Plant Cell Rep (2009) 28:1747–1758 Photo-afﬁnity labelling using 5-azido 7-[3H]-indole acetic acid In order to determine if the putative moss ABP1 homo- logues with a size of 31–33 and 46 kDa identiﬁed in this study are capable of binding auxin, photo-afﬁnity labelling using 5-azido-7-3H-IAA was carried out with the micro- somal membrane fractions under the conditions described in ‘‘Materials and methods’’. Binding of the radioactive azido-auxin to the microsomal fractions of moss also revealed the presence of two major polypeptides at 31–33 and 46 kDa (Fig. 6). The speciﬁcity of this binding was evaluated by increasing the amounts of non-radioactive IAA in the reaction mixture prior to UV cross-linking as by such means the high-afﬁnity binding sites would be out- competed. The proteins were separated under denaturing conditions and signals from the gel were detected by ﬂuorography. Strikingly, polypeptides of identical molec- ular masses, i.e. 31–33 and 46 kDa, were intensively labelled by azido-auxin, although with different afﬁnities (Fig. 6, lane 1). Upon competition with increasing amounts of non-radioactive IAA (5–10 lM) in the reaction mixture (lanes 2–5) and prior to UV cross-linking, there was a complete disappearance of the azido-IAA labelling (Fig. 6, lanes 4, 5). Phylogenetic analysis and sequence comparison of ABP1 in land plants The topology of the phylogenetic tree for the 14 ABP1 proteins from 13 land plants (Fig. 7) conﬁrms the evolu- tionary conservation of the protein sequence among mosses, as the ABP1 from P. patens and the one from C. purpureus Fig. 6 Proteins from the acetone-washed microsomal vesicles of moss were solubilised in buffer as described in ‘‘Materials and methods’’. Equal amount of proteins from both buffer-soluble fraction and insoluble pellet were incubated with 0.33 lm azido-IAA in the presence of increasing amount of non-radioactive IAA for 20 min under red safe light (lanes 1–5 with 0 nM, 100 nM, 500 nM, 2.5 lM and 1 lM, respectively). The contents were exposed to UV light for 30 s, denatured in SDS sample buffer, separated using 14% SDS- PAGE. The gels were washed, ﬂuorographed and autoradiographed for 25 days at -70 C. Note the highest labelling of the 28 kDa polypeptide in the particulate fraction 1753 cluster together. These species lack the classical C-terminal KDEL ER retention sequence that is found in all other land plants, neither do the moss sequences encode a derived ER retention signal [Prosite pattern PS00014, according to Andres et al. (1990)] (Fig. 8). The sequences for the ABP1 proteins from S. moellendorfﬁi, as well as one from P. tremula (Poptr_ABP1–2) and from G. max are likely to be fragmentary which might explain why they do not depict the KDEL sequence. Discussion From an evolutionary viewpoint bryophytes (mosses and liverworts) are considered to be the earliest known land plants (Qiu et al. 1998, 1999; Lang et al. 2008). The study of hormone signalling in these organisms might hence be gaining attention in the scientiﬁc community as research on them might be the key to understand the possible origin of a primitive auxin sensing machinery (Johri 2004; Paponov et al. 2009). Protonema cells of Funaria were chosen in this study because they are highly responsive to physio- logically relevant concentrations of exogenously applied auxin. In moss both developmental steps, chloronema proliferation and the differentiation to caulonema are under the regulation of auxin. Hence, as part of these processes basipetal transport as well as auxin-binding sites might be involved (Reski 1998). The present investigation of ABPs in Funaria using microsomal membrane fractions of this moss showed the presence of speciﬁc auxin-binding sites. The derived Km of IAA to its binding sites in Funaria and corn was comparable being in a physiological range of 3– 5 lM (Hertel et al. 1972). There are several ABPs described in different plants. The classical ER localised ABP1 of corn has drawn far more attention over the years. A small fraction of ABP1 (ca. *2%) is localised to the plasma membrane where it has been shown to function in one of the early effects of auxin, i.e. hyper-polarisation of the membrane (Henderson et al. 1997; Barbier-Brygoo et al. 1989). Several lines of evidence is accumulating that suggest ABP1 might func- tion in the plasma membrane and control a variety of events such as cell polarity, cell cycle, ion channels and sense cytoplasmic auxin gradients. These assign ABP1 as a possible candidate for a bonaﬁde auxin receptor (Kramer 2009). Among mosses, the C. purpureus ABP1 homologue possesses most of the conserved domains, however, it lacks the C-terminal ER retention sequence (Napier et al. 2002). The analysis of sequence data from the moss P. patens also leads to the identiﬁcation of an ABP1 (Phypa_151546) although it does also not contain any ER retention sequence as well. The use of standard computational prediction 123