reSeArcH Article
Together, our results demonstrate that moca1 mutants are severely
defective in major early salt signalling events ([Ca^2 +]i spikes or waves
and SOS1 activation) as well as showing attenuated growth and devel-
opment in response to salt stress, implying that salt detection might be
disrupted in this mutant.
MOCA1 encodes a glucuronosyltransferase for GIPCs
As the T-DNA insertion in moca1 was lost, we carried out positional
mapping^17. Segregation analysis showed that the moca1 phenotype
was caused by a recessive mutation in a single nuclear gene (Extended
Data Fig. 7a). We crossed moca1 to the Wassilewskija ecotype to gen-
erate mapping lines, and mapped moca1 to the upper arm of chromo-
some 5 (Extended Data Fig. 7b). Through fine mapping and candidate
gene sequencing, we identified a 12-nucleotide deletion in a gene
(At5g18480; Extended Data Fig. 7c, d; Fig. 4a), that encodes inositol
phosphorylceramide glucuronosyltransferase 1 (IPUT1)^23. IPUT1 is
classified as glycogenin-like starch initiation protein 6 (PGSIP6) in the
glucuronosyltransferase subfamily 8 (GT8)^24. Hydrophobicity analyses
predicted that MOCA1 is an integral membrane protein with six trans-
membrane (TM) α-helices and a long stretched GT8 domain between
TM1 and TM2, and the moca1 mutant has a four-amino-acid-residue
deletion in TM6 (Extended Data Fig. 7d, e; Fig. 4a).
To confirm that MOCA1 is the relevant gene, we found that expres-
sion of MOCA1 from the endogenous promoter (MOCA1 moca1) or
overexpression driven by the 35S promoter (MOCA1ox moca1) could
complement the moca1 Ca^2 + phenotype (Fig. 4b, c; Extended Data
Fig. 8a, b). The salt hypersensitivity phenotypes in root growth and
viability were also complemented to wild-type levels (Extended Data
Fig. 8c–e; P > 0.05). We tried to generate homozygous moca1-2 and
moca1-3 T-DNA insertion mutants, but these mutations were lethal
(as seen in iput1-1 and iput1-2 mutants^23 ). Note that moca1 is moca1-1
in this study.
Analysis of transgenic plants expressing a GUS (β-glucuronidase)
reporter gene driven by the MOCA1 promoter (pMOCA1::GUS)
showed that MOCA1 is broadly expressed, particularly in cotyledons
and roots, consistent with the tissues where Ca^2 + phenotypes and phys-
iological salt sensitivity were observed (Fig. 4d; Extended Data Fig. 8f).
We analysed fluorescence in pMOCA1::MOCA1–GFP lines that con-
tained a Golgi marker (mCherry–Golgi) and observed punctate patterns
co-localized with the Golgi marker in the cytosol in root epidermal
cells (Fig. 4e), consistent with PGSIP6 (MOCA1)–GFP localization and
Golgi proteome analyses^24 ,^25. The four-amino-acid-residue deletion did
not affect the subcellular localization of mutant MOCA1 (mMOCA1)
(Extended Data Fig. 8g). The remaining question was how MOCA1
governs salt-induced increases in [Ca^2 +]i.Na+ ions bind to GIPC sphingolipids
IPUT1 transfers a glucuronic acid (GlcA) residue from UDP–GlcA
to inositol phosphorylceramide (IPC) to form GIPCs^23 (Extended
Data Fig. 8h). The iput1 mutant rescued by expressing pollen-specific
promoter-driven IPUT1 contains low levels of GIPCs and is a severe
dwarf^26. We measured GIPCs and IPCs, and found that moca1 plants
contained lower levels of GIPCs but higher levels of IPCs than the wild
type (Fig. 5a–d). moca1 and wild-type plants grown in agar plates and
soil were indistinguishable throughout their life cycle (Figs. 1b, 2c, 4b;
Extended Data Fig. 2a–g), in contrast to the severe phenotypes seen in
iput1 and iput1-rescued lines^23 ,^26. Thus, the GIPC levels might be above
the threshold required for normal growth and development in moca1
plants, while being low enough to compromise salt sensing.
GIPCs are a major class of lipids in fungi, protozoans, and plants,
but not in animals, and are abundant in the plasma membrane (about
25% of total lipids)^27 –^30. GIPCs have very long saturated acyl chains;
and have been proposed to be located in the outer leaflet of the plasma
membrane and enriched in raft-like lipid micro-domains^27 ,^29 –^31.
Although several hypotheses have been proposed regarding the role
of GIPCs, including cell wall anchoring, lipid moieties for protein
anchoring, cell-surface recognition, and precursors of signalling mol-
ecules, their exact roles remain unclear^27 ,^28. Conversely, in animals,
sphingolipids play a central role in cell signalling^29 ,^31 , and pertur-
bations of sphingolipids lead to various human diseases^32. The
negatively charged GIPCs are structural homologues of animal gan-
gliosides^27 ,^28 , which regulate receptors and ion channels as well as Ca^2 +
homeostasis^31 ,^32.
On the basis of this information, we hypothesized that the negatively
charged GIPCs could provide Na+-binding sites on the cell surface and
gate Ca^2 + influx channels in plants, as seen in the regulation of channels
in animals^33. We first measured changes in cell-surface potentials in
response to Na+ treatment. In wild-type protoplasts, increases in NaCl
concentration led to increases in the ζ potentials, which largely repre-
sent cell-surface potentials (Fig. 5e). In moca1 protoplasts, the ζ poten-
tials did not respond to NaCl treatment, and were decreased slightly
(Fig. 5e). In the absence of NaCl, ζ potentials were consistently lower
in moca1 protoplasts than in wild-type protoplasts, further demon-
strating the marked alteration in electric charges in the plasma mem-
brane. Treatment with up to 15 mM NaCl did not significantly affect
the integrity of wild-type protoplasts (survival rate ∼85%; Fig. 5f), but
lowered moca1 survival rates to about 25%. Without NaCl treatment,
the survival rates for wild-type and moca1 protoplasts were about 85%
and 65%, respectively (Fig. 5f), showing that moca1 protoplasts were
hypersensitive to salt stress. These results suggested that GIPCs might
directly detect Na+ levels in the apoplastic space.6493 – 496 (LMVG)*
TM1 TM2 TM3TM4 TM5TM6Glycosyltransferase family 8Bright light1
0
[Ca2+]i (aequorin)abec
pMOCA1::GUSpMOCA1::MOCA1–GFP mCherry–Golgi Merge01 0.5 .0 1.5
[Ca2+]i (μM)WTmoca1MOCA1
moca1
MOCA1ox
moca1*
***
*****dmoca1WT
MOCA1
moca1MOCA1ox
moca1
MOCA1ox
moca1MOCA1
moca1 moca1WT***Fig. 4 | MOCA1 encodes a glucuronosyltransferase. a, The predicted
topology of MOCA1. TM, transmembrane domain; asterisk, Δ493–496
(LMVG), the four amino acid residues that are deleted in the mutant
protein product of moca1. b, c, Complementation of moca1 Ca^2 +
phenotype by expressing MOCA1 driven by its own promoter (MOCA1
moca1) or 35S promoter (MOCA1ox moca1). Aequorin images of seedlings
treated with 200 mM NaCl are shown (b), and increases in [Ca^2 +]i
resulting from experiments as in b are quantified (c; mean ± s.d.; n = 40
pools (30 seedlings per pool); Student’s t-test, ***P < 0.001). d, Expression
patterns of pMOCA1::GUS in the seedling, leaf, and root. Similar results
were seen in more than ten independent experiments. e, Golgi membrane
localization of MOCA1 in root epidermal cells co-expressing MOCA1
promoter-driven MOCA1–GFP (pMOCA1::MOCA1–GFP) and a Golgi
marker tagged with mCherry (mCherry–Golgi). Similar results were seen
in more than ten independent experiments.
344 | NAtUre | VOl 572 | 15 AUGUSt 2019