SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188 NATURE PLANTS | www.nature.com/natureplants 1 Peipei Wang 1,2, *, Hong Liao 1,2, *, Wengen Zhang 1,2, *, Xianxian Yu 1,2 , Rui Zhang 1 , Hongyan Shan 1 , Xiaoshan Duan 1,2 , Xu Yao 1,2 , Hongzhi Kong 1 1 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China 2 University of Chinese Academy of Sciences, Beijing 100049, China * These authors contributed equally to this work Author for correspondence: Hongzhi Kong Tel: +86 010 62836489 Email: [email protected]This PDF file includes: Supplementary Methods Supplementary Tables 1 to 10 Supplementary Figures 1 to 12 Flexibility in the structure of spiral flowers and its underlying mechanisms
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Floral organ identity determination in Nigella damascena … · 2016-01-06 · Floral organ identity determination in Nigella damascena (Ranunculaceae), a species with spiral flowers
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1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany,
Chinese Academy of Sciences, Beijing 100093, China 2University of Chinese Academy of Sciences, Beijing 100049, China *These authors contributed equally to this work
AmtrAGL2 AY936232.1 — aTAIR: The Arabidopsis Information Resource, https://www.arabidopsis.org/ bNCBI: National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ cPhytozome v10.2.1: http://phytozome.jgi.doe.gov/pz/portal.html
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10. Galimba, K. D. et al. Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant. Proc. Natl. Acad. Sci. USA 109, E2267-E2275 (2012).
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14. Shan, H. Y. et al. Conservation and divergence of candidate class B genes in Akebia trifoliata (Lardizabalaceae). Dev. Genes Evol. 216, 785-795 (2006).
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SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
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Supplementary Figure 1 Correlation between the number of floral organs per flower
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Supplementary Figure 1. Correlation between the number of floral organs per flower and the size of floral meristem in Nigella damascena. a, An individual plant at anthesis. b, A diagram showing the structure of the plant, in which the 1st, 2nd and 3rd bloomed flowers are indicated. c, Correlation between the total number of floral organs and the height and width of the floral receptacle in mature flowers (stage 16). A total of 20 flowers were investigated. The Spearman correlation coefficient (ρ) is shown for each chart. The regression equation for the bottom chart is height = -0.0254 + 0.4852*width. d-e Scanning electron microscopy (SEM) images of flower buds at stage 4 showing thee, Scanning electron microscopy (SEM) images of flower buds at stage 4, showing the difference in the size of floral meristem. f-i, SEM images of flower buds at stages 4, 5, 6 and 8, respectively. Br, bract; S, sepal; P, petal; St, stamen; C, carpel. The numbers indicate the forward (in f-h) or reverse (in i) order of the organ initiated. Scale bar = 100 µm.
Supplementary Figure 2. Classification and phylogenetic analysis of floral MADS-box genes. a, An unrooted tree showing the classification of the N. damascena genes into lineages. b-f, Phylogenetic trees of each gene lineage. Bootstrap values higher than 50% are indicated for each node. Yellow stars indicate inferred gene duplication events.
Supplementary Figure 3. Expression patterns of 12 Nigella damascena genes as revealed by qRT-PCR analyses. Flowers at stage 12 were assayed, and the relative
i l l f th li d t th t f NdAP3 3 i t l E bexpression levels of other genes were normalized to that of NdAP3-3 in petals. Error bars indicate the collective standard deviations of three biological replicates and three technical replicates. B, bract; F, flower bud; S, sepal; P, petal; St, stamen; C, carpel.
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
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Supplementary Figure 4. Expression patterns of 12 Nigella damascena genes as revealed by RNA in situ hybridization. a, NdFL1. b, NdFL2. c, NdAP3-1. d, NdAP3-2. e,NdPI1. f, NdPI2. g, NdAG1. h, NdAG2. i, NdSEP1. j, NdSEP2. k, NdSEP3. l, NdAGL6. The stages of the flowers are shown in the top left corners of the images br bract; se sepal;stages of the flowers are shown in the top left corners of the images. br, bract; se, sepal; pe, petal; st, stamen; ca, carpel. Scale bar = 100 µm.
SUPPLEMENTARY INFORMATION DOI: 10.1038/NPLANTS.2015.188
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Supplementary Figure 5. Expression patterns of 13 Nigella damascena genes as revealed by digital gene expression profiling (DGE). Flowers at stages 6-7, 9, 12 and 16 were used, and the relative expression levels of floral MADS-box genes were normalized to those of NdAP3-3 in petals at stage 12. Error bars indicate the collective standard deviations of three biological replicates. S, sepal; P, petal; St, stamen; C, carpel.
Supplementary Figure 6. VIGS construction. a, Schematics showing the positions of the primers used to amplify fragments for VIGS construction. Rectangles and lines indicate the coding regions and untranslated regions (UTRs), respectively. M, I, K and C stand for the sequences that encode the MADS, I, K and C regions of the proteins, respectively. Wavy lines indicate the regions that were used for the VIGS assays. The relative positions of the
i d f RT PCR d i it h b idi ti l i di t d b fill d dprimers used for qRT-PCR and in situ hybridization are also indicated by filled and open triangles, respectively. b, A simplified diagram of the tobacco rattle virus PYL156 vector. Red fonts indicate the restriction enzyme recognition sites used in this study. c, Schematics of VIGS constructs.
Supplementary Figure 8. Phenotypes of the flowers in which AP3- and PI-lineage members were knocked down. a-x, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) five organs, which display different degree of petal-to-sepal transformation in TRV2–NdAP3-3–treated flowers with moderate phenotypic changes, are shown in (g); 2) a petal of TRV2–NdAP3-1–NdANS–treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2–NdAP3-treated flower with strong phenotypic changes is shown in (p); 3) a petal of TRV2 NdAP32–NdANS–treated flower with strong phenotypic changes is shown in (q); and 4) two petals of TRV2–NdAP3-1–NdAP3-2–treated flowers with moderate phenotypic changes are shown in (r). y, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively.
Supplementary Figure 9. Phenotypes of the flowers in which AG-lineage members were knocked down. a-p, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Note that: 1) three organs, which display different degree of stamen-to-petal transformation in TRV2–NdAG1–NdAG2–treated flowers with moderate phenotypic changes, are shown in (m); and 2) two organs showing different degree of carpel-to-sepal transformation in TRV2–NdAG1–NdAG2–treated flowers with strong and moderate phenotypes are shown in (n). q, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers, respectively, and stars (*) indicate the P values obtained with a Mann-Whitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.
Supplementary Figure 10. Phenotypes of the flowers in which AP1-, AGL6- and SEP-lineage members were knocked down a t Flowers and floral organs For each flowerlineage members were knocked down. a-t, Flowers and floral organs. For each flower, both side and top views are shown, and “strong” and “moderate” indicate the flowers with strong and moderate phenotypic changes, respectively. Two sepals of TRV2–NdAGL6–treated flowers with moderate (left) and weak (right) phenotypic changes are shown in (i). Three petals of TRV2–NdAGL6–treated flowers with nearly strong (left), moderate (middle) and weak (right) phenotypic changes are shown in (j). u, Numbers of floral organs in the aforementioned flowers. 1st, 2nd and 3rd represent the first, second and third flowers respectively and stars (*) indicate the P values obtained with a Mann-third flowers, respectively, and stars ( ) indicate the P values obtained with a MannWhitney U test. *, P<0.05; **, P< 0.01; ***, P< 0.001.
Supplementary Figure 11. Interactions among proteins of floral MADS-box genes as revealed by using Y2H assays a Test for auto activation b Test for interaction Left panelrevealed by using Y2H assays. a, Test for auto-activation. b, Test for interaction. Left panel, colonies grown on the SD-LW medium. Middle panel, colonies grown on the selective medium (SD-LWHA + 5 mM 3-AT). Right panel, lacZ test results of the positive colonies on the selective medium. Positive colonies in left panel indicate the co-existence of two vectors in the cells; positive colonies in middle panel and blue coloration in right panel indicate the capability of auto-activation in (a) and the interaction of the paired proteins in (b). ΔC indicates the truncation of the C-terminal regions in NdAP3-1 and NdSEP2, and the C-terminal truncated versions of NdAP3-1 and NdSEP2 were inserted into the pGBKT7the C terminal truncated versions of NdAP3 1 and NdSEP2 were inserted into the pGBKT7 vectors to avoid auto-activation in (b). L, leucine; W, tryptophan; H, histidine; A, adenine; 3-AT, 3-amino-1,2,4-triazole.