Genes That Are Uniquely Stress Regulated in Salt Overly Sensitive (sos) Mutants

Genes That Are Uniquely Stress Regulated in Salt OverlySensitive (sos) Mutants1

Zhizhong Gong2, Hisashi Koiwa, Mary Ann Cushman, Anamika Ray, Davi Bufford, Shin Kore-eda,Tracie K. Matsumoto, Jianhua Zhu, John C. Cushman, Ray A. Bressan, and Paul M. Hasegawa*

Center for Plant Environmental Stress Physiology, 1165 Horticulture Building, Purdue University, WestLafayette, Indiana 47907–1165 (Z.G., H.K., T.K.M., J.Z., R.A.B., P.M.H.); Department of Biochemistry MS200,University of Nevada, Reno, Nevada 89557–0014 (M.A.C., S.K., J.C.C.); and Department of Biochemistry andMolecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078 (A.R., D.B.)

Repetitive rounds of differential subtraction screening, followed by nucleotide sequence determination and northern-blotanalysis, identified 84 salt-regulated (160 mm NaCl for 4 h) genes in Arabidopsis wild-type (Col-0 gl1) seedlings. Probescorresponding to these 84 genes and ACP1, RD22BP1, MYB2, STZ, and PAL were included in an analysis of salt responsivegene expression profiles in gl1 and the salt-hypersensitive mutant sos3. Six of 89 genes were expressed differentially inwild-type and sos3 seedlings; steady-state mRNA abundance of five genes (AD06C08/unknown, AD05E05/vegetative storageprotein 2 [VSP2], AD05B11/S-adenosyl-l-Met:salicylic acid carboxyl methyltransferase [SAMT], AD03D05/cold regulated6.6/inducible2 [COR6.6/KIN2], and salt tolerance zinc finger [STZ]) was induced and the abundance of one gene (AD05C10/circadian rhythm-RNA binding1 [CCR1]) was reduced in wild-type plants after salt treatment. The expression of CCR1,SAMT, COR6.6/KIN2, and STZ was higher in sos3 than in wild type, and VSP2 and AD06C08/unknown was lower in themutant. Salt-induced expression of VSP2 in sos1 was similar to wild type, and AD06C08/unknown, CCR1, SAMT, COR6.6/KIN2, and STZ were similar to sos3. VSP2 is regulated presumably by SOS2/3 independent of SOS1, whereas the expressionof the others is SOS1 dependent. AD06C08/unknown and VSP2 are postulated to be effectors of salt tolerance whereas CCR1,SAMT, COR6.6/KIN2, and STZ are determinants that must be negatively regulated during salt adaptation. The pivotalfunction of the SOS signal pathway to mediate ion homeostasis and salt tolerance implicates AD06C08/unknown, VSP2,SAMT, 6.6/KIN2, STZ, and CCR1 as determinates that are involved in salt adaptation.

High soil salinity, which is caused typically byNaCl, results in ion toxicity and hyperosmotic stressleading to numerous pathologies including genera-tion of reactive oxygen species (ROS) and pro-grammed cell death (Niu et al., 1995; Zhu et al., 1997;Hasegawa et al., 2000b). Salt tolerance determinantsare categorized either as effectors that directly mod-ulate stress etiology or attenuate stress effects, or asregulatory molecules that are involved in stress per-ception, signal transduction, or modulation of effec-tor function. Enzymes that catalyze rate-limitingsteps in the biosynthesis of compatible solutes orosmoprotectants, e.g. sugar alcohol, quaternary am-monium, and tertiary sulfonium compounds, are cat-egorical examples of osmotic stress tolerance effec-tors (Hanson et al., 1994; Ishitani et al., 1997; Yoshibaet al., 1997; Nelson et al., 1998; Hasegawa et al.,2000b). Other effectors include proteins that protectmembrane integrity, control water or ion homeosta-sis, and ROS scavenging (Bray, 1994; Ingram and

Bartels, 1996; Hasegawa et al., 2000b). Determinantfunction of some effectors has been confirmed be-cause expression in transgenic plants enhances salttolerance sufficiency (Hasegawa et al., 2000b).

Regulatory determinants include transcription fac-tors and signal pathway intermediates that posttran-scriptionally activate effectors (Hasegawa et al.,2000b). Basic Leu zipper motif, MYB and MYC, andzinc finger transcription factors, including rd22BP1(MYC), AtMYB2 (MYB), DREB1A, and DREB2A (AP2domain), and ALFIN1 (zinc finger), interact with pro-moters of osmotic-regulated genes (Abe et al., 1997;Liu et al., 1998; Hasegawa et al., 2000b). The osmoticstress tolerance function of DREB1A in Arabidopsis(Kasuga et al., 1999) and ALFIN1 in alfalfa (Medicagosativa; Winicov, 2000) has been confirmed by ectopicexpression in transgenic plants. Regulatory interme-diates that modulate plant salt stress responses in-clude SOS3 (Ca21-binding protein), SOS2 (Suc non-fermenting-like) kinase, Ca21-dependent proteinkinases, and mitogen-activated protein kinases(Sheen, 1996; Halfter et al., 2000; Kovtun et al., 2000).Additional signal intermediates have been impli-cated in the plant response to salt (Hwang and Good-man, 1995; Mizoguchi et al., 1996; Mikami et al., 1998;Piao et al., 1999; Hasegawa et al., 2000b).

The Zhu laboratory recently has pioneered identi-fication of salt tolerance determinants using forward

1 This work was supported by a National Science FoundationPlant Genome award (no. DBI–9813360). This is Purdue UniversityAgricultural Experiment Station paper no. 16427.

2 Present address: Department of Plant Sciences, University ofArizona, Tucson, AZ 85721.

* Corresponding author; e-mail [email protected];fax 765–494–0391.

Plant Physiology, May 2001, Vol. 126, pp. 363–375, www.plantphysiol.org © 2001 American Society of Plant Physiologists 363

genetics in the plant model Arabidopsis (Hasegawaet al., 2000a; Sanders, 2000; Zhu, 2000). This effort hasidentified three complementation groups of ion hy-persensitive (salt overly sensitive [sos1-sos3]) mu-tants. Genetic and physiological data indicate thatSOS3, SOS2, and SOS1 are components of a signalpathway that regulates ion homeostasis and salt tol-erance and their functions are Ca21 dependent. Po-sitional cloning revealed that SOS1 encodes a puta-tive plasma membrane Na1/H1 antiporter, SOS2encodes a Suc non-fermenting-like (SNF) kinase, andSOS3 encodes a Ca21-binding protein with sequencesimilarity to the regulatory subunit of calcineurinand neuronal Ca21 sensors (Liu and Zhu, 1998; Liu etal., 2000; Shi et al., 2000). Molecular interaction andcomplementation analyses indicate that SOS3 is re-quired for activation of SOS2 that regulates SOS1transcription (Halfter et al., 2000; Shi et al., 2000),further confirming that the order of the signal path-way is SOS3 ¡ SOS2 ¡ SOS1 (Hasegawa et al.,2000a; Sanders, 2000; Zhu, 2000).

Herein is described a functional dissection of theplant salt stress response by analysis of gene expres-sion controlled by the SOS signal pathway. Differen-tial subtraction and northern analysis identified 84salt-regulated genes, the majority of which have notbeen annotated to be salt responsive in Arabidopsis.Comparison of salt regulation in wild type and sos3identified six genes that are controlled by the SOSsignal pathway. Transcription of one gene (VSP2) iscontrolled as an output from the SOS3/SOS2 path-way, similar to SOS1 (Shi et al., 2000), whereas reg-ulation of five other genes (AD06C08, CCR1, SAMT,COR6.6/KIN2, and STZ) is dependent on both SOS3and SOS2 as well. Because SAMT, COR6.6/KIN2, andSTZ are induced by NaCl, negative control by SOS3indicates that the SOS pathway functions to reducethe numerous signals induced by salt to those thatfunction more specifically, to mediate processes likeion homeostasis.

RESULTS

Identification of Expressed Sequence Tags (ESTs)Corresponding to Salt-Regulated Genes

A combination of approaches was used to identifystress-regulated transcriptional outputs from the SOSpathway on the premise that these are salt tolerance

determinants. A population of ESTs representingsalt-regulated transcripts was identified by screen-ing, through three rounds, a subtracted cDNA libraryprepared from wild-type (Col-0 gl-1) seedlingstreated for 4 h without or with 160 mm NaCl. Differ-ential dot-blot hybridization, and sequence andnorthern-blot analyses identified unique ESTs (salt-regulated ESTs [SREs]) that detected salt-regulatedtranscripts (Table I). The second and third rounds ofscreening eliminated highly repetitive clones fromthe initial subtracted library, including those ESTsidentified during the first round of screening. Thedifferential subtractive chain selection protocol wasused in the third round (Luo et al., 1999) to furtherenrich for less abundant cDNAs in the library. Thedifferent rounds of screening led to identification of84 nonredundant ESTs that hybridize to salt-regulated transcripts based on northern-blot analysis(Table II). The SREs represent the greatest numberthat has been used in one study to define the tran-scriptional response of plants to salt stress. Theseresults identify genes whose expression is most likelycontrolled by transcriptional activation, althoughother factors such as salt stress-dependent mRNAstability might contribute also to steady-state tran-script abundance.

Database comparisons of SREs using Blast pro-grams determined that the corresponding encodedproteins included those involved in primary metab-olism, cell wall synthesis or degradation, other cellu-lar functions, transport or nutrient assimilation, sig-naling, and defensive responses (Table II). The SREswere compared with Arabidopsis genome data usingBlastn. Blastp analysis was performed on any SREORF, without predicted function, that was identifiedin an Arabidopsis database. Blastx/Blastp analysiswas performed on SRE sequences that were unanno-tated as an ORF.

Several of the salt-responsive genes identified inthis evaluation encode components of octadecanoidsignaling through jasmonic acid (Table II, lipid sig-naling responses). The plant hormone appears to bederived from hydrolysis of membrane phospholipids(Koiwa et al., 1997). Triacyl glycerol lipase (AD03B08)can release free linolenic acid from phospholipids thatis then oxidized by lipoxygenase (AD04G12) and cy-clized by allene oxide synthase and allene oxide cy-clase (AD04D07) to 12-oxo-phytodienoic acid. Two

Table I. Arabidopsis (Col-1 gl1) SREs identified after successive rounds of differential subtractionSalt regulation was based on detection of altered steady-state transcript abundance after NaCl

treatment.

Round Dot Blot Sequenced Unique Clones Salt Regulated

First 1,000 137 34 25Second 6,000 157 91 42Third 3,000 320 80 17

Total 10,000 614 205 84

Gong et al.

364 Plant Physiol. Vol. 126, 2001

Tabl

eII

.A

rabi

dops

is(C

ol-0

and

gl1)

ESTs

corr

espo

ndin

gto

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resp

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ium

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ith16

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for

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ted

toB

last

nan

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ustr

ated

are

anno

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form

atio

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nom

ese

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rom

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ctio

non

chro

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omes

2an

d4,

orB

AC

clon

eid

entif

icat

ion

no.(

for

chro

mos

omes

1,3,

and

5),a

ndge

ne/p

rote

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cess

ion

nos.

Hig

hlig

hted

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ldar

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Esth

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tect

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ript

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ntia

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ted

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1an

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s3.

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scri

ptab

unda

nce

isra

ted

base

don

the

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ated

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Ban

kA

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sion

No.

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No.

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som

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Salt and SOS-Regulated Genes

Plant Physiol. Vol. 126, 2001 365

Tabl

eII

.C

ontin

ued

Gen

Ban

kA

cces

sion

No.

EST

No.

Chr

omo-

som

eB

AC

Clo

neN

o.ge

neID

prot

einI

DA

nnot

atio

non

Ara

bido

psis

Gen

e/H

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oga

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eE

Val

ueC

omm

ents

gl-1

Con

trol

gl-1

Salt

bits

BE8

4512

1A

D06

H02

46

AT4

g023

30C

AB

8072

6.1

Stro

ngsi

mila

rity

tosi

mila

rto

pect

ines

-te

rase

,co

ntai

nspe

ctin

este

rase

sign

a-tu

res

AA

407-

414

424

e-11

81

11

1

Ves

icle

/pro

tein

traf

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046

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06A

055

F17I

14F1

7I14

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CA

B89

377.

1Pe

riax

in-l

ike

prot

ein

856

0.0

PDZ

dom

ain

11

11

BE8

4504

8A

D06

A07

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3N19

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19.7

AA

F195

50.1

Sim

ilar

tova

cuol

arpr

oces

sing

enzy

me

385

e-10

6Pr

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se–

1B

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064

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06C

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189

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2740

1.1

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4490

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112

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9e-

173

Tran

spor

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11

BE8

4505

8A

D06

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8933

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Puta

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sim

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amin

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ansp

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prot

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r–

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r23

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ansp

orte

r–

11

1

Gong et al.


Tabl

eII

.C

ontin

ued

Gen

Ban

kA

cces

sion

No.

EST

No.

Chr

omo-

som

eB

AC

Clo

neN

o.ge

neID

prot

einI

DA

nnot

atio

non

Ara

bido

psis

Gen

e/H

omol

oga

Scor

eE

Val

ueC

omm

ents

gl-1

Con

trol

gl-1

Salt

bits

BE8

4534

7A

D10

C07

5M

UG

13A

B02

1934

BA

A74

589.

1N

icot

iana

min

esy

ntha

se54

1e-

152

Iron

upta

ke–

11

1B

E845

350

AD

10C

113

F4P1

2F4

P12_

210

CA

B67

658.

1A

BC

tran

spor

ter-

like

prot

ein

5866

0.0

Tran

spor

ter

–1

Sign

alin

gG

ener

alsi

gnal

com

pone

nts

BE8

4489

4A

D04

B01

210

6A

t2g1

8190

AA

D31

347.

1Pu

tativ

eA

AA

-typ

eA

TPas

e88

40.

01

11

1B

E844

899

AD

04B

071

T10O

24T1

0O24

.2A

AD

3958

2.1

Hyp

othe

tical

prot

ein,

dbjIB

AB

1155

4.1I

(AB

0114

79),

cont

ains

sim

ilari

tyto

bHLH

DN

A-b

indi

ngpr

otei

n:ge

ne_i

d:M

NA

5.5

(Ara

bido

psis

)a

1085

36.7

2.9e

-185

0.16

Tran

scri

p-tio

n–

11

1

BE8

4497

3A

D05

B05

5M

JC20

MJC

20.1

1B

AB

0843

4.1

Con

tain

ssi

mila

rity

toun

know

npr

otei

n(A

F117

897)

,ra

b11-

bind

ing

prot

ein

(Bos

taur

us)a

507

187

e-14

24e

-46

WD

repe

at1

11

1

BE8

4501

2A

D05

F04

3M

MM

17M

MM

17.7

BA

B01

914.

1C

asei

nki

nase

-lik

epr

otei

n35

34e

-96

11

11

11

1B

E845

028

AD

05G

103

T5P1

9T5

P19_

160

CA

B88

054.

1Pu

tativ

epr

otei

n:si

mila

rity

toTA

TA-

bind

ing

prot

ein-

bind

ing

prot

ein,

AB

T1:

Mus

mus

culu

s,EM

BL

AB

0218

60

291

e-77

Tran

scri

p-tio

n1

11

BE8

4505

2A

D06

A11

5M

QN

23M

QN

23.2

3B

AB

1166

4.1

Gpr

otei

n-co

uple

dre

cept

or-l

ike

prot

ein

377

e-10

31

11

11

11

1

BE8

4507

7A

D06

D03

3F2

4M12

F24M

12.1

70C

AB

6263

5.1

Puta

tive

prot

ein:

sim

ilari

tyto

lin-1

0pr

otei

n:R

attu

sno

rweg

icus

,PI

R:

JE02

39

268

2e-7

0R

ecep

tor

targ

etin

g1

11

BE8

4508

2A

D06

D08

5M

AC

9M

AC

9.10

BA

B10

078.

1Tr

ansc

ript

ion

fact

or-l

ike

prot

ein

317

2e-8

5Tr

ansc

rip-

tion

11

11

BE8

4510

6A

D06

F11

1F2

4B9

F24B

9.4

AA

F750

68.1

Con

tain

ssi

mila

rity

toa

prot

ein

kina

segb

lD88

207

139

9e-3

31

11

1

BE8

4523

4A

D08

G06

3F2

6F24

F26F

24.8

AA

F869

97.1

Hyp

othe

tical

prot

ein,

embl

CA

B92

072.

1I(A

L121

575)

,dJ

914N

13.2

.1(c

ofac

tor

requ

ired

for

Sp1

tran

scri

ptio

nal

activ

atio

n,su

b-un

it3;

130k

d;C

RSP

130,

DR

IP13

0,SU

R2,

and

KIA

A12

16;

isof

orm

1)(H

omo

sapi

ens)

274

84.3

2e-7

29e

-15

–1

BE8

4524

3A

D08

H07

277

At2

g137

90A

AD

2831

8.1

Puta

tive

rece

ptor

-lik

epr

otei

nki

nase

184

7e-4

6–

11

BE8

4539

4A

D10

H07

3M

GL6

MG

L6.1

0B

AB

0006

9.1

Tran

slat

iona

llyco

ntro

lled

tum

orpr

o-te

inlik

e55

9e-

157

Ca2

1bi

ndin

g1

11

11

11

1

Lipi

dsi

gnal

ing/

resp

onse

BE8

4484

6A

D03

B08

3T8

P19

T8P1

9.20

0C

AB

6235

8.1

Puta

tive

prot

ein,

splP

2448

4ILI

P2_M

OR

SPLI

PASE

2(T

RIA

CY

LGLY

CER

OL

LIPA

SE)a

1176

58.6

0.0

9e-0

8Li

pase

11

11

BE8

4491

7A

D04

D07

3K

13N

2K

13N

2.11

BA

A95

764.

1U

nkno

wn,

embl

CA

B95

731.

1I(A

J272

026)

alle

neox

ide

cycl

ase

(Ly-

cope

rsic

ones

cule

ntum

)a

472

249

e-13

23e

-65

JAsy

nthe

sis

11

11

1

BE8

4495

0A

D04

G12

3T1

4D7

T14D

3.80

CA

B72

152.

1Li

poxy

gena

seA

tLO

X2

432

e-12

01

11

11

1.

BE8

4500

1A

D05

E05

5?

AB

0067

78B

AA

3344

7.1

Vsp

2ge

nefo

rve

geta

tive

stor

age

prot

ein

509

e-14

3–

11

1



Tabl

eII

.C

ontin

ued

Gen

Ban

kA

cces

sion

No.

EST

No.

Chr

omo-

som

eB

AC

Clo

neN

o.ge

neID

prot

einI

DA

nnot

atio

non

Ara

bido

psis

Gen

e/H

omol

oga

Scor

eE

Val

ueC

omm

ents

gl-1

Con

trol

gl-1

Salt

bits

BE8

4509

1A

D06

E08

5M

TI20

MTI

20.3

BA

B08

850.

1Li

pid

tran

sfer

prot

ein;

glos

sy1

hom

olog

414

e-11

4–

11

1B

E845

100

AD

06F0

53

T02O

04T0

2O04

.11

AA

B63

638.

1Ja

smon

ate-

indu

cibl

epr

otei

nis

olog

,m

yros

inas

ebi

ndin

gpr

otei

nlik

e46

6e-

130

11

11

11

Hor

mon

ere

late

dB

E844

978

AD

05B

113

F28D

10F2

8D10

_50

CA

C03

536.

1A

tPP-

like

prot

ein:

prok

aryo

ticm

em-

bran

elip

opro

tein

lipid

atta

chm

ent

site

AA

199-

209

(AF1

3305

3),

S-ad

-en

osyl

-L-m

ethi

onin

e:sa

licyl

icac

idca

rbox

ylm

ethy

ltran

sfer

ase

(Cla

rkia

brew

eri)a

706

129

0.0

6e-2

9SA

–1

1

BE8

4540

1A

D05

C05

??

AF1

8382

7A

AF2

2295

.1B

eta-

gluc

osid

ase

hom

olog

(BG

1)35

150.

0–

11

11

BE8

4508

4A

D06

D10

3T1

0D17

T10D

17_9

0C

AB

8899

8.1

Nitr

ilase

220

2e-

501

11

1C

ell

deat

hB

E844

851

AD

03C

065

MQ

L5M

QL5

.19

BA

A97

167.

1Pa

lmito

yl-p

rote

inth

ioes

tera

sepr

ecur

-so

rlik

e42

2e-

117

Ant

i-ap

opto

sis?

11

1

BE8

4495

8A

D04

H10

3F2

8D10

F28D

10_7

0C

AC

0353

8.1

Leth

alle

af-s

pot

1ho

mol

ogLl

s1:

con-

tain

spr

okar

yotic

mem

bran

elip

opro

-te

inlip

idat

tach

men

tsi

teA

A15

0-16

0

472

e-13

2D

ioxy

gena

se1

11

Def

ense

resp

onse

Def

ense

prot

eins

BE8

4487

5A

D03

G09

1F2

2K20

F22K

20.1

9A

AC

0062

5.1

Alc

ohol

dehy

drog

enas

e48

6e-

136

11

11

BE8

4489

6A

D04

B03

5M

SG15

MSG

15.3

BA

B11

043.

1M

ande

loni

trile

lyas

e-lik

epr

otei

n51

9e-

146

Cya

noge

ne-

sis

–1

1

BE8

4492

6A

D04

E06

3M

DB

19M

DB

19.5

BA

B02

775.

1C

onta

ins

sim

ilari

tyto

endo

-1,3

-1,4

-be

ta- D

-glu

cana

sege

ne_i

d:M

DB

19.5

414

e-11

51

11

1

BE8

4497

2A

D05

B03

1F5

F19

F5F1

9.6

AA

D12

691.

1Si

mila

rto

gblY

0943

7m

yros

inas

ebi

nd-

ing

prot

ein

from

B.

napu

s19

48e

-49

–1

BE8

4499

0A

D05

D04

1F2

4B9

F24B

9.34

AA

F750

98.1

Met

allo

thio

nein

605

e-17

21

11

11

11

11

BE8

4540

3A

D06

E03

1F1

5K9

F15K

9.17

AA

C72

119.

1St

rong

sim

ilari

tyto

gblD

1455

0ex

tra-

cellu

lar

derm

algl

ycop

rote

in(E

DG

P)pr

ecur

sor

from

Dau

cus

caro

ta

745

0.0

11

1

BE8

4511

7A

D06

G10

1F1

4N23

F14N

23.2

5A

AD

3288

7.1

Sim

ilar

togl

utat

hion

eS-

tran

sfer

ase

TSI-

1(g

il219

0992

)76

30.

0C

yano

gene

-si

s1

11

11

1

BE8

4515

2A

D07

F07

223

6A

t2g4

3620

AA

B64

044.

1Pu

tativ

een

doch

itina

se54

9e-

154

11

11

1B

E845

153

AD

07F0

85

MO

J9M

OJ9

.4B

AB

1114

5.1

Poly

gala

ctur

onas

e-in

hibi

ting

prot

ein

509

e-14

2–

11

11

Cor

/RD

prot

eins

BE8

4485

0A

D03

C02

473

AT4

g306

50C

AB

7978

3.1

Stro

ngsi

mila

rity

tolo

wte

mpe

ratu

rean

dsa

lt-re

spon

sive

prot

ein

LTI6

A,

Ara

bido

psis

383

e-10

51

11

1

BE8

4485

4A

D03

D05

5F1

N13

F1N

13_1

10C

AC

0179

6.1

Col

d-re

gula

ted

prot

ein

CO

R6.

6(K

IN2)

341

2e-9

2–

11

11

1.

BE8

4485

5A

D03

D11

2F1

4N22

At2

g425

40A

AD

2299

9.1

Col

d-re

gula

ted

prot

ein

cor1

5apr

ecur

sor

519

e-14

–1

11

11

.

BE8

4486

5A

D03

F02

5K

24M

7D

1304

4B

AA

0237

6.1

Des

icca

tion-

resp

onsi

verd

29A

371

e-10

1–

11

11

1.

BE8

4502

2A

D05

G04

5T1

4C9

ATH

RD

22B

AA

0154

6.1

rd22

Gen

e44

8e-

124

–1

11

11

BE8

4504

9A

D06

A08

1F1

5M15

F5M

15.2

1A

AF7

9613

.1Si

mila

rto

cold

-reg

ulat

edpr

otei

nco

r47

127

4e-2

8?

?

Gong et al.


Tabl

eII

.C

ontin

ued

Gen

Ban

kA

cces

sion

No.

EST

No.

Chr

omo-

som

eB

AC

Clo

neN

o.ge

neID

prot

einI

DA

nnot

atio

non

Ara

bido

psis

Gen

e/H

omol

oga

Scor

eE

Val

ueC

omm

ents

gl-1

Con

trol

gl-1

Salt

bits

Ant

hocy

anin

synt

hesi

sB

E844

845

AD

03B

065

MO

P10

MO

P10.

14B

AB

1154

9.1

Leuc

oant

hocy

anid

indi

oxyg

enas

e-lik

epr

otei

n66

40.

0–

11

11

1.

BE8

4489

2A

D04

A11

220

6A

t2g3

8240

AA

C27

173.

1Pu

tativ

ean

thoc

yani

din

synt

hase

203

4e-8

7–

11

11

BE8

4510

1A

D06

F06

4T2

9A15

AT4

g275

60C

AB

3826

8.1

Stro

ngsi

mila

rity

toU

DP

rham

nose

-an

thoc

yani

din-

3-gl

ucos

ide

rham

no-

syltr

ansf

eras

e,Pe

tuni

ahy

brid

a

837

0.0

11

1

Ant

ioxi

datio

nB

E844

884

AD

04A

025

F5O

24A

B03

5137

BA

A86

999.

1B

lue

copp

er-b

indi

ngpr

otei

n49

4e-

138

Ant

i-ox

idat

ion

11

11

1

Unk

now

nB

E844

860

AD

03E0

83

T19D

11–

–N

otan

nota

ted,

dbjlB

AB

0281

9.1I

(AB

0240

36),

dbjlB

AA

8793

6.1:

gene

_id:

MQ

C12

.15,

sim

ilar

toun

know

n

135

46.1

4e-3

15e

-05

–1

11

BE8

4499

2A

D05

D06

5K

3M16

K3M

16_3

0C

AC

0189

0.1

Hyp

othe

tical

prot

ein

777

0.0

–1

11

1B

E845

016

AD

05F0

93

F14O

13F1

4O13

.28

BA

B03

026.

1Si

mila

rto

unkn

own

prot

ein

476

e-13

31

11

BE8

4506

9A

D06

C07

1F2

2O13

F22O

13.2

9A

AF9

9773

.1U

nkno

wn

prot

ein

690

0.0

11

1B

E845

070

AD

06C

083

F16J

14–

–N

otan

nota

ted

910

0.0

11

1em

blC

AB

8642

2.1I

(AL1

3864

8)pu

ta-

tive

prot

ein

(Ara

bido

psis

)a78

.84e

-14

BE8

4509

7A

D06

F02

??

––

Not

anno

tate

d1

11

11

BE8

4535

5A

D10

D05

1F2

4J8

––

Not

anno

tate

d77

50.

0Tr

ansp

orte

r–

11

11

gblA

AF2

8474

.1IA

F173

553_

1(A

F173

553)

V-A

TPas

e11

0-kD

inte

-gr

alm

embr

ane

subu

nit

(Man

duca

sext

a)a

29.7

16

BE8

4537

6A

D10

F07

1T2

7G7

––

Not

anno

tate

d57

9e-

164

11

11

splQ

0818

0IIC

CR

_DR

OM

EIR

REG

ULA

RC

HIA

SMC

-RO

UG

HES

TPR

OTE

INPR

ECU

RSO

R(IR

REC

PRO

TEIN

)a

32.1

3.7

aB

last

pan

alys

isw

aspe

rfor

med

onan

yA

rabi

dops

isop

enre

adin

gfr

ame

(OR

F)th

atco

rres

pond

edto

anSR

Eth

atis

not

func

tiona

llyan

nota

ted.

Any

SRE

sequ

ence

that

did

not

mat

chan

Ara

bido

psis

OR

Fw

assu

bjec

ted

toB

last

x/B

last

pan

alys

is.



SREs were annotated previously as jasmonic acid reg-ulated, VSP2 (AD05E05) and AD06F05. Furthermore,the genes of a number of SREs are involved in plantdefense and may be regulated by the octadecanoidsignal pathway. Some of these genes have been shownto express upon dehydration in tomato (Lycopersiconesculentum; Reymond et al., 2000). Several abscisic acid(ABA)-responsive SREs are included in Table II andthe plant hormone is a potentiator of octadecanoidsignaling.

Genes Differentially Regulated by Salt in WildType and sos3

The salt regulated expression profile of SRE tran-scripts, as well as that of previously characterizedstress-regulated genes (ACP1, RD22BP1, MYB2, STZ,and PAL), revealed that most are controlled similarlyin wild type and sos3. Six of the salt-responsivegenes, including STZ (Lippuner et al., 1996), weredifferentially regulated in wild type and sos3 (Fig. 1).Transcript abundance of two was lower (AD06C08/unknown and AD05E05/vegetative storage protein2[VSP2]) and of four was higher (encoding AD05C10/cold-circadian rhythm-RNA binding1 [CCR1], STZ/salt tolerance zinc finger [not shown], AD05B11/S-adenosyl-l-Met: salicyclic acid carboxyl methyl-transferase [SAMT], AD03D05/cold regulated/coldinducible [COR6.6/KIN2]) in the salt-sensitive mutant.SAMT and COR6.6/KIN2 transcript abundance wasslightly elevated in sos3 but the steady-state mRNAlevels were hyper-induced by salt treatment. CCR1was the only gene for which transcript abundance isreduced by salt treatment.

Salt regulation of VSP2 is similar in wild type andsos1 indicating that transcriptional activation is not

dependent on SOS1 (Fig. 2). Methyl jasmonate(MeJA) induces VSP2 transcript abundance in wildtype and sos3 (not shown). The SOS3/2 pathway andthe hormone seem to regulate VSP2 independently.Signal pathways often converge to regulate transcrip-tion of key effectors involved in cellular adaptation toenvironmental perturbation (Rep et al., 2000). VSP2 isa member of a two-gene family (87% nucleotide se-quence identity over the coding region) that encodesa protein with similarity to soybean VSPs (Berger etal., 1995; Utsugi et al., 1998), which are vacuolar-localized glycoproteins with acid phosphatase activ-ity (Mason and Mullet, 1990). These proteins arepresumed to be amino acid sinks during water deficitbut are important reduced nitrogen sources afterstress relief (Mason and Mullet, 1990).

CCR1, STZ, SAMT, COR6.6/KIN2, and AD06C08/unknown have similar expression profiles in sos1,sos2, and sos3, implicating these as transcriptionaloutputs requiring all components of the SOS path-way. CCR1 encodes a Gly-rich RNA-binding proteinimplicated in posttranscriptional regulation. CCR1and CCR2 comprise a two-gene family, and theirexpression is regulated by a diurnal circadian clock(Carpenter et al., 1994; Heintzen et al., 1997; Krepsand Simon, 1997). CCR1 or CCR2 expression nega-tively regulates either gene and this feedback looppresumably facilitates diurnal oscillation controlledby the master circadian clock (Heintzen et al., 1997).CCR1 and CCR2 steady-state mRNA levels are in-duced by cold but CCR1 transcript is negatively reg-ulated by ABA and dehydration, whereas CCR2 ex-pression is induced by dehydration (Carpenter et al.,1994). CCR1 transcript abundance was down-regulated in sos3 indicating that the SOS pathway isat least another negative regulator that controls CCR1expression downstream of the circadian rhythmclock (Heintzen et al., 1997; Kreps and Simon, 1997).These results indicate that control of circadian oscil-lations may be required during the salt stressresponse.

COR6.6//KIN2 and SAMT are both implicated inplant stress responses. COR6.6/KIN2 is linked in tan-dem to its homolog KIN1 (95% nucleotide sequenceidentity in the coding region) and both encode hy-drophilic peptides that are boiling soluble but ofunknown function (Thomashow, 1999). KIN1 and 2transcript abundance is cold and ABA induced (Kur-kela and Franck, 1990; Kurkela and Borg-Franck,1992; Thomashow, 1999) but KIN2 and not KIN1 ex-pression is modulated positively by drought and salt(Kurkela and Borg-Franck, 1992). SAMT catalyzes theformation of methylsalicylate from salicylic acid us-ing S-adenosyl-l-Met as the methyl donor (Ross etal., 1999). The volatile ester is implicated as a polli-nator attractant and a signal in plant defense medi-ated by salicylic acid (Ross et al., 1999; Dudareva etal., 2000).

Figure 1. Salt-responsive gene expression that is dependent on theSOS pathway in Arabidopsis. Genes that are differentially regulatedin wild-type (Col-0 gl1) and sos3. Total RNA (40 mg) from seedlingsgrown in liquid culture (14 d) and treated without or with 160 mM

NaCl for 4 h. The northern blot was hybridized with 32P-labeledprobe corresponding to: COR6.6/KIN2, SAMT, CCR1, VSP2, andAD06C08 (unknown). AtGAPDH (glyceraldehyde-3-phosphate-dehydrogenase) is the control.

Gong et al.


DISCUSSION

Many salt-regulated genes are responsive also toother biotic or abiotic perturbations, indicating thatthese stresses have common etiologies, e.g. waterdeficit, and cold are both osmotic stresses, occursimultaneous in the environment or elicit similar pa-thologies (Bohnert et al., 1995; Shinozaki andYamaguchi-Shinozaki, 1997; Zhu et al., 1997; Hase-gawa et al., 2000b). Furthermore, osmotic and ionicstresses induce secondary cellular perturbations thatarise from ROS, elicitors from degradation of cellwall and plasma membrane macromolecules, orwounding, which initiate signal transduction path-ways that modulate other plant defensive processes(Hasegawa et al., 2000b). In fact, many of the proteinsencoded by the 84 salt-responsive genes identified inthis study can be categorized as functional outputsfrom these different signal cascades (Table II). Mostof these genes were not known previously to bemodulated as a part of the Arabidopsis salt response.It is interesting that even after subtractive hybridiza-tion only approximately 13% of the ESTs (84/614)were unique and detected salt-regulated transcripts.

Six of 89 genes examined were differentially re-sponsive to NaCl in wild type and sos3, implicatingthe SOS pathway in their transcriptional regulation.AD06C08/unknown and VSP2 are induced andCCR1, STZ, SAMT, and COR6.6/KIN2 are controllednegatively by SOS3. CCR1 expression was lower inwild type after NaCl treatment, whereas the messageabundance of the others was salt induced. From theseresults, a model for the SOS pathway regulation ofthese genes is illustrated in Figure 3 (Zhu, 2000). Saltregulated expression of VSP2 is the same in wild typeand sos1 defining this gene as a transcriptional out-put from the SOS pathway that does not require

SOS1. This supports the premise that SOS3 and SOS2are signal intermediates and SOS1 is an effector ofNa1 homeostasis (Shi et al., 2000). However, saltregulation of AD06C08/unknown, CCR1, STZ, SAMT,and COR6.6/KIN2 is dependent on SOS2 and SOS1,perhaps implicating a signaling function for SOS1.Genes encoding an enzyme catalyzing the penulti-mate step in Pro biosynthesis (P5CS) and a putativetranscription factor (AtMYB) are hyper-induced by

Figure 2. Comparative expression of genes dependent on the SOS pathway in wild type (Col-0 and gll) and sos1, sos2, andsos3. Illustrated is the northern blot of steady-state mRNA levels of COR6.6/KIN2, SAMT, CCR1, and VSP2 in plants without(0 h) or 160 mM NaCl (24 h). Ethidium bromide staining was used to monitor RNA loading.

Figure 3. Illustrated is a model depicting the SOS pathway regulationof salt responsive genes. Hypersaline conditions activate the SOS(SOS3 ¡ SOS2 ¡ SOS1) signal pathway (Zhu, 2000) and transcriptabundance of AD0608 (unknown), VSP2 (vegetative storage protein2, AD05E05), SAMT (S-adenosyl-L-Met:salicylic acid carboxyl meth-yltransferase, AD05B11), COR6.6/KIN2 (cold regulated 6.6/induc-ible 2, AD03D05 [COR6.6/KIN2], and STZ [salt tolerance zincfinger], Lippuner et al., 1996) increase and of CCR1 (circadianrhythm-RNA binding1, AD05C10) decreases in Arabidopsis seed-lings. Positive (2) or negative (') regulation by the SOS pathway isindicated.



salt in sos1 compared with wild type (Liu and Zhu,1997). Furthermore, some transport proteins includesensor domains or function in association with sen-sors (Ozcan et al., 1998; Heinisch et al., 1999; Sabirovet al., 1999) as could the putative Na1/H1 antiporterSOS1 (Shi et al., 2000).

The experimental evidence presented here indi-cates that the SOS pathway controls expression ofonly a few salt stress-specific tolerance determinantgenes among the numerous genes (six of 89 in thisstudy) that are regulated in the plant response toNaCl treatment (Zhu et al., 1997). This is similar tothe paradigm that has been established recently forthe salt stress response of the unicellular eukaryoteyeast (Saccharomyces cerevisiae). Genome-wide arrayanalysis determined that osmotic upshock causes arapid and multi-fold increase in mRNA of between186 and 1,359 genes and reduced transcript abun-dance of more than 100 genes depending on theseverity of osmotic shock, the osmotic agent (NaCl orsorbitol), and time after treatment (Posas et al., 2000;Rep et al., 2000; J. Yale and H.J. Bohnert, unpublisheddata). Salt-induced expression of most is either par-tially or completely controlled by the high osmolarityglycerol and mitogen-activated protein kinase path-way. The yeast calcineurin pathway is analogous tothe Arabidopsis SOS pathway, controls ion ho-meostasis and is essential for salt tolerance in yeast(Mendoza et al., 1994, 1996), and affects expression ofmany fewer osmotic responsive genes (T.K. Matsu-moto, unpublished data). Like the SOS pathway, cal-cineurin regulates expression of genes that encodesalt tolerance effectors such as Na1 efflux transport-ers (Mendoza et al., 1994; Shi et al., 2000; Matusmotoet al., unpublished data).

It is conceivable that signal transduction throughthe SOS pathway that mediates salt tolerance mayhave a substantial component that involves posttran-scriptional activation of salt tolerance effectors, par-ticularly over the time span (minimum of 4 h) of thesalt treatment used in experiments reported here.Plant survival in severe stress likely requires veryimmediate cellular responses, whereas transcrip-tional regulation may be sufficient for stress recoveryand adaptation. Notwithstanding, salt induces tran-scriptional activation of genes in yeast within min-utes (Posas et al., 2000; Rep et al., 2000). Genes thatare transiently induced or weakly expressed furthercomplicate inference of function from expressionprofile analysis. The majority of yeast genes inducedby mild salt shock exhibit transient expression (Posaset al., 2000). Determinant gene transcript abundancedifferences in wild type and sos3 may be insufficientfor the resolution limits of the subtraction protocols,yet are biologically meaningful to salt stressadaptation.

It is interesting that the SOS pathway negativelycontrols the expression of four salt-regulated genes(SAMT, C0R 6.6/KIN2, STZ, and CCR1), three (SAMT,

C0R6.6/KIN2, and STZ) of which are induced byNaCl treatment. So, salt tolerance determinants in-clude genes that must be repressed, at least tempo-rally, during the plant stress response. Negativelyregulated genes may include those that contribute togrowth arrest necessary during the period of adjust-ment or ameliorate other etiologies that occur coin-cidentally with salt in the native environment of theorganism. CCR1 and COR6.6/KIN2 are cold induced,whereas SAMT is implicated in plant defense againstpathogens indicating their principal function is not insalt adaptation. Together, this suggests that a func-tion of the SOS pathway is to discriminate against themyriad of stress signals that are elicited by salt and tofocus the capacity of the plant to cope with the prin-cipal etiology; in this instance, ion dis-equilibrium.Furthermore, the SOS pathway may coordinate tem-poral gene expression to focus the availability ofeffectors, as required, during stress perception, ame-lioration, or adaptation. Confirmation that the genesidentified in this study encode tolerance determi-nants awaits molecular genetic confirmation by loss-or gain-of-function experimentation.

MATERIALS AND METHODS

Plant Material

Arabidopsis (ecotype Columbia-0 gl1) and sos1, sos2, andsos3 were wild-type and salt-hypersensitive genotypes, re-spectively (provided by Dr. Jian-Kang Zhu, University ofArizona). Seeds were surface disinfected and stratified for2 d at 4°C. Seeds were germinated in liquid medium (Mu-rashige and Skoog salts [Murashige and Skoog, 1962] and3% [w/v] Suc [pH 5.8]) in 250-mL flasks on a gyratoryshaker (80–100 rpm) under low-intensity Cool White fluo-rescent illumination (light/dark:16/8 h daily) at 22°C to24°C. After 14 d, seedlings were transferred to fresh me-dium without or with 160 mm NaCl for the time intervalindicated. Seedlings were harvested, frozen in liquid nitro-gen, and stored at 280°C.

Construction of Subtraction Libraries

Total RNA was isolated as described by Gong et al.(1997). mRNA was isolated using the Poly(A)1 RNA puri-fication kit (CLONTECH, Palo Alto, CA). The PCR-SelectcDNA subtraction kit (K1804-1, CLONTECH) was used toobtain subtracted cDNA libraries.

Subtraction of cDNAs Obtained from mRNA of Salt-Treated sos3 and gl1

Subtractive hybridization was used to identify cDNAscorresponding to salt regulated genes differentially ex-pressed in sos3. Both forward and reverse subtractive hy-bridizations were performed with salt-treated (160 mmNaCl, 4 h) seedlings. The forward subtraction used testercDNA obtained from mRNA of gl1 and driver cDNA fromsos3. In the reverse subtraction, the tester cDNA was ob-

Gong et al.


tained from sos3 and driver cDNA from gl1. Driver cDNAsare the reference and are targets for elimination duringsubtraction leaving unique tester cDNAs. Forward andreverse subtractive hybridization was meant to identifysalt-responsive genes that are specifically regulated in sos3.

Subtraction of cDNAs Obtained from gl1 afterTreatment without or with NaCl

This subtraction was intended for identification of genesdifferentially regulated by salt in gl1. The forward subtrac-tion tester cDNA was obtained from mRNA of gl1 seed-lings 4 h after transfer to medium with 160 mm NaCl anddriver cDNA from gl1 plants grown in medium withoutsalt. In the reverse subtraction, tester and driver cDNA wasobtained from gl1 grown without and with 160 mm NaCl,respectively.

The subtracted libraries were subjected to two rounds ofPCR amplification, the second using nested primers foradaptors 1 and 2R (CLONTECH). The PCR products wereligated into pT-Adv (CLONTECH), and transformed intoEscherichia coli. The white colonies were isolated and insertsamplified by PCR.

Enrichment of Unique Salt-Regulated cDNAs in theSubtraction Library

A modification of the differential subtraction chainmethod (Luo et al., 1999) was used to enrich the subtractedlibrary for unique salt-regulated cDNAs. The driver for thissubtraction (driver 2) is a mixture of second PCR productfrom reverse subtraction (100 mL; tester DNA:gl1 withoutsalt, driver DNA:gl1 with salt) and PCR products amplifiedfrom highly repetitive salt-induced cDNAs isolated fromprevious rounds of screening (0.5 mL of each). To removeadaptor sequences from driver 2, the mixture was digestedat the restriction sites of adaptors 1 (SmaI and RsaI) and 2R(EagI and RsaI) by consecutive restriction digestion usingSmaI, and then EagI 1 RsaI. The digested DNA was ex-tracted with phenol and phenol/chloroform, and then pre-cipitated with ethanol. The precipitate was redissolved in100 mL of water and passed through the Microcon YM-30column (Amicon, Beverly, MA) to separate the DNA fromAdaptor fragments. Driver 2 DNA was recovered in 20 mLof water and used as driver in the following PCR subtrac-tion. About 10 times excess amount of driver 2 DNA (2 mL)was mixed with 1mL of forward subtraction library (driverand tester 5 gl1 grown without and with 160 mm NaCl,respectively) in hybridization buffer {5 mm HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-HCl (pH8.3), 12 mm NaCl, and 0.05 mm EDTA} in a total volume of12 mL. The hybridization solution was denatured at 94°Cfor 5 min, and incubated at 72°C for 12 h.

After hybridization, the mixture was precipitated withethanol, and digested with mung bean nuclease (Promega,Madison, WI) to remove adaptor sequence from productsof the driver-tester hybridization. Adaptor sequences intester-tester hybridization products are aligned withmatching ends and these are not digested by mung bean

nuclease. The mung bean nuclease digested mixture waspassed through the Microcon column and purified DNAwas rehybridized at 72°C. The mung bean nuclease diges-tion and DNA purification procedures were repeated. TheDNA was amplified by PCR using nested primers for 1 and2R, 11 cycles. The cloning of PCR products was as de-scribed as above.

Dot-Blot Analysis

For dot-blot analysis, cDNA inserts of forward subtrac-tion library clones were individually amplified by PCR and2 mL of PCR product was mixed with 2 mL of 0.6 m NaOH.Two microliters of the mixture was blotted onto each oftwo duplicate nylon membrane filters. Probes for dot-blotanalysis were either forward or reverse subtraction prod-ucts or cDNA of RNA from plants, as indicated. The prod-ucts of forward and reverse subtraction were digested withSmaI, RsaI, and EagI to remove the adapter sequences andlabeled with 32P using the Ready-To-Go kit (AmershamPharmacia Biotech, Piscataway, NJ). The labeled forwardprobes were hybridized to one membrane and the reverseprobes to the duplicate.

Template Preparation, DNA Sequencing, andData Analysis

Plasmid templates were prepared from selected bacterialcolonies by 96-well alkaline lysis minipreps according tothe manufacturer’s instructions (Edge BioSystems, Inc.,Gaithersburg, MD).

DNA sequencing reactions were conducted usingDyeDeoxy “Terminator PRISM” mix (Perkin-Elmer-ABI,Foster City, CA) according to the manufacturer’s instruc-tions in a multiplate thin-wall 96-well microplate on an MJResearch PTC-100-96 (MJ Research, Watertown, MA) pro-grammable thermal controller using the following profile:96°C for 30 s, 45°C for 15 s, and 60°C for 4 min for 49 cycles.Unincorporated dye terminators were removed by passingreactions over a 96-well gel filtration block (Edge BioSys-tems). Recovered sequencing reaction products were ana-lyzed on either an ABI 373A-XL Stretch or an ABI 3700capillary array automated DNA sequencing system(Perkin-Elmer Applied BioSystems). Raw sequence datawas analyzed using PHRED (Ewing and Green, 1998; Ew-ing et al., 1998) and Cross match to removal vector se-quences. Additional vector sequence removal and editingwas done manually using FACTURA software (Perkin-Elmer Applied BioSystems). Polished EST sequence fileswere assembled into singleton and contig files usingPHRAP (P. Green, unpublished data). EST identities weredetermined by sequence comparison to the nonredundantGenBank database using BLASTN (BLAST 2.0) using de-fault parameters (Altschul et al., 1997). In instances wherean unannotated match was obtained, BLASTX searcherswere conducted and sequence homology information wasused to assign putative identities. All EST sequencesreported here have been deposited in dbEST and can bebrowsed and retrieved from the NCBI website(http://www.ncbi.nlm.nih.gov) under accession numbersBE844684 through BE845405.



Northern-Blot Analysis of Putative Clones

Total RNA was isolated from seedlings and 40 mg fromeach sample was separated on 1.2% (w/v) agarose formal-dehyde gels and transferred to Hybond-N nylon mem-branes (Amersham) as previously described (Gong et al.,1997). The cDNA insert of each clone was amplified by PCRusing nested primers that hybridize to adaptors 1 and 2R,and purified from agarose gels using the Qiagen Gel Puri-fication kit. The probes for RD22BP1, MYB2, PAL, STZ, andACP1 were obtained by PCR amplification using cDNAobtained from mRNA of salt-treated gl1 as template;RD22BP1 (AB000875), forward primer: 59-ATGACGCTGT-TGATGAGGAG-39 and reverse primer: 59-TTTCGGATT-CTGGGTCTGAG-39 (0.56 kb); MYB2 (D14712), forwardprimer: 59-GAAATGGAAGATTACGAGCG-39 and reverseprimer: 59-TTAATTATACGAATACGATGTC-39 (1.0 kb);PAL (L33677), forward primer: 59-ATGGAGATT-AACG-GGGCACAC-39 and reverse primer: 59-ACGT-TCACCG-TTGGGACCAG-39 (1.1 kb); STZ (X95573), ORF; and ACP1(AF009228), forward primer: 59-CAA-AAGCCATTTTT-CAAATTTCAAACTCAG and reverse primer 59-GTTTT-CAATGATAGTGAAGAAAGATG-TAC-AAC (0.83 kb).The purified PCR products were labeled using 32P dCTPusing the Ready-To-Go kit. Blot-blot hybridization andwashes were as described (Gong et al., 1997). The blotswere stripped by boiling in 0.5% (w/v) SDS solution for 3min and were rehybridized with another probe.

ACKNOWLEDGMENTS

The authors would like to thank Sue Ann Hudiburg andJanet Rogers of the Oklahoma State University Recombi-nant DNA/Protein Resource Facility for providing oligo-nucleotide synthesis and automated DNA sequencing anal-ysis services.

Received September 29, 2000; returned for revision Decem-ber 15, 2000; accepted February 9, 2001.

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Genes That Are Uniquely Stress Regulated in Salt Overly Sensitive (sos) Mutants

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Transcript of Genes That Are Uniquely Stress Regulated in Salt Overly Sensitive (sos) Mutants