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 ADPglucose 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 a-1,6 bonds into these glucans.
The random localisation of branches does not fit the dense packing of a-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 bamylases 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 b-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.
25
22
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
H. sapiens 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
A. thaliana cy1
O. sativa cy
P. trichocarpa cy
S. tuberosum cy
P. sativum cy
99
91
92
44
99
100
91
0.5 substitution per site
V. cholerae
E. coli
Y. pestis
S. usitatus
M. gilvum
Synechocystis sp. PCC6803
T. vaginalis
E. histlytica
17
A. fumigatus 2
S. cerevisiae 2
100
V. carteri 2
C. reinhardtii 2
T. cruci
X.
tropicalis
2
38
G. gallus 2
M. musculs 2
H. sapiens 2
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.
V. carteri sBE2b
C. reinhardtii sBE2b
V. carteri sBE2a
C.
reinhardtii sBE2a
56
O. lucimarinus sBE2
O. tauri sBE2
88
M. pusilla CCMP1545 sBE2
M. pusilla RCC299 sBE2
A. thaliana sBE2a
97
A. thaliana sBE2b
S. tuberosum sBE2
P. trichocarpa sBE2
46
O. sativa sBE2b
O. sativa sBE2a
O. sativa sBE1
P. trichocarpa sBE1
S. tuberosum sBE1
O. lucimarinus sBE1
76
O. tauri sBE1
56
M. pusilla RCC299 sBE1
M. pusilla CCMP1545 sBE1
C. reinhardtii sBE1
V. carteri sBE1
H. sapiens
S. cerevisiae
A. fumigatus
59
C. elegans
D. melanogaster
M. pusilla RCC299 sBE3
100
M. pusilla CCMP1545 sBE3
100
77
100
33
0.5 substitution per site
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
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.
C. reinhardtii
V. carteri
58
M. pusilla CCMP1545
M. pusilla RCC299
O. lucimarinus
93
D-enzymes
O. tauri
O. sativa
99
S. tuberosum
P. trichocarpa
A. thaliana
A. variabilis
Synechocystis
Cyanobacterial glucanotransferases
C. watsonii
G. variabilis
P. marinus
O. sativa
41
A. thaliana
P. trichocarpa
S. tuberosum
68
O. tauri
O. lucimarinus
M. pusilla RCC299
M. pusilla CCMP1545
100
Transglucosidases
V. carteri
C. reinhardtii
D. discoideum
0.5 substitution
E. histolytica 1
per site
26
E. histolytica 2
T. vaginalis 1
T. vaginalis 2
T. vaginalis 3
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.
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
Chlamydomonas
Volvox
Arabidopsis
AT5G51820.1
AT1G23190.1
AT1G70730.1
AT5G48300.1
AT1G05610.1
Phosphoglucomutase
-
estExt_GenewiseW_1.C_380007
Chlre2_kg.scaffold_1000037
estExt_Genewise1Plus.C_10675
fgenesh4_pg.C_scaffold_27000118
ADPGPyrophosphorylase
Small
estExt_gwp_1W.C_140184
estExt_Genewise1.C_570073
Large
estExt_fgenesh2_pg.C_20112
e_gwW.14.62.1
estExt_Genewise1.C_260077
gw1.66.6.1
fgenesh4_pg.C_scaffold_14000178
AT5G19220.1
AT1G27680.1
AT4G39210.1
AT2G21590.1
fgenesh1_pm.C_scaffold_32000005
e_gwW.3.57.1
e_gwH.40.7.1
e_gwW.31.195.1
estExt_Genewise1.C_30360
AT1G32900.1
fgenesh4_pg.C_scaffold_85000033
fgenesh4_pg.C_scaffold_75000028
AT5G24300.1
SSII
fgenesh2_kg.C_scaffold_14000006
estExt_fgenesh4_pg.C_40144
AT3G01180.1
SSIII
estExt_fgenesh2_pg.C_260084
MHS_fgenesh1_pg.C_scaffold_2000364
e_gw1.11.115.1
fgenesh4_pg.C_scaffold_98000001
AT1G11720.1
Pullulanase
estExt_fgenesh4_pg.C_1170022
estExt_fgenesh4_pg.C_540009
estExt_Genewise1Plus.C_500076
estExt_Genewise1Plus.C_10629
e_gw1.31.3.1
estExt_fgenesh4_pg.C_330004
e_gw1.95.23.1
fgenesh4_pg.C_scaffold_15000043
fgenesh5_synt.21__4
fgenesh5_synt.47__11
AT4G18240.1
AT5G65685.1
"SBE3"
Isa1
Isa2
Isa3
-
estExt_fgenesh2_kg.C_670012
Chlre2_kg.scaffold_67000057
e_gwW.26.5.1
Chlre2_kg.scaffold_3000101
fgenesh1_est.C_scaffold_10000004
estExt_fgenesh2_pg.C_210207
fgenesh1_pm.C_scaffold_34000011
fgenesh1_pg.C_scaffold_13000279
estExt_gwp_1H.C_470047
fgenesh1_pg.C_scaffold_33000008
Glucano-Transferases
D-enz
estExt_gwp_1W.C_820038
e_gw1.4.241.1
AT5G64860.1
DPE2
e_gwW.62.1.1
estExt_fgenesh4_pg.C_650060
AT2G40840.1
Phosphorylases
Chlre2_kg.scaffold_50000042
estExt_gwp_1W.C_200107
fgenesh4_pg.C_scaffold_13000247
estExt_fgenesh4_pg.C_730071
AT3G29320.1
AT3G46970.1
Glucan dikinases
GWD
SKA_Chlre2_kg.scaffold_6000249
fgenesh2_pg.C_scaffold_10000246
fused estExt_fgenesh2_kg.C_100091
estExt_fgenesh2_pg.C_290060
fgenesh5_synt.41__18
fgenesh5_synt.110__3
PWG
estExt_fgenesh2_pg.C_320003
fgenesh4_pg.C_scaffold_15000185
estExt_fgenesh4_pg.C_160002
AT5G26570.1
Beta-amylases
-
estExt_fgenesh2_kg.C_120039
e_gwW.3.506.1
estExt_gwp_1H.C_10432
estExt_fgenesh4_pg.C_40269
e_gw1.71.76.1
AT4G15210.1, AT5G45300.1
AT5G18670.1, AT2G45880.1
AT2G32290.1, AT5G55700.1
AT3G23920.1, AT4G17090.1
AT4G00490.1
Alpha-amylases
-
e_gwW.21.36.1
estExt_GenewiseH_1.C_290028
fgenesh2_pg.C_scaffold_21000020
estExt_Genewise1Plus.C_420085
estExt_Genewise1.C_230010
AT4G25000.1
AT1G76130.1
AT1G69830.1
Mex1
Sex4
-
SKA_fgenesh2_pg.C_scaffold_11000277
Chlre2_kg.scaffold_27000025
fgenesh5_synt.9__81
estExt_Genewise1Plus.C_170055
AT5G17520.1
AT3G52180.1
Starch synthases
gbss
SSI
Branching enzymes
SSIV
SSV
SBE1
SBE2
Isoamylases
AT2G36390.1
AT5G03650.1
AT3G20440.1
AT2G39930.1
AT1G03310.1
AT4G09020.1
AT5G04360.1
AT1G10760.1
AT4G24450.1
Table 1. PART 2
Ostreococcus
Tauri
Lucimarinus
Phosphoglucomutase
-
fgenesh1_pm.C_Chr_15.0001000049
e_gwEuk.14.55.1
ADPGPyrophosphorylase
Small
e_gw1.07.00.151.1
e_gwEuk.7.142.1
Large
estExt_gwp_GeneWisePlus.C_Chr_20.00010037
e_gw1.07.00.135.1
e_gwEuk.19.26.1
eugene.0700010350
gbss
estExt_gwp_GeneWisePlus. C_Chr_06.00010052
estExt_Genewise_ext. C_Chr_60515
SSI
fgenesh1_pm.C_Chr_13.0001000019
e_gwEuk.13.68.1
SSII
estExt_genewise1.C_Chr_16.00010070
gwEuk.15.82.1
SSIV
SSV
SBE1
e_gw1.16.00.2.1
estExt_gwp_GeneWisePlus.C_Chr_13.00010266
e_gw1.06.00.85.1
estExt_gwp_GeneWisePlus.C_Chr_04.00010531
e_gwEuk.15.9.1
e_gwEuk.13.15.1
e_gwEuk.6.95.1
estExt_Genewise_ext.C_Chr_10716
eugene.0400010396
SBE2
300010084
estExt_GenewiseEukaryote.C_Chr_30051
Pullulanase
"SBE3"
Isa1
Isa2
Isa3
-
e_gw1.14.00.51.1
fgenesh1_pg.C_Chr_02.0001000307
1200010031
100010303
e_gwEuk.20.34.1
eugene.0200010431
estExt_fgenesh1_pm.C_Chr_120008
e_gwEuk.1.22.1
Glucano-Transferases
D-enz
fgenesh1_pg.C_Chr_02.0001000209
estExt_GenewiseEukaryote.C_Chr_20118
DPE2
gw1.11.00.47.1
estExt_fgenesh1_pm.C_Chr_04.00010030
fgenesh1_pm.C_Chr_11.0001000017
e_gw1.11.00.95.1
e_gwEuk.11.22.1
fgenesh1_pg.C_Chr_4000150
e_gwEuk.11.36.1
estExt_fgenesh1_pg.C_Chr_110020
GWD
estExt_gwp_GeneWisePlus.C_Chr_13.00010209
gw1.08.00.13.1
gw1.16.00.18.1
ost_13_007_009
estExt_fgenesh1_pg.C_Chr_150146
estExt_Genewise_ext.C_Chr_80084
PWG
fgenesh1_pg.C_Chr_04.0001000273
e_gw1.08.00.3.1
ost_04_018_105
eugene.0800010117
-
300010319
0200010698
fgenesh1_pg.C_Chr_3000249
gwEuk.2.152.1
Alpha-amylases
-
fgenesh1_pg.C_Chr_16.0001000029
1000010026
fgenesh1_pm.C_Chr_07.0001000030
fgenesh1_pg.C_Chr_15000021
e_gwEuk.7.210.1
estExt_GenewiseEukaryote.C_Chr_100021
e_gwEuk.2.224.1
Mex1
Sex4
-
900010316
fgenesh1_pg.C_Chr_02.0001000254
eugene.0900010288
eugene.0200010086
Starch synthases
SSIII
Branching enzymes
Isoamylases
Phosphorylases
Glucan dikinases
Beta-amylases
Table 1. PART 3
Micromonas
RCC299
CCMP1545
Phosphoglucomutase
-
e_gw2.01.168.1
estExt_fgenesh1_kg.C_20035
ADPGPyrophosphorylase
Small
estExt_fgenesh2_kg.C_Chr_070019
estExt_fgenesh1_kg.C_10041
Large
e_gw2.01.322.1
estExt_Genewise2Plus.C_Chr_020427
estExt_Genewise1Plus.C_20473
e_gw1.4.863.1
gbss
estExt_fgenesh2_kg.C_Chr_020003
estExt_fgenesh1_kg.C_110003
SSIV
SSV
SBE1
estExt_fgenesh2_kg.C_Chr_060061
gw2.11.73.1
e_gw2.15.329.1
e_gw2.07.522.1
estExt_fgenesh2_kg.C_Chr_150005
estExt_fgenesh2_kg.C_Chr_060062
e_gw2.08.94.1
estExt_fgenesh2_kg.C_Chr_030028
e_gw2.08.666.1
estExt_fgenesh1_pg.C_50151
gw1.8.77.1
estExt_Genewise1.C_140367
fgenesh1_pm.C_scaffold_9000005
estExt_Genewise1Plus.C_50295
estExt_fgenesh1_pg.C_140161
e_gw1.11.141.1
estExt_fgenesh1_pm.C_170017
e_gw1.8.541.1
SBE2
estExt_Genewise2Plus.C_Chr_010412
estExt_fgenesh1_kg.C_20047
Pullulanase
"SBE3"
Isa1
Isa2
Isa3
-
e_gw2.08.666.1
estExt_fgenesh2_pg.C_Chr_130076
EuGene.0100010130
estExt_fgenesh2_kg.C_Chr_150010
fgenesh2_pm.C_Chr_07000017
fgenesh1_pm.C_scaffold_5000096
estExt_Genewise1Plus.C_70118
est_orfs.2_3198_4270363:1
estExt_fgenesh1_pm.C_140015
estExt_Genewise1.C_10891
Glucano-Transferases
D-enz
estExt_Genewise2.C_Chr_010539
estExt_Genewise1.C_20623
DPE2
e_gw2.12.29.1
estExt_fgenesh2_kg.C_Chr_110012
estExt_fgenesh2_kg.C_Chr_120004
e_gw2.12.509.1
e_gw1.10.23.1
estExt_fgenesh1_kg.C_80004
estExt_Genewise1Plus.C_100032
estExt_Genewise1Plus.C_100176
GWD
e_gw2.04.10.1
fgenesh2_kg.C_Chr_06000057
e_gw2.12.513.1
estExt_fgenesh1_pg.C_50208
estExt_fgenesh1_pg.C_90205
estExt_fgenesh1_pg.C_100126
PWG
estExt_fgenesh2_kg.C_Chr_050017
e_gw2.09.6.1
estExt_fgenesh2_kg.C_Chr_060065
est_orfs.6_8624_4273671:1
e_gw1.9.75.1
EuGene.0000050241
EuGene.0000050242
-
est_cluster_kg.Chr_04__16__3199517:1
fgenesh2_kg.C_Chr_11000018
e_gw1.12.72.1
e_gw1.8.113.1
Alpha-amylases
-
fgenesh2_kg.C_Chr_14000003
fgenesh2_kg.C_Chr_02000034
e_gw2.02.334.1
fgenesh2_kg.C_Chr_06000003
fgenesh2_pm.C_Chr_09000077
EuGene.0600010408
EuGene.1100010582
gw2.15.105.1
gw1.12.147.1
estExt_Genewise1.C_40491
estExt_Genewise1.C_40669
estExt_Genewise1.C_90048
fgenesh1_pg.C_scaffold_18000135
Mex1
Sex4
-
estExt_fgenesh2_kg.C_Chr_140010
gw2.05.501.1
EuGene.0000030121
estExt_fgenesh1_pg.C_30481
Starch synthases
SSI
SSII
SSIII
Branching enzymes
Isoamylases
Phosphorylases
Glucan dikinases
Beta-amylases
Supplementary Table 2. Transit peptide predictions and EST survey.
ESTs
Phosphoglucomutase 1
Phosphoglucomutase 2
ADP-glucose pyrophosphorylase S
ADP-glucose pyrophosphorylase L1
ADP-glucose pyrophosphorylase L2
Granule bound starch synthase 1
Granule bound starch synthase 2
Soluble starch synthase 1a
Soluble starch synthase 1b
Soluble starch synthase 2a
Soluble starch synthase 2b
Soluble starch synthase 3a
Soluble starch synthase 3b
Soluble starch synthase 3c
Soluble starch synthase 4
Soluble starch synthase 5
Branching enzyme 1
Branching enzyme 2a
Branching enzyme 2b
Branching enzyme 3
Isoamylase 1
Isoamylase 2
Isoamylase 3
Pullulanase
D-enzyme
Transglucosidase
Phosphorylase 1
Phosphorylase 2
Phosphorylase 3
Glucan water dikinase 1
Glucan water dikinase 2
Glucan water dikinase 3
Phosphoglucan water dikinase 1
Phosphoglucan water dikinase 2
Phosphoglucan water dikinase 3
Phosphoglucan water dikinase 4
b-amylase 1
b-amylase 2
b-amylase 3
a-amylase 1
a-amylase 2
a-amylase 3
a-amylase 4
a-amylase 5
a-amylase 6
a-amylase 7
a-amylase 8
Maltose exporter
Sex4
Chlamydomonas
~ 50
Y
Y
2
Y
~ 40
N
1
N
~ 60
Y
N
1
Y
N
~ 20
N
n/a
n/a
~ 15
N
N
n/a
n/a
~ 30
Y
8
N
N
4
N
~ 50
N
3
Y
2
Y
Y
~ 15
Y
N
~ 10
Y
N
~ 50
N
~ 20
N
n/a
n/a
~ 10
N
~ 10
N
1
N
1
Y
n/a
n/a
n/a
n/a
n/a
n/a
~ 40
N
2
N
10
N
~ 15
N
~ 20
N
~ 10
Y
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
4
Y
10
Y
Transit peptide
Micromonas
Volvox
N
N
Y
Y
N
Y
n/a
N
N
Y
n/a
N
Y
n/a
Y
N
N
N
N
Y
N
N
N
N
N
N
Y
Y
n/a
N
N
n/a
N
N
n/a
n/a
N
N
n/a
Y
N
n/a
n/a
n/a
n/a
n/a
n/a
N
N
CCMP 1545
Y
n/a
Y
Y
Y
Y
n/a
N
Y
N
Y
N
N
N
N
n/a
N
Y
n/a
Y
N
N
N
N
Y
N
Y
Y
N
N
N
Y
N
N
Y
Y
N
N
n/a
N
N
N
N
N
n/a
n/a
n/a
N
N
RCC299
N
n/a
N
N
N
N
n/a
N
N
N
Y
N
N
N
N
n/a
Y
N
n/a
N
N
Y
Y
Y
N
N
N
N
N
N
N
N
N
N
Y
n/a
N
N
n/a
N
N
N
N
N
N
N
N
N
N
Ostreococcus
tauri
lucimarinus
N
n/a
N
N
N
N
n/a
N
n/a
N
n/a
N
N
N
n/a
n/a
Y
N
n/a
n/a
Y
N
N
N
Y
N
Y
N
N
N
Y
N
N
N
n/a
n/a
N
N
n/a
N
N
N
n/a
n/a
n/a
n/a
n/a
N
N
N
n/a
N
N
N
N
n/a
N
n/a
N
n/a
N
Y
N
N
n/a
N
N
n/a
n/a
Y
N
N
Y
N
N
N
N
N
N
N
N
N
N
n/a
n/a
N
N
n/a
N
N
N
N
n/a
n/a
n/a
n/a
N
N
In the first column, under the heading"ESTs", we have listed numbers that correspondto the number of different ESTs
found in the extensive databasesgeneratedfor Chlamydomonasexclusively.In the other columns, we have symbolise
by "Y" and "N" respectively the presence or abscence of a putative transit peptide as detected using ChloroP
(http://www.cbs.dtu.dk/services/ChloroP/).We insist that this detection is meaningless as many plastidials proteins
detected by proteomicslack transit peptides and because some gene that contain very clear targeting sequencesmay
not at all be located in plastids.A good example in starch metabolismis afforded by the ArabidopsisDPE2 protein wich
contains a transit peptide and yet is exclusively located in the cytosol.