Functional conservation between CRABS CLAW orthologues from widely diverged angiosperms

Abstract

Background and aims: CRABS CLAW (CRC) encodes a transcription factor of the YABBY family that plays important roles in carpel and nectary development in Arabidopsis thaliana. Combined evolutionary and developmental studies suggest an ancestor of the CRC gene to have controlled carpel development in the last common ancestor of the angiosperms. Roles for CRC orthologues in leaf development and carpel specification in rice, and in nectary development in core eudicots, have accordingly been interpreted as derived. The aim of this study was to assess the capacity of CRC orthologues from a basal angiosperm and from rice to complement CRC mutants of arabidopsis. These experiments were designed to test the hypothesized ancestral role of CRC in the angiosperms, and to indicate whether putatively novel roles of various CRC orthologues resulted from changes to their encoded proteins, or from other molecular evolutionary events.

Methods: The crc-1 mutant of arabidopsis was genetically transformed with the coding sequences of various CRC orthologues, and with paralogous YABBY coding sequences, under the control of the arabidopsis CRC promoter. The phenotypes of transformed plants were assessed to determine the degree of complementation of the crc-1 mutant phenotype in carpel fusion, carpel form and nectary development.

Key results: The CRC orthologue from the basal angiosperm Amborella trichopoda partially complemented the crc-1 mutant phenotype in carpels, but not in nectaries. The CRC orthologue from rice partially complemented all aspects of the crc-1 mutant phenotype. Though most non-CRC YABBY coding sequences did not complement crc-1 mutant phenotypes, YABBY2 (YAB2) proved to be an exception.

Conclusions: The data support a hypothesized ancestral role for CRC in carpel development and suggest that novel roles for CRC orthologues in monocots and in core eudicots resulted principally from molecular changes other than those affecting their coding sequences.

Fig. 1.

GUS and GFP reporter gene expression under the control of the CRC gene promoter. (A) GUS staining in the gynoecium and nectaries (ne) of a flower from a proCRC::GUS transformant at developmental stage 11–12 (Smyth et al., 1990), showing the expected CRC-like pattern of reporter gene expression. (B) GFP localization in the gynoecium of a flower from a proCRC::GFP transformant at developmental stage 10–11 (Smyth et al., 1990), showing the expected CRC-like pattern of reporter gene expression. (C) GUS staining in leaf tissue of a proCRC::GUS transformant showing reporter gene activity outside of the normal zone of CRC expression. (D) GUS staining in mature flower tissues (se = sepal, st = stamen) in a proCRC::GUS transformant showing reporter gene activity outside of the normal zone of CRC expression.

Fig. 2.

Complementation of the crc-1 mutation by transformation with various YABBY coding sequences under the control of the CRC promoter. (A, D, G, J, M, P) Silique apices showing degrees of carpel fusion in wild-type plants (A), crc-1 mutants (D) and crc-1 mutants transformed with the YABBY coding sequences CRC (G), AmbCRC (J), DL (M) and YAB2 (P). Scale bars = 1 mm. (B, E, H, K, N, Q) Fully elongated siliques in wild-type plants (B), crc-1 mutants (E) and crc-1 mutants transformed with the YABBY coding sequences CRC (H), AmbCRC (K), DL (N) and YAB2 (Q). Scale bars = 5 mm. (C, F, I, L, O, R) The base of the third floral whorl, showing the presence or absence of nectaries (ne), in mature flowers of wild-type plants (C), crc-1 mutants (F) and crc-1 mutants transformed with the YABBY coding sequences CRC (I), AmbCRC (L), DL (O) and YAB2 (R).

Fig. 3.

Phenotypic effects and expression of DL in leaves and other organs in a proportion of proCRC::DL transformants. (A) A mature plant transformed with proCRC::DL (corresponding to plant 1 in Fig. 3G), showing normal development of leaves and inflorescence architecture. (B) A seedling transformed with proCRC::DL, showing aberrant leaf development. (C) A mature plant transformed with proCRC::DL (corresponding to plant 9 in Fig. 3G), showing aberrant leaf development and plant architecture. (D) The underside of a leaf from the proCRC::DL transformant shown in (C). (E) A pro35S::CRC transformant showing a leaf phenotype similar to that of some proCRC::DL transformants. (F) Mature rosette leaves, showing the effect of overexpression of CRC and DL transgenes in leaf tissue. (G) Northern hybridization of a DL cDNA probe to RNA from leaves and inflorescences of three proCRC::DL transformants (plants 1, 3 and 5) that showed normal leaf development, and to RNA from leaves of three proCRC::DL transformants (plants 9, 13 and 33) that showed aberrant leaf development. High levels of DL expression in leaves correlate with aberrant leaf phenotypes.