JrGA20ox1 -transformed rootstocks deliver drought response signals to wild-type scions in grafted walnut

Abstract

Targeted regulation using transgrafting technology has become a trend. However, the mechanisms of transgene-derived signal communication between rootstocks and scions remain unclear in woody plants. Here, we grafted wild-type (WT) walnut (Juglans regia L.) on WT (WT/WT), JrGA20ox1 (encodes a gibberellin 20-oxidase)-overexpressing (WT/OE), and JrGA20ox1-RNAi transformation (WT/RNAi) walnut in vitro. We aimed to elucidate the mechanisms of JrGA20ox1-derived signal communication under PEG-simulated drought stress between rootstocks and scions in walnut. We demonstrated that JrGA20ox1-OE and JrGA20ox1-RNAi rootstocks could transport active gibberellins (GAs) and JrGA20ox1-RNAi vector-produced sRNAs to WT scions under PEG-simulated drought stress, respectively. The movement of sRNAs further led to a successive decline in JrGA20ox1 expression and active GA content. Meanwhile, unknown mobile signals may move between rootstocks and scions. These mobile signals reduced the expression of a series of GA-responsive and GA-non-responsive genes, and induced ROS production in guard cells and an increase in ABA content, which may contribute to the drought tolerance of WT/RNAi, while the opposite occurred in WT/OE. The findings suggest that JrGA20ox1-derived rootstock-to-scion movement of signals is involved in drought tolerance of scions. Our research will provide a feasible approach for studying signal communication in woody plants.

Acknowledgements

This work was supported by the National Key R&D Program of China (2023YFD2200305), Zhejiang Agriculture New Variety Breeding Major Science and Technology (2021C02066-12), the National Natural Science Foundation of China (NSFC) (32101557; 31971672; 32330069), and the Scientific Research Startup Fund Project of Zhejiang A&F University (2018FR028). We thank Professor Shaojian Zheng from Zhejiang University for technical support.

Author contributions

Q.Z. and H.L. conceived this project. Q.Z., J.H., J.W. (Jiasheng Wu), and H.L. supervised this project. F.W., J.Z., G.W., J.W. (Jingjing Wei), and H.H. performed experiments. Y.L., K.W., Z.W., Y.H., and D.P. contributed comments. H.L. and Q.Z. analyzed the data. H.L. and Q.Z. wrote the manuscript.

Data availability

All the data supporting the findings of this study are available in the paper and supplementary data.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary data

Supplementary data are available at Horticulture Research online.

References

[1] Aloni B, Cohen R, Karni L. et al. Hormonal signaling in rootstock-scion interactions. Sci Hortic. 2020; 127:119-26

[2] Gonçalves B, Moutinho-Pereira J, Santos A. et al. Scion-rootstock interaction affects the physiology and fruit quality of sweet cherry. Tree Physiol. 2006; 26:93-104

[3] Koepke T, Dhingra A. Rootstock scion somatogenetic interactions in perennial composite plants. Plant Cell Rep. 2013; 32:1321-37

[4] Martinez-Ballesta MC, Alcaraz-Lopez C, Muries B. et al. Physiological aspects of rootstock-scion interactions. Sci Hortic. 2010; 127:112-8

[5] Gaut BS, Miller AJ, Seymour DK. Living with two genomes: grafting and its implications for plant genome-to-genome interactions, phenotypic variation, and evolution. Annu Rev Genet. 2019; 53:195-215

[6] Warschefsky EJ, Klein LL, Frank MH. et al. Rootstocks: diversity, domestication, and impacts on shoot phenotypes. Trends Plant Sci. 2016; 21:418-37

[7] Albacete A, Martínez-Andújar C, Martínez-Pérez A. et al. Unravelling rootstock × scion interactions to improve food security. J Exp Bot. 2015; 66:2211-26

[8] Lu X, Liu W, Wang T. et al. Systemic long-distance signaling and communication between rootstock and scion in grafted vegetables. Front Plant Sci. 2020; 11:460

[9] Song GQ, Walworth AE, Loescher WH. Grafting of genetically engineered plants. J Am Soc Hortic Sci. 2015; 140:203-13

[10] Lemgo GNY, Sabbadini S, Pandolfini T. et al. Biosafety considerations of RNAi-mediated virus resistance in fruit-tree cultivars and in rootstock. Transgenic Res. 2013; 22:1073-88

[11] Artlip TS, Wisniewski ME, Arora R. et al. An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy. Hortic Res. 2016; 3:16006

[12] Zhao D, Song GQ. Rootstock-to-scion transfer of transgene-derived small interfering RNAs and their effect on virus resistance in nontransgenic sweet cherry. Plant Biotechnol J. 2014; 12:1319-28

[13] Rugini E, Silvestri C, Cristofori V. et al. Ten years field trial observations of ri-TDNA cherry colt rootstocks and their effect on grafted sweet cherry cv Lapins. Plant Cell Tiss Org. 2015; 123:557-68

[14] Nagel AK, Kalariya H, Schnabel G. The Gastrodia antifungal protein (GAFP-1) and its transcript are absent from scions of chimeric-grafted plum. HortScience. 2010; 45:188-92

[15] Liu X, Walawage SL, Leslie CA. et al. In vitro gene expression and mRNA translocation from transformed walnut (Juglans regia) rootstocks expressing DsRED fluorescent protein to wild-type scions. Plant Cell Rep. 2017; 36:877-85

[16] Song GQ, Walworth A, Lin T. et al. VcFT-induced mobile florigenic signals in transgenic and transgrafted blueberries. Hortic Res. 2019; 6:105

[17] Sidorova T, Miroshnichenko D, Kirov I. et al. Effect of grafting on viral resistance of non-transgenic plum scion combined with transgenic PPV-resistant rootstock. Front Plant Sci. 2021; 12:621954

[18] Smolka A, Li XY, Heikelt C. et al. Effects of transgenic rootstocks on growth and development of non-transgenic scion cultivars in apple. Transgenic Res. 2010; 19:933-48

[19] Flachowsky H, Tränkner C, Szankowski I. et al. RNA-mediated gene silencing signals are not graft transmissible from the rootstock to the scion in greenhouse-grown apple plants Malus sp. Int J Mol Sci. 2012; 13:9992-10009

[20] Duan X, Zhang W, Huang J. et al. PbWoxT 1 mRNA from pear (Pyrus betulaefolia) undergoes long-distance transport assisted by a polypyrimidine tract binding protein. New Phytol. 2016; 210:511-24

[21] Gentile A, Frattarelli A, Nota P. et al. The aromatic cytokinin meta-topolin promotes in vitro propagation, shoot quality and micrografting in Corylus colurna L. Plant Cell Tiss Org. 2017; 128:693-703

[22] Behrooz A, Vahdati K, Rejali F. et al. Arbuscular mycorrhiza and plant growth-promoting bacteria alleviate drought stress in walnut. HortScience. 2019; 54:1087-92

[23] Vahdati K, Lotfi N, Kholdebarin B. et al. Screening for drought-tolerant genotypes of Persian walnuts (Juglans regia L.) during seed germination. HortScience. 2009; 44:1815-9

[24] Hooley R. Gibberellins: perception, transduction and responses. Plant Mol Biol. 1994; 26:1529-55

[25] Band LR, Nelissen H, Preston SP. et al. Modeling reveals posttranscriptional regulation of GA metabolism enzymes in response to drought and cold. Proc Natl Acad Sci USA. 2022; 119:e2121288119

[26] Shohat H, Cheriker H, Kilambi HV. et al. Inhibition of gibberellin accumulation by water deficiency promotes fast and long-term ‘drought avoidance’ responses in tomato. New Phytol. 2021; 232:1985-98

[27] Song X, Zhao Y, Wang J. et al. The transcription factor KNAT2/6b mediates changes in plant architecture in response to drought via down-regulating GA20ox1 in Populus alba × P. glandulosa. J Exp Bot. 2021; 72:5625-37

[28] Hedden P, Phillips AL. Gibberellin metabolism: new insights revealed by the genes. Trends Plant Sci. 2000; 5:523-30

[29] Israelsson M, Mellerowicz E, Chono M. et al. Cloning and overproduction of gibberellin 3-oxidase in hybrid aspen trees. Effects on gibberellin homeostasis and development. Plant Phys-iol. 2004; 135:221-30

[30] Carrera E, Bou J, Garcia-Martinez JL. et al. Changes in GA 20-oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants. Plant J. 2000; 22:247-56

[31] Colebrook EH, Thomas SG, Phillips AL. et al. The role of gibberellin signalling in plant responses to abiotic stress. J Exp Bot. 2014; 217:67-75

[32] Regnault T, Davière JM, Wild M. et al. The gibberellin precursor GA12 acts as a long-distance growth signal in Arabidopsis. Nat Plants. 2015; 1:15073

[33] Binenbaum J, Weinstain R, Shani E. Gibberellin localization and transport in plants. Trends Plant Sci. 2018; 23:410-21

[34] Camut L, Regnault T, Sirlin-Josserand M. et al. Root-derived GA12 contributes to temperature-induced shoot growth in Arabidopsis. Nat Plants. 2019; 5:1216-21

[35] Fischer-Kilbienski I, Miao Y, Roitsch T. et al. Nuclear targeted AtS40 modulates senescence associated gene expression in Arabidopsis thaliana during natural development and in darkness. Plant Mol Biol. 2010; 73:379-90

[36] Hong D, Jeon BW, Kim SY. et al. The ROP2-RIC7 pathway negatively regulates light-induced stomatal opening by inhibiting exocyst subunit Exo70B1 in Arabidopsis. New Phytol. 2016; 209:624-35

[37] Epple P, Mack AA, Morris VR. et al. Antagonistic control of oxidative stress-induced cell death in Arabidopsis by two related, plant-specific zinc finger proteins. Proc Natl Acad Sci USA. 2003; 100:6831-6

[38] Gao H, Cui J, Liu S. et al. Natural variations of ZmSRO1d modulate the trade-off between drought resistance and yield by affecting ZmRBOHC-mediated stomatal ROS production in maize. Mol Plant. 2022; 15:1558-74

[39] Turnbull CG, Booker JP, Leyser HO. Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant J. 2002; 32:255-62

[40] Holbrook NM, Shashidhar VR, James RA. et al. Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying. J Exp Bot. 2002; 53:1503-14

[41] Omid A, Keilin T, Glass A. et al. Characterization of phloem-sap transcription profile in melon plants. J Exp Bot. 2007; 58:3645-56

[42] Zhang WN, Duan XW, Ma C. et al. Transport of mRNA molecules coding NAC domain protein in grafted pear and transgenic tobacco. Biol Plantarum. 2013; 57:224-30

[43] Thieme CJ, Rojas-Triana M, Stecyk E. et al. Endogenous Arabidop-sis messenger RNAs transported to distant tissues. Nat Plants. 2015; 1:15025

[44] Ota R, Ohkubo Y, Yamashita Y. et al. Shoot-to-root mobile CEPD-like 2 integrates shoot nitrogen status to systemically regulate nitrate uptake in Arabidopsis. Nat Commun. 2020; 11:641

[45] Hedden P, Thomas SG. Gibberellin biosynthesis and its regulation. Biochem J. 2012; 444:11-25

[46] 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

[47] Loreti E, Perata P. Mobile plant microRNAs allow communication within and between organisms. New Phytol. 2022; 235:2176-82

[48] Tuna AL, Kaya C, Dikilitas M. et al. The combined effects of gibberellic acid and salinity on some antioxidant enzyme activities, plant growth parameters and nutritional status in maize plants. Environ Exp Bot. 2008; 62:1-9

[49] Omena-Garcia RP, Martins AO, Medeiros DB. et al.Growth and metabolic adjustments in response to gibberellin deficiency in drought stressed tomato plants. Environ Exp Bot. 2019; 159:95-107

[50] Achard P, Renou JP, Berthomé R. et al. Plant DELLAs restrain growth and promote survival of adversity by reducing the levels of reactive oxygen species. Curr Biol. 2008; 18:656-60

[51] Nir I, Moshelion M, Weiss D. The Arabidopsis GIBBERELLIN METHYL TRANSFERASE 1 suppresses gibberellin activity, reduces whole-plant transpiration and promotes drought tolerance in transgenic tomato. Plant Cell Environ. 2014; 37:113-23

[52] Hua D, Wang C, He J. et al. A plasma membrane receptor kinase, GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis. Plant Cell. 2012; 24:2546-61

[53] Song X, Xi S, Zhang J. et al. ‘Zhong Ning Sheng’: a new distant hybrid cultivar of walnut. HortScience. 2019; 54:2257-9

[54] Zhang Q, Walawage SL, Tricoli DM. et al. A red fluorescent protein (DsRED) from Discosoma sp. as a reporter for gene expression in walnut somatic embryos. Plant Cell Rep. 2015; 34:861-9

[55] Koca H, Bor M, Özdemir F. et al. The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ Exp Bot. 2007; 60:344-51

[56] Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant Soil. 1973; 39:205-7

[57] Chen Y, Chen Y, Shi C. et al. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Giga-science. 2018; 7:1-6

[58] Langmead B, Trapnell C, Pop M. et al. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10:R25-34

[59] Nawrocki EP, Eddy SR. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics. 2013; 29:2933-5