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First published online 22 March 2006
doi: 10.1242/dev.02335

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NUBBIN and JAGGED define stamen and carpel shape in Arabidopsis

¹ Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA.
² Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA.
³ Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany.

View larger version (134K): [in a new window]   Fig. 1. NUB expression during lateral organ development. (A-K) NUB expression detected by in situ hybridization using an anti-sense probe on wild-type tissue. (A-C) Transverse (A,B) and longitudinal (C) cross sections of vegetative shoots. NUB expression starts in leaf primordia during initiation (A) and quickly becomes restricted to the adaxial side (B). NUB expression is not detected in the shoot apical meristem (C). (D) NUB expression is not detected in the inflorescence meristem, early stages of flower meristem development, cryptic bract or sepal primordia. (D-G) NUB expression in stamen primordia is apparent just prior to organ initiation, and soon becomes restricted to the adaxial side after initiation. (E) Stage 5 flower showing NUB expression throughout early stamen primordia. During late stage 5 (F) and stage 6 (G), NUB expression becomes restricted to the adaxial side of stamen primorida. (H) During stage 7, NUB expression becomes restricted to a group of cells in between the two sets of microsporangia (black arrowhead). (G) During gynoecium development, NUB is initially expressed in all cell layers of the valves at stage 6. During stage 7 (H,I), NUB expression becomes restricted to the adaxial side of the valves and is maintained there until stage 8 (not shown). (J) During early stage 9, NUB expression can be detected in ovule primordia. (K) NUB expression is briefly detected in petal primordia at stage 6. ab, abaxial; ad, adaxial; cb, cryptic bract; fm, floral meristem; im, inflorescence meristem; ov, ovule; pe, petal; r, replum; sam, shoot apical meristem; se, sepal; st, stamen; v, valve. Scale bars: 50 µm.

View larger version (32K): [in a new window]   Fig. 2. The nub-1 insertion allele. (A) Diagram of the nub-1 T-DNA insertion allele. (B) Expression of NUB and TUBULIN (TUB) detected by reverse transcription followed by polymerase chain reaction (RT-PCR), using RNA isolated from Col-0 and jag nub inflorescence tissue. (C,D) Phenotypes observed in AP1::NUB-RNAi jag-1 transgenic plants. (C) The valve tissues are often shredded near the apical region of the gynoecium, resulting in exposure of the ovules. (D) Microsporangia development is absent in the anther (compare with Fig. 3D). ov, ovule. Scale bars: 200 µm in C; 50 µm in D.

View larger version (67K): [in a new window]   Fig. 3. Flower and anther development in jag nub mutants. SEM images are of stamens taken from stage 12 flowers. (A-C) Wild type (A), jag (B) and jag nub (C) flowers. Sepal and petal development is similar between jag and jag nub mutant flowers; however, anthers are reduced to stubs of tissue and the distal region of the jag nub gynoecium is abnormal. (D-F) SEM images of the adaxial side of anthers from wild-type (D), jag (E) and jag nub (F) stamens. Four elongated sacs of tissue (microsporangia) are apparent in wild type and jag mutants. In jag nub mutants, however, a small stub of tissue develops instead, with epidermal characteristics of the connective in the apical region and cells of uncertain identity in the basal region. (G) SEM image of the abaxial side of a wild-type anther revealing the connective that develops in between the microsporangia. (H,I) SEM images of epidermal cells taken from the connective region of wild-type anthers (H) and the connective-like region of jag nub anthers (I). (J) SEM image of a stamen from a jag nub+/- mutant showing partial development of the microsporangia. (K-M) Plastic-thin sections (3 µm) of wild type (K), jag (L) and jag nub (M) anthers. (K) In wild-type anthers, four locules develop on the adaxial side separated by the connective on the abaxial side. Black arrowheads indicate dehiscence zones. (L) In jag mutants, two locules appear to develop and will split through a dehiscence zone that develops down the middle of the microsporangia (black arrowheads). (M) In jag nub mutants, no locule or pollen development is apparent. Inset is the same magnification as K and L. an, anther; co, connective; fi, filament; lo, locule; ms, microsporangia. Scale bars: 0.5 mm for A-C; 100 µm for D-G,J; 25 µm for H,I; 50 µm for K-M.

View larger version (66K): [in a new window]   Fig. 4. Characterizing jag nub patterning defects in the anthers using FIL, PHB and SPL expression. (A-D) Expression of FIL detected by in situ hybridization using an anti-sense probe. (A) During wild-type stamen development, FIL is initially expressed throughout the abaxial side at stage 6. (B) As the adaxial microsporangia proliferate, FIL-expressing tissues develop into the connective and grow more slowly in comparison to the adaxial region. Similar to wild type (C), FIL expression in stage 6 jag nub stamens (D) is observed throughout the abaxial half. Although the FIL expression domain is soon dwarfed by the proliferation of the microsporangia in wild type, in jag nub anthers, no microsporangia proliferation is apparent and the FIL expression domain extends towards the adaxial side. (E,F) Expression of PHB detected by in situ hybridization using an anti-sense probe. (E) In wild type, PHB expression is detected in cells marking the dehiscence zone in between the two microsporangia in each pair (black arrowhead), as well as in the vasculature (red arrowhead). (F) In jag nub mutants, PHB expression is only detected in the vasculature. (G-K) SPL expression monitored using the SPL::GUS reporter. In wild type, SPL-reporter activity marks two domains in the anther that develop into the two sets of microsporangia (G, stage 6 shown, white arrowheads) and is maintained in the anthers until anthesis (H, stage 12 shown). (I) In jag nub mutants, the SPL reporter is activated in a similar spatial pattern to wild type. SPL-reporter activity is maintained in jag nub anthers until about stage 11 (J), but then disappears by anthesis (K, stage 12 shown). ab, abaxial side; ad, adaxial side; co, connective; gy, gynoecium; ms, microsporangia. Scale bars: 50 µm.

View larger version (70K): [in a new window]   Fig. 5. Abaxial NUB expression promotes connective growth. (A-D) SEM images of the abaxial side (A-C) and adaxial side (D) of anthers from plants transformed with a FIL::NUB transgene. (A,B) The JD148-B transgenic line develops moderate defects in anther morphology. Anthers from this line develop enlarged connectives (A) that can also be elongated (B). (C,D) The JD148-A transgenic line develops strong defects in anther morphology. Anthers from this line develop enlarged and elongated connectives, as well as extended sheets of abaxial tissue that extend apically beyond the normal limits of the anther (white arrowhead). (E) Plastic-thin section through a stage 10 anther from a FIL::NUB transgenic line that develops strong defects in anther development. Ectopic tissue proliferation can be seen in the connective region (black arrowhead). No pollen development occurs in the ectopic tissue, indicating that this tissue does not have microsporangia identity. (F) FIL expression detected by in situ hybridization using an anti-sense probe. FIL expression can be seen in this stage 7 flower throughout the abaxial side. ad, adaxial; ab, abaxial; co, connective; fi, filament; lo, locules; ms, microsporangia. Scale bars: 50 µm.

View larger version (117K): [in a new window]   Fig. 6. Gynoecium development in wild type and jag nub mutants. (A-H) SEM images of gynoecia from stage 7 (A,B), stage 10 (C,D), stage 11 (E,F) and stage 12 (G,H) flowers from wild type (A,C,E,G) and jag nub mutants (B,D,F,H). Defects in valve growth in jag nub mutants are initially observed as small notches in the gynoecial cylinder (B, white arrowhead). Later, as the gynoecium grows apically, the notches widen and elongate, leading to the exposure of the normally internal ovules (D,F,H). an, anther; gy, gynoecium; r, replum; st, style. Scale bars: 50 µm for A,B; 100 µm for C,D; 200 µm for E-H.

View larger version (88K): [in a new window]   Fig. 7. Valve histogenesis is disrupted in jag and jag nub mutants. (A-C) Plastic-thin sections of valves from gynoecia of stage 13 flowers from wild type (A), jag (B) and jag nub (C) mutants. (A) Wild-type valves develop six tissue layers: an outer epidermal layer (yellow arrowhead), three layers of mesophyll (blue arrowheads), one layer of enb (red arrowhead) and a modified inner epidermal layer termed the ena (green arrowhead). (C) In jag nub mutants, one layer of mesophyll is often missing, and the enb and ena layers lack the characteristics that differentiate them from other tissue layers. (D) Graph showing the effects of the jag and jag nub mutations on cell-layer number in the medial and lateral valve regions. (E) Graph showing the effects of the jag and jag nub mutations on the average cell width in the ena and enb layers of the valves. Error bars indicate s.d. Scale bar: 25 µm.

View larger version (81K): [in a new window]   Fig. 8. Effects of the FIL::NUB transgene on valve growth and cell layer proliferation. (A,B) SEM images of stage 13 gynoecia from the JD148-A transgenic line taken from the replum side (A) and valve side (B). Note the extension of valve tissue into the style region (B, white arrowhead). (C-H) Plastic-thin sections of gynoecia (C,D), sepals (E,F) and petals (G,H) from wild type (C,E,G) and the FIL::NUB transgenic line JD148-A (D,F,H). Tissue layer proliferation is seen in the medial region of the valves (brackets), in irregular locations in sepals (arrowheads) and throughout the petals of JD148-A flowers. Scale bars: 50 µm.

View larger version (83K): [in a new window]   Fig. 9. Leaf phenotypes of jag nub mutants. (A-D) Fourth or fifth leaves from wild-type (A), jag (B) and jag nub (C,D) mutants. (C) Arrowheads point to notches in a jag nub leaf. (E) In jag nub mutants, leaves often develop radial outgrowth (enations, white arrowhead) on the abaxial side. These enations often develop near the tips of leaves and serrations. (F,G) SEM images of an enation emerging from the abaxial mid-vein of a jag nub leaf. Scale bars: 2 mm for A-D; 1 mm for E.

View larger version (13K): [in a new window]   Fig. 10. The effect of abaxial NUB expression on the timing of trichome development in rosette leaves. Trichome number was counted on the abaxial side of leaves from plants that had just bolted. T2 segregating transgenic plants were scored. Four leaves were counted for each leaf class. Trichomes that developed overlying the main vascular bundles were not counted. Col-0, wild type; JD148-A, FIL::NUB transgenic line. Error bars indicate s.d.