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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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