ORIGINAL PAPER Effects of Yariv dyes, arabinogalactan-protein binding reagents, on the growth and viability of Brazilian pine suspension culture cells Juliana Bello Baron Maurer • Adaucto Bellarmino Pereira-Netto • Filomena Angela Pettolino • Yolanda Maria Gaspar • Antony Bacic Received: 24 July 2009 / Revised: 6 December 2009 / Accepted: 23 December 2009 / Published online: 20 January 2010 Ó Springer-Verlag 2010 Abstract Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several aspects of plant growth and devel- opment. (b-D-glucosyl) 3 Yariv phenylglycoside (b-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures of Araucaria angustifolia, the Brazilian pine, with b-GlcY and observed inhibition of biomass increase in a culture medium with 50 lM b-GlcY. However, the growth was not inhibited by (a-D-galactosyl) 3 Yariv phenylglycoside (a-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that b-GlcY severely affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic cell death, were not observed in b-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic shrinkage and condensation were observed in b-GlcY- treated cells. In addition, callose accumulation, which is another marker of PCD, was also observed in b-GlcY- treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture medium did not reverse the growth inhibition caused by b-GlcY. These data indicate that the b-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven by necrosis. Keywords Araucaria angustifolia Á Caspase-6-inhibitor Á Gymnospermae Á Phenyl methyl sulphonyl fluoride Á Programmed cell death Á Yariv phenylglycoside Introduction Arabinogalactan-proteins (AGPs), a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs), are major components in the extracellular space of all plant tissues (Nothnagel 1997; Darjania et al. 2002). AGPs are widely distributed in plant species and are located at the plasma membrane and cell wall, and secreted into the media of cell cultures (Fincher et al. 1983; Komalavilas et al. 1991). These proteoglycans are typically composed of at least 90% (w/w) carbohydrate. The sugar moieties are composed of (1 ? 3)-b-D-galactan backbones and (1 ? 6)-b-D-galactan side chains with terminal sugars of arabinose or glucuronic acid (Nothna- gel 1997). Although their biological function has not been precisely identified, there is an evidence that AGPs play important roles in various aspects of plant growth and development, including cell proliferation, cell expansion, cell differentiation, regulation of somatic embryogenesis, pollen tube growth and programmed cell death (reviewed in Tang et al. 2006). Communicated by K. Klimaszewska. J. B. B. Maurer Department of Biochemistry and Molecular Biology, SCB, Parana Federal University, P.O. Box 19046, Curitiba, PR 81531-990, Brazil A. B. Pereira-Netto (&) Department of Botany, SCB, Parana Federal University, P.O. Box 19031, Curitiba, PR 81531-970, Brazil e-mail: apereira@ufpr.br F. A. Pettolino Á Y. M. Gaspar Á A. Bacic Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, Melbourne, VIC 3010, Australia 123 Trees (2010) 24:391–398 DOI 10.1007/s00468-009-0408-y