BLADE-ON-PETIOLE-dependent signaling controls leaf and floral patterning in Arabidopsis

Abstract

NONEXPRESSOR OF PR GENES1 (NPR1) is a key regulator of the plant defense response known as systemic acquired resistance. Accumulation of the signal molecule salicylic acid (SA) leads to a change in intracellular redox potential, enabling NPR1 to enter the nucleus and interact with TGACG sequence-specific binding protein (TGA) transcription factors, which in turn bind to SA-responsive elements in the promoters of defense genes. Here, we show that two NPR1-like genes, BLADE-ON-PETIOLE1 (BOP1) and BOP2, function redundantly to control growth asymmetry, an important aspect of patterning in leaves and flowers. Phenotypes in the double mutant include leafy petioles, loss of floral organ abscission, and asymmetric flowers subtended by a bract. We demonstrate that BOP2 is localized to both the nucleus and the cytoplasm, but unlike NPR1, it is highly expressed in young floral meristems and in yeast interacts preferentially with the TGA transcription factor encoded by PERIANTHIA (PAN). In support of a biological relevance for this interaction, we show that bop1 bop2 and pan mutants share a pentamerous arrangement of first whorl floral organs, a patterning defect that is retained in bop1 bop2 pan triple mutants. Our data provide evidence that BOP proteins control patterning via direct interactions with TGA transcription factors and demonstrate that a signaling mechanism similar to that formally associated with plant defense is likely used for the control of developmental patterning.

Figure 1.

Phylogenetic Tree and Structural Motifs for the NPR1 Protein Family in Arabidopsis. (A) Phylogenetic tree for the NPR1 protein family. Arabidopsis Genome Initiative numbers are as follows: NPR1, At1g64280; BOP1, At3g57130; and BOP2, At2g41370. (B) Conserved structural motifs in NPR1-like proteins. BTB/POZ and ankyrin motifs are represented by boxes marked BTB and ANK, respectively. Cys1 and Cys2 indicate conserved Cys residues that mediate redox control of NPR1 oligomerization (Cys-82 and Cys-262, respectively) (Mou et al., 2003). (C) Alignment of the predicted BTB/POZ domains for NPR1-like proteins. The asterisk indicates Cys-82 in NPR1, which mediates redox control. (D) Alignment of the predicted ankyrin repeats for NPR1-like proteins.

Figure 2.

Expression of BOP1 and BOP2 in the Wild Type and bop1 bop2 Mutants. (A) Analysis of BOP1 and BOP2 expression in wild-type (WT) and bop1 bop2 seedlings as monitored by RT-PCR. The gene amplified in each set of reactions is indicated at left of each gel. BOP1, 40 cycles; BOP2, 30 cycles; glyceraldehyde-3-phosphate dehydrogenase (GAPC) control, 30 cycles. A small amount of BOP1 transcript was detected in bop1 bop2 double mutants. (B) RT-PCR analysis of tissue-specific expression of BOP1 and BOP2 in wild-type plants. The indicated tissues (top) were harvested from 6-week-old soil-grown plants, and the gene amplified in each set of reactions is indicated at left of each gel. RT-PCR conditions and cycle numbers were as in (A). (C) to (H) In situ localization of CER6 (control) or BOP2 mRNA in the inflorescence apex and flowers of wild-type plants. Hybridization is indicated by the presence of a purple precipitate. (C) Central section of an inflorescence meristem (im) and young floral buds hybridized with an L1-specific CER6 control probe. (D) to (F) Central longitudinal sections showing inflorescence meristems and young flower buds hybridized with a BOP2 probe. BOP2 mRNA is first detected in the floral anlagen (P0) on the flanks of the inflorescence meristem. Arrows in (D) and (F) indicate a strong band of expression at the base of stage 1 (P1) and stage 2 (P2) floral primordia in the abaxial region of these primordia corresponding to the cryptic bract. (G) Cross section of an inflorescence meristem and young floral buds hybridized with a BOP2 probe. Arrows indicate expression in the developing sepal primordia of a stage 3 floral primordium (P3). (H) Cross section of a mature flower showing strong bands of expression at the bases of anthers and petals (arrows). The inset shows detail at the base of the anther. Bars = 100 μm, except 50 μm in (H).

Figure 3.

Leaf and Inflorescence Phenotypes for Wild-Type, bop1, bop2, and bop1 bop2 Plants and Complementation of bop1 bop2 with BOP2. (A) and (B) Twenty-eight-day-old plants are shown for the wild type (A) and bop1 bop2 (B). Bar = 2 cm for (A) and (B). (C) Leaf series from 3-week-old wild-type (top row) and bop1 bop2 (bottom row) plants. (D) Cauline leaves from wild-type (left) and bop1 bop2 (middle and right) plants. (E) Inflorescences from wild-type (left) and bop1 bop2 (right) plants. Arrows indicate lack of floral organ abscission. The inset shows wild-type (left) and bop1 bop2 (right) siliques. (F) Leaf series showing representative rosette leaves from a wild-type plant (left), a bop1 bop2 plant (middle), and a bop1 bop2 plant complemented by a fragment of genomic DNA containing the BOP2 gene (right). (G) Inflorescence of a bop1 bop2 plant partially complemented for floral organ abscission by a fragment of genomic DNA containing the BOP2 gene.

Figure 4.

Comparison of Wild-Type and bop1 bop2 Floral Phenotypes. (A) Morphology of mature flowers. (a) Wild type. (b) bop1. (c) bop2. (d) bop1 bop2 abaxial side. (e) Adaxial side. (f) Abaxial view of a flower with abaxial first whorl organs removed. (g) Abaxial view of a young flower showing retarded growth of abaxial organs in the first whorl. (h) Example of fused abaxial first whorl organs with sepal-petal characteristics. Bars = 1 mm. (i) to (k) Examples of bracts and bract-like structures that subtend flowers. Bar = 0.5 mm. (B) Scanning electron micrographs of wild-type and bop1 bop2 inflorescence apices depicting inflorescence meristems, floral meristems, and flowers. (a) Wild-type inflorescence apex. The asterisk marks the abaxial sepal. (b) bop1 bop2 inflorescence apex. Arrows indicate floral bracts. (c) Stage 2 floral primordia with subtending bract (arrow). FM, floral meristem. (d) Stage 5 flower with subtending bract and five evenly spaced organs in the sepal whorl such that the adaxial sepal is in the same position as in the wild type. An extra stamen primordia occurs between the two abaxial sepal whorl organs (asterisks). (e) Stage 7 flower showing retarded development of sepals. Smaller abaxial first whorl organs are marked by asterisks. (f) Example of a flower with abaxial first whorl organs that are half-sepal (bumpy cells) and half-petal (smooth inner cells). Bars = 50 μm except 100 μm in (e) and 500 μm in (f). (C) Floral diagrams for wild-type and bop1 bop2 flowers. The adaxial side is marked with a closed circle and indicates the position of the stem. The wild-type flower shows radial symmetry (left), and the bop1 bop2 flower shows bilateral symmetry (right).

Figure 5.

Subcellular Localization of BOP2-GFP Protein. (A) Confocal image of GFP fluorescence in root cells of a 35S:BOP2-GFP transgenic seedling. (B) Protein gel blot of whole cell extracts from leaves of 35S:GFP and 35S:BOP2-GFP transgenic plants. Blots were probed with an anti-GFP antibody and deliberately overexposed to reveal the presence of minor bands. No major degradation bands in the form of free GFP were seen. An arrow denotes the position of a band corresponding to the full-length BOP2-GFP fusion protein. An asterisk denotes the position of a band corresponding to free GFP.

Figure 6.

Analysis of Genetic and Biochemical Interactions between BOP and TGA Transcription Factors. (A) Yeast two-hybrid interactions between NPR1, BOP1 and BOP2, and TGA transcription factors. Left, X-Gal filter-lift assay. Bait proteins are indicated at top and prey proteins are indicated at left. Accumulation of blue precipitate indicates β-galactosidase activity. Right, quantitative determination of reporter gene expression for each corresponding interaction shown at left, presented in chart format as β-galactosidase activity units. All data shown were corrected for self-activation of reporter gene expression by bait protein. (B) Phylogenetic tree for the Arabidopsis TGA transcription factor family (Jakoby et al., 2002). Arabidopsis Genome Initiative numbers are as follows: PAN, At1g68640; TGA5, At5g06960; TGA2, At5g06950; TGA6, At3g12250; AtbZIP21, At1g08320; AtbZIP65, At5g06839; TGA3, At1g22070; TGA7, At1g77920; TGA1, At5g65210; TGA4, At5g10030. (C) to (H) Comparison of pan-3 and bop1 bop2 pan-3 mutant floral phenotypes. (C) Typical pan-3 mutant flower with pentamerous arrangement of floral organs: five sepals, five petals, and five stamens (abaxial view). (D) Scanning electron micrograph of a stage 5 pan-3 flower with pentamerous arrangement of sepals. Abaxial sepals are marked with asterisks. (E) Floral diagram of an average pan-3 flower. The adaxial side is marked with a closed circle. (F) Typical bop1 bop2 pan-3 flower with pentamerous arrangement of first whorl organs (lateral view). (G) Scanning electron micrograph of a stage 5 bop1 bop2 pan-3 flower that retains a pentamerous arrangement of first whorl organs. Abaxial organs are marked with asterisks. (H) Floral diagram of an average bop1 bop2 pan-3 triple mutant flower. The adaxial side is marked with a closed circle. Bars = 1 mm for (C) and (F) and 100 μm for (D) and (G).