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Auxin Binding Protein 1: A Red Herring After All?

2015; Elsevier BV; Volume: 8; Issue: 8 Linguagem: Inglês

10.1016/j.molp.2015.04.010

1674-2052

Myckel E. J. Habets, Remko Offringa,

Plant nutrient uptake and metabolism

The natural auxin indole-3-acetic acid is the first hormone identified in plants, and since it plays such a central role in plant growth and development, auxin has been the subject of intensive studies. A central question has been how the auxin signal is perceived by plant cells. The earliest experiments showed the presence of auxin binding particles at the plasma membrane (PM) and in the endoplasmic reticulum (ER) (Hertel et al., 1972Hertel R. Thomson K.-S. Russo V.E.A. In-vitro auxin binding to particulate cell fractions from corn coleoptiles.Planta. 1972; 107: 325-340Crossref PubMed Scopus (201) Google Scholar). Screens for PM-localized auxin binding activities have led to the photo-affinity labeling and purification of Auxin Binding Protein 1 (ABP1) from maize coleoptile cells (Löbler and Klämbt, 1985Löbler M. Klämbt D. Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization.J. Biol. Chem. 1985; 260: 9848-9853PubMed Google Scholar). Despite observations in different laboratories that ABP1 localized to the PM where it seemed to mediate rapid electrophysiological and cell physiological responses to auxin, the auxin community remained skeptical about the role of ABP1 as auxin receptor for a long time, in part because of its predominant localization in the ER (reviewed by Napier et al., 2002Napier R. David K. Perrot-Rechenmann C. A short history of auxin-binding proteins.Plant Mol. Biol. 2002; 49: 339-348Crossref PubMed Scopus (136) Google Scholar). At some point, ABP1 was even jokingly referred to as a potential red herring in the search for the auxin receptor (Venis, 1995Venis M.A. Auxin binding protein 1 is a red herring? Oh no it isn't!.J. Exp. Bot. 1995; 46: 463-465Crossref Scopus (24) Google Scholar). However, after the first Arabidopsis abp1-1 loss-of-function allele pointed to a key role for ABP1 in cell elongation and division, the auxin community has adopted this abundantly expressed 22-kDa protein as extracellular auxin receptor (reviewed by Napier et al., 2002Napier R. David K. Perrot-Rechenmann C. A short history of auxin-binding proteins.Plant Mol. Biol. 2002; 49: 339-348Crossref PubMed Scopus (136) Google Scholar). Especially in recent years, the role of ABP1 in development has become more firmly established, in part as modulator of clathrin-mediated endocytosis and microtubule orientation through its action on the Rho of Plants (ROP) family of GTPases (Robert et al., 2010Robert S. Kleine-Vehn J. Barbez E. Sauer M. Paciorek T. Baster P. Vanneste S. Zhang J. Simon S. Čovanová M. et al.ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis.Cell. 2010; 143: 111-121Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, Chen et al., 2012Chen X. Naramoto S. Robert S. Tejos R. Löfke C. Lin D. Yang Z. Friml J. ABP1 and ROP6 GTPase signaling regulate clathrin-mediated endocytosis in Arabidopsis roots.Curr. Biol. 2012; 22: 1326-1332Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, Chen et al., 2014Chen X. Grandont L. Li H. Hauschild R. Paque S. Abuzeineh A. Rakusova H. Benkova E. Perrot-Rechenmann C. Friml J. Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules.Nature. 2014; 516: 90-93Crossref PubMed Scopus (65) Google Scholar) but also as regulator of auxin-responsive gene expression (Tromas et al., 2013Tromas A. Paque S. Stierlé V. Quettier A.-L. Muller P. Lechner E. Genschik P. Perrot-Rechenmann C. Auxin-Binding Protein 1 is a negative regulator of the SCFTIR1/AFB pathway.Nat. Commun. 2013; 4: 2496Crossref PubMed Scopus (60) Google Scholar). Recent evidence that auxin-bound ABP1 docks on the extracellular domain of the TRANSMEMBRANE KINASE1 (TMK1) finally linked its apoplastic localization to signaling by the PM-associated ROPs. TMK1 belongs to a small subfamily of four leucine-rich-repeat receptor-like kinases and the quadruple tmk1234 loss-of-function mutant shows several auxin-related phenotypes (Dai et al., 2013Dai N. Wang W. Patterson S.E. Bleecker A.B. The TMK subfamily of receptor-like kinases in Arabidopsis display an essential role in growth and a reduced sensitivity to auxin.PLoS One. 2013; 8: e60990Crossref PubMed Scopus (68) Google Scholar, Xu et al., 2014Xu T. Dai N. Chen J. Nagawa S. Cao M. Li H. Zhou Z. Chen X. De Rycke R. Rakusová H. et al.Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling.Science. 2014; 343: 1025-1028Crossref PubMed Scopus (205) Google Scholar). In addition, auxin-mediated activation of ROP2 and ROP6 and the downstream effects on the actin and microtubule cytoskeleton, respectively, are largely abolished in this mutant (Xu et al., 2014Xu T. Dai N. Chen J. Nagawa S. Cao M. Li H. Zhou Z. Chen X. De Rycke R. Rakusová H. et al.Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling.Science. 2014; 343: 1025-1028Crossref PubMed Scopus (205) Google Scholar, Grones and Friml, 2015Grones P. Friml J. Auxin transporters and binding proteins at a glance.J. Cell Sci. 2015; 128: 1-7Crossref PubMed Scopus (95) Google Scholar). The strong defects observed for the Arabidopsis abp1-1null allele, which were seemingly confirmed by the later identified abp1-1s allele (Table 1), have considerably hampered ABP1 research. In the homozygous state, abp1-1 causes arrest of cell division, thereby blocking embryogenesis at the globular stage (Chen et al., 2001Chen J.-G. Ullah H. Young J.C. Sussman M.R. Jones A.M. ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis.Genes Dev. 2001; 15: 902-911Crossref PubMed Scopus (248) Google Scholar). In the heterozygous state, various weaker auxin-related defects have been reported, such as altered gravitropic and phototropic responses, changes in hypocotyl length, and changes in expression of early auxin-induced genes (Effendi et al., 2011Effendi Y. Rietz S. Fischer U. Scherer G.F.E. The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin-regulated genes.Plant J. 2011; 65: 282-294Crossref PubMed Scopus (57) Google Scholar). The strong phenotype of the abp1-1 allele has triggered the isolation of a weaker allele (abp1-5) with a point mutation in the auxin binding pocket, and the generation of knockdown lines by the inducible expression of either antisense ABP1 RNA or antibodies directed against ABP1 (Table 1). In a recent publication, ABP1 mutant versions with amino acid substitutions in the auxin binding pocket were expressed in the abp1-1 background (Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar). A central aspect of all these mutant lines is that they show a weak reduction in auxin sensitivity similar to heterozygous abp1-1 mutant plants (Effendi et al., 2011Effendi Y. Rietz S. Fischer U. Scherer G.F.E. The heterozygous abp1/ABP1 insertional mutant has defects in functions requiring polar auxin transport and in regulation of early auxin-regulated genes.Plant J. 2011; 65: 282-294Crossref PubMed Scopus (57) Google Scholar). Interestingly, over expression of an ABP1 deletion version lacking the KDEL ER-retention signal also led to auxin-related phenotypes but frequently also to more severe phenotypes such as seedling lethality or sterile development (Robert et al., 2010Robert S. Kleine-Vehn J. Barbez E. Sauer M. Paciorek T. Baster P. Vanneste S. Zhang J. Simon S. Čovanová M. et al.ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis.Cell. 2010; 143: 111-121Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar).Table 1abp1 Loss-of-Function Alleles and ABP1 Overexpression or Inducible Knockdown Lines.AlleleTypeDescriptionPhenotypesReferenceabp1-c15 bp deletionCRISPR/CAS generated 5 bp deletion 107 bp downstream from ATGWild-type(Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar)abp1-TD1T-DNA insertT-DNA insert 27 bp downstream from ATGWild-type(Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar)abp1-1T-DNA insertT-DNA insert 51 bp downstream from ATGEmbryo lethal(Chen et al., 2001Chen J.-G. Ullah H. Young J.C. Sussman M.R. Jones A.M. ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis.Genes Dev. 2001; 15: 902-911Crossref PubMed Scopus (248) Google Scholar)abp1-s1T-DNA insertT-DNA insert in the 5′ UTR of BSM/RUG2Embryo lethal(Tzafrir et al., 2004Tzafrir I. Pena-Muralla R. Dickerman A. Berg M. Rogers R. Hutchens S. Sweeney T.C. McElver J. Aux G. Patton D. et al.Identification of genes required for embryo development in Arabidopsis.Plant Physiol. 2004; 135: 1206-1220Crossref PubMed Scopus (394) Google Scholar)abp1-5Point mutationTILLING selected point mutant: substitution in the auxin binding pocketPavement cell (PC) defects, auxin insensitive(Xu et al., 2010Xu T. Wen M. Nagawa S. Fu Y. Chen J.-G. Wu M.-J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar)SS12SKnockdownInducible antibody against tobacco ABP1Cotyledon defects, growth delay/arrest, sterility(Braun et al., 2008Braun N. Wyrzykowska J. Muller P. David K. Couch D. Perrot-Rechenmann C. Fleming A.J. Conditional repression of AUXIN BINDING PROTEIN1 reveals that it coordinates cell division and cell expansion during postembryonic shoot development in Arabidopsis and tobacco.Plant Cell. 2008; 20: 2746-2762Crossref PubMed Scopus (141) Google Scholar, Tromas et al., 2009Tromas A. Braun N. Muller P. Khodus T. Paponov I.A. Palme K. Ljung K. Lee J.-Y. Benfey P. Murray J.A.H. et al.The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.PLoS One. 2009; 4: e6648Crossref PubMed Scopus (107) Google Scholar)SS12KKnockdownInducible antibody against tobacco ABP1Cotyledon defects, growth delay/arrest, sterility, reduced auxin sensitivity(Braun et al., 2008Braun N. Wyrzykowska J. Muller P. David K. Couch D. Perrot-Rechenmann C. Fleming A.J. Conditional repression of AUXIN BINDING PROTEIN1 reveals that it coordinates cell division and cell expansion during postembryonic shoot development in Arabidopsis and tobacco.Plant Cell. 2008; 20: 2746-2762Crossref PubMed Scopus (141) Google Scholar, Tromas et al., 2009Tromas A. Braun N. Muller P. Khodus T. Paponov I.A. Palme K. Ljung K. Lee J.-Y. Benfey P. Murray J.A.H. et al.The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.PLoS One. 2009; 4: e6648Crossref PubMed Scopus (107) Google Scholar, Tromas et al., 2013Tromas A. Paque S. Stierlé V. Quettier A.-L. Muller P. Lechner E. Genschik P. Perrot-Rechenmann C. Auxin-Binding Protein 1 is a negative regulator of the SCFTIR1/AFB pathway.Nat. Commun. 2013; 4: 2496Crossref PubMed Scopus (60) Google Scholar)ABP1ASKnockdownInducible ABP1 antisense RNACotyledon defects, growth delay/arrest, PC defects, auxin insensitivity(Braun et al., 2008Braun N. Wyrzykowska J. Muller P. David K. Couch D. Perrot-Rechenmann C. Fleming A.J. Conditional repression of AUXIN BINDING PROTEIN1 reveals that it coordinates cell division and cell expansion during postembryonic shoot development in Arabidopsis and tobacco.Plant Cell. 2008; 20: 2746-2762Crossref PubMed Scopus (141) Google Scholar, Tromas et al., 2009Tromas A. Braun N. Muller P. Khodus T. Paponov I.A. Palme K. Ljung K. Lee J.-Y. Benfey P. Murray J.A.H. et al.The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.PLoS One. 2009; 4: e6648Crossref PubMed Scopus (107) Google Scholar, Xu et al., 2010Xu T. Wen M. Nagawa S. Fu Y. Chen J.-G. Wu M.-J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar)ABP1ΔKDEL-GFPOverexpressionOverexpression of ABP1-GFP fusion lacking the KDEL domainReduced auxin sensitivity, seedling lethality, sterility(Robert et al., 2010Robert S. Kleine-Vehn J. Barbez E. Sauer M. Paciorek T. Baster P. Vanneste S. Zhang J. Simon S. Čovanová M. et al.ABP1 mediates auxin inhibition of clathrin-dependent endocytosis in Arabidopsis.Cell. 2010; 143: 111-121Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar)XVE >> ABP1 OEOverexpressionEstradiol-inducible overexpression of ABP1-GFPEnhanced auxin-induced microtubule re-orientation(Chen et al., 2014Chen X. Grandont L. Li H. Hauschild R. Paque S. Abuzeineh A. Rakusova H. Benkova E. Perrot-Rechenmann C. Friml J. Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules.Nature. 2014; 516: 90-93Crossref PubMed Scopus (65) Google Scholar)abp1-8Overexpressionabp1-1 overexpressing tagged ABP1 with substitution in auxin binding pocketReduced auxin sensitivity, PC defects(Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar)abp1-9Overexpressionabp1-1 overexpressing tagged ABP1 with substitution in auxin binding pocketReduced auxin sensitivity, PC defects, reduced auxin transport(Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar)abp1-10Overexpressionabp1-1 overexpressing tagged ABP1 with substitution in auxin binding pocketReduced auxin sensitivity, PC defects, reduced auxin transport(Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar)abp1-11Overexpressionabp1-1 overexpressing tagged ABP1Near wild-type phenotypes, reduced auxin transport(Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar) Open table in a new tab In an attempt to study the role of ABP1 in flower development, Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar designed an elegant CRISPR-CAS-based strategy to obtain mutant lines that become homozygous for an abp1 null mutation at the onset of flower development. For this purpose, the ABP1 gene-specific guide RNA was expressed under the constitutive 35S promoter and the CAS9 endonuclease was expressed under the APETALA 1 promoter. To their surprise, the authors did not obtain T1 plants with mutant phenotypes, and when they recovered a T2 plant homozygous for a 5 base pair (bp) deletion in the first exon (named abp1-c1), this plant also showed a wild-type appearance. Sequencing of RT-PCR-derived ABP1 cDNA from this plant line confirmed that the 5 bp deletion is present in mRNA transcripts and causes a frame shift generating a premature stop codon. Western blot analysis using anti-ABP1 antibodies showed that the ABP1 protein is not detectably expressed and that abp1-c1 is likely a null allele. To confirm their results, the authors obtained a T-DNA insertion line from the Arabidopsis stock center. RT–PCR and Western blot analysis indicated that this mutant allele (abp1-TD) is also a null mutant with the same wild-type appearance as the abp1-c1 allele. This led the authors to conclude that ABP1 is not required in plant development, at least not under the growth conditions tested. The article by Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar presents the auxin community with a dilemma. Do we trust the data accumulated by many different laboratories during 40 years of ABP1 research or do we accept the rather convincing evidence presented by Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar that ABP1 is not important for plant development? There are several aspects that should be considered before drawing a final conclusion. First, the analysis performed by Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar makes it very likely that the new mutants represent null alleles but it does not fully exclude that the mutant alleles produce a low level of functional ABP1, undetectable on Western blot, but sufficient to obtain a wild-type phenotype. The 5 bp deletion in the abp1-c1 allele is close to the first intron and a small part of the mutant transcripts could be rescued by alternative splicing, which has been shown to occur for the ABP1 gene (Wang and Brendel, 2006Wang B.-B. Brendel V. Genomewide comparative analysis of alternative splicing in plants.Proc. Natl. Acad. Sci. USA. 2006; 103: 7175-7180Crossref PubMed Scopus (425) Google Scholar), e.g. by using a possible cryptic splice acceptor site a few base pairs upstream of the mutation (AGGA). It would therefore be interesting to know if more T2 lines with larger deletions in the ABP1 gene were rescued from the CRISPR-CAS approach. Moreover, the abp1-TD allele has an activation tag T-DNA, containing four tandem 35S promoters on the right border (Robinson et al., 2009Robinson S.J. Tang L.H. Mooney B.A.G. McKay S.J. Clarke W.E. Links M.G. Karcz S. Regan S. Wu Y.-Y. Gruber M.Y. et al.An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches.BMC Plant Biol. 2009; 9: 101Crossref PubMed Scopus (52) Google Scholar), inserted close to the translation start of the ABP1 gene. While RT–PCR and Western blot analysis exclude that ABP1 is detectably produced in this line, it is still possible that a truncated transcript is produced that leads to low-level expression of a functional ABP1 protein. For both new alleles, the mutation is located in the region coding for the signal peptide, which does not require strong conservation (Martoglio and Dobberstein, 1998Martoglio B. Dobberstein B. Signal sequences: more than just greasy peptides.Trends Cell Biol. 1998; 8: 410-415Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar, Napier et al., 2002Napier R. David K. Perrot-Rechenmann C. A short history of auxin-binding proteins.Plant Mol. Biol. 2002; 49: 339-348Crossref PubMed Scopus (136) Google Scholar). Mutant ABP1 versions with a few amino acid deletions or substitutions in their signal peptide are therefore likely to be functional. We have to note here that this is an extremely unlikely scenario. However, if this scenario is true, this would still imply that the phenotypes observed for the ABP1AS antisense line (Braun et al., 2008Braun N. Wyrzykowska J. Muller P. David K. Couch D. Perrot-Rechenmann C. Fleming A.J. Conditional repression of AUXIN BINDING PROTEIN1 reveals that it coordinates cell division and cell expansion during postembryonic shoot development in Arabidopsis and tobacco.Plant Cell. 2008; 20: 2746-2762Crossref PubMed Scopus (141) Google Scholar, Tromas et al., 2009Tromas A. Braun N. Muller P. Khodus T. Paponov I.A. Palme K. Ljung K. Lee J.-Y. Benfey P. Murray J.A.H. et al.The AUXIN BINDING PROTEIN 1 is required for differential auxin responses mediating root growth.PLoS One. 2009; 4: e6648Crossref PubMed Scopus (107) Google Scholar, Xu et al., 2010Xu T. Wen M. Nagawa S. Fu Y. Chen J.-G. Wu M.-J. Perrot-Rechenmann C. Friml J. Jones A.M. Yang Z. Cell surface- and Rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis.Cell. 2010; 143: 99-110Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) are not caused by the reduced, but still detectable, ABP1 expression. Second, it would be good to analyze the different abp1 mutant alleles (including abp1-5 and abp1-1 and abp1-1S) by genome sequencing to know the exact nature of the mutations and to exclude the occurrence of gene duplications or second site mutations. In the most likely situation that the abp1-c1 and abp1-TD alleles are true null mutants, the strong phenotypes of the abp1-1 and abp1-1s alleles could be explained by a second site mutation in another gene. In fact, the T-DNA insertion in the embryo lethal abp1-1s allele is located in the 5′ untranslated region of the inversely oriented BELAYA SMERT/RUGOSA2 (BSM/RUG2) gene located upstream of ABP1 (Babiychuk et al., 2011Babiychuk E. Vandepoele K. Wissing J. Garcia-Diaz M. De Rycke R. Akbari H. Joubès J. Beeckman T. Jänsch L. Frentzen M. et al.Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family.Proc. Natl. Acad. Sci. USA. 2011; 108: 6674-6679Crossref PubMed Scopus (109) Google Scholar, Quesada et al., 2011Quesada V. Sarmiento-Mañús R. González-Bayón R. Hricová A. Pérez-Marcos R. Graciá-Martínez E. Medina-Ruiz L. Leyva-Díaz E. Ponce M.R. Micol J.L. Arabidopsis RUGOSA2 encodes an mTERF family member required for mitochondrion, chloroplast and leaf development.Plant J. 2011; 68: 738-753Crossref PubMed Scopus (62) Google Scholar). Interestingly, the bsm mutant allele shows embryo arrest at the late globular stage (Babiychuk et al., 2011Babiychuk E. Vandepoele K. Wissing J. Garcia-Diaz M. De Rycke R. Akbari H. Joubès J. Beeckman T. Jänsch L. Frentzen M. et al.Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family.Proc. Natl. Acad. Sci. USA. 2011; 108: 6674-6679Crossref PubMed Scopus (109) Google Scholar) and the fact that the BSM/RUG2 promoter region partly overlaps with the ABP1 coding region suggests that the embryo lethality observed for abp1-1 and abp1-1s might be caused by disruption of the BSM/RUG2 promoter function, which for the abp1-TD allele might be overcome by the presence of the 35S enhancer sequences on the activation tag T-DNA. In any case, it will be essential to reevaluate the abp1-1 complementation experiments presented in previous publications (Chen et al., 2001Chen J.-G. Ullah H. Young J.C. Sussman M.R. Jones A.M. ABP1 is required for organized cell elongation and division in Arabidopsis embryogenesis.Genes Dev. 2001; 15: 902-911Crossref PubMed Scopus (248) Google Scholar, Effendi et al., 2015Effendi Y. Ferro N. Labusch C. Geisler M. Scherer G.F.E. Complementation of the embryo-lethal T-DNA insertion mutant of AUXIN-BINDING-PROTEIN 1 (ABP1) with abp1 point mutated versions reveals crosstalk of ABP1 and phytochromes.J. Exp. Bot. 2015; 66: 403-418Crossref PubMed Scopus (9) Google Scholar). For the phenotypes observed in the ABP1 antisense or antibody lines Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar suggested that they could be caused by off target knockdown of other genes. It is important to note here that these off target genes could still encode redundantly acting, yet unidentified auxin receptors that may compensate for the loss of ABP1 in the abp1-c and abp1-TD alleles. The publication by Gao et al., 2015Gao Y. Zhang Y. Zhang D. Dai X. Estelle M. Zhao Y. Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development.Proc. Natl. Acad. Sci. USA. 2015; 112: 2275-2280Crossref PubMed Scopus (245) Google Scholar provides food for thought. Can plant life proceed without a PM-localized auxin receptor? If not ABP1, are there other (ABP1-related) auxin binding proteins at the PM that (by interacting with the TMKs) mediate the previously observed rapid cellular responses to auxin, such as elevated cytosolic calcium levels, changes in pH, or ROP-dependent changes in cytoskeleton localization or orientation (Napier et al., 2002Napier R. David K. Perrot-Rechenmann C. A short history of auxin-binding proteins.Plant Mol. Biol. 2002; 49: 339-348Crossref PubMed Scopus (136) Google Scholar, Shishova and Lindberg, 2010Shishova M. Lindberg S. A new perspective on auxin perception.J. Plant Physiol. 2010; 167: 417-422Crossref PubMed Scopus (31) Google Scholar, Monshausen et al., 2011Monshausen G.B. Miller N.D. Murphy A.S. Gilroy S. Dynamics of auxin-dependent Ca2+ and pH signaling in root growth revealed by integrating high-resolution imaging with automated computer vision-based analysis.Plant J. 2011; 65: 309-318Crossref PubMed Scopus (168) Google Scholar, Chen et al., 2014Chen X. Grandont L. Li H. Hauschild R. Paque S. Abuzeineh A. Rakusova H. Benkova E. Perrot-Rechenmann C. Friml J. Inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules.Nature. 2014; 516: 90-93Crossref PubMed Scopus (65) Google Scholar, Xu et al., 2014Xu T. Dai N. Chen J. Nagawa S. Cao M. Li H. Zhou Z. Chen X. De Rycke R. Rakusová H. et al.Cell surface ABP1-TMK auxin-sensing complex activates ROP GTPase signaling.Science. 2014; 343: 1025-1028Crossref PubMed Scopus (205) Google Scholar)? It is still too early to rewrite the text books, as one can be sure that several laboratories are currently investigating whether ABP1 has been a red herring after all or not. It has been suggested to "re-examine previous data, down to the lab bench level" (Liu, 2015Liu C.-M. Auxin Binding Protein 1 (ABP1): a matter of fact.J. Integr. Plant Biol. 2015; 57: 234-235Crossref PubMed Scopus (13) Google Scholar). In our opinion, the most important issue is to unequivocally determine which of the reported abp1 alleles are true nulls and whether there are undetected off-site mutations or unexpected effects of the known mutations that explain the observed differences between the earlier "reference" alleles and the new abp1 alleles that show wild-type phenotypes. M.E.J.H. was supported by the Chemical Sciences Division of the Netherlands Organization for Scientific Research (NWO-CW TOP 700.58.301 to R.O.).