Grafting: a potential method to reveal the differential accumulation mechanism of secondary metabolites

Abstract

Plant secondary metabolites make a great contribution to the agricultural and pharmaceutical industries. Their accumulation is determined by the integrated transport of target compounds and their biosynthesis-related RNA, protein, or DNA. However, it is hard to track the movement of these biomolecules in vivo. Grafting may be an ideal method to solve this problem. The differences in genetic and metabolic backgrounds between rootstock and scion, coupled with multiple omics approaches and other molecular tools, make it feasible to determine the movement of target compounds, RNAs, proteins, and DNAs. In this review, we will introduce methods of using the grafting technique, together with molecular biological tools, to reveal the differential accumulation mechanism of plant secondary metabolites at different levels. Details of the case of the transport of one diterpene alkaloid, fuziline, will be further illustrated to clarify how the specific accumulation model is shaped with the help of grafting and multiple molecular biological tools.

References

[1] Ramakrishna A, Ravishankar GA . Influence of abiotic stress signals on secondary metabolites in plants. Plant Signal Behav. 2011; 6: 1720-31.

[2] Khare S, Singh NB, Singh A et al. Plant secondary metabolites synthesis and their regulations under biotic and abiotic constraints. J Plant Biol. 2020; 63: 203-16.

[3] Bartwal A, Mall R, Lohani P et al. Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses. J Plant Growth Regul. 2012; 32: 216-32.

[4] Newman DJ, Cragg GM . Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 2012; 75: 311-35.

[5] Carter GT . Natural products and Pharma 2011: strategic changes spur new opportunities. Nat Prod Rep. 2011; 28: 1783-9.

[6] Yazaki K . Transporters of secondary metabolites. Curr Opin Plant Biol. 2005; 8: 301-7.

[7] Nour-Eldin HH, Halkier BA . The emerging field of transport engineering of plant specialized metabolites. Curr Opin Biotechnol. 2013; 24: 263-70.

[8] Dawson RF . Accumulation of nicotine in reciprocal grafts of tomato and tobacco. Am J Bot. 1942; 29: 66-71.

[9] Shitan N, Dalmas F, Dan K et al. Characterization of Coptis japonica CjABCB2, an ATP-binding cassette protein involved in alkaloid transport. Phytochemistry. 2013; 91: 109-16.

[10] Bartusch K, Melnyk CW . Insights into plant surgery: an overview of the multiple grafting techniques for Arabidopsis thaliana . Front Plant Sci. 2020; 11: 613442.

[11] Molnar A, Melnyk CW, Bassett A et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science. 2010; 328: 872-5.

[12] Ali EM, Kobayashi K, Yamaoka N et al. Graft transmission of RNA silencing to non-transgenic scions for conferring virus resistance in tobacco. PLoS One. 2013; 8: e63257.

[13] Notaguchi M, Daimon Y, Abe M et al. Graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering. Plant Signal Behav. 2009; 4: 123-5.

[14] Chilukamarri L, Ashrafzadeh S, Leung DWM . In-vitro grafting - current applications and future prospects. Sci Hortic. 2021; 280: 109899.

[15] Deng WW, Han J-Y, Fan Y et al. Uncovering tea-specific secondary metabolism using transcriptomic and metabolomic analyses in grafts of Camellia sinensis and C. oleifera . Tree Genet Genomes. 2018; 14: 1-16.

[16] Xu D, Yuan H, Tong Y et al. Comparative proteomic analysis of the graft unions in hickory (Carya cathayensis) provides insights into response mechanisms to grafting process . Front Plant Sci. 2017; 8: 676.

[17] Kyriacou MC, Rouphael Y, Colla G et al. Vegetable grafting: the implications of a growing agronomic imperative for vegetable fruit quality and nutritive value. Front Plant Sci. 2017; 8: 741.

[18] Nawaz MA, Imtiaz M, Kong Q et al. Grafting: a technique to modify ion accumulation in horticultural crops. Front Plant Sci. 2016; 7: 1457.

[19] Loupit G, Cookson SJ . Identifying molecular markers of successful graft union formation and compatibility. Front Plant Sci. 2020; 11: 610352.

[20] Goldschmidt EE . Plant grafting: new mechanisms, evolutionary implications. Front Plant Sci. 2014; 5: 727.

[21] Mudge K, Janick J, Scofield S et al. A history of grafting. In: Hortic Rev.Vol. 35. 2009, 437-93.

[22] Habran A, Commisso M, Helwi P et al. Rootstocks/scion/nitrogen interactions affect secondary metabolism in the grape berry. Front Plant Sci. 2016; 7: 1134.

[23] Duan X, Bi HG, Li T et al. Root characteristics of grafted peppers and their resistance to Fusarium solani . Biol Plant. 2017; 61: 579-86.

[24] Proietti S, Rouphael Y, Colla G et al. Fruit quality of mini-watermelon as affected by grafting and irrigation regimes. J Sci Food Agric. 2008; 88: 1107-14.

[25] Tsutsui H, Notaguchi M . The use of grafting to study systemic signaling in plants. Plant Cell Physiol. 2017; 58: 1201-91.

[26] Thomas HR, Frank MH . Connecting the pieces: uncovering the molecular basis for long-distance communication through plant grafting. New Phytol. 2019; 223: 582-9.

[27] Chen A, Komives EA, Schroeder JI. An improved grafting technique for mature Arabidopsis plants demonstrates long-distance shoot-to-root transport of phytochelatins in Arabidopsis. Plant Physiol. 2006;141:108-20.

[28] Regnault T, Daviere JM, Achard P . Long-distance transport of endogenous gibberellins in Arabidopsis . Plant Signal Behav. 2016; 11: e1110661.

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

[30] Kakizaki T, Kitashiba H, Zou Z et al. A 2-oxoglutarate-dependent dioxygenase mediates the biosynthesis of glucoraphasatin in radish. Plant Physiol. 2017; 173: 1583-93.

[31] Symons GM, Ross JJ, Jager CE, et al. Brassinosteroid transport. J Exp Bot 2008; 59: 17-24.

[32] Osugi A, Kojima M, Takebayashi Y et al. Systemic transport of trans-zeatin and its precursor have differing roles in Arabidopsis shoots. Nat Plants. 2017; 3: 17112.

[33] Paultre DSG, Gustin MP, Molnar A et al. Lost in transit: long-distance trafficking and phloem unloading of protein signals in Arabidopsis homografts. Plant Cell. 2016; 28: 2016-25.

[34] Lucas WJ, Yoo BC, Kragler F . RNA as a long-distance information macromolecule in plants. Nat Rev Mol Cell Biol. 2001; 2: 849-57.

[35] Corbesier L, Vincent C, Jang S et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis . Science. 2007; 316: 1030-3.

[36] Takahashi F, Suzuki T, Osakabe Y et al. A small peptide modulates stomatal control via abscisic acid in long-distance signalling. Nature. 2018; 556: 235-8.

[37] Fuentes I, Stegemann S, Golczyk H et al. Horizontal genome transfer as an asexual path to the formation of new species. Nature. 2014; 511: 232-5.

[38] Stegemann S, Keuthe M, Greiner S, et al. Horizontal transfer of chloroplast genomes between plant species. Proc Natl Acad Sci USA. 2012; 109: 2434-8.

[39] Stegemann S, Bock R . Exchange of genetic material between cells in plant tissue grafts. Science. 2009; 324: 649-51.

[40] Lu Y, Stegemann S, Agrawal S et al. Horizontal transfer of a synthetic metabolic pathway between plant species. Curr Biol. 2017; 27: 3034-41.

[41] Kim M, Canio W, Kessler S et al. Developmental changes due to long-distance movement of a homeobox fusion transcript in tomato. Science. 2001; 293: 287-9.

[42] Rasool A, Mansoor S, Bhat KM et al. Mechanisms underlying graft union formation and rootstock scion interaction in horticultural plants. Front Plant Sci. 2020; 11: 590847.

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

[44] Robe K, Conejero G, Gao F et al. Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process. New Phytol. 2021; 229: 2062-79.

[45] Mothes K. Pflanzenphysiologische Untersuchungen über die Alkaloide. I. Das Nikotin im Stoffwechsel der Tabakpflanze. Zeitschr Biol. 1928; 5: 563-615.

[46] Dawson RF . A titrimetric method for the determination of nicotine in tobacco tissues. Proc Indiana Acad Sci. 1939; 49: 91-3.

[47] Vickery HB, Pucher GW . Chemical Investigations of the tobacco plant . Bull Connecticut Agric Exp Sta 1933, No. 323

[48] Yasinok AE, Sahin FI, Eyidogan F et al. Grafting tomato plant on tobacco plant and its effect on tomato plant yield and nicotine content. J Sci Food Agr. 2009; 89: 1122-8.

[49] Ruiz JM, Blasco B, Rivero RM et al. Nicotine-free and salt-tolerant tobacco plants obtained by grafting to salinity-resistant rootstocks of tomato. Physiol Plant. 2005; 124: 465-75.

[50] Ellerbrock BLJ, Kim JH, Jander G . Contribution of glucosinolate transport to Arabidopsis defense responses. Plant Signal Behav. 2007; 2: 282-3.

[51] Manzi M, Lado J, Rodrigo MJ et al. Root ABA accumulation in long-term water-stressed plants is sustained by hormone transport from aerial organs. Plant Cell Physiol. 2015; 56: 2457-66.

[52] Zhao J, Ding B, Zhu E et al. Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins. Plant Physiol. 2021; 186: 2111-23.

[53] Liu YS, Wang QL, Li BY . New insights into plant graft hybridization. Heredity. 2010; 104: 1-2.

[54] Sidorov V, Armstrong C, Ream T et al. “Cell grafting”: a new approach for transferring cytoplasmic or nuclear genome between plants. Plant Cell Rep. 2018; 37: 1077-89.

[55] Gurdon C, Svab Z, Feng Y et al. Cell-to-cell movement of mitochondria in plants. Proc Natl Acad Sci USA. 2016; 113: 3395-400.

[56] Dobrogojski J, Adamiec M, Luciński R . The chloroplast genome: a review. Acta Physiol Plant. 2020; 42: 1-13.

[57] Zhang LX, Guo QS, Chang QS et al. Chloroplast ultrastructure, photosynthesis and accumulation of secondary metabolites in Glechoma longituba in response to irradiance. Photosynthetica. 2015; 53: 144- 53.

[58] Kicinska A, Jarmuszkiewicz W . Flavonoids and mitochondria: activation of cytoprotective pathways? Molecules. 2020; 25: 3060.

[59] Li X, Chai Y, Yang H et al. Isolation and comparative proteomic analysis of mitochondria from the pulp of ripening citrus fruit. Hortic Res. 2021; 8: 1-16.

[60] Haroldsen VM, Szczerba MW, Aktas H et al. Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Front Plant Sci. 2012; 3: 39.

[61] Uthup TK, Karumamkandathil R, Ravindran M, et al. Heterografting induced DNA methylation polymorphisms in Hevea brasiliensis . Planta 2018; 248: 579-89.

[62] Neves DM, da Hora Almeida LA, Santana-Vieira DDS et al. Recurrent water deficit causes epigenetic and hormonal changes in citrus plants. Sci Rep. 2017; 7: 13684.

[63] Avramidou E, Kapazoglou A, Aravanopoulos FA et al. Global DNA methylation changes in Cucurbitaceae inter-species grafting. Crop Breed Appl Biotechnol. 2015; 15: 112-6.

[64] Wu R, Wang X, Lin Y et al. Inter-species grafting caused extensive and heritable alterations of DNA methylation in Solanaceae plants. PLoS One. 2013; 8: e61995.

[65] Cao L, Yu N, Li J et al. Heritability and reversibility of DNA methylation induced by in vitro grafting between Brassica juncea and B. oleracea . Sci Rep. 2016; 6: 27233.

[66] Yang D, Huang Z, Jin W et al. DNA methylation: a new regulator of phenolic acids biosynthesis in Salvia miltiorrhiza . Ind Crops Prod. 2018; 124: 402-11.

[67] Chitarra W, Perrone I, Avanzato CG et al. Grapevine grafting: scion transcript profiling and defense-related metabolites induced by rootstocks. Front Plant Sci. 2017; 8: 654.

[68] Spano R, Mascia T, Kormelink R et al. Grafting on a non-transgenic tolerant tomato variety confers resistance to the infection of a sw5-breaking strain of tomato spotted wilt virus via RNA silencing. PLoS One. 2015; 10: e0141319.

[69] Han Y, Grierson D . Enhancement of post-transcriptional gene silencing by grafting. Plant Signal Behav. 2008; 3: 30-3.

[70] Luo L, Zheng Y, Gao Z et al. Grafting improves drought stress memory by increasing the P5CS1 gene expression in Brassica rapa . Plant Soil. 2020; 452: 61-72.

[71] Liu N, Yang J, Fu X et al. Genome-wide identification and comparative analysis of grafting-responsive mRNA in watermelon grafted onto bottle gourd and squash rootstocks by high-throughput sequencing. Mol Gen Genomics. 2016; 291: 621-33.

[72] Kehr J, Kragler F . Long distance RNA movement. New Phytol. 2018; 218: 29-40.

[73] Harada T . Grafting and RNA transport via phloem tissue in horticultural plants. Sci Hortic. 2010; 125: 545-50.

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

[75] Huang NC, Yu TS . The sequences of Arabidopsis GA-INSENSITIVE RNA constitute the motifs that are necessary and sufficient for RNA long-distance trafficking. Plant J. 2009; 59: 921-9.

[76] 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.

[77] Xu H, Zhang W, Li M et al. Gibberellic acid insensitive mRNA transport in both directions between stock and scion in Malus . Tree Genet Genomes. 2010; 6: 1013-9.

[78] Pant BD, Buhtz A, Kehr J et al. MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis. Plant J. 2008; 53: 731-8.

[79] Pant BD, Musialak-Lange M, Nuc P et al. Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing. Plant Physiol. 2009; 150: 1541-55.

[80] Martin A, Adam H, Díaz-Mendoza M et al. Graft-transmissible induction of potato tuberization by the microRNA miR172. Development. 2009; 136: 2873-81.

[81] Dunoyer P, Brosnan CA, Schott G et al. An endogenous, systemic RNAi pathway in plants. EMBO J. 2010; 29: 1699-712.

[82] Menke FL, Champion A, Kijne JW et al. A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP-domain transcription factor, ORCA2. EMBO J. 1999; 18: 4455-63.

[83] Shitan N, Sugiyama A, Yazaki K . Functional analysis of jasmonic acid-responsive secondary metabolite transporters. Methods Mol Biol. 2013; 1011: 241-50.

[84] Abbasi BH, Stiles AR, Saxena PK et al. Gibberellic acid increases secondary metabolite production in Echinacea purpurea hairy roots. Appl Biochem Biotechnol. 2012; 168: 2057-66.

[85] van der Fits L, Memelink J . ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science. 2000; 289: 295-7.

[86] Schluttenhofer C, Yuan L . Regulation of specialized metabolism by WRKY transcription factors. Plant Physiol. 2015; 167: 295-306.

[87] Patra B, Schluttenhofer C, Wu Y et al. Transcriptional regulation of secondary metabolite biosynthesis in plants. Biochim Biophys Acta. 2013; 1829: 1236-47.

[88] Zheng X, Zhao Y, Shan D et al. MdWRKY9 overexpression confers intensive dwarfing in the M26 rootstock of apple by directly inhibiting brassinosteroid synthetase MdDWF4 expression. New Phytol. 2018; 217: 1086-98.

[89] Deng WW, Li M, Gu C-C et al. Low caffeine content in novel grafted tea with Camellia sinensis as scions and Camellia oleifera as stocks . Nat Prod Commun. 2015; 10: 789-92.

[90] Mo Z, He H, Su W, et al. Analysis of differentially accumulated proteins associated with graft union formation in pecan (Carya illinoensis) . Sci Hortic. 2017; 224: 126-34.

[91] Xu Y, Yuan Y, Du N et al. Proteomic analysis of heat stress resistance of cucumber leaves when grafted onto Momordica rootstock. Hortic Res. 2018; 5: 53-3.

[92] Aguero CB, Uratsu SL, Greve C et al. Evaluation of tolerance to Pierce’s disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene . Mol Plant Pathol. 2005; 6: 43-51.

[93] Golecki B, Schulz A, Thompson GA . Translocation of structural P proteins in the phloem. Plant Cell. 1999; 11: 127-40.

[94] Notaguchi M, Abe M, Kimura T et al. Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering . Plant Cell Physiol. 2008; 49: 1645-58.

[95] Lin MK, Belanger H, Lee Y-J et al. FLOWERING LOCUS T protein may act as the long-distance florigenic signal in the cucurbits. Plant Cell. 2007; 19: 1488-506.

[96] Lu KJ, Huang NC, Liu YS et al. Long-distance movement of Arabidopsis FLOWERING LOCUS T RNA participates in systemic floral regulation. RNA Biol. 2012; 9: 653-62.

[97] Shen Y, Liang WJ, Shi YN et al. Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids. Nat Prod Rep. 2020; 37: 763-96.

[98] Zhao D, Shen Y, Shi Y et al. Probing the transcriptome of Aconitum carmichaelii reveals the candidate genes associated with the biosynthesis of the toxic aconitine-type C₁₉-diterpenoid alkaloids. Phytochemistry. 2018; 152: 113-24.

[99] Shi Y, Zhao Y, Qian J et al. Aconitum diterpenoid alkaloid profiling to distinguish between the official traditional Chinese medicine (TCM) fuzi and adulterant species using LC-qToF-MS with chemometrics . J Nat Prod. 2021; 84: 570-87.

[100] Notaguchi M, Kurotani K-I, Sato Y et al. Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases. Science. 2020; 369: 698-702.

[101] Lv H, Li J, Wu Y et al. Transporter and its engineering for secondary metabolites. Appl Microbiol Biotechnol. 2016; 100: 6119-30.

[102] 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.

[103] Tang M, Bai X, Wang J et al. Efficiency of graft-transmitted JcFT for floral induction in woody perennial species of the Jatropha genus depends on transport distance. Tree Physiol. 2021; 42: 189-201.