reSeArcH Article
growth and developmental phenotypes; 2) the Ca^2 + phenotype was
confirmed using another Ca^2 + indicator; 3) the Ca^2 + phenotype was
shown to be specific to ionic stress; 4) physiological responses to salt
stress were compromised; 5) the biochemical function of the mutated
gene was directly related to Ca^2 +-associated salt sensing. Here, we
report on the most affected mutant, monocation-induced [Ca^2 +]i
increases 1 (moca1).
The moca1 seedlings did not display morphological, growth, or
developmental phenotypes throughout their lifecycle (Extended Data
Fig. 2a–g; Fig. 1b). Changes in [Ca^2 +]i in response to water treatment
were similar in moca1 and wild-type plants, whereas moca1 plants
showed much lower levels of [Ca^2 +]i in response to treatment with
200 mM NaCl than did wild-type plants, in both leaves and roots
(Fig. 1b, c; Extended Data Fig. 2h, i). We analysed the kinetics of [Ca^2 +]i
elevation and detected lower peaks in moca1 plants (Fig. 1d). Increases
in [Ca^2 +]i induced by concentrations of NaCl up to 200 mM were lower
in moca1 (Fig. 1e; Extended Data Fig. 2j, k), but increases in [Ca^2 +]i
induced by sorbitol up to 400 mM were similar in moca1 and wild-type
plants (Fig. 1f; Extended Data Fig. 2l, m).
We confirmed the moca1 phenotype using the GFP-based yellow
cameleon 3.6 (YC3.6) Ca^2 + indicator^7 ,^17 ,^18 ; NaCl-induced increases in
[Ca^2 +]i were lower in moca1 roots (Fig. 2a, b). To test whether moca1
and osca1 are specific to ionic and osmotic sensing, respectively, we
observed that with respect to NaCl-induced increases in [Ca^2 +]i, osca1
and wild-type seedlings grown side-by-side were similar; whereas with
respect to sorbitol-induced increases in [Ca^2 +]i, moca1 and wild-type
seedlings were similar (Extended Data Fig. 3a–e), showing that moca1
and osca1 differ and that moca1 has ionic-specific effects.The moca1 mutant is hypersensitive to salt stress
Salt treatment inhibited both leaf and root growth in moca1 plants
(Fig. 2c, d). Under mild salt stress of 60 mM NaCl and low medium
Ca^2 + concentrations, the survival rate of moca1 plants was reduced
(Fig. 2c, e). We investigated whether moca1 affects the SOS pathway;
that is, whether salt-induced increases in [Ca^2 +]i are necessary and
sufficient for triggering the SOS3 → SOS2 → SOS1 signalling relay^5 ,^7 ,^8.
Although Na+/H+ antiporter activity was similar in moca1 and wild-
type plants, treatment with NaCl enhanced the activity in wild-type but
not moca1 plants (Fig. 2f; Extended Data Fig. 3f). As the concentra-
tion of NaCl in the medium increased, the Na content in moca1 plants
increased more, whereas the K content decreased more than these in
wild type (Fig. 2g, h), consistent with the mutant’s hypersensitivity to
salt and reduced Na+/H+ antiporter activity. These results fill the gap
in Ca^2 + elevation between NaCl stress and the SOS pathway^5 ,^7 , show-
ing that moca1 is upstream of SOS. Nevertheless, seedling growth in
response to osmotic stress and abscisic acid was not different from the
wild type in moca1 plants (Extended Data Fig. 4a–d), revealing that
moca1 does not have pleiotropic defects in stress response.moca1 lacks cation-evoked Ca^2 + spikes and waves
To determine whether moca1 is specific to ionic stress as compared
to other stimuli known to trigger increases in [Ca^2 +]i^5 ,^7 ,^8 ,^19 , we meas-
ured increases in [Ca^2 +]i in response to H 2 O 2 , cold temperature, and
high external Ca^2 +. These responses were nearly identical in moca1
and wild-type plants (Fig. 3a–c; Extended Data Fig. 5a–f). Increases in
[Ca^2 +]i in response to other monovalent cations, such as K+ and Li+,
were reduced in moca1 plants, and the substitution of Cl− for NO 3 − did
not affect the moca1 Ca^2 + phenotype (Fig. 3d–f; Extended Data Fig. 5g–
l), showing that moca1 is mainly selective for monovalent cations. Note
that KCl and LiCl stresses inhibited the growth of wild-type and moca1
plants to the same degree (Extended Data Fig. 4e–h), suggesting that
although the initial Ca^2 +–SOS signalling mechanism could be identical
for Na+, K+, and Li+, the selectivity of SOS1 for Na+ over K+ and Li+
might explain the attenuated growth of moca1 only under Na+ stress.
In plants, long-distance signalling—such as plasma membrane
potential depolarization and [Ca^2 +]i waves—has been proposed to be
a response to environmental stresses, including salt stress^18 ,^20. Localized
salt stress at the root tip causes increases in [Ca^2 +]i, which initiate a
[Ca^2 +]i wave that propagates to distal shoot tissues^21 ,^22. Using YC3.6
fluorescence resonance energy transfer (FRET)-based Ca^2 + imaging,
we observed that the NaCl-triggered [Ca^2 +]i wave was almost com-
pletely absent in moca1 roots (Fig. 3g–i; Supplementary Video 1).
As expected, the Ca^2 + channel inhibitor La^3 + blocked the [Ca^2 +]i
wave in wild-type roots (Extended Data Fig. 6; P < 0.01), confirming
that Ca^2 + signals are propagated from the root tip^21 ,^22. Previous studies
have shown that the speed but not the initiation of the Ca^2 + wave is
altered in the tpc1 mutant^21 ,^22 , placing TPC1 downstream of MOCA1.WTRootmoca1–NaCl
+NaCl01.20.80.41.6WT moca1Leaf ******
***0 100 200 300 400 500200 400 600 800[NaCl] (mM)NaClSorbitol03.02.01.0[Ca2+
] (iμM)4.0[Sorbitol] (mM)01 ,00001.20.80.41.60 400
Sorbitol (mM)100 200 300WT
moca1f01.20.80.41.60 200
[NaCl] (mM)50 100 150WTmoca1Time (s)0310 20 0 4001.00.51.5NaClmoca1WTa010 mM NaCl200 mM NaClWT moca1Bright lightbcde[Ca2+](μiM)10[Ca2+
] (i
μM)[Ca2+
] (iμM)[Ca2+
] (iμM)Fig. 1 | Isolation of moca1 mutant defective in NaCl-induced
increases in [Ca^2 +]i. a, Elevation in [Ca^2 +]i in wild-type (WT) plants
expressing aequorin plotted as a function of applied concentrations
of NaCl and sorbitol. Data from four representative experiments are
shown (mean ± s.d.; n = 32 seedlings; two-way ANOVA, P < 0.001).
b, Aequorin imaging of NaCl-induced increases in [Ca^2 +]i in plants treated
with water or 200 mM NaCl. [Ca^2 +]i is shown on a pseudo-colour scale
(middle, bottom). Similar results were seen in more than 50 independent
experiments. c, Quantification of increases in [Ca^2 +]i in leaves and roots
from experiments similar to those in b. Data from five representative
experiments are shown (mean ± s.d.; n = 32 seedlings; ***P < 0.001).
d, Time-course analysis of NaCl-induced increases in [Ca^2 +]i. Seedlings
were treated with 200 mM NaCl, and bioluminescence was recorded at
intervals of 1 s. Data from four representative experiments are shown
(mean ± s.d.; n = 18 seedlings; two-way ANOVA, P < 0.001). e, Averaged
increases in [Ca^2 +]i plotted as a function of applied [NaCl]. Data from four
separate experiments are shown (mean ± s.d.; n = 32 seedlings; two-way
ANOVA, P < 0.001). f, Increases in [Ca^2 +]i plotted as a function of applied
[sorbitol] (mean ± s.d.; n = 32; two-way ANOVA, P = 0.079).
342 | NAtUre | VOl 572 | 15 AUGUSt 2019