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First published online 19 October 2005
doi: 10.1242/dev.02067

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Recruitment of CRABS CLAW to promote nectary development within the eudicot clade

¹ Section of Plant Biology, University of California Davis, Davis, CA 95616, USA
² Department of Biology, Duke University, Durham, NC 27708, USA
³ Department of Plant Sciences, University of California Davis, Davis, CA 95616, USA

View larger version (45K): [in a new window]   Fig. 1. Phylogenetic distribution of nectaries in angiosperms. Non-eudicot angiosperms have floral nectaries in the perianth, or nectary tissues are not conspicuously developed. In eudicots, floral nectaries are mostly associated with reproductive organs, although some examples of perianth nectaries exist. The angiosperm phylogeny is based on that of Soltis et al. (Soltis et al., 2000). Orders with extrafloral nectaries are marked bold with an asterisk. Extrafloral nectaries are found only within eudicots and monocots. The extrafloral nectary distribution is based on that of Elias (Elias, 1983) and locations of floral nectaries are primarily based on those of Brown (Brown, 1938) and others (Endress, 1986; Endress, 2001; Proctor et al., 1996; Ronse DeCraene, 2004; von Balthazar and Endress, 2002). Groups reported as not having nectaries are indicated (–). Austrobaileyaceae and Amborellaceae were marked with a question mark, as the possible nectar secretion on the tip of carpels has not been characterized. Taxa used in this study and the positions of nectaries in these taxa are indicated. ax, leaf axil (Capparis flexuosa); le, abaxial midvein of leaf (Gossypium hirsutum); se, sepal (Gossypium hirsutum); pe, petal (Aquilegia formosa); st, stamen (Arabidopsis thaliana, Lepidium africanum, Cleome sparsifolia); ca, carpel (Petunia hybrida, Nicotiana benthamiana, Grevillea robusta).

View larger version (35K): [in a new window]   Fig. 2. Phylogenetic analysis of YABBY genes. Bayesian phylogenetic tree based on the nucleotide sequences of the zinc finger and YABBY domains of the YABBY family gene members. Posterior probabilities for clades are shown above branches.

View larger version (113K): [in a new window]   Fig. 3. Nectaries of the Brassicales. Nectaries and expression of CRC orthologs in Lepidium africanum (A-D), Cleome sparsifolia (E-G) and Capparis flexuosa (H-P). (A) Floral nectaries (n) of Lepidium africanum are located between stamens and petals (p). Nectary glands flank each of the two stamens, such that each flower has four glands. (B) Nectary glands have apical stomata (arrow). (C,D) LaCRC is expressed in all cells of the nectary glands (arrow in C), from inception beyond anthesis. (E,F) Cleome sparsifolia floral nectaries (arrow) form a ring at the abaxial bases of the stamens. (G) CsCRC is expressed at high levels throughout nectaries. (H,I) Extrafloral nectaries of Capparis flexuosa (arrows) are located in leaf axils and are covered with scales, dissected away in I. (J) Longitudinal section of an extrafloral nectary reveals the internal anatomy (arrow). An axillary bud is adjacent and below the extrafloral nectary. (K) The covering sheath is composed mostly of thickened collenchyma cells and fibers, and the nectary has stomatal openings on its surface (arrows). (L) Nectaries comprise mostly parenchyma cells with phloem traces containing sieve tube members and companion cells. (M-P) Before nectary tissue develops inside the sheath, CfCRC expression is not observed (M, the dark spot is not signal). As nectary tissue initiates development, CfCRC expression increases dramatically and levels remain high throughout the nectary (N,O). As nectary growth ceases CfCRC expression decreases and only a minimal expression level remains (P).

View larger version (69K): [in a new window]   Fig. 4. The extrafloral nectaries of Gossypium. (A) Extrafloral nectaries are located on midveins on the abaxial sides of the leaves. (B) The secretory structure is composed of trichomes that densely populate the concave region of the midvein. (C,D) Secretory trichomes are multicellular and small cytoplasmically dense parenchyma cells are located below the trichomes. (E) Expression of GhCRC in an involucral bract nectary localized by in situ PCR with a green signal representing the fluorescence signal from in situ PCR products of GhCRC. (F) GhCRC is highly expressed in the papillar cells and subtending cells in a leaf nectary. (G) Expression of GhCRC during extrafloral nectary development as monitored by semi-quantitative RT-PCR. 1-29, G. hirsutum; 30-32, G. tomentosum; left lane, actin; right lane, GhCRC (or GtCRC) amplified from same cDNA for actin under the same PCR condition. The upper bands in lanes 27-29 are from genomic DNA; the lower bands represent cDNA. 1, 3 mm whole leaf; 2, 5 mm whole leaf; 3, 1 cm whole leaf; 4, 1.4 cm leaf blade; 5, 1.4 cm leaf vein; 6, 2.8 cm leaf blade; 7, 2.8 cm leaf vein; 8, 2.8 cm leaf nectary; 9, 7 cm leaf blade; 10, 7 cm leaf vein; 11, 7 cm leaf nectary; 12, 12 cm leaf blade; 13, 12 cm leaf vein; 14, 12 cm leaf nectary; 15, bract nectary of 1.5 cm flower; 16, rest of bracts; 17, sepals; 18, petals; 19, stamens; 20, carpels; 21, bract nectary of 6 cm long flower; 22, rest of bracts; 23, sepals; 24, petals; 25, stamens; 26, carpels; 27, root of seedlings; 28, hypocotyl of seedlings; 29, apex of seedlings; 30, root of seedlings; 31, hypocotyl of seedlings; 32, apex of seedlings.

View larger version (108K): [in a new window]   Fig. 5. Floral nectaries of Petunia hybrida and Nicotiana benthamiana. (A) The Petunia hybrida nectary develops as a disc at the base of the ovary but the gland (arrows) is more pronounced where the carpels are fused. (B-D,F-H,J) Longitudinal sections; (E,I,K) transverse sections. (B) PhCRC expression (blue-purple color) initiates in carpel primordia, specifically in carpel walls. s, sepal; pe, petal; st, stamen; pl, placenta; cw, carpel wall. (C) PhCRC expression is abaxial in both carpel walls and placenta. (D) Abaxial PhCRC expression in the carpel wall and placenta is stronger in the basal regions. PhCRC expression is also observed at the tips of the ovary walls, from which the style and stigma will develop. (E) Abaxial expression is present in the carpel wall and placenta, but no expression is detected in margins of fusion of the two carpels, similar to CRC expression in Arabidopsis. (F) As the style and stigma begin to develop PhCRC is expressed at the tip of the ovary. Abaxial expression in the basal region of carpel walls persists (arrows) but expression in the placenta has diminished. (G) PhCRC expression has ceased except at the base of the carpel walls (arrow) where the nectary will develop in this stage 0 flower (Izhaki et al., 2002). (H) Expression patterns in stage 1 flowers are similar to that of stage 0 flowers with higher expression at the base of ovary wall (arrow). (I) PhCRC expression is present throughout developing nectaries in stage 4 flowers. The two lobes of the nectary are indicated by arrows. (J) PhCRC expression is very strong and specific to the nectary (arrows) in stage 6 flowers. (K) PhCRC expression levels decrease after anthesis but persist throughout the nectary in stage 8 flowers. (L) VIGS-mediated silencing of CRC results in a loss of nectaries in Nicotiana benthamiana. In wild type (flower on left), nectaries are clearly visible as an orange ring of tissue at the base of the ovary (arrow). When CRC function is reduced, a progression of severity in phenotype is observed, with mildly affected flowers having some nectary tissue (second flower from left, arrow) and severely affected flowers having little or no nectary tissue (two flowers on right). (M) VIGS-mediated silencing of CRC results in a loss of flower meristem determinacy in Petunia hybrida flowers, but little visible effect on nectary development (arrow).

View larger version (111K): [in a new window]   Fig. 6. Change versus conservation of CRC regulation. (A-E) Expression of pGhCRC(8kb)::GUS in Arabidopsis. GUS expression is detected in the vasculature of the hypocotyl and cotyledons in 1-week-old seedlings (A), in rosette leaves (B) and in cauline leaves (C) of older plants. GUS expression is also detected in the pedicels and receptacles of flowers (D), and the styles and stigma of gynoecia (E). (F) pLaCRC::GUS in the Arabidopsis nectary in a stage 15 flower. (G) pAtCRC::GhCRC is sufficient to rescue the nectary and carpel phenotypes in a crc-1. GUS expression in the nectary is provided by a pAtCRC::GUS transgene. (H) pAtCRC::GUS in Nicotiana tabacum flower. GUS expression is in the nectary disc (arrow) and style of the gynoecium.

View larger version (99K): [in a new window]   Fig. 7. Nectaries and carpels of Aquilegia formosa flowers. (A) Owing to growth in the pedicel, the nectar spurs of Aquilegia formosa flowers, which develop from the abaxial side of the petals, point upwards (arrow). (B) As carpel (c) primordia emerge, AfCRC expression (B-F) is restricted to the abaxial side as seen in this flower. st, stamen; p, petal; se, sepal. (C) AfCRC expression precedes the emergence of carpels. As carpel primordia begin to elongate, AfCRC expression is excluded from the vasculature and restricted to cells adjacent to the vasculature (D,E). In developing carpels, AfCRC expression is specifically localized to a ring of cells surrounding the vasculature in the abaxial regions (E,F). (G) Anatomy of the carpel. Each of the five carpels is separate with ovules initiating from the margins of the carpels. AfCRC expression is excluded from the vasculature (arrow) but is expressed in cells surrounding it. (H,I) Anatomy of the nectar spur. The tip of the developing nectar spur consists of densely cytoplasmic parenchymatous cells, characteristic of nectary tissue (arrow in H). (J) No detectable AfCRC expression was observed in developing nectar spurs (arrow).