The Arabidopsis HOBBIT gene encodes a CDC27 homolog that links the plant cell cycle to progression of cell differentiation

Abstract

In plant meristems, dividing cells interpret positional information and translate it into patterned cell differentiation. Here we report the molecular identification of the Arabidopsis HOBBIT gene that is required for cell division and cell differentiation in meristems. We show that it encodes a homolog of the CDC27 subunit of the anaphase-promoting complex (APC). HOBBIT partially complements a yeast nuc2/cdc27 mutant. Unlike other CDC27 homologs in Arabidopsis, its transcription is cell cycle regulated. Furthermore, hobbit mutants show a reduction in DR5 :: GUS auxin reporter gene expression and accumulate the AXR3/IAA17 repressor of auxin responses. HOBBIT activity may thus couple cell division to cell differentiation by regulating cell cycle progression in the meristem or by restricting the response to differentiation cues, such as auxin, to dividing cells.

Figure 1

The HBT gene is required for progression of cell differentiation. Marker gene expression and shoot apical meristem development in wild-type (A,C,E,G,I,K,M,O) and hbt mutant (B,D,F,H,J,L,N,P) plants. (A,B) Sections of globular-stage embryos with GUS staining of promoter trap 267-1 in suspensor and at position of hypophysis. (C,D) Whole-mount view of early-heart-stage embryos expressing GFP driven by SCR promoter at QC position (arrow). (E,F) Idem, early torpedo stage, expression extends in ground tissue layers (arrow). (G,H) Whole-mount view of seedlings expressing GUS driven by SCR promoter, strongest reduction at QC position (arrow). (I,J) Whole-mount view of GFP expression from J1092 enhancer trap in mature embryos. (K,L) Scanning electron micrographs show arrested leaf primordia in hbt with bloated, incorrectly differentiated epidermal cells (arrowheads) and the absence of a shoot apical meristem. (M,N) Astra-blue-stained sections of seedlings reveal the absence of a shoot apical meristem and aberrant cell division planes (arrow) in young leaf primordia of hbt. (O,P) Whole-mount view of seedlings expressing GUS driven by KNAT2 promoter. Bars: A–J, 20 μm; K,L, 100 μm; M,N, 25 μm.

Figure 2

The HBT gene encodes a CDC27/Nuc2 homolog that can act in the Schizosaccharomyces pombe anaphase promoting complex. (A) Map-based cloning and structure of the HBT gene. The position of the markers sti, phyB, mi148, mi238, and er are given in centimorgans. The HBT gene location is shown relative to a contig of four BAC clones (F3P11, F6F22, T2G17, and F11A3). Triangles represent CAPS markers (from left to right, F27F23/91, F3P11/100, F6F22/64, T2G17/34, F11A3/45, and F11A3/25). The numbers in italics above the markers correspond to the recombinants that have a recombination breakpoint between the marker and the HBT locus. Structure of the HBT gene: The black boxes represent the exons. Nucleotide sequence changes have been described for 10 hbt alleles (see Table 1). (B) Expression analysis of the HBT gene. Northern blot (upper panel) with RNA from seedling (Se), siliques (Si), roots (R), and cotyledons (C). PolyA, mRNA purified from seedling RNA. RNAse protection analysis (lower panel) showing two potential transcription starts. Probe, probe used for RNAse protection without hybridization with the plant RNAs. (C) Amino acid sequence of the HBT gene-encoded protein. The sequences of the TPR domains are represented in bold letters. The TPR domain specific for CDC27 orthologs is boxed. (D) Protein similarity tree of the TPR-containing APC components and alignment of CDC27-specific TPR domain (boxed in C). The predicted sequences of the TPR-containing APC components from S. pombe (Sp), Aspergillus nidulans (An), Saccharomyces cerevisiae (Sc), Homo sapiens (Hs), and A. thaliana (At) were used to generate a phylogenic tree using Megalign software. Conserved TPR domains characteristic of CDC27 orthologs are aligned for different species. Identical amino acids are boxed, and the consensus of the TPR domain is shown in bold. (E) S. pombe strain nuc2^ts complementation with the coding region of HBT cDNA. Growth at permissive (25°C) or restrictive (37°C) temperature of a nuc2^ts strain carrying an empty vector as control (pREP3) or expressing the HBT gene (HBT) is followed by optical density at 700 nm (O.D.₇₀₀) measurements (in arbitrary units, a.u.).

Figure 3

The HBT transcript is cell cycle-regulated in contrast to transcripts from other APC-related genes. HBT expression during embryogenesis in wild-type (A–D) and in hbt²³¹¹ mutant (E,F) plants, and postembryonically (M–O) in wild-type roots using antisense (A,B,D,E,M,N) or sense (C,F,O) probe. (D, insert) Detail of boxed region. Double labeling of AtCYCB2;2 and HBT in a single section: Embryos were hybridized first with AtCYCB2;2 antisense (G) and then with HBT antisense (H) probes. As control, a double labeling was done with AtCYCB2;2 antisense and HBT sense probes in the same section (I). In both cases, the AtCYCB2;2 antisense probe was detected using the fast red substrate, the alkaline phosphatase was inactivated, and HBT transcripts were detected using NBT/BCIP. AtCDC27 expression in wild-type embryos (J–L) and seedlings (P–R) with antisense (J,K,P,Q) or sense (L,R) probes. Arrowheads show punctuate HBT expression. Bars: A–F,J–L, 25 μm; G–I, 50 μm; M,P, 10 μm; O,Q, 20 μm.

Figure 4

Reduced auxin sensitivity and auxin reporter gene expression in hbt seedlings. Vascular pattern is abnormal in hbt as xylem elements are not complete compared with wild-type (A,B). hbt, axr1, bodenlos, and tir1 do not form an apical hook (C; from left to right: WT, bodenlos, axr1-3, tir1-1, and hbt¹⁶¹¹). hbt¹⁶¹¹ root growth is induced by IAA. (D) Root growth is inhibited in wild-type and axr1-3 plants, whereas an initial stimulation is negated only at higher IAA concentrations in hbt mutants (E). DR5 :: GUS expression in wild-type (F) and hbt²³¹¹ (G) embryos, and in wild-type roots (H) and hbt²³¹¹ seedlings (I,J). Three d.p.g. seedlings were transferred to 1/2 GM medium (H,I) and to medium containing 5 × 10⁻⁷ M 2,4-D (J) and incubated for another 3 d. Bars: B, 50 μm; F,G, 10 μm; H, 20 μm; I,J, 100 μm.

Figure 5

Elevated AXR3/IAA17 protein levels in hbt. Western blot analysis of AXR3/IAA17 expression in wild-type (Col-0) and hbt background (hbt²³¹¹ and hbt⁹⁶²⁰; A); and in the hbt²³¹¹/axr3^GT3958 double mutant (B). Proteins were extracted from whole plants (10 d.p.g.) and quantified, and 1 μg was separated on a 15% polyacrylamide gel. After transfer, membranes were incubated first with anti-AXR3/IAA17 antibody (upper blot), and then with anti-actin antibody (lower blot) as control of protein loading. The numbers indicate size in kilodaltons. (C) RT-PCR analysis of AXR3expression in wild-type (Col-0) and hbt background (hbt⁹⁶²⁰ and hbt⁵⁷²²). RNA was extracted from wild-type and mutant seedlings. After cDNA synthesis, PCR was performed using AXR3-specific primers and ubiquitin (UBQ) as constitutive control. (D) Western blot analysis of HY5 expression in wild-type and hbt²³¹¹ mutant. The experiment was carried out under the same conditions as A and B.