This article isThis article by protected copyright. All rightsreserved. 10.1111/tpj.13761 differences thisbetween andVersion version the ofRecord.c Please the through copyediting, typesetting, pagination and proofreading process, to whichmay lead This article has been accepted for publication and undergone full peer review but has not been 1. e d c b a Matleena Punkkinen Barajas Juan deDios tolerance Upstream kinases of plant SnRKs are involved in salt stress Article Original : Article type ID :0000 (Orcid FUJII DR HIROAKI

Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Cientificas, Cientificas, de Superior Investigaciones Consejo Fotosíntesis, y Bioquímica Vegetal de Instituto Academy of Sciences, Shanghai 200032, China 200032, Shanghai ofSciences, Academy USA. Spain; Sevilla, 41092 Cientificas, 410 Finland Molecular Plant Biology Unit, Department of Biochemistry, University Turku, University of Biochemistry, of Department Unit, Biology Plant Molecular Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Sciences, Chinese Biological for Institutes Shanghai StressBiology, Plant for Center Shanghai Department of Horticulture and Landscape Architecture, andLandscape ofHorticulture Department deSevilla, y Agrobiología Naturales Recursos de Instituto Accepted addressed; be should correspondence To whom Article

12

Sevilla, Spain; Sevilla,

-

a Lopez

, Jian - a Kang Zhu Kang , Jose Ramon Moreno Ramon Jose

- 0002 d, d, e , Francisco J. Quintero J. Francisco - 0013 -

5891) b , Francisco M.Gamez , Francisco

Purdue Univers Purdue Consejo c

, Hiroaki Fujii , Hiroaki

Superior de Investigaciones Investigaciones de Superior - Arjona ite ite this asdoi: article ity, West Lafayette, IN, Lafayette, ity, West a, 1

b ,

20014, Turku, Turku, 20014, Jose M.Pardo Jose c ,

This article isThis article by protected copyright. All rights reserved. thaliana Arabidopsis J. Quintero Francisco Jian Matleena Punkkinen M.Pardo: Jose Gamez M. Francisco Moreno Ramon Jose Barajas Juan deDios Fujii: Hiroaki K R Email ey Acceptedunning Article

- words Kang Zhu Kang

addresses

title Molecular Plant Biology Unit, Department of Biochemistry, University of Turku, Turku, of University Biochemistry, of Unit, Department PlantBiology Molecular +358 Phone: FI Turku, 6thFloor, 6A Tykistökatu

GRIKs, SnRKs, SOS2, upstream kinases, salinity, sugar, phosphorylation, stress, stress, phosphorylation, salinity,sugar, kinases, upstream SOS2, SnRKs, GRIKs,

ORCID Upstream kinases of SnRKs in salt stress tolerance instress SnRKs of salt kinases Upstream :

[email protected] [email protected]

of

other :

- -

: : 0000 Arjona : Lopez:

[email protected] [email protected] - [email protected] 2 - 333

authors - 0002 :

[email protected] [email protected] - [email protected] 7915, Fax:+358 7915, - 0013

-

5891

-

20520

-

29 - 450 , Finland

-

5040 (main for the university), theuniversity), (mainfor 5040 , Email: Email:

[email protected]

This article isThis article by protected copyright. All rights reserved. inthe response areinvolved (AMPKs) kinases change toenvironmental responds pro Reversible pathways oftheSnRK other members thatGRIK indicate results Our yeast. system in reconstituted T168 bywild levels to normal restored was phosphorylate tosalinity. response the of mediator critical isa subfamily, ofthe SnRK3 member indicat addition of stress GRIKs under of the function allele condition sensing Rep to be Although andSnRK3. SnRK2, SnRK1, clades: three familyhas This responses. Sucrose Introduction Summary

Accepted Article SnRK1.1 - I

nteracting nteracting

residue phosphorylated by GRIK1. Activation of SOS2 by GRIK1 was also also was GRIK1 by SOS2 of Activation GRIK1. by phosphorylated residue activated by upstream kinases, the overall mechanism remains obscure. Geminivirus Geminivirus obscure. remains mechanism theoverall kinases, upstream by activated of ing that GRIKs are also involved in involvedin arealso GRIKs that ing , and -

gene N to high sensitivity to glucose to to high sensitivity s

on in vivo in . was In this study, we established an established we thisIn study,

- t

F he GRIK1 tein phosphorylation is one of the most important mechanisms by which cell signa cell by which mechanisms important most isoneofthe tein phosphorylation d ermenting

reduced

K SOS2 SOS2 .

inase

( grik1 - in vitr in

1 ( GRIK)

, grik2 1 but not eliminated but not - - related kinases (SnRKs) are important for plant growth and stress stress and plant growth for important are (SnRKs) kinases related protein 2 3 o

grik2

, resulting in elevated elevated , resultingin 1 and GRIK2 1 and family and family and - 1

double mutant double - condition 1 s . )

Sucrose , the that that - type SOS2, but not by a mutated form of SOS2 lacking the SOS2 lacking of form amutated not by but type SOS2, that they that salinity

grow phosphorylate SnRK1s, which are involved in sugar/energy in involved sugar/energy which are SnRK1s, phosphorylate grik1 other , suggesting that that , suggesting s

. s non In the the In

to energy depletion in yeasts and m yeasts and in depletion to energy

s - show

2

similarly signalling pathways. Salt pathways. signalling Arabidopsis -

grik2 fermenting 1 fermenting play important roles in multiple signalling signalling in multiple roles play important kinase kinase grik1 s

growth retardation und growth retardation - 1

to the wild type to thewild mutant was sensitive to highsalt, sensitiveto was mutant - activity 2

s grik2

the

mutant mutant

phosphorylate and activate SnRK1 and and activate SnRK1 and phosphorylate

(SNF1)/AMP grik1 -

of SOS2 of 1

double mutant, phosphorylation phosphorylation mutant, double line harbouring harbouring line - 2

muta , enabling us to evaluate to evaluate us , enabling . The salt tolerance of salt tolerance The . O verly - plant plant activated protein protein activated tion demonstrated in a in demonstrated er er regular growth regular S GRIK1 GRIK1

ensitive (SOS) is SnRKs ammals (Kemp (Kemp et ammals

a weak allele a weak a different different a are thought are

2, a sos2 l . In ling

This article isThis article by protected copyright. All rights reserved. Rep yeastS of homologs identified as been of similar tothat kinases upstream these encoding kinases, regulatory activation inthe Phosphorylation beenproposed. have other , with binding to interactions In addition 2007 comprise S diversity combinatorial calcium al., 2004). et (Cheng antiporter CAX1 K tonoplast as the such targets vacuolar Na membrane SOS2 pathway, the SOS In 2000). Liuetal., etal.,1998, (Zhu tolerance 2/SnRK3.11 B calcineurin characteri best ofthe One Luan,2009). 2010, et al., Umezawa 2003, al., et (Hrabak in respectively, members, 10 and25 al., 2007). (Baena signalling energy in roles play important SnRK1s homology, on based expected KIN10as and known (also 1.2 and SnRK1.1 including proteins, SnRK1 SnRK3. and SnRK2 SnRK1, three subfamilies: comprise al., 2003 C

Accepteda Article BP8/CBL10 - I ). nteracting nteracting

- binding protein binding SAK ,

Hardie, 2004). The plant homologs of SNF1, the SNF1 ofSNF1, homologs Theplant Hardie, 2004). an autoinhibitory domain autoinhibitory an On the other hand, hand, the other On mechanism of SnRK proteins (Hanks and Hunter 1995, 1995, andHunter (Hanks SnRK proteins of mechanism / 1 CIPK24), is a Ser/Thr protein kinase Ser/Thr protein isa CIPK24), - / li + P

ke proteins /H after AK K  inases +

1, T snf1 antiporter SOS1 (Shi et al., 2000; Quan et al., 2007 Quan etal., et al., 2000; (Shi SOS1 antiporter

bind OS

(

(Halfter et al., 2000, Kim et al., 2000). Kim et al., et al., 2000, (Halfter , (GRIK) 1 and GRIK2 because because GRIK2 1 and (GRIK) Hong et al., 2003, Sutherland et al., 2003). In In etal.,2003). Sutherland al.,2003, et Hong

3 a.k.a. Calcineurin B Calcineurin a.k.a. ing

-

11) of interacting protein kinases protein interacting and

to the FISL motif (a.k.a. NAF domain) (a.k.a. motif the FISL to SnRK2 and SnRK3 proteins proteins SnRK3 and SnRK2 ELM Arabidopsis thalian Arabidopsis abolishes the catalytic activity of S of activity catalytic the abolishes , in a Ca , in 1, phosphorylate S phosphorylate 1, Arabidopsis SnRK3s bind to the to bind SnRK3s AK + ( 1, T Na 2+ - + - like/CBL), with varying degrees of specificity degreesof with like/CBL), varying OS )/ dependent manner (Halfter et al., 2000, etal., (Halfter manner dependent H 3 and E and 3

+ acting ,

exchangers N exchangers and and a their expression is induced by isinduced expression their , ,

are involved in are involved

NF

have the highest similarity to SNF1. As As SNF1. to similarity highest the have CIPK in the SOS pathway SOS the in - LM regulatory mechanisms of the SnRK thefamily of SnRK mechanisms regulatory loop (a.k.a. T (a.k.a. loop calcium 1 are 1. These proteins were named named were proteins These 1. in vitro in s ) , SOS2 ( , SOS2 plant specific subfamilies consisting of of consisting specific subfamilies plant SOS2 is activated by SOS3 or SOS3 by activated is SOS2

- HX related protein kinases (SnRK) kinases protein related - binding proteins, proteins, binding

, and , Cutler et al., 2010 etal., Cutler s (Qiu et al., 2004) and theH and etal.,2004) s (Qiu of SOS2 of NF S - diverse ; Quintero et al., et al., ; Quintero loop) is an important important isan loop) alt alt Arabidopsis activa 1 and causes a phenotype aphenotype andcauses 1 the the O , which verly

that triple deletion of genes of triple deletion tes

signalling pathways pathways signalling

is required for salt salt for is required S the plasma the plasma ensitive

, is thought to to is thought two kinases have kinases two z SCaBPs (SOS3 SCaBPs geminivirus geminivirus ). ed SnRK3s ed In yeast, t yeast, In

Quan et Quan al., - 2011 González et González

G and and eminivirus eminivirus )

and ,

+ (a.k.a. (a.k.a. hree hree /Ca

- like 2+

This article isThis article by protected copyright. All rights reserved. characterized previously ofthe phenotype thestrong Because phenotype the inspected T kinase upstream activation inthe phosphorylation 2002a et (Gong al., other SnRK3s on observed effects similar and et al., 2004), (Guo to salt more tolerant are mutation Thr168→Asp enzym ofthe activity activation Chaves activation sites inthe activation proteins SnRK1 of theactivation Resembling (Glab etal., al., (Glabet seeds not produce onsugar be rescued could mutant double mutant large 2010). A Crozet etal. theactivation phosphorylate activation Ext functionally and Hanley to they bind and infection o investigate Accepted Articleract s - Sanjuan etal2014 Sanjuan was

from yeast cells expressing GRIK1 or or GRIK1 cells yeastexpressing from - - - loop make SOS2 constitutively active (Guo et al., 2001). Three candidate phosphorylation phosphorylation Three candidate etal.,2001). (Guo active constitutively makeSOS2 loop of loop loop of SOS2 with an Asp residue to mimic phosphorylation significantly increases the increases significantly phosphorylation tomimic Aspresidue with an ofSOS2 loop -

Bowdoin, 2006). GRIK1 or GRIK2 GRIK1 or 2006). Bowdoin, 2017 complement the complement embry

the the s involved ). Thus, GRIK1 and GRIK2 play essential roles playessential GRIK2 GRIK1 and ). Thus, the possible possible onic lethal (Bolle et al. 2013), but a but al. 2013), (Bolleet lethal onic e - loop are conser loop SnRK1 in vitro of of ). Replacement of the Ser the Replacement of ). geminivirus geminivirus - . role of GRIK1 and GRIK2 and GRIK1 role of scale study of double mutants mutants double of scale study Arabidopsis

- clade yeast loop of SnRK1.1 and 1.2 and ofSnRK1.1 loop

(Gong et al., 2002 et (Gong 2017 - loop activates SOS2, raising the question of the identity of the ofthe identity the of question raising the SOS2, activates loop

(Hey et al., 2007). In addition, recombinant GRIK1 and GRIK2 GRIK1 and recombinant In addition, etal.,2007). (Hey elm1 sak1 tos3 sak1 elm1 ). Phosphorylation of SnRK1s was reduced in the double mutant mutant double reducedinthe was SnRK1s of Phosphorylation ). ved among the SnRK3 family theSnRK3 among ved replication protein AL1 protein replication

plants bearing mutations in genes genes in mutations plants bearing - supplemented medium, albeit albeit medium, supplemented , phosphorylation ( GRIK a.k.a. SnRK1 a.k.a. , 2002c b ). Transgenic plants plants ). Transgenic 2 phosphorylate a peptide corresponding to the to corresponding apeptide phosphorylate 2

triple

- as upstream regulators of SnRK3s, we have have we SnRK3s, regulators of upstream as 156, Thr 156, ). These results strongly strongly sug results ).These ,

recent report showed that the the that showed recent report activating

mutant (Shen andHanley (Shen mutant suggested activating kinase activating - 168 or Tyr or 168

- in sugar/energy signalling. signalling. sugar/energy in ( grik1 mimicking mutations in the inthe mutations mimicking Kong and Hanley and Kong

t hem hem

(Gong et al., 2002 (Gong et al., -

1 that the that over

grik2 in vitro in - 175 residue in the residue in 175 plants were plants expressing SOS2 with the with the SOS2 expressing GRIK1 , Hey et2007 , Hey al., - 1 grik1

double mutant could could mutant double (Shen et al., 2009, 2009, etal., (Shen - Bowdoin 2002, Shen 2002, Bowdoin gest that gest that

and - - Bowdoin, 2006). Bowdoin, 1

b, grik2 grik1 small anddid small GRIK2 have been have

- - 1 1 )

. double double

grik2 can can

- 1

This article isThis article by protected copyright. All rights reserved. possibility it mutant sterile. and stunted plant was mature the yet supplementation, sugar al. 2013 genessuggested paralogous GRIK2 RT by andconfirmed respectively, , and tenth lines the mutant a and identified mutant lines difference no significant GABI knock Fig. 1 either To investigate andphosphorylation theamount affect and GRIK1 2 in plants. tolerance that We salt show sensitive. alleles. The mutant of combination different a we conditions, used stress toenvironmental responses mask the Results

Acceptedwas Article - a 713C09

-

possible that that possible ). the the is expressed in the double mutant (Fig. 1 (Fig. mutant double in the is expressed out line characterized so far, and and far, so characterized line out

). Recently, Glab et al( et Glab ). Recently, used here used For this study, we will denote these mutant alleles as alleles mutant these will denote study,we this For

. GRIK1 that the T the that Thus, GRIK1 and GRIK2 coordinate the responses to metabolic and environmental stresses stresses andenvironmental tometabolic theresponses coordinate GRIK2 GRIK1 and Thus,

grik1

mutant reported previously (Bolle et al., 2013; Glab et Glab al., 2013; etal., (Bolle previously reported mutant

the function of GRIK1 and GRIK2 GRIK2 and ofGRIK1 function the

- grik1 or or 2 grew grew grik2 GRIK2 grik1 - DNA insertion in insertion in DNA - 2 and reproduced reproduced and

s - and -

1 2 be

gene

double mutant double is a weak allele is aweak

tween the wild tween the GRIK1 and that the that grik2 grik1 2017 s

was - - 1 2 ) have shown that the that shown ) have

used in this study harbour T harbour study this in used

grik2 disrupted (Salk_142938 disrupted grik1 the the first

GRIK2 phosphorylate GRIK2 phosphorylate grik1 normally under our grow our under normally

- - used here grew normally under regular conditions but was but was conditions underregular grew normally here used

1 and not a complete complete aloss not and 1 - type, type,

double mutant in the F2 generation. We F2generation. the in mutant double grik2 - 2 yielded intron

b in vivo in because this isa this because ). A large ). A grik1 - - 1 PCR that no full that no PCR

d ouble mutant mutant ouble state state , we analysed T , weanalysed - 2

and and - grik1 scale study of double mutants mutants ofdouble study scale of SnRK1.1

a truncated form of GRIK1 in the GRIK1of inthe form truncated a and activate and for grik2

grik2 - 1

- grik1 grik1

DNA insertions in the f inthe DNA insertions grik2 th - - - - length mRNA for either for mRNA length 1 of - 1 was room conditions ( conditions room

plants -

since this is the only GRIK2 is theonly this since -

DNA insertion lines in which inwhich lines insertion DNA The The allele different to the tothe different allele function and Salk_015230 and - .

1

2017 embryonic lethal (Bolle et (Bolle lethal embryonic

mutant could be rescued by be rescued could mutant SOS2 grik1 , we crossed the two the crossed , we ). . To . Because we observed weobserved Because

- to increase salt salt to increase 2 g investigate the investigate rik2 veri Fig. 1 Fig. -

1 irst intron irst for of of

fied that that fied double double

GRIK1 c grik2 ). Thus, ). Thus, ;

or or This article isThis article by protected copyright. All rights reserved. phosphorylation was (Fig. al sucrose, with plates 1% (MS) medium Skoog the lower in were SnRK1.2 and SnRK1.1 ( AMPK against phosphorylated 201 activation inthe phosphorylation the mutant in GRIKs of targets theprimary SnRK1s are plant development. ofGRIKs in the role from g grik1 resu N peptides encode the type in the wild sequence as the fused the to pertaining type the wild size to endogenous of downstream grik1 Baena rik2 - Accepted Article truncated terminal 7 second second lts and the phenotype of theof thephenotype lts and also also 1 ). - - - 2 1 2 f to ) To evaluate the phosphorylation rate phosphorylation the evaluate To -

. double mutant double could mutant performed was blotting northern mutant, González et al., 2007, Cho et al., 2016 et Cho al., etal.,2007, González

Western blotting with the anti with blotting Western the first intron and the and intron the first reduced in the reduced in and GRIK1 the third third and 7 grik1 -

, 11 transcript in the wild typewild the in transcript T in the in -

were correctly spliced out, the out, spliced correctly were introns amount amount provide residual provide DNA insertion DNA GRIK1 -

- allowed the inspection of stress related responses, which could be separated beseparated whichcould related responses, ofstress theinspection allowed 2 , and , and grik1

allele allele grik1

protein starting at starting protein 59 - of SnRK1 of 2 protein that recognizes the that recognizes protein revealed that there was a was that there revealed - grik2 - second residues long residues 1 downstream downstream grik1

mutant (Fig. mutant . - In the In the 1

GRIK1 function -

- proteins 2

double mutant (Fig. (Fig. mutant double SnRK1.1 antibody showed that the total amount of SnRK1.1 ofSnRK1.1 amount total thatthe showed antibody SnRK1.1 exon of grik1

mutant suggest that that suggest mutant Arabidopsis grik1 in the in

- - ).

, the longest , the longest 2 loop of SnRK1.1 was almost eliminated (Glabetal., eliminated almost was ofSnRK1.1 loop was detected, while no band was detected was band no while detected, was 3´end region. 3´end though phosphorylation was not entirely eliminated entirelyeliminated not was phosphorylation though

Met Results showed that the amounts of phosphorylated phosphorylated of that the amounts showed Results 1d). 1d).

grik2 GRIK1 are reduced in the in are reduced - 2 using asprobe

mutant, a mutant, grik1 - Sequencing the ofthe the Sequencing 156 - . 1

in the 5´end region of region 5´end in the Importantly, t Importantly,

(Shen et al., 2009). et (Shen al., mutant mutant

- in the wild in the n 2

mRNA containing containing mRNA Arabido ORF ORF grik2 1 While several sh several While remaining remaining

f hybridizing ). These results suggest that that the suggest results ).These

this compared to WT to compared in the in -

1 the the

truncated GRIK1 expressed expressed GRIK1 truncated mutant psis - grik1 type protein he robust growth the of growth robust he middle region of of region middle grik1

SnRK1 proteins SnRK1 mRNA may have the same thesame have may mRNA -

band band 2 , we used an antibody anantibody used , we

RT In the In the - grik2 2 the the ort ORFs could possibly possibly could ORFs ort

grik1 - transcript transcript PCR stro - , T 1 o grik1 (Fig. 1e). -

- n Murashige and and Murashige n amplicon amplicon DNA left border border left DNA mutant ng 2

mRNA er -

GRIK1 1

than the encod

grik2 with .

These These . Because Because , grik1 - ed

in the in the similar 1

an an - 2 This article isThis article by protected copyright. All rights reserved. similarly to mutant double ofthe thesensitivity we Next, assessed The the specifically type and glucose ( wild type and o MS of growth hand, the other On type. to thewild MS On phenotype. conditions. the tested under functions important mediatephysiologically to is sufficient SnRK1s of phosphorylation the residual (Supplemen dark in the significant detected no differences phenotypic manifested activity SnRK1.1 were ( roles playimportant whichSnRK1s in conditions, ofthe phenotype the we Next, examined The Supplemental Supplemental Accepted the between ingreening difference no observed 4%),we (total sucrose 3%additional n plates with Article

plates with plates with grik1 grik1 grik1 kept and sucrose supplemented supplemented sucrose and - - sized 3 sized the - - 1

2 2 in the dark or submerged in water for 60h water for in dark orsubmerged in the

function inhigh function grik2 grik2 grik2 double mutant mutant double the Fi 1% sucrose 1% sucrose – - g. g. to - - 1 double mutant, although the wild type tended to grow slightly faster growslightlyfaster to type tended wild the although mutant, double 1 1

mutant (Glab etal,201 (Glab mutant S -

plates with 1% sucrose, sucrose, with plates 1% double double double mutant is sensitive tohighconcentration mutant sensitive is double 4 1 - c day ). tal Fig. tal growth growth On - old seedlings were transferred to MS agar plates supplemented with 50 with 50 plates supplemented MS agar to were transferred seedlings old - and

mutant is sensitive to glucose during post during to glucose sensitive mutant is glucose conditions glucose 3% sorbitol plates, 3% sorbitol

( By contrast Supplemental Supplemental S

difference between the wild type and and wildtype the between difference supplemented supplemented 1 a ) or under submergence (Supplemental Fig. Fig. (Supplemental submergence under ) or plates, we observed no difference in growth in growth difference we no observed plates, , the grik1 7 grik1 ) Fig. Fig. .

grik1 , which is consistent with the with consistent is , which with 3% glucose ( glucose 3% with - the in 2 S -

1 2 grik2 which the the which Baena c

- double mutant was arrested before greening on greening before arrested was mutant double grik2 2 ). These results indicate that GRIK1 and GRIK2 GRIK2 GRIK1 and that indicate results ). These with with , conditions in which mutants with altered withaltered mutants which in , conditions grik2 - 1 - -

1 double mutant under low energy energy underlow mutant double the wild type the wild etal.,2007) González environmental - showed post showed 1 osmolar

double mutant had had mutant double

Supplemental Supplemental ity was identical to that that to ity identical was grik1 s -

( germination growth germination of NaCl. of - Cho et al., 2016 etal., Cho germination growth germination

stresses. When stresses. - 2

reported phenotype of phenotype reported grik2 S Fig. Fig. . Four . 1 a glucose b

between the wild the between ), suggesting that that ), suggesting - S 1 1

- double mutant mutant double c week ). ). ). We By contrast, contrast, By - - sensitive sensitive old plants plants old

of theof

similar This article isThis article by protected copyright. All rights reserved. 3 GRIK1 whereas activity, autophosphorylation had GRIK1 that revealed kinase assays ATP in proteins recombinant we experiments, these For SOS2. phylo theSnRK belongs to SOS2, which phosphorylates GRIK1 sodium resultsindicate these Together, salt The etal.,2012). (Barragan evapotranspiration maximises to GRIK2 GRIK1 and Hence, w SOSpathway. the with inconcert toare likely function ofsalinity and component ionic tothe tolerance inmounting instrumental are GRIK2 that GRIK1and wild mutant and tes the ofstrains any length among root in differences significant no wedetected mMmannitol, mM300 200 or with plates supplemented (Fig.2 plates mM NaCl on100 survive grik1 grik1 100m mM or a

Accepted). kinase A Article - - sensitive phenotype at 20 mM NaCl, but was butwas NaCl, 20mM at phenotype sensitive binding residue Lys137 was changed to Arg, was also produced as a negative control. control. asanegative produced also was to Arg, changed was Lys137 binding residue - - genetically related to SnRK1s to related genetically 2 2

grik2 or tolerance of tolerance grik2 ionic - - 1 dead type of SOS2, in SOS2, in type dead of M NaCl, the roots of the double mutant were shorter than those of the wild type, type, ofthewild those than were shorter mutant ofthedouble roots the M NaCl,

double mutant mutant double - - 1 - sensitive phenotypes due due to phenotypes sensitive type plants to 3% sorbitol (165 mM; Supplemental Fig. Fig. (165 mM; Supplemental sorbitol to 3% plants type

(Fig. 2 (Fig. the salt the salt Arabidopsis Thr168 inthe activation Thr168 a E. coli E. ); on regular MS plates, the double mutant had slightly shorter roots. The roots. shorter slightly mutant had double the MS plates, regular ); on tolerance of of tolerance . A kinase . A was

that that . 3

produced

.

su not as sensitive to NaCl as NaCl to sensitive as not which Lys40 was mutated to Gln (SOS2 to mutated was Lys40 which Hence GRIK1 b a family ). On the other hand, when seedlings were transferred to MS transferred seedlings were hand,when theother ). On - ted dead type mutant protein of GRIK1 (G ofGRIK1 protein type mutant dead Arabidopsis , we investigated whether GRIKs could GRIKs could whether investigated , we

, in keeping with the similar sensitivity of the double double the of sensitivity similar the with keeping , in and GRIK2 play redundant yet important roles in the rolesin yet important playredundant GRIK2 and g

lutathione S lutathione and the grik1 - loop of loop

enhanced plays an important role in salt tolerance in salt role animportant plays still still -

in hydroponic culture with LAK medium, which which medium, LAK culturewith hydroponic in 2 less sensitive than the than the sensitive less grik2 SOS2 - transferase (GST) transferase

sodium sodium - 1

.

the

double mutant had a significant had asignificant mutant double e also evaluated the contribution of contribution the evaluated e also

sos2 uptake uptake - 2

S mutant, which could not whichcould mutant, 1d - - KN; Gong et KN; Gong linked to to linked fused GRIK1 GRIK1 fused ) sos2 . These results indicate indicate results These RIK1 - also also 2

mutant (Fig. (Fig. 2 mutant - - KR), in which the which KR), in KR KR phosphorylate phosphorylate

al. 2002 al. did not did not , is , is In vitro In (Fig. b ),

b ).

This article isThis article by protected copyright. All rights reserved. to wild expressing plants ofthe those than shorter SOS2 high well under grow could M the in expressed SOS2 were activity site thephosphorylation whether examined We next of phenotype sensitive thesalt cannotrescue SOS2 of mutant The T168A vitro. Fig.S2 SOS2 whereas auto assay. SOS2 SOS2 mutants (single to Ala were changed residues phosphorylated Chaves mimic phosp ofthe the members (Ser sites candidate three at the SO in site phosphorylation GRIK1 the To identify SOS2 GRIK1 phosphorylates that indicate GRIK1, but not by phosphorylated was osaic osaic

Acceptedplates the NaCl Article - - -

phosphorylation. phosphorylation. ). These results suggest that GRIK1 phosphor GRIK1 that resultssuggest ).These Y175A in the in Y175A Y175A; These mutations mutations These -

V of Sanjuan etal2014 Sanjuan irus (35S promoter) irus

SOS2 under salt stress stress salt under SOS2 horylation significantly increase significantly horylation and - S156A andSOS2 S156A triple mutant SOS2 mutant triple

among more than 10 10 more than among sos2 Arabidopsis Phosphorylation ofSOS2 Phosphorylation - of SOS2 of 2

background could survive on 100 mM NaCl plates NaCl 100mM on survive could background ). -

salt conditions salt conditions We generated SOS2 mutants, in which one or all threeof all one or in mutants, which SOS2 generated We sos2 . Transgenic plants expressing wild expressing plants . Transgenic - - Y175A were Y175A were combined with the K40N mutation to abrogate to abrogate mutation theK40N with combined were

SnRK3 subfamily and and SnRK3 subfamily 156, Thr 156, - in vivo 2 background under the control of the 35S promoter 35S the of the control under background - AAA), and used them as them as used and AAA), by by . For this purpose, phosphorylation site phosphorylation purpose, Forthis . independent independent

- in vitro 168 andTyr168 GRIK1 (Fig. 4) d still still

the activity of activity the - - - S2, we introduced mutations inthe activation mutations introduced S2, we T168A andSOS2 T168A type type . phosphorylated by GRIK1 (Fig. 3 (Fig. by GRIK1 phosphorylated KR (Fig. (Fig. 3 KR . Transgenic plants expressing SOS2 expressing plants Transgenic .

ylates Thr168 in theactivation in Thr168 ylates

lines in the activation in the SOS2 - replacement of these residues with with theseof residues replacement 175) a for . , because , because , Supplemental , Supplemental The The SOS2

GRIK1 GRIK1 each each - type SOS2 in the inthe SOS2 type lines presenting the the presenting lines - AAA

in vitro in transgene - S156A, S S156A, substrates in an in substrates they are fully conserved in all in conserved arefully they - loop is essential for essential for is loop by GRIK1 was not detected, notdetected, was GRIK1 by

(Gong etal.,2002 (Gong Fig. Fig. sos2 , albeit OS2

were S2 - - mutated forms of of mutated forms 2 b - - sos2 ). These results ).These results . loop ofSOS2 loop T168A T168A , Supplemental , Supplemental

t selected heir roots were roots heir best best in - - the S156A or or S156A 2 of Cauliflower Cauliflower of

vitro

background background and resistan putative putative Asp to Asp the the b,

for for kinase kinase

- loop loop in ce This article isThis article by protected copyright. All rights reserved. Supplemental by SOS2 SOS1 (SOS2 SOS3 (SOS2 SOS2 of aminoacids 138 activates SOS2 GRIK1 by phosphorylation we Next, whether asked vitroIn stress. salt under survival for isessential inparticular Thr168 that and salt stress under SOS2 of thefunction involvedin are SOS2 of Tyr175 and Thr168 Ser156, expressing inplants retardation root growth for the reason not were expression transgene in that differences demonstrating wild the wild the by complemented plants transgenic S inactive to of accumulation wild expressing wild thanthoseof abundant more weresubstantially line transgenic SOS2 of transcripts revealed that blot analysis SOS2 expressing these experiments

Accepted Articlethe SOS2 - type type - (SOS1 - independent form of the kinase (Guo et al., 2001). (Guoet al., kinase the of form independent

known upregulation ofthe upregulation known ∆

activation of activation SOS 308 - ∆ - OS2 mutants would suffer from acute salinity stress compared to the wild the to compared stress salinity fromacute suffer would mutants OS2 S156A, SOS2 S156A, SOS2 308 to a greater d toagreater 308 - / CT, Fujii and Zhu, 2009) 2009) andZhu, Fujii CT, T168A) was used as a negative control. control. anegative as used T168A) was Fig. Fig.

- transgene transgene type or endogenous orendogenous type - SOS2 T168A in the in T168A S2

in ). Fig. 4. Fig. -

- S156A, SOS2 S156A, Little phosphorylation of SOS1 was observed when GRIK1 was combined with with combined was GRIK1 when observed was SOS1 of phosphorylation Little T168A and SOS2 and T168A 2 byGRIK1 showed showed On the other hand, hand, theother On egree in the presence of GRIK1 than in the presence ofGRIK1 thepresence in than GRIK1 of presence the in egree sos2 -

∆ SOS2 308) removes the autoinhibitory domain of SOS2, yieldinga ofSOS2, domain theautoinhibitory removes 308)

SOS2 similar complementation results complementation similar - - 2 T168A and SOS2 and T168A were

SOS2

background died on the 100 mM mM NaCl the 100 diedon background - S156A orSOS2 S156A

transcript by salt stress and and stress by salt transcript - Y175A

used as used (Fig. 4 - type type - S156A, SOS2 S156A,

transcripts at levels commensurate with the with commensurate levels at transcripts all the b ). a SOS2

The reason for this disproportionate thisdisproportionate for reason The substrate substrate The - - Y175A Y175A. Together, t Together,Y175A. 12 lines 12 lines

gene.

GST The T168A The -

T168A andSOS2 T168A transcript -

fused C fused for In any case, transgenic lines expressing expressing lines any case,transgenic In tested tested SOS2. SOS1 SOS2. in vitro

- that that (Supplemental Fig. S3), Fig.S3), (Supplemental mutated form ofSOS2 form mutated - - type type terminal 148 amino acids amino 148 terminal of T2 transgenic plants plants transgenic T2 of s

is unclear, but but is unclear, transgenic plants expressing expressing plants transgenic hese results indicate that that resultsindicate hese . Deletion of the C of . Deletion SOS2

plates - - Y175A in each in Y175A each CT was phosphorylated phosphorylated was CT

in the transgenic line transgenic in the

(Fig. 4). Northern Northern (Fig. 4). - type and and type could be due due be could - KR (Fig.5 KR - ∆

- terminal terminal 308 sos2

of of - 2 ,

This article isThis article by protected copyright. All rights reserved. Acceptedet al., 2004). inserted nhx1 GRIK1 stre yeast strain pathway The SOS Fig (Supplemental cell the yeast fully active in thedead not GRIK1by but thenon media with growthin of tos3Δ SNF1, innon grow kinase The SNF1 yeast Article activate GRIK1 GRIK1 anucleo shows SOS2 Although cyto interact GRIK1 and that SOS2 C Fluorescence SOS2 GRIK1 and of interaction physical the To confirm activate SOS2 the ss ( ss SOS2 plasmic : : :: . - KanMX Arabidopsis SOS2 complex was detected inside thenuclei. inside detected was complex SOS2 TRP1 Quintero et al., 2002, 2011 2002, et al., Quintero For this study, we used strain YP890, a derivative of AXT3K (∆ ofAXT3K aderivative YP890, strain used study,we this For chromosomally - ∆ 308 s

- ) rims around nuclei, in in nuclei, around rims

fermentable carbon sources if SNF1 is inactive. To test the ability of GRIKs to activate to activate GRIKs Totest of the ability isinactive. if SNF1 sources carbon fermentable , which , This strain This strain that ) (Quintero et al., 2002) in which a in which al.,2002) et ) (Quintero in vitro / s omplementation (BiFC) experiments (BiFC) omplementation T168A

SOS2

, comprising the Na/H exchanger SOS1, SOS2 and SOS3, andSOS3, SOS2 SOS1, exchanger Na/H the , comprising lack all major sodium efflux transporters and transporters sodium efflux major all lack

GRIK1 was expressed int expressed was GRIK1

lack .

in yeast is essential for for is essential protein. protein. expresses the endogenous SNF1 theendogenous expresses

s to provide moderate and constitutive expression of of expression constitutive and moderate to provide

all thre all - kinase mutant bearing the mutation K137R demonstrated that GRIK1 that demonstrated K137R themutation bearing kinase mutant

at least least at

- These results indicate that phosphorylation of Thr168 by GRIK1 can byGRIK1 ofThr168 that phosphorylation indicate results These cytoplasmi - e up fermentable carbon sources glycerol and ethanol by wild the ethanol and glycerol sources carbon fermentable ). cytosol We used used We stream regulatory kinases of SNF kinasesof regulatory stream in the cytosol (Fig6). the cytosol in carbon utilization, as utilization, carbon - c distribution when expressed alone (Kim et al., 2007),no (Kim et al., alone whenexpressed c distribution filled he he this system to analyse to analyse system this .

yeast yeast S 4 transvacuolar strand transvacuolar PGK1 ).

in in strain YPDahl55 ( YPDahl55 strain

prom

N. benthamiana in planta - : upstream kinases kinases upstream SOS1 BiFC fluorescence was visible in the visiblein was fluorescence BiFC evidenced by evidenced

: are CYC1 , we conducted Bimolecular conducted , we

exceedingly sensitive to sodic to sensitive exceedingly the ter ena1 s sak1Δ , and in cyto , andin

1 (Ye et al., 2008) etal., (Ye 1 . The resu The . expression cassette cassette was expression in vivo in

the inability of yeast to yeast of the inability can be reconstituted in reconstituted be can :: HIS3 SAK1/PAK1 :: the KanMX elm1Δ KanMX

activation of activation of :: lts ena4 SOS1 protein (Guo protein SOS1 plasmic

demonstrated demonstrated , , nha1 ELM1 . Restoration . Restoration

pockets. pockets. :: : SOS2 by SOS2 KanMX KanMX :

LEU2 and and - type type

was was , This article isThis article by protected copyright. All rights reserved. t reports, that previous of identical to cotyledon the beyond grow to ofsugar needed supplementation or etal.,2013) (Bolle germination before growth vegetative we In thisstudy, yeast. in SOS2 can activate GRIK1 salt toconfer SOSproteins ofthe capacity essentially inactive in was module the SOS2 that indicating alone, SOS1 by conferred to that identicalwas SOS proteins ( concentrations NaCl glucose salt theof components in tolerance SAK1/PAK1 wegenerated Next, kinases. upstream endogenous by activated (Fig. 7b). rem SOS2 cascade, we phosphorylation GRIK1/SOS2/SOS1 ho are thefungal TOS3, which AcceptedDiscussion Article - oval of the autoinhibitory domain of SOS2 (SOS2 SOS2 domainof autoinhibitory ofthe oval sensitive phenotype (Fig. (Fig. phenotype sensitive - AAA AAA - supplemented AP medium due to the complementation of of to thecomplementation due medium AP supplemented These results indicate that, in contrast towild incontrast that, resultsindicate These - stage (Glab et al., etal., (Glab stage in

, ELM1 S. cerevisiae S.

strain strain successfully produced successfully

. Previous data had indicated that the that had indicated data . Previous and and YP890 Arabidopsis

strain Δ3K4E, strain 10 TOS3, TOS3, 0 mM NaCl), the residual salt tolerance imparted by the full complement of complement thefull by imparted salttolerance residual the mM NaCl), 0 , had been deleted been , had . SOS2 2017 and the ENA1 and the 7c) mologues of GRIKs (Shen et (Shen et al., 2006) ofGRIKs mologues

SOS pathway (SOS1, SOS2 and SOS3) failed to complement the complement to failed SOS3) SOS2 and (SOS1, pathway SOS - AAA was unable to activate SOS1 in in SOS1 unabletoactivate was AAA . ). Whereas the ).Whereas Expression of GRIK1 in ofGRIK1 Expression in in

Δ3K4E (Fig. (Fig. Δ3K4E which which he mutant lines harbouring T harbouring lines mutant he

a

gri - tolerance tolerance - encoding the genes . Transformation of Δ3K4E cells with the cellswith Δ3K4E of Transformation . 4 sodium pumps that are the major determinants ofNa determinants themajor thatare pumps 4 sodium

k1 - 7c 2 first tested first ). However, co ). However, grik2 grik2 - to AAA - - 1 1

- Δ3K4E cells. These results demonstrate that that demonstrate results cells. These Δ3K4E type SOS2, the SOS2 SOS2, type

grik1 double mutant double mutant line used here (Salk_015230) is (Salk_015230) here lineused mutant Δ3K4E Δ308, Fig. Δ308,

the non -

1

- the three the

expression of GRIK1 of expression cells improved growth in in growth improved cells grik2 - DNA insertions in DNA insertions . For reconstitution of the the of . Forreconstitution sak1 elm1 t elm1 sak1 - 7a phosphorylatable phosphorylatable this system this - 1 )

double mutant was lethal lethal was mutant double or the or with near wild near with fungal fungal - AAA mutant cannot be be cannot mutant AAA co os3 upstream kinases kinases upstream , irrespective of theof irrespective , - expression of SOS3 of expression

mutations. mutations. core - increased SOS3 regulatory regulatory SOS3 GRIK1 - mutant mutant type type

were

At low At low the the +

This article isThis article by protected copyright. All rights reserved. o activity SnRK1.1 compromised disproportional SnRK1.1 Since grik2 the SnRK1.1 in of the amount decrease in GRIK1 intact 201 ( 2016) etCho al., Fig. (Supplemental grik1 phosphorylation the by encoded protein putative that the activation 156 starting at methionine full blotting theT bearing Salk_142938 line used we have T with the line713C09 GABI used the studies Previous different. Accepted Supplemental Article - 7 length length ), indicating that the that ), indicating - - 1 2

double mutant, mutant, double grik2 and RT and would would f SnRK1.1 in the f SnRK1.1 in - loop was reduced wasreduced loop GRIK1 . - 1 Lack of GRIKs might have multiple effects on SnRK1 on effects multiple mighthave GRIKs Lack of

in both double mutants double both in degradation is strictly dependent on its activity on dependent isstrictly degradation

Fig. double mutant did not exhibit any defect any exhibit didnot mutant double - l occur occur PCR PCR y stable . On the other hand, the the hand, the other . On

of SnRK1s SnRK1s of transcript

S S 1 1 data in c a )

and and

t

as as grik1 ( he amount of SnRK1.1 was similar was ofSnRK1.1 amount he

Crozet et al., 2016) etal., Crozet grik1 (Fig. (Fig. 1 truncated truncated previously previously .

may be may be b but Our results showing that showing results Our but ), in which SnRK1s play important roles ( roles important play SnRK1s which ), in - 2 grik2 - 2 d allows

, not eliminated not background e ) also also

suggest that in that suggest protein encoded by the bythe encoded protein - 1 shown shown

compared to the wild type to the wild compared the expression of of expression the

compared to the compared sufficient to maintain SnRK1 maintain to sufficient grik1 grik1 grik1 , it is likely that , itislikely for . -

DNA insertion at the1 at insertion DNA - - 2 - 2

2 the in the in

grik2 allele retains some level of activity some level of retains allele grik2

grik1

grik1

phosphory -

grik1 1 - 1

s - double mutant was sensitive to high glucose highglucose to sensitive was mutant double a m grik1

2 - double mutant (Fig. (Fig. mutant double

under dark or submergence conditions conditions orsubmergence dark under 1 the T the to that that to - the the grik2 RNA RNA grik1 2

-

and inactive and grik2 1 grik2 synthesis of SnRK1.1 is ofSnRK1.1 synthesis lation of SnRK1s atthe ofSnRK1s lation - - - DNA insertion disrupts the disrupts DNA insertion DNA insertion in the8 in insertion DNA - , encoding - 1 mediated pathways, including including pathways, mediated

2 whereas of

- double

allele cannot fully replace fully replace allele cannot 1 st -

mediated pathways, as the as pathways, mediated the wild type (Glab et al., 2017). etal., (Glab type the wild

- Baena double mutant double (Fig. 1 intron 1 mutant mutant

SnRK1.1 variants are are variants SnRK1.1 a truncated form of GRIK1 of form a truncated mutant (Glab et al., etal., (Glab mutant higher degradation degradation higher - González et al., 20 etal., González 1 f ) due to the residual theresidual due to .

In the In a . The remaining . Theremaining ).

(Fig. (Fig. Northern Northern somehow somehow th

grik1

intron, and intron, 1 f )

- suggest suggest 1 the 07, of of

This article isThis article by protected copyright. All rights reserved. Accepted residues these although growth, stress theactivation T168in that show Our data upstream to not limited ofGRIKs roles are that the theactivation the abilitytophosphorylate has SOS interact in physically SOS2 GRIK1 and target SnRK2. of exception the (with in SnRK2s T168 to due may be the difference alternatively, T168/T169; o the reduction to mask degree sufficient to a sites other on phosphorylated (T168A SOS2 conditions, their Under unclear. al.remains Shenet of indeed SOS2 in disappeared Article theactivation phosphorylate wild ofthe to that (T168A SOS2 of form mutated the of intensity phosphorylation that observation 3 (Fig. todetectable be enough GRIK1wasstrong by SOS2 of phosphorylation toph none corresponding but SOS2, phosphorylated their However, SOS2. phosphorylate SnRK1s. than other substrates might have indicate The grik1 pathway in ayeast pathway

residue of SOS2 is conserved among SnRK3s (Gong al.,2002 et (Gong SnRK3s among SOS2isconserved of residue ed in vivo

phosphorylate d

by

- that GRIKs play important roles in several signalling pathways signalling pathways in roles several playimportant GRIKs that 2

grik2 GRIKs. In our hands, GRIK1 phosphorylated and activated SOS2 activated and phosphorylated GRIK1 hands, In our GRIKs. (Fig. 5) - 1 - type

double mutant was sensitive to high NaCl (Fig. 2). The effects on salt sensitivity saltsensitivity on effects The (Fig.2). high NaCl sensitive to was mutant double . Mutations . Mutations - T168A andSOS2 T168A s

-

SOS2 at T168. The reason for the discrepancy between our results and those those resultsand our between the discrepancy for reason The atT168. SOS2 SOS2, Shen et al. also concluded that GRIK1 and GRIK2 could not could GRIK2 and GRIK1 that concluded also etal. Shen SOS2, reconstituted system (Fig. (Fig. system reconstituted - loop of SOS2. We observed that the phosphorylated the that observed We ofSOS2. loop at

were not essential for plant survival under under plant survival not essentialfor were S159 and Y175 also affected salt tolerance, as reflected by root root by reflected as tolerance, affected salt and Y175also S159 in - N. benthamiana N. AAA (Fig. 3 (Fig. AAA - vitro 8). This suggests that SnRK3s, but not SnRK2s, butnot SnRK2s, thatSnRK3s, 8). Thissuggests - Shen et al. (2009) concluded that GRIK1 and GRIK2 do not donot GRIK2 GRIK1and that concluded (2009) etal. Shen loop of SOS2 is important for function function isimportant for ofSOS2 loop

kinase assay detected bands detected bands kinase assay - loop of SOS2 and to activate activate to ofSOS2and loop b 7 , Supplemental Fig. S2c , Supplemental regulation of SnRK1s. of regulation ). Taken together, our results indicate that GRIK1 GRIK1 that indicate ourresults ).Taken together, osphorylated SnRK2.4. In our experiments, experiments, Inour SnRK2.4. osphorylated

(Fig. 6). (Fig. 6). unintended

In addition, GRIK1 could activate the could activate GRIK1 addition, In

, effect b T169A) may have been havebeen may T169A) ), whereas this residue is aSer is this residue ), whereas and

s possibly possibly ), indicating that GRIK1 that ), indicating

of the fused tags. The tags. fused the of - this kinase this ∆ acute acute suggest f phosphorylation on phosphorylation f 308 308 a ). Based on the on ). Based , of of T169A) was similar was T169A) in vitro

salt stress (Fig. 5). stress salt corresponding to corresponding SOS2 SOS2 under salt under SOS2 ed may , suggesting , suggesting

that they that

band band

(Fig. 4). 4). (Fig.

be be This article isThis article by protected copyright. All rights reserved. min, 30x 5 1x95°Cfor conditions: following the under (Takara) polymerase KOD with (PCR) chainreaction bypolymerase amplified reverse and with oligo(dT) seedling of 10 days from prepared Total RNA mutagenesis site directed and Construction but al well SnRK the membersof andactivate phosphorylate GRIKs can In summary, t upon depending induces salt when stress that case, In activation. al., 2007 SOS3 by activated SOS2 isalso residues phosphorylating by perhaps ofSOS2, the full to activation contributing kinase upstream another e.g. resistance, forsalt responsible mechanisms parallel there exist SOS2 the than to salt sensitive less was however, function forSOS2 important salinity under SOS2 activation T168 on phosphorylation an effect on the for be mightimportant two sites These E Acceptedxperimental Article - known targets targets known in vivo in so in in so ). -

GRIK loop the SOS the , then either ,

procedures

- such as phosphorylation by additional kinases contributing to the full activation of the to activation full contributing kinases additional by asphosphorylation such mediated phosphorylation may affect the competence of SOS2 for Ca for ofSOS2 competence the mayaffect phosphorylation mediated he sugar/energy information gating by GRIKs by gating information sugar/energy he . pathway SnRK1s, and SnRK1s, In any case, our data demonstrate data case, our In any the putative protein encoded by the by encoded protein the putative

- transcribed with the Superscript II RT (Invitrogen). (Invitrogen). IIRT Superscript with the transcribed

for salt salt tolerance for in vitro in

and they they

SC (95 °C for 2 °C for (95

and in the yeast syste in theyeast and play important roles not only in the sugar signalling pathway signalling pathway sugar inthe notonly roles important play a ,

BP8 or or for for

in aCa local local sos2

another another Arabidopsis thaliana Arabidopsis

in vivo 0 s, 55 °C 20 s, 70 °C 2 min). 2min). 70°C 55°C20s, 0 s,

- Ca molecular conformation of SOS2, thereby exerting exerting thereby SOS2, of conformation molecular 2 2+

mutant. If GRIKs are the upstream kinases of kinases arethe upstream GRIKs If mutant. 2+ - depe .

signalling, SOS3 or SC or signalling, SOS3 unknown

that phosphorylation of T168 by GRIK1 is GRIK1 T168 by of that phosphorylation ndent manner (Halfter et2000, (Halfter al., manner ndent m . The . The

grik1

(Fig.8) mechanism involving the involving mechanism grik1

Columbia - 2

. allele works allele works

- 2 3 grik2

a Primers used to obtain obtain toa used Primers family in addition to in addition family BP8 may activate SOS2 mayactivate BP8 - 0 ecotype was primed ecotype was 0 GRIK1 - 1

S159 and S159 double mutant, mutant, double to some extent to some

cDNA was cDNA was 2+ - dependent dependent /or

(s) (s) Quan et Y175. their

or or This article isThis article by protected copyright. All rights reserved. RT for templates as wereused above Accepted from10 cDNAs purified Supplemental 1min) 70°C 55°C20s, 20s, °C for (95 min, 35x 5 1x95°Cfor were used: PCR Resour Biological T of The seeds Arabidopsis et al., 1995) version truncated pRT105. derived from 35S promoter oftheCaMV thecontrol under (pCAMBIA1200) vector abinary were cloned into ( pGEX4T1 into cloned was Article conditions; following s). 70 °C90 firstPCR: forthe used 1were Table Supplemental SOS2 into mutations the point To introduce 2002;2011) et al., (Quintero elsewhere beendescribed have pathway oftheSOS components core inof yeast expression the for yeast. Plasmids in expression GRIK1 sequenced. between vector into pGEX4T1 the encoding bp 1207 fragment

following the instruction following

cDNA was inserted into the vector p425GPD into thevector inserted cDNA was

Using the PCR products as templates, second amplification was performed under the under performed was amplification second astemplates, products Using the PCR .

T - - Table 1. Table DNA insertion lines DNA insertion DNA insertion lines (Salk_142938 and Salk_015230) were obtained from Arabidopsis Arabidopsis from were obtained Salk_015230) and (Salk_142938 lines DNA insertion c e Center (Alonso et al., 2003). Homozygous Homozygous 2003). etal., (Alonso e Center SOS2 1 x 95 °C for 5 min, 30x (95 °C for 20 s, 51 °C 20 s, 70 °C 90 s). 70°C90 20 s, 51°C 20s, °Cfor 30x(95 5min, °Cfor x 95 1

For expression in yeast, t yeast, in expression For The insertion sites were identified by sequencing the amplicons. sequencing wereidentified by sites insertion The - - AAA day BamHI - ∆ old seedlings of Col of seedlings old s 308

EcoRI (http://signal.salk.edu/cgi GRIK1 -

EcoRI w ere

and and - PCR. PCR.

are in Supplemental Table 1. The PCR product was subcloned subcloned was product 1. ThePCR Table are inSupplemental

subcloned sites for sites for XhoI The following conditions conditions following The - pGEX4T1 (Guo et al., 2001), primer pairs in pairsin primer 2001), (Guoet al., pGEX4T1

sites. Inserted fragments of all constructs were were constructs ofall fragments Inserted sites. he he - 0 plants and T and 0 plants SOS2

1 x 95 °C for 5 min, 20x (95 °C for 20 s, 51 °C 20 s, 51°C20s, 20s, °C for (95 min, 20x 5 x 95 °Cfor 1 full length mutant mutant length allele full as

(Mumberg et al., 1995) et (Mumberg Bam ). For expressio ).For - bin/tdnaexpress). The following conditions following The bin/tdnaexpress). HI/ . Eco

- DNA insertion lines as mentioned mentioned as lines insertion DNA insertion lines were identified with with identified were lines insertion RI fragment were used: were n in A. thaliana A.

with primers with

SOS2 as s

Bam in p414GPD

1 x 95° 1 x - AAA HI/ The final product product final The , fragments , fragments

Eco and the the and

C for 5min, C for

described RI for RI for

(Mumberg (Mumberg

in in

This article isThis article by protected copyright. All rights reserved. incub μg/ml lysozyme), 100 EDTA and 1mM NaCl, mM pH8.0, 150 inpre were resuspended thepellets and min, 4ºC), xg,5 (5,000 beta isopropyl mM 0.2 by induced was expression protein Recombinant hour. 1 cultured for further media, and ofLuria fresh20xvolume to transferred 37ºC, overnight at weregrown Single colonies GST encoding The constructs of Purification and Expression concentrations. NaCl attheindicated treatment bysalt followed mM Fe MgSO 1mM KH follows: wasas basesolution ofthe LAK final composition the maximize Na mMK with 1 solution mineral Ashton Long modified A et(2012). al. Barragan culture, hydroponic in salt For treatment medium LAK with culture Hydroponic by amplified fragm (2009). Zhu Fujii and in described as performed was analysis blot Northern Table1. in Supplemental with primers sfor 20 20s,58°C °Cfor 35x (95

Accepted Article+

and NH and ent digested with PstI (248 PstI ent digested with 4 , 30 mM mM H , 30 2+

as 4 Sequestrene Sequestrene +

RT

(LAK medium) was used for hydroponic cultures. This medium was designed to to designed was medium This cultures. hydroponic for used was (LAK medium) toxicity of Na toxicity of - D - PCR PCR - 3 thiogalactopyranoside for 4h at 37ºC. The cells were harvested by centrifugation centrifugation by harvested cellswere The 37ºC. 4hat for thiogalactopyranoside BO with primers with 3 , 10 mM MnSO , 10 138 +

- ions, while minimizing the osmotic effects of supplemental NaCl. The The NaCl. supplemental effectsof osmotic the minimizing while ions, -

fu Fe, pH 5.3. Seedlings were grown in LAK medium for one week week one for medium LAK in were grown Seedlings 5.3. Fe, pH GST GST sed proteins were transformed into into transformed were proteins sed - GRIK1 889 of ORF) was used as a probe. a as used was ofORF) 889 given given Fusion Protein Fusion 4 , 1 mM 1 mM ZnSO ,

and in Supplemental Table 1 Table in Supplemental seeds were directly grown on hydroponics as described by described as hydroponics on directlygrown were seeds GRIK2 s

4 or 54 °C 20 s for tubulin, respectively, 70 °C 45 s) 45s) °C 70 tubulin,respectively, sfor 54 °C20 or

, 1 mM 1 mM CuSO , in E. in

coli

- , followed bysequencing. , followed 4 chilled lysis buffer (10 mM Tris mM (10 buffer lysis chilled , 0.03 mM (NH 0.03mM ,

ated on ice for 15 min. After 15min.After onice for ated

The mRNA of of mRNA The 2 E. coli PO 4 , 2 mM Ca(NO mM , 2

Ro

For For s +

etta cells (Novagen). (Novagen). ettacells and nominally free of free nominally and GRIK1 4 ) grik1 6 Mo , 7 - 3 the O - 2 ) Bertani Bertani 2 24

, 1 mM mM , 1 was was ,

cDNA cDNA

and 100 and This article isThis article by protected copyright. All rights reserved. AcceptedMgCl 40mM mM or 10 7.2), Tris(pH mM 20 contained mixture theof reaction microliters Twenty some modification. In vitro vitroIn program Studio Image with LiCor evaluated were strengths band CA,USA), and (Advansta, ECL Westernbright Tween 0.05% and mM NaCl, pH 7.5,150 inTTBS made were dilutions antibody All with agitation. slow temperature withHRP incubated anti anti either with were incubated i were run and samples Article heat and 1970) (Laemmli, buffer to sample Laemmli added were tissue samples The nitrogen. inliquid ground collected and Two Blotting Western EDTA). 1mM mM NaCl, with pre times six were washed Thebeads at 4ºC. least 1 hour superna to the added were (Sigma) glutathione Then, at4ºC. min 5 xgfor at 30,000 wascentrifuged the suspension X Triton and (1mM) fluoride phenylmethanesulfonyl mM), (50 dithiothreitol - - SnRK1.1 antibody (Agrisera, Sweden) Sweden) over (Agrisera, antibody SnRK1.1 week

phosphorylation assays were performed as described previously (Fujii and Zhu, 2009) with 2009) with Zhu, and (Fujii previously described as wereperformed assays phosphorylation Kinase Assays Kinase (LiCor, UK). Experiments were replicated with five biological samples. biological with five replicated were UK). Experiments (LiCor, - old Col old

- 0 and 0 and - 2 conjugated anti conjugated , 10 , 10

n SDS n grik1 μM -

treated (10 min at 65 °C). Solid material was removed by centrifugation, centrifugation, removed by was material Solid min 65 °C). at treated (10

- ATP, 5 5 ATP, polyacrylamide gel electrophoresis followed by western blot. The blots blots The blot. by followed western electrophoresis gel polyacrylamide - 2 - phospho grik2 tant and the mixture were incubated with gentle agitation forat gentle agitation with incubated were mixture the and tant μ - rabbit antibody (GE Healthcare, UK) for 2 h at room hat room UK) 2 for (GEHealthcare, antibody rabbit Ci [γ - 1

seedlings, seedlings, - 20). Signals from b the from Signals 20). - - T172 32 P] ATP and 2 mM dithiothreito 2mM and P] ATP - - night at 4 °C with slow agitation. All blot agitation. slow at 4°Cwith night AMPK grown on MS media with 1% sucrose, were were sucrose, 1% MS mediawith on grown - α antibody (Cell Signaling, MS, USA) or USA) MS, (Cell Signaling, α antibody

- chilled buffer (10 mM Tris pH8.0, 150 150 mM TrispH8.0, (10 buffer chilled lots were detected using using were detected lots - l. Reaction mixtures were mixtures Reaction l. buffer (20 mM mM Tris (20 buffer - 100 (1.5%) were added, added, (1.5%) were 100

- agarose beads beads agarose s were then s were - HCl This article isThis article by protected copyright. All rights reserved. NaCl of concentrations various distilled water. in decimally diluted and resuspended cells were harvest, KCl.After mM with1 supplemented mediu by substituted was innon growth To test 2%glucose). 2%peptone, yeast extract, lithium Δ3K4E. wasdenoted strain resulting tos3Δ Next, diagnostic PCR. by confirmed was followed disrupte GRIK by SOS2 activation of etal.,2008) (Ye ( al., 2004) YP890 Yeast strain Yeast bySDS followed 40min. for at30°C incubated Accepted Articlesak1Δ

:: strains and media. and strains m (Rodriguez :: TRP1

acetate KanMX elm1Δ KanMX d by transformation with an with transformation d by

by was used to test the function of wild of thefunction test usedto was

selection of of selection w as isolated by isolated as – Five - was used as the starting biological material to produce a strain suitable to test test to strainsuitable a produce to biological material starting the as used was polyethylene glycol meth glycol polyethylene polyacrylamide gel electrophoresis. gel polyacrylamide (∆ena1 3% - - μL a Navarro and Ramos 1984) andRamos Navarro ::

ethanol ethanol KanMX tos3Δ KanMX liquots were spotted onto AP platessuppleme AP were onto spotted liquots uracil

:: 1 HIS3 . The . The

counter The reaction was stopped by the addition of Laemmli’s sample buffer, buffer, sample ofLaemmli’s theaddition by wasstopped reaction The

protot , 2% and grown for 3 to 4 3to for and grown : :ena4, nha1 :ena4, ENA1

glycerol. The ability of yeast cells yeast abilityof The glycerol. - T ena1 :: r select ophs and sodium and ophs ransformation of of ransformation TRP1 - ENA4 a loss od. Yeast cells were propagated in rich YPD medium (1% medium YPD inrich propagated cells were Yeast od. :: ion hisG ) lacking all three upstream regulatory kinases of SNF1 SNF1 kinasesof regulatory allupstream lacking three ) ::

gene tandem tandem gene - LEU2, nh LEU2,

of - . Strains were cultured overnight in liquid AP medium medium liquid AP overnight in cultured were . Strains with 5 with type and mutant SOS2. mutant and type :: - URA3 function mutant of of function mutant

- - d at 28°C. fluoroanth :: x1 - S. cerevisiae sensitive transformants. Gene replacement Genereplacement transformants. sensitive hisG :: KanMX, KanMX, array array :: ena4

- fermentable carbon sources, glucose glucose sources, carbon fermentable

ranilic acid (Toyn et al., 2000). The The al.,2000). et (Toyn ranilic acid encoding Na encoding

PGK1 gene replacement casset gene replacement

was performed using a using performed was to grow in salt was tested onAP wastested insalt to grow the nted with 1 mM KCl and KCland 1mM with nted The The prom TRP1 yeast yeast :: AtSOS1 +

gene gene - ATPases was was ATPases strain YPDahl55 YPDahl55 strain marker in :: CYC1 ter standard standard te ) (Guo et ) (Guo , This article isThis article by protected copyright. All rights reserved. was respectively multiplecomparison, single and T Student Statistics objective. with m μmol humidity, 150 h day/8 (16 chamber growth controlled keptina plants were 3 of The et(Silhavy al.,2002). silencing carrying suspension Agrobacterium ofan amounts final OD toa adjusted 5. MES pH with in resuspended Pellets were min. xgfor10 15000 at and centrifuged tumefaciens Agrobacterium promoter. the35S of the control under pSPYNE(R)173 C the 2008) using the full assays, BiFC For benthamiana. inNicotiana BiFC experiments - Accepted Article terminal - adopted

a to 4 FluoView FV1000 Confocal Microscope (Olympus) using a using (Olympus) Microscope Confocal FV1000 FluoView -

- week 6, 10 mM MgCl 10 mM 6, The The test and o test and moiety of YFP. The N YFP. of moiety

with

Olympus FluoView 4.2 FluoView Olympus has been described before (Waadt et al 2008) al (Waadt et before described been has - old old Xba

0.5 as 0.5 N. benthamiana benthamiana N. I and ne 600nm - 2 - -

way s len

a priori a - Sma 1 2

, 0. PAR) for 3 days until anal until for3days PAR) of 0.2. Appropriate combinations of cultures were mixed with equal equal mixedwith were ofcultures combinations 0.2.Appropriate of gth cDNA of of gth cDNA

ANO (strain GV3101). The bacterial cultures were grown at 28ºC overnight overnight at 28ºC weregrown cultures bacterial The GV3101). (strain 1 mM acetosyringone) and kept at RT for5 atRT kept and 1 mM acetosyringone) I sites to create to create I sites -

probability >1. of probability terminal fusion of SOS2 to the N to ofSOS2 fusion terminal VA followed by Tukey's multiple comparison test comparison multiple byTukey's followed VA Agrobacterium plants as described (Marillonnet et al., 2005). The infiltrated infiltrated The et al., 2005). (Marillonnet described plants as software was used to analys used to was software

The resulting plasmids The resulting GRIK1 . For normalized values, non values, . Fornormalized

a

was transferred to the pSPYCE(M) the pSPYCE(M) to transferred was C - termi

suspensions were then infiltrated into t into then were infiltrated suspensions the ysis by confocal microscopy. by confocal ysis

nal translational fusion of GRIKof fusion translational nal p19 suppressor ofpost p19 suppressor . All the YFP fusions wereexpressed fusions . Allthe YFP

h night, 25/22ºC, 60 night,25/22ºC, h

were electroporated into into electroporated were

488 - terminal moiety of YFP in in YFP moiety of terminal e the images. - nm Ar/ArKr laser and 60x 60x laserand nm Ar/ArKr

- h. Cell cultures were were Cellcultures h. parametric parametric filtration buffer (10 buffer filtration - transcriptional gene transcriptional

I vector (Waadt et al (Waadt etal vector mages were taken were mages -

was performed performed was 70% relative relative 70% binomial test binomial 1 to the 1 to he leaves leaves he

mM

for for

This article isThis article by protected copyright. All rights reserved. the in (YA) Y175A (TA), and 3 Figure Supplemental 2 Figure Supplemental 1 Figure Supplemental thatthereis declare The authors Korea of Republic the from additional support FJQ BFU2015 by grants JZ and to R01GM059138) Health (Grant 292763 271832, 263853, 259169, number (Projects Finland bythe wassupported work theT providing for Resource Center Biological Arabidopsis the We thank grik1 GRIK1 AcceptedShort Conflict ArticleAcknowledgement Accession from the Spanish Ministry of Economy and Competitiveness, co Competitiveness, and Economy of Ministry Spanish the from -

2 : S : Salk_142938, : Salk_142938, AT3G45240, AT3G45240, upporting

of

numbers interest

. I

nformation

GRIK grik2 . . .

Control experiments for the for the experiments Control Transgenic lines with similar with lines similar Transgenic O 2: 2: ther phenotypes phenotypes ther - AT5G60550, SOS2: AT5G35410 SOS2: AT5G60550, sos2

Turku Collegium for Science and Medicine and by the Academy of Academy the by Medicineand and Science for Collegium Turku 1 SSAC grant PJ0110510 grant SSAC : Salk_015230

Legends - 2 no conflict of interest. of no conflict .

of of

grik1 - 5 2 grik2 2

from the from in vitro in expression expression

- , SOS1: AT2G01980, SOS3: AT5G24270, SOS3: AT5G24270, AT2G01980, , SOS1: - 1 ,

64671 to JMP and BIO2015 and toJMP 64671 kinase assays. kinase

307335 lines. lines.

Rural Development Administration, Administration, Development Rural leve

- ) to HF, by National Institutes of Institutes National ) HF,by to financed by FEDER by financed ls of SOS2 of - DNA insertion mutants. This This mutants. insertion DNA

- S159A (SA) S159A - , and 70946 , T168A T168A ,

with - R to R to This article isThis article by protected copyright. All rights reserved. B. andWeisshaar, D. Leister, K., G., Mayer, Haberer, N., Kleinbölting, Huep, Bolle, G., C., Pardo, and B. J.A.,Cubero, A. De,Fernandez, L.,Luca, Z.,Rubio, Andres, E.O., V.,Leidi, Barragan, Baena Leisse, T.J., A.N., Stepanova, Alonso, J.M., 1 Table Supplemental use source carbon 4 Figure Supplemental Accepted ArticleReferences thaliana GABI F Stomatal Turgor and Regulate Cell J.M. sign energy and stress plant networks in transcription thaliana. Arabidopsis C.C L., Berry, Crosby, W. A., D.,Zeko, Brogden, E., Risseeuw, T., E., Marchand, D. Carter, D., Weigel, M., Schmid, Aguilar P., Guzman, I., R.,Schmidt, C., Ndubaku, Mulholland, M., Karnes, Y., Ansari, L., H., Prednis, Parker, C. C., E.,Meyers, Koesema, A., Jeske, Heller, C., C., R.,Gadrinab, P., Cheuk, J., Barajas, Zimmerman, - González, E., Rolland, F., Thevelein, J.M. and Sheen, J. and Sheen, J.M. Thevelein, F., E., Rolland, González,

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. Plant Plant .

J. , .

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S., Quintero, F.J., Pardo, J.M., Ohta, M., Zhang, C., Schumaker, K.S. and Zhu, Zhu, J. and K.S. Schumaker, C., M.,Zhang, Ohta, J.M., Pardo, F.J., S., Quintero, f the Function of an Arabidopsis PKS Protein Kinase. Kinase. PKSProtein anArabidopsis of the Function f -

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(2003) (2003) – ,

8.

This article isThis article by protected copyright. All rights reserved. AcceptedHanley and M.I. Shen, W., Reyes, Hanley and Shen, W. Baena Rolland, F., Rodríguez Article Martinez F.J., Quintero, J. Shi,H.,Zhu, M., Ohta, F.J., Quintero, I., Mendoza, Lin,H., Quan, R., J.K. Zhu, and K.S. Schumaker, J.M., Pardo, F.J., Y., Guo, Quintero, Qiu, Q.S., specifically activate SnRK1 by phosphorylating its activation loop. loop. activation its by phosphorylating activate SnRK1 specifically Plant Physiol. toSNF1 yeast related kinases protein Arabidopsis Mechanisms. and Novel Conserved cerevisiae. domain. Salt antiporter Yun, D. Na for pathway signaling SOS Arabidopsis Stress. from Salt Shoots Arabidopsis to Protect SOS2 Guo, Y. Biol. Chem. Na Vacuolar - Navarro, A. and Ramos, J. Ramos, and A. Navarro, -

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Plant Cell Plant

(2002) Reconstitution in yeast in yeast Reconstitution (2002)

Proc. Natl. Acad. Sci. Natl.Acad. Proc. , 57 - Y., Ali, Z., Fujii, H., Mendoza, I., H., Mendoza, Ali,Z.,Fujii, Y., , 675 - - Overly regulates the expression of two of theexpression regulates , 19 – Plant Physiol. Plant , 1415 in kinases GRIK1 and GRIK2 GRIK1and in kinases transport in Saccharomyces inSaccharomyces transport 709. - Sensitive (SOS) Pathway. (SOS)Pathway. Sensitive

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- role of potassium nutrition. nutrition. potassium role of stranded RNAs. RNAs. stranded , 553 - Shinozaki, K. Shinozaki,

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EMBO J. EMBO (1998) Genetic analysis of salt tolerance in salt of tolerance analysis Genetic (1998) –

, (2010) Molecular basis of the core regulatory network in ABA in network regulatory thecore of basis Molecular (2010) 2826. + 13

an , 12

tiporter. tiporter. (2008) The pathway by which the yeast pro yeast the by which pathway (2008) The Plant J. Plant

, 99 21 – Plant Cell Plant , 3070

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, 505 – 80. Plant CellPhysiol. Plant , 10

– Bock, R. and Kudla, andJ. Bock, R. 16. , 1181 , T., Tanokura, M., Shinozaki, and M., Shinozaki, K. , T., Tanokura,

- generated, 21 generated, – 1191. -

fluoroanthranilic acid resistance. acidresistance. fluoroanthranilic

, 51 , 1821 , , A - Saccharomyces cerevisiae Saccharomyces 97

rabidopsis. Evidence a for rabidopsis.

to 25 (2008) Multicolor Multicolor (2008) , 6896 –

tein kinase Snf1p Snf1p tein kinase 1839. (2000) A (2000) - nucleotide nucleotide – 6901.

(2002) A viral (2002) A viral

This article isThis article by protected copyright. All rights reserved. test). comparison ( type wild from significant differences to 12 taken to weretransferred plate MS agar ( Figure 2 ofSnRK1.1. amounts from ( amounts protein show used to stainingwas Coomassie with blot anti Western ( an N ( of region themiddle with blotting (d) Northern type and ofthewild (c) Phenotype grik1 (b) RT of diagram (a)Schematic Figure 1 Figure a f e ) Amount and phosphorylation status of SnRK1s in the wild type and the type in wild ofSnRK1s status phosphorylation Amount and ) ) Seedlings of the wild type (Col thetype of wild ) Seedlings ) Accepted concentr indicated with MS plates agar Article cDNA -

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This article isThis article by protected copyright. All rights reserved. control. aloading used as was stained) bromide (ethidium rRNA mM100 NaCl. SOS2 sos2 ( platesNaCl transfer. d after 12 weretaken NaCl. Photographs (a) the rescued Figure 4 in SDS ( SDS kinase ( Figure 3 test) (P<0.01, type wild from differences significant indicate Asterisks ofthe experiment. the end n=7) ±at (mean S.E., plants dry weightof 4 for grown were Plants indicated. withNaClas supplemented ( b b a b ) Accepted blot for Northern ) GST ) Article ) Seedlings grown on MS agar medium were transferred to transferred were medium MS agar on grown Seedlings A Hydroponic culture of Col cultureof Hydroponic - - PAGE followed by Coomassie staining (left) and autoradiograph (right). autoradiograph (left) and staining by Coomassie followed PAGE

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- and and -

PAGE followed byC followed PAGE transgenic plants expressing expressing plants transgenic dead ( dead - fused fused . . Expression of SOS2 of Expression vitro In non

- were dead SOS2 SOS2 dead sos2 K137R mutant mutant - transformed transformed

-

phosphorylation of SOS2 byGRIK1 SOS2 of phosphorylation selected selected 2

under salt stress condition. stress salt under ) of GRIK1 ofGRIK1 ) SOS2 T168A of SOS2 ofSOS2 T168A fused to GST to fused

for this test for oomassie staining (left) and autoradiograph (right). (right). autoradiograph and staining(left) oomassie transcript was performed with total RNA extracted from wild type (WT), (WT), type wild from RNA extracted with total performed was transcript sos2

- mutants mutants 0, (GRIK1 grik1 - 2 . Total RNA was purified purified was RNA . Total

in one in the (SOS2 - - was KR) . S159A 2

, S159A

grik2

also fused to GST (arrowhead) to GST fused also incubated with GST incubated - - way ANOVA followed by Tukey's multiple comparison comparison multiple by Tukey's followed ANOVA way KN

(SA) -

1 ,

(SA), (SA),

, arrow grik1

and Y175A and The lines presenting the presenting lines The . T168A

) was incubated with GST with ) incubated was - 2 grik2

(TA) and (TA) and 12h after transfer to MSagarpla to transfer after 12h

(YA) f - -

resh fused GRIK1. Proteins were separated separated were GRIK1.Proteins fused 1 - weeks. The weeks.

and and , but not T168A butnot ,

plates with or without 100 mM mM 100 without or with plates Y175A sos2 . Proteins were separated in separated were . Proteins - 2

(YA) bar graph bar best resistan best mutants in LAK medium LAKmedium in mutants

-

mutant forms of of mutant forms fused GRIK1 fused

(TA)

, partially partially , represents the the represents ce to the ce

or or tes with the

This article isThis article by protected copyright. All rights reserved. μm. show control negative and and(d) (c) panels strandin transvacuolar light) transmitted with overlay images; right: fluorescence fr Panels Figure 6. ( differences indicate significant Asterisks n=6). ±S.E., (mean SOS1 intensity of achieve GRIK1 amountof thesmall that Note (right). andautoradiograph (left) staining phosphorylation version mutation The Figure 5 SOS2 ±S.E.) (mean ( and threetimes repeated was ( d c ) Survival rates of the seedlings on MS agar plate agar MS on ratesofthe seedlings ) Survival Accepted Article Primar )

GST

( co P

< of GRIK1of (GRIK1 . - the - om om 0.05, in one 0.05, fused SOS3 fused transformation with transformation

SOS2 vitro In in y

root length of seedlings 12d after transfer to to after transfer 12d of seedlings length root activation of activation SOS2 ( . a Asterisks indicate significant differences from differences significant indicate Asterisks ) -

GRIK to

with activation ofSOS2 activation - substrate of SOS2. Proteins were separated SDS in separated Proteinswere SOS2. of substrate - CT bands bands CT

∆ ( f 308 ) 1 -

- independent independent show show

way ANOVA followed by Tukey's multiple comparison test). test). comparison multiple Tukey's by ANOVA followed way interaction interaction GRIK were - KR). KR). SOS2, even though GRIK1 bands were not observed. werenot observed. GRIK1bands though even SOS2, 1 in several images of of images several was

The The incubated with incubated SOS2

mean mean pSPYCE(M

normalized normalized visualised SOS1 C SOS1 form

- in pSPYNE(R)173 and the empty vector and theempty in pSPYNE(R)173 ∆ values 308 byGRIK1 308

of SOS2(SOS2 of - ) and the empty vector pSPYNE(R)173. P pSPYNE(R)173. vector theempty and ) terminal fragment (SOS1 fragment terminal

to the to the signal (± S.E.) are shown. shown. S.E.) are (± by BiFC.

reconstitut GST

nuclei - fused GRIK1 or GST GRIK1or fused s .

with or without 100 mM NaCl. The NaCl. mM 100 without with or in panels (e) and (f) (e) and panels in - ∆ MS agar plates with or without 100 mM NaCl 100 without or with MS plates agar

ed 308 from .

BiFC by GRIK1 by BiFC Arrows , sos2

P arrowhead)

<0.05, <0.05, the SOS2 the

- 2 -

CT indicate the presence of presence the indicate transformed with the wild type wild with the transformed - PAGE followed by Coomassie Coomassie by followed PAGE binomial test binomial - , fused kinase fused

arrows) was added as a as added was arrows) - . Panels . ∆ , - 308WT/GRIK1

SOS2 interaction SOS2 or or pSPYCE(M the combined combined the The r The used ( g anels ). )

-

dead (K137R) (K137R) dead elative band band elative and and

was enough enough was ) . Scale bar: 10 bar: Scale .

experiment experiment ( - ( i KR KR lane h )

) and

show show ( T168A T168A a left: left: ( j )

a to This article isThis article by protected copyright. All rights reserved. st Figure 8 tolerance. restoresalt ableto was SOS1 SOS3 and GRIK1, SOS2, tos3 also was GRIK1 When indicated, lacks ( SOS1. to activate SOS3 with complex formaproductive to isunable that SOS2AAA of concentration with increasing co also SOS3 was in the expressed ( SOS2. ofNaCl indicated amounts the with supplemented plates inAP startingcultures of dilutions decimal byspotting analysed The integration. chromosomal from a SOS1 expressing (SOS2 phosphorylation putative intheactivation mutation T168D thephosphomimic either C the lacking ofSOS2 activeform constitutively (a)The Figure 7 c b atus and atus and andSOS3 ) SOS2 SOS1, )

Accepted Article The full The

mutations with regard to SOS2 activation. Results indicated that only the full complement of complement the full thatonly indicated Results activation. SOS2 to regard with mutations t

- he AAA∆308) were expressed in the yeast yeast strain in the expressed were AAA∆308) . .

three three M A ctivation of SOS2 by GRIK1 byGRIK1 ofSOS2 ctivation - salinity odel odel length SOS2 protein and the triple mutant the and protein SOS2 length SNF1 of of

yeast strain YP890 harbouring a chromosomal integration of SOS1. When indicated, indicated, When ofSOS1. integration chromosomal a YP890 harbouring strain yeast - expressed. The expressed. stress signalling stress the placement of the placement - activating activating were expressed, in various combination as indicated,as combination in expressed, various were sites by GRIK kinases (S156/T168/Y175) GRIKkinases by sites .

Growth inNaCl Growth kinases

co

NaCl as described above described as NaCl salt salt .

- expressed to test for the ability to complement the the to thecomplement ability test for to expressed GRIK tolerance in

SAK1, ELM1 and TOS3 and ELM1 SAK1,

yeast

kinases at the interface between sensing of the energy ofthe energy between sensing the interface at kinases - supplemented media reported the activation of SOS1 by by ofSOS1 the activation reported media supplemented .

of the transformants the transformants of YP890 bearing mutations mutations bearing YP890

S1

- salt sensitivity salt sensitivity terminal autoinhibitory domain and with with and domain autoinhibitory terminal 56A/T168A/Y175A 56A/T168A/Y175A .

Failure to convey salt tolerance indicated indicated tolerance to conveysalt Failure - loop (SOS2 loop

in addition addition in

converted to alanine residues residues alanine to converted was was of all transformants transformants all of - T/D∆308) or with the three the with or T/D∆308) t analysed in AP medium medium inAP analysed o (SOS2 the ena1

in strain in

Na -

AAA) + -

4 nha1 nhx1 4 nha1 pumps 

sak1 elm1 sak1 were were 3K4E, which which 3K4E, wa

ENA s 1

and and - 4 . This article isThis article by protected copyright. All rights reserved. Accepted Article SnRK1 theactivity of reciprocally status Sugar/energy through ofSOS2 the activation induces Salt stress is transmitted is s.

through through SOS2 phosphorylation by GRIK1/2 by phosphorylation SOS2 calcium/CBL (SOS3 and SCaBP8) binding SCaBP8) and (SOS3 calcium/CBL

, which also control control whichalso , . This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article

This article isThis article by protected copyright. All rights reserved. Accepted Article