www.sciencemag.org/cgi/content/full/315/5817/1423/DC1

Supporting Online Material for

Coupling Diurnal Cytosolic Ca2+ Oscillations to the CAS−IP3 Pathway in

Arabidopsis

Ru-Hang Tang, Shengcheng Han, Hailei Zheng, Charles W. Cook, Christopher S. Choi, Todd E. Woerner, Robert B. Jackson, Zhen-Ming Pei*

*To whom correspondence should be addressed. E-mail: zpei@duke.edu

Published 9 March 2007, Science 315, 1423 (2007)

DOI: 10.1126/science.1134457

This PDF file includes:

Materials and Methods SOM Text Figs. S1 to S4 References

− S1 −

Coupling Diurnal Cytosolic Ca2+ Oscillations

to the CAS−IP3 Pathway in Arabidopsis

Ru-Hang Tang, Shengcheng Han, Hailei Zheng, Charles W. Cook, Christopher S. Choi, Todd E. Woerner, Robert B. Jackson, Zhen-Ming Pei

Supporting Online Material

Contents

Materials and Methods S1

Supporting Text S7

CAS null mutant

CAS expression patterns and Ca2+ uptake and distribution

The IP3 pathway in plants

The soil Ca2+ signaling might be physiologically and evolutionarily relevant

fig. S1: The abundance of cytoplasmic aequorin protein in wild-type and CASas S10

fig. S2: The abundance of extracellular aequorin protein in wild-type and CASas S10

fig. S3: Plasma membrane localization of PHPLCδ-GFP in Arabidopsis S11

fig. S4: The working model S11

References S12

Materials and Methods

Plant material and growth conditions

The Arabidopsis thaliana genotypes used here are in Col-0 background. CASas1 to CASas6 are

CAS antisense lines as described (1). CASas is CASas1 in this report unless described elsewhere.

Plants were grown in the soil (Scotts Metro-Mix 200) where Ca2+ levels were measured with a

Ca2+-selective electrode as ~1 mM with saturated water, and also in petri dishes (100 x 15 mm)

containing ½ Murashige and Skoog salts (MS; Gibco), 1.5% (w/v) sucrose (Sigma), and 0.8%

− S2 −

(w/v) agar (Becton Dickinson) in controlled environmental rooms at 21 ± 2°C. The fluence rate

of white light was ∼110 μmol m-2 sec-1. The photoperiods were 16 h light/8 h dark cycles for

long days. Seeds were sown on soil or MS media, placed at 4°C for 4 days in the dark, and then

transferred to growth rooms.

Lines expressing intracellular and extracellular aequorin

Wild-type plants constitutively expressing intracellular aequorin (pMAQ2, a kind gift from Dr.

M. Knight) (2, 3) were generated by the floral dip method (4). Independent homozygous

transformants carrying a single insertion in the T3 generation were isolated, and aequorin

luminescence was examined. The single wild-type line with high aequorin expression was

crossed into CASas1 to CASas3. The expression of aequorin in these lines has been stable for 8

and 6 generations for wild-type and CASas, respectively. Additionally, the aequorin

luminescence among these CASas lines was very consistent (see next section; fig. S1). The wild-

type line expressing apoplastic aequorin was obtained from Dr. C. Plieth (5) and crossed into

CASas1 and CASas2. The expression of apoplastic aequorin in the two homozygous CASas lines

has been stable for 5 generations, and their luminescence was similar (fig. S2). The aequorin

protein abundance was similar in plants grown for one to three weeks. We observed no

differences in growth and development between wild-type, CASas and their aequorin-expressing

lines, respectively.

Western blot analysis

Seedlings grown under long days were homogenized in the solution containing 2% (w/v) SDS,

5% (v/v) glycerol, 1 mM DMSF, 1 mM DTT, and 100 mM Tris-HCl, pH 7.4. Thirty micrograms

of crude protein was separated on SDS-PAGE gel and blotted onto a polyvinylidene difluoride

membrane. Immunodetection of aequorin was performed using a polyclonal antiserum against

aequorin (Novus) as primary antibody (diluted 1:2,000) and alkaline phosphatase–coupled anti-

rabbit antiserum (Sigma) as a secondary antibody (diluted 1:10,000) as described (1).

Aequorin bioluminescence imaging

The aequorin bioluminescence-based Ca2+ imaging is the best technique available for monitoring

the resting level and diurnal oscillations of [Ca2+]i in plants (2, 3, 6). [Ca2+]i and [Ca2+]o were

− S3 −

measured using plants expressing aequorin as described (2, 3, 5). Seedlings were applied evenly

with 120 µL for cytoplasmic aequorin and 200 µL for apoplastic aequorin, of 10 µM

coelenterazine (Prolume) per 7 mm petri dish (~30 seedlings), 24 h before imaging. Aequorin

luminescence imaging was performed using a ChemiPro HT system equipped with a light-tight

box, a cryogenically cooled and back-illuminated CCD camera, and a liquid nitrogen autofiller

(Roper Scientific). The camera was controlled by WinView/32 (Roper), Imaging Workbench 5.1

(Indec Biosystems), or Meta Morph 6 (Universal Imaging). Nine petri dishes were imaged

simultaneously using seedlings grown under long days for 2 to 3 weeks. Bright-field images

were taken before aequorin imaging. The aequorin luminescence was recorded for 20 min every

3 h after switch from the light to the dark for 5 min as described (3). The first 5 min imaging

contained the autofluorescence of chlorophylls, and was discarded. The total aequorin was

estimated by discharging with 0.9 M CaCl2 in 10% (v/v) ethanol (3). The relative luminescence

intensities were calculated as the ratio of aequorin luminescence intensity and discharged

aequorin luminescence intensity. Experiments were carried out at room temperature (22–24°C).

Stomatal conductance analysis

Stomatal conductance was measured on plants grown in soil under long days for 3 to 4 weeks

using a photosynthesis system (Li-Cor LI-6400) (7). The shoot was inserted into a Magenta

vessel that was home made, and teflon tape was used to seal the top of the pot to completely

separate the shoot from the soil. Conditions inside the chamber were controlled by the LI-6400 at

a constant level for the entire period of measurement. CO2 was controlled to the ambient level of

370 μmol mol-1, and temperature to 22°C. The relative humidity was maintained at 72%, so that

leaf-to-air vapor pressure deficits (0.63 kPa) were at levels that permitted the measurement of

near maximum stomatal conductance. Leaf area was measured using a Li-Cor Model 3100 Area

Meter.

45Ca uptake analysis

Plants were grown in soil for 12 long days, and transferred to the ½ MS nutrient solution with air

bubbled for 3 days. After addition of 45Ca (PerkinElmer) at 10 µM to the solution at dawn, leaves

were collected every 2 h for two days, and digested with 70% nitric acid. Note that, roots were in

the solution with 45Ca, while the shoots/leaves were free from the radioactive solution. The

− S4 −

radioactive half-life of 45Ca is 163 days (PerkinElmer Catalogue) (8), and the calculated decay of 45Ca radioactivity is 0.61% for two days, which should not affect 45Ca supply in the liquid media.

In addition, the water loss in the growth media resulted from evaporation as well as plant

transpiration was small compared to the total volume of nutrient solution, which ensured more or

less constant 45Ca supply in the liquid media. The radioactivity of 45Ca was measured by liquid

scintillation counting (Beckman LS 6000SC) (1), and normalized by leaf area. The radioactivity

was increased with increases in time. To calculate the relative 45Ca uptake rate at time t, the

normalized 45Ca level at time t (h) was subtracted by the normalized 45Ca level at time (t – 2) (h),

i.e. the immediate previous data point, as the net increase in the radioactivity within two hours.

Data are presented as raw data values, counts per minute/leaf area (cpm/cm2).

Media Ca2+ Treatment and Ca2+ content analysis

Seedlings were grown in the MS-based media containing MS minor salts (Sigma), vitamins

(Sigma), 1.5% sucrose (Sigma), 0.8% agar (Becton Dickinson), and MS major salts (Sigma) with

Ca2+ supplemented to 0 mM to 40 mM CaCl2 as indicated. Each Magenta vessel and petri dish

contained 50 mL and 25 mL media, respectively (1). For Ca content analysis, seedlings were

grown under these media Ca2+ concentrations for 16 long days. Total leaves were collected and

digested with 70% nitric acid. The total Ca content was determined using an atomic absorption

spectrometer (Perkin Elmer) (1).

Histochemical GUS activity analysis

The histochemical staining for GUS activity using the CAS promoter-driven GUS (CAS::GUS)

transgenic T3 lines carrying a single insertion were performed as described (1). Seedlings grown

in MS media or the soil under long days were collected at the two-leaf stages, and used for the

histochemical staining (9). Data represent 12 independent lines examined, which displayed

similar staining patterns.

Imaging of [Ca2+]i in HEK293 cells

HEK293T cells were grown and maintained in DMEM medium supplemented with 10% fetal

bovine serum, 0.1% penicillin and streptomycin in a CO2 incubator at 37°C. For transfection,

cells were seeded onto poly-lysine-coated eight-well chambered coverglasses (Nunc). After 24 h,

− S5 −

cells were washed in OptiMEM medium and transfected with CAS plasmid DNA using

LipofectAMINE 2000 reagent (Invitrogen) as described (1). Cells were loaded with the Ca2+

sensitive dye Fura-2-AM (5μM; Sigma). A Fura-2-based Ca2+ imaging assay was performed in

the HEK293 cells 18 to 24 h after transfection for Ca2+-induced [Ca2+]i increase (CICI) using a

fluorescence microscope (Axiovert 200; Zeiss) equipped with two filter wheels (Lambda 10-2;

Sutter Instruments), and a cooled CCD camera (CoolSNAPƒχ; Roper Scientific)(1). Cells were

incubated in a solution containing 0.1 mM Ca2+. After addition of Ca2+ at a final concentration of

2.5 mM, emission ratiometric images (F340 nm/F380 nm) were collected using Imaging Workbench

5.1 software (Indec Biosystems) (1). Experiments were carried out at room temperature (22–

24°C).

Imaging of [Ca2+]i in guard cells and intact leaves

Arabidopsis plants were grown in soil with a 16 h light/8 h dark cycle. Cytosolic Ca2+

measurements using the GFP-based cameleon, YC2.1 (10), were conducted on guard cells as

described (11). Epidermal peels of rosette leaves carrying a cameleon construct were placed in a

microwell chamber containing 50 μM CaCl2, 5 mM KCl, 10 mM MES-Tris, pH 6.15.

Ratiometric Ca2+ imaging was performed using the fluorescence microscope described above.

Excitation was provided at 440 nm, and emission images at F535 nm and F480 nm were collected

separately. Ratiometric images (F535 nm/F480 nm) were monitored and analyzed using the Image

Workbench 5.1 software (1, 12). Experiments were carried out at room temperature. We also

detected CICI using intact whole-leaves expressing aequorin described above, although we could

only record two time points, before and after addition of 10 mM Ca2+, due to the low temporal

resolution of aequorin imaging.

IP3 measurements

For IP3 content in HEK293 cells, cells grown in petri dishes were transfected with CAS plasmids

for 24 h, and incubated in a solution containing 0.1 mM Ca2+ for 1 h prior Ca2+ treatment. Cells

were then treated with 2.5 mM CaCl2, and harvested at the indicated times by addition of liquid

nitrogen for the IP3 content assay. For IP3 content in Arabidopsis, rosette leaves from 4-week-old

plants were floated in solutions containing 50 μM CaCl2, 5 mM KCl, and 10 mM MES-Tris, pH

6.15 for 2 h. After treatment with 10 mM CaCl2, at the indicated times at room temperature the

− S6 −

leaves were immediately frozen in liquid nitrogen. The IP3 content of these samples was

measured using an [3H] IP3 radioreceptor assay kit (NEN Life Science) as described (13, 14).

Imaging of IP3 in guard cells

The pleckstrin-homology domain of the PLC-δ1 (PHPLCδ) binds to the precursor of IP3,

phosphatidylinositol-4,5-bisphosphate, which is associated with the plasma membrane, but

translocates to the cytosol after the increases in IP3 synthesis mediated by phospholipase C (15,

16). Tagging the PHPLCδ domain with green fluorescent protein (GFP) allows this process to be

visualized and the fractional increase in cytosol fluorescence can be used as an index of IP3

production (17-19). The full-length PHPLCδ-GFP (Hirose et al., 1999) was subcloned into the

binary vector pBin19, which contains a CaMV 35S promoter. Arabidopsis wild-type plants

constitutively expressing the PHPLCδ-GFP construct were generated by the floral dip method (4).

Independent homozygous transformants carrying a single insertion in the T3 generation were

isolated, and GFP fluorescence was examined. The single wild-type line expressing PHPLCδ-GFP

was crossed into CASas1 to CASas2. Arabidopsis plants were grown for about 4 weeks in soil

under long days. Epidermal peels of rosette leaves carrying the PHPLCδ-GFP construct were

placed in a microwell chamber containing 50 μM CaCl2, 5 mM KCl, 10 mM MES-Tris, pH 6.15

under light for 2 h. The guard cells that showed a clear membranous distribution of PHPLCδ-GFP

were selected for imaging. The real-time visualization of changes in the subcellular distribution

of PHPLCδ-GFP was performed using the Zeiss Axiovert 200 fluorescence microscope described

above. Excitation was provided at 485 nm, and emission images at 510 nm were collected. The

regions of the plasma membrane and the cytosol were selected manually using tool kits in Image

Workbench 5.1 software. Apart from region selection, which could not be performed

automatically by Image Workbench software, changes in cytosolic and plasma membrane GFP

fluorescence were quantified automatically using the built-in tool kits in Image Workbench 5.1

The fluorescence changes were calculated by taking the ratio (ΔF/F0) of the changes in

fluorescence signal at time t (Ft) in respect to the initial signal (F0) (ΔF = Ft-F0) to the initial level

(F0) in the same region. Note that, the proportion of PHPLCδ-GFP protein in the cytosol is high in

guard cells compared to that in epidermal cells (fig. S3). It seemed also that guard cells are a

better system for analyzing PHPLCδ-GFP fluorescence compared to mesophyll cells that contain

− S7 −

high levels of fluorescence from chlorophylls. Guard cells showing both a decrease in the plasma

membrane GFP fluorescence and an increase in cytosolic fluorescence in response to elevated

Ca2+ were considered as positive in IP3 increases. The response was quantified as the number of

positive guard cells in respect to the total number of guard cells examined in addition to the

average of all cells examined (Fig. 4C). Experiments were carried out at room temperature.

Statistical analysis

The statistical analysis was performed using EXCEL 10 software (Microsoft). Data were

presented as mean ± sd/sem as described (12). To analyze the difference between wild-type and

CASas, two-way analysis of variance (ANOVA) was carried out using SAS 9.1 software (SAS

Institute). Values of p < 0.05 were considered statistically significant.

Supporting Text

CAS null mutant

In general, due to the tissue preferential expression of the 35S promoter and the potential non-

specific nature of the antisense approach, the resulting phenotypes might not be at all related to

the targeted gene. Ideally we might obtain even more conclusive results from a CAS null mutant.

We have attempted extensively to isolate/obtain CAS null mutants using several resources, such

as the Salk Arabidopsis T-DNA insert population (20) and the TILLING project (21) without

success. For the SALK_070416 line, we confirmed that a T-DNA is inserted in the first intron of

CAS, but full-length CAS mRNA and CAS protein were detected (data not shown). Because CAS

plays a vital role in Arabidopsis, and CAS is a single copy gene in the genome (1), it is highly

possible that CAS null mutants might be lethal. It seems that the residual CAS activity in CASas

lines may be critical for plant survival. Therefore, CASas lines may provide a valuable tool for us

to genetically dissect the molecular mechanism for [Ca2+]i oscillations and for their function in

plant growth and development.

− S8 −

CAS expression patterns and Ca2+ uptake and distribution

In roots, Ca2+ moves rapidly through the cortical extracellular space (apoplast) by diffusion and

together with water enter the xylem in the root apical region. Ca2+ is primarily transported from

roots to shoots through apoplastic xylem and more than 90% of the Ca2+ that roots absorb is

transported to shoots (22, 23). In contrast, the symplast is not an effective pathway for Ca2+

uptake and transport, as [Ca2+]i (∼0.1 μM) is extremely low compared to [Ca2+]o (∼2 mM) (22,

24). Thus, [Ca2+]o in leaves likely represents the Ca2+ status in the soil. The distribution of Ca2+

in the plant is affected by the rate of water transport and evaporation (transpiration). Ca2+ moves

in relatively large amounts to highly transpiring older leaves, but much less to weakly transpiring

young leaves.

To analyze whether the expression patterns of CAS are correlated approximately to the

Ca2+ distribution in the plant, we used CAS promoter-driven GUS expression lines (CAS::GUS)

(1). We found that CAS was expressed mainly in leaves, particularly in veins and mesophyll

cells, while the expression was low in roots, shoot apical meristems, and young leaves (Fig. 2F),

consistent with our previous mRNA analysis (1). The expression patterns fit well with the route

of Ca2+ transport and distribution in the shoot, and also suggest that CAS may function as a

sensor for soil Ca2+.

The IP3 pathway in plants

In animals, hydrolysis of phosphoinositide lipids is a major mechanism for transmembrane

signalling in response to various stimuli such as hormones, growth factors, and neurotransmitters

(25). Some of the components have been identified in plants (26-28). For instance, increases in

IP3 have been detected in response to ABA (14), auxin (29), light (30), as well as salt stress (31-

33). PLCs are widely present and their activities are enhanced by these stimuli (26-28). High-

affinity IP3 binding sites, sharing features common to animal IP3R have been demonstrated (34,

35), although animal IP3R homologues have not yet been identified in plants. Finally, IP3 causes

[Ca2+]i increases by triggering Ca2+ releases in plant cells (34, 36). Moreover, for stomatal guard

cells, they contain IP3, and stimulus induces its rapid turnover (37, 38). Blocking PLC activity is

reported to attenuate the effects of ABA on K+ release from guard cells and [Ca2+]i oscillations

(39, 40). Application of IP3 triggers [Ca2+]i increases in guard cells and stimulates stomatal

closure (41). Based on these findings, putative cell-surface receptors have long been postulated

− S9 −

(26), however, their molecular nature remains unknown. Thus, identification of CAS may shed

light upon the perception of other stimuli via the IP3 signaling pathway in plants.

CAS differs from numerous cell-surface receptors identified in the IP3 pathway in

animals, which belong to G protein-coupled receptors (GPCRs), receptor tyrosine kinases

(RTKs), or receptors associated with protein tyrosine kinases (PTK) (25, 42). In mammals, 11

PLC isoforms are grouped into 4 subfamilies (β, γ, δ, and ε) (42). PLC-β is activated by

heterotrimeric G proteins; PLC-γ by PTKs; PLC-δ by Ca2+ and Gh; and PLC-ε by Gα12 or Ras

(42). Considering that plant PLC is activated by Ca2+ and that CAS mediates Ca2+-induced IP3

production in both HEK293 cells and plant cells (Figs. 3 and 4), it is likely that CAS might

activate PLC-δ in HEK293 cells. Arabidopsis CAS functions similarly to its mammalian

counterpart CaR, a GPCR (43, 44), however, shows homology neither to CaR nor to RTKs (25,

42). Therefore, CAS may represent a unique cell-surface receptor in the IP3 pathways although

how CAS activates PLC need to be determined in the future.

The soil Ca2+ signaling might be physiologically and evolutionarily relevant

Unlike animals, which can escape from unfavorable conditions, plants compensate for their

sessile lifestyle through programming growth and development. In nature, soil Ca2+ levels

fluctuate greatly throughout the year. The [Ca2+] at the root-soil interface ranges typically from

0.1 mM to 1 mM (45). At the same sites, Ca2+ levels have been observed to fluctuate between 10

μM to 600 μM during the year due to changes in water status, pH, and Al3+ levels (46-48). Thus,

it is essential for plants to sense soil Ca2+ changes and adjust growth and development, which

implies the physiological importance of the soil Ca2+ signaling pathway.

This soil Ca2+ pathway may be also relevant evolutionarily. Plants living in Ca-rich soils

tend to have high Ca content, and vice versa, and the shoot Ca content displays phylogenetic

variations (49-51). In addition, although soil Ca2+, which ranges from 0.5 mM to 38 mM (52), is

not a limiting factor for plant growth and development, acid depletion of soil Ca2+ increasingly

causes problems. For instance, some soils have lost ~75% of their original Ca over 250 years

(48). Thus, Ca fertilization has become a common practice in agriculture and forests (53, 54),

which suggests a practical implication of the soil Ca2+ signaling pathway.

− S10 −

fig. S1. The abundance of cytoplasmic aequorin protein in wild-type and CASas.

(A) Western-blot analysis of aequorin protein in wild-type (WT) and CASas seedlings grown

under long days for 2 to 3 weeks. AQ, aequorin; *, a nonspecific band detected by aequorin

antibodies, which can serve as a loading control (also see D); WT-AQ, WT expressing AQ;

CASas-AQL1 and CASas-AQL2, line 1 and 2, respectively, of CASas expressing AQ.

(B) Western-blot analysis of aequorin protein over a long day in 2- to 3-week-old WT and

CASas plants. Control, plants without expressing aequorin; Time, in h starts after dawn.

fig. S2. The abundance of extracellular aequorin protein in wild-type and CASas.

Western-blot analysis of apoplastic aequorin in WT and CASas plants grown for 2 to 3 weeks.

WT-AQo, WT expressing apoplastic AQ; CASas-AQoL1 and CASas-AQoL2, line 1 and 2,

respectively, of CASas expressing AQo.

− S11 −

fig. S3. Plasma membrane localization of PHPLCδ-GFP in Arabidopsis.

The fluorescence of PHPLCδ-GFP protein in the leaf epidermal from an intact leaf was analyzed

using a laser scanning confocal microscope. The PHPLCδ-GFP fluorescence was localized mainly

to the plasma membrane, and to a less extent to the cytosolic regions possibly close to the

nucleus. Scale bar, 25 μm.

fig. S4. The working model.

We propose that [Ca2+]i oscillations are coupled to [Ca2+]o oscillations via CAS-mediated Ca2+

release and a molecularly unknown ICa (Ca2+ channel)-mediated influx. The amplitude of [Ca2+]i

oscillations is controlled by soil Ca2+ levels and stomatal conductance, while the phase and

period largely by stomatal conductance oscillations that are determined further by the clock.

− S12 −

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