A novel plant-specific family gene, ROOT PRIMORDIUM DEFECTIVE 1, is required for the maintenance of active cell proliferation

Abstract

Hypocotyl segments of Arabidopsis (Arabidopsis thaliana) produce adventitious roots in response to exogenously supplied auxin. root primordium defective 1 (rpd1) is a temperature-sensitive mutant isolated on the basis of impairment in this phenomenon. This study describes further phenotypic analysis of the rpd1 mutant and isolation of the RPD1 gene. When adventitious root formation was induced from the rpd1 explants at the restrictive temperature, cell proliferation leading to root promordia formation was initiated at the same time as in wild-type explants. However, development of the root primordia was arrested thereafter in the mutant. Temperature-shift experiments indicated that RPD1 exerts its function before any visible sign of root primordium formation. The expression patterns of the auxin-responsive gene DR5:beta-glucuronidase and the cytodifferentiation marker gene SCARECROW suggest that the rpd1 mutation interferes with neither axis formation nor cellular patterning at the initial stage of root primordium development. Taken together with the effect of the rpd1 mutation on callus cell proliferation, these data imply a role for RPD1 in prearranging the maintenance of the active cell proliferation during root primordium development. Positional cloning of the RPD1 gene revealed that it encodes a member of a novel protein family specific to the plant kingdom. Disruption of the RPD1 gene by a T-DNA insertion caused embryogenesis arrest at the globular to transition stages. This phenotype is consistent with the hypothesized function of RPD1 in the maintenance of active cell proliferation.

Figure 1.

Time course of adventitious root formation as influenced by the rpd1 mutation. Hypocotyl explants of the wild type (A) and rpd1-1 mutant (B) were cultured at 22°C or 28°C for the indicated days. Bar = 20 μm.

Figure 2.

Effects of temperature shift on adventitious root formation in rpd1-1. Hypocotyl explants of the wild type (A–F) and rpd1-1 mutant (G–L) were cultured on RIM with the indicated temperature programs. A and G, Twenty-two degrees Celcius, 6 d. E and K, Twenty-eight degrees Celcius, 6 d. B to D and H to J, Temperature shift up. Hypocotyl explants were cultured at 22°C, followed by culture at 28°C. F and L, Temperature shift down. Hypocotyl explants were cultured at 28°C, followed by culture at 22°C. Bar = 200 μm.

Figure 3.

Effects of the rpd1 mutations on expression patterns of DR5:GUS and SCR:GUS (END199) during adventitious root formation. Hypocotyl explants of the DR5:GUS (A) and END199 (B) reporter lines with or without the rpd1 mutations were cultured at 28°C on RIM for the indicated days and stained for GUS activity. Bars = 100 μm.

Figure 4.

Effect of temperature shift on the growth rate of the rpd1-1 primary root. Seedlings of the wild type and rpd1-1 were grown at 22°C on GMA. After 7 d of culture, half of them were transferred to 28°C. Growth rate was measured by marking the position of the root tip every day. Symbols represent averages of data from eight to 15 primary roots for each condition. Vertical lines indicate sds.

Figure 5.

Time course of callus formation as influenced by the rpd1 mutation. Hypocotyl explants of the wild type (A) and rpd1-1 mutant (B) were cultured at 22°C or 28°C for the indicated days. Bars = 50 μm.

Figure 6.

Map-based cloning of RPD1. A, Chromosomal position of RPD1. B, Alignment of the amino acid sequences of full-length RPD1 and related proteins. Identical amino acid residues are highlighted on a black background. Positions of the rpd1-1 and rpd1-2 mutations are marked with asterisks. An N-terminal peptide retained in the T-DNA insertion mutant (rpd1-3) is shadowed. Predicted nuclear localization signals are underlined. The full-length amino acid sequence of TK59A01 is deduced from the genomic sequence around the EST, TK59A01 (accession no. CO048598; Katari et al., 2005), which is located on chromosome IV between At4g01030 and At4g01040.

Figure 7.

Semiquantitative RT-PCR analysis of RPD1 expression. A, Expression of RPD1 mRNA in various organs of 14-d-old wild-type seedlings. Seedlings were divided into cotyledons, hypocotyls, roots, and the remaining shoot apex parts with true leaves. B, Expression of RPD1 mRNA upon adventitious root induction in wild-type hypocotyl explants. ACT2 was used as the control.

Figure 8.

Effect of RPD1 knockout on embryogenesis. A, Opened silique of RPD1/rpd1-3. B, Opened silique of RPD1/RPD1. C and D, Seeds in the mature RPD1/rpd1-3 silique. E, Higher magnification of embryo in D. Bars = 5 mm (A and B) or 50 μm (C–E).

Figure 9.

Possible structural features of RPD1 and related proteins. A, Putative structural units of RPD1. Positions of three K/RH/YPXXF motifs are shown by black boxes with corresponding amino acid sequences. Uppercase letters indicate residues that match to the motif consensus and lowercase letters indicate residues that differ from the consensus. Domains A and B are shown by gray boxes. B, Sequence alignment of domains A and B of RPD1 and related proteins. Residues predicted to form α helix and β strand are written in blue and red, respectively. Consensus between domain A and domain B is given above the domain A sequence of RPD1. Lowercase h indicates hydrophobic residues. C, Sequence alignment of winged helix proteins. Residues in α helix and β strand are written in blue and red, respectively. LexA, DNA-binding domain of Escherichia coli LexA repressor, Protein Data Bank (PDB) accession 1LEA (Fogh et al., 1994); ADAR1, Zα domain of human ADAR1, PDB accession 1QBJ (Schwartz et al., 1999); GH5, chicken linker histone H5, PDB accession 1HST (Ramakrishnan et al., 1993); E2F4, DNA-binding domain of human E2F4, PDB accession 1CF7 (Zheng et al., 1999); Rap30, DNA-binding domain of Rap30 subunit of human TFIIF, PDB accession 2BBY (Groft et al., 1998); APC2, WH-B domain of Saccharomyces cerevisiae APC2, PDB accession 1LDD (Zheng et al., 2002); DLM-1, Zα domain of mouse DLM-1, PDB accession 1J75 (Schwartz et al., 2001); HNF-3g, DNA-binding domain of rat HNF-3γ, Swissprot accession P32183 (Clark et al., 1993); TFIIEB, central core domain of human TFIIEβ, PDB accession 1D8K (Okuda et al., 2000). Asterisks indicate residues that constitute the hydrophobic cores of DLM-1, TFIIEβ, and HNF-3γ. Secondary structures are illustrated at the bottom. Yellow shadings show that the positions of conserved hydrophobic residues of RPD1 and related proteins correspond to those of the hydrophobic core residues of the winged helix fold.