BIG: a calossin-like protein required for polar auxin transport in Arabidopsis

Abstract

Polar auxin transport is crucial for the regulation of auxin action and required for some light-regulated responses during plant development. We have found that two mutants of Arabidopsis-doc1, which displays altered expression of light-regulated genes, and tir3, known for its reduced auxin transport-have similar defects and define mutations in a single gene that we have renamed BIG. BIG is very similar to the Drosophila gene Calossin/Pushover, a member of a gene family also present in Caenorhabditis elegans and human genomes. The protein encoded by BIG is extraordinary in size, 560 kD, and contains several putative Zn-finger domains. Expression-profiling experiments indicate that altered expression of multiple light-regulated genes in doc1 mutants can be suppressed by elevated levels of auxin caused by overexpression of an auxin biosynthetic gene, suggesting that normal auxin distribution is required to maintain low-level expression of these genes in the dark. Double mutants of tir3 with the auxin mutants pin1, pid, and axr1 display severe defects in auxin-dependent growth of the inflorescence. Chemical inhibitors of auxin transport change the intracellular localization of the auxin efflux carrier PIN1 in doc1/tir3 mutants, supporting the idea that BIG is required for normal auxin efflux.

Figure 1

(A) CAB expression is increased in dark-grown tir3-1 seedlings. Five micrograms of total RNA from 5-day-old dark-grown seedlings was probed with a CAB2 cDNA. To confirm equal loading, the blot was stripped and re-probed with an 18S rRNA gene. (B) doc1-1 inflorescence stems are deficient in polar auxin transport. Twenty-five millimeters of stem was excised, and the apical end was placed in nutrient solution containing 1 μM ¹⁴C-IAA ± NPA for 18 h. The basal end of the inverted segments was placed in nutrient solution where indicated. The amount of radioactive IAA (CPM) transported to the basal end of the stem was assayed by liquid scintillation. Each column represents the mean of 10 stem segments. The average of three stem segments was used for the control columns. Bar, SEM.

Figure 2

Suppression of overexpression of light-regulated genes in dark-grown doc1 by auxin overproducing yucca. (A) Four-day-old dark-grown wild type, doc-1, yucca, and doc1-1 yucca double mutant. (B) Expression levels of some light-regulated genes in dark-grown seedlings analysed by microarray. The Affymetrix accession nos. for CAB(A), CAB(B), psaE1(A), psaE1(B), lhcb2, Elip, and psaH2 are 16004-s-at, 13213-s-at, 18088-i-at, 18089-r-at, 15153-at, 16637-s-at, and 18081-at, respectively. The CAB(A) oligos were designed based on the genomic sequence of the CAB gene (GenBank AL049655), and CAB(B) oligos were based on the mRNA sequence of the CAB gene (GenBank X56062). Both sets of oligos for the CAB gene may also pull out other homologous genes such as CAB2 in the experiments shown here. Oligos for both psaE1(A) and psaE1(B) were designed based on the mRNA sequence of the photosystem-I subunit IV precursor (GenBank AJ245908). Both sets of psaE1 oligos were designed for detecting the same gene, but neither of them is perfect based on Affymetrix rules for oligo design. There are <15 oligos in the set for psaE1(A), whereas some rules for oligo design were dropped for the psaE1(B). Microarray experiments were performed using instructions provided by Affymetrix.

Figure 3

The tir3 mutation confers a severe phenotype in combination with auxin transport and response mutants. Plants were photographed after 45 d of growth in potting soil (see Materials and Methods). All bars represent 3 cm. (A) wild type; (B) tir3-1; (C) pin-formed1-1; (D) tir3-1 pin-formed1-1; (E) pinoid1-3; (F) tir3-1 pinoid1-3 (insert: a primary inflorescence dissected from a tir3 pid1 plant; the arrow points to a terminal pin-shaped organ); (G) from left to right: wild type, tir3-1, axr1-12, and tir3-1 axr1-; (H) larger image of tir3-1 axr1-12 plant showing severe reduction in elongation between flowers.

Figure 4

(A) Physical mapping and cloning of the BIG gene. The BIG gene was mapped in a ∼260-kb region on the top arm of chromosome 3 by identifying three recombinant chromosomes at marker nga32 and one recombinant chromosome at marker 17D8LE. This region was covered by YACs and BACs, cosmids, and large-insert plasmids. A T-DNA insertion in the doc1-3 allele identified by RFLP analysis in the ∼2-kb right-end fragment from the YAC yUP6B2 served to clone the BIG gene. (B) Structure of the BIG gene. Scale diagram of the BIG gene consisting of 14 exons (shaded boxes) and 13 introns confirmed by sequencing overlapping cDNA clones and RT–PCR-generated DNA fragments. Positions of the putative Zn-finger domains (solid black boxes) are shown. Molecular analysis of mutations in three doc1 and two tir3 alleles and their relative positions is shown. (C) BIG protein. The similarity between BIG, Calossin/Pushover (CalO), a large fragment of human calossin (hCALO; KIA0462 protein; accession no. AB007931) and a Caenorhabditis elegans predicted calossin (cCALO, AF003140) extends over >3000 amino acids at the C-terminal part of the protein (shaded gray). BIG is >60% identical to CALO at two cysteine-rich regions (shaded in black). The first cysteine-rich domain (CRD-1) has similarity to a domain present in ubiquitin ligases. HYD indicates hyperplastic discs L14644; Progestin-ind prot, human Progestin-induced protein AF006010; and unknown (C.e.) C. elegans putative protein Z81077. The second cysteine-rich domain (CRD-2) conserved among the pushover family of proteins, shows some similarity to Zn-finger domains in eukaryotic transcription factors. A third domain not conserved in the Calossin-like proteins is the ZZ domain, a Zinc-binding domain seen in both chromatin and cytoskeletal proteins. HERC2 indicates mouse HERC2 protein (AAD08658); p62, human phosphotyrosine-independent ligand p62B for the Lck SH2 domain B-cell isoform (U46752); ADA2, Saccharomyces cerevisiae Transcriptional Adaptor 2, (NP_010736); CBP (human), human CREB-binding protein, (AAC51331); DYSTROPHIN, human dystrophin (AAA53189).

Figure 5

(A) BIG expression in Arabidopsis organs. RT–PCR analyses of tissue samples of 4-week-old plants grown in soil (leaves, stems, and flowers) or vertical plates (roots). (B) RT–PCR analyses of 7-day-old seedlings grown under continuous light (CL) or continuous dark (CD) or treated with 50 μM 2,4-D (CL+2,4-D) or 1 μM Brassinolide (CL+BR) as described (Gil et al. 1994). Radioactive signals were quantified and normalized using elongation factor EIF-4A expression as an internal standard. Relative BIG transcript levels are shown in black and levels of the auxin-inducible gene SAUR-AC1 in gray.

Figure 6

Localization of PIN1 in the stele region of 4-day-old Arabidopsis wild-type and tir3-1 seedling root tips after treatment with 150 μM NPA for 3 h. Immunocytochemical analysis was performed using primary antibodies raised against PIN1 and a FITC-conjugated secondary antibody. Fluorescent staining was imaged by laser-confocal microscopy. Indirectly visualized signals of the FITC-conjugated antibody are indicated by green color. The tissue autofluorescence indicated by red color of stele tissue is overlaid with the green immunofluorescence channel, facilitating the visualization of PIN1.