1748 fungi, auxin is produced and detectable as secondary metabolite, although its function in these organisms is not yet known. The role of auxin as plant hormone in early land plants has been documented starting from the bryophytes (Cooke et al. 2002; Johri 2004, 2008; Paponov et al. 2009; Sztein et al. 1999, 2000). The ﬁlamentous, tip-growing protonemata of the closely related mosses Funaria hy- grometrica and Physcomitrella patens have been extensively studied to understand the role of phytohormones in cell differentiation (Johri 1974; Decker et al. 2006). The proto- nema consists of two distinct cell types, the chloronema and the caulonema. Chloronema cells have more chloroplasts and are separated by a straight septum whereas caulonema cells have fewer plastids and oblique cross walls (Johri 1974; Reski 1998). In addition, chloronema cells are mainly arrested in the G2/M phase of the cell cycle, whereas caulonema cells are mainly arrested in G1/S (Schween et al. 2003). Similar to ﬂowering plants, an auxin gradient exists in the moss protonema with maxima in the most actively dividing cells (Bierfreund et al. 2003). Auxin evokes two responses in Funaria protonema: at a low level it inhibits chloronema proliferation, while at higher levels it enhances secondary caulonema differenti- ation (Johri and Desai 1973). Both responses are antag- onised by the anti-auxin p-chlorophenoxyisobutyric acid (PCIB) (Johri and D’Souza 1990). PCIB reduces polar, basipetal auxin transport in Funaria rhizoids (Rose and Bopp 1983) and was reported to impair the auxin-signal- ling pathway by reducing the stability of auxin/indole-3- acetic acid (Aux/IAA) gene transcripts in Arabidopsis roots (Oono et al. 2003). Thus, both basipetal transport and IAA- binding sites seem to be involved during caulonema dif- ferentiation and chloronema inhibition (Johri 2004). In addition, cell division is inhibited, if auxin efﬂux from Physcomitrella protoplasts is blocked by napthylphtalamic acid (NPA) (Bhatla et al. 2002). It is known from ﬂowering plants that parts of the multiple auxin responses are mediated by the nuclear auxin receptor TRANSPORT INHIBITOR RESPONSE 1 (TIR1) (Dharmasiri et al. 2005; Kepinski and Leyser 2005) which, upon binding of auxin, degrades AUXIN/INDOLE-3- ACETIC ACID (Aux/IAA)-proteins (Dharmasiri and Estelle 2002), releasing the inhibitory effect on auxin response factors (ARFs), transcription factors that in turn regulate auxin responsive gene expression (Quint and Gray 2006; Benjamins and Scheres 2008). It is, however, evident that not all auxin responses are regulated via this nuclear receptor (Badescu and Napier 2006). Thus, another important mediator of auxin action may be ABP1, an auxin-binding protein involved in cell expansion (Jones et al. 1998), and subsequently found in a variety of seed plants by afﬁnity labelling (Christian et al. 2003; Napier et al. 2002). ABP1 is a 22–24 kDa protein 123 Plant Cell Rep (2009) 28:1747–1758 from corn which is localised predominantly in the endo- plasmic reticulum (ER), as it possesses a C-terminal KDEL ER retention sequence (Henderson et al. 1997; Jones and Herman 1993; Woo et al. 2002). A smaller fraction of this protein is secreted outside and located in the outer leaﬂet of the plasma membrane (Jones and Herman 1993; Oliver et al. 1995). ABP1 has been shown to mediate the hyper- polarization response and stomatal opening stimulated by auxin (Barbier-Brygoo et al. 1992; Christian et al. 2003, Leblanc et al. 1999; Gehring et al. 1998) and to be involved in cell cycle control (David et al. 2007). Other investiga- tions have proposed a role of ABP1 in directional root growth (Shimomura 2006). The current study aimed at characterising proteins in the protonema of moss F. hygrometrica homologous to ABP1 of Zea mays. To this end a radioactive IAA-binding assay was employed to determine if moss microsomes contained speciﬁc auxin-binding sites. To further investigate speciﬁc ABPs, biochemical, immunological and photo-afﬁnity labelling approaches were used. The results presented here reveal the presence of speciﬁc ABPs in Funaria. Antibodies speciﬁc to corn ABP1 could detect polypeptides of 31–33 and 46 kDa, respectively. However, unlike ABP1 of corn, both these polypeptides did not contain epitopes of the classical KDEL ER retention sequence. Similar results were obtained when nucleotide sequences from other mosses homologous to the ABP1 of maize were compared as shown for P. patens and Ceratodon purpureus. We therefore pro- pose that the non-ER localised ABPs of Funaria, and probably in all mosses, play a role in the regulation of auxin responses during the differentiation of moss protonema. Materials and methods Plant material The protonema of F. hygrometrica Hedw. (cell line J-2) and dark grown coleoptiles of corn (Z. mays L.) were used in the present study. Chloronema cells of Funaria were grown in liquid suspension cultures as described earlier (Johri 1974). Cells grown in minimal medium supplemented with glu- cose (MMG) were harvested at a cell density of 4–5 mg/ml and used fresh or kept frozen until used. Hybrid corn seeds (variety MMH 65, from Maharastra Hybrid Seeds, Mumbai, India) were germinated on moist, autoclaved vermiculite and the coleoptiles (approximately 1.5 cm from the tips) were harvested and used for the experiment. Microsomes for [3H]-IAA binding Two grams of freshly harvested protonema cells were homogenised in an ice-cold mortar and pestle in the