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First published online 28 January 2009
doi: 10.1242/dev.031625

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The NAC-domain transcription factor GOBLET specifies leaflet boundaries in compound tomato leaves

Yael Berger^1,*, Smadar Harpaz-Saad^1,*, Arnon Brand^1,*, Hadas Melnik¹, Neti Sirding¹, John Paul Alvarez², Michael Zinder¹, Alon Samach¹, Yuval Eshed² and Naomi Ori^1,

¹ The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture and The Otto Warburg Minerva Center for Agricultural Biotechnology, Faculty of Agriculture, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
² Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel.

View larger version (49K): [in this window] [in a new window]   Fig. 1. GOBLET affects leaflet initiation and separation. (A) Diagram of the compound wild-type tomato leaf, indicating the terminology of the leaf parts. (B) GOBLET (GOB) gene structure, showing coding exons (rectangles), introns (black lines), the conserved NAC domain (gray box), the miR164 recognition site, and nucleotide and amino acid changes in the gob loss- and gain-of-function alleles. (C) Sequence of SlmiR164 (Pilcher et al., 2007) and of the miR164 recognition site in the wild-type and Gob-4d GOB alleles, with mismatches represented in red and blue, respectively, and identical nucleotides represented by hyphens. (D-G) Nine-day-old seedlings of wild type (WT) (D), gob-3 (E; arrowhead points to cotyledon fusion) and Gob-4d (F,G). (H,I) Scanning electron micrographs (SEMs) of embryos fixed 10 (H) or 18 (I) days after anthesis. (J,K) SEM of a wild-type shoot apical meristem (SAM) (J) and a split Gob-4d SAM (K). (L-P) The fifth leaf from plants of the indicated genotypes. Arrowheads point to the fusion between the gob-3 terminal leaflet and the adjacent lateral primary leaflets. (Q,R) Terminal (Q) and primary (R) leaflets from the ninth leaf. (S-U) Mature fruits. gob-3 leaves and leaflets were formed on a seedling recovered following cotyledon removal. Scale bars: 1 cm in D-G,Q,R,S-U; 100 µm in H,I; 500 µm in J,K; 5 cm in L-P.

View larger version (89K): [in this window] [in a new window]   Fig. 2. GOB is expressed in tomato leaf and leaflet boundaries in a complementary pattern with miR164. In situ hybridization with GOB or miR164 probes. Probes are indicated at the top right corner and genotypes at the bottom left corner of each panel. (A-C) Transverse sections of the SAM. (D-F) Longitudinal sections of the SAM. (G,H) Longitudinal sections of primary leaflets. Insets indicate the context of the portion shown within the leaf primordia, and arrows point to the leaflet shown in the enlarged section. (I) Longitudinal sections of leaf primordia. (J-L) Whole-mount in situ hybridization of the SAM (J,L) or a primary leaflet (K). The inset in K indicates the context of the primary leaflet, and the red area marks the leaflet shown. Asterisks mark the SAM, P indicates the plastochron number, and arrowheads point to stripes of GOB expression (D,E,G,H,J,K), reduced miR164 expression between leaflets (I) or elevated miR164 expression in initiating leaflets (L). Scale bars: 100 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Leaf-specific overexpression of miR164 results in simple leaflets. (A-D) In situ hybridization of longitudinal leaf primordia sections. Probes are indicated at the top right corner and genotypes at the bottom left corner of each panel. Asterisk, SAM; P, plastochron number; arrowhead, stripe of GOB expression. (E,F) Fully expanded fifth leaves. Scale bars: 100 µm in A-D; 5 cm in E,F.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Dramatic leaf shape alterations are caused by ectopic GOB expression. (A,B) Expression pattern of the FIL promoter in the FIL>>GOBm SAM (A) and leaf primordia (B), using an op:RFP reporter. (C,D) In situ hybridization performed on longitudinal sections of a FIL>>GOBm SAM (C) and leaf primordia (D) with the GOB probe. (E-M) Mature fifth leaf (E-H), terminal leaflet from the ninth leaf (I-L) and cotyledons (M) of tomato plants expressing the different GOB versions through the FIL promoter. (N,O) A 35S>>GOB4d seedling (N) and SEM of ectopic meristems developed on a 35S>>GOB4d seedling apex (O). Asterisk, SAM; P, plastochron number. Scale bars: 200 µm in C,D,O; 500 µm in A,B; 1 cm in I-N; 5 cm in E-H.

View larger version (51K): [in this window] [in a new window]   Fig. 5. The effect of altering GOB expression on early leaf and leaflet development. (A-C) Early leaf development (P3-6) in wild-type (A), FIL>>GOBm (B) and FIL>>miR164 (C) tomato plants. Arrowheads point to primary leaflet primordia according to the order of their initiation: red, blue, yellow and pink, respectively. (D-O) SEMs of P3 (D-F), P4 (G-I) and P5 (J-O). K, M and O are magnifications of J, L and N, respectively, demonstrating the appearance of intercalary leaflets. (P) Schematic illustrating the context of the secondary leaflet shown in the SEM image in Q and R. The red area marks the leaflet shown. (Q,R) SEM of the secondary leaflet of wild type (Q) and FIL>>miR164 (R). Scale bars: 500 µm in A-C; 200 µm in D-O,Q,R.

View larger version (71K): [in this window] [in a new window]   Fig. 6. Effect of miR164 overexpression on Cardamine hirsuta compound-leaf development. (A) Leaves removed from a wild-type Cardamine hirsuta plant and displayed in acropetal sequence from left to right. Cauline leaves formed following the transition to flowering are bracketed. (B) Leaves removed from an intermediate line of 35S::AtmiR164b C. hirsuta and arranged in acropetal sequence from left to right. (C) Leaves dissected from a strong line of 35S::AtmiR164b C. hirsuta and placed in acropetal sequence from left to right. Probable fusion events between adjacent leaves are marked by arrowheads; regions of the rachis where torn leaf tissue exists from a disrupted congenital fusion event are marked with an asterisk. Scale bar: 1 cm.

View larger version (74K): [in this window] [in a new window]   Fig. 7. Interaction between GOB and ENTIRE. (A) Structure of the tomato ENTIRE (E) gene. Exons (white boxes), introns (black lines), conserved IAA domains I-IV (gray stripes), and the nucleotide and amino acid changes in the e-3 and e-4 alleles are indicated. The e-2 mutant, used for the genetic interaction and in situ hybridization shown in B and C, was confirmed as an e allele but sequence analysis of the genomic region spanning the entire ORF could not identify the causative mutation. (B-F) A fifth leaf from each of the indicated single and double mutants. (G-I) In situ hybridization of longitudinal SAM sections with the GOB probe. Genotypes are indicated at the bottom left corner. Scale bars: 5 cm in B-F; 100 µm in G-I.

View larger version (33K): [in this window] [in a new window]   Fig. 8. A proposed model of GOB function. (A) The roles of GOB in early stages of tomato leaf development. The GOB expression pattern is indicated in red. In early P3 primordia, GOB is expressed at the leaf margin prior to leaflet initiation and inhibits maturation in the adjacent area, enabling future leaflet initiation. During primary leaflet formation, restricted GOB expression in space and time allows proper leaflet separation. In the terminal leaflet, GOB expression enables the development of lobes and serrations. In plastochron 5 (P5), stripes of GOB expression in the primary leaflet flanks enable initiation and separation of secondary leaflets. (B) Diagrams of the fifth leaf of wild-type, gob-3 and Gob-4d plants, demonstrating the relatively minor effect of alterations in GOB activity on primary leaflet initiation. The rectangle indicates the primary leaflets shown in C. (C) The effect of fluctuations in GOB expression on secondary-leaflet initiation and separation. (Top) Schematics of relative spatial GOB expression in the developing leaflets of wild type and Gob-4d, and of GOB activity in gob-3. (Bottom) Diagrams of the resulting primary lateral leaflet. In the wild type, a sharp boundary between a narrow region of high GOB expression and an adjacent region with no expression enables initiation and development of a distinct secondary leaflet (sl1, sl2, arrows). In gob-3, elimination of GOB activity results in a faliure to form a boundary and in the lack of initiation of secondary leaflets, leading to the development of a primary leaflet with smooth margins. In Gob-4d, GOB expression is elevated and expanded relative to the wild type. The boundary is less sharp, and adjacent GOB expression domains are closer to each other than in the wild type. This results in lobed primary leaflets owing to fusion of adjacent leaflet primordia (sl1, sl2, arrows), and the lack of distinct secondary leaflets with petiolules.

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