# 1

# 2

E

ATG

T-DNA

1047 bp

TGA

Actin2CAP1

cap1-1 WT # 1 # 2

A

B

D

cap1-1

Supplemental Figure 1 online. The CAP1 T-DNA insertion mutant has a lowercytoplasmic calcium concentration and BLAST results of cap1-1 and over-expressiontranscripts. (A) Location of T-DNA insertion. The structure of CAP1 is shown schematically;this gene has no intron. The vertical arrow indicates the T-DNA insertion site in the mutantlines. (B) RT-PCR analysis of CAP1 expression in T-DNA insertion mutant and over-expression lines (#1 and #2). CAP1 cDNA was amplified (~650 bp) for 35 PCR cycles; thepositive control Actin2 was amplified with 24 PCR cycles, and amplified fragments wereseparated on a 1.0% agarose gel (left panel). Smaller amplified fragments (~250 bp) of CAP1across the deletion region were separated on the 4% agarose gel (right panel). WTtranscripts display relatively slower mobility. Three biological replicates showed the sameresults. (C) Resting [Ca2+]cyt in seedlings. Values were averaged and plotted (WT = 31; cap1= 21). (D) Sequence BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of cDNA fragmentsderived from cap1-1. A sequence deletion from 1422 bp to 1449 bp occurred in the mutant.Query seq = WT CAP1 cDNA sequence. (E) Sequence BLAST of cDNA fragment derivedfrom the two over-expression lines (#1 and #2). The deletion has been rescued in these lines.

C

WTcap1-1

1.0% Agarose

4.0% Agarose

1421 1450

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

1

0

30

60

90

WT cap1-1 Com-1 Com-2

Roo

t hai

r len

gth

(mm

)

A B

WT CAP1-RNAi1

CAP1-RNAi2

CAP1-RNAi3

C

Roo

t hai

r num

ber

Supplemental Figure 2 online. Root hair phenotype was complemented by CAP1driven by the wild-type promoter in mutants and suppression of CAP1 expressionin Arabidopsis RNAi knock-down lines. (A) WT, cap1-1, and complementation lines(Com-1 and Com-2, mutant cap1-1 transformed with CAP1 driven by the WT promoter)grow vertically on 1.2% MS medium for 7 d. Root hairs were recovered in bothtransformation lines. (B) Lengths and numbers of root hairs; means ± SE are indicated(n = 100 root hairs for WT and cap1-1; n = 60 for the Com-1 and Com-2 lines). (C)CAP1-silenced Arabidopsis roots at 7 d post germination.

0

0.2

0.4

0.6

0.8

WT cap1-1 Com-1 Com-2

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

2

B

C

WT

cap1-1

Dcap1-1

10 μm

Ca2+ (nM)

310

110

20

Supplemental Figure 3 online. FRET-based YC3.6 shows levels of [Ca2+]cyt and

oscillation of tip-focused Ca2+ gradients only in wild-type root hairs. (A) Cytosolic

Ca2+ levels of root hair cells (a) and their adjacent epidermal cells (b) in WT and cap1-1.

Tip-focused Ca2+ gradients oscillated in growing WT root hairs (B), but no oscillation was

detected in cap1-1 root hairs (C). Cytosolic Ca2+ levels were pseudocolor-coded

according to the scale at the left. Images were taken every 15 s for WT and every 10 s for

cap1-1. (D) The Ca2+ gradient and oscillation in root tips were recovered in cap1 when

seedlings were grown in ammonium-deficient medium. Quantitative analysis of cytosolic

Ca2+ oscillations in the root hair is shown on the right of each panel. Ca2+ levels were

measured in 10 µm2 regions of interest along the root hair length. An increase in the

FRET/CFP ratio reflects an increase in cytoplasmic Ca2+ level. Bars = 10 µm.

Ca2

+ -de

pend

ent r

atio

[FR

ET/C

FP fl

uore

scen

ce]

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60 70 80 90

Time (s)

0 s 15 s 30 s

45 s 60 s 75 s 90 s

0.4

0.6

0.8

1

1.2

1.4

0 15 30 45 60 75 90

5 μm10 μm15 μm20 μm

0.2

0.4

0.6

0.8

1

1.2

0 20 40 60 80 100 120

0 s 10 s 20 s

30 s 40 s 50 s 60 s

0 s 10 s 20 s

30 s 40 s 50 s 60 s

70 s 80 s 90 s 100 s

A

WT cap1-1

a

b

ab

a

b

ab

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

3

base tip

WT

cap1-1

WT

cap1-1/CAP1

Supplemental Figure 4 online. Ion-selective vibration microelectrode

recording of Ca2+ fluxes at root hairs surfaces in 7-d-old seedlings. Red

arrows show the positions corresponding to the root hair apex (tip) and below the

hair's midpoint (base). Bar = 50 µm.

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

4

MS-Fe(Fe deprivation)

MS-P(P deprivation)

Supplemental Figure 5 online. Effects of auxin (IAA), ethylene (ETH), various

nutrients, and pH on the growth of root hairs in wild-type and cap1-1 plants.

Seedlings were grown on the indicated medium for 7 d. Representatives of 30

seedlings for each genotype on different media in three separate experiments are

presented. Bars = 0.5 mm.

MS-K (K deprivation)MS

WT cap1-1

1 mg/L ETH0.1 µM IAA

WT cap1-1 WT cap1-1 WT cap1-1 WT cap1-1

pH = 5 pH = 5.5 pH = 6 pH = 7.0 pH = 6.5

WT cap1-1

WT cap1-1 WT cap1-1 WT cap1-1 WT cap1-1 WT cap1-1

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

5

y = 0.2838e0.1957x

R² = 0.996

0

0.2

0.4

0.6

0.8

1

1.2

1.4

5 5.5 6 6.5 7 7.5 8

410

nm /

470

nm ra

tio

pH value

Supplemental Figure 6 online. Standard curve of the calibrated 410 nm/470 nm

ratio in different pH solutions. Black line indicates the standard calibration curve,

and red line is the trendline generated by Microsoft Excel. Root hair cells of

Arabidopsis were used, bars represent means ± SE (n = 6).

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

6

20 min0 minTransmission

Supplemental Figure 7 online. High levels of NH4+ enhances root hair pHc.

Fluorescence was monitored before and 20 min after 100 mM NH4+ was added

to WT root hairs. Representative figures are shown (n = 6). Bars = 10 µm.

5.0

pH8.5

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

7

Control Ba2+

500

200

-100-200

E (mV)-200 -100 100

I (pA)

10 mM Ba2+control

Control La3+

500

200

-100-200

E (mV)-200 -100 100

I (pA)

1 mM La3+control

500 ms100 pA

Supplemental Figure 8 online. Characterization of ammonium currentrecording on the whole-cell model using Ba2+ and La3+. (A) Typical time-dependent currents recorded in WT root hair cell protoplasts in normal bathingsolution (left, control) and bathing solution with 10 mM Ba2+ (middle) and thecurrent-voltage relationships for the control (n = 4) and 10 mM Ba2+ (n = 5) inthe bathing solutions (I-V curve, right) are shown. I-V curves plot means ± SE.(B) The calcium-channel inhibitor La3+ (1 mM) showed no influence on thecurrent. WT root hair protoplasts were used. Control, n = 6; La3+-treated, n = 5.

A

B

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

8

pH = 5.5

pH = 7.0

WTcap1-1Com-1Com-2

Supplemental Figure 9 online. cap1-1 was more sensitive to high levels ofammonium than WT and the complementary lines. WT, cap1-1, and twocomplementary lines (com-1, 2) were sown on media with increasing ammonium(NH4Cl) levels (MS [NH4

+] = 20.6 mM, concentration of ammonium in MS medium).At pH 5.5, cap1-1 growth was inhibited more severely than that of other lines onmedia with increasing NH4

+ levels; while seedlings grew more slowly on higher pH(7.0) medium, inhibition of cap1-1 growth was more severe than in other lines athigh NH4

+ levels. Results from one of the three repeated experiments are shownhere. Photographs were taken after vertical growth in a growth chamber for 9 d.

1×

3×

5×

6×

1×

3×

5×

6×

MS [NH4+]

Supplemental Data. Bai et al. (2014). Plant Cell 10.1105/tpc.114.124586

9