The Arabidopsis thaliana SOS2gene encodes a protein kinase that is required for salt tolerance

Jiping Liu* ,Manabu Ishitani ,Ursula Halfter,Cheol-Soo Kim,and Jian-Kang Zhu

Department of Plant Sciences,University of Arizona,Tucson,AZ85721

Communicated by Steven D.Tanksley,Cornell University,Ithaca,NY,January24,2000(received for review November8,1999)

In Arabidopsis thaliana,the Salt Overly Sensitive2(SOS2)gene is required for intracellular Na and K homeostasis.Mutations in SOS2cause Na and K imbalance and render plants more sensitive toward growth inhibition by high Na and low K environments. We isolated the SOS2gene through positional cloning.SOS2is predicted to encode a serine threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase.Sequence analyses of sos2mutant alleles reveal that both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2are functionally essential.The steady-state level of SOS2transcript is up-regulated by salt stress in the root.Auto-phosphorylation assays show that SOS2is an active protein kinase. In the recessive sos2-5allele,a conserved glycine residue in the kinase catalytic domain is changed to glutamate.This mutation abolishes SOS2autophosphorylation,indicating that SOS2protein kinase activity is required for salt tolerance.

C ontrol of intracellular ion homeostasis is essential for all

cellular organisms.Most cells maintain relatively high K and low Na concentrations in the cytosol.In plants,this is achieved through coordinated regulation of transporters for H , K ,and Na .At the plasma membrane,a family of P-type H -ATPases serves as the primary pump that generates a protonmotive force driving the active transport of other solutes, including K and Na (1).Several plant K channels and transporters have been molecularly characterized.The inward rectifying K channel AKT1is essential for root K uptake in Arabidopsis thaliana(2,3).Expression characteristics indicate that the KAT1channel is involved in K influx in Arabidopsis guard cells(4,5).Recently,an outward rectifying K channel has been shown to be essential for unloading K into the Arabidopsis root xylem(6).The wheat HKT1gene product functions as a high-affinity K transporter(7).In addition,a family of KUP genes exists in Arabidopsis.At least one of them,KUP1,encodes

a protein that can function as a dual-affinity K transporter(8,

9).Na enters plant cells passively,presumably through K transport systems(10).Unlike animals or fungi,plants do not seem to possess Na K -ATPases or Na -ATPases.Na efflux is achieved through the activities of Na H antiporters on the plasma membrane.Much of the Na that enters the cell is compartmentalized into the vacuole through the action of vacuolar Na H antiporters(11,12).The driving force for the vacuolar transporters is the protonmotive force created by vacuolar V-type H -ATPases and the H -pyrophosphatase(1, 13).Although there has been great progress in the character-ization of K and Na transporters in plants,little is currently known about their regulation.

In the trophic chain,plant roots play pivotal roles by taking up mineral nutrients from soil solutions.Plant roots experience constant fluctuations in soil environments.A frequent variant in the soil solution is Na concentration(14).Na is not an essential ion for most plants.In fact,the growth of the majority of plants,glycophytes,is inhibited by the presence of high concentrations of soil Na .External Na causes K deficiency by inhibiting K uptake into plant cells(15).Na accumulation within the cell is toxic to many cytosolic enzymes.In contrast, many cellular enzymes are activated by K ,which is the most abundant cation in the cytoplasm.Certain cytoplasmic enzymes are especially prone to Na inhibition when K concentration is reduced(16).Therefore,maintaining intracellular K and Na homeostasis to preserve a high K Na ratio is important for all cells and especially critical for plant cells.

A family of Arabidopsis sos(salt overly sensitive)mutants defective in the regulation of intracellular Na and K ho-meostasis was recently characterized(15,17,18).The sos mutants are specifically hypersensitive to inhibition by high concentrations of external Na or Li (17,18).In response to high Na challenge,the sos2and sos3mutants accumulate more Na and retain less K than wild-type plants(18).The mutants are also unable to grow when the external K concentration is very low(17,18).These phenotypes suggest that the mutant plants are defective in the regulation of K and Na transport (18).The SOS3gene was recently cloned and shown to encode an EF hand-type calcium-binding protein that shares significant sequence similarities with animal neuronal calcium sensors and the yeast calcineurin

B subunit(19).In yeast,calcineurin is a central component in the signaling pathway that regulates Na and K homeostasis(20,21).Loss-of-function mutations in calcineurin B cause increased sensitivity of yeast cells to Na or Li stress.

We report here the positional cloning of the SOS2locus.SOS2 is predicted to encode a serine threonine type protein kinase with an N-terminal catalytic domain highly similar to those of yeast SNF1and mammalian AMPK kinases.Sequence analyses of several sos2mutant alleles point to a functional requirement of both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2.SOS2is expressed in both the root and shoot.In the root,SOS2mRNA is up-regulated by salt stress. Autophosphorylation assays demonstrate that SOS2is an active protein kinase.Furthermore,a mutation that abolishes SOS2 autophosphorylation renders plants hypersensitive to salt stress, indicating that SOS2protein kinase activity is necessary for salt tolerance.This demonstrates that a protein kinase is essential for intracellular Na and K homeostasis and plant salt tolerance. Materials and Methods

Genetic and Physical Mapping.Genetic mapping with restriction fragment length polymorphism and PCR-based markers was as described(19).Construction of yeast artificial chromosome (YAC)and bacterial artificial chromosome(BAC)clone contigs (19)was partly based on information available at http:

Abbreviations:SOS,salt overly sensitive;YAC,yeast arti cial chromosome;BAC,bacterial arti cial chromosome;GST,glutathione S-transferase.

Data deposition:The sequence reported in this paper has been deposited in the GenBank database(accession no.AF237670).

*Current address:Department of Plant Breeding,Cornell University,Ithaca,NY14853.

J.L.and M.I.contributed equally to this work.

To whom reprint requests should be addressed.E-mail:jkzhu@ag.arizona.edu.

The publication costs of this article were defrayed in part by page charge payment.This article must therefore be hereby marked“advertisement”in accordance with18U.S.C.§1734solely to indicate this fact.

Article published online before print:Proc.Natl.Acad.Sci.USA,10.1073 pnas.060034197. Article and publication date are at www.wendangwang.com cgi doi 10.1073 pnas.060034197

3730–3734 PNAS March28,2000 vol.97 no.7