[J]. The Plant Cell, 2018, 30(11): 2813-2837.

[126]Signora L, De Smet I, Foyer C H, et al. ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis[J]. The Plant Journal, 2001, 28(6): 655-662.

[127]Sivasankar S, Oaks A. Nitrate assimilation in higher plants: the effect of metabolites and light[J]. Plant Physiol Biochem, 1996, 34(5): 609-620.

[128]Sonoda Y, Ikeda A, Saiki S, et al. Distinct expression and function of three ammonium transporter genes (OsAMT1;1-1;3) in rice[J]. Plant & Cell Physiology, 2003, 44(7): 726-734.

[129]Su H, Wang T, Ju C, et al. Abscisic acid signaling negatively regulates nitrate uptake via phosphorylation of NRT1.1 by SnRK2s in Arabidopsis[J]. Journal of Integrative Plant Biology, 2021, 63(3): 597-610.

[130]Sun H, Qian Q, Wu K, et al. Heterotrimeric G proteins regulate nitrogen-use efficiency in rice[J]. Nature Genetics, 2014a, 46(6): 652-656.

[131]Sun H, Tao J, Bi Y, et al. OsPIN1b is involved in rice seminal root elongation by regulating root apical meristem activity in response to low nitrogen and phosphate[J]. Scientific Reports, 2018, 8(1): 13014.

[132]Suenaga A, Moriya K, Sonoda Y, et al. Constitutive expression of a novel-type ammonium transporter OsAMT2 in rice plants[J]. Plant & Cell Physiology, 2003, 44(2): 206-211.

[133]Taochy C, Gaillard I, Ipotesi E, et al. The Arabidopsis root stele transporter NPF2.3 contributes to nitrate translocation to shoots under salt stress[J]. The Plant Journal: for cell and molecular biology, 2015, 83(3): 466-479.

[134]Tang Z, Fan X, Li Q, Sonoda Y, et al. Knockdown of a rice stelar nitrate transporter alters longdistance translocation but not root influx[J]. Plant Physiology, 2012, 160(4): 2052-2063.

[135]Trotochaud AE, Jeong S, Clark SE. CLAVATA3, a multimeric ligand for the CLAVATA1 receptor-kinase[J]. Science, 2000, 289(5479): 613-617.

[136]Tabuchi M, Sugiyama K, Ishiyama K, et al. Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1; 1, acytosolic glutamine synthetase1;1[J]. The Plant Journal: for cell and molecular biology, 2005, 42(5): 641-651.

[137]Tamura W, Kojima S, Toyokawa A, et al. Disruption of a novel NADH-glutamate synthase2 gene caused marked reduction in spikelet number of rice[J]. Frontiers in Plant Science, 2011, 2: 57.

[138]Tsay YF, Schroeder JI, Feldmann KA, Crawford NM. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter[J]. Cell, 1993, 72(5):705-713.

[139]Vega A, Fredes I, O'Brien J, et al. Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture[J]. EMBO Reports, 2021, 22(9): e51813.

[140]Vidal E A, Álvarez J M, Gutiérrez R A. Nitrate regulation of AFB3 and NAC4 gene expression in Arabidopsis roots depends on NRT1.1 nitrate transport function[J]. Plant Signaling & Behavior, 2014, 9(3): e28501.

[141]Vidal EA, Araus V, Lu C, et al. Nitrate-responsive miR393/AFB3 regulatory module controls root system architecture in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(9): 4477-4482.

[142]Vidal EA, Moyano TC, Riveras E, Contreras-Lopez O, Gutierrez RA. Systems approaches map regulatory networks downstream of the auxin receptor AFB3 in the nitrate response of Arabidopsis thaliana roots[J]. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(31): 12840-12845.

[143]Wallsgrove RM, Turner JC, Hall NP, et al. Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis[J]. Plant Physiology, 1987, 83(1): 155-158.

[144]Wang D, Tangqian Xu, Yin Z, et al. Overexpression of OsMYB305 in rice enhances the nitrogen uptake under low-nitrogen condition[J]. Frontiers in Plant Science, 2020, 11: 369.

[145]Wang J, Lu K, Nie H, et al. Rice nitrate transporter OsNPF7.2 positively regulates tiller number and grain yield[J]. Rice, 2018a, 11(1):12.

[146]Wang L, Gu X, Xu D, et al. miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis[J]. Journal of Experimental Botany, 2011, 62(2): 761-773.

[147]Wang M, Takahiro H, Makoto H, et al. OsNLP4 is required for nitrate assimilation gene expressions and nitratedependent growth in rice[J]. BioRxiv, 2020, 2: 19.

[148]Wang Q, Nian J, Xie X, et al. Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice[J]. Nature Communications, 2018b, 9(1): 735.

[149]Wang R, Crawford NM. Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants[J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(17): 9297-9301.

[150]Wang R, Liu D, Crawford NM. The Arabidopsis CHL1 protein plays amajor role in high-affinity nitrate uptake[J]. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(25): 15134-15139.

[151]Wang W, Hu B, Yuan D, et al. Expression of the nitrate transporter gene OsNRT1.1A/OsNPF6.3 confers high yield and early maturation in rice[J]. The Plant Cell, 2018c, 30(3): 638-651.

[152]Wang Y, Yuan Z, Wang J, et al. The nitrate transporter NRT2.1 directly antagonizes PIN7-mediated auxin transport for root growth adaptation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2023, 120(25): e2221313120.

[153]Wang YY, Cheng YH, Chen KE, Tsay YF. Nitrate transport, signaling, and use efficiency[J]. Annual Review of Plant Biology, 2018d, 69: 85-122.

[154]Wang YY, Hsu PK, Tsay YF. Uptake, allocation and signaling of nitrate[J]. Trends in Plant Science, 2012, 17(8): 458-467.

[155]Wang YY, Tsay YF. Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport[J]. The Plant Cell, 2011, 23(5), 1945-1957.

[156]Wei J, Zheng Y, Feng H, et al. OsNRT2.4 encodes a dual-affinity nitrate transporter and functions in nitrate-regulated root growth and nitrate distribution in rice[J]. Journal of Experimental Botany, 2018, 69(5): 1095-1107.

[157]Wilkinson JQ, Crawford NM. Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2[J]. The Plant Cell, 1991, 3(5): 461-471.

[158]Wilkinson JQ, Crawford NM. Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2[J]. Molecular & General Genetics, 1993, 239(1-2): 289-297.

[159]Wu F, Chi Y, Jiang Z, et al. Hydrogen peroxide sensor HPCA1 is an LRR receptor kinase in Arabidopsis[J]. Nature, 2020b, 578(7796): 577-581.

[160]Wu K, Wang S, Song W, et al. Enhanced sustainable green revolution yield via nitrogen-responsive chromatin modulation in rice[J]. Science, 2020a, 367(6478): eaaz2046.

[161]Xia X, Fan X, Wei J, et al. Rice nitrate transporter OsNPF2.4 functions in low-affinity acquisition and long-distance transport[J]. Journal of Experimental Botany, 2015, 66(1): 317-331.

[162]Xu G, Fan X, Miller AJ. Plant nitrogen assimilation and use efficiency[J]. Annual Review of Plant Biology, 2012, 63: 153-182.

[163]Xu Q, Zhao M, Wu K, Fu X, Liu Q. Emerging insights into heterotrimeric G protein signaling in plants[J]. Journal of Genetics and Genomics, 2016b, 43(8): 495-502.

[164]Yamaya T, Kusano M. Evidence supporting distinct functions of three cytosolic glutamine synthetases and two NADH-glutamate synthases in rice[J]. Journal of Experimental Botany, 2014, 65(19): 5519-5525.

[165]Yang X, Nian J, Xie Q, et al. Rice ferredoxin-dependent glutamate synthase regulates nitrogen-carbon metabolomes and is genetically differentiated between japonica and indica subspecies[J]. Molecular Plant, 2016, 9(11): 1520-1534.

[166]Yan M, Fan X, Feng H, Miller AJ, Shen Q, Xu G. Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges[J]. Plant, Cell & Environment, 2011, 34(8): 1360-1372.

[167]Yao S, Sonoda Y, Tsutsui T, et al. Promoter analysis of OsAMT1;2 and OsAMT1;3 implies their distinct roles in nitrogen utilization in rice[J]. Breeding Science, 2008, 58: 201-207.

[168]Yu C, Yihua L, Aidong Z, et al. MADS-box transcription factor OsMADS25 regulates root development through affection of nitrate accumulation in rice[J]. PLoS ONE, 2015, 10(8): e0135196.

[169]Yu L, Miao Z, Qi G, et al. MADS-box transcription factor AGL21 regulates lateral root development and responds to multiple external and physiological signals[J]. Molecular Plant, 2014, 7(11): 1653-1669.

[170]Yu P, Eggert K, Von Wiren N, Li C, Hochholdinger F. Cell type-specific gene expression analyses by RNA sequencing reveal local high nitrate-triggered lateral root initiation in shoot-borne roots of maize by modulating auxin-related cell cycle regulation[J]. Plant Physiology, 2015, 169(1): 690-704.

[171]Yu J, Xuan W, Tian Y, et al. Enhanced OsNLP4-OsNiR cascade confers nitrogen use efficiency by promoting tiller number in rice[J]. Plant Biotechnology Journal, 2021, 19(1): 167-176.

[172]Yuan L, Loqué D, Kojima S, et al. The organization of high-affinity ammonium uptake in Arabidopsis roots depends on the spatial arrangement and biochemical properties of AMT1-type transporters[J]. The Plant Cell, 2007, 19(8), 2636-2652.

[173]Cao Y, Fan XR, et al. Effect of nitrate on activities and transcript levels of nitrate reductase and glutamine synthetase in rice[J]. Pedosphere, 2008, 18(5): 664-673.

[174]Zhang HM, Forde BG. An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture[J]. Science, 1998, 279(5349): 407-409.

[175]Zhang K, Novak O, Wei Z, et al. Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins[J]. Nature Communications, 2014, 5: 3274.

[176]Zhang S, Zhu L, Shen C, et al. Natural allelic variation in a modulator of auxin homeostasis improves grain yield and nitrogen use efficiency in rice[J]. The Plant Cell, 2021, 33(3): 566-580.

[177]Zhou C,Lin Q, Lan J, et al. WRKY transcription factor OsWRKY29 represses seed dormancy in rice by weakening abscisic acid response[J]. Frontiers in Plant Science, 2020, 11: 691-706.