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An Arabidopsis Minute-like phenotype caused by a semi-dominant mutation in a RIBOSOMAL PROTEIN S5 gene

Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands

View larger version (96K): [in a new window]   Fig. 1. GUS expression in the 826 line is confined to cell division foci. (A) Expression of the GUS gene in 826 seedlings is found in the apical regions of both shoot and root. (B) GUS activity is found in the SAM and leaf primordia, but not the surrounding tissues. (C) The division zone of the main root, but not the quiescent center (QC), is marked by strong GUS expression. (D) Lateral root primordia are marked by GUS activity. (E) GUS expression in the vascular tissue of cotyledons.

View larger version (174K): [in a new window]   Fig. 2. Embryo development is delayed in 826 hemizygotes and arrests prematurely in 826 homozygotes. Whole-mount analysis of developing seeds from plants hemizygous for the 826 T-DNA. (A-C) Representative seeds from three siliques in subsequent stages of development. Indices (i-iii) indicate the different classes of embryos found in the same silique. When development of wild-type embryos is at the torpedo (Ai), bent-cotyledon (Bi) or mature stage (Ci), 826 hemizygous embryos have only reached the heart (Aii), torpedo (Bii) or bent-cotyledon stage (Cii). At all later time points, homozygous 826 embryos remain arrested at the globular stage (Aiii, Biii, Ciii). The arrested embryos display storage structures typical for late wild-type embryo development (D). Histochemical staining of seeds from the same silique reveals that the GUS activity correlates with the delay in embryo development. GUS expression is absent in mature embryos (Ei), whereas weak and strong expression is observed in respectively delayed torpedo stage (Eii) and arrested globular stage embryos (Eiii).

View larger version (112K): [in a new window]   Fig. 3. Vascular and floral defects in hemizygous 826 plants. (A) Abnormal venation pattern in a cotyledon of a plant hemizygous for the 826 T-DNA. Note that the central vein is short and a secondary vein is interrupted. (B) Vascular pattern complexity in the most advanced cotyledon of wild-type (GUS negative, white bars) and hemizygous 826 (GUS positive, black bars) seedlings. The y-axis shows the percentage of seedlings falling into the categories represented on the x-axis. (C) Hemizygous 826 flowers carry only four to five stamens, as clearly shown in a cross-section of a flower bud from an 826 hemizygous plant. (D) The floral defect originates from the floral meristem, as indicated by the absence of one vascular strand (compare top with bottom arrow) in a cross-section through the receptacle of a young flower.

View larger version (41K): [in a new window]   Fig. 4. Molecular characteristics of the Arabidopsis RPS5 gene family. (A) Alignment of the extreme C terminus of RPS5 proteins from different species shows a strong conservation of primary structure. AtRPS5A, GenBank ID N37913; AtRPS5B, GenBank ID H35978; CaRPS5, Cicer arietinum (GenBank ID AJ005346); ScRPS5, Saccharomyces cerevisiae (GenBank ID X89368); CeRPS5, Caenorhabditis elegans (GenBank ID P49041); DmRPS5, Drosophila melanogaster (GenBank ID U48394); and HsRPS5, Homo sapiens (GenBank ID MN001009). The fusion of RPS5A and GUS in line 826 (aml1) is shown in the bottom row with the connecting border sequence in italics and the GUS sequence in bold. The number between brackets indicates the position in the protein relative to the N terminus. The asterisks indicate plant-specific residues. (B) Gene structure of AtRPS5A and the aml1 allele. The AtRPS5A gene contains five exons, interrupted by four introns. Arrows I, II and III indicate the positions of PCR-primers. (C) Complementation of the aml1 mutant with a wild-type RPS5A gene copy. The top panel shows the result of a duplex-PCR to detect the wild-type (1.3 kb) and aml1 (0.8 kb) alleles of the RPS5A gene in seedlings. From left to right: C24 wild-type plants, the aml1 mutant and ten independent primary transformants with the MOG-RPS5A construct. Plant 1 is homozygous for the aml1 allele. Bottom panel shows the presence of the NPTII gene located on the MOG-RPS5A construct. (D) Intron and exon sizes (numbering is in the 5' to 3' direction of the genes) are extremely conserved between AtRPS5A and AtRPS5B. (E) RT-PCR analysis using AtRPS5 and AtRPS5B gene-specific primer sets. AtRPS5A primers (left panel) amplify a 0.4 kb fragment from seedling RNA (R) to higher levels than do AtRPS5B specific primers (R, right panel). Control PCR reactions using chromosomal DNA (C) indicate equal efficiency of both primer pairs.

View larger version (174K): [in a new window]   Fig. 5. Localisation of AtRPS5A mRNA by in situ hybridisation. Five-day-old wild-type seedlings were hybridised with an AtRPS5A-specific antisense (A-F) or sense (G) probe. Untreated seedlings (A-E,G) as well as NAA-treated seedlings (F) were used. Expression is detected in young leaf primordia (A). (B) A close-up of the apical shoot region of a seedling. Expression is confined to the leaf primordia (lp1 and lp2) and the shoot apical meristem (sam). A significant signal is detected in the vasculature of cotyledons (C), and the primary root tip (D). (E) A close-up of a root apical region. A strong signal is present in all dividing cells, but not in the quiescent center (qc). NAA-induced lateral root primordia stain strongly for the AtRPS5A signal (F). (G) The absence of a signal in a sense-probe control on an untreated seedling root.

View larger version (129K): [in a new window]   Fig. 6. AtRPS5A and AtRPS5B are differentially expressed in seedlings. Four-day-old seedlings from pAtRPS5A::GUS (A-C) and pAtRPS5B::GUS (D-F) plants were stained for GUS activity during 10 minutes and 2 hours, respectively. The AtRPS5A promoter is strongly active in the division zone of the primary root tip (A) and the SAM and leaf primordia (C), and is weaker in the pericycle of the root (B). pAtRPS5B is active in the epidermal cells of the primary root tip (D), in a zone more distal from the tip than pAtRPS5A. Activity is almost absent in the root pericycle (E), but present in lateral root primordia (not shown). GUS activity is also found in the distal, differentiating cells of the shoot primordium, but absent from the SAM (F). AtRPS5B (H), but not AtRPS5A (G) is expressed in root hairs.

View larger version (93K): [in a new window]   Fig. 7. The AtRPS5A and AtRPS5B genes are differentially expressed during embryogenesis. Siliques of pAtRPS5A::GUS (A-E) and pAtRPS5B::GUS (G-K) plants were stained for GUS activity for 2 and 8 hours, respectively. (A) In pAtRPS5A::GUS lines, GUS activity is already observed at the 2-cell stage in both embryo and endosperm. GUS activity remains strong through the octant (B) and globular stage (C), and decreases after the transition (D) and late torpedo stage (E). Activity is strongest in the peripheral cell layer (C-E). Expression of pAtRPS5B::GUS in embryos is weak until the globular stage (G,H), but increases during the late globular (I), heart (J) and late torpedo (K) stages. Note that expression is strongest in the inner cell layers (I-K), and is visible in the provascular tissue (K). (F) Schematic representation of the differential expression pattern of AtRPS5A (yellow) and AtRPS5B (blue). Places where both genes are expressed are marked with green. (i) 2-cell, (ii) octant, (iii) globular, (iv) transition, (v) heart and (vi) torpedo stage.