Evolutionarily conserved repressive activity of WOX proteins mediates leaf blade outgrowth and floral organ development in plants

Abstract

The WUSCHEL related homeobox (WOX) genes play key roles in stem cell maintenance, embryonic patterning, and lateral organ development. WOX genes have been categorized into three clades--ancient, intermediate, and modern/WUS--based on phylogenetic analysis, but a functional basis for this classification has not been established. Using the classical bladeless lam1 mutant of Nicotiana sylvestris as a genetic tool, we examined the function of the Medicago truncatula WOX gene, STENOFOLIA (STF), in controlling leaf blade outgrowth. STF and LAM1 are functional orthologs. We found that the introduction of mutations into the WUS-box of STF (STFm1) reduces its ability to complement the lam1 mutant. Fusion of an exogenous repressor domain to STFm1 restores complementation, whereas fusion of an exogenous activator domain to STFm1 enhances the narrow leaf phenotype. These results indicate that transcriptional repressor activity mediated by the WUS-box of STF acts to promote blade outgrowth. With the exception of WOX7, the WUS-box is conserved in the modern clade WOX genes, but is not found in members of the intermediate or ancient clades. Consistent with this, all members of the modern clade except WOX7 can complement the lam1 mutant when expressed using the STF promoter, but members of the intermediate and ancient clades cannot. Furthermore, we found that fusion of either the WUS-box or an exogenous repressor domain to WOX7 or to members of intermediate and ancient WOX clades results in a gain-of-function ability to complement lam1 blade outgrowth. These results suggest that modern clade WOX genes have evolved for repressor activity through acquisition of the WUS-box.

Fig. 1.

Phenotypes of lam1 mutant and expression level-dependent complementation by STF. (A–H) Phenotypes of tobacco WT and lam1 plants at different developmental stages. (A and B) One-wk-old WT and lam1 seedlings showing cotyledons and the emerging first leaves. (Scale bars: 1 mm.) (C and D) Cross-sections of first leaves from One-wk-old WT and lam1 plants. (Scale bars: 100 μm.) (E and F) Six-wk-old vegetative-stage WT and lam1 plants. (Scale bars: 1 cm.) (G and H) Flowering-stage WT and lam1 plants. (Scale bars: 10 cm.) (I–K) Leaf phenotypes of different STF::STF-complemented lam1 lines. (Scale bars: 1 cm.) (L–N) Petal phenotypes of transgenic lines corresponding to those in I–K. (Scale bars: 1 cm.) (O–Q) Female floral organs of transgenic lines corresponding to those in I–K. (Scale bars: 1 cm.) (R) Quantitative RT-PCR showing expression levels of STF in transgenic lines corresponding to I–K, normalized to the expression of Ubiquitin. Error bars represent mean ± SD (n = 3). *P < 0.05; **P < 0.01 (one-way ANOVA, Dunnett’s test).

Fig. 2.

STF acts mainly as a repressor in blade outgrowth. (A) Constructs used in transient expression assay. (B) Amino acid sequence of the WUS-box in STF and mutations introduced into the WUS box (STFm1), indicated by frames. (C) Relative luciferase activities using STF or STFm1 as effector compared with GAL4-DB control. Error bars indicate SD (n = 3). **P < 0.01 (one-way ANOVA, Dunnett’s test). (D) Constructs used for complementation of lam1 mutant. (E–L) Transgenic lam1 plants complemented with STF::STF (E and I), STF::STFm1 (F and J), STF:: SRDX-STFm1 (G and K), and STF::STFm1-VP16 (H and L). (Scale bars: 5 cm.)

Fig. 3.

Functional complementation of lam1 by WOX family genes. (A–L) Complementation of the lam1 mutant with Arabidopsis WOX family genes. Six-wk-old whole plants and leaves of lam1 mutant after transformation with STF::WUS (A), STF::WOX1 (B), STF::WOX2 (C), STF::WOX3 (D), STF::WOX4 (E), STF::WOX5 (F), STF::WOX6 (G), STF::WOX7 (H), STF::WOX9 (I), STF::WOX11 (J), STF::WOX13 (K), and STF::GUS (L). (Scale bars: 5 cm.) (M) Amino acid sequence of the WUS-box in WUS. Mutations (L to A amino acid substitutions) introduced into the WUS box (WUSm1) are indicated in red. (N) Schematic representation of the chimeric constructs used for transformation. (O–Q) Plant and leaf phenotypes of lam1 complemented with STF::WUSm1 (O), STF::SRDX-WUSm1 (P), and STF::WUSm1-VP16 (Q). (Scale bars: 5 cm.) (R–T) Transverse sections through the leaves of transgenic lam1 transformed with STF::GUS (R), STF::WUSm1-VP16 (S), and STF::WOX9 (T). (Scale bars: 100 μm.)

Fig. 4.

WUS-box and repressive activity required for leaf blade outgrowth are conserved only in the modern clade WOX family members. (A) Sequence alignment of the homeodomain and WUS-box of STF and Arabidopsis WOX proteins. The conserved WUS-box core sequence is shown below the alignment. (B) Phylogenetic analysis of STF and Arabidopsis WOX proteins using homeodomain sequences. The three evolutionary clades are statistically supported by bootstrap values. (C) Relative luciferase activities in Arabidopsis leaf protoplasts using the indicated genes driven by the 35S promoter as effector plasmids. Error bars indicate SD (n = 3). **P < 0.01 (one-way ANOVA, Dunnett’s test). (D) Schematic representation of the chimeric constructs used for transformation. (E–K) Phenotypes of transgenic lam1 plants and leaves complemented with STF::WOX7-WB (E), STF::SRDX-WOX7 (F), STF::WOX9-WB (G), STF::SRDX-WOX9 (H), STF::WOX13-WB (I), STF::SRDX-WOX13 (J), and STF::GUS (K). WB represents WUS-box. (Scale bars: 5 cm.)