DEV026708 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. The zou gain-of-function phenotype is due to ectopic expression of At1g49770. (A) 22-day-old plants grown in long-day conditions. Plants carrying the zou-1D mutation are smaller than wild type and have narrow leaves that curl upwards along the leaf margin. zou-1D homozygotes have smaller, more severely curled leaves, than heterozygotes, and are also more dwarfed and less fertile. (B) 35S::ZOU transgenic plant grown in tissue culture. To confirm that the leaf curling phenotype was due to overexpression of ZOU, we made transgenic plants that mis-expressed the At1g49770 cDNA under control of the constitutive CaMV 35S promoter. These plants recapitulated the zou-1D phenotype (12/23 primary transformants). (C) ZOU::ZOU-GFP transgenic plant showing ZOU gain-of-function leaf curling phenotype. When a ZOU::ZOU-GFP reporter gene fusion (described in main paper) was introduced into wild-type plants, a low proportion of plants (two out of 32 primary transformants) showed a leaf curling phenotype. These plants expressed ZOU-GFP in leaves, whereas the majority of transformants had normal leaf morphology and did not express the reporter in leaves. Scale bars: 5 mm.

• Supplemental Figure S2 -

  Fig. S2. Alignment of ZOU homologues from diverse land plants. Alignment of ZOU protein sequences from Picea sitchensis (Sitka Spruce, Accession Number ABK22833), Arabidopsis thaliana (ZOU, Accession Number NM 103864), Vitis vinifera (Grape, Accession Number CAN74162), Medicago truncatula (Medicago, Accession Number Q1SLX1), Oryza sativa (Rice, Accession Number BAF14724), Selaginella moellendorffii (Selaginella, contig 529.1). The bHLH region is indicated below the alignment with a black line below the alignment. The conserved region at the C-termini of the proteins is indicated by a red line below the alignment. Numerous other dicotyledenous and gymnosperm homologues were detected on plant genome databases; only representatives of major groups are shown here.

• Supplemental Figure S3 -

  Fig. S3. Endosperm is abnormally persistent in zou mutant seed. Imbibed wild-type (A,B) and zou (C-E) seed, from which testa has been removed. Seed are observed under bright field (A,C,E) or UV (B,D) illumination. Autofluorescence under UV illumination allows the cell structure of the mature endosperm to be observed. In wild-type seed (A,B), the embryo (e) separates from the single-layered endosperm (arrow). In zou seed, the endosperm adheres strongly to the stunted embryo, making it difficult to separate the two tissues. The endosperm forms a sac-like structure of persistent endosperm in the uninvaded chalazal zone of the seed (C,D). When this structure is dissected away from the embryo (E), a paste-like tissue is extruded from the endosperm sac (arrowheads).

• Supplemental Figure S4 -

  Fig. S4. Real-time PCR analysis of ALE1 and AtSuc5 expression in zou mutant seed. Expression of AtSUC5 (A) and ALE1 (B) in siliques from zou2 and ZOU+ plants. Two independent biological samples were prepared from plants of each genotype. Each measurement is an average from three technical replicates. Expression levels were normalised by comparison to the expression of the eukaryotic translation initiation factor 4A (EiF4A). The expression levels are relative to the first of the two wild-type samples, arbitrarily designated 1.0.

• Supplemental Figure S5 -

  Fig. S5. Real-time PCR analysis of ALE1 and ZOU expression in zou-1D mutants. Expression of ALE1 (A) and ZOU (B) in seedlings of zou1-D and ZOU+ plants. Three independent biological samples were prepared from bulked seedlings of zou1-D plants. Each measurement is an average from three technical replicates. Expression levels were normalised by comparison to the expression of the eukaryotic translation initiation factor 4A (EiF4A). The expression levels are relative to that in wild-type siliques (Ws SLQ), arbitrarily designated 1.0. The level in wild-type seedlings (Ws) is also shown.

• Supplemental Figure S6 -

  Fig. S6. Invasive growth of zygotic embryos into nutritive tissues in angiosperms, gymnosperms and Selaginales. Schematic representations of developing embryos within their surrounding nutritive tissues. Zygotic embryos are indicated in red, diploid maternal sporophytic tissues (integuments and nucellus of angiosperm and gymnosperm seeds) are shown in yellow, haploid maternal gametophytic tissues are shown in blue, and the angiosperm endosperm is shown in orange. (A) Developing wild-type Arabidopsis seed at torpedo stage. The embryo has separated from the endosperm at this stage. See also Fig. 1I. (B) Developing Pinus embryos invading the megagametophyte tissue. The embryo grows into a ‘corrosion cavity’ formed by programmed cell death of megagametophyte cells surrounding the developing embryo and benefits from the stored nutrients (Filonova et al., 2000; Filonova et al., 2002). Diagram adapted, with permission, from Chamberlain (Chamberlain, 1966). (C) Megagametophye of Selaginella denticulata showing invasive growth of the embryo into the megagametophyte tissue. The megagametophyte is surrounded by the megaspore wall (darker blue) and absorbs nutrients and water through rhizoids (arrow). Diagram adapted, with permission, from Foster and Gifford (Foster and Gifford, 1959).
  References
  Chamberlain, C. J. (1966). Gymnosperms: Structure and Evolution. New York: Dover Publications.
  Filonova, L. H., Bozhkov, P. V., Brukhin, V. B., Daniel, G., Zhivotovsky, B. and von Arnold, S. (2000). Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce. J. Cell Sci. 113, 4399-4411.
  Filonova, L. H., von Arnold, S., Daniel, G. and Bozhkov, P. V. (2002). Programmed cell death eliminates all but one embryo in a polyembryonic plant seed. Cell Death Differ. 9, 1057-1062.
  Foster, A. S. and Gifford, E. M. (1959). Comparative Morphology of Vascular Plants. San Francisco, London: WH Freeman.