Article reSeArcH
Methods
Plant material and growth conditions. Arabidopsis thaliana ecotype Col-0
and Arabidopsis thaliana Col-0 constitutively expressing the intracellular Ca^2 +
indicator aequorin (pMAEQUORIN2; a gift from M. Knight; Col-0 (aequorin))^9 ,^10
and YC3.6 (a gift from S. Gilroy)^51 were used. Two Arabidopsis T-DNA insertion
lines for At5g18480, SALK_131321 (moca1-2) and GK-856G03 (moca1-3), were
obtained from the Arabidopsis Biological Resource Center (ABRC). Note that
moca1 is moca1-1 in this study. Arabidopsis seedlings were grown in soil (Scotts
Metro-Mix 200) or in Petri dishes in ½ MS (Sigma), 1.5% (w/v) sucrose (Sigma),
and 0.6% (w/v) agar (Sigma) unless otherwise described in controlled environmen-
tal rooms or plant growth chambers (Percival Scientific) at 21 ± 2 °C. The fluency
rate of white light was ∼ 110 μmol photons m–2 s–1. The photoperiods were 16 h
light/8 h dark cycles. Seeds were sown on soil or MS medium, placed at 4 °C for 3
days in the dark, and then transferred to growth rooms or chambers.
Aequorin bioluminescence-based Ca^2 + imaging. Cytosolic free Ca^2 + concen-
tration ([Ca^2 +]i) was measured using plants expressing aequorin as described
previously^9 ,^17. Arabidopsis seedlings were applied evenly with 3.3 ml of 10 μM
coelenterazine (Prolume) per 150 mm × 15-mm Petri dish 12 h before imag-
ing and placed in the dark. Aequorin bioluminescence imaging was performed
using either a cryogenically cooled and back-illuminated CCD camera ChemiPro
HT system equipped with a light-tight box (Roper Scientific) or a newer version
Lumazone system (Pylon1300B, Roper Scientific) equipped with the H-800
light-tight controlled environmental box (Bio-One Scientific Instrument). A
liquid nitrogen autofiller (Roper Scientific or Bio-One Scientific Instrument) was
attached to the imaging system to provide constant cooling. The cameras were
controlled by WinView/32 (Roper) and bioluminescence images were analysed
using MetaMorph 6.3 or MetaMorph Basic Acquisition for Microscope (Molecular
Devices). The recording of luminescence (L) was started 10 s before treatments
and lasted for 3 min unless otherwise described. Bright-field images were taken
after aequorin imaging. The total remaining aequorin luminescence (Lmax) was
estimated by discharging with 0.9 M CaCl 2 in 10% (v/v) ethanol^9 ,^17. The calibration
of [Ca^2 +]i measurements was performed as described previously (pCa = 0.6747
× (−log k) + 5.3177, where k is a rate constant equal to luminescence counts (L)
divided by total remaining counts (Lmax)^17. All treatments were carried out in
the dark, and the experiments were carried out at room temperature (22–24 °C).
Generation of T-DNA insertion-mutagenized Arabidopsis populations. The
T-DNA insertion-mutagenized Arabidopsis populations for genetic screens^52 were
generated using wild-type Arabidopsis Col-0 expressing aequorin^9. Initially, we
used the activation tagging pSKI015 vector^53 and the flower dipping method^54 to
transform aequorin-expressing Arabidopsis, and generated T-DNA insertion popu-
lations of over 100,000 lines. Unfortunately, a very high percentage (>30%) of these
T-DNA lines did not show a wild-type level of aequorin bioluminescence signals
by discharging with 0.9 M CaCl 2 , suggesting that the aequorin 35S promoter was
largely silenced, as reported previously^55. Therefore, these populations could not
be used to screen for mutants defective in stimulus-induced increases in [Ca^2 +]i.
Subsequently, we decided to use a different vector without the 35S promoter, the
pBIB-BASTA vector (a gift from J. Li)^56. We did not find a high percentage of
aequorin silencing in these aequorin-expressing Arabidopsis lines transformed
with pBIB-BASTA. Then, we regenerated the T-DNA insertion populations, and
collected the seeds for over 120,000 independent T2 transgenic lines in pools.
Screens for mutants defective in salt-induced increases in [Ca^2 +]i (moca). The
aequorin Ca^2 +-imaging-based genetic screens for mutants defective in monoca-
tion-induced [Ca^2 +]i increases (moca) were carried out largely as described pre-
viously for the reduced hyperosmolality-induced [Ca^2 +]i increases (osca) mutants^17.
In brief, T3 seeds were sterilized, and individual seeds were planted evenly using a
template in 150 mm × 15-mm Petri dishes (260 seeds per Petri dish), and grown
in ½ MS medium for seven days. Aequorin bioluminescence images were acquired
for the salt treatment, that is, addition of 100 ml 200 mM NaCl solution into Petri
dishes via a home-made device. A total of ∼86,000 T3 seeds was screened in the
first round, and ∼10,000 seedlings that showed weaker [Ca^2 +]i increases were
picked up. These seedlings were then transferred to soil, and collected individually
for seeds. From the second- to the fourth-round screens, individual lines were
analysed for the moca phenotype and six putative mutants with a stable moca phe-
notype were isolated. To ensure that the moca phenotype was not caused by defects
related to aequorin-based Ca^2 + imaging, we sequenced the aequorin transgene in
these putative mutants to eliminate those lines with T-DNA insertion or mutation
in aequorin. We also analysed the total aequorin signals by discharging to eliminate
those lines in which aequorin expression was silenced. In addition, we paid extra
attention to those lines with growth and developmental phenotypes, such as small
in size and dark leaf colour, and in general selected those with no obvious growth
and developmental phenotypes for further studies.
Yellow Cameleon-based [Ca^2 +]i imaging in root cells. The moca1 mutant was
crossed into wild-type plants constitutively expressing the Ca^2 + sensor YC3.6, and
GFP FRET-based Ca^2 + imaging was carried out as described^17 ,^51 ,^57. More than ten
homozygous lines were generated and analysed. Cameleon-based measurements of
[Ca^2 +]i spikes and waves in root cells were conducted as described previously^21 ,^22.
In brief, Arabidopsis seedlings expressing YC3.6 were grown under in a thin layer
(~2 mm) of 1.0% (w/v) agar and ½ MS medium on a cover glass for five days under
light. A small window (~1 mm × 1 mm) was removed from the agar gel at the tip
of the root to expose the apical ~500 μm of the root and allow accurate application
of NaCl solution. For inhibitor treatments, a small window (~1 mm × 1 mm) was
made in the gel in the middle region of the root (Fig. 3g). Five to ten microlitres of
50 μM LaCl 3 was added in the middle gel window about 30 min before salt treat-
ment. Ratiometric Ca^2 + imaging was performed using a fluorescence microscope
(Axiovert 200/Axio Observer 3; Zeiss) equipped with two filter wheels (Lambda
10-2/10-3; Sutter Instruments), and a cooled CCD camera or CMOS (CoolSNAPfx/
Prime sCMOS; Roper Scientific)^17. Excitation was provided at 440 nm and emis-
sion ratiometric (F535 nm/F485 nm) images were collected using MetaFluor software.
Growth responses to salt stress. For the salt tolerance assay, wild-type and moca1
mutant Arabidopsis seeds were sterilized, kept in darkness at 4 °C for three days,
and then placed in low calcium (0.2 mM CaCl 2 ) ½ MS agar medium as described^58 ,
containing different concentrations of NaCl. The ½ MS agar medium contained
major salts (NH 4 NO 3 , MgSO 4 , KH 2 PO 4 , and KNO 3 ; Sigma), vitamin solution
(Sigma), 0.5% sucrose (Sigma), 0.6% agar (Sigma), 0.05% MES (Sigma), and
calcium supplemented to 0.2 mM CaCl 2. Seedlings were grown for 12 days, and
seedling weight, root length, and survival rate were analysed.
Na and K content analysis by ICP-MS. The content of cations was analysed
using inductively coupled plasma mass spectrometry (ICP-MS) as described
previously^59 –^61. Arabidopsis seedlings were grown in Petri dishes under several
NaCl concentrations for two weeks as described above, and harvested for the
analysis of Na and K contents. Seedlings were dried overnight at 65 °C and weighed
before digestion. Samples were digested in 75% nitric acid and 25% hydrogen per-
oxide for 3 h at 180 °C in a microwave digestion system (Ethos One, Milestone).
Each sample was diluted to 10.0 ml with 18 MΩ water and measured using a
NexIon300X ICP-MS Spectrometer (PerkinElmer, USA).
Na+/H+ exchange assay. The activities of Na+/H+ exchange were analysed
as described previously^62. Arabidopsis seedlings were grown in flasks in liquid
medium (low Ca^2 + and ½ MS) on a shaker for 12 d. One day before harvest-
ing, the original medium was replaced with fresh medium supplemented with or
without 100 mM NaCl. Plasma membrane-enriched vesicles were prepared using
aqueous two-phase (Dextron-PEG3350) partitioning^62. Inside-out vesicles were
produced by adding 0.05% (w/v) Brij58 to the medium^63. The protein content
was determined by Bradford’s method using BSA as a standard^64. The membrane
identity and transport competence of the vesicles were assessed by measuring the
H+-transport activity of the plasma membrane H+-ATPase^62. Na+/H+ exchange
activity was measured as a Na+-induced dissipation of ΔpH^62 ,^65. When the max-
imum ΔpH was formed (reached steady state), NaCl was added to initiate Na+
transport. To determine initial rates of Na+/H+ exchange (change in fluorescence
per minute, Δ%F/min), changes in relative fluorescence were measured 15 s after
the addition of Na+. Specific activity was calculated by dividing the initial rate
by the mass of plasma membrane protein in the reaction (Δ%F/min per mg of
protein)^62.
Genetic analysis and physical mapping. We could not identify the T-DNA inser-
tion in the moca1 mutant by either adaptor ligation-mediated PCR or thermal
asymmetric interlaced PCR (Tail PCR). We also found that the moca1 mutant had
lost Basta resistance and could not survive the Basta selection in the same way as
Col-0 (aequorin). We back-crossed moca1 to aequorin-expressing Col-0 (aequorin)
plants. The homozygous moca1 lines in the Col-0 (aequorin) background, which
showed a 1:3 mutant:wild-type ratio, were crossed to the ecotype Wassilewskija
(Ws) followed by self-pollinating F 1 progeny to yield an F 2 population as described
previously for physical mapping^17. For moca1 mapping, a total of ∼5,200 F 2 seed-
lings (out of ∼6,800 seeds) grown on Petri dishes that showed kanamycin resistance
(aequorin transgene) were transferred to soil. We then genotyped aequorin using
PCR, and aequorin homozygous lines were harvested individually for F 3 seeds.
These F 3 lines were analysed individually for the moca1 phenotype using aequorin
imaging. Eventually, homozygous moca1 lines with homozygous aequorin were
obtained as the mapping population. Linkage analysis of F 2 plants revealed that
the moca1 locus was located on chromosome 5. Markers for fine mapping were
searched from the databases of https://www.arabidopsis.org and http://archive.is/
amp.genomics.org.cn/. These markers were used to perform PCR and to isolate the
interval that flanks the mutation as described previously^17. Finally, we fine-mapped
the mutation into the narrowest interval, then sequenced open reading frames
(ORFs) in the interval, and identified a 12-base-pair deletion in an ORF in moca1.
Transmembrane α-helical spanners of MOCA1 were predicted by various models
using Aramemnon (http://aramemnon.botanik.uni-koeln.de)^66.
DNA constructs and transgenic lines. Gateway cloning^67 was used to construct
p35S::MOCA1, p35S::MOCA1–GFP, pMOCA1::GUS, pMOCA1::MOCA1–GFP,
and pMOCA1::mMOCA1–GFP. The MOCA1 full-length complementary or