1756 storage mechanism in order to ensure a continuous supply of this to the plasma membrane. This suggestion is sup- ported by the observation that ABP1 does not bind auxin in the lumen of the ER despite this being the predominant sub-cellular location (Tian et al. 1995). It is also interesting in this respect to note that two distinct signalling pathways of auxin action have been described in the epidermal cells of pea (Pisum sativum): the ABP1-dependent pathway, which has higher sensitivity to NAA and is independent of extracellular Ca2? and the ABP1-independent pathway, which shows higher sensi- tivity to IAA and is dependent on extracellular Ca2? (Yamagami et al. 2004). Since Funaria responds to IAA much better than NAA and grows better in calcium rich soil, it is tempting to suggest that the ABP1-independent mechanism seems to have evolved earlier than the ABP1- dependent one. The plasma membrane bound ABPs have been found in plants earlier with the ones of Zucchini displaying a molecular mass of 40–42 kDa (Hicks et al. 1993) and showing to be photo-afﬁnity labelled by azido- auxin (Hicks et al. 1989). Therefore, the putative ABPs detected in the microsomal fraction appear to be associated with the plasma membrane (Fig. 4b, d). However, more work, like in vivo targeting of GFP-fusion proteins (Schaaf et al. 2004), is required to establish their precise localisa- tion as it is also conceivable that plants have variable amounts of classical ABP1 proteins. The lack of detection of the classical 22 kDa ABP1 protein with an ER retention signal in Funaria does not appear to be a result of lower expression or abundance. Even after loading about 15–20 times more protein from moss than from corn, and even after several fold enrichment by subcellular fractionation, a polypeptide comparable to ABP1 was not detectable in our recent study. The 28 kDa polypeptide detected in the P3 sub-cellular fraction of Funaria (Fig. 3 marked with an arrow) could be most likely the result of protein degrada- tion speciﬁc to P3 fraction. In summary, evidence is provided here for the presence of additional immunologically related ABP1 homologues in the moss Funaria that do not show the presence of the classical ER retention KDEL signal sequence. As sequence comparisons with other mosses conﬁrm the lack of the retention signal in the ABP1 of mosses it is postulated that such a signal might have evolved during later stages of evolution in land plants, a feature that has recently been described for nuclear auxin signalling (Paponov et al. 2009) and for gibberellin signalling (Vandenbussche et al. 2007). Acknowledgments We thank Prof. Mike A. Venis and Dr. Richard M. Napier, HRI, UK for providing the anti-ABP1, anti-KDEL and anti-HDEL antibodies. Additionally, acknowledge Dr. Karen Hicks and Dr. Amaury de Montaigu for discussions on the results. More- over, we are thankful to Dr. Jacinta S. D’Souza for maintaining the 123 Plant Cell Rep (2009) 28:1747–1758 cell line and Dr. Mark von Stackelberg for providing some moss F. hygrometrica. Special thanks to Prof. Eberhard Schaefer and Prof. George Coupland for their continued support. This research was supported by Deutsche Forschungsgemeinschaft (grant Re837/10) and by the Excellence Initiative of the German Federal and State Gov- ernments (EXC 294). Substantial work for this publication was car- ried out by K. P. as graduate student in the Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 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