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First published online 13 March 2008
doi: 10.1242/dev.012807

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The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis

Sarah M. McKim¹, Grethe-Elisabeth Stenvik², Melinka A. Butenko², Wenche Kristiansen², Sung Ki Cho³, Shelley R. Hepworth^1,*, Reidunn B. Aalen² and George W. Haughn^1,

¹ Department of Botany, University of British Columbia, Vancouver, British Columbia, V6T 1Z4, Canada.
² Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, N-0316 Oslo, Norway.
³ Division of Biological Sciences, 303 Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.

View larger version (48K): [in this window] [in a new window]   Fig. 1. Abscission phenotype and petal breakstrength of bop1 bop2. (A) Inflorescences of wild-type Arabidopsis plants (Col-0, left) abscise floral organs while bop1 bop2 plants (right) retain their floral organs. (B) Abscission in wild-type flowers occurs between positions 5 and 7. (C) Floral organs of bop1 bop2 never abscise. (D) Petal breakstrength measurements of wild-type flowers (black bars) decrease with floral age, while bop1 bop2 flowers (grey bars) show no change in force over time. Error bars represent s.d. n=30 per genotype.

View larger version (166K): [in this window] [in a new window]   Fig. 2. Morphology of floral AZs in wild type (Col-0) and bop1 bop2. (A,B) Scanning electron micrographs of Arabidopsis petal AZs. The fracture plane on the receptacle (arrow) was observed following removal or natural abscission of petals in wild-type (A) and bop1 bop2 (B) flowers at positions 2, 4, 6 and 12. (A) Wild-type fracture planes demonstrate progression from broken cells (position 2) to rounded AZ cells (position 6) to protective surface cells (position 12). (B) Fracture planes of bop1 bop2 petals from all positions show broken cells. (C-J) Toluidine Blue-stained sections from flowers at anthesis. (C-E) AZ cells at the stamen filament-receptacle (C), petal-receptacle (D) and sepal-receptacle (E) junctions in wild type (arrows). (F) Longitudinal section along the adaxial and abaxial faces of wild-type flower. (G-I) Receptacle-organ boundaries of stamen filaments (G), petals (H) and sepals (I) of bop1 bop2 lack cells displaying AZ anatomy (arrows). (J) Abaxial petalloid sepal of bop1 bop2 lacks an organ-receptacle boundary. f, stamen filament; p, petal; s, sepal; ad, adaxial; ab, abaxial. Scale bars: 400 µm in A,B; 35 µm in C-E,G-I; 70 µm in F,J.

View larger version (211K): [in this window] [in a new window]   Fig. 3. Morphology of vestigial AZs in wild type (Col-0) and bop1 bop2. (A-F) Scanning electron micrographs of stem fracture planes in Arabidopsis following removal of cauline leaves. (A) Fracture plane in wild type shows ruptured cells following removal of green leaves. (B) Removal of 50% yellow leaves reveals a few rounded AZ cells. (C) Fracture planes from yellow leaves often show many rounded AZ cells. Stipules always flank the fracture plane (A, arrow). (D-F) Corresponding fracture planes of bop1 bop2 from green (D), 50% yellow (E) and yellow (F) cauline leaves show no development of rounded AZ cells or evidence of flanking stipules. (G,H) A furrow of narrowed cells (arrows) flanked by stipules (H, arrowhead) is observed between the abaxial surface of the cauline leaf and the stem in wild type. (I) Toluidine Blue-stained longitudinal sections show darkened files of cells that mark the vestigial AZ (arrow) at the adaxial boundary between the cauline leaf and stem in wild type and at the branching point with the primary inflorescence stem (arrowhead). The leaf-steam boundary furrow is also apparent (asterisk). (J,K) bop1 bop2 cauline leaves lack stipules and leaf-stem boundary furrow. (L) No vestigial AZs are visible in bop1 bop2 sections. as, axillary shoot; cl, cauline leaf; st, primary inflorescence stem. Scale bars: 600 µm in A-H,J,K; 35 µm in I,L.

View larger version (139K): [in this window] [in a new window]   Fig. 4. Expression of BOP1::GUS. (A-I) Whole mounts of BOP1::GUS (Col-0) transgenic Arabidopsis plants stained for GUS activity. (A) Expression is restricted to the base of developing organs in stage 9 and 10 flowers. (B) Expression at the base of floral organs and in lateral nectaries of a stage 12 flower (arrow). (C) GUS activity at the base of floral organs, sepal vasculature, style and pollen of stage 14 flowers. (D) Newly abscised petal and stamen show expression in their AZs and filaments. (E) Position 9 siliques show AZ expression in the receptacle AZ. (F) Young pedicel-stem junctions have an abaxial GUS signal that spreads around the attachment point in older pedicels (G). (H) BOP1::GUS bop1 bop2 plant with GUS activity at the base of the ectopic bract (arrow). (I) Cauline leaves show GUS activity at their base. (J-N) Sections of stained BOP1::GUS flowers. (J) Stage 5/6 flower showing GUS activity in stamen primordia (asterisk) and sepals. (K) Staining of a stage 7/8 flower is restricted to the base of stalked stamens (asterisk) and outgrowing sepals. (L) Section through the same stage 7/8 flower showing staining throughout the petal primordia (asterisk). (M,N) Mature flowers with GUS activity through the AZ (M) and nectaries (N). Scale bars: 250 µm in A,B,D-F; 0.5 mm in C; 1 mm in G-I; 35 µm in J-N.

View larger version (84K): [in this window] [in a new window]   Fig. 5. Suppression of 35S::IDA phenotype in bop1 bop2. (A-D) Arabidopsis flowers from positions 3, 7 and 10. (A) Wild type (C24) shows floral organ abscission by position 7. (B) 35S::IDA (C24) with premature abscission of floral organs by position 3. (C) bop1 bop2 lacks floral organ abscission. (D) 35S::IDA bop1 bop2 (Col-0) showing no floral organ abscission. (E) Cauline leaves do not abscise in bop1 bop2. (F) Cauline leaves of 35S::IDA show ectopic abscission. (G) 35S::IDA bop1 bop2 cauline leaves resemble bop1 bop2. (H) RT-PCR from cDNA derived from rosette leaves, buds and flowers from bop1 bop2 and 35S::IDA bop1 bop2. Upper panel shows RT-PCR with IDA primers. IDA is expressed in bop1 bop2 flowers only. 35S::IDA bop1 bop2 plants show expression in rosette leaves, buds and flowers. lower panel, RT-PCR of ACTIN2-7 (ACT) as a positive control. Lane 1 is a genomic PCR control (g). l, leaves; b, buds; f, flowers.

View larger version (126K): [in this window] [in a new window]   Fig. 6. Expression of IDA::GUS, BAC::GUS and CHIT::GUS in wild type and bop1 bop2. Arabidopsis floral positions are indicated in the upper right of panels. (A) Wild-type (C24xCol-0) flowers at position 3 show very weak IDA::GUS activity in the base of floral organs that intensifies throughout position 4 and 5. (B) IDA::GUS expression in bop1 bop2 (Col-0xC24) is detected at position 4 and more strongly in position 5. (C,D) Dark-field microscopy of stained IDA::GUS flowers in wild type (C) and bop1 bop2 show staining throughout the proximal sepal. (E,F) BAC::GUS in wild type (Col-0) or bop1 bop2 at positions 1, 3-6 stained for GUS activity. (E) BAC::GUS plants show strong staining at the base of all floral organs, especially concentrated in the vasculature of position 1 and 3 flowers. Activity persists in position 4 and 5 flowers during abscission and is visible in single AZ cells (arrow). (F) BAC::GUS bop1 bop2 flowers at position 1 show weak staining at the bases of petals and stamens, and strengthens and enlarges by position 3. Very slight expression is detected on the adaxial side of sepals in position 4. (G,H) CHIT::GUS expression from flowers in wild type (Col-0) or bop1 bop2 at positions 3-7. (G) CHIT::GUS position 3 and 4 flowers demonstrate weak GUS staining, while positions 5 and 6 exhibit very strong AZ staining. (H) CHIT::GUS bop1 bop2 position 3 and 4 floral organs show weak GUS activity at their base. GUS activity is stronger in position 5. Staining is maintained in position 6 flowers extending further into the base of these floral organs. Arrowheads indicate expression. f, stamen filament; p, petal; s, sepal. Scale bars: 0.5 mm in A,B,E-H; 35 µm in C,D.

View larger version (88K): [in this window] [in a new window]   Fig. 7. Nectary morphology and CRC::GUS activity in wild type (Col-0) and bop1 bop2. (A,B) Sepals and petals in Arabidopsis were removed from wild-type (A) and bop1 bop2 (B) position 4 flowers. Lateral nectary protrusions are obvious in wild type (arrow) but not in bop1 bop2. (C) Pair of lateral nectary glands with secretory stomata (arrow) in wild type. (D) Lateral nectary glands absent in bop1 bop2. (E-H) Toluidine Blue-stained sections from position 3 flowers. (E) Wild-type transverse section shows paired lateral nectary glands with distinct epidermal and starch-containing parenchymal regions, a ridge of connecting nectary tissue (arrow) and a medial nectary gland. (F) Corresponding bop1 bop2 cross-section shows two primordia (arrows) in the lateral nectary position. (G) A lateral nectary gland visible in longitudinal section of wild type. (H) Corresponding longitudinal section of bop1 bop2 shows a small outgrowth (arrow) in the lateral nectary position. (I-N) CRC::GUS plants stained for GUS activity. (I-K) CRC::GUS in wild type (Col-0xLer). (I) Expression in the nectary anlagens of a stage 7/8 flower (arrow). (J) Strong nectary gland expression in a mature flower. (K) Position 7 flower with GUS staining nectaries and the ring of connecting nectary tissue. (L-N) CRC::GUS activity in bop1 bop2 (Col-0xLer). (L) Expression in stage 7/8 flowers in the nectary anlagens (arrow). (M) Strong expression in the nectary gland region of a mature flower. (N) GUS staining in position 7 flowers shows strong staining in the lateral nectary gland region and weaker staining in the medial and connecting nectary tissue regions. ln, lateral nectary; ls, lateral stamen; mn, medial nectary; p, petal; s, sepal. Scale bars: 500 µm in C,D; 70 µm in E-H,J,K,M,N; 35 µm in I,M.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Model of abscission. BOP1 and BOP2 act early to promote the development of AZ specific anatomy of small cytoplasmically dense cell files in Arabidopsis. Later in flower development, a suite of enzymes involved in middle lamella degradation are expressed specifically in the AZ although with differing temporal patterns. Transcription of these enzymes is independent of BOP-driven formation of AZ anatomy. Abscission occurs following the expression of IDA in the AZ, which promotes cell separation. Expression of IDA is also driven independently from BOP1/2 activity.

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