Supporting Figure S1A

Supporting Figure S1B: AtNCL 0.1 Supporting Figure S1. (a): Sequence alignment of AtNCL. Alignment of Arabidopsis AtNCL with proteins from various species was performed with ClustalW ( Deduced AtNCL protein sequence (NP_564623), rice (Oryza stiva) EF hand proteins BAF08346 (Os02g , NP_ ), BAF04290 (Os01g , NP_ ), 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_ ), Micromonas (Micromonas sp. RCC299) CAX protein (XP_ ), Physcomitrella (Physcomitrella patens) CAX protein XP_ , Dictyostelium (Dictyostelium discoideum AX4) CAX protein (XP_642144), and poplar (Populus trichocarpa) predicted protein XP_ 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 (

Supporting Figure S2 TM11TM10 TM7 TM9 TM8 TM10 21% 18% 19% 25% AtNCL 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.

AtNCL Δ EF CAX1 AtNCL NCX1 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 ( Supporting Figure S3

Supporting Figure S4 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 CaCl 2. 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. 500 mM NaCl50 mM CaCl 2 Vector HA-AtNCLΔEF HA-AtNCL AtNCL-GFP YPD (b) (a) 75 kDa 45 kDa 95 kDa 115 kDa 207 kDa 30 kDa 17 kDa Free GFPControl AtNCL-GFP Vector HA- AtNCLΔEF HA-AtNCL 45 kDa 75 kDa (c)

Supporting Figure S5 (b) * * * * (d) (a) 30 mM CaCl 2 AP-Ura 20 mM NaCl AXT3 Vector AtNCLΔEF AtNCL K667 (c) Ca in Vacuole (ppm) Concentration of Na 2 VO 4 (mM)

Figure S5. Ca 2+ /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 CaCl 2 (K667). (b) 20μl of the third dilutions were inoculated into 48-well plates containing YPD medium supplemented with NaCl and CaCl 2 at various concentrations. OD 600nm 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). Ca 2+ release by vacuolar membrane vesicles was assayed in uptake reaction buffers containing various concentrations of Na 2 VO 4. 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) Ca 2+ uptake by whole yeast cells. The K667 cells expressing AtNCL, AtNCLΔEF, or vector were used for 45 Ca 2+ uptake assay. Asterisks indicate statistically different (single asterisk P<0.05, double asterisk P <0.01, Student ’ s t test)

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 CaCl 2 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. Supporting Figure S6

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 = S::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. Supporting Figure S7