b C scion, epitope present in the walls of the dividing cells (full arrows), in the cell compartments (arrow), and on the surface of the callus cells protruding outside the scion (arrowhead). cellular compartments of graft union cells (arrows), no epitope observed in extracellular material on the surface of graft union (arrowheads). C C, Calcofluor White. E C epitope detected in walls of some graft union cells (arrows), apart from extracellular material on the surface of graft union (arrowhead). E E, Calcofluor White. F C strong fluorescence signal in cell wall of sieve tubes (arrows). G C epitope absent from graft union cells (arrows) and from extracellular material (arrowheads). G G, Calcofluor White. c Calcofluor White. Scale bars: A, A, C, C, E, E, G, and G?=?50?m; B, D, and F?=?10?m. (JPG 2868 kb) 12870_2019_1748_MOESM2_ESM.jpg (2.8M) GUID:?DFC8F4CA-35D4-42FD-BF56-96D89207C100 Additional file 3: Figure S3. Immunohistochemistry of grafted hypocotyl sections C extensins (JIM12 and LM1 epitopes) and AGPs (JIM13, JIM8, and LM2 epitopes). A C epitope present in some of the cortical cells (full arrow) and graft union area (arrowheads), intensive fluorescence signal detected in the outer periclinal cell walls and cuticle of the epidermis (arrow); intensive fluorescence signal detected in the outer periclinal cell walls and cuticle of the epidermis (arrow). B C epitope detected in the cell wall (arrow) and on the outside of the cell (arrowhead). C C epitope present in the cytoplasmic compartments of cortical cells near the graft union area (arrow). D C occurrence of epitope in the cells of the regenerated vascular bundle (arrows), in some endodermal cells (arrowhead), and peripheral cells of the graft union (arrowhead), no fluorescence signal detected on the cell surface (full arrow). E C epitope present in the cytoplasm and/or plasmolemma of the graft union cells located peripherally (arrowheads), no fluorescence signal detected on the cell surface (arrow). F and C weak labeling in the cytoplasmic compartments of the peripheral cells (arrowheads), no fluorescence signal detected on the cell surface (arrows). c Calcofluor White, ep epidermis. Scale bars: A, D and hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological IQ-1 and cellular changes that accompany autografting of hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union. Results During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either closed or open. Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), IQ-1 JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well CAGL114 as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration. Conclusions To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectinCextensin interaction but also due to its origin. The findings presented here IQ-1 implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material. Electronic supplementary material The online version of this article (10.1186/s12870-019-1748-4) contains supplementary material, which is available to authorized users. hypocotyl, we observed the formation of a layer covering the surface of the graft union. As this phenomenon has not been described so far, we focused on the exterior area of a graft union instead of the adhesion zone, which has been the subject of numerous studies. The aims of this study were 1) to describe the histological and cellular changes that occur IQ-1 during the process of regeneration in autografted hypocotyls and IQ-1 2) to perform preliminary chemical and structural analyses of the material that extracellularly deposits and finally seals the graft union. Results Formation of the graft union C morphological features Three time frames were chosen to evaluate the process of regeneration of the hypocotyls during grafting based on the occurrence of dominant cellular events (Fig.?1, section I). The first time frame,.