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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).
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