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First published online 7 July 2004
doi: 10.1242/dev.01216

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KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium

Commonwealth Scientific and Industrial Research Organization, Division of Plant Industry, Horticulture Unit, PO Box 350, Glen Osmond, SA 5064, Australia

View larger version (95K): [in a new window]   Fig. 1. Features of wild-type and knuckles gynoecia and siliques from plants grown at 25°C. (A) Inflorescences. (B) Drawing depicting wild-type anthesis-stage gynoecium, lateral and medial views, and cross-sectional view through single locule of the ovary. Inset: comparison of wild-type and knu gynoecia at anthesis. (C) Wild-type (left) and knu siliques (right) with examples of protruding ectopic structures (far right). (D) Longitudinal section through immature knu silique showing ectopic stamen and gynoecium. (E) Partially dissected dehiscent knu siliques showing a pellet-like knuckle at left with a teased out structure at right where a secondary ectopic gynoecium is emerging from the first. The arrowhead indicates stem-like umbilicus that attaches knuckle to the replum and pedicel. (F) Replum of dehiscent knu silique with knuckle dissected to show desiccated ovules of primary iteration and ectopic stamens of the secondary iteration. (G) Cross-section through tricarpelloid knuckles silique showing knuckle with arrows indicating at least three iterations of carpel tissue. Scale bars: 10 mm in A; 1 mm in B,G; 5 mm in C,F; 100 µm in D; 200 µm in E. ab, abaxial; ad, adaxial; eg, ectopic gynoecium; eo, ectopic ovule; ep, ectopic primordia/um; es, ectopic stamen; g, gynophore; lat, lateral; med, medial; ov, ovule; pd, pedicel; p, placenta; re, reiteration; r, replum; sp, septum; s, style; tt, transmitting tract; v, valve; wt, wild-type.

View larger version (152K): [in a new window]   Fig. 2. Ectopic floral organs arise from the placentae. (A-E) Longitudinal sections of floral buds, whereas F and G are whole-mounts where images were captured using Nomarski optics. Stages of floral development are indicated in the bottom-left of each panel. (A) Early gynoecium initiation in a wild-type Ws flower bud. (B) Wild-type Ws gynoecium with ovule primordia. (C) knu gynoecium showing ectopic primordium developing from placental tissue. (D) Developing ectopic organs inside the basal one-third of a primary knu pistil. (E) Ectopic primordium interspersed with developing ovules inside a primary knu pistil. (F) Ectopic floral primordium developing inside primary knu pistil. (G) Ectopic carpel and stamen primordia developing inside primary knu pistil. Scale bars: 25 µm in A,E; 50 µm in B-D,F,G. a, anther; ecp, ectopic carpel primordium; ep, ectopic primordium; es, ectopic stamen; fp, floral primordium; gy, gynoecium; op, ovule primordium; se, sepal; st, stamen.

View larger version (66K): [in a new window]   Fig. 3. Pollen development is restored and anthers dehisce in knuckles plants grown at 16°C. (A) Morphological comparison of wild-type and knu anthers at the time of flower opening taken from plants grown at 25°C and 16°C. Lateral and adaxial views are shown. (B) Section through knu anther from an early stage 10 flower taken from a plant grown at 25°C, showing post-tetrad microspores and a semi-intact tetrad. At this stage knu anthers and developing pollen were indistinguishable from wild-type. (C) Cross-section through knu anther taken from the open flower of a plant grown at 25°C. Stamen is non-dehiscent and mature pollen grains are absent. Scale bars: 50 µm in B,C. dpg, degenerating pollen grains; lc, locule; ms, microspore; mit, microspore tetrad.

View larger version (74K): [in a new window]   Fig. 4. Comparison of basal regions of wild-type and knuckles siliques grown at 25°C. Whole green wild-type and knu siliques are shown far left in vertical orientation. (A,B) Details of the basal portions of two wild-type siliques, and C and D show details of two knu siliques in plants grown at 25°C, in horizontal orientation. knu siliques show exaggerated nectary tissue relative to wild-type. The gynophore is extended in D and the green silique has three valves. Scale bars: 500 µm in A-D. g, gynophore; n, nectaries; pd, pedicel; r, replum; v, valve.

View larger version (74K): [in a new window]   Fig. 5. Wild-type and knuckles silique phenotypes at 16°C. (A) Wild-type and knu replae from dehiscent siliques grown at 16°C. (B) Features of a single 16°C-grown dehiscent knu silique. Left, one valve removed to expose bulging septum of un-knuckled side of the replum, abaxial (ab) side of valve visible. Right, both valves removed, with knuckled side of replum and inner (adaxial, ad) faces of spoon-like valves visible. Knuckle (kn) indicated by arrow. Note that seeds have been removed. Scale bars: 5 mm in A,B.

View larger version (29K): [in a new window]   Fig. 6. Motif sequence comparisons between KNUCKLES and other Arabidopsis C2H2 zinc finger proteins. (A) Alignment of C2H2 zinc fingers and adjacent basic residues. The first line consensus shown in red was taken from Evans and Hollenberg (Evans and Hollenberg, 1988). Cysteine and histidine residues responsible for zinc binding are highlighted in yellow. The sub-motif QALGGH is present in a majority of plant zinc finger proteins (Takatsuji, 1999). At5g48890.1, a protein of unknown function, is 77% identical to KNUCKLES through the aligned region. AtZFP10 has been studied by Dinkins et al. (Dinkins et al., 2002), and is part of a family of approximately 30 small, single-fingered proteins identified by those authors and Tague and Goodman (Tague and Goodman, 1995). (B) Alignment of EAR and EAR-like motifs. ZAT11 contains a consensus EAR motif (L/FDLNL/FXP) as defined by Ohta et al. (Ohta et al., 2001), whereas the others are EAR-like as described by Hiratsu et al. (Hiratsu et al., 2002). Residues conserved in all five proteins shown in blue and indicated below alignment by (*); 5/5 conservative by (5), and 4/5 conserved or conservative by (4), 3/5 conserved or conservative by (3). Numbers at right indicate position of motif relative to total protein length. Accession numbers as follows: KNUCKLES (NP_196905), SUPERMAN (S60325), At5g48890.1 (NM_124266), AtZFP10 (AAC23644), ZAT11 (F84792).

View larger version (28K): [in a new window]   Fig. 7. RT-PCR demonstrates that KNUCKLES is transcribed at low levels in green flower buds but not in anthesis-stage flowers. Ethidium bromide-stained 3% TAE agarose gel. First row, 869 bp fragment of ß-tubulin transcript cDNA amplified after 30 cycles. Second row, 332 bp KNU transcript cDNA fragment amplified after 40 cycles, loaded 2:1 relative to volumetric proportion of 50 µL ß-tubulin reaction (ßtub). Positive control amplifications from plasmid templates are denoted (+). Tissue sampled included: floral apices, including green buds up to floral stage 11 (FA), stage 12 buds with petals showing (PS), and open flowers at stage 13 with dehisced anthers (AN), in addition to floral apices from the knuckles mutant (knu).

View larger version (169K): [in a new window]   Fig. 8. KNUCKLES mRNA localization in developing wild-type Arabidopsis flowers. (A-E) Sectioned flower buds or floral organs from buds at the stages indicated at bottom-left of each panel were probed with the short (3') antisense KNU probe, except the section inset in A, which was probed with sense KNU probe as a control. Hybridization of KNU transcripts and antisense probe is indicated by formation of a blue-purple precipitate. Scale bars: 20 µm in A, inset; 50 µm in B,C; 100 µm in D,E. a, anther; cw, carpel wall; f, filament; gy, gynoecial primordium or gynoecium; ov, ovule; op, ovule primordium; pd, pedicel; pe, petal; pn, pollen; se, sepal; st, stamen primordium or stamen; sg, stigma; s, style.

View larger version (28K): [in a new window]   Fig. 9. Complementation and GUS reporter constructs and their effects in transgenic Arabidopsis. (A) Complementation constructs. Plasmids listed far left. Binary T-DNA parent plasmids shown in parentheses. Centre, inserts are depicted as coloured bars. KNU coding sequences are shown in red, and flanking genomic sequences are shown in yellow. Far right, hygromycin-resistant knu transgenics were scored as being fully or partially complemented or uncomplemented based on restoration of fertility and morphological criteria. (B) GUS reporter constructs. The uidA gene is shown as a blue bar. Far right, cartoons representing stage 6 and 9 flower buds, early ovules, and leaf and stem are shaded blue to approximate patterns of GUS staining at the stages depicted in multiple independent lines containing each construct. Scale in kilobases (Kb). H, HindIII; N, NcoI; P, PstI; X, XbaI; Xm, methylation-sensitive XbaI site.

View larger version (119K): [in a new window]   Fig. 10. Expression of KNU:GUS constructions in wild-type and knu genetic backgrounds. The three GUS constructions shown in Fig. 9 were introduced into Arabidopsis wild-type (A-M), and knu mutant backgrounds (N-P). Patterns of GUS activity were examined in whole-mounts or thin sections (D-F,M). (A-C) Inflorescences. (D-G) Representative expression in developing flower buds. (H) Detail of an anther; I-L show details of developing ovules. Images B,D-I,K were taken from primary transgenics, and the remainder from homozygous plants. The introduced construct is named in the top right-hand corner of each panel, and the developmental stage in the bottom-left corner (where relevant). Scale bars: 25 µm in D-F,H-L,M,N; 50 µm in O,P; 100 µm in G; 1 mm in A-C, inset. a, anther; ecp, ectopic carpel primordium; ep, ectopic primordium; esp, ectopic stamen primordia; ems, embryo sac; gy, gynoecium or gynoecial primordium; ii, inner integument of ovule; mmc, megaspore mother cell; met, megaspore tetrad; mit, microspore tetrad; op, ovule primordium; ov, ovules; pe, petal; se, sepal; st, stamen or stamen primordium.