Integrated multi-omics analysis reveals the molecular mechanism underlying poplar 107 rootstock–mediated regulation of Populus tomentosa scion growth

Abstract

Grafting is widely used for asexual propagation and enhancing plant productivity; however, the molecular mechanisms underlying rootstock–scion interactions remain largely unclear. In this study, Populus × euramericana cv. ‘Neva’ (poplar 107) served as rootstock and Populus tomentosa ‘Yixian’ (P. tomentosa) as scion for grafting. By integrating physiological measurements, transcriptome sequencing [messenger RNA (mRNA) and long non-coding RNA (lncRNA)], widely targeted metabolomics, and mobile RNA identification revealed that heterografted scions had increased metabolites related to carbon fixation and metabolism, decreased metabolites associated with respiration and defense, and enhanced net photosynthetic rate, potentially promoting height growth; differential expression lncRNAs may represent an important molecular basis for accelerated scion growth; in scions, mobile mRNA-associated differential mRNAs primarily involved amino acid biosynthesis, while mobile lncRNA-associated ones focused on energy metabolism; no target mRNAs of mobile lncRNAs were found in the rootstock; both mRNAs and lncRNAs were transported in full-length and fragmented forms, and downward RNA transport showed a significant correlation with transcript abundance; in recipient tissues, mobile mRNAs were less abundant while mobile lncRNAs were more abundant than in donor tissues; the flavonoid biosynthesis pathway associated with mobile RNAs played an important role in rootstock-mediated scion growth, and kaempferol, as a key metabolite, reduced reactive oxygen species–induced damage, increased net photosynthetic rate, and promoted growth in Nicotiana benthamiana. This study provides an initial insight into the transport and regulatory patterns of mRNAs and lncRNAs in woody grafted plants, offering a theoretical basis for the rational application of grafting technology.

References

[1] Melnyk CW, Meyerowitz EM . Plant grafting. Curr Biol. 2015; 25: R183-8

[2] Li W, Chen X, Zhao S, et al. Effect of grafting on the growth and flowering of sprays chrysanthemums. Sci Hortic. 2022; 291: 110607

[3] Liu R, Jia J, Wang C, et al. Transcriptomic and primary metabolic profiles reveal the mechanism of development and maturation of Fuji apple grafted onto different dwarfed intermediate rootstocks. Sci Hortic. 2025; 343: 114060

[4] Zhu B, Guo P, Wu S, et al. A better fruit quality of grafted blueberry than own-rooted blueberry is linked to its anatomy. Plants. 2024; 13: 625

[5] Liu W, Wang Q, Zhang R, et al. Rootstock-scion exchanging mRNAs participate in the pathways of amino acid and fatty acid metabolism in cucumber under early chilling stress. Hortic Res. 2022; 9: uhac031

[6] Wang L, Yang M, Akinnagbe A, et al. Bacillus thuringiensis protein transfer between rootstock and scion of grafted poplar . Plant Biol. 2012; 14: 745-50

[7] Miao C, Zhang Y, Cui J, et al. An enhanced interaction of graft and exogenous SA on photosynthesis, phytohormone, and transcriptome analysis in tomato under salinity stress. Int J Mol Sci. 2024; 25: 10799

[8] Li Q, Gao Y, Wang K, et al. Transcriptome analysis of the effects of grafting interstocks on apple rootstocks and scions. Int J Mol Sci. 2023; 24: 807

[9] Li W, Chen S, Liu Y, et al. Long-distance transport RNAs between rootstocks and scions and graft hybridization. Planta. 2022; 255: 96

[10] Ham BK, Lucas WJ . Phloem-mobile RNAs as systemic signaling agents. Annu Rev Plant Biol. 2017; 68: 173-95

[11] Haywood V, Yu TS, Huang NC, et al. Phloem long-distance trafficking of gibberellic acid-insensitive RNA regulates leaf development. Plant J. 2005; 42: 49-68

[12] Zhang M, Liu WQ, Wang CC, et al. Root-to-shoot mobile mRNA CmoKARI1 promotes JA-Ile biosynthesis to confer chilling tolerance in grafted cucumbers. Nat Commun. 2025; 16: 7782

[13] Mattick JS, Amaral PP, Carninci P, et al. Long non-coding RNAs: definitions, functions, challenges and recommendations. Nat Rev Mol Cell Biol. 2023; 24: 430-47

[14] Numan M, Sun YG, Li GL . Exploring the emerging role of long non-coding RNAs (lncRNAs) in plant biology: functions, mechanisms of action, and future directions. Plant Physiol Biochem. 2024; 212: 108797

[15] Zhang ZL, Zheng Y, Ham BK, et al. Plant lncRNAs are enriched in and move systemically through the phloem in response to phosphate deficiency. J Integr Plant Biol. 2019; 61: 492-508

[16] Cheng SLH, Xu HY, Ng JHT, et al. Systemic movement of long non-coding RNA ELENA1 attenuates leaf senescence under nitrogen deficiency. Nat Plants. 2023; 9: 1598-606

[17] Lv X, Sun Y, Hao P, et al. RBP differentiation contributes to selective transmissibility of OPT3 mRNAs. Plant Physiol. 2021; 187: 1587-604

[18] Zhang FC, Zhong HX, Zhou XM, et al. Grafting with rootstocks promotes phenolic compound accumulation in grape berry skin during development based on integrative multi-omics analysis. Hortic Res. 2022; 9: uhac055

[19] Balfagón D, Rambla JL, Granell A, et al. Grafting improves tolerance to combined drought and heat stresses by modifying metabolism in citrus scion. Environ Exp Bot. 2022; 195: 104793

[20] Conesa A, Madrigal P, Tarazona S, et al. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016; 17: 13

[21] Gabaldón T, Koonin EV . Functional and evolutionary implications of gene orthology. Nat Rev Genet. 2013; 14: 360-6

[22] Liu Z, Liu X, Wang S, et al. Comparative transcriptome analysis of Arabidopsis seedlings under heat stress on whole plants, shoots, and roots reveals new HS-regulated genes, organ-specific responses, and shoots-roots communication. Int J Mol Sci. 2025; 26: 2478

[23] Lee J, He K, Stolc V, et al. Analysis of transcription factor HY5 genomic binding sites revealed its hierarchical role in light regulation of development. Plant Cell. 2007; 19: 731-49

[24] Jiang N, Lee YS, Mukundi E, et al. Diversity of genetic lesions characterizes new Arabidopsis flavonoid pigment mutant alleles from T-DNA collections. Plant Sci. 2020; 291: 110335

[25] Haldrup A, Naver H, Scheller HV . The interaction between plastocyanin and photosystem I is inefficient in transgenic Arabidopsis plants lacking the PSI-N subunit of photosystem. Plant J. 1999; 17: 689-98

[26] Kneeshaw S, Keyani R, Delorme-Hinoux V, et al. Nucleoredoxin guards against oxidative stress by protecting antioxidant enzymes. Proc Natl Acad Sci USA. 2017; 114: 8414-9

[27] Gao Y, Dai X, Aoi Y, et al. Two homologous INDOLE-3-ACETAMIDE (IAM) HYDROLASE genes are required for the auxin effects of IAM in Arabidopsis. J Genet Genomics. 2020; 47: 157-65

[28] Li W, Chu C, Li H, et al. Near-gapless and haplotype-resolved apple genomes provide insights into the genetic basis of rootstock-induced dwarfing. Nat Genet. 2024; 56: 505-16

[29] Liu W, Xiang C, Li X, et al. Identification of long-distance transmissible mRNA between scion and rootstock in cucurbit seedling heterografts. Int J Mol Sci. 2020; 21: 5253

[30] Wang Y, Wang L, Xing N, et al. A universal pipeline for mobile mRNA detection and insights into heterografting advantages under chilling stress. Hortic Res. 2020; 7: 13

[31] Li S, Wang X, Xu W, et al. Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts. Nat Plants. 2021; 7: 50-9

[32] Wu Y, Luo D, Fang L, et al. Bidirectional lncRNA transfer between Cuscuta parasites and their host plant. Int J Mol Sci. 2022; 23: 561

[33] Zhao X, Li F, Ali M, et al. Emerging roles and mechanisms of lncRNAs in fruit and vegetables. Hortic Res. 2024; 11: uhae046

[34] Huang D, Chen R, Zhang D . The role of lncRNAs in flowering time regulation in rice. Rice Genom Genet. 2025; 16: 4

[35] Fahad M, Tariq L, Muhammad S, et al. Underground communication: long non-coding RNA signaling in the plant rhizosphere. Plant Commun. 2024; 5: 100927

[36] Jia Y, Zhao H, Niu Y, et al. Identification of birch lncRNAs and mRNAs responding to salt stress and characterization of functions of lncRNA. Hortic Res. 2023; 10: uhac277

[37] Li B, Feng C, Zhang W, et al. Comprehensive non-coding RNA analysis reveals specific lncRNA/circRNA-miRNA-mRNA regulatory networks in the cotton response to drought stress. Int J Biol Macromol. 2023; 253: 126558

[38] Jha UC, Nayyar H, Roychowdhury R, et al. Non-coding RNAs (ncRNAs) in plant: master regulators for adapting to extreme temperature conditions. Plant Physiol Biochem. 2023; 205: 108164

[39] Zhang X, Wang W, Zhu W, et al. Mechanisms and functions of long non-coding RNAs at multiple regulatory levels. Int J Mol Sci. 2019; 20: 5573

[40] Herman AB, Tsitsipatis D, Gorospe M . Integrated lncRNA function upon genomic and epigenomic regulation. Mol Cell. 2022; 82: 2252-66

[41] Calderwood A, Kopriva S, Morris RJ . Transcript abundance explains mRNA mobility data in Arabidopsis thaliana . Plant Cell. 2016; 28: 610-5

[42] Xia C, Zheng Y, Huang J, et al. Elucidation of the mechanisms of long-distance mRNA movement in a Nicotiana benthamiana/tomato heterograft system. Plant Physiol. 2018; 177: 745-58

[43] Yang Y, Mao L, Jittayasothorn Y, et al. Messenger RNA exchange between scions and rootstocks in grafted grapevines. BMC Plant Biol. 2015; 15: 251

[44] Heeney M, Frank MH . The mRNA mobileome: challenges and opportunities for deciphering signals from the noise. Plant Cell. 2023; 35: 1817-33

[45] Faulkner C . Plasmodesmata and the symplast. Curr Biol. 2018; 28: R1374-8

[46] Kirk P, Benitez-Alfonso Y . Plasmodesmata structural components and their role in signaling and plant development. Methods Mol Biol. 2022; 2457: 3-22

[47] Oparka K, Prior D, Cruz SS, et al. Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J. 1997; 12: 781-9

[48] Fu M, Xu Z, Ma H, et al. Characteristics of long-distance mobile mRNAs from shoot to root in grafted plant species. Hortic Plant J. 2024; 10: 25-37

[49] Li X, Wang C, Chen Y, et al. m5C and m6A modifications regulate the mobility of pumpkin CHOLINE KINASE 1 mRNA under chilling stress. Plant Physiol. 2025; 197: kiae511

[50] Kehr J, Morris RJ, Kragler F . Long-distance transported RNAs: from identity to function. Annu Rev Plant Biol. 2022; 73: 457-74

[51] Feng S, Yao YT, Wang BB, et al. Flavonoids are involved in salt tolerance through ROS scavenging in the halophyte Atriplex canescens. Plant Cell Rep. 2024; 43: 5

[52] Arikan B, Yildiztugay E, Ozfidan-Konakci C . Protective role of quercetin and kaempferol against oxidative damage and photosynthesis inhibition in wheat chloroplasts under arsenic stress. Physiol Plant. 2023; 175: e13964

[53] Watkins JM, Chapman JM, Muday GK . Abscisic acid-induced reactive oxygen species are modulated by flavonols to control stomata aperture. Plant Physiol. 2017; 175: 1807-25

[54] Gao G, Lv Z, Zhang G, et al. An ABA-flavonoid relationship contributes to the differences in drought resistance between different sea buckthorn subspecies. Tree Physiol. 2021; 41: 744-55

[55] Mahdavian K. Effects of ultraviolet radiation on plants and their protective mechanisms. Russ J Plant Physiol. 2024; 71: 184

[56] Vidović M, Morina F, Milić S, et al. Ultraviolet-B component of sunlight stimulates photosynthesis and flavonoid accumulation in variegated Plectranthus coleoides leaves depending on background light. Plant Cell Environ. 2015; 38: 968-79

[57] Song C, Huang Y, Wu CC, et al. Scion organ removal alters hormone levels and gene expression associated with adventitious root development in grafted watermelon seedlings. Plant Signal Behav. 2025; 20: 2556300

[58] Harrison N, Barber-Perez N, Pennington B, et al. Root system architecture in reciprocal grafts of apple rootstock-scion combinations. Acta Hortic. 2016; 1130: 409-14

[59] Wu J, Lv S, Zhao L, et al. Advances in the study of the function and mechanism of the action of flavonoids in plants under environmental stresses. Planta. 2023; 257: 108

[60] Luo X, Huang Q . Relationships between leaf and stem soluble sugar content and tuberous root starch accumulation in cassava. J Agric Sci. 2011; 3: 64

[61] Bradford MM . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54

[62] Chen W, Gong L, Guo Z, et al. A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics. Mol Plant. 2013; 6: 1769-80

[63] Fraga CG, Clowers BH, Moore RJ, et al. Signature-discovery approach for sample matching of a nerve-agent precursor using liquid chromatography−mass spectrometry, XCMS, and chemometrics. Anal Chem. 2010; 82: 4165-73

[64] Bolger AM, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30: 2114-20

[65] Trapnell C, Pachter L, Salzberg SL . TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25: 1105-11

[66] Kang YJ, Yang DC, Kong L, et al. CPC2: a fast and accurate coding potential calculator based on sequence intrinsic features. Nucleic Acids Res. 2017; 45: W12-6

[67] Sun L, Luo H, Bu D, et al. Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res. 2013; 41: e166-6

[68] Wang Y, Chen YJ, Ding LP, et al. Validation of reference genes for gene expression by quantitative real-time RT-PCR in stem segments spanning primary to secondary growth in Populus tomentosa . PLoS One. 2016; 11: e0157370

[69] Wu JH, Chen XH, Wang SJ, et al. Analyses of multicopy insertions and unintended responses to drought stress in dual Bt gene transgenic poplar. Ind Crop Prod. 2025; 223: 120247