DEV031625 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Alignment of the GOBLET deduced amino acid sequence with other NAC-domain proteins. The tomato GOB deduced amino acid sequence is aligned with its closest homologs: the Arabidopsis CUC2 (AtCUC2; BAA19529/AT5G53950, GenBank/TAIR) and petunia NAM (PhNAM; CAA63101, GenBank). The conserved NAC domain and miR164 binding site are shown.

• Supplemental Figure S2 -

  Fig. S2. Effect of gob mutations and FIL>>miR164 on GOB expression. Relative expression of intact GOB mRNA was analyzed by quantifying RT-PCR products obtained with primers spanning the miR164 binding site. The results were normalized to TUBULIN expression and standardized by the wild-type value. (A) Reactions were performed with RNA from 9-day-old seedling apices. (B) Reactions were performed with RNA from 15-day-old seedling apices.

• Supplemental Figure S3 -

  Fig. S3. miR164-directed GOB cleavage products. (A) RLM-RACE PCR product of cleaved GOB mRNA from wild-type and Gob-4d plants, as compared with TUBULIN expression. (B) (Above) Sequence of the miR164 binding site in GOB. Arrow indicates the miR164 cleavage site. (Below) RLM-RACE products of wild type (WT), Gob-4d/+, Gob-4d and FIL>>GOB^m were cloned and sequenced. The ratios indicate the miR164-directed cleavage product of GOB mRNA from total sequenced RLM-RACE products, for each genotype. In heterozygous Gob-4d and FIL>>GOB^m, the ratio is provided separately for the wild-type (wt) and mutant (m) alleles.

• Supplemental Figure S4 -

  Fig. S4. Phenotypes of gob-3 and Gob-4d. (A,B) Whole-plant architecture. (C) Development of ectopic meristems on the Gob-4d mature leaf. (D) Mature late leaf of a recovered gob-3 plant. (E-G) GOB effect on flower development. The gob-3 flower displays elongated and fused sepals (F), as compared with the wild type (E), whereas the Gob-4d flower produces numerous unfused stamens (G). (H,I) gob-3 sepals enclose the fruitlet as a result of sepal fusion (I); compare with wild type (H). Scale bars: 5 cm in A-D; 1 mm in E-G; 1 cm in H,I.

• Supplemental Figure S5 -

  Fig. S5. Sequences of miR164 and the miR164 binding site in the GOB forms used in the transgenic plants. GOB^wt, wild-type GOB form; GOB^4d, a GOB form identical to the Gob-4d gain-of-function allele; GOB^m, a GOB form that carries nine silent mutations in the miR164 recognition site. In blue are natural mismatches between miR164 and the wild-type GOB; in red are mutations introduced into the miR164 recognition site. The encoded amino acid sequence is shown beneath, in the middle of each codon.

• Supplemental Figure S6 -

  Fig. S6. Flower phenotypes of transgenic plants expressing GOB forms with decreased sequence match to miR164. (A-C) Open flowers of tomato plants expressing the different GOB forms, as detailed in Fig. S5, through the FIL promoter. Scale bars: 1 mm.

• Supplemental Figure S7 -

  Fig. S7. Early leaf development of Gob-4d and gob-3. (A-D) Analysis of early development of the fifth leaf of Gob-4d in the P3-6 stages, as indicated. (E,F) SEM of a Gob-4d P4 primordium (E) and a gob-3 leaf primordium from a recovered SAM (F). (G) Schematic of P6 and the initiation of secondary leaflets. The primary leaflet is colored red and provides the context of the SEMs in H and I. (H,I) Primary leaflets. The arrowhead points to secondary leaflet initiation in Gob-4d. Scale bars: 500 µm in A-D; 200 µm in E,F,H,I

• Supplemental Figure S8 -

  Fig. S8. Effect of miR164 overexpression on Cardamine hirsuta seedling and flower development. (A-D) Wild-type C. hirsuta plant and seedlings. (A) Aerial parts of a C. hirsuta plant after bolting. (B) C. hirsuta seedling showing cotyledons and first leaf. (C) Older C. hirsuta seedling after growth of the second leaf away from the plant axis. (D) Higher magnification of the petioles and apex of the seedling shown in C, demonstrating the lack of fusion between the cotyledons and leaves at their base. (E-G) 35S::AtmiR164b C. hirsuta seedlings. (E) Strong 35S::AtmiR164b C. hirsuta line, where the cotyledons have fused and the apical meristem is absent. (F) Intermediate 35S::AtmiR164b C. hirsuta line, where the base of the leaf petiole is fused to that of the cotyledon. (G) Higher magnification of the fused leaf and cotyledon petioles of the seedling shown in F. (H) Mature flower of wild-type and intermediate 35S::AtmiR164b C. hirsuta plants, showing fusion between the sepals of flowers overexpressing miR164b. Arrowheads mark fusion events.