Evidence that CRABS CLAW and TOUSLED have conserved their roles in carpel development since the ancestor of the extant angiosperms

Abstract

The carpel is the female reproductive organ specific to flowering plants. We aim to define the genes that controlled carpel development in the common ancestor of this group as a step toward determining the molecular events that were responsible for the evolution of the carpel. CRABS CLAW (CRC) and TOUSLED (TSL) control important aspects of carpel development in the model plant, Arabidopsis thaliana. The basal angiosperm species Amborella trichopoda and Cabomba aquatica very likely represent the two most early diverging groups of flowering plants. We have identified putative orthologues of CRC and TSL from A. trichopoda and C. aquatica, respectively. We demonstrate the expression patterns of these genes in carpels to be very highly conserved, both spatially and temporally, with those of their Arabidopsis orthologues. We argue that CRC and TSL in Arabidopsis are likely to have conserved their respective roles in carpel development since the common ancestor of the living flowering plants. We conclude that a divergent role shown for the CRC orthologue in rice, DROOPING LEAF, most probably arose specifically in the monocot lineage. We show that, in addition to its expression in carpels, the TSL orthologue of C. aquatica is expressed in tissues that contribute to buoyancy and argue that its role in these tissues may have arisen later than its role in carpel development.

Fig. 1.

Comparisons of predicted protein domains in CRC and AmbCRC (A) and TSL and CabTSL (B). CC, coiled-coil domain; CAT, protein kinase catalytic domain; NLS, nuclear localization signal; Q, glutamine-rich region; Y, YABBY (DNA-binding) domain; Zf, zinc-finger domain.

Fig. 2.

Phylogenetic analysis of AmbCRC with other predicted YABBY proteins. The tree shown was constructed by the neighbor joining method (37) from an alignment corresponding to amino acid residues 19–55 (the zinc-finger domain) and 110–157 (the YABBY domain) of Arabidopsis CRC. Arabidopsis sequences are underlined, and percentage support for nodes in 500 bootstrap replicates are encircled. Unpublished sequence accession numbers are as follows: AY451399 (Antirrhinum), AY703987 (Lepidium), AY071845 (Nicotiana), and AF545436 (wheat).

Fig. 3.

Northern and virtual Northern blot hybridizations of AmbCRC, CabTSL, and their Arabidopsis orthologues. Hybridizations are to virtual Northern blots of A. trichopoda and polyadenylated RNA Northern blots of C. aquatica and A. thaliana. Hybridizations to a cDNA encoding GAPDH from each species are included to demonstrate equivalent loading of tracks. F, female flowers; I, inflorescences; L, leaves; M, male flowers; RL, rosette leaves; and V, vegetative tissues (submerged leaves and stems).

Fig. 4.

Nonradioactive in situ hybridizations showing expression of AmbCRC in A. trichopoda and CabTSL in C. aquatica, with accompanying images. In situ hybridization signals appear blue or violet. Very dark material in tepals of A. trichopoda is natural coloration. All gene expression signals referred to were specific to antisense-strand riboprobes and were not observed by using negative control sense-strand riboprobes on serial sections from the same tissue blocks (results not presented). (A) A female flower of A. trichopoda. (B) In situ hybridization to a longitudinal section (l.s.) of an A. trichopoda female flower bud showing AmbCRC expression in the carpel wall. (C) SEM of a female A. trichopoda flower bud with the perianth removed (stage as for B). (D) SEM of a mature A. trichopoda female flower. (E) In situ hybridization to an l.s. of an A. trichopoda male flower bud showing AmbCRC expression in the stamen filaments. (F) In situ hybridization to an l.s. of a C. aquatica flower bud showing expression of CabTSL in the style and stigma. (G) SEM of the C. aquatica gynoecium (stage as for F). (H) In situ hybridization to a transverse section (t.s.) of a 3-mm-long C. aquatica flower bud showing expression of CabTSL in the style and stigma. (I) In situ hybridization to an l.s. of a 4-mm-long C. aquatica flower bud showing expression of CabTSL in the stigma. (J) SEM of the stigma and upper style of C. aquatica (stage as for I). (K) In situ hybridization to a t.s. of a C. aquatica flower bud showing expression of CabTSL in the anther wall. (L) SEM of a C. aquatica flower bud showing the stamens (stage as for K). (M) A flower and dimorphic leaves of C. aquatica.(N) In situ hybridization to a t.s. of a C. aquatica flower peduncle showing expression of CabTSL in cells surrounding air canals. (O) In situ hybridization to an l.s. of a C. aquatica floating leaf showing expression of CabTSL in cells surrounding air canals in the petiole. a, anther; ac, air canal; al, anther loculus; c, carpel; d, dehiscence zone; f, stamen filament; fb, floating leaf blade; o, ovary; ov, ovule; p, perianth; pd, peduncle; pe, petiole; r, receptacle; sc, stylar canal; sm, staminode; st, stigma; su, submerged leaves; sy, style; t, tepal.

Fig. 5.

Mapping of CRC and TSL expression in gynoecium tissues onto the phylogenetic tree of the angiosperms. The Amborella and Cabomba (Nymphaeales) lineages are placed in equally basal positions to reflect uncertainty in their order of branching (2).