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Supplemental Figures
B
8
µg
StuI+SacI
6
NotI
std1
4
std1
StuI+SacI
8
XmaI
WT
NotI
WT
XmaI
A
C
Con. Acc.
Degr.
Acc.
Degr.
WT
std1
std1::STD1-1
std1::STD1-2
Figure S1. Southern blot analysis and complementation of the std1 mutant.
(A) Southern blot analysis indicates single insertion of the antibiotic resistance cassette in the genome
of std1. NotI-, XmaI or StuI/SacI-restricted genomic DNA of wild-type and std1 mutant cells was
loaded on an agarose gel, Southern blotted and hybridized with a probe against the AphVIII gene
(paromomycin resistance cassette). Loaded amount of DNA per lane is indicated in µg.
(B) Vector construct used for the complementation of the std1 mutant. Genomic wild-type DNA
coding for STD1 was amplified by PCR and cloned into the pSL-Hyg vector harbouring the psaD
promoter and terminator, and a hygromycin resistance marker allowing selection of positive
transformants.
(C) Starch degradation phenotype of the std1 mutant and of two complemented lines. Intracellular
starch levels of wild-type strain (black), std1 mutant (white) and two complemented strains
(std1::STD1-1 and -2, grey) were analyzed in response to nutrient deprivation and resupply. Cultures
were grown in TAP medium (Con. = Control), subjected for two days to nitrogen (TAP-N) or sulfur
starvation (TAP-S), which induced starch accumulation (Acc.). Subsequently, the starved cells were
transferred to minimal medium (MM) and kept for 8 or 24 hours in the dark. Starch was catabolized
(Degr.) in MM (comprising N but no C) in the dark. Starch values are the means ± SD (n ≥ 3).
1
A
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
DH box
908aa--YHMDEQGNVLYEYDPDYIDRKYEVFELRVIHRRHRTGFEETKDFPIRLNDLIAG
57aa--EDEEDAAVQEHTRRIRAEEEEFETFDLKIIHRKNRTGFEEDKDFPVVINAVVAG
783aa--DDEEAVAVQEQVRQIKAQEEEFETFDLKIVHRKNRTGFEEEKNFNVVLNSVIAG
782aa--EDEEAAAVQEQVRQIKVQEEEFETFNLKIVHRKNRTGFEEDKNFHVVLNSVIAG
1486aa--EDEEAAAVQEQVRQIKAQEEEFETFNLKIVHRKNRTGFEEDKNFHVVLNSVIAG
746aa--DDEEAAALQEQVRQIKAQEEEFETFNLKIVHRKNRTGFEEDKNFHVVLNSVIAG
361aa--LKAEEDAETNIDLALSDFHREYEVFELRIIHRKNRTGFEENKDFPIVLNSVVAG
964
413
839
838
1542
802
417
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
I
II
III
RYQVMDFLGSAAFSRAVQALDIKTGQLVCLKIIKNHKDYFDQSLDEIKLLKYVNTMDPND
RYHVTEYLGSAAFSKAIQAHDLHTGMDVCMKIIKNNKDFFDQSLDEIKLLKYINKHDPGD
RYHVTEYLGSAAFSKAIQAHDLQTGMDVCIKIIKNNKDFFDQSLDEIKLLKYVNKHDPAD
RYHVTEYLGSAAFSKAIQAHDLHTGMDVCVKIIKNNKDFFDQSLDEIKLLKYVNKHDHAD
RYHVTEYLGSAAFSKAIQAHDLHTGMDVCVKIIKNNKDFFDQSLDEIKLLKFVNKNDPAD
RYHVTEYLGSAAFSKAIQAHDLHTGMDVCVKIIKNNKDFFDQSLDEIKLLKYVNKHDPAD
RYYITEYLGSAAFSKVVQAHDLRTGMDVCLKIIKNDKDFFDQSLDEIKLLKFVNKYDPLD
1024
473
899
898
1602
862
477
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
IV
V
VIa
EYAIVRLYDFFYYKEHLFLVCELLRANLYEFQKYNKESGDPAYFTNARIQRIARQALRSL
KHHLLRLYDYFYHKEHLFIVCELLRANLYEFHKYNRESGGEVYFTMPRLQSIARQCLESL
KYHLLRLYDYFYYREHLLIVCELLKANLYEFHKFNRESGGEVYFTMPRLQSITIQCLESL
KYHILRLYDYFYYREHLLIVCELLKANLYEFHKFNRESGGEVYFTMPRLQSITTQCLEAL
KYHILRLYDYFYYREHLLIVCELLKANLYEFHKFNRESGGEVYFTMPRLQSITIQCLEAL
KYHLLRLYDYFYYREHLLIVCELLKANLYEFQKFNRESGGEVYFTMPRLQSIAIQCLEAL
EHHVLRLYDYFYHQEHLFIVTELLRANLYEFQKYNQESGGEVYFTLPRIQVIARQCLEAL
1084
533
959
958
1662
922
537
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
VIb
VII
VIII
AFLHSLGLIHSDLKPENILIKSYSRCEVKVIDLGSSCFITDQLSSYVQSRSYRAPEVILG
EFIHGLGLIHCDLKPENILVKSYSRCEIKVIDFGSSCFQTDHLFSYVQSRSYRAPEVIVG
QFLHGLGLIHCDLKPENILVKSYSRCEIKVIDLGSSCFETDHLCSYVQSRSYRAPEVILG
QFLHGLGLIHCDLKPENILVKSYSRCEVKVIDLGSSCFETDHLCSYVQSRSYRAPEVILG
QFLHGLGLIHCDLKPENILVKSYSRCEVKVIDLGSSCFETDHLCSYVQSRSYRAPEVILG
QFLHGLGLIHCDLKPENILVKSYSRCEVKVIDLGSSCFETDHLCAYVQSRSYRAPEVILG
VYLHRLRIIHCDLKPENILIKSYSRCEIKVIDLGSSCFLTDNLCLYVQSRSYRAPEVILG
1144
593
1019
1018
1722
982
597
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
IX
X
LPYDYKVDVWSLGCILAELSSSFVLFQNDSLSTLLARLEGILGPVPEWMLHKGRYAHRFY
LPYDQKIDMWSLGCILAELCSGNVLFQNDSLATLLARVVGILGPIDPELLTKGRDTHKFF
LPYDKKIDVWSLGCILAELCTGNVLFQNDSPASLLARVMGIVGSFDNEMLTKGRDSHKYF
HPYDKKIDVWSLGCILAELCTGNVLFQNDSPATLLARVIGIIGPIDQSMLAKGRDTYKYF
LPYDKKIDVWSLGCILAELCTGNVLFQNDSPATLLARVIGIIGSIDQGMLAKGRDTYKYF
LPYDKKIDMWSLGCILAELCTGNVLFQNDSPATLLARVMGIIGSIEQAMLAQGRETYKYF
LPYDQRIDIWSLGCILSELYTGEVLFPNEPVSVMLAQMIGITGPIDMEMLELGQETQKYF
1204
653
1079
1078
1782
1042
657
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
XI
TRSGMLYERNATTQKYDMLQPKRTSLRHRMPDADEGLLEFVGHLLTVDPRKRPTAAEALK
TKNHMLYERNQDSDQLEYLLPKKTSLAHRLPMGDKGFVEFVEHLLNINPLLRPTASEALK
TKNRMLYERNQESNRLEYLIPKRTSLRHRLPMGDQGFTDFVAHLLEINPKKRPSAAEALK
SKNHMLYERNQDTSRLEYLIPKKTSLRHRLPMGDQGFIDFVSHLLEVNPKKRPSASEALK
TKNHMLYERNQDTNRLEYLIPKKTSLRHRLPMGDQGFIDFVSHMLEINPKKRPSASEALK
TKNHMLYERNQESNRLEYLIPKKTSLRHRLPMADQGFIEFVAYLLEVNPKKRPSASEALK
TDDYDLFTENEETDQLEYLIPEKSSLRQHIQCPDSEFVDFLSYLLQINPRKRPTADEALQ
1264
713
1139
1138
1842
1102
717
CrDYRKP1
PpDYRKP1
AtDYRKP1
PtDYRKP4
VvDYRKP1
OsDYRKP2
ZmDYRKP6
HPWLQQEYPSLDSM
HPWLSYLYEPISSHPWLSYPYEPISAHPWLSYPYEPISAHPWLSYPYEPISSHPWLSFPYEPISSHPWLSFSY------
1278
726
1152
1151
1855
1115
B
Becker and Joost, 1999
DYRK1
DYRK2
YAK1
DYRKP-A
DYRKP-B
DYRKP-algae
C
N
N
N
N
N
N
H
X —
D/H —
X —
D/N —
R T
R T
R/K T
G Y/F D D D/E
G Y D D D
G Y/F D D D/E
G X D N E
G F E E D/E
G F E E N
G F E E S
N
N
R
N
K
K
K
X D
H/Y D
G D/S
X D
— N/D
— D/E
— D/E
Y
X
X X
X
X D/E
Y
I
V K/R N/S G
E
Y
X
X V P/L H/G/R D
Y/L
I
L X
V N/G/H D/X
F H/P/N V V
L
N S/A
F/L
P
I
V
X N/D S/T
F
P
V R/K — G/N D
DH KINASE
CreYak1
AtYak1
CreDYRKP
AtDYRKP-1
Figure S2. Conserved sequence features of DYRKP kinases.
(A) Alignment of amino acid sequences from the C-terminal kinase domain of Chlamydomonas
DYRKP, five higher plant DYRKP homologs and one DYRKP homolog from moss. The sequences
were aligned using ClustalW and shaded using BoxShade (http://www.ch.embnet.org). Amino acids
2
highlighted in black are perfectly conserved, and similar residues are indicated by a grey background.
Arrows above the alignment indicate the DYRK homology (DH) box that precedes the conserved
catalytic kinase domain and the kinase subdomains I-XI according to Aranda et al., 2011. Subdomains
I and II respectively harbour the ATP anchor and the phosphate anchor. The catalytic loop is found
within subdomain VIb, the cation binding site is located within subdomain VII, and subdomain VIII
contains the activation loop and the P+1 loop. The conserved tyrosine in the activation loop (“YxY”)
was found to be autophosphorylated and is marked by a triangle. The segment between subdomains X
and XI of the protein is known as the CMGC insert. For details see Aranda et al., 2011.
(B) Consensus sequence of the DH-box of DYRKs according to (A), additional file 1: Figure S8,
Figure S9, and Table S1. Below the published DH consensus sequence from (Becker and Joost, 1999),
consensus sequences of DYRK1 (7 sequences), DYRK2 (22 sequences), and Yak1 (21 sequences) are
shown. Three minor subgroups were distinguished within the DYRKP subgroup, DYRKP-A (12 land
plant sequences including moss), DYRKP-B (11 vascular plant sequences), DYRKP-algae (7
sequences). Multiple sequence alignments of DH-box motifs were performed using the program
WebLogo (http://weblogo.berkeley.edu/logo.cgi).
(C) Schematic illustration of protein domain organization in two Yak1 and two DYRKP
representatives from C. reinhardtii (CreYak1 and CreDYRKP) and A. thaliana (AtYak1 and
AtDYRKP-1). DH, DYRK-homology box; Kinase, kinase domain.
3
WT
std1::STD1-1
std1
std1::STD1-2
Figure S3. Persistently high starch levels were observed in the std1 mutant during
photoautotrophic S deprivation conditions.
Intracellular starch was measured in wild-type (black), std1 mutant (white), and complemented lines
(grey) during photoautotrophic S deprivation under medium light (100 µmol photons m-2 s-1). Starch
values are the means of 5 biological replicates ± SD.
4
A
B
C
D
E
F
G
H
J
WT
std1
std1::STD1-1
std1::STD1-2
Figure S4. Total cellular volume data and chlorophyll contents for the kinetic
experiments in nitrogen deprivation shown on Figure 2.
(A, B, C) Total cellular volume measurements in µm3/ml were recorded by Multisizer™ 3
Coulter Counter® (Beckman). Cells were kept photoautotrophically at low light (LL, 35 µmol
photons m-2 s-1) (A) or medium light (100 µmol photons m-2 s-1) (B) supplemented with 2%
CO2 (A, B) or mixotrophically (TAP) under medium light (C) as in Figure 2. Values are
means ± SD (n ≥ 3).
(D, E, F) Chlorophyll contents during N deprivation in photoautotrophic or mixotrophic
conditions. Chlorophyll was extracted by methanol, and chlorophyll a and b were determined
by measuring the absorbance at 653, 666 and 750 nm using UV-VIS spectrophotometer
(SAFAS UVmc2 with the software SP2000). Chlorophyll content was calculated using the
formula Concentration (µg/ml) = (A1-A3)*19.71+(A2-A3)*4.44. Chlorophyll concentrations
are means ± SD of ≥ 3 experiments, duplicate samples were taken.
5
(G, H, J) Cell or particle concentrations per ml were recorded by Multisizer™ 3 Coulter
Counter® (Beckman) as in (A, B, C).
WT
std1
std1::STD1-1
std1::STD1-2
Figure S5. Oil accumulate in std1 mutant following N deprivation.
TAG content in cells grown photoautotrophically under medium light (100 µmol photons m-2
s-1) supplemented with 2% CO2. A representative experiment is shown with means from three
technical replicates.
6
Figure S6. The std1 mutant forms cell aggregates enclosed by the mother cell wall.
(A) Bright field and differential interference contrast images of wild-type (WT), std1 mutant and
complemented (std1::STD1-1 and -2) strains grown in minimal medium (MM) supplemented with 2%
CO2 and then subjected to N deprivation for 0, 2 or 6 days. Arrows indicate mother cell walls. Scale
bars, 10 µm.
(B) Particle numeration (left panels) and total cellular volumes (right panels) as a function of the
particle diameter, determined in cultures of WT (red), std1 mutant (green) and two complemented
strains std1::STD1-1 (blue) and -2 (black) at 0, 2 or 6 days in MM-N/2% CO2. Data are from the same
experiment as in (A).
(C, D) Cell counting during nitrogen starvation following treatment with the cell wall-degrading
enzyme autolysin. Particle concentration (C) and particle diameter (D) were determined in WT (red),
std1 mutant (green) and complemented lines (blue) during nitrogen deprivation in photoautotrophic
condition (MM-N/2% CO2). Data are from before (solid lines) and after treatment with autolysin
(dashed lines) and represent means ± SD (n = 4). Autolysin treatment allowed determination of std1
cell number during deprivation kinetics (C) and revealed an increase in the volume of each single
mutant cell, while the volume of wild-type cells decreased during nutrient starvation (D).
7
A
std1
WT
0
1
2
3
0
1
2
3
RbcL
Cyt f
PsbD
PsaC
AtpB
COXIIb
std1
WT
B
0
1
2
3
0
1
2
3
250
150
100
75
50
37
25
20
15
10
Figure S7. Protein levels in wild-type and std1 mutant cells during photoautotrophic N
deprivation as determined by immunodetection.
(A) Provided antibody names include RbcL, Rubisco large subunit; Cyt f, cytochrome f; PsbD, D2
photosystem II subunit; PsaC, PSI-C core photosystem I subunit; AtpB, beta subunit of ATP synthase;
and COXIIb, cytochrome oxidase subunit II b. 10 µg of total protein were loaded from N starved cells
and harvested at the indicated time points after N depletion. Days in MM-N are indicated.
(B) Coomassie brilliant blue coloration. Molecular weight marker units are in kDa.
8
ZmDYRKP-3/4
DYRK1
DYRKP
VcaDYRK2
PhypaDYRKP-1/2/3
DYRK2
Yak1
Figure S8. Phylogenetic tree of the DYRK protein family.
The complete tree obtained by the Neighbour-Joining (NJ) method as in Figure 1d, before
combination of branches from Populus trichocarpa, Zea mays, and two species of Micromonas and
Ostreococcus. Bootstrap values are indicated.
9
A
DYRKP
DYRK2
AtDYRKP-4
OsDYRKP-2 AtDYRKP-1/2
DYRK2/3
dDYRK3
CreDYRKP
Phypa
DYRK-1/2/3
VcaDYRKP
DYRK4
VivDYRKP-1
ChlNCDYRKP
VivDYRKP-2
dDYRK2
ChlNC-DYRK2
AtDYRKP-3
MicpuDYRKP
OsDYRKP-1
CreDYRK2
VcaDYRK2
OstDYRKP
OsDYRKP-3
PhypaDYRK2
MicpuDYRK2
Pom1
Ppk5p
DdDYRK2
Minibrain
DYRK1B
DdDYRK1
DYRK1A
DYRK1
YakA
Yak1
PhypaYak
1/2/3/4/5
Yak1p
AspYak1
B
OsYak1/2
MicpuYak1
OstYak1
CreYak1 VcaYak1
Ppk15
VivYak1
AtYak1
DYRKP
OsDYRKP-3 OsDYRKP-1
VivDYRKP-2
VcaDYRKP
CreDYRKP
AtDYRKP-3
ChlNC-DYRKP
AtDYRKP-1/2
VivDYRKP-1
OsDYRKP-2
DYRK2
Phypa
DYRKP-1/2/3
DYRK2/3
dDyrk3
AtDYRKP-4
Ppk5p
MicpuDYRKP
OstDYRKP
Pom1
DYRK4
dDyrk2
ChlNC-DYRK2
VcaDYRK2
CreDYRK2
MicpuDYRK2
DdDyrk2
DdDyrk1
Minibrain
DYRK1A
PhypaDYRK2
Dyrk1b
DYRK1B
YakA
DYRK1
Yak1p
AspYak1
Ppk15p
OstYak1
PhypaYak
1/2/3/4/5
MicpuYak1
CreYak1
VcaYak1
OsYak1/2
Yak1
VivYak1 AtYak1
Figure S9. Phylogenetic tree of the DYRK protein family by using the Maximum
Likelihood (ML) or the Parsimony (Pars) approach. Phylogenetic tree obtained by the ML (A)
or the Pars (B) method. Branches were combined as displayed in Figure 1d.
10
Table S1. Accession numbers of the sequences used for the phylogenetic tree in Figure
1D.
Sequences in grey were not utilized for the alignment. Definitively incomplete gene models are
indicated by an asterisk. When the predicted number of amino acids differed between two compared
genome databases, the longer version was typically selected. Several genes display different splice
variants, e.g. “ZmDYRKP3”, which harbours three transcripts at this locus. For Danio rerio and
Xenopus laevis, not all existing DYRK genes were provided for the alignment. Data were obtained
from
NCBI
and
the
http://rice.plantbiology.msu.edu,
following
genome
websites:
http://www.phytozome.org,
http://www.arabidopsis.org,
http://www.maizesequence.org,
http://www.cosmoss.org, http://genome.jgi.doe.gov/.
11
Table S1. Accession numbers of the sequences used for the phylogenetic tree in Figure
1D, continued.
Alternative names of gene models: 1LOC_Os02g47410; 2LOC_Os04g51370; 3LOC_Os01g61620;
4
LOC_Os03g51020, AAT77851, GI:50540694; 5LOC_Os05g39080, EEE64022, GI222631890.
12
Table S1. Accession numbers of the sequences used for the phylogenetic tree in Figure
1D, continued.
Predictions concerning the subgroup of incomplete gene models (indicated by an asterisk) from
Ostreococcus and Micromonas species are difficult due to missing information about the N-terminal
part including the DH-box.
13
Table S2. List of primers used in this study.
Name
Sequence
AphORF_For
CGAAGCATGGACGATGCGTT
Aph_tail3
CGAGACTGCGATCGAACGGACA
GSP1
CTGGTGCTGCGCGAGCTGGCCCACGAGGAG
GSP2
TGGTTCGGGCCGGAGTGTTCCGCGGCGTT
XbaG4forHyg
GTCTAGAATGTCGCTCCGCCTGAACCGATG
XbaG4RevHyg
GTCTAGACTACATGCTGTCGAGCGAGG
Std1UTR1
CATAGTGCTCAGCAGGGGACAAGGC
Std1P3rev
AGCGTGCCAGAGGTTTCGCCGTC
Std1FW2
CCGCGGACGGCGAAACCTCTGGCAC
G4rev14
GATCTCGTCCAGCGACTGGTCAAAGTAG
ACG4_FW3
GCGGATCCGACGAGCAGGGCAACGTGCTG
ACG4_Rev1
CGGCAAGCTTCTACATGCTGTCGAGCGAGG
Actin_FW
AATCGTGCGCGACATCAAGGAGAA
Actin_Rev
TTGGCGATCCACATTTGCTGGAAGGT
14
Supplemental references
Aranda, S., Laguna, A., and de la Luna, S. (2011). DYRK family of protein kinases:
evolutionary relationships, biochemical properties, and functional roles. Faseb J 25,
449-462.
Becker, W., and Joost, H.G. (1999). Structural and functional characteristics of Dyrk, a novel
subfamily of protein kinases with dual specificity. Prog. Nucl. Acid. Res. Mol. Biol.
62, 1-17.
15