A B Supplemental data: Figure S1 CHX14 CHX17 CHX23 CHX13

Figure S1. Phylogenetic comparison of Arabidopsis CHXs and yeast transporters Nha1p and Kha1. (A) Non-rooted neighbor-joining phylogenetic tree of several functionally characterized Arabidopsis CHXs and yeast K+ transporters. The phylogenetic tree was generated by using ClustaW. GenBank accession numbers are CHX13 (EF571901), CHX14 (EF571900), CHX17 (AY926473), CHX20 (NP_190940), CHX21 (NP_180750), CHX23 (AY926477), and yeast plasma membrane Na + (K + )/H + transporter Nha1p(NP_013239) and endomembrane K+/H+ transporter Kha1p (EEU06761). (B) Proposed topology of CHX transporters as determined by the TMHMM2 program in SMART (

0 mM K 100 mM K 0 1d 2d 4d CHX14 ACTIN1 B Figure S2. CHX14 expression in young Arabidopsis seedlings. (A) qRT-PCR analysis of CHX14 expression in different tissues. Four-day-old Col-0 seedlings were transferred to one half-strength MS media supplemented with 100 mM KCl or to the modified one half-strength MS without KCl. The seedlings were collected at specified time points as indicated for RNA isolation. ACTIN2 was used as internal control. (B) Semiquantative RT-PCR analysis of CHX14 expression. One-week-old Col-0 seedlings were transferred to one half-strength MS media supplemented with 100 mM KCl, or to the modified one half-strength MS without KCl. The seedlings were collected at specified time points as indicated for RNA isolation. ACTIN1 was used as internal control. A Supplemental data Figure S2

Supplemental data Figure S3 Expression level

Figure S3. Arabidopsis root cell-specific expression patterns for various K+ transporter genes. Data are extracted from microarray profiling data (Brady et al., 2007). Comparison are made between CHX13, CHX14, KUP1, AKT1, and SKOR, in order to reveal their putative functions in K+ uptake, homeostasis, and translocation. Note that CHX14 is expressed at very low levels compared to KUP1.

Supplemental data Figure S4 AB C D Figure S4 CHX14::GUS staining in vascular tissue in root and leaves and meristem tissues of young Arabidopsis seedlings. (A) Cotyledon in 2-days old seedlings. (B) Primary root staining of 7days old seedlings. Vascular tissue expresses strong GUS activity (C) GUS staining of 7-days old CHX14::GUS transgenic seedling grown in 1/2MS supplemented with 100 mM KCl. (D) Staining of 7 day-old CHX14::GUS seedlings treated with 100 mM KCl for 3 days. (E) Cross-section of 7-days old CHX14::GUS seedlings grown in 1/2MS with 100 mM KCl. Symbols in E, X- xylem; P- phloem; E- endodermis; C- cortex. X P C E

Figure S5. GUS staining of CHX14::GUS seedlings grown under different K + concentrations. (A) CHX14::GUS plant seeds germinated in 1/2MS plate (10 mM K), seedlings show normal growth and GUS expression in root vascular tissues. (B) CHX14::GUS plant seeds germinated in modified 1/2MS plate containing 2 mM K, seedlings show abnormal growth and slightly enhanced GUS expression in root vascular tissues. (C) In 1/2MS containing 100 mM KCl CHX14::GUS expression was strongly induced; GUS expression extended to most cotyledon and hypocotyls’ issues. Germination was severely affected. All are 9-days old seedlings. CAB Supplemental data Figure S5

C pH 4.3 pH 5.6 pH mM K2 mM K Vector35S::CHX14Vector35S::CHX14 A B 10 mM K pH 4.3 pH 5.6 pH 7.5 Col-0chx14-1chx14-2 pH 4.3 pH 5.6 pH mM K Col-0chx14-1chx14-2 Supplemental data: Figure S6

Figure S6 Effects of K-deficiency on growth of seedlings with altered CHX14 expression. (A) CHX14 knockout mutant and wild-type Col-0 seedlings germinated and grown in 1/2MS plate (10 mM K). (B) CHX14 knockout mutants germinated and grown on plates containing 2 mM KCl. The chx14 mutant lines grown better than others. (C) Although 35S::CHX14 and vector control grow similarly well on one half-strength MS medium with 10 mM K, under 2 mM K conditions 35S::CHX14 (CHX14OE) lines grew worse than wild-type Col-0. All shown are 9-days old seedlings.

Leaf Stem Root-shoot junctions Root Figure S7. Drawing of parts of Arabidopsis seedlings for sampling gene expression and K + accumulation.

100 mMKCl Vector CHX mMKCl Vector CHX14 5 mMKCl Vector CHX14 Vector CHX14 10 mMKCl 50mMKCl Vector CHX14 Figure S8. CHX14 expression makes 59m15 (MATa leu2 ura3 trp1::trk1Δtrk2Δ::His3) more sensitive to K+ deficiency Yeast mutant 59m15 (MATa leu2 ura3 trp1::trk1Δtrk2Δ::His3) cells expressing empty vector or CHX14 were grown overnight in selection media (SC-Ura) supplemented with 100 mM KCl. Saturated cultures were brought to a similar cell density and 5-fold serial dilutions were spotted on SC-Ura medium supplemented with various concentrations of KCl as indicated. Photos were taken after 4 days of growth at 30ºC. Photos shown are representative of three independent experiments. Supplemental data: Figure S8

1 MKCl -Ura 2 MKCl 1.5 MKCl YPD CHX14 Vector CHX14 Vector CHX14 Vector CHX14 Vector CHX14 Vector Supplemental data: Figure S9 Figure S9. CHX14 expression in wild type yeast W303A causes the tolerance to excess K. W303A expressing CHX14 show better growth in K-stress conditions. Saturated cultures were brought to a similar cell density and serial dilutions were spotted on SC- Ura medium supplemented with various concentrations of KCl. Photos were taken after 2-3 days of growth at 30ºC.

Supplemental data: Figure S10 Figure S10. GFP-tagged CHX14 function in yeast suppression assays. Yeast mutant LMM04 (ena 1-4 ∆ nha1 ∆ trk1 ∆ trk2 ∆ kha1 ∆ tok1 ∆) cells expressing vector, CHX14-GFP, free GFP, or KAT1 were grown overnight in selection media (SC-Ura) supplemented with 100 mM KCl. Saturated cultures were brought to a similar cell density and 5-fold serial dilutions were spotted on SC-Ura medium at pH 4 and various concentrations of KCl as indicated. Photos were taken after 4 days of growth at 30ºC. Photos shown are representative of three independent experiments Vector CHX14-GFP KAT1 Free GFP CHX14 LMM04 pH mM K20 mM K5 mM K