A Developmental Framework for Graft Formation and Vascular Reconnection in Arabidopsis thaliana

•Phloem reconnects first, followed by root growth resuming, and then xylem reconnection•Auxin and cytokinin response are strongly enhanced in pericycle and vascular cambium•Cutting drives tissue asymmetry in cellular processes; grafting restores symmetry•Certain auxin response genes, including ALF4, are required specifically below the graft Plant grafting is a biologically important phenomenon involving the physical joining of two plants to generate a chimeric organism. It is widely practiced in horticulture and used in science to study the long-distance movement of molecules. Despite its widespread use, the mechanism of graft formation and vascular reconnection is not well understood. Here, we study the dynamics and mechanisms of vascular regeneration in Arabidopsis thaliana during graft formation when the vascular strands are severed and reconnected. We demonstrate a temporal separation between tissue attachment, phloem connection, root growth, and xylem connection. By analyzing cell division patterns and hormone responses at the graft junction, we found that tissues initially show an asymmetry in cell division, cell differentiation, and gene expression and, through contact with the opposing tissue, lose this asymmetry and reform the vascular connection. In addition, we identified genes involved in vascular reconnection at the graft junction and demonstrate that these auxin response genes are required below the graft junction. We propose an inter-tissue communication process that occurs at the graft junction and promotes vascular connection by tissue-specific auxin responses involving ABERRANT LATERAL ROOT FORMATION 4 (ALF4). Our study has implications for phenomena where forming vascular connections are important including graft formation, parasitic plant infection, and wound healing. Plant grafting is a biologically important phenomenon involving the physical joining of two plants to generate a chimeric organism. It is widely practiced in horticulture and used in science to study the long-distance movement of molecules. Despite its widespread use, the mechanism of graft formation and vascular reconnection is not well understood. Here, we study the dynamics and mechanisms of vascular regeneration in Arabidopsis thaliana during graft formation when the vascular strands are severed and reconnected. We demonstrate a temporal separation between tissue attachment, phloem connection, root growth, and xylem connection. By analyzing cell division patterns and hormone responses at the graft junction, we found that tissues initially show an asymmetry in cell division, cell differentiation, and gene expression and, through contact with the opposing tissue, lose this asymmetry and reform the vascular connection. In addition, we identified genes involved in vascular reconnection at the graft junction and demonstrate that these auxin response genes are required below the graft junction. We propose an inter-tissue communication process that occurs at the graft junction and promotes vascular connection by tissue-specific auxin responses involving ABERRANT LATERAL ROOT FORMATION 4 (ALF4). Our study has implications for phenomena where forming vascular connections are important including graft formation, parasitic plant infection, and wound healing. The majority of plants possess the ability to adhere tissues and reconnect their vasculature after severing of the vascular strands by wounding. The ability to heal the vascular tissue is particularly important, as this tissue transports water, nutrients, and signaling molecules throughout the plant [1Lough T.J. Lucas W.J. Integrative plant biology: role of phloem long-distance macromolecular trafficking.Annu. Rev. Plant Biol. 2006; 57: 203-232Crossref PubMed Scopus (392) Google Scholar]. It is also horticulturally relevant, as plant grafting involves the severing and rejoining of vascular strands from different plant species or varieties to introduce resistance to abiotic and biotic stresses, to propagate plants, or to change plant size [2Melnyk C.W. Meyerowitz E.M. Plant grafting.Curr. Biol. 2015; 25: R183-R188Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]. Parasitic plants also in a sense “graft,” as they join elements of their vasculature to the host vasculature upon infection [3Musselman L.J. The biology of Striga, Orobanche, and other root-parasitic weeds.Annu. Rev. Phytopathol. 1980; 18: 463-489Crossref Google Scholar]. Despite this biological and horticultural relevance, the mechanism of vascular tissue regeneration remains poorly understood. Previous work describes a hierarchy of events that occurs at the graft junction of Arabidopsis and other plants. After cutting, ruptured cells collapse to form a necrotic layer at the graft junction, and cells from opposing tissues, termed the scion and rootstock, adhere to each other. Through cell proliferation above and below the graft junction, a mass of pluripotent cells, termed callus, is formed. Lastly, it is thought that these callus cells differentiate into vascular tissue to reconnect the phloem and xylem across the graft junction [4Flaishman M.A. Loginovsky K. Golobowich S. Lev-Yadun S. Arabidopsis thaliana as a model system for graft union development in homografts and heterografts.J. Plant Growth Regul. 2008; 27: 231-239Crossref Scopus (75) Google Scholar, 5Moore R. Walker D.B. Studies of vegetative compatibility-incompatibility in higher-plants 0.1. a structural study of a compatible autograft in Sedum telephioides (Crassulaceae).Am. J. Bot. 1981; 68: 820-830Crossref Google Scholar, 6Jeffree C.E. Yeoman M.M. Development of intercellular connections between opposing cells in a graft union.New Phytol. 1983; 93: 491-509Crossref Scopus (105) Google Scholar]. A common theme to plant wound responses is the involvement of plant hormones, which are critical regulators of growth and development. Injury by wounding prompts organs to divide and differentiate [7Sugimoto K. Gordon S.P. Meyerowitz E.M. Regeneration in plants and animals: dedifferentiation, transdifferentiation, or just differentiation?.Trends Cell Biol. 2011; 21: 212-218Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar]. Part of the wound response is mediated by the plant hormone cytokinin and by the WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) pathway [8Iwase A. Mitsuda N. Koyama T. Hiratsu K. Kojima M. Arai T. Inoue Y. Seki M. Sakakibara H. Sugimoto K. Ohme-Takagi M. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis.Curr. Biol. 2011; 21: 508-514Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar]. WIND1 is strongly upregulated upon wounding, and overexpression of this gene results in excess callus formation [8Iwase A. Mitsuda N. Koyama T. Hiratsu K. Kojima M. Arai T. Inoue Y. Seki M. Sakakibara H. Sugimoto K. Ohme-Takagi M. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis.Curr. Biol. 2011; 21: 508-514Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar]. In cut Arabidopsis inflorescence stems, the plant hormone auxin promotes the division of pith cells and wound healing [9Asahina M. Azuma K. Pitaksaringkarn W. Yamazaki T. Mitsuda N. Ohme-Takagi M. Yamaguchi S. Kamiya Y. Okada K. Nishimura T. et al.Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2011; 108: 16128-16132Crossref PubMed Scopus (119) Google Scholar]. Ethylene and jasmonic acid are also involved in the wound-healing response and promote the expression of the RAP2.6L and ANAC071 transcription factors around a cut site [9Asahina M. Azuma K. Pitaksaringkarn W. Yamazaki T. Mitsuda N. Ohme-Takagi M. Yamaguchi S. Kamiya Y. Okada K. Nishimura T. et al.Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis.Proc. Natl. Acad. Sci. USA. 2011; 108: 16128-16132Crossref PubMed Scopus (119) Google Scholar]. In particular, the plant hormone auxin plays a pivotal role in vascular development [10Sachs T. The control of the patterned differentiation of vascular tissues.Adv. Bot. Res. 1981; 9: 151-262Crossref Scopus (530) Google Scholar, 11Scarpella E. Marcos D. Friml J. Berleth T. Control of leaf vascular patterning by polar auxin transport.Genes Dev. 2006; 20: 1015-1027Crossref PubMed Scopus (599) Google Scholar]. Classical experiments demonstrated that the patterns of auxin flow through a tissue determine the sites of vein formation [12Sachs T. Polarity and the induction of organized vascular tissues.Ann. Bot. (Lond.). 1969; 33: 263-275Google Scholar]. Similarly, when auxin is added to callus, it promotes the formation of xylem and phloem [13Wetmore R.H. Rier J.P. Experimental induction of vascular tissues in callus of angiosperms.Am. J. Bot. 1963; 50: 418-430Crossref Google Scholar]. In Arabidopsis, normal vein patterning depends on polar auxin transport and can be modified by auxin transport inhibitors such as 1-N-naphthylphthalamic acid (NPA) or mutations in genes coding for auxin transport proteins [11Scarpella E. Marcos D. Friml J. Berleth T. Control of leaf vascular patterning by polar auxin transport.Genes Dev. 2006; 20: 1015-1027Crossref PubMed Scopus (599) Google Scholar, 14Sieburth L.E. Auxin is required for leaf vein pattern in Arabidopsis.Plant Physiol. 1999; 121: 1179-1190Crossref PubMed Scopus (229) Google Scholar, 15Mattsson J. Sung Z.R. Berleth T. Responses of plant vascular systems to auxin transport inhibition.Development. 1999; 126: 2979-2991PubMed Google Scholar]. Auxin signals by binding the TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) receptors that target the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins for degradation [16Kepinski S. Leyser O. The Arabidopsis F-box protein TIR1 is an auxin receptor.Nature. 2005; 435: 446-451Crossref PubMed Scopus (1294) Google Scholar, 17Dharmasiri N. Dharmasiri S. Weijers D. Lechner E. Yamada M. Hobbie L. Ehrismann J.S. Jürgens G. Estelle M. Plant development is regulated by a family of auxin receptor F box proteins.Dev. Cell. 2005; 9: 109-119Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar]. These proteins are negative regulators of many auxin response factors (ARFs), so that the presence of auxin ultimately promotes ARF activity and therefore auxin-regulated transcription [18Benjamins R. Scheres B. Auxin: the looping star in plant development.Annu. Rev. Plant Biol. 2008; 59: 443-465Crossref PubMed Scopus (440) Google Scholar]. Many mutant Arabidopsis lines that block auxin signaling have been identified. The AUXIN-RESISTANT 1 (AXR1) gene is required for normal TIR1 function and, when mutated, changes the stabilization dynamics of the Aux/IAA proteins [19Gray W.M. Kepinski S. Rouse D. Leyser O. Estelle M. Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins.Nature. 2001; 414: 271-276Crossref PubMed Scopus (1044) Google Scholar]. Mutations in the TIR1/AFB binding DII domain of Aux/IAAs render these proteins insensitive to auxin and can therefore keep ARFs and auxin signaling repressed. Mutants in several auxin signaling genes, including MONOPTEROS (MP/ARF5), BODENLOS (BDL/IAA12), and CULLIN 1 (CUL1/AXR6), perturb vascular patterning [20Przemeck G.K. Mattsson J. Hardtke C.S. Sung Z.R. Berleth T. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization.Planta. 1996; 200: 229-237Crossref PubMed Scopus (375) Google Scholar, 21Hamann T. Mayer U. Jürgens G. The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo.Development. 1999; 126: 1387-1395PubMed Google Scholar, 22Berleth T. Jurgens G. The role of the monopteros gene in organising the basal body region of the Arabidopsis embryo.Development. 1993; 118: 575-587Google Scholar, 23Hobbie L. McGovern M. Hurwitz L.R. Pierro A. Liu N.Y. Bandyopadhyay A. Estelle M. The axr6 mutants of Arabidopsis thaliana define a gene involved in auxin response and early development.Development. 2000; 127: 23-32PubMed Google Scholar]. Mutation of ARF6 and ARF8 reduces cell division in the pith cells upon cutting [24Pitaksaringkarn W. Ishiguro S. Asahina M. Satoh S. ARF6 and ARF8 contribute to tissue reunion in incised Arabidopsis inflorescence stems.Plant Biotechnol. 2014; 31: 49-53Crossref Scopus (27) Google Scholar]. Alternatively, increasing auxin in plants by exogenous applications promotes the formation of callus from xylem pole pericycle cells, the cells that give rise to lateral roots [25Atta R. Laurens L. Boucheron-Dubuisson E. Guivarc’h A. Carnero E. Giraudat-Pautot V. Rech P. Chriqui D. Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro.Plant J. 2009; 57: 626-644Crossref PubMed Scopus (292) Google Scholar, 26Sugimoto K. Jiao Y. Meyerowitz E.M. Arabidopsis regeneration from multiple tissues occurs via a root development pathway.Dev. Cell. 2010; 18: 463-471Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar]. One gene required for callus formation in plant tissue culture and for lateral root formation is ABERRANT LATERAL ROOT FORMATION 4 (ALF4; AT5G11030) [26Sugimoto K. Jiao Y. Meyerowitz E.M. Arabidopsis regeneration from multiple tissues occurs via a root development pathway.Dev. Cell. 2010; 18: 463-471Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, 27Celenza Jr., J.L. Grisafi P.L. Fink G.R. A pathway for lateral root formation in Arabidopsis thaliana.Genes Dev. 1995; 9: 2131-2142Crossref PubMed Scopus (375) Google Scholar, 28DiDonato R.J. Arbuckle E. Buker S. Sheets J. Tobar J. Totong R. Grisafi P. Fink G.R. Celenza J.L. Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation.Plant J. 2004; 37: 340-353Crossref PubMed Scopus (121) Google Scholar]. ALF4 mutants are resistant to auxin [27Celenza Jr., J.L. Grisafi P.L. Fink G.R. A pathway for lateral root formation in Arabidopsis thaliana.Genes Dev. 1995; 9: 2131-2142Crossref PubMed Scopus (375) Google Scholar], and ALF4 is hypothesized to act downstream of auxin to maintain the xylem pole pericycle cells in a mitotically competent state [28DiDonato R.J. Arbuckle E. Buker S. Sheets J. Tobar J. Totong R. Grisafi P. Fink G.R. Celenza J.L. Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation.Plant J. 2004; 37: 340-353Crossref PubMed Scopus (121) Google Scholar]. The ALF4 gene is expressed throughout the plant, and the protein is nuclear localized but contains no similarities to proteins in other families, and its precise role is unknown [27Celenza Jr., J.L. Grisafi P.L. Fink G.R. A pathway for lateral root formation in Arabidopsis thaliana.Genes Dev. 1995; 9: 2131-2142Crossref PubMed Scopus (375) Google Scholar, 28DiDonato R.J. Arbuckle E. Buker S. Sheets J. Tobar J. Totong R. Grisafi P. Fink G.R. Celenza J.L. Arabidopsis ALF4 encodes a nuclear-localized protein required for lateral root formation.Plant J. 2004; 37: 340-353Crossref PubMed Scopus (121) Google Scholar]. The research reported here characterizes vascular reconnection during graft formation using a well-established Arabidopsis hypocotyl grafting method [29Turnbull C.G. Booker J.P. Leyser H.M. Micrografting techniques for testing long-distance signalling in Arabidopsis.Plant J. 2002; 32: 255-262Crossref PubMed Scopus (301) Google Scholar]. By analyzing cell division, cell differentiation, and gene expression changes, we identify a process whereby contact with the opposing tissue reduces gene expression asymmetries between the rootstock and scion. Furthermore, we identify several genes involved in vascular reconnection and demonstrate that these genes are required specifically in the rootstock near the graft junction. We propose that these genes act as part of a mechanism that senses the opposing tissue by perceiving transported auxin, thereby promoting wound healing and vascular formation. Hallmarks of successful graft formation are the attachment of scion to rootstock (Figure S1) and the resumption of root growth. We used previously described Arabidopsis grafting protocols [29Turnbull C.G. Booker J.P. Leyser H.M. Micrografting techniques for testing long-distance signalling in Arabidopsis.Plant J. 2002; 32: 255-262Crossref PubMed Scopus (301) Google Scholar, 30Melnyk C.W. Molnar A. Bassett A. Baulcombe D.C. Mobile 24 nt small RNAs direct transcriptional gene silencing in the root meristems of Arabidopsis thaliana.Curr. Biol. 2011; 21: 1678-1683Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar] and found that scion and rootstock attached within 2 days after grafting (DAG), as assayed by adherence of scion and rootstock when plants were lifted (Figure 1B) . Grafting initially arrested root growth, but at 5 DAG, the majority of roots resumed growth (Figure 1B). We then asked whether the vasculature connected prior to or after the resumption of root growth. Previous analyses have used the water-soluble dye carboxyfluorescein diacetate (CFDA) to monitor movement through the phloem and xylem [31Botha C.E. Aoki N. Scofield G.N. Liu L. Furbank R.T. White R.G. A xylem sap retrieval pathway in rice leaf blades: evidence of a role for endocytosis?.J. Exp. Bot. 2008; 59: 2945-2954Crossref PubMed Scopus (52) Google Scholar, 32Oparka K.J. Duckett C.M. Prior D.A.M. Fisher D.B. Real-time imaging of phloem unloading in the root-tip of Arabidopsis.Plant J. 1994; 6: 759-766Crossref Scopus (197) Google Scholar]. CFDA is a non-fluorescent compound until it is taken up by a cell, whereupon the acetate group is cleaved off, creating a fluorescent molecule. We reasoned that we could use CFDA to monitor phloem and xylem connectivity at the graft junction. CFDA application on the cotyledons would be expected to allow detection of phloem connectivity, as phloem transport can occur from shoot to root, whereas application to the roots would be expected to allow detection of xylem connectivity, since xylem transports from root to shoot (Figure 1A) [1Lough T.J. Lucas W.J. Integrative plant biology: role of phloem long-distance macromolecular trafficking.Annu. Rev. Plant Biol. 2006; 57: 203-232Crossref PubMed Scopus (392) Google Scholar]. Consistent with these expectations, we observed fluorescence in the hypocotyl phloem poles after CFDA was applied to the cotyledons and observed fluorescence in the tissues surrounding the hypocotyl xylem after CFDA was applied to the roots of ungrafted plants (Figure S1). Upon treatment of the scion with CFDA, we observed fluorescence in the rootstock of grafted individuals 3 DAG, and by 4 days, nearly all individuals fluoresced (Figure 1C). CFDA treatment to the rootstock in grafted individuals produced fluorescence in the scions 6 DAG, and by 7 days, the scions from the majority of individuals fluoresced (Figures 1D and S1). As a second test of vascular reconnection, we grafted scions carrying the pSUC2::GFP transgene [33Imlau A. Truernit E. Sauer N. Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.Plant Cell. 1999; 11: 309-322Crossref PubMed Scopus (447) Google Scholar], which express free GFP in the phloem companion cells, to non-transgenic rootstocks (Figure S1), an assay previously used to monitor phloem connectivity [34Yin H. Yan B. Sun J. Jia P. Zhang Z. Yan X. Chai J. Ren Z. Zheng G. Liu H. Graft-union development: a delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.J. Exp. Bot. 2012; 63: 4219-4232Crossref PubMed Scopus (131) Google Scholar]. We monitored individuals daily, and we observed that at 3 DAG, the root vasculature showed GFP fluorescence similar to ungrafted pSUC2::GFP plants (Figures 1C and S1), consistent with previously published reports [34Yin H. Yan B. Sun J. Jia P. Zhang Z. Yan X. Chai J. Ren Z. Zheng G. Liu H. Graft-union development: a delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.J. Exp. Bot. 2012; 63: 4219-4232Crossref PubMed Scopus (131) Google Scholar]. We also exposed grafted individuals to a low-humidity environment to test water transport across the graft junction. Newly grafted individuals wilted under these conditions. However, when transferred to low humidity 7 DAG, a substantial number of individuals could take up sufficient water from the medium to remain turgid (Figure 1E). Our results point to a scenario in which attachment occurs first, followed by phloem reconnection at about 3 DAG, root growth at approximately 5 DAG, and xylem reconnection at around 7 DAG. To characterize further vascular formation and cell differentiation at the graft junction, we visualized xylem by clearing the tissue using a previously described method [35Malamy J.E. Benfey P.N. Organization and cell differentiation in lateral roots of Arabidopsis thaliana.Development. 1997; 124: 33-44PubMed Google Scholar]. New xylem vessels formed above the graft junction 4–5 DAG, whereas new xylem formed below the graft junction 5–6 DAG (Figures 2A and 2B) . Since xylem is composed of dead cells, it appears that xylem precursor cells differentiated and underwent programmed cell death to form new xylem vessels. Even in grafts where the old xylem vessels appeared aligned, the new xylem often took an indirect route to reconnect below the cut site (Figure 2A), possibly avoiding damaged xylem elements. Xylem formation appeared to be promoted by an apically process, consistent with our observation that xylem formation occurred in cut shoots, but not cut roots (Figure 2C, Figure S2). To test whether a similar phenomenon occurred with the phloem, we grafted pSUC2::GFP scions to two segments of Col-0 to form a three-segment or interstock graft (Figure S1). The top junction connected before the lower junction (Figure 2D), consistent with a process that begins in apical tissues also driving phloem reconnection. To understand better cell growth during vascular connection, we grafted Arabidopsis expressing different fluorescent reporters regulated by the constitutive promoters CaMV 35S and Ubiquitin10 (Table S1) on a microscope coverslip (Figure S1) and monitored fluorescence over 7 days. Contact between the scion and rootstock promoted vascular formation but suppressed the cell expansion and cell division that occurred at the cut surface of ungrafted shoots (Figure S2). Within 24 hr of grafting, epidermal cells above and below the graft junction expanded to fill the graft junction (Figure 2F). In some individuals, vascular cells expanded and proliferated across the graft junction into the adjoining tissue (Figures 2E and S2). To assay cell division, we monitored the endodermis and observed cell divisions above the graft junction 3 DAG (Figure 3A) , which correlated with the expression of a gene involved in Casparian strip formation in the endodermis, CASP1 [36Roppolo D. De Rybel B. Dénervaud Tendon V. Pfister A. Alassimone J. Vermeer J.E. Yamazaki M. Stierhof Y.D. Beeckman T. Geldner N. A novel protein family mediates Casparian strip formation in the endodermis.Nature. 2011; 473: 380-383Crossref PubMed Scopus (255) Google Scholar]. We observed pCASP1::NLS-GFP expression at the graft junction 4 DAG, and this expression was substantially higher than in cut or ungrafted controls (Figures 3C and S3). CASP1 expression appeared below the graft junction 1 to 2 days later than above the graft junction (Figures 3D and S3), indicating an asymmetry in cell differentiation at the graft junction between the scion and rootstock. We also observed the presence of lignin, consistent with the reformation of the Casparian strip (Figures 3B and S3) [37Naseer S. Lee Y. Lapierre C. Franke R. Nawrath C. Geldner N. Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin.Proc. Natl. Acad. Sci. USA. 2012; 109: 10101-10106Crossref PubMed Scopus (293) Google Scholar]. To assess cell division patterns during graft formation in more detail, we performed in situ hybridizations to detect mRNA expression of Histone H4, a marker of S phase. We observed a higher level of Histone H4 expression in the vascular tissue of scions than in rootstocks 2 DAG (Figures 3E and 3F). At 3 DAG, Histone H4 expression was present in the rootstock vascular tissue at levels similar to those in the scion (Figures 3E and 3F). Histone H4 expression was detected close to the graft junction and occasionally was present in the outer cell layers, where new vasculature may be forming (Figure 3F), but was not present in the hypocotyls of ungrafted individuals (Figure S3). To see whether markers associated with wounding would be upregulated during grafting, we tested the wound-induced gene, WIND1 [8Iwase A. Mitsuda N. Koyama T. Hiratsu K. Kojima M. Arai T. Inoue Y. Seki M. Sakakibara H. Sugimoto K. Ohme-Takagi M. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis.Curr. Biol. 2011; 21: 508-514Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar], and found that pWIND1::GFP was highly upregulated 3 DAG in the vasculature and epidermis of the scion’s hypocotyl, but not in the rootstock (Figures 3G and S3). Expression of pWIND1::GFP was at a similar level below and above the graft junction 6 DAG and decreased by 10 DAG (Figure 3G). Cut but ungrafted shoots showed a similar wound response, but we did not observe a WIND1 response in cut roots or ungrafted controls (Figure S3), indicating that the response in grafted rootstocks was promoted by the presence of the scion. These results point toward coordinated gene expression, cell division, and cell expansion driven by contact between the scion and rootstock. Auxin and cytokinin are two key hormones implicated in vascular differentiation [38Aloni R. Differentiation of vascular tissues.Annu. Rev. Plant Physiol. 1987; 38: 179-204Crossref Google Scholar, 39Bishopp A. Help H. El-Showk S. Weijers D. Scheres B. Friml J. Benková E. Mähönen A.P. Helariutta Y. A mutually inhibitory interaction between auxin and cytokinin specifies vascular pattern in roots.Curr. Biol. 2011; 21: 917-926Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar], so we sought to understand their roles in vascular reconnection. We used the auxin-responsive promoter DR5 [40Ulmasov T. Murfett J. Hagen G. Guilfoyle T.J. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements.Plant Cell. 1997; 9: 1963-1971Crossref PubMed Scopus (1599) Google Scholar] and, as a positive control, observed activation at the graft junction upon exogenous auxin treatment after 6 hr (Figure S4). We did not observe a strong increase in auxin response 1 or 2 DAG (Figure S4), but at 3 DAG, there was a response (Figure S4). The delayed hormone response may be due to sensitivity of the reporters, or it may be a response driven by a wound and vascular reconnection pathway rather than the expected accumulation of auxin at the cut surface due to basipetal transport. The pDR5rev::GFP-ER [41Friml J. Vieten A. Sauer M. Weijers D. Schwarz H. Hamann T. Offringa R. Jürgens G. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis.Nature. 2003; 426: 147-153Crossref PubMed Scopus (1418) Google Scholar] response peaked above and below the graft junction at 5 days (Figures 4A and S4) and diminished by 10 days (Figures 4A and S4). To identify which cells respond, we hand sectioned above and below the graft junction (Figure 4B) and observed auxin response in the pericycle cells on both sides of the graft junction (Figures 4C and 4D). Notably, the response below the graft junction was strongest in the pericycle cells adjacent to the xylem (the xylem pole pericycle cells) and was often asymmetric (Figure 4D). Ungrafted controls did not show this strong response (Figure S4). Cut but ungrafted roots had no response, and cut shoots had a strong auxin response throughout the tissue (Figure S4). Auxin response in the rootstock was specific to grafting, and could result from auxin transported from the scion. We also tested the cytokinin-responsive promoters ARR5 and TCSn [42Zürcher E. Tavor-Deslex D. Lituiev D. Enkerli K. Tarr P.T. Müller B. A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta.Plant Physiol. 2013; 161: 1066-1075Crossref PubMed Scopus (201) Google Scholar, 43D’Agostino I.B. Deruère J. Kieber J.J. Characterization of the response of the Arabidopsis response regulator gene family to cytokinin.Plant Physiol. 2000; 124: 1706-1717Crossref PubMed Scopus (454) Google Scholar, 44Yanai O. Shani E. Dolezal K. Tarkowski P. Sablowski R. Sandberg G. Samach A. Ori N. Arabidopsis KNOXI proteins activate cytokinin biosynthesis.Curr. Biol. 2005; 15: 1566-1571Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar] and, as a control, observed activation at the graft junction upon exogenous cytokinin treatment after 6 hr (Figure S4). In grafted individuals, pARR5::GFP showed a response at 4 DAG and peaked at 6 DAG (Figures 4E, 4F, and S4), whereas pTCSn::GFP-ER showed a response at 5 DAG and peaked at 7 DAG (Figures 4I and S4). With both reporters, the response occurred above and below the junction (Figures 4F and 4I) and was present in the pericycle cells and vascular cambium (Figures 4G–4H and 4J–4K). Ungrafted plants had a slight response in the phloem poles, and cut shoots showed no response (Figure S4). Cut roots showed strong pARR5::GFP and pTCSn::GFP expression throughout the tissue (Figure S4). Cytokinin response in the scion was specific to grafting and could resu