Auxin binding protein 1 (ABP1) is not required for either auxin signaling or Arabidopsis development

Abstract

Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.

Keywords: ABP1; CRISPR; auxin; plant development; receptor.

Fig. 1.

Generation of a null allele of abp1 mutant using the ribozyme-based CRISPR gene editing technology. (A) A schematic description of the CRISPR construct that contains a Cas9 expression cassette and a CaMV 35S promoter controlled gRNA production unit. (B) A 5-bp deletion was detected in genomic DNA of abp1-c1 mutants. The intron sequences are in lowercase and in red. (C) The abp1-c1 cDNA also contained the same 5-bp deletion. (D) There was no detectable ABP1 protein in abp1-c1 as shown in this Western blot image.

Fig. 2.

The abp1-c1 and WT plants display no significant differences at various developmental stages. (A) Seven-day-old seedlings on regular MS plates. (B and C) Hypocotyl length and root length of 7-d-old WT and abp1-c1 seedlings. Shown are average ± SD (n = 50). (D and E) Root cell shape of 7-d-old WT and abp1-c1. (F, G, and I) Phenotype of WT and abp1-c1 at juvenile stage (F), floral transition stage (G), and mature plant stage (I). (H) Flowers and floral organs of WT and abp1-c1.

Fig. 3.

Pavement cell development in abp1-c1 and WT. Confocal images of cotyledon pavement cells of WT (A and C) and abp1-c1 (B and D) with auxin (C and D) and without auxin (A and B) treatments. Five-day-old light-grown seedlings were transferred to MS plates with or without 25 nM NAA for 2 d. Samples were treated with 5 μg/mL FM1-43 (Life Technologies; F-35355) for 30 min before confocal imaging. (E) Quantification of pavement cell lobes. One hundred fifty cells for each treatment and each genotype were quantified. Images were gridded to 25 of 20,000 μm² squares by using ImageJ before counting. Error bars are SD.

Fig. 4.

Effects of auxin treatments on abp1-c1 root elongation. Quantification of root elongation of WT and abp1-c1 with various concentrations of IAA (A) or NAA (B) for 2 d. Shown are average ± SD (n = 50).

Fig. 5.

AUX/IAA transcripts abundance in abp1-c1 with NAA treatments. Light grown, 7-d-old seedlings were treated with or without 1 μM NAA for 2 h and were collected for RNA extraction. For each genotype and treatment, five biological replicates were performed. Expression of IAA3, IAA7, IAA14, and IAA17 with reduced y axis are shown as inset. Error bars are SD.

Fig. 6.

Identification of a T-DNA insertion null allele of abp1. (A) Schematic presentation of the T-DNA insertion site in abp1-TD1. The T-DNA insertion is 27 bp downstream of ATG start codon of the first exon. (B) abp1-TD1 is viable and does not have obvious developmental defects. (C) abp1-TD1 is fertile and similar to WT in size. (D) RT-PCR results indicate that abp1-TD1 plants do not produce ABP1 mRNA. The A5P and A3P pair amplifies the full length ABP1 cDNA from the start codon to the stop codon. The A2E and A3P primers amplify the ABP1 cDNA that does not contain the sequences of the first exon. The positions of the PCR primers are schematically indicated in the panel A. The RT-PCR products were amplified with 45 saturated cycles and loaded onto 1.2% agarose gel. (E) A Western blot image indicates that abp1-TD1 lacked ABP1 protein. The band between 25 and 20 kDa in WT lane is ABP1, which has a predicted size of 22 kDa.