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First published online 28 September 2005
doi: 10.1242/dev.02062

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A genetic framework for fruit patterning in Arabidopsis thaliana

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, 72076 Tübingen, Germany

View larger version (75K): [in a new window]   Fig. 1. Wild-type fruit development. (A) Bright-field image of a stage 17 fruit. (B,C) SEM of the apical region (B) and basal region (C) of a fruit. (D) Diagram of a cross section of a fruit with the various tissues indicated. (E) Safranin O- and Alcian Blue-stained cross section near the valve margin. The separation layer stains light blue and the lignified layer stains pink. (F,G) Phloroglucinol-stained cross sections of the valve margin (F) and valve (G). This stain is lignin specific and marks the lignified layer of the valve margin, the enb layer and cells of the vascular bundle. v, valve; r, replum; vm, valve margin; st, style; gy, gynophore; SL, separation layer; LL, lignified layer; vb, vascular bundle. Scale bars: 1 mm (A), 200 µm (B,C), 50 µm (E-G).

View larger version (97K): [in a new window]   Fig. 2. The fil yab3 double mutant has defects in valve margin patterning. (A) Expression of FIL detected by in situ hybridization in a stage-8 wild-type gynoecium. (B-H) The fil yab3 fruits develop a complex arrangement of valve margin tissues in which the apical regions lack valve margin, while the basal regions develop ectopic valve margin. (B) SEM of a whole fil yab3 fruit. Brackets mark regions that develop no visible valve margin (–vm) and that develop ectopic valve margin (++vm). The gynophore is also expanded in fil yab3 mutants. (C) SEM of the apical region of a fil yab3 fruit. Note the lack of creases that normally mark the location of the valve margin. (D,E) Phloroglucinol-stained cross sections of the apical region of a fil yab3 fruit in the replum/valve margin region (D), or valve (E) region. The fruit lack lignin in cells normally found at the valve margin and enb layer. (F) SEM of the basal region of a fil yab3 fruit. A large stripe of valve margin-like tissue (evm) is shown passing through the middle of a valve. (G,H) Phloroglucinol-stained cross sections of the basal region of a fil yab3 fruit in the replum/valve margin (G) and valve (H) regions showing the expansion of lignified layer tissues into the valves. r, replum; v, valve; st, style; –vm, loss of valve margin; ++vm, ectopic valve margin; evm, ectopic valve margin; gy, gynophore; EL, ectopic lignification. Scale bar: 50 µm (A,D,E,G,H), 1 mm (B), 200 µm (C), 100 µm (F).

View larger version (94K): [in a new window]   Fig. 3. FIL and YAB3 are necessary for proper SHP2 and FUL expression. GUS expression driven by the SHP2 promoter (A-E) or the ful-1 enhancer trap line (F-I). All plants stained for FUL GUS activity are heterozygous for ful-1. (A) In wild-type fruit, the SHP2::GUS reporter is active in the valve margins throughout the fruit at stage 14. (C) In cross section, reporter activity is visible in the ovules as well as the valve margins. (B,D,E) In fil yab3 fruit, SHP2 reporter activity is lost in the apical region of the fruit (B –,D) while ectopic reporter activity is present in the valves of the basal portion (B ++,E). (F,G) FUL GUS staining of a stage 12 wild-type gynoecium showing expression in the two valves as well as in the vasculature of the style (F) and replum (G). (H,I) In the fil yab3 gynoecium, reporter activity is absent from the valves but remains in the vasculature. Note the presence of a supernumerary carpel in the fil yab3 gynoecium (I). (J-N) Lignin staining of valve cells in mature fruit (stage 17). (J) 35S::FUL valves develop a lignified enb layer similar to wild type. (K) 35S::FUL is able to suppress most of the ectopic lignification that occurs at the base of fil yab3 mutants and is able to rescue enb layer lignification in the apical region (L). Note some ectopic lignification is still present in the basal region. (M) In ful mutants, valve mesophyll cells become ectopically lignified as a result of the expanded expression of valve margin identity genes. (N) In the apical region of ful fil yab3 fruits, however, all cells of the valves are unlignified including the enb layer, suggesting that the valve margin identity genes are not active. v, valve; vm, valve margin; r, replum; ov, ovule; st, style; vb, vascular bundle; gy, gynophore; EL, ectopic lignification. Scale bar: 0.5 mm (A), 1 mm (B), 100 µm (C,D,E), 200 µm (F,H), 50 µm (G,I-N).

View larger version (109K): [in a new window]   Fig. 4. JAG acts redundantly with FIL and YAB3 to control valve margin patterning. (A) Expression of JAG detected by in situ hybridization in a stage 8 wild-type gynoecium. JAG is expressed in the valves and presumptive valve margin region and is expressed in all cell layers. (B) SEM of the apical region of a jag fruit. The shoulders of the valves are sloped downwards in jag. (C) SEM of a jag fil fruit. The style/ovary (white arrowhead) and gynophore/ovary (black arrowhead) boundaries have shifted, with the style and gynophore becoming enlarged. (D) jag fil fruit develop a stripe of valve margin tissue that runs down the middle of the valves. (E) In cross sections stained with Safranin O and Alcian Blue, separation layer and lignified layer cells are present overlying the vascular bundle. (F) SEM of jag fil yab3+/– fruit showing further expansion of style and gynophore into the ovary region. (G) SEM of jag fil yab3+/– ER fruit. The erecta mutation suppresses the growth of the style as it does in ful mutants (Ferrándiz et al., 2000a). (H) Close-up SEM of a jag fil yab3+/– replum. The normally narrow ridge of tissue has expanded in width and twists throughout the length of the fruit similar to the replum of ful mutants (J). (I) SEM of a patch of small cells that form in the valves of jag fil yab3+/– fruit. These cells appear very similar to the ectopic valve margin cells that replace the valves in ful mutants (K). (L) SEM of a jag fil yab3 mutant flower. All the floral organs have patterning defects. Sepals, petals and stamens are replaced by filamentous organs with no apparent floral characteristics. (M) SEM of a fertilized jag fil yab3 fruit. No valve margin development is present. (N) Epidermal cells of jag fil yab3 valves are elongated with stomata interspersed (white arrowheads), similar to wild type. Occasionally, however, cells with an irregular shape can be seen. (O,P) Cross sections at the base of a jag fil yab3 fruit stained with Safranin O and Alcian Blue. All cell types necessary for dehiscence, including the lignified layer, separation layer and enb layer are absent. Some enb lignification may be present internally to the vascular bundle. r, replum; v, valve; vm, valve margin; LL, lignified layer; SL, separation layer; vb, vascular bundle. Scale bar: 50 µm (A,E,I,K,N,O,P), 200 µm (B,L), 1 mm (C,G,M), 100 µm (D,H,J), 500 µm (F).

View larger version (88K): [in a new window]   Fig. 5. JAG promotes FUL and SHP expression redundantly with FIL and YAB3. (A-D) FUL GUS reporter activity in the gynoecium using the ful-1 enhancer trap line. (E-N) SHP2::GUS reporter activity in stage 15-16 fruit (E-J) and stage 12 gynoecia (K-N). (A,B) Whole mount (A) and cross section (B) of FUL GUS staining in a stage 12 jag fil yab3+/– gynoecium showing reporter activity restricted to small islands corresponding to the valves. (C,D) Whole mount (C) and cross section (D) of FUL GUS staining in a stage 12 jag fil yab3 gynoecium. Reporter activity is completely absent from the valves. (E,F) Frontal (E) and side (F) views of SHP2::GUS expression in wild type. (G) In jag mutant fruit, a small wedge of ectopic reporter activity is detected in the apical region of the valves (black arrowhead). (H) In fil mutant fruit, a stripe of ectopic reporter activity can be seen extending through the center of the valve, overlying the vasculature. (I) In fil yab3+/– mutant fruit, the intensity of reporter activity in the valves is stronger than in fil single mutants. (J) In jag fil mutant fruit, strong reporter activity is present in a stripe in the valves with weaker staining elsewhere. (K,L) Cross section of a jag fil yab3+/– gynoecium showing strong ectopic reporter activity in the valves. Using dark-field microscopy, strong GUS staining appears purple, whereas weak staining appears pink/orange. (L) The same section as in K viewed under bright-field microscopy. (M,N) Cross section of a jag fil yab3 gynoecium showing weak reporter activity in the valves (M). Using bright-field microscopy (N) to image the section in M, the loss of SHP2::GUS reporter activity in the valves and valve margins is even more apparent, compared to the strong GUS staining seen in the valve regions of jag fil yab3+/– gynoecia (L). v, valve; r, replum; ov, ovule and st, style. Scale bars: 200 µm (A,C); 100 µm (B,D); 0.5 mm (E-J); 100 µm (K-N).

View larger version (116K): [in a new window]   Fig. 6. RPL promotes the formation of two valve margins by negatively regulating JAG and FIL activity. The loss-of-function JAG allele jag-1, and the activation-tagged allele jag-5D. (A,B,D-F,H,I,K,L) Cross sections of stage 17 fruit from various mutants stained with Alcian Blue and Saffranin O. (A) jag-5D fruit. The replum is replaced by lignified layer and separation layer-like cell types. (B) Example of a strongly affected jag-5D fruit showing the fusion of the lignified layer to form a `lignified bridge'. Note that the two valves have detached from the rest of the fruit as a single unit. (C) SHP2::GUS reporter activity in a stage 13 jag-5D fruit expands into the replum region. Note that the SHP2::GUS reporter is not activated as highly in the vascular bundle where FUL is expressed (white arrowhead). (D) shp1,2 fruit lack separation and lignified layer development. The cross section is taken near the base of the fruit where the shp mutant phenotype is strongest. (E) jag-5D shp1,2 fruit near the base. The ectopic development of valve margin tissues is suppressed, indicating that SHP is necessary for the replumless phenotype of jag-5D mutants. (F) rpl fruit have patterning defects similar to jag-5D. (G) In situ hybridization of JAG in a stage 8 rpl mutant gynoecium. Expression of JAG is not detectable in the replum region. (H) jag-1 fruit are similar to wild type. (I) jag-1 rpl fruit showing a rescue of replum development. Note that replum rescue is variable and this is a strong example. (J) Expression of FIL in a stage 8 rpl gynoecium expands into the replum region (black arrowhead). (K) fil mutant fruit. The replum tends to be larger than in wild type. (L) fil rpl fruit display a consistent and strong rescue of replum development. LL, lignified layer; SL, separation layer; r, replum; v, valve; o, ovule. Scale bars: 50 µm (in A for A,B,D-F,H,I,K,L; in C; and in G for G,J).

View larger version (36K): [in a new window]   Fig. 7. Model for fruit patterning in Arabidopsis. JAG, FIL and YAB3 function redundantly to promote the expression of the valve margin identity genes and FUL. These activities are negatively regulated by RPL in the replum.

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