Supporting Figure
S1A
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
AtNCL1
Supporting Figure S1B:
CAX2
AtNCL
AtNCL1 XP_001762095
XP_002500016
NP_002298903
XP_642144
CAX1
NP_001042378
NP_001046432
At1g29020
At2g34020
HsNCX1
0.1
Supporting Figure S1.
(a): Sequence alignment of AtNCL. Alignment of Arabidopsis AtNCL with proteins from
various species was performed with ClustalW (http://www.ebi.ac.uk/clustalw/). Deduced
AtNCL protein sequence (NP_564623), rice (Oryza stiva) EF hand proteins BAF08346
(Os02g0247800, NP_001042378), BAF04290 (Os01g0212400, NP_001042378),
Arabidopsis thaliana unknown proteins At2g29020 (BAF00582), At2g34020 (BAC42052),
CAX1 (NP_181352), CAX2 (NP_566452), and amoeba (Dictyostelium discoideum) protein
(XP642144), Human(Homo sapiens) HsNCX1(NP_001106273), Micromonas (Micromonas
sp. RCC299) CAX protein (XP_002500016), Physcomitrella (Physcomitrella patens) CAX
protein XP_001762095, Dictyostelium (Dictyostelium discoideum AX4) CAX protein
(XP_642144), and poplar (Populus trichocarpa) predicted protein XP_002298903. The
underlined regions are putative transmembrane domains (TMDs), as determined by
hydropathy plot. EF-hand motifs were shown in box. Identical and similar residues between
these proteins are blacked and marked as gray color, respectively.
(b): Alignment and neighbor-joint phylogenetic tree was generated with ClustalW
(http://www.ebi.ac.uk/clustalw/).
Supporting Figure S2
AtNCL
TM7
TM8
AtNCL
TM8
TM9
TM10
21%
18%
19%
25%
25%
AtNCL
TM10
TM11
Supporting Figure S2. Alignment of C-terminal sequences of AtNCL and CAXs from Arabidopsis.
Deduced AtNCL C-terminal protein sequence (NP_564623) compared with Arabidopsis proteins
CAX1 (NP_181352), CAX2 (NP_566452), CAX3 (BAF00582), CAX4 (NP180949). The underlined
regions are putative transmembrane domains (TMDs), as determined by hydropathy plot. Identical
and similar residues between these proteins are blacked and marked as gray color, respectively.
Supporting Figure S3
NCX1
AtNCL
AtNCLΔEF
CAX1
Supporting Figure S3. Topological analysis of AtNCL, AtNCLΔEF, CAX1 (NP_181352), and
NCX1 (AAD26362). The transmembrane domain (TMD) regions indicated by black lines were
determined by TMHMM2 program (http://smart.embl-heidelberg.de).
Supporting Figure S4
(a)
YPD
500 mM NaCl
50 mM CaCl2
Vector
HA-AtNCLΔEF
HA-AtNCL
AtNCL-GFP
(b)
(c)
207 kDa
115 kDa
75 kDa
45 kDa
95 kDa
75 kDa
45 kDa
30 kDa
17 kDa
Supporting Figure S4. Functional expression of epitope-tagged AtNCL and AtNCLΔEF in yeast cells. (
a). K667 expressing HA-AtNCL, HA-AtNCLΔEF, AtNCL-GFP, or empty vector were grown in SC
medium overnight for complementary test. Five-fold serial dilutions were spotted on YPD medium
supplemented with indicated concentrations of NaCl or CaCl2. Plates were incubated in 30°C for 3 days.
(b) Expression of HA tagged AtNCL and AtNCLΔEF, 10 µg proteins from protein extracts of K667 cells
were resolved in SDS-PAGE for western blotting and detected with HA antibody. （c）Western blot
detection of AtNCL-GFP fusion expressed in yeast at a predicted size of about 86 kDa. The existence of a
higher band suggests that AtNCL may form homodimers that are often observed with membrane
transporters.
Supporting Figure S5
(a)
AXT3
20 mM NaCl
AP-Ura
Vector
K667
30 mM CaCl2
AtNCLΔEF
AtNCL
(c)
5
Ca
*
Na
*
4
3
*
2
*
1
AtNCL
AtNCL∆EF
360
270
180
90
0
0
0
Vector
(d)
450
Ca in Vacuole (ppm)
Ion contents (mg/g DW)
(b)
AtNCL
AtNCL ΔEF
5
10
15
20
Concentration of Na2VO4 (mM)
Figure S5. Ca2+/Na+ transport activity of AtNCL and EF-hand function.
(a) Yeast strains K667 and AXT3 expressing AtNCL, AtNCLΔEF, or empty vector were
grown overnight for drop tests on AP medium containing NaCl (AXT3) or YPD medium
containing CaCl2 (K667).
(b) 20μl of the third dilutions were inoculated into 48-well plates containing YPD medium
supplemented with NaCl and CaCl2 at various concentrations. OD600nm was monitored. After
24 h of growth, yeast were collected for ion content assay.
(c) Na-dependent Ca release by AtNCL and truncated AtNCL at EF hand motif
(AtNCLΔEF). Ca2+ release by vacuolar membrane vesicles was assayed in uptake reaction
buffers containing various concentrations of Na2VO4. Uptake was stopped at 6 min of
incubation, and vacuole-enriched membranes were washed and used for measuring Ca.
Vacuole membrane-rich fractions were prepared from overnight cultures of WX1 yeast cells
expressing AtNCL, AtNCLΔEF, or vector in YPD medium.
(d) Ca2+ uptake by whole yeast cells. The K667 cells expressing AtNCL, AtNCLΔEF, or
vector were used for 45Ca2+ uptake assay.
Asterisks indicate statistically different (single asterisk P<0.05, double asterisk P <0.01,
Student’s t test)
Ion contents (ppm)
3
2.5
Na content (ppm)
Ion levels (mg/g dry weight )
Supporting Figure S6
Ca
2
1.5
1
0.5
0
6000
5000
Na
4000
3000
2000
1000
0
800
600
Zn
Fe
400
200
0
Supporting Figure S6. K667 cells expressing AtNCL, AtNCLΔEF, or vector were
grown in 5 ml SC-His medium overnight. Saturated cultures were transferred to YPD
medium supplemented with or without 20 mM CaCl2 or 100 mM NaCl. After overnight
growth, yeast cultures (OD 650 about 1.9) were collected and dried for ion analysis.
Data from three independent experiments are presented as means ±SD.
Flowering rate (% bolted)
Supporting Figure S7
100
Day 24
Day 28
80
60
40
20
0
Figure S7. Quantification of the flowering timing of wild-type, atncl mutants, and
35S::AtNCL lines under LD conditions. Data show the percent of bolted buds in all
flower buds in wild-type, atncl mutants, and 35S::AtNCL lines at 24 days and 28 days
under LDs. The graph is representative of three experiments with n = 12.
35S::AtNCL lines all flowered later than wild type or atncl mutants. At 24 days, more
than 55% wild-type and 80 % of atncl mutants had flowered, whilst only 25 % of
35S::AtNCL lines had flowered. At day 28, most of wild type and atncl knockouts
flowered. While more than only 50 % of 35S::AtNCL lines flowered. These results
showed that 35S::AtNCL lines flowered averagely up to 3-5 days late than wild type, but
atncl mutants early flowered.