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First published online 4 August 2004
doi: 10.1242/dev.01320

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MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems

Laboratoire de Biologie Cellulaire, Institut J. P. Bourgin, INRA, 78026 Versailles Cedex, France

View larger version (67K): [in a new window]   Fig. 1. miR164 overexpression phenocopies the cuc1 cuc2 double mutant. (A) The 2x35S::miR164A and 2x35S::miR164B constructs contain 1049 bp and 1021 bp of genomic DNA centred on the two predicted pre-miR164 precursors, respectively (black box) (Reinhart et al., 2002) under the control of the Cauliflower mosaic virus double 35S promoter and upstream of the Cauliflower mosaic virus 35S terminator. The 2x35S::erGFP construct was used as a control. (B) Floral phenotype of wild-type and transgenic lines. (1) Wild-type plants have unfused sepals and expanded petals and stamens. (2,3) 2x35S::miR164A or 2x35S::miR164B flowers have fused sepal margins. (3) In lines with strong phenotypes, petals and stamens did not expand. (4) However, dissection of the flower reveals petals and stamens which cannot grow out of the fused sepals, resembling cuc1 cuc2 double mutant flowers (Aida et al., 1997). The degree of sepal fusion (5) and the petal number (6) was scored in 2x35S:: erGFP, 2x35S::miR164A and 2x35S::miR164B primary transformants. The degree of fusion is expressed on a scale ranging from 0 for normal sepals (as represented in 1) to 16 for the strongest sepal fusion phenotype (as shown in 3). Data represent mean values for 10 flowers per line. (C) Seedling phenotype of wild-type and transgenic lines. (1) Wild-type seedlings have aligned cotyledons. (2-5) 2x35S::miR164A or 2x35S::miR164B seedlings have misaligned (2), partially fused cotyledons (3) or cup-shaped cotyledons (4) and petiole fusion revealed by dissection (5). (D) miR164 is overexpressed in 2x35S::miR164A and 2x35S::miR164B lines. (1) MIR164A and MIR164B predicted hairpin precursors (Reinhart et al., 2002). In each precursor, the nucleotides corresponding to miR164 are red and those corresponding to the other strand resulting from RNase III-mediated cleavage, miR164A* or miR164B*, are blue. (2) Detection of miR164 and control 5S RNA in apices of wild-type plants, 2x35S::erGFP and representative 2x35S::miR164A and 2x35S::miR164B lines showing either a weak (W), intermediate (I) or strong (S) flower phenotype. The normalised ratio between miR164 and 5S RNA expression level is indicated. The migration of 21 and 24 nucleotides DNA primers is indicated. (3) Detection of miR164A*, miR164B* and miR164 and control 5S RNA, in wild type and in representatives of strong 2x35S::erGFP, 2x35S::miR164A and 2x35S::miR164B lines. Each inset (right) represents the hybridization signal obtained under identical conditions with 100 pg of DNA oligonucleotides corresponding to miR164A*, miR164B* and miR164.

View larger version (43K): [in a new window]   Fig. 2. Expression of the five predicted miR164 targets and of CUC3 in 2x35S::miR164 lines. (A) RT-PCR analysis of CUC1, CUC2 and CUC3 mRNA accumulation in inflorescence apices of wild-type plants, 2x35S::erGFP and representative 2x35S::miR164A and 2x35S::miR164B lines showing either a weak (W), intermediate (I) or strong (S) flower phenotype. APT was used as a control. (B) RT-PCR analysis of At5g61430, At5g07680 and NAC1 mRNA accumulation in inflorescence apices. Histograms show quantification of the target expression level relative to the APT control.

View larger version (43K): [in a new window]   Fig. 3. Importance of miR164-mediated regulation of CUC2 in planta. (A) Partial sequence of CUC2 mRNA showing the region complementary to miR164. Note the three mismatches. One or four additional mismatches were introduced into the binding site of miR164 in CUC2 (CUC2-m1 and CUC2-m4). Similar mutations were introduced as controls outside the miR164-binding site (CUC2-c1 and CUC2-c4). All the mutations are silent at the protein level. (B) Ubiquitous overexpression of wild-type CUC2 and CUC2-m4. (1) The two CUC2 forms were cloned under the control of the double 35S promoter. (2-4) 2x35S::CUC2 and 2x35S::CUC2-m4 transgenic lines show mild and severe growth reduction, respectively, compared with wild type. (4-6) Both 2x35S::CUC2 and 2x35S::CUC2-m4 lines have wrinkled leaves. (C) Strategy used to obtain inducible expression of the wild-type and modified CUC2s in the STM-expressing domain. The alcA::CUC2s constructs were introduced into a STM::ALCR alcA::erGFP line. The ALCR transcription factor is expressed under the control of STM regulatory sequences and can be activated by ethanol induction. It will then activate simultaneously the expression of the reporter erGFP and the different CUC2s under the control of the alcA promoter. (D) Expression of miR164-resistant CUC2s leads to abnormal seedling development. No leaf (1, right seedling), asymmetrical leaves (2, right seedling) or small leaves (3, right seedling) were observed in 10-day-old seedlings expressing miR164-resistant CUC2s, in contrast to what is observed in wild-type plants (left seedling in all panels). (E) Quantification of leaf development in the progeny of 15 transgenic lines expressing the different alcA::CUC2s. Ten-day-old seedlings were scored as having normal leaves (blue), abnormal leaves (deep red; such as those shown in D2,3) or no leaf (yellow; such as that shown in D1). At least 100 T2 plants were analysed per line. (F) Phenotype of mature STM::ALCR-alcA::erGFP (1,3) or STM::ALCR-alcA::erGFP alcA::CUC2-m4 (2,4) flowers that have been ethanol-induced for 6 days. In control lines (1,3), the margins of two adjacent sepals (arrowheads) are next to each other, hiding the petal insertion point. In STM::ALCR-alcA::erGFP alcA::CUC2-m4 lines (2,4), the spacing between the sepals is increased, revealing the insertion point of the petal. Petals are coloured in red in (3) and (4).

View larger version (26K): [in a new window]   Fig. 4. miR164-binding is required for CUC2 mRNA cleavage. (A) RT-PCR analysis of the CUC2s expression levels in STM::ALCR-alcA::erGFP alcA::CUC2-wt (left) and STM::ALCR-alcA::erGFP alcA::CUC2-m4 (right) transgenic lines. Ten-day-old seedlings were sampled after overnight ethanol induction of the CUC2-wt or CUC2-m4 gene. The primers used amplified RT products of the endogenous CUC2 gene and the CUC2-wt or CUC2-m4 transgenes. The same lines as in Fig. 3E were analysed and are plotted in the same order. (B) A cleavage product of CUC2 is detected in lines overexpressing CUC2 (2x35S::CUC2, arrow) and is absent in lines overexpressing the miR164-resistant CUC2-m4 (2x35S::CUC2-m4).

View larger version (18K): [in a new window]   Fig. 5. Restoration of normal flower phenotype upon expression of CUC2-m4 in miR164 overexpressing lines. Sepal fusion was scored in the F1 progeny of a cross between a 35S::miR164 line and STM::ALCR-alcA::erGFP alcA::CUC2-m4 line and is shown here for a representative plant that was ethanol-induced for 5 days (circles) or not induced (triangles). The degree of fusion is expressed on a scale ranging from 0 for fully separated sepals to16 for the strongest sepal fusion phenotype. Scoring on successive flowers was carried out between day 15 and 28 following induction start.

View larger version (154K): [in a new window]   Fig. 6. Expression of miR164-resistant CUC2-m4 leads to progressive boundary enlargement. Expression of erGFP under the control of STM regulatory sequences in developing flowers following 6 days of induction. At the end of the induction period, erGFP was expressed in a strip two or three cells wide between the sepals of a stage 4 STM::ALCR-alcA::erGFP flower (arrow, 1). This domain was enlarged to six to seven cells wide in a STM::ALCR-alcA::erGFP alcA::CUC2-m4 flower at similar stage (arrow, 2). Six days later, one or two cells between the sepals expressed erGFP in a stage 6-7 STM::ALCR-alcA::erGFP flower (arrow, 3). In STM::ALCR-alcA::erGFP alcA::CUC2-m4 flowers, this domain was enlarged to about 10 cells (arrow, 4). In order to realise the observations of (3) and (4), the plants had been induced again overnight to activate erGFP expression. Scale bar: 100 µm.

View larger version (144K): [in a new window]   Fig. 7. The dcl1, hen1 and hyl1 mutants show boundary enlargement. Spacing of the sepals is increased in the mutants (arrows in F,J,N) compared with wild type (B). This defect is already visible at stage 5 of hyl1-1 and dcl1-9 mutants (arrows in G,K). The expression domain of a boundary marker (STM::ALCR-alcA::erGFP) is enlarged in stage 6-7 flowers of hyl1-1 (H) and dcl1-9 (I) mutants compared with wild type (D). Scale bars: 100 µm.

View larger version (57K): [in a new window]   Fig. 8. Proliferation in the sepal boundaries. (A) An alcA::HistoneH4GFP (alcA::H4GFP) construct was introduced into a STM::ALCR alcA::GUS line (1) or into a LFY::ALCR alcA::GUS line (2) in order to obtain inducible H4GFP expression in the boundary domain or in the entire meristem, respectively. As STM-driven expression of the H4GFP extended towards the centre of stage 2 meristems, we considered as part of the boundary only the two marked cells files closest to the primordium (between broken and unbroken blue lines in 1). (3) The mitotic index (MI) in the sepal boundary domain (black bars) and in the entire meristem (white bars) was calculated for floral meristems before (stage 2) during (stage 3) or just after (stage 4) sepal primordia initiation. The four outermost cell layers were analysed and the number of cell counted for each class is indicated below the bars. (B) The sepal boundaries of stage 2-4 flowers were subdivided into boundaries between two sepals (S-S) or between sepals and the meristem (S-M) (1). (2) The MI of these two domains was calculated. The four outermost cell layers were analysed and the number of cells counted for each class is indicated below the bars. (C) The orientation of the division axis of dividing cells relative to the axis of the boundary was measured for the cells of the outermost layer. The division axis was defined as the axis joining the two future daughter cells and is perpendicular to the axis of the metaphase plate or the new cell wall (1,2). The orientation of a S-M dividing cell was calculated relative to the boundary axis (1). The boundary axis was defined as a line tangent to the outer limit of the boundary domain (recognisable as the limit between GFP-expressing and non-expressing cells). The orientation of a S-S dividing cell was calculated relative to the two adjacent boundaries (2). The insets (1,2) show magnifications of the dividing cell. (3) The number of dividing cells was plotted against the orientation of the division axis. Orientations with a high angle value (see 1) are perpendicular to the boundary whereas low values (see 2) are parallel. m, meristem centre; s, sepal primordium.

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