Supplemental Figure S1. The number of hydathodes is reduced in the as2-1 rev-1

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1 Supplemental Data Supplemental Figure S1. The number of hydathodes is reduced in the as2-1 rev-1 and kan1-11 kan2-5 double mutants. A, The numbers of hydathodes in different leaves of Col-0, as2-1 rev-1, and kan1-11 kan2-5 plants. B, The numbers of GUS staining spots in leaf margins (excluding the leaf tips) of different leaves in pyuc4::gus/col-0, pyuc4::gus/as2-1 rev-1, and pyuc4::gus/kan1-11 kan2-5 transgenic plants. n = 20 for the first-pair leaves and n = 10 for the other leaves, bars show s. e. *, P < **, P <

2 Supplemental Figure S2. Contributions of YUC genes in leaf margin development and leaf adaxial-abaxial polarity establishment. To determine contributions of the four YUC genes in margin development and blade outgrowth, we analyzed leaf phenotypes of different yuc combinations. A to E, The 26-DAG seedlings of Col-0 (A), yuc1 yuc4 (B), yuc1 yuc2 yuc4 (C), yuc1 yuc4 yuc6 (D), and yuc1 yuc2 yuc4 yuc6 (E). F, The sixth mature leaves of Col-0 and different yuc combination mutants, with both adaxial (leaf) and abaxial (right) views. G, The number of the cell patches was examined from one-side leaf margin of the sixth leaves of Col-0 and different yuc combination mutant plants. n = 10. Bars show s. e. **, P < Leaves of yuc1 yuc4 were slightly narrow (F), and the number of cell patches was increased along the yuc1 yuc4 margin with no hydathodes (G). yuc1 yuc2 yuc4 and yuc1 yuc4 yuc6 enhanced the narrow-leaf phenotype of yuc1 yuc4 (F), with further increased cell patches (G). The yuc1 yuc2 yuc4 yuc6 quadruple mutant exhibited the most severe defect in leaf expansion (F). These results, plus previous data that yuc1, yuc2, yuc4, yuc6, yuc1 yuc2, yuc1 yuc6, yuc2 yuc4, and yuc4 yuc6 are all phenotypically normal, and yuc2 yuc6, yuc1 yuc2 yuc6, and yuc2 yuc4 yuc6 do not significantly affect blade outgrowth (Cheng et al., 2006), indicate that YUC1 and YUC4 are the major contributors for margin development and blade outgrowth, while YUC2 and YUC6 act redundantly with YUC1 and YUC4. H and I, Transverse sections of the leaf midvein (H) and blade (I) of the 2

3 sixth leaf of wild-type Col-0 plants. J and K, Transverse sections of the leaf midvein (J) and blade (K) of the sixth leaf of a yuc1 yuc2 yuc4 yuc6 plant. Because yuc1 yuc2 yuc4 yuc6 shows normal xylem-phloem arrangement and palisade-spongy tissue arrangement, the YUC genes may not be directly involved in leaf adaxial-abaxial polarity establishment, and thus may not directly regulate the leaf polarity genes. yuc14, yuc1 yuc4; yuc124, yuc1 yuc2 yuc4; yuc146, yuc1 yuc4 yuc6; yuc1246, yuc1 yuc2 yuc4 yuc6. Scale bars: 5 mm in A-F; 50 μm in H; 100 μm in I and K; 20 μm in J. 3

4 Supplemental Figure S3. Expression patterns of pyuc1::gus, pyuc2::gus, pyuc4::gus, and pdr5::gus in later-appearing leaves, and phenotypes of as2-1 rev-1, as2-1 rev-1 yuc1 yuc2 yuc4/+ and as2-1 rev-1 yuc1 yuc4 yuc6. A to D, GUS staining of the sixth leaf of 24-DAG pyuc1::gus (A), pyuc2::gus (B), pyuc4::gus (C) and pdr5::gus (D). E and F, 30-DAG as2-1 rev-1 (E) and as2-1 rev-1 yuc1 yuc2 yuc4/+ (F). G, The 27-DAG as2-1 rev-1 and as2-1 rev-1 yuc1 yuc4 yuc6 seedlings. yuc124/+, yuc1 yuc2 yuc4/+; yuc146, yuc1 yuc4 yuc6. Scale bars: 1 mm in A-D; 5 mm in E-G. 4

5 Supplemental Figure S4. Formation of leaf protrusions in the pfil::as2 transgenic plants is dependent on the YUC activity. pfil::as2 was constructed by fusing the AS2 cdna to the promoter of the FILAMENTOUS FLOWER (FIL) gene (Watanabe and Okada, 2003). Since FIL is specifically expressed in the abaxial leaf domain and the endogenous AS2 gene is expressed in the adaxial leaf domain (Sawa et al., 1999; Iwakawa et al., 2002; Watanabe and Okada, 2003; Iwakawa et al., 2007), the pfil::as2 transgenic plants make AS2 ectopically expressed in the abaxial leaf domain. Therefore, this results in the new adaxial-abaxial juxtaposition on the abaxial leaf side to form protrusions. A, The 26-DAG pfil::as2/col-0 and pfil::as2/yuc1 yuc4 yuc6 seedlings. B, The abaxial surface of a sixth leaf of pfil::as2/col-0. C, The abaxial surface of a sixth leaf of pfil::as2/yuc1 yuc4 yuc6, showing that the protrusions were absent. Yellow arrowheads show ectopic protrusions. Ab, the abaxial leaf side. yuc146, yuc1 yuc4 yuc6. Scale bars: 5 mm in A; 200 μm in B and C. 5

6 Supplemental Figure S5. GUS staining of YJ158 in the wild type. A, YJ158 is a transposon insertion line in which GUS reporter is specifically expressed in the leaf margin (Eshed et al., 2004). B and C, GUS staining was detected in the tip of an immature hydathode (B), but became weakened in a mature hydathode (C). D and E, cell patches closed to hydathodes contained GUS staining. F, a cell patch apart from hydathodes also showed GUS staining. Arrows indicate hydathodes, and arrowheads indicate cell patches. Scale bars: 50 μm. LITERATURE CITED Cheng Y, Dai X, Zhao Y (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in Arabidopsis. Genes Dev. 20: Eshed Y, Izhaki A, Baum SF, Floyd SK, Bowman JL (2004) Asymmetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities. Development 131: Iwakawa H, Iwasaki M, Kojima S, Ueno Y, Soma T, Tanaka H, Semiarti E, Machida Y, Machida C (2007) Expression of the ASYMMETRIC LEAVES2 gene in the adaxial domain of Arabidopsis leaves represses cell proliferation in this domain and is critical for the development of properly expanded leaves. Plant J. 51: Iwakawa H, Ueno Y, Semiarti E, Onouchi H, Kojima S, Tsukaya H, Hasebe M, 6

7 Soma T, Ikezaki M, Machida C, Machida Y (2002) The ASYMMETRIC LEAVES2 gene of Arabidopsis thaliana, required for formation of a symmetric flat leaf lamina, encodes a member of a novel family of proteins characterized by cysteine repeats and a leucine zipper. Plant Cell Physiol. 43: Sawa S, Ito T, Shimura Y, Okada K (1999) FILAMENTOUS FLOWER controls the formation and development of arabidopsis inflorescences and floral meristems. Plant Cell 11: Watanabe K, Okada K (2003) Two discrete cis elements control the Abaxial side-specific expression of the FILAMENTOUS FLOWER gene in Arabidopsis. Plant Cell 15: