Article
https://doi.org/10.1038/s41586-019-1449-zPlant cell-surface GIPC sphingolipids
sense salt to trigger Ca
2 +
influxZhonghao Jiang1,2,3,4, Xiaoping Zhou3,8, Ming tao1,8, Fang Yuan2,3,8, lulu liu2,3,8, Feihua Wu1,2,3,8, Xiaomei Wu^3 , Yun Xiang^2 ,
Yue Niu^2 , Feng liu^2 , chijun li^2 , rui Ye^2 , Benjamin Byeon^2 , Yan Xue^2 , Hongyan Zhao^3 , Hsin-Neng Wang4,5,
Bridget M. crawford4,5, Douglas M. Johnson^6 , chanxing Hu^2 , christopher Pei^2 , Wenming Zhou^1 , Gary B. Swift^6 ,
Han Zhang^7 , tuan Vo-Dinh4,5, Zhangli Hu^1 , James N. Siedow^2 & Zhen-Ming Pei^2
Salinity is detrimental to plant growth, crop production and food security worldwide. Excess salt triggers increases in
cytosolic Ca^2 + concentration, which activate Ca^2 +-binding proteins and upregulate the Na+/H+ antiporter in order to
remove Na+. Salt-induced increases in Ca^2 + have long been thought to be involved in the detection of salt stress, but the
molecular components of the sensing machinery remain unknown. Here, using Ca^2 +-imaging-based forward genetic
screens, we isolated the Arabidopsis thaliana mutant monocation-induced [Ca^2 +]i increases 1 (moca1), and identified
MOCA1 as a glucuronosyltransferase for glycosyl inositol phosphorylceramide (GIPC) sphingolipids in the plasma
membrane. MOCA1 is required for salt-induced depolarization of the cell-surface potential, Ca^2 + spikes and waves,
Na+/H+ antiporter activation, and regulation of growth. Na+ binds to GIPCs to gate Ca^2 + influx channels. This salt-sensing
mechanism might imply that plasma-membrane lipids are involved in adaption to various environmental salt levels, and
could be used to improve salt resistance in crops.More than 6% of the world’s total land area and about 20% of irrigated
land (which produces one-third of the world’s food) are increasingly
affected by salt buildup^1. Excessive salt is detrimental to plant growth
and development, and causes agricultural loss and severe deterioration
of plant ecosystems^1 ,^2. Sodium chloride is the most soluble and wide-
spread salt found in soils. Sodium is not an essential nutrient in plants,
and plants have evolved mechanisms to reduce intracellular sodium
buildup^1 ,^3. In plants, high salinity triggers early short-term responses
for perceiving and transducing the stress signal, and subsequent long-
term responses for remodelling the transcriptional network to regulate
growth and development. Although several molecular components in
the early signalling pathway have been identified, plant salt sensors
remain unknown^3 –^8.
Salt stress triggers increases in cytosolic free Ca^2 + concentration
([Ca^2 +]i)^9 ,^10 , and the expulsion of excess intracellular Na+ involves the
Ca^2 +-related salt-overly-sensitive (SOS) pathway^3 ,^5. The SOS pathway
comprises the Ca^2 + sensor SOS3 (a calcineurin B-like protein (also
known as CBL4)), the protein kinase SOS2 (also known as CIPK24),
and the Na+/H+ antiporter SOS1. Although salt-induced increases in
[Ca^2 +]i are thought to act as a detection mechanism, the molecular
components involved in these increases are unknown^3 –^8 ,^11. In ani-
mals, sodium is an essential nutrient, and dedicated mechanisms have
evolved to detect attractive low salt and aversive high salt conditions^12.
Notably, several ion channels act as salt-sensing taste receptors^13 –^16.
Sodium also triggers [Ca^2 +]i spikes that are mediated by these salt-
sensing channels. However, homologues of these channels do not exist
in sequenced plant genomes.
High salinity increases both osmotic pressure and ionic strength, so
salt can exert two stress effects: osmotic and ionic^1 ,^4. Ca^2 +-imaging-
based forward genetic screens have previously been used to isolate
Arabidopsis mutants defective specifically in osmotic stress-induced
Ca^2 + increases, resulting in cloning of the osmosensing OSCA1 Ca^2 +
channel^17. Here we have optimized experimental conditions for similar
Ca^2 +-imaging-based genetic screens to distinguish the ionic effect from
the osmotic effect of salt stress. In this way, we isolated Arabidopsis
mutants defective specifically in ionic stress-induced increases in
[Ca^2 +]i. Analysis of a mutant identified through these screens revealed
that plant-specific GIPC sphingolipids are involved in sensing salt-
associated ionic stress in the plasma membrane.moca1 is defective in salt-induced Ca^2 + spikes
We attempted to identify ion-specific sensing mechanisms by using the
same genetic approaches that were used to identify the osmosensing
osca1 mutant^17. First, we needed to establish conditions under which
the ionic effect of NaCl on [Ca^2 +]i elevation was large whereas its effect
on osmotic [Ca^2 +]i elevation was minimal. We analysed the dose-
dependent [Ca^2 +]i increases induced by NaCl (ionic + osmotic effects)
and sorbitol (osmotic effects only) using aequorin-based Ca^2 + imag-
ing. Throughout the range of concentrations tested, NaCl was more
potent in triggering [Ca^2 +]i increases than sorbitol at a similar osmo-
lality (Fig. 1a; Extended Data Fig. 1). We reasoned that a threshold of
200 mM NaCl, at which the ionic effect was the highest and the osmotic
effect was negligible, could be used to screen for mutants impaired in
increases in [Ca^2 +]i induced by ionic but not osmotic stresses.
Because it was difficult to physically map ethyl methanesulfonate
(EMS)-induced mutations for the identification of aequorin-express-
ing osca1^17 , we generated aequorin-expressing Arabidopsis populations
mutagenized by transfer DNA (T-DNA) insertions using the vector
pBIB-BASTA. We screened around 86,000 T2 seeds, and recovered
about 10,000 seedlings in which increases in [Ca^2 +]i in response to
200 mM NaCl were low. These lines were retested individually for
four generations, and six individual lines with stable phenotypes were
isolated as putative mutants. We then analysed several phenotypes to
prioritize further characterization: 1) the plants did not have apparent(^1) College of Life Sciences and Oceanography, Longhua Innovation Institute for Biotechnology, Shenzhen University, Shenzhen, China. (^2) Department of Biology, Duke University, Durham, NC, USA.
(^3) College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, China. (^4) Fitzpatrick Institute for Photonics, Duke University, Durham, NC, USA. (^5) Department of Biomedical
Engineering, Duke University, Durham, NC, USA.^6 Department of Physics, Duke University, Durham, NC, USA.^7 College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China.
(^8) These authors contributed equally: Xiaoping Zhou, Ming Tao, Fang Yuan, Lulu Liu, Feihua Wu. *e-mail: [email protected]; [email protected]
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