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First published online 8 April 2004
doi: 10.1242/dev.01096

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The HALTED ROOT gene encoding the 26S proteasome subunit RPT2a is essential for the maintenance of Arabidopsis meristems

Minako Ueda¹, Keisuke Matsui^1,*, Sumie Ishiguro^1,, Ryosuke Sano², Takuji Wada², Ivan Paponov³, Klaus Palme³ and Kiyotaka Okada^1,2,4,

¹ Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
² Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
³ Institut für Biologie II, Zellbiologie, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Germany
⁴ Core Research of Science and Technology (CREST) Research Project

View larger version (116K): [in a new window]   Fig. 1. The halted root (hlr) mutant is defective both in roots and shoots. Phenotypes of wild type (A,B,D,F,H) and hlr-1 (A,C,E,F,G,I). (A) Ten-day-old seedlings of wild-type (WT; left), hlr-1 mutant (center) and the hlr-1 mutant possessing a genomic fragment of the wild-type HLR gene (right). (B,C) Primary roots of 12-day-old wild-type (B) and hlr-1 (C) seedlings. Brackets indicate the area of the root cap. (D,E) Root tips of one-month-old seedlings. White and black arrows indicate the lateral root and the lateral root primordium, respectively. (F,G) Aerial parts of 42-day-old plants. Red arrowhead indicates the secondary inflorescence without cauline leaves, and white arrowheads indicate lateral organs with irregular internode length (higher magnification is shown in G). (H,I) Aerial parts of one-month-old plants. The number on each leaf indicates age order, from young to old leaves. (J) Elongation of primary roots. Each point represents the mean of 100 seedlings. Error bars indicate s.e. (n=103 for wild type; n=100 for hlr-1). Scale bars: 1 cm in A; 500 µm in B,C; 50 µm in D,E.

View larger version (121K): [in a new window]   Fig. 2. The HLR gene is required for post-embryonic meristems. Structures of embryonic and post-embryonic meristems in wild type (A,C,E,G) and hlr-1 mutants (B,D,F,H). (A-D) Whole-mount preparations of the root apical meristem (RAM) in mature embryos (A,B), and in PI-stained root tips of 36-hour-old seedlings (C,D). White arrowheads indicate the quiescent center (QC). (E-H) Longitudinal sections of the shoot apical meristem (SAM) in mature embryos (E,F) and in 8-day-old seedlings (G,H). Black lines and arrowheads indicate the boundaries between cell layers and the irregular cell division planes, respectively. Scale bars: 25 µm.

View larger version (165K): [in a new window]   Fig. 3. QC identity is lost in the hlr mutant after germination. Expression of QC-specific markers in wild type (A,C,E) and hlr-1 mutants (B,D,F). (A-D) Expression of QC184 in mature embryos (A,B) and in 36-hour-old seedlings (C,D). (E,F) Expression of QC46 in 7-day-old seedlings. Scale bar: 25 µm.

View larger version (75K): [in a new window]   Fig. 4. The identities of differentiated tissues in the RAM remain in the hlr mutant. Cell arrangement and cell-type-specific markers in the post-embryonic RAM of wild type (A,C,E,G,I) and hlr-1 mutants (B,D,F,H,J). Arrowheads indicate the QC. (A,B) PI-stained root tips of 6-day-old seedlings. (C,D) Lugol-stained root tips. Starch granules, a marker for the root cap, are visualized. (E,F) Expression of J1092, a marker for the lateral root cap, in 6-day-old seedlings. (G,H) Expression of SHRp::GFP, a marker for the stele, in 6-day-old seedlings. (I,J) Expression of SCRp::GFP, a marker for the QC, cortex/endodermal initial cells and endodermis, in 6-day-old seedlings. Scale bar: 25 µm.

View larger version (102K): [in a new window]   Fig. 5. The expression pattern of the WUSCHEL (WUS) gene is disturbed in the mutant SAM. Longitudinal sections of the SAM in 11-day-old seedlings of wild type (A) and hlr-1 mutants (B,C) hybridized with an antisense (A,B) or sense (C) WUS probe. Arrows (B) indicate WUS mRNA accumulation in some groups of cells in abnormal places. Scale bar: 25 µm.

View larger version (72K): [in a new window]   Fig. 6. The HLR gene encodes RPT2a, a homolog of the 26S proteasome subunit. Map-based cloning of the HLR gene. (A) Physical and genetic map of the HLR gene on chromosome 4. Molecular markers and their positions are shown above the line. Values under the lines indicate the frequency of recombinants. The bipolar arrow indicates the 45 kb region sequenced to find the mutation site in hlr-1. (B) Schematic structure of the HLR gene. Black and white rectangles indicate the protein coding regions and untranslated regions (UTRs), respectively. The 13 bp deletion found in hlr-1 is shown. (C) Alignment of the deduced amino acid sequence of HLR with selected homologous proteins. Identical residues are shown in white on a black background. Major motifs and mutation sites found in the hlr mutant are shown. Orthologous proteins are from Oryza sativa (PRS4-ORYSA), Drosophila melanogaster (PRS4-DROME), Homo sapiens (PRS4-HUMAN) and Saccharomyces cerevisiae (PRS4-YEAST).

View larger version (157K): [in a new window]   Fig. 7. The HLR gene is expressed both in the RAM and in the SAM. (A-C) Longitudinal sections of roots in 36-hour-old wild-type seedlings hybridized with an antisense (A,B) or sense (C) HLR probe. Arrowheads indicate the QC. (D,E) Longitudinal sections of the SAM in 11-day-old (D) and 32-day-old (E) seedlings hybridized with the antisense HLR probe. The SAM in D and E is in vegetative and reproductive phase, respectively. Scale bars: 25 µm.

View larger version (64K): [in a new window]   Fig. 8. Proteasome activity is reduced in the hlr mutant. GUS staining of HS::AXR3 NT-GUS, a marker for the degradation of AUX/IAA proteins, in wild type (A,C) and in the hlr-1 mutant (B,D). (A-D) 36-hour-old seedlings stained for 0 (A,B) or 60 minutes (C,D) after the end of heat-shock induction. (E) GUS activity in the absence or presence of the proteasome inhibitor MG132. Relative activity was calculated as a percentage of the number of seedlings with GUS staining in the roots per total number of seedlings examined. Scale bar: 25 µm.

View larger version (75K): [in a new window]   Fig. 9. Disruption of RAM organization in the hlr mutant is not caused by the defective degradation of cyclin B1 or PIN4. Marker gene expression and localization of putative target proteins for proteasome-mediated degradation in 36-hour-old (A,B,E,F,I,J) and 10-day-old (C,D,G,H,K,L) seedlings of wild type (A,C,E,G,I,K) and hlr-1 mutants (B,D,F,H,J,L). (A-D) GUS staining of cycB1::GUS, a marker for the degradation of cyclin B1. (E-H) Whole-mount in situ immunolocalization of the PIN4 protein, a putative auxin efflux carrier. (I-L) Expression of DR5::GFP, a marker for auxin accumulation. Scale bars: 25 µm.