Volume 26, Issue 7, Pages 903-909 (April 2016) The INDETERMINATE DOMAIN Protein BROAD LEAF1 Limits Barley Leaf Width by Restricting Lateral Proliferation  Moritz Jöst, Götz Hensel, Christian Kappel, Arnis Druka, Adrien Sicard, Uwe Hohmann, Sebastian Beier, Axel Himmelbach, Robbie Waugh, Jochen Kumlehn, Nils Stein, Michael Lenhard  Current Biology  Volume 26, Issue 7, Pages 903-909 (April 2016) DOI: 10.1016/j.cub.2016.01.047 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Phenotype of the blf1-1 Mutant (A–D) Three-week-old Bonus (A and C) and blf1-1 plants (B and D) photographed from above (A and B) or from the side (C and D). (E) Mature leaf 3 blades from blf1-1, the blf1-1 introgression line BW58, two blf1 TILLING lines, and their respective background cultivars Bonus, Bowman (Bow), and Barke. (F and G) Cross sections of the third mature leaf blade from Bonus (F) and blf1-1 (G) at 50% leaf length. Arrowheads mark the midvein. Scale bars represent 10 cm in (A)–(E) and 0.5 mm in (F) and (G). See also Figure S1 and Table S1. Current Biology 2016 26, 903-909DOI: (10.1016/j.cub.2016.01.047) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Quantification of Leaf Width and Meristem Size (A) Leaf-width profiles of mature leaf 3 blades. Open symbols represent Bonus; closed symbols represent blf1-1 mutants throughout (A–H). (B–G) Leaf-width profiles of leaves 4–9/plastochron stages 6–1 (L4/P6 to L9/P1) at 7 days after germination. Dashed lines indicate the average length of the corresponding Bonus leaves; dotted lines indicate the average length of the blf1-1 leaves. Average leaf lengths between the genotypes were not significantly different based on Student’s t test at any stage. (H) Average cross section area from shoot apical meristems (SAM) at 7 days after germination. Values are mean ± SEM from ten plants. See also Figure S2 and Table S1. Current Biology 2016 26, 903-909DOI: (10.1016/j.cub.2016.01.047) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Identification of BLF1 (A) Mapping summary. Green numbers are recombinant chromosomes for the respective markers. The sequenced FPCs and genes tested for absence/presence are in blue. Genes deleted in blf1-1 are in red. (B) Gene and protein structure of BLF1 with mutant alleles and predicted effects. For the gene, boxes show exons, lines show introns, and dashed lines are UTRs. (C) Maximum blade width of leaf 3 from blf1 mutant alleles. Values are mean ± SD from ≥12 plants. (D) Expression level of BLF1 in leaves of different stages at 7 days after germination. The leaf 4 sample also includes the next younger leaves and the meristem. Values are mean ± SEM from three biological replicates. The scale bars represent 1 mm (left), 1 cm (middle), and 10 cm (right). (E) Maximum blade width of leaf 3 from BLF1 transformants (BLF1TG) and BLF1-vYFP transformants (BLF1-vYFP) relative to the non-transgenic blf1-1 siblings from the same family. F2GPxBo are plants from the BC2F2 used for transformation. Values are mean ± SEM from greater than or equal to five plants. Percent decrease in width of plants carrying the BLF1 allele relative to the respective blf1-1 mutant control is shown. (F) Transcript levels of BLF1 gene relative to the housekeeping gene ADP in meristems and young leaf primordia of BLF1-containing genotypes in (E) as determined by qRT-PCR. WT indicates BLF1 plants from the BC2F2 population. Two biological replicates were analyzed. Asterisks indicate significant differences after Student’s t test at p < 0.05 (∗), p < 0.01 (∗∗), and p < 0.001 (∗∗∗). See also Figures S3 and S4. Current Biology 2016 26, 903-909DOI: (10.1016/j.cub.2016.01.047) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Expression Pattern of BLF1 Confocal images from pBLF1::BLF1-vYFP expressing blf1-1 mutants. Green channel: vYFP signal; red channel: FM4-64 staining. The scale bars represent 50 μm. A non-transgenic control is shown in Figure S4. (A) 3D projection of dissected shoot apex. The meristem (asterisk), P2, and P3 primordia are indicated. (B) Optical section of (A) to the left of the meristem, indicating BLF1-vYFP expression in the epidermis and subepidermal cells of P2 and P3 (arrowhead). (C) Dissected shoot apex. BLF1-vYFP is expressed throughout the meristem (asterisks), at the bases of P2 and P3 (arrowhead), and toward the tip of P3. (D) Dissected shoot apex. BLF1-vYFP expression is weaker in P1 than in the meristem (asterisk). (E and F) Dissected inflorescence meristems. BLF1-vYFP is expressed above and below developing spikelet primordia (arrowheads). Asterisk in (E) indicates inflorescence meristem. (G and H) Young ∼1.5-mm-long P5/P6 leaf primordium. BLF1-vYFP is expressed at low levels throughout the epidermis with a stronger signal along developing veins. (I) Optical section through putative developing vein. BLF1-vYFP expression is seen in the epidermis and within developing vein tissue (arrowhead). See also Figure S3. Current Biology 2016 26, 903-909DOI: (10.1016/j.cub.2016.01.047) Copyright © 2016 Elsevier Ltd Terms and Conditions