Comparative Study
doi: 10.1093/emboj/cdf614.Cell numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene
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- PMID:12426376
- PMCID: PMC137206
- DOI: 10.1093/emboj/cdf614
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Comparative Study
Cell numbers and leaf development in Arabidopsis: a functional analysis of the STRUWWELPETER gene
Daphné Autran et al. EMBO J..
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Abstract
The struwwelpeter (swp) mutant in Arabidopsis shows reduced cell numbers in all aerial organs. In certain cases, this defect is partially compensated by an increase in final cell size. Although the mutation does not affect cell cycle duration in the young primordia, it does influence the window of cell proliferation, as cell number is reduced during the very early stages of primordium initiation and a precocious arrest of cell proliferation occurs. In addition, the mutation also perturbs the shoot apical meristem (SAM), which becomes gradually disorganized. SWP encodes a protein with similarities to subunits of the Mediator complex, required for RNA polymerase II recruitment at target promoters in response to specific activators. To gain further insight into its function, we overexpressed the gene under the control of a constitutive promoter. This interfered again with the moment of cell cycle arrest in the young leaf. Our results suggest that the levels of SWP, besides their role in pattern formation at the meristem, play an important role in defining the duration of cell proliferation.
Figures
Fig. 1. General plant phenotype and mature leaves inswp mutant. (A) Sixteen-day-old wild-type andswp plants grownin vitro. (B) swp mature plant grown in soil. Mature first rosette leaves of 4-week-old wild-type (C andE) andswp (D andF) plants were visualized using SEM. Althoughswp leaves displayed a severely reduced size (D) compared with wild-type ones (C, same magnification as in D), adaxial epidermal cells are larger (compare E with F, same magnification), showing that cell number is reduced inswp (F). (G–I) Petal size and cell size. Mutant petals inset in G are much smaller than wild-type petals (G). This difference in overall size is not compensated by extra cell expansion, as the cells in the mutant petal (H) are even somewhat smaller than those in the wild type (I). Scale bars: 1.5 mm (B); 1 mm (C and D); 200 µm (E and F); 500 µm (G); 20 µm (H and I).
Fig. 2.In vivo analysis of cell proliferation inswp leaf primordia and organ size in earlyswp primordia. (A) Epidermal cell divisions were followed by analysing reconstructions of serial sections of living primordia, made every 12 h. (B) Detail showing divisions undergone within 36 h by a group of four epidermis cells. (C) The rate of cell proliferation in wild-type andswp primordia was evaluated by scoring the number of cells that had divided zero times, one time, two times or three times, 12, 24 and 36 h after the start of the experiment, at 6 days after sowing. (D) The mean primordia area measured on median longitudinal sections in 2-day-old wild-type andswp seedlings is significantly different betweenswp and wild-type (Student’st-test,p < 0.05). Scale bars: 40 µm (A); 5 µm (B).
Fig. 3. Kinematic analysis ofswp leaf development. Kinematic analysis was performed on the first leaf pair of wild-type (WS) (open circles) andswp plants (black diamonds). Cells of abaxial epidermis were analysed. (A) Average leaf blade area. (B) Average cell area. (C) Average number of cells. (D) Cell division rate. (E) Stomatal index.
Fig. 4. Ploidy level measurements in matureswp leaves. Cell flow cytometric profiles of wild-type (A) andswp (B) leaves of 4-week-old plants. Quantification of ploidy distribution is represented (C) as mean ± SD of three independent measurements involving different sets of plants. (D) and (E) show a projection of several confocal sections through the epidermis of full-grown wild-type (D) and mutant (E) leaves stained for DNA with propidium iodide. Note that the spindle-form nuclei of the pavement cells (arrows) are bigger in the mutant. The nuclei of stomata have the same size. Bars: 125 µm.
Fig. 5. Shoot apical meristem organization and maintenance in theswp mutant. (A–D) Histological sections of meristems stained with toluidine blue. In 6-day-old seedlings in the progeny ofswp heterozygous plants, the SAM structure is indistinguishable from the wild type [(A) representative of the 60 individual plants examined]. Thirteen-day-old wild-type seedlings present a characteristic dome-shaped SAM (B). In contrast, theswp SAM at this stage is flat, with only a few irregular meristematic cell layers (C). The 20-day-oldswp SAM region is enlarged and shows groups of vacuolated cells adjacent to meristematic type cells (D). (E–H) STM expression pattern in wild-type (E and G) andswp (F and H) meristems. From 8 days after germination onward (F), STM expression inswp becomes irregular. This situation increased in 16-day-old plants (H). Note that downregulation of STM at the level of the incipient leaf primordia is systematically observed inswp. (I andJ) ANT expression in wild-type (I) andswp (J) primordia anlagen. (K andL) WUS expression pattern. In 10-day-old wild-type SAM, WUS is expressed in a central group of inner cells of the SAM (K). Inswp SAM at the same stage, WUS is ectopically expressed in an irregular pattern across the SAM (L).
Fig. 6. TheSWP gene: gene structure, phenotypic restoration and similarity to Med150/RGR1-like proteins. (A) TheSWP gene (SWP1) contains six introns (lines) and seven exons (boxes). The SWP cDNA has the potential to encode a 1703 aa polypeptide from the initiating methionine (arrowhead) situated 250 bp downstream of the 5′end of the cDNA. Also shown is the position of the T-DNA insertion in the swp1 mutant line. A 1.3 kb promoter region has been shown to be sufficient to drive SWP expression in phenotypic restoration experiments (see text) using the D3-9.7 genomic clone that is represented. The two regions of the SWP1 putative protein showing significant similarity to Med150/RGR1-like proteins are shown (light grey bars). (B–D) Fourteen-day-old seedlingsin vitro grown fromswp homozygote plants containing a transgenic copy of the SWP gene (D3-9.7 genomic clone represented above) and exhibiting a wild-type phenotype (B, line 8) or a partially restored phenotype (C, line 23), as compared with non-transgenicswp seedlings segregating in the same line (line 8, D). (E) The N-terminal domain of SWP protein (SWP; F7018.23 protein; AAF04900.1; residues 39–442) was aligned with similar regions (N-terminal domains) of the human hMed150/TRAP170/hRGR1/DRIP150/EXLM1 protein (hRGR1; gb AAD24360.1; residues 77–327),Drosophila dMed150/dTRAP170/RGR1 protein (dRGR1; AAG02462 GadFly identifier: CG12031; residues 63–466), mouse mRGR1 protein (mRGR1; dbj BAA76610.1; residues 83–333), putativeC.elegans 157 kDa protein C38C10.5 (cRGR1; pir S28289; residues 1–264) and yeast (S.cerevisae) sMed150/RGR1 protein (RGR1; dbj BAA14104.1; residues 123–286). The number of amino acids for the full proteins is indicated. (F) A C-terminal domain of SWP (residues 1232–1335) was aligned with a conserved domain of theDrosophila protein dRGR1 (residues 1418–1524, C-terminal position). All alignments were performed using the Clustal_W and Boxeshade softwares at Infobiogen (http://www.infobiogen.fr/services/analyses ).
Fig. 7. Consequences of T-DNA insertion onSWP expression inswp mutant. (A) Northern blot analysis ofSWP expression in theswp mutant showing overproduction of aberrant transcripts in swp. A northern blot with ∼2 µg of poly(A)+ RNA fromswp homozygote plants (swp) or wild-type plants (ws) was hybridized with aSWP RNA-specific probe corresponding to the 3′ end of the cDNA (represented in C). RNA size was estimated using a RNA ladder run on the same gel. (B) RT–PCR experiment showing fusion ofSWP mRNA with T-DNA left border region inswp mutant. Total RNA fromswp plants (swp) or wild-type plants (ws) were reverse transcribed and amplified using primers designed to the 5′ end of theSWP cDNA (swpUp) and to the T-DNA left border (LB1) (represented in C). A 0.7 kb fragment could be amplified fromswp RNA (lane 4) and not from WS RNA (lane 3). Amplification using primers from theSWP cDNA only (swpUp/swpLow; see C) provides the loading control (lanes 1 and 2). SWP1 and SWP2 primers are located downstream and upstream of the first intron of theSWP gene, respectively (see C), excluding genomic contamination of the reverse transcribed RNA samples, which would have led to an 1 kb amplification product as shown in lane 6. (C) Schematic representation of the T-DNA insertion site in theswp mutant line. The T-DNA caused a 6 bp deletion at the predicted transcription start site of theSWP gene (arrow, WT transcription start). Inswp, initiation of transcription may occur at upstream functional sites existing within the T-DNA and could be driven by the cauliflower mosaic virus promoter (35S) as suggested by the high expression level detected inswp. The sequence of the region of fusion between theSWP mRNA and T-DNA left border (LB) is given. In the T-DNA LB sequence (underlined), several putative start codons (bold letters) are detected that could compete with the wild-typeSWP start codon, potentially leading to transcriptional attenuation inswp mutant, as suggested by the recessive nature of theswp mutation. bar, basta (ammonium glufosinate) herbicide resistance gene; tnos, nos gene terminator.
Fig. 8.SWP RNA expression pattern.SWP mRNA was detected usingin situ hybridization. (A) A STM antisense control probe showing SAM-specific expression in a young torpedo stage embryo. (B) A torpedo stage embryo in the same experiment showingSWP mRNA expression in all cells. (C) In the 8-day-old seedling apex, strongSWP expression was detected in the primordia (P) and weakly in the SAM. (D) In the inflorescence meristem (IM)SWP is also detected, but more mRNA was present in the lateral floral meristems (FM), and in young organ primordia of developing flowers (SP: sepal primordia). (E) SWP mRNA was also present in meiocytes and tapetum within the anthers (arrowhead). (F) The expression was maintained in ovules (arrowhead) within the gynoecium in later stages of flower development.
Fig. 9. Overexpression ofSWP. (A) Control plant (ecotype Ler) grown in soil. (B) 35S::SWP primary transformant (line 145; ecotype Ler) grown in soil. (C) Over-accumulation ofSWP transcript in 21-day-old 35S::SWP plants, line 31 (Ler), 145 (Ler) and 2.9 (WS) using semi-quantitative PCR. Controls:SWP levels in a line transformed with the empty binary plasmid (pv, WS) in a line that did not present a phenotype (Ler 154) and in the wild type (WS). APT1 was used as a loading control. (D–G) Epidermis of mature first rosette leaves of 3-week-old control (D and F) and35S::SWP (line 145) (E and G) plants was visualized using SEM (adaxial epidermis; D and E) or tubu drawings (abaxial epidermis; F and G). Average leaf area was measured (H), average cell area (I) was estimated using the tubu drawings as described for the kinematic analysis of leaf growth, then average cell number per leaf (J) was calculated. Scale bars: 0.5 cm in (A) and (B); 125 μM in (D) and (E).
Fig. 10. Clustering of cells on leaves overexpressingSWP. (A) Cell area distribution in wild-type (Ler) and 35S::SWP leaves. Cell area was measured on tubu images of the abaxial epidermis of the first leaves of 28-day-old wild-type (292 cells measured on five independent leaves) and 35S::SWP plants (line 145, 1077 cells measured, eight independent leaves). Guard cells were excluded from the measurements. The number of cells in each size class was scored. (B) ‘Cluster’ index. The clustering of cells was determined in the leaves of 21 35S::SWP leaves and 11 wild-type leaves. Per leaf, at least a surface of 100 cells was analysed. A cluster of cells was defined as a group of two or more small cells with an area <500 µm2. The cluster index is defined as the number of cell clusters in a population of 100 cells. (C) Average number of cells per cluster. The number of cells was scored in each cluster of wild-type (46 clusters in 11 leaves) and overexpressing leaves (298 clusters in 21 leaves). (D) Distribution of cell number per cluster.
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