1. Supplementary Fig.1. Starch metabolism in green algae and land plants. Schematic representation of the major steps of the starch metabolism pathway. All of these steps, which are strictly conserved from green algae to cereals, are supported numerous biochemical and genetic studies. WSP : Water soluble polysaccharide. MOS : Maltooligosaccharide. STARCH-(P)n : Poly-phosphorylated starch. Synthesis starts within the plastid stroma by the major rate limiting step: the synthesis of ADP-glucose by the ADP- glucose pyrophosphorylase. This step is under an allosteric regulation sensitive to 3-PGA (the product of Rubisco) as activator and orthophosphate as inhibitor). ADP-glucose is the substrate of soluble starch synthases which elongate oligosaccharides through the synthesis of a-1,4 linkages. Branching enzymes insert  -1,6 bonds into these glucans. The random localisation of branches does not fit the dense packing of  -1,6 branches displayed at the root of the mature amylopectin clusters. The misplaced branches are removed through the action of isoamylase. This step is critical to obtain aggregation of the polymer into the starch granule (plant mutants defective for isoamylase revert to the synthesis of smaller amounts of glycogen). MOS produced by the isoamylase action are recycled by a glucanotransferase: DPE1 (also named D-enzyme). Amylose is synthesized through the sole action of GBSS within the starch granule. The first step of the amylopectin degradation resides in the phosphorylation of the glucose residues through the action glucan water dikinases (GWD and PWD). This phosphorylation facilitate and engage the action of the b- amylases which release maltose into the stroma. The maltose is exported into the cytoplasm by MEX where it is cleaved by a transglucosidase named DPE2 which transfers one of the glucose residues from  -maltose onto an acceptor heteroglycan. Recent studies support that this heteroglycan is degraded through the action of a cytoplasmic phosphorylase. The first WSP displayed above is a pre-existing branched glucan used as a primer by starch synthases. WSP1 is the same glucan with added glucose residues. WSP2 is a branched glucan with additional branches formed by the action of a branching enzyme. WSP3 is a less branched polysaccharide, missing branches are released by the isoamylase into free soluble MOS. The thick arrow toward starch symbolizes aggregation into insoluble semi-crystalline material.

2. 0.5 substitution per site E. histolytica A. tumefaciens N. punctiforme Cyanothece CCY0110 P. marinus S. elongatus N. crassa A. fumigatus S. cerevisiae 1 G. gallus 1 X. tropicalis 1 M. musculus 1 V. carteri 1 C. reinhardtii 1 O. tauri M. pusila RCC299 M. pusila CCMP1545 O. sativa ch A. thaliana ch S. tuberosum ch P. sativum ch P. trichocarpa ch D. dictyostelium A. thaliana cy2 O. sativa cy P. trichocarpa cy S. tuberosum cy P. sativum cy V. cholerae E. coli Y. pestis S. usitatus M. gilvum Synechocystis sp. PCC6803 T. vaginalis E. histlytica A. fumigatus 2 S. cerevisiae 2 V. carteri 2 C. reinhardtii 2 T. cruci X. tropicalis 2 G. gallus 2 M. musculs 2 H. sapiens 2 H. sapiens 1 A. thaliana cy1 100 91 17 38 91 44 99 92 22 25 99 Supplementary Fig.2. Plant phosphoglucomutases were duplicated from a single eukaryotic gene. Maximum likelihood unrooted tree inferred for phosphoglucomutases enzymes. The scalebar represent the branch length corresponding to 1 substitution per site. Bootstrap values are indicated at corresponding nodes. Three subgroups can be clearly observed. Two subgroups at the bottom of the tree are a bacterial subgroup and a eukaryotic subgroup. Two isoforms of Chlamydomonas and Volvox can be observed in this latter. Enzymes form which involvement in starch meatbolism is experimentaly proven are all related within the upper subgroup. Inferring the tree with only these sequences support the Eukaryotic origin of the plant phosphoglucomutases.

3. 0.5 substitution per site V. carteri sBE2b C. reinhardtii sBE2b V. carteri sBE2a C. reinhardtii sBE2a O. lucimarinus sBE2 O. tauri sBE2 M. pusilla CCMP1545 sBE2 M. pusilla RCC299 sBE2 A. thaliana sBE2a A. thaliana sBE2b S. tuberosum sBE2 P. trichocarpa sBE2 O. sativa sBE2b O. sativa sBE2a O. sativa sBE1 P. trichocarpa sBE1 S. tuberosum sBE1 O. lucimarinus sBE1 O. tauri sBE1 M. pusilla RCC299 sBE1 M. pusilla CCMP1545 sBE1 C. reinhardtii sBE1 V. carteri sBE1 H. sapiens S. cerevisiae A. fumigatus C. elegans D. melanogaster M. pusilla RCC299 sBE3 M. pusilla CCMP1545 sBE3 B. subtilis G. violaceus P. marinus Synechocystis Cyanothece CCY0110 1 C. watsonii 1 C. watsonii 2 Cyanothece CCY0110 2 A. tumefaciens E. coli Y. pestis Cyanothece CCY0110 3 C. wasonii 3 P. trichocarpa sBE3 A. thaliana sBE3 59 88 56 97 76 46 56 100 33 100 77 Supplementary Fig.3. Plant branching enzymes are of eukaryotic origin. Maximum likelihood unrooted tree inferred for starch branching enzymes. Bootstrap values are indicated at corresponding nodes. Every plant and green alga contain one type I and at least one type 2 branching enzymes. The existence of a bacteria-related branching enzyme in Populus and Arabidopsis was already reported (Han et al. 2007), this enzyme is probably not involved in starch metabolism (Dumez et al. 2005). A third subgroup of undefined phylogeny can be distinguished in Micromonas species. The scalebar represent the branch length corresponding to 0.5 substitution per site. 100

4. C. reinhardtii V. carteri M. pusilla CCMP1545 M. pusilla RCC299 O. lucimarinus O. tauri O. sativa S. tuberosum P. trichocarpa A. thaliana A. variabilis Synechocystis C. watsonii G. variabilis P. marinus O. sativa A. thaliana P. trichocarpa S. tuberosum O. tauri O. lucimarinus M. pusilla RCC299 M. pusilla CCMP1545 V. carteri C. reinhardtii D. discoideum E. histolytica 1 E. histolytica 2 T. vaginalis 1 T. vaginalis 2 T. vaginalis 3 0.5 substitution per site 93 68 26 41 58 100 99 D-enzymes Cyanobacterial glucanotransferases Transglucosidases Supplementary Fig.4. Two kind of alpha-glucanotransferase are detected in every plants. Maximum likelihood unrooted tree inferred for alpha-1,4-glucanotransferases. Bootstrap values are indicated at corresponding nodes. This tree supports the phylogenetic divergence between the two kinds of glucanotransferase activities. D-enzymes (DPE1) are clearly related to bacterial glucanotransferase (which catalyse the same reaction) while transglucosidases (DPE2) seem only to exist in eukaryotic cells. The scalebar represent the branch length corresponding to 0.5 substitution per site.

5. Supplementary Table 1. Complete references of the annotated genes used in this work. These sequences can be retrieved using the gene name as search criteria on the JGI website pages (http://genome.jgi-psf.org/) of each genome. PART 1

6. Table 1. PART 2

7. Table 1. PART 3

8. Supplementary Table 2. Transit peptide predictions and EST survey.