Cytokinin and auxin metabolism mediation of elevated [CO₂]-enhanced shoot growth under different nitrogen conditions in perennial grass

Abstract

Elevated atmospheric [CO ₂] and nitrogen (N) availability are critical determinants of plant growth. This study investigated the underlying mechanisms of hormones in mediating elevated [CO ₂]-promoted shoot growth and leaf elongation under different N conditions in tall fescue ( Festuca arundinacea). Plants were grown under low N (LN, 0.25 mM) and moderate N (MN, 4 mM) conditions. Subsequently, the plants from each N treatment were divided and immediately transferred to ambient (400 μmol mol ⁻¹) or elevated [CO ₂] (800 μmol mol ⁻¹). Elevated [CO ₂] promoted plant growth under both LN and MN conditions through affecting cell division and cell elongation, with a more pronounced effect under MN supply levels. Elevated [CO ₂]-induced shoot growth and leaf elongation were associated with increased cytokinin level under LN and with enhanced cytokinin and auxin under MN conditions. Exogenous cytokinin inhibitor (lovastatin) and auxin inhibitor (2,3,5-triiodobenzoic acid) altered elevated [CO ₂]-enhanced growth in tall fescue regardless of N conditions. Elevation of [CO ₂]-enhanced growth by modulating cell growth-related genes OsCycD2, OsPCNA, and OsEXPA10 was counteracted and reduced in FaCKX11-OE lines under LN and MN conditions, respectively. However, this enhancement was counteracted in FaDAO-OE lines under MN but not under LN conditions. These results demonstrated that elevated [CO ₂]-enhanced shoot growth in perennial grass species could be primarily mediated by cytokinin under LN conditions, while both cytokinin and auxin were involved in regulating elevated [CO ₂]-enhanced growth under MN conditions.

Acknowledgements

The study was supported by the National Natural Science Foundation of China (32471773, 32401474).

Author contributions

Z.Y. and J.Y. designed the study. N.F. and T.H. performed all experiments. Q.L., D.W., and P.Y. analyzed the data. N.F. and J.Y. wrote the manuscript and provided funding. N.F., J.Y., and Z.Y. reviewed and edited the manuscript.

Data availability

All the data generated or analyzed underlying this article are available within the article and its supplementary data files.

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Lan X, Tans P, Thoning K . Trends in globally-averaged CO₂ determined from NOAA global monitoring laboratory measurements. Version Wednesday, 31-Dec- 2025.

[2] Roy S, Mathur M . Delineating the mechanisms of elevated CO₂ mediated growth, stress tolerance and phytohormonal regulation in plants. Plant Cell Rep. 2021; 40: 1345-65.

[3] Liang F, Abdelhakim OL, Hegelund J, et al. ABA-mediated regulation of leaf and root hydraulic conductance in tomato grown at elevated CO₂ is associated with altered gene expression of aquaporins. Hortic Res. 2019; 6: 104.

[4] Yu J, Fan N, Li R, et al. Proteomic profiling for metabolic pathways involved in interactive effects of elevated carbon dioxide and nitrogen on leaf growth in a perennial grass species. J Proteome Res. 2019; 18: 2446-57.

[5] Zhuang L, Yang Z, Fan N, et al. Metabolomic changes associated with elevated CO₂-regulation of salt tolerance in Kentucky bluegrass. Environ Exp Bot. 2019; 165: 129-38.

[6] Nikolopoulou A, Nektarios P, Aivalakis G, et al. Effects of rootzone CO₂ and O2 levels on seed germination and stolon growth of Cynodon dactylon . Acta Agric Scand Sect B Soil Plant Sci. 2012; 62: 53-61.

[7] Xu Q, Fan N, Zhuang L, et al. Enhanced stolon growth and metabolic adjustment in creeping bentgrass with elevated CO₂ concentration. Environ Exp Bot. 2018; 155: 87-97.

[8] Foyer C, Noctor G . Redox homeostasis and signaling in a higher-CO₂ world. Annu Rev Plant Biol. 2020; 29: 157-82.

[9] Huang B, Xu Y . Cellular and molecular mechanisms for elevated CO₂-regulation of plant growth and stress adaptation. Crop Sci. 2015; 55: 1405-24.

[10] Laza H, Bhattarai B, Mendu V, et al. Peanut leaf transcriptomic dynamics reveals insights into the acclimation response to elevated carbon dioxide under semiarid conditions. Front Plant Sci. 2025; 15: 1407574.

[11] Zhu K, Wang C, Li Y, et al. Exogenous spraying of naphthaleneacetic acid (NAA) and sodium diethyldithiocarbamate trihydrate (DIECA) increased the female to male ratio and seed production of buffalo grass. Grass Res . 2024; 4: e014.

[12] Cohen I, Halpern M, Yermiyahu U, et al. CO₂ and nitrogen interaction alters root anatomy, morphology, nitrogen partitioning and photosynthetic acclimation of tomato plants. Planta. 2019; 250: 1423-32.

[13] Fan N, Hao T, Yang Z, et al. Effects of elevated [CO₂] on leaf elongation under different nitrogen conditions mediated by auxin in tall fescue. Plant Physiol Biochem. 2025; 228: 110282.

[14] Reich P, Hobbie S . Decade-long soil nitrogen constraint on the CO₂ fertilization of plant biomass. Nat Clim Chang. 2013; 3: 278-82.

[15] Cui H, Shi Z, Cai J, et al. Effects of atmospheric CO₂ concentration enhancement and nitrogen application rate on wheat grain yield and quality. Chin J Appl Ecol. 2011; 22: 979-84.

[16] Wang W, Xu X, Zhu C, et al. Elevated CO₂-induced changes in cytokinin and nitrogen metabolism are associated with different responses in the panicle architecture of two contrasting rice genotypes. Plant Growth Regul. 2019; 89: 119-29.

[17] Gamage D, Thompson M, Sutherland M, et al. New insights into the cellular mechanisms of plant growth at elevated atmospheric carbon dioxide concentrations. Plant Cell Environ . 2018; 6: 1233-46.

[18] Yu J, Li M, Li Q, et al. Reallocation of soluble sugars and IAA regulation in association with enhanced stolon growth by elevated CO₂ in creeping bentgrass. Plants (Basel). 2022; 11: 1500.

[19] Fan N, Yang Z, Hao T, et al. Differential effects of elevated atmosphere CO₂ concentration on root growth in association with regulation of auxin and cytokinins under different nitrate supply. Environ Exp Bot. 2022; 201: 104943.

[20] Kiba T, Takebayashi Y, Kojima M, et al. Sugar-induced de novo cytokinin biosynthesis contributes to Arabidopsis growth under elevated CO₂ . Sci Rep. 2019; 9: 7765.

[21] Zhang J, Peer W . Auxin homeostasis: the DAO of catabolism. J Exp Bot. 2017; 68: 3145-54.

[22] Ashikari M, Sakakibara H, Lin S, et al. Cytokinin oxidase regulates rice grain production. Science. 2005; 309: 741-5.

[23] Liu Z, Lv Y, Zhang M, et al. Identification, expression, and comparative genomic analysis of the IPT and CKX gene families in Chinese cabbage (Brassica rapa ssp. pekinensis) . BMC Genomics. 2013; 14: 594.

[24] Zhang W, Ma C, Du L, et al. Two DNA-binding one zinc finger transcription factors, MdCDOF3 and MdDOF3.6, accelerate leaf senescence by activating cytokinin oxidase MdCKX7 in response to sorbitol signaling in apple . Hortic Res. 2025; 12: 8.

[25] Suter D, Frehner M, Fischer B, et al. Elevated CO₂ increases carbon allocation to the roots of Lolium perenne under free-air CO₂ enrichment but not in a controlled environment . New Phytol. 2002; 154: 65-75.

[26] Aranjuelo I, Erice G, Sanz-Sáez A, et al. Differential CO₂ effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.) . Plant Cell Environ. 2015; 38: 2780-94.

[27] Krapp A, Truong H . Regulation of C/N interaction in model plant species. J Crop Improv. 2006; 15: 127-73.

[28] Lv X, Zhang Y, Hu L, et al. Low-nitrogen stress stimulates lateral root initiation and nitrogen assimilation in wheat: roles of phytohormone signaling. J Plant Growth Regul. 2021; 40: 436-50.

[29] McMurtrie R, Norby R, Medlyn B, et al. Why is plant-growth response to elevated CO₂ amplified when water is limiting, but reduced when nitrogen is limiting? A growth-optimisation hypothesis. Funct Plant Biol. 2008; 35: 521-34.

[30] Luo S, Yang L, Liu X, et al. Elevated ambient CO₂ promotes auxin-modulated NO3 ⁻ uptake and growth of Brassica napus L. under low nitrogen availability . Environ Exp Bot. 2024; 224: 105815.

[31] Stitt M, Krapp A . The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular background. Plant Cell Environ . 1999; 22: 583-621.

[32] Wang Y, Liu H, Xin Q . Genome-wide analysis and identification of cytokinin oxidase/dehydrogenase (CKX) gene family in foxtail millet (Setaria italica) . Crop J. 2014; 2: 244-54.

[33] Porco S, Pěnčík A, Rashed A, et al. Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis . Proc Natl Acad Sci USA. 2016; 113: 11016-21.

[34] Mellor N, Band L, Pěnčík A, et al. Dynamic regulation of auxin oxidase and conjugating enzymes AtDAO1 and GH3 modulates auxin homeostasis. Proc Natl Acad Sci USA . 2016; 113: 11022-7.

[35] Zhao Z, Zhang Y, Liu X, et al. A role for a dioxygenase in auxin metabolism and reproductive development in rice. Dev Cell. 2013; 27: 113-22.

[36] Teng N, Wang J, Chen T, et al. Elevated CO₂ induces physiological, biochemical and structural changes in leaves of Arabidopsis thaliana . New Phytol. 2006; 172: 92-103.

[37] Wei H, Gou J, Yordanov Y, et al. Global transcriptomic profiling of aspen trees under elevated [CO₂] to identify potential molecular mechanisms responsible for enhanced radial growth. J Plant Res. 2013; 126: 305-20.

[38] Inzé D, De Veylder L . Cell cycle regulation in plant development. Annu Rev Genet. 2006; 40: 77-105.

[39] Hu Q, Zhang S, Huang B . Strigolactones promote leaf elongation in tall fescue through upregulation of cell cycle genes and downregulation of auxin transport genes in tall fescue under different temperature regimes. Int J Mol Sci. 2019; 20: 1386.

[40] Velappan Y, Signorelli S, Considine M . Cell cycle arrest in plants: what distinguishes quiescence, dormancy and differentiated G1? Ann Bot. 2017; 120: 495-509.

[41] Cockcroft E, den Boer B, Healy J, et al. Cyclin D control of growth rate in plants. Nature. 2000; 405: 575-9.

[42] Kinsman EK, Lewis C, Davies M, et al. Elevated CO₂ stimulates cells to divide in grass meristems: a differential effect in two natural populations of Dactylis glomerata . Plant Cell Environ. 1997; 20: 1309-16.

[43] García-Gómez C, Gordillo F, Palma A, et al. Elevated CO₂ alleviates high PAR and UV stress in the unicellular chlorophyte Dunaliella tertiolecta . Photochem Photobiol Sci. 2014; 13: 1347-58.

[44] Eklöf JM, Brumer H . The XTH gene family: an update on enzyme structure, function, and phylogeny in xyloglucan remodeling . Plant Physiol. 2010; 153: 456-66.

[45] Wang T, Chen Y, Tabuchi A, et al. The target of β-expansin EXPB1 in maize cell walls from binding and solid-state NMR studies . Plant Physiol. 2016; 172: 2107-19.

[46] Bordoloi K, Dihingia P, Krishnatreya D, et al. Genome-wide identification, characterization and expression analysis of the expansin gene family under drought stress in tea (Camellia sinensis L.) . Plant Sci Today. 2021; 8: 32-44.

[47] Li F, Xing S, Guo Q, et al. Drought tolerance through overexpression of the expansin gene TaEXPB23 in transgenic tobacco . J Plant Physiol. 2011; 168: 960-6.

[48] Che J, Yamaji N, Shen R, et al. An Al-inducible expansin gene, OsEXPA10 is involved in root cell elongation of rice . Plant J. 2016; 88: 132-42.

[49] Fan N, Xu Q, Yang Z, et al. Identification of expansin genes as promoting or repressing factors for leaf elongation in tall fescue. Physiol Plant. 2023; 175: e13861.

[50] Chun J, Wan M, Guo H, et al. Cytokinin-mediated enhancement of potato growth and yield by Verticillium dahliae effector VDAL under low temperature stress . BMC Plant Biol. 2024; 24: 1115.

[51] Tang Y, Luo K, Bai J, et al. Development of an in vitro bulbil-induction system for Lilium lancifolium and the impact of exogenous auxin on bulbil formation . Sci Hortic. 2025; 340: 113885.

[52] Li R, Wang R, Li M, et al. Auxin mediated elevated CO₂-induced stolon growth and soluble sugar accumulation in creeping bentgrass. Environ Exp Bot . 2024; 217: 105567.

[53] Zhang X, Xing R, Ding Y, et al. Overexpression of gibberellin 2-oxidase 4 from tall fescue affected plant height, tillering and drought tolerance in rice. Environ Exp Bot. 2023; 205: 105118.

[54] Toki S . Rapid and efficient Agrobacterium-mediated transformation in rice . Plant Mol Biol Report. 1997; 15: 16-21.

[55] Toki S, Hara N, Ono K, et al. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice . Plant J. 2006; 47: 969-76.