Genome Sequencing of Arabidopsis abp1-5 Reveals Second-Site Mutations That May Affect Phenotypes

Abstract

Auxin regulates numerous aspects of plant growth and development. For many years, investigating roles for AUXIN BINDING PROTEIN1 (ABP1) in auxin response was impeded by the reported embryo lethality of mutants defective in ABP1. However, identification of a viable Arabidopsis thaliana TILLING mutant defective in the ABP1 auxin binding pocket (abp1-5) allowed inroads into understanding ABP1 function. During our own studies with abp1-5, we observed growth phenotypes segregating independently of the ABP1 lesion, leading us to sequence the genome of the abp1-5 line described previously. We found that the abp1-5 line we sequenced contains over 8000 single nucleotide polymorphisms in addition to the ABP1 mutation and that at least some of these mutations may originate from the Arabidopsis Wassilewskija accession. Furthermore, a phyB null allele in the abp1-5 background is likely causative for the long hypocotyl phenotype previously attributed to disrupted ABP1 function. Our findings complicate the interpretation of abp1-5 phenotypes for which no complementation test was conducted. Our findings on abp1-5 also provide a cautionary tale illustrating the need to use multiple alleles or complementation lines when attributing roles to a gene product. INTRODUCTIONThe plant hormone auxin regulates cell division and cell expansion to affect all aspects of plant growth (reviewed in PerrotRechenmann, 2010). Auxin regulates a wide range of developmental processes, and tight control of auxin response is maintained by multiple modes of regulation. These include regulating auxin biosynthesis and metabolism, transport, and signaling (reviewed in Enders and Strader, 2015). To date, nuclear auxin signaling components have been well characterized (reviewed in Chapman and Estelle, 2009;Salehin et al., 2015). In addition, a nontranscriptional auxin response pathway has been proposed, with AUXIN BINDING PROTEIN1 (ABP1) acting in the apoplast as an auxin receptor and transmitting a cytoplasmic signal to regulate auxin responses such as auxin transport and cytoskeletal rearrangements (reviewed in Shi and Yang, 2011;Sauer et al., 2013).Study of ABP1 has a long, complicated history. Experiments demonstrating auxin binding activity for ABP1 were published as early as the 1980s (reviewed in Jones, 1994). Reverse genetics proved to be a complicated approach to study ABP1 function in Arabidopsis thaliana, as two independently generated abp1 mutants, presumed to be null alleles, appeared to be embryo lethal (Chen et al., 2001;Tzafrir et al., 2004;Meinke et al., 2008;Sassi et al., 2014). Knockdown lines of ABP1 provided some ability to study ABP1 function by reverse genetics. Arabidopsis lines expressing an inducible ABP1 antisense transcript or an inducible single-chain fragment variable scFv12 (David et al., 2007) from a monoclonal antibody raised to ABP1 (Leblanc et al., 1999), targeted to either the apoplast (SS12S) or the endoplasmic reticulum (SS12K), led to phenotypes consistent with decreased auxin activity (Braun et al., 2008;Tromas et al.,...

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis

“…Surprisingly, two null Arabidopsis abp1 mutants generated by the authors using ribozyme-based CRISPR and T-DNA insertion technologies do not show phenotype defects described in the case of other knockdown/knockout abp1 alleles. In addition, it has been demonstrated that phenotypes observed in the Arabidopsis abp1-1 and abp1-5 alleles might not be caused by the disruption of ABP1 Enders et al, 2015). These findings call into question the results of previous publications describing a prominent role of ABP1 in embryogenesis, growth, cell division, cell expansion, and auxin signaling [see Liu (2015) and Strader and Zhao (2016) for a commentary].…”

Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis

“…This model, however, does not preclude a role for other factors that may be required for the auxin regulation of clathrin function during CME. Indeed, the recent finding that abp1 mutants show no gross growth defects (Enders et al, 2015;Gao et al, 2015) warrants further study of the role of ABP1 in the auxin-dependent regulation of CME. Recent studies showing that SA inhibition of plant CME is independent of SA receptor-mediated transcription (NONEXPRESSER OF PR GENES3/4; Du et al, 2013) support the SA inhibition of CME via an untranscriptional mechanism.…”

On growth and formins

“…14,16 However, genuine null mutations in this gene have normal pavement cells, 19 and the abp1-5 line carries numerous additional mutations outside the ABP1 locus, obviously contributing to its phenotype. 20 Nevertheless, pavement cell lobing is also altered in auxin biosynthesis mutants (reviewed in ref. 14), and the ROP-dependent pathway, mediated by ICR1 and the exocyst complex subunit SEC3, acts at least in part through modulation of membrane trafficking, 12 which important not only for cell expansion (see ref.…”

“…27 Intriguingly, the Arabidopsis abp1-5 line carries several mutations inactivating the FH17 gene, encoding a yet uncharacterized Class II formin. 20 It remains to be seen if this locus contributes to the abp1-5 pavement cell lobing defect that was originally attributed to impairment of auxin signaling.…”