Fig. 2. Non cell-autonomous effects of CLV3 can be suppressed by CLV1. (A-D) In situ hybridizations to seedling meristems. Control hybridizations using corresponding sense riboprobes did not produce any specific staining (not shown). (A,B) LhG4 (A) and WUSGR (B) expression in ATML1::LhG4; pOp::WUS-GR plants is restricted to the epidermis of the SAM (arrow) and young leaf primordia. (C) The endogenous WUS gene is expressed in the centre of the SAM in wild-type seedlings, underneath the outermost three cell layers (arrow). (D) Endogenous CLV3 expression is detected in the presumed stem cells of the SAM in the outermost three cell layers (arrow). (E-J,L-Q) Light micrographs of live plants (E-J,L-O) and GUS-stained, cleared inflorescences (P,Q). (E,F) wus-1 mutant (E) and ATML1::CLV3-expressing clv3-1 mutant (F) seedlings 2 weeks after germination. In both cases, the SAM has terminated (arrow) after the formation of two true leaves. (G) Terminated inflorescence of a wus-1 mutant plant showing a flower that lacks stamens and carpels (arrow). (H) Inflorescence of an ATML1::CLV3-expressing clv3-1 mutant plant. The meristem has terminated (white arrow) after the formation of several flowers which lack the central gynoecium (black arrow). (I) ATML1::CLV3hetSP-expressing seedling with terminated meristem. (J) ATML1::CLV3w/oSP-expressing seedling. Meristem function is unaffected. (K) Sequence alignment of the translated cDNAs for the endogenous CLV3 (CLV3), the CLV3 gene lacking its signal peptide (CLV3w/oSP) and the CLV3 gene fused to the signal peptide of Purple acid phosphatase1 (CLV3hetSP). Identical amino acids are shaded black, similar amino acids are shaded grey. Note the weak sequence similarity between the endogenous CLV3 and the heterologous Purple acid phosphatase1 signal peptides. The lengths of the predicted signal peptides were determined using TargetP [http://www.cbs.dtu.dk (Emanuelsson et al., 2000)]. The arrow indicates the predicted site of cleavage of the signal peptide for CLV3 and CLV3hetSP. (L) ATML1::CLV3; ATML1::CLV1 coexpressing seedlings are indistinguishable from wild type. (M) ATML1::CLV3; ATML1::clv1-4 coexpressing seedling. The meristem has terminated as in E. (N,O) Inflorescences of (N) ATML1::CLV3; ATML1::CLV1- and (O) ATML1::CLV3; ATML1::clv1-4-expressing plants. In both cases, the inflorescence meristem is self-maintaining, however, some flowers in O lack a gynoecium (arrow). (P,Q) Inflorescences of ATML1::CLV3 (P) and ATML1::CLV3; ATML1::CLV1 (Q)-expressing plants with strong GUS staining from the ATML1::GUS reporter that is linked to the ATML1::CLV3 gene. (R,S) CLSM images. Signal from GFP fluorescence is shown in green, chlorophyll autofluorescence is in red. (R) ATML1::CLV3-GFP plant with an even gradient of fluorescence extending from the epidermis to the centre of the meristem. (S) ATML1::CLV3-GFP; ATML1::CLV1 coexpressing plant with strong fluorescence in the epidermis of the inflorescence meristem, yet only very weak signal in the underlying cell layer. Note that in (R) and (S), strong GFP fluorescence is only visible in shoot and floral meristems, even though the ATML1 activator is expressed in the epidermis throughout the aerial part of the plant (compare with P,Q). This lack of a signal could either be due to weaker ATML1 promoter activity or to a post-transcriptional regulation of CLV3 expression outside of the SAM. Scale bars: 50 µm (A-D,R,S): 1 mm (E-J,L-Q).

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